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General occurrence of exophylactic and necrophylactic periderms and non-suberized impervious tissues… Soo, Benjamin Vui Ling 1977

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GENERAL OCCURRENCE OF EXOPHYLACTIC AND NECROPHYLACTIC PERIDERMS.AND NON-SUBERI ZED IMPERVIOUS TISSUES IN WOODY PLANTS by BENJAMIN VUI LING SOO B. Sc. (Hons.) Agr., National Taiwan University, 1967 M. Sc. Forestry, University of Toronto, 1969 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES i n the Faculty of Forestry We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA September, 1977 (c^ Benjamin Vui Ling Soo, 1977 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Dean of the Faculty of Forestry or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Faculty of Forestry The University of British Columbia Vancouver, B.C. V6T 1W5 Canada Date: September 27, 1977 ABSTRACT The intent of this thesis is to determine whether the new concepts of necrophylactic and exophylactic periderms proposed by Mullick from studies of four species of conifers are generally valid in woody plants, and whether the non-suberized impervious tissue (NIT) recently discovered by him is a pre-requisite to the formation of necrophy1 actic periderms. Studies of 10 species of conifers with repre-sentatives from a l l coniferous families indigenous to North America and 5 species of deciduous trees by cryofixation technique of Mullic showed the general validity of the necrophylactic and exophylactic periderm concept, the general occurrence of NIT and the invariable presence of NIT prior to necrophylactic periderm formation, establishing NIT as a pre-requisite to necrophylactic periderm format ion. Characterization of periderms in the 15 species showed that, within a species, they could be classified into two categories and that when so categorized, a l l periderms within a species f u l f i l l e d the c r i t e r i a for either the exophylactic or necrophylactic category, thus establishing the general validity of the concept. Non-suberized impervious tissue (NIT) was found to be present in each species studied. Developmental studies of NIT in P i nus contorta and Lar ix occidental is, and characterization of NIT in a l l species, revealed common features in NIT development and features at a l l sites of its formation in a l l species studied. Necrophylactic periderm was invariably associated with NIT and was only found after NIT formation. Investigation of the chemical nature of NIT indicated that gymnosperm lignin, or closely related compounds are most likely responsible for the imperviousness. Implications of the role of NIT and necrophylactic periderm formation in pathogenesis as proposed by Mulliek are discussed in relation to the now widely established occurrence of them. - i v -TABLE OF CONTENTS D Page ABSTRACT i i TABLE OF CONTENTS iv LIST OF TABLES vi i i LIST OF FIGURES ix ACKNOWLEDGEMENTS xxvi 1. INTRODUCTION 1 2. LITERATURE REVIEW . . . ' 3 2.1 Theory of Periderm Formation 3 2.2 Classical and Modern Concepts of Periderms. . k 2.3 Recent Concepts of Wound Periderm Formation in Conifers..7 2.4 Process of Phellogen Restoration at Rhytidome 9 2.5 An Overview of Recent Concepts and Terminology / of Periderms 12 2.6 General Developmental Features of NIT Formation . . . . 13 2.7 The Role of Non-suberized Impervious Tissue (NIT) and Necrophylactic Periderm (NP) in Pathogenesis. . . . 14 2.8 Cryofixation -- a New Technique for Studying Developmental Processes in Plants 18 3. MATERIALS AND METHODS 20 3.1 Exophylactic and Necrophylactic Periderms 20 3.1.1 Collection of Samples of FEP, SEP and NP Abutting Rhytidome 20 3.1.2 Samples of Necrophylactic Periderms at Healed Sites of Mechanical Injuries, Abscission Zones, Resin Blisters and Pathogenesis 22 - v -3-1 -3 Cryof ixation Technique 23 3-1-3-1 Photomicrography Techniques and Their Limitations . . . . . . . . . . . 24 3-1.4 Histochemica 1 Tests . 26 3.2 Non-suberized Impervious Tissue (NIT) 27 3.2.1 Detection of NIT by F-F Test . . . . . 28 3.2.1.1 F-F Test Through Wound Surface 28 3.2.1.2 F-F Test Through Cambial Surface . . . .29 3.2.1.3 Detection of NIT by Cryofixation Characteristics 29 3.2.2 Histochemical Tests 30 4. RESULTS .32 4.1 Investigation of Exophylactic and Necrophylactic Periderms in Gymnosperms . . . . 32 4.1.1 Pinaceae 32 4.1.1.1 Picea glauca (Moench) Voss 32 Picea engelmanni ? Parry 32 Picea sitchensis (Bong) Carr 32 4.1.1.2 Pinus contorta Dougl 36 P i nus mont icola Dougl 36 h. 1.1.3 Pseudotsuga menziesii (Mirb.) Fr. . . .40 4.1.1.4 Lar ix occidental i s Nutt 43 4.1.2 Cupressaceae 45 4.1.2.1 Cupressus macrocarpa Hartw. . . . . . . 45 4.1.3 Taxodiaceae . . . . . . . . 46 4.1.3-1 Sequoia sempervirens End!. . . 46 - vi -4.1.4 Taxaceae .48 4.1.4.1 Taxus brevifol ia Nutt 48 4.2 Investigation of Exophylactic and Necrophylactic Periderms in Angiosperms. 51 4.2.1 Aceraceae 51 4.2.1.1 Acer macrophyl1um Pursh. . . . 51 4.2.2 Leguminosae 54 4.2.2.1 Robinia pseudoacacia L. 54 4.2.2.2 Gleditsia triacanthos L. Inermis, . . . 56 4.2.3 Rosaceae 59 4.2.3.1 Pyrus species 59 4.2.4 Malvaceae . 61 4.2.4.1 Hibiscus syriacus L. Hamabo 61 4.3 Investigation of NIT in Gymnosperms and Angiosperms . . £>2 4.3.1 General Survey and Characteristics of NIT at Injuries . . . .63 4.3.2 Developmental Study of NIT in Pinus contorta and Larix occidental is by Cryofixation 64 4.3.3 General Features of NIT at Injuries Using Cryofixation and F-F Test . 68 4.3.4 General Features of NIT at Rhytidome Using Cryofixation and F-F Test 70 4.3.5 General Features of NIT at Diseased Sites and Abscission Zones Using Cryofixation and F-F Test 71 - v i i -4.3.6 Histochemica1 Characteristics of NIT- . . . . . .72 4.3.7 Association of NIT with Necrophylactic Periderms (NP) 75 5. DISCUSSIONS .76 5.1 The Validity of the Concepts of Exophylactic and Necrophylactic Periderms. . . . . . . . . . . . . . . . 76 5.2 The General Occurrence of the NIT. . . . . . . . . . . .85 6. SUMMARY AND CONCLUSIONS . . . . . . . . . . . .92 TABLES -95 FIGURES .102 ABBREVIATIONS USED TO LABEL FIGURES . . . . . . . . . . . . . . . 103 LITERATURE CITED. 312 APPENDIX 318 - v i i i -LIST OF TABLES T a b l e Page 1 Age o f t r e e r e g i o n and d a t e o f ba r k c o l l e c t i o n o f FEP samples . 96 2 Age o f t r e e r e g i o n and d a t e o f ba r k c o l l e c t i o n o f NP and SEP 97 3 Usual t r e e stem age o f i n i t i a l r h y t i d o m e f o r m a t i o n . .98 c k C r y o f i x a t i o n c h a r a c t e r i s t i c s o f t h e c o n t e n t s and w a l l s o f e x o p h y l a c t i c p h e l l e m 99 5 C r y o f i x a t i o n c h a r a c t e r i s t i c s o f t h e c o n t e n t s and w a l l s o f n e c r o p h y l a c t i c p h e l l e m 100 6 Time t o NIT f o r m a t i o n f o l l o w i n g i n j u r y , made on June 11, 1974 101 - i x -LIST OF FIGURES F i g u r e Page 1 T h i s f i g u r e shows t h e p a t t e r n and s i t e o f p h e l l e m ( c o r k ) , p h e l l o g e n ( c o r k cambium), p h e l l o d e r m , and c o r t e x f o r m a t i o n i n woody p l a n t s ( c o u r t e s y o f Mul1 i c k 1977) 104 2 T h i s f i g u r e shows t h e mechanism o f c e l l d i v i s i o n i n v o l v e d i n c l a s s i c a l p e r i d e r m f o r m a t i o n . Note t h a t t h e p h e l l e m ( d a r k c e l l s ) i s produced towards th e o u t s i d e o f t h e p h e l l o g e n ( g r a y c e l l ) w h i l e t h a t o f t h e p h e l l o d e r m ( w h i t e c e l l s ) towards t h e i n s i d e ( c o u r t e s y o f M u l l i c k 1977) 105 3 T h i s f i g u r e shows the a p p r o x i m a t e l o c a t i o n s where f i r s t p e r i d e r m , sequent p e r i d e r m and r h y t i d o m e can be l o c a t e d i n woody p l a n t s ( c o u r t e s y o f Mul1ick 1977) 106 4 The s p l i t s i n e p i d e r m i s t o g e t h e r w i t h the a d h e r i n g n e e d l e bases o r p e t i o l e from the 5th i n t e r n o d a l a r e a o f P i c e a g1auca r e s u l t i n roughened appearance o f t h e b a r k s u r f a c e . S i m i l a r f e a t u r e s were o b s e r v e d i n P i c e a engelmanni? and P i c e a s ? t c h e n s i s 107 5 Roughened appearance o f b a r k from the 6th i n t e r n o d a l a r e a i n P i c e a g l a u c a . Needles were shed b e f o r e t h e n e e d l e bases were s l o u g h e d 107 6, 7, 8 These f i g u r e s show th e t h i n and t h i c k w a l l e d p h e l l e m and 9 c e l l s o f P i c e a g l a u c a i n c r y o f i x e d s e c t i o n u s i n g v a r i o u s modes o f i l l u m i n a t i o n s 109 10 A c r o s s s e c t i o n o f FEP o f P i c e a e n g e l m a n n i i a t the 5th i n t e r n o d e c o l l e c t e d on June 12, 1974. The t h i n -and t h i c k - w a l l e d p h e l l e m c e l l s , and t h e remains o f t h e s l o u g h e d e p i d e r m i s a r e shown. Note the un-d i s t o r t e d r e c t a n g u l a r shape o f t h e newly formed t h i n - w a l l e d phel 1 em'(8'5X) 112 11,"12, Those f i g u r e s show NPs found next t o r h y t i d o m e i n 13 and 14 P i c e a s i t c h e n s i s under v a r i o u s modes o f i l l u m i n a t i o n i n c r y o f i x e d s e c t i o n s 114 15 A thawed s e c t i o n o f NP w i t h t h i n - and t h i c k - w a l l e d p h e l l e m o f SEP a b u t t i n g i t a t r h y t i d o m e o f P i c e a  s i t c h e n s i s a f t e r t r e a t m e n t w i t h a b s o l u t e e t h a n o l . The d a r k and p i n k c o n t e n t s o f t h e n e c r o p h y l a c t i c p h e l l e m had d i s a p p e a r e d and t h e p h e l l e m had assumed a f a i r l y u n i f o r m shape ( t b f , 85X) 117 - x -16 C r y o f i x a t i o n c h a r a c t e r i s t i c s o f a NP a t an a b s c i s s i o n s c a r a t the 3rd i n t e r n o d a l a r e a i n P i c e a g l a u c a . Only t h i n - w a l l e d p h e l l e m a r e p r e s e n t . The t h i n - w a l l e d p h e l l e m o f SEP (not c l e a r l y seen here) was found a b u t t i n g the NP, but the t h i c k - w a l l e d SEP p h e l l e m was not o b s e r v e d (1/53, 85X) 119 17 NP i n P i c e a s i t c h e n s i s a t the s i t e o f a t t a c k by P ? ssodes s i t c h e n s i s Hopk? ns as r e v e a l e d by c r y o f i x a t i o n . The p e r i d e r m was i d e n t i c a l t o t h o s e found a t r h y t i d o m e (1/53, 135X) 119 18 A NP o f P i c e a s i t c h e n s i s found a t rh y t i d o m e (1/53, 210X) . . .. 121 19 and T h i n - and t h i c k - w a l l e d p h e l l e m o f FEP i n P i n u s c o n t o r t a 20 i s shown. S i m i l a r p h e l l e m was o b s e r v e d i n FEP o f P i n u s  moht i c o l a . Note t h a t some o f t h e t h i c k - w a l l e d p h e l l e m had t h i c k o u t e r and t h i n i n n e r t a n g e n t i a l w a l l s ( t b f , 135X)- • • • 121 21 A FEP o f P i n u s c o n t o r t a t r e a t e d w i t h Sudan 111. Only t h i n - w a l l e d p h e l l e m was s t a i n e d r e d ; t h i c k - w a l l e d p h e l l e m d i d not s t a i n . The brown pigmented c o n t e n t s s t i l l remained i n t h e p h e l l e m even a f t e r many washings w i t h 85% e t h y l e n e g l y c o l f o r s e v e r a l h o u r s . S i m i l a r r e s u l t s were o b t a i n e d w i t h FEP o f P i n u s m o n t i c o l a ( t b f , 135X) 124 22 NP found a t f r e s h l y f o r m i n g r h y t i d o m e i n P i nus c o n t o r t a c o l l e c t e d on November 25, 1974. The p h e l l e m i s t h i n -w a l l e d and s l i g h t l y d i s t o r t e d (1/53, 210X) 124 23 and A 35 day and a 210 day o l d m e c h a n i c a l i n j u r y o f P i h u s 24 c o n t o r t a made on June 17, 1974 and A p r i l 11, 1974 and c o l l e c t e d on J u l y 17, 1974 and November 9, 1974, r e s p e c t i v e l y as r e v e a l e d by c r y o f i x a t i o n . The n e c r o p h y l a c t i c p h e l l e m c e l l s i n t h e e a r l y s t a g e s o f development a r e u n d i s t o r t e d and r o u g h l y r e c t a n g u l a r l y shaped and have no pigment. Note pigment had a l r e a d y d e v e l o p e d i n the f i r s t p h e l l e m ( a r r o w ) . In t h e o l d m e c h a n i c a l i n j u r i e s t he n e c r o p h y l a c t i c p h e l l e m c e l l s have pigment and t h e c e l l s a r e f l a t t e n e d and compressed. In both i n j u r i e s SEP was not p r e s e n t (1/53, 135X) 126 25 A 2 month o l d m e c h a n i c a l i n j u r y showing a NP i n P i n u s  m o n t i c o l a . S l i g h t d i s t o r t i o n o f t h e p h e l l e m c e l l s i s e v i d e n t w h i c h were not y e t pigmented (1/53, 85X) 127 26 A NP found i n t h e stem o f P i n u s m o h t i c o l a a t t h e s i t e o f an i n f e c t i o n o f C r o n a r t i u m r i b i c o l a as r e v e a l e d by c r y o f i x a t i o n . The t r e e was about 25 y e a r s o l d ( I V / 4 1 , 135X). . 130 - xi -27 A NP found in a diseased site on a branch of P i nus  monticola attacked by Cucurbidoth?s pi thyophi1 a as revealed by cryofixation. The tree was about 30 years old (IV/41, 135X) 130 28 Three NPs found in successively deeper regions of bark without the development of a SEP at rhytidome in Pinus contorta. The sample was collected from a region of old rhytidome where no scaling was observed ( 1 / 5 3 , 85X) 132 29 Typical, appearance or bark surface of Pinus contorta showing scaling of dead rhytidome tissues. The bark surface appeared very rough from the sloughing 132 30 and An exposed SEP of Pihus contorta rhytidome showing 31 thin- and thick-walled phellem. There are more layers of thick-walled phellem cells than thin-walled phellem cells (tbf, 85X; tbf, 35X) 134 32 An unexposed SEP of P i nus mont icola rhytidome showing thin- and thick-walled phellem abutting the necrophylactic hellem which was deteriorating. Thin-walled phellem was found external to the thick-walled phellem which are more in number (1/53, 135X) 136 33 A NP with SEP abutting it found in a diseased branch infected wiith Cucurb i doth i s p i thyoph i 1 a in P i nus mont i col a treated with phlorog1ucinol and concentrated hydrochloric acid. The NP phellem contents have disappeared, but the brown contents in the thin-walled cells of SEP remained. All phellem walls stained f a i r l y pink. Similar results were obtained with NPs and SEPs of Pinus contorta (tbf, 135X) . 136 34 and Cryofixation shows FEP found below the epidermis in 35 Pseudotsuga menziesii. The phellem had brown contents, and thin-walls which fluoresced pale blue (IV/41, 135X; IV/41, 210X) I 3 8 36 and NP of Pseudotsuga menziesii collected in the year of its 37 formation at rhytidome, macroscopically and microscopically, respectively. The NPs appear dark gray in the frozen block and the phellem cells at this early stage of development are undistorted and rectangular in shape (1/53, 85X) 140 3& NPs in rhytidome of Pseudotsuga menziesi i collected at the end of the growing season [macroscopically) . .. . 142 - x i i -39 and Seasonal renewal of necrophylactic phellem at 40 rhytidome of Pseudotsuga menziesii are shown. These seasonal renewals of NP were denoted by the phellogen activity marker cells (PAMC). The PAMC have thickened walls and stronger birefringence, and they fluoresced brighter blue (TV/41) and brighter yellow (1/53) than the abutting necrophylactic phellem c e l l s . They are (PAMC) usually 1 to 2 cells thick (fcp, 210X; 1/53, 35X) • • .144 41 and NP of Pseudotsuga menziesi i collected at the end of the 42 growing season as revealed by cryofixation. The phellem and the phellogen activity marker cells (PAMC) are shown (1/53, 135X; IV/41, 135X) 146 43 NP found at resin blister in Pseudotsuga menziesi i . The NP is similar to those found at rhytidome (Table 5) The phellogen activity marker cells (PAMC) are evident ( I V / 4 1 , 135X) 148 44 Rhytidome NP of Pseudotsuga menziesii after treatment with Sudan I I I . All necrophylactic phellem cell walls are stained red. NPs of similar characteristics were also found around beetle holes (tbf, 135X) 148 45 PAMC found at abscission scar in Pseudotsuga menziesi ? ( I V / 4 1 , 135X) 150 46 FEP was found 2 to 3 cells below the epidermis in Larix occidental is (1/53, 135X) 150 47 The smooth and rough bark surfaces in Larix occidental is. The rough lower portion of the stem shows sloughing of dead rhytidome tissues. The tree was approximately twenty years old -1 52 48 Phellem of FEP in young stem of Larix occidental is. Under IV/41 fluorescence f i l t e r combination, the phellem contents appeared dark brown; while the phellem walls fluoresced pale blue ( I V / 4 1 , 210X) 152 49 A FEP in old stem of Larix occidental is. Note the presence of some thick-walled phellem interspersed in the thin-walled phellem of the FEP. Under IV/41 f i l t e r combination the thick-walled phellem fluoresced khaki ( I V / 4 1 , 210X) . • • • 154 50 NP found at rhytidome in Larix occidentalis as revealed cryofixation under 1/53 f i l t e r combination. Note that the phellem content fluoresced dull red and the cell walls fluoresced pale green. The phellem cells are also compressed and distorted (1/53, 135X) 154 - x i i i -51 NP found a t a 33 day o l d i n j u r y i n L a r i x o c c i d e n t a l i s by c r y o f i x a t i o n under 1/53 f i l t e r c o m b i n a t i o n a f t e r F-F t e s t . Note t h a t d u l l red pigment has formed i n the f i r s t NP p h e l l e m . The o u t e r p h e l l e m i s f l a t t e n e d and t h e i n n e r p h e l l e m i s t u r g i t (.1/53, 135X) 156 52 M a c r o s c o p i c view o f FEP, NPs and SEP a t r h y t i d o m e o f L a r i x o c c i d e n t a l i s . FEP and SEP a r e brown. NPs a r e r e d d i s h p u r p l e . The dead rhyti d o m e t i s s u e s between NPs a r e b r o w n i s h and l i v i n g t i s s u e s below a r e g r a y i s h . . . . 156 53 An a i r d r i e d c r y o f i x e d s e c t i o n o f L a r i x o c c i d e n t a 1 i s showing NP and SEP a t r h y t i d o m e was t r e a t e d w i t h ph1orog1ucina1-HC1. The d i s t o r t e d and compressed NP p h e l l e m and SEP p h e l l e m was not s t a i n e d . Note t h a t the brown c o n t e n t s i n t h e SEP p h e l l e m s t i l l remained i n the phe11 em whi1e t h a t i n t h e NP phel1 em had d i s a p p e a r e d ( t b f , 135X) 158 54 and NPs found a t r h y t i d o m e and a m e c h a n i c a l i n j u r y 55 ( f i v e months o l d ) r e s p e c t i v e l y i n Cupressus macrocarpa (.1/53, 135X) . • • 160 56 A FEP found at the 6 t h i n t e r n o d e o f Sequoia s e m p e r v i r e n s . The p e r i d e r m was brown and has compressed and f l a t t e n e d t h i n - w a l l e d p h e l l e m ( t b f , 21 OX) = 162 57 NP found a t r h y t i d o m e i n Sequoia s e m p e r v i r e n s . The p e r i d e r m a l s o r e p r e s e n t t h o s e formed a t i n j u r i e s . The NP a r e 2 t o 3 c e l l s t h i c k . T h i s NP formed a t a 1974 r h y t i d o m e c o l l e c t e d i n l a t e November, 1974, no SEP was o b s e r v e d a b u t t i n g the NP (1/53, 135X) 162 58 I n i t i a t i o n o f NP a t r h y t i d o m e i n Sequoia s e m p e r v i r e n s . Note t h e development o f t h e NP below t h e s e m i - l u n a r o r l e n s - s h a p e d b e i g e c o l o r bark t i s s u e s . • • • . . 164 59 The t i s s u e s above t h e NPs became brown to dark brown at the end o f t h e g r o w i n g season i n Sequoia sempervi rens 1 64 60, 61 FEP d e v e l o p e d i n t h e e p i d e r m i s below t h e c u t i c l e i n and 62 Taxus b r e v i f o l i a i s shown under t h r e e d i f f e r e n t modes o f i l l u m i n a t i o n s . The p e r i d e r m i s brown and c o n s i s t e d o f v e r y few t h i n - w a l l e d and p h e l l e m c e l l s . These f i g u r e s show t h e s h e d d i n g o f t h e FEP and the f o r m a t i o n of t h e f i r s t NP a t r h y t i d o m e (1/53, 210X; mbf, 210X, t b f , 210X) 166 - x i v -63 MPs found at mechanical, injury in Taxus brev? f o l i a . SEP has not developed abutting the NP at injury. The injury was about 4 months old (tbf, 135X) 168 64 SEP abutting NP found at rhytidome iri Taxus  brevi f o l i a as revealed by cryofixation under tbf. The NP phellem has reddish purple contents and the SEP phellem, like the FEP, has brown contents (tbf, 135X) 170 65 Small whitish and light brownish spots (arrows) appearing in the f i r s t and, second internodes of an Acer macrophyl 1 urn stem 170 66 The development of dark brown patches from spots at the fourth internode is shown. Intense dark patches appearing on the south side are evident (Acer macrophyl 1 urn) 172 67, 68 Site of FEP initiation in tissues abutting the and 69 epidermis below the spots of various sizes in Acer macrophy11 urn by cryofixation. When observed wTth IV/41 (Fig. 67) and 1/53 (Fig. 68) f i l t e r combinations, the tissues in which the initiation of FEP are taking place fluoresced pale blue and yellow respectively. Figure 69 shows the formation of FEP under fcp (IV/41, 85X; 1/53, 210X; fcp, 135X) 172 70 Cryofixation shows the FEP has developed below the dark brown or grayish patches. FEP has not developed below the green strips of bark in Acer macrophy11 urn; only the epidermis is present TT753, «5X) . . . 175 71 and Anatomical features as well as cryofixation 72 characteristics of FEP in Acer macrophyllum are shown by cryofixation under f i l t e r combinations 1/53 and IV/41 respectively (1/53, 210X; IV/41, 210X) 175 73 The bark surface appears rough as long longitudinal cracks appeared at the base of the tree resulting from rhytidome development in Acer macrophyllum 178 74 NPs developed at rhytidome in Acer macrophyllum as revealed by cryofixation under f i l t e r com-bination IV/41. SEP has formed abutting the NP (IV/41, 210X) 178 - xv -75 C r y o f i x a t i o n shows t h a t a NP d e v e l o p e d a t a 167 -d a y - o l d m e c h a n i c a l i n j u r y i n A c e r macrophy11 urn under mbf. Note t h a t t h e phel1 em e e l Is a re compressed and d i s t o r t e d (mbf, 210X) 180 76 C r y o f i x e d s e c t i o n o f a m e c h a n i c a l i n j u r y c o l l e c t e d 35 days a f t e r t he i n j u r y was made. Note t h a t t he NP p h e l l e m c e l l s a r e r e c t a n g u l a r and u n d i s t o r t e d i n A c e r macrophy11 urn under f l u o r e s c e n c e f i l t e r c o m b i n a t i o n 1/53- A f t e r 167 days t h e p h e l l e m c e l l s a r e compressed c o n s i d e r a b l y , see F i g u r e 75 (1/53, 135X) 180 77 No SEP d e v e l o p s a b u t t i n g t he NP formed i n t h i s y e a r ' s r h y t i d o m e i n A c e r macrophy11 urn under t b f in a c r y o f i x e d s e c t i o n ( t b f , 135X) , '. 182 78 A thawed c r y o f i x e d s e c t i o n from a rh y t i d o m e sample o f A c e r macrophy11 urn shows t h a t t h e NP p h e l l e m c o n t e n t s has f l o w e d away but t h a t t he c o n t e n t s o f t h e e x o p h y l a c t i c p h e l l e m remained i n t a c t ( t b f , 135X) 182 79 Development o f FEP i n t i s s u e two t o t h r e e c e l l s below t h e e p i d e r m i s a t the second i n t e r n o d e o f R o b i n i a p s e u d o a c a c i a as r e v e a l e d by c r y o f i x a t i o n (1/53, 210X) ]8k 80 C r y o f i x a t i o n shows FEPs development i n R o b i n i a  p s e u d o a c a c i a . Seasonal renewal o f FEPs, i n d i c a t e d by t h e p r e s e n c e o f t h e p h e l l o g e n a c t i v i t y marker c e l l s , i s shown. The p h e l l em i n t h e r e c e n t l y d e v e l o p e d FEP was r e c t a n g u l a r and u n d i s t o r t e d b u t t h o s e i n t h e o l d p e r i d e r m a r e compressed (IV/41 , 210X) 184 81 A c r y o f i x e d s e c t i o n o f FEP i n R i b i n i a p s e u d o a c a c i a shows t h a t t he p h e l l e m was compressed and s t r e t c h e d and s e a s o n a l renewal o f FEPs ( c a . 4) i n d i c a t e d by t h e p r e s e n c e o f t h e PAMC (arrows) w h i c h have t h i c k e n e d w a l l s (mbf, 210X) 186 82 and C r y o f i x e d s e c t i o n s o f R o b i n i a pseudoacac.i a 83 showing NP d e v e l o p e d a t rhytid o m e under f l u o r e s c e n c e f i l t e r c o m b i n a t i o n IV/41 and 1/53 r e s p e c t i v e l y . S e a sonal renewal o f t h e NP p h e l l e m was i n d i c a t e d by the PAMC ( F i g . 82). P r e v i o u s l y formed p h e l l e m was s t r e t c h e d and compressed and r e c e n t l y d e v e l o p e d p h e l l e m was u n d i s t o r t e d ( I V / 4 1 , 85X; 1/53, 210X) 186 - xvi 84 A FEP of Robinia pseudoacacia.treated wi th Sudan I I I in ethylene glycol. The phellem including the PAMC are stained red, Simi1ar positive results.were obtained when other fat stains were used (tbf, 135X) 189 85 A cryofixed section of Robihi a pseudoacacia showing NP developed a f t e r 2 8 days following injury under fluorescence f i l t e r combination 1/53 0 / 5 3 , 135X). 189 8 6 A cryofixed section shows the initiation of FEP in tissues two to three cells below the epidermis in the second internode in Gleditsia triacanthos under fluorescence f i 1 ter combination 1/53 ( 1 / 5 3 , 21 OX). 191 87 and A cryofixed section of FEP in G l e d i t s i a tr iacanthos 88 consisting of approximately ten seasonal activities as indicated by the phellogen activity marker cells under pop and fluorescence f i l t e r combination IV/41 respectively. The phellem contained brown pigment and was thin-walled. Weathered FEP phellem on the bark surface was sloughing (pep, 210X; IV/41 , 210X) 191 89 NPs developed at rhytidome of Gleditsia triacanthos as revealed by cryofixation under fluorescence f i l t e r combination IV/41 (IV/41, 21 OX). Three seasonal activities of the NPs as indicated by the phellogen activity marker cells are shown. No SEP was observed abutting the NP phellem which are considerably dis-torted and compressed 194 90 A NP formed at a 21 day old mechanical injury in Gledits ia  triacanthos as revealed by cryofixation under fluorescence f i l t e r combination IV/41 (210X) 194 91 Air dried cryostat sections from this year's ( 1 9 7 4 ) rhytidome of Gleditsia triacanthos were treated with Sudan III and examined with fluorescence f i l t e r combination 1/53 to enhance contrast and sensitivity ( 1 / 5 3 , 8 5 X ) . Phellem of FEP and NP are stained red 196 92 Serial air-dried cryostat sections to Fig. 91 of Gleditsia triacanthos were treated with phlorog1ucino 1 -HCL. FEP and NP phellem cell walls are stained pink. The pigmented contents in the NP phellem had disappeared but that in the FEP phellem remained intact (tbf, 85X) I 9 6 xvi i -93 A cryofixed section of FEP of Pyrus specles under tbf (tbf, 135X). The phellem had brown contents and its tangentia 1 wa11s were thicker than its radia 1 wal1s . 198 94 A cryofixed section of FEP of Pyrus species is shown under f1uorescence fi1ter combination IV/41 (135X). The phellem contents appeared black and the phellem walls fluoresced blue. . - I 9 8 95 A partially developed NP during rhytidome formation in Pyrus species is revealed by cryofixation under fluorescence fi1ter- combination 1/53- The phellem cells were small and thin-walled. No SEP had developed abutting the periderm. (I/53V 135X) 200 96 A cryofixed section of NPs formed in the years previous to rhytidome formation in Pyrus species under tbf. SEPs abut a l l NPs. NPs usually have fewer phellem cells than SEPs (tbf, 85X) . . . 200 97 NP developed in a one-year-old rhytidome in Pyrus species as revealed by cryofixation under fluores-cence f i l t e r combination 1/53- A SEP has developed abutting this NP (1/53, 135X) 202 98 A cryofixed section of SEP from a rhytidome sample of a Pyrus species where sloughing was taking place. The SEP was partial ly exposed (tbf, 35X). 202 99 and Initiation of FEP occurred in tissue abutting the 100 epidermis in the f i r s t and second internodes of Hibiscus syriacus respectively as revealed by cryofixed sections using fluorescence f i l t e r combination IV/41 (135X; 135X) 204 101 and A cryofixed sectiomof FEP in Hibiscus syriacus 102 developed at about the f i f t h internode using fluorescence f i l t e r combination IV/41 and 1/53 respetively. Brown pigmented contents formed only in one layer of phellem (arrows) on the outside of FEP close to the surface. The phellem cells are thin-walled (IV/41, 135X; 1/53, 135X) 206 103 A cryofixed section showing NP formed during rhytidome formation in Hibiscus syriacus with fluorescence f i l t e r combination IV/41. the phellem was thin-walled and usually rectangular at the early stage of development (IV/41, 85X) 208 - xvi i i -104 Air-dried crye-star section containing a NP formed during rhytidome formation i n H i b i scus syr i acus, treated with Sudan III. All NP phellem stained red. Similar results were obtained of FEP (1/53, 1-35X) . 208 105 Cryofixed section of a five-hour-old injury of bark sample collected from Pinus contorta following F-F test. Deep penetration of the bark sample by the F-F solutions is evident 210 106 Deep F-F permeation in an injury bark sample of Picea glauca colleeted on the 18th day after wounding as revealed by cryofixation (tbf, 85X) 210 107 Cryofixation under tbf revealed the depth of F-F permeation of Robinia Pseudoacacia injured bark samples collected on the 7th day after wounding. Wounded zone, as usual, becomes much deeper blue than the living zone (tbf, 135X) 212 108 A 28-day-old injured sample of Taxus brevi f o l i a was F-F tested through the wound surface; The permeation of the test solutions was stopped at , the non-suberized impervious tissue (NIT). Only the brown dead tissues are colored blue. Similar results were observed in a l l species after NIT format ion 212 109 The extent of permeation of the test solutions in the F-F tested samples of Picea glauca 32 days after wounding is shown by cryofixation with fluorescence f i l t e r combination 1/53- NIT has formed at this time (1/53, 135X) . 214 110 A cryofixed section of a F-F tested 50-day injured sample of P i cea s i tchens i s is shown with fluorescence f i l t e r combination IV/41. Note that NIT and NP phellem had developed (IV/41, 135X) 214 111 The extent of penetration by the test solutions in the F-F tested sample of Pinus contorta 30 days after injury is shown by cryofixation using tbf. NIT had formed and the NP phellem was just forming (tbf, 135X) 1 216 112 A cryof ixed section of. a F-F tested 18-day-old injured sample of Ciipressus macrocarpa is shown with fluorescence f i l t e r combination 1/53- NIT had formed and NP phellem had not developed at this time (1/53, 85X) 216 - x i x -113 The depth o f p e r m e a t i o n by the t e s t s o l u t i o n s i n the F-F t e s t e d samples o f Taxus b r e v i f o l i a 28 days a f t e r i n j u r y i s shown by c r y o f i x a t i o n under f l u o r e s c e n c e f i l t e r c o m b i n a t i o n IV/41.. NIT i s e v i d e n t and NP has not d e v e l o p e d (IV/41 , 85X) 218 114, 115 C r y o f i x e d . s e c t i o n s from a. F-F t e s t e d sample o f and 116 R o b i n i a p s e u d o a c a c i a a f t e r 21 days i n j u r y shown w i t h t b f and f l u o r e s c e n c e - f i l t e r c o m b i n a t i o n 1/53 and IV/41, r e s p e c t i v e l y . N o t e . t h a t t h e F-F s o l u t i o n s had s t o p p e d e x t e r n a 1ly a b u t t i n g t h e NIT. Tes t was c a r r i e d out from t h e wound s u r f a c e . NP i s shown d e v e l o p i n g ( t b f , 135X; 1/53, 135X; IV/41, 135X) 218 117 I n j u r e d b a r k sample c o l l e c t e d two days a f t e r i n j u r y from L a r i x o c c i d e n t a l i s shown by c r y o f i x a t i o n and f l u o r e s c e n c e f-i 1 t e r c o m b i n a t i o n 1/53- The brown 'dead' zone shows fa d e d f l u o r e s c e n c e o f c e l l c o n t e n t s , and d u l l g r e e n i s h f l u o r e s c e n c e o f w a l l s (1/53, 8 5 * ) . . • 221 118 A c r y o f i x e d s e c t i o n from a two-day i n j u r e d bark sample o f P i n u s c o n t o r t a i s shown w i t h f l u o r e s c e n c e f i l t e r c o m b i n a t i o n 1/53 (85X) . 221 119 I n j u r e d bark samples c o l l e c t e d on t h e f o u r t h day a f t e r wounding from P i n u s c o n t o r t a i s shown by c r y o f i x a t i o n w i t h f l u o r e s c e n c e f i l t e r c o m b i n a t i o n 1/53 (85X) 223 120 I n j u r e d b a r k sample c o l l e c t e d on t h e s e v e n t h day a f t e r wounding from P i n u s c o n t o r t a i s shown by c r y o f i x a t i o n and f l u o r e s c e n c e f i l t e r c o m b i n a t i o n 1/53 (85X). C e l l s a b u t t i n g t h e 'dead' brown t i s s u e s had l o s t t h e f l u o r e s c e n c e o f t h e i r c o n t e n t s w h i c h had become r e t i c u l a t e d and f l u o r e s c e d p a l e g r e e n . A m e r i s t e m a t i c zone o f t i s s u e s i s e v i d e n t 223 121 and C r y o f i x e d s e c t i o n s o f i n j u r e d bark samples c o l l e c t e d 122 on t h e e l e v e n t h and f o u r t e e n t h days a f t e r wounding from P i n u s c o n t o r t a a r e shown w i t h f l u o r e s c e n c e f i l t e r c o m b i n a t i o n 1/53 (85X, 85X). Pronounced t i s s u e m o d i f i c a t i o n s a r e o b s e r v e d compared t o t h a t o f t h e s e v e n t h day ( F i g . 120) 225 123 C r y o f i x e d s e c t i o n o f i n j u r e d bark sample c o l l e c t e d on the s e v e n t h day a f t e r wounding from L a r i x o c c i d e n t a l i s shown under f l u o r e s c e n c e f i l t e r c o m b i n a t i o n 1/53 (35X). Note t h a t the f l u o r e s c e n c e o f the c o n t e n t s o f the c e l l s a b u t t i n g t h e 'dead' brown t.issues was c h a n g i n g from y e l l o w t o y e l l o w i s h green 227 - xx -124 Cryofixation.sect ion of injured bark sample collected on the ninth day after wounding from La r ix occ i dental i s*-. shown with fluorescence f i l t e r combination 1/53 (35X) 229 125, 126 Cryofixed sections of injured bark samples and 127 collected on the eleventh, fourteenth, and sixteenth day respectively after wounding from Larix occidental is shown using fluorescence f i l t e r combination 1/53- 229 128, 129 Cryofixed sections of injured bark samples and 130 collected on the sixteenth (tbf, 135X) eighteenth (1/53, 135X) and twenty-fifth (1/53, 135X) day after wounding from Pinus contorta, respectively. The formation of the NIT is evident. Sporadic formation of NIT as indicated by the sporadic F-F color in the living bark is shown in Fig. 128 '. .• • -232 131, 132 Injured bark samples collected on the eighteenth, 133 & 134 twenty-first, twenty-fifth and t h i r t y - f i f t h day after wounding from Larix occidental is respectively as revealed by cryofixation using fluorescence f i l t e r combination 1/53 (85X). Note that NIT had developed. NIT cells had yellowish green cell contents and greenish cell walls. Abutting NIT was the dark band of meristematic tissue • • 234 135 Cryofixed section of a freshly formed NIT in a 32-day-old injury of Picea glauca under fluorescence f i l t e r combination 1/53- 237 136 and Cryofixed section of a 36-day-old F-F tested 137 injury of Picea sitchensis•showing NIT under fluorescence f i l t e r combination IV/41 and 1/53 respectively 239 138 A 60-day-old injury of Picea engelmannii showing NIT which had formed-after 32 days and the initiation of the NP. NIT consisted of enlarged cells of varying shapes and sizes with irregularly thickened wa11s as revealed by cryofixation under fluorescence f i l t e r combination IV/41 (85X) 241 139 Cryofixed section of a 21-day-old injury of Picea  contorta showing the formation of NIT in 21 days under fluorescence f i I t e r combination IV/41 (135X). Sporadic formation of NP phellem is shown abutting the NIT • 241 - xx i -140 and Cryofixed sect ion of a 60-day-old i nj ury of Pi nus 141 mont icola showing NIT wh ich had developed in 2 8 days and the initiation of the NP is taking place in tissue interna1ly abutting NIT as seen under fluorescence fi1ter combination 1/53 and IV/41 respectively (135X) 243 142 and Freshly formed NIT in a 27-day-old injury of 143 Pseudotsuga menziesi i . NIT had,developed in 23 days and the differentiation of NP has not occurred as revealed by cryofixation under fluorescence f i l t e r combination 1/53 and IV/41 respectively (85X) 245 144 and Cryofixed section of a 23-day-old injury of Lar ix 145 occidenta1 is showing the formation of NIT which is located in between the brown dead tissue and the 1 i v i ng ba rk. . . 247 146, 147 Cryofixed section of a 17~day-old and a 24-day-old and 148 injuries of Cupressus macrocarpa showing the formation ,of NIT respectively as revealed under fluorescence f i l t e r combination IV/41, 1/53 and IV/41 respectively. . . . 2h3 149 Cryofixed section of a 23-day-old injury of Sequoia  sempervirens showing the freshly formed NIT in between the brown 'dead' tissue and the living bark under fluorescence f i l t e r combination IV/41 (85X) NIT has developed in 21 days. NP differentiation is not shown. 252 150 Cryofixed section of a 28-day-old F-F tested injury of Taxus brevi f o l i a showing the development of NIT in between the 'dead1 tissue and the living bark under fluorescence f i l t e r combination 1/53 (85X) 252 151 Cryofixed section of a 14-day-old injury of Acer  macrophy11 urn showing the freshly formed NIT in between the 'dead' tissue and the living bark under fluorescence f i l t e r combination IV/41 (135X) 254 Cryofixed section of a 21-day-old F-F tested injury of Robinia pseudoacacia showing the forming of NIT which consists of enlarged cells of varying shapes and sizes with irregularly thickened walls 254 154 Cryofixed section of a freshly formed NIT in a 21-day-old F-F tested injury of Gled i ts ia tr iacanthos under fluorescence f i l t e r combination IV/41 (210X) ". . . . . 257 152 and 153 - xx i i -155 Cryofixed section of a 15 -day-old F-F tested injury of Hibiscus syriacus showing the freshly formed NIT in between the 'dead' tissue and the 1iving bark under fluorescence f i 1 t e r combination IV/41. NIT had formed in 15 days. No sign of NP initiation was detected (85X) 257 156 Cryofixed section of a 36-day^old F-F tested injury °f Picea sitchens is.showing NIT and the formation of the NP in tissue interna-ly abutting NIT under fluorescence f i I t e r combination 1/53 (210X). 259 157 Cryofixed section of a 60-day-old injury of Picea  engelmanni i showing completion of NIT development and the initiation of NP in tissue internally abutting NIT under fluorescence f i l t e r combination IV/41 (85X) . . . .259 158 Cryofixed section of a 60-day-old injury of P i nus  monticola showing NIT has developed and the formation of the NP in tissue internally abutting the NIT under fluorescence f i l t e r combination IV/41 (85X) 261 159 and Cryofixed section of a 30-day-old F-F tested injury 160 of Pinus contorta. NIT had formed in 21 days and the initiation of the NP in the internal abutting tissue has occurred as shown with fluorescence f i l t e r combination 1/53 and IV/41 respectively (135X) . . 261 161 A 4lxday-old injury of Sequoia sempervirens showing the formation of NIT and the NP in tissue internally abutting NIT as revealed by cryofixation under fluorescence f i l t e r combination 1/53 and IV/41 respectively (85X) • • •• . .264 162,163 Cryofixed section of a 28-day-old injury of Rob in ia and 164- pseudoacacia showing completion of NIT development and the formation of the NP in tissue internally abutting the NIT under tbf and fluorescence f i l t e r combinations 1/53 and IV/41, respectively. 264 165 Cryofixed section of a 33-day-old injury of Larix  occ i denta1i s showing NIT has developed and the formation of the NP in tissue internally abutting NIT under fluorescence f i l t e r combination IV/41 267 166 Cryofixed section of NIT.formed at rhytidome of P icea  glauca examined with fluorescence f i l t e r combination IV/41. NIT consisted of a zone of enlarged cells of varying shapes and sizes with thickened walls. NP had formed internally abutting NIT (135X) 267 - xx i i i -167,168 Cryofixed section of NIT developed at rhytidome of and 169 Picea engelmanni i and P icea s i tchens is exami ned under f i l t e r combinations IV/41 , tbf and 1/53 respectively. 269 170 and Cryofixed sections of NIT formed at rhytidome of 171 P i nus contorta and P i nus mont icola respect ively examined with f1uorescence fi1ter combination 1/53-In both species the NP has formed abutting NIT internally. The cell walls of NIT fluoresced consistently yellow with very l i t t l e greenish tinge and most eel 1 contents have disappeared 272 172 and Cryofixed section of NIT formed at rhytidome of. 173 Pseudotsuga menziesi i revealed under fluorescence f i l t e r combination IV/41 and 1/53 respectively. 274 174 Cryofixed section of NIT formed at rhytidome of Larix occidental is observed under fluorescence f i l t e r combination 1/53- Sporadic initiation of the NP is seen abutting NIT (85X) .276 175 Cryofixed section of NIT developed at rhytidome in Cupressus macrocarpa revealed under fluorescence f i l t e r combination IV/41 (85X). Initiation of the NP was observed at the dark band of meristematic tissues abutting the NIT 278 176 Cryofixed section of NIT formed at rhytidome in Sequoia sempervirens observed under IV/41 (135X) . . . . . . 278 177 Cryofixed section of NIT formed at rhytidome of Taxus brevifolia observed with tbf. NIT consisted of a zone of enlarged cells of irregular shapes and sizes with thickened walls 280 178 and Cryofixed section of NIT developed at rhytidome of 179 Acer macrophyllum examined with 1/53 f i l t e r combination at different magnifications 280 180 Cryofixed section of NIT formed at rhytidome in Robinia pseudoacacia observed under fluorescence f i l t e r combination IV/41 (210X). NIT and NP have developed and NP was found abutting NIT internally 283 181 Cryofixed section of NIT developed at rhytidome in Gleditsia triacanthos examined under fluorescence f i l t e r combination 1/53 (135*). . 283 - xx i v -182 NIT found at rhyt idome inH i b i s c u s s y r i a c u s a s revealed by cryofixation under fluorescence fi1ter combination 1/53 (85X). NIT developed prior to NP and consisted of a zone of enlarged cells of varying shapes with thickened eel 1 walls. NP had formed in tissue internally abutting NIT 285 I83 and Cryofixed section of a leader of P icea s i tchens i s 184 attacked by P issodes•: Strobi • Hopkins showing the presence of NIT and NP under fluorescence f i l t e r combination IV/41. and 1/53 (135X) 285 185 Air-dried cryostat section from a beetle hole bark sample of Pseudotsuga menz i es ? i treated with Sudan III in tbf (135X). NIT and NP had developed. Only NP phel lem eel lwal Is were stained red. 288 186 Cryofixed section of NIT and NP found at a resin blister of Pseudotsuga menziesii under fluorescence f i l t e r combination I/53 (135X) NIT always located externally abutting the NP 288 187 and Cryofixed sections from the branch and stem of 188 Pi nus mont icola attacked by Cucurbidothis  pithyophila Fr. and Cronartium ribicola F. respectively, under fluorescence f i l t e r combination 1/53 (135X) 290 189 Cryofixed section of an abscission scar collected at the third internode of Picea glauca showing the presence of NIT and NP under fluorescence f i I t e r combination 1/53 (85X) 292 190 A thawed cryofixed section from an 25_day-old F-F tested injury of Picea glauca treated with aniline sulfate-HCL is shown in tbf. . • 294 191 A thawed cryofixed section from an 18-day-old F-F tested injury of P icea engelmanni i , treated with phlorogl uci nol -HCL is shown in tbf 294 192 and Sections from a 40-day-old injury of Picea sitchensis 193 stained with Sudan I I I and ammonia hydroxide-crystal violet are shown in these two figures respectively with fluorescence f i l t e r combination 1/53 (135X) 296 194 A section from a 33"day-old injury of Larix  occidental is showing NIT and NP treated with phosphene 3R i s shown 298 - xxv -195 A section from a 40-day-old injury of Picea  s ? tchens is showi ng NIT and NP treated with methylene blue is shown. > . 300 196 Air-dried'cryostat section from a 33-day-old injury of Larix occ i denta1i 5 treated with phlorog1ucinol-HCL is shown under tbf. 300 197 Air-dried cryostat section from a 21-day-old injury of Acer macrophyllum treated with phloroglucinol-HCL is shown under tbf. NIT and sclereids were stained bright red. NP had not developed (85X) 302 198 Air-dried cryostat sections from a F-F tested 21- x day-old injury of Rob inia pseudoacacia treated with phloroglucinol-HCL is shown under tbf 302 199 Air-dried cryostat sections from a 4l-day-old injury of Taxus brev i fo1i a treated with aniline sulfate-NCL is shown under tbf 304 200 Air-dried cryostat sections from a F-F tested 21-day-old injury of Robinia pseudoacacia treated with aniline sulfate-NCL in tbf is shown 304 201 and Air-dried cryostat sections from an abscission 202 zone of Picea glauca and a diseased site of P i nus mont icola attacked by Cucurb i doth ? s p i thyoph i1 a Fr. respectively, and stained with phloroglucinol-HCL are shown in tbf 306 203 Air-dried cryostat sections from a 40-day-old injury of P icea s i tchens i s treated with Maule reaction is shown in tbf . . 308 204 Air-dried cryostat from a F-F tested 21-day-old injury of Acer macrophyllum treated with Maule reaction is shown in tbf 310 205 Air-dried cryostat sections from a 35"day-old injury of Acer macrophyllum after treatment with diethyl ether for seven days was stained with phloroglucinol-HCL under tbf. NIT and sclereids s t i l l showed positive test (85X) 310 - xxv i -ACKNOWLEDGMENTS I dedicate this thesis to my parents, Mr. and Mrs. Joseph Soo, for without their guidance, encouragement and sacrifice I would not have been able to complete my education. I wish to express my gratitude to Dr. D.B. Mullick for his advice, supervision, support and encouragement given during this study, and for access to and frequent discussions of unpublished data and interpretations of his research which greatly aided in interpretation of my findings. Appreciation is extended to my graduate committee: Dr. J.A.F. Gardner, Dean, Faculty of Forestry, University of British Columbia, Dr. B.J. van der Kamp, Faculty of Forestry, University of British Columbia, Dr. K. Graham, now retired, Faculty of Forestry, University of British Columbia, Dr. T. Bisalputra, Department of Botany, University of British Columbia, Vancouver, B.C., and Dr. R.W. Meyer, Department of Material Science and Engineering, Washington State University, Pullman, Washington, U.S.A. It is with deepest feeling that I express my gratitude to Mr. G.D. Jensen for the invaluable help he gave while the research was being done, and for his assistance in preparing the thesis. A special thank goes to Mrs. E. Jensen for letting me to stay with them while in Victoria, B.C. in preparing the manuscript. - xxv i i -Dr. R. Hunt, Dr. L.H. McMullen, and Dr. D.M. Shrimpton, research scientists, Pacific Forest Research Centre, Canadian Forestry Service, Victoria, B.C. for providing some of the samples and trees used in this study are gratefully acknowledged. Special thanks to Mr. M.H. Drinkwater and Mr. D.R. MacDonald, Director and Deputy Director, respectively, Pacific Forest Research Centre, Canadian Forestry Service, Victoria, B.C. for allowing me free access to the laboratory, and to University of British Columbia Research Forest, Maple Ridge, B.C. for free access to the forest. Financial aid provided by the Department of Environment of Canada through graduate fellowships and assistantships and University of British Columbia, Faculty of Forestry teaching and'summer research assistantships are gratefully acknowledged. I would like to thank my wife for her continued support through the completion of this work. . - 1 -1. INTRODUCTION Periderm is a vital skin-like tissue for maintaining the internal environment of plants by insulating them from harmful external factors. The physiology of periderms, in contrast to human skins, had remained obscure until a series of studies on periderm published from Mul1ick's laboratory (Mullick 1969a; 1969b; 1971; 1975; 1977; Mullick and Jensen 1973a; 1973b; 1976; Puritch and Mullick 1975). These studies based on several conifers have f i l l e d in major gaps in our understanding of periderms. They show that periderms belong to two basic categories termed exophylactic and necrophylactic. exophylactic periderm (FEP) and sequent exophylactic periderm (SEP) "which protect living tissues from external environmental factors constitute the exophylactic (exo, external; phylaca, a guard) periderms. The periderms which arise whenever dead tissues occur and regeneration of new periderm for which continuing protection is essential constitute the necrophylactic (necrus, a corpse; phylaca, a guard) periderms. Their prime function appears to be protection of living tissues from the adverse effect associated with death of c e l l s " . impervious tissue (NIT) was a prerequisite to the formation of necrophylactic periderms (NP) at sites of pathogenesis as well as in the absence of injury and pathogenesis, e.g. necrophylactic periderm abutting rhytidome. These and other studies of Mullick (1977, in preparation) showed that the process of NIT, and therefore of NP According to Mullick and Jensen (1973b) the f i r s t Mullick (1975) found that formation of a non-suber i zed - 2 -formation, is set in motion whenever phellogen, a tissue vital in seasonal renewal of phellem for accommodating circumferential growth, is damaged on penetration by any pathogens, or becomes non-functional regardless of the cause. Our understanding of plant diseases has remained stalemated largely because of the lack of knowledge of the host component in host-pathogen" interactions. The above studies show that the process of NIT formation constitutes a major host component in disease interactions that involve damage to the f i r s t layer of living c e l l s , the phellogen. In susceptible responses, the pathogen appears either to avoid damage to phellogen, or to interfere with the process of NIT formation, resulting either in the absence or considerable delay in restoration of phellogen. The direct significance of these findings in pathogenesis raised the question as to whether similar processes might occur in other plants, and thus represent a universal phenomenon in plants. As a f i r s t step towards that understanding, this thesis attempts to determine, in f i r s t , the validity of the new concepts of types of periderm formation and, in second, whether indeed NIT formation is a prerequisite to formation of a l l necrophylactic periderms. Confirmation of the above concepts is essential to development of a coherent view of the nature of the host component in host-pathogen interactions in woody plants. * pathogen is defined as any agent (microbes, insects, mites, nematodes, and parasitic plants) that causes chronic physiological disorders i.e., disease, in the host (Treshow 1970; Mullick 1975)-- 3 -2. LITERATURE REVIEW 2.1 Theory of Periderm Formation Periderm is found in almost a l l dicotyledonous and some monocotyledonous plants. In general, periderm replaces the epidermis on the spring shoots of woody plants during or after the f i r s t year of growth. Detai1ed,descriptions of periderm formation have been given by Srivastava (_1964) , Esau (1965) and Fahn (. 1967) -Briefly, periderm consists of 3 tissues: phellem (cork), phellogen (cork cambium) and phelloderm in most plants (Figure 1). The theory of periderm formation was put forth some 125.years ago by von Mohl (see Srivastava 1964). According to this theory both phellem and phelloderm are produced de novo by the lateral meristem, the phellogen. Depending upon the species of plant, periderm may arise either in the epidermis or cells immediately below the epidermis, or in several cells below the epidermis. The mechanism of cell division involved in periderm formation is illustrated in Figure 2. The phellogen cell divides into two, of which an outer cell matures into phellem, while the inner remains as the phellogen. The inner cell may divide again, the outer cell again becoming the phellem, the inner remaining as phellogen. This process of cell division leads to formation of a radial f i l e of phellem c e l l s . The phellogen cell at times may divide giving rise to a cell toward the inside while the outer cell - it -remains as the phellogen. The inner cell is referred to as a phelloderm c e l l . The phellogen may again divide inward giving rise to more phelloderm c e l l s . In general phellem cells outnumber phelloderm cells in most plants. The phellem cells on maturation die while phellogen and phelloderm cells remain alive. Phelloderm cells are similar to underlying cortical cells except they are generally smaller and are in radial f i l e s . Phellem walls in the course of maturation develop suberin, a substance that makes the phellem impervious to water. In some plants phelloderm may not form at a l l , but even then the tissue is referred to as periderm. The same process occurs in wound periderm formation, except that in some instances, the wounded surfaces may develop suberin and gum prior to periderm formation. 2.2 Classical and Modern Concepts of Periderms Prior to the discoveries by Mullick ( 1 971 ) , 'natural' periderms or the so-called f i r s t and 'usual' sequent periderms abutting rhytidomes, were believed to be alike. They were considered different only on the basis of time of origin. The differences in site of origin pertain only to the f i r s t periderm because in some plants i t arises from the epidermis, in others below the epidermis, and in st i 1 1'.others , in deeper layers of bark (Fig. 3) (Srivastava 1963, 1964; Esau 1965; Fahn 1 9 6 7 ) -- 5 -The wound periderm was believed to differ from natural periderms, because i t is induced by a stimulus of injury, or by factors other than those responsible in the origin of natural periderms (Srivastava 1964; Akai 1959; Bloch 1941, 1952, 1953). The wound periderms, sometimes referred to as pathological periderms, were believed to be induced under the stimulus of microbial toxins and insect saliva (Akai 1959; Oechssler 1962; Carter 1962; Hare 1966), hormones and auxins (Bloch 1941; Davies 1949). As early as 1953, Bloch cautioned pathologists that induction of specific responses could have resulted from chemical substances released from death or breakdown products of host eel Is alone rather than pathogen-introduced substances in the host-pathogen interaction zones. Periderms developing under normal physiological stimuli, as for example during abscission of plant parts, such as leaves and branches (Neger and Fuchs 1915; Eames and MacDaniels 1947; Facey 1956; Esau 1965; Kozlowski 1973) were referred to generally as secondary protective layers, and occasionally as cork, or simply as periderms. Distinctions in the above periderms prevailed in the literature seemingly because of differences in site, time and causal factors in thei r origin. A series of studies on periderm (Mullick 1969a, 1969b, 1971; Mullick and Jensen 1973a, 1973b) led to the discovery of equivalence, within a species, of the 1 usual 1 sequent periderm abutting rhytidomes and wound periderms regardless of cause, biotic or abiotic, and with periderms at abscission sites, and in Abies species at old resin - 6 -blisters. These periderms comprised a single category and were termed by Mullick as necrophylactic periderm (NP) . Mullick (1971) also discovered a second type of sequent periderm which developed internally abutting the phellem of NP, which is identical to the f i r s t periderm in a l l respects except site of origin. This periderm constitutes another category, the exophylactic periderm, because the cells differ distinctly from the NP category in anatomy and chemistry. For clarity, Mullick N (1975) refers to them as f i r s t exophylactic periderm (FEP) and sequent exophylactic periderm (SEP), respectively. Mullick (1971) found that SEP was associated with en masse sloughing. His overall observations have led him to the view that periderms in the exophylactic category are analogous to the normal human skin and those in the necrophylactic category to scar skin in humans. The difference is that whereas in humans the scar skin is not normally replaced by normal skin, some species of trees seemingly possess the mechanism whereby the NP'(scar skin) is eventually replaced by SEP (normal skin). In some trees SEP develops quickly, resulting in rapid sloughing of scales and dead bark and hence in a return to smooth bark, while in others, SEP develops late or not at a l l , resulting in adherence of dead bark and hence to varied morphological appearance of the bark (Mullick, unpubli shed). It is clear from the above that the concepts based on site and time of origin were only descriptive. There are such distinctions but not in the sense of the earlier literature. For example, differences in time of origin would apply between FEP and SEP but not to NP, which can develop even ahead of the FEP, e.g. at a site of injury to epidermis prior to FEP development. Differences in site of origin do apply to NP, which develops specifically in tissues internally abutting a newly discovered tissue in the bark of conifers, the hon-suberized impervious tissue (NIT) (Mullick 1975)- It is however, not the site or time of origin which is important. The importance lies in the new focus on the dynamic physiological role of periderm as functional skins rather than the age-old concepts of dead phellem serving merely as a protective cover i ng.' (Mul l i ck 1977) • 2.3 Recent Concepts of Wound Periderm Formation in Conifers Most of the studies on wound periderm formation have been carried out on agricultural crop plants such as potato, sweet potato and sugar beet, where the injured surfaces become suberized and periderm may form below the suberized surface within one to two days of injury. Mullick (1975) found that in conifers the wound periderm development is initiated usually after 3 to 4 weeks in the growing season and that the injured surfaces, contrary to literature belief remained non-suberized prior to the initiation of periderm. - 8 -However, during his studies on the development of necrophylactic periderms Mullick (1975) did find non-periderm impermeability. He sought to determine (a)> the nature of this non-periderm impermeability, (b) the fate of the several types of bark tissues such as cortical and phloem parenchyma, ray parenchyma, sclereids, and sieve elements, in the period prior to periderm initiation, (c) which cells below the injury transform to phellogen and (d) how to recognize them. . These pursuits led him to delineation of a non-suberized impervious tissue (NIT) by development of an F-F test and cryofixation technique (see Materials and Methods). NIT is an integral part of NP formation regardless of cause. The development of NIT is essential because it provides the necessary environment in tissues internally abutting it for the initiation of the meristematic activity leading to NP formation. Mullick (1975) described the sequence of visible macro changes that occur in the process of NIT formation, seen when injured bark is cut through, the injury indicating that a series of consistent changes occurred in NIT formation. In 1977, he presented an outline of a cryofixation study of NIT formation at rhytidome giving the highlight of cryofixation features through NP formation. NIT forms several cells away from the site of injury and usually is concave near the margins of an injury. NIT cells are of varied shapes, sizes and have non-uniform highly irregular walls. These cells are derived not from any meristematic activity but from stepwise hypertrophic dedifferentiation and redifferentiation of various pre-existing - 9 -tissues below t h e injury. The process is initiated soon after injury, and entails extensive biochemical and structural alterations as revealed by fluorescent characteristics using the cryofixation technique. This discovery of NIT provides for the f i r s t time a clear-cut understanding that phellogen type activity develops only in tissues internally abutting NIT. The rates of NIT formation vary on the same tree with the changing environment at different times of the year. In Abies amabilis (Dougl.) Forbes, rates varied from 14 days in June, slowing gradually to 35 days in fa 11, virtua1ly ceasing in winter and resuming slowly (70 days) in spring. This cyclical pattern in rates of NIT formation was also found in Tsuga heterophylla (Raf.) Sarg. and Thuja  pl i cata Donn (Mullick and Jensen 197b). 2.4 Process of Phellogen Restoration at Rhytidome The term rhytidome l i t e r a l l y means wrinkled dead bark, and it is invariably bounded by the usual sequent periderm. I t has been variously defined by plant anatomists. Esau (1965) included the last-formed sequent periderm as part of the rhytidome, while Fahn (1967) included only the phellem of the usual sequent periderm as part of the rhytidome, while s t i l l others e.g. Chang (1954a, 1954b) and Srivastava (1964) did not include the periderm as part of the rhytidome. Mullick and Jensen (1973b) reasoned that the living plant must have functional s k i n , e.g. periderm. Therefore they - 10 -considered the latest sequent periderm as part of the living bark and excluded it from the definition of rhytidome. The periderm according to the new terminology is referred to as necrophylactic periderm abutting rhytidome. It is we 11 known that rhytidome formation is initiated around a certain age depending upon the species. For example, in Abies amabi1i s (Dougl.) Forbes, the FEP is replaced by NP during rhytidome formation after 35 to 40 years, i n Tsuga heterophylla (Raf.) Sarg.. after 12 years and in Abies holophylla after only 2 to 3 years. It was believed in the literature that depending upon the age, the f i r s t periderm (FEP) is replaced by sequent periderm (NP) which cut off the outer tissues of bark. These tissues undergo cellular modifications and finally die (Esau 1965; Srivastava 1964; Fahn 1967). Thus development of the NP was the cause of the death of bark external to the NP and therefore the formation of rhytidome. Although injury could be justified as a cause for NP formation at sites of wounding, pathogenesis and even at abscission, i t could not account for the formation of NP at the rhytidome which develops, in the absence of injury and disease, as an inherent autonomous physiological response. Mullick (1975) was therefore interested in understanding the common causal factors in the origin of NP at varied sites of their occurrence. He found that published views on the rhytidome formation were only conjectural because no criterion for detection of rhytidome initiation was known, leave alone developmental studies on rhytidome formation. - 11 -Mullick (1977), after defining criteria for detecting initiation of rhytidome development, devised techniques for studying the process of rhytidome formation in Tsuga heterophylla. It is well-known that phellem of a l l periderms dies soon after differentiation. He found that the FEP phellogen becomes active largely early in summer and renews the FEP phellem for accommodating cireumferentia1 growth. Without this renewal, the dead phellem layer would rupture, leaving underlying living tissues unprotected. So long as the FEP phellogen remains functional, the FEP phellem is renewed year after year and the bark surface remains smooth. In different species, at certain ages which are characteristic for each species, regions of the FEP phellogen become non-functional and are therefore unable to renew FEP phellem which is vital to protection of the plant. The tree therefore sets in motion a complex physiologica1 process of phellogen restoration so that new phellem can be produced for continuing protection of the tree in subsequent years. This restorative process invariably leads to NIT formation and then periderm is formed abutting i t . This periderm is always of the necrophylactic category. Thus, contrary to literature belief, NP is not the cause of death and therefore rhytidome formation, but it is the non-functionality of the phellogen that results in NP formation and rhytidome formation, i.e. dead bark results largely after the process of NIT formation has been completed. - 12 -The process of NP formation abutting rhytidomes therefore represents an inherent endogeneous physiological process of phellogen restoration in the absence of injuries and pathogens. 2.5 An Overview of Recent Concepts and Terminology of Permiderms Mullick illustrates his concepts of periderms by comparing plant periderms (plant 'skins') with human skins. In this comparison, FEP is equivalent to normal skin. Normal skin at small injuries is restored by the division of dermal tissue surrounding the injured dermis. The phellogen cells surrounding the injury do not divide to cover the injured zone. Therefore, plants lack the abili t y to regenerate FEP directly. When the f i r s t living cell (i.e. a phellogen or epidermis) is killed (injury) or is even functionally damaged (rhytidome formation - see Section 2.4), eel Is below the injury undergo dedifferentiation and lead to formation of NIT and finally to that of NP and not FEP. NP is analogus to scar tissue arising as a result of dedifferentiation at surgical injuries.to humans. Unlike animals, trees always f i r s t form a 'scar skin' following injury. In trees however, SEP which is in a l l ways equivalent to FEP, the 'normal skin' may replace NP, the 'scar skin'. So although trees always f i r s t form a scar skin regardless of the size of the injury they have the abil i t y returning to a normal state by replacing the scar tissue with normal 'skin' (SEP). Humans lack the - 13 -a b i l i t y to replace scar tissue with normal skin. Mullick believes that the abil i t y of trees to replaces NP (e.g. scar skin) with normal skin (e.g. SEP) should be reflected in gross aspects of bark morphology. From the findings in the three conifers, he suggested that sloughing of rhytidome tissue involves SEP formation and it occurs at the boundary between NP phellem and SEP. From this he reasoned that SEP development plays a key role in bark "morphology, since if SEP forms soon after NP, sloughing should occur quickly and the bark surface would remain smooth while if SEP forms slowly, infrequently, or not at a l l , the rhytidome tissue would adhere, giving a rough external appearance to the bark. This suggested role of SEP with its effect on bark morphology is based on results from a limited number of species and requires more detailed investigation. 2.6 General Developmental Features of NIT Formation Developmental studies of NIT formation at rhytidome in Tsuga heterophylla and at injuries i h Ab ies amab i l i s , Abi es grand i s and Tsuga heterophylla (Mul1ick 1975, 1977, personal communication) are summarized below. Following the i n i t i a l k i l l i n g of superficia 1.eel 1s at the time of injury internally abutting cells continued to die and progressive modification of tissues internally'occurred including - ]k -cell hypertrophy, culminating in NIT and NP formation. Anatomical and biochemical modifications of tissues were indicated in part by the changes in fluorescence and other characteristics of the cells and their contents in the cryofixed state. These particular observations were unique to the cryofixation technique. Cell division was not observed during NIT development, but it was manifest in NP formation. The process of NIT development, which involved only enlargement of pre-existing c e l l s , is referred to as hypertrophic dedifferentiation and that of the NP formation which involved both cell enlargement and cell division is referred to as meristematic differentiation. In general, NIT consisted of en 1arged eel 1s with thickened walls of varying sizes and shapes. These eel Is appeared yellowish brown and intercellular spaces did not exist between these eel 1s. Histological testsshowed that NIT imperviousness was not due to suberin or callose. 2.7 The Role of Non-suberized Impervious Tissue (NIT) and Necrophylactic Periderm (NP) in Pathogenesis Despite extensive researches on host-pathogen interactions, progress in understanding the process of disease, as well as resistance and susceptibi1ity to disease has remained stalemated because of the lack of knowledge of the host component in host-pathogen interactions. - 15 -Such processes had remained unknown for lack of techniques for studying physiological processes. Understanding the sequence of metabolic and anatomical alterations in physiological processes would be greatly enhanced if it were possible to compare these alterations by direct observations, beginning with the normal state of c e l l s . To avoid losses of chemicals caused by usual histological techniques, Mullick used the cryofixation technique which involves freezing of tissues under appropriate conditions, cutting frozen sections and examining them while s t i l l frozen, using fluorescence and other optical microscopy techniques. The technique yielded sequential chemical and anatomical land-marks which provided new insights in understanding the physiology of plant development. Mullick ( 1 9 7 7 ) presented "evidence and supportive rationale to show that whenever functioning of tissues essential to a tree is affected, regardless of cause, non-specific autonomous processes are triggered for their restoration". These processes involve dynamic metabolic and anatomical alterations, and once triggered, occur automatically in pre-existing totipotent ce l l s . Phellogen, as discussed above, is one of the tissues essential to trees because i t accommodates circumferential growth of bark through seasonal renewal of the impervious outer covering , the phellem. Phellogen is the f i r s t 1iving tissue affected during penetration of the stem by pathogens. Through use of the cryofixation technique it was shown that whenever phellogen becomes non-functional, - 16 -regardless of cause, the autonomous process of phellogen restoration, constituting a major non-specific host component in host-pathogen interactions, is initiated. The process of phellogen restoration entails the formation of periderms of the necrophylactic category, including wound periderms. Wound periderm has been assigned only a passive role in defense partly because it is found several cells away from the site of pathogen containment. This seemed to be a logical inference at a time when the process of its formation prior to the establishment of phellogen had remained l i t t l e understood, (see Section 5.6). Mullick found that phellogen restoration, involving necrophylactic periderm formation, is an active process in defense interactions, since it is triggered as soon as the functionality of the f i r s t living layer of cells is affected. If, however, phellogen is not affected during a defense interaction, then the process of its restoration is not triggered, and may lead to susceptibility. It is well known that stresses resulting from fluctuations in environment, e.g. temperature, water, light, site and soil may weaken trees and predispose them to outbreak of disease and epidemic . Since we now.have an understanding for the f i r s t time of a physiological process which constitutes a host component in host-pathogen interactions, it has now become possible to measure how this physiological process is affected by various factors of stress. Puritch and Mullick (1975) showed that water stress greatly retards the rate of NIT formation after mechanical injury. The resumption of the normal rate - 17 -of NIT development was observed when the stress signs were alleviated on watering. Mullick and Jensen (1976) found the rates of development of NIT vary on the same tree with the changing environment at different times of the year. Fluctuations in the environment occasioned by year-to-year within-season variations appear to affect the physiological process of NIT formation. Intraspecific variations in.the rates of NIT formation were also observed. Observations on thi rteen Abies  amabi1i s showed intraspecific .variation, in the rate of NIT formation. Defects in the process of NIT formation at points of mechanical injuries contribute to the slowness of NIT formation. Such defects could be partly responsible for the intraspecific variations (Mullick and Jensen 1976). Rates of NIT formation were also found consistently faster on the resistant than on the susceptible Abies amabi1is heavily infested with balsam woolly aphid. Here, the infestation stress and defects in NIT formation, singly or together, may be responsible for the differences in the rates of NIT formation. Since health is the normal, and disease is the abnormal, yet any individual may become diseased and either recover or succumb to i t , one may assume that certain factors of stress may predispose it to attack by a specific pathogen (Mullick and Jensen 1976). It is therefore necessary to know how various environmental factors which promote a given pathogenic condition affects this process. Since the involvement of phellogen restoration in pathogenesis is non-specific, it appeared desirable for the purposes of this thesis to determine the general validity of the exophylactic and necrophylactic - 18 -peridermal processes in other woody plants. 2.8 Cryofixation - a New Technique for Studying Developmental Processes in Plants The cryofixation method of tissue preparation, recently developed by Mullick (1971) was used for the purpose of interpreting exophylactic and necrophylactic periderms. The significance of the cryofixation technique (Mullick 197') in studying developmental processes in plants has been discussed by Mullick (1977)- He summarized the problems inherent in the usual histological techniques such as fixation artifacts and removal of soluble constituents. He pointed out that under appropriate conditions, the contents of a cell could be fixed by freezing thereby retaining cytological conditions approximately unchanged from the living state, and avoiding problems inherent in classical techniques. The cryofixation technique consists of fixation by freezing, cutting cryostat sections and observing the frozen section on a freezing stage with a variety of optical techniques, including fluorescence in place of conventional histochemical tests. Comparable samples may be studied by conventional light or electron microscopy. The technique has an additional advantage of rapid sample processing, as desired for extensive surveys of cellular changes. The technique has "revealed many natural characteristics of c e l l s , obtained without staining or other (chemical) treatment which permits physiological interpretations of anatomical aspects of growth and development" - 19 -as shown by t h e de v e l o p m e n t a l s t u d i e s r e p o r t e d by M u l l i c k ( 1 977 ) . F u r t h e r , i t r e v e a l s s t e p w i s e c h e m i c a l changes, i n d i c a t e d by f l u o r e s c e n c e changes i n c e l l w a l l s and c o n t e n t s t h a t o c c u r under v a r i o u s c o n d i t i o n s , e.g. t h o s e . f o l l o w ! n g wounding. Through d e v e l o p m e n t a l s t u d i e s , c h a r a c t e r i s t i c p a t t e r n s o f c e l l u l a r m o d i f i c a t i o n can be d e t e r m i n e d and landmarks e s t a b l i s h e d , s u c h as f o r i n s t a n c e , t h e s t a g e a t w h i c h f l u o r e s c e n t r e t i c u l a t e d c o n t e n t s d e v e l o p i n c e r t a i n h y p e r t r o p h i e d c e l l s p r i o r t o NIT f o r m a t i o n and the f l u o r e s c e n t c e l l w a l l s o f p h e l l o g e n as markers o f t h e non-f u n c t i o n a l s t a t e o f p h e l l o g e n ( M u l 1 i c k 1977)- These and o t h e r parameters ( M u l l i c k , u n p u b l i s h e d ) p r o v i d e the c r i t e r i a f o r i n t e r -p r e t a t i o n s i n the s t u d i e s p r e s e n t e d h e r e . - 20 -3. MATERIALS AND METHODS 3.1 Exophylactic and Necrophylactic Periderms The concepts of exophylactic and necrophylactic periderms resulted from detailed studies on one of the four families of Coniferales, namely the Pinaceae (Mullick 1971, Mullick and Jensen 1973b). In this thesis, 15 species with representatives from a l l coniferous families and a few families of Angiosperms (Tables 1 and 2) were investigated to examine the general validity of the new concepts of periderms. Some of these species were selected because of their economic importance, others because of their convenient availability. Healthy trees of various ages (Tables 1 and 2) growing outdoors were used, except Hibiscus syriacus which was maintained in a greenhouse. 3.1.1 Collection of Samples of FEP, SEP and NP Abutting Rhytidome As a f i r s t step, necrophylactic and exophylactic periderms can often be differentiated by microscopic examination of their pigments in cut bark samples. This differentiation is ascertained best by stereomicroscopic examination of the sample. The pigments are found in phellem, having developed usually during the final stages of phellem maturation. While examination of phellem at early stages of its maturation is informative and useful, it cannot be used for comparative purposes because the distinguishing features change during maturation (Mullick - personal communication). - 21 -In most woody trees studied, epidermis in the newly flushed leader and branch tips is generally replaced by FEP either about the end of the f i r s t year's growth or early in the following growing season. In Acer macrophy11 urn the replacement is in patches and the epidermis surrounding the patches remains functional for several years (Figs. 66 and 70')'. The epidermis is either sloughed off at the time of FEP formation or remains attached to the bark for a few years after the development of FEP, resulting in a smooth or slightly roughened appearance of the bark in the crown of the tree. In general, after the epidermis is sloughed off, the surface of the FEP becomes smooth and remains as such until formation of rhytidomes. The rhytidome appears when the tree has reached a certain age range, which usually is characteristic of * the species (Table 3)» for example, in the Pyrus spec ies studied, its formation is initiated after approximately 6 years, and in Pseudotsuga menziesii after 16 years (Table 3)- Thus, in Pyrus, the FEP samples were collected from internodal areas 2 to 6 and NP samples abutting rhyti-domes from internodal areas 6 to 10 years old. Similarly in Pseudotsuga menziesii, FEP was collected from second to sixteenth and NP from sixteenth to twentieth internode" respectively. The latter region generally contained only a single layer of rhytidome. * Current year's terminal growth designated as internode 1. - 22 -Although NP can be obtained from regions of bark with several layers of rhytidome, processing and sectioning of such samples encounters d i f f i c u l t i e s . To avoid sectioning d i f f i c u l t i e s , samples of NP were collected generally from zones where it had formed recently, and consisted of only a single layer. Extensive searches were necessary to obtain appropriate samples of SEP partly because factors associated with SEP development are unknown and partly because its development cannot be observed in the inner regions of bark by external visual examination. In general, regions of needle abscission scars, and rhytidome showing a slight tendency towards sloughing and scaling reveals the presence of SEP. 3.1.2 Samples of Necrophylactic Periderms at Healed Sites of Mechanical Injuries, Abscission Zones, Resin Blisters and Pathogenesis Shallow mechanical injuries (ca. 1/5th of bark thickness), made with a scalpel on April 11, 1974 and June 17, 1974, were collected on October 4, 1974 for examining the features of NP in al l species studied. Samples of abscission scars were col 1ected from the 4th to 6th internodes. of Picea glauca and those of resin blisters from Pseudotsuga menziesi i and Picea si tchensis. Samples of NP at healed sites of pathogensis were obtained from leaders of Picea sitchensis attacked by Pissodes strobi. These trees of about 30 and 20 years were found at U.B.C. Research - .23 " F o r e s t a t Maple Ridge and a t t h e p l a n t a t i o n o f the P a c i f i c F o r e s t Research C e n t r e (PFRC) o f t h e Canadian F o r e s t r y S e r v i c e , V i c t o r i a , B.C. ( C o u r t e s y o f Dr. L.H. M c M u l l e n ) . B e e t l e h o l e s on t h e stem and from branches o f Pseudotsuga m e n z i e s i i ( a p p r o x i m a t e l y 35 y e a r s o l d ) i n f e c t e d w i t h C a l i c i o p s i s pseudotsugae F i t z . , from P i n u s c o n t o r t a (about 25 y e a r s o l d ) a t t a c k e d by E n d o c r o n a r t i urn harkness i i ( J . P . Moore) Y. H i r a t s u k a and from branches and stems o f P i nus mont i c o l a ( a p p r o x i m a t e l y 20 y e a r s o l d ) i n f e c t e d by C r o n a r t i u m r i b i c o l a F. and Cucurb i doth i s  p i t h y o p h i l a F r . were i d e n t i f i e d by Dr. R. Hunt, PFRC, V i c t o r i a , B.C. 3.1.3 C r y o f i x a t i o n Technique Samples were brought to t h e l a b o r a t o r y i n p l a s t i c bags over i c e i n a dewar f l a s k f o r p r o c e s s i n g . D e t a i l e d c h a r a c t e r i z a t i o n s o f the two c a t e g o r i e s o f p e r i d e r m s were done by the c r y o f i x a t i o n t e c h n i q u e ( M u l l i c k 1971). In g e n e r a l , 10 urn t h i c k s e c t i o n s were c u t w i t h an I n t e r n a t i o n a l C r y o s t a t Model C T l , p i c k e d up on a p r e -c h i l l e d s l i d e and mounted i n p r e e h i l l e d c r y o s t a t o i l ( C r y o - c u t M icrotome L u b r i c a n t , A m e r i c a n O p t i c a l C o r p o r a t i o n ) c o v e r e d by a p r e e h i l l e d c o v e r g l a s s , a l l a t a p p r o x i m a t e l y -20C. The p r e p a r e d s l i d e w i t h the mounted f r o z e n s e c t i o n s was examined on a C a r l Z e i s s f r o z e n s t a g e c o o l e d t o about -35C. The c o n t e n t s of p e r i d e r m i n c r y o f i x e d s e c t i o n s were examined w i t h t u n g s t e n i l l u m i n a t i o n b r i g h t - 24 -field (tbf) and mercury bright f i e l d (mbf). Cell walls were observed with polarized light with partially (pep) or fully crossed polarizing (fcp) f i l t e r s . Fluorescent materials in cell contents and cell walls were examined with the combination of Carl Zeiss exciter and barrier f i Iters 1/53 and IV/41. In addition, the nature of modifications occurring in phellem contents when cryofixed sections mounted in o i l were allowed to thaw were examined with the microscope (see Appendix). 3.1.3-1 Photomicrography Techniques and Their Limitations Since cryofixed sections are not permanent, the only way to obtain a large permanent body of data is to use extensive photomicrography. Photomicrography of cryofixed sections (and sections following histochemica1 testing, Section 3-1.4) was done on a Carl Zeiss Photoscope II using the built-in 35 mm camera and High Speed Ektachrome - Tungsten transparency film (EHB-135~36, Eastman Kodak Co.). For a l l tbf, mbf, pep and fcp photomicrographs, the automatic exposure mode was used. A modified procedure, developed in Mullick's laboratory, was used for fluorescence photomicrography. This was required because of the relatively very low light level of the fluorescence which necessitated long exposure times and because, with the Photoscope II, only half the light gathered by the objective is delivered to the film. By use of a cable release, al1 the light - 25 -can be diverted to the film, but then automatic monitoring of the exposure is not possible. Therefore, a calibration curve as prepared by Mr. G.D. Jensen, was used for calculating exposure time for film having a speed rating of DIN 40 (ASA 8000).. The conversion factor for 1/53 photomicrographs was an actual exposure of 22 seconds for one second indicated exposure time, and the factor for IV/41 was 90 seconds. Care must be taken to eliminate any light entering the eyepieces as this would shorten the indicated exposure and result in an underexposure. With this procedure, exposure time is cut in half but accuracy of correct exposure is maintained. This results in a significant time saving, with average exposures reduced to an average range of 4 to 8 minutes from 8 to 16 minutes. These long exposure times produce d i f f i c u l t i e s in rendering accurate color in photomicrographs because of reciprocity failure of the film. Thus, for c r i t i c a l color comparisons, photo-micrographs of close to the same exposure time must be used. Even with this limitation however, photomicrographs are s t i l l essential, providing the only permanent record of characteristics and the only opportunity for objective, direct comparison between samples. With the above proviso, photomicrograph transparencies proved to be a good record. The greatest problem in accurate color reproduction lies in the preparation of color prints. Because no internal reference color exists in the photomicrographs comparable to fleshtones or an included color bar injuore conventional color photography, commercial printers experience great d i f f i c u l t y in establishing appropriate'printing - 26 -c o n d i t i o n s f o r n e g a t i v e s . Here, t r a n s p a r e n c i e s a r e p r e f e r a b l e t o n e g a t i v e s i n t h a t t h e i r p r o c e s s i n g i s f u l l y a u t o m a t i c , r e q u i r i n g no o p e r a t o r judgement, and i f they a r e s u b s e q u e n t l y used t o o b t a i n p r i n t s , t he p r i n t e r has an o b j e c t i v e r e f e r e n c e ( the c o l o r t r a n s p a r e n c y ) t o w h i c h t o work, a l t h o u g h such "hand p r i n t i n g " i s e x p e n s i v e . Even so, e x p e r i e n c e has shown t h a t c o l o r , r e p r o d u c t i o n i n p r i n t s f o r f i g u r e s i s a t b e s t never e x a c t . T h e r e f o r e , p r i n t s cannot be used f o r d e t a i l e d c o m p a r i s o n s and a r e used o n l y t o show g e n e r a l f e a t u r e s t o o r i e n t t h e r e a d e r . 3-1.4 Histochemica1 T e s t s S u b e r i n i n p h e l l e m was d e t e c t e d by t r e a t i n g a i r - d r i e d c r y o s t a t s e c t i o n s w i t h Sudan III i n e t h y l e n e g l y c o l (Gurr 1965), phosphene 3R, methylene b l u e (Gurr 1956), and ammoniurn h y d r o x i d e -c r y s t a l v i o l e t ( T i s o n 1899) as d e s c r i b e d by M u l l i c k (1975). These t e s t s were p a r t i a l l y d e v e l o p e d by the a u t h o r . A i r - d r i e d s e c t i o n s on s l i d e s were t r e a t e d w i t h 2% phiorog1ucinol i n a b s o l u t e e t h a n o l f o r 2 minutes f o l l o w e d by a d d i n g 2-3 drops o f c o n c e n t r a t e d h y d r o c h l o r i c a c i d on t h e s e c t i o n s and ob s e r v e d i m m e d i a t e l y under t h e m i c r o s c o p e . The same sequence o f pr o c e d u r e s was f o l l o w e d w i t h a n i l i n e s u l f a t e ( s a t u r a t e d aqueous s o l u t i o n ) and c o n c e n t r a t e d h y d r o c h l o r i c a c i d . These t e s t s were used t o s t a i n. 1 1 i g n i f i e d 1 t i s s u e s (.Purv i s e t a 1 . 1966) . - 27 -Differences in solubility of pigmented contents in phellem of FEP, SEP and NP were studied with a microscope by mounting air-dried cryostat sections separately in absolute ethanol, petroleum ether, diethyl ether, and also after soaking such sections for 7 days at room temperature in the three solvents. 3-2 Non-Suberized. Impervious Tissues (NIT) The species studied which were the same as those used in periderm studies, are included in Table 1. Shallow mechanical injuries 1 to 2 mm deep were made on June 11, 1974 with a scapel on the smooth areas of bark. Samples were collected on alternate days until the development of NIT and initiation or, in some cases completion, of periderm formation. A 4 x 4 cm bark sample was removed to the depth of the vascular cambium making sure that the injury was in the centre of the sample. Care was necessary in removing the samples, particularly during and after NIT formation so that the samples remained intact. The samples were brought to the laboratory in plastic bags over ice in a dewar. A large number of rhytidome samples were collected from June to August, 1974, from each species so that recently formed NIT structures could be examined. Needle scars and weevil (P i ssodes strobi) attacked leaders were obtained from Picea glauca and Picea  sitchensis respectively. Old resin. blisters and beetle holes were collected from Pseudotsuga menziesi i . Bark samples were also collected from - 28 -diseased branches and stems of Pinus monticola attacked by Cucurbidothis pithyophila Fr, and Cronartium ribicola F. respectively. In addition, prepared slides of necrophylactic periderms from the previous section were re-examined for the presence of NIT. 3 . 2 . 1 Detection of NIT by F-F Test The average bark thickness of the species studied was about 5 to 6 mm. F-F test (Mullick 1975 ) as described below, was used for detecting impermeability resulting from NIT at mechanical injuries and rhytidome. The test can be carried out in two ways. 3 . 2 . 1 . 1 F-F Test Through Wound Surface A wax well was built around the injury by f i r s t depositing layers of smoking hot paraffin wax and then placing the sample on a piece of polyurethane foam in a petri dish containing water to ensure the bark remained hydrated. The well was f i l l e d with 2% ferric chloride. After 3 days, the ferric chloride solution was removed, the well was rinsed with water, r e f i l l e d with k% potassium ferricyanide and returned to the petri dish for another 3 days. Then, after removal of the wax and rinsing with water, the samples were.cut open to determine the depth of permeation as indicated by the blue color produced by the reagents. - 29 -Rhytidomes, a b s c i s s i o n s c a r s , r e s i n b l i s t e r s and d i s e a s e d samples were t e s t e d 'through a wounded s u r f a c e ' by mec h a n i c a l removal o r s c r a t c h i n g o f t h e p e r i d e r m from t h e c e n t r e so as t o p e r m i t p e n e t r a t i o n o f t h e t e s t s o l u t i o n s t o t h e zone o f NIT f o r m a t i o n . 3.2.1.2 F-F T e s t Through Cambial S u r f a c e The edges o f t h e bark sample were s e a l e d w i t h smoking hot wax a p p l i e d w i t h a P a s t e u r p i p e t . The sample was then p l a c e d on p o l y u r e t h a n e foam i n a p e t r i d i s h , and 2% f e r r i c c h l o r i d e added u n t i l t h e sample was about t o f l o a t . The p e t r i d i s h was c o v e r e d , and the s o l u t i o n a l l o w e d t o d i f f u s e t h r o u g h t h e b a r k sample toward t h e wound s u r f a c e f o r 3 days. The c a m b i a l s u r f a c e o f the sample was then r i n s e d w i t h water and kept f o r 3 days i n k% p o t a s s i u m f e r r i c y a n i d e s o l u t i o n i n a p e t r i d i s h . The r i n s e d , b l o t t e d sample was c u t open w i t h a v e r y s h a r p s c a l p e l t o r e v e a l t he e x t e n t o f pe r m e a t i o n . Minimum p r e s s u r e was a p p l i e d when the t e s t e d samples were b e i n g c u t so t h a t e r r o n e o u s i n t e r p r e t a t i o n r e s u l t i n g from smearing o f the b l u e c o l o r c o u l d be reduced. 3-2.1.3 D e t e c t i o n o f NIT by C r y o f i x a t i o n C h a r a c t e r i s t i c s A l l b a r k samples d e s c r i b e d i n S e c t i o n 3-2 were examined f o r c r y o f i x a t i o n c h a r a c t e r i s t i c s as d e s c r i b e d by Mul l i c k (.1975, 1977) -C r y o s t a t s e c t i o n s from t h e s e samples were a l s o used f o r d e t e r m i n i n g c h e m i c a l c h a r a c t e r i s t i c s o f NIT as d e s c r i b e d n e x t . - 30 -3.2.2 H i s tochemica1 T e s t s The f o l l o w i n g t e s t s were c a r r i e d out on a i r - d r i e d c r y o s t a t s e c t i o n s to d e t e r m i n e the p r o b a b l e chemica l n a t u r e o f the s u b s t a n c e o r s u b s t a n c e s a s s o c i a t e d w i t h NIT i m p e r m e a b i l i t y . T e s t s f o r s u b e r i z a t i o n o f NIT c e l l w a l l s were s e l e c t e d from tho se d e s c r i b e d by M u l l i c k (1975). Sudan III in e t h y l e n e g l y c o l was used (Gurr 1965) but s e c t i o n s were not dehydra ted w i t h e t h y l e n e g l y c o l as recommended. No d i f f e r e n c e was found between a i r - d r i e d s e c t i o n s w i t h or w i t h o u t p r e t r e a t m e n t w i t h e t h y l e n e g l y c o l . Phosphene 3R (0.1%) and methy l ene b l u e (1%) in d i s t i l l e d water were used (Gurr 1956) and s e c t i o n s were examined under UV i l l u m i n a t i o n w i t h the Ca r l Z e i s s f l u o r e s c e n c e f i l t e r c o m b i n a t i o n IV/41, w i t h which s u b e r i z e d c e l l w a l l s f l u o r e s c e d s i l v e r and l i g h t b l u e a f t e r t rea tment w i t h phosphene 3R and methy lene b l u e r e s p e c t i v e l y . A m o d i f i e d ammonium h y d r o x i d e - c r y s t a l v i o l e t ( T i s o n 1899) was a l s o employed. The s t a i n s o l u t i o n was a lways f r e s h l y p r e p a r e d . S e c t i o n s a f t e r t r ea tmen t w i t h Sudan III and ammonium h y d r o x i d e - c r y s t a l v i o l e t were a l s o examined w i t h f l u o r e s c e n t f i l t e r c o m b i n a t i o n s 1/53 and IV/41. With e i t h e r s t a i n , s u b e r i z e d c e l l w a l l s f l u o r e s c e d red w i t h bo th 1/53 and IV/41. The a n i l i n e b l u e - v i s i b l e l i g h t method, a n i l i n e b l u e -f l u o r e s c e n c e method, and r e s o r c i n b l u e were used as t e s t s f o r the p r e s e n c e o f c a l l o s e (Jensen 1962), a p o s s i b l e s u b s t a n c e o f NIT impermeab i1 i t y . - 31 -Phloroglucinol [2% in absolute ethanol) and concentrated hydrochloric acid, aniline sulfate (saturated aqueous solution) and concentrated hydrochloric acid (Purvis et_ a_l_. 1966), and Maule reaction (Rawlins 1933) were employed as lignin tests. These tests were also used for the detection of wound gum (Hewitt 1938). According to Hewitt, wound gum reacts positively to most lignin tests but does not color with the Maule reaction. Cryostat sections containing only NIT or both NIT and necrophylactic phellem were exposed to fumes of concentrated aqueous ammonium hydroxide ( 2 8 . 0 to 30.0%). Colorless plant tissues containing flavonoids with free phenolic groups (e.g. flavones, flavonols) turn yellow and yellow tissues deepen in shade (Craft and Audia 1962) with this reagent. Sections after soaking in either absolute ethanol, diethyl ether, petroleum ether, benzene, or xylene for seven days at room temperature were treated with phloroglucinol-HCl and aniline sulfate-HCl to see if the stainable substance or substances contained in the NIT cell Walls were extractable. - 32 -4. RESULTS 4.1 Investigation of Exophylactic and Necrophylactic Periderms in Gymnosperms In general, the characteristic features of FEP, SEP, and NP in the four families of gymnosperm as revealed by cryofixation technique are summarized in Tables 4 and 5, and are presented below species by species within a family. Approximate ages of rhytidome initiation in species studied are shown in Table 3-4.1.1 Pi naceae 4.1.1.1 Picea glauca (Moench) Voss. (White spruce) Picea engelmannii Parry (Engelmann spruce) Picea sitchensis (Bong) Carr. (Sitka spruce) (i) First exophylactic periderm (FEP). : In these species, FEP was found 2 to 3 cells below the epidermis of the stem around the end of the f i r s t year of growth. Needles are shed much earlier than the petioles. During the sloughing of the epidermis and needle bases in subsequent years of growth, the bark surface appears f a i r l y rough in a l l species and may be confused for rhytidome formation (Figs. 4 and 5)- In Picea - 33 -g1auca and Picea engelmanni i sloughing of the needle bases were from the sixth and eighth internodal area respectively, while in Picea sitchensis sloughing of this tissue was from the third internodal area. The FEPs consisted of alternating layers of thin-and thick-walled phellem, the contents of these phellem cells were brown (tbf). Brown pigmented contents were always found in the thin-walled phellem c e l l s . Thin-walled phellem was always found abutting the epidermis. The phellem contents of FEP in Picea glauca and Picea engelmannii appeared light brown and dark brown, but in Picea sitchensis they appeared dark brown and black in cryofixed sections under fluorescence f i l t e r combinations 1/53 and IV/41 respectively. Under f i l t e r s IV/41, walls of the thin-walled phellem fluoresced pale blue and those of the thick-walled phellem blue-green, however, under f i l t e r s 1/53, the thin and thick phellem walls of the three species fluoresced yellowish green (e.g. Figs. 6 to 9 ) • Early in the growing season, the thick-walled phellem at the time of its formation is turgid and rectangular in transverse section (Fig. 10), but becomes distorted and compressed towards the end of the growing season because of pressure of growth. When the cryofixed sections mounted in o i l were thawed, the phellem contents did not flow out and the cellular configurations remained compressed and distorted almost identical to that seen in the cryofixed state. - 34 -(ii) Necrophylactic periderm (NP) In P_. glauca and P_. s i tchens i s rhyti domes develop after approximately 15 years and in P_. engelmanni i after approximately 17 years (Table 3 ) • Cryofixation characteristics of NPs found adjacent to rhytidome in Picea glauca, Picea engelmannii and Picea  sitchensis are shown in Table 4. Some of the features of NP found next to rhytidome in Picea sitchensis are shown in Figs. 11 to 14. The necrophylactic phellem was thin-walled in a l l species. The contents of the phellem in cryofixed sections of Picea sitchensis were pink while those of Picea glauca and Picea engelmannii were colorless (tbf) but in the whole sample block appeared cream colored under a stereomicroscope. The mature NP phellem, like the mature FEP, are compressed and distorted (Figs. 11 to 14) but in early stages of maturation, they were found to be turgid and undistorted. When cryofixed sections mounted in o i l were thawed the contents of the phellem disappeared (see Fig. 15). Under fluorescence, the phellem contents in both P_. glauca and P_. engelmanni i appeared dark (1/53, IV/41) while in P_. s i tchens is they appeared dull red (1/53) and pale blue (IV/41). The phellem walls of the Picea species fluoresced greenish-yellow (1/53) and blue (IV/41). NP at needle abscission scars in P_. glauca and around spruce weevil attack sites in P_. sitchensis are shown in - 35 -Figs. 16 and 17, respectively. Within a species these periderms as well as those found at injuries (e.g. Fig. 18) and resin blisters were similar to those at rhytidomes as judged by a l l of the cryofixation characteristics studied (Table 5 ) . Thus within a species periderms at abscission scars, resin blisters, wounds and sites of pathogen attack were found to be identical to one of the normal periderms, referred to in the earlier literature as the sequent periderm i.e. the NP at rhytidomes according to the new concepts of periderms. Exfoliation of dead tissues was observed commonly in these species at old sites of rhytidomes, injuries and abscission scars. Breaks usually occurred in the necrophylactic phellem leaving a few layers of it adhering to the SEP phellem. The adhering NP phellem weathers away with time so as to expose the SEP. ( i i i ) Sequent exophylactic periderm (SEP) SEP in Picea species develops abutting the entire unexposed NP, and constitutes the outer surface of the bark following sloughing of dead tissues at rhytidomes, injuries, diseased sites and abscission scars. SEP was most commonly found in bark where there was sloughing or scaling of dead tissue. These observations support the belief of Mullick (1971) that SEP is associated with en masse sloughing or scaling. - 36 -SEP was always found abutting the NP at rhytidomes, injuries, abscission scars except in the year NP formed. Within a species, SEP were identical to FEP on the basis of a l l cryofixation characteristics (Table 4 ) . The f i r s t SEP cells were always thin-walled, like the FEP. Histochemical tests showed that the NP phellem and the thin-walled phellem of FEP and SEP were suberized, but the thick-walled phellem was positive to 'lignin' sta i ns. 4 . 1 . 1 . 2 Pinus contorta Dougl. (Lodgepole pine) Pinus monticola Dougl. (Western white pine) (i) First exophylactic periderm (FEP) FEPs of P i nus contorta and P i nus monticola were found 2 to 3 cells below the epidermis. The epidermal surface of Pinus contorta remains rough for 3 to 4 years, as in the Picea species, because of the adhering needle bases. In general, in Pinus contorta the epidermis and the needle bases begin to shed at the 4 t h internode from the tip and their sloughing becomes pronounced at the 5 t h and 6 th internodes. Thus, the bark surface appears f a i r l y rough up to the 6 t h internode, but becomes smooth from the 7 th internode until rhytidome formation. In contrast to Pinus contorta, in Pinus monticola the epidermis began shedding at the 2nd and 3 rd internodes. The bark surface became smooth at the 4 t h internode until rhytidome - 37 -f o r m a t i o n . The c o n t i n u e d p r e s e n c e o f an i n t a c t c o n t i n u o u s l a y e r o f p h e l l e m d u r i n g stem d i amete r i n c r e a s e , and t r a c e s o f s l oughed p h e l l e m on the o u t e r s u r f a c e in both s p e c i e s i n d i c a t e sea sona l renewal o f FEP to accommodate the i n c r e a s e in c i r c u m f e r e n t i a l g rowth . The FEP o f P inus c o n t o r t a and P inus m o n t i c o l a c o n s i s t s o f a l t e r n a t i n g l a y e r s o f t h i n - and t h i c k - w a l l e d phe l l em w i t h brown c o n t e n t s , each o f wh ich was s e v e r a l c e l l s t h i c k . Whereas in P inus m o n t i c o l a the t h i c k - w a l l e d p h e l l e m had u n i f o r m l y t h i c k e n e d w a l l s , in P inus c o n t o r t a t h e i r o u t e r t a n g e n t i a l w a l l s were u s u a l l y t h i c k e r than the i n n e r w a l l s ( F i g s . 19 and 2 0 ) . With f l u o r e s c e n c e , the c o n t e n t s o f both t h i n - and t h i c k - w a l l e d p h e l l e m in the two P inus s p e c i e s appeared brown (1/53) and da rk brown ( I V / 4 1 ) . A l l p h e l l e m w a l l s f l u o r e s c e d y e l l o w i s h g reen (1/53), but w i t h IV/41, the t h i n - and t h i c k - w a l l e d p h e l l e m f l u o r e s c e d b l u e and g r e e n i s h b l u e r e s p e c t i v e l y ( Tab le 4 ) . C h a r a c t e r i s t i c s o f p h e l l e m under o t h e r modes o f l i g h t i n g a r e summarized in T a b l e 4. H i s t o c h e m i c a1 t e s t s showed t h a t o n l y the t h i n - w a l l e d p h e l l e m was s u b e r i z e d ( F i g . 2 1 ) . T h i c k - w a l l e d phe l l em was p o s i t i v e to 1 1 i g n i n 1 s t a i n s . ( i i ) N e c r o p h y l a c t i c p e r i d e r m (NP) In Pi nus c o n t o r t a and P i nus monti c o l a r h y t i domes deve l oped on the main stem u s u a l l y a f t e r 18 and 14 y e a r s r e s p e c t i v e l y . NPs at rhy t idomes c o n s i s t e d o f o n l y t h i n - w a l l e d p h e l l e m in both s p e c i e s . - 38 -Whereas the phellem contents in Pinus contorta were yellow (tbf) and birefringent, they were yellowish brown (tbf) and non-birefringent in Pinus monticola. With fluorescence microscopy, the phel1 em contents in Pinus contorta appeared dull red (1/53) (Fig. 22) and dark red ( I V / 4 1 ) , wh i1e in Pi nus mont i cola pale brownish yellow (1/53) and pale blue (IV/41) (Table 5 ) . The phellem walls in both Pinus species fluoresced greenish yellow (1/53) and pale blue (IV/41) (Table 5 ) . Some features of NP phellem at 35 and 210 day old mechanical injuries in Pinus contorta are seen in Figs. 23 and 24, respectively. At the early stages of development the necrophylactic phellem was turgid, rectangular, and without any pigment (Fig. 2 3 ) . The necrophylactic phellem at rhytidome as well as at healed sites of attack by Endocronartiurn harknessii (J.P. Moore) Y . Hiratsuka showed similar features indicating early stages of development. However, towards the end of the growing season the phellem became distorted and compressed, apparently from pressure of growth (e.g. Fig. 2 4 ) . The NPs from three Pinus monticola found at a 2-month old mechanical injury, at a site attacked by Cronartiurn  ribicola and at a site attacked by Cucurbidothis pithyophila Fr. are shown in Figs. 25, 26 and 27 respectively. Within a species the anatomical features and fluorescence characteristics of NPs found at injuries, blister rust and diseased areas in Pinus contorta and Pinus monticola were identical to those found at rhytidome. - 39 -Thus, the non-specificity of NP development regardless of the causal factor (Mullick and Jensen 1973b) was further substantiated. ( i i i ) Sequent exophylactic periderm (SEP) In Pinus contorta and Pinus monticola, the search for SEP was very d i f f i c u l t . SEP was not found abutting NP at regions of the oldest available injury (210 days), and generally in rhytidome samples even where scaling of dead tissue was taking place. Usually several NPs developed in successively deeper regions of bark without the development of the SEP (Fig. 28). This situation is similar to other conifers (Mullick and Jensen 1973a). However, SEPs could occasionally be found abutting NPs in samples collected from regions of scaling (Figs. 29, 30, 31, 32). As in the Picea species, although less frequently, SEPs in Pinus contorta and Pinus monticola could also be found abutting the entire unexposed NPs (Fig. 32) and also at exposed surfaces following sloughing of dead rhytidome tissues (Fig. 31). The exfoliation usually occurred in the necrophylactic phellem. » SEP, like FEP, consisted of several alternating layers of thin- and thick-walled phellem. However, the SEP usually had more thick-walled than thin-walled phellem (Figs. 30 and 32). In both species, thin-walled, not thick-walled, SEP phellem was always found f i r s t abutting the necrophylactic phellem (Fig. 33). Within a species, SEP was identical to FEP in a l l the cryofixation characteristics stud ied (Table k). - 40 -Thick-walled phellem of FEP and SEP was positive to 'lignin' stains. The thin-wa11ed phel1 em of FEP and SEP as well as the NP phellem was positive to suberin tests. 4.1.1.3 Pseudotsuga menziesii (Mirb.) Franco. (Douglas-fir) (i) First exophylactic periderm (FEP) This species usually retained its epidermis for one year. It was shed the following year at which time the FEP was found abutting it (Figs. 34 and 35). Features of the phellem geometry in immature and mature states were similar to those of the species already described. The seasonal renewal of the phellem occurs for many years and the bark surface remains smooth for approximately 17 years, until initiation of rhytidome. The phellem contents were brown and the walls were thin and birefringent. Thick-walled phellem was rarely observed in the approximately 40 samples examined. When examined under 1/53 and IV/41 f i l t e r combinations, the phel1 em contents appeared light brown and dark brown, while the phellem walIs fluoresced greenish yellow and pale blue respectively (Table 4). Histochemica1 tests showed that the phellem was suberized. - 41 " (ii) Necrophylactic periderm (NP) In Pseudotsuga menziesii rhytidome development is initiated usually after 17 years (Table 3 )-The NP at a newly forming rhytidome is shown in Figs. 36 and 37- The phellem geometry was undistorted and roughly rectangular as expected for the early stage in NP formation. The samples were collected on July 26, 1974. In cryofixed sections the NP phellem contents were dark gray (tbf) and walls were thin and birefringent. Under fluorescence, the phellem contents appeared pale green (1/53) (Fig. 37), and blue (IV/41) respectively. Samples collected at the end of the growing season showed that seasonal activity of phellem renewal ends with a distinct layer of special phellem cells which differed from the normal NP phellem (Figs. 38, 39 40, 41 and 42). Similar cells were referred to as phellogen activity marker cells (PAMC) in other conifers (Mullick and Jensen 1973a). This layer, usually 1 to 2 cells thick, had brown contents and thicker walls exhibiting stronger birefringence, and also fluorescing a stronger blue (IV/41).. (Fig. 42) and brighter yellow (1/53) (Fig. 40) than the normal NP phellem. Some rhytidome samples showed that the NP renewal occurs for a number of seasons as evidenced by the presence of a number of layers of PAMC (Figs. 39 and 40). On thawing, only the brownish contents of the latest layers of these cells flowed out; that in older periderm layers remained and could not be removed by soaking in absolute ehtanol and petroleum ether. - kl -This indicated that the contents in the PAMC had undergone changes either by desiccation or polymerization of the contents. It was observed that after several seasonal renewals of the NPs, new NP frequently developed in inner bark tissues giving rise to ever thickening layers of rhytidome characteristic of this species. The amount of phellem produced varied both within and between seasonal a c t i v i t i e s . The number of phellem cells produced per seasonal activity varied from 15 to kO eel 1s. Features of NPs at resin blisters and beetle holes are shown in Figs. k3 and kk respectively. These periderms were similar to those found at rhytidomes and mechanical injuries in a l l characteristics as revealed by cryofixation and other techniques (Table 5). Histochemical tests showed that the phellem was suberized, e.g. by Sudan III (Fig. kk). ( i i i ) Sequent exophylactic periderm (SEP) Over kO rhytidome samples obtained from several trees were processed and examined. No SEP was observed abutting the NPs. - 43 -Examination of abscission scars did not reveal any SEP abutting NP at needle scars (Fig. 45), which was referred to as the secondary protective layer in the earlier literature. Further investigations to determining the presence of SEP in Pseudotsuga menziesii are necessary. 4.1.1.4 Larix occidental is Nutt. (Western larch) (j) First exophylactic periderm (FEP) FEP (Fig. 46) was found 2 to 3 cells below the epidermis which began to slough off after development of brown FEP about the end of the f i r s t growing season. The sloughing was markedly increased the following season. Thereafter, the bark surface became smooth and remained so for about 14 years until rhytidome development (Fig. 47). The FEP renewal occurred every season and old outer phellem weathered away gradually. The FEP phellem in its early stages of development was turgid and rectangular, whereas later in the growing season it became stretched and compressed as usual. The phellem in the young trees was always thin-walled (Fig. 48), but in some trees thick-walled phellem occasionally was interspersed sporadically in patches in the thin-walled phellem (Fig. 49). The phellem contents under fluorescence appeared brown (l/53) and dark brown (IV/41), and a l l the phellem walls fluoresced yellowish green (1/53) but the thin- and occasional thick- phellem walls fluoresced blue and khaki under IV/41 (Fig. 49), respectively. - kk -(ii) Necrophylactic periderm (NP) In La r ix occ i denta1i s rhytidomes developed after approximately ]k years. Some features of NP abutting rhytidomes and mechanically caused injuries are shown in Figs. 50 and 51. The NP phellem (Figs. 50, 51 and 52), . readily distinguishable in the cryofixed state from FEP by pigments, had reddish-purple contents, thin walls and occurred in layers usually 5 to 6 cells thick. Similar to the phellem of FEP, these NP phellem cells were turgid and rectangular in the early stages of formation but had become stretched and compressed in the later stages of development (Figs. 50 and 5 1 ) . The NP phellem content fluoresced dull red (1/53) and with f i l t e r combination (IV/41) i t appeared black, while the walls fluoresced pale green (1/53) and pale blue (IV/41) (see Figs. 50 and 51 for 1/53 characteristics). Other features of the NP phellem found at rhytidomes and injuries were alike as revealed by cryofixation and other techniques (Table 5 ) . NP phellem was positive to a l l suberin tests. Similar results were found also in FEP phellem. NP phellem was not stained with the 'lignin' stains (e.g. Fig. 53)-( i i i ) Sequent exophylactic periderm (SEP) SEP in,Larix occidentalis does not occur as frequently as in the Picea species. SEP was not always found abutting the NP. NPs develop in successively deeper regions of bark for - 45 -several years without the development of a SEP, a feature commonly found in other conifers (Mullick and Jensen 1973a). SEP was found either abutting the entire unexposed NP or was at exposed surfaces following sloughing of dead rhytidome tissues. Features of SEP were identical to FEP in a l l respects as revealed by cryofixation and other techniques, but they differed from NP (Table 4 ) . 4.1.2 Cupressaceae 4.1.2.1 Cupressus macrocarpa Hartw. (Monterey cypress) (i) First exophylactic periderm (FEP) Unlike the previously described species, FEP develops in patches, generally appearing as brown spots in between needle bases in the 2nd and 3rd internodes (see also sections 4.1.3-1; and 4.1.4.1) FEP was found generally 2 to 3 cells below the epidermis. At the junction of the FEP with the epidermis, the latter disappears, suggesting thereby that the epidermis took part in the formation of the FEP at the junction zone. The spots gradually coalesce and spread over the entire surface. The FEP phellem cells had brown contents and thin walls; under mbf the contents appeared khaki. When examined under fluorescence f i l t e r combinations 1/53 and IV/41, the phellem contents appeared brown and dark brown, and the phellem walls fluoresced - 46 -y e l l o w i s h g reen and p a l e b l u e , r e s p e c t i v e l y ( Tab le 4). ( i i ) N e c r o p h y l a c t i c p e r i d e r m (NP) The NPs found a t rhy t idome ( F i g . 54) and mechan i ca l i n j u r i e s ( F i g . 55) were i d e n t i c a l . The p h e l l e m c o n t e n t o f these NPs was r e d d i s h - p u r p l e and c o n s i s t e d o f o n l y 3 t o 5 l a y e r s o f t h i n - w a l l e d c e l l s . The p h e l l e m w a l l s f l u o r e s c e d b r i g h t y e l l o w and b l u e , and the p h e l l e m c o n t e n t s appeared d u l l red and b l a c k under 1/53 and IV/41 r e s p e c t i v e l y ( Tab le 5). ( i i i ) Sequent e x o p h y l a c t i c p e r i d e r m (SEP) A l t h o u g h many rhy t idome samples f rom v a r i o u s r e g i o n s o f t he stem where s l o u g h i n g had o c c u r r e d were s t u d i e d , the p r e s e n c e o r ab sence o f SEP c o u l d not be c o n f i r m e d , l a r g e l y because o f the l i m i t e d a v a i l a b i l i t y o f a p p r o p r i a t e t r e e s . 4.1.3 T a x o d i a c e a e 4.1.3.1 Sequo ia s e m p e r v i r e n s E n d l . (Redwood) ( i ) F i r s t e x o p h y l a c t i c p e r i d e r m (FEP) FEP was found in sma l l pa t ches u s u a l l y 2 to 3 c e l l s below the e p i d e r m i s below brown spo t s in the 2nd i n t e r n o d e . The spo t s - 47 -gradually coalesced so that the entire stem surface appeared brown at about the 3 rd internode. The FEP phellem had brown contents and thin walls (Fig. 5 6 ) - At the early stages of FEP formation the phellem cells were undistorted and turgid, but became distorted and compressed during the later stages of development. Seasonal renewal of FEP was observed. Under fluorescence f i l t e r combination 1/53 and IV/41, the phellem contents fluoresced brown and dark brown, and the phellem walls fluoresced green and pale blue respectively. Under mbf the phellem contents appeared khaki brown (Table 4 ) . ( i i ) Necrophylactic periderm (NP) In this species rhytidome formation began usually in 7 _to -8-year-old regions of the stem. Sloughing of dead rhytidome tissues was uncommon and the dead bark accumulated on the tree. The reddish brown bark appeared rough and fibrous, and was spongy to the touch. The dead rhytidome tissues adhered strongly to the underlying tissues. At rhytidomes, as well as at mechanical injuries, the NPs were readily distinguishable from FEPs by their reddish-purple pigmentation (tbf) and had only 2 to 3 layers of thin-walled phellem. Under fluorescence f i l t e r combinations 1/53 (Fig. 57) and IV/41, the phellem contents appeared rusty red and black and the phellem walls fluoresced greenish yellow and blue, respectively. - 48 -Under mbf, the phellem contents appeared dark. In over 20 samples from the only available tree, no seasonal renewal of the NP was observed. New rhytidomes with the usual concave shape occurred in some areas of the stem below the previously formed periderms. Normally, the bark tissues were pink, but in the early stages of NP formation, a lens-shaped zone of beige colored tissues appeared below the previously formed periderms and the NP developed abutting this tissue (Fig. 58). Later in the growing season this beige tissue changed progressively to dark brown (Fig. 59). ( i i i ) Sequent exophylactic periderm (SEP) SEPs were found abutting the NP phellem at the rhytidomes. No SEP was observed abutting NP within the year of rhytidome formation. Thus it seems that SEPs were formed the season following the formation of the NPs. SEPs appeared identical to FEPs in every respect except that they consisted of less phellem, usually 1 to 2 eel 1s. 4.1.4 Taxaceae 4.1.4.1 Taxus brevifolia Nutt. (Pacific yew) (i) First exophylactic periderm (FEP) - h3 -Epidermis after the f i r s t year remains functional in several areas of the bark up to at most 6 years. The f i r s t sign of FEP formation was observed in the 2nd internode under small brown or dark spots in between the needle bases. The small brown spots of FEP formation increased in size and number and at about the 4th internode almost the whole stem became dark brown excepting small patches of green living epidermis. Cryofixation showed that the FEP developed from the epidermis. The FEP phellem was thin-walled, its contents brown and was only 1 to 2 layers thick. The.phellem walls fluoresced yellowish green and pale blue, and its content appeared brown and dark brown when observed with fluorescence f i l t e r combination 1/53 (Fig. 60) and IV/41 respectively. (ii) Necrophylactic periderm (NP) Rhytidome development is somewhat unusual because FEP does not develop uniformly but develops in patches and replaces the epidermis over a period of about six years. The FEPs were replaced by the NPs in the year following FEP formation, a phenomenon not commonly observed in other conifers studied (Figs. 60, 61 and 62). Rhytidomes develop beneath the dark brown patches where FEP has replaced epidermis. It develops in approximately 3-year-old regions. New NPs arise more frequently than in the other species studied. Rhytidomes, formed in previous years, were shed after new, deeper - 50 -periderms had developed. The bark surface appeared rough and flaky because of the facile exfoliation of old rhytidome tissues. The features of NPs found at rhytidomes are shown in Figs. 60 to 62 and at mechanical injuries in Fig. 6 3 - Cryofixation and other techniques showed that these NPs were alike. They were reddish purple and consisted of about 2 to 4 layers of thin-walled phellem cells. When examined with fluorescence f i l t e r combinations 1 / 5 3 and IV/41, the phellem contents appeared red and black and the phellem walls fluoresced green and blue respectively. Under mbf the contents appeared dark red (Table 5 ) . ( i i i ) Sequent exophylactic periderm (SEP) Over 20 samples of rhytidome from the one available tree were studied. SEPs were found internally abutting most NPs but they were absent when the NP had formed in the same year that they were sampled. Careful study of rhytidomes in the year they were formed showed SEP was absent even in samples collected in late November, 1974. Further SEPs were not found abutting NPs at mechanical injuries in the year the injuries were made. Thus it seems that the SEPs developed abutting the NPs the following year. SEPs were brown and consisted of 1 to 2 layers of thin-walled phellem eel 1s abutting NPs (Fig. 6 4 ) . Cryofixation and other techniques showed that SEPs were identical to FEPs in a l l respects. The pigmented contents of NP phellem were lost when cryofixed sections were allowed to thaw, but the brown pigmented contents in f i r s t and sequent exophylactic phellem remained intact. - 51 -4.2 Investigation of Exophylactic and Necrophylactic Periderms in Angiosperms 4.2.1 Aceraceae 4.2.1 .1 Acer macrophyllum Pursh. (Broad-leaved maple) (i) First exophylactic periderm (FEP) Field and macroscopic observations based on only 2 Acer macrophy11 urn, showed that small spots of various sizes, varyi in color from whitish, light brown to dark brown, appeared over the entire bark surface from the tip of the leader to where rhytidomes (zone age 10 years; Table 3) had developed. In the f i r s t internode the whitish spots existed individually. They increased in size and appeared light brown in the 2nd internode (arrows Fig. 65)- In the 3rd internode these spots became dark brown and coalesced to form small dark patches. Such dark patches were observed from the 4th ) internode downwards particularly on the south side (Fig. 66). examined by cryofixation, the initiation of FEP was observed in tissues abutting the epidermis at these spots (Figs 67, 68 and 69) This process of periderm formation in spots and patches continued in the stem for about 9 to 10 years and greenish living strips of epidermis were observed in between these patches of periderm. The When these spots from various internodes were - 52 -FEP in cryofixed sections was i n i t i a l l y formed below the dark patches. The FEP phellem contents are brown but become grayish on weathering. Only epidermis was observed in the greenish strips of bark. Below the epidermis and the FEP the outer cortex consisted of closely packed small cortical c e l l s . The contents of these cells below the epidermis appeared more reddish, while that below the FEP appeared more greenish or reddish green when examined with fluorescence f i l t e r combination 1/53 (Fig. 70). This FEP was brown and consisted of phellem cells which had walls of intermediate thickness when compared to the thin-walled and thick-walled FEP phellem of Pinus contorta and Picea species. The phellem contents appeared dark brown and black, and the phellem walls fluoresced green and blue when observed with fluorescence f i l t e r combinations 1/53 and IV/41 respectively (Figs. 71 and 72, and Table 4 ) . (ii) Necrophylactic periderm (NP) Rhytidome tissue was found at the base of the 2 trees just above ground. The bark surface at these regions was rough, and longitudinal cracks had appeared (Fig. 73). The features of NPs at rhytidome and a mechanical injury are-shown in Figs. 74, 75 and 76. When the necrophylactic phellem cells were f i r s t laid down they were:turgid and rectangular (Fig. 76) but later in the growing season they were distorted and compressed (Figs. 74 and 75). - 53 -Seasonal renewal of the NP phellem was not observed in the year after their formation and SEP had formed. Cryofixation and other techniques revealed that NPs which developed at rhytidomes and injuries were identical (Table 5). These periderms were rusty red (tbf) with thin-walled phellem (Fig. 7 7 ) . With 1/53 (e.g. Fig. 76) and- IV/41 (e.g. Fig. 74) fluorescence f i l t e r combinations, the phellem walls fluoresced yellowish green and pale blue, and their contents appeared yellowish brown and dull khaki respectively. ( i i i ) Sequent exophylactic periderm (SEP) Samples of the rhytidomes as well as of mechanically injured tissues mentioned above, were examined for SEP. SEP abutted a l l NPs in rhytidomes (e.g. Fig. 7 4 ) . No SEP was observed abutting the NPs in rhytidome formed in 1974 (Fig. 7 7 ) . This suggested that SEPs were not developed in the same year as the NPs but were formed in the subsequent year(s). Similarly within one growing season, SEP was absent in the oldest mechanical injuries collected (age 170 days) . SEPs were identical to FEPs in a l l respects (Table 4 ) . When cryofixed sections from rhytidome were thawed, the necrophylactic phellem contents flowed away slowly, whereas the cell contents in the sequent exophylactic phellem remained intact (Fig. 7 8 ) . - 54 -4.2.2 Leguminosae 4.2.2.1 Robinia pseudoacacia L. (Locust, Yellow locust) (i) First exophylactic periderm (FEP) FEP was present in the 2nd and 3rd internodes 2 to 3 cells below the epidermis (Fig. 79). Seasonal renewal of FEP, as indicated by the phellogen activity marker cell s , was observed every year. The bark surface, until rhytidome formation, appeared quite smooth. FEP phellem cells have brown contents and thin walls, and are rectangular in shape in the early stages of formation. These cells became flattened and stretched at the end of each growing season, presumably from stresses of growth (Figs. 8 0 and 81). Under fluorescence f i l t e r combinations 1/53 and IV/41, the phellem contents appeared light brown and dark brown, and the phellem walls fluoresced greenish yellow and pale blue, respectively. The phellem layer was usually 7 to 9 cells thick, the inner two of which con-stituted phellogen activity marker cells (Figs. 80 and 81). In contrast to the rest of the phellem cel l s , these marker cells had thicker walls which fluoresced deeper blue (IV/41) (Fig. 82 ) and green (1/53) (Fig. 83). These cell s , like the rest of the phellem, reacted positively to a l l tests for suberin (Fig. 84). - 55 -(ii) Necrophylactic periderm (NP) Rhytidome samples were collected from rough bark regions of stem (approximately 8 years old) showing deep furrows. Mechanical injuries (28 days old) were also obtained. The features of NPs at a rhytidome (Figs. 82 and 83) and an injury (Fig. 85) are shown. Cryofixation and other techniques revealed the identity of these periderms. These NPs were identical in a l l respects and were yellowish gray (tbf). They consisted of thin-walled phellem cel l s . Seasonal renewal of necrophylactic phellem was evident by the occurrence of the phellogen activity marker cell layers which were commonly two cells thick. The number of seasonal renewals of the NPs rarely exceeded more than 3 or 4 (Fig. 82). The number of phellem cells produced per seasonal activity varied from 4 to 10 c e l l s . These cells were rectangular in the early stages of formation, becoming flattened at the end of the growing season. Under 1/53 and IV/41 fluorescence f i l t e r combinations, the phellem contents appeared pale green and black while the phellem walls fluoresced yellowish and greenish blue, respectively. The phellogen activity marker cells had black contents (tbf) and thickened walls which fluoresced green and deeper blue than the normal phellem cells under f i l t e r combinations 1/53 and IV/41 respectively. - 56 -( i i i ) Sequent exophylactic periderm (SEP) Among the rhytidome samples and the oldest available mechanical injuries (167 days) investigated, no SEP was found abutting any of the NPs. In i.deep furrowed bark samples the periderm(s) exposed by the cracking of the bark was found to be the NP. This phenomenon was also observed in Pseudotsuga menziesii. The occurrence of SEP remains to be ascertained. 4.2.2.2 Gleditsia triacanthos L. Inermis (Honey locust) (i) First exophylactic periderm (FEP) FEP was found in tissues 2 to 3 cells below the epidermis (Fig. 86) in the second internode. The epidermis was shed in small white flakes after the FEP had developed. The bark surface appeared very smooth in a l l trees studied except in the older ones of age approximately 20 years. At breast height these had developed a few cracks on the stem just above ground. The annual renewal of FEP was indicated by the presence of the PAMC (Figs. 87 and 88), and by the fact that the FEP remain intact during stem expansion. Old outer layers of phellem on the bark surface were shed and weathered away very slowly as evidenced by the extensive build-up of phellem layers (Figs. 87 and 88) . - 57 -The FEP phellem was thin-walled and had brown contents. The phellogen activity marker ce l l s , which were also thin-walled, appeared beige. The number of phellem cells and phellogen activity marker cells produced per seasonal activity varied both within and between seasonal ac t i v i t i e s . The former varied from 4 to 6, while the latter varied from 2 to 4 c e l l s . Generally the PAMC had stronger birefringence than the other phellem. Under fluorescence f i l t e r combinations 1/53 and IV/41, the phellem contents appeared dark brown and black, and the phellem walls fluoresced yellowish green and pale blue respectively. The cell walls of the phellogen activity marker cells fluoresced green (1/53) and, relative to the rest of the phellem, stronger blue (IV/41). (ii) Necrophylactic periderm (NP) Samples of NPs at rhytidome were obtained from regions of the stem where cracks had developed. Rhytidome formation usually begins f i r s t in the 6 _year~old regions of the stem (Table 3)- Samples of mechanical injuries were collected 21 days and 167 days after injuries were made. Cryofixation and other techniques showed that the periderms which developed at rhytidomes (Fig. 89) and, 21 day old injuries (Fig. 90) were identical in a l l respects. The cryofixation characteristics of these periderms are presented in Table 5. - 58 -Similar to FEP, seasonal renewal of the NP phellem, as indicated by the phellogen activity marker ce l l s , was observed." However, such renewal activities occurred only 2 to 3 times (Fig. 89). The amount of phellem produced varied considerably both within and between ac t i v i t i e s . The number of layers of phellogen activity marker cells varied from 2 to k cells usually (Fig. 89). The phellem cells in the early stage of development were rectangular and as in other tree species, later became flattened (Fig. 90 and 89 respectively). The NP phellem had orange contents (tbf) and thin walls. The phel1 em wa11s when observed under f i l t e r combinations 1/53 and IV/41 fluoresced green and blue, and their contents appeared orange red and black, respectively. The phellogen activity marker cells also were thin-walled. They were identical to those found at FEPs in every respect (e.g. Figs. 8.7, 88 and 89). ( i i i ) Sequent exophylactic periderm (SEP) The occurrence of SEP was observed only in a few rhytidome samples. SEPs were found to be identical to FEPs as revealed by cryofixation and other techniques (Table 4). When air-dried cryostat sections containing FEP and NP in a rhytidome formed in 1974 were treated with Sudan III and phloroglucinol-HCl a l l phellem cell walls stained red with the former and faintly pink with the latter (Figs. 91 and 92 respectively). The remnants of the pigmented necrophylactic phellem contents were - 59 -removed but that in the f i r s t exophylactic phellem remained intact (Fig. 92). A similar result was observed with the SEP phellem. 4.2.3 Rosaceae 4.2.3.1 Pyrus species (Pear tree) (i) First exophylactic periderm (FEP) FEP developed in the epidermis in the 1st internode of the leader giving rise to patchy appearance. Differences in the fluorescence characteristics distinguished the epidermis from the outer cortex before and during the formation of FEP. Information was obtained on FEP development from several samples which showed different stages of FEP formation. The epidermis differentiated into phellem, and the cortical cells abutting i t also were undergoing changes, becoming enlarged and their cell walls fluorescing yellowish green (1/53) and blue (IV/41). The phellem which developed from the epidermal cells and the modifying cortical cells with fluorescent walls stained red with Sudan III, indicating suberization. Other suberin stains also showed positive reactions. Similar observations on modification of cortical cells have been made in conifers (Mullick, personal communication). - 60 -The phellem had brown contents and usually had thicker tangential than radial walls (Fig. 93). Under fluorescence f i l t e r combinations 1/53 and IV/41 (Fig. 94), the phellem contents appeared dark brown and black, while the phellem walls fluoresced green and blue, respectively. Seasonal renewal of FEPs occurred until they were replaced by the NPs during rhytidome formation which f i r s t occurred in regions of stem approximately 5 to 6 years old. Phellogen activity marker cells were not present. (ii) Necrophylactic periderm (NP) The development of NPs was investigated only in rhytidomes. The tree studied was on private land and only minimal sampling with no injury experiments was permitted. Some features of freshly forming NP are shown in Fig. 95, and of old NPs at rhytidome in Fig. 96 and 97. The NPs were pale gray or colorless and consisted of thin-walied phellem usually 4 to 6 cells thick. Under fluorescence f i l t e r combinations 1/53 and IV/41, the phellem walls fluoresced yellow and pale blue while their contents appeared dark and black, respectively. ( i i i ) Sequent exophylactic periderm (SEP) Samples of SEPs were obtained from various regions of the stem where rhytidome had developed either with or without - 61 -signs of sloughing of dead rhytidome tissues. SEP phellem appeared identical to FEP phellem as revealed by cryofixation and other techniques. They differed only in time and site of origin. SEPs were found to be associated with sloughing of dead rhytidome tissues (Fig. 98). They were found at exposed surfaces following shedding of dead rhytidome tissues and also abutting the entire unexposed NPs (e.g. Fig. 96). 4.2.4 Malvaceae 4.2.4.1 Hibiscus syriacus L. Hamabo. (i) First exophylactic periderm (FEP) Initiation of FEP occurred in tissues abutting the epidermis in the f i r s t internode (Figs. 99 and 100). Epidermis was sloughed off in small grayish strips and flakes generally soon after the initiation of FEP. FEP consisted of thin-walled rectangular phellem cel l s . Brown contents usually developed only in one layer of phellem closest to the surface. Under fluorescence f i l t e r combinations 1/53 (Fig. 102) and IV/41 (Fig. 101) the phellem walls fluoresced yellow and pale blue, and their brown contents appeared brown and dark brown respectively. - 62 -(ii) Necrophylactic periderm (NP) Investigation of NPs at rhytidomes (Fig. 103) as well as injuries (Fig. 104) showed that these periderms were alike as revealed by cryofixation (Table 5). Under tbf the phellem contents were gray or colorless and the phellem was thin-walled. Under 1/53 and IV/41 fluorescence f i l t e r combinations, the phellem contents fluoresced pale green and pale blue while the phellem walls fluoresced yellowish green and blue, respectively (Table 5). When air-dried cryostat sections were treated with suberrn stains, the NP phellem showed positive tests. Similar results were observed with FEP except that their brown phellem contents remained intact while that of the NP phel1 em-was gone. ( i i i ) Sequent exophylactic periderm (SEP) Although rhytidome formation had occurred in the -species studied, the presence of SEP was not observed, possibly because the plants were too young for SEP to have formed. The only plants available were potted seedlings growing in a green house. The occurrence of SEP in Hibiscus syriacus needs further investigation. 4.3 Investigation of Non-Suberized in Gymnosperms and Angiosperms Impervious Tissues (NIT) - 63 - i 4.3.1 General Survey and Characteristics of NIT at Injuries Injuries on the bark of Picea glauca, Picea engelmanni i , P icea s i tchens i s, P i nus contorta, P i nus mont i cola, Pseudotsuga  menziesi i , Larix occidenta1 is, Cupressus macrocarpa, Sequoia  sempervi rens, Taxus brevi f o l i a , Rob i nia pseudoacacia, Gledi ts ia  triacanthos, Acer macrophy11 urn and Hi biscus syriacus, made on June 11, 1974, were F-F tested thrice a week for dye penetration through the wound surface. The F-F test solutions continued to penetrate, coloring bark tissues including the injured zone blue (e.g. Fig. 105), until the 12th day after wounding in'A_. macrophy 11 urn; 13th day in H_. syriacus; 15th day in C_. macrocarpa ; 19th day in P_. s i tchens is , P_. contorta, S_. sempervi rens , L_. occi denta 1 i s , R_. pseudoacac ia and G_. tr iacanthos ; 21st day in P_. menz ies i i ; 26th day in P_. monticola and J_. brevi fol ia; and 30th day in P_. glauca and P_. engelmanni i . The deep permeation of the test solutions prior to NIT formation after wounding occurs in gymnosperms and angiosperms and can be seen in cryofixed sections v (Figs. 106 and 107) • When the next samples, collected two days later than those described above, were F-F tested through the wound surface, only the brown 'dead' tissues were colored blue. The penetration of the test solutions was stopped at the semilunar zone abutting the dead tissues in a l l species (e.g. Fig. 108). A few of these samples were also F-F tested through the cambial surface, the permeation of the test solutions was stopped at the boundary of a thin concave - 64 -zone abutting the brown 'dead' tissues. Newly forming phellem was pervious to the test solutions. Two to three weeks after injury the phellem had become impervious. The aforementioned observations were consistent in a l l coniferous and deciduous species, and suggested that the semi-lunar zone of tissues lying between the brown 'dead' tissues and the living bark was the tissue responsible for the imperviousness, and in conjunction with the cryofixation characteristics (Sections 4.3-3, 4.3.4, and 4.3-5) and histochemica1 test (Section 4.3-6) was identified as the non-suberized- impervious tissue (NIT). The cryofixed sections of F-F tested and non F-F tested samples after NIT formation showed the extent of the F-F permeation, (F-F clearly seen only in tbf, less clearly seen in 1/53), (e.g. Figs. I l l and 114) and the location and structure of NIT in each of the species (e.g. Figs. 109 and 116). The time taken for the formation of NIT in each species is shown in Table 6. 4.3.2 Developmental Study of NIT in Pinus contorta and Larix occ i denta1i s by Cryofixation Cryofixed sections of the injured bark from the two species two days after wounding showed that a zone of tissues abutting the wound surface had become brown (tbf) and in Larix occidental is the yellow fluorescence of their cell contents had faded. The i n i t i a l yellow fluorescence of the cell contents in living tissues abutting - 65 -the brown 'dead' tissues was also disappearing (Fig. 117). In Pinus contorta, the red fluorescence of the chloroplasts in the injured zones was diminishing. The cells abutting the 'dead' tissues had green fluorescent walls, and in contrast to Larix  occidentalis, most of the parenchyma cells which normally showed l i t t l e fluorescence had developed yellow fluorescent contents (Fig. 118). At this stage the walls of the cells adjacent- to the dead tissues in Larix occidental is were not fluorescent. By the fourth day after injury in both species, more cells had become brown or 'died' in tissues adjacent to the wound surface. In Pinus contorta the cells abutting the 'dead' brown tissues became slightly enlarged and the intensity of the yellow fluorescence had increased. The red fluorescence of the chloroplasts had faded cons iderably (Fig. 119)- In Lar ix occ i denta1i s the eel 1s abutting the 'dead' brown tissues retained the yellow fluorescence of their content but the cells abutting them were losing the yellow fluorescence of their contents, becoming enlarged with reticulated, pale green fluorescent contents. Their cell walls remained non-fluorescent. The appearance of fluorescent reticulum is similar to that reported for other conifers (Mullick 1977). By the seventh day, more cells had 'died' and become brown in both species. In Pinus contorta, the cells abutting the 'dead' brown tissues lost the yellow fluorescence of their contents which at this stage had become reticulated and fluoresced pale green. The cell walls now fluoresced greenish yellow (Fig. 120). These - 66 -modifications were increasingly pronounced in samples collected on the eleventh and fourteenth days (Figs. 121 and 122 respectively). In Larix occidental is the fluorescence of the contents of the cells abutting the 'dead' brown tissues was changing from yellow to yellowish green. The cells internally adjacent to these cells had become enlarged with reticulated contents that fluoresced pale green with fiIters 1 / 5 3 (Fig. 1 2 3 ) . By the ninth day, the 'death' of cells (zone a) had increased very l i t t l e in Larix occidenta1is. Contents and walls of cells abutting the 'dead' brown tissues had yellow fluorescent contents and the cell walls fluoresced yellowish green (zone b). Further cell enlargement in this zone was not apparent. Cells internally adjacent to this zone had dark green reticulated fluorescent contents and were considerably enlarged (zone c) (Fig. 124). Abutting zone c was the living bark (zone d). Such classificat ion of tissues could also be noticed in the samples collected on the seventh day but was not easily evident. These modifications became progressively more pronounced in samples collected on the eleventh, fourteenth, and sixteenth days (Figs. 125, 126 and 127 respectively). During these stages, the tissues below the wound surface could be divided into four zones: (a) brown 'dead' tissues, (b) tissues consisting of cells with ye 11ow f1uorescent contents, (c) tissues consisting of cells with pale green to dark green fluorescent contents, and (d) the living normal tissues. The cells in zone (b) and (c) had become enlarged and the green fluorescent cell contents in zone (c) were considerably recticulated. - 67 -In Pinus contorta the sporadic formation of NIT was observed in cells internally abutting the 'dead' tissues in the sixteenth and eighteenth day samples. The NIT cells were large with strongly yellow fluorescent, thickened walls, (Figs. 128, 129 and 130). Tissues internally abutting the NIT by this time had lost their fluorescence and appeared as a dark band. Mullick (1977) has designated this as a zone of meristematic activity. NP phellem differentiates in this zone. In Larix occidental is on the eighteenth day, the cells abutting the 'dead' tissues showed a reduction in the intensity of the yellow fluorescence of their contents and very l i t t l e cell enlargement. But cells abutting them (zone c) had become considerably enlarged and their recticulated cell contents were very pronounced. The cell walls had become detectable under 1/53 • fluoresced yellowish green (Fig. 131). These cells later differentiated into NIT with yellowish green fluorescent cell contents and yellow fluorescent thickened cell walls by the 2 1 s t day. Complete NIT formation and subsequent changes in samples collected on the 2 1 s t , 2 8 t h , and 35th days are shown in Figs. 132, 133 and 13** respectively. The dark band of meristematic tissue also developed abutting the NIT. In P i nus contorta and La ri x occ i denta1i s NIT formed on the eighteenth and twenty-first days respectively after wounding as revealed by the F-F test. - 68 -4.3-3 General Features of NIT at Injuries using Cryofixation and the F-F Test Freshly formed NIT at an injury of Picea glauca, P_. s i tchensi s , P_. engelmanni i , Pinus contorta, P_. monticola, Pseudotsuga  menz ies i i , Lar ix occ i dentalis, Cupressus macrocarpa, Sequoia sempervi rens, Taxus brev i f o l i a , Acer macrophyllum, Robinia pseudoacac ia, G1ed i ts ia triacanthos, and Hibiscus syriacus are shown in Figs. 135 to 155 respectively. The NIT in these species consisted of a zone of enlarged cells of varying shapes and sizes with irregularly thickened walls located between the brown 'dead' tissue and the living bark. This was the only characteristic that differed between species. The characteristics of the cel l contents and cel l walls in cryofixed sections in general were similar. Typical characteristics of NIT in cryofixed sections are shown in Figs. 11, 12, 13 and 14. In tbf (Fig. 11), the NIT cell wa11s appeared colorless to yellowish brown. When the NIT cell walls were examined under fluorescence f i l t e r combinations 1/53 (Fig. 13) and IV/41 (Fig. 14) they fluoresced consistently yellow with a slightly greenish tinge and pale blue . respectively. These cell walls were birefringent (Fig. 12) when observed with pep. The cell contents of NIT varied from colorless, through gray and brown to black in tbf. The cell contents usually fluoresced yellow to pale green under f i l t e r combination 1/53, and they fluoresced pale blue to black under IV/41. Enlarged cells as reported by Mullick (1975, 1977) in the 'dead' zone were observed - 69 -externally abutting the NIT. These cells showed clear distinction from the NIT cells in untreated cryofixed sections because they do not possess wall fluorescence. However, F-F test was the reliable test for distinguishing the NIT c e l l s . The differentiation of NP phellem at the time of NIT formation usually had not begun, although sporadic initiation was sometimes observed below local patches of NIT but prior to complete NIT formation (Figs. 14-1 and 154). This observation was consistent in a l l species. In the species studied, the development of NP generally began only after the completion of NIT formation and in no case was the development of phellem observed prior to at least partial NIT formation and in such cases development was confined to areas internally abutting the patches of NIT. Figures 156 to. 165 show that a few NP phellem had formed in tissues internal to NIT in samples collected 15> 2 8 , 3 2 , 9 , 2 0 , 7 and 12 days after the impermeability (Table 6) was f i r s t detected in P_. s i tchens i s , P_. engelmann i i , P_. monticola, R_. pseudoacac ia , P. contorta, S_. semperv i rens , and L_. occidental is respectively. Ear 1 ier i njur ies F-F tested through the cambia1 surface and examined by cryofixation showed that the newly formed NP phellem was pervious. At this early stage, NP phellem gave a negative test to a l l ^ suberin stains used. The developmental features of NIT as revealed by cryofixation showed that NIT was a complex tissue which formed from pre-existing cells through tissue modifications, particularly through hypertrophic dedifferentiation. The cell types involved in NIT - 70 -formation are cortical parenchyma, ray parenchyma, sieve c e l l s , sclereids, epithelial cells and any type of cells in the living bark tissue which l i e in the path of NIT formation. 4.3.4 General Features of NIT at Rhytidome using Cryofixation and F-F Test Features of NIT, in cryofixed sections from rhytidomes of a l l the species studied, are shown in Figures 166 to 182. These figures were taken from samples which had not been F-F tested. In F-F. tested samples the test solutions permeated only the brown 'dead' tissue and stopped at the NIT. Within a species the cryofixation characteristics of NIT at rhytidomes were identical to those of NIT at injuries. The cell walls of NIT consistently fluoresced yellow with a slightly greenish tinge and pale blue under fluorescence f i l t e r combination 1/53 and IV/41, respectively. When NP phellem was present it was always found internally abutting NIT. In a l l rhytidome samples investigated, NP was never present without the presence of NIT. As with mechanical injuries, the site of NP formation was in the tissue internally abutting NIT. NIT formation at rhytidomes also involved tissue modifications of pre-existing cells of any cell types in the region of NIT formation. - 71 -4.3.5 General Features of NIT at Diseased Sites and Abscission Zones using Cryofixation and the F-F Test Features of NIT at healed sites of weevil attack in leaders of Picea sitchensis, beetle holes and old resin blisters of Pseudotsuga menziesii, healed sites of diseases in Pinus monticola attacked by canker and blister rust, and abscission scars of Picea  glauca are shown in Figures 183 to 189 respectively. In F-F tested samples, the test solutions permeated the brown dead tissues and stopped at the NIT. At regions where NIT had deteriorated and cracked the test solutions were stopped at the NP phellem. NPs were always found internally abutting a zone of compressed and distorted enlarged cells which constituted the NIT. The cryofixation characteristics for detection of NIT gradually become obscure and NIT cells become highly compressed and distorted. It is therefore essential that samples for studies be procured soon after formation of NIT (Mullick 1977). Extensive search was necessary to obtain samples of diseased sites at the appropriate stage of NIT maturation. For this reason, only a few diseases were studied. Nevertheless, the figures clearly show that the NIT cell walls consistently fluoresced yellow and pale blue when examined under f i l t e r combinations 1/53 and IV/41, respectively. They were yellowish brown (tbf) and birefringent (fcp). The cell contents usually fluoresced yellowish brown and dark brown under 1/53 and IV/41 f i l t e r combinations respectively. In tbf the cell contents were dark brown. 1 - 72 -W i t h i n a s p e c i e s , c r y o f i x a t i o n and o t h e r c h a r a c t e r i s t i c s o f NIT a t t h e s e s i t e s were b a s i c a l l y i d e n t i c a l t o those o f NIT a t i n j u r i e s and r h y t i d o m e . NIT was found between the brown ' d e a d ' t i s s u e s and the l i v i n g bark in d i s e a s e d samp le s . In a b s c i s s i o n z o n e s , NIT d e v e l o p e d o n l y a round the v a s c u l a r bund le and was seen between the s u b e r i z e d p r i m a r y p r o t e c t i v e l a y e r and the s u b e r i z e d seconda ry p r o t e c t i v e l a y e r , the l a t t e r b e i n g the n e c r o p h y l a c t i c p e r i d e r m . In a l l t he se s i t e s , NP was a lways found i n t e r n a l l y a b u t t i n g the NIT. 4.3.6 H i s t o c h e m i c a l C h a r a c t e r i s t i c s o f NIT C r y o s t a t s e c t i o n s showing NIT o r NIT and NP p h e l l e m from F -F and non F -F t e s t e d samples o f i n j u r e d ba rk , r h y t i d o m e , d i s e a s e d s i t e s , and a b s c i s s i o n zones were s t a i n e d w i t h Sudan II I, ammonium h y d r o x i d e - c r y s t a l v i o l e t , phosphene 3R, and methy lene b l u e f o r s u b e r i n . On ly p h e l l e m o f n e c r o p h y l a c t i c and e x o p h y l a c t i c pe r i de rms showed p o s i t i v e s t a i n i n g . In t ho se e x o p h y l a c t i c pe r i de rms which had t h i n - and t h i c k - w a 1 1 e d p h e l l e m , o n l y the t h i n - w a l l e d p h e l l e m r e a c t e d p o s i t i v e l y . These r e s u l t s were f u r t h e r c o n f i r m e d by e x a m i n a t i o n o f Sudan III and c r y s t a l v i o l e t s t a i n e d s e c t i o n s under f l u o r e s c e n c e w i t h f i l t e r c o m b i n a t i o n 1/53 and IV/41, a p r o c e d u r e t h a t enhances c o n t r a s t and s e n s i t i v i t y o f these s t a i n s . NIT showed n e g a t i v e s t a i n i n g c o n s i s t e n t l y and i t s c e l l w a l l s f l u o r e s c e d y e l l o w o r y e l l o w i s h g reen ( 1/53) and w h i t i s h b l u e ( I V / 4 1 ) . T h e r e f o r e , the - 73 -imperviousness of the NIT was not due to suberin (e.g. Figures 192 to 195). NIT did not show a positive test to aniline blue and resorcin blue which were specific tests for callose (Jensen 1962). The impermeability of the NIT, therefore, was also not due to callose. Phloroglucinol-HC1 and aniline sulfate-HCl tests showed positive staining of NIT only at injuries (e.g. Figs. 196 to 200). However, these tests were unsuccessful at rhytidomes because they stained the entire rhytidome tissues including NIT (e.g. Fig. 92). These tests also showed positive reaction with evidently older phloem, sclereids, and thick-walled exophylactic phellem. Furthermore, sections of samples collected prior to NIT formation showed a zone of enlarged distorted cells that reacted positively to phloroglucinol-HC1. When cryostat sections from 25 day and 18 day old F-F tested injuries of Picea glauca and Picea engelmannii were treated with phloroglucinol-HCl or ani1ine sulfate-HC1, a semilunar band of tissues at some distance from the wound surface reacted positively to the tests. These tissues were stained red with phlorog1ucinol-HC1 and stained yellow with aniline sulfate-HCl. In studies of NIT development the latter reacted positively to these tests. This indicated that the concave band of tissues was probably the region of NIT differentiation- in the bark (e.g. Figs. 190 and 191). Thus, while this is added information about NIT development, the test alone cannot establish the presence of NIT. As expected, these - Ik -tests were non-specific for NIT detection (e.g. Figs. 192 to 202). They are reactions of specific organic groups, not compounds. Some of the substituents of lignin are well known to give positive results. The phlorog1ucinol-HC1 test also stained tissues other than NIT at diseased sites and abscission scars (e.g. Figs. 201 and 202). Therefore, the test is valid only at sites of mechanical injuries. After subjection to the Maule reaction, NIT cell walls of both coniferous and deciduous species turned yellowish brown or pale brown. Similar results were observed for sieve and parenchyma eel Is externally abutting NIT, thick-walled exophylactic phellem, and a l l sclereids of the conifers. In the deciduous species these cell types also stained pale brown and only the sclereids stained red, a positive test for angiosperm lignin (e.g. Figs. 203 and 204). Upon exposure to ammonia gas and hydrogen chloride, NIT cell walls turned yellow and pinkish red respectively. The reactions were reversible. This method was also non-specific for NIT detection because sclereids showing this reaction were noted. NIT and other tissues continued to react positively to ph1orog1ucinol-HC1 and aniline sulfate-HCl after sections were treated with absolute ethanol and diethyl ether for seven days at room temperature. Therefore, the substance(s) which was stainable with these reagents and was a candidate(s) for the NIT imperviousness, was non-extractable with absolute ethanol and diethyl ether. - 75 -Similar results were obtained from sections treated with petroleum ether, benzene, and xylene (e.g. Fig. 205). 4.3.7 Association of NIT with Necrophylactic Periderms (NP) Results presented in the previous sections show the general occurrence of NIT at the typical sites of NPs, such as mechanical injuries (Figs. 154 to 165), rhytidome (Figs. 166 to 182), diseased sites (microbes and insects) (Figs. 183 to 188) and abscission zones (Fig. 1 8 9 ) . NIT always developed prior to NP formation and NIT was always found externally abutting the NP. NIT was not found in association with the two types of exophylactic periderms, the f i r s t and sequent exophylactic periderms, in any of the coniferous and deciduous species studied. These observations firmly substantiate that NIT is always associated with the NPs and provide the specific environment for NP formation. - 76 -5. DISCUSSION 5.1 The V a l i d i t y o f the Concepts o f E x o p h y l a c t i c and N e c r o p h y l a c t i c P e r i d e r m s The FEP i n Taxus b r e v i f o l i a and Pyrus s p e c i e s d e v e l o p e d from t h e e p i d e r m i s i t s e l f . In Pseudotsuga menz i e s i i , A cer macrophy!Turn, and H i b i s c u s s y r i a c u s , i t was found i n t i s s u e s a b u t t i n g the e p i d e r m i s and i n P i c e a g l a u c a , P i c e a e n g e l m a n n i i , P i c e a s i t c h e n s ? s, P i nus c o n t o r t a , P i nus m o n t i c o l a , Lar i x occ i d e n t a l i s,  Cupressus m a c r o c a r p a , Sequo i a semperv ? r e n s , Rob i n i a pseudoacac i a , and G l e d i t s i a t r i a c a n t h o s , i t d e v e l o p e d i n t i s s u e s two t o t h r e e c e l l s below t h e e p i d e r m i s . The r e s u l t s o f t h e p r e s e n t s t u d y a r e c o n s i s t e n t w i t h t h e e a r l i e r s t u d i e s on t h e f o r m a t i o n o f t h e f i r s t p e r i d e r m (FEP) (DeBarry 1884; M e t c a l f e and C h a l k 1950; Change 1954a, 1954b; S r i v a s t a v a 1963, 1964; Esau 1965; Fahn 1967, and A r z e e e t a l . 1968) . The f i r s t p e r i d e r m s u s u a l l y formed i n the stem d u r i n g t h e f i r s t y e a r o f growth, a f t e r e x t e n s i o n and p r i m a r y d i f f e r e n t i a t i o n had o c c u r r e d . P r i o r t o M u l l i c k ( 1971 ) , d i s t i n c t i o n o f t h e s e p e r i d e r m s from the ' u s u a l ' sequent p e r i d e r m s was r e p o r t e d o n l y on the b a s i s of t h e s i t e and time o f o r i g i n and not on t h e b a s i s o f fundamental d i f f e r e n c e s i n t h e i r anatomy and c h e m i s t r y . A l t h o u g h t h e v a r i a t i o n o f p e r i d e r m p i g m e n t a t i o n between s p e c i e s has been r e c o r d e d (e.g. Chang 1954a, 1954b; P r o t s e n k o e t . a l . I 9 6 0 ) , p i g m e n t a t i o n as a b a s i s f o r fundamental d i s t i n c t i o n s between - 77 -periderms within a species was not recognized. Mullick (1971) differentiated periderms originally by macroscopic differences observed in forty species of conifers from thirteen genera. Greatly refined distinctions between periderms was made on the basis of characteristics provided by cryofixation including color differences and differences in the chemistry of the pigments. This led to the new concepts of necrophylactic and exophylactic periderms (Mullick and Jensen 1973b) . It was clear from these studies that cryofixation could be used alone for dependable differentiation of the two categories of periderms, and as such it was the major technique used in this study. The f i r s t and sequent exophylactic periderms in the conifers and deciduous species studied always had brown or orange brown contents (tbf). With fluorescence, the cell contents of these brown periderms appeared various shades of brown (1/53) and dark brown or black ( I V / 4 1 ) . The necrophylactic periderms varied from colorless through shades of yellow and orange to reddish purple (tbf). Under fluorescence conditions with f i l t e r s 1/53 the most commonly recorded color was dull red (six species) but ranged from black through yellows and greens. With f i l t e r s IV/41 the contents appeared black in the majority of species, but dull khaki and pale blue were also recorded. Under mbf the brown contents of the exophylactic phellem appeared khaki green to khaki brown (Table h) and the contents of the necrophylactic phellem varied (Table 5 ) . - 78 -Distinction in fluorescence of the phellem walls of the exophylactic and necrophylactic periderms within a species is shown in Tables k and 5. Such distinction between the exophylactic and necrophylactic phellem cell walls within a species have been reported for three species of conifers (Mullick 1971; Mullick and Jensen 1973a). Differences in fluorescence characteristics of the cell walls and cell contents of the exophylactic and necrophylactic phellem were major fundamental distinctions between the necrophylactic and exophylactic periderms (Tables k and 5). When cryostat sections were thawed, air-dried, and then treated with absolute ethanol, diethyl ether, petroleum ether, ethylene glycol, hydrochloric acid, or sulfuric acid solutions, the brown contents in the exophylactic phellem remained intact, whereas the contents in the necrophylactic phellem were lost on thawing and (or) dissolved. This indicated that the phellem contents in these two types of periderms were chemically distinct. Similar distinctions between the classes of periderms were reported by Mullick (1971) and Mullick and Jensen (1973a, 1973b). SEP was found in a l l the species studied except in Cupressus macrocarpa, Pseudotsuga menziesii, Robinia pseudoacacia, and Hibiscus syriacus. FEP was present in these species and it was distinct from the NPs in a l l cryofixation characteristics (Tables k and 5)• Although SEP was not observed in these species, its existence cannot be ruled out. For some species, very limited material was available. In species where SEP was observed, it always - 79 -i n t e r n a l l y a b u t t e d the NPs. The f r e q u e n c y o f i t s o c c u r r e n c e v a r i e d c o n s i d e r a b l y between s p e c i e s . For example, in L a r i x o c c i d e n t a l i s and P inus c o n t o r t a , NPs d e v e l o p e d s u c c e s s i v e l y in deeper r e g i o n s o f bark o v e r a p e r i o d o f y e a r s w i t h o u t the deve lopment o f a SEP. S i m i l a r o b s e r v a t i o n s were r e p o r t e d in p r e v i o u s s t u d i e s o f pe r i de rms in o t h e r c o n i f e r s ( M u l l i c k 1971, M u l l i c k and Jensen 1973a, 1973b). I n the P i c e a s p e c i e s , Sequo ia semperv ? ren s , Taxus b r e v i f o l i a , Ace r  macrophyl1 urn, and G l e d i t s i a t r i a c a n t h o s , SEP was found a b u t t i n g a l l NPs excep t in the y e a r o f NP f o r m a t i o n . The NPs and SEPs d i d not d e v e l o p in the same y e a r in a l l s p e c i e s . It was e v i d e n t t h a t w i t h i n a s p e c i e s the f i r s t and the ' u s u a l ' sequent pe r i de rms in c o n i f e r s as w e l l as in d e c i d u o u s t r e e s were c l e a r l y d i s t i n c t f rom one a n o t h e r . SEP was i d e n t i c a l t o FEP and a lways d i s t i n c t f rom NP as r e v e a l e d by c r y o f i x a t i o n . Pe r i de rms a t s i t e s o f a b s c i s s i o n ( e . g . P i c e a g l a u c a and Pseudotsuga m e n z i e s i i ) , wound and p a t h o l o g i c a l pe r i de rms r e g a r d l e s s o f the c a u s a l a gen t s o r s i t e o f o c c u r r e n c e , were i d e n t i c a l t o the ' u s u a l ' sequent p e r i d e r m s a t rhy t idomes which a r o s e w i t h o u t any d i s e a s e o r t rauma. T h i s f u r t h e r s u b s t a n t i a t e d the s u g g e s t i o n o f s i m i l a r i t y in the deve lopmenta l p r o c e s s e s o f t he se NPs as w e l l as the c a u s a l f a c t o r s in t h e i r o r i g i n ( M u l l i c k and J e n s e n 1973b, M u l l i c k 1977). In Taxus b r e v i f o l i a , Sequo ia s e m p e r v i r e n s , and Cupressus m a c r o c a r p a , e x o p h y l a c t i c and n e c r o p h y l a c t i c pe r i de rms had o n l y t h i n - w a l l e d phe l l em c e l l s . In Pseudotsuga m e n z i e s i i o n l y the NPs were e n t i r e l y composed o f t h i n - w a l l e d p h e l l e m c e l l s . A l t h o u g h - 8 0 -the p h e l l e m i n t h e FEP and SEP o f Pseudotsuga m e n z i e s i i was m a i n l y t h i n - w a l l e d , s p o r a d i c a l l y d i s t r i b u t e d t h i c k - w a l l e d p h e l l e m was p r e s e n t . W i t h i n a s p e c i e s d i s t i n c t fundamental d i f f e r e n c e s were ob s e r v e d between t h e e x o p h y l a c t i c and n e c r o p h y l a c t i c p e r i d e r m s ( T a b l e s 4 and 5 ) . A l t h o u g h Chang (1954b, T a b l e 1) r e p o r t e d t h a t t h e p e r i d e r m s i n t h e s e s p e c i e s c o n s i s t e d o f 'mainly t h i n - w a l l e d p h a l l e m c e l l s ' , no d i s t i n c t i o n was r e p o r t e d among t h e p e r i d e r m s and, s e e m i n g l y , t h e y were c o n s i d e r e d a l i k e . The s p o r a d i c a l l y d i s t r i b u t e d t h i c k - w a l l e d p h e l l e m c e l l s , w h i c h were o c c a s i o n a l l y o b s e r v e d i n the FEP o f Pseudotsuga m e n z i e s i i were a l s o r e p o r t e d (Chang 1954b). Chang (1954b T a b l e 1) showed t h a t the p e r i d e r m s i n Lar i x o c c i d e n t a l i s , P i c e a s p e c i e s , P i n u s c o n t o r t a , and P i n u s m o n t i c o l a had 'bands o f t h i n - w a l l e d and t h i c k - w a l l e d c e l l s ' . R e s u l t s r e p o r t e d i n t h i s t h e s i s c l a r i f y t h a t Chang's o b s e r v a t i o n s p e r t a i n e d o n l y t o t h e FEP o r SEP and not t h e NPs. The FEP p h e l l e m i n the young bark o f L a r i x o c c i d e n t a l i s i s a lways t h i n - w a l l e d . Chang-. (1954b) r e p o r t e d t h a t t h e p e r i d e r m s i n .Pinus m o n t i c o l a were 'composed o f t h i n - w a l l e d o r d i n a r y c o r k c e l l s e n t i r e l y o r o f t h i n - w a l l e d and t h i c k - w a l l e d c e l l s i n a l t e r n a t e bands'. The f i n d i n g s o f t h i s s t u d y show t h a t the former ' o r d i n a r y c o r k c e l l s ' were t h e n e c r o p h y l a c t i c p h e l l e m , and t h e ' a l t e r n a t e bands o f t h i n -and t h i c k - w a l l e d c e l l s ' were the sequent e x o p h y l a c t i c p h e l l e m . The o c c u r r e n c e o f t h e n e c r o p h y l a c t i c and e x o p h y l a c t i c p e r i d e r m s i n P i n u s m o n t i c o l a and P i c e a s i t c h e n s i s was e s t a b l i s h e d by D o p p e l r e i t e r (1973) and S c h e l l e n b e r g (1974) r e s p e c t i v e l y . The v a l i d i t y o f t h e i r f i n d i n g s was c o n f i r m e d i n t h i s s t u d y . - 81 -Chang (1954a) found that periderms in Acer saccharum had alternate bands of thin- and thick-walled phellem c e l l s . However, distinction between the f i r s t and the 'usual' sequent periderms, or between periderms developed in 'young and mature bark' was not reported. This study shows that in Acer macrophyllum the periderms which developed in rhytidomes and around injuries were identical. These were the NPs and, similar to Acer saccharum, they also had thin- and thick-walled phellem c e l l s . The thin-walled phellem was the phellem of the NP, and the abutting thick-walled phellem, which was identical to the phellem of FEP, was the phellem of the SEP. Samples of bark, injured June 11, 1974 and collected in late November, 1974 had only NP. This suggested that the thin- and thick-walled phellem cells which belonged to the NPs and SEPs respectively do not form in the same year. Instead, the thin-walled phellem cells of NP form during one year's activity, and the thick-walled phellem cells of SEP develop in the following year. Similar results were observed in rhytidome formation. The thin-walled phellem of the NPs did not have SEP abutting it in the year of its formation. The phellogen activity marker c e l l s , which were suggested to denote the end of.a phase of phellogen activity in NP phellem formation, were f i r s t reported in Abies species and Tsuga  heterophylla (Mullick and Jensen 1973a, 1973b). Such phellogen activity marker cells (PAMC) were found in Pseudotsuga menziesi ?, Robinia pseudoacacia, and Gleditsia triacanthos in the necrophylactic phellem. Variations in seasonal renewal of the necrophylactic phellem formation revealed by the PAMC were observed within and between species. - 82 -In Robinia pseudoacac ia and G1ed i tsia tr iacanthos such renewal of the necrophylactic phellem occurred usually two to four times. In Pseudotsuga menziesii renewals of the necrophylactic phellem were observed. The number of necrophylactic phellem layers produced by one seasonal activity of phellogen varied within and between seasonal activities. In addition, PAMC were also found in the exophylactic phellem of FEP in Rob in ia pseudoacacia and Gled i ts ia tr iacanthos. These PAMC were identical seemingly to those in the necrophylactic phellem as revealed by cryofixation. The occurrence of these PAMC in the FEP had never been reported. Although seasonal renewal of FEP was observed in other species studied, PAMC were not detected. In Taxus brev?folia no seasonal renewal of FEP was observed. The FEP was replaced soon after formation by the NPs. The present study has substantiated the existence and distinctness of three periderms in woody plants namely FEP, SEP and NP. The f i r s t two periderms are identical in a l l anatomical characteristics, differing only in time and place of origin. The FEP replaces the epidermis, and usually renews its e l f for a number of years, depending on the species and environment, as it contends with the stem circumferential growth. During the period of FEP renewal, the old phellem is sloughed off slowly and the bark surface remains smooth without rhytidomes. When the FEPs are replaced by the NPs during rhytidome formation, cracks appear on the bark surface and, as more NPs developed over a period of years, more rhytidome is produced and the outer dead bark becomes thicker. Its surface becomes fissured and rough. Sloughing of rhytidome tissues in some species becomes - 83 -apparent when SEP has formed internally abutting the necrophylactic phellem. The tree has restored its normal 'skin' when SEP has developed inasmuch as SEP is identical to FEP. Thus, as pointed out by Mullick ( 1 9 7 7 ) , when trees' normal 'skin', FEP, becomes non-functional, new 'skin', SEP may eventually be restored, but before a new 'skin' is produced, the tree must provide the appropriate environment in the internal tissues for its restoration by the formation of the NP. This is because SEP is invariably found internally abutting the NP. When SEP has developed, rhytidome tissues often are sloughed off either as scales, for example i n P i cea, P ? nus, Lar ix, and Pyrus spec ies; or in strips as in Cupressus, Taxus, and ACer species. The outer appearance of bark and the sloughing of rhytidome tissues either in the form of scales or strips, can in part, be related to the nature and pattern (DeBarry 1 8 8 4 ; Muhldorf 1 9 2 5 ; Pfeiffer 1 928 ) of the NPs and the internally abutting SEPs. Scale bark is observed when NPs and SEPs are formed in overlapping scale-like layers. But when NPs and SEPs are developed in long overlapping strips, strip-bark is produced. These observations support the suggestion of the association SEP with en masse sloughing of dead bark tissue (Mul1ick 1 9 7 1 ) • However, the involvement of periderms insloughing or lack of i t is more complex than this. Although no SEP develops abutting the NPs in Cupressus macrocarpa, exfoliation of rhytidome tissues is observed seemingly due to the presence of cells with - 84 -t h i c k e n e d w a l l s a b u t t i n g t h e n e c r o p h y l a c t i c p h e l l e m . These c e l l s d i f f e r from t h e p h e l l e m o f FEP as r e v e a l e d by c r y o f i x a t i o n and o t h e r t e c h n i q u e s . In Seq u o i a s e m p e r v i r e n s and G l e d i t s i a t r i a c a n t h o s , SEPs abut, a l l NPs, y e t s l o u g h i n g o f r h y t i d o m e t i s s u e s i s not a p p a r e n t , p r o b a b l y due t o t h e s t r o n g a d h e s i o n o f the p e r i d e r m c e l l s and the s u c c e e d i n g l a y e r s o f r h y t i d o m e (Esau 1965). The r h y t i d o m e t i s s u e s i n Seq u o i a s e m p e r v i r e n s can be p e e l e d i n t o f i b r o u s s t r i p s (Chang 1954b) and a r e r e f e r r e d t o as f i b r o u s bark (Esau 1965). In Pseudotsuga m e n z i e s i i and R o b i n i a p s e u d o a c a c i a , no SEP was found a b u t t i n g t h e NPs, and no s l o u g h i n g o f r h y t i d o m e t i s s u e s i s e v i d e n t . Bark i s c r a c k e d and f i s s u r e d w i t h a c c u m u l a t i o n o f rhy t i d o m e t i s s u e s . Loss o f r h y t i d o m e t i s s u e s s e e m i n g l y o c c u r s s l o w l y by d e t e r i o r a t i o n and w e a t h e r i n g . The r e l a t i o n s h i p o f the n a t u r e and p a t t e r n o f NP and SEP f o r m a t i o n t o t h e form o f s l o u g h i n g o f r h y t i d o m e t i s s u e s r e q u i r e s f u r t h e r i n v e s t i g a t i o n . The new c o n c e p t s o f n e c r o p h y l a c t i c and e x o p h y l a c t i c p e r i d e r m s were e s t a b l i s h e d i n t h i s s t u d y f o r r e p r e s e n t a t i v e s p e c i e s from a l l o f t h e c o n i f e r o u s f a m i l i e s and a l s o f o r a number o f d e c i d u o u s s p e c i e s . T h i s s u g g e s t s t h a t the e x o p h y l a c t i c and n e c r o p h y l a c t i c p e r i d e r m s a r e o f g e n e r a l o c c u r r e n c e i n woody p l a n t s . The e x t e n s i v e s a m p l i n g s c a r r i e d out i n t h i s s t u d y and the l a r g e body of d a t a from i t f i r m l y e s t a b l i s h t h e g e n e r a l i t y o f the new c o n c e p t s . Combined w i t h some o f t h e e a r l i e r works on p e r i d e r m s , i n t e r p r e t e d i n terms o f t h e new c o n c e p t s , a b e t t e r u n d e r s t a n d i n g o f the p h y s i o l o g y o f p e r i d e r m s s h o u l d now be p o s s i b l e . - 85 -5.2 The General Occurrence of the Non-suberized Impervious Tissues (NIT) The F-F test solutions invariably stopped at the external and internal boundaries of NIT when tests were carried out either through wounds or the cambial surface, or through both surfaces simultaneously. Mullick (1975) presented supportive evidence and rationale for the validity of the F-F test in detection of an impervious barrier in bark tissue. The present findings in al l samples from fourteen species were consistent with those reported by Mul1ick (ibid), reinforcing his conclusion on the the F-F test va 1 id i ty. Detailed developmental studies were made of NIT in Pinus contorta and Larix occidental is and characterization of NIT was made for the other species studied. The sequence of changes observed were consistent with the earlier reports of Mullick (1975, 1977, personal communication). Following injury internally abutting cells continued to die and progressive changes of tissues internally occurred including cell hypertrophy, culminating in NIT and NP formation. Anatomical and biochemical changes of tissues were shown in part by the changes in fluorescence and other characteristics of the cells and their contents in the cryofixed state. During NIT development cell division was not observed, but was present in NP formation. The process of NP formation which involved cell enlargement and cell division of pre-existing cells abutting NIT is referred to as meristematic differentiation and that of NIT which involved only enlargement of pre-existing c e l l s , is referred to as hypertrophic - 86 -de-differentiation. Thus the processes of NIT and NP formation were fundamentally distinct. Generally, NIT consists of enlarged cells with thickened walls of varying shapes and sizes. These cells appear yellowish brown in thin sections under tbf, and intercellular spaces do not exist between these c e l l s . Usually the external boundary of NIT in cryostat sections was delineable only by the blue coloring of the test solutions. Without the test the boundary was not easily recognizable because cells externally abutting NIT appear quite similar, although fluorescence characteristics of NIT cells were distinctive. Histological tests substantiate that the imperviousness of NIT is not due to suberization. They further showed that this imperviousness is distinct from the often-mentioned suberization of injured surfaces, prior to wound periderm formation, in agricultural plants, e.g. in potato tubers (Artschwager 1927; Esau 1965)- The non-suberization of NIT was further confirmed from sections treated with Sudan III and ammonia-crystal violet examined with f i l t e r combinations 1/53 and IV/41. Only the cell walls of necrophylactic phellem fluoresced red, a positive test for suberization (Mullick 1975)- NIT cell walls showed neither red fluorescence nor any red fluorescent lining on the inside surfaces of its cell walls. Thus, NIT was also distinct from the primary protective layer reported by Facey (1956) which possessed a thin lining of suberin in the c e l l s . The tests of NIT for xallose.were also negative. - 8 7 -These findings from the developmental studies combined with the characterization of NIT in the other species studied strongly support the idea that NIT formation is a process of general occurrence in coniferous and in deciduous trees. In coniferous and deciduous species, NIT showed positive staining to phiorog1ucinol-HC1 and aniline sulfate-HCl, stains commonly used for detecting lignin. Thus, the test results alone do not permitpositive identification of NIT because in addition to NIT, sclereids, thick-walled phellem of exophylactic periderm, cortical parenchyma and sieve cells externally abutting NIT, particularly in rhytidome, were also stained uniformly. Even identification of NIT on the basis of positive tests in conjunction with its characteristic morphology was not possible, since positive tests were obtained in tissues which probably were destined to become NIT prior to impermeability formation. Similar results were obtained of NIT in sections treated with the Maule reaction which were used to distinguish gymnosperm lignin from that of the angiosperm. The former yields a yellow or pale brown color while the latter gave a red color (Panshin and de Zeeuw 1 9 7 0 ) . In conifers, NIT and other tissues which reacted positively to the usual lignin stains were stained yellow to pale brown, which could be interpreted as a positive detection for gymnosperm lignin. In deciduous species, the results were similar, except for sclereids and xylem cells which stained red, a positive staining for angiosperm lignin. - 88 -The observations of the Maule reaction in normal deciduous tree tissues is interesting by i t s e l f , since, in angiosperm, thick-walled phellem of FEP, cortical parenchyma and sieve cells react positively to a l l lignin tests except the Maule reaction. It indicates either that the Maule reaction is not specific for angiosperms lignin or there are at least two distinct types of lignin in angiosperms, one unique to angiosperms (Maule positive) and one closely related to or identical to gymnosperm lignin (Maule negative). Further, Hewitt (1938) distinguished 'wound gum' in angiosperm from lignin indicated by Maule reaction. According to him wound gum 'reacts positively to most lignin tests but does not color with the Maule reaction'. It is generally agreed (Panshin and de Zeeuw 1970) that the basic structure unit of lignin is the phenyl propane skeleton. It is well reported that angiosperms exhibit both guaiacyl and syringyl units in their lignin, while gymnosperms contain only guaiacyl. Thus one possible explanation is that the Maule 'positive' tests (red color) indicate the presence of syringyl units while Maule 'negative' tests (yellow or pale brown) indicates only guaicyl units. It appears that sclereids and xylem cells in angiosperms contain lignin of the angiosperm type while NIT and other cell types in gymnosperms and angiosperms contain lignin only of the gymnosperm type. NIT cell walls turned yellow and pinkish red upon exposure to ammonia and hydrogen chloride gases respectively. The former were used for the detection of flavonoids with free phenolic - 89 -groups (flavones, flavonols) in plant-tissue sections (Craft and Audia 1962; Harborne 1964). This finding further suggested that the NIT cell walls may contain abundant polyphenolic compounds of the C,-C_ skeleton because flavonoids as well as lignins are o 5 Cg-C^ compounds. NIT and the other tissues which give positive staining to the usual lignin tests, remain stainable after sections are treated with absolute ethanol, diethyl ether, petroleum ether, benzene and xylene. This indicates that the compound or compounds which are stainable with the lignin tests are non-extractable with these solvents. Although the chemical nature of NIT has been cla r i f i e d somewhat, the substance or substances which contribute to its imperviousness remained unknown. However, if the usual lignin tests are valid, it seems reasonable to conclude at this stage that gymnosperm lignin or closely related compounds are involved in NIT imperviousness. The findings here confirm those of Mullick (1975) that NIT formation always precedes the development of NP. Whenever NP is found, NIT is always present. NIT is never observed in association with exophylactic periderm formation. The invariable association of NIT with NP and its absence at exophylactic periderms supports the statement that the presence or absence of NIT is a distinguishing characteristic between the two categories of periderms (Mullick 1975)• NP always developed specifically from tissues internally abutting NIT. IF NIT did not develop, NP did not form. NIT was often seen without NP, but NP was never found without NIT. Thus it is concluded that Mullick's 1975 statement that the specific site of a l l NP formation must be the tissue internally abutting NIT is generally valid. - 90 -The equivalence of NIT within a species at injuries, diseased sites, resin blisters, rhytidome formation, and abscission zones observed in this thesis confirms the findings of Mullick (1975) that the process of NIT formation, like that of NP, was also non-specific. The non-specificity of NIT formation in the absence and presence of injury and disease agents showed that its induction, as reported by Mullick (1977)> results from a common cause rather than from the various stimuli which were reported to induce the formation of wound and pathological periderms (Bloch 19^ 1, 1952, 1953; Davies 1945; Struckmeyer and Riker 1951; Oechssler 1962; Carter 1962; Hare 1966)-The invariable presence and equivalence within a species of NIT at injuries, diseased sites, resin blisters, rhytidome formation, and abscission zones suggested that the processes of its formation and causal factors of origin at the various sites were similar. Further, the data obtained from 14 species fully supports the suggestion by Mullick (1977) that whenever the phellogen becomes non-functional for whatever cause, biotic or abiotic, it is the initiation of the non-specific process of phellogen regeneration that results in NIT and NP formation. This information provides a new insight in understanding the physiology of wound healing and also provides an opportunity for clear-cut isolation of the host component in host-parasite interactions. On the basis of the facts that NP does not develop in the absence of NIT and that NP is always found internally abutting NIT, Mullick (1975) concluded that NIT provides the specific site and appropriate environments in tissue internally abutting it for NP formation. The present studies fully support this conclusion. - 91 -In susceptible Pinus mont icola, NP did not develop at Cronartium  ribicola F. infection sites (Struckmeyer and Riker 1951)- Similarly, in Abies species under heavy attack by balsam woolly aphids (Adelges  piceae Ratz.), NP failed to form, or formed only partially, presumably because the processes of NIT formation were affected (Mullick 1975). Water stress, a factor in promoting some diseases has been shown to affect adversely NIT formation (Puritch and Mullick 1975). Mullick and Jensen (1976) have demonstrated that environmental factors associated with seasonal changes can greatly slow the rates of NIT formation to a virtual halt in winter and to lesser degrees at other times of the year. Thus, if the processes of NIT formation becomes adversely affected by any factor, biotic or abiotic, phellogen regeneration leading to NP formation would not occur. Consequently, the tree, then incapable of healing its wound and protecting it s e l f would provide unopposed entry for invading organisms. - 92 -6. SUMMARY AND CONCLUSIONS Mullick (1973) recently reported the discovery of a non-suberized impervious tissue (NIT) which is a pre-requisite to the formation of necrophylactic periderms but is never found in association with exophylactic periderms in three species of conifers. The purpose of this thesis has been to determine whether necrophylactic and exophylactic periderms and their association with NIT are of general occurrence not only in a l l coniferous families but also in a few families of Angiosperms. 1. The concept of exophylactic and necrophylactic periderms was found to be valid in a l l conifers studied and i t is reasonable to extrapolate that the concept is valid for conifers. Although the concept was found to be valid for the 5 deciduous species studied, more data on additional species are needed before its general validity for this group can be claimed. 2 . The non-specific occurrence of non-suberized impervious tissue (NIT) was demonstrated in a l l 15 species studied, and always provided the essential environment for NP formation. NIT can be found without NP prior to completion of the process of phellogen restoration but NP was never found without NIT. NIT was never found in association with the exophylactic periderms. This general occurrence of NIT in association with NP only further strengthens the exophylactic and necrophylactic periderm concept. - 93 -In a l l s p e c i e s s t u d i e d t h e e x o p h y l a c t i c p e r i d e r m s w i t h i n a s p e c i e s may c o n s i s t e i t h e r o f o n l y t h i n - w a l l e d p h e l l e m c e l l s o r both t h i n - w a l l e d and t h i c k - w a l l e d p h e l l e m c e l l s a r r a n g e d i n a l t e r n a t e l a y e r s o f v a r i o u s t h i c k n e s s . T h i n -w a l l e d p h e l l e m c e l l s a r e always formed f i r s t . The p h e l l e m o f n e c r o p h y l a c t i c p e r i d e r m s i n a l l s p e c i e s s t u d i e d c o n s i s t s o f o n l y t h i n - w a l l e d p h e l l e m c e l l s . H i s t o l o g i c a l s t a i n i n g s show t h a t the i m p e r v i o u s n e s s o f NIT i s not due t o s u b e r i n o r c a l l o s e . However, i f the u s u a l l i g n i n t e s t s a r e v a l i d , i t seems r e a s o n a b l e t o c o n c l u d e t h a t gymnosperm l i g n i n o r c l o s e l y - r e l a t e d compounds (C^-C^) a r e i n v o l v e d i n NIT i m p e r v i o u s n e s s . Seasonal renewal o f NP p h e l l e m denoted by t h e p h e l l o g e n a c t i v i t y marker c e l l s (PAMC) was found i n Pseudotsuga menz i es i i , R o b i n i a pseudoacac i a , and G l e d ? t s i a t r i a c a n t h o s . The number o f p h e l l e m c e l l s produced per s e a s o n a l phel1ogen a c t i v i t y v a r i e d w i t h i n and between s e a s o n a l a c t i v i t i e s and s p e c i e s . The p r e s e n c e o f PAMC a b u t t i n g the p h e l l e m c e l l s o f FEP i n Gled i t s i a t r i a c a n t h o s and Rob i n i a pseudoacac i a was o b s e r v e d and r e p o r t e d f o r the f i r s t t i m e . S l o u g h i n g , o f dead r h y t i d o m e t i s s u e s i n t h e form o f s c a l e s , s t r i p s , and f i s s u r e s seemed t o be r e l a t e d t o the n a t u r e and p a t t e r n o f t h e NP and SEP. - Sh -9. Process of phellogen restoration constitutes a major host-component in host-pathogen interactions. Therefore, detailed studies of the process of NIT formation and how environmental factors affect it are essential to understanding the nature of diseases of bark. - 95 -T A B L E S (1 " 6) - 96 -Table 1. Age of tree region and date of bark collection of FEP samples. Age of No.of Age regions Date of Species trees in studied Collection studied y e a r s ( y e a r s ) (1974) Picea glauca (Moench) Voss. 4 4,6,13,26 1 to 7 June 12 Picea engelmanii Parry 4 4,7,15,28 1 to 8 June 12 Picea sitchensis (Bong) Carr. 3 11,15,37 > 1 to 6 June 12 Pinus contorta Dougl. 4 4,4,19,24 to 6 June 12,26 Pinus monticola Dougl. 3 11,14,32 to 6 July 12 Pseudotsuga menziesii (Mirb) Franco 3 4,16,50 to 5 July 26 Larix occidentalis Nutt 5 4,4,14,19,28 to 6 Aug. 8 Feb. 6 Cupressus macrocarpa Hartw. 1 36 to 6 June 17 Aug. 8 Sequoia sempervirens Endl . 1 22 to 5 June 18,20 Taxus brevi'folia Nutt. 3 45,52,68 to 7 June 18,25 Acer macrophyllum Pursh. 2 11,13 to 10 June 13 Robinia pseudoacacia L. 4 10,14,25,46 to 8 May 20 June 3 Gleditsia triacanthos Inermis 6 6,14-20 to 10 Aug. 8 Pyrus species 1 4,6 1 to 4 Oct. 1 Hibiscus syriacus Hamabo. 2 to 3 May 23 - 97 -Table 2. Age of tree region and date of bark collection of NP and SEP. Age of Species regions stud ied (years) Picea glauca (Moeneh) Voss. 12,24. June 12, Aug. 14 Picea engelmannii Parry 14,18 June 12 Picea sitchensis (Bong) Carr. 14,30 June 12 Pinus contorta Dougl. 11,18 June 12, July 26 Pinus monticola Dougl. 11,13,26 June 12 Pseudotsuga menziesii (Mirb) 16,30,42 July 26 Larix occidentalis Nutt. 12,15,25 June 19, Aug. 8 Cupressus macrocarpa Hartw. 7,26,30 June 17, Aug. 8 Sequoia sempervirens Endl. 7,20 June 20, July 25 Taxus brevifolia Nutt. 8,40 July 25, Sept. . 3 Acer macrophyllum Pursh. 10,12 June 13, June 20 Robinia pseudoacacia L. 10,20,32 May 6, June 10 Gleditsia triacanthos Inermis 13,18,19 Aug. 8 Pyrus species 6,16,19 Oct. 9 Hibiscus syriacus Hamabo. 6 May 23, Aug. 8 Date of col lection (1974) - 98 -Table 3. Usual tree stem age of i n i t i a l rhytidome formation. Spec i es Approximate age of stem at rhytidome formation Ci n years) Picea glauca (Moench) Voss. Picea engelmanni i Parry Picea sitchensis (Bong) Carr. P i nus contorta Doug 1. Pinus monticola Dougl. Pseudotsuga menziesii (Mirb) Franco. Larix occi denta1i s Nutt. Cupressus macrocarpa Hartw. Sequoia sempervi rens Endl. Taxus brevifolia Nutt. Acer macrophyllum Pursh. Robinia pseudoacacia L. Gleditsia triacanthos Inermis Pyrus species Hibiscus syriacus Hamabo. 15 17 15 18 ik 17 14 7 7 3 10 6 6 7 6 - 99 " T a b l e 4. C r y o f i x a t i o n c h a r a c t e r i s t i c s o f t h e c o n t e n t s and w a l l s o f e x o p h y l a c t i c p h e l l e m . P h e l l e m c o n t e n t s P h e l l e m w a l l s S p e c i e s t b f mbf 1/53 IV/41 1/53 IV/41 P i c e a g l a u c a (Moeneh) V o s s . Brown P i c e a s i t c h e n s i s (Bong) C a r r . Brown P i c e a e n g e l m a n n i i P a r r y Brown P i n u s c o n t o r t a D o u g l . Brown P i nus mont ? c o l a Doug 1. Brown P s e u d o t s u g a m e n z i e s i i ( M i r b ) F. Brown L a r i x o c c i d e n t a l i s N u t t . Orange brown Cup re s su s m a c r o c a r p a H a r t w . Brown S e q u o i a s empe r v i r en s End 1. Brown Taxus b r e v ? f o l i a N u t t . Brown A c e r mac rophy l ! u rn P u r s h . Orange b rown R o b l n i a p s e u d o a c a c i a L. Brown G l e d i t s i a t r i a c a n t h o s I n e r m i s Brown Py r u s s p e c i e s Orange u . . . . „ , brown H i b i s c u s s y r i a c u s Hamabo. Orange brown Khak i L i g h t Dark Y e l l o w i sh TN:PB g r een brown brown g r e e n TK:BG Khak i Dark B l a c k Y e l l o w i sh TN: PB g r e e n brown g r een TK:BG Khak i L i g h t Dark Y e l l o w i s h TN: PB g r e e n brown b rown g r e e n TK:BG Khak i Brown Dark Y e l l o w i sh T N : B l u e g r e e n brown g r e e n TK:GB Khak i Brown Dark Y e l l o w i sh TN:GB g r een brown g r e e n TK:GB Khak i L i g h t Dark G r e e n i s h P a l e g r een brown brown y e l l o w b l ue Khak i L i g h t Dark Y e l l o w i s h T N : B l u e g r e e n brown brown g r e e n T K : K h a k i Khak i Brown Dark Y e l l o w i sh P a l e brown brown g r e e n b l ue Khak i Brown Dark Green P a l e brown brown b l ue Khak i Brown Dark Y e l l o w i sh P a l e brown brown g r e e n b l u e Khak i Dark B l a c k B l u e brown brown Khak i L i g h t Dark Y e l l o w i sh P a l e g r e e n brown brown g r e e n b l ue Khak i Dark B l a c k Y e l l o w i s h P a l e brown brown g r e e n b l u e Khak i Dark B l a c k G reen B l u e brown brown Khak i L i g h t Dark Y e l l o w P a l e brown brown brown b l u e N o t e : TN: t h i n - w a l l e d p h e l l e m TK: t h i c k - w a l l e d p h e l l e m PB: p a l e b l u e BG; b l u e g r e e n GB: g r e e n i s h b l u e t b f : t u n g s t e n b r i g h t f i e l d mbf: m e r c u r y b r i g h t f i e l d 1/53: f l u o r e s c e n c e c o m b i n a t i o n o f Z e i s s e x c i t e r f i l t e r I and b a r r i e r f i I t e r 53 IV/41: f l u o r e s c e n c e c o m b i n a t i o n o f Z e i s s e x c i t e r f i l t e r IV and b a r r i e r f i I t e r 41 - 100 -T a b l e 5- C r y o f i x a t i o n c h a r a c t e r i s t i c s o f t h e c o n t e n t s and w a l l s o f n e c r o p h y l a c t i c p h e l l e m . P h e l l e m c o n t e n t s Phe l1 em wa11s e c i e s . t b f mbf 1/53 IV/41 1/53 IV/41 P i c e a q l a u c a (Moench) V o s s . No c o l o r No c o l o r B l a c k B l a c k Green B l u e ye 1 low P i c e a s i t c h e n s i s (Bonq) C a r r . P i n k P a l e Dul 1 P a l e Green B l u e g r a y r ed b l ue ye 11ow P i c e a e n g e l m a n n i i P a r r y No c o l o r No c o l o r B l a c k B l a c k Green B l u e ye1 low P i n u s c o n t o r t a Doug l . Go 1 den Y e l l o w D u l l Dark Green P a l e P i n u s m o n t i c o l a Doug l . ye 11ow g r e e n red red ye 1 low b l ue Y e l l o w Brown P a l e Green P a l e b rown ye 11ow b 1 ue ye 11ow b l u e P s eudo t s uga m e n z i e s i i ( M i r b ) F. Dark Brown P a l e P a l e Y e l l o w B l u e g r a y g r e e n g r e e n b l ue g r e e n L a r i x o c c i d e n t a l i s N u t t . R e d d i s h B l a c k Dul 1 B l a c k P a l e P a l e p u r p l e r ed g r een b l ue Cup re s su s m a c r o c a r p a H a r t w . R e d d i s h Dark Dul 1 B l a c k B r i g h t B l u e p u r p l e red r e d ye 1 low S e q u o i a s e m p e r v i r e n s E n d l . R e d d i s h B l a c k R u s t y B l a c k Green B l u e p u r p l e r e d ye1 low Taxus b r e v i f o l i a N u t t . R e d d i s h Dark Red B l a c k G reen B l u e p u r p l e red A c e r m a c r o p h y l l u m P u r s h . Ru s t y Dark Y e l l o w D u l l Y e l l o w P a l e r e d brown brown k h a k i g r e e n b l u e R o b i n i a p s e u d o a c a c i a L. Y e l l o w Gray P a l e B l a c k Y e l l o w G ree r g r a y g r e e n g r e e n b l u e G l e d i t s i a t r i a c a n t h o s I n e r m i s Orange P a l e Orange B l a c k G reen B l u e o r a n g e r e d P y r u s s p e c i e s P a l e L i r . h t B l a c k B l a c k Y e l l o w P a l e g r a y g r a y b l u e H i b i s c u s s y r i a c u s Hamabo. Gray Brown P a l e P a l e Y e l l o w B l u e g r a y g r e e n b l u e g r e e n Note: TN t h i n - w a l l e d p h e l l e m t b f : TK t h i c k - w a l l e d p h e l l e m mbf: PB p a l e b l u e 1/53: BG b l u e g r e e n GB g r e e n i s h b l u e IV/41 t u n g s t e n b r i g h t f i e l d m e r c u r y b r i g h t f i e l d f l u o r e s c e n c e c o m b i n a t i o n o f Z e i s s e x c i t e r f i l t e r I and b a r r i e r f i I t e r 53 : f 1 u o r e s c e n c e c o m b i n a t i o n o f Z e i s s e x c i t e r f i l t e r IV and b a r r i e r f i I t e r 4 1 . - 101 -Table 6. Time to NIT formation following injury, made on June 11, 1974. Species Days Picea glauca (Moeneh) Voss. 32 Picea sitchensis (Bong) Varr. 21 Picea engelmanni i Parry 32 Pinus contorta Dougl. 21 P i nus'mont icpla Doug 1 . 28 Pseudotsuga menziesii (Mirb) Franco. 23 Larix occidental is Nutt. 21 Cupressus macrocarpa Hartw. 17 Taxus brevifolia Nutt. 28 Sequoia sempervirens Endl. 21 Robinia pseudoacacia L. 21 Gleditsia triacanthos Inermis 21 Acer macrophyllum Pursh. ]k Hibiscus syriacus Hamabo. 15 - 102 -F I C U R E S (1 - 205) - 103 -ABBREVIATIONS.USED TO LABEL FIGURES b bark dt dead bark tissues dz de-differentiation zone epi epidermis FEP f i r s t exophylactic periderm F-F permeation of ferric chloride and potassium ferricyanide permeability test solutions lt living bark tissues mt meristematic tissues NIT non-suberized impervious tissues NP necrophylactic periderms OCT optium cutting temperature compound manufactured by Ames Company, Division of Miles Lab. Inc., Indiana, U.S.A. PAMC phellogen activity marker cells SEP sequent exophylactic periderm s i t semi-lunar tissues spl secondary protective layer Tk thick-walled phellem cells Tn thin-walled phellem cells ws wound surface x xy1 em - 104 -F i g u r e 1 T h i s f i g u r e shows the p a t t e r n and s i t e o f p h e l l e m ( c o r k ) , p h e l l o g e n ( c o r k cambium), p h e l l o d e r m , and c o r t e x f o r m a t i o n in woody p l a n t s ( c o u r t e s y o f M u l l i c k 1977). - 105 -IELLEM FORMATION PHELLODERM FORMATION Figure 2 This figure shows the mechanism of cell division involved in classical periderm formation. Note that the phellem (dark cells) is produced towards the outside of the phellogen (gray cell) while that of the phelloderm (white cells) towards the inside (courtesy of Mullick 1977). - 106 -Figure 3 • This figure shows the approximate locations where f i r s t periderm, sequent periderm and rhytidome can be located in woody plants (courtesy of Mullick 1977). - 107 -Figure 4 The splits in epidermis together with the adhering needle bases or petiole from the 5th internodal area of Picea glauca result in roughened appearance of the bark surface. Similar features were observed in Picea engelmannii and Picea sitchensis. Figure 5 Roughened appearance of bark from the 6th internodal area in Picea glauca. Needles were shed before the needle bases were sloughed. - 108 -- 109 -Figures 6, 7, 8 and 9 These figures show the thin and thick walled phellem cells of Picea glauca in cryofixed section using various modes of illuminations. Figure 6 is under tbf Figure 7 is pcp/mbf Figure 8 is 1/53 and Figure 9 is IV/41. Figure 9a is mbf. Note the sloughing of the epidermis (21 OX). Similar findings were observed in Picea engelmannii and Picea sitchensis. - 110 -- I l l -- 112 -Figure 10 A cross section of FEP of P i cea engelmanni i at the 5th internode collected on June 12, 1974. The thin- and thick-walled phellem cells, and the remains of the sloughed epidermis are shown. Note the undistorted rectangular shape of the newly formed thin-walled phellem (85X). - 113 --114 -F i g u r e s 11, 12, 13 and 14 Those f i g u r e s show NPs found next t o rhytidome i n P i c e a s i t c h e n s i s under v a r i o u s modes o f i l l u m i n a t i o n i n c r y o f i x e d s e c t i o n s . F i g u r e 11 i s t b f F i g u r e 12 i s pep F i g u r e 13 i s 1/53 and F i g u r e 14 i s IV/41 r e s p e c t i v e l y . Note t h a t the p h e l l e m i s t h i n - w a l l e d . The p h e l l e m c o n t e n t s appear dark and p i n k ( t b f ) and f l u o r e s c e d u l l red and p a l e b l u e when examinedwith f i l t e r c o m b i n a t i o n s 1/53 and IV/41 r e s p e c t i v e l y . A l l p h e l l e m w a l l s f l u o r e s c e g r e e n i s h y e l l o w (1/53) and b l u e (IV/41) (135X). - 115 -- 116 -- 117 -Figure 15 A thawed section of NP with thin- and thick-walled phellem of SEP abutting it at rhytidome of Picea sitchensis after treatment with absolute ethanol. The dark and pink contents of the necrophylactic phellem had disappeared and the phellem had assumed a f a i r l y uniform shape (tbf, 85X) . - 1 1 9 -Figure 16 Cryofixation characteristics of a NP at an abscission scar at the 3rd internodal area in Picea glauca. Only thin-walled phellem are present. The thin-walled phellem of SEP (not clearly seen here) was found abutting the NP, but the thick-walled SEP phellem was not observed (1/53, 85X). Figure 17 NP in P i cea s i tchens i s at the site of attack by P i ssodes s i tchens i s Hopkins as revealed by cryofixation. The periderm was identical to those found at rhytidome (1/53, 135X). - 120 -- 121 -Figure 18 A NP of Picea sitchensis found at rhytidome (1/53, 210X). Figures 19 and 20 Thin- and thick-walled phellem of FEP in Pinus contorta is shown. Similar phellem was observed in FEP of Pinus monticola. Note that some of the thick-walled phellem had thick outer and thin inner tangential walls (tbf, 135X). - 123 -- 124 -Figure 21 A FEP of Pinus contorta treated with Sudan 111. Only thin-walled phellem was stained red; thick-walled phellem did not stain. The brown pigmented contents s t i l l remained in the phellem even after many washings with 85% ethylene glycol for several hours. Similar results were obtained with FEP of Pinus monticola (tbf, 135X). Figure 22 NP found at freshly forming rhytidome in Pinus contorta collected on November 25, 1974. The phellem is thin-walled and slightly distorted (1/53, 210X). - 126 -Figures 23 and 24 A 35 day and a 210 day old mechanical injury of Pi nus contorta made on June 17, 1974 and April 11, 1974 and collected on July 17, 1974 and November 9, 1974, respectively as revealed by cryofixation. The necrophylactic phellem cells in the early stages of development are undistorted and roughly rectangularly shaped and have no pigment. Note pigment had already developed in the f i r s t phellem (arrow). In the old mechanical injuries the necrophylactic phellem cells have pigment and the cells are flattened and compressed. In both injuries SEP was not present (1/53, 135X). - 128 -A 2 month old mechanical Slight distortion of the not yet pigmented (1/53, Figure 25 injury showing a NP in Pinus monticola. phellem cells is evident which were 85X). - 130 -Figure 26 A NP found in the stem of Pinus monticola at the site of an infection of Cronartium ribicola as revealed by cryofixation. The tree was about 25 years old (IV/41, 135X). Figure 27 A NP found in a diseased site on a branch of Pinus monticola attacked by Cucurbidothis pithyophila as revealed by cryofixation. The tree was about 30 years old (IV/41, 135X). - 131 - 132 -Figure 28 Three NPs found in successively deeper regions of bark without the development of a SEP at rhytidome in Pinus contorta. The sample was collected from a region of old rhytidome where no scaling was observed (1/53, 85X). Figure 29 Typical appearance or bark surface of Pinus contorta showing scaling of dead rhytidome tissues. The bark surface appeared very rough from the sloughing. - 133 -- 134 -Figures 30 and 31 An exposed SEP of Pinus contorta rhytidome showing thin- and thick-walled phellem. There are more layers of thick-walled phellem cells than thin-walled phellem cells (tbf, 85X; tbf, 35X). - 135 -- 136 -Figure 32 An unexposed SEP of Pinus monticola rhytidome showing thin- and thick-walled phellem abutting the necrophylactic phellem which was deteriorating. Thin-walled phellem was found external to the thick-walled phellem which are more in number (1/53, 135X). Figure 33 A NP with SEP abutting it found in a diseased branch infected with Cucurbidothis pithyophila in Pinus monticola treated with phloroglucinol and concentrated hydrochloric acid. The NP phellem contents have disappeared, but the brown contents in the thin-walled cells of SEP remained. All phellem walls stained f a i r l y pink. Similar results were obtained with NPs and SEPs of Pinus contorta (tbf, 135X). - 138 -F i g u r e s 34 and 35 C r y o f i x a t i o n shows FEP found below the e p i d e r m i s i n Pseudotsuga  menz i e s i i . The p h e l l e m had brown c o n t e n t s , and t h i n - w a l l s w h i c h f l u o r e s c e d p a l e b l u e ( I V / 4 1 , 135X; IV/41, 210X). - 139 -- 140 -Figures 36 and 37 NP of Pseudotsuga menziesi i collected in the year of i ts format ion at rhytidome, macroscopically and microscopically, respectively. The NPs appear dark gray in the frozen block and the phellem cells at this early stage of development are undistorted and rectangular in shape (1/53, 85X). - l i t l -- 142 -F i g u r e 38 NPs i n r h y t i d o m e o f Pseudotsuga m e n z i e s i i c o l l e c t e d a t the end o f the g r o w i n g season ( m a c r o s c o p i c a l l y ) . - U,3 -- 144 -F i g u r e s 39 and 40 Seasona l renewal o f n e c r o p h y l a c t i c p h e l l e m a t rhy t idome o f Pseudotsuga m e n z i e s i i a r e shown. These seasona l renewals o f NP were denoted by the p h e l l o g e n a c t i v i t y marker c e l l s (PAMC). The PAMC have t h i c k e n e d w a l l s and s t r o n g e r b i r e f r i n g e n c e , and they f l u o r e s c e d b r i g h t e r b l u e ( IV/41) and b r i g h t e r y e l l o w (1/53) than the a b u t t i n g n e c r o p h y l a c t i c p h e l l e m c e l l s . They a r e (PAMC) u s u a l l y 1 t o 2 c e l l s t h i c k ( f c p , 210X; 1/53, 35X). - 146 -Figures 41 and 42 NP of Pseudotsuga menziesii collected at the end of the growing season as revealed by cryofixation. The phellem and the phellogen activity marker cells (PAMC) are shown. (1/53, 135X; IV/41, 135X). - 147 -- 148 -Figure 43 NP found at resin blister in Pseudotsuga menziesii. The NP is similar to those found at rhytidome (Table 5)- The phellogen activity marker cells (PAMC) is evident (IVA1, 135X). Figure 44 Rhytidome NP of Pseudotsuga menziesii after treatment with Sudan III. All necrophylactic phellem cell walls are stained red. NPs of similar characteristics were also found around beetle holes (tbf, 135X). - 149 -- 150 -F i g u r e 45 PAMC found a t a b s c i s s i o n s c a r i n P seudo t suga m e n z i e s i i ( I V /41 , 135X) . F i g u r e 46 FEP was f ound 2 t o 3 c e l l s be low the e p i d e r m i s i n L a r i x o c c i d e n t a l i s ( 1 / 5 3 , 1 3 5 X ) . - 152 -Figure 47 The smooth and rough bark surfaces in Larix occidentalis. The rough lower portion of the stem shows sloughing of dead rhytidome tissues. The tree was approximately twenty years old Figure 48 Phellem of FEP in young stem of Larix occidental is. Under IV/41 fluorescence f i l t e r combination, the phellem contents appeared dark brown; while the phellem walls fluoresced pale blue (IV/41, 210X). - 153 -- 154 -Figure 49 A FEP in old stem of Larix occidental is. Note the presence of some thick-walled phellem interspersed in the thin-walled phellem of the FEP. Under IV/41 f i l t e r combination the thick-walled phellem fluoresced khaki (IV/41, 210X). Figure 50 NP found at rhytidome in Larix occidental is as revealed by cryofixation under 1/53 f i l t e r combination. Note that the phellem content fluoresced dull red and the cell walls fluoresced pale green. The phellem cells are also compressed and distorted (1/53, 135X). - 155 -- 1 5 6 -Figure 51 NP found at a 33 day old injury in Larix occidental is by cryofixation under 1/53 f i l t e r combination after F-F test. Note that dull red pigment has formed in the f i r s t NP phellem. The outer phellem is flattened and the inner phellem is turgid (1/53. 135X). Figure 52 Macroscopic view of FEP, NPs and SEP at rhytidome of Larix occidentalis• FEP and SEP are brown. NPs are reddish purple. The dead rhytidome tissues between NPs are brownish and living tissues below are grayish. - 157 -- 158 -Figure 53 An air dried cryofixed section of Larix occidental is showing NP and SEP at rhytidome was treated with phloroglucina1-HCL. The distorted and compressed NP phellem and SEP phellem was not stained. Note that the brown contents in the SEP phellem s t i l l remained in the phellem while that in the NP phellem had disappeared (tbf, 135X). - 159 -- 160 -F i g u r e s 54 and 55 NPs found a t rhyti d o m e and a m e c h a n i c a l i n j u r y ( f i v e months o l d ) r e s p e c t i v e l y i n Cupressus macrocarpa ( 1 / 5 3 , 135X). - 161 -- 162 -Figure 56 A FEP found at the 6th internode of Sequoia sempervirens. The periderm was brown and has compressed and flattened thin-walled phellem (tbf, 21 OX). Figure 57 NP found at rhytidome in Sequoia sempervirens. The periderm also represent those formed at injuries. The NP are 2 to 3 cells thick. This NP formed at a 1974 rhytidome collected in late November, 1974, no SEP was observed abutting the NP (1/53, 135X). - 163 -- 164 -Figure 58 Initiation of NP at rhytidome in Sequoia sempervirens. Note the development of the NP below the semi-lunar or lens-shaped beige color bark tissues. Figure 59 The tissues above the NPs became brown to dark brown at the end of the growing season in Sequoia sempervirens - 165 -- 166 -Figures 60, 61 and 62 FEP developed in the epidermis below the cuticle in Taxus brevi f o l i a is shown under three different modes of illuminations. The periderm is brown and consisted of very few thin-walled phellem c e l l s . These figures show the shedding of the FEP and the formation of the f i r s t NP at rhytidome (1/53, 21 OX; mbf, 210X, tbf, 21 OX) . - 167 -- 168 -Figure 63 NPs found at mechanical injury in Taxus brevi f o l i a . SEP has not developed abutting the NP at injury. The injury was about k months old (tbf, 135X). - 169 -- 170 -Figure 6k SEP abutting NP found at rhytidome in Taxus brevifolia as revealed by cryofixation under tbf. The NP phellem has reddish purple contents and the SEP phellem, like the FEP, has brown contents (tbf, 135X). Figure 65 Small whitish and light brownish spots (arrows) appearing in the f i r s t and second internodes of an Acer macrophyllum stem - 172 -Figure 66 The development of dark brown patches from spots at the fourth internode is shown. Intense dark patches appearing on the south side are evident (Acer macrophyllum) Figures 67, 68 and 69 Site of FEP initiation in tissues abutting the epidermis below the spots of various sizes in Acer macrophyllum by cryofixation. When observed with IV/41 (Fig. 67) and 1/53 (Fig. 68) f i l t e r combinations, the tissues in which the initiation of FEP are taking place fluoresced pale blue and yellow respectively. Figure 69 shows the formation of FEP under fcp (IV/41, 85X; 1/53, 210X; fcp, 135X). - 173 -- 174 -- 175 -Figure 70 Cryofixation shows that FEP has developed below the dark brown or grayish patches. FEP has not developed below the green strips of bark in Acer macrophyllum; only the epidermis is present (1/53, 85X). Figures 71 and 72 Anatomical features as well as cryofixation characteristics of FEP in Acer macrophyllum are shown by cryofixation under f i l t e r combinations 1/53 and IV/41 respectively (1/53, 210X; IV/41, 21 OX). - 176 -- 178 -Figure 73 The bark surface appears rough as long longitudinal cracks appeared at the base of the tree resulting from rhytidome development in Acer macrophyllum. Figure ~jk NPs developed at rhytidome in Acer macrophyllum as revealed by cryofixation under f i l t e r combination IV/41. SEP has formed abutting the NP (IV/41, 210X). - 179 -- 180 -Figure 75 Cryofixation shows that a NP developed at a l67 -day-old mechanical injury in Acer macrophy11 urn under mbf. Note that the phellem cells are compressed and distorted (mbf, 21 OX). Figure 76 Cryofixed section of a mechanical injury collected 35 days after the injury was made. Note that the NP phellem cells are rectangular and undistorted in Acer macrophyllum under fluorescence f i l t e r combination 1/53- After 167 days the phellem cells are compressed considerably, see Figure 75 (1/53, 135X). - 181 -- 182 -Figure 77 No SEP develops abutting the NP formed in this year's rhytidome in Acer macrophyllum under tbf in a cryofixed section (tbf, 135X). Figure 78 A thawed cryofixed section from a rhytidome sample of Acer macrophyllum shows that the NP phellem contents has flowed away but that the contents of the exophylactic phellem remained intact (tbf, 135X)-- 183 -- 184 -Figure 79 Development of FEP in tissue two to three cells below the epidermis at the second internode of Robinia pseudoacacia as revealed by cryofixation (1/53, 210X). Figure 80 Cryofixation shows FEPs development in Robinia pseudoacacia. Seasonal renewal of FEPs, indicated by the presence of the phellogen activity marker ce l l s , is shown. The phellem in the recently developed FEP was rectangular and undistorted but those in the old periderm are compressed (IV/41, 210X). - 185 -- 186 -Figure 81 A cryofixed section of FEP in Robinia pseudoacacia shows that the phellem was compressed and stretched and seasonal renewal of FEPs (ca. 4) indicated by the presence of the PAMC (arrows) which have thickened walIs (mbf, 210X). Figures 82 and 83 Cryofixed sections of Robinia pseudoacacia showing NP developed at rhytidome under fluorescence f i l t e r combination IV/41 and 1/53 respectively. Seasonal renewal of the NP phellem was indicated by the PAMC (Fig. 82). Previously formed phellem was stretched and compressed and recently developed phellem was undistorted (IV/41, 85X; 1/53, 210X). - 188 -- 189 -Figure 84 A FEP of Robi nia pseudoacac i a treated with Sudan III in ethylene glycol. The phellem including the PAMC are stained red. Similar positive results were obtained when other fat stains were used (.tbf, 1 3 5 X ) . Figure 85 A cryofixed sect ion of -Robi n ia after 28 days following injury combination 1/53 ( 1 / 5 3 , 135X). pseudoacacia showing NP developed under fluorescence f i l t e r - 190 -- 191 -Figure 86 A cryofixed section shows the initiation of FEP in tissues two to three cells below the epidermis in the second internode in Gleditsia triacanthos under fluorescence fi1ter combination 1/53 (1/53, 210X). Figures 87 and 88 A cryofixed section of FEP in Gleditsia triacanthos consisting of approximately ten seasonal activities as indicated by the phellogen activity marker cells under pep and fluorescence fi1ter combination IV/41 respectively. The phellem contained brown pigment and was thin-walled. Weathered FEP phellem on the bark surface was sloughing (pep, -21 OX; IV/41, 210X). - 192 -- 193 -- 194 -Figure 89 NPs developed at rhytidome of Gleditsia triacanthos as revealed by cryofixation under fluorescence f i l t e r combination IV/41 (IV/41, 21 OX). Three seasonal activities of the NPs as indicated by the phellogen activity marker cells are shown. No SEP was observed abutting the NP phellem which are considerably distorted and compressed. Figure 90 A NP formed at as revealed by IV/41 (210X). a 21 day old cryofixation mechanical injury in Gleditsia triacanthos under fluorescence f i l t e r combination - 195 -- 196 -Figure 91 Air dried cryostat sections from this year's (1974) rhytidome of Gleditsia triacanthos were treated with Sudan III and examined with fluorescence f i l t e r combination 1/53 to enhanced contrast and sensitivity (1/53, 85X). Phellem of FEP and NP are stained red. Figure 32 Serial air-dried cryostat sections to Fig. 91 of Gleditsia triacanthos were treated with phloroglucinal-HCL. FEP and NP phellem cell walls are stained pink. The pigmented contents in the NP phellem had disappeared but that in the FEP phellem remained intact (tbf, 85X). - 197 -- 198 -Figure 93 A cryofixed section of FEP of Pyrus species under tbf (tbf, 135X) . The phellem had brown contents and its tangential walls were thicker than its radial walls. Figure 3k A cryofixed section of FEP of Pyrus species is shown under fluorescence fi1ter combination IV/41 (135X). The phellem contents appeared black and the phellem walls fluoresced blue. - 199 -- 200 -Figure 95 A partially developed NP during rhytidome formation in Pyrus species is revealed by cryofixation under fluorescence f i l t e r combination 1/53- The phellem cells were small and thin-walled. No SEP had developed abutting the periderm ( 1 / 5 3 , 135X) . Figure 96 A cryofixed section of NPs formed in the years previous to rhytidome formation in Pyrus species under tbf. SEPs abut a l l NPs. NPs usually have fewer phellem cells than SEPs (tbf, 85X) . - 201 -- 202 -F i g u r e 97 NP d e v e l o p e d i n a o n e - y e a r - o l d r h y t i d o m e i n Pyrus s p e c i e s as r e v e a l e d by c r y o f i x a t i o n under f l u o r e s c e n c e f i l t e r c o m b i n a t i o n 1/53. A SEP has d e v e l o p e d a b u t t i n g t h i s NP (1/53, 135X)., F i g u r e 98 A c r y o f i x e d s e c t i o n o f SEP from a r h y t i d o m e sample o f a Pyrus s p e c i e s where s l o u g h i n g was t a k i n g p l a c e . The SEP was p a r t i a l exposed ( t b f , 35X). - 203 -- 204 -Figures 99 and 100 Initiation of FEP occurred in tissue abutting the epidermis in the f i r s t and second internodes of Hibiscus syriacus respectively as revealed by cryofixed sections using fluorescence f i l t e r combination IV/41 ( 1 3 5 X ; 1 3 5 X ) . - 205 -- 206 -Figures 101 and 102 A cryofixed section of FEP in Hibiscus syriacus developed at about the f i f t h internode using fluorescence f i l t e r combination IV/41 and 1/53 respectively. Brown pigmented contents formed only in one layer of phellem (arrows) on the outside of FEP close to the surface. The phellem cells are thin-walled (IV/41, 135X; 1/53, 135X). - 208 -Figure 103 A cryofixed section showing NP formed during rhytidome formation in Hibiscus syriacus with fluorescence f i l t e r combination IV/41. The phellem was thin-walled and usually rectangular at the early stage of development (IV/41, 85X) . Figure 104 Air-dried cryostat section containing a NP formed at a 60-day-old mechanical injury in Hibuscus syriacus, treated with Sudan I I I . All NP phellem stained red. Similar results were obtained of FEP (1/53, 135X). - 209 -- 210 -F i gure 105 C r y o f i x e d s e c t i o n o f a f i v e - h o u r - o l d i n j u r y o f ba rk sample c o l l e c t e d from P inus c o n t o r t a f o l l o w i n g F -F t e s t . Deep p e n e t r a t i o n o f the bark sample by the F^F s o l u t i o n s i s e v i d e n t . F i g u r e 106 i Deep F -F pe rmea t i on in an i n j u r y bark sample o f P i c e a g l a u c a c o l l e c t e d on the 18th day a f t e r wounding as r e v e a l e d by c r y o f i x a t i o n ( t b f , 85X). - 211 -- 212 -Figure 107 Cryofixation under tbf revealed the depth of F-F permeation of Robinia pseudoacacia injured bark samples collected on the 7 t h day after wounding. Wounded zone, as usual, becomes much deeper blue than the living zone (tbf, 135X). Figure 108 A 28-day-old injured sample of Taxus brevifolia was F-F tested through the wound surface. The permeation of the test solutions was stopped at the non-suberized impervious tissue (NIT). Only the brown dead tissues are colored blue. Similar results were observed in a l l species after NIT formation. - 213 -- 214 -F i g u r e 109 The e x t e n t o f p e r m e a t i o n o f t h e t e s t s o l u t i o n s i n t h e F-F t e s t e d samples o f P i c e a g l a u c a 32 days a f t e r wounding i s shown by c r y o f i x a t i o n w i t h f l u o r e s c e n c e f i l t e r c o m b i n a t i o n 1/53. NIT has formed a t t h i s t i m e (1/53, 135X). F i g u r e 110 A c r y o f i x e d s e c t i o n o f a F-F t e s t e d 50-day i n j u r e d sample of P i c e a  s i t c h e n s i s i s shown w i t h f l u o r e s c e n c e f i l t e r c o m b i n a t i o n IV/41. Note t h a t NIT and NP p h e l l e m had dev e l o p e d ( I V / 4 1 , 135X). - 216 -F i g u r e 111 The e x t e n t o f p e n e t r a t i o n by t h e t e s t s o l u t i o n s i n t h e F-F t e s t e d sample o f P i n u s c o n t o r t a 30 days a f t e r i n j u r y i s shown by c r y o f i x a t i o n u s i n g t b f . NIT had formed and the NP p h e l l e m was j u s t f o r m i n g ( t b f , 135X). F i g u r e 112 A c r y o f i x e d s e c t i o n o f a F-F t e s t e d 18-day-old i n j u r e d sample o f Cupressus macrocarpa i s shown w i t h f l u o r e s c e n c e f i l t e r c o m b i n a t i o n 1/53- NIT had formed and NP p h e l l e m had not d e v e l o p e d a t t h i s t ime (1/53, 85X) . - 2 1 7 -- 218 -Figure 113 The depth of permeation by the test solutions in the F-F tested samples of Taxus b r e v i f o l i a 28 days after injury is shown by cryofixation under fluorescence f i l t e r combination IV/41. NIT is evident and NP has not developed (IV/41, 85X). Figures 114, 115 and 116 Cryofixed sections from a F-F tested sample of Robinia pseudoacacia after 21 days injury shown with tbf and fluorescence f i l t e r combination 1/53 and IV/41, respectively. Note that the F-F solutions had stopped externally abutting the NIT. Test was carried out from the wound surface. NP is shown developing (tbf, 135X; 1/53, 1 35X; IV/41, 135X) . - 220 -- 221 -Figure 117 Injured bark sample collected two days after injury from Larix  occidental is shown by cryofixation and fluorescence f i l t e r combination 1/53- The brown 'dead' zone shows faded fluorescence of cell contents, and dull greenish fluorescence of walls (1/53, 85X). Figure 118 A cryofixed section from a two-day injured bark sample of P i nus  contorta is shown with fluorescence f i l t e r combination 1/53 (85X). - 222 -- 2 23 " Figure 119 Injured bark samples collected on the fourth day after wounding from Pinus contorta is shown by cryofixation with fluorescence f i l t e r combination 1/53 C85X) . Cells abutting the 'dead' brown zone have become enlarged and the intensity of their content's fluorescence has increased. The red fluorescence of the chloroplasts had disappeared. At this stage, the tissues below the would surface can be divided into four zones: (a) Brown dead tissue, (b) Tissue consisting of c e l l s with yellow fluorescent contents, (c) Tissue consisting of c e l l s with greenish fluorescent contents, and (d) l i v i n g normal tissues. Figure 120 Injured bark sample collected on the seventh day after wounding from Pinus contorta is shown by cryofixation and fluorescence f i l t e r combination 1/53 (85X). Cells abutting the 'dead' brown tissues had lost the fluores-cence of their contents which had become reticulated and fluoresced pale green. A meristematic zone of tissues is evident. - 224 -- 2 2 5 -Figures 121 and 122 Cryofixed sections of injured bark samples collected on the eleventh and fourteenth days after wounding from Pinus contorta are shown with fluorescence f i l t e r combination 1/53 (85X, 85X). Pronounced tissue modifications are observed compared to that of the seventh day (Fig. 1 2 0 ) . - 226 -- 227 " Figure 123 Cryofixed section of injured bark samp 1e co11ected on the seventh day after wounding from Lar ix occidental is shown under fluorescence f i l t e r combination 1/53 (35X). Note that the fluorescence of the contents of the cells abutting the 'dead' brown tissues was changing from yellow to yellowish green. - 228 - 229 " Figure 124 Cryofixation section of injured bark sample collected on the ninth day after wounding from Larix occidental is shown with fluorescence f i l t e r combination 1/53 (35X). At this stage the tissues below the wound surface can be c l a s s i f i e d into four zones: (a) Brown dead tissue, (b) Tissue consisting of c e l l s with yellow fluorescent contents, (c) Tissue consisting of c e l l s with greenish fluorescent contents, and (d) The l i v i n g normal tissues. Figures 125, 126 and 127 Cryofixed sections of injured bark samples collected on the eleventh, fourteenth, and sixteenth day respectively after wounding from Lari x  occidental is shown using fluorescence f i l t e r combination 1/53- At this stage of development the tissues below the injured surface can be easily divided into four zones: (a) Brown dead tissue, (b) Tissue consisting of c e l l s with yellow fluorescent contents, (c) Tissue consisting of c e l l s with pale green to dark green fluorescent contents, and (d) The l i v i n g normal tissues. (35X). - 231 -- 232 -Figures 128, 129 and 130 Cryofixed sections of injured bark samples collected on the sixteenth (tbf, 135X) eighteenth (1/53, 135X) and twenty-fifth (1/53, 135X) day after wounding from Pinus contorta, respectively. The formatioi of the NIT is evident. Sporadic formation of NIT as indicated by the sporadic F-F color in the living bark is shown in Fig. 128. - 233 -- 234 -Figures 131, 132, 133 and 134 Injured bark samples collected on the eighteenth, twenty-first, twenty-fifth and t h i r t y - f i f t h day after wounding from Larix occidental is respectively as revealed by cryofixation using fluorescence f i l t e r combination 1/53 (85X). Note that NIT had developed. NIT c e l l s had yellowish green c e l l contents and greenish c e l l walls. Abutting NIT was the dark band of meristematic tissue. - 235 -- 237 -Figure 135 Cryofixed section of a freshly formed NIT in a 32-day-old injury of Picea glauca under fluorescence f i l t e r combination 1/53- NIT had developed in 32 days and was located in between the brown 'dead' tissue and the living bark and it consists of enlarged cells of varying shapes and sizes with irregularly thickened walls. Tissue modification is shown in the living bark tissue internally adjacent to the NIT. No sign of NP phellem formation is observed (1/53 » 135X). - 238 -- 239 -Figures 136 and 137 Cryofixed section of a 36-day-old F-F tested injury of Picea sitchensis showing NIT under fluorescence f i l t e r combination and 1/53 respectively. NP is developing in tissue internally abutting the NIT which is located in between the brown 'dead' tissue and the living bark. NIT had formed in 21 days (IV/41, 135X; 1/53, 135X). - 2 4 0 -- 241 -Figure 138 A 60-day-old injury of Picea engelmannii showing NIT which had formed after 32 days and the initiation of the NP. NIT consisted of enlarged cells of varying shapes and sizes with irregularly thickened walls as revealed by cryofixation under fluorescence f i l t e r combination IV/41 (85X). Figure 139 Cryofixed section of a 21-day-old injury of Picea contorta showing the formation of NIT in 21 days under fluorescence f i l t e r combination IV/41 (135X). Sporadic formation of NP phellem is shown abutting the NIT. - 242 -- 243 -Figures 140 and 141 Cryofixed section of a 60-day-old injury of Pinus monticola showing NIT which had developed in 28 days and the initiation of the NP is taking place in tissue internally abutting NIT as seen under fluorescence fi1ter combination 1/53 and IV/41 respectively (135X). - 2kk -- 245 -Figures 142 and 143 Freshly formed NIT in a 27-day-old injury of Pseudotsuga menziesii. NIT had developed in 23 days and the differentiation of NP has not occurred as revealed by cryofixation under fluorescence f i l t e r combination 1/53 and IV/41 respectively (85X). - 246 -- 247 -Figures 144 and 145 Cryofixed section of a 23-day-old injury of Larix occidental is showing the formation of NIT which is located in between the brown dead tissue and the l i v i n g bark. NIT which had formed in 21 days consisted of enlarged c e l l s of varying shapes and sizes with thickened walls as seen under fluorescence f i l t e r combination 1/53 and tbf respectively (85X). No sign of NP formation is shown. A dark band of meristematic tissue had developed abutting the NIT. - zks -Figures 146, 147 and 148 Cryofixed section of a 17 _day-old and a 24-day-old injuries of Cupressus macrocarpa showing the formation of NIT respectively as revealed under fluorescence f i l t e r combination IV/41, 1/53 and IV/41 respectively. Many types of c e l l s are taking part in NIT formation, e.g. sieve c e l l s , c o r t i c a l parenchyma, ray parenchyma, and e p i t h e l i a l c e l l s . NIT has developed in 17 days. The formation of the NP has not occurred (IV/41, 85X; 1/53, 85X; IV/41, 8 5 X ) . - 250 -- 251 -- 252 -Figure 149 Cryofixed section of a 23-day-old injury of Sequo i a semperv i rens showing the freshly formed NIT in between the brown 'dead' tissue and the l i v i n g bark under fluorescence f i l t e r combination IV/41 (85X). NIT has developed in 21 days. NP d i f f e r e n t i a t i o n is not shown.' Figure 150 Cryofixed section of a 28-day-old F-F tested injury of Taxus brevi f o l i a showing the development of NIT in between the 'dead' tissue and the l i v i n g bark under fluorescence f i l t e r combination 1/53 (85X). NIT consisted of a zone of enlarged c e l l s of varying shapes and sizes with irregu l a r l y thickened wa11s. The NIT c e l l walls fluoresced d i s t i n c t l y from the internally or externally adjacent c e l l s . NIT had formed in 28 days. Formation of the NP has not occurred. - 253 -- 254 -Figure 151 Cryofixed section of a 14-day-old injury of Acer macrophyllum showing the freshly formed NIT in between the 'dead' tissue and the living bark under fluorescence f i l t e r combination IV/41 (135X). NIT had formed in 14 days between two rows of scereids. The formation of NP has not occurred. Figures 152 and 153 Cryofixed section of a 21-day-old F-F tested injury of Robi nia  pseudoacacia showing the forming of NIT which consists of enlarged cells of varying shapes and sizes with irregularly thickened walls. The cell walls f1uoresced yellow and pale blue when examined with fluorescence f i l t e r combinations 1/53 and IV/41 respectively. NIT had formed in 21 days. Formation of NP has not occurred. A dark band of meristematic tissue has developed abutting the NIT (210X). - 256 -- 257 -Figure 154 Cryofixed section of a freshly formed NIT in a 21-day-old F-F tested injury of Gleditsia triacanthos under fluorescence f i l t e r combination IV/41 (210X). The F-F permeation is stopped at the NIT which consists of enlarged cells of varying shapes with thickened walls. NIT had formed in 21 days. Initiation of the NP has developed sporadically in tissue internally abutting NIT. F i g u r e 155 C r y o f i x e d s e c t i o n o f a 15 -day-old F-F t e s t e d i n j u r y o f H i b i s c u s  s y r i a c u s showing t h e f r e s h l y formed NIT i n between the 'dead' t i s s u e and t h e l i v i n g b ark under f l u o r e s c e n c e f i l t e r c o m b i n a t i o n IV/41. NIT had formed i n 15 days. No s i g n o f NP i n i t i a t i o n was d e t e c t e d (85X). - 258 -- 259 -Figure 156 Cryofixed section of a 36-day-old F-F tested injury of Picea sitchensis showing NIT and the formation of the NP in tissue internally abutting NIT under fluorescence f i l t e r combination 1/53 (21 OX) . Figure 157 Cryofixed section of a 60-day-old injury of Picea engelmannii showing completion of NIT development and the i n i t i a t i o n of NP in tissue internally abutting NIT under fluorescence f i l t e r combination IV/41 (85X). - 260 -- 261 -Figure 158 Cryofixed section of a 60-day-old injury of Pinus monticola showing NIT has developed and the formation of the NP in tissue internally abutting the NIT under fluorescence f i l t e r combination IV/41 (85X). Figures 159 and 160 Cryofixed section of a 30-day-old F-F tested injury of Pinus contorta. NIT had formed in 21 days and the initiation of the NP in the internal abutting tissue has occurred as shown with fluorescence f i l t e r combination 1/53 and IV/41 respectively (135X). - 263 -- 264 -F i g u r e 161 A 4 l - d a y - o l d i n j u r y o f S e q u o i a s e m p e r v i r e n s showing t h e f o r m a t i o n o f NIT and t h e NP i n t i s s u e i n t e r n a l l y a b u t t i n g NIT as r e v e a l e d by c r y o f i x a t i o n under f l u o r e s c e n c e f i l t e r c o m b i n a t i o n 1/53 and IV/41 r e s p e c t i v e l y (85X). Figures 162, 163 and 164 Cryofixed section of a 28-day-old injury of Robinia pseudoacacia showing completion of NIT development and the formation of the NP in tissue internally abutting the NIT under tbf and fluorescence f i l t e r combinations 1/53 and IV/41, respectively. The NIT cell walls showed clear distinctions from the NP phel1 em eel 1 walls. NIT cell walls fluoresced yellow and pale blue when examined with 1/53 and IV/41 f i l t e r combinations, and the NP phellem cell walls fluoresced yellowish green and blue respectively. NP phellem cells are arranged in radial f i l e (135X). - 267 -Figure 165 Cryofixed section of a 33 -day-old injury of Larix occidental is showing NIT has developed and the formation of the NP in tissue internally abutting NIT under fluorescence f i l t e r combination IV/41. Newly formed NP phellem c e l l s were turgid and uncompressed, while those formed e a r l i e r had become stretched and distorted (135X). F i g u r e 166 C r y o f i x e d s e c t i o n o f NIT formed a t r h y t i d o m e o f P i c e a g l a u c a examined w i t h f l u o r e s c e n c e f i l t e r c o m b i n a t i o n IV/41. NIT c o n s i s t e d o f a zone o f e n l a r g e d c e l l s o f v a r y i n g shapes and s i z e s w i t h t h i c k e n e d w a l l s . NP had formed i n t e r n a l l y a b u t t i n g NIT (135X). - 268 -- 269 -F i g u r e s 167, 168 and 169 C r y o f i x e d s e c t i o n o f NIT d e v e l o p e d at rhy t idome o f P i c e a engelmanni i and P i c e a s i t c h e n s i s examined under f i l t e r c o m b i n a t i o n s IV/41, t b f and 1/53 r e s p e c t i v e l y . C r y o f i x a t i o n c h a r a c t e r i s t i c s o f NIT i n t h e s e two s p e c i e s were i d e n t i c a l t o those o f NIT in P i c e a g l a u c a . The s i t e o f NP f o r m a t i o n was the i n t e r n a l t i s s u e a b u t t i n g NIT ( IV /41, 1 35X ; t b f , 210X; 1/53, 135X r e s p e c t i v e l y ) . - 270 -- 272 -Figures 170 and 171 Cryofixed sections.of•NIT formed at rhytidome of Pinus contorta and Pinus monticola respectively examined with fluorescence f i l t e r combination 1/53- In both species the NP has formed abutting NIT internally. The cell walls of NIT fluoresced consistently yellow with very l i t t l e greenish tinge and most cell contents have disappeared (I/53, 135X; 1/53, 135X). - 273 -- 274 " F i g u r e s 172 and 173 C r y o f i x e d s e c t i o n o f NIT formed a t r h y t i d o m e o f Pseudotsuga m e n z i e s i i r e v e a l e d under f l u o r e s c e n c e f i l t e r c o m b i n a t i o n IV/41 and 1/53 r e s p e c t i v e l y . NP was f o r m i n g a t t h e dark band o f m e r i s t e m a t i c t i s s u e s i n t e r n a l l y a b u t t i n g t h e NIT (85X). - 275 -- 276 -Figure 17^ Cryofixed section of NIT formed at rhytidome of Larix occidenta1is observed under fluorescence f i l t e r combination 1/53- Sporadic i n i t i a t i o n of the NP is seen abutting NIT (85X). - 277 -- 278 -Figure 175 Cryofixed section of NIT developed at rhytidome in Cupressus  macrocarpa revealed under fluorescence f i l t e r combination IV/41 (85X). I n i t i a t i o n of the NP was observed at the dark band of meristematic tissues abutting the NIT. Figure 176 Cryofixed section of NIT formed at rhytidome in Sequoia sempervirens observed under IV/41 (135X). - 280 -F i g u r e 177 C r y o f i x e d s e c t i o n o f NIT formed a t rhytid o m e o f Taxus b r e v i f o l i a o b s e r v e d w i t h t b f . NIT c o n s i s t e d o f a zone o f e n l a r g e d c e l l s o f i r r e g u l a r shapes and s i z e s w i t h t h i c k e n e d w a l l s . NIT was always found e x t e r n a l l y a b u t t i n g t h e NP wh i c h c o n s i s t e d o f one t o t h r e e l a y e r s o f p h e l l e m c e l l s c o n t a i n i n g r e d d i s h - p u r p l e c o n t e n t s ( t b f , 135X). F i g u r e s 178 and 179 C r y o f i x e d s e c t i o n o f NIT d e v e l o p e d a t examined w i t h 1/53 f i l t e r c o m b i n a t i o n c o n s i s t e d o f a zone o f e n l a r g e d - e e l Is w i t h t h i c k e n e d w a l l s and i t d e v e l o p e d 1/53, 1 3 5 X ) . r h y t i d o m e o f A c e r macrophyllum a t d i f f e r e n t m a g n i f i c a t i o n s . NIT o f i r r e g u l a r shapes and s i z e s p r i o r t o NP f o r m a t i o n (1/53, 85X; - 281 -- 282 -- 283 -Figure 180 Cryofixed section of NIT formed at rhytidome in Robinia pseudoacacia observed under fluorescence fi1ter combination IV/41 (210X). NIT and NP have developed and NP was found abutting NIT internally. Note that the PAMC had also developed internally abutting the NP phellem which had become distorted and compressed at this stage. Figure 181 Cryofixed section of NIT developed examined under fluorescence f i l t e r NIT and NP had formed. at rhytidome in Gledi ts ia combination 1/53 (135X). triacanthos Note that - 285 -Figure 182 NIT found at rhytidome in Hibiscus syriacus as revealed by cryofixation under fluorescence f i l t e r combination 1/53 (85X). NIT developed prior to NP and consisted of a zone of enlarged eel Is of varying shapes with thickened eel 1 walls. NP had formed in tissue internally abutting NIT. Figures 183 arid 184 Cryofixed section of a leader of Picea sitchensis attacked by P issodes Strob i Hopkins showing the presence of NIT and NP under fluorescence fi1ter combination IV/41 and 1/53 (135X). NP developed internally abutting NIT which eonsisted of a zone of enlarged cells of varying shapes and sizes with thickened walls. Under f i l t e r combinations 1/53 and IV/41 the NIT cell walls fluoresced yellow and pale blue and the cell contents fluoresced brown and dark brown respectively. - 286 -- 287 -- 288 -F i g u r e 185 A i r - d r i e d c r y o s t a t s e c t i o n f rom a b e e t l e h o l e ba rk sample o f Pseudotsuga m e n z i e s i i t r e a t e d w i t h Sudan II I in t b f (135X) . NIT and NP had d e v e l o p e d . Only NP p h e l l e m c e l l w a l l s were s t a i n e d r e d . F i g u r e 186 C r y o f i x e d s e c t i o n o f NIT and NP found, at a r e s i n b l i s t e r o f P seudotsuga m e n z i e s i i under f l u o r e s c e n c e f i l t e r c o m b i n a t i o n 1/53 (135X). NIT a lways l o c a t e d e x t e r n a l l y a b u t t i n g the NP. - 290 -Figures 187 and 188 Cryofixed sections from the branch and stem of Pinus monticola attacked by Cucurbidothis pithyophila Fr. and Cronartiurn ribicola F. respectively, under fluorescence f i l t e r combination 1/53 ( 1 3 5 X ) . In both eases, NIT and NP had developed. NP always formed internally abutting NIT which consisted of a zone of distorted and compressed enlarged eel 1s of varying shapes with thickened cell walls. These NIT cells were identical to those seen at injuries and rhytidome described in the text. 1 - 291 -- 292 -Figure 189 Cryofixed section of an abscission scar collected at the third internode of-Picea glauca showing the presence of NIT and NP under fluorescence fi1ter combination 1/53 (85X). NIT surrounding the vascular bundle is clearly seen between the suberized primary protective layer (PPL) and the phellem of the NP, i.e., the suberized secondary protective layer. Cryofixation characteristics and anatomical features of NIT at abscission zone were identical to those of NIT at injuries and rhytidome in Picea glauca. - 293 -) - 294 -Figure 190 A thawed cryofixed section from an 25-day-Old F-F tested injury of Picea glauca treated wi th ani1ine sulfate-HCL is shown in tbf. A semi-lunar band of tissues some distance from the injured surface reacted positively to the test. This test was not specific for NIT. Thick-walled phellem of the exophylactic periderm and the sclereids are also reacted positively to the test (tbf, 135X). F i g u r e 191 A thawed c r y o f i x e d s e c t i o n from an 18-day-old F-F t e s t e d i n j u r y o f Picea engelmanni i , t r e a t e d w i t h phlorog1ucinol-HCL i s shown i n t b f . A s e m i - l u n a r band o f t i s s u e s i n t e r n a l t o t h e wound s u r f a c e was s t a i n e d r e d . S c l e r e i d s and t h i c k - w a l l e d p h e l l e m o f t h e e x o p h y a c t i c p e r i d e r m were a l s o s t a i n e d r e d . The.depth o f F-F p e r m e a t i o n i s shown by the b l u e c o l o r ( t b f , 85X). - 296 -Figures 192 and 193 Sections from a 40-day-old injury of Picea sitchensis stained with Sudan I I I and ammonia hydroxide-crystal violet are shown in these two figures respectively with fluorescence f i l t e r combination 1/53 (135X). Only NP phellem and not NIT reacted positively to these tests-NP phellem fluoresced red. - 297 -- 298 -Figure 194 A section from a 33 -day-old injury of Larix occidental is showing NIT and NP treated with phosphere 3R is shown. Only NP phellem fluoresced silver under fluorescence f i l t e r combination IV/41. NIT fluoresced brownish yellow while 'dead' tissues externally abutting the NIT appeared yellowish brown (21.0X) . - 299 -k W - 300 -Figure 195 A section from a 40-day-old injury of Picea sitchensis showing NIT and NP treated with methylene blue is shown. Only NP phellem fluoresced pale blue under f1uorescence fi1ter combination IV/41. NIT fluoresced dark blue. (135X). Figure 196 Air-dried cryostat section from a 33-day-old injury of Larix occidental is treated with phloroglucinol - HCL is shown under tbf. NIT cell walls were stained bright red. Sieve and parenchyma cells externally abutting NIT also showed positive staining. Mature NP phellem cell walls were faintly stained (1 3 5 X ) . - 301 -- 302 -Figure 197 Air-dried cryostat section from a 21-day-old injury of Acer macrophyllum treated with phloroglucinol - HCL is shown under tbf. NIT and sclereids were stained bright red. NP had not developed (85X). Figure 198 Air-dried cryostat sections from a F-F tested 21-day-old injury of Robinia pseudoacacia treated with phloroglucinol - HCL is shown under tbf. The test solution is shown stopped at NIT. NIT and sclereids were stained bright red. Mature NP phellem cell walls were faintly stained (85X). - 303 -- 304 -Figure 199 Air-dried cryostat sections from a 4l-day-old injury of Taxus  brevifolia treated with aniline sulfate-HCL is shown under tbf. NIT cell walls are stained yellow. External to NIT, sieve cel l s , parenchyma, and sclereids were also stained. Internal to NIT, only sclereids were stained. Usually NP was not stained (85X). Figure 200 Air-dried cryostat sections from a F-F tested 21-day-old injury of Robinia pseudoacacia treated with aniline sulfate-HCL in tbf is shown. NIT cell walls are stained yellow. External to NIT, sclereids, sieve and parenchyma cells were also stained. Internal to NIT, only sclereids were stained. NP phellem was not stained (135X). - 306 -Figures 201 and 202 Air-dried cryostat sections from an abscission zone of Picea glauca and a diseased site of Pinus monticola attacked by Cucurb? doth i s  pithyophila Fr. respectively, and stained with phloroglucinol-HCL are shown in tbf. NIT cell walls are stained red. Note that the NP cell walls are faintly stained and the cells are void of any contents. In Pinus monticola, thin-walled phellem of the sequent exophylactic periderm (SEP) is shown and they are f i l l e d with brown contents which remained intact even after concentrated hydrochloric acid (HCL) treatment (85X, 135X). v - 308 -F i g u r e 203 A i r - d r i e d c r y o s t a t s e c t i o n s f rom a 4 0 - d a y - o l d i n j u r y o f Pi cea s i t chens i s t r e a t e d w i t h Maule r e a c t i o n i s shown in t b f . NIT c e l l w a l l s were s t a i n e d p a l e brown o r brown. S i e v e and parenchyma c e l l s e x t e r n a l l y a b u t t i n g NIT t u r n e d da rk brown w h i l e s c l e r e i d s t u rned p a l e brown. I n t e r n a l l y t o NIT, s c l e r e i d s were a l s o s t a i n e d . NP was not s t a i n e d . These o b s e r v a t i o n s were common in a l l c o n i f e r s (85X) . - 309 -- 310 -Figure 204 Air-dried cryostat from a F-F tested 21-day-old injury of Acer  macrophyl1 urn treated,with Maule reaction is shown in tbf. NIT cell walls were stained brown. Externally to NIT, sclereids were stained red while sieve and parenchyma eel 1s turned down. Internally to NIT, sclereids were also stained red; sieve and parenchyma eel 1s were not stained. NP phellem was not stained (135X). Figure 205 Air-dried cryostat sections from a 35-day-old injury of-Acer macrophy11 urn after treatment with diethyl ether for seven days was stained with phloroglucinol-HCL under tbf; NIT and sclereids s t i l l showed positive test (85X). - 311 -- 312-LITERATURE CITED 1. Akai, S. 1959- Histology of defense in plants. In plant pathology. Vol. I. Edited by J.G. Horsfall and A.E. Diamond. Academic Press, New York. pp.392-467. 2. 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University of British Columbia, Faculty of Forestry, Vancouver. BSF Thesis. 44 . Srivastava, L.M. 1963. Secondary phloem in the pinaceae. Univ Calif. Berkeley Press, pp.142. 45. Srivastava, L.M. 1964. Anatomy, chemistry and physiology of bark. In: J.A.'Romberger and P. Mikola, eds. International Review of Forestry Research. Vol. 1. Academic Press, New York, pp. 203-277. - 317 -Struckmeyer, B.E. and A.J. Riker. 1951• Wound-periderm formation in white pine trees resistant to b l i s t e r rust. Phytopathology 41: 276-281. Tison, M.A. 1899- Methode nouvelle de coloration des tissus subereux. Association Francaise pour Lavancement des Compte Rendus de la Session 28(2): 454-456. Treshow, M. 1970. Environment and plant response. McGraw-Hill, New York. - 318 -APPENDIX Freshly removed bark samples were embedded and frozen in Lab-Tek O.C.T. compound using the quick-freeze attachment of the 'International' cryostat Model CTI. The embedded samples were sectioned to 8 to 10 microns (urn) in the cryostat chamber prechilled at -20C. The sections were transferred from the microtome knife by pressing gently against glass slides which had been prechilled in the chamber. The sections were then mounted quickly in prechilled cryostat o i l , Cryo-cut Microtome Lubricant, American Optical Corporation, using a prechilled cover glass. The frozen section was now ready for microscopic examination at ~30C on an improved freezing stage system described below. A Carl Zeiss Heating and Cooling Stage mounted on a Leitz Heating and Cooling Stage 80 was modified to f i t a Zeiss Photoscope II and adapted to cooling to below -kOC by pumping methanol, cooled to ~55C by mechanical refrigeration to the stage via heavily insulated copper tubing. A steady-state temperature of the stage was achieved by adjusting the speed of the Masterflex (Cole-Palmer, 7425 North Oak Park Ave., Chicago, 111., U.S.A.) pump equipped with cryogenic tubing. The pump head does not require insulation. The pump was placed downstream from the stage so the warming of the methanol, during passage through the pump, occurred after i t passed through the stage. - 3 1 9 -Frosting of the stage was minimized by custom f i t t i n g a plexiglass glove box (ca. 3^ x 36 x 45 cm with an inclined panel to accommodate eye pieces) around the front of the microscope so that the heat-generating lamp sources are excluded. The usual glove box gloves, which are normally very expensive, were unsuitable for the delicate manipulations. In l i e u of these gloves, inexpensive p l a s t i c bags (60 cm long x 15 cm diameter) with the open end mounted on the glove port c o l l a r s by e l a s t i c bands, proved highly satisfactory. A bag with the free end sealed serves ideally as a glove for the left-hand port. The free end of the right-hand one was cut open to serve as a sleeve through which the frozen s l i d e could be transferred to the cold stage. A s u f f i c i e n t l y dry atmosphere in the box was maintained by c i r c u l a t i n g a i r which passed through two 8 cm diameter cylinders 30 cm long connected in series which were f i l l e d with indicating s i l i c a gel. The cryostat, after the frozen sections were readied for examination, was wheeled to the microscope and the frozen s l i d e was quickly mounted on the cold stage through the sleeved glove port. The stage temperature was monitored with a telethermometer (Mullick 1971). 

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