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Some observations on germination of Pseudotsuga menziesii (Mirb.) Franco pollen in vitro Ho, Ronghui 1968

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SOME OBSERVATIONS ON GERMINATION OF PSEUDOTSUGA MENZIESII (MIR33.) FRANCO POLLEN IN VITRO fey RONGHUI HO B. A., National Taiwan University, 1963 M. S., National Taiwan University, 1966 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in the Faculty of Forestry We accept this thesis as conforming to the.required standard THE UNIVERSITY OF BRITISH COLUMBIA October, 1968 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e 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 r e f e r e n c e and S t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . It i s u n d e r s t o o d t h a t c o p y i n g o r 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 g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . o f Forestry  The U n i v e r s i t y o f B r i t i s h C o l u m b i a V a n c o u v e r 8, Canada Date October 15. 1968 i i ABSTRACT Pollen morphology, p o l l e n germination, and the e f f e c t s of nutrients during pollen germination of Pseudotsuga  menziesii (Mirb.) were studied. Pollen extraction was done at room temperature and the pollen morphology was studied using a st a i n i n g technique. Several types of substances were introduced to the medium ( d o u b l e - d i s t i l l e d water) to culture the pollen. The substances were boric acid, calcium n i t r a t e , potassium s a l t of g i b b e r e l l i e a c i d , i n d o l e a c e t i c a c i d , indolebutyric a c i d , naphthaleneacetic a c i d , thiamin, water extracts of Douglas-fir seeds, sucrose and stock sol u t i o n . Incubation of p o l l e n grains was c a r r i e d out i n the growth chamber where l i g h t i n t e n s i t y was about 3500 foot-candles during the 12-hour l i g h t period each day and the temperature was maintained at 20°G at night and 25°C during the day time. The r e l a t i v e humidity was kept at about 40 percent. Pollen grains were checked d a i l y . Mature p o l l e n grains were at the two-celled stage with two degenerated p r o t h a l l i a l c e l l s . When dry, the grains were cup-shaped, while tu r g i d grains were spherical or e l l i p t i c a l without any furrows or sacs. The exine was t h i n being about 2 microns, and quite smooth. The i n t i n e was about 8 microns thick and was of uniform hyaline appearance. The pores i n the exine were about 2 microns i n diameter; those i n the i n t i n e were enclosed with a membrane. Boron and i i i calcium ions were very important to the pollen germination and elongation. Pollen germination was stimulated by growth-promoting substances, but was inhibited by fungicide and bactericide. Sucrose solution of 10 to 15 percent was recommended for the osmotic milieu and for nutrient pur-poses. Stock solution (boron 0 . 1 g., calcium nitrate 0 . 3 g., and double-distilled water 100 ml.) i s the best for pollen germination and elongation. Pollen grains cultured in the medium containing stock solution B, 10 ppm IM, and sucrose v/ere found in four-celled stages (tube c e l l , two sperm cells and stalk cell) after five days. The actual germination of the Douglas-fir pollen in vitro was accomplished in this study. These may be of practical value in ensuring a uni-formly high rate of seed production in Douglas-fir seed orchards, but f i e l d studies ( a r t i f i c i a l pollination) are needed to obtain further information. i v ACKNOWLEDGEMENTS The w r i t e r wishes to express h i s s incere a p p r e c i a t i o n to Dr . Oscar S z i k l a i , F a c u l t y of F o r e s t r y , the U n i v e r s i t y of B r i t i s h Columbia, f o r suggesting the problem, superv i s ing the work, h i s va luable advice and o f f e r i n g a l l the f a c i l i t i e s needed f o r the completion of h i s work. Thanks are a lso due to Dr. K. Cole , Department of Botany, the U n i v e r s i t y of B r i t i s h Columbia, f o r c o n t r i b u t i n g her va luable advices and f o r reading the manuscript . Grate fu l acknowledgement i s extended to Dr . E . B. Tregunna, Department of Botany, the U n i v e r s i t y of B r i t i s h Columbia, f o r h i s reading the manuscript . V TABLE OF CONTENTS INTRODUCTION 1 MATERIALS AND METHODS. 4 RESULTS 9 DISCUSSION 33 A. Pollen Size 33 B. Dry and Turgid Pollen Grains 34 C. Effects of Nutrients during Pollen Germination 39 (a) Roles of Boron and Calcium 39 (b) Roles of Growth-promoting Substances 42 (c) Roles of Sucrose 42 (d) Effects of Fungicide and Bactericide ....... 44 (e) Effects of Seed Extracts 46 (f) Effects of Ultraviolet Radiation ... 46 D. Germination and Growth of Pollen Grain 47 SUMMARY 56 REFERENCES 58 INTRODUCTION The development o f male and female gametophyte and f e r t i l i z a t i o n o f P seudot suga m e n z i e s i i M i r b . F r a n c o ( D o u g l a s - f i r ) have been d e s c r i b e d by Lawson (1909) . He gave d e t a i l s o f p o l l e n grown i n v i v o and p r e s e n t e d d iagrams o f the development of g l o b u l a r p o l l e n g r a i n s f rom the t ime o f p o l l i n a t i o n to the t h r e e - n u c l e a t e s tage ( s t a l k c e l l , body c e l l and tube c e l l ) . But the a c t u a l d i v i s i o n o f the body c e l l to two sperm c e l l s was n o t o b s e r v e d ; i t seems t h i s d i v i s i o n took p l a c e b e f o r e the t i p o f the p o l l e n tube r e a c h e d the n u c e l l u s . A c c o r d i n g to A l l e n (1943) , the p o l l e n remains i n the m i c r o p y l e about t h r e e weeks b e f o r e i t g e r m i n a t e s . A f t e r f i v e weeks ' g e r m i n a t i o n , the t i p o f the p o l l e n tube r e a c h e s the n u c e l l u s , and a week l a t e r i t p e n e t r a t e s the a r c h e g o n i u m . F e r t i l i z a t i o n o f the egg n u c l e u s o c c u r s about n i n e weeks a f t e r p o l l i n a t i o n . A l l e n a l s o p r e s e n t e d photographs o f the p o l l e n g r a i n a t the t h r e e -n u c l e a t e s tage but n o t the f o r m a t i o n o f the sperm c e l l s . C h i n g and C h i n g (1959) s t u d i e d e x t r a c t s o f D o u g l a s - f i r p o l l e n and e f f e c t s o f g i b b e r e l l i c a c i d on i t s g e r m i n a t i o n . T o t a l p o l l e n p r o d u c t i o n was h i g h e s t under s t a n d a r d room c o n d i t i o n . Treatment w i t h the p o t a s s i u m s a l t o f g i b b e r e l l i c a c i d caused s i g n i f i c a n t l y more p o l l e n tube growth and i n d i c a t e d the p o s s i b i l i t y o f i n c r e a s i n g seed s e t t i n g by such a t r e a t m e n t . The t h r e e - n u c l e a t e s tage o f the p o l l e n g r a i n o c c u r r e d a f t e r 46 hour i n c u b a t i o n under 100 ppm 2, g i b b e r e l l i c a c i d t r e a t m e n t . The f o u r - n u c l e a t e s tage and f o r m a t i o n o f the p o l l e n tubes o f D o u g l a s - f i r p o l l e n g r a i n s i n v i v o were p r e s e n t e d i n photographs by B a r n e r and C h r i s t i a n s e n (1962) , when the p o l l e n g r a i n comes i n t o c o n t a c t w i t h the n u c e l l u s t o p , the body c e l l d i v i d e s f o r m i n g two male c e l l s . S i m u l t a n e o u s l y , a p o l l e n tube i s formed a t the p o l e o f the p o l l e n g r a i n i n c o n t a c t w i t h the n u c e l l u s . S i n c e the b e g i n n i n g of t h i s c e n t u r y , the q u e s t i o n whether the p o l l e n tubes i n v i v o and i n v i t r o u t i l i z e e x t e r n a l l y s u p p l i e d n u t r i e n t s o r n o t , has a t t r a c t e d much a t t e n t i o n . The growth o f p o l l e n tubes o f gymnosperms i s e x t r e m e l y s low. The p o l l e n g r a i n s o f gymnosperms u s u a l l y have a low v i t a m i n c o n t e n t , whereas those o f angiosperms a re r i c h i n v i t a m i n s o f the B-group (Lunden , 1954) . The i m p o r t a n c e o f a u x i n s , v i t a m i n s and t r a c e e lements on the growth o f p l a n t s and p o l l e n tube e l o n g a t i o n i s now g e n e r a l l y r e c o g n i z e d ( S t i l e s , 1964; S c h o p f e r , 1949; Audus , 1953; L e o p o l d , 1955 ) . However the e v i d e n c e i n r e g a r d to the p o s s i b l e use o f such s u b s t a n c e s i n e n s u r i n g a u n i f o r m l y h i g h r a t e o f p o l l i n a t i o n and f e r t i l i z a t i o n o f D o u g l a s - f i r i n r e p r o d u c t i v e phase appears to he s c a n t y . I t i s , t h e r e f o r e , c o n s i d e r e d n e c e s s a r y to s tudy the g e r m i n a t i o n o f D o u g l a s - f i r p o l l e n and t o de te rmine the type o f s u b s t a n c e s e i t h e r a u x i n s , v i t a m i n s , t r a c e e lements o r a n t i b i o t i c s , t h a t s t i m u l a t e g e r m i n a t i o n and the r a t e o f e l o n g a t i o n o f the p o l l e n tube 3. i n v i t r o . Such stimulation may be of practical value in ensuring a uniformly high rate of f i l l e d seed production i n Douglas-fir seed orchards,. 4. MATERIAL AND METHODS Male cones of Douglas-fir were collected from trees at Caycuse clone bank of B. C. Forest Products Ltd., Vancouver Island on May 2, 1967. Pollen extraction was done at room temperature. The pollen was stored in a v i a l at 0°G to 2°C in the refrigerator. Pollen grains collected from eight trees (Tree N 0. BCFP 25, 37, 44, 133, 135, 166 and 189) were placed i n d i s t i l l e d water for about 2 minutes. 30 grains were mea-sured in random f i e l d under the microscope from each tree. Dry pollen was directly fixed and stained with fast green and observed under the microscope to study pollen shape. Identification of the pollen pore was carried out using basic fuchsin glycerine j e l l y technique (Wodehouse, 1959). In this technique, dry and turgid pollen grains was used. The procedure was as follows: 1. Prepare basic fuchsin glycerine j e l l y according to Wodehouse (1959). 2. Place pollen on the slide. 3. Add one to three drops of 100 per cent alcohol and allow to p a r t i a l l y evaporate. As the alcohol eva-porates, i t spreads out and leaves an o i l ring. 4. Wipe off the o i l ring with cotton 5. 5. Add one drop hot b a s i c f u c h s i n g l y c e r i n e j e l l y and s t i r . 6 . Pass a c o v e r g l a s s t h r o u g h a f lame s e v e r a l t imes and c o v e r the s l i d e . 7 . C o o l ( o r s e a l w i t h l a c q u e r f o r permanence) . P o l l e n g r a i n s c o l l e c t e d f rom t r e e BCFP 189 were chosen f o r the g e r m i n a t i o n t e s t . F i v e k i n d s o f medium were p r e p a r e d as f o l l o w s : 1. A s e r i e s o f s o l u t i o n s were p r e p a r e d : 0 . 0 1 , 0 . 1 , 1, 5, 10 , 100 , and 1000 ppm o f b o r i c a c i d , c a l c i u m n i t r a t e , p o t a s -sium s a l t o f g i b b e r e l l i c a c i d (10$ G A ) , i n d o l e a c e t i c a c i d ( I A A ) , i n d o l e b u t y r i c a c i d ( I B A ) , n a p h t h a l e n e a c e t i c a c i d (NAA) , and t h i a m i n . 2. Water e x t r a c t was p r e p a r e d from seeds from w h i c h the seed c o a t s were p e e l e d . Crushed seeds were b o i l e d i n d i s t i l l e d water f o r h a l f an h o u r . One p r e p a r a t i o n i s one gram p e r 100 m l . o f d i s t i l l e d w a t e r ; the o t h e r 10 gram per 100 m l . o f d i s t i l l e d w a t e r . 3 . Sucrose s o l u t i o n was t e s t e d to the o smot i c m i l i e u o f p o l l e n g r a i n s i n a c o n c e n t r a t i o n s e r i e s 1 1, 5 » 8 , 10 , 15 and 20 p e r c e n t . 4. A s t o c k s o l u t i o n , b a s i c a l l y d e s c r i b e d by Brewbaker and Kwack (1963) , was p r e p a r e d as f o l l o w s : S tock s o l u t i o n A H^BO^ 0 .1 g . C a ( N 0 3 ) 2 . 4 H 2 0 0.3 g . 6. M g S O r 7 H 2 0 0 .2 g . K N 0 5 0 ,1 g . i n d i s t i l l e d water 100 m l . S tock s o l u t i o n B s t o c k s o l u t i o n A 1 m l . d i s t i l l e d water 9 m l . 5 . F u n g i c i d e ( A r a s a n : a c t i v e i n g r e d i e n t - t i r a m " t e t r a m e t h y l -t h i u r a m d i s u l f i d e " , 7 5 $ ; i n e r t i n g r e d i e n t , 2 5 $ ) , and b a c t e r i c i d e ( f o r t i m y c i n ) i n d i f f e r e n t c o n c e n t r a t i o n s The pH o f the c u l t u r e s o l u t i o n was a d j u s t e d to about 7. D e t a i l s o f p r o c e d u r e are as f o l l o w s : 1. There are t h r e e p o s s i b l e methods f o r s t e r i l i z i n g the P e t r i d i s h e s and two c a v i t y s l i d e s : (a) S o a k i n g f o r 12 hour s i n 3N HC1, r i n s i n g 5 t imes i n d i s t i l l e d w a t e r , and t h e n e o v e r . i n g to p r o t e c t from d u s t . (b) W i p i n g w i t h 70$ a l c o h o l . (c ) A u t o c l a v i n g under 15 l b p r e s s u r e f o r 20 m i n u t e s . 2. The e x p e r i m e n t a l s t a n d was wiped w i t h 70$ a l c o h o l . 3. F i l t e r paper was p l a c e d i n the P e t r i d i s h e s and a t h i n l a y e r o f d i s t i l l e d water was added . 4. S l i d e w i t h t w o - c a v i t y was p l a c e d i n the P e t r i d i s h . 5 . Two o r t h r e e d rops o f c u l t u r a l medium were added to the s l i d e c a v i t i e s . were i n t r o d u c e d to the c u l t u r e medium. 7. 6 0 Pollen grains were immediately dusted on the medium. They were not immersed in the medium or covered with a cover s l i p , as free access to a i r i s necessary for germination. 7. A l l above steps were done i n the transfer chamber. 8. Then the Petri dishes were kept in the growth chamber where light intensity was about 3500 foot-candles during the 12-hour light period each day. The temperature was 20°C at night and 25°C during the day time, and relative humidity about 40$. 9. After 24 hours' incubation, pollen grains were picked up with a sterile micro spatula, transferred to the slides and then covered with a cover s l i p . Sometimes fixation and staining were used after a cover s l i p was put on the slide. 10. Pollen grains were measured under the microscope. Over one thousand pollen grains were counted under each experimental condition. F i f t y or more, randomly selected, were measured. From pollination to f e r t i l i z a t i o n , i t takes about nine weeks (Allen 1943). Pollen grains grow very slowly in vivo. In vitro i t should take a long time of culture to get the 4-nucleate stage. The solution for this culture i s prepared from the above information which shows elongation and growth. The culture solution i s combined with stock solution B, 8. 10 ppm IAA and a ser i e s of 5, 8, 10, 15 and 20 per cent sucrose. Dry p o l l e n gra ins were i r r a d i a t e d with u l t r a v i o l e t l i g h t frome one hour to eight hours and then tested f o r germination rate i n stock s o l u t i o n B. A 30 watt u l t r a -v i o l e t l i g h t source was used at a dis tance of 50 cm from the p o l l e n . Incubation and measurement were done under the same condi t ions as mentioned above. When p o l l e n gra ins begin to elongate, pressure i s exerted on the exine. When the exine can no longer sus ta in the t ens ion , i t breaks and i s cast o f f gradua l ly or q u i c k l y . Methods of breakage were observed and counted to e s t a b l i s h which was the more frequent method. •9. RESULTS Measurements of the sizes of tu r g i d pollen from the eight selected trees are summarized i n Table 1. Table 1 Mean and standard deviation of the sizes of turg i d p o l l e n grains from eight selected trees. Tree No. Width (micron) Length (micron) BCFP 25 94.71+ 4.79 119.65+12.35 37 93.99+16.90 146.91+25.25 44 95.70+ 3.48 129.09+11.39 55 95.59+ 4.93 131.92+19.82 133 95.85+ 3.52 128.27+16.31 135 94.40+ 1.91 143.25+16,64 166 91.13+ 4.06 127.79+12.74 189 93.22+ 6.14 105.02+16.05 Average 94.32+ 5.74 128.99+16.32 Pollen ranged from 91.13+4.06 to 95.85+3.52 microns i n width and from 105.02+16.05 to 146.91+25.25 microns i n length. The average of the t o t a l 240 pol l e n grains was 94.32+5.72 by 128.99+16.32 microns. The v a r i a t i o n of each p o l l e n grain i n width and i n length i s from 80.24 to 106.02 microns, and from 82.60 to 194.70 microns, r e s p e c t i v e l y (Pig. 1). Dry p o l l e n grains appeared to be cup shaped (Fi g s . 2, 3). The pol l e n grains are round from the polar view with 10 the nucleus in the middle (Pig. 3). Prom the bottom view, there are two shaded areas around the dry pollen and a light part i s between them. A nucleus can he found i n the middle of the light area. The shaded area i s due to the part of cup-shaped pollen grain shadowed on i t (Pig. 4)» The turgid grains are spherical or e l l i p t i c a l , without a trace of bladders or furrow. The exine i s thin, measuring only about 2 microns i n thickness. The surface i s quite smooth (Pig. 5), with no sculpturing. The intine measures about 8 microns i n thickness and i s of uniform hyaline appearance (Wodehouse, 1959) (Pig. 6). A protuberance at the proximal pole (Pig. 7) where two degenerated p r o t h a l l i a l c e l l s are placed can sometimes be observed. The leptoma (the area with thin exine) at the d i s t a l pole described by Erdtman (1957) was not found in this study. In the basic fuchsin glycerine j e l l y technique only the exine of the pollen grains stains, leaving the intine and c e l l contents uncolored. The pore (Pigs. 8, 19) of about 2 microns can be observed when the focus i s being adjusted. Data obtained from pollen germination i n auxins, vitamins and trace elements are compiled in Table 2. It i s shown that pollen germination i n trace elements, boric acid and calcium nitrate, i s proportional to concentration. Prom " t " Table for significance test, both of them stimulate the pollen germination at 1000 ppm. Boric acid proportionally increases the pollen growth, but calcium nitrate does not 11. s i g n i f i c a n t l y a f f e c t the pollen growth. GA enehances both p o l l e n germination and elongation. IAA, IBA, and NAA increases the p o l l e n germination and elongation i n low concentration, but completely i n h i b i t s both at high concentration, 1000 ppm. Thiamin stimulates the pollen germination to a considerable extent at 10 ppm, but s l i g h t l y i n h i b i t s i t at concentration above 100 ppm. With regard to the e f f e c t of thiamin on pol l e n elongation, i t seems that high concentration (1000 ppm) s t i l l promotes p o l l e n elongation. Seed extracts were used to culture the p o l l e n . Two' concentrations were introduced as media. The r e s u l t s are presented i n Table 3. It i s shown that extracts not only increased the elongation but also increased the p o l l e n germination. Sucrose supplies the pollen grains both nutrients and osmotic m i l i e u . Osmotic^pressure of the medium i s very important during germination. I f i t i s not balanced, the p o l l e n bursts or shrinks. Extent of germination i n sucrose s o l u t i o n i s summarized i n Table 4 and extent of v i a b i l i t y i n Table 5. Sucrose aids p o l l e n germination, but not p o l l e n growth. As to the concentration f o r the culture, i t i s recommended that 15$ i s the best when only sucrose i s used. Pollen i s cultured i n the two stock solutions A and B. Table 6 shows that both solutions give the highest germination percentage and longest elongation of a l l media used. Also stock s o l u t i o n e f f e c t i v e l y prevents pollen grains from bursting or shrinking. F i g . 1. Turgid p o l l e n grains from BCFP 189. (250X). F i g . 2. Dry p o l l e n grains (cup shape). (2001). F i g . 3. Polar view (upper two grains) and side view (lower two) of dry-pol l e n grains. (240X). F i g . 4 . Bottom view of dry pollen grains. (2001). 14 Fig-. 6. (1) thin, exine, about 2 microns (2) thi c k , uniform hyaline i n t i n e , about 8 microns. (3000X). 15 F i g . 7. Protuberance at the proximal pole where one p r o t h a l l i a l c e l l i s placed. (1000X). F i g . 8. Pore, about 2 microns, shown i n the exine of p o l l e n grain. (2000X). 16 Table 2 Per cent of germination and the size (mean and standard deviation) of width and length of elongated pollen grains i n microns. Boric Acid Width Length 0.01 ppm 38.85 85.90+4.21 160.13+16.48 0.1 40.11 86.73+3.98 165.88+16.74 1.0 42.97 86.85+2.63 174.29+15.23 5.0 43.43 88.03+2.12 170.63+17.44 10.0 47.89 85.31+2.54 179.92+16.23 100.0 48.70 87.44+2.46 183.34+15.48 1000.0 40.76 88.13+3.12 177.12+20.31 Calcium Nitrate 0.01 ppm 41.97 87.44+1.43 160.27+14.28 O o l 38.18 88.03+5.24 159.77+12.21 1.0 42.86 88.62+3.29 153.05+18.32 5.0 43.21 85.67+6.71 160.24+17.79 10.0 43.08 86.61+2.18 158.59+17.55 100.0 47.52 86.26+3.52 165.59+16.84 1000.0 39.62 87.36+3.68 179.36+21.49 GA 0.01 ppm 40.32 86.62+2.47 151.39+14.12 0.1 44.94 87.44+2.36 150.09+10.02 1.0 49.03 88.85+4.51 160.01+13.27 5.0 51.34 87.32+3.69 162.84+13.45 17. (Table : 2 continued) GA Width Length 10.0 53.33 89.44+5.01 161.66tl2.09 100.00 50.77 87.56+2.43 153.40+19.83 1000.00 44.79 86.73+2.96 140.03+23.11 IAA 0.01 ppm 45.80 91.33+3.03 167.91+16.49 0.1 47.01 88.62+2.38 164.41+17.56 1.0 55.46 90.51+2.16 154.11+19.94 5.0 46.72 88.03+5.21 168.86+19.33 10.0 49.30 87.67+5.40 157.94+25.83 100.0 46.96 88.06+3.24 151.40+21.14 1000.0 8,03 IBA 0.01 ppm 37.21 87.20+3.45 160.83+14.78 0.1 50.95 88.62+2.93 161.66+14.42 1.0 46.40 90.30+2.89 156.38+15.63 5.0 45.73 86.85+3.56 170.75+26.31 10.0 45.00 89.09+3.74 157.32+20.05 100.0 50.53 89.21+3.80 154.34+19.92 1000.0 4.31 NAA 0.01 ppn 34.74 85.00+6.83 158.86+14.65 0.1 48.76 87.08+2.52 169.33+17.59 1,0 48.95 89.21+3.09 161.37+17.77 5.0 54.14 92.16+2.21 169.21+16.12 18. (Table ; 2 continued) NAA Width Length 10.0 45.04 86.61+7.35 159.89+18.12 100.0 30.58 87.08+5.20 160.95+20.13 1000.0 4.49 Thiamin 0.01 ppm 46.97 88.38+3.70 157.18+15.43 0.1 44.04 90.15+2.98 162.20+16.30 1.0 49.06 88.97+2.69 168.03+16.47 5.0 49.83 88.15+2.34 157.06+19.83 10.0 51.33 88.74+5.21 158.59+21.04 100.0 37.00 86.49+8.27 157.41+21.33 l O O O o O 35.67 87.30+5.36 174.32+24.23 Control 0.00 ppm 38.17 87.30+1.17 150.48+10.58 Table 3 Per cent of germination and the size (mean and standard deviation) of width and length of elongated p o l l e n grains i n microns i n seed extracts. Concentration $ Width Length 1 g./lOO ml. 42.36 86.74+3.77 160.62+25.48 10 g./lOO ml. 45.61 89.20+8.02 167.43+28.55 19 Arasan which i s u s u a l l y used to s t e r i l i z e the seeds was introduced to the medium f o r the pollen germination t e s t . The r e s u l t s are shown i n Tables 7 and 8. In high con-centrations (more than 500 ppm), Arasan i n h i b i t s the germina-t i o n and growth of p o l l e n . The lower concentrations ( l e s s than 500 ppm) of Arasan do not have much e f f e c t on germination and on growth. Stock s o l u t i o n B with Arasan makes no diff e r e n c e to the p o l l e n germination and elongation as compared to stock s o l u t i o n only. Fortimycin (133333 IU) was d i l u t e d to various concentrations f o r ther germination t e s t . The r e s u l t s are shown i n Tables 9 and 10. In low concentrations, i t seems fo r t i m y c i n does enhance the germination and growth of p o l l e n , but high concentration decreases the germination and growth. After 54 hours of incubation, i t i s shown that f o r t i m y c i n i n h i b i t s the growth of p o l l e n . When stock s o l u t i o n B i s com-bined with a low concentration of fortimycin, i t enhances pol l e n germination and growth improvement. Pol l e n grains were incubated i n a s o l u t i o n con-t a i n i n g stock s o l u t i o n B, 10.0 ppm IAA, and a series of 5, 8, 10, 15, 20 per cent sucrose. The r e s u l t s are shown i n Table 11. A f t e r 24 hours 1 incubation, the two c e l l stage could be found: the generative c e l l and the tube c e l l (Pig. 9). After two days, the three c e l l stage, i . e . , the tube c e l l , body c e l l and s t a l k c e l l could be seen i n the elongated pollen where the body c e l l i s between the other 20. two c e l l s . Stalk c e l l i s at the end of the pollen where two degenerated p r o t h a l l i a l cells are cut off (Fig. 10). In five days' incubation, the body c e l l divides to form two sperm c e l l s where two overlap and remain in the original position (Fig. 11). Ultraviolet radiated pollen grains were incubated in stock solution B for the germination test. It seems ultraviolet light (UV) does not give any v i t a l effects under 8 hours 1 irradiation. It i s shown i n Table 12. When the grains are moistened, they swell and begin to germinate; the exines s p l i t open and are subse-quently thrown off gradually or a l l at once. The exine usually s p l i t s i n the following two appearances: 1) the exine separates into two cups, equal or one larger than the other (Fig. 12); 2) the exine s p l i t s wide open (Fig. 13, 14), the cast off exines have a tendency to curl tightly inward. The po s s i b i l i t y of dehiscence in the two ways in 15$ sucrose medium i s compiled as follows: No. of Pollen $ Counted Exine separates in two 777 75.73 Exine s p l i t s wide open 249 24.27 Total 1026 100.00 21. F i g . 9. Two-nucleate stage, ( l ) f i r s t p r o t h a l l i a l c e l l (2) second p r o t h a l l i a l c e l l (3) generative c e l l (4) tube c e l l . (600X). F i g . 10. Three nucleate stage. (1) s t a l k c e l l (2) body c e l l (3) tube c e l l . (250X). 22. Pig. 1 1 - 1 . (200X). Pig. 1 1 . Pour nucleate stage, ( l ) s t a l k c e l l (2) (3) sperm c e l l s . (4 ) tube c e l l (5) f i r s t p r o t h a l l i a l c e l l . ( 6 ) second p r o t h a l l i a l c e l l . (N) nucleus. 23. P i g . 11 - 2 . ( 2 5 0 X ) . 24 F i g . 11-3. (300X). 25 F i g . 12. Exine separates i n two parts. (2501). 26 Table 4 Per cent of germination and the size (mean and standard deviation) of width and length of elongated p o l l e n grains i n microns i n sucrose s o l u t i o n . Concen-t r a t i o n $ Width Length 5$ 57.02 87.67+5.78 145.18+20.53 8$ 56.65 87.79+4.60 146.79+23.24 10$ 57.75 88.21+4.48 145.24+30.90 15$ 56.08 86.33+3.12 152.22+15.22 20$ 63.83 88.25+2.86 150.29+18.17 Average 58.26 87.86+4.28 147.53+23.75 Control 40.56 89.45+6.34 : Table 5 158.35+24.42 Per cent of v i a b i l i t y of p o l l e n grains i n sucrose s o l u t i o n Concen-t r a t i o n 8$ non-germination 43.35 Germination viable bursting shrinkage 24.84 24.29 7.25 10$ 36.49 28.19 25.12 10.09 15$ 43.91 48.87 0.75 6.47 20$ 36.17 52.26 0.35 11.04 27 Table 6 Per cent of germination and the size (mean and standard deviation) of width and length of elongated pollen grains i n microns i n stock s o l u t i o n . Stock Solution Width Length A 59.82 87.67+4.01 183.61+27.84 B 54.08 85.90+6.84 188.68+27.61 Average 56.95 86.78+5.42 186.14+27.73 Control 34.85 87.79+6.31 157.06+14.93 Table 7 Per cent of germination and the size (mean and standard deviation) of width and length of elongated p o l l e n grains i n microns i n Arasan s o l u t i o n . Concen-t r a t i o n Width Length 5 ppm 40.80 85.67+3.41 152.14+23.36 10 35.19 85.90+5.02 158.88+19.47 50 38.29 86.73+4.18 157.47+17.46 100 40.25 88.50+2.25 153.87+17.81 500 36.01 87.33+2.13 147.11+19.47 1000 28.04 88.64+4.32 142.63+16.75 Control 37.96 87.79+6.38 152.37+20.17 28. Table 8 Per cent of germination and the size (mean and standard deviation) of width and length of elongated pollen grains i n microns i n stock s o l u t i o n with Arasan. Concentration * Width Length 1 ppm+stock B 60.59 87.44+2.39 161.07+17.34 5 +stock B 66.47 85.67+4.41 164.46+15.28 10 +stock B 58.94 85.90+4.38 179.12+18.40 50 +stock B 60.10 86.73+4.63 180.18+25.70 100 + s t o c k B 60.20 88.50+3.12 159.30+30.08 Control 39.36 89.04+7.67 157.77+16.52 Table 9 Per cent of germination and the size (mean and standard deviation) of width and length of elongated p o l l e n grains i n microns i n fort i m y c i n s o l u t i o n . Concen-t r a t i o n Width Length 2 4 Hrs 54 Hrs 10.44 ITJ 42.67 86.14+4.15 160.95+17.43 171.34+21.52 20.83 44.95 87.56+2.45 169.21+14.59 170.51+20.13 41.66 43.96 89.44+2.76 156.33+15.48 163.56+23.45 83.33 43.77 88.26+3.51 146.08+16.78 152.78+21.03 166.66 47.30 88.77+1.83 142.93+15.62 155.64+18.48 333.33 37.47 89.56+2.03 142.31+20.43 142.24+15.23 666.66 32.60 88.26+4.21 132.08+19.21 143.83+14.45 1333.33 30.02 88.85+2.53 136.33+19.35 134.93+15.85 2 9 . Table 10 Per cent of germination and the size (mean and standard deviation) of width and length of elongated pollen grains i n microns i n stock s o l u t i o n B with fortimycin. Concen-t r a t i o n $ Width Length 10.44 IU+stock B 59.91 82.84+6.24 192.10+20.36 20.83 +stock B 60.00 84.24+4.19 175.58+14.98 41.66 +stock B 54.96 88.50+2.38 200.60+25.48 83.33 +stoek B 55.25 86.14+5.06 168.27+14.42 166.66 +stoek B 56.95 90.39+2.41 176.06+21.13 Table 11 Per cent of germination and the size (mean and standard deviation) of width and length of elongated p o l l e n grains i n microns i n the following s o l u t i o n . Contents of Solution 5$ sucrose 10 ppm IAA stock B °/o sucrose 10 ppm IAA stock B 10$ sucrose 10 ppm IAA stock B 15$ sucrose 10 ppm IAA stock B 20$ sucrose 10 ppm IAA stock B Control 24 Hours $ Length 67.31 174.99+26.58 65.48 185.99+22.61 63.13 168.74+18.43 66.88 183.59+17.68 69.57 173.39+25.74 38.92 168.49+15.38 48 Hours $ Length 66.54 205.44+19.48 67.69 207.68+27.55 70.56 218.30+23.39 70.86 208.61+29.34 68.74 217.31+20.27 40.95 189.29+20.31 30. The number of cases i n which the exine s p l i t s wide open i s one third of those in which the exine separates in two. There are also two other methods i n the dehiscence of exine but they can seldom be found under the microscope. 1) Two small ends of exine are cut:;off and the remaining ribbon-like exine i s around the middle of the growing pollen (Fig. 15). 2) One end of exine i s cut off and the germinating pollen seems to be squeezed out from the exine (Fig. 16). Sometimes the exine s p l i t s open and gives way to the growing pollen (Fig. 17). Table 12 Per cent of germination and the size (mean and standard deviation) of width and length of elongated pollen grains irradiated by ¥V in microns i n stock solution B. Time of * Irradiation Width Length 1 hr 66.08 86.85+4.60 158.71+15.73 2 57.57 88.15+3.50 157.06+14.15 3 59.89 86.97+4.25 164.59+16.31 4 64.53 86.26+2.58 156.11+15.48 5 61.16 87.56+3.01 157.88+16.73 6 58.33 . 87.56+3.21 167.79+20.18 7 64.31 87.79+2.35 173.97+11.23 8 64.31 85.19+4.12 163.19+14.58 Control 36.62 87.44+6.46 154.95+18.67 F i g . 14. Exine s p l i t s wide open i n the middle of the po l l e n and then the growing pollen tears the exine apart. (250X). F i g . 15. Two ends of exine were cut o f f and one ribbon-l i k e part of exine formed a r i n g around the po l l e n . (200X). 32. Pig. 16. Abortive pollen grains. One end of exine i s cut o f f and pollen grains are squeezed out. (200X). Pig. 17. One end of exine i s cut o f f and the part l e f t s p l i t s open. (250X). 33 DISCUSSION A. P o l l e n Size The p o l l e n grains of the Pseudotsuga species have been described by several authors. The measurements of diameter are summarized i n Table 13. Table 13 The diameter of Pseudotsuga pollen g r a i n s i z e s . Authors Years Species Size (microns) Bunnell 1965 P. menziesii 91.07 Eisenhut 1961 P. menziesii 84.8x81.1 Erdtman 1943 P. menziesii 80x93 S z i k l a i 1964 P. menziesii 91.08-99.19 Van Campo • 1950 P. menziesii 140 Wodehouse 1935 P. mucronata 90-100 Erdtman 1965 P. rhederi 75-115 Ueno 1958 P. japonica 85 Yamazaki 1962 P. japoniea 80-85 The average size reported by Wodehouse (1935) u t i l i z i n g stained preparation was 90 to 100 microns. Van Campo Duplan obtained an average size of 140 microns from f r e s h Douglas-f i r p o llen.. S z i k l a i (1964) reported the diameter of p o l l e n grains varied considerably between four trees investigated, from 91.08 to 99.19 microns. The average diameter calcu-l a t e d from 1000 measurements by Bunnell (1965) was 91.07 microns, ranged from 67.7 to 137.8 microns. Turgid p o l l e n appears spherical or oval, usually an oval. Therefore the 34 measurement of width and length i s necessary. In this study, the average of fresh pollen from 240 grains was 94.32+5.72 by 128.99+16.32 microns ranging from 80.24 to 106.20 microns i n width, and from 82.60 to 194.70 microns in length. The minimum and maximum values of width appeared to be 70.80 and 108.56 microns, respectively. From the above information, the measurement of pollen size varied one from the others. Wodehouse gave the lowest values but Van Campo Duplan, the highest one. The reason Wodehouse got the lowest values was due to use of stained preparation. In this study, i t was found the pollen grains of stained preparation gave values of about 20 microns lower than that of fresh preparation. Van Campo Duplan obtained an average 140 microns in fresh preparation. It i s believed that this i s the average length of pollen grains, since in the present study,"the length of pollen grains of BCFP 37 and 135 i s over 140 microns. B. Dry and Turgid Pollen Grains The dry, l i v i n g pollen grains which were collected at room temperature and stored i n a v i a l at 0°C to 2°C in the refrigerator are relatively small. The exine i s contracted and the furrow appears to make the grain cup-shaped (Fig. 2, 3). The exine of dry pollen grains i s i n network-like appearance (Fig. 18). 35 It i s believed that i n recent investigations the so-called apertures are not really open but are covered by extremely thin parts of exine (Larson and Lewis, 1961). Eisenhut (1961) reported.the presence of an extraordinarily large pore in Douglas-fir pollen and supported his claim with photographs. He described the typical or average pore as being somewhat oval-shaped, 56 by 70 microns in size and 18.6 microns deep. Wodehouse (1959) presented a sche-matic optical cross-section of a Douglas-fir pollen grain without an aperture. In this study Eisenhut's claim was not supported and i t was found that Douglas-fir pollen has pores in the exine. The average pollen size i s 94.32+5.72 by 128.99+16.32 microns and thickness of exine and intine i s about 10 microns. But i t i s impossible that Douglas-fir pollen has an oval-shaped pore, 56 by 70 microns in size, and 18.6 microns deep, almost the same size as pollen. However, the thickness of exine and intine i s about 10 microns. Eisenhut presented a photograph of Douglas-fir pollen in which the pollen grains seem to be in a dry state, the same as in Fig. 3 (the two.lower pollens). The furrow of dry pollen i n cup shape seems to be a pore from the side view. Maybe Eisenhut had mistaken the furrow of dry pollen as his so-called pore. The exine consists of one of the most extraordina-r i l y resistant material known i n this organic world. The more differentiated, thick exine shows various layers and 3 6 . c o n s i s t s of s p o r o l l e n i n , a high-molecular terpene (Zetschke, 1928, 1932). Also the exine channels remain evident as do the c y t o p l a s m a t i c strands through the i n t i n e . These channels are suggested to he the route f o r m a t e r i a l used d u r i n g the development of exine moving from the p r o t o -plasm to the exine (Larson, 1961). In a r e l a t i v e l y dry s t a t e the p o l l e n g r a i n i n v a g i n a t e s i n t o the proximal p a r t pro-ducing a convex or cup shape. However, the t u r g i d g r a i n appears s p h e r i c a l or o v a l . A pore can he seen by u s i n g b a s i c f u c h s i n g l y c e r i n e j e l l y techniques ( F i g . 8, 19). Comparing two apertures i n F i g . 19, we can e a s i l y recognize t h a t one i s the breakage of exine of germination p o l l e n , the other i s the p o l l e n pore. I t was found t h a t Douglas-f i r p o l l e n does not grow by p o l l e n tube but p o l l e n e l o n g a t i o n . So i t seems th a t the pores i n the p o l l e n are the p o i n t s where exines s p l i t . With regard to the pore i n the i n t i n e , I can c o n f i r m Barner and C h r i s t i a n s e n ' s statement (1962). There i s ail aperture at the proximal pole of the p o l l e n g r a i n s ( F i g . 20, 23), where two s m a l l d i s c s - the two degenerated p r o t h a l l i a l c e l l s , a f l a t one and the other concave are found near the pore. I t seems t h a t the o r i f i c e of the pore i s enclosed by a membrane ( F i g . 21). The t u r g i d p o l l e n i s s p h e r i c a l or e l l i p t i c a l , u s u a l l y i n an e l l i p t i c a l shape, which i s f r e e of sacs or 37 19. (1) The breakage o f the e x i n e of g e r m i n a t i n g p o l l e n g r a i n . (2) The pore i n the e x i n e o f p o l l e n g r a i n . ( 3000X) . 3 8 . 39. f u r r o w s . The e x i n e i s t h i n a n d q u i t e s m o o t h , a b o u t 2 m i c r o n s t h i c k . The i n t i n e i s t h i c k a n d i s o f u n i f o r m h y a l i n e a p p e a r a n c e , a b o u t 8 m i c r o n s t h i c k ( P i g . 5 , 6 ) . The c o l o r o f D o u g l a s - f i r p o l l e n i s y e l l o w o r y e l l o w i s h . I t i s c a u s e d b y p i g m e n t m a t t e r l o c a l i z e d i n t h e c a v e s a n d h o l e s o f e x i n e i n t h e f o r m o f o i l a n d f a t , ( P r e y t a g , 1 9 5 9 ) . C h a n g e s i n t h e c o l o r o f t h e o i l d u r i n g s t o r a g e seem t o be a c c o m p a n i e d b y a l o s s o f g e r m i n a t i o n p o w e r ( P f e i f f e r , 1 9 5 5 ) . P r o t e c t i o n a g a i n s t u l t r a v i o l e t r a d i a t i o n i s a f u n c t i o n o f t h i s p i g m e n t ( A s b e e k , 1 9 5 4 ) . C . E f f e c t s o f N u t r i e n t s D u r i n g P o l l e n G e r m i n a t i o n a . R o l e s o f B o r o n a n d C a l c i u m On t h e b a s i s o f p r e s e n t e v i d e n c e , t h e r o l e o f b o r o n i n p o l l e n g e r m i n a t i o n a n d p o l l e n t u b e g r o w t h may be t h r e e -f o l d ( V a s i l , I 9 6 0 ) : " ( a ) i t p r o m o t e s a b s o r p t i o n a n d m e t a b o l i s m o f s u g a r s b y f o r m i n g s u g a r - b o r a t e c o m p l e x e s , ( b ) i t i n c r e a s e s o x y g e n u p t a k e , a n d ( c ) i t i s i n v o l v e d i n t h e s i y n t h e s i s o f p e c t i c m a t e r i a l f r o m t h e w a l l o f t h e a c t i v i t y o f t h e e l o n g a t i n g p o l l e n " . I n t h i s e x p e r i m e n t , b o r o n i n c r e a s e d n o t o n l y t h e g e r m i n a t i o n p e r c e n t a g e b u t a l s o t h e p o l l e n e l o n g a t i o n . I t s t i m u l a t e d t h e g e r m i n a t i o n a n d g r o w t h t o a c o n s i d e r a b l e e x t e n t . The s t i m u l a t i o n o f p o l l e n i s d u e t o t h e p r e s e n c e o f b o r o n w h i c h i n c r e a s e s i t v i a b i l i t y a n d p r e v e n t s b u r s t i n g . I t p l a y s a v e r y i m p o r t a n t r o l e i n t h e m e t a b o l i s m . 40. P o l l e n g r a i n s are low i n calc i u m content, averaging about 0.03$ (Todd and B r e t h e r i c k , 1942). Gymnosperm p o l l e n appears to have lower calcium contents than angiosperm p o l l e n , ranging between 0.03$ and 0.04$ f o r the three s p e c i e s s t u d i e d by Todd and B r e t h r i c k . Calcium p l a y s many important r o l e s i n p l a n t growth to which p e c t i n seems to be the most p e r t i n e n t to the p o l l e n s t u d i e s . Calcium r e l a t e s p r i m a r i l y to the b i n d i n g of calcium i o n to pectate c a r b o x y l groups along the p o l l e n w a l l which i s i n c o r p o r a t e d non-m e t a b o l i c a l l y , i n exchangeable form (Brewbaker and Kwack, 1963). I t was found that the f a c t o r to poor germination and growth i n small p o l l e n p o p u l a t i o n s , but e x c e l l e n t i n l a r g e p o l l e n p o p u l a t i o n s , i s shown to be the calcium i o n s (Brewbaker and Kwack, 1963). Also d e f i c i e n c y of calc i u m may p l a y an important r o l e i n n a t u r a l p o l l e n mutation r a t e s , as w e l l as i n the i n c r e a s i n g r a t e s of mutation d u r i n g prolonged p o l l e n storage (Brewbaker and Kwack, 1963). This experiment shows the p o p u l a t i o n e f f e c t can be overcome by the calc i u m i o n s . Germination and growth of sm a l l p o l l e n p o p u l a t i o n s i n s o l u t i o n w i t h calcium i o n s are s i m i l a r to th a t of the l a r g e p o p u l a t i o n . Also the r e l a t i o n of calcium to the ger-m i n a t i o n r a t e i s determined by i t s c o n c e n t r a t i o n . The hi g h e r the c o n c e n t r a t i o n the b e t t e r the germination, but not the p o l l e n e l o n g a t i o n . The l e n g t h of p o l l e n e l o n g a t i o n i s not a f f e c t e d very much, by the calc i u m c o n c e n t r a t i o n . For p r e v e n t i n g p o l l e n from b u r s t i n g , b o r i c a c i d 41. works more e f f i c i e n t l y than calcium. Calcium improves the g e r m i n a t i o n percentage and e l o n g a t i o n , hut not the v i a b i l i t y . B o r i c a c i d does not e x a c t l y prevent the p o l l e n g r a i n s from b u r s t i n g , but the percentage of b u r s t i n g i n v e r s e l y r e l a t e s to the c o n c e n t r a t i o n . Rapid e l o n g a t i o n and b u r s t i n g at the t i p under osmotic s t r e s s , responds markedly to exogenous c a l c i u m i o n and borate and g i v e s no c o n v i n c i n g response to auxins. Stock s o l u t i o n c o n t a i n i n g b o r i c a c i d , calcium n i t r a t e , magnesium s u l f a t e and potassium n i t r a t e not only i n c r e a s e s the germination percentage and p o l l e n e l o n g a t i o n , but a l s o e f f e c t i v e l y prevents the p o l l e n g r a i n s from b u r s t -i n g . P o l l e n germination percentage i s 56.95 i n stock s o l u t i o n and p o l l e n l e n g t h , 186.14 microns. The balance of osmotic pressure between the cytoplasm i n the p o l l e n and the o u t s i d e medium depends upon stock s o l u t i o n . There are three p o s s i b l e r o l e s t h a t b o r i c and calcium i o n s have pla y e d , (a) the borate could r e a c t w i t h sugar and pass through the c e l l u l a r membranes as the i o n i z e d sugar-borate complexes u n t i l such time as a c e l l u t i l i z e s t h i s complex and l i b e r a t e s the boron i o n , (b) the borate a s s o c i a t e d w i t h the c e l l u l a r membranes r e a c t c h e m i c a l l y w i t h sugar mole-c u l a r f a c i l i t a t i n g i t s passage through the membrane. The sugar i s r e l e a s e d i n s i d e the c e l l by a second r e a c t i o n (G-anch and Duggar, 1953)» (c) b o r i c a c i d ions may p l a y a 42. r o l e t o m e m b r a n e p o r e b y c o n t r o l l i n g t h e w a t e r a n d i o n s i n a n d o u t t h e c e l l u l a r m e m b r a n e . B o r a t e d o e s i n c r e a s e t h e r a t e o f c a l c i u m i o n u p t a k e a n d i n a n e x c h a n g e a b l e f o r m . C a t i o n i c e q u i l i b r i u m i n v o l v i n g c a l c i u m i s r e q u i s i t e t o t h e n o r m a l p e r m e a b i l i t y a n d s e l e c t i v i t y p r o p e r t i e s . I n a d d i t i o n , t h e p r o b l e m o f p o p u l a t i o n e f f e c t d o e s n o t e x i s t i n t h e s t o c k s o l u t i o n . b . R o l e s o f G r o w t h - P r o m o t i n g S u b s t a n c e s P o l l e n g e r m i n a t i o n w a s s t i m u l a t e d b y g r o w t h - p r o m o t -i n g s u b s t a n c e s , b u t p o l l e n e l o n g a t i o n w a s n o t m a r k e d l y a c c e l e r a t e d . T h e g e r m i n a t i o n p e r c e n t a g e w a s d i r e c t l y r e l a t e d t o t h e c o n c e n t r a t i o n o f G A , I A A , I B A , NAA a n d t h i a m i n u p t o a c e r t a i n c o n c e n t r a t i o n a f t e r w h i c h a d e c r e a s e b e c a m e e v i d e n t . T h e c o n c e n t r a t i o n o f GA a n d t h i a m i n a t 1000.0 ppm g a v e a l i t t l e i n h i b i t i o n t o t h e p o l l e n g e r m i n a t i o n , w h i l e I A A , I B A a n d NAA c o m p l e t e l y i n h i b i t e d t h e g e r m i n a t i o n o f p o l l e n . A f e w p o l l e n g r a i n s g e r m i n a t e d b u t b u r s t a f t e r w a r d s . No v i a b l e p o l l e n c o u l d be m e a s u r e d . G r o w t h - p r o m o t i n g s u b -s t a n c e s s t i m u l a t e d t h e g e r m i n a t i o n a n d i m p r o v e d t h e g r o w t h , b u t c o u l d n o t p r e v e n t t h e p o l l e n f r o m b u r s t i n g . T h e v i a b i l i t y o f p o l l e n i s n o t h i g h . c . R o l e s o f S u c r o s e P o l l e n g r a i n s d o n o t c o n t a i n c h l o r o p h y l l a n d a r e d e p e n d e n t o n i n t e r n a l a n d e x t e r n a l s o u r c e s f o r t h e s u p p l y o f e s s e n t i a l n u t r i e n t s . T h e y o n l y c o n t a i n some r e s e r v e f o o d 43. which i s u t i l i z e d during the i n i t i a l stages of germina-t i o n , but i s not enough f o r continued growth. The reserve food may support growth u n t i l i t i s consumed. However, i n the culture medium, the nutri e n t s stimulate and improve the germination and elongation of pol l e n grains. When pol l e n grains of Douglas-fir were cultured i n deionized water f o r 24 hours, the germination percentage was 40.56 and the size was 89.45+6.34 by 154.35+24.42 microns. .If some sucrose was added, the average germina-t i o n percentage increased to 58.26 and the size decreased to 87.86+4.28 by 147.53+23.75 microns. Even though the range of pol l e n length i n the sucrose medium was lower than the c o n t r o l , the germination percentage of the former was rather higher than that of the l a t t e r . The exter n a l l y supplied sucrose i n the medium improved the germination of po l l e n . P o l len grains are cultured i n a series of sucrose medium 8, 10, 15 and 20 per cent f o r v i a b i l i t y t e s t . I t was found that 15 percent sucrose was the best f o r Douglas-f i r p o l l e n f o r eithe r nutrient purposes or osmotic m i l i e u . When the concentration of sucrose was over 15 percent, the cytoplasm i n the elongated p o l l e n shrank and was misshapen (Pig. 21). However when the concentration was below 15 percent the osmotic pressure of medium was so low that the elongated pollen hburstd a f t e r germination (Pig. 22). I t served as a nutrient material f o r the growing po l l e n . 44. Apart from the nutrient purpose, i t creates favourable osmotic condition f o r germination and growth. Using p o l l e n grains, incubated i n stock s o l u t i o n plus 10 ppm IAA and a s e r i e s of sucrose f o r two days, i t was found that solutions containing 15 percent sucrose were optional f o r germination, growth and osmotic m i l i e u . The osmotic role was performed by sucrose, stock s o l u t i o n , and IAA. A f t e r one-day culture i n stock s o l u t i o n plus 10 ppm IAA and 15 percent sucrose, the germination percentage was 66.88, and length 183.59+17.68 microns; two-day culture, the extent of germination was 70.96 percent and length, 208.62+ 29.34 microns. Bursting i n 10 percent sucrose and shrinkage i n 20 percent sucrose solutions were higher than those i n 15 percent sucrose s o l u t i o n . Ching and Ghing (1959) germinated Douglas-fir p o l l e n on a basic medium containing 10 percent sucrose and one percent agar. He got a high germination percentage of 88.4 and growth, 209.09 microns i n length f o r 46 hours. Therefore, 10 to 15 percent sucrose w i l l be recommended to make pol l e n suspension s o l u t i o n f o r a r t i f i c i a l p o l l i n a t i o n . d. E f f e c t s of Fungicide and Bactericide Arasan was tested i n t h i s experiment. In low concentration, Arasan did not i n h i b i t the germination and growth of poll e n , but could not keep the p o l l e n from burst-ing. In high concentration, Arasan i n h i b i t s not only the germination percentage but the elongation of po l l e n . When 45 Arasan combined w i t h s tock s o l u t i o n B , i t had no e f f e c t on p o l l e n g e r m i n a t i o n and g rowth . Johnson (1963) r e p o r t e d t ha t l a b s t u d i e s showed tha t v i a b i l i t y decreased w i t h i n c r e a s i n g c o n c e n t r a t i o n of i n s e c t i c i d e (G-uthion and Sevin) and d u r a t i o n of immers ion . A s e r i e s of f o r t i m y c i n was t e s t e d i n p o l l e n g e r -m i n a t i o n . C o n c e n t r a t i o n h i g h e r than 333.33 IU caused the decrease of p o l l e n g e r m i n a t i o n and e l o n g a t i o n . Concen t ra -t i o n under 166.66 IU seemed to enhance b o t h . T h i s may be due to f o r t i m y c i n c o n t a i n i n g p e n i c i l l i n which i s con tami -na ted w i t h o ther c h e m i c a l s . I t i s known tha t s t r e t o m y c i n , contaminated w i t h p h e n y l a c e t i c a c i d i n the medium, improves the g e r m i n a t i o n and growth of p o l l e n ( P u l v e r t a f t , 1946; V a s i l , 1958, I960; Sen, I 9 6 0 ) . D o u g l a s - f i r p o l l e n grows v e r y s l o w l y . As a r e s u l t we face the con tamina t ion of f u n g i and b a c t e r i a i n l o n g -term c u l t i v a t i o n i n v i t r o . A l l of the s t e r i l i z e d ins t rument s became contaminated . T h i s i s due to the p o l l e n i t s e l f . The bes t way to o b t a i n g e r m i n a t i n g p o l l e n wi thou t m i c r o b i a l con tamina t ion i s by the method d e s c r i b e d by P e t r u et a l . (1964) , or to add f u n g i c i d e and b a c t e r i c i d e to c u l t u r e medium to prevent c o n t a m i n a t i o n . B u t , low c o n c e n t r a t i o n of f u n g i -c i d e and b a c t e r i c i d e d i d not prevent con tamina t ion of mic ro -o rgan i sm and b u r s t i n g of p o l l e n . In h i g h c o n c e n t r a t i o n i t i n h i b i t e d the g e r m i n a t i o n and growth . Use of f u n g i c i d e and b a c t e r i c i d e i n c u l t u r e medium i n p h y s i o l o g i c a l exper iments r e q u i r i n g l ong - t e rm c u l t i v a t i o n of p o l l e n under 46. a r t i f i c i a l conditions i s not recommended. e. E f f e c t s of Seed Extracts Water extraction of seed raised the germination percentage and growth. However, i t did not show any considerable improvement but had the same e f f e c t s of boron and other growth-promoting substances. The e f f e c t of extracts from one gram of dry seeds per 100 ml. of water were not much d i f f e r e n t from that of 10 gram per 100 ml. of water on germination and elongation. The l a t t e r did r e t a i n the balance of osmotic pressure i n and out of the p o l l e n cytoplasm and kept the p o l l e n from bursting. f . E f f e c t s of U l t r a v i o l e t Radiation It i s known that u l t r a v i o l e t r a d i a t i o n f o r as l i t t l e as 8 hours i s s u f f i c i e n t to cause an important decrease of germination capacity (Asbeck, 1954, 1955). It can be shown that a r e l a t i v e change i n the color of p o l l e n takes place depending on the i n t e n s i t y of u l t r a v i o l e t r a d i a t i o n . The pigment protects against u l t r a v i o l e t r a d i a t i o n . According to the chemical nature of the pigment, t h i s f i l t e r works eithe r by chromatic absorption alone or with fluorescence (Asbeck, 1954, 1955). Douglas-fir p o l l e n was i r r a d i a t e d by 30 watt u l t r a v i o l e t l i g h t at a distance of 50 cm. f o r as long as 8 hours and then incubated i n the stock s o l u t i o n B. It gave the same r e s u l t s as the pollen without i r r a d i a t i o n . It seems that the pigment i n exine serves 47. quite well as u l t r a v i o l e t f i l t e r D. Germination and Growth of Pollen Grain Dry p o l l e n stained with "basic fuchsin glycerine j e l l y can r e a d i l y be detected i n that some have one degenerated p r o t h a l l i a l c e l l l y i n g close to the spore, and the other about to divide (Pig. 9, 23). Some have no degenerated p r o t h a l l i a l c e l l with the d i v i d i n g one. This gives evidence that a f t e r the reduction d i v i s i o n of the p o l l e n mother c e l l and the formation of four pollen grains, another d i v i s i o n begins and gives the f i r s t and second degenerated p r o t h a l l i a l c e l l . Lawson (1909) found that two degenerated p r o t h a l l i a l c e l l s were recognizable within the p o l l e n . A l l e n (1943) observed no d i v i s i o n of the p r o t h a l l i a l c e l l s i n the Douglas-fir po l l e n . In t h i s study Lawson's claim was supported that two degenerated c e l l s were detected. After the po l l e n becomes tu r g i d , growth begins. The exines become tense f i r s t and begin to break. Eisenhut (1961) described one way of dehiscence of pollen exine s i m i l a r to Larix and gave the diagram of the castoff exine. In t h i s study, the dehiscence of exine s p l i t s i n four ways. Separation of exine i n two (Pig. 12) i s the more frequent method; two halves are on the ends of po l l e n grains or thrown o f f gradually or at once. The second method i s that the exine s p l i t s wide open on one end (Pig. 13) or i n the middle of the po l l e n g r a i n and the exine i s not cast o f f , 48. the elongating p o l l e n g r a i n w i l l tear the exine apart and leave a ribbonlike exine across i t (Pig. 14). The t h i r d method i s seldom found. The two ends of the exine are cut o f f and one ribbon-like exine i s l e f t around the middle of the germinating p o l l e n (Pig. 15). The l a s t method shows one t i p of exine cut o f f and the germinating po l l e n seems to be squeezed out of the remaining exine (Pig. 16). Pollen which germinates i n t h i s way i s i n v i a b l e . The reason i s that the exine mechanically i n h i b i t s the growth of pollen. Sometimes the remaining exine s p l i t s open and follows the second method (Pig. 17). Following the s p l i t of exine, the f i r s t and second p r o t h a l l i a l c e l l s were divided from the po l l e n (Pig. 23). The f i r s t p r o t h a l l i a l c e l l was cast o f f immediately, the second one gradually. Sometimes the second p r o t h a l l i a l c e l l stocks to the pollen g r a i n and does not leave the elongating p o l l e n . At t h i s stage the po l l e n c e l l has divided to form the tube c e l l and the generative c e l l (Pig. 9). The length of elongated po l l e n i s about 160 microns. The diameter of the generative c e l l i s about 45 microns, and that of the tube nucleus, about 13 microns under carmine st a i n i n g . The tube nucleus i s at the opposite end to the abortive p r o t h a l l i a l c e l l s . After two days, three c e l l s (Pig. 10) i . e . , tube c e l l , body c e l l and s t a l k c e l l , can be found i n the elon-gated pollen. The generative c e l l has divided to form two d i s t i n c t c e l l s , presumably the body c e l l and the s t a l k c e l l . 50. The body c e l l i s between the tube c e l l and the stalk c e l l . The body c e l l i s l a r g e r than the stalk c e l l . The tube c e l l i s considerably l a r g e r than these two c e l l s at t h i s stage. The diameter of the stalk nucleus i s almost the same as the tube nucleus. In f i v e days, i t r e s u l t s i n the organization of four c e l l s , i . e . , tube c e l l , two sperm c e l l s , and stalk c e l l , within the elongated pollen from the culture medium of stock solution..B, 10 ppm IAA and sucrose ( F i g . 11). Each c e l l i s completely surrounded by a t h i n but sharply defined c e l l membrane. Two sperm c e l l s appear to be i n the elongating Douglas-fir po l l e n , but not two n u c l e i which happens i n some gymnosperm species. At t h i s stage, the tube c e l l occupies almost two t h i r d s of the elongated pollen. The two sperm c e l l s are roughly the same s i z e , but the stalk c e l l i s f a r smaller than the other three c e l l s . The two sperm c e l l s and the stalk c e l l remain i n t h e i r o r i g i n a l p o s i t i o n s while the tube c e l l descends with the t i p of the growing p o l l e n . In carmine sta i n i n g , the whole sperm c e l l absorbs the s t a i n , but only the n u c l e i of the s t a l k c e l l and the tube c e l l absorb the s t a i n . It was observed that the rupture of the exine and the protrusions of the elongating p o l l e n always takes place at the side of the pollen grain d i r e c t l y opposite to that occupied by the two degenerated p r o t h a l l i a l c e l l s , i . e . , on the side nearest the tube nucleus. A protuberance at the proximal pole ( F i g . 7) where two degenerated p r o t h a l l i a l ( 51. c e l l s are placed can sometimes be observed. The t h i c k n e s s of the exine of the protuberant part i s the same as the other p a r t . Erdtman (1957) des c r i b e d t h a t a protuberance was at the d i s t a l pole w i t h t e n u i t a s ( c a l l e d leptoma which i s the t h i n sporoderm area, f u n c t i o n i n g as an a p e r t u r e ) . This was not confirmed i n t h i s study. When the p o l l e n tube emerges or p o l l e n elongates from the p o l l e n i t grows by a d d i t i o n of w a l l m a t e r i a l at i t s apex (Schoch-Bodmer, 1945). The p o l l e n tube w a l l c o n t a i n s c e l l u l o s e and i s c u t i n i z e d (Frey-Wyssling, 1959). During the e l o n g a t i o n of p o l l e n g r a i n , no e x t e n s i o n of the w i d t h of p o l l e n could be observed. Even though the p o l l e n g r a i n s have elongated to 400 microns, there i s no growth i n width of p o l l e n g r a i n . I t remains i n the same s t a t e as when the p o l l e n begins to germinate. F i g u r e 15 shows tha t the two ends of the exine are cut o f f and one r i b b o n - l i k e exine i s l e f t around the middle of the p o l l e n . As the growth of the p o l l e n c o ntinues, no t e n s i o n i s exerted on the e l o n g a t i n g p o l l e n g r a i n by the remaining r i b b o n - l i k e exine. P rotoplasmic streaming was not seen i n the elongated p o l l e n of D o u g l a s - f i r . In gymnosperms, where the growth of the p o l l e n tube i s extremely slow, the streaming of the cytoplasm has never been desmonstrated (Takeuchi, 1953). In e o n t r a g t , the p o l l e n tubes of most angiosperms show r a p i d p r otoplasmic streaming; the r a t e of protoplasmic streaming v a r i e s from p l a n t to planti.and i s d i r e c t l y p r o p o r t i o n a l to 52. the rate of elongation of the tube ( Y a s i l , 1958, I960). During penetration of the nucellus t i s s u e , the body-c e l l divides to form the male n u c l e i or c e l l s of unequal s i z e . F e r t i l i z a t i o n of the egg nucleus occurs about nine weeks a f t e r p o l l i n a t i o n ( A l l e n , 1943). The actual d i v i s i o n of the body c e l l was not observed, but a f t e r a study of the chromatic and the cytoplasm immediately surrounding the nucleus, i t seemed quite c e r t a i n that t h i s d i v i s i o n takes place before the t i p of the pollen tube reaches the nucellus (Lawson, 1909). Ching and Ching (1959) i n t h e i r study on e x t r a c t i o n of Douglas-fir pollen and e f f e c t s of GA on i t s germination presented a picture of p o l l e n grain containing three n u c l e i - the tube c e l l , the body c e l l and the s t a l k c e l l - with 100 ppm GA treatment a f t e r 46 hours' germination. At t h i s stage no p o l l e n tube protrusion i s shown but there i s the elongation of p o l l e n . No one gives any d e t a i l s of the d i v i s i o n of body c e l l to two sperm c e l l s . Lawson (1909) observed there was an e a r l y d i s i n t e g r a t i o n of the tissue of the apex of the nucellus correlated with the p o s i t i o n of p o l l e n tubes i n Pseudotsuga. The c e l l s i n t h i s region of the nucellus separate from one another i n places, and there appears to be a general breaking-down or d i s s o l v i n g of the t i s s u e i n advance of the descending p o l l e n tubes. These l a t t e r structures, therefore f i n d no obstructions i n t h e i r path. There i s no f i r m tissue to penetrate before reaching the archegonial chambers. At the time of f e r t i l i z a t i o n , the 5 3 . apex of the nucellus i s completely broken down, and i t takes an appearance very unlike that of Pinus and the majority of other Coniferales where the nucellus p e r s i s t s f o r some time of f e r t i l i z a t i o n . A l l e n (1946) stated that nothing substantiated Lawson's account of a breakdown of the nuclear apex (the nucellus top) i n advance of the pol l e n tube; apart from the mucilaginous l a y e r on the nucellus top, the tissue i s normal. Barner and Christiansen (1962) confirmed Allen's f' statement and reported the pollen was deposited on the nucellus top. Wherever the pollen grains came into contact with the nucellus top, the body c e l l divided forming two male c e l l s . The one nearest the d i s t a l pole was smaller than the other. Simultaneously, a p o l l e n tube i s formed e i t h e r from the membrane of the body c e l l or from an inner layer of i n t i n e . The p o l l e n tube i s always formed at the top of the pollen g r a i n which i s i n contact with the nucellus top. In t h i s study i n v i t r o , the four-nucleate stage was obtained but pol l e n tube protrusion was not observed, only the elongated pollen. Although Barner and Christiansen (1962) found actual germination of the p o l l e n i n v i t r o was unattainable i t was accomplished i n t h i s study. Barner and Christiansen (1962) report that the pol l e n tube i s always formed at the top of the pollen grain which i s i n contact with the nucellus top (either d i s t a l or proximal pole). In t h i s study the protrusion of the elon-54. gating p o l l e n always takes place at the side of the po l l e n g r a i n d i r e c t l y opposite to that occupied by the two pro-t h a l l i a l c e l l s ( d i s t a l pole). I f the proximal pole were i n contact with the nucellus top, how could the pollen tube be formed at t h i s pole? I would suggest that the d i s t a l pole of the elongated p o l l e n might c u r l down into the nucellus when the proximal pole was i n contact with the nucellus top. This i s due to chemotropism between the d i s t a l pole and the nucellus. In v i t r o the c u r l i n g , elongated p o l l e n ( F i g . 10-b) can sometimes be observed. The development of po l l e n germination of angiosperms i s d i f f e r e n t from that of gymnosperms. In angiosperms, the pol l e n tube comes through the pore. In gymnosperms, the germinating p o l l e n breaks the exine and protrudes the tube ( ? ) . P o l l e n grains of some species (Douglas-fir, larch) discard t h e i r exine, some (pine, spruce, hemlock etc.) do not. The germinated Douglas-fir p o l l e n grains discarded t h e i r exine, and the elongated pollen was cigar-shaped. It seems that pollen produces no tube but a protrusion. I f the germinated pine p o l l e n cast o f f t h e i r exine, should we c a l l i t a pollen tube or a protrusion? We c a l l the elongated protrusion of pine p o l l e n a pol l e n tube. Then we c a l l the elongated part of Douglas-fir pollen a pollen tube, but a c t u a l l y i t i s an elongated p o l l e n g r a i n (not a pollen tube). In Barner and Christiansen's report (1962), the photographs showed that the pol l e n tube was a thinner part of the d i s t a l 55. pole. Figure 11-2 showed t h e i r so-called p o l l e n tube. Laboratory studies showed that development of Douglas-fir p o l l e n from two-nucleate stage to four-nucleate stage could be stimulated by trace elements and auxins. The actual germination of the p o l l e n i n v i t r o i s possible. Also the above information t e l l s us that stock s o l u t i o n i s the best f o r p o l l e n germination and elongation. It may be of p r a c t i c a l value i n ensuring a uniformly high rate of seed production i n Douglas-fir seed orchards, but i t needs f i e l d studies ( a r t i f i c i a l p o l l i n a t i o n ) to get further information. 56. SUMMARY A study was made of the p o l l e n morphology and the p o l l e n germination of Pseudotsuga menziesii (Mirb.) Franco (Douglas-fir) i n v i t r o , and the e f f e c t s of nutri e n t s during p o l l e n germination. 1. Po l l e n was extracted at room temperature. The width and length of p o l l e n grains were measured under the microscope. 2. Dry pollen i s cup shaped. Turgid pollen i s spherical or oval, u s u a l l y oval. 3. The exine i s t h i n and smooth; the i n t i n e i s thick and of uniform hyaline appearance. 4. Basic fuchsin glycerine j e l l y techniques were used to i d e n t i f y the pore i n the pol l e n . Pores i n exine and i n t i n e are v i s i b l e . 5. Several types of substances were introduced to the medium to culture the pol l e n . It was found that boric acid and calcium ions were very important to the po l l e n germina-t i o n and elongation. Pollen germination was stimulated by growth-substances, but pollen elongation was not markedly accelerated. 1000.0 ppm of IAA, IBA, and NAA completely i n h i b i t e d the germination and growth of p o l l e n . Fungicide and bactericide improved the germination and growth of po l l e n i n low concentration, but not i n high concentration. 57 6. 10 to 15 percent of sucrose i s recommended to be used f o r the osmotic m i l i e u and n u t r i e n t . 7. U l t r a v i o l e t r a d i a t i o n f o r 8 hours has not any e f f e c t s on p o l l e n germination i n stock s o l u t i o n . 8. Breakage of the exine can occur by four methods. The exine separated i n two. The exine s p l i t wide open. The two ends of exine were cut o f f and one ribb o n - l i k e exine l e f t around the pollen. The l a s t method with one t i p of exine cut o f f and the germinating pollen seemed to be squeezed out of the remaining exine. 9. Pollen grains cultured i n stock s o l u t i o n , 10 ppm IAA, and sucrose f o r one day were found i n two nucleate stage (tube c e l l , generative c e l l ) with two degenerated pro-t h a l l i a l c e l l s . The three nucleate stage (tube c e l l , body c e l l and s t a l k c e l l ) could be found i n two days' incubation. In f i v e days, the body c e l l had divided to form two sperm c e l l s . Therefore actual germination of the p o l l e n i n v i t r o proved a t t a i n a b l e . 58 REFERENCES 1. A l l e n , G. S. 1943. The embryogeny of Pseudotsuga  t a x i f o l i a . Am. J . Bot. 30:655-661. 2. 1946. Embryogeny and development of the a p i c a l meristems of Pseudotsuga. 1. F e r t i l i z a t i o n and e a r l y embryogeny. Am. J. Bot. 33:666-667. 3. Asbeck, F. 1954a. Sonnenlicht und Biogenese.Strahlen-therapie. 93:602-609-4. 1954h Proc. Intern. Photobiol. Congr., B i o l . Sect. 328-31. 5. 1955. Fluoreszierender Blutenstaub. Naturwiss. 42:632. 6. Audus, L.J. 1953. Plant growth substances. Leonard H i l l . p. 169-170. 7. Barner, H. and Christiansen, H. 1962. The formation of pol l e n , the p o l l i n a t i o n mechanism, and the determination of the most favorable time f o r c o n t r o l l e d p o l l i n a t i o n i n Pseudotsuga menziesii. Silvae Genet. Band 11 (4):89-102. 8. Brewbaker, J . L. and Emery, G. C. 1961. Pollen radio-botany. Radiation Bot. 1:101-154. 9. and Kwack, B. H. 1963. The e s s e n t i a l r o l e of calcium ions i n p o l l e n germination and pollen tube growth. Am. J. Bot. 50:859-65. 10. Bunnell, F. L. 1965. V a r i a t i o n i n po l l e n morphology of selected conifers native to B. C. (B. S. F. Thesis). U. B. C. 11. Ching, K. K. and T. M. Ching. 1959. Extrac t i n g Douglas-f i r p o l l e n and e f f e c t s of GA on i t s germination. For. Scie. 5(l):74-80. 12. E b e l l , J . P. and R. L. Schmidt. 1964. Meteorological f a c t o r s a f f e c t i n g c o n i f e r p o l l e n d i s p e r s a l on Vancouver Island. Can. Dept of For. p u b l i c a t i o n . No. 106, 28 pp. tt 13. Eisenhut, G. 1961. Untersuchungen uber die Morphologie und Skologie der Pollenkorner heimischer und fremlandis-cher Waldbaume. Forstwissenschaftliche und Forschungen. Heft 15:68. 59. 14. Erdtman, G. 1957. Pollen and spore morphology and plant taxonomy. Almqvist and W i k s e l l , Stockholm, Sweden, & N. W. p. 40. 15. 1943. An introduction to p o l l e n a n a l y s i s . Chronica Botanica Co., Waltham, Mass. U.S.A. p. 142. 16. Preytag, K. 1959. Z. Bot. 47:113-20. 17. Prey-Wyssling, A. 1959. Die P f l a n z l i c h e Zellwand. B e r l i n e , Springer-Verlag. p. 85. 18. Ganch, H. G. and Dugger, W. M. J r . 1953. The r o l e i n the t r a n s l o c a t i o n of sucrose. Plant P h s i o l . 28:457-466. 19. Johnson, N. E. 1963. Compatibility of p o l l e n with i n s e c t i c i d e s used i n c o n t r o l l i n g insects i n Douglas-f i r cones. For. Res. Note Weyerhaeuser Co., C e n t r a l i a , Wash. No. 52, 10 pp. 20. Larson, D. A. and Lewis, C. W. 1961. Pine structure of Parkinsonia aculeata pollen. 1. The pollen w a l l . Am. J. Bot. 48:934-43. 21. Lawson, A. A. 1909. The gametophytes and embryos of Pseudotsuga d o u g l a s i i . Ann. Bot. 23:163-180. 22. Leopold, A. C. 1955. Auxins and plant growth. Univ. of C a l i f , press, chap. 6. 2 3 . Lunden, R. 1954. A short introduction to the l i t e r a t u r e of p o l l e n chemistry. Svensk Kern. Tidskr. 66:201-213. 24. Petru, E., Hrabetova, E., and Tupy, J . 1964. The techniques of obtaining germinating p o l l e n without microbial contamination. B i o l . Plantarm (Praha) 6 (l):68-69. 25. P f e i f f e r , N. E. 1955. Contrib. Boyce Thompson Inst., 18:153-58. 26. P u l v e r t a f t , R. J . V. 1946. E f f e c t of a n t i s e p t i c s on the germination of pollen grains. Nature (London) 157:301-302. JI 27. SchochrrBodmer, H. 1945. Uber das Spitzenwachstum der Pollenschlanche. Schovliz. Bot. G e s e l l . Ber. 55:154-168. 28. Schopfer, W. H. 1943. Plant and Vitamins. Chronica Botanica Co., Waltham, Mass., U. S. A. 60. 29. Sen, S. K. I960. On the e f f e c t s of a n t i b i o t i c on the rate of pollen tube growth i n Qorchorus o l i t o r r i u s L, s t r a i n G., i n v i t r o . Indian Agr. 4:59-62. 30. S t i l e s , W. 1946. Trace elements i n plants and animals. Cambridge Univ. Press, p.113-114. 31. S z i k l a i , 0. 1964. V a r i a t i o n of inheritance of some ph y s i o l o g i c a l and morphological t r a i t s i n Pseudotsuga  menziesii (Mirb.) Franco var. menziesii. Ph.D. th e s i s . U. 1. C. 32. Takeuchi, M. 1953. Studies on the germination of the po l l e n grains.,conifers 1. Jan. J . Bot. 14:13-21. 33. Todd, F. E. and Bretherick, 0. 1942. The composition of pollens. J. Econ. Entomol. 35:312-317. 34. Tulecke, W. 1959. The pollen culture of C. D. LaRue: a tissue from the pol l e n of Taxus. B u l l . Torrey Bot. Club 85:283-289. 35. Ueno, J. 1958. Some palynologieal observations of Pinaceae. J . Inst. Polytechn. Osaka City Univ., Ser. D. 9:163-177. 36. Van Campo Duplan, M. 1950. Recherches sur l a Phylogenie des Abietienees d'apres leurs grains de pol l e n . Trav. Lab. For. de Toulouse, t. 11, IV, Art 1:9-37. V a s i l , I. K. 1958. The c u l t i v a t i o n of excised anthers and the culture and storage of po l l e n grains. Ph. D. Thesis, Delhi Univ., India. Proc. Delhi Univ. Seminar Modem Developments i n Plant P h y s i o l . 123-126. 38. V a s i l , I. K. I960. Studies on pol l e n germination of c e r t a i n Cucurbitaceae. Am. J. Bot. 47:239-247. 39. Wodehouse, R. P. 1959. Pollen Grains. Hafner Pub. Co., N. Y. p. 266. 40. Yamazaki, T. and Takeoka, M. 1962. Electron-Microscope Investigations of the f i n e d e t a i l s of the pol l e n g r a i n surface i n Japanese gymnosperms. Grana Palynol. 3 (2):3-12. 41. Zetsehke, F. and Huggler, K. 1928, Justus L e i b i g s . Ann. Chem. 461:86-108. 42 

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