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Reduced ferility in red raspberry (Rubus Idaeus L.). Virdi, Bessie Violet 1971

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REDUCED FERTILITY IN RED RASPBERRY(RUBUS IDAEUS L). by BESSIE VIOLET VIRDI B.Sc.(Agr), University of British Columbia. 1969 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in the Department of Plant Science We accept this thesis as conforming to the required standard University of British Columbia August, 1971. In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia , I agree that the library shall make i t freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Plant Science University of British Columbia Vancouver 8, Canada. August, 1971. 1 ABSTRACT Mtcrosporogenesis, embryo sac development Including fertilization, and fruit set involving cross pollinations were studied in the highly self-fertile red raspberry' (Rubus"i'daeus L.) cultivar Mailing Jewel and the partially male sterile United States Department of Agriculture -Oregon State University selection Ore-US 1314 from the United States Department of Agriculture-Oregon State University red raspberry breeding program. Pollen was stained with aceto-carmine to determine pollen abnormality. Anthers of different filament lengths differed in the percent of normal pollen produced in the Ore-US 1314. Percent normal pollen produced by this selection is also much less than that, produced by Mailing Jewel. Longitudinal and cross sections of the anthers of both clones were examined and no direct involvement of the tapetum was observed to be contributing to the production of abnormal microspores in either clone. Anther squashes showed that only 41.8% of the cells' of Ore-US 1314 underwent normal meiosis as compared to 93% in Mailing Jewel. Lagging chromosomes at meiosis I were also observed in Ore-US 1314. Cytogenetic factors are thought to be involved in causing increased pollen abortion and thus male sterility in Ore-US 1314. Examinations of embryo sac development under open pollination showed that while Ore-US 1314 did riot have a retarded embryo sac development, i t had a high percentage of degenerate embryo sacs. Degeneration is believed to be a result of pollen of poor quality, incompatible pollen, the collapse of the egg cell due to failure of pollen to fertilize i t at the critical time, or the involvement of all three. The pollination experiments involved self, open, and cross-pollinations of the two clones. The results obtained from the druplet set suggest that Ore-US 1314 could be both partial and cross-incompatible. Druplet set in Ore-US 1314 when open pollinated increased above that when selfed suggesting a positive reaction to pollen mixtures. Mailing Jewel, although not self-incompatible, was found to be partially cross-incompatible with Ore-US 1314. iv ACKNOWLEDGEMENTS I would like to thank Dr. G. W. Eaton, Associate Professor, Department of Plant Science, University of British Columbia, under whose supervision this project was undertaken, and for his encouragement and guidance in the writing of this thesis. Acknowledgement is also gratefully extended to the members of my thesis committee, Dr. V. C. Runeckles, Dr. V. C. Brink, Dr. C. A. Hornby all of the Department of Plant Science and especially to Dr. H. A. Daubeny, Canada Department of Agriculture for his interest in this work and for providing the material used in this project. My sincere thanks to Miss S. Lee for her time and effort spent in the typing of this thesis. V TABLE OF CONTENTS Page INTRODUCTION 1 REVIEW OF THE LITERATURE 3 MATERIALS AND METHODS . . 11 Pollen s t a i n a b i l i t y . 1 1 Meiotic study 12 Anther tapetum study 12 Megagametogenesis. 13 Po l l i na t i on studies . . . . 14 RESULTS 17 Pollen s t a i n a b i l i t y 17 Meiotic study 25 Anther tapetum study . 29 Megagametogenesis. . 33 Po l l i na t i on studies . 47 DISCUSSION 51 Pollen study . . . . . . . . . . . 51 Embryo sac study . . . 54 Po l l i na t i on studies . . . . ; . . . . 56 SUMMARY AND CONCLUSIONS . . . . . . . . . 58 LITERATURE CITED 60 APPENDIX 65 vi LIST OF'FIGURES Figure Figure Figure -Figure 1. 2. 3. 4. Figure 5, Figure Figure Figure 8. Figure 9, Figure 10. Figure 11, Figure 12, Figure 13, Figure 14. Figure 15. Figure 16. Figure 17. Figure 18. Figure 19. Page Pollen grains of the cu l t i v a r Mai l ing Jewel. . 22 Pollen grains of the se lect ion Ore-US 1314 . . . 22 Contents of a s t e r i l e anther of Ore-US 1314. . . 24 Cytoplasmic connections between normal and abnormal pollen grains of Ore-US 1314. . . . . 24 Cytoplasmic connections between normal pollen grains only of Ore-US 1314 24 Contents of a s t e r i l e anther of Ore-US 1314. . . 24 Sample of pollen grains of Ore-US 1314 24 Thick walled pollen grains of Ore-US 1314. . . . 24 Anaphase I in Ore-US 1314 with lagging chromosomes - 28 Anaphase I in Ore-US 1314 with a chromosome bridge. 28 Uneven chromosome d i s t r i bu t i on in Ore~US 1314 . . 28 In complete tetrads of Ore-US 1314 28 Diakinesis in Ore-US 1314 with the normal number of seven chromosomes 28 Metaphase II in Ore-US 1314 with the normal number of seven chromosomes at each pole 28 Longitudinal section of an anther of Ore-US 1314 p r i o r to anthesis . 32 Longitudinal section of a p a r t i a l l y s t e r i l e anther of Ore-US 1314 at anthesis. 32 Cross section of an anther of Ore-US 1314. . . . 32 Embryo sac of the cu l t i v a r Willamette at anthesis 44 Embryo sac of the c u l t i v a r Willamette at two days a f te r anthesis . 4 4 VI1 LIST OF FIGURES (continued) Page Figure 20. Embryo sac of the cultivar Willamette at four days after anthesis 44 Figure 21. The egg apparatus of the female gametophyte of the cultivar Willamette at two days after anthesis. 44 Figure 22. A degenerated embryo sac of Ore-US 1314 at six days after anthesis 46 vi i i LIST OF TABLES Page Table 1. Percent normal pollen from counts of 500 pollen grains 18 Table 2. Analysis of variance of the percentage of normal pol len from four filament lengths of ten randomly selected flowers of Mai l ing Jewel. . . . . . . 19 Table 3. Analysis of variance of the percentage of normal pol len from four fi lament lengths of ten randomly selected flowers of Ore-US 1314. . 20 Table 4. Percent of anther ce l l s at varying meiotic stages. . 26 Table 5. Percent of anthers at anthesis having v i s i b l e differences between locules as to pollen qua l i t y and instance of cytoplasmic connections 30 Table 6. Description of embryo sac categories used. . . . 37 Table 7. Percentage of embryo sacs in each stage of development • within the ovules of the normal clone of the Sumner c u l t i v a r in 1970. 38 Table 8. Percentage of embryo sacs in each stage of development with in the ovules of the crumbly clone of the Sumner cu l t i v a r in 1970 39 Table 9. Percentage of embryo sacs in each stage of development within the ovules of the cu l t i v a r Willamette in 1970. 40 Table 10. Percentage of embryo sacs in each stage of development within the ovules of Ore-US 1314 in 1970 41 Table l i . Percentage of embryo sacs in each stage of development with in the ovules of Ore-US 1314 in, 1969. . . . . . 42 Table 12. Percent druplet set and berry weight of Mai l ing Jewel and Ore-US 1314 from s e l f p o l l i n a t i o n , open po l l i na t i on and cross po l l i na t i on with each other 49 Table 13. S i gn i f i cant ef fects at the one percent leve l from s ingle degree freedom comparisons. . . . . . . . 50 - 1 -INTRODUCTION ' A high level of s e l f - f e r t i l i t y i s an essent ial feature of any red raspberry c u l t i v a r since a s ingle vegetatively propagated clone i s often planted in large acreages. Under these conditions pollen from other clones i s not ava i l ab le . Reduction in s e l f - f e r t i l i t y in raspberry i s usually express-ed by f r u i t crumbliness. The few druplets of such f r u i t may separate when picked due to poor cohesion. Reduction in s e l f - f e r t i l i t y has been found to ex i s t in clones of some s e l f - f e r t i l e cu l t i va r s (Crandal l , 1964; Daubeny, et al_., 1967; Jennings, 1967), and th i s f a i l u r e for f r u i t set has been at -t r ibuted to factors such as virus i n fect ion (Braun and Keplinger, 1969; Chamberlain, et al_., 1964; Converse and Endo, 1969; Daubeny. et^ al_., 1970; Keplinger, et al_., 1968; S late, et al_., 1942; Taylor, et al_., 1965), n u t r i -t iona l imbalance, f ros t i n ju ry , and tox ic sprays in bloom (Crandal l , 1964), somatic mutations which resu l t in pa r t i a l s t e r i l i t y (Daubeny, e_t c fL , 1967), and s e l f i ncompat ib i l i t y of the clone (Daubeny, 1971). Freeman, e_t al_.r(1969) reported increased male s t e r i l i t y as a resu l t of virus i n fect ion in four red raspberry c u l t i v a r s . Jennings (1967) stated that the meiotic i r r e g u l a r i t y he observed in the pollen mother ce l l s of two red raspberry cu l t i va r s could be coincidental or perhaps a resu l t of -virus i n fec t i on . Daubeny (1969) observed a pa r t i a l male s t e r i l e condition of genotypic o r i g i n associated with s e l f - f e r t i l i t y in the United States De-partment of Agriculture-Oregon State Univers ity se lect ion Ore-US 1314 from the United States Department of Agriculture-Oregon State Univers ity red raspberry breeding program. The present study was undertaken to determine the causes of -2 -reduced f e r t i l i t y in Ore-US 1314 the clone previously described by Daubeny (1969). Aspects of micro- and macrosporogenesis were studied. In addit ion po l l i na t i on studies were done to determine i f c ro s s -po l l i na t ion might a l l e v i a t e some or a l l of the reduced s e l f - f e r t i l i t y observed. -3-REVIEW OF THE LITERATURE Literature on the following subject is reviewed:- i) methods for the determination of pollen viability, i i ) microsporogenesis in rela-tion to pollen fertili t y and i i i ) megagametogenesis in relation to reduced fer t i l i t y . i ) Methods'of pollen viability determination. The more commonly used methods of estimating pollen viability are by (a) the use of stains or by (b) in vitro germination of pollen grains. (a) Staining. One of the most commonly used stains is aceto-carmine. The pollen grains are stained and the deeply colored grains, are considered normal and therefore viable, where as the weakly and non-stain-ing grains are considered abnormal, and therefore non-viable. King (1960) however, stated such tests gave results which could not be associated with viability, and that at best only indicate the degree of pollen maturity. Also, germination tests were unreliable due to the erratic behaviour of pollen when germinated in an artificial environment. Instead;, he recommends a vital test in preference to the ordinary staining and germination tests, involving the oxidation of benzidine by peroxidase in the presence of hy-drogen peroxide. Hauser and Morrison (1964) however, concluded that -for a large number of species, a test based on the cytochemical reduction of nitro blue tetrazolium was preferable. Ostapenko (1956a) on the other hand, felt that for the older pollen germination in a ten percent sucrose solution gave a better indication of pollen viability than staining with aceto-carmine or the peroxidase determination and that these two methods were to be regarded as only of relative value in determining pollen viability (b) In vitro tests. In vitro tests for the determination of pollen viability involve the germination of pollen on an artificial -4-medium. Ostapenko (1956b) comparing results of germination te s t s , obtained lower values from germinating pol l en'of various f r u i t trees in sucrose so lu -tions than by aceto-carmine sta in ing or by the method of estimating the per-oxidase, espec ia l l y in stored pol len. Eaton (1961) not only obtained low germination values but also var iat ion between values in two d i f fe rent exper-iments using a sample of commercial cherry pol len. Ga l le t ta (1959) also reported var iat ion in results obtained from germination tests and concluded that s t a i n a b i l i t y was more ind ica t i ve of differences in pollen than in v i t r o tes t s . Whelan (1965) suggested that the var ia t ion in results obtained could be due to e r r a t i c pollen response as a resu l t of factors such as chemotropism (Mascarenhas and Machlis, 1962; Rosen, 1961), population e f fec t s , (Brewbaker and Kwack, 1963; Brewbaker and Majumder, 1961), calcium ion concentration-(Brewbaker and Kwack, 1963), boron and sugar concentrations and the pH of the germinating media ( V a s i l , I960), not to mention the vigor and genotype of the plant sampled or the duration and the temperature and humidity con-dit ions at the time of and fol lowing anthesis. i i ) Male s t e r i l i t y Male s t e r i l i t y according to Gabelman (1956) may take many forms, some of these being pol len s t e r i l i t y , staminal s t e r i l i t y , and po s i t i on -al or s t ructura l s t e r i l i t y , with pollen s t e r i l i t y being the resu l t of any one of the fol lowing f ac to r s : - a) gene act ion , b) cytoplasmic factors or c) the combination of both factors . Williams (1964) and Lewis (1941) further stated that interact ions between cytoplasmic and nuclear factors which resu l t in male s t e r i l i t y have been most frequently reported. Male s t e r i l i t y caused as a resu l t of the interact ion between nuclear and cytoplasmic factors re -sults in pollen abortion by upsetting the meiotic behavior and or pollen development (A l l a rd , 1960). Abortion of pol len may occur in various de-grees, from a number of c e l l s with abnormalities at meiosis I to abortion of a l l the pollen mother c e l l s within an anther. -5-Two common causes frequently emphasized in studies on male s t e r i l i t y are i r r e g u l a r i t i e s in the meiotic process, whether there i s complete i nh i b i t i o n of meiosis(Singh and Rhodes, 1961), or breakdown during the process(Rick, 1948) and abnormalities associated with the tapetal complex(Alam and Sandal, 1967; Dubey and Singh,1965; F i l i o n and Chr i s te , 1966; Joppa, et al .,1966 ; Singh and Hadley,lB61; Webster and Singh, 1964; Zenkteler, 1962; Brooks, et a l . , 1966). Francis and Bemis(1970) divided the abnormalities associated with the tapetum into three categor ies: -a) the formation of an i r regu la r Plasmodium, b) the premature breakdown of the tapetal c e l l s and c) the persistence of the tapetum, Davis(1966) stated that the anthers of Rosaceous flowers are tetrasporangiate with a res i s tant epidermis, and that the two or three middle layers are ephemeral with the c e l l s of the tapetum becoming two to eight nucleate as the anther develops. She also reported that simultaneous cytokinesis in the microspore mother c e l l follows meiosis, that the microspore tetrads are tet rahedra l , and the pollen grains two-ce l led when shed. Singh and Hadley(1961) observed that the development of anthers was approximately the same in both the f e r t i l e and male s t e r i l e l ines of sorghum with the exception of the behavior of the microspores and tapetal c e l l s a f te r meiosis. Meiosis and the formation of tetrads in both l ines were reported to be normal; however, the pollen from male s t e r i l e plants had re t i cu la te exines while the grains from the f e r t i l e l i n e were smooth. The authors also reported that in the, s t e r i l e material most of the microspores remained uninucleate while in the f e r t i l e plants., the pollen grains at time of shedding had a tube nucleus and two -6-male gametes. They also noted endomitosis in the tapetal c e l l s and the persistence of the tapetal layer in the s t e r i l e l i n e . This associat ion between the post meiotic behavior of the tapetum and the v i a b i l i t y of the pol len grains has been observed by Artschwager (1947), Chang (1954), and Monosmith (1928). This associat ion indicated to the authors that the cytoplasmic e f fec t i s mediated through the sporophytic t i s sue, and that since the ro le of the tapetum i s generally considered as nu t r i t i v e to the microspores, the abnormal behaviour of the tapetum probably upsets the regular supply of nutrients going to the microspores. Reports of pol len abortion a f te r meiosis in hybrids have been made by Juel (1900), T i sch ler (1906), and Osawa (1912). Francis and Bemis (1970) also observed normal development in the ear ly stages of microsporogenesis in a male s t e r i l e mutant of Cucurbita maxima. They noted hypertrophy of the tapetal c e l l s and the s h r i v e l l i n g of the microspores. This suggests that the microspores of male, s t e r i l e plants are ' s t a r ved ' . Although researchers have presented cytochemical evidence pertinent to the role of the tapetum in the nuclei acid metabolism of the microsporocytes, only speculative conclusions have been poss ib le. Cooper (1952) advanced the theory that the ro le of the tapetum was not only a source of nu t r i t i on for d i v id ing pollen mother ce l l s but was also a t issue that secreted substances that were necessary for the maturation of microspores a f te r meiosis. Saini and Davis (1969), reporting on male s t e r i l i t y in Al1iurn species gave strong evidence supporting the idea that microspore degeneration a f te r quartet formation was a re su l t of def ic iency of -7 -RNA and DNA. Furthermore, they suggested that the breakdown products from deter iorat ing tapetal c e l l s might be used in the resynthesis of RNA and DNA necessary for microspore development. The same conclusion was made by Vas i l (1959). However, no f i rm evidence has been shown to ex i s t fo r tapetal function in th i s way during meiosis (Alam and Sandal, 1967; Kosan, 1959). Foster and Stern (1959) demonstrated that there were sharp temporal increases in the amount of soluble deoxyribosidic material present in Li 1 iurn anthers j u s t before the respective DNA synthesis in microsporocytes, microspores and po l len. They also noted that the amount of DNA in the anther declines over a period when. DNA was in fact being synthesized in the microspores and pollen grains. This observation suggested to the authors that a large amount of DNA was broken down in the anther and that some of i t might serve as precursor material for the DNA synthesis in the microspores. Takats(1962), however, reported on the catabolism of tapetal DNA to a level not u t i l i z a b l e in synthesis, and of the presence of a non-tapetal pool of DNA precursors. That male s t e r i l i t y results from hybr id izat ion has been pointed out by several workers (Knight, 1821; Hoar, 1916; Lewis, 1941). Lewis (1941) also reported that cytoplasmic male s t e r i l i t y i s the commonest form of male s t e r i l i t y in species hybrids and that the degree of s t e r i l i t y was related to the compat ib i l i ty between the parents (Longley and Darrow, 1924; Knight, 1821; Hoar, 1916). Brainerd (1914) reported that the genus Rubus was notorius for interbreeding and thus producing new forms. Hoar (1916) in an attempt at c l a s s i f y i n g the species of Rubus concluded that while hybrids of d i s t i n c t species were very s t e r i l e , crosses of va r ie t ie s more c lose ly related may give almost no ind icat ion of t he i r hybrid o r i g i n . -8-Longley and Darrow (1924). found unequal d i s t r ibut ions of chromosomes during tetrad formation, lagging chromosomes and the presence of univalents at both anaphase I and I I„ in a number of d i p l o i d and poly-p lo id forms of raspberries. S imi lar chromosomal i r r e g u l a r i t i e s were reported by Craig (I960) for R_. canadensis L. a t r i p l o i d , and by Thompson (1962) for some polyploid forms of Rubus. Jennings (1967) observed a lagging chromosome at anaphase in a crumbly clone of the red raspberry var iety 'Norfolk Giant ' a d i p l o i d , and concluded that th i s meiotic i r r e -gu la r i t y could perhaps be the cause of the crumbly phenomenon of some raspberry c u l t i v a r s . Meiotic i r r e g u l a r i t i e s re su l t ing in male s t e r i l i t y have also been reported by Mulford (.1937) in chrysanthemum. He suggested that these i r r e g u l a r i t i e s which led to unequal segregation of chromosomes and thus probably a v a r i a b i l i t y in s i ze of pollen grains could have resulted from structura l hyb r id i t y , polyplo idy, or unfavorable environmental con-d i t i on s . Hull and Br i t ton (1958) also observed var ia t ion in s ize of pollen grains in one of t he i r polyplo id seedlings of Rubus and assumed that th i s was due to varying chromosome number. Grun (1952) working with meiosis in some species and hybrids of Poa found that while some plants were not affected by the environment, others were strongly af fected. He further observed that weakly growing plants frequently had a greater number of univalents during meiosis than did plants which made vigorous growth. i i i ) Megagametogenesis. Crane (1940) stated that sexual reproduction was generally the rule in d ip l o id species of Rubus, and that even for apomixis to take place po l l i na t i on was necessary. Thus, he reported that seed development in Rubus was dependent upon p o l l i n a t i o n , rather than independent of i t . -9-This i s in agreement with the findings of Gustafsson (1930), and Darrow and Waldo (1933). Although d ip l o id species of "Rubus were reported to behave sexually (Darrow and Waldo, 1933), occasional unreduced germ c e l l s were found to take part in f e r t i l i z a t i o n giving r i se to polyploid forms, Thomas (1940) reported that in the sexually reproducing . d i p l o i d R. rustlcanus, meiosis occurred in the embryo sac mother c e l l a l -most as soon as in the anthers with the embryo sac forming very qu ick ly. The polar nuclei fused to form the fusion nucleus at least two days before the flower opened and no departure from the 'normal ' or Polygonum type of development was observed in th i s species. Furthermore, he pointed out that exceptional behavior in embryo sac development such as fusion of haploid nuclei other than that of the male nucleus with the polar nuclei or egg c e l l , the presence of two d i f fe rent i a ted egg c e l l s and one synergid, or the presence of three undifferent iated nuclei at the micropylar end may occur with in sexual embryo sacs. Sexual reproduction with normal embryo sac development i s reported for clones of _R. spectab i l i s a d i p l o i d , by V i rd i and Eaton (1969). Pratt and Einset (1955) also reported that d ip l o id species of Rubus reproduced sexual ly and that embryo sac development in. these species was of the 'normal ' or Polygonum type. Markarian and Olmo (1959) working with R. procerus a te t rap lo id species, obtained results which demonstrated that in th i s species, embryo sac development was s im i l a r to that described by Pratt and Einset (1955) for d i p l o i d species of Rubus and was therefore normal and reproduction was concluded to be sexual. Pratt and Einset (.1955) in a survey of the development of the embryo sac of seven species of the American blackberry found that the Rubus carpel has two anatropous, unitegmic, c rass inuce l late ovules. The ovules v/ere found to be attached one above the other to the placenta with -10-the one above aborting early in the development. Apospory was observed to occur only very rare ly in d i p l o i d species ofRubus while i t occurred more commonly in the odd-ploid forms of the species. The authors also reported that the odd-ploid forms were characterized by the presence of young stages of embryo sac development in large buds and open flowers and the occurrence of mult ip le sacs per ovule. While these authors found no instance of the development of any but the chalazal megaspore into a sac i n a number of species studied, and reported that by the time the sac had attained a two-nucleate stage the megaspore was no longer d i s t ingu ishable. Markarian and Olmo (1959) stated that i t was d i f f i c u l t to estab l i sh c e l l differences be-tween sporogenous and somatic t issues at the time of megaspore formation, but that l a t e r stages gave no evidence that the nucel lar t issue had generative a b i l i t y . Kerr (1954) however, in describing seed development in three ' f a c u l t a t i v e pseudogamous v a r i e t i e s ' and three 'syngamic v a r i e t i e s ' of t e t rap lo id American b lackberr ies , reported that while the chalazal megaspore usually gave r i se to the sac, any one of the other megaspores occas ional ly did so. Davis (1966) in describing embryo sac development for the family Rosaceae stated that cytokinesis in the megaspore mother c e l l accompanied meiosis and that^the chalazal megaspore of a l i nea r tetrad developed into a Polygonum type embryo sac, but that in Rubus any megaspore could function to form the sac. She further stated that in Rubus i f the megaspore mother c e l l degenerates i t i s replaced by one or more secondary megaspore mother c e l l s which undergo meiosis and give r i se to embryo sacs in the usual way. - 1 1 -' MATERIALS AND METHODS Pollen s t a i n a b i l i t y Pollen s t a i n a b i l i t y was determined for the c u l t i v a r , Mai l ing Jewel and the se lect ion Ore-US 1314. Mai l ing Jewel is described as highly s e l f - f e r t i l e by Topham (1967) and Ore-US 1314 as lacking f u l l s e l f -f e r t i l i t y by Daubeny (1969). Both the c u l t i v a r and se lect ion were supplied by the Canada Department of Agr iculture Small Fruits Substation at Abbotsford, B r i t i s h Columbia. Both clones were free of known viruses and of the same physiological age. Blossoms were randomly co l lected ju s t p r io r to anthesis from plants of both clones and f ixed immediately in a 1:3 acet ic ac id -ethanol solut ion for forty eight hours. Anthesis i s here used to describe that stage of f l o r a l development where the petals have expanded ju s t enough to expose the stigmas. After f i x a t i o n , the samples were stored in 70% ethanol in a re f r i ge ra to r un t i l they were observed a month l a t e r . From the samples thus preserved, ten flowers from each clone were randomly picked and four anthers of varying lengths were removed from each flower. Each of the removed anthers was designated as ' a 1 , ' b 1 , ' c ' , and ' d ' , with s a ' having the longest filament followed by ' b ' , ' c ' , and ' d ' . Each anther was then squashed and stained with aceto-carmine. To ensure the removal of a l l the pollen grains from the anther sacs, the anthers were cut in ha l f and the pollen grains r o l l ed out with a needle. A drop of g lycer in was added on the s l i de with the s ta in as i t allowed even d i s t r i bu t i on of the pol len grains by l i m i t i n g the movement of the aborted and l i g h t e r grains to the edges of the cover glass (Marks, 1954). The s l ides were then l e f t overnight on a warming table at 40 C to in tens i fy the s ta in . Five separate counts of 100 pollen grains each were observed -12-fo r each anther and three types of pol len grains normal, abnormal, and intermediate were recorded. Those grains which, were large and t r iangu lar were considered normal, while those that were lacking cytoplasm and there-fore non-staining were c l a s s i f i e d as abnormal. Intermediates were those grains which did not f i t into e i ther of these categories. An analysis of variance was car r ied out on the data obtained for both clones. Meiosis Buds measuring between 2.0 and 4.0 mm, of the c u l t i v a r Mai l ing Jewel and the se lect ion Ore-US 1314 were co l lected from virus free plants at the Small F r u i t Substation of the Canada Department of Agr icu l ture at Abbotsford. Upon c o l l e c t i o n , the buds were immediately f ixed in a 1:3 acet ic acid-ethanol solut ion for for ty eight hours. A f ter f i x a t i o n the buds were stored in 70% ethanol and ref r igerated unt i l use. Anthers were dissected out of the buds, put into pre-warmed v i a l s and hydrolyzed with IN HC1 at 60 C for t h i r t y minutes. Individual anthers were then removed, stained with aceto-carmine and squashed by tapping the edges of the cover glass with the end of a penc i l . S l ides were sealed with a mixture of paraf f in wax and gum arabic and observed. Five anthers per bud and two buds each of s im i l a r s i ze were sampled. Anther tapetum study Five flowers at each of the fol lowing three stages: p r io r to anthesis, anthesis, and two days a f te r anthesis, of the c u l t i v a r Mai l ing Jewel and the advanced se lect ion Ore-US 1314 were randomly picked and from -13-each flower seven anther were removed and f i xed and stored as for the meiotic study. Following an e thano l - te r t i a ry bjjtanol ser ies as described by Johansen [1940), the anthers were embedded in paraplast and se r i a l sections of 10 u thickness were cut and mounted on s l ides coated with Haupt's adhesive (1930). The s l ides were then stained with haematoxylin (Johansen, 1940) and mounted in Canada Balsam. Megagametogenesis The red raspberry cu l t i va r s Wil lamette, Sumner ( including both the normal and crumbly clones) and the se lect ion Ore-US 1314 were studied. Two plants each of the above clones were brought into the green-house at UBC from the Small F ru i t Substation at Abbots ford in the winter of 1969. The plants blossomed in the f i r s t week of March 1970. The se lect ion Ore-US 1314 was the l a t e r bloomer. F lora l stages co l lected for study were anthesis, two s four, and s i x days a f te r anthesis, These cu l t i va r s Willamette i s included in th i s study as i t i s highly s e l f -f e r t i l e and has been used by Daubeny (1971) as a standard for s e l f -f e r t i l i t y . The normal and crumbly clones of Sumner were included as the crumbly clone has been reported to have a retarded embryo sac development (Daubeny, et a l . , 1967). Twenty four blossoms of each c u l t i v a r were tagged and s i x blossoms from each c u l t i v a r were removed at anthesis. Removal of s i x more tagged blossoms from each of the cu l t i va r s followed a f te r two, four and s i x days. A larger number of blossoms per f l o r a l stage could not be obtained as the plants became infected with spider mites. Tedion sprays were used to keep the in fes ta t ion at a minimum. A co l l e c t i on of flowers of Ore-US 1314 was also made at anthesis in the summer of 1969 from plants grown in the f i e l d at -14-Abbotsford. To summarize, the co l lec t ions made for the. study of the development of the embryo sac in the red raspberry-were*,~ Cu l t i va r " Year ' f l o r a l stages Ore-US 1314 Summer 1969 [ F i e l d ] * Anthesis Ore-US 1314 Spring 1970 (GH)* Anthesis, +2, +4, +6 Willamette Spring 1970 (GH) Anthesis, +2, +4, +6 Crumbly Sumner Spring 1970 (GH) Anthesis, +2, +4, +6 Normal Sumner Spring 1970 (GH) . Anthesis, +2, +4, +6 * F i e l d refers to plants grown at the substation in Abbots ford. *GH refers to the plants grown in the greenhouse at UBC, Blossoms were immediately f ixed in S e l l i n g ' s Modified Navashin's f l u i d and embedded in paraplast a f te r fol lowing an ethanol-t e r t i a r y butanol series as described by Johansen (1940). Ser ia l sections were then cut as for anther tapetum study. A few s l ides were stained with hematoxylin but since a modified t r i p l e s ta in technique of Gerlach's (1969) gave comparable results and was much shorter than the sta in ing procedure with hematoxylin, the t r i p l e sta in technique (Appendix I) was used throughout the remainder of the study. After s ta in ing , the s l ides were permanently mounted in Canada Balsam. Po l l i na t i on studies Po l l inat ions were carr ied out in the summer of 1970 at the Small F ru i t Substation of the Canada Department of Agr iculture at Abbots ford. The experiment consisted of the fol lowing treatments: Select ion or Cu l t i va r Treatment  i ) Ore-US 1314 Open po l l inated i i ) Ore-US 1314 Selfed i i i ) Ore-US 1314 X Mai l ing Jewel ' Select ion or Cu l t i va r "Treatment iy\ Mai l ing Jewel Open po l l inated v) Mai l ing Jewel Selfed v i ) Mai l ing Jewel X Ore-USJ.314 For s e l f i n g , ten flower c lus ter s of each, clone s t i l l i n the bud stage and having at least seven buds were marked and bagged with 9 x 18cm. glassine bags. S im i l a r l y , for each of the clones ten other adjacent c lusters were marked and these ware l e f t to be open po l l i na ted. These open po l l inated clusters were l e f t undisturbed while the sel fed clusters.had the i r bags removed every other day and the flowers manually po l l inated by brushing the pol len from the anthers on to the stigmas with the thumb. This procedure was car r ied out over a period of about three weeks un t i l petal f a l l . For c ro s s -po l l i na t i on treatments, f i f t een flower c lusters of each clone s t i l l in the bud stage were marked and buds measuring between 7.5 and 8.0mm across the base were emasculated and bagged. The emasculated buds were po l l i nated two days a f te r emasculation as described by Eaton, et al_. ,(1967). Since Ore-US 1314 did not produce a large number of buds of s im i l a r s i ze at the same time, bags were removed every other day and emasculation and po l l i na t i on carr ied out as the buds reached the desired stage. As Ore-US 1314 was much l a t e r blooming than Mai l ing Jewel, pol len from th i s c u l t i v a r was co l lected and stored un t i l ready to be used. Pollen was obtained by co l l e c t i n g flowers at anthesis or ju s t before, removing the anthers and drying them at 43 C for approximately two hours. To keep the treatments uniform, pol.len from Ore-US 1314 was treated the same way. Pol len was applied to the stigmas with a camel-hair brush. The glassine bags were l e f t in place un t i l the berries -16-r were r ipe. A f ter removal t berry-weight and counts of the number of the normal and aborted druplets were made for each, berry. A s ingle degree of freedom test was carr ied out on the .• percentage of druplets set and the berry weight for each of the treatments, -17-RESULTS Pol len s t a i n a b i l i t y Ma i l ing Jewel had a very high percent of normal-appearing pollenCTable 1) ( f i g . 1) with a c lear d i s t i n c t i o n betv/een the normal and abnormal pollen grains. The analysis of variance (Table 2) showed that in th i s c u l t i v a r there were no s i gn i f i c an t differences in the percentage of normal pol len produced by anthers having d i f f e ren t fi lament lengths. However, highly s i g n i f i c an t differences were found to ex i s t among the flowers sampled in the percent of normal pollen produced. Also, s i g n i f i c an t f i lament by flower interact ions were found in th i s c u l t i v a r (Appendix I I ) . Ore-US 1314 showed a very high degree of pollen s t e r i l i t y (Table 1) (F ig . 2), with pol len s t e r i l i t y ranging from shrunken pollen grains having no cytoplasm at a l l (F ig. 7) to th ick walled pol len grains with stainable cytoplasm (Fig. 8). In many cases ent i re anthers were found to be s t e r i l e (Figs. 3 and 6). An analysis of variance showed that unl ike Mai l ing Jewel, highly s i g n i f i c an t differences in the percent normal pollen produced were found to ex i s t in the anthers of d i f f e ren t lengths (Table 3) the shorter filaments being associated with greater s t e r i l i t y . Like Mai l ing Jewel, s i g n i f i c an t differences were found to ex i s t among the flowers sampled in the percent normal pollen produced. S i gn i f i cant fi lament length by flower interact ions were also present in the Ore-US 1314 (Appendix I I I ) . - 1 8 -Table 1. Percent normal pollen from counts of 500 pollen grains Cu l t i va r or se lect ion 1970 1970* Ore-US 1314 11,05 23.40 Mai l ing Jewel 91.42 Willamette - 67.14 *These values were kindly supplied by Dr. H.A.Daubeny of the Canada Department of Agr iculture at Agassiz, B r i t i s h Columbia. The values were obtained from counts of 300 grains The value for the cu l t i v a r Willamette i s included in th i s table to serve as a comparison since i t i s used as a standard by Daubeny (1971). ' ' -19-Table 2. An analysis of variance of the percentage of normal pol len from four fi lament lengths from ten randomly selected flowers of the c u l t i v a r Mai l ing Jewel. Source df SS MS F" Flower 9 7451.7 827.96 26.38** Filament length 3 261.3 87.10 2.46 n s Filament length X flowers 27 6077.8 255.10 7.20** Error 160 4997.4 31.23 -Total 199 28788.2 _ _ * * S i gn i f i cant at the 1 percent l e v e l . n s Not s i gn i f i c an t at the 5 percent level -20-Table 3. An analysis of variance of the percentage of normal pollen from four fi lament lengths from ten randomly selected flowers of the se lect ion Ore-US 1314. Source df SS MS Flower Filament length Filament length X flowers Error Total 9 3 27 160 199 34327.1 6615,4 25097.0 5028.0 71067.5 3814.12 2205.13 929.40 31.32 121.07** 70.18** 29.50** * * S i gn i f i cant at the 1 percent l e v e l . - 2 1 -Figure 1. A sample of pollen grains of the c u l t i v a r Mai l ing Jewel showing a random f i e l d of pollen grains. Note the absence of abnormal pollen grains. 209X Figure 2. A sample of pollen grains of the Ore-US 1314 showin a random f i e l d . Note the d i s t r i bu t i on of normal (N and abnormal (Ab) pol len grains. 209X -22--23-Contents of a completely s t e r i l e anther of Ore-US 1314. Pollen grains f a i l to form as shown by the mass of sporo-genous t i s sue, b i t s of t i ssue broken away from the mass f a i l in t he i r attempt at forming pollen grains by having no c e l l w a l l . 900X Longitudinal section of an anther of Ore-US 1314 showing cytoplasmic connections between normal (N) and abnormal (Ab)' pol len grains. 328X Longitudinal section of an anther of Ore-US 1314 showing cytoplasmic connections between normal grains. 300X Contents of a p a r t i a l l y s t e r i l e anther of the advanced se lect ion Ore-US 1314 showing clumping of abnormal pollen grains. 328X A sample of pollen grains of Ore-US 1314 showing normal (N), and intermediate (I) pollen grains. The intermediate pol len grains were considered non-viable. 900X A sample of pollen grains of Ore-US 1314 demonstrating contorted c e l l walls with shrunken cytoplasm wi th in . 328X -25-Meiosis Buds measuring betv;een 2.5 and 3.0 mm across the base of both the c u l t i v a r Mai l ing Jewel and the se lect ion Ore-US 1314 were found to have d iv id ing microspores (Appendix VI). There was no synchrony in d i v i s i on espec ia l l y in the.se lect ion Ore-US 1314 and i t was possible to f ind c e l l s at telophase II next to c e l l s s t i l l at anaphase I (Appendix VI). In th i s se lect ion irregular meiosis (both of meiosis I and meiosis II) was observed. Lagging chromosomes (F ig . 9 ) , chromosome bridges (F ig. 10), and i r regu la r chromosome numbers (F ig. 11), were observed in these c e l l s . Larger buds of th i s se lect ion had incomplete tetrads, that i s there were less than the usual four meiospores within the pollen mother c e l l wall (F ig . 12)(Table 4). Normal meiosis in th i s se lect ion i s shown in Figs.13 and 14 with the meiocytes having the normal number of n-7 chromosomes, The same bud s ize in the c u l t i v a r Mai l ing Jewel produced anthers which had c e l l s a l l at meiosis II unl ike the se lect ion Ore-US 1314 which had 46.2% of the c e l l s observed at meiosis I (Table 4 ) , and only 53.8% at meiosis I I. The c u l t i v a r Mai l ing Jewel demonstrated c e l l s w i th -out c e l l wa l l s . The reason for f a i l u r e of c e l l wal l formation was not invest igated. Unlike the se lect ion Ore-US 1314, no i r r egu la r chromosome numbers, lagging chromosomes, or chromosome bridges were found in the samples pbserved fo r the c u l t i v a r Mai l ing Jewel. -26-Table 4. Percentage of anther c e l l s at varying meiotic stages from f i ve anthers from each of two buds measuring 2.7 mm of the c u l t i v a r Mai l ing Jewel and the se lect ion Ore-US 1314. Cu l t i va r or Meiotic stages * Total no. se lect ion JJ TT T i l I r r . o f c e l 1 s Mail ing Jewel 0 0 0 93 5 0 2 10,500 Ore-US 1314 1.9 8.2 36.1 41.8 11.5 0.5 0 7,500 *AI Anaphase I I r r . A I rregular anaphase I • TI Telophase I TII Telophase II with four, three, or two microspores with in the pol len mother c e l l w a l l . I r r . I rregular c e l l s having no c e l l walls -27-Figure 9. A c e l l from an anther of a 2.7 mm bud of Ore-US 1314 at anaphase I, with lagging chromosomes (arrows). 3328X Figure 10. Anaphase I of Ore-US 1314 with a chromosome bridge. 3328X Figure 11. A c e l l from an anther of a 2.7 mm bud of Ore-US 1314 showing uneven chromosome d i s t r i b u t i on . 3328X Figure 12. Incomplete tetrads of Ore-US 1314 from an anther of a 3.7 mm bud. 1178X Figure 13. Diakinesis in the se lect ion Ore-US 1314 with the normal number of seven chromesomes. 3328X Figure 14. Metaphase II in the se lect ion Ore-US 1314 with the normal number of seven chromosomes at each pole. 3328X -28--29-Anther tapetum study Examination of both longitudinal and cross sections of anthers of the advanced se lect ion Ore-US 1314 showed no involvement of the tapetum in pollen abortion (F ig . 15, 16, and 17). The tapetum in a l l the anthers observed had dis integrated pr io r to anthesis. Differences in the quantity and qua l i t y of pollen existed between the locules of the same anther (Fig. 17). This was noted in 34.2% of the anthers observed (Table 5). P a r t i a l l y s t e r i l e anthers had locules that were almost empty with both the normal and abnormal pollen grains clumped together (F ig . 16). Cytoplasmic connections between pollen grains were found in 5.7% c f the anthers of th i s se lect ion (Table 5). The con-nections were between normal grains (F ig. 5) as well as between normal and abnormal pol len grains (F ig.4). No cytoplasmic connections were observed in the anthers of the c u l t i v a r Mai l ing Jewel. The two locules of an anther of th i s c u l t i v a r were s im i l a r in the quantity and qua l i t y of pol len produced. Like 0re~US 1314, the anthers of Mai l ing Jewel showed complete d i s i n -tegrat ion of the tapetum pr i o r to anthesis. -30-Table 5. Percent of anthers at anthesis having visible differences between locules as to pollen quality and the instance of cytoplasmic connections in Mailing Jewel and Ore-US 1314. Cultivar or selection Percent of un!i ke anthers with locules Percent of anthers with cytoplasmic ....connections Mailing Jewel , 7.6 0.0 Ore-US 1314 34.2 5.7 Observations were made from seven anthers from each of five flowers of each of the clones at anthesis. -31-Figure 15. Longitudinal section of an anther of the advanced se lec-- . t ion Ore-US 1314 p r i o r to anthesis showing normal darkly s ta in ing pollen grains (N), and the absence of a per-s i s t i n g tapetum/ 283X Figure 16. Longitudinal section of a p a r t i a l l y s t e r i l e anther of the advanced se lect ion Ore-US 1314 at anthesis. Note the reduced number and clumping of pollen grains within the l o cu le s 9 and lack of a pers istant tapetum. Taken under phase contrast. 283X Figure 17. Cross section of the anther of the advanced se lect ion Ore~US 1314 at anthesis showing var ia t ion in the qua l i t y and quantity of pollen produced by the two locules. Again the tapetum i s not present. Taken under phase contrast. 283X -32--33-Megagametogenesis ' Normal Sumner At anthesis a l l the sacs observed in normal Sumner had reached the d i f fe rent i a ted eight-nucleate stage (Table 7). The two obvious stages of embryo sac development here are that of the d i f fe rent i a ted egg c e l l with d i f fe ren t i a ted polar nuclei and that of the d i f fe rent i a ted egg c e l l with a fusion nucleus. At two days a f te r anthesis several sacs s t i l l exh ib i t ing stages with an undifferent iated egg c e l l were found. Nevertheless, the majority of the sacs observed at th i s stage had a d i f fe rent i a ted egg c e l l with a fusion nucleus. The presence of a pollen tube was observed in a number of sacs four days a f te r anthesis and in some suchembryo sacs the egg c e l l had began to lose i t s crescent shape and become' rounded. In such embryo sacs i t was d i f f i c u l t to t e l l whether or not these egg c e l l s had been f e r t i l i z e d and were in fact zygotes or whether they were merely under-going metabolic a c t i v i t i e s due to the presence of the pollen tube in the sac. As a resu l t of th i s uncertainty, these sacs were c l a s s i f i e d in two separate categories (E)PP and (E)(PP) (see Table 6 fo r deta i l of sac categor ies). Developing embryos were also abserved at four days a f te r anthesis. There did not seem to be much dif ference in development between the embryo sacs at four and s i x days a f te r anthesis and in both these stages undifferent iated egg c e l l s had disappeared and embryos were present. The most frequent stage in the development of the embryo sacs was that of the d i f fe ren t i a ted egg c e l l with the fusion nucleus. -34-This stage E(PP) predominated throughout the ent i re examination period and ranged from 80% at anthesis to 54% s i x days l a t e r . Crumbly Sumner Development of the embryo sac in the crumbly clone of the Sumner c u l t i v a r was d i f fe rent from that of the normal Sumner clone in that i t exhibited a high percentage of degenerated embryo sacs (Table 8). Degeneration of the sacs was due e i ther to the col lapse of the egg apparatus alone or to the degeneration of the ent i re contents of the s a c . Retarded embryo sac development was noticed in th i s clone with the undif ferent iated eight-nucleate stage being present in the ovules in stages a f te r anthesis. Also, a l l types of sac stages p r i o r to that of a d i f fe ren t i a ted egg c e l l with a fusion nucleus (a common stage of sac development with in the ovules of the normal clone of th i s c u l t i v a r ) , were found to ex i s t in th i s clone up t i l l four days a f te r anthesis. This retarded development changed pace a f te r four days a f te r anthesis and by s i x days a f te r anthesis most of the sacs observed were degenerated (Table 8). Those sacs c l a s s i f i e d as i r regu la r e i ther had mult ip le sacs or extra c e l l s at the antipodal or chalazal ends of the embryo sac (F ig. 19). Willamette Most of the Willamette embryo sacs observed at anthesis had a d i f f e ren t i a ted egg c e l l with a fusion nucleus (Table 9) (Fig. 18 and 19). The e a r l i e s t sac.stage observed was that of sacs d i f fe rent i a ted by the presence of migrating polar nuclei but having no d i f fe rent i a ted egg eel 1. The entry of a pol len tube (F ig. 20 and 21) was noticed at -35-two days a f te r anthesis with more than ha l f the embryo sacs demonstrating th i s phenomena (stage E(PP)pt, (E)(PP), (E)PP, and Z). A noteworthy feature observed f i r s t at two days a f te r anthesis and present t i l l s i x days a f te r anthesis in th i s c u l t i v a r was the presence of unfused polar nuclei in embryo sacs which had already been entered by the pollen tube (embryo sac stage (E)PP, Table 9). Although not frequent, there were instances of micro nuclei in the egg c e l l (F ig. 21). Embryo sac development at four and s i x days a f te r anthesis were very s im i l a r and d i f fe red from the development at two days a f te r anthesis by having embryos. No i r regu la r sacs were ever found in th i s c u l t i v a r . Ore-US 1314 Embryo sac development of Ore-US 1314 at anthesis (Table 10) i s s im i l a r to that of the c u l t i v a r Willamette and the normal clone of the Sumner c u l t i v a r for anthesis with the majority of the sacs exh ib i t ing a d i f f e ren t i a ted egg c e l l and fusion nucleus. The i r regu la r sacs at th i s stage and at two days a f te r anthesis were cases of mult ip le sacs with in one ovule. Development at two days a f te r anthesis showed a great increase in the percentage of degenerating sacs. Here degeneration was due e i ther to-the collapse of the egg apparatus or the fusion nucleus. Unlike the crumbly clone of Sumner no undifferent iated egg c e l l s were found, and by four days a f te r anthesis embryos had formed. Six days a f te r anthesis s i x t y - fou r percent of the sacs observed were degenerated, and the degeneration at th i s stage was due mainly to the breakdown of the ent i re contents of the sac (F ig. 22). -36-Embryo sac development- in the se lect ion Ore-US 1314 at anthesis in 1969 was not very d i f fe rent from that at anthesis in 1970 (Table 11). The majority of the sacs observed had a d i f fe ren t i a ted egg c e l l with a fusion nucleus. The degeneration observed here was due to the col lapse of the egg apparatus. -37-Table 6, Description of sac categories used in Tables 7 to 11. 8-n An eight-nucleate undifferentiated embryo sac. epp Embryo sac differentiated by migrating polar nuclei but s t i l l having an undifferentiated egg cell. e(pp) Embryo sac differentiated by the fusion of the polar nuclei but s t i l l having an undifferentiated egg eel 1. Epp Embryo sac having a differentiated egg cell with migrating polar nuclei. E(PP) Embryo sac having a differentiated egg cell with the polar nuclei fused. E(PP)pt Embryo sac as above with pollen tube. (E)PP Embryo sac with migrating polar nuclei and a rounded, darkly staining differentiated egg cell. (E)(PP) Embryo sac with fused polar nuclei and a rounded, darkly staining differentiated egg-cell. Z Embryo sac with a.zygote. EMB Embryo sac with a young embryo. Deg Degenerated embryo sacs. The degeneration is due either to the collapse of the egg apparatus or of the fusion nucleus, or degeneration of the entire sac content. I Irregular ovules, either having more than one embryo sac per ovule or having extra cells either at the antipodal or chalazal ends of the embryo sacs. Table 7. Percentage of embryo sacs in each stage of development wi th in the ovules of the normal clone of the Sumner cu l t i v a r i n ' 1970. Days a f te r SAC CATEGORIES Total no. of anthesis 8-n ePP e(PP) EPP E(PP) E(PP)pt (E)(PP) (E)PP Z EMB Deg I ovule 0 2 0 0 18 80 0 0 0 0 0 0 0 64 2 0 10 0 10 80 0 0 0 0 0 0 0 71 4 0 0 0 0 70 . 9 ; 11 0 5 r-0 0 0 132 6 0 0 0 0 54 13 22 0 0 11 0 0 83 Total no. of ovules 19 245 23 16 0 0 350 Table 8. Percentage of embryo sacs in each stage of development with in the ovules of the crumbly clone of the Sumner cu l t i v a r in 1970. Days a f te r SAC CATEGORIES Total no. of anthesis 8-n ePP e(PP) . EPP E(PP) E(PP)pt (E)(PP) (E)PP Z EMB Deg T ovules 0 17 10 6 19 38 0 .0 0 0 0 10 0 141 2 5 0 /> L 7 26 • 8 29 0 7 0 16 0 42 4 3 2 1 8 35 15 18 0 0 3 12 3 120 6 • 0 0 0 . 0 0 ' 20 22 0 0 11 47 0 68 Total no. of ovules 30 11 39 106 35 46 11 67 371 Table 9. Percentage of embryo sacs in each stage of development with in the ovules of the c u l t i v a r Willamette in 1970. a ^ SAC CATEGORIES T o t ^ n 0 anthesis 8-n ePP e(PP) EPP E(PP) E(PP)pt (E)(PP) (E)PP 7. EMB Deg I ovules 0 0 8 0 14 71 0 o- 0 0 0 7 0 107 2 0 0 0 8 35 6. 40 4 5 0 2 0 103 4 0 C 0 2 12 0 57 5 8 5 1 0 104 6 0 0 0 0 14 6 53 5 0 12 0 0 54 Total no. of ovules 0 9 0 25 132 9 145 12 13 12 10 0 357 Table 10. Percentage of embryo sacs in each stage of development w i th in the ovules of the advanced select ion Ore-US 1314 in 1970. SAC CATEGORIES Total no. a f t e r of anthesis 8-n ePP e(PP) EPP E(PP) E(PP)pt (E)(PP) (E)PP Z EMB Deg I ovules 0 0 0 3 7 86 0 0 0 0 0 2 2 68 2 0 .0 3 38 6 20 0 o •J 0 28. 2 65 4 0 0 0 0 20 8 8 o 4 10 50 0 50 6 0 ' 0 0. 0 0 10 9 0 0 17 64 0 65 Total no. of ovules 0 0 2 7 93 14 23 0 4 16 86 3 248 Table 11 . -Percentage of embryo sacs in each stage of development with in the ovules of the advanced se lect ion Ore-US 1314 in 1969. Days SAC CATEGORIES Total no. a f t e r of anthesis 8-n ePP e(PP) EPP E(PP) E(PP)pt (E)(PP) (E)PP Z EMB Deg I ovules 0 0 0 6 *2 80 0 0 0 0 0 2 0 70 Total no. of ovules 0 0 4 8 57 0 0 0 0 0 1 0 70 -43-Figure 18. Embryo sac of the c u l t i v a r Willamette at anthesis. The egg c e l l , E, the two polar n u c l e o l i , PP, with in a s ing le nuclear membrane and two of the three a n t i -podal s (a) are present. 1040X Figure 19. Embryo sac of the c u l t i v a r Willamette at two days a f te r anthesis. The egg ce l l i s s t i l l crescent shaped and the polar nuclei have not yet fused although the embryo sac has lengthened. 1040X Figure 20. Embryo sac of the c u l t i v a r Willamette at four days a f te r anthesis. The egg c e l l i s now rounded, E, the polar nuclei have fused (PP) and heavy s ta in ing of the cytoplasm around the egg apparatus i s obvious. 1040X Figure 21. The egg apparatus of the female gametophyte of the c u l t i v a r Willamette at two days a f te r anthesis. The egg nucleus (EN) .with two micro nuclei (arrows), the heavi ly s ta in ing pollen tube (p t ) , and a synergid (S) are shown. 1800X -45-Degenerated embryo sac ES of Ore-US 1314 at s i x days a f te r anthesis. 1040X -46--47-Po l l i na t i on studies. Druplet set: The results obtained for the percent normal druplet set showed that Mai l ing Jewel set f r u i t well when selfed and thus demonstrated i t s high level of s e l f f e r t i l i t y . The results also showed that i t i s highly f e r t i l e when open po l l i na ted. However, Ore-US 1314 pollen reduced the percent of normal druplets that set (Table 12). Ore-US 1314 also showed a high level of f e r t i l i t y when open po l l i na ted. However, there was a great reduction in the percent normal druplet set when i t was se l fed. Nevertheless, when po l l inated with pollen from the c u l t i v a r Mai l ing Jewel there was an increase in the percent normal druplet set from that when se l fed. However, th i s increased percent was s t i l l less than that obtained when open po l l inated (Table 12). The s ingle degree of freedom contrasts (Table 13) showed highly s i g n i f i c an t differences in the percent normal druplet set be-tween the c u l t i v a r Mai l ing Jewel and the se lect ion Ore-US 1314 (c lones), between the open po l l inated treatments when compared with the s e l f and cross po l l inated treatments for both clones, and between the s e l f and cross po l l i na t i on s . A highly s i g n i f i c an t in teract ion between the clones and the s e l f vs. cross po l l i na t i on comparison was also present (Table 13). Berry weight: Single degree of freedom contrasts (Table 13) showed several highly s i g n i f i c an t ef fects on berry weight. In comparisons with open po l l i na t i on berry weight in Mai l ing Jewel was decreased by s e l f po l l i na t i on and further by crossing with Ore-US 1314 (Table 12). -48-Reductions in berry weight also occurred in.Ore-US 1314 when sel fed or crossed with Mai l ing Jewel. However, unl ike Mai l ing Jewel, crossing gave an increase in berry weight over s e l f i n g in th i s se lect ion. -49-Table 12. Percent druplet set and berry weight of Mai l ing Jewel and Ore-US 1314 from s e l f p o l l i n a t i o n , open po l l i na t i on and cross po l l i na t i on with each other. Cu l t i va r or se lect ion Type of po l l i na t i on Se l f Open Cross Percent, druplet set Mai l ing Jewel 74.24 92.98 30.19 Ore-US 1314 2 2 ' 7 6 9 3 - 5 0 3 7 ' 3 5 Berry weight (gm) Mai l ing Jewel • 2.37 3.68 1.23 Ore-US 1314 0.64 3.89 1.48 -50-Table 13. S i gn i f i cant ef fects at the one percent level from s ingle degree of freedom comparisons.* Variable S i gn i f i cant e f fects Percent normal druplet set Clones Open vs others Se l f vs cross Clones X (Self vs cross) 0.7148 Berry weight Open vs others Clones X (Sel f vs cross) 0.6797 * See Appendix VIII fo r subdivis ion of degrees of freedom and Appendicies IX and X for graphs of in teract ions . -51-DISCUSSION Pollen s t a i n a b i l i t y The reduced percentage of normal pollen produced by Ore-US 1314 (Table 1) i s in accordance with reports made by Daubeny (1969) on the pol len production of th i s se lect ion. The dif ference in value between his observation and that obtained in th i s study for the percent of normal pol len produced could be due to technique, To obtain his values Daubeny made f i ve ind iv idual counts of three hundred pol len grains each from a squash preparation of several anthers. In th i s study, f i ve ind iv idual counts of one hundred pollen grains each were made from a squash preparation of one anther. This procedure was repeated on four other anthers at the same stage. Thus, f i ve anthers per stage per flower were sampled from ten randomly chosen flowers. I t i s f e l t that in th i s way (one anther per squash preparation) p a r t i a l l y and completely s t e r i l e ind iv idual anthers could be noted instead of being masked by the presence of other more f e r t i l e anthers as would be the case where several anthers are used to make up a squash preparation. Also, in the se lect ion Ore-US 1314 a great var ia t ion among flowers and among anthers of varying lengths (Table 3) in the s ta in ing a b i l i t y of the pollen grains was noted. An aceto-carmine s t a i n a b i l i t y tes t was used as the method of estimating pol len f e r t i l i t y in th i s study although i t has been c r i t i c i z e d (King, 1960; Hauser and Morrison, 1964) s ince, i t has been shown to be a repeatable method of estimating pol len f e r t i l i t y in sweet cherry (Whelan, 1965) and apple ( B a r r i t t , 1966) when s u f f i c i e n t pollen grains were included in the estimate. I t i s also a quick method and works well on f ixed mater ia l . The average percent normal pol len from each of four fi lament -52-length categories were recorded (Appendix IV). This was done since the se lect ion has been reported to be p a r t i a l l y male s t e r i l e (Daubeny, 1969) . and varying lengths of anthers are found with in a flower of th i s se lec-t i o n , while the highly s e l f f e r t i l e c u l t i v a r Mai l ing Jewel has anthers with almost s im i l a r f i lament lengths with in the same flower. It i s therefore possible that fi lament length and thus f l o r a l structure could be p a r t i a l l y contr ibut ing to the observed pa r t i a l male s t e r i l i t y in Ore-US 1314. There was in Mai l ing Jewel no s i g n i f i c an t difference in the percentage of normal pollen produced by anthers of d i f f e ren t fi lament lengths (Table 2) while there was a highly s i gn i f i c an t difference between such anthers in Ore-US 1314 (Table 3). A Duncan's New Mult ip le Range Test (Appendix V) carr ied out on the means of percent normal pollen produced by anthers of four d i f fe rent lengths showed no s i gn i f i c an t difference in the percent of normal pollen produced by anthers having long filaments (filaments designated as ' a ' and ' b ' ) . A l so, these long filaments subtended anthers which produced s i g n i f i c a n t l y more pollen than those anthers subtended by the shorter filaments ' c ' and ' d ' . Thus, there i s a suggestion that s t ructura l s t e r i l i t y could be involved in the observed pa r t i a l s t e r i l i t y of the se lect ion . Observations of the anther tapetum showed that i t was not involved in contr ibut ing to the male s t e r i l i t y observed in Ore-US 1314. The i r regu la r meiosis present in th i s se lect ion could very well lead to the varying stages of intermediate and abnormal pollen grains. The work of Saini and Davis (1969) strongly supported the idea that microspore degeneration a f te r quartet formation was a resu l t of de f i c ient RNA and DNA. The results of the present study indicate that most of the degeneration of the microspores occured before quartet formation (Table 4) in the -53-p a r t i a l l y s t e r i l e anther. Again, i f the DNA- formed as a resu l t of tapetum breakdown i s involved i s serving as precursor material for the DNA synthesis in the microspores as suggested by these workers, then in Ore-US 1314 the precursor for microspore DNA synthesis should be present since the tapetum both in th i s se lect ion and Mai l ing Jewel i s broken down p r i o r to anthesis. Thus, the f a i l u r e of microspores to form in some of the anthers could perhaps be due to the absence of a non-tapetal pool of DNA precursors and not to the absence of tapetal DNA precursors. This further suggests that cytoplasmic factors could be playing a part in confering male s t e r i l i t y in th i s se lect ion . No experiments were carr ied out to test i f cytoplasmic factors were indeed involved with male s t e r i l i t y . That only 41.8% of.the microspores of the se lect ion Ore-US 1314 reached the normal quartet stage as compared to the 93% in the c u l t i v a r Mai l ing Jewel and the observation that percent normal pollen production varied with anther length suggests also that genetic factors may also be involved in contr ibut ing to male s t e r i l i t y in th i s se lec t i on . One of the cu l t i va r s used in the development of Ore-US 1314 i s Mt. Mi tchel l a clone of Rubus idaeus subspecies str igosus. Thus, i t would seem to fol low that.the pa r t i a l male s t e r i l i t y observed in Ore-US 1314 could come from th is clone since s t e r i l e 'b lood ' has been associated with i t . The occurrence of intermediates (pollen grains of varying s ize) in large numbers in the Ore-US 1314 i s f e l t to be due to the un-equal segregation of the chromosomes observed at anaphase I and II. The cytoplasmic connections between the pollen grains of Ore-US 1314 could perhaps be an a r t i f a c t formed as a resu l t of in jury -54-to the anthers p r i o r to or during f i x a t i on and handling. I t was observed only in those anthers sampled from flowers at anthesis and was found in only 5.7% of the samples. The cytoplasmic connections cha rac te r i s t i c of Prunus (Whelan, 1965) and other angiosperm species (Heslop-Harrison, 1966) are between meiocytes at meiosis I which disappear with cytok ines i s . The cytoplasmic connections observed in th i s study were between microspores a f te r cytokinesis and ju s t p r io r to shedding. Also, only one f i x i n g so lut ion was used and thus the nature of the observed cytoplasmic connections could not be v e r i f i e d . No deviation from the Polygonum type of development was observed in the ovules of any of the clones examined. As previously reported by Daubeny et al.,(1967) and supported by the present work the crumbly clone of Sumner was found to have retarded embryo sac develop-ment (Table 8 and Appendix VII). Both th i s clone and the se lect ion Ore-US 1314 showed a high percentage of degenerated embryo sacs. In both these clones and the c u l t i v a r Willamette f e r t i l i z a t i o n took place at two days a f te r anthesis while in the normal f e r t i l i z a t i o n of the egg c e l l took place at four days a f te r anthesis. I f the sac categories EPP and E(PP) are grouped together and considered as the l a te s t embryo sac stages before f e r t i l i z a t i o n then results at anthesis showed that normal Sumner had 98%, crumbly Sumner 67%, Ore-US 1314.93% and.Willamette.55% of the i r sacs at th i s u n f e r t i l i z e d stage. The percentage of embryo sacs at th i s stage decreased with time and a pa ra l l e l increase i n the percent of f e r t i l i z e d sacs was observed most c l ea r l y in Willamette (Appendix VII). This implies that in Willamette a high percentage of the sacs formed were f e r t i l i z e d . Daubeny (1971) used th i s c u l t i v a r as a standard of comparison in a f e r t i l i t y study of red raspberries. -55-Since a high percentage of degeneration was observed a f te r anthesis in the embryo sacs of Ore-US 1314, degeneration of the sacs could be due to col lapse of the egg c e l l p r io r to f e r t i l i z a t i o n , collapse of the egg c e l l apparatus a f te r being f e r t i l i z e d , or col lapse of the ent i re sac as a resu l t of not being f e r t i l i z e d . Probably there was no pol len ava i lab le ot the desired time to f e r t i l i z e the reasonable number of mature egg c e l l s . As to degeneration observed l a t e r i t could be that the ava i lab le pollen i s p a r t i a l l y incompatible with the female gametophyte, with incompat ib i l i t y perhaps ranging from incompatible reactions between pollen grains and the st igmatic surface to incom-p a t i b i l i t y between egg and sperm cytoplasm. Jennings (1971) att r ibuted the f a i l u r e to set large pyrenes part ly to an adverse material i n t e r -action caused by poor balance between the contr ibut ion of the material and paternal parents, Daubeny (1971) reported that reductions in druplet set from open po l l inated flowers probably involved reduced numbers of functional ovaries and not incompat ib i l i ty since in his study flowers under open po l l i na t i on were exposed to pol len from various sources. In Ore-US 1314 then, th i s suggestion would hold for the reduced number of normal embryo sacs observed before f e r t i l i z a t i o n arid not for those observed at the time of or a f te r f e r t i l i z a t i o n . The lagging chromosomes at meiosis I observed in th i s study has been previously reported by Jennings (1967) as a possible cause of the crumbly phenomenon of some red raspberries. I f crumbliness (reduction in s e l f - f e r t i l i t y ) and pol len s t e r i l i t y associated (Daubeny, et al_., 1967; B a r r i t t and Eaton, 1965; Eaton, 1968) then the observed s e l f - i n f e r t i l i t y in part could be related with male s t e r i l i t y due to lagging chromosomes. However, Topham (1967) and Daubeny (1971) report that pollen qua l i t y i s usually not associated with reductions in s e l f - f e r t i l i t y ; A re lat ionsh ip between pol len qua l i ty and reduction in s e l f - f e r t i l i t y seems to ex i s t in Ore-US 1314. - 5 6 -Th e po l l i na t i on experiments showed that druplet set in Ore-US 1314 increased when the flowers were po l l inated with Mai l ing Jewel pol len (Table 12, Appendix IX). The reduction both in the percent druplet set and berry weight in the two clones when se l fed and crossed-compared to the open po l l inated (control) suggest that these reduction could in part be due to the e f fect of bagging (Daubeny, 1968), however, since both the c lusters that were s e l f - po l l i n a ted and cross -po l l inated were bagged, the reduced druplet l e t set and berry weight of Mai l ing Jewel when cross-po l l inated compared to se l f -po l1 inated (Table 2; Appendix IX) suggests not so much a bagged ef fect but qua l i t y of Ore-US 1314 pol len. The poor qua l i ty of Ore-US 1314 pollen has been shown previously by Daubeny (1969) and th i s study. Ore-US 1314 pol len may also be incompatible with Mai l ing Jewel. When cross -po l l inated with Mai l ing Jewel both druplet set and berry weight in Ore-US 1314 increased compared to s e l f - po l l i n a t ed . Again, th i s suggests a pollen e f f e c t . Jennings and Topham (1971) repotted that pol len d i l u t i o n in red raspberries leads to a reduction in the number of germinating pollen grains, thus reducing f r u i t set. However, the maternal genotype influenced pol len germination. The d i l u t i on of the pollen was f e l t to lower the supply of hormones with consequences both for druplet set and for other aspects of development determined soon af ter f e r t i l i z a t i o n . Besides i t s e l f and Mai l ing Jewel no other c u l t i v a r was used as maternal parent to study the e f fec t of Ore-US 1314 as being influenced by maternal genotype. Thus, Ore-US 1314 as a female could be p a r t i a l l y both s e l f and cross incompatible with Mai l ing Jewel. The reduction in druplet set of Mai l ing Jewel when po l l inated with pol len from Ore-US 1314 suggests po l l i n a t i on with pollen of poor qua l i t y . I t also suggests -57-pa r t i a l cross incompat ib i l i t y between Mai l ing Jewel and Ore-US 1314. The results from the study of pol len development mega-gametogenesis and po l l i na t i on suggest that pa r t i a l male s t e r i l i t y and perhaps pa r t i a l s e l f and cross - incompat ib i l i ty (from incompatible r e -actions between pollen grains and stigmatic surface to unbalanced male and female gametic interact ions)together account more in causing reduced f e r t i l i t y in red raspberries than pa r t i a l female or male s t e r i l alone. -58-SUMMARY AND CONCLUSIONS Percent normal pollen production, megagametogenesis and f r u i t set in the highly s e l f - f e r t i l e c u l t i v a r Ha i l ing Jewel and the p a r t i a l l y male s t e r i l e se lect ion Ore-US 1314 were studied. Pol len qua l i t y and development. A pollen s t a i n a b i l i t y test was used to determine the per-cent normal pollen produced by the two clones. Although results obtained have been c r i t i c i z e d , •„ i t was employed in the present study as i t gives quick repeatable re su l t s , and works well with f ixed mater ia l . It was found that squashes of ind iv idual anthers gave a better picture of s t e r i l i t y should i t ex i s t than squashes made from a number of anthers. The se lect ion Ore-US 1314 produced a much lower percent, of normal pollen than did Mai l ing Jewel. Microsporogenesis studies indicated that the tapetum was not involved in contr ibut ing to the pa r t i a l male s t e r i l i t y observed in th i s se lect ion. However, perhaps non-tapetal DNA i s involved. A cytochemical examination i s required to ver i f y t h i s . I t i s also suggested that i f the tapetal DNA present was not e f fec t i ve in bringing about normal microspore formation then other cytoplasmic factors contr ibut ing to male s t e r i l i t y could be operating. The i r r e g u l a r i t i e s observed at meiosis (lagging chromosomes, uneven chromosomal d i s t r i bu t i on and chromosome bridges) are believed to lead to incomplete tetrads, pollen grains of varying s i ze and reduced number of normal pollen grains a l l of which are typ ica l of pol len pro-duction of Ore-US 1314. S i gn i f i can t var ia t ion present in the percent of normal -59-pollen produced by anthers having d i f fe rent fi lament lengths in Ore-US 1314 and not in Mai l ing Jewel suggests s t ructura l s t e r i l i t y since the anthers of Mai l ing Jewel are of almost uniform length. Anthers with the longer fi lament lengths in Ore-US 1314 produced less s t e r i l e pollen than those with shorter f i laments. I t i s felt, that th i s observed pa r t i a l male s t e r i l i t y i s inher i ted by the se lect ion from R. idaeus subspecies str igosus. Thus, pa r t i a l male s t e r i l i t y in Ore-US 1314 may be due to both cytoplasmic and genetic factors. Embryo sac development A high percentage of degenerated sacs was present in crumbly Sumner and Ore-US 1314. The causes of the degeneration were f e l t to be due mainly to incompatible reactions between the male and female gametophytes rather than to female s t e r i l i t y alone. Po l l i na t i on studies The results obtained show that perhaps Ore-US 1314 i s p a r t i a l l y sel f - incompatible and cross -incompatible with Mai l ing Jewel. Druplet set was extremely high in Ore-US 1314 when open po l l inated suggesting a pos i t i ve reaction to a mixture of pollen grains. . . Male s t e r i l i t y , pa r t i a l s e l f and cross - incompat ib i l i ty and unbalanced interact ions between the male and female gametes to -gether seem to play a greater part in causing reduced f e r t i l i t y than female s t e r i l i t y or male s t e r i l i t y per se. - 6 0 -LITERATURE CITED Alam,S. and P.C.Sandal. 1967. 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M.S.A. thes i s . Univers ity of B r i t i s h Columbia. Williams,W. 1964. Genetical p r inc ip les and plant breeding. Blackwell S c i e n t i f i c Publ icat ions, Oxford. Zenkteler,M. 1962. Microsporogenesis and tapetal development in normal and male s t e r i l e carrots(Daucus carota). Amer. J . Bot. 49:241-248. Appendix I ' . Modi f icat ion of Ger lach's (1969) t r i p l e s ta in procedure. 1. Xylene 2. Xylene 3. Xylene 4. Xylene:EtOH (1:1) 5. 99% EtOH 6. 70% EtOH 7. 50% EtOH 8. 30% EtOH 9. D i s t i l i e d water 10. 3% Safranin in 50% EtOH 11. Rinse in running tap water 12. 1% Aqueous Gentian v i o l e t 13. Rinse in d i s t i l l e d water 14. 90% EtOH 15" 90% EtOH 16. 0.5% Light Green in 90% EtOH 17. 99% Isopropanol 18. 99% Isopropanol 19. 99% Isopropanol:Xylene (1:1) 20. Xylene 21. Xylene 5 nn'n 5 min 5 min 5 min 5 nn'n 5 min 5 min 5 min • 5 min 1 hr 5 min 2 min 3 changes 2 min with frequent 2 min ag i ta t ion Dip s l ides 3-4 times un t i l the purple stops running 2 min 2 min 5 min 10 min 10 min -66-Appendix II FILMENT X FLOWER INTERACTION MALLING JEWEL z: LU o CL cn O CJ cn L U 100 80 60 0 a FILAMENT LENGTH -67-Appendix III F I L A M E N T X F L O W E R I N T E R A C T I O N IN O R E - U S 1314 -i 1 — 1 fr a b e d F I L A M E N T L E N G T H -68-Appendi x IV The average percentage of normal pollen produced by f i ve ind iv idual anthers o f . four d i f fe rent filament lengths of Mai l ing Jewel and Ore-US 1314. Flower no. Fi lament Lengths  a b e d Mai l ing Jewel 1. 88 .8 92. 8 93 .8 89 .6 2. 85 .8 86. 2 87 .6 84 .2 3. 95 .4 94. 6 94 .8 94 .2 4. 96 .4 95. 8 94 .8 97 .8 5. 96 .0 64. 6 97 .3 95 .4 6. 95 .4 99. 2 94 .6 95 .6 7. 76 .4 94. 0 .95 .4 88 .2 8. 80 .8 72. 8 66 .2 85 .2 9. 96 .6 95. 2 93 .2 96 .8 ' 10. 98 .0 96. 2 94 .8 96 .2 Ore-US 1314 1. 48 .2 57. 4 0 .0 11 .4 2. 0 .0 0. 0 0 .0 0 .0 3. 0 .0 0. 0 10 .2 0 .0 4. 43 .8 37. 2 17 .2 4 .2 5. 0 . 0. 0 0 .0 5 .4 6. 20 .0 8. 2 0 .6 3 .6 7. 0 .0 5. .0 25 .2 0 .8 8. 0 .0 0. 0 0 .0 0 .0 9. 54 .2 63. 6 6 .8 21 .0 10. 0 .0 0. 0 0 .0 0 .0 -69-Appendix V A Duncan's New M u l t i p l e Range Test on the means of percent normal p o l l e n produced by anthers o f the four f i l amen t lengths ' a 1 , ' b ' , ' c 1 , and ' d ' i n Ore-US 1314. S_ = 1.77 y Means 84.7 83.1 30.0 23.2 % normal p o l l e n b a c d f i l a m e n t length Means not unde r l i ned by the same l i n e were s i g n i f i c a n t l y d i f f e r e n t at the 5% l e v e l . -70-Appendix VI Meiotic stages found in Jewel and Ore-US 1314. Clone Mail ing Jewel buds measuring between 2. Bud s ize 2.0 mm 2.7 mm 0 and 4.0 mm of Mail ing Meiotic stages Too ear ly to note any chromosomal a c t i v i t y . Telophase I I. 3.7 mm Individual pollen grains and tetrads within the pol len mother c e l l w a l l . 4.0 mm Individual pollen grains Ore-US 1314 2.0 mm loo early to note any chromosomal a c t i v i t y . 2.7 mm Anaphase I, telophase I, and telophase II. 3.7 mm Individual pol len grains -and tetrads within the pol len mother c e l l w a l l . 4.0 mm Individual pollen grains. Appendix VII Percentage of embryo sacs in each stage of development within and crumbly clones of Sumner and Ore-US 1314 in' 1970. the ovules of Willamette, the normal Days after anthesis 8-n ePP e(PP) EPP E-(PP) E(PP)pt CE)(PP) -CE)P.P.. z. • -EMB--..Deg... ...I.... Total no. of . ovules.. Anthesis Normal S. 2 0 0 18 80 0 0 0 0 0 0 0 64 Crumbly S. 17 10 6 19 38 0 0 0 0 0 10 0 141 Ore-US 1314 0 0 3 : 7 36 0 0 0 0 0 2 2 68 Willamette 0 8 0 14 71 0 0 0 0 0 7 0 107 Anthesis + 2 Normal S. 0 10 0 10 80 0 0 0 0 0 0 0 71 Crumbly S. 5 0 2 7 26 8 29 0 7 0 16- 0 42 Ore-US 1314 0 0 0 3 38 6 20 0 3 0 28 2 65 Wi11amette 0 0 ' 0 8 35 6 40 .4 5 0 2 0 .102 Anthesis + 4 Normal S. . 0 o • 0 0 70 9 11 0 5 5 0 0 132 Crumbly S. 3 2 . 1 8 35 15 18 0 0 3 12 3 120 Ore-US 1314 0 0 0 0 20 p> 8 0 4 10 50 0 50 Wi11amette 0 0 0 2 12 0 67 5 8 5 I .0 104 Anthesis t 6 Normal S. 0 0 0 0 54 13 22 0 0 11 0 0 83 Crumbly S. 0 0 0 0 • 0 20 22 0 0 11 47 0 68 Ore-US 1314 0 0 0 0 0 10 9 0 0 17 . 64 0 65 Willamette 0 0 0 0 14 6 63 0 12 0 0 54 -72-Appendix VIII D i s t r ibut ion of degrees of freedom for s ingle degree freedom contrasts. Source CI ones Open vs Others Se l f vs Cross Clones X Open vs Others Clones X Se l f vs Cross Error Total -73-Appendix IX CLONES X SELF vs CROSS INTERACTION FOR DRUPLET SET _j _ — — _ i _ OPEN SELF CROSS P O L L I N A T I O N T R E A T M E N T S -74-Appendix X _ _ 4 _ _ _ _ , , — j - , , .—{ OPEN SELF CROSS P O L L I N A T I O N T R E A T M E N T S 

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