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Some factors affecting pollen viability in a tomato breeding program. Charles, Winston Benson 1962

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SOME FACTORS AFFECTING POLLEN VIABILITY IN A TOMATO BREEDING PROGRAM. by WINSTON BENSON CHARLES A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF'SCIENCE IN AGRICULTURE i n the Department of H o r t i c u l t u r e We a c c e p t t h i s t h e s i s as conforming to the standard r e q u i r e d from candidates f o r the degree of MASTER OF SCIENCE IN AGRICULTURE THE UNIVERSITY OF BRITISH COLUMBIA November, 1962. I n 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 a n a d v a n c e d d e g r e e a t t h e 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 m a k e 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 a n d 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 m a y b e g r a n t e d b y t h e H e a d o f m y D e p a r t m e n t o r b y h i s r e p r e s e n t a t i v e s . I t 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 b e a l l o w e d w i t h o u t m y w r i t t e n p e r m i s s i o n . D e p a r t m e n t o f -P&&?s&t cc^^Ct-c^, . T h e 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, C a n a d a . D a t e ABSTRACT In the tomato-growing areas i n southern Canada, i t i s d e s i r a b l e to have commercial v a r i e t i e s having the c h a r a c t e r of being a b l e to s e t f r u i t a t r e l a t i v e l y c o o l temperatures. Puck a non-commercial v a r i e t y s e t s f r u i t a t temperatures between 55° and 65°F. Experiments attempting to i d e n t i f y the mechanism of the d e s i r a b l e c h a r a c t e r of Puck i n terms of p o l l e n v i a b i l i t y were done. The tomato v a r i e t i e s , Puck, Bonny Best, E a r l i a n a ^ 9 8 , and t h e i r r e c i p r o c a l c r o s s e s , -were grown both i n the f i e l d and i n greenhouses. V a r i o u s f a c t o r s a f f e c t i n g v a r i a b i l i t y i n p o l l e n samples and p o l l e n v i a b i l i t y d e t e r m i n a t i o n s , were s t u d i e d . E xperimental r e s u l t s i n d i c a t e d t h a t a sample of p o l l e n taken a t a n t h e s i s c o n s i s t s of mature, immature, and empty g r a i n s . The r e l a t i v e p r o p o r t i o n s of the three c l a s s e s of g r a i n s depended upon the method of c o l l e c t i o n used, the time of c o l l e c t i o n and the l o c a t i o n of the p o l l e n source on the p l a n t . The g r e a t e r the v a r i a b i l i t y i n the sample, the l a r g e r i s the r e p r e s e n t a t i v e sample s i z e r e q u i r e d f o r m i c r o s c o p i c examination a t a cnosen degree of t o l e r a n c e . The s t a i n i n g technique gave the h i g h e s t percentages of v i a b l e p o l l e n . Low v i a b i l i t y percentages -were obtained i n v i t r o , and these are a t t r i b u t e d to b u r s t i n g of some of the normal mature g r a i n s d u r i n g p o l l e n tube i n i t i a t i o n . A q u a n t i t a t i v e i n v i v o procedure of counting p o l l e n tubes by c a l -l o s e f l u o r e s c e n c e under u l t r a v i o l e t l i g h t , was developed. Var-i a t i o n s i n the i n v i v o r e s u l t s can be a s c r i b e d to v a r i a t i o n i n i i the c o n c e n t r a t i o n of p o l l e n growth f a c t o r (PGF) -which appeared to vary w i t h the number and d e n s i t y of g r a i n s used i n p o l l i n -a t i o n s . R e s u l t s showed i n c r e a s e d germination percentages when the number of g r a i n s used was i n c r e a s e d . The most s u i t a b l e number and d e n s i t y f o r maximum group e f f e c t has not been i n v e s t -i g a t e d . The v a r i e t y Puck gave higher v i a b i l i t y percentages than Bonny Best i n most cases. F u r t h e r i n v e s t i g a t i o n s of the problem would r e q u i r e more p r e c i s e c o n t r o l of environmental f a c t o r s d u r i n g p o l l e n development, and exposure of p o l l e n f o l l o w i n g a n t h e s i s . i i i ACKNOWLEDGEMENTS The w r i t e r wishes to express h i s thanks to Dr. C A . Hornby, A s s o c i a t e P r o f e s s o r of H o r t i c u l t u r e and Chairman of- the t h e s i s Committee, f o r s u p e r v i s i o n of the experimental work and f o r h i s v a l u a b l e a s s i s t a n c e i n the p r e p a r a t i o n of the t h e s i s . Acknowledgement i s giv e n to the other members of the t h e s i s Committee; Dean B.A. E a g l e s , Dr. V.C. B r i n k , P r o f e s s o r of Agron-omy and Chairman of the D i v i s i o n of P l a n t S c i e n c e ; Dr. G.H. H a r r i s , P r o f e s s o r of H o r t i c u l t u r e ; Dr. N.A. Mac Lean, A s s i s t a n t P r o f e s s o r of P l a n t Pathology; Dr. A.J.Renney, A s s o c i a t e P r o f e s s o r of Agron-omy, and Dr. G.E. Rouse, I n s t r u c t o r , Department of B i o l o g y and Botany, f o r t h e i r i n t e r e s t and concern w i t h the p r o j e c t and p r e p a r a t i o n of the t h e s i s . I t i s wished to o f f e r s p e c i a l t h i n k s to Dr. D.P. Ormrod, A s s i s t a n t P r o f e s s o r of Agronomy, and member of the t h e s i s Com-mit t e e f o r h i s i n v a l u a b l e help i n the s t a t i s t i c a l a n a l y s e s used i n e v a l u a t i n g data d u r i n g the p r e p a r a t i o n of the manuscript. The w r i t e r i s indebted f o r f i n a n c i a l a s s i s t a n c e d u r i n g the course of the work, to the D i v i s i o n of P l a n t S c i e n c e . v i i TABLE OF CONTENTS. Page INTRODUCTION 1 LITERATURE REVIEW 3 MATERIALS AND METHODS A. M a t e r i a l s 16 B. Methods 18 RESULTS I. M o d i f i c a t i o n of the s t a i n i n g procedure 39 I I , Experiments on sampling (a) Sample s i z e . . . . ho Experiment 1 (b) Methods of p o l l e n c o l l e c t i o n h6 ( i ) Methods of p o l l e n e x t r a c t i o n *+6 Experiment 1.... *+6 Experiment 2.., *+6 Experiment 3 • 9^ Experiment h. 50 ( i i ) Time of p o l l e n c o l l e c t i o n i n r e l a t -i o n to age of flo w e r 51 (1) P o l l e n m a t u r i t y a t d i f f e r e n t bud and f l o w e r stages 51 Experiment 5 51 (2) P o l l e n v i a b i l i t y as a f f e c t e d by l o n g e v i t y of the flo w e r 51 Experiment 6 51 (§) V a r i a t i o n due to d i f f e r e n c e s i n l o c a t i o n of p o l l e n sources on p l a n t s 5*+ i v . Page ( i ) P o l l e n v i a b i l i t y a t d i f f e r e n t zones of the same stamen 5^ Experiment 1 5*+ Experiment 2... . 5*+ ( i i ) P o l l e n v i a b i l i t y of d i f f e r e n t stamens of the same f l o w e r 58 Experiment 3 58 ( i i i ) P o l l e n v i a b i l i t y of d i f f e r e n t f l o w e r s on the same t r u s s . . . . . . . . . . . . . . . . . . . 58 Experiment ^ e o o o . . . . o o o » 0 . . . o . . . a o o « . 6 . » . 58 ( i v ) P o l l e n v i a b i l i t y of d i f f e r e n t t r u s s e s on the same p l a n t 60 III.Comparison of methods of p o l l e n v i a b i l i t y d e t e r m i n a t i o n s . . 62 (a) Comparison of s t a i n i n g and i n v i t r o Experiment 1 63 (b) Experiments on i n v i v o germination 63 Experiment 1. 63 Experiment 2<,.... 65 Experiment 3 66 E x p e r i m e n t s 67 Experiment 5 68 Experiment 6............ • 69 Experiment 7 70 (c) Comparison of the s t a i n i n g , i n v i t r o and i n v i v o techniques used i n p o l l e n v i a b i l -V P a g e Experiment 1 71 DISCUSSION 72 SUMMARY AND CONCLUSIONS 79 LITERATURE CITED .' 83 INTRODUCTION It i s desirable to have commercial varieties of tomato that w i l l set f r u i t under the relatively cool temperatures which prevail in the early spring and occasionally in the early summer months in Canada. Existing commercial varieties, when grown under these conditions frequently f a i l to set f r u i t on the f i r s t and sometimes second trusses, resulting in a reduction in potent-i a l yield of early f r u i t . Early fruit-set in tomatoes grown in Canada i s important for at least three reasons? 1. The f r u i t which sets in early May and throughout June brings the highest price on the early market. 2. In most of the northern tomato growing regions, the f i r s t k i l l i n g frost often occurs early enough that a significant portion of the tomato crop i s not harvested (Kerr, 1955? and Seelig, 1956). Early fruit-set would allow time for the ripening of a greater bulk of f r u i t s before the f i r s t k i l l i n g frost. 3. Early fruit-set would enable the extension of commercial tomato production into areas where the short season i s the li m i t -ing factor at present. The non-commercial variety Puck was found to have the charac-ter of setting f r u i t in England, Oregon and British Columbia, at temperatures 55° - 65°F. which are too low for commercial tomato production, (Crane, 19^8, Heydecker, 19*4-8, Frazier, 1951, and Daubeny, 1955). Fluctuating temperature conditions in the f i e l d make i t impossible to rely on identification of the low temperature -2-character for any given plant. Thus the breeding program requires a controlled environment to ensure identification of the desired germ plasm. In previous work, some evidence has indicated a correlation between the percentage of good pollen and the early fruit-setting character of Puck (Daubeny, 1955). If such a correlation could be established in terms of pollen v i a b i l i t y , i t would permit the rapid evaluation in greenhouses or growth chambers of crosses and breeding lines possessing the desired characteristic. This would reduce the time required to bring plants to the fruiting stage. Also, this procedure would permit the handling of larger populations for making selections, and this would hasten the breeding program for originating a new, true-breeding variety. Daubeny (1955) and Guccione (1959) working with Bonny Best and Puck tomato pollen, reported results which showed tremendous variab i l i t y in pollen v i a b i l i t y among samples taken from each of the two varieties. Hence, further investigation of the causes which may have been responsible for the variab-i l i t y in pollen, has remained desirable. In the present investigation, experiments were designed to study methods of sampling pollen along with a comparative study of the techniques used in determining pollen v i a b i l i t y . The objective was to evaluate the suitability of the methods for use in the identification of the desired low-temperature character of Puck variety and in segregating populations. -3-LITERATURE REVIEW The review of literature i s reported under the following three parts: (1) sampling (2) methods used in pollen v i a b i l i t y determination (3) use of pollen as a means of plant selection. 1. Sampling. It is surprising to find that so l i t t l e attention has been paid to sampling procedures and techniques used in making pollen counts in the literature dealing with pollen studies. Several reports dealing with pollen v i a b i l i t y and abortion, have expres-sed percentages without stating the number of grains examined. Even where mentioned, the precise method of extracting samples, along with the method of selection of a microscopic f i e l d for obtaining a random sample, are not reported in detail. (a) Microscopic f i e l d . Few reports have mentioned how the spread of pollen grains on the slide affect the microscopic f i e l d from which counts are made. Oberle and Goertzen (1952) used a haemacytometer for making counts in f r u i t varieties. Marks (195*0 stated that empty grains being lighter tend to move towards the edges of the coverslip when i t is lowered so that a random distribution of viable and non-viable grains i s not obtained. (b) Sample size. It is necessary to know the minimum sample size that is required to give a true estimate of the pollen v i a b i l i t y of a sample. Among the s e v e r a l i n v e s t i g a t o r s who have expressed percent-ages o f v i a b l e p o l l e n without s t a t i n g the sample s i z e used a re Sandsten (1908), Nebel (1939), Hough (1939) working w i t h p o l l e n from d i f f e r e n t f r u i t v a r i e t i e s , and Poole (1932) working w i t h C r e n l s p o l l e n . The c h o i c e of sample s i z e appears to be a r b i t r a r i l y chosen i n the f o l l o w i n g r e p o r t s , except i n the case o f F l o r y and Tomes (19^3). Becker (1932) working w i t h Prunus used 3,000 to 8,000 p o l l e n g r a i n s f o r most plum h y b r i d s but o n l y hOO and 500 i n c e r t a i n cases. Jaranowski (1961) used s e v e r a l thousand p o l l e n g r a i n s i n a M e l i l o t u s i n t e r s p e c i f i c h y b r i d to determine the percentage of normal p o l l e n . F l o r y and Tomes (19^3) r e p o r t e d from a s t a t i s -t i c a l t e s t f o r homogeneity t h a t 1,000 g r a i n samples from w e l l -mixed p o l l e n from many f l o w e r s on the same p l a n t of f r u i t t r e e s were s u f f i c i e n t l y l a r g e to g i v e a r e l i a b l e v a r i e t a l index. Oberle and Goertzen (1952) evaluated the number of g r a i n s per anther by suspending the contents of 100 anthers i n 2,5 ml. s o l u t i o n c o n t a i n i n g 5 grams of calgomite to 100 ml. of water. Hence i t was assumed t h a t the contents of each anther were sus-pended i n 0,025 ml, o f s o l u t i o n , and one of the h corner squares of the c o u n t i n g chamber occupied 16 areas each 0,25 m i l l i m e t e r square. Smith (19*4-2) used 370-379 g r a i n s i n p o l l e n of A n t i r r h i n u m and over 1,000 g r a i n s i n p o l l e n of Brypphyllum to base h i s germination percentages. He emphasized t h a t s i z e of p o l l e n g r a i n s determines the number of g r a i n s t h a t would be presen t - 5 -i n the microscopic f i e l d . Daubeny (1955) working with tomato pollen used three samples of 100 grains each, to determine per-centages. Guccione (1959) working also with tomato pollen based percentages on samples of 200 grains. Dorsey (191*0 used 200 grains to determine percentages i n each grape variety, and f i v e years l a t e r , without stating any reason f o r his choice, based counts on l e s s than 100 grains of several plum v a r i e t i e s . Martin (1913) used 107-209 grains i n Tr i f o l i u m pratense and calculated v i a b i l i t y percentages on the number of pollen grains that germ-inated. V a s i l (I960 a) used f o r each germination t e s t on Cucur-bitaceae. random counts of 100-200 grains ( i n groups of 25 or more from d i f f e r e n t microscopic f i e l d s on the same sl i d e ) i n order to determine the percentage germination. King (i960) working with tomato and potato pollen stated that " v i a b i l i t y percentages were taken from an average of at l e a s t 100 grains counted i n each of two areas i n a pollen culture"• ? (c) Method of c o l l e c t i n g pollen. ( i ) Method of extraction:- Rarely does the l i t e r a t u r e state how pollen samples are extracted from flowers. Among the several workers who have f a i l e d to state precisely how pollen samples were a c t u a l l y removed from flowers, are Sands-ten (1908), Becker (1932), Judkins (1939), Nebel (1939), Hough (1939), Smith (19*+2), Oberle and Watson (1953), Kurtz and L i v e r -man (1958), and V a s i l (I960 a ) . Martin (1913) working with Tr i f o l i u m pratense reported that the keel of the flower was sprung with a s c a l p e l , and as the pollen was thrown from the anthers, i t was collected on the i n s -- 6 -trument and spread on the medium. ' Anthony and Harlan (1920) dusted barley pollen from already dehisced anthers, on a s l i d e , Poole (1932) working with Crepis species removed 6-8 new f l o r e t s d a i l y with a pair of forceps and placed them i n a drop of aceto-carmine into which the grains immediately settled of t h e i r own accord. The f l o r e t s were then removed and the drop sealed. Daubeny (1955) and Guccione (1959) extracted tomato pollen from f r e s h l y dehisced anthers by means of a dissecting needle. Brew-baker and Majumber (1959) transferred small samples of Petunia pollen with a dissecting needle d i r e c t l y from dehiscing anthers to hanging drops of 0,01 ml. stock solution (10$ sucrose + 100 ppm, H3BO3 i n deionized d i s t i l l e d water). y ( i i ) Time of c o l l e c t i o n . Several workers used anthesis as a guide f o r c o l l e c t i n g pollen samples. Sisa (1932) observed that pollen grains germin-ated more r e a d i l y the day preceding anthesis and l e s s r e a d i l y a f t e r noon on the day of anthesis i n Cucurblta species. Judkins (1939)» without stating the reason f o r his choice, carried out p o l l i n a t i o n f o r i n vivo studies i n the tomato i n the early a f t e r -noon. F l o r y and Tomes (19*+3) gathered buds just p r i o r to anthesis when working with plum pollen. V a s i l (i960 a ) working with Cucur-b i t a pollen collected pollen samples soon a f t e r dehiscence of the anthers early i n the morning, and observed that c o l l e c t i o n s made on a warm day, even a few hours a f t e r dehiscence, became desic-cated and l o s t v i a b i l i t y . Jaranowski (1961) working with the i n t e r s p e c i f i c hybrid Melllotus palonica X M. alba collected sam-ples from recently dehisced anthers. - 7 -(d) Variability of pollen at different sources. As early as l8*+9 and 1869, Schleiden and Van Tieghem respec-tively reported that in vitro germination results varied from species to species and also from variety to variety. Valleau (1918) observed flower-to-flower variations between five straw-berry flowers from a single plant which had 9.9 to 25.5 percent of aborted pollen. Further investigation showed that the percen-tage of pollen abortion varied from 17.09 to 50 in nine different anthers of a single flower. Brink (1921+ b) reported that pollen from different flowers or even from different anthers of a flower gave variable results and suggested that differences in exposure to light may be responsible. Smith (19h2) also noted anther-to-anther pollen variation in the same flower of Antirrhinum and Bryophyllum. Oberle and Goertzen (1952) reported that the number of grains per anther in f r u i t varieties appears to vary from year to year for the same variety of f r u i t , and attributed this variation to fluctuating environmental or nutritional conditions which exist during blossom development. Jaranowski (1961) also reported flower-to-flower variation in the percentage of normal grains taken from the same Melilotus hybrid. Kubo (195*0, and Kurtz and Liverman (1958) also reported flower-to-flower variation and anther-to-anther variation in tomato and cocklebur. Wanscher (19*H) reported on the effect of flower position on pollen abortion in plums and showed that the percentages of stainable grains were highest in flowers at the base of the twigs, the values being gradually lower and usually reaching zero at the twig tips. - 8 -2. Methods used i n p o l l e n v i a b i l i t y d e t e r m i n a t i o n s . I t i s apparent from the l i t e r a t u r e t h a t great v a r i a b i l i t y e x i s t s among the three methods used i n p o l l e n v i a b i l i t y d e t e r -m i n a t i o n s . The standard methods which have been used f o r determining p o l l e n v i a b i l i t y a r e : (a) s t a i n i n g - examination of p o l l e n u s i n g s t a i n to show d i f f e r -ences w i t h i n sample, (b) i n v i t r o - the germination of p o l l e n on a r t i f i c i a l media, (c) i n v i v o - the germination of p o l l e n i n n a t u r a l s t a t e on f l o w e r p i s t i l . (a) S t a i n i n g . The s t a i n i n g procedures have been questioned as a v a l i d measure of p o l l e n v i a b i l i t y . J o h r i and V a s i l (1961) s t a t e d t h a t the commonest and most r e l i a b l e methods a r e the i n v i t r o and i n v i v o . The major o b j e c t i o n to the s t a i n i n g method i s t h a t the s t a i n i n g c a p a c i t y of the c ontents of the p o l l e n g r a i n s may not be i n d i c a t i v e of v i a b i l i t y . L e i t n e r (1938) r e p o r t e d t h a t p o l l e n of L o l l u m perenne taken from herbarium m a t e r i a l s t a i n e d normally a l t h o u g h known to l o s e v i a b i l i t y one day a f t e r a n t h e s i s . S e v e r a l workers c o n s i d e r both the i o d i n e and acetocarmine methods which a r e i n d i c a t i v e of the presence o f s t a r c h to be c o r -r e l a t e d w i t h p o l l e n v i a b i l i t y . Daubeny (1955), and Guccione (1959) used i o d i n e - p o t a s s i u m i o d i d e and acetocarmine f o r determining p o l l e n v i a b i l i t y i n tomato. Marks (195*+) used acetocarmine j e l -l y f o r e s t i m a t i n g p o l l e n v i a b i l i t y of Splanum sp. B a j p a i and L a i (1958), a l s o considered p o l l e n of f r u i t t r e e s and v e g e t a b l e s - 9 -to be viable as long as they are stainable with acetocarmine. King (I960) used the peroxidase method and reported r e l i a b l e r e s u l t s from the t e s t . This method i s dependent on the oxidation of benzidine by peroxidase (present i n the pollen grain) i n the presence of hydrogen peroxide. Ostapenko (1956) used the acetocarmine and peroxidase methods f o r determining v i a b i l i t y of pollen i n apple, peach, apri c o t , plum and pear and regarded the methods as unreliable i n determining pollen v i a b i l i t y . Mattson et a l (19l+,7) reported on the use of triphenyl t e t r a -zolium chloride (T.Z.) as an indicator of v i a b i l i t y . In the pre-sence of viable tissue the colourless solution of 2,3,5 - triphenyl tetrazolium chloride forms the insoluble red triphenyl formazan. V i e i t z e v (1952) using triphenyl tetrazolium chloride f o r testing pollen v i a b i l i t y reported that the method i s only a p p l i c -able where the pollen possesses a t h i n and colourless exine. Ober-l e and Watson (1953) working on pollen v i a b i l i t y of f r u i t trees compared t h i s method of determination with the i n v i t r o method and found that a l l v a r i e t i e s showed a higher percentage of viable pollen with the tetrazolium s a l t . I t was further observed that pollen samples heated at 80°C. f o r 15 minutes gave negative i n v i t r o t e s t , but p o s i t i v e T.Z. reaction, but when heated at 110°C. both tests gave negative r e s u l t s . The r e s u l t s suggested that temperatures over 100°C. were necessary to i n a c t i v a t e the enzyme. I t was then concluded that the 2,3,5 - triphenyl tetrazolium chloride i s of l i t t l e value as an indicator of pollen germinab-i l i t y f o r c e r t a i n f r u i t trees. - 10 -There seems to be some c o n t r o v e r s y over the nature o f the food r e s e r v e s i n p o l l e n g r a i n s a t f u n c t i o n a l m a t u r i t y . Maheshwari (1950) working on angiosperm p o l l e n r e p o r t e d t h a t , as f u n c t i o n a l p o l l e n g r a i n s matured they sy n t h e s i z e d s t a r c h . Daubeny (1955)» and Guccione (1959) working on Maheshwari 1s a s -sumption regarded Puck and Bonny Best tomato p o l l e n to be v i a b l e i only when they were s t a i n e d a dark c o l o u r by i o d i n e - p o t a s s i u m i o d i d e s o l u t i o n . L e s l e y and L e s l e y (1939) used d i l u t e i o d i n e -potassium i o d i d e s o l u t i o n to s t a i n tomato p o l l e n and concluded from the i n t e n s i t y of s t a i n i n g t h a t s t a r c h i s hydrolysed to sugars as f u n c t i o n a l g r a i n s matured. Iwanami (1959) r e p o r t e d on the disappearance o f s t a r c h i n p o l l e n g r a i n s j u s t p r i o r to dehiscence of the anthers and considered the disappearance of s t a r c h to be due to i t s c o n v e r s i o n i n t o sugars. (b) In v i t r o germination. The l i t e r a t u r e on the i n v i t r o technique i s so e x t e n s i v e t h a t no attempt i s made to g i v e a complete review, s i n c e t h a t work has been done by J o h r i and V a s i l (1961). C o n f l i c t s o f o p i n i o n e x i s t on the r e l i a b i l i t y of the i n v i t r o d e t e r m i n a t i o n of p o l l e n v i a b i l i t y . Howlett (1936) r e p o r -ted t h a t tomato p o l l e n g r a i n s t h a t appeared normal i n shape and e x t e r n a l appearance d i d n o t always germinate i n v i t r o . King (1955) r e p o r t e d cases when stored potato p o l l e n d i d not germinate i n v i t r o but produced s a t i s f a c t o r y f r u i t s e t i n v i v o . He s t a t e d f u r t h e r t h a t f a i l u r e to get i n v i t r o germination i s n o t ne c e s s a r -i l y an i n d i c a t i o n of dead p o l l e n , thus the method i s not a r e l -i a b l e p r e p o l l i n a t i o n estimate o f the potency of p o l l e n i n the f i e l d . - 11 -Several suggestions have been made with respect to the failure of stored pollen to germinate in vitro. Johri and Vasil (1961) stated that the loss of certain v i t a l systems during storage are later compensated by the stigmatic tissue at the time of germination. Nebel and Ruttle (1937) believed that desiccation, and u t i l i z a t i o n of reserve food and inactivation of the enzymes may cause failure of the metabolic processes responsible for germination. On the contrary there are cases where stored pollen gave a moderate in vitro germination but a correspondingly poor f r u i t set in vivo (Sandsten, 1908; and Knowlton, 1922). Medium Guccione (1959) reported on choice of medium for germin-ation of tomato pollen, and confirmed the common procedure of using 10$ sucrose solution containing 30 ppm. of boron. Temperature Temperature effects on stored pollen have been observed in the in vitro procedure. The pollen of several species remains viable for longer periods at lower temperatures than at higher (Sandsten, 1908). Knowlton (1922) stored the pollen of Antirrhinum at five temperatures and concluded that the lower the temperature the longer the v i a b i l i t y . Sexsmith and Fryer (19*+3) found that a linear relation-ship existed between pollen tube growth and temperature in Medlcago sativa. The correlation between temperature and pollen germination in apple was found to be represented by an optimum curve (Roberts and Struckmeyer, 19*+8; Visser, 1955)» and maximum tube length - 12 -i s obtained between 20° and 30°C. in pollen of V i t i s vlnifera (Winkler, 1926) and Irish potato, (King and Johnston, 1958). At sub-optimal temperatures satisfactory tube lengths can be obtained only i f the period of growth i s not a limiting factor. Adams (1916) reported 90-100 per cent germination in apple at lH-°C. after 2h hours. Ostlind (19^5) observed only 10-36 per cent germination in apple after 69 hours at 2°C, but there was a marked increase to 90-100 per cent after 119 hours. Smith (19^2) reported that the diameter of the pollen tubes in Antirrhinum ma .jus and Bryophyllum d a e g r emon t ianum increases with an increase in temperature. Sutton and Wilcox (1912) found that the optimum temperature required for tomato pollen germination was 93°F« Guccione (1959) found that the optimum temperature required for the in vitro germination of tomato varieties of Puck and Bonny Best pollen was 20°C. Irrespective of temperature the growth curve of the tube remains sigmoid (Vasil, 1958, aj Vasil and Bose, 1959). Bursting A drawback to the in vitro procedure i s the bursting of pollen grains and tubes after placement in the media. Brink (192*f a) commented on the role of sugar as an osm-otic agent and stated that "A source of no l i t t l e annoyance and perplexity to those who have attempted to cultivate pollen a r t i f i c i a l l y has been the bursting of pollen and pollen tubes". There are several conflicting reports on the cause of bursting. Van Tieghem (1869), Molisch (1893), and Lidfors (1896) have stated that bursting cannot be due to an osmotic phenomenon. Lloyd (1918), Anthony and Harlan (1920), Brink - 13 -(192*f c) and Vasil (1958 a,b; i960 a) attributed bursting to be due to an osmotic phenomenon, and showed experimentally that bursting of the pollen of Cucumis melo var. utilissimus and Mormordica charantia invariably decreased with an increase in the osmotic concentration of the medium. (c) In vivo germination of pollen. In vivo procedures have been very d i f f i c u l t to use and have depended on k i l l i n g , sectioning, and staining material (Smith and Cochran 1935). Procedures, l i k e those used by Buchholtz (193D, Chandler (193D, Nebel (193D and, Pandey and Henry (1959), are successful in plants having large p i s t i l s and relatively large pollen tubes. Linskens and Esser (1957) and Martin (1959) have reported tracing pollen tube extension down the styles of tomato and petunia respectively by fluorescent stain. The method enabled them to examine small pollen grains, and the tiny slender tubes in the small p i s t i l s by means of the aniline blue dye resulting in fluorescent staining of callose tissue under ultra violet l i g h t . They used the procedure for descriptive and qualitative work only. Collose plugs. It has been observed that as the pollen tube elongates in vitro certain plug-like structures are formed at regular inter-vals along i t s length and divide into small segments. These plugs are made up of callose, a substance very similar to cellu-lose (Currier, 1957)e -Callose i s a polyglucoside derived from glucose but i t s exact chemical nature i s s t i l l uncertain* Its formation depends - l l * -on the limited capacity of pollen tubes to u t i l i z e the gluco-pyranose component of the metabolised sucrose during respiration (Tupy, I960). This explains the increased accumulation of cal-lose in incompatible and slow growing tubes (Tupy, 1 9 5 9 ; Vasil, I960 a). Brink (192*4- d) and Iwanami ( 1 9 5 9 ) have demonstrated exper-imentally that the tips of pollen tubes cut off from the older portions by callose plugs, are capable of independent growth, and that the plugs give mechanical strength to the tube and restricts protoplasmic streaming to the actively growing region. Group effect or Pollen Growth Factor (PGF). Several reports have shown that the aggregation of pollen grains in culture media has a marked stimulatory effect on number of grains germinating and on the ultimate length of tubes. Brink (192*+ d), Kuhn ( 1 9 3 8 ) , Visser ( 1 9 5 5 ) , Savelli and Caruso (19*+0) have demonstrated convincingly "the grouping effect" on the increase of germination percentage and pollen tube growth. Visser ( 1 9 5 5 ) suggested that the substance with the "germ-ination promotion" properties which diffuses from the grains is neither boric acid nor an enzyme but perhaps a glucoside. Brewbaker and Majumber ( 1 9 5 9 ) have shown that the pollen growth factor (PGF), was dialyzable, insoluble in ether, relat-ively heat stable, and was not replaceable by kinetin or auxin. It was suggested that lack of pollen tube growth in incompatible styles i s due to insufficient (PGF). 3. Use of pollen as a means of plant selection. The use of a pollen character as a basis for plant selection - 15 -to s e p a r a t e . p l a n t s of d i f f e r e n t genotypes has n o t been used w i t h much succ e s s . Oberle and Goertzen (1952) s t r e s s e d t h a t u n f a v o u r a b l e e n v i -ronmental or n u t r i t i o n a l c o n d i t i o n s may account f o r unfavourable blossom development and some of the v a r i a t i o n i n number of normal g r a i n s per anther which may vary from year to year f o r the same v a r i e t y of f r u i t . Bennett (1959) used a r t i f i c i a l p o l l e n germination of D a l l i s grass to e v a l u a t e d i f f e r e n c e s between h i g h and low f e r t i l i t y l i n e s , but found the method to be u n r e l i a b l e as a b a s i s of s e l e c -t i o n f o r improved seed p r o d u c t i o n . Attempts to use p o l l e n s i z e as an i n d i c a t o r of p o l y p l o i d y or as a taxonomic c h a r a c t e r i s t i c , have f a i l e d due to the v a r -i a t i o n s i n p o l l e n s i z e brought about by the environmental f a c t o r s . Schoch - Bodmeur (19*+0) rep o r t e d t h a t a p o s i t i v e c o r r e l a t i o n e x i s -ted between a v a i l a b l e moisture and p o l l e n s i z e i n Lythrum s a l i c a -r i a . B e l l (1959) showed t h a t m i n e r a l n u t r i t i o n a f f e c t s the amount of v a r i a b i l i t y of p o l l e n s i z e . He observed t h a t the l a r g e s t p o l l e n s i z e obtained i n tomato, came from p l a n t s t r e a t e d w i t h n i t r o g e n . Blanco (1951) has r e p o r t e d t h a t i n maize a c o r r e l a t i o n has been found between p o l l e n s i z e and combining a b i l i t y . No doubt the l i m i t e d u s e f u l n e s s of p o l l e n as a s e l e c t i o n c h a r a c t e r has been l i m i t e d by the v a r i a t i o n produced by e n v i r o n -mental c o n d i t i o n s . - 16 -MATERIALS AND METHODS. A. Materials. The plant materials used in the experiments consisted of the tomato varieties, Puck, Bonny Best, Earliana h9& and their recip-rocal crosses. Puck. Puck was introduced in England in 19^8 by M.B. Crane (19^8). He described i t as a dwarf bush variety that produced an early and a heavy crop. Heydecker (19^8) described the f r u i t of Puck as medium in size and possessing the uniform colour gene. The f r u i t wall i s differentiated from the placenta and at maturity lacks the fleshy character of commercial varieties. As reported by Daubeny (1955)> the variety requires a greater number of days between blossoming and ripening than some of the commercial varieties grown in British Columbia. The leaves are dark green, thick, with characteristic roughness of surface. The plant has a determinate growth habit which is synonymous with self-pruning and i s controlled by a single recessive gene sp. located In the sixth chromosome and belongs to the fourth linkage group (Yeager, 1927; Rick and Butler, 1956). The stem i s woody, and i s strong enough to bear the weight of the f r u i t s in the early period of growth. The main stem terminates in a leaf and carries a double inflorescence in the last internode. Axillary side-shoots develop, and are determinate in growth habit. These axillary shoots carry single clusters but in the last internode double clusters occur. The variety was selected from the F2 of a cross between the - 17 -American bush type v a r i e t y , V i c t o r , and an u n i d e n t i f i e d dwarf type v a r i e t y r e l e a s e d from R u s s i a , a t the end of World War I I . The v a r i e t y has the o u t s t a n d i n g f e a t u r e of s e t t i n g f r u i t a t temperatures between 55° and 65°F., ( F r a z i e r , 1951, Smith and Cochran, 1935), which makes i t a v a l u a b l e source of germ plasm f o r improving commercial v a r i e t i e s so as to i n c r e a s e p r e s e n t day commercial tomato p r o d u c t i o n . Bonny Best. Bonny Be s t was developed from a s i n g l e p l a n t s e l e c t i o n of C h a l k 1 s E a r l y Jewel, ( B o s w e l l , 1933). I t i s an o l d and well-known commercial v a r i e t y . I t has been e x t e n s i v e l y c u l t i v a t e d as a second e a r l y v a r i e t y on the North American c o n t i n e n t s i n c e the e a r l y p a r t of t h i s century subsequent to i t s i n t r o d u c t i o n to P h i l a d e l p h i a by the f i r m of Johnson and Stokes. Boswell (1933) observed t h a t the f r u i t s e t of Bonny Best was c o n s i d e r a b l y delayed under low temperature c o n d i t i o n s . The v a r i e t y has f l e s h y , c oloured f r u i t s , medium i n s i z e , and s l i g h t l y oblong i n shape. The p l a n t has indeterminate growth h a b i t and medium v i n e growth. There a r e f o u r or f i v e f l o w e r s per c l u s t e r , w i t h two or t h r e e f r u i t s being s e t per c l u s t e r . Although there a r e e a r l i e r commercial v a r i e t i e s , Bonny Best was chosen i n p r e f e r e n c e to others s i n c e more i s known about the e f f e c t of temperature on i t s p o l l e n germination. E a r l i a n a . E a r l i a n a was one of the e a r l i e s t important tomato v a r i e t i e s grown i n B r i t i s h Columbia. The number of f l o w e r s per c l u s t e r i s seven to n i n e w i t h normally three or f o u r f r u i t s being s e t per - 18 -c l u s t e r ( B o s w e l l , 1 9 3 3 ) . T h i s v a r i e t y i s regarded as a popular e a r l y commercial tomato v a r i e t y and thus was chosen f o r comparison w i t h Puck, The s t r a i n of E a r l i a n a used i n t h i s work was Morse's S p e c i a l S98 and w i l l subsequently be r e f e r r e d to as S 9 8 . H y b r i d s . The h y b r i d s used i n t h i s work a r e l i s t e d as f o l l o w s , showing the maternal parent f i r s t . The l i s t of c r o s s e s shows corresponding symbols which w i l l be used f o r f u t u r e r e f e r e n c e . 1 . (Bonny Best x Puck) F-^ (BxP) F X 2 . (Puck x'Bonny Best) F-j_ (PxB) F-L 3 . (Bonny Best x Puck) F 2 (BxP) F 2 (Puck x Bonny Best) F 2 (PxB) F 2 5 . (Bonny Best x Puck) F ^ (BxP) F3 6 . (Puck x Bonny Best) F ^ (PxB) F ^ 7. (Puck x E a r l i a n a S 9 8 ) F3 (Px*f98) F 3 B. Methods. 1 . C u l t u r e of p l a n t s . The experiments were conducted both i n the f i e l d and green-houses of the D i v i s i o n of P l a n t S c i ence a t the U n i v e r s i t y of B r i t i s h Columbia, i 9 6 0 - 1 9 6 2 . S i x l o t s of p l a n t s were grown. L o t 1 . Seeds of Puck and Bonny Best were sown October 2 0 , I 9 6 0 , and p l a n t s were grown i n two benches i n both warm ( 6 0 ° -7 0 ° F . ) and c o o l ( 5 0 ° - 6 0 ° F . ) greenhouses. Lo t 2 . Seeds of Puck, Bonny Best, E a r l i a n a U-98 along w i t h t h e i r r e c i p r o c a l c r o s s e s were sown A p r i l 2 1 , 1 9 6 1 , and p l a n t s were grown i n the f i e l d d u r i n g the s p r i n g and summer, i T h i s was an e x c e l l e n t season f o r the tomato crop and - 1 9 -plants were in a vigorous state a l l the time. Lot 3. Seeds of Puck, Bonny Best, Earliana *f98 along with their reciprocal crosses were sown August 20, 1961 and plants were grown in the south benches of the warm and cool green-houses. Lot A set of plants similar to those in l o t 3 was started October 10th, 1961 and were grown in the benches on the northern sides in both warm and cool greenhouses. Lot 5. Plants of Bonny Best and Puck were started March 10, 1962 and were grown in the southern benches in both houses. Lot 6. A set of plants similar to those i n lo t 5 was\ started April 16, 1962, and was' planted in the northern benches in both houses. The plants in lots 5 and 6 were essentially subjected to similar temperatures since the differential between cool and warm temperatures became less pronounced as the spring advanced. In general, seedlings were pricked out two weeks after seed sowing and set in 2" x 2" veneer bands in f l a t s . The plants were transplanted two weeks later to benches of sterilized s o i l in the greenhouses or to the f i e l d . In the greenhouses the exper-iments were planted in a simple randomized block design with 15 plants of each variety, Puck and Bonny Best, randomized within a single block. When the 10 varieties were grown the experimental design used was 3 randomized blocks with 10 treatments or single plant plots per block. A l l plants in benches were spaced at 20 inches apart between rows and approximately 15 inches apart within rows. In the f i e l d the experimental design consisted of 8 random-ized blocks with 10 treatments per block,with single plant plots - 20 -spaced 3 feet apart within and between rows. Prior to planting, f e r t i l i z e r was applied at the rate of 800 lbs per acre of *f:10:10. The plants were sprayed periodically with 5 percent malathion to protect them from aphids and white f l i e s in the greenhouses. In the f i e l d 5 percent D.D.T. dust was put in a circular band around each plant soon after planting to prevent cutworm damage. The benches used for the greenhouse experiments were parallel to the north-south walls in both houses. In both cool and warm houses, temperatures were recorded by thermographs. The temperature in the cool house was kept as closely as possible between the range of 50°F.- 60°F. and in the warm house between 60°F.- 70°F. A l l seedlings were kept in the warm house un t i l transplanting time. During the winter 1960-61 the temperature in the cool house went above 65°F. on eleven days and lasted for a total period of hours. For the greater part of the time, temperatures were between 55°F. and 60°F. In the warm house, temperatures went a l i t t l e below 65°F. at night, and only five times below 60°F. for very short periods lasting between two to three hours. During the winter 1961-62 the temperature in the cool house went above 65°F. on eighteen days and for a cumulative total period of 6h hours. For the greater part of the time, temperatures were between 55° and 60°F. In the warm house, temperatures went below 65°F. at night, and sixteen times below 60°F., for very short periods. Supplementary lighting was used during the course of the experiments to provide extra-illumination in the morning and - 21 -e v e n i n g u n d e r t h e s e a s o n a l c o n d i t i o n s o f low l i g h t i n t e n s i t y and s h o r t p h o t o p e r i o d . An e i g h t e e n hour p h o t o p e r i o d was t h e n e n s u r e d . A l l p l a n t s e x c e p t t h o s e grown i n t h e f i e l d , were pruned t o a s i n g l e stem and s t a k e d . P o l l e n samples were t a k e n f r o m f l o w e r s , w h i c h were marked p r i o r t o o p e n i n g , when stamens had r e a c h e d f u l l a n t h e s i s , e x c e p t i n e x p e r i m e n t ¥ , on methods o f p o l l e n c o l l e c t i o n . S a m p l i n g was done on t h e second day a f t e r the f l o w e r o p e n e d . P o l l e n samples were examined and g r a i n s counted under the m i c r o s c o p e u s i n g a m a g n i f i c a t i o n o f 100X. C o u n t s were made on t h e t o t a l number o f g r a i n s ("which u s u a l l y r a n g e f r o m between S O O - ^ O O _ g r a i n s ) , o c c u p y i n g the 2h s q u a r e s o f the o c u l a r m i c r o -m e t e r . I t was n o t p o s s i b l e to compare p o l l e n v i a b i l i t y o f the two v a r i e t i e s , Puck and Bonny B e s t under c o o l house c o n d i t i o n s b e c a u s e o f l a c k o f Bonny B e s t p o l l e n . When p o l l e n was produced by Bonny B e s t i n t h e c o o l h o u s e , t h e r e was no g u a r a n t e e t h a t the p o l l e n was formed w i t h i n t h e e x p e r i m e n t a l t e m p e r a t u r e r a n g e o f 55°-65°F. s i n c e s h o r t e x p o s u r e s t o h i g h t e m p e r a t u r e s d u r i n g sunny days i n t h e w i n t e r months were a d e q u a t e t o cause p o l l e n development i n the v a r i e t y . 2. L a b o r a t o r y m e t h o d s . (1) S t a i n i n g . A f t e r e v a l u a t i n g the f o u r s t a i n i n g m e t h o d s , namely the a c e t o -c a r m i n e , t h e p e r o x i d a s e t e s t , t e t r a z o l i u m s a l t , and s t a n d a r d i o d i n e -p o t a s s i u m i o d i d e ( I - K I ) , r e p o r t e d i n the l i t e r a t u r e f o r - - d e t e r m i n i n g p o l l e n v i a b i l i t y , o n l y t h e i o d i n e - p o t a s s i u m i o d i d e p r o c e d u r e was s a t i s f a c t o r y f o r t h e work a f t e r i t had been m o d i f i e d . Daubeny (1955) - 22 -and Guccione (1959) reported on the customary use of I-KI solution in which darkly stained grains were considered viable. Experiments were carried out dealing with the concentration of I-KI solution and stages of maturity of pollen. Pollen samples stained with dilute I-KI (one drop of a 1% solution of I-KI i n lOcc of water) showed different staining intensities for the 3 flower stages. In vitro and in vivo experiments were done using mature and immature pollen samples to confirm the results of the staining. As the staining experiments were conducted, pollen was classified as (1) viable - large and l i g h t l y stained grains (2) im-mature - darkly stained grains and (3) non viable - empty grains which were unstained. (2) In vitro germination. Considering Hewlett*s (1936) report that tomato pollen grains which appear normal in shape and external appearance are often non-viable, i t was considered desirable to compare the germination percentages of pollen in vitro with those obtained from staining. As a result in vitro experiments were run concurrently with the staining experiments in several cases throughout the work by using subsamples from samples for both pollen v i a b i l i t y determinations. The medium used for germinating pollen grains contained 10$ sugar, 0*5% agar, and 30 ppm. boron, (Guccione, 1959)• A small drop of medium was placed on a slide at room tempera-ture, and the sample of pollen was stirred into the drop. The slide was then inverted over well-slides containing two drops of water. The slide was then incubated in an oven at a temperature of 20°C. On one occasion in the summer of 1962 germination was , o poor due to high temperatures of 24- C. in the laboratory. Sub-- 23 -sequently by placing a beaker of water in the oven, germination percentages were back to normal. It i s l i k e l y that the increase in humidity at the higher temperature, or that a lowering of temperature created the favourable conditions for germination. After eleven hours, counts were made of the three classes: (1) viable (2) burst and (3) empty grains. (3) In vivo germination. The determination of pollen v i a b i l i t y in vivo is a problem in plants with a relatively small stigma, e.g. the tomato. Several attempts were made to observe pollen germinating and traversing the style by staining. None of the methods used by Buchholtz (193D, Chandler (193D etc., proved successfull. Martin's (1959) qualit-ative procedure of observing pollen tubes by fluorescence was adapted, and yielded results. Several preliminary experiments were carried out to test the suitability of Martin's method for giving a quantitative expression of pollen v i a b i l i t y in vivo. The d i f f i c u l t i e s encountered during the course of the early investigation were as follows. 1. Finding a procedure to have fluorescent streaks of clustered bands of germinated pollen grains distinct enough to count accur-ately under the dissecting microscope. 2. The plants on which preliminary in vivo germination studies were carried out were in a declining state of vigour and the pollen samples had extreme variabi l i t y in the amount of functional pollen. More uniform germination results were obtained when flowers from vigorous plants of Puck and Bonny Best had come into flower. 3 . It was d i f f i c u l t to arrive at an appropriate number of grains - 2k -necessary to give a germination percentage on the stigma because of the pollen group effect. The procedure developed was as follows. Flowers were emas-culated and tagged before the petals were reflexed, and this operation was done with a pair of tweezers, so that the anthers were removed as a cone. Pollen grains were counted under a dissecting microscope, picked up by means of a single camel hair, and transferred to the stigmatic surfaces of the emasculated flowers at the time when the petals became reflexed and flu i d was noticeable on the stig-matic surface. Care was taken to place the grains in the areas where there was an accumulation of stigmatic f l u i d . These areas occurred in small pockets on the stigma. After pollen was trans-ferred to the stigmatic surface, the camel hair was inspected under the microscope. If pollen grains persisted the camel hair was passed along the stigmatic surface repeatedly until free of grains. Pollen grains were allowed to germinate for *f8 hours, after which the stigmas were removed. The styles were fixed in formalin-acetic-acid- Q0% alcohol (1:1:8) for kQ hours, the time being double that used by Martin (1959) so as to attain satisfac-tory J-peneitratioriv The styles were washed for 1.5 hours then softened and cleared in a strong 8N sodium hydroxide solution for 2k hours. They were then washed again for 2 hours and stained with a 0,1% solution of water soluble aniline blue dye dissolved in 0.1N K^PO^. Stain was allowed to penetrate over a k hour period. Then styles were smeared by pressing them between a cover-sli p and a microscope slide, and examined under a dissecting microscope of kOX, using direct illumination with ultraviolet light - 25 -of a wavelength of about 356-myu* Counts were made in a dark room of the number of grains which had germinated. Under these condit-ions the callose in germinated pollen tubes appeared fluorescent with a bright-yellow-green colour and contrasted strongly with the bluish or greyish colour of the stylar tissue. The germin-ated pollen tubes were outlined by a callose lining and irregular-l y spaced callose plugs. 3. Experiments on sampling. Experiments on sampling pollen were conducted to ascertain (1) the number of pollen grains which must be examined to give a reliable estimate of pollen v i a b i l i t y , (2) the variability between methods of extracting or collecting pollen for examin-ation and ( 3 ) the variab i l i t y between pollen from different sources. Both the staining and in vitro germination of pollen were employed for v i a b i l i t y determinations. (a) Sample size. Experiment 1. The experiment was conducted to determine the number of grains necessary to give a reliable estimate of the pollen v i a b i l i t y of a sample. Pollen samples were collected in small glass vials by means of a pollen vibrator, using each of three single flowers from two plants of Bonny Best variety. Each sample was taken on a different date, namely March 3, 1962, May 5, and May 7, 1962. From these three samples the pollen v i a b i l i t y of ten subsamples per sample was examined using the staining procedure. Results were obtained by counting (1) the number of grains occupying the 24- squares on a graded occular micrometer (Method A); and (2) in the same microscopic f i e l d for the same subsample, 100 grains were counted in squares taken at random in the microscopic - 26 -f i e l d (Method B). The data were converted to percentages. Only data on mature pollen grains were analyzed s t a t i s t i c a l l y . Sample 1 used in the experiment was taken from a flower on a truss of a Bonny Best plant of l o t which was approximately five months old, and already laden with f r u i t . The vigour of the plant for the production of new flowers was low. Samples 2 and 3 were taken from single flowers from the f i r s t and second clusters respectively on the same three-month-old Bonny Best plant of l o t 5. The plant was in a relatively high state of vigour. Results are presented in table 1. (b) Methods of pollen collection. It was realized that a pollen sample taken from a f u l l y reflex-ed tomato flower would not contain grains of equal and uniform maturity, but would consist of pollen at different stages of maturity which could be distinguished by the intensity of stain-ing. It was decided vto study further the maturity of pollen as i t affected v i a b i l i t y measurements, especially since i t was found experimentally that immature pollen does not germinate in vitro. Hence age of pollen or stage of maturity was considered a major source of variation in pollen v i a b i l i t y determinations. The methods of pollen collection were: (1), the effect of different methods of pollen extraction on the percentage of mature and immature pollen in samples; and (2), the effect of time of sampling on the degree of maturity and v i a b i l i t y of pollen grains in a sample. (i) Methods of pollen extraction. - 27 -Experiment 1. The experiment was conducted to observe the effect of two methods of collection of pollen on maturity and v i a b i l i t y in two varieties, Puck and Bonny Best. Pollen v i a b i l i t y determinations were started December 1, 1961, on plants in l o t 1, using the staining and in vitro procedures concurrently. In the f i r s t method, a dissecting needle was used for collec-ting pollen samples. The point of the needle was inserted into the anther and the needle was then moved along the anther groove on one side of the stamen. Each of four stamens from the same flower of truss 1 wasc examined, and one plant of each variety was sampled. Thus 2 flowers were used to provide 8 stamens for pollen samples. In the second method, a vibrator was used to collect pollen samples in glass vials. Each sample was taken from a single flower. Two flowers on truss 1, and two on truss 2 were used from each variety. Thus 8 flowers were used to provide 8 samples. Three subsamples per sample were examined on the second day of anthesis. The plants were young and vigorous in appearance. Results are presented in table 3. Experiment 2 . The objectives of the experiment were identical with those of experiment 1, except that the effects of the two methods of extraction were studied on the ten varieties of plants in l o t 1. The needle method of extraction was modified by taking pollen samples from the surface of a single stamen of a single flower of each variety since i t was thought that collection from the surface would give a more uniform sample of mature grains than - 28 -extraction from the i n t e r i o r . The experiment was started on January 5, 1961. The plants at the time of sampling were advanced i n age and laden with f r u i t s . Results are presented i n table Experiment 3« A preliminary experiment was conducted to test further the differences i n pollen grain maturity. Samples were obtained from 3 stamens per flower of each of the two v a r i e t i e s , Puck and Bonny Best. Sampling was performed by: (1), passing the needle i n the i n t e r i o r of the anther along one side of the anther groove; and (2) , by passing the needle over the surface of the anther; and (3) > by passing the anther through water to suspend the pollen. Five subsamples per anther f o r each method of extraction were examined f o r one flower of each of Puck and Bonny Best plants of l o t 3. Pollen v i a b i l i t y was determined by staining. The experiment was begun on December 20, 1961. Results are presented i n table 5» Experiment k-. A further experiment was conducted to obtain information on the percentage of immature grains associated with ten sub-samples from each pollen sample derived from each of four methods of c o l l e c t i o n . The four methods used f o r obtaining pollen samples weres 1. from a single flower by means of the pollen vibrator; 2. from a single stamen of a flower by passing the needle over the surface; 3. from a single flower by tapping i t over a drop of s t a i n on the s l i d e ; - 2 9 -h, from each of 5 stamens of a s i n g l e f l o w e r , by p a s s i n g each stamen twice through water. V i a b i l i t y d e t e r m i n a t i o n s were made by the s t a i n i n g technique. The f l o w e r s used were obtained by t a k i n g one f l o w e r from the f i r s t t r u s s on each of f o u r h e a l t h y Puck p l a n t s of l o t 5» which were young and v i g o r o u s . The experiment was begun on May 10, 1962. R e s u l t s a r e presented i n t a b l e 6. ( i i ) Time of p o l l e n c o l l e c t i o n i n r e l a t i o n to age of f l o w e r . (1) P o l l e n m a t u r i t y a t d i f f e r e n t bud and f l o w e r stages. Experiment 5» I t was considered d e s i r a b l e to study the e f f e c t of stage of m a t u r i t y of buds and f l o w e r s on the m a t u r i t y of p o l l e n g r a i n s . C l u s t e r s were chosen on p l a n t s i n l o t 2, to p r o v i d e f o u r f l o w e r s or buds a t d i f f e r e n t stages of m a t u r i t y on each c l u s t e r , namely (1) a f l o w e r w i t h p e t a l s f u l l y r e f l e x e d and a t f u l l a n t h e s i s , (2) a bud b e g i n n i n g to open, w i t h s e p a l s s t a r t i n g to spread, (3) a bud a t an e a r l i e r stage than number 2, (h) a very young bud a t an e a r l i e r stage than number 3. Such c l u s t e r s were chosen on a s i n g l e p l a n t of each of ten v a r i e t i e s , and there were two r e p l i c a t i o n s . Thus e i g h t y buds or f l o w e r s were used i n the experiment. A l l p o l l e n samples were taken by i n s e r t i n g a d i s s e c t i n g needle i n t o anthers on J u l y 7> 1961. M a t u r i t y and v i a b i l i t y were determined by the s t a i n i n g procedure. A l l f l o w e r s were on second t r u s s e s of s e l e c t e d p l a n t s i n the two b l o c k s . - 30 -R e s u l t s are presented i n t a b l e 7. (2) P o l l e n v i a b i l i t y as a f f e c t e d by l o n g e v i t y of the f l o w e r . Experiment 6 . A f t e r c o n s i d e r i n g the e f f e c t of immature p o l l e n on the v i a b i l i t y r e s u l t s i n samples taken from f l o w e r s a t d i f f e r e n t ages on the same c l u s t e r , i t was considered d e s i r a b l e to o b t a i n i n f o r m a t i o n on p o l l e n m a t u r i t y and v i a b i l i t y i n f l o w e r s over the l i f e p e r i o d beginning a t f u l l a n t h e s i s , which occurred on the second day a f t e r opening, to the time p e t a l s w i l t e d . T h i s span was f i v e days under the c o n d i t i o n s f o r the experiment. P r e v i o u s workers (Daubeny, 1955> and Guccione, 1959)> used the f u l l y r e f l e x e d f l o w e r as a guide to time of p o l l e n sampling. However, a f u l l y r e f l e x e d f l o w e r could cover a p e r i o d of probab-l y 5 days and v a r i a b i l i t y i n p o l l e n v i a b i l i t y could be a f u n c t i o n of t h i s t i n e . I t was considered d e s i r a b l e to check t h i s assump-t i o n . A s i n g l e f l o w e r from each of ten v a r i e t i e s i n p l a n t s of l o t 2 , was s e l e c t e d , and one stamen per f l o w e r was examined on each of f i v e s u c c e s s i v e days. Thus f i v e of the s i x stamens i n each f l o w e r were sampled. Each stamen had to be removed before e x t r a c t i n g a p o l l e n sample, r e s u l t i n g i n a d a i l y removal from each f l o w e r . Sampling was s t a r t e d on August 8 , 1 9 6 1 , and the f i e l d - g r o w n p l a n t s were very v i g o r o u s . Removal of anthers was a d e l i c a t e o p e r a t i o n , but there was no apparent damage to remain-in g anthers and other f l o r a l p a r t s . R e s u l t s are presented i n t a b l e 8 . - 31 -(c) Variation due to differences in location of pollen sources on plants. The inconsistent results obtained by Daubeny (1955)? and Guccione (1959)> on pollen v i a b i l i t y determinations in tomato pollen suggested variability in pollen samples. No mention was made in these reports of the location of the flowers on the plant from which pollen samples were obtained. It was desirable to check more carefully the variability in pollen v i a b i l i t y which may have resulted from: (i) different zones of the same stamen; ( i i ) different stamens of the same flower; ( i i i ) different flowers on the same truss and; ( (iv) different trusses on the same plant. (i) Pollen v i a b i l i t y at different zones of the same stamen. Experiment 1. The experiment was designed to determine the v i a b i l i t y of pollen of the two varieties, Puck and Bonny Best, at three zones, namely, tip, middle, and base, of each of three relatively long stamens. Pollen samples were taken by means of an inserted needle, and pollen v i a b i l i t y determinations were made by staining and in vitro techniques. One flower from a plant of each of the two varieties in l o t was used. Three stamens from each flower were divided into zones and three subsamples were examined per zone. The experiments were started January 17, 1962, on flowers on the third trusses of the varieties. Results are presented in table 9. Experiment 2. The experiment was started July 15, 1961. The objectives - 3 2 -•were the same as experiment 1, except that a single stamen from a single flower of the 10 v a r i e t i e s of plants i n l o t 2, was used. Results are presented i n table 10. ( i i ) Pollen v i a b i l i t y of d i f f e r e n t stamens of the same flower. Experiment 3« The experiment was intended to determine the pollen v i a b i l i t y of s i x stamens of each of four flowers of Puck. Four pollen samples were obtained by the needle method of extraction from the i n t e r i o r of the stamen. Pollen v i a b i l i t y was determined by staining and i n v i t r o methods. Samples were taken on July 13, 1961, from flowers on the f i r s t truss of four Puck plants of l o t 2. The plants were young and vigorous. Results are presented i n table 11. ( i i i ) Pollen v i a b i l i t y of d i f f e r e n t flowers on the same truss. Experiment 4-. The experiment was designed to determine the pollen v i a b i l i t y of d i f f e r e n t flowers on the same truss. The samples were three flowers from one truss per plant, on three plants from each of two v a r i e t i e s i n two blocks. Thus a single pollen sample was taken from each of 36 flowers by means of the vibrator, and each sample was subdivided to give 3 subsamples and each of the three subsamples was stained and cultured i n v i t r o . Pollen samples were taken on January 2 3 , 1961, from flowers on each of the two v a r i e t i e s , Puck and Bonny Best i n l o t 1. Results are presented i n table 1 2 . - 33 -( i v ) P o l l e n v i a b i l i t y of d i f f e r e n t t r u s s e s on the same p l a n t . Experiment 5 . The experiment was designed to determine d i f f e r e n c e s i n p o l l e n v i a b i l i t y among each of 3 t r u s s e s of Puck. Samples were c o l l e c t e d on June 28, 1961, from t r u s s 1, on June 30, 1961 from t r u s s 2, and J u l y 1961 from t r u s s 3> on the same p l a n t of l o t 2. Four f l o w e r s per t r u s s were used and one sample was c o l -l e c t e d from each of the h f l o w e r s by the v i b r a t o r . P o l l e n v i a b i l -i t y was determined on 3 subsamples per sample by the s t a i n i n g technique. The p l a n t was young and v i g o r o u s a t the time sampling was done. R e s u l t s are presented i n t a b l e 13. l f . Comparison of methods of p o l l e n v i a b i l i t y d e t e r m i n a t i o n s . I t was considered i m p e r a t i v e to study more c l o s e l y the v a r i a t -i o n s i n p o l l e n v i a b i l i t y which may be a t t r i b u t e d to the d i f f e r e n t methods used i n measuring t h i s v i a b i l i t y namely, the s t a i n i n g , the i n v i t r o and i n v i v o techniques. Throughout the course of the r e s e a r c h both the s t a i n i n g and i n v i t r o methods of p o l l e n v i a b i l i t y d e t erminations were used and t h e i r v a l u e s compared. The experiments on the comparison of methods were conducted i n t hree p a r t s . (a) Comparison of the s t a i n i n g and i n v i t r o techniques. (b) Experiments on i n v i v o germination. (c) Comparison of s t a i n i n g , i n v i t r o and i n v i v o techniques. (a) Comparison of s t a i n i n g and i n v i t r o techniques. Experiment 1. The experiment was designed to compare the techniques of s t a i n i n g and i n v i t r o germination f o r r e v e a l i n g the p o l l e n v i a b -i l i t y of ten v a r i e t i e s . A s i n g l e f l o w e r a t f u l l a n t h e s i s from each of the 10 v a r i e t i e s of p l a n t s of l o t 3» was used. P o l l e n samples were c o l l e c t e d on November 24-, 1961, w i t h the v i b r a t o r . S i x subsamples per sample were used f o r p o l l e n v i a b i l i t y determ-i n a t i o n s . The p l a n t s were young and v i g o r o u s . R e s u l t s are presented i n t a b l e 14-. (b) Experiments on i n v i v o germination. In the p r e v i o u s experiments the s t a i n i n g and i n v i t r o determ-i n a t i o n s of p o l l e n v i a b i l i t y gave w i d e l y d i f f e r i n g r e s u l t s . These d i f f e r e n c e s r a i s e the q u e s t i o n as to which method most c l o s e l y r e v e a l s the potency of p o l l e n under n a t u r a l c o n d i t i o n s . Consequent-l y , i t was d e s i r a b l e to i n v e s t i g a t e the r e l a t i o n s h i p between p o l l e n v i a b i l i t y d e t e r m i n a t i o n s made i n v i v o (under n a t u r a l c o n d i t i o n s ) w i t h those determined a r t i f i c i a l l y , namely the s t a i n i n g and i n v i t r o t e c hniques, u s i n g the same p o l l e n sample f o r the three methods. Experiment 1. The e x t e n s i o n of p o l l e n tubes i n tomato s t y l e s was c l e a r l y v i s i b l e i n v i v o when M a r t i n ' s (1959) f l u o r e s c e n t technique was employed. A mass of p o l l e n was used f o r p o l l i n a t i o n . However, the r e s u l t i n g t h i c k f l u o r e s c e n t bands of aggregated tubes, made i t i m p r a c t i c a b l e to count the number of g r a i n s which had germinated i n the f i r s t samples examined. - 35 -The f i r s t m o d i f i c a t i o n towards the development of a q u a n t i t -a t i v e procedure was to decrease the sample s i z e used i n p r e v i o u s p o l l i n a t i o n s . In a p r e l i m i n a r y experiment, an a r b i t r a r y number of 10 g r a i n s was used to p o l l i n a t e each of s i x stigmas of each of the v a r i e t i e s , Puck and Bonny Best. The experiment was done on p l a n t s of l o t and isas s t a r t e d on March 7j 1962. These p l a n t s were i n a r e l a t i v e l y low s t a t e of v i g o u r . Experiment 2 . The f a c t t h a t no germination was observed when 10 g r a i n s were used f o r the i n v i v o germination i n experiment 1, neces-s i t a t e d r e p e a t i n g the experiment u s i n g an in c r e a s e d number of g r a i n s . An a r b i t r a r y number of 100 g r a i n s was used to p o l l i n a t e each of f i v e stigmas of each of the v a r i e t i e s , Puck and Bonny Best, p l a n t s of l o t k, and the p l a n t s were i n a low s t a t e of v i g o u r . The experiment was s t a r t e d on March 12, 1962. R e s u l t s are presented i n t a b l e 15. Experiment 3« Having obtained s u c c e s s f u l germination i n experiment 2, w i t h i n l i m i t of counting p o l l e n tubes i n the s t y l e s , an experiment was designed to study the e f f e c t of number of g r a i n s on germin-a t i o n on stigmas of the v a r i e t i e s , Puck and Bonny Best. Thus 15, 5 0 , 100 and 200 p o l l e n g r a i n s were a p p l i e d r e s p e c t i v e l y to f o u r stigmas of each of the two v a r i e t i e s . Three r e p l i c a t i o n s were used. A t o t a l of 2k separate f l o w e r s were p o l l i n a t e d . The f i r s t f l o w e r on a t r u s s was used i n each case i n order to employ l a r g e - 36 -v i g o r o u s buds. The experiment was begun on A p r i l 15, 1962, u s i n g p l a n t s i n l o t k. R e s u l t s a re presented i n t a b l e 16. Experiment k. Lack of germination of p o l l e n on three Bonny Best stigmas when 50 g r a i n s were used f o r p o l l i n a t i o n s i n experiment 2, sug-gested the e x i s t e n c e of a v a r i e t a l d i f f e r e n c e i n germination bet-ween the two v a r i e t i e s , Puck and Bonny Best. The i n d i c a t i o n of t h i s v a r i e t a l response was not considered c o n c l u s i v e beeause there were only 3 f l o w e r s used. Consequently an experiment was designed to i n v e s t i g a t e f u r t h e r the e f f e c t on- i n v i v o germination when 50 and 100 g r a i n s were a p p l i e d r e s p e c t i v e l y to two stigmas of each of the two v a r i e t i e s . S i x r e p l i c a t i o n s g i v i n g a- t o t a l of 2k separate f l o w e r s were used. The experiment was s t a r t e d on A p r i l 22, 1962, us i n g p l a n t s of l o t k. R e s u l t s are presented i n t a b l e 17. Experiment 5. The experiment was designed to i n v e s t i g a t e the e f f e c t of s e l f - and c r o s s - p o l l i n a t i o n s on p o l l e n v i a b i l i t y d e t e r m i n a t i o n s , s i n c e i t was considered d e s i r a b l e to see whether v a r i e t a l d i f -f e r e n c e s i n p o l l e n and stigma could a f f e c t the percentage germin-a t i o n of p o l l e n i n v i v o . Consequently s e l f - and c r o s s - p o l l i n a t i o n s were c a r r i e d out on each of two stigmas r e s p e c t i v e l y of each of the two v a r i e t i e s , Puck and Bonny Best. Two d e n s i t i e s of p o l l e n , namely 50 and 100 g r a i n s , were used i n both s e l f - and c r o s s -p o l l i n a t i o n s . Two r e p l i c a t i o n s g i v i n g a t o t a l of 16 separate - 37 -f l o w e r s were used. The experiment was begun on A p r i l 23, 1962, u s i n g p l a n t s of l o t h. R e s u l t s are presented i n t a b l e 18. The p l a n t s of l o t ht from which f l o w e r s were obtained f o r the p r e v i o u s i n v i v o experiments, were i n a low s t a t e of v i g o u r a t the time the experiments were conducted. As a consequence two f u r t h e r experiments, 6 and 7, were c a r r i e d out u s i n g f l o w e r s from young vigorous p l a n t s of l o t 6, to study (1) the e f f e c t of number of g r a i n s on i n v i v o germination of p o l l e n from each of the two v a r i e t i e s , Puck and Bonny Best, and (2) the e f f e c t of v a r i e t a l d i f f e r e n c e s of p o l l e n and stigma on germination of p o l l e n i n v i v o . Experiment 6. In t h i s experiment 50 and 100 g r a i n s were used to p o l l i n a t e each of two stigmas r e s p e c t i v e l y of each of the two v a r i e t i e s . Twelve r e p l i c a t i o n s were used, g i v i n g a t o t a l of hQ separate f l o w e r s . Two stigmas of the f i r s t two f l o w e r s on t r u s s three on each of the 2h p l a n t s of l o t 6 were used. The remaining fl o w e r buds on the t r u s s a l o n g w i t h the f i r s t two c l u s t e r s were removed from the p l a n t s . R e s u l t s are presented i n t a b l e 19. Experiment 7* Sin c e the e f f e c t s of 50 and 100 g r a i n s on germination were a l r e a d y i n v e s t i g a t e d i n experiment 5, i t was d e s i r a b l e to f i n d out the e f f e c t of l a r g e r numbers of g r a i n s on germination. R e c i p -r o c a l c r o s s - p o l l i n a t i o n was compared with s e l f - p o l l i n a t i o n , u s i n g 200 g r a i n s on each of 2h stigmas of each of the two v a r i e t i e s , - 38 -Puck and Bonny Best. The experiment was s t a r t e d on July 24-, 1962, u s i n g v i g o r o u s p l a n t s of l o t 6. R e s u l t s a r e presented i n t a b l e 20. (c) Comparison of the s t a i n i n g , i n v i t r o and i n v i v o t e c h -niques used i n p o l l e n v i a b i l i t y d e t e r m i n a t i o n s . Experiment 1. The p r e v i o u s i n v i v o experiment made p o s s i b l e the comparison of a r t i f i c i a l p o l l e n v i a b i l i t y d e t e r m i n a t i o n s ( s t a i n i n g and a r t -i f i c i a l g e r m i n a t i o n ) , w i t h n a t u r a l p o l l e n v i a b i l i t y d e t e r m i n a t i o n made by i n v i v o germination. The i n v i v o r e s u l t s from the Puck and Bonny Best s e l f - p o l l i n a t i o n s i n experiment 7 , were a l s o used f o r t h i s experiment. Twelve subsamples of Puck and Bonny Best p o l l e n used f o r the p o l l i n a t i o n s i n the i n v i v o experiment 7 , were used to o b t a i n data on the s t a i n i n g and i n v i t r o p o l l e n v i a b i l i t y d e t e r m i n a t i o n s . The experiment was s t a r t e d J u l y 24-, 1962. R e s u l t s a r e presented i n t a b l e 21. The s t a t i s t i c a l methods of a n a l y s i s of data v a r i e d w i t h the experiment. The data were analyzed as simple randomized block experiments, f a c t o r i a l experiments, and a combination of f a c t o r i a l w i t h nested c l a s s i f i c a t i o n as o u t l i n e d i n S t e e l and T o r r i e (I960). - 39 -R E S U L T S I. M o d i f i c a t i o n o f t h e s t a i n i n g p r o c e d u r e . T h e r e s u l t s o f s t a i n i n g p o l l e n , f r o m f l o w e r s a t d i f f e r e n t s t a g e s , w i t h d i l u t e i o d i n e - p o t a s s i u m i o d i d e s o l u t i o n showed t h a t d i f f e r e n t s t a g e s o f p o l l e n d e v e l o p m e n t e x h i b i t e d a v a r i a t i o n i n t h e i n t e n s i t y o f s t a i n i n g w h i c h was r e l a t e d t o s t a g e o f m a t u r i t y o f t h e f l o w e r s . P o l l e n f r o m f l o w e r s a t t h e v e r y e a r l y bud s t a g e g a v e l i t t l e s t a i n i n g i n d i c a t i n g l i t t l e o r n o s t a r c h . I n t h e i n t e r m e d i a t e b u d s t a g e p r i o r t o a n t h e s i s , w h e n t h e a n t h e r showed a g r e e n i s h t o v e r y l i g h t y e l l o w c o l o u r , p o l l e n s a m p l e s g a v e a v e r y h i g h i n t e n s i t y o f s t a i n i n g . T h e s t a i n i n g was s o i n t e n s e t h a t t h e t y p i c a l d e e p b l u e c o l o u r was o b s e r v e d u n d e r t h e m i c r o s c o p e . T h i s s t a g e o b v i o u s l y m a r k s t h e p o i n t o f g r e a t e s t a c c u m u l a t i o n o f s t a r c h i n t h e p o l l e n c e l l . I n p o l l e n s a m p l e s t a k e n f r o m f l o w e r s a t a n t h e s i s t h e m a j o r i t y o f p o l l e n g r a i n s showed l i t t l e t o n o s t a i n i n g w h i c h i s i n d i c a t i v e o f l i t t l e o r n o s t a r c h . D a r k l y s t a i n e d g r a i n s s i m i l a r t o t h o s e f o u n d i n t h e i n t e r m e d i a t e b u d s t a g e , w e r e a l s o p r e s e n t among t h e l i g h t l y - s t a i n e d m a t u r e , f u n c t i o n a l p o l l e n g r a i n s . T h e d a r k l y s t a i n e d g r a i n s w e r e r e g a r d e d a s i m m a t u r e g r a i n s . N o n - v i a b l e o r e m p t y g r a i n s w e r e a l s o p r e s e n t i n t h e s a m p l e s . I n v i t r o g e r m i n a t i o n t e s t s o f t h e s e d a r k i m m a t u r e g r a i n s showed t h a t t h e y a b s o r b e d l e s s w a t e r a n d w e r e l e s s d i l a t e d t h a n t h e v i a b l e m a t u r e g r a i n s w h e n t h e l a t t e r w e r e p l a c e d i n t h e m e d i a . N o n e o f t h e s e i m m a t u r e g r a i n s g a v e a n y s i g n o f p o l l e n t u b e i n i t i a t i o n . - S o -l s vivo germination tests using immature grains showed no germination. On this basis i t was considered j u s t i f i e d to use the three classes (1) mature (2) immature and (3) empty or non-viable grains i n a l l subsequent staining of pollen samples. I I . Experiments on sampling. (a) Sample size. Experiment 1. The d i f f i c u l t i e s i n chosing the number of pollen grains to be examined and counted f o r a r e l i a b l e estimate of v i a b i l i t y i s i l l u s t r a t e d i n the data of table 1. Table 1. Percentages of mature pollen i n samples examined by two procedures; A= 24- squares of the microscopic f i e l d , and B= 100 grains taken at random i n squares. _ -— Subsamples 1 2 3 4- 5 6 7 8 9 10 x x 1 A 69 69 60 68 69 63 67 69 61 68 663 586 B 55 64- 28 21 71 65 k7 68 3^ 56 509 2 A 82 85 87 89 90 95 92 91 95 92 898 874-B 65 83. 75 90 84- 9k 89 90 93 87 850 3 A 7k 89 82 7k 94- 89 91 90 83 89 855 838 B 80 93 82 66 74- 91 76 9k 79 86 821 Analysis of variance. Source D.F. M.S. F Total 59 - — Treatments 5 224-1.8 — Methods (M) 1 928.0 10.82** Samples (S) 2 4-975.0 53.84-** M x S 2 215.5 2.33 Error 54- 92.4-Duncan's t e s t . Samples 1 3 2 x 58.6 8^ .8 87.4-- 4-1 -The analysis shows that the differences between the two factors, choice of counting procedure and source of samples, are each highly significant. The interaction of method x sample was non-significant which suggested that these factors acted independently. The results indicated that the method A, (count-ing the total number of grains in 24- squares of the ocular micro-i, meter), gave significantly higher percentages of mature pollen than method B, (counting a total of 100 grains in squares at random in the microscopic f i e l d ) . When the three sample means of 58.6, 83.8, and 87.4- were compared by Duncan's test, i t was noted that the differences between the percentages of mature pollen in samples 2 and 3 were significantly greater than in sample 1. The inference is drawn that samples 2 and 3 were taken from the same population, the young vigorous plants (lot 6), whereas sample 1 was taken from another population, namely, the older plants bearing many frui t s (lot 40. Each sample was considered separately in the calculations of required sample size. The ten subsamples of sample 1 showed standard deviations of + 3.56 and + 17.70 for methods A and B respectively. The data from both methods of microscopic determin-ation are comparable because their standard deviations are derived from the same number of subsamples. It should be noted that the standard deviation for method A is almost five times less than that for B. It i s evident that less variability existed between subsamples in the former case than in the latter. The number of subsamples required to give a reliable estimate - k2 -would vary with the degree of tolerance chosen. Consequently table 2, was calculated from the data of table 1 to give a range from 1-50 percent degree of tolerance at the 95% l e v e l . Table 2. Values of n= number of subsamples, when degree of tolerance (D) ranges from 1% to 50$ at 9% l e v e l , i n application to data i n table 1. Samples 1 2 \ Methods A B A B A B D n n n n n n 1 1203.39 67.4-0 200.78 189.06 311.52 2 12.1*4- 300.8*4- 16.85 50.19 *+7.26 77.88 3 5.39 133.35 7.*f8 22.28 20.98 3k. 57 *4- 3.0*4- 75.22 *t.21 12.5*4- 11.81 19.*+7 5 1.9k *f 8.1*4- 2.69 8.03 7.56 12.*+6 6 1.3"+ 33.33 1.86 5.56 5.23 8.62 7 0.99 2*f. 55 1.37 *f.09 3.85 6.35 8 0.75 18.77 1.05 3.13 2.9*+ *f.85 9 0.59 1*4-. 80 0.83 2.*+7 2.33 3.8*4-10 0.*4-8 12.00 0.67 2.*4-0 1.89 3.11 11 0.*f*f 10.09 0.61 1.82 1.71 2.83 12 0.*f0 10.02 0.61 0.67 1.57 2.59 2k- 0.20 5.01 0.28 0.83 0.78 0.12 50 0.09 0.60 0.13 0.*+2 0.37 0.62 The calculations were performed as follows. I f x = sample mean M - true mean s = standard deviation s_ = standard error of the mean n = required number of subsamples x-y" = p the chosen percent degree of tolerance. Then t^ = x = x --^ s- s DC "™ Solving for n \ 2 - S3 -When n = 1 x -M- = t ^ x s. Example. Suppose the degree of tolerance was a r b i t r a r i l y chosen to give a deviation about the true mean of not more than + 5 at the 95% l e v e l . Then the c a l c u l a t i o n f o r sample 1 would be. n f o r A = ( 1 , 9 6 x ^ t 5 6 j 2 = I . 9 S n f o r B = (l.96 x 17 . 7 o f = S 8 . 1 S Consequently with a 5$ degree of tolerance the number of sub-samples to be examined f o r method A would be approximately 2 , and f o r method B approximately S 9 . On the basis of th i s formula, the degree of tolerance f o r the two methods of the 3 samples examined when n = 1 , would be as follows. Sample 1 A = 3 - 5 6 x l . 9 6 = 6 . 9 7 % degree of tolerance B = 17 . 70 x 1 . 9 6 = 3 S . 6 9 % " " " Sample 2 A = S . 1 9 x 1 . 9 6 = 8 . 2 1 % « « « B = 7 . 2 3 x 1 . 9 6 = 1 S . 1 7 % " 1 1 " Sample 3 A = 7 . 0 2 x 1 . 9 6 = 1 3 . 7 5 % " " " B = 9 . 0 1 x 1 . 9 6 = 1 7 . 6 5 % " " " The standard deviations of the subsamples of A and B f o r sample 2 were + S . 1 9 and + 7 . 2 3 respectively. Once again method A showed l e s s v a r i a t i o n between subsamples than method B. In sample 3 , the standard deviations f o r A and B were + 7 . 0 2 and + 9 . 0 1 respectively. Again greater v a r i a b i l i t y existed i n the l a t t e r . I t i s of i n t e r e s t to note that the number of grains to be counted would depend upon the degree of tolerance chosen and would be i n d i c a t i v e of the more e f f i c i e n t method i n saving time. - kk -The average number of grains counted i n the 2k squares per subsample i n method A for samples 1, 2 and 3 was 36*+.6, 3*+*+»5 and 359.8 respectively. The average number of grains counted per subsample f o r B for the three samples -was 100. An example of the use of table 2 f o r computing actual pollen sample size needed f o r obtaining a r e l i a b l e estimate of v i a b i l i t y , i s the comparison of two a r b i t r a r y choices of l e v e l of tolerance, 2% and 10$ i n the v a r i a b i l i t y . The number of grains required f o r counting by the two methods allowing a 2% tolerance i s as follows. Sample 1 A = 36l+.6 x 12.Ik = k,k26.2k grains to be counted B = 100 x 300.8*+ = 30,08*f.OO " " " " Sample 2 A = 3^.5 x 16.85 = 5,80*+.82 " » » « B = 100 x 50.19 = 5,019.00 " » " » Sample 3 A = 359.8 x *+7.26 = 17,00*+.1*+ » " » « B = 100 x 77.88 = 7,788.00 » « " «» The number of pollen grains required when a 10$ tolerance was permitted would be. Sample 1 A = 36*+.6 X 0.*+8 — 175.00 grains to be counted B = 100 X 12.00 1,200.00 ti tt » tt Sample 2 A = 3^.5 X 0.67 230.81 H tt tt tt B = 100 X 2.1+0 = 2*f0.00 it tt tt it Sample 3 A = 359.8 X 1.89 680.02 n tt 11 ti B = 100 X 3.11 - 311.00 tt tt tt 11 From the calculations above i t i s seen that the degree of t o l e r -ance desired would determine the labour involved i n counting. For example i n sample 1 where 2% tolerance was allowed, using table 2, the number of subsamples required to give a r e l i a b l e estimate f o r B was 300.8*+, and where the degree of tolerance was % the number of subsamples would be 12. This difference i s - 4-5 -magnified when transposed to 30,084- and 1,200 p o l l e n g r a i n s . O b v i o u s l y over 28,000 more g r a i n s would need to be counted to reduce the t o l e r a n c e from 10% to 2%, By f u r t h e r i n s p e c t i o n of the c a l c u l a t i o n s i n both cases i t can be seen t h a t f o r sample 1, method A i s more e f f i c i e n t than B, s i n c e fewer g r a i n s need to be counted. In sample 2, the v a l u e s f o r n i n both i n s t a n c e s a r e higher f o r B than A, method B appears to be more e f f i c i e n t a t the 2% t o l e r a n c e because approximately 800 g r a i n s l e s s need to be counted than f o r A. At the 10% t o l e r a n c e l e v e l , both methods r e q u i r e almost i d e n t i c a l numbers of g r a i n s , and thus could be considered e q u a l l y e f f i c i e n t . S i m i l a r l y i n sample 3, method B was more e f f i c i e n t a t the 2% and 10% degree of t o l e r a n c e . The f o r e g o i n g c o n s i d e r a t i o n s of the l i m i t e d data emphasize t h a t tomato p l a n t s may be very v a r i a b l e i n producing v i a b l e p o l l e n . A l a r g e r number of g r a i n s was r e q u i r e d to g i v e a r e l i a b l e estimate f o r the o l d e r p l a n t s of sample 1 under the growing c o n d i t i o n s f o r p l a n t s of l o t 4-. In c o n t r a s t , the young vig o r o u s p l a n t s of l o t 6 provided samples 2 and 3> which had s i m i l a r h i g h percentages of mature p o l l e n . The former p l a n t s could be assessed by method A, counting g r a i n s i n the m i c r o s c o p i c f i e l d per subsample, -whereas the l a t t e r p l a n t s could be c o n f i d e n t l y assessed u s i n g method B of choosing 2 or 3 groups of 100 g r a i n s g i v i n g a range of 200 -300 g r a i n s . Obviously counts of 100 g r a i n s per sample would be inadequate f o r these s t u d i e s . Thus percentages g i v e n here are based on counts ra n g i n g between 300-4-00 g r a i n s per subsample. - 1+6 -(b) Methods of p o l l e n c o l l e c t i o n . Among the s e v e r a l sources c o n t r i b u t i n g to the i n a c c u r a t e , estimate of p o l l e n v i a b i l i t y , d i f f e r e n c e s i n stages of m a t u r i t y of p o l l e n g r a i n s i n samples could account f o r gre a t v a r i a t i o n i n p o l l e n v i a b i l i t y d e t e r m i n a t i o n s . ( i ) Methods of e x t r a c t i o n . The methods employed i n e x t r a c t i n g p o l l e n samples i n f l u e n c e the r e l a t i v e amounts of immature and mature g r a i n s p r e s e n t i n samples, as shown i n the data of t a b l e s 3 to 6 i n c l u s i v e l y . Experiment 1 9 A n a l y s i s of data i n t a b l e 3, shows a l l i n t e r a c t i o n s to be s i g n i f i c a n t . The d i f f e r e n c e s i n p o l l e n v i a b i l i t y between the needle and v i b r a t o r methods of p o l l e n c o l l e c t i o n were a f f e c t e d by the s t a i n i n g and i n v i t r o techniques. A l s o the d i f f e r e n c e s i n p o l l e n v i a b i l i t y between the s t a i n i n g and i n v i t r o methods v a r i e d w i t h each of the two v a r i e t i e s , Puck and Bonny Best. I n s p e c t i o n of the data shows c o n s i s t e n t l y lower percent ages of mature p o l l e n f o r the v i b r a t o r than the needle methods when the s t a i n i n g technique was used. Experiment 2. The two methods of p o l l e n e x t r a c t i o n were then t e s t e d on a l l ten v a r i e t i e s as shown i n the data of t a b l e h. A n a l y s i s of data shows a l l i n t e r a c t i o n s to be s i g n i f i c a n t . The d i f f e r e n c e s i n p o l l e n v i a b i l i t y between the needle and v i b r a -t o r methods of p o l l e n c o l l e c t i o n were a f f e c t e d by (1) the s t a i n i n g and i n v i t r o techniques and (2) the v a r i a t i o n i n v i a b i l i t y r e s u l t s of a l l 10 v a r i e t i e s , due to g e n e t i c c o n s t i t u t i o n of the tomato l i n e s . - 4-7 -Table 3. V i a b i l i t y percentages of p o l l e n c o l l e c t e d by (1) needle i n the i n t e r i o r of the anther (NI) and (2) the p o l l e n v i b r a t o r , of Puck (P) and Bonny Best ( B). Needle V i b r a t o r V a r i e t i e s T e s t s Sub- Stamens Flowers samples 1 2 3 4 - 1 2 3 4-S t a i n i n g 1 2 95 80 53 81 4-7 4-7 26 31 75 89 67 83 4-7 4-7 33 32 16 93 76 58 4-6 4-0 33 29 P X X 61.9 87.2 65.2 73.9 4-6,6 44.6 30,6 30.6 72.1 38.1 In v i t r o 1 2 3 59 63 78 25 32 4-5 28 58 63 61 67 32 27 4-8 30 66 64- 61 72 56 2 9 4-1 19 56 X X 61.9 61.6 72.2 37.6 29.3 44.6 25.6 59.9 58.3 39.9 S t a i n i n g 1 2 3 87 83 73 77 12 53 50 36 95 79 81 77 15 52 51 31 73 81 72 67 15 4-9 53 35 B X X 74-.9 80.9 75.2 73 . 5 13.9 51.2 51.2 33.9 78.7 37.6 In v i t r o 1 2 _1 59 66 51 57 67 64- 71 63 75 55 60 52 66 61 69 59 73 61 54- 27 55 62 75 65 X X 68.9 60.6 54-.9 4-5.2 62.6 62.2 71.5 62.2 57.S 64-. 7 A n a l y s i s of v a r i a n c e . Source D.F. M.S. F T o t a l 95 -Exp. U n i t s 31 861.96 6.22 Treatments 7 304-5.85 13.53 Methods (M) 1 11180.00 4-9.68** V a r i e t i e s (V) 1 1350.00 5.99* Tests (T) 1 57.00 n.s + 2.36 M x V 1 533.00 M x T 1 5012.00 22.27** V x T 1 14-77.00 6.56* M x V x T 1 1712.00 7.60* Exp. E r r o r 24- 225.00 1.30 Sampling E r r o r 64- 172.22 + n.s. i s used to denote F val u e s of l e s s than 1 and thus non-s i g n i f i c a n t . Table V i a b i l i t y percentages of p o l l e n c o l l e c t e d by (1) the needle on the s u r f a c e of the stamen (N) and (2) the v i b r a t o r (V), u s i n g a s i n g l e f l o w e r of each of the two v a r i e t i e s , Puck (P) and Bonny Best (B), and t h e i r , r e c i p r o c a l c r o s s e s . T e s t s B Methods N V N V V a r i e t i e s (BxP)F (PxB)F (BxP)F (PxB)F (BxPF (PxB)F E 498 (PxM-98)F N N V N V I T \V: H V N V N N Sub-samples 1 63 33 70 35 79 35 78 4-9 79 4-8 65 21 71 9 31 14- 79 12 31 39 57 39 69 4-2 79 29 73 4-8 57 50 58 15 74- 6 34- 14- 77 15 70 35 73 66 65 30 70 27 77 4-7 80 51 62 22 70 6 26 16 4-7 14- 52 4-1 64-3 4-59 679 356 759 303 759 4-79 719 4-96 616 193 716 69 303 14-6 676 136 509 383 551 518 531 619 608 404- 393 224- 4o6 446 S t a i n i n g 2 3 x In v i t r o 1 2 3 x X 53 55 17 33 9 21 16 20 17 4-2 35 34- 14- 8 13 26 36 19 30 17 61 57 18 39 18 23 16 21 15 46 23 33 37 6 18 30 36 17 29 16 61 53 19 3,3 21 32 16 21 22 4-7 33 28 4-6 7 15 29 4-3 19 23 18 583 54-9 179 34^ 9 159 253 179 206 179 449 303 316 323 69 153 283 383 183 2?3 169 566 264- 206 193 314- 309 196 218 283 221 A n a l y s i s of variance, Source D.F. M.S. F T o t a l 119 -Treatments 39 - -V a r i e t i e s (V) 9 609.7 12.7** Methods (M) 1 9048.0 189.2** T e s t s (T) 1 11175.0 233.7** V x M 9 932.0 19. *+** V x T 9 772.3 16.1** M x T 1 7906.0 165.3** V x M x T 9 154-.4- 3.2** E r r o r 80 4-7.8 - h9 ~ Inspection of the data shows consistently lower percentages of viable pollen in the staining technique for the vibrator than for the needle methods of collection. The same trend exists in the in vitro technique except in the Bonny Best variety and the (BxP)F]_, (BxP)F2, (PxB)F]_, and (PxB)F3 crosses. The inconsistency in results caused by the interactions of factors in experiments 1 and 2 led to further work, and experiments 3 and h were done to obtain further information on methods of extrac-tion. Experiment 3. Table 5. Vi a b i l i t y percentages of pollen collected by (1) water (W), (2) the needle in the interior of the stamen (NI) and (3) the needle on the surface of the stamen (NS); v i a b i l i t y being determined by staining. Puck Bonny Best Methods W NI NS W NI NS 1 86 81 96 81 78 89 2 95 97 80 79 93 3 91 89 91 75 79 82 h Sh 91 91 77 80 79 5 92 89 81 8*f 85 90 X 91. 89.0 91.2 79.*+ 80.2 86.6 X 90.5 82.0 Analysis of variance. Source D.F. M.S. F Total 29 Treatments 5 Varieties (V) 1 538.0 2*+.9** Methods (M) 2 52.5 2.*+ M x V 2 3^.0 1.5 Error 2*+ 21.6 Analysis of data in table 5 shows the interaction, variety x method, i s non-significant. No significant differences in v i a b i l i t y - 50 -e x i s t e d between the jthree methods of e x t r a c t i o n , but there was a s i g n i f i c a n t d i f f e r e n c e a t t r i b u t e d to v a r i e t y . Puck had a g r e a t e r percentage v i a b l e p o l l e n than Bonny Best. Experiment h. R e s u l t s of experiment 3 f a i l e d to show any d i f f e r e n c e s between the 3 methods of e x t r a c t i n g p o l l e n samples by the needle. Conse-quently f o u r methods of p o l l e n e x t r a c t i o n s were st u d i e d i n experiment k. To emphasize the e f f e c t of the methods of e x t r a c t i o n , data are rep o r t e d only on the percentages of immature p o l l e n g r a i n s obtained i n the samples. Table 6 . Percentages of immature g r a i n s of Puck u s i n g h methods of e x t r a c t i o n , v i a b i l i t y b e i n g determined by s t a i n i n g . Subsamples Methods 1 2 3 if 5 6 7 a 9 10 X V i b r a t o r Needle (NS) Tapping Water 10.0 0.5 6.0 7.0 1.2 2.0 h.O 7.0 0.5 h.o h.o 9.0 O.h 5.0 2.0 8.0 7.0 0.5 1.5 h.o 5.0 3.0 2.0 12.0 0.3 *f.O 5.0 9.0 7.0 1.2 0.3 3.0 *f.O 5.0 3.0 8.0 0.3 *f.O 3'° 8.h 0.6 h.l 2.h X h.ti 3.5 3.B h.l 3.8 3.8 5.3 h.5 3.5 3.8 A n a l y s i s of v a r i a n c e . Source D.F, M.S. F T o t a l Subsamples Methods E r r o r 39 9 3 27 1.37 102A5 1.30 n.s 78.80** Duncan's t e s t . Methods Needle Water Tapping V i b r a t o r x 0.67 3.'+0 *+.10 8.»+0 In t a b l e 6, i t can be seen t h a t h i g h l y s i g n i f i c a n t d i f f e r e n c e s e x i s t e d between the methods of sampling. Duncan's t e s t r e v e a l e d f u r t h e r t h a t the v i b r a t o r gave s i g n i f i c a n t l y higher percentages of immature p o l l e n than any of the remaining three methods. No s i g n i f -i c a n t d i f f e r e n c e s were found between the water and tapping methods, - 51 -both of which gave s i g n i f i c a n t l y g r e a t e r percentages of immature p o l l e n than the needle method. Thus p a s s i n g a d i s s e c t i n g needle on the s u r f a c e of the stamen appears to be the most d e s i r a b l e procedure. ( i i ) Time of p o l l e n c o l l e c t i o n i n r e l a t i o n to age of f l o w e r . The age a t which the f l o w e r i s sampled can a f f e c t the per-centages of immature g r a i n s i n p o l l e n samples, as shown i n t a b l e s 7 and 8. (1) P o l l e n m a t u r i t y a t d i f f e r e n t bud and f l o w e r stages. Experiment 5» Four f l o w e r s were sampled s i m u l t a n e o u s l y , thus p r o v i d i n g f l o w e r s i n a sequence of m a t u r i t y from the a n t h e s i s stage to i n c r e a s i n g l y immature buds. The e f f e c t s of t h i s g r a d i e n t i n maturation are shown i n t a b l e 7. There were h i g h l y s i g n i f i c a n t d i f f e r e n c e s between the per-centages of immature p o l l e n i n the f o u r f l o w e r s on the same t r u s s . The Duncan's t e s t showed t h a t the f i r s t two f l o w e r s had l e s s immature p o l l e n than the l a s t two which were e s s e n t i a l l y buds, and t h i s d i f f e r e n c e was h i g h l y s i g n i f i c a n t . (2) P o l l e n v i a b i l i t y as a f f e c t e d by l o n g e v i t y or l i f e -span of the f l o w e r . Experiment 6 . The time a t which p o l l e n samples a r e taken d u r i n g the p e r i o d the f l o w e r remains open appears to have gr e a t i n f l u e n c e on p o l l e n v i a b i l i t y as shown i n t a b l e 8. Table 7. Percentages of immature grains from four flowers at different stages on tjae same truss of each of 10 varieties, v i a b i l i t y determined by staining. , 131 nstlj- Varieties -CLOCK r JLOwerS P - B (BxP)F (PxB)F (BxP)F (PxB)F (BxP)F (PxB)F 1+98 (Px!+98)F _x 1 1>+ 12 8 9 15 7 15 9 2*+ 9 12.2 x 2 58 10 9 89 22 15 97 16 32 6 35.*+ X 3 99 93 6*+ 95 99 22 99 83 77 97 82.8 h 99 99 99 96 98 97 99 98 90 99 97.7 X 67.0 53.5 ^5,03 72.2 58.5 35.2 77.5 51.5 55.7 52.7 1 20 21 21 16 39 32 9 13 39 25 23.5 II 2 16 19 10 7 10 31 21 16 68 36 23.!+ 3 25 86 97 7 95 92 86 93 98 *+7 72.6 i+ ? 8 95 97 95 99 ql+ 98 95 9§ 55 88.1+ X 29.7 55.2 56.2 31.2 60.7 62c2 53.5 5^ .2 75.7 *+0.7 Analysis of variance. Source D.F. M.S. F Total 79 — — Blocks 1 1+95.0 n.s Treatments 39 - -Flowers (F) 3 27331.0 1+8.2** Varieties (V) 9 379.2 n.s F x V 27 21+1+.1 n.s Error 39 567.0 Duncan's test. Flowers 1 2 3 1+ x 17.8 2 9 A 77.7 92.9 Table 8. Percentages of viable pollen from five stamens of each of 10 varieties with one stamen from each flower being examined successively over a period of 5 days. Tests Days p g ( B x P ) F ( P x B ) F (BxP)F (PXB)F (BXP)F ( PX B ) F 4-98 (PX4-98TF Staining In vitro 1 95 85 75 85 91 81 S9 65 71 86 78.3 2 91 71 56 23 96 76 23 4-5 12 79 57.2 3 81 65 S5 89 83 71 65 6 29 66 60.0 4- 93 5H- 67 33 69 56 71 18 26 61 54-.8 5 P 61 52 . 67 74- 56 7 7 64- 4-9.6 X 87.0 66.0 59.0 59.4- 82.6 63.4- £2.8 28.2 29.0 71.2 1 69 74- 4-9 3 7 6 6 56 6 5 S5 4-1 71 57.3 2 7 5 6 5 3 8 21 86 73 51 65 19 53 54-. 6 3 4-9 31 51 5 70 23 4-6 15 23 61 37.4-4-38 6 2 9 21 9 10 25 5 12 13.7 5 51 3 11 15 50 11 12 16 6 19 19.4-x 56A Analysis of variance. Source D.F. M.S. F Treatments 99 — — Tests (T) 1 13806.0 91.36** Days (D) 4- 4-129.5 27.32** Varieties (V) 9 24-04-. 1 15.91** V x D 239.2 1.58 T x D 4- 999.8 6.66** T x V 9 538.6 3.56** Error 36 151.1 - 5*f -A n a l y s i s of data i n t a b l e 8 shows main e f f e c t s and i n t e r a c t i o n s i n v o l v i n g T (method of e s t i m a t i n g v i a b i l i t y ) to be h i g h l y s i g n i f -i c a n t . The d i f f e r e n c e s between s t a i n i n g and i n v i t r o had e f f e c t s s i m i l a r to those r e p o r t e d i n p r e v i o u s experiments. The d i f f e r e n c e s between stamens on days of sampling a given f l o w e r were h i g h l y s i g n i f i c a n t , w i t h decreases i n the amount of v i a b l e p o l l e n a s s o c -i a t e d w i t h i n c r e a s i n g age. Thus, i t appears t h a t v a r i a b i l i t y i n r e s u l t s of p o l l e n counts could be due to d i f f e r e n c e s i n the time of c o l l e c t i o n when the f l o w e r had been opened, even i f the f l o w e r s appeared to be f r e s h or i n good c o n d i t i o n . Maximum v i a b i l i t y would be assured i f p o l l e n was sampled when the f l o w e r reached f u l l a n t h e s i s , i . e second day a f t e r f i r s t r e f l e x i n g of p e t a l s . Although, a l l 10 v a r i e t i e s showed a d e c l i n e i n v i a b i l i t y , t h ere were s i g n i f i c a n t d i f f e r e n c e s among v a r i e t i e s w i t h i n c r e a s i n g time. (c) V a r i a t i o n due to d i f f e r e n c e s i n l o c a t i o n of p o l l e n sources on p l a n t s . D i f f e r e n c e s i n l o c a t i o n of p o l l e n sources on the p l a n t may account f o r some of the v a r i a b i l i t y i n p o l l e n v i a b i l i t y determ-i n a t i o n s . ( i ) P o l l e n v i a b i l i t y a t d i f f e r e n t zones of the same stamen. Experiments 1 and 2. The e f f e c t of zonal d i f f e r e n c e s of p o l l e n samples, taken from the same stamen, on p o l l e n v i a b i l i t y d e t e r m i n a t i o n s , i s shown i n the data of t a b l e s 9 and 10. Table 9. P o l l e n v i a b i l i t y a t 3 zones i n the same stamen of each of 3 stamens i n a s i n g l e f l o w e r of each of the two v a r i e t i e s , Puck (P) and Bonny Best ( B ). V i a b i l i t y determined by s t a i n i n g and i n v i t r o . V a r i e t i e s Tests Stamen ] L Stamen < 3 Stamen 3 Dampj.es Tip Mid Base Ti p Mid Base T i p Mid Base 1 99 97 97 99 97 96 95 97 97 2 98 99 94 95 93 98 99 94 94 S t a i n i n g 3 89 95 97 98 96 93 98 9? 96 X 9 5 . 2 y o . y 95.9 97.2 95.7 97.2 y4 .p X 95.1 95.1 94.9 P 1 51 50 52 60 55 50 31 27 41 2 56 49 31 51 56 52 58 43 45 In v i t r o 3 16 49 52 hJ 55 53 40 31 27 X 40.9 {+9.2 44 .9 53.2 55.2 51.6 "V2.9 33.6 37.6 X 44.6 52.9 37 .7 B 1 86 90 77 84 78 77 91 82 85 2 94 85 89 95 96 72 78 83 86 S t a i n i n g 3 90 79 85 §9 74 8 l 87 94 79 x W79 8475 5375 $%2 8 2 . 5 7675 B5T2 86T2 837T x 85 .2 82 .0 84 .1 1 58 53 31 47 52 53 19 44 53 2 49 52 18 36 47 43 51 26 25 In v i t r o 3 51 27 41 24 19 23 17 25 19 x 5 2 3 4379 2979 3T7S 3972 397S 2879 31 .6 32 .3 x 41.8 37.8 30 .6 - 56 -A n a l y s i s of v a r i a n c e . Source D.F. M.S. F T o t a l 107 — — Rep. (Stamens) 5 718.6 11.31** Treatments 11 — — V a r i e t i e s (V) 1 26U-0.0 U-1.57** T e s t s (T) 1 6H-827.0 1020.89** Zones (Z) 2 113.0 1.77 V x T 1 59.0 n.s V x Z 2 60.0 n.s T x Z 2 10.0 n.s V x T x Z 2 3.0 n.s E r r o r 91 63.5 Duncan's t e s t . Stamens 3 B 2 B 1 B 3 P I P 2 P X 57.9 60.5 6k.1 66.9 70.6 7kf7 A n a l y s i s o f data i n t a b l e s 9 and 10, shows no s i g n i f i c a n t d i f f e r e n c e s between the v i a b i l i t y of p o l l e n from the three zones ( t i p , middle, and base) of the same stamen. Duncan's t e s t i n t a b l e 9» r e v e a l e d t h a t s i g n i f i c a n t d i f f e r -ences e x i s t e d between stamens 3 and 2, and stamens 3 and 1, i n the same f l o w e r from the v a r i e t i e s of Puck and Bonny Best r e s p e c -t i v e l y . Puck gave s i g n i f i c a n t l y higher percentages of v i a b l e p o l l e n than Bonny Bes t . The s t a i n i n g technique gave s i g n i f i c a n t l y higher and more uniform percentages of v i a b l e p o l l e n than the i n v i t r o germination. I t was n o t p o s s i b l e from the data i n t a b l e 10 to d i f f e r e n t i a t e between the e f f e c t s of p o l l e n v i a b i l i t y which are due to v a r i e t y and those which are due to method of v i a b i l i t y d eterminations s i n c e both f a c t o r s i n t e r a c t e d . Table 10. Pollen viability at 3 zones in one stamen of a single flower of each of ten varie-ties; viability determined by staining and in vitro. Block Tests Zones P B (BxP)F (PxB)F (BxP)F (PxB)F (BxP)F (PxB)F 4-98 (Px4-98)F X Tip 89 78 75 96 89 4-7 75 87 69 77 78.2 Mid 88 66 73 86 94- 87 71 73 89 65 84- 80.5 Staining Base 9k 88 72 86 82 71 82 76 84- 80.6 X 90.3 77.3 73.3 89.3 88.3 68.3 72.9 85.9 69.9 81.6 I Tip k9 36 69 ^9 67 56 77 86 39 64- 59.2 TT%** 4 j» Mr* Mid k9 k5 66 79 67 69 77 87 67 67 67.3 m v i t r o Base 63 4-1 54- 51 83 75 59 82 ^7 66 61.1 X *3.6 40.6 62.9 59.6 72.2 66.6 70.9 84-. 9 4-7.6 65.6 Tip 9k 69 81 89 87 38 78 86 72 87 78.1 C? ^  r\ *t V N 4 v\ /•» Mid 83 66 79 87 81 58 79 91 79 85 78.8 beaming Base 88 67 84- 82 89 71 79 78 81.8 -r -r X 88.3 67.3 81.3 85.9 85.6 55.6 83.9 87.3 76.6 83.3 11 Tip 59 62 4-2 k8 78 69 78 59 67 73 63.5 s %*  Mid 65 61 67 52 89 57 76 83 61 81 69.2 ±n v i t r o Base 63 61 70 53 40 75 70 78 69 75 65.H-£ 2 7 2 6 1 7 2 5976 50V9 6879 6679 W*5 7372 6576 7672 - 58 -Analysis of variance. Source Total Blocks 119 1 M . S . 81.0 n.s F Treatments Varieties (V) Zones (Z) Tests (T) 59 9 2 567.0 178.0 7099.0 72.5 388.0 122.5 69.6 102.7 5.5** 1.7 69.1** V x Z x T Error V x Z V x T Z x T 1 18 9 2 18 59 n.s n.s 3.7** 1.2 ( i i ) Pollen v i a b i l i t y of different stamens of the same flower. Experiment 3« No variation in pollen v i a b i l i t y was found to be due to zonal sources of pollen, hence a more detailed study of the variation in pollen v i a b i l i t y between stamens of the same flower was made, and data are shown in table 11. Analysis of data in table 11 shows a l l interactions to be significant. Hence i t was not possible to evaluate the difference in pollen v i a b i l i t y between the stamens within a flower, because the pollen v i a b i l i t y results were affected by the variability due (1) to the staining and in vitro methods and (2) the four flowers from which the samples were taken. Also by inspection i t is clear that there i s great variation from stamen to stamen within a flower. ( i i i ) Pollen v i a b i l i t y of flowers on the same truss. Experiment 4-. The effect on v i a b i l i t y of pollen samples taken from different flowers on the same truss, i s shown in the data of table 12. Table 11. Pollen v i a b i l i t y percentages of six stamens from the same flower of Puck; p o l l e n v i a b i l i t y determined by staining and in vitro. Staining In vitro Flowers Samples Stamens Stamens 1 2 3 4 J 6 1 2 3 4 6 1 6k 81 93 88 59 77 55 50 75 66 61 59 2 57 77 95 86 54 73 58 65 59 80 63 1 3 69 78 98 88 61 78 49 65 42 34 71 75 4 82 97 83 62 71 ft 72 66 71 86 61 X 65,2 79.5 95.7 86.2 59.0 74.5 50.0 61.2 62.0 57.5 74.5 64.5 X 76.7 61.6 1 48 59 81 89 95 6eJ 33 63 19 71 34 21 2 49 75 94 77 87 56 65 29 44 65 81 19 2 3 56 7? 97 78 91 51 4l 45 53 49 63 35 4 54 64 95 67 93 41 52 61 31 52 43 18 X 51.7 68.2 91.7 77.7 91.5 54.0 47.5 49.5 36.7 59.2 55.2 23.2 X 72.4 45.2 1 98 96 93 78 64 71 30 43 16 42 49 54 2 79 95 81 69 72 68 44 61 21 37 78 46 3 3 83 95 96 81 69 65 54 21 51 65 43 3h 4 81 94 75 71 61 46 ?9 33 ,36 55 61 X U9? 91.7 91.0 75.7 69.0 66.2 43.5 41.0 30.2 45.O 56.2 48.7 X 84.0 44.1 1 89 69 78 91 47 85 65 45 32 68 61 45 2 94 68 95 82 71 78 34 45 23 65 42 18 4 3 87 69 82 89 86 87 36 29 61 57 19 56 4 71 52 91 56 77 21 33^  28 50 25 51 X 89.7 69.2 76.7 88.2 65.0 81.7 39.0 38.0 36.0 60.0 36.7 42.5 X 78.4 42.0 - 6 0 -Analysis of variance. Source D.F. Total Treatments Tests (T) Flowers (F) Stamens (S) T x F T x S F x S T x F x S Error 191 47 1 3 5 3 5 15 15 144 M.S. 39704.0 •1037.0 547.8 1214.3 488.6 610.4 539.8 124.0 320.1** 8.36** 4.41** 9.79** 3.94** 4,92** 4.35** Data in table 12 were not analysed s t a t i s t i c a l l y due to the complexity involving factorials, split-plot, subsampling and non-interpretable interactions. Inspection of the data, however, revealed no appreciable differences in pollen v i a b i l i t y among the three flowers examined from the same truss. The in vitro v i a b i l i t y determinations are comparatively lower than the staining. Puck pollen showed consis-tently higher v i a b i l i t y percentages than Bonny Best. (iv) Pollen v i a b i l i t y of different trusses of the same plant. Experiment 5. The variation in pollen v i a b i l i t y of different trusses on the same plant could influence the results of v i a b i l i t y determinations as shown in table 13. Analysis of data in table 13 showed that some highly significant differences existed between the subsamples taken from a single flower. Although the F-test did not show differences among the pollen viab-i l i t y of the three trusses, the Duncan's test revealed significant differences among them. This test showed the differences in pollen v i a b i l i t y of truss 1, and truss 3» to be significant, but differences between truss 1 and 2, and also between 2 and 3 were non-significant. Table 12. Via b i l i t y percentages of pollen of Puck (P) and Bonny Best (B) of different flowers on the same truss. Staining In vitro  Varieties Plant No. Flowers Block I Block II Block I Block II Subsamples Subsamples Subsamples Subsamples  1 2 3 1 2 3 1 2 3 1 2 3 x 1 93 94 91 95 91 98 71 74 56 31 78 53 77.0 1 2 95 92 84 97 93 95 62 49 31 44 78 64 73.6 3 86 90 93 89 93 97 57 53 53 53 21 62 70.5 1 80 98 97 87 95 93 61 64 54 45 76 53 75.2 P 2 2 92 97 98 97 91 96 36 78 44 44 42 29 70.3 3 93 95 86 94 99 98 57 76 78 19 27 27 70.7 1 96 94 97 91 95 97 44 60 45 31 35 60 70.4 3 2 96 92 95 87 93 99 49 51 75 58 24 1 5 6 9.4 3 91 91 93 92 92 98 29 48 61 27 71 62 71.2 1 89 8 3 92 99 95 94* 31 34" 41 51 2 l ~ 64.9 1 2 83 89 86 93 93 95 21 62 59 24 19 61 65.4 3 93 90 96 97 91 87 65 58 45 18 23 51 67.8 1 95 95 92 84 93 94 4 l 56 47 16 19 4l 64.4 B 2 2 85 89 87 88 81 85 57 45 62 44 35 23 65.0 3 93 93 93 86 82 79 3 5 50 41 61 21 62 66.3 1 94 86 89 93 93 92 53 4 7 53 3 6 3 7 29 66.8 3 2 87 89 89 89 92 93 28 61 46 35 46 60 67.9 3 94 87 91 91 87 91 31 33 33 47 60 43 65.6 - 62 -Table 13. V i a b i l i t y percentages of p o l l e n from three t r u s s e s of the same Puck p l a n t , determined by s t a i n i n g . Truss 1 Truss 2 Truss 3  Flowers 1 2 3 M- 1 1 2 3 M- 1 2 3 k 1 91 85 78 87 88 81 73 91 65 90 6k 75 , S 2 89 82 87 89 73 86 69 86 66 87 51 67 •§o. 3 93 9k 82 dk 89 83 71 Qk 73 88 57 65 w s x 90.9 86.9 82.5 86.5 83.2 83.2 70.9 86.9 67.9 88.2 57.2 68.9 8 x 86.7 81.1 70.6 A n a l y s i s of v a r i a n c e . Source D.F. M.S. F T o t a l 3l? - = Exp. U n i t s 11 332.09 Samp. E r r o r 2k 22.08 Trusses 2 800.00 3.50 Exp. E r r o r 9 228.11 10.33** Duncan's t e s t . Trusses 3 2 1 x 70.6 81.1 86.7 A trend can be observed t h a t the percentage v i a b i l i t y f e l l o f f from t r u s s 1 to t r u s s 3. T h i s s i t u a t i o n may not always be the case s i n c e the v i g o u r of the p l a n t may determine the extent of decreases i n p o l l e n v i a b i l i t y . I I I . Comparison of methods of p o l l e n v i a b i l i t y d e t e r m i n a t i o n s . One of the g r e a t e s t d i f f i c u l t i e s encountered i n the e s t i m a t i o n of p o l l e n v i a b i l i t y of f l o w e r s taken from a s i n g l e p l a n t i s the extreme v a r i a b i l i t y which e x i s t s i n p o l l e n samples. The v a r i a b l e r e s u l t s are due not only to v a r i o u s l o c a t i o n s from which the sample i s taken, but a l s o to the v a r i a b i l i t y r e s u l t i n g from the techniques used i n determining the v i a b l e count. - 6 3 -(a) Comparison of the s t a i n i n g and i n v i t r o techniques. Experiment 1. D i f f e r e n c e s between the s t a i n i n g and i n v i t r o procedures as seen i n data i n t a b l e lh are h i g h l y s i g n i f i c a n t because the per-centages of v i a b l e p o l l e n determined by s t a i n i n g are c o n s i s t e n t l y very much higher than the r e s u l t s from the i n v i t r o t e s t s on the same samples. The a n a l y s i s of data shows a s i g n i f i c a n t i n t e r a c t i o n between v a r i e t y x v i a b i l i t y , i n d i c a t i n g t h a t the d i f f e r e n c e s bet-ween the two techniques were not the same f o r a l l v a r i e t i e s . These r e s u l t s c o n f i r m the o b s e r v a t i o n s of previous experiments, where the two methods were employed s i m u l t a n e o u s l y , and these ex-periments r a i s e the q u e s t i o n as to which procedure i s the more r e l i a b l e f o r e s t i m a t i n g the p o l l e n which would be f u n c t i o n a l under n a t u r a l c o n d i t i o n s f o r r e p r o d u c t i o n on the tomato p l a n t . (b) Experiments on i n v i v o germination of p o l l e n . The v a l i d i t y of the s t a i n i n g and i n v i t r o techniques could only be a s c e r t a i n e d by comparison w i t h the n a t u r a l c o n d i t i o n s f o r p o l l i n a t i o n and growth of p o l l e n i . e examination i n v i v o . To count the number of t i n y tubes from germinated p o l l e n on the p i s t i l of a tomato p l a n t , i n order to e s t a b l i s h an i n v i v o q u a n t i t a t i v e measure, i s very d i f f i c u l t as shown by the f o l l o w i n g experiments. Experiment 1. P r e l i m i n a r y t r i a l s u s i n g M a r t i n ' s (1959) i n v i v o procedure i n d i c a t e d the f e a s i b i l i t y of the t e s t , but when l a r g e amounts of p o l l e n were used the r e s u l t i n g t h i c k f l u o r e s c e n t bands of aggreg-ated p o l l e n tubes prevented reasonable counts of the germinated p o l l e n w i t h i n the s t y l e . Table 14-. Pollen v i a b i l i t y percentages determined by staining and i n v i t r o germination on a single flower from each of 10 v a r i e t i e s . Sub- Va r i e t i e s l e s t s samples P a (BxP)F (PxB)F vBxP)F" (PxB)F (BxP)F (PxB)F 4-98 ( P x W ) F 1 86 73 80 85 88 82 88 84- 73 79 Staining 2 89 78 76 86 75 76 70 82 71 6 5 3 8? 74- 79 83 86 79 81 82 70 67 X 8 7 . 2 74-. 9 7 8 . 2 84-. 5 82 .9 7 8 . 9 7 9 . 5 82.5 7 1 . 2 7 0 . 2 1 53 4-6 4-0 57 77 6 9 33 37 4-2 S 5 In v i t r o 2 33 ? 6 25 44 62 S9 S 9 4-2 5S 4-8 51 3 4? 4-3 60 63 38 4-6 4-1 4-9 X 4-7.2 3 9 . 9 4-2.2 5 9 . 6 62 .9 5 8 . 9 37.6 4-5.6 4-3.6 4-8.2 Analysis of variance. Source D.F. M.S. F Total -59 - -Treatments 19 - — Tests (T) 1 1 3 9 2 3 . 0 2 8 7 . 6 * * V a r i e t i e s (V) 9 2 2 1 . 0 4 . 5 * * V x T 9 1 0 7 . 6 2 . 2 * Error 40 48 .4 - 65 -The i n vivo q u a l i t a t i v e test -would have been easiest when a. small number of grains were used; and i n the f i r s t experiment, 10 pollen grains were placed on each of the stigmatic surfaces. No germination occurred on any of the 12 stigmas used i n experiment 1. Experiment 2 . The s u r p r i s i n g l y negative r e s u l t s i n experiment 1, and the successful germination of masses of pollen i n preliminary work, required a study between these extremes. An a r b i t r a r y choice of 100 grains resulted i n the germination data i n table 15. Table 15. Percentage pollen germination i n vivo on 5 stigmas of each of two v a r i e t i e s , Puck (P) and Bonny Best (B), using 100 grains per stigma. V a r i e t i e s Stiemas X 1 2 3 4 5 P B 98 21 57 9 35 hi 30 23 12 17 46.4 22.2 Analysis of variance. Source D .P. M.S. F Total V a r i e t i e s Error 9 1 8 1465.00 614.25 2 .38 Data i n table 15> show that successful germination was ob-tained, when 100 grains were used as compared with 10 grains used in experiment 2. The density of fluorescent pollen tubes was well within l i m i t s of counting. Inspection of the data suggests that Puck gave higher ger-mination percentages than Bonny Best, but the difference i s not s i g n i f i c a n t . - 6 6 -Experiment 3« The f a c t t h a t number of g r a i n s appeared to have an e f f e c t on germination i n v i v o suggested t h a t f u r t h e r i n v e s t i g a t i o n was warranted. Table 16. P o l l e n v i a b i l i t y percentages of Puck (P) and Bonny Best (B) v a r i e t i e s u s i n g 15» 50, 100,and 200 g r a i n s i n p o l l i n a t i o n s . f* *n o *t v\ "ftT _ V a r i e t i e s _ u r a i n wo. P B P B P B X 15 0 0 0 0 0 0 0.0 50 6 0 13 14- o 5.5 100 32 18 17 18 20 6 18.5 200 73 7 18 5 20 19 23.6 A n a l y s i s of v a r i a n c e on data transformed by the angular method (A Source D.F. M.S. F T o t a l 23 - -Treatments 7 — — V a r i e t i e s (V) 1 74-3.26 12.08** G r a i n No. (N) 3 1014,96 16.50** V x N 3 129.08 2.09 E r r o r 16 61.48 Duncan's t e s t . G r a i n No. 15 50 100 200 x . 0 , 0 5sl 18.5 23,6 The more comprehensive experiment 3 confirmed the r e s u l t s of experiments 1 and 2. Data i n t a b l e 16, i n d i c a t e t h a t no germination occurred w h e n V t h e r r e l a t i v e l y s m a l l number of 15 g r a i n s was used per stigma, but t h a t some germination occurred on a l l stigmas when 100 g r a i n s were a p p l i e d . The in t e r m e d i a t e number of 50 shows a d i s t i n c t v a r i e t a l response i n t h a t Puck p o l l e n germinated but none of the Bonny Best p o l l e n germinated. The v a r i e t a l response was n o t con-s i s t e n t when the number of p o l l e n g r a i n s was in c r e a s e d to 200, Thus the a n a l y s i s d i d not show a s i g n i f i c a n t i n t e r a c t i o n of p o l l e n g r a i n number and v a r i e t y . - 6 7 -Puck gave a highly s i g n i f i c a n t l y greater percentage of pollen germination i n vivo than Bonny Best. Duncan's test revealed no s i g n i f i c a n t differences between the germination of 15 and 100 grains, or between 100 and 200 grains, but the l a t t e r pair gave s i g n i f i c a n t -l y higher germination percentages than the former pai r . Experiment 4 . The i n d i c a t i o n of a d i s t i n c t v a r i e t a l response i n experiment 3, when 50 grains were used, was very i n t e r e s t i n g , but due to the limited sample of three Bonny Best flowers used, the experiment was repeated using a larger number of flowers. Table 17. Percentage germination i n vivo of Puck and Bonny Best using 50 and 100 grains per stigma. Pi r a i n Nn. Puck flowers Bonny Best flowers V 1 2 3 4 5 6 1 2 3 4 5 6 50 grains 100 grains 26 24 30 20 52 4-1 51 46 28 54-30 46 6 14 46 63 12 0 16 0 58 25 48 51 17.1 48 .4 Analysis of variance. Source D.F. M.S. F Total 23 Treatments 3 V a r i e t i e s (V) 1 495.0 7.63* Grain No. (N) 1 5859.0 9 0 . 4 1 * * V x N 1 523.0 8.07* Error 20 64.8 Analysis of data i n table 17, showed the inte r a c t i o n (V x N) to be s i g n i f i c a n t . This indicated that the differences between the v i a b i l i t y percentages of 50, and 100 grains, varied with the two v a r i e t i e s , Puck and Bonny Best. Although the percentages of viable pollen i n Bonny Best were lower than Puck, Bonny Best showed a higher germination percentage - 68 -increase in vivo when the number of grains used in pollination was increased from 50 to 100 grains. Experiment 5. The experiment was designed to investigate the effects of varietal differences of pollen and stigma, on pollen v i a b i l i t y determinations in vivo, resulting from controlled self- and cross-pollinations of the two varieties, Puck and Bonny Best. Table 18. Pollen germination percentages in vivo from reciprocal pollinations and self-pollinations of Puck~TP) and Bonny Best (B). Pollen P P B B P P B B Stigmas P B B P P 3 B ; P 50 grains 32 36 4-0 14- 38 4-4- 12 6 27.75 100 grains 71 76 68 64- 69 63 58 13 60.25 Analysis of variance, Source D.F. M.S. F Total 15 _ Treatments 7 _ _ Grain No. (N) 1 4-225.0 16.98** Pollen (P) 1 14-82.0 5.95* Stigmas (S) 1 506.0 2.03 N x P 1 0.0 n. s N x S 1 102.0 n. s P x S l 224-. 0 n.s N x P x S 1 50.0 n.s Error 8 24-8.8 Analysis i D f data in table 19, showed no significance for the interactions. There were no significant differences in • varietal effects on germination, between stigmas of the varieties, Puck and Bonny Best. One hundred grains applied to stigmas gave significant-ly higher germination percentages than f i f t y grains. Puck pollen gave significantly higher percentages germination in vivo than Bonny Best pollen regardless of stigma, thus suggesting the differences are inherent i n the pollen only. - 69 -Experiment 6. The p l a n t s of l o t 4 , from which f l o w e r s were obtained f o r the p r e v i o u s i n v i v o experiments, were i n a r a t h e r low s t a t e of v i g o u r a t the time the i n v e s t i g a t i o n s were made. Consequently two a d d i t i o n a l experiments (6 and 7,) were performed on f l o w e r s from young vi g o r o u s Puck and Bonny Best p l a n t s of l o t 6. T h i s was done i n order to compare the e f f e c t s of v i g o u r of p l a n t s on p o l l e n germination which had p r e v i o u s l y been found i n experiments 4- and 5. The p r e c i s i o n of experiments 6 and 7 was i n c r e a s e d by u s i n g twice the number of r e p l i c a t i o n s t h a t had been used i n experiments 4- and 5. Table 19* Percentage germination i n v i v o of Puck ( P ) , and Bonny Bes t ( B ) , u s i n g 50 and 100 g r a i n s . V a r i e t i e s G r a i n Subsample s — X No. 1 2 ,3 4- 5 6 7 8 9 10 11 12 P 50 100 76 92 78 65 56 80 50 56 70 88 94-97 73 73 4-2 55 i+6 82 72 91 56 80 84- 68.80 71 75.05 B 50 100 4-2 4-6 98 83 66 51 70 65 50 66 52 82 67 71 81 85 46 92 68 81 70 91 38 57.89 56 76.80 A n a l y s i s of v a r i a n c e . Source D.F. M.S. F T o t a l s 47 - I Treatments 3 - -V a r i e t i e s (V) 1 252.0 1.19 G r a i n No. (N) 1 1900.0 8.99** V x N 1 4-81.0 2.27 E r r o r 44 211.3 A n a l y s i s of data i n t a b l e 19, showed no s i g n i f i c a n c e f o r the i n t e r a c t i o n . There were no v a r i e t a l d i f f e r e n c e s i n p o l l e n germin-a t i o n between Puck and Bonny Best p o l l e n . Germination percentages were s i g n i f i c a n t l y g r e a t e r from 100 g r a i n s than 50 g r a i n s . - 70 -Germination percentages were considerably improved when f l o -wers from vigorous plants were used. Experiment 7 . The choice of 2 0 0 grains f o r carrying out s e l f - and cross-p o l l i n a t i o n s i n the experiment, was used since the effects of number of grains using 5 0 , and 1 0 0 grains, were already evident from the r e s u l t s i n experiment 5 . Table 2 0 . Pollen percentage germination i n vivo from r e c i p r o c a l p o l l i n a t i o n s of Puck (P) and Bonny Best ( 1 7 , 2 0 0 grains being applied to each stigma. Pollen Stigma Stigmas 1 2 3 4 $ 6 7 8 9 10 11 12 X B B P B B P P P 86 76 8 0 91 3 7 61 8 0 96 60 56 64 74 58 6 8 82 86 9 5 80 78 44 93 6 5 78 90 60 76 73 82 8 5 92 58 78 2 5 58 61 8 1 82 9 5 7 5 97 81 36 73 62 61 52 8 5 70 7 3 . 0 7 1 . 8 Analysis of variance. Source D.F. M.S. F Total Treatments Pollen (P) Stigmas (V) P x V Error 4 7 3 1 1 1 4 4 1 5 1 9 . 0 1 6 . 0 7 . 0 2 5 8 . 3 • 5."88* n.s n.s Analysis of data i n table 2 0 , showed no significance for the int e r a c t i o n . There were no s i g n i f i c a n t differences i n v a r i e t a l e f f e c t s on germination between stigmas of the v a r i e t i e s , Puck, and Bonny Best. When 2 0 0 grains were used i n p o l l i n a t i n g flowers on vigorous plants, the variety Puck gave s i g n i f i c a n t l y higher v i a b i l i t y percentages than Bonny Best. f - 71 -(c) Comparison of the s t a i n i n g and i n v i v o techniques used i n p o l l e n v i a b i l i t y d e t e r m i n a t i o n s . Experiment 1. The development of a q u a n t i t a t i v e procedure f o r counting p o l l e n tubes i n s t y l e s made i t p o s s i b l e to compare the a r t i f i c i a l methods of p o l l e n v i a b i l i t y d e t e r m i n a t i o n s , namely the s t a i n i n g and i n v i t r o techniques, w i t h the i n v i v o method. The comparisons are shown i n t a b l e 21. Table 21. P o l l e n v i a b i l i t y percentages determined by the s t a i n i n g , i n v i t r o % and i n v i v o techniques. V a r i e t i e s Tests 1 2 Stigmas  3 4- 5 6 7 8 9 10 11 12 S t a i n i n g 92 96 86 93 90 99 89 95 87 82 92 95 91.2 In v i t r o 75 67 32 4-5 58 64- 72 78 63 31 6k 6k 59.3 75.9 In v i v o 91 6k 7k 86 kh 73 82 78 81 97 85 70 77.0 B S t a i n i n g 85 83 94- 87 93 83 91 93 92 89 86 79 87.8 In v i t r o 60 55 27 31 5k 63 57 k6 28 51 4-1 30 4-5.2 66.7 In v i v o 86 37 62 58 95 93 65 85 25 82 81 36 67.O A n a l y s i s of v a r i a n c e . Source T o t a l Treatments V a r i e t i e s (V) T e s t s (T) V x T E r r o r Duncan's t e s t . T e s t s x D.F. 71 5 1 2 2 66 In v i t r o 52l3~~ M.S. 1552.0 8354-.0 161.0 20£.0 In v i v o 72.0 7.57** 40.75** S t a i n i n g 89.6 A n a l y s i s of data i n t a b l e 21, showed no s i g n i f i c a n c e f o r the i n t e r a c t i o n . Puck v a r i e t y gave h i g h l y s i g n i f i c a n t l y g r e a t e r d i f -f e r e n c e s i n p o l l e n v i a b i l i t y than Bonny Best v a r i e t y . - 72 -There were h i g h l y s i g n i f i c a n t d i f f e r e n c e s between the three methods of p o l l e n v i a b i l i t y d e t e r m i n a t i o n s . Duncan's t e s t r e v e a l e d t h a t the three methods were s i g n i f i c a n t l y d i f f e r e n t from each other w i t h the s t a i n i n g technique g i v i n g the h i g h e s t percentage v i a b l e p o l l e n , f o l l o w e d by the i n v i v o and i n v i t r o techniques. DISCUSSION The l i t e r a t u r e c o n t a i n s many r e f e r e n c e s to v a r i a b i l i t y i n p o l l e n v i a b i l i t y d e t e r m i n a t i o n s . T h i s v a r i a b i l i t y has made i t d i f f i c u l t to o b t a i n c o n s i s t e n t p o l l e n v i a b i l i t y measurements,and to have c o n f i d e n c e i n the r e s u l t s of e v a l u a t i n g p o l l e n samples. In order to p r o v i d e more i n f o r m a t i o n , three important sources of v a r i a b i l i t y were s t u d i e d i n the f o r e g o i n g experiments. These were (1), methods of sampling ( 2 ) , the p l a n t m a t e r i a l i t s e l f and, ( 3 ) methods used i n p o l l e n v i a b i l i t y d e t e r m i n a t i o n s . (1) Methods of sampling. The c h o i c e of a number of g r a i n s necessary to give a r e p r e s -e n t a t i v e sample s i z e , v a r i e s w i t h the f o l l o w i n g : (1), the degree of t o l e r a n c e chosen a t a f i x e d c o n f i d e n c e l i m i t ; and ( 2 ) , the amount of v a r i a b i l i t y e x i s t i n g i n the sample. In g e n e r a l , i t was found t h a t the l a r g e r the degree of t o l e r a n c e chosen, the s m a l l e r was the sample r e q u i r e d . A sample of p o l l e n taken from a f l o w e r on an o l d p l a n t a l r e a d y l a d e n w i t h f r u i t , showed tremendous v a r i a b i l i t y among measurements of v i a b i l i t y of subsamples. Such extreme v a r i a b i l i t y n e c e s s i t a t e d the use of a l a r g e sample to r e p r e s e n t the p o p u l a t i o n . In c o n t r a s t , p o l l e n taken from f l o w e r s - 73 -on young v i g o r o u s p l a n t s showed more u n i f o r m i t y among subsamples, and consequently a small sample s i z e was adequate to give r e l i a b l e r e s u l t s . I t i s reasonable to expect t h a t a young vig o r o u s p l a n t would have enough food r e s o u r c e s to develop l a r g e q u a n t i t i e s of v i a b l e p o l l e n a t a n t h e s i s , and t h a t p l a n t s i n an advanced f r u i t i n g stage would be l i k e l y to have more undeveloped or abnormal p o l l e n . T h i s c o n d i t i o n i s a p p a r e n t l y normal, c o n s i d e r i n g the l i f e c y c l e and morphology of the tomato p l a n t , i n which f r u i t s a l r e a d y s e t and allowed to develop have p r i o r i t y on the food resources (Mur-neek, 1926, Mc Collum, 1934, arid Dearborn, 1936). Apart from age of p l a n t , s e v e r a l f a c t o r s i n the environment appear to a f f e c t the v a r i a b i l i t y i n p o l l e n samples, among which a r e : s o i l c o n d i t i o n s , temperature, l i g h t , r e l a t i v e humidity and m a t u r i t y of f l o w e r a t ' the time of sampling. I t i s probable t h a t v i g o u r of p l a n t may have g r e a t e r e f f e c t s on v a r i a b i l i t y i n p o l l e n samples than age of p l a n t . The c h o i c e of sample s i z e of 300-400 p o l l e n g r a i n s used i n the present experiments can be considered w i t h i n l i m i t s of d e s i r e d r e l i a b i l i t y i n cases where p o l l e n was obtained from vigorous p l a n t s . T h i s number i s of l e s s value where p o l l e n was obtained from p l a n t s i n a non-vigorous s t a t e , s i n c e v a r i a b i l i t y would be measured a t a l a r g e degree of t o l e r a n c e . E x p e rimental r e s u l t s on sample s i z e show how much conf i d e n c e should be placed i n p o l l e n v i a b i l i t y measurements made by p r e v i o u s workers, r e f e r r e d to i n the l i t e r a t u r e , where v i a b i l i t y percentages are expressed (1) without s t a t i n g the number of g r a i n s used and (2) by u s i n g an a r b i t r a r y number of g r a i n s as a r e p r e s e n t a t i v e sample s i z e r e g a r d l e s s of source and growing c o n d i t i o n s . - 7k -Experimental r e s u l t s have a l s o i n d i c a t e d t h a t the extent of v a r i a b i l i t y i n a s i n g l e p o l l e n sample i s i n p a r t dependent on the method used i n i t s c o l l e c t i o n . I t was observed t h a t when-ever the v i b r a t o r was used f o r p o l l e n c o l l e c t i o n , l a r g e q u a n t i t i e s of immature g r a i n s were obtained. T h i s mixture "was not expected, but a p p a r e n t l y v i b r a t i o n d i s l o d g e s a c o n s i d e r a b l e q u a n t i t y of immature g r a i n s i n samples, and the q u a n t i t y probably v a r i e s w i t h the d u r a t i o n of v i b r a t i o n of the f l o w e r . In s t r i k i n g c o n t r a s t to t h i s s i t u a t i o n , the procedure of p a s s i n g the needle along the s u r f a c e of the stamen gave the l e a s t amount of immature g r a i n s . I t was g e n e r a l l y observed t h a t the method of c o l l e c t i o n which employed the l e a s t mechanical f o r c e r e s u l t e d i n the h i g h e s t per-centages of f u n c t i o n a l p o l l e n . I t has a l s o been shown t h a t the time a t which a tomato flo w e r i s sampled may account f o r l a c k of c o n s i s t e n c y of p o l l e n v i a b i l i t y measurements. T h i s s i t u a t i o n r e s u l t s from the d i f f e r e n t stages of p o l l e n m a t u r i t y a s s o c i a t e d w i t h d i f f e r e n t bud and f l o w e r stages. Hence p o l l e n sampled a t e a r l y bud stages would give lower v i a b i l i t y percentages j u s t b efore or a t a n t h e s i s , due to the h i g h immature g r a i n content. R e s u l t s on sampling the same fl o w e r over s u c c e s s i v e days i n d i c -ated t h a t more v i a b l e p o l l e n i s p r e s e n t on the f i r s t 2 days a f t e r f u l l a n t h e s i s occurs than sampling on subsequent p e r i o d s d u r i n g the time the f l o w e r remains open. I t i s apparent t h a t l a c k of the c o n s i d e r a t i o n of sample s i z e , methods of p o l l e n c o l l e c t i o n , and time of c o l l e c t i n g samples, could e x p l a i n some of the d i s c r e p a n c i e s encountered i n p r e v i o u s p o l l e n v i a b i l i t y d e t e r m i n a t i o n s . - 75 -(2) The p l a n t m a t e r i a l . When d e a l i n g w i t h a p l a n t l i k e the tomato, which bears a s u c c e s s i o n of f l o w e r s , i t i s q u i t e p o s s i b l e t h a t d i f f e r e n c e s i n environmental c o n d i t i o n s under which separate f l o w e r s a re i n i t -i a t e d and developed, could have subsequent e f f e c t s on p o l l e n development and v i a b i l i t y a t d i f f e r e n t l o c a t i o n s on the same p l a n t . C o n s i d e r i n g the stamen as the s m a l l e s t u n i t of d i f f e r e n t l o c a t i o n of p o l l e n on a p l a n t , i t was found t h a t no d i f f e r e n c e s i n p o l l e n v i a b i l i t y e x i s t e d w i t h i n three zones of the r e l a t i v e l y l o n g stamens s t u d i e d . These r e s u l t s suggested t h a t the r a t e s of p o l l e n development were the same i n the three zones. In c o n t r a s t , p o l l e n v i a b i l i t y d i f f e r e d g r e a t l y from stamen to stamen i n the same f l o w e r . I t i s p o s s i b l e t h a t the v a r i o u s f a c t o r s i n the environment a c t i n g on a dev e l o p i n g f l o w e r , have g r e a t e r i n f l u e n c e on p o l l e n development among stamens r a t h e r than w i t h i n a s i n g l e stamen. T h i s v a r i a b i l i t y from stamen to stamen i s q u i t e p o s s i b l e when i t i s r e a l i s e d t h a t there may be d i f f e r e n c e s i n q u a n t i t i e s of n u t r i e n t s t r a n s l o c a t e d , and other e f f e c t s such as l i g h t i n t e n -s i t y , temperature and r e l a t i v e humidity. I t i s a l s o p o s s i b l e t h a t the f l u c t u a t i n g environmental f a c t o r s r e s u l t e d i n a hig h v a r i a t i o n i n p o l l e n v i a b i l i t y among stamens of the same f l o w e r , and may account f o r the l a c k of s i g n i f i c a n t d i f f e r e n c e s among f l o w e r s . D i f f e r e n c e s i n p o l l e n v i a b i l i t y among t r u s s e s on the same p l a n t may be a g a i n a t t r i b u t e d to the v a r y i n g environmental c o n d i t -i o n s under which each of the three t r u s s e s was i n i t i a t e d and dev-eloped. The f a c t t h a t p o l l e n v i a b i l i t y d e c l i n e d from t r u s s 1 to - 76 -t r u s s 3 would suggest t h a t p l a n t food was being u t i l i z e d by dev-e l o p i n g f r u i t on o l d e r t r u s s e s and t h a t , under the p r e v a i l i n g environmental c o n d i t i o n s , p l a n t growth l i m i t e d the food s u p p l i e d to d e v e l o p i n g p o l l e n i n the upper t r u s s e s . The r e s u l t s show the v a r i a b i l i t y t h a t may be obtained when p o l l e n samples are taken from (1) s i n g l e stamens by needle, (2) f l o w e r to f l o w e r on the same t r u s s and a l s o (3) d i f f e r e n t t r u s s e s of the sample p l a n t . T h i s v a r i a b i l i t y could account f o r the ap-parent l a c k of c o n s i s t e n c y experienced by other workers. (3) Methods used i n p o l l e n v i a b i l i t y d e t e r m i n a t i o n s . E x p e rimental r e s u l t s have i n d i c a t e d t h a t p o l l e n v i a b i l i t y determined by d i f f e r e n t procedures, shows v a r i a t i o n w i t h i n the same p o l l e n sample. T h i s s i t u a t i o n was n o t i c e a b l e when the s t a i n i n g t e c h -nique showed comparatively higher v i a b i l i t y percentages than the i n v i t r o technique. The m o d i f i c a t i o n of the io d i n e - p o t a s s i u m i o d i d e procedure f o r p o l l e n v i a b i l i t y d e t e r m i n a t i o n s was based on the nature of the food r e s e r v e r e v e a l e d by s t a i n i n g . Maheshwari (1950) r e p o r t e d t h a t s y n t h e s i s of s t a r c h occurs as f u n c t i o n a l g r a i n s mature i n angiosperm p o l l e n . Daubeny (1955) and Guccione (1959) regarded tomato p o l l e n to be v i a b l e only when they s t a i n e d a dark c o l o u r by i o d i n e - p o t a s s i u m i o d i d e s o l u t i o n . C u r r e n t i n v e s t i g a t i o n ( u s i n g d i l u t e i o d i n e - p o t a s s i u m i o d i d e s o l u t i o n to s t a i n p o l l e n from d i f -f e r e n t bud and flow e r stages) suggested t h a t (1) tomato p o l l e n g r a i n s a re n o n - v i a b l e a t t h e i r h i g h e s t peak of s t a r c h content which occurs a t the in t e r m e d i a t e bud stage and (2) f u n c t i o n a l mature g r a i n s g i v e l i t t l e to no s t a i n r e a c t i o n w i t h d i l u t e i o d i n e -- 7 7 -potassium i o d i d e s o l u t i o n , i n d i c a t i n g the presence of l i t t l e or no s t a r c h . T h i s d i f f e r e n c e i s no doubt due to the c o n v e r s i o n of s t a r c h to sugars as g r a i n s mature, and confirms r e p o r t s of L e s l e y and L e s l e y (1939) and Iwanami (1959) . Consequently a sample of p o l l e n taken from a f u l l y r e f l e x e d tomato f l o w e r a t a n t h e s i s , i s found to c o n s i s t of a mixture of p o l l e n g r a i n s which vary i n stages of m a t u r i t y . "These stages are a p p a r e n t l y c o r r e l a t e d w i t h the degree of c o n v e r s i o n of s t a r c h to sugars w i t h i n the g r a i n s . Not a l l o f the normal mature g r a i n s germinate when placed i n a r t i f i c i a l medium. The f a i l u r e to germinate i s a t t r i b u t e d to (1), f r e q u e n t b u r s t i n g of mature g r a i n s d u r i n g p o l l e n tube i n i t -i a t i o n and (2), a decrease i n the group s t i m u l a t i o n e f f e c t on germ-i n a t i o n , caused by small l o c a l i z e d areas of uneven d e n s i t y . T h i s r e s u l t s from uneven d i s t r i b u t i o n when p o l l e n samples are being s t i r r e d i n t o the medium. B u r s t i n g of mature g r a i n s occurs a f t e r the f i r s t f i f t e e n minutes of germination. B u r s t i n g of tubes can occur a t any time d u r i n g p o l l e n tube e l o n g a t i o n . The reason f o r b u r s t i n g i s unknown. V a s i l (I960 a) rep o r t e d t h a t b u r s t i n g of p o l l e n g r a i n s decreases w i t h an i n c r e a s e i n the osmotic c o n c e n t r a t i o n of the medium. Experimental r e s u l t s have i n d i c a t e d t h a t the gr e a t e r the stage of p o l l e n m a t u r i t y , the higher i s the sugar c o n c e n t r a t i o n w i t h i n the g r a i n . When g r a i n s a re placed i n a germinating medium of f i x e d c o n c e n t r a t i o n , the amount of water absorbed by each g r a i n i s a f f e c t e d by i t s osmotic c o n c e n t r a t i o n . Consequently g r a i n s having higher osmotic c o n c e n t r a t i o n s than the surrounding medium w i l l absorb g r e a t e r volumes of water than those of lower osmotic c o n c e n t r a t i o n s . Hence b u r s t i n g w i l l r e s u l t when the volume of - 78 -water absorbed e x e r t s a p r e s s u r e beyond the mechanical s t r e n g t h of the i n t i n e d u r i n g i t s i n i t i a l stage of p r o t r u s i o n through the germ pore. I t i s q u i t e p o s s i b l e t h a t the mechanical s t r e n g t h of the i n t i n e may be g e n o t y p i c a l l y c o n t r o l l e d . The i n c o n s i s t e n t behaviour of samples of Bonny Best p o l l e n and a few r e c i p r o c a l c r o s s e s , i s q u e s t i o n a b l e s i n c e the g e n e r a l trend r e s u l t i n g from experiments on the comparison of s t a i n i n g w i t h i n v i t r o d e t e r m i n a t i o n s , showed higher v i a b i l i t y percentages f o r the former technique. I t i s p o s s i b l e t h a t the p o l l e n samples of these v a r i e t i e s are very e a s i l y a f f e c t e d by s l i g h t d i f f e r e n c e s i n d e n s i t y of g r a i n s d u r i n g germination. The r e l a t i v e value of p o l l e n v i a b i l i t y determined under a r t i f -i c i a l and n a t u r a l c o n d i t i o n s , was only assessed a f t e r a q u a n t i t a t -i v e i n v i v o procedure was developed. The phenomenon of the mutual s t i m u l a t i o n caused by i n c r e a s i n g d e n s i t y of p o l l e n g r a i n s was obvious throughout the course of the i n v i v o work. Both Puck and Bonny Best showed an i n c r e a s e d germ-i n a t i o n percentage when the number of g r a i n s used f o r p o l l i n a t i o n s was i n c r e a s e d . Although the i n v i v o technique shows great promise as a v a l i d t e s t of p o l l e n v i a b i l i t y , f u r t h e r i n v e s t i g a t i o n i s r e q u i r e d to a s c e r t a i n the sample s i z e t h a t would give the most a p p r o p r i a t e d e n s i t y f o r the group s t i m u l a t i o n e f f e c t on germination. T h i s p o l l e n growth f a c t o r (PGF) has been repo r t e d by v a r i o u s workers f o r i n v i t r o c u l t u r e s , but i t s occurrence i n nature has not been p r e v i o u s l y r e c orded. The p r e s e n t i n v e s t i g a t i o n has r e v e a l e d v a r i e t a l d i f f e r e n c e s - 79 -i n p o l l e n between Puck and Bonny Best tomato v a r i e t i e s , w i t h Puck y i e l d i n g higher v i a b i l i t y percentages than Bonny Best i n most cases. I t i s p o s s i b l e t h a t d i f f e r e n c e s between the two types of p o l l e n might be g e n e t i c . I t i s a l s o p o s s i b l e t h a t Puck may be more e f f i c i e n t i n c a r r y i n g out pho t o s y n t h e s i s a t a g r e a t e r r a t e than Bonny 3 e s t . Subsequently, g r e a t e r amounts of carbohydrates are made a v a i l a b l e to maturing p o l l e n i n Puck p l a n t s . F l u c t u a t i n g environmental c o n d i t i o n s , both i n the f i e l d and under greenhouse c o n d i t i o n s when p o l l e n was produced, probably obscured g e n e t i c d i f f e r e n c e s among the v a r i e t i e s , " P u c k , Bonny Best, E a r l i a n a 4-98, and t h e i r r e c i p r o c a l c r o s s e s . I t i s p o s s i b l e t h a t i f p l a n t s are developed i n growth chambers, the environmental e f f e c t s on p o l l e n development would be more uniform. As a r e s u l t , i t would be p o s s i b l e to assess g e n e t i c d i f f e r e n c e s i n the l a b o r a t o r y , and use such d i f f e r e n c e s as the b a s i s f o r s e l e c t i o n of p l a n t s c a r r y i n g the d e s i r e d g e n e t i c c o n s t i t u t i o n . SUMMARY AND CONCLUSIONS Experiments on p o l l e n v i a b i l i t y i n the tomato v a r i e t i e s Puck, Bonny Best, E a r l i a n a 4-98, and t h e i r r e c i p r o c a l h y b r i d s were done to a s c e r t a i n s u i t a b i l i t y of u s i n g p o l l e n t e s t s as a b a s i s of s e l e c -t i o n i n a breeding program aiming to O r i g i n a t e a new, tr u e - b r e e d i n g commercial v a r i e t y p o s s e s s i n g the c h a r a c t e r i s t i c of s e t t i n g f r u i t a t c o o l temperatures (below 65°F.) i n southern Canada. P l a n t s were grown i n the f i e l d and under two l e v e l s of temp-e r a t u r e i n greenhouses. The temperatures i n the warm house were r e g u l a t e d a t 65°-75°F., and i n the c o o l house 55°-65°F. A l l p o l l e n - 8 0 -s t u d i e s u t i l i z e d p o l l e n from p l a n t s i n the f i e l d and warm green-houses. The experiments i n c l u d e d a study of the sources of v a r i a b i l -i t y which a f f e c t p o l l e n v i a b i l i t y d e t e r m i n a t i o n s . The sources st u d i e d were (1) methods of sampling (2) the p l a n t m a t e r i a l i t -s e l f and (3) the methods used i n p o l l e n v i a b i l i t y d e t e r m i n a t i o n s . The experimental r e s u l t s showed t h a t a sample of p o l l e n taken from a tomato f l o w e r a t f u l l a n t h e s i s possesses v a r i a b i l i t y , which i s due to the presence of mature, immature and empty g r a i n s . D i f -f e r e n t p o l l e n samples showed d i f f e r e n t p r o p o r t i o n s of these c l a s s e s . The v a r i a b i l i t y of p o l l e n sampled, i s a f f e c t e d by method of c o l l e c t i o n , the time of c o l l e c t i o n and the l o c a t i o n of the p o l l e n source on the p l a n t . P o l l e n samples c o l l e c t e d from the s u r f a c e s of stamens w i t h a d i s s e c t i n g needle, gave h i g h e s t percentages of v i a b l e p o l l e n . P o l l e n samples taken on the f i r s t day when the f l o w e r a t t a i n e d maximum a n t h e s i s gave higher percentages of mature p o l l e n than samples c o l l e c t e d subsequently d u r i n g the p e r i o d the f l o w e r p e t a l s were open. P o l l e n samples taken from d i f f e r e n t l o c a t i o n s on the same p l a n t , showed no d i f f e r e n c e s i n p o l l e n v i a b i l i t y between p o l l e n taken from zones of the same stamen. The great v a r i a t i o n i n p o l l e n v i a b i l i t y between s i n g l e stamens w i t h i n a f l o w e r could account f o r the v a r i a b i l i t y between f l o w e r s on the same c l u s t e r and a l s o between c l u s t e r s on the same p l a n t . The v a r i a b i l i t y of p o l l e n which r e s u l t s from methods of sampling, has subsequent e f f e c t s on the r e p r e s e n t a t i v e sample s i z e . The g r e a t e r the v a r i a b i l i t y , the l a r g e r i s the number of g r a i n s r e q u i r e d to give a r e l i a b l e sample s i z e a t a chosen degree of t o l e r a n c e . - 8 1 -The s t a i n i n g technique showed higher percentages of v i a b l e p o l l e n than the i n v i t r o and .in v i v o techniques, f o r the same sample of p o l l e n . Reduction i n the v i a b l e percentages i n v i t r o i s b e l i e v e d to be due to b u r s t i n g of some mature p o l l e n g r a i n s . V a r i a t i o n s i n germination percentages i n v i v o are r e l a t e d to the d e n s i t y and number of g r a i n s used i n p o l l i n a t i o n . Both v a r i e t i e s , Puck and Bonny Best showed response to group s t i m u l a t i o n s i n c e i n c r e a s e d germination percentages were obtained by i n c r e a s i n g the number and d e n s i t y of g r a i n s . Although the p o l l e n v i a b i l i t y r e s u l t s of the two v a r i e t i e s , Puck and Bonny Best, l a c k e d c o n s i s t e n c y , there was some i n d i c a t i o n t h a t Puck gave higher v i a b l e percentages than Bonny Best. I t i s recommended t h a t f u r t h e r work d e a l i n g w i t h p o l l e n v i a b -i l i t y as a means of tomato p l a n t c h a r a c t e r i d e n t i f i c a t i o n should be done i n r e l a t i o n to a c o n s i d e r a t i o n of sources ac c o u n t i n g f o r v a r i a b i l i t y i n p o l l e n samples. 1. Method of p o l l e n c o l l e c t i o n - p o l l e n samples should be c o l l e c t e d by p a s s i n g a needle over the s u r f a c e of an anther u s i n g as l i t t l e mechanical f o r c e as p o s s i b l e . 2. Time of p o l l e n c o l l e c t i o n - p o l l e n samples should be c o l l e c t e d on the f i r s t day when the f l o w e r ' a t t a i n s maximum a n t h e s i s . 3. L o c a t i o n of p o l l e n source - sampling of p o l l e n should be r e s t -r i c t e d only to f l o w e r s developed under s i m i l a r environmental cond-i t i o n s . 4. Sample s i z e - samples p o s s e s s i n g a h i g h percentage of mature g r a i n s are d e s i r a b l e , s i n c e sample s i z e i s e a s i l y assessed when i t i s p o s s i b l e to use a s m a l l number of g r a i n s a t a s m a l l degree of t o l e r a n c e . - 82 -5» I S v i v o - v i a b i l i t y d e t e r m i n a t i o n s i n v i v o appear to be the most v a l i d measure of p o l l e n v i a b i l i t y , but f u r t h e r i n v e s t i g a t i o n of number and d e n s i t y of g r a i n s on group s t i m u l a t i o n e f f e c t on germination i s d e s i r a b l e . 6 , F u r t h e r i n v e s t i g a t i o n of the s u b j e c t would c a l l f o r more p r e c i s e c o n t r o l i n the environmental c o n d i t i o n s d u r i n g p o l l e n development, and exposure of p o l l e n f o l l o w i n g a n t h e s i s . - 83 -LITERATURE CITED 1. Adams, J . 1916. On the germination of the p o l l e n g r a i n s of apple and other f r u i t t r e e s . 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