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Leaf and stem anatomy of several apple cultivars, their compact mutants, and alar treated plants. Liu, Alice Chen-Miao 1970

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LEAF AND STEM ANATOMY OF SEVERAL APPLE CULTIVARS, THEIR COMPACT MUTANTS, AND ALAR TREATED PLANTS by ALICE CHEN-MIAO LIU B.S.A., Taiwan P r o v i n c i a l Chung-Hsing University, 1967 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN THE DEPARTMENT of PLANT SCIENCE We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA March, 1970 In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study . I f u r t h e r agree tha permiss ion fo r e x t e n s i v e copying o f t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r e p r e s e n t a t i v e s . I t i s understood that copying or p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l ga in s h a l l not be a l lowed without my w r i t t e n p e r m i s s i o n . Department The U n i v e r s i t y of B r i t i s h Columbia Vancouver 8, Canada ABSTRACT Natu r a l l y occurring compact (spur type) apple trees (Malus s y l v c s t r i s L.) were compared with standard and A l a r treated trees. Stem anatomy received s p e c i a l a t t e n t i o n because no comparisons had been done of the stem anatomy i n standard and compact apple trees. Anatomical studies of leaves i n compact mutants and i n A l a r treated Red D e l i c i o u s are more d e t a i l e d than i n e a r l i e r reports. Four c u l t i v a r s were used i n the studies. They were Harrold Red D e l i c i o u s , a compact mutant of Red D e l i c i o u s , namely Starkrimson, and standard Golden D e l i c i o u s , and i t s compact mutant Starkspur. There were three main studies i n t h i s i n v e s t i g a t i o n . In the f i r s t study, anatomical examinations were made on the four c u l t i v a r s without A l a r treatment. Starkspur Golden D e l i c i o u s was found to have the thi c k e s t leaf and palisade parenchyma among the four c u l -t i v a r s studied. The compact type was found to have a thicker l e a f , palisade parenchyma and greater mean palisade number when compared with the standard type. The e f f e c t of A l a r at concentrations of 0 and 1000 ppm on the same c u l t i v a r s was investigated i n the second study. The suppression of terminal growth by A l a r v a r i e d among the c u l t i v a r s . The response to A l a r was greatest with Starkspur and 50 per cent i n h i b i t i o n of shoot growth was observed. Starkrimson was not a f f e c t e d by A l a r t r e -atment. Microscopic examination revealed that there were no s i g n i f i -cant d i f f e r e n c e s i n c e l l length of collenchyma, parenchyma and p i t h i i c e l l s or i n c e l l diameters and tissue thickness when the samples were taken from the f i r s t internode under the growing t i p . In study three, the e f f e c t of A l a r and i t s i n t e r a c t i o n with g i b b e r e l l i c a c i d on Red and Golden D e l i c i o u s were considered. In t h i s study, comparisons were also made with the untreated compact mutants. A l a r treatments of Red D e l i c i o u s were found to increase thickness of t o t a l l e a f , spongy parenchyma and the length of palisade c e l l s . The l a t t e r two accounted f o r the increase i n t o t a l thickness of A l a r -treated Red D e l i c i o u s leaves. G i b b e r e l l i c a c i d stimulated the shoot growth of Golden D e l i c i o u s and Starkspur by 29 per cent, but t h i s stimulating e f f e c t was preven-ted by A l a r . ACKNOWLEDGEMENTS I wish to express my deepest gratitude to Dr. G. W. Eaton, Associate Professor, Department of Plant Science, University of B r i t i s h Columbia, under whose supervision t h i s project was undertaken, f o r h i s technical advice during the research, and for h i s guidance i n the preparation of t h i s t h e s i s . Sincere appreciation i s e s p e c i a l l y expressed to Dr. N. E. Looney, Pomologist, Canada Department of Ag r i c u l t u r e Research Station, Summerland, B r i t i s h Columbia, who kindly provided many of the plant materials used i n t h i s project and who also gave valuable counsel and assistance i n several ways. Also appreciation i s extended to Drs. K. Beamish, C. A. Hornby, and V. C. Runeckles for t h e i r h e l p f u l suggestions during t h i s endeavor. This Research was supported by NRCC Operating Grant A2023 awarded to Dr. G. W. Eaton. V TABLE OF CONTENTS Page I. INTRODUCTION 1 I I . LITERATURE REVIEW .' 3 I I I . MATERIALS AND METHODS 12 IV. RESULTS 20 Experiment 1: Comparisons of Untreated Compact and Standard Apple C u l t i v a r s . . . 20 Experiment 11: E f f e c t s of A l a r on Compact and Standard Apple C u l t i v a r s 30 Experiment 111 (a): Comparisons of Compact with A l a r treated Red Del i c i o u s 38 Experiment 111 (b): E f f e c t s of A l a r and G i b b e r e l l i c A c i d on Compact and Standard Golden D e l i c i o u s 43 V. DISCUSSION 48 VI. SUMMARY 53 BIBLIOGRAPHY 55 APPENDIX 59 v i LIST OF TABLES Table Page 1. Thickness of Leaf Tissues of Four C u l t i v a r s 21 2. Thickness of Leaf Tissues of.Red Delicious and Golden Delicious . 25 3. Thickness of Leaf Tissues of Standard and Compact Types 26 4. C e l l Lengths of Stem Collenchyma, Parenchyma and P i t h of Four Apple C u l t i v a r s 29 5. Thickness of Stem Cortex, Vascular Tissue, Diameters of P i t h and Stem of Four Apple C u l t i v a r s 31 6. C e l l Diameters of Stem Collenchyma, Parenchyma and P.ith of Four Apple C u l t i v a r s 32 7. E f f e c t of A l a r on Shoot Growth of Red Del i c i o u s and Starkrimson 33 8. E f f e c t of A l a r on Leaf Number of Red Del i c i o u s and Starkrimson 34 9. E f f e c t of A l a r on Average Leaf Length and Width of Red De l i c i o u s and Starkrimson 35 10. Influence of A l a r on Shoot Growth of Starkspur and Golden D e l i c i o u s .a 3§ 11. E f f e c t of A l a r on Leaf Number of Golden D e l i c i o u s . . . 37 12. Influence of A l a r on Thickness of Leaf Tissues of Four Apple C u l t i v a r s 39 v i i Table Page 13. Influence of A l a r on the C e l l Lengths of Collenchyma, Parenchyma and P i t h of Four Apple C u l t i v a r s 40 14. Influence of A l a r on the Thickness of Leaf Tissues of Red Deliciou s 41 15. E f f e c t of A l a r on Shoot Growth of Starkrimson and Red D e l i c i o u s 45 16. Influence of A l a r and G i b b e r e l l i c A c i d on Thickness of Leaf Tissues of Starkspur and Golden D e l i c i o u s . . . 46 17. Influence of A l a r and G i b b e r e l l i c Acid on Mean Shoot Growth of Starkspur and Golden Delicious . . . . 47 v i i i LIST OF FIGURES Figure Page 1. Thickness of Leaf Tissues of Four Apple C u l t i v a r s . . . . 22 2. The Leaf Cross Section of Starkspur Showing Longer Palisade C e l l s and Greater Mean Palisade Number Than i n the Standard Golden Delicious 23 3. The Leaf Cross Section of Starkrimson Showing Greater Leaf Thickness Than i n Red De l i c i o u s 24 4. Longitudinal Section of Red De l i c i o u s Stem Showing Collenchyma, Parenchyma, Vascular Tissue and Part of Pi t h 27 5. Cross Section of Red Delicious Stem Showing Part of Collenchyma, Parenchyma, Vascular Tissue and Pi t h . . . 28 6. Red Delicious Leaves Treated with A l a r at 4000 ppm Were Thicker Than Untreated Leaves 42 INTRODUCTION Due to the cost of labor i n pruning and harvesting f r u i t grown on large trees, c o n t r o l l i n g the s i z e of f r u i t trees has held the intermittent i n t e r e s t of pomologists for hundreds of years. A recent approach to a t t a i n i n g tree s i z e control has involved chemical growth retardants. One showing considerable promise for use on f r u i t trees i s s u c c i n i c a c i d 2,2-dimethyl hydrazide commercially known and hereafter r e f e r r e d to as A l a r . The f i r s t published report of the use of this chemical as a growth retardant on plants was made by R i d d e l l et a l . (31) i n 1962. Studies on the movement and fate of A l a r i n apple seedlings have been reported (25). Other studies have shown that A l a r applied to apple trees caused many desirable e f f e c t s without a major suppression of root growth ( 3 ) . A l a r suppresses terminal growth (3, 4, 14, 16), delays bloom one to three days and has also been reported to increase f r u i t set (14), improve fr u i t - k e e p i n g q u a l i t y (14), reduce pre-harvest drop (14), and increase f r u i t color (21, 23, 33). Treatments often r e s u l t i n smaller and firmer f r u i t (5) and greener and thicker leaves (14, 16, 17). In s p i t e of such wide-spread at t e n t i o n on a d i v e r s i t y of subjects, there i s l i t t l e d e t a i l e d information on anatomical effects of Al a r (16) or anatomical comparisons between standard and compact types. The object of the present study was to further compare compact and standard types and the ef f e c t s of a l a r on the morphology . 2 and anatomy of apple leaves and stems using standard and compact st r a i n s of Red Delicious and Golden Delicious apples. LITERATURE REVIEW The development of plant growth retardants has been very rapid since the report i n 1949 that a new class of chemicals, the nico-tiniums, reduced stem elogation of bean plants without other formative changes. The most a c t i v e compound was 2,4-dichlorobenzyInicotinium. When applied i n one per cent l a n o l i n paste, the f i r s t internode of the treated plants was found to be one-quarter of the length of the co n t r o l (28). One year l a t e r , Wirwile and M i t c h e l l (39) reported that a number of quaternary ammonium carbamates retarded the growth and development of a broad v a r i e t y of plant species without the develop-ment of malformed leaves, stems, roots and flowers. 4-Hydroxy-5~iso~ propyl-2-methylpheny1 trimethyl ammonium ch l o r i d e , 1-piperidine car-boxy late (Amo-1618) was found to be the most a c t i v e compound i n this group of chemicals tested. In 1958, Preston and Link (30) found that 2,4-dichlorobenzyl-tributylphosphonium chloride (Phosfon) a f f e c t e d the growth of more widely d i f f e r e n t species than did Amo-1618. Then i n 1960 (2-chloroethy1) trimethylammonium chloride (CCC) was found to retard the growth of a larger number of species than any of the e a r l i e r compounds (36). In 1962, R i d d e l l et a l . (32) reported that sprays of N-dimethylamino maleamic a c i d (CO 11) retarded the growth of legumes, vine crops, potatoes and ornamental plants. However, whereas C011 was found to be unstable i n aqueous s o l u t i o n , i t s analogue, N-dimethylaminosuccinamic a c i d (B995), was stable and retarded the growth of the same species as did C011 (13). B995 was the o r i g i n a l experimental code number given by the discoverer, Uniroyal, then the Naugatuck Chemical D i v i s i o n of the United States Rubber Company. Later the name was shortened to B-Nine, B-9, DMAS and A l a r . O r i g i n a l l y the material was intended for use on ornamentals and sold for t h i s purpose under the name of B-Nine. The l a t e r commercial preparation, Alar-85, i s registered for use on several species of f r u i t crops. The chemical structure i s as follows: 0 II CHo - C I CH 2 - C II 0 Among the growth retardants tested, A l a r seems very promising, and has been studied by many in v e s t i g a t o r s . The movement and fate of A l a r i n sweet cherries, apple seedlings and the short-day plant, P h a r b i t i s n i l , have been studied (33, 26). Ryugo reported r e s i d u a l A l a r was found i n new leaves of the sweet cherry, Prunus avium, i n the spring following a l a t e f a l l a p p l i c a t i o n (33). With radioactive B995, Zeevaart was able to demonstrate the mobility and persistence of t h i s growth re-tardant i n P h a r b i t i s plants (40). Also, by using labeled A l a r , Martin et al. (26) were able to follow the movement of A l a r i n apple seedlings. From chemical analysis they concluded that i t was r e s i s t a n t to breakdown i n the plant and was absorbed and translocated r a p i d l y i n the t r a n s p i r a t i o n stream. Due to i t s rapid absorption and - NH - N' CHr; - OH 5, high m o b i l i t y , coverage should be of less importance and a more casual approach to a p p l i c a t i o n may be i n order. This i s an advantage of Ala r over many other chemical sprays. Once within the plant, A l a r causes a number of e f f e c t s . The e f f e c t on root growth of one year old apple trees was studied by Barden (3) who reported that the merit of Ala r over some other growth retardants i s that A l a r causes many desirable e f f e c t s on the above ground portions of an apple tree without a major supression of root growth. Other workers have studied the e f f e c t of A l a r on shoots. Zeevaart reported that treatment of the short-day plant P h a r b i t i s with B995 resulted i n short, thick internodes (40). The e f f e c t of Alar on the shoot diameter of apple trees has also been noted. Halfacre et a l . (16) reported that A l a r treatment increased stem radius of both Golden Delicious and York Imperial apples. The increased radius i n the former was due to an increase i n r a d i a l thickness of the p i t h , phloem and cortex. For York Imperial, i t was due to p i t h and cortex being thi c k e r . Longitudinal sections of Al a r - t r e a t e d plants of both apple c u l t i v a r s had fewer and shorter c e l l s per internode. C e l l d i v i s i o n was affected more than c e l l expansion transversely and l o n g i t u d i n a l l y . Several workers have noted e f f e c t s of A l a r on flowering of f r u i t trees. Batjer et al. (4) reported that apple and cherry trees sprayed with A l a r i n the early summer of 1962 produced more flowers i n 1963 than unsprayed trees. However, i n the short day plant P h a r b i t i s n i l , " V i o l e t " , flower formation was i n h i b i t e d by the a p p l i c a t i o n of A l a r 6 via the roots f o r a period of 24 hours p r i o r to one inductive long night (40). Edgerton et a_l. reported that flower bud formation was promoted on three year o ld Delicious trees sprayed with B995. The pre-bloom a p p l i c a t i o n of B995 on mature trees delayed bloom one to three days and resulted i n higher f r u i t set as compared with unsprayed control when f r o s t s occurred following the treatments (14). Several reports indicated that A l a r also affected keeping q u a l i t y of the f r u i t . Edgerton _et aj.. found that sprays of B995 on three year old Del i c i o u s trees early i n the growing season reduced f r u i t size at harvest (14) and pre-harvestsprays of B995 to more mature Mcintosh apple trees resulted i n firmer f r u i t than on untreated plants. Other workers have demonstrated the e f f e c t s of Ala r on enhancing apple qu a l i t y at harvest and a f t e r storage (15). When A l a r was applied at a concentration of 2000 ppm to sweet cherry Prunus avium, early production of the anthocyanin pigments i n the f r u i t was observed and Ryugo (33) concluded that although Al a r enhanced the biosynthesis of anthocyanins, i t did not measurably advance the p h y s i o l o g i c a l maturity of cherries. Looney, however, reports that early season a p p l i c a t i o n of Ala r promotes several para-meters of sweet cherry maturity (24). The e f f e c t of post-bloom a p p l i c a t i o n of A l a r on apple f r u i t ripening has also been studied. Looney (21) reported that the amount of chlorophyll i n peel and f l e s h of apple was lower through the season when a spray of 4000 ppm of A l a r was applied i n mid-May, two weeks a f t e r bloom but he concluded that A l a r did not noticeably advance or delay maturity. He also studied the respiratory behavior of apples under storage conditions and found that a mid-July a p p l i c a t i o n of 2000 ppm A l a r s i g n i f i c a n t l y reduced r e s p i r a t i o n of stored f r u i t at 0° C, whereas, a mid-May 2000 ppm spray, did not (21). In a l a t e r report from the same laboratory, the.ripening of Mcintosh apples was delayed by treatments of A l a r applied two weeks a f t e r bloom, and t h i s i n h i b i t o r y e f f e c t of A l a r was counteracted by 100 ppm of ethy-lene. Looney suggests that A l a r suppressed ethylene biosynthesis within the f r u i t and t h i s suppression.may not be r e l a t e d to f r u i t maturity (23). One of the most noteworthy e f f e c t s of A l a r i s i t s excellent control" of pre-harvest drop. Batjer _et aJL. reported that the a p p l i c a t i o n of A l a r to D e l i c i o u s and Winesap apple trees reduced pre-harvest drop and delayed the development of watercore. Treated f r u i t s were firmer and somewhat lower i n soluble solids' (5). Blanpied et aJL. thought A l a r might have c e r t a i n s p e c i f i c e f f e c t s rather than a general e f f e c t on the f r u i t (8) because they found that the a p p l i c a t i o n of A l a r to apple trees increased f r u i t firmness and delayed best harvest date for three v a r i e t i e s i n Ireland, but did not do so for Mcintosh i n New York. They a t t r i b u t e the c o n f l i c t i n g r e s u l t s to differences i n the i n t e r a c t i o n of v a r i e t y , season, l o c a t i o n and the s p e c i f i c e f f e c t s of the material. Various e f f e c t s of A l a r on leaf c h a r a c t e r i s t i c s have been re-ported. Edgerton and Hoffman reported that A l a r applied as a f o l i a r spray to three-year-old Delicious trees i n mid-June produced leaves 8 normal and, i n some cases, larger i n s i z e . The leaves appeared darker green and thicker i n texture, than the untreated leaves (14). Halfacre and Barden investigated the leaf anatomical responses of one year old trees of Golden D e l i c i o u s and York Imperial apple treated with A l a r at various concentrations. They found the leaves of treated plants were thicker as a r e s u l t of longer palisade c e l l s and a looser arrangement of the spongy parenchyma c e l l s . In Golden D e l i c i o u s leaves, the lower concentrations of A l a r stimulated transverse palisade c e l l production and expansion whereas Alar i n h i b i t e d c e l l d i v i s i o n and expansion at a l l concentrations used on York Imperial (16). Later, the same workers reported that A l a r treatment decreased leaf area on York Imperial apple trees and also fresh and dry weights per unit area of leaf t i s s u e (17). E f f e c t s of A l a r on leaf anatomy should be studied i n other c u l t i v a r s such as Red D e l i c i o u s . A l l the above findings may have s i g n i f i c a n c e i n the orchard because of the intimate r e l a t i o n s h i p between the leaf structure and i t s maximum photosynthetic rate. McClendon studied the leaves of twenty-three d i f f e r e n t species of plant and found that the photosyn-, 2 t h e t i c rate was a function of t h e i r density thickness (g/cm fresh weight) (27). Beakbane concluded that the number of palisade c e l l s per unit leaf surface was r e l a t e d to the photosynthetic and r e s p i r a -tory a c t i v i t y of the palisade mesophyll and also related to the growth po t e n t i a l of apple rootstocks (7). The e f f e c t of a growth stimulant, g i b b e r e l l i c a c i d (GA), on stem elongation i n plants has often been investigated. As there was no evidence of c e l l elongation for at least 72 hours a f t e r a p p l i c a t i o n of g i b b e r e l l i n to the vegetative, plants of the b i e n n i a l short-day Hyoscyamus and of the long-day plant Salmolus, Sachs _et al. concluded that the i n i t i a l increase i n stem length was due s o l e l y to an increase i n c e l l number (34). Beakbane suggested that GA might be used as a possible expanding agent i n leaf t i s s u e subjected to shade conditions (6), because i n apple leaf d i s c s treated i n a basic medium plus 0.5 ppm GA, great expansion of the epidermal c e l l s was observed. The d i s t r i b u t i o n and shape of mesophyll c e l l s were also affected by GA. Several workers have examined the i n t e r a c t i o n of A l a r with GA i n plants. Zeevaart found the i n h i b i t i o n of flower formation i n P h a r b i t i s n i l by A l a r could be completely overcome by a p p l i c a t i o n of g i b b e r e l l i n A3 to the plumule before the long night (40). By using cucumber seedlings, Moore (29) was able to demonstrate that 1 / i g of GA3 applied to the shoot t i p was s u f f i c i e n t to completely n u l l i f y the e f f e c t of 25/ug of A l a r applied simultaneously (29). When three-year-old D e l i c i o u s apple trees were sprayed with a mixture of A l a r at 1000 ppm and of potassium g i b b e r e l l a t e (KGA) at 200 ppm, the shoot growth was reduced to less than 50 per cent of that made by shoots treated with KGA alone (14). However, the r e a l mechanism of i n t e r -a c t i o n between A l a r and the g i b b e r e l l i n s s t i l l remains unknown. Further studies are needed to elucidate the r e l a t i o n s h i p between A l a r and GA i n the apple. The d i f f e r e n c e i n growth habit, chemical content, and leaf com-po s i t i o n between Starking Delicious and the natural compact mutant 10 Starkrimson have been studied extensively. In general, when compared with Starking, the mutant i s reported to have shorter shoots, fewer l a t e r a l shoots, more f r u i t i n g spurs, more nodes per foot and thicker leaves with a greater depth of palisade parenchyma, more dry weight, c h l o r o p h y l l , N, and Ca (1, 37, 38). Before i t i s possible to g e n e r a l l i z e about the anatomical differences between standard and compact types, comparisons must be made i n other c u l t i v a r s such as Golden Delicious and i t s compact mutant, Starkspur. Only Arasu (1) has con-sidered these l a t t e r two str a i n s and he only reports data for t o t a l leaf and palisade parenchyma thickness. Stem anatomy comparisons between compact and standard growing types have not been made i n any c u l t i v a r . Since the natural compact mutants have many merits over the standard growing str a i n s and since A l a r i s reported to cause s i m i l a r changes i n standard apple trees, a c r i t i c a l comparison of the d i f f e -rences and s i m i l a r i t i e s between these two approaches to s i z e control i s required. I n t r i e r i reported that spur type trees and those treated with retardants showed many analogies i n morphology, physiology, bio-chemistry and anatomy (19). This was based on an extensive l i t e r a t u r e review and not c r i t i c a l comparisons within any s i n g l e experiment. This report therefore must be regarded as s e t t i n g up a number of hypotheses which demand i n v e s t i g a t i o n . Likewise, Looney reported there were simi-l a r i t i e s between compact mutants and standard Delicious apple trees treated with A l a r (22). He found that the net a s s i m i l a t i o n rate of Starkrimson was approximately twelve per cent higher than that of Starking Delicious and a s i m i l a r difference was revealed between Starkspur and Golden Delicious (22). According to the l i t e r a t u r e , A l a r i s the most promising growth retardant studied to date. While i t s e f f e c t s on c e r t a i n characteris-t i c s such as growth habit, external appearance, morphology and biochemical function have been studied, there i s no information at a l l concerning the e f f e c t of A l a r on the natural compact mutants. The h o r t i c u l t u r a l importance of both growth retardants and geneticccom-paction as tools for s i z e c o n t r o l would indi c a t e that any possible i n t e r a c t i o n merits c a r e f u l study. Two main points a r i s i n g from this l i t e r a t u r e review w i l l be investigated for the f i r s t time i n this study; e f f e c t s of A l a r on leaf and stem anatomy i n Red Delicious and detailed anatomical comparisons of leaf and stem i n standard and compact types of Red and Golden D e l i c i o u s . While some anatomical e f f e c t s of A l a r on Golden Delicious and comparisons of leaf anatomy i n compact and standard Golden and Red Delicious have already been reported, these w i l l be included i n the present study to allow more d i r e c t comparison with the r e s u l t s of e a r l i e r s t u d i e s . Previous workers have not studied stem anatomy i n any compact mutant and leaf anatomy studies to date have been rather s u p e r f i c i a l . With this information i t should be possible to generalize about the anatomical e f f e c t s of A l a r or natural compaction upon apple v a r i e t i e s . MATERIALS AND METHODS Experiment I Comparisons of Untreated Compact and Standard C u l t i v a r s The leaf and shoot samples of apple trees were obtained from the orchard of the Canada Department of A g r i c u l t u r e Research Station at Summerland. Two v a r i e t i e s , Red D e l i c i o u s and Golden D e l i c i o u s , each with standard and compact types, i . e . Harrold Red D e l i c i o u s (standard), Starkrimson (compact), Golden D e l i c i o u s (standard) and Starkspur (com-pact), were used i n t h i s experiment. On June 11, 1968, three nine year old trees were randomly chosen from each c u l t i v a r . One shoot from the North and South side of each tree was sampled by taking two neighboring leaves from the middle part of the current year's shoot. Each leaf was sampled by taking discs from three d i f f e r e n t p o s i t i o n s , a p i c a l , middle and basal on each side of the midrib. From the same shoot, the f i r s t internode below the shoot t i p was taken. Each internode was cut transversely into two parts for both cross and l o n g i t u d i n a l sections. The leaf and shoot tissues were fi x e d i n B e l l i n g s Modified Nava-shin F l u i d (20) for which the formula i s as follows: Solution A: Chromic a c i d c r y s t a l s 5g G l a c i a l a c e t i c acid 500cc D i s t i l l e d water 320cc Solution B: Formalin 200cc D i s t i l l e d water 175cc Saponin 3g A f t e r f i x a t i o n , the samples were washed with running tap water, dehy-drated i n an ethanol series and embedded i n paraplast according to the procedure of Johansen (20) which i s shown below: Dehydration: 1. 5%, ethyl alcohol . . . . . . . . .2 hours 2. ll%'.;ethyl alcohol 2 hours 3. 18% ethyl alcohol 2 hours 4. 30% ethyl alcohol 2 hours 5. approximate 50% alcohol 2 hours or longer D i s t i l l e d water 5 parts 95% ethyl alcohol 4 parts T e r t i a r y b u t y l alcohol 1 part 6. approximate 70% alcohol overnight or longer D i s t i l l e d water 3 parts 9570 ethyl alcohol 5 parts T e r t i a r y butyl alcohol 2 parts 7. approximate 85% alcohol at least 1 hour D i s t i l l e d water 3 parts 95% ethyl alcohol 10 parts T e r t i a r y b u t y l alcohol 7 parts 8. approximate 95% alcohol at l e a s t 1 hour 95% et h y l alcohol 9 parts T e r t i a r y butyl alcohol 11 parts 9. approximate 100%-:alcbh6l. . . . . at least 1 hour T e r t i a r y b u t y l alcohol 3 parts 14 100% ethyl alcohol 1 part 10. T e r t i a r y b u t y l alcohol 3 changes (one of which should remain overnight) I n f i l t r a t i o n : 1. mixture of equal parts of p a r a f f i n o i l and T e r t i a r y b u t y l alcohol at l e a s t 1 hour 2. f i l l a v i a l three-fourth f u l l of melted paraplast and l e t the paraplast s o l i d i f y but not cool completely. 3. put the material on top of the s o l i d i f i e d paraplast, j u s t cover with the b u t y l a l c o h o l - p a r a f f i n o i l mixture and place the container i n the oven at once. 4. about 1 hour a f t e r the material has sunk to the bottom of the v i a l , pour o f f the e n t i r e mixture of p a r a f f i n o i l and what traces of alcohol remain and replace with pure melted paraplast. 5. repeat the process twice during the next 6 hours or so, discar-ding each change of paraplast. 6. f i n a l l y replace with pure melted paraplast and the material w i l l be ready for embedding within the next 30 minutes. Embedding: 1. remove the v i a l from the oven, shake the material to get i t off the bottom and quickly pour into the p l a s t i c mold. 2. add more melted paraplast from the stock container i f necessary. 3. with a needle heated s l i g h t l y i n the flame, quickly dispose the pieces of material into an orderly arrangement. 4. as soon as the mould can be moved without d i s t u r b i n g the 15 arrangement of the pieces of material, transfer tbsa vessel of cold water. 5. l e t the mold f l o a t u n t i l the surface of the paraplast becomes s u f f i c i e n t l y f i r m to permit plunging the mold slowly beneath the surface of the water. 6. leave the molds i n the water for half an hour or u n t i l tho-roughly cooled. A f t e r embedding, a l l the leaf and stem samples were cut with a rotary microtome at ten microns thickness. Sections were a f f i x e d to s l i d e s with Haupt's adhesive (18), and stained with safranin and f a s t green according to the schedule described by Johansen (20) and shown below: Staining: 1. Xylene - 10 to 15 minutes. 2. Xylene - 100% ETOH (1-1) - 5 minutes. 3. 95% ETOH - 5 minutes. 4. 70% ETOH - 5 minutes. 5. 50% ETOH - 5 minutes. 6. water - wash we l l . 7. Stockwell's so l u t i o n - at l e a s t 24 hours, depending on material. 8. water - wash we l l . 9. tannic acid - 10 to 15 minutes. 10. water - wash we l l . 11. f e r r i c chloride - several minutes, depending on blacking. 12. water - wash w e l l . 16 13. safranin - at l e a s t overnight; usually 24 hours. 14. water - wash w e l l . 15. 95% ETOH with 1/2% p i c r i c a c i d - no more than 10 seconds. 16. 95% ETOH with 4-5 drops ammonia per 100 cc - 2 minutes. 17. 100% ETOH - about 30 seconds. 18. f a s t green staini n g solution - s t a r t i n g with a 10 second immersion. 19. clove o i l (to stop a c t i o n of f a s t green) - a few seconds. 20. clear f o r one or two minutes i n : clove o i l - 50 cc 100% ETOH - 25 cc Xylene - 25 cc 100 cc 21. Xylene - at l e a s t 10 minutes. The s l i d e s were then examined microscopically. An ocular micrometer was used for measuring the c e l l length and r a d i a l diameter of stem and leaf t i s s u e s . The expected mean squares f or the analysis of variance of t h i s experiment are shown i n the Appendix, Tables 1 to 4. Experiment 11 E f f e c t s of Ala r on Compact and Standard C u l t i v a r s (A) Apple trees of Golden D e l i c i o u s and Starkspur on EM VII rootstocks were grown i n a growth chamber at Summerland. In December 1968, A l a r at 0 and 1000 ppm was applied to each c u l t i v a r . At t h i s time Golden De l i c i o u s had an average shoot length of 14.25 cm and Starkspur had 10.6 cm. There were s i x trees of each treatment and c u l t i v a r . A l a r sprays and measurements of shoot length and leaf number were made at weekly i n t e r v a l s . The 'compact' Starkspur grew very vigorously and i n f a c t , was not noticeably d i f f e r e n t from Golden D e l i c i o u s when A l a r was not applied. However, A l a r appeared to reduce shoot growth more on Starkspur than on Golden D e l i c i o u s . Shoot lengths were measured from the base of the new shoot to the base of the t i p leaf c l u s t e r , and leaves were counted from the base up, inc l u d i n g half-opened ones at top of the shoot. The treatments were terminated on February 4, 1969. Samples were co l l e c t e d at that same time. Sampling and preparation procedures were e s s e n t i a l l y the same as i n experiment I, but the second and t h i r d f u l l y expanded leaves below the shoot apex were chosen for leaf sampling to ensure that the leaves sampled were i n i t i a t e d well a f t e r the A l a r treatments were begun. In each tree one or two shoots were used, and there were s i x trees of each c u l t i v a r randomly assigned to each of the treatments and the con-t r o l s . (B) A t o t a l of twenty-four trees of Harrold Red D e l i c i o u s and Stark-rimson were used i n t h i s experiment, also at Summerland. Technical grade A l a r at 0 or 1000 ppm was applied to s i x trees of each c u l t i v a r . When f i r s t treated, the average shoot length of Harrold was 13 cm and of Starkrimson, 8 cm. Ala r treatments, shoot length and leaf number determinations were done once a week for eight weeks beginning A p r i l 8, 1969 with Harrold and A p r i l 22, 1969 with Starkrimson. Sampling and preparation procedures were the same as described i n 18 experiment I. The expected mean squares f or the analysis of variance of t h i s experiment are shown i n the Appendix, Tables 5 to 13. Experiment III (a) Comparisons of Compact with A l a r treated Red Delicious In A p r i l , 1969, the scions of Red D e l i c i o u s , and Starkrimson were grafted on EM 11 rootstocks planted i n p l a s t i c pots. A l l l a t e r a l shoots were removed and only one shoot was allowed to develop on each tree. Twenty-one Red De l i c i o u s and seven Starkrimson trees were used i n t h i s experiment. On June 12, the Red De l i c i o u s trees had an ave-rage shoot length of 35 cm. Technical grade A l a r with a small amount of T r i t o n added, was applied at 0, 1000 and 4000 ppm. There were seven r e p l i c a t e s within each treatment. The seven Starkrimson trees with an average shoot length of 14.3 cm were l e f t untreated for com-parison. A l l twenty-eight trees were randomly arranged within one block. Shoot lengths were measured at weekly i n t e r v a l s . The experi-ment was terminated f i v e weeks a f t e r treatment. Experiment III (b) E f f e c t s of A l a r and G i b b e r e l l i c Acid on Compact and Standard  Golden Delicious Four trees each of Golden D e l i c i o u s and Starkspur were randomly arranged wi t h i n each of two blocks. These trees were also on EM 11 rootstocks grafted i n A p r i l , 1969 and prepared as described above. On June 12, 1969, Ala r at 0 and 1000 ppm, GA at 1000 ppm, and GA at 1000 ppm combined with A l a r at 1000 ppm were applied i n d i v i d u a l l y to single trees within the same block. Shoot lengths were measured once 19 weekly f or s i x consecutive weeks. Leaf and shoot samples were c o l -lected at the end of the experiment. The same c o l l e c t i o n and preparat-i o n procedures were used as i n experiment I . The one exception was that only two discs along the midrib of each l e a f were taken because i n experiment I , no s i g n i f i c a n t d i f f e r e n c e s among the d i f f e r e n t posi-tions on a leaf had been found. The expected mean squares for the anal y s i s of variance of t h i s experiment are shown i n the Appendix, Tables 14 to 17. RESULTS Experiment I Comparisons of Untreated Compact and Standard Apple C u l t i v a r s There were no differences among Harrold Red D e l i c i o u s , Starkrimson, Golden Delicious and Starkspur i n the thickness of the upper and lower epidermis and spongy parenchyma. The Starkspur had a thicker palisade parenchyma than the other three c u l t i v a r s (Table 1, F i g . 1). The i n -creased thickness of the palisade parenchyma was found to be due to greater mean palisade c e l l layer number and length (Table 1, F i g . 2). Although the analysis of variance showed no differences i n the t o t a l leaf thickness between Harrold Red D e l i c i o u s and Starkrimson, d i s c r i -minant function analysis showed that the c u l t i v a r s ( F i g . 3) d i f f e r e d s i g n i f i c a n t l y at the f i v e per cent l e v e l . Red and Golden De l i c i o u s were compared with respect to leaf tissue thickness, pooling the com-pact and standard types. The thicker palisade parenchyma of Golden Delicious was probably due to both the greater mean number of layers and the length of palisade c e l l s (Table 2). Also compact and standard types were compared pooling the two c u l t i v a r s . Standard type was found to have thinner leaves, thinner palisade parenchyma and smaller mean number of palisade c e l l layers than the compact mutant (Table 3). In order to inv e s t i g a t e the cause for the thicker shoots and shorter internodes i n the compact apple mutants, measurements were made of c e l l size i n the p i t h and cortex, the thickness of vascular tissue as w e l l as stem thickness. Figures 4 and 5 show l o n g i t u d i n a l TABLE 1 THICKNESS OF LEAF TISSUES OF FOUR CULTIVARS IN MICRONS Harrold Red Delicious Starkrimson Golden D e l i c i o u s Starkspur Significance Level Lower Epidermis 12a X 12a 12a 13a 0.1566 Spongy Parenchyma 75a 75a 75a 82a 0.1999 Palisade Parenchyma 84c 90bc 100b 112a 0.0028 Upper Epidermis 14a 14a 14a 15a 0.5218 Total 186b 191b 202b 222a 0.0047 Number of Palisade C e l l Layer 3.0b 3.1b 3.0b 3.3a 0.0015 Average Palisade C e l l Length 29 b 29 b 33ab 34a 0.0413 within a row, means having a l e t t e r i n common are not s i g n i f i c a n t l y d i f f e r e n t at the 5 per cent leve l by Duncan's Multiple Range Test. Leaf thickness in microns FIGURE 2 The l e a f cross section of Starkspur showing longer palisade c e l l s and greater mean palisade number ( l e f t ) than i n the standard Golden D e l i c i o u s ( r i g h t ) . (381X) u> The leaf cross section of Starkrimson ( l e f t ) showing greater l e a f thickness than Red Delicious ( r i g h t ) . (300X) TABLE 2 THICKNESS OF LEAF TISSUES OF RED DELICIOUS AND GOLDEN DELICIOUS IN MICRONS Red D e l i c i o u s x Golden D e l i c i o u s x Significance L e v e l Lower Epidermis 12 13 0.0456 Spongy Parenchyma 75 78 0.2317 Palisade Parenchyma 87 106 0.0008 Upper Epidermis 14 15 0.2107 Total 188 212 0.0018 Number of Palisade C e l l Layer 3.02 3.19 0.0039 Average Palisade C e l l Length 29 33 0.0078 xmean of standard and compact types. TABLE 3 THICKNESS OF LEAF TISSUES OF STANDARD AND COMPACT TYPES IN MICRONS Standard Compact x x S i g n i f i c a n c e Level Lower Epidermis 12 12 0.6533 Spongy Parenchyma 75 79 0.1923 Palisade Parenchyma 92 101 0.0350 Upper Epidermis 14 14 0.7804 Tota l 195 206 0.0345 Number of Palisade C e l l Layer 3.0 3.2 0.0013 Average Palisade C e l l Length 31 32 0.3983 ^ e a n s of the Red Delicious and Golden D e l i c i o u s . 'means of the Starkrimson and Starkspur. 27 COLLENCHYMA PARENCHYMA VASCULAR TISSUE PITH FIGURE 4 Lon g i t u d i n a l section of Red D e l i c i o u s stem showing collenchyma, parenchyma, vascular t i s s u e , and part of p i t h . (118 X) COLLENCHYMA) ) CORTEX PARENCHYMA ) VASCULAR TISSUE PITH FIGURE 5 Cross section of Red D e l i c i o u s stem showing part of collenchyma, parenchyma, v a s c u l a r . t i s s u e and p i t h . (60 X) [S3 0 0 TABLE 4 CELL LENGTHS OF STEM COLLENCHYMA, PARENCHYMA AND PITH OF FOUR APPLE CULTIVARS IN MICRONS Cul t i v a r Collenchyma Parenchyma Pit h Harrold 48a X 61a 41a Starkrimson 44a 67a 38a Golden 45a 57a 37a Starkspur 41a 51a 38a W i t h i n a column, means having a l e t t e r i n common are not s i g n i f i c a n t l y d i f f e r e n t at the 5 per cent l e v e l by Duncan's Multiple Range Test. 30 and cross sections respectively of Red Delicious shoot t i s s u e . There were no s i g n i f i c a n t differences i n c e l l length of collenchyma, paren-chyma or p i t h c e l l s among the four c u l t i v a r s (Table 4). In stem cross sections, Starkrimson had a thicker vascular tissue than the other c u l t i v a r s (Table 5), but the thickness of the cortex, p i t h and t o t a l stem were not s i g n i f i c a n t l y d i f f e r e n t i n the c u l t i v a r s . The c e l l dia-meter of the collenchyma of Red Delicious and Starkrimson Red Delicious was greater than that of Golden Delicious and Starkspur. Starkspur had a smaller p i t h c e l l diameter than the other three c u l t i v a r s , but the mean diameter of the parenchyma c e l l s did not d i f f e r among c u l t i -vars (Table 6). The data presented i n Table 5 were measured as though each tissue were c i r c u l a r . Experiment 11 E f f e c t s of A l a r on Compact and Standard Apple C u l t i v a r s The shoot growth of Red D e l i c i o u s was not s i g n i f i c a n t l y affected by A l a r u n t i l s i x weeks a f t e r treatment, but growth rate suppression was f i r s t suspected four weeks a f t e r treatment (Table 7). A l a r at 1000 ppm did not show any i n h i b i t i n g e f f e c t on the shoot growth of Starkrimson (Table 7). A l a r did not s i g n i f i c a n t l y a f f e c t leaf number (Table 8), leaf length or width i n either Red D e l i c i o u s or Starkrimson (Table 9). How-ever, i n Golden D e l i c i o u s , s i g n i f i c a n t i n h i b i t i o n on shoot length was observed from the second week a f t e r A l a r treatment. The d i f f e r e n c e due to treatment increased gradually t i l l the termination of the experi-ment (Table 10). The e f f e c t of A l a r on Starkspur shoot growth was TABLE 5 THICKNESS OF STEM CORTEX, VASCULAR TISSUE, DIAMETERS OF PITH, AND STEM OF FOUR APPLE CULTIVARS IN MICRONS Cult i v a r Cortex Vascular Tissue P i t h Diameter Shoot Diameter Harrold Red Delicious X 256a 248b 1073a 2160a Starkrimson 263a 414a 9 73a 2273a Golden Delicious 233a 327ab 908a 2006a Starkspur 288a 279b 939a 1950a within a column, means sharing the same l e t t e r are not s i g n i f i c a n t l y d i f f e r e n t at the 5 per cent l e v e l by Duncan's M u l t i p l e Range Test. TABLE 6 CELL DIAMETERS OF STEM COLLENCHYMA, PARENCHYMA AND PITH OF FOUR APPLE CULTIVARS IN MICRONS Cultivar Collenchyma Parenchyma Pi t h Harrold Red Delicious 20a X 29a 35a Starkrimson 21a 27a 33a Golden Delicious 18b 25a 30a Starkspur 18b 25a 26b W i t h i n a column, means sharing the same l e t t e r are not s i g n i f i c a n t l y d i f f e r e n t at the 5 per cent l e v e l by Duncan's M u l t i p l e Range Test. TABLE 7 EFFECT OF ALAR ON SHOOT GROWTH OF RED DELICIOUS AND STARKRIMSON (CM) Cu l t i v a r A l a r Concentration (ppm) 1 2 Weeks Aft e r Treatment 3 4 5 6 7 8 Red Delicious 0 1000 8h X 8h 15g 15g 23f 21f 29 e 28e 35d 32de 43b 37cd 50a 41b c 55a 44b Significance Level 0.0135 Starkrimson 0 1000 12a 14a 18a 24a 26a 30a 33a 38a 40a 42a 45a 45a 45a 47a Significance Level 0.9389 x w i t h i n each c u l t i v a r , means having a l e t t e r i n common are not s i g n i f i c a n t l y d i f f e r e n t at the 5 per cent l e v e l by Duncan's Multiple Range Test. TABLE 8 EFFECT OF ALAR ON LEAF NUMBER OF RED DELICIOUS AND STARKRIMSON Weeks Aft e r Treatment 3 4 5 6 7 8 6 8 11 14 16 18 21 23 6 8 11 14 16 18 20 22 Significance Level 0.5581 Starkrimson 0 8 12 15 18 20 22 23 1000 10 13 17 19 20 23 24 Significance Level 0.7543 Co •P-C u l t i v a r A l a r Concentration (ppm) Red Delicious 0 1000 TABLE 9 EFFECT OF ALAR ON AVERAGE LEAF LENGTH AND WIDTH OF RED DELICIOUS AND STARKRIMSON (CM) Cul t i v a r A l a r Concentration (ppm) Leaf Length Leaf Width Red Delicious 0 10.1 5.3 1000 10.0 4.9 Significance Level 0.8042 0.2430 Starkrimson 0 12.4 6.1 1000 12.0 5.7 Significance Level 0.2673 0.1551 TABLE 10 INFLUENCE OF ALAR ON SHOOT GROWTH OF STARKSPUR AND GOLDEN DELICIOUS (CM) Cu l t i v a r A l a r Concentration (ppm) Weeks A f t e r Treatment 2 3 4 5 6 Golden Delicious 0 1 6 i X 20h 26f 33d 40c 46b 51a 1000 141 16i 19h 23g 27f 30e 33d Significance Level 0.0000 Starkspur 0 13hij 16gh 22ef 31d 41c 47b 52a 1000 9 i l l i j 14hi 16hi 21fg 24ef 26de Significance Level 0.0000 within each c u l t i v a r , means having a l e t t e r i n common are not s i g n i f i c a n t l y d i f f e r e n t at the 5 per cent l e v e l by Duncan's Mult i p l e Range Test. Co TABLE 11 EFFECT OF ALAR ON LEAF NUMBER OF GOLDEN DELICIOUS Cu l t i v a r Alar Concentration (ppm) 1 Weeks A f t e r 2 Treatment 3 4 Golden Delicious 0; 14f X 16.6d 19b 22a 1000 12g 15e 17cd 17.6b Significance Level 0.0007 ^Sneans having a l e t t e r i n common are not s i g n i f i c a n t l y d i f f e r e n t at the 5 per cent l e v e l by Duncan's Multiple Range Test. 38 quite d i f f e r e n t from expectation. The retardation e f f e c t was noted during the second week a f t e r treatment, but less retardation was found during the fourth week. However, si x weeks a f t e r treatment, A l a r i n -h i b i t e d shoot growth by 50% (Table 10). Four weeks a f t e r treatment, A l a r 1000 ppm s i g n i f i c a n t l y reduced Golden D e l i c i o u s leaf number by 25% (Table 11). A l a r did not show any s i g n i f i c a n t e f f e c t on the leaf number of Starkspur. Microscopic examination of Harrold and Starkrimson leaf lamella sections revealed no s i g n i f i c a n t differences between treatments i n any one of the seven v a r i a b l e s measured. In Golden D e l i c i o u s , the treated plants had thinner lower epidermis and fewer palisade c e l l s than the c o n t r o l . Treated Starkspur had thinner spongy parenchyma, fewer p a l i -sade c e l l s and less t o t a l leaf thickness (Table 12). The data for c e l l length i n the stem l o n g i t u d i n a l sections of four c u l t i v a r s , each with two d i f f e r e n t treatments i s shown i n Table 13. Apparently the A l a r treated Starkrimson plants had s i g n i f i -cantly shorter collenchyma c e l l s but the length of parenchyma c e l l i n cortex and p i t h were not s i g n i f i c a n t l y shorter than the untreated ones. Starkspur Golden Delicious plants showed longer parenchyma c e l l i n cortex as compared with the treated plants. The c e l l lengths i n treated Harrold and Golden Delicious were not s i g n i f i c a n t l y d i f f e r e n t from the untreated plants. Experiment III (a) Comparisons of Compact with A l a r treated Red D e l i c i o u s A l a r was found to increase the thickness of t o t a l l e a f , spongy TABLE 12 INFLUENCE OF ALAR ON THICKNESS OF LEAF TISSUES OF FOUR APPLE CULTIVARS (MICRONS) C u l t i v a r Alar Concentration (ppm) Lower Epidermis Spongy Parenchyma Palisade Parenchyma Upper Epidermis T o t a l Pa 1 i sade Avera ge•-• Number Palisade Red Delicious 0 12 76 124 16 226 3 41 1000 11 73 114 15 213 3 39 Starkrimson 0 11 81 100 16 208 3 33 1000 11 90 113 15 229 3 36 Golden Delicious 0 11 77 96 14 198 3 32 1000 10** 81 90 14 195 2.8* 33 Starkspur 0 11 80 97 14 203 3 33 1000 11 71* 89 14 184* 2.9* 31 -•significant at 5 per cent l e v e l . * * s i g n i f i c a n t at 1 per cent l e v e l . TABLE 13 INFLUENCE OF ALAR ON THE CELL LENGTHS OF COLLENCHYMA, PARENCHYMA AND PITH OF FOUR APPLE CULTIVARS (MICRONS) Alar Concentration (ppm) Red Delicious Starkrimson Golden D e l i c i o u s Starkspur 0 33 46 38 42 1000 35 31** 43 33 0 59 54 50 56 1000 53 50 54 42* P i t h 0 35 37 37 41 1000 38 34 39 32 -'significant at 5 per cent l e v e l . * * s i g n i f i c a n t at 1 per cent l e v e l . o TABLE 14 INFLUENCE OF ALAR ON THE THICKNESS OF LEAF TISSUES OF RED DELICIOUS IN MICRONS Cu l t i v a r Alar Concentration Lower Spongy Palisade Upper Total Palisade Average (ppm) Epidermis Parenchyma Parenchyma Epidermis Number Palisade x Red D e l i c i o u s 0 11a 63ab 78b 16a 168b 3a 26b Red D e l i c i o u s 1000 10a 71a 87a 16a 184a 3a 30a Red D e l i c i o u s 4000 10a 69a 90a 16a 186a 3a 30a Starkrimson 0 10a 59b 75b 16a 161b 3a 27b Significance Level 0.8043 0.0342 0.0039 0.7688 0.0062 0.0907 0.0085 W i t h i n each column, means having a l e t t e r i n common are not s i g n i f i c a n t l y d i f f e r e n t at the 5 per cent l e v e l by Duncan's Multiple Range Test. 43 parenchyma and length of palisade c e l l i n Harrold Red D e l i c io us (Table 14). The r e s u l t s from two concentrations i . e . 1000 and 4000 ppm, were not d i f f e r e n t from each other. The t o t a l leaf thickness, palisade parenchyma and average palisade parenchyma of Starkrimson were not d i f f e r e n t from untreated Harrold Red D e l i c i o u s , although s l i g h t l y thicker spongy parenchyma was found i n the l a t t e r . The upper and lower epidermis and palisade number were not d i f f e r e n t from treat-ment to treatment, hence the thicker leaf of A l a r treated Red D e l i c i o u s resulted from an increase i n the thickness of spongy parenchyma and palisade parenchyma ( F i g . 6). A l a r i n h i b i t e d shoot growth of Red D e l i c i o u s apple trees and the i n h i b i t i n g e f f e c t was greater at the higher concentration, i . e . at 4000 ppm (Table 15). The treatment e f f e c t was noticeable during the second week's growth and was s t i l l present at the termination of the experiment. A f t e r two weeks, the growth rate of A l a r - t r e a t e d Red De-l i c i o u s was less than that of Starkrimson Red D e l i c i o u s although Star-krimson was s t i l l smaller i n t o t a l s i z e . Experiment I I I (b) E f f e c t s of A l a r and G i b b e r e l l i c A c i d on Compact and Standard  Golden D e l i c i o u s There were no s i g n i f i c a n t differences between means for the thick-ness of Golden D e l i c i o u s leaf t i s s u e s as a r e s u l t of treatment with A l a r or GA (Table 16). However, GA increased mean shoot growth of Golden D e l i c i o u s and Starkspur Golden D e l i c i o u s by 29 per cent by the end of the experiment (Table 17). A l a r at 1000 ppm or the A l a r and GA 44 combination did not a f f e c t the shoot growth of Golden D e l i c i o u s and Starkspur Golden Delicious (Table 17). Apparently A l a r n u l l i f i e d the stimulating e f f e c t of GA. TABLE 15 EFFECT OF ALAR ON SHOOT GROWTH OF STARKRIMSON AND RED DELICIOUS (CM) Cult i v a r Alar Concentration (ppm) 1 Weeks A f t e r Treatment 2 3 4 5 6 Red Delicious 0 36h x 47ef 54c 59b 63a 66a Red Delicious 1000 36h 45 gh 48ef 50de 52cd 54c Red Delicious 4000 34i 42gh 45fg 46f 47ef 48ef Starkrimson 0 14m 211 25kl 28jk 31j 33i Significance Level f o r Interaction 0.0000 ^ e a n s having a l e t t e r i n common are not s i g n i f i c a n t l y d i f f e r e n t at the 5 per cent l e v e l by Duncan's Multiple Range Test. TABLE 16 INFLUENCE OF ALAR AND GIBBERELLIC ACID ON THICKNESS OF LEAF TISSUES OF STARKSPUR AND GOLDEN DELICIOUS* C u l t i v a r Treatment (ppm) Lower Epidermis Spongy Parenchyma Palisade Parenchyma Upper Epidermis T o t a l Number of Palisade Layer Average Pa l i s a d e C e l l Length Golden D e l i c i o u s c o n t r o l 11 70 93 15 189 3 32 A - 1000 11 65 89 15 180 3 33 GA - 1000 11 54 85 16 166 3 28 A GA - 1000, - 1000 10 58 86 15 169 3 31 Starkspur c o n t r o l 11 66 90 14 182 3 31 A - 1000 11 60 88 15 174 3 31 GA - 1000 12 61 87 15 175 3 29 A GA - 1000, - 1000 12 72 94 14 191 3 34 S i g n i f i c a n c e Level 0.4403 0.2.187 0.6475 0.8584 0.1668 0.9695 0.4339 There was no s i g n i f i c a n t e f f e c t of any of these treatments on le a f t i s s u e s thickness of ei t h e r c u l t i v a r . TABLE 17 INFLUENCE OF ALAR AND GIBBERELLIC ACID ON MEAN SHOOT GROWTH OF STARKSPUR AND GOLDEN DELICIOUS (CM) Treatment (ppm) 1 2 Weeks Aft e r 3 Treatment 4 5 6 Control X 23h 31g 33fg 35cdefg 38cdef 40cd A l a r - 1000 24h 34efg 38cdef 39cdef 39cdef 41c GA - 1000 23h 34efg 41c 48b 56a 58a A l a r - 1000, GA - 1000 19 h 32g 35defg 38cdef 40cd 41c Significance Level of Interaction 0.0001 means having a l e t t e r i n common are not s i g n i f i c a n t l y d i f f e r e n t at the 5 per cent l e v e l by Duncan's Multiple Range Test. DISCUSSION Starkspur was found to have a thicker l e a f , palisade parenchyma and a greater mean palisade number and size than the other three c u l -t i v a r s . These r e s u l t s agree with the conclusions of Westwood (37), Arasu (1) and Westwood and Z i e l i n s k i (38) that the spurtype mutants have thicker leaves and palisade t i s s u e . They also found the mutant to have shorter internodes, greater leaf surface per foot of shoot, fewer 2 side branches but more spurs, and greater chlorophyll content per cm of l e a f . These a t t r i b u t e s of spurtypes favor them with regard to l i g h t d i s t r i b u t i o n , bearing surface, photosynthetic e f f i c i e n c y and f r u i t - b e a r i n g p o t e n t i a l . The present study has demonstrated the thicker leaf and palisade tissues of the spurtype mutants by c a r e f u l examination of the leaf anatomy of the spurtype mutants. The increase i n thickness of the palisade parenchyma was found due both to longer palisade c e l l s and a greater mean palisade layer of them. The Star-krimson leaves were only s l i g h t l y thicker than those of Red D e l i c i o u s and t h i s difference was a t t r i b u t e d to thicker palisade parenchyma. By comparing the thickness of leaf tissues of Red D e l i c i o u s with Golden D e l i c i o u s (Table 2), i t i s evident that the l a t t e r was 11.3 per cent thicker i n terms of t o t a l leaf thickness. Apparently there were no s i g n i f i c a n t d ifferences i n thickness of lower epidermis, upper epi-dermis and spongy parenchyma between these two v a r i e t i e s . The d i f -ference i n t o t a l leaf thickness i s a t t r i b u t a b l e to d i f f e r e n c e s i n p a l i -sade parenchyma which was 18 per cent thicker i n Golden D e l i c i o u s than 49 i n Red D e l i c i o u s . Looney reported that Golden Delicious had an 18 per cent higher net a s s i m i l a t i o n rate than Red Delicious (22). Compact mutants have been reported to have a thicker stem and shorter internodes than standard v a r i e t i e s (37, 38). However, i n this study i t was found that there were no s i g n i f i c a n t differences i n c e l l length of collenchyma, parenchyma and p i t h c e l l s among the four c u l t i -vars studied (Table 4). In stem cross sections, the ..thickness of cor-tex, p i t h and t o t a l stem of mutants were also not d i f f e r e n t from the standard (Table 5). Furthermore, i n c e l l diameter of d i f f e r e n t tissues (Table 6), standard types were not d i f f e r e n t from the spur types. These results represent the f i r s t comparisons between compact and standard types with respect to stem anatomy. Since the shoot material used i n this experiment was taken from the f i r s t internode under the growing t i p , manifestation of the reasons for the reported thicker and shorter shoots may not occur i n this region of the shoot. Future work should consider mature t i s s u e s . I t i s quite possible that the e f f e c t s of A l a r treatment have not shown up completely on this premature t i s -sue . In experiment I I , Red Delicious treated with A l a r at 1000 ppm did not show any s i g n i f i c a n t difference i n leaf thickness when compared with the c o n t r o l (Table 12). However, i n experiment I I I , A l a r at 1000 ppm was found to increase the thickness of t o t a l leaf, spongy paren-chyma and length of palisade c e l l s (Table 15). These c o n f l i c t i n g r e s u l t s might be due to the differences of season, location and the s p e c i f i c e f f e c t of the material used i n the experiment. These are the 50 f i r s t r e s u l t s reported on the e f f e c t s of A l a r on the anatomy of e i t h e r compact or standard Red D e l i c i o u s . In Golden Delicious (Table 12), the leaves of treated plants had thinner lower epidermis and fewer palisade c e l l s and the treated Stark-spur Golden Delicious had thinner spongy parenchyma, a smaller t o t a l leaf thickness, and fewer palisade c e l l s . Thus c u l t i v a r might i n f -luence the e f f e c t of A l a r treatment. Halfacre (17) also reported that the Golden Delicious and York Imperial responded to A l a r d i f f e r e n t l y when treated at the same concentration. The present study also revealed that suppression of terminal growth by A l a r v a r i e d among the c u l t i v a r s used. I t was found that of the four c u l t i v a r s used i n experiment I I , Starkrimson trees were not a f f e c t e d by A l a r treatment (Table 7, F i g . 7) and Starkspur was the most susceptible to the A l a r treatment (Table 10). Besides differences among c u l t i v a r s , the above r e s u l t s could be explained i f i n c o r r e c t l y labeled material had been used i n the experiment. However, even though the Starkspur trees sampled grew very vigorously before the app-l i c a t i o n of A l a r , they responded to the treatment much d i f f e r e n t l y than the Golden Delicious trees. Neither leaf number, length or width of Red Delicious and Star-krimson were af f e c t e d by A l a r treatment. These findings do not agree with those of Halfacre (16), and the findings need v e r i f i c a t i o n . In Golden Delicious A l a r at 1000 ppm was found to reduce the leaf number by 19 per cent (Table 11), but no e f f e c t was observed on Starkspur. GA stimulated shoot growth of Golden Delicious and Starkspur but when GA was applied i n combination with A l a r , the stimulating e f f e c t was r 51 cancelled completely by A l a r . Edgerton and Hoffman (14) also found that the stimulating e f f e c t of GA on Red Delicious apple trees could be cancelled by A l a r . Since the natural compact mutants have many merits over the stan-dard growing s t r a i n s , i t may be desirable to induce s i m i l a r changes i n standard apple trees or i n t e n s i f y the desirable t r a i t s of the compacts by means of treatment with growth regulators such as A l a r . However, there are only a few workers studying the relation sh ip s among the natural compact mutants and the chemically induced compact habit. One worker, I n t r i e r i (19), reported, upon reviewing the l i t e r a t u r e , that spur type treesrand standard trees treated with A l a r showed many simi-l a r i t i e s i n morphology and anatomy. A l a r treatment increased leaf thickness of Harrold Red Delicious by 9.5 per cent. Apparently the increase i n t o t a l thickness of leaf t i s s u e a f t e r A l a r treatment was due to an increase i n thickness of the palisade parenchyma. Hence i t seems that this t i s s u e i s the primary a c t i v e s i t e within the leaf t i s s u e which responds to the growth r e t a r -dant. A f t e r the treatment with A l a r 4000 ppm, a 16 per cent increase i n thickness of palisade parenchyma was noted. These anatomical f i n -dings help explain the report of Edgerton and Hoffman (14) that A l a r treated apple trees of several c u l t i v a r s had thicker leaves. A l a r i n h i b i t e d the growth rate of Red Delicious apple shoots. This i n h i b i t o r y e f f e c t was immediate and lasted for more than s i x weeks a f t e r the a p p l i c a t i o n of A l a r . When A l a r was applied at concen-trations of 1000 ppm and 4000 ppm, the i n h i b i t i o n s of the shoot growth of Harrold Red Delicious were found to be 18 per cent and 27 per cent 52 r e s p e c t i v e l y . This r e s u l t concurs with the findings of other workers (3, 4, 14, 15, 16, 17) that A l a r i n h i b i t s the terminal growth of apples. I n t e r e s t i n g l y , A l a r did not appear to influence the length of any of the stem c e l l types examined i n the current study. As discussed e a r l i e r , t h i s may have been due to the sampling procedure but i t also may support the findings of Martin jit a l . (24) who found that A l a r had a greater e f f e c t on c e l l d i v i s i o n i n apple f r u i t s than on c e l l s i z e . Untreated Harrold Red Delicious shoots have a tendency to grow continuously. This would r e s u l t i n a t a l l e r tree. However, i n the Starkrimson and A l a r treated Harrold Red Delicious the shoot growth curves are quite s i m i l a r and become l e v e l at about the fourth week. These re s u l t s are consistent withtthe review of I n t r i e r i (19) who con-cluded that standard apple trees treated with A l a r and spur type trees are quite s i m i l a r i n morphology and anatomy. This present study has contributed to the understanding of the e f f e c t s of A l a r on apple trees. Apparently both the nature of the c u l t i v a r and the concentration of A l a r are important factors when considering the use of this growth retardant. SUMMARY A two year study was conducted to investigate the morphological and anatomical changes i n apple leaf and stem t i s s u e s . Factors ana-lyzed were le a f anatomy, leaf number, le a f length and width, shoot anatomy and shoot length. There i s no previous anatomical comparisons of stems between standard and compact apple trees or between Ala r treated and untreated trees of Red D e l i c i o u s . Detailed measurements of spongy parenchyma thickness and numbers of palisade layers i n compact mutants or i n A l a r treated Red D e l i c i o u s are the f i r s t reported. In 1968, two v a r i e t i e s , Red D e l i c i o u s and Golden D e l i c i o u s , each with standard and compact types, were studied without A l a r treatments. In the second phase of the experiment A l a r at the concentrations of 0 and 1000 ppm was applied to a d d i t i o n a l trees of the same c u l t i v a r s . From A p r i l to June 1969, concentrations of 0, 1000 and 4000 ppm of A l a r were applied to c u l t i v a r s which had been grafted on EM II root-stocks . Without Al a r treatment, i t was found that Starkspur had a thicker l e a f and thicker palisade parenchyma than the other three c u l t i v a r s studied and Red D e l i c i o u s was found to have less t o t a l l e a f thickness than Golden D e l i c i o u s . Results also indicated that compact apple mutants had on average thicker leaves and palisade parenchyma and greater mean palisade number as compared with standard types. The compact mutants have been reported to have thicker stems and shorter internode. However, microscopic examination of samples taken 54 from the f i r s t internode under the growing t i p revealed no s i g n i f i c a n t d i f f e r e n c e s i n c e l l length of collenchyma, parenchyma, p i t h c e l l s , c e l l diameter or thickness i n the same t i s s u e s . The suppression of terminal growth by A l a r varied.among c u l t i -vars. The response to A l a r was greatest with Starkspur where an i n -h i b i t i o n of shoot growth by 50 per cent was observed. Starkrimson was not a f f e c t e d by A l a r treatment i n the same experiment. In the t h i r d phase of the experiment, Al a r treated l e a f blades of plants were found to increase i n t o t a l thickness, i n thickness of spongy parenchyma and i n the length of palisade c e l l s . The r e s u l t s from two concentration of A l a r , i . e . 1000 and 4000 ppm, were not found to d i f f e r from each other. These data i n d i c a t e that the s i t e which i s most a f f e c t e d by A l a r i s i n the palisade parenchyma c e l l s of the leaf t i s s u e . GA stimulated the shoot growth of Golden D e l i c i o u s and Starkspur by 29 per cent, but t h i s stimulating e f f e c t was prevented by A l a r . BIBLIOGRAPHY 1. Arasu, N. T. 1968. Spur-Type sports i n Apples. Ann. Rept. E. M a i l i n g Res. Stn. for 1967. 113-119. 2. Ashby, D. L. and N. E. 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(2-chloroethyl) trimethylammonium chloride and re l a t e d compounds as plant growth substances. J . B i o l . Chem. 235:475-479. 37. Westwood, M. N. 1963. Some diff e r e n c e s i n growth, chemical com-p o s i t i o n and maturity between a spur mutant and standard growing D e l i c i o u s apple. Proc. 59th Ann. Meeting Wash. Hort. 58 Assoc. Dec. 2, 3, 4. Wenatehee, Wash. 38. and Q. B. Z i e l i n s k i . 19 66. Comparative growth habit and leaf composition of a compact mutant and standard D e l i c i o u s apple. Proc. Amer. Soc. Hort. S c i . 88:9-13. 39. Wirwille, J. W. and J. W. M i t c h e l l . 1950. Six new plant-growth-i n h i b i t i n g compounds. Bot. Gaz. 111:491-494. 40. Zeevaart, J . A. D. 1966. I n h i b i t i o n of stem growth and flower formation i n P h a r b i t i s n i l with N, N-dimethylaminosuccinamic a c i d (B-995). Planta (Berl.) 71:68-80. Table 1 Line Number APPENDIX Analysis of Variance Models (Table numbers correspond to those i n text) 59 5 6 7 8 9 10 E Source of Variation Clone Trees within Clones Shoots within Trees Leaves within Shoots Po s i t i o n P o s i t i o n x Clone P o s i t i o n x Trees within Clones P o s i t i o n x Shoots within Trees P o s i t i o n x Leaves within Shoots Disc Measurements within Discs Total Degrees of freedom Line number of F denominator (c-1) = 3 c( t - l ) = 8 ct(s- l ) = 12 cts(e-l) = 2<4 p-1 = 2 (p-D(c- l ) = 6 c ( p - l H t - l ) = 16 c t ( p - l X s - l ) = 24 cts(p- l ) ( l - l )= 48 ctspl(d-l) = 144 ctspld(m-l) = 864 ctspldm-1 =1151 Expected mean squares 10 10 o 2 E + 2 o 2 D ( T C S L P ) + 6 ° 2 L ( T C S ) + 1 2 a 2 S ( T C ) + 2 4 o 2 T ( C ) + 7 2 e 2 C ° 2 E + 2 a 2 D ( T C S L P ) + 6° 2L(TCS) +1 2 o 2 S ( T C ) + 2 l* o 2T(C) 2 2 2 2 0 E + 2 ° D(TCSLP) + 6° L ( T C S ) + 1 2 ° S(TC) 2 2 2 0 E + 2 ° D(TCSLP) + 6° L(TCS) 2 2 2 2 2 2 0 E + 2 ° D(TCSLP) + 2° PL(TCS) + 1* a PS(TC) + 8° T P ( C ) + 9 6 6 P. 2 2 + 2o +4o + 8a D(TCSLP) PL(TCS) PS(TC) TP(C) .•24o' PC 2 2 0 E + 2 ° ,+l(o D(TCSLP) ' PL(TCS) PS(TC) 2 2 2 2 0 E + 2 ° D(TCSLP) + 2° PL(TCS) + 4° PS(TC) 2 2 2 ° E + 2 ° D(TCSLP) + 2° PCLCTCS) + 8o TP(C) 2 2 0 E + 2 D(TCSLP) 60 Table 2, 3 Line Number Source of Variation 1 Vari e t y 2 Type 3 Variety x Type 4 Tree with Variety and Type E Measurements with Variety, Type Line number Degrees of of F freedom denominator (v-1) = 1 (t-1) = 1 ( v - D ( t - l ) = 1 v t ( r - l ) = 8 vtr(m-l) =1140 Expected mean squares o 2 E + 9 6 ° 2 R ( V T ) + 5 7 6 6 2 V ° 2 E + 9 6 ( j 2 R ( V T ) + 5 7 6 e 2 T ° 2 E + 9 6 < j 2 R ( V T ) + 2 8 8 ° 2 V T ° 2 E + 9 6 ° 2 R ( V T ) Total vtrm-1 = 1151 61 Table U, 6 Line Number Source of Variation Variety Tree within Variety Shoot within Tree Section within Shoot Measurements within Shoot Total Degrees of freedom (v-1) = 3 v ( t - l ) = 8 v t ( s - l ) = 12 v t s ( c - l ) = 24 vtsc(m-l) = 192 vtscm-1 = 239 Line number of F denominator Expected mean squares ° 2 E + 5 o 2 C ( V T S ) + 1 0 o 2 S ( V T ) + 2 0 o 2 T ( V ) + 6 0 e 2 V a 2 E + 5 o 2 C ( V T S ) + 1 0 o 2 S ( V T ) + 2 0 ° 2 T ( V ) a 2 E + 5 a 2 C ( V T S ) + 1 0 ° 2 S ( V T ) aV 5° 2C<VTS) Table 5, 9 Line Number Source of Variation 1 Variety 2 Tree within Variety 3 Shoot within Tree E Measurements within Shoot T o t a l Line number of F denominator Expected mean squares 2 0 V 3 o 2 S ( V T ) + 6 ° 2 T ( V ) + 1 8 9 2 V 2 2 2 3 0 E + 3 ° S(VT) + 6° T(V) 2 2 E 0 E + 3 ° S(VT> Table 7, 8, 10, 11 Line Number Source of Variation 1 Treatment 2 Shoot within Treatment 3 Date 4 Date x Treatment E Date x Shoot T o t a l 63 . Line number Degrees of of F freedom denominator Expected mean squares (t-1) = 1 2 a 2 E + 8 ° 2 S ( T ) + 4 8 6 2 T t ( s - l ) = 1 0 E ° 2 E + 8 ° 2 S ( T ) (d-1) = 7 E c 2 E + 1 2 6 2 D ( d - D ( t - l ) = 7 E a 2 E + 1 2 a 2 D T t ( d - l M s - l ) = 70 o 2 E tds-1 = 95 Table 12 Line Number Source of Va r i a t i o n Degrees of freedom Treatment (t-1) Shoot within Treatment t ( s - l ) Leaf within Shoot t s ( l - l ) 10 Disc within Leaf t s l ( d - l ) = 100 Section within Disc t s l d ( c - l ) = 120 Measurements within Section tsldc(m-l) -240 Total tsldcm-1 :479 64 Line number of F denominator Expected mean squares  2 o 2 E + 2 ° 2 C ( T S L D ) + 4 o 2 D ( T S L ) + 2 4 o 2 L ( T S ) + 4 8 o 2 S ( T ) + 2 1 , 6 2 T 3 a 2 E + 2 ° 2 C ( T S L D ) + 4 o 2 D ( T S L ) + 2 4 o 2 L ( T S ) + 4 8 o 2 S ( T > 4 ° 2 E + 2 o 2 C ( T S L D ) + 4 o 2 D ( T S L ) + 2 4 ° 2 L ( T S ) 2 2 2 5 0 E + 2 a C(TSLD) + 4° D(TSL) 2 2 E 0 E + 2 ° C(TSLD) 2 0 E Table 13 Line Number Source of Variation Degrees of freedom Line number of F denominator Expected mean squares Treatment Shoot within Treatment Section within Shoot Measurements within Section (t-1) s ( t - l ) s t ( c - l ) stc(m-l) 10 80 aV 5° 2C(TS) + 1 0 a 2 S ( T ) + 5 0 8 2T ° 2 E + 5 o 2 C ( T S ) + 1 0 ° 2 S ( T ) ° 2 E + 5 ° 2 C ( T S ) Total stcm-1 99 66 Table 14 Line Number Source of Variation Treatments Tree within Treatment Leaf within Tree Disc within Leaf Section within Disc Measurement within Section T o t a l Degrees of freedom Line number of F denominator (t-1) = 3 t ( r - l ) = 16 t r ( l - l ) = 20 t r l ( d - l ) = "»0 t r l d ( c - l ) = 80 trldc(m-l) = 160 trldcm-1 = 319 Expected mean squares 2 2 2 2 2 2 0 E + 2 ° S ( T R L D ) + 4 a D(TRL) + 8° L ( T R ) + 1 6 ° R ( T ) + 8 0 6 T 2 4 . 1 2 o r + 2 o E " " S(TRLD) + 4° D(TRL)* o u L(TR) 2 2 2 2 a E + 2 ° S(TRLD) + 4° D(TRL) + 8° L(TR) , + 8o 2 2 . h 2 0 E S(TRLD) 0 D(TRL) + 16o' R(T) 2 . - 2 ° E S(TRLD) 67 Table 15 Line Number Source of Var i a t i o n Treatment Tree within Treatment Date Date x Treatment Date x Tree within Treatment T o t a l Degrees of freedom (t-1) t ( r - l ) d-1 3 24 5 Line number of F denominator 2 E E Expected mean squares ( d - l ) ( t - l ) = 15 t ( d - l ) ( r - l ) = 120 tdr-1 = 167 a V 6 < j 2 R ( T ) + 4 2 9 2 T 2 2 ° E + 6 ° R(T) 2 2 « E + 2 8 ° D 2 2 a E+7o D T 68 Table 16 Line Number Source of Var i a t i o n Block Variety Treatment Varie t y x Treatment Black x Varie t y and Treatment Leaf within Treatment P o s i t i o n within Leaf Section within P o s i t i o n Measurement within Section T o t a l Degrees of freedom Line number of F denominator ( r - l ) = 1 (v-1) = 1 (t-1) = 3 ( v - l X t - 1 ) = 3 ( r - D ( v t - l ) = 7 v t r ( l - l ) = 16 v t r l ( p - l ) = 32 v t r l p ( s - l ) = 64 vtrlps(m-l) = 128 vtrlpsm-1 = 255 Expected mean squares ° 2 E + 2 c 2 S ( V T R L P ) + '* o 2p(VTRL) + 8 (' 2L(VTR) + 1 2 8 o 2 R 2 2 2 2 2 0 E + 2 a S(VTRLP) +"° P(VTRL) + 8° L ( V T R ) + 1 2 8 8 V 2 2 2 2 2 0 E + 2 ° S(VTRLP) +l t a P(VTRL) +8 ° L(VTR) +6 1 4 8 T 2 2 2 2 2 2 0 E + 2 a S(VTRLP) + "*° P(VTRL) + 8° L ( V T R ) + 1 5 ° R V T + 3 2 ° VT 2 2 a ,-+2a v + 4 a + 8o S(VTRLP) ° P(VTRL) u L(VTR) >+16o' RVT 2 2 2 2 0 E + 2 a S(VTRLP) + 4° P(VTRL) + 8° L(VTR) 2 2 2 0 E + 2 ° S(VTRLP)* 1* 0 P(VTRL) 2 2 0 E + 2 ° S(VTRLP) Table 17 Line Number Source of V a r i a t i o n  1 Block 2 Variety 3 Treatment H Variety x Treatment 5 Block x Treatment, Variety 6 Date 7 Date x Varie t y 8 Date x Treatment 9 Date x Treatment x Variety E Date x Block within Treatment Total 69 Line number Degrees of of F freedom denominator Expected mean squares (b-1) = 1 E o 2 E+48o 2 B (v-1) = 1 E o 2 E * U 8 e 2 v (t-1) = 3 E c 2 E + 2 4 8 2 T ( t - D ( v - l ) = 3 5 o 2 +6o 2 „+12o 2 ( b - D ( t v - l ) = 7 E o 2 r.+6o 2 (d-1) = 5 E o 2 r.+16e 2 ( d - D ( v - l ) = 1 5 E o 2 *8o 2 ( d - l X t - l ) = 1 5 E o 2 • t o 2 ( d - l ) ( t - l ) ( v - l ) a l S E o 2 +2o 2 v t ( d - l X b - l ) = HO o 2 vtdb-1 = 95 E T B V T V E + 6 ° T B V E + 1 6 E 2 D E + 8 ° 2 D V E ' ^ D T  2 „ 2 E 1 0 D T V E 

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