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Investigation of the requirements and androgenesis in cherry (Prunus avium) and peach (Prunus persica) Lane, William David 1971

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AN INVESTIGATION OF THE REQUIREMENTS FOR ANDROGENESIS IN ' CHERRY (Prunus avium) AND PEACH (Prunus persica). BY WILLIAM DAVID LANE B.Sc. (Agr), University of B r i t i s h Columbia, 1969 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN THE DEPARTMENT OF PLANT SCIENCE We accept this,.as'conforming to the required standard. UNIVERSITY OF BRITISH COLUMBIA A p r i l 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 it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the 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• The University of British Columbia Vancouver 8, Canada Date May, 1971. i ABSTRACT Haploid plants are p o t e n t i a l l y valuable for the breeding of many crops p a r t i c u l a r l y those with long reproductive cycles. In t h i s i n v e s t i g a t i o n an attempt was made to determine the requirements of androgenesis of peach.and cherry, a technique which has been used to produce large numbers of haploids i n other species. A procedure used s u c c e s s f u l l y with tobacco was v e r i f i e d . A preliminary survey of apple, cranberry and rose showed that they do not have the same requirements as tobacco. In most experiments with cherry and peach immature p o l l e n was used. However the lack of d i f f e r e n t i a l responces of p o l l e n to treatment made i n t e r p e t a t i o n d i f f i c u l t . Hence responces of anthers and the growth of c a l l i from somatic flower parts was used to assess d i f f e r e n t treatments instead of p o l l e n growth. C a l l i emerged from cherry anthers when anthers were cultured on Nitsch's medium containing naphthalene a c e t i c acid (NAA) and coconut milk and incubated i n the dark at 30°C. Because these c a l l i contained both d i p l o i d and t e t r a p l o i d c e l l s and because of the lack of abnormal po l l e n development i n the anthers i t was concluded that the c a l l i probably originated from anther connective tissue rather than p o l l e n . In experiments with mature cherry p o l l e n several d i f f e r e n t types of abnormal p o l l e n growth were observed including m u l t i c e l l u l a r p o l l e n grains. A comparison of the requirements for species i n which ! ' androgenesis has been demonstrated i s discussed. In some species components making up the requirements appear to form a pattern which i f v e r i f i e d by reports from other specLes w i l l be valuable i n future i n v e s t i g a t i o n s . Suggestions are made as- to the d i r e c t i o n which further i n v e s t i g a t i o n s of the requirements f o r androgenesis should take. i v ACKNOWLEDGEMENTS I would l i k e to;, .thank s i n c e r e l y my supervisor, Dr. G.W.Eaton, associate professor of plant science, D.B.C; f o r h i s advice throughout a l l phases of the research and w r i t i n g of t h i s thesis as well as the other help he has given me. My s p e c i a l thanks also f o r the suggestions from the other members of my committee: Dr. V.C.Runeckles (chairman)Department of Plant Science, U.B.C. Dr. P.M.Townsley Department of Food Science U.B.C. Dr. K.O.Lapins Canada Department of Ag r i c u l t u r e R e s e a r c h ^ .. Station, Summerland, B.C. Dr. E.B.Tregunna Department of Botany, U.B.C. The discussions I had with Dr. Townsley were most enlightening. Dr. Lapins supplied most of the plant material and h i s suggestions at the onset of the reseach as w e l l as the discussions I had with him and h i s encouragement are a l l very much appreciated. Thanks also go to a l l the other people; my parents, friends and fellow graduate students f o r t h e i r moral support and patience through-out the l a s t two years. Support f o r t h i s work was from NRC operating grant A-2023 1 awarded to Dr. Eaton. V TABLE OF CONTENTS PAGE INTRODUCTION .-. • - 1 3 LITERATURE REVIEW— — — , 3 Some uses of haplbiSls Ways , to obtain haploid plants P r o l i f e r a t i o n of gymnosperm pollen —--: — -Haploid plant production using the anther culture method 10 METHODS AND MATERIALS — " " 16 Tobacco I (i) - - " ' ~ ~ ~ 21 Cranberry I I ( i ) — — : 22, Apple I T I ( i ) • 2 3 Rose IV(i) — — 24 IV ( i i ) — : " 24 Cherry V(i) ~ " " — -25 V ( i i ) " " " " 25 V ( i i i ) " — 25 V(iv) " " " 26 V(v) " 26 V(vi) " " 27 V( v i i ) : " " — 27 V ( v i i i ) — 27 V(ix) 28 V(x) -— " " " 28 V(xi) — .29 ! V ( x i i ) —- 29 V ( x i i i ) —-. 29 : PAGE Peach VI(i) •-- — 31 VI ( i i ) — - — — — 31 V l ( i i i ) — — — : 31 VI(iv) — 31 VI (v) -- . 31 VI (vi) 32 32 ,VI(vii). ----- — — VI ( v i i i ) — — : 32 RESULTS — — — — _ . ;: _ _____ 39 Tobacco I(i) — 3 9 Cranberry II (i) : 40 Apple III(i) ~ — 41 Rose IV(i) — : • 41 IV(ii) : 41 Cherry V(i) 42 V(ii) —-- — '• 42 V ( i i i ) — 42 V(iv) = 43 V(v) — — : 43 V(vi) — — — — _ _ _ ;__ 44 V(vii) — 44 V(vii i ) — — 45 V(ix) • 45 V(x) — - — — — : 46 V(xi) -r 47 V(xii) 47-V(xiii) - — 48 v i i PAGE Peach Vlfl) — — 4 9 v i ( i i ) — — - — — ~ — - 4 9 v i ( i i i ) — - — — — — _ 4 9 VI (iv) - — — 5 0 VI ( v ) - — — — 5 1 . VI (Vi) — ' :~ " 5 1 VI ( v i i ) — — 5 2 V l ( v i i i ) - ~ — 5 3 DISCUSSION -T 5 4 Special d i f f i c u l t i e s with cherry and peach —; ^ Requirements f o r androgenesis — -.56 D i f f i c u l t i e s i n determining the requirements for cherry and peach :- • 60 Developmental c h a r a c t e r i s t i c s associated with androgenesis • — 63 Discussion of r e s u l t s — : — — -65 The d i r e c t i o n of further i n v e s t i g a t i o n s . .67 SUMMARY -. — — 6 9 LITERATURE CITED - — : 70 APPENDIX-1 :— - - — — 7 3 II — — ^ .74 II I —- 75 IV 76 v i i i LIST OF TABLES . PAGE Table I. Percentage of anthers producing p l a n t l e t s i n treatment I ( i ) 39 Table I I . The requirements for androgenesis r - 55 i x PAGE LIST OF FIGURES Figure 1. Tobacco embryoids emerging from an anther • — 33 Figure 2. D e t a i l of tobacco embryoids —-—-• • : 33 Figure 13. Degrees of anther enlargement 34 Figure 4. A d i c e l l u l a r pollen grain ---- 34 Figure 5 . A m u l t i c e l l u l a r pollen grain ~ 35 Figure 6 . Abnormal germination — r 35 Figure 7. Germination anomalies 36 Figure. 8. P r o l i f e r a t i o n i n a germ tube ' • 36 Figure 9. A giant pollen grain 37 Figure 10. Types of pollen enlargement :- 37 Figure.11. Callus emerging from anthers 38 Figure 12 A dividing callus c e l l 38 1 INTRODUCTION Haploid plants are very i n t e r e s t i n g . Rather than possessing p a i r s of s i m i l a r chromosomes the t o t a l number of chromosomes i s reduced by one-half and only one member of each p a i r i s present i n a c e l l . Several methods are a v a i l a b l e which can be used to duplicate the chromosomes i n haploid c e l l s thus r e s t o r i n g the d i p l o i d s i t u a t i o n i n which p a i r s of chromosomes are present. There i s however one important d i f f e r e n c e between the pai r s of chromosomes i n doubled haploid c e l l s and those i n most other p l a n t s . In doubled haploid c e l l s the chromosomes i n each' chromosome p a i r are i d e n t i c a l to each other. Such a plant during reduction d i v i s i o n produces, c e l l s i n which a l l gametes are i d e n t i c a l . Because of the increasing i n t e r e s t i n the u t i l i z a t i o n of haploid plants both i n physio-l o g i c a l studies and i n plant breeding programs (Melchers and Labib, 1970) the demand for them w i l l undoubtedly increase i n the future. In cherry and peach breeding programs the use of haploids i s p o t e n t i a l l y more valuable than i n most other crops,obecause of the longer time between seed germination and flowering i n peach and cherry. Breeders of these crops are w e l l aware of the increases i n e f f i c i e n c y i f haploids could be used i n t h e i r programs. However extensive use of haploids has been r e s t r i c t e d because very few peach and no cherry haploid plants are a v a i l a b l e . Recently i t has been shown that i n several species p o l l e n can be cultured i n . v i t r o while enclosed i n the anther and induced to grow in t o haploid plants. With these species t h i s anther culture method can be used to obtain large numbers of haploids e a s i l y and cheaply. This i n v e s t i g a t i o n was undertaken because the natural occurence of cherry and peach haploids i s of low frequency and because there i s ther p o t e n t i a l f o r a large increase i n the e f f i c i e n c y of Breeding programs i f a method can be found by.which large numbers of haploids become a v a i l a b l e . An attempt.was made to adapt the anther culture method to achieve t h i s goal e i t h e r i n whole or i n part. 3 LITERATURE REVIEW SOME USES OF HAPLOIDS A haploxd plant was- f i r s t described by Blakeslee et a l . (1922) i n Datura stramonium (jimson weed). Since that time there have been many reports and de s c r i p t i o n s of haploid plants. Two reviews, those of Kimber and R i l e y (1963) and Magoon and Khanna (1963) have summarized most of these. Haploid a r i s e i n some natural populations but t h e i r occurence i s rare i n most species. In the crop plant species which have been studied the frequency of haploidsoccurence i s us u a l l y less than one i n a thousand. The frequence of haploid occurence i s considered to be under genetic c o n t r o l l at l e a s t p a r t i a l l y and i s often correlated with polyembryony. Haploid plants have been used i n p h y s i o l o g i c a l studies but to date have beenii used more often i n plant breeding programs. There are;, variow types of studies and uses f o r which haploids are desirable i f not e s s e n t i a l . Haploids can be used to study the genetic c o n s t i t u t i o n of some species. For example the r e l a t i o n s h i p between d i f f e r e n t species of SPlanum (potato) has been nearly completely worked out, many of the re l a t i o n s h i p s being elucidated because.of the chromosomal associations during meiosis i n polyhaploids. A summary of the r e s u l t s of this work i s given by Urgent (1970). Haploids can also be used to advantage i n many plant breeding programs. Nei (1963) has calculated what frequency of haploid occurence i s necessary f o r the doubling of haploids to be more e f f i c i e n t than the method of.repeated s e l f i n g of d i p l o i d s to a t t a i n the desired homozygous genotype. He concluded that the haploid method i s more e f f i c i e n t when the • 4 number of genes In the. desired combination is- large and the frequency of favourable a l l e l e s i n the population i s small, even i f r e l a t i v e l y small numbers of haploids are a v a i l a b l e . Another example of haploids- being u s e f u l i n breeding programs i s i n crops with long reproductive c y c l e s such as f r u i t f o r e s t and palm trees. Here homozygous l i n e s could be developed from haploids i n one or two generations with a saving of many years when compared to the repeated s e l f i n g method. In crops such as the date,coconut or o i l palm which have both, long reproductive cycles and are d i f f i c u l t to propagate vegetatively i t would be pos s i b l e to f i x the desired genotype by crossing the homozygous plants possessing the desired c h a r a c t e r i s t i c s to produce uniform hybrids. At present, palm plantations are grown from seed the genotype of which i s the r e s u l t of random combinations of gametes from a v i r t u a l l y unselected gene pool. Many crops are self-incompatible and cannot be repeatedly s e l f e d to a t t a i n homozygosity. This i s necessary f o r the production of y commercially valuable F^ hybrids. Doubled haploids could also be used to a t t a i n homozygosity i n these crops. However i n cherry and peach there i s l i t t l e p o s s i b i l i t y of .obtaining plants more.vigorous than present v a r i e t i e s by using F^ hybrids (Dodds 1955) because these crops have probably already been selected f o r hybrid v i g o r . Because doubled haploids are completely homozygous a l l the gametes from a plant are the same thus the same genes are transferred with every p o l l e n grain. A hybrid of two homozygous plants would produce i d e n t i c a l F^ progeny. In a crop such as Asparagus t h i s technique could be used to produce e n t i r e f i e l d s of female plants rather than the random frequency of haploid occurrence. PerliapstBe onos-t common of these i s ' to make i n t e r s p e c i f i c crosses, the progeny of which are sometimes haploid. This introduction of an a l i e n nucleus into maternal cytoplasm sometimes gives quite large proportions of haploids, in one case 53% of the progeny I(Kihara and Tsunewaki, 1962).: Delayed pollination-and X - i r r a d i a t i o n have also been used to increase the frequency of haploids. Two less common practices are colchicine treatment and temperature shock at the time of p o l l i n a t i o n . These practices are not used extensively i n the breeding of most crops because i t i s too 1 ()difficult to obtain the required number of haploids to make i t worthwhile. However there are several important exceptions. Because the occurrence of haploids i s 'under genetic control i t would be expected that the frequency of haploid occurrence would vary. A most encouraging discovery was made by Hougas et a l . (1963); a l l the progeny of crosses of tetraploid Solarium tuberosum with d i p l o i d S. phurej a were haploid. In 1970 another i n t e r s p e c i f i c crosswas also found to produce almost exclusively haploid progeny (Kasha and Kao, 1970). These resulted when the induced autotetraploid Hordium vulgare (2n 28) was crossed with the natural te t r a p l o i d iH. biilbosum (2n 28). The progeny a l l resembled R. vulgare,the commercially important species. This discovery prompted sim i l a r crosses to be made with the d i p l o i d forms of these species, again using H. vulgare as the female parent. Again, a l l the progeny were of the H. vulgare type and were haploid. To avoid abortion of the haploid embryo i t was'necessary to remove the top half of the f r u i t eight to ten days after p o l l i n a t i o n and to place the bottom half on an embryo culture medium. Observation of the developing embryo showed that f e r t i l i z a t i o n did occur but that the H. bulbdsum chromosomes 5 mixture of female and unproductive male plants^which i& now an inconvenience to asparagus growers. Because haploids do not have pairs of chromosomes but rather one chromosome from each pair, a gene is not masked by.a corresponding gene in the chromosome of the other member of the pair,as is the case with diploids.- This makes i t possible to determine the power of gene expression or the dosage effect of genes. Haploids would also make i t much easier to obtain auxotrophic mutants (Tulecke 1960.and Carlson 1970). Potentially such mutants are of commercial value as well as being very powerful tools for studing the metabolism of higher plants. Spontaneous or induced mutations-•are usually recessive and because of this are often not expressed i n diploid plants unless they are homozygous but i n haploids mutations are expressed immediately. Thus, i f mutants are sought, inducing mutations i n haploids would be an efficient method for their selection. Once obtained, doubling the chromosome number would make i t possible.to combine the mutation with other desired characteristics by making the appropriate cross. WAYS TO OBTAIN HAPLOID PLANTS The reason haploids have not been used extensively for the purposes suggested above is because they do not occur frequently. When they do occur they may have originated in a number of different ways. Kimber and Riley (1963) and Magoon and Khanna (1963) have tabulated how most of the haploids reported up to 1963 originated. Most arose as one twin in polyembryonic situations, normally as the haploid member of a haploid-diploid pair. A number of different things can be done to increase the are lost tin the. f i r s t few mitotic divisions of : the embryo (Kasha and Kao, .1970). A distinctly different- technique with which i t is possible to obtain large numbers of haploids- from some species quickly and relatively easily has been developed in recent years. This technique is the anther culture method.1 Anthers containing pollen are cultured in,vitro on a tissue culture medium thus altering the normal metabolism of the pollen and causing i t to proliferate and form either haploid embryoids or haploid callus which can be induced to regenerate haploid plantlets. The f i r s t observation which suggested that pollen could proliferate in' an unusual way was made by Nemec:(1898).• He observed pollen grains i n the petaloid anthers of. Hyacinthus brieritalis containing eight nuclei spaced within the pollen grain in the same relative positions as the nuclei i n an embryosac. Stowe (1930) observed similar pollen in the anthers of the same species and also reported some pollen with as many as fifteen nuclei. PROLIFERATION OF GYMNOSPERM POLLEN In 1941 LaRue cultured the female gametophyte of Zamia  floridana in vitro (LaRue, 1948) and was able to induce roots and shoots to grow from a tissue originating from i t . He thought at the time that C . i t was haploid but this was shown not to be the case; However, he -continued his experiments culturing the pollen of thirty-six angiosperm and seventeen gymnosperm species, hoping to obtain a haploid tissue from them. This work was reported by LaRue's student Tulecke (1959). About 1952 he succeeded i n growing a tissue from the pollen of Taxus (probably Taxus bfevifblia) a gymnosperm (Tulecke, 1959).' 8 The cells of the tissue mass were elongated wl;th a concentration of cytoplasm and the nucleus- at one end. In this regard the cells were similar to pollen germ tubes. The tissue grew irery slowly and when division occurs red the new c e l l was much smaller than the original at f i r s t but soon i t elongated. Sometimes the divisions-resulted in branching from the youngest c e l l s . Althoughtelongated filamentous cells were the predominent type, aggregates of compact cells with dense nuclei were sometimes seen. It is f a i r l y certain although only from indirect evidence that the tissue spontaneously Increased Its chromosome number.and became polyploid. The Taxus tissue originated growth on a medium based on that of White (1943), supplemented with 0.6 ppm 2,4-dichlorophenoxyacetic acid (2,4-D) and 15% coconut milk. Later i t was found that i t grew nearly as we well on a completely defined medium containing 100 ppm 1-arginine HC1,100 ppm NaR^PO^.H^O, 0.1 ppm naphthalene acetic acid and 1.0 ppm calcium pantothenate (Tulecke, 1959). Tulecke has reported growing tissues which originated from proliferating pollen of two other gymnosperms Ginkgo bilbba (Tulecke, 1953) and Torreya nucifera (Tulecke, 1963). The cells of the tissue from Ginkgo were parenchymous and showed no signs'of^differentiation. This tissue grew about ten times faster than that of Taxus and c e l l division appeared normal although there were many multinucleate cells i n i t . Like the Taxus tissue theGinkgo tissue spontaneously became Hiploid. Tulecke found that about four percent of the inoculations developed into tissue masses when the completely defined medium which supported the growth of Taxus was used. Sometime after growth was initiated an interesting green sector arose as a spontaneous mutation from the normally white tissue and i t was maintained through a number of subcultures'(Tulecke, 1959). 9 In contrast to the Ginkgo tissue the haploid t i s s u e from the p o l l e n of Torreya was- slow growing. The medium, which was based on that of Wood and Braun (Tulecke, 1963) was.inoculated with p o l l e n at the stage of development at which i t i s normally.shed. In v i t r o some of the p o l l e n developed normally to the four^nucleate stage but abnormal devel-opment, u s u a l l y by budding rather than normal c e l l d i v i s i o n , was also seen. Many c e l l s i n t h i s tissue were multinucleate. Haploid t i s s u e has been grown fromtathe p o l l e n of a fo u r t h gymnosperm. Konar (1963a) picked cones from Ephridas f o l i a t a , removed the microsporangia, surface s t e r i l i z e d them and cultured them on a medium based on 1 that of Reinert (Konar, 1963b) containing 2% sucrose, 15% coconut milk and 1.5 ppm 2,4-D. Within twenty-four days a f t e r ^ i n o c u l a t i o n a ti s s u e began to grow from the p o l l e n grains. Development from the p o l l e n was of two types. In the f i r s t long filaments developed from the p o l l e n . At f i r s t these filaments divided transversly followed by elongation of the new c e l l . However, v e r t i c a l d i v i s i o n s ensued and the tissuecbecame an unorganized mass of c e l l s . In the second growth pattern the p o l l e n grains enlarged considerably then d i v i s i o n s began and a mass of t i s s u e was formed. Once growth.was w e l l under way tissues d i f f e r i n g i n formative growth could not be. distinguished. The tissue was f a s t grow-ing and maintained the haploid number of chromosomes f o r at l e a s t seven months (Konar 1963a). In contrast to Ginkgo,tissue developed from s i x t y percent of the inoculations rather than four percent. A p r o l i f e r a t i o n of the megagametophytic tissue of t h i s species has also been reported (Sankhla et a l . 1967). P r o l i f e r a t i o n from both the megagametes and microspores of Pinus resinosa has also been reported (Bonga and Towler, 1970). Only s m a l l c a l l ! were obtained from megagametophytic t i s s u e . However, more vigorous c a l l u s growth..was.obtained from t i s s u e o r i g i n a t i n g from . m i c r o s p o r a n g i a t e . s t r o b i l i . o r s p o r o p h y l l s which were c u l t u r e d on Brown and Lawrence's (1968) medium. I t was not p o s s i b l e to i n i t i a t e c a l l u s growth from p o l l e n when i t was seeded onto the medium. As w i t h the other h a p l o i d gymnospefm c a l l i , i t has not been p o s s i b l e to regenerate p l a n t s from the Pinus t i s s u e ; ' There are few examples of the regeneration of p l a n t l e t s from gymnosperm c a l l u s ; thus i t i s not s u r p r i z i n g that.no p l a n t l e t s have been obtained from h a p l o i d t i s s u e s grown from the p o l l e n of Taxus,T Ginkgo  Torreya, Ephridas or Pinus. Angiosperms have a much higher c a p a c i t y f o r the regeneration of p l a n t l e t s from c a l l u s and i t i s t h i s c h a r a c t e r i s t i c which i s p a r t i a l l y r e s p o n s i b l e f o r the s u c c e s s f u l development of the anther c u l t u r e technique f o r some angiosperms. ANGIOSPERM HAPLOID PLANT PRODUCTION USING THE ANTHER CULTURE METHOD The d i s c o v e r y that p o l l e n can be t o t i p o t e n t was an a c c i d e n t a l one made by Guha and Maheshwari (1964). They had c u l t u r e d i n v i t r o the anthers of Datura i n n o x i a to study the f a c t o r s which c o n t r o l meiosis and c e l l d i v i s i o n . Two months a f t e r seeding mature anthers on e i t h e r H e l l y e r ' s medium supplemented w i t h 2% sucrose plus e i t h e r 15% coconut —6 m i l k , 10 M k i n e t i n or plum j u i c e , embryoids emerged from l o n g d i t u d i n a l s p l i t s i n the anthers.,The embryoids developed i n t o mature p l a n t s and were v e r i f i e d as being h a p l o i d and as having o r i g i n a t e d from p o l l e n .(Guha and Maheshwari, 1966 and Guha and Haheshwari, 1967). The second r e p o r t of embryoids o r i g i n a t i n g from p o l l e n of an angiosperm species was that of Tanaka and Nakata from N i c o t i a n a tabacum.(tobacco) ( c i t e d by Kamayama and Tanaka, 1969). EmbryoIds were obtained from the pollen.of three -varieties^ Bright yellow, Hicks 103 and White Daruma (Nakata and Tanaka, 1968).' The haploid embryoids grew i f anthers were placed on the medium when po l l e n was at the uninucleate stage j u s t a f t e r i t s release from the tetradeand incubation was at 25°C with an i l l u m i n a t i o n of 3500-4000 lux. Androgenesis occured when a se r i e s of three media were used, the f i r s t based on Hildebrant's (1962) "C" medium i n which the Fe o(C.H.0,)' / H H O had been replaced by Na£ EDTA plus TeSO^.H^O, thiamirier-HC 1 was increased to 0.4 mg/1 and glycine and calcium pantothenate were omitted. A f t e r eighty days on t h i s medium the anthers were transferred to a modified RM-1964 medium CLinsmaier and Skoog,,1965) which contained three times the amount of NK^NO^ and K^PO^ s p e c i f i e d by Linsmaier and Skoog. This second medium also contained 4 mg/1 k i n e t i n and 2 mg/1 indole a c e t i c acid (IAA). The optional constituents- were not added. Rootless embryoids emerged from s i x percent of the anthers f i f t e e n days af t e r they had been transferred to t h i s medium. To encourage root formation anthers with emerging embryoids were transferred to a modified White's medium (1943) containing 2 mg/1 IAA, 0.05 mg/1 k i n e t i n and 100 mg/1 ii i y o - i n o s i t o l . The embryoids then formed roots and could be transferred to s o i l - f i l l e d pots i n the greenhouse. Sixty days a f t e r .transfer to the RM-1964 medium, c a l l u s which had grown from the base of some embryoids d i f f e r e n t i a t e d forming roots and shoots. Although the c a l l u s grew from haploid embryoids the p l a n t l e t s which grew from the c a l l u s were d i p l o i d (Nakata and Tanaka, 1968). C y t o l o g i c a l observations i n d i c a t e that the tobacco embryoids developed from p o l l e n grains i n much the same way as those of Datura. Homozygous d i p l o i d plants were obtained by doubling 12 the chromosome complement with.c o l c h i c i n e (Tanaka and Nakata, 1968). Concurrent w i t h Tanaka and Nakata's f i r s t report, Bourgin and ' " N i t s c h (1967) reported s i m i l a r f i n d i n g s . This report was l a t e r expanded upon (Nitsch and Nitsch, .1969 and Nitsch, 1969). N i t s c h (1969) investigated twelve species of Nicotiana and obtained haploid plants from f i v e . They were N. a l t a , a t e t r a p l o i d . s t r a i n of N_. glutinosa, N. f u s t i c a , N. s y l v e s t r i s and N. tabacum. The medium used by Ni t s c h (see Appendix; I) induced haploid embryoids to grow; from as manyas 67% of the anthers. S a t i s f a c t o r y r e s u l t s were obtained.with s o l i d i f i e d mineral s o l u t i o n plus 2% sucrose, with or without the add i t i o n of vitamins.and 100 ^ug/1 IAA. The minimal medium necessary for the formation of tobacco embryoids i s a s o l u t i o n of 2% sucrose s o l i d i f i e d with agar. On t h i s medium they do not develope past the globular stage but development does continue i f minerals, p a r t i c u l a r l y iron,.are added to the medium (Nitsch, 1969). Small , differences i n the composition of the medium were not nearly as important as the developmental stage of the p o l l e n when incubation began. By far. the most s u i t a b l e were anthers containing p o l l e n at the uninucleate stage s l i g h t l y before the m i t o t i c d i v i s i o n i n the microspore. Sunderland (1970) claims that i t i s the vegetative c e l l of the p o l l e n grain which divides to form the ti s s u e mass. Although the generative nucleus may d i v i d e the l i m i t i s one d i v i s i o n . Nitsch (1969) has found that the embryoids bear a strong resemblence to zygotic embryos and has suggested that embryoids developing from tobacco p o l l e n would be a s u i t a b l e system for studing embryonic development. Although i t i s possible to use c o l c h i c i n e to double haploids perhaps a better way i s to take advantage of endomitosis as suggested by N i t s c h (1969). He grows c a l l u s from haploid plants and has found that 13 substituted diphenylureas with cytokinin a c t i v i t y are.more e f f i c i e n t than other cytokinins i n inducing the production of d i p l o i d plants from haploid c a l l u s . This method avoids the problem of chimera formation which often a r i s e s when c o l c h i c i n e i s used. In 1968 anther cultures of Orysa s a t i v a (rice) yeilded haploid p l a n t l e t s ( N i i z e k i and Oono, .1968 and papers c i t e d by Katayama and Tanaka, 1969), the f i r s t report f o r Graminaceae. Anthers were c o l l e c t e d two days before heading and were cultured on Blaydes' (1966) medium supplemented 'with 1-2 mg/1 IAA, 1-2 mg/1 2,4-D and 1-2 mg/1 k i n e t i n and incubated at 28° C i n the dark. Anthers of twelve r i c e c u l t i v a r s were cultured but haploid plants were obtained from only s i x . Three weeks a f t e r i n o c u l a t i o n the anthers turned black and at four to eight weeks pale yellow c a l l u s emerged from the anthers. I t was possible to induce•the regeneration of p l a n t l e t s from c a l l u s by leaving 2,4-D out of the medium and adjusting the concentration of IAA to 2 mg/1 and k i n e t i n to 2-4 mg/1 and incubating the cultures i n white fluorescent l i g h t rather than the dark. Four weeks-after subculturing i n t h i s medium haploid plants were d i f f e r e n t i a t e d . About 0.57% of the anthers produced haploid c a l l u s and although most of the p l a n t l e t s were normal some were albino. The requirements necessary f o r the production of haploid plants from the p o l l e n of two other species were reported i n 1970. Nitzsche (1970) cultured 488 nearly mature anthers obtained from the F^ hybrid produced by crossing Lolium multiflorum with Festuca arundinacea. Haploid c a l l u s developed from the p o l l e n i n one of these anthers. Four p l a n t l e t s were regenerated from i t , three albino and one normal. P e t a i l s of the medium and the incubation conditions were not given but c y t o l o g i c a l r observations are reported. Twenty-four m u l t i c e l l u l a r p o l l e n grains were 14 found as'well'as as number of embryosac-like p o l l e n grains. The low y i e l d of.haploid c a l l u s i s perhaps because most of the p o l l e n i n the anthers of the hybrid was s t e r i l e . The requirements for Brassica oleracea and an F^ hybrid of. I3_. oleracea X 15. alboglabra have also been reported (Kameya and Hinata, 1970). This i s the f i r s t report of successfullanther culture i n an out breeding crop. Haploid c a l l i grew when Nitsch's medium was used plus the addition of the following growth regulators: e i t h e r 0.5 ppm 2,4-D or 1.0 ppm 2,4-D plus 1.6 ppm k i n e t i n or 2.0 ppm,2,4-D plus 1.6^ 'ppm k i n e t i n or 4 ppm 2,4-D or 10% coconut milk. Nearly mature anthers were used and incubation was i n d i f f u s e l i g h t at 20°C. Two of the three v a r i e t i e s of B_. oleracea as w e l l as the F^ hybrid produced haploid c a l l i . The percentage. of anthers producing c a l l u s was 1.6% - 6.-7% for B_. oleracea. and 8.2% for the hybrid. Shoots grew from the c a l l i seven weeks a f t e r subculturing on to Nitsch's medium containing e i t h e r 10% coconut milk or 0.5 ppm, to 1.0 ppm NAA plus 1.0 ppm k i n e t i n (Kameya and Hinata, ,1970). Kameya and Hinata (1970) were also able to able to grow haploid c e l l c l u s t e r s from hanging drop suspensions of p o l l e n . These from the p o l l e n of the hybrid and one of the three B_. oleracea c u l t i v a r s . The p o l l e n i n the suspensions only grew when the medium contained 10% coconut milk and between 10% and 15% sucrose which was included t o ' prevent the p o l l e n from bursting. They were not able to regenerate plants from t h i s haploid t i s s u e . There have been no reports of the production of haploid plants from megagametophytes. However because of the organization of the tissue surrounding the megaspore and t h e i r p r e d i s p o s i t i o n f o r the nurishment of developing embryos there i s a p o s s i b i l i t y that i n the future : t h i s may 15 be a u s e f u l method f o r the production of l a r g e numbers of h a p l o i d s . 1 6 MATERIALS AND METHODS Although the primary objective of t h i s study was to determine the requirements f or the anther culture of cherry and peach, rose,, cranberry and apple were used in.some preliminary experiments. These experiments w i l l be described f i r s t followed by a d e s c r i p t i o n of the in v e s t i g a t i o n s with cherry and peach; Rose,cranberry and apple were used because they were conveniently a v a i l a b l e at a time when cherry and peach anthers were not. I t i s not d i f f i c u l t to remove the anthers from large flowers such as tobacco and cranberry. A f t e r removal the anthers were surface s t e r i l i z e d by tying a reasonable number of them i n the center of a square of cheese c l o t h and immersing them f o r about three minutes i n a s o l u t i o n of sodium hypochlorite ( 0.525% avalable chlorine) to which a.drop of the surfactant Tween-80.had been added. This was followed by a rinse i n d i s t i l l e d water f o r another three minutes. The cheese c l o t h and the anthers were then spread out on a s t e r i l e paper towel to absorb the excess moisture and the anthers were then transferred to the medium. Care was taken to ensure that the number of anthers s t e r i l i z e d i n each batch was small enough so that they didinot dry out before they were transferred to the medium. . With small flowers such as cherry and peach i t i s d i f f i c u l t to handle the anthers i n d i v i d u a l l y so the procedure was a l t e r e d s l i g h t l y from the above. Here whole flowers were s t e r i l i z e d and rinsed. The i n d i v i d u a l anthers,were.removed f or the transfer to the medium by removing the basal end of the flower bud with a s t e r i l e needle and then gently squeezing the remaining part between forefinger.and thumb to,force the 17 anthers through the cut end. The anthers were; collected on a s t e r i l e needle as.they were squeezed out and then transferred d i r e c t l y to the medium. A s t e r i l e glove was used throughout this: operation so as not to contaminate the anthers. In order to be able to use pollen at the desired stage of development at any time of the year i t was necessary to store plant material i n the cold to prolong dormancy. Five eight-year-old seedling cherry trees were transplanted into pots and stored i n a cold dark room at 3°C and 100% r e l a t i v e humidity.. Some of these.were moved into the greenhouse throughout the year so that fresh material would be available out of season. Both cherry and peach prunings were collected i n February and stored i n the same.cold room as the potted trees. When flowers were needed growth was forced at room temperature. From February u n t i l May fresh material could be collected from the orchard although towards the end of this period only the more advanced stages of anther development were available. Unless otherwise stated Nitsch's medium (Appendix I) was used. In some of.the l a t e r experiments the concentration of iron was increased by a factor of ten. This was because of an unfortunate misprint i n a description of the medium (see Nitsch, .1969 for the correct medium-. ' r _ containing the increased concentration of i r o n ) . Media were prepared by f i r s t mixing together appropriate volumes of stock solutions. Each chemical (macro-nutrients, micro-nutrients, vitamins and growth regulators) ,vwas- i n a separate stock solution with the exception of iron with was combined with Na EDTA. The sucrose was 2 then disolved i n the medium and the pH adjusted to 5.5. Normally agar was added to the medium after this and was disolved by heating the mixture to 90 C for ten minutes. The medium was then pipetted into either test tubes (75mm X 16mm) containing 5.0 ml or Erlenmeyer flasks (125ml or 250ml capacity) containing 50 or 60 ml respectively.. Test tubes were then capped with p l a s t i c "Bacti-capalls" and the Erlenmeyer flasks were stoppered with foam.rubber plugs. The tops of the plugged flasks were wrapped i n aluminum f o i l and steam s t e r i l i z e d i n an autoclave at f i f t e e n pounds pressure for f i f t e e n minutes. As soon as they were removed from the autoclave they were quickly taken to a laminar a i r flow table to ensure that as cooling proceeded only s t e r i l e a i r was drawn into the fl a s k s . I t was often convenient to hasten s o l i d i f i c a t i o n by placing the flasks i n an ice bath. The number of anthers incubated i n each test tube slant varied with the size of the anthers; with large anthers such as tobacco and cranberry about ten were used; with small anthers such as cherry and peach about t h i r t y were used, a l l of which were from the same flower. Incubation was either i n a light:dark cycle of. 16:8 hours and 28:22°C respectively (henceforth referred to as the l i g h t regime) or i n continuous darkness at 30°C (dark regime). The period of incubation was at least s i x t y days unless otherwise stated. In many experiments coconut milk was used. This was made up. of a mixture of coconut water and a water extract of the meat (coconut , "milk") and was prepared as follows: Reasonably fresh coconuts (Cocos nucifera) were obtained from the l o c a l food market. Those containing the most.coconut water were selected and this l i q u i d was decanted and saved. The coconut meat (s o l i d endosperm) from these same 3 coconuts was diced into about 1-cm cubes. The cubes were then put i n a blender and d i s t i l l e d water added u n t i l the cubes were submerged and 19 the blender turned.onto the'fastest speed' for f i v e minutes'. The l i q u i d portion coconut ("milk") was separated from the coconut meat by f i l t r a t i o n through several layers of cheese cloth. Both.the previously collected ~ coconut water and the coconut "milk" were then autoclaved separately, then cooled to about 4°C. The cooling made i t easier to decant the coconut-water from the precipitated protein. The decanting procedure was repeated with coconut."milk", this time pouring i t through'several layers of cheese cloth. I t was then added to the coconut water so that the solution contained one part coconut water to two part coconut "milk". This solution was considered to be'30% coconut milk by volume and was added to media to give the desired concentration. Deionised double g l a s s . d i s t i l l e d water was the only water used i n preparing media. Glassware was cleaned by vigorous scrubbing with an organic soap. This was followed immediately by a rinse i n tap water and then a further rinse i n d i s t i l l e d water. Previously rinsed glassware was always washed immediately prior to use. While the stage of pollen development could be ascertained by microscopic examination i t was impractical to examine every flower i n d i v i d u a l l y so two charateristics of flowers which were thought to be correlated with the stage of development of the.anthers was often used. The f i r s t was the size of the flower bud and the second the color of the anthers. Both cherry and peach anthers turn a bright yellow before, the pollen i s mature and the .-intensity of this color was used to select flowers which were at the desired stage of development. Aceto-carmine was routinely used when pollen was examined microscopically. The procedure of Wittmann (1965) was used to examine callus c e l l s and to make chromosome counts. The choice of which treatments to include i n an experiment was governed p a r t i a l l y by the r e s t r i c t i o n s of the f a c t o r i a l arrangement which was used whenever.possible. The more basic choice of what type of experiment to do i n the i n v e s t i g a t i o n was somewhat a r b i t r a r y . Most involved t r i a l s with one or more of the following c u l t u r a l compohnents-which have been shown to have an influence.on plant tissues grown i n culture: S p e c i f i c growth regulators, s p e c i f i c vitamins, u n s p e c i f i c growth regulators and vitamins contained i n such things as coconut milk, yeast extract and casein hydrolysate, organic and inorganic nitrogen sources, carbohydrate sources, minerals, pH, l i g h t i n g and temperature. The developmental stages of the anthers which were used was varied because a l l stages are not equally s u i t a b l e for successful anther culture and because the most s u i t a b l e stage appears to be species s p e c i f i c but to vary from species to species. I d e a l l y v a r i a t i o n s i n each of the areas mentioned should be investigated i n combination with every other v a r i a t i o n i n a l l the areas Because of the extremely large number of experiments theis would require, such an approach was not p o s s i b l e . In t h i s i n v e s t i g a t i o n only a few of the many possible combinations were t r i e d . Results obtained throughout this i n v e s t i g a t i o n and those i n published reports as well as i n t u i t i o n a l l contributed to the choice of which ones. Following each experimental number the c u l t u r a l componnents which were varied i n that experiment are stated followed by a d e t a i l e d d e s c r i p t i o n of the experiment. 21 TOBACCO (Nicotiana tabacum,. cv. Haranova) I ( i ) . ( Two mineral solutions; four flower treatments) The anther culture method of obtaining haploid tobacco plants reported by Nitsch and Nitsch (1969) was repeated for practice and to v e r i f y the method.' Flowers were'obtained from plants^ grown i n the greenhouse. Nitsch and Nitsch's procedure was followed as closely as possible but several variations of this procedure were also t r i e d . The f i r s t v a r i a t i o n was increasing the concentration of MgSO^ by 1.6 times. The other three variations were used to determine the effect of storing flowers i n the refrigerator and l i q u i d nitrogen. Some flowers were picked at the stage.of development successfully used by Nitsch and: Nitsch and were stored i n the dark at 4°C for from three to ten days. Others were stored for fourteen days but otherwise handled i n the same way. A t h i r d c o l l e c t i o n was made; the anthers were removed from the flowers and were stored i n l i q u i d nitrogen for fourteen days. Flowers which had been handled i n each of these three ways and freshly picked flowers were used withrthe basal medium and the modified basal medium to give eight treatments. About 2oo anthers were used i n each treatment of anthers which had been stored i n the r e f r i g e r a t o r , about f i f t y with treatments of anthers which had been stored i n l i q u i d nitrogen. A l l the treatments were incubated i n the l i g h t regime. 22 II CRANBERRY (Vaccinium macrocarpon,. cv. McFarlin) • I I ( i ) . (Two flower stages; four,mineral so l u t i o n s ; vitamins and IAA alone and combined) Cranberry flowers were c o l l e c t e d and sorted into two groups depending upon the stage of development of the flowers. The group contain containing the most advanced anthers were flowers within a few days of anthesis. Anthesis was expected to occur i n the l e a s t advanced group i n about a weeks time. The macro and micro-mineral constituents of four media were used. They were Nitsch's medium (Appendix I ) , PRL-4 medium (Appendix I I ) , M.S. medium (Appendix III) and Wolter's medium (Appendix IV).. Three v a r i a t i o n s were superimposed on each of these mineral solutions to give a t o t a l of twelve d i f f e r e n t media. The three v a r i a t i o n s were the addition of a) 10% coconut milk b).vitamins and growth regulators normally used i n Nitsch's medium and c) both vitamins and growth regulators and 10% coconut milk. In each treatment three t e s t tubes contained anthers from the-l e a s t advanced flowers and seven test tubes anthers from more.advanced anthers. A l l were incubated i n the l i g h t regime. 23 I I I APPLE (Pyrus malus, cv. Red Delicious) I I I ( i ) . (Three flower ;stages; IAA, vitamins and coconut milk alone and combined) Flowers were collected and sorted into three groups depending upon their stage of development. The youngest flowers were i n a group, i n which the petals-were v i s i b l e but not f u l l y expanded. The middle group consisted of flowers which were s t i l l a week from anthesis and the most advanced were expected (based on their size) to reach anthesis i n less than a week. Pollen even in.the least advanced group was f u l l y expanded,although not mature. Nitsch's mineral solution supplemented with four different combinations of organics was used. They, were a) IAA alone b) IAA and 10% cocbnut.milk. . c).IAA.and vitamins.as suggested by Nitsch (1969) and d) IAA, vitamins and 10% coconut(;.milk. Flowers fromeach of the three stages of flower development were incubated i n each medium making a t o t a l of twelve treatments of ten test tubes each. Incubation was i n the l i g h t regime. 24 IV ROSE (Rosa multiflora, cv. Queen Elizabeth)' IV(i). (A gradient of anther stages; four mineral solutions; vitamins and IAA, casein hydrolysate and yeast extract.alone and combined) Tight flower buds were picked and as there was.'*.a gradient of stages of anther development within each bud the anthers were not sorted. Normally .the most advanced rose antherscontained nearly mature 1 pollen the- least.advanced anthers, tetrads. In this experiment, the mineral constituents of four mediae-were ca-used (see Appendices I, II, III, IV). Six different additions to each.of these four made a, total of twenty-four .treatments,.' The additions were a) casein hydrolysate, b) casein hydrolysate plus the organics from ' Nitsch's medium, c) yeast extract, d)yeast extract plus the organics from Nitsch's medium, e) casein hydrolysate plus yeast extract, f) casein hydrolysate plus yeast extract.plus the organics from Nitsch's medium. Casein hydrolysate and yeast extract were always at a concentration of 3 mg/1. Ten test tubes' of each were incubated in the light regime. I V ( i i ) . (A gradient of anther stages; IAA and seven vitamins in every combination) ' Anthers'":were collected and sorted as in rose experiment IV(i-) . Nitsch's mineral solution was used in this experiment. The vitamins suggested by Nitsch (1969) were added in every combination. A l l thirty-two treatments containing both glycine and IAA. In the second part of this experiment the same thirty-two combinations were again prepared; this time glycine was added to a l l treatments.but IAA was excluded. Ten tubes of •'/$:.•••• each medium were prepared and a l l were incubated in,the light regime. 25 V CHERRY (Pramus avium). V ( i ) . ( l i q u i d coconut milk) Lambert anther flowers were collected one 1 day before anthesis and the anthers were removed from the flowersV? The medium consisted e n t i r e l y of 15% coconut milk with the precipitated protein included. No agar was added and the l i q u i d medium was poured into p e t r i dishes The anthers were placed i n it;b u t were not submerged. Incubation was i n thedark at 28°C and the period of incubation varied because contamination took longer to reach some dishes than others. V ( i i ) . (Three flower stages; IAA and vitamins and coconut milk alone and combined) Van flowers were collected and sorted into three groups < depending upon thei r stage of development. In the group containing the most mature flowers anthesis could be expected to occur within a day, . i n the second group within f i v e days and i n the th i r d group within ten days. Four media were prepared a l l of which contained Nitsch's minerals. These were a) which contained i n addition the organics used by Nitsch including IAA, b) 15% coconut milk, c) 15%coconut milk plus IAA and d) 15% coconut milk plus IAA plus the vitamins used by Nitsch; Incubation was i n the l i g h t regime. V ( i i i ) . (Four mineral solutions; IAA and vitamins and coconut milk alone and i n combination) Lambert flowers were collected one day before expected anthesis. They were removed from.the flowers and surface s t e r i l i z e d , dried and allowed 26 to dehisce overnight i n a sealed s t e r i l i z e d container 5.' The next.day the p o l l e n was suspended i n a 2% sucrose s o l u t i o n and germination was allowed to proceed for two hours. 0.5 ml of t h i s suspension was then transferred with a syringe to the surface of the.prepared media. The twelve media used were based on four.mineral solutions (see Appendices I, I I j •III, IV) and the addition of three combinations of organics. The f i r s t combination was the vitamins used by Nitsch plus IAA, the second 15% coconut milk and the t h i r d 15% coconut milk plus the vitamins used by Nitsch and IAA. Incubation was i n the l i g h t regime. V ( i v ) . (Five mineral sol u t i o n s ; l i g h t and dark) Flowers were obtained from a potted seedling tree which had been brought into the greenhouse to break dormancy. The p o l l e n in-these flowers was barely past the tetrad stage and the anthers had not yet developed any yellow color. Five media were prepared the f i r s t Nitsch's medium. The remaining four were v a r i a t i o n s of the f i r s t treatment a l t e r e d by increasing the concentration of f o u r . s a l t s 1.5 times. The four s a l t s were KN0 o, MgSO., NH.N0„ and KH.PO.. Ten test tube slants 3 4 4 3 2 4 of each treatment were incubated i n the l i g h t regime and ten i n continuous dark at 17°C. • V(v). ( s o l i d coconut milk) Flowers were obtained from a potted seedling tree which had been brought i n t o the greenhouse to break dormancy. ' The p o l l e n from these Vr. i • flowers was.barely past the tetrad stage. Nitsch's medium plus 15% coconut milk was the only treatment i n t h i s experiment. 27 V ( v i ) . (Nitsch's medium or coconut milk alone or combined; kinetinand, coconut milk alone or combined with Nitsch's medium)-Lambert flowers which were expectdd to reach anthesis within two days were used i n this experiment.' Five media were used: a) 10% coconut milk, b) Nitsch's medium, c) Nitsch's medium plus 10 k i n e t i n dj.^ • 10%' coconut milk plus 10 "*M k i n e t i n and e) Nitsch's medium.plus 10% coconut milk and 10."*M k i n e t i n . Twenty test "tubes of each of cthe f i v e media were incubated i n the l i g h t regime. V ( v i i ) . (2,4-D, kinetin. and IAA -alone and combined; NAA,-kinetin-and IAA alone and combined) Lambert flowers were picked from prunings which had been o gathered during dormancy and stored in.the cold room.(3 C). The pollen . was just past the tetrad stage of development. Eight media-were prepared a l l of which had the mineral and vitamin constituents of Nitsch!s medium. The differences i n the media were i n the growth regulators used. The combinations were: a) 2,4-D, b) 2,4-D plus IAA c) 2,4-D plus k i n e t i n , d) 2;4-D plus IAA and k i n e t i n , e) NAA, f) NAA plus IAA, g) NAA plus k i n e t i n and h) NAA plus IAA and k i n e t i n . Each of the growth regulators was atta concentration of.10 M^. Incubation was.in the l i g h t regime. V ( v i i i ) . (Nitsch's medium plus either k i n e t i n or coconut milk; f i v e temperatures) Lambert flowers were obtained from pruning stored i n a cold . room. The anthecs were past the tetrad stage of development and were ;; beginning to turn yellow. Two media were used, Nitsch's plus 10~5M ki n e t i n and Nitsch's plus 10% coconut milk. Fifteen tubes were incubated 28 i n the dark at each.of the following temperatures: 12, 15, 17, • 23 and 31°C. V ( i x ) . (Five pH's) Lambert ^flowers , were obtained from pruning which had been' stored i n the dark for four months. The pollen was past the tetrad stage of development.and the anthers had begun to develop a yellow color. Five aliquots of Nitsch's medium, containing in.addition 10 NAA and 10 k i n e t i n but no IAA, were each adjusted to a different pH. The pH's were 5.0, 6.0, 6.5, 7.0 and 7.5. Five more media i d e n t i c a l to the above but with the addition of 10% coconut milk were also prepared. A l l ten treatments were incubated i n the dark regime. V(x). (Eight nitrogen sources) S t e l l a flowers were picked from prunings which had been stored i n the dark at 3 _.• The pollen was past the tetrad stage of development and the anthers3," were a pale yellow color. Eight media based on Nitsch's medium were prepared. NAA was substituted for IAA and the nitrogen source was, varied. In the f i r s t medium urea was the only nitrogen source, i n the second n i t r a t e and i n the t h i r d ammonium. In the fourth the nitrogen source was equal molar concentrations of urea and n i t r a t e j i n the f i f t h equal molar concentrations of ammonium and n i t r a t e . The t o t a l amount of nitrogen i n each of the above treatments was the same. The concentrations of the other s a l t s with the exception of chlorine was the same as i n Nitsch's medium. This was done by.adding appropriate amounts of KC1, NH^Cl or NaNO and thus manipulating the nitrogen.source while maintaining the 29 concentrations of other s a l t s at levels as close as possible to those i n Nitsch's medium. The seventh medium was Nitsch's medium with NAA • - • ..; substituted for IAA plus 0.1 gm/1 peptone and the eight the same.as the seventh plus 0:lgm/l bovine serum albumin. Incubation was i n the dark regime. V ( x i ) . (IAA or NAA plus various•concentrations.of coconut milk) Royal Ann flowers were picked from prunings which had been stored i n the cold room. Four media were prepared a l l based on Nitsch's medium. Two of these contained IAA the f i r s t , i n addition, 10% coconut milk and the second 15% coconut milk. In the t h i r d and.fourth NAA was • substituted for IAA. The t h i r d contained in.addition 10% coconut milk and the fourth 20% coconut milk. There were fourty tubes of each treatment a l l l o f which were incubated i n the dark regime. V ( x i i ) . (Mannitol, mannose.and sucrose combined at varbus concentrations) Royal Ann,flowers were taken from pruning which had been stored i n the cold room. ' Eight media,were prepared a l l based on Nitsch's medium but containing in.addition 10% coconut milk and 10 k i n e t i n . The f i r s t medium contained 2% sucrose plus 2% mannitol, the second 2% mannose, the t h i r d 3% mannitol, the fourth 4% mannitol, the f i f t h 1% mannitol, the s i x t h 1% sucrose plus 1% mannitol, >the seventh 1% mannitol plus 1% mannose and the eight 1% mannose. Ten tubes of each treatment were a l l incubated i n the l i g h t -regime. V ( x i i i ) . (Five v a r i e t i e s ) In this experiment prunings were again used as the pollen sources 30 The experiment was designed to test the growth response of f i v e c u l t i v a r s cultured on the same medium. Nitsch's medium was altered by leaving out IAA but including 10 NAA and 10% coconut milk. The f i v e c u l t i v a r s used were S t e l l a , Lambert, 5G-9-11, 5G-29-8 and Royal Ann. The numbered selections were obtained from the Canada Department of Agriculture Research Station, Summerland, B.C. The anthers which were used had been i n the cold (3°C) for approximately eight months and the flower buds showed varing degrees of browning. S t e l l a flowers appeared brown on the outside but fresh on the inside of the bud. Lambert appeared i n the best condition of the f i v e c u l t i v a r s but was not as good as the fresh material.• 5G-9-11 showed general browning of the flower bud on both the inside and the outside. 5G-29-8 apppeared fresh on the outside of the flower bud but the anthers were beginning to turn brown. Royal Ann flowers were brown on the outside near the base of the bud but fresh otherwise. A l l the anthers after being i n storage eight months had developed a d i s t i n c t yellow color. Because of the poor condition of most of the buds the smallest and freshest looking were used. Fifteen tubes of each treatment were prepared and a l l were incubated i n teh dark regime. 31 VI PEACH' (Prunus-persica cv; "Valiant) VI (<i) . ( s o l i d medium; coconut milk) This experiment was the same as cherry experiment V(v). In th i s experiment the peach anthers were j u s t beginning to turn yellow. V I ( i i ) . (Nitsch's medium or coconut milk alone or combined; k i n e t i n plus-coconut milk alone or combined with Nitsch's medium) This experiment. was the same as cherry experiment V(vi).. The flowers were picked from prunings which had been stored i n the cold. The anthers were pale green ( the color of th'e. anthers before they become yellow) and they contained p o l l e n which had developed past the tetrad stagg. V I ( i i i ) . ( 2, 4-D, k i n e t i n and IAA, alone or combined; NAA, k i n e t i n and IAA, alone or combined) This experiment was the same as cherry experiment V ( v i i ) . The flowers were.picked from prunings which had been stored i n the cold and the anthers were j u s t begining to turn yellow. V I ( i v ) . (Nitsch's medium plus either k i n e t i n or coconut milk; f i v e temperatures. This experiment was the same as cherry experiment V ( v i i i ) . The anthers were j u s t begining to turn yellow. VI'<v) . ( Five d i f f e r e n t pH's) This experiment was the same as cherry experiment V5(ix) . The young anthers which were used were yellow. 32 V I ( v i ) . (Eight nitrogen sources) This•experiment was the same as cherry experiment V(x). The anthers were a d i s t i n c t yellow and were begining to enlarge. V I ( v i i ) . (Mannitol,.mannose and sucrose combined at various concentrations) This experiment was the same as cherry experiment V ( x i i ) . The anthers which were used were t h f ' s a m e maturity as those i n experiment V I ( v i ) . V I ( v i i i ) . • In this experiment peach c a l l i obtained i n experiment VI(vi) were transferred to the same type of medium on which their growth Was ' i n i t i a t e d . This time 0.1% peptone was added rather than 0.1 gm/1 and the pH was adjusted to 6.5 rather than 5.5. Two Erlenmeyer flasks containing the transferred c a l l i were incubated i n a light:dark cycle of 14:10 hours at a continuous temperature of,30°C, two similar flasks were incubated i n the dark regime. 33 Figure 1. One half of a tobacco anther with embryoids emerging from i t . Photographed four weeks after seeding. X 14 Figure 2. Part of figure 1. Numerous embryoids most of which are at the globular stage of development are v i s i b l e . X 46. 34 Figure 3; Relative sizes of anthers as described i n the. text. From l e f t to right cherry anthers which have enlarged a) considerably b) s l i g h t l y c) very s l i g h t l y d) not at a l l . Bar i s 2 mm. Figure 4. D i c e l l u l a r cherry pollen grain. Two c e l l s surrounded by the exine (E). X 525. 35 Figure 5. Cherry pollen grain containing two or three c e l l s , surrounded by the exine. X 400. Figure 6. Abnormal germination i n cherry. A. X 510. B. X 710. 36 Figure 7. Cherry pollen with an accumulation of cytoplasm at the tip of the germ tube (G), two types of protrusions (P ) and (P ) and normal pollen (N). A. X 200. B. X 200. Figure 8. Proliferation in a cherry germ tube. X 375. 37 Figure 10. Four hypertrophied cherry p o l l e n grains and two normal p o l l e n grains (N). A. X 330. B. X 200. 38 Figure 11. A. Four c a l l i growing from within cherry anthers, photographed three weeks after inoculation. X 2.6. B. Callus emerging from both locules of a cherry anther. X 25. Figure 12. Dividing c e l l found i n a cherry callus s i m i l a r to those i n figure 11. More than the haploid number of eight chrom chromosomes are v i s i b l e . X 250. 39 RESULTS I TOBACCO I'(i) . (Four flower treatments; two mineral solutions) Nitsch and Nitsch's method of r a i s i n g tobacco plants was successfully repeated. The f i r s t plants emerged i n twenty-three days. About seventy percent of the t o t a l had emerged before day t h i r t y but plants continued to emerge u n t i l about day si x t y y ( F i g . 1 and 2). TABLE I Percentage of anthers producing plantlets i n experiment I ( i ) . FLOWER TREATMENT MEDIUM New Medium Old Liquid Nitrogen Nitsch 14 2 7 0 MgS04 X 1.6 15 1 31 0 The number of anthers producing plantlets (Table I) i s not a true indication of the t o t a l number of plantlets produced because although most anthers produced more than one pl a n t l e t the range was from one to about one hundred. In each treatment the number of anthers producing many plantlets was about,the same. Those which emerged towards the end of the incubation period were oftemchlorotic and usually did not produce roots. The proportion of anthers i n each treatment which produced this type of plan t l e t was also the same. In one Erlenmeyer, f i f t y anthers were incubated and plantlets emerged from 36% of them. A week after transferring the anthers containing plantlets with roots to Nitsch and Nitsch's medium minus IAA the number of plantlets 40 •: per anther had been reduced to a maximum of s i x because of competition between plants as they grew larger. F i n a l l y the-transfer to s o i l resulted i n a further loss of about 10% of those transferred. The anthers began turning dark colored soon after the beginning of incubation and had soon become black. Concomitantly with this t h e - . 4 ' , anthers opened a s ' i f they were dehiscing. The anthers enlarged s l i g h t l y during the f i r s t week of incubation but after this time did not become larger. Plantlets were grown.to maturity i n the greenhouse. They were considered to be haploid after examination of the pollen because most of i t was s t e r i l e . A further indication of the.haploid nature Sf these plants was their height which was about 'two-thirds that of d i p l o i d plants. After three weeks of incubation anthers were collected and the pollen examined microscopically. Manyttabnormal pollen grains were observed , which were similar to those noted by Nakata and Tanaka (1968). I I CRANBERRY I I ( i ) . (Two flower stages; four mineral solutions; vitamins and IAA alone and combined) The results of this experiment were negative. Examination at the end of.the incubation period revealed only degenerated pollen. During the experiment the anthers enlarged s l i g h t l y and changed from their o r i g i n a l dark brown color to a pale grey. The loss of color was accompanied by loss of turgor. 4 1 . III APPLE I I I ( i ) . (Three flower stages; IAA, vitamins and coconut milk alone and combined) The r e s u l t s of t h i s experiment were negative. Examination at the end of'the incubation period revealed only degenerate p o l l e n . No other abnormalities were observed. The anthers were a pale yellow at the beginning of the experiment and became darker as the experiment progressed becoming brown by. the end of the incubation period. The anthers became s l i g h t l y enlarged during the experiment. IV ROSE I V ( i ) . (A gradient of anther stages; four mineral so l u t i o n s ; vitamins and IAA, Casein hydrolysate and yeast extract, alone and i n combination) An i n t e r e s t i n g abnormality was observed when the pol l e n from t h i s experiment was examined. Some.of the p o l l e n had enlarged and accumulated: "starch ( F i g . 10a). This type of abnormality was observed•'. frequently i n rose and was c h a r a c t e r i s t i c of a l l the treatments i n t h i s experiment. The rose anthers were pale yellow at the beginning of the experiment but a f t e r a week of incubation they had developed a brown color which i n t e n s i f i e d throughout the incubation period. I V ( i i ) . (A gradient of flower stages; IAA and seven vitamins i n every combination) P o l l e n development i n t h i s experiment was s i m i l a r to that i n experiment I V ( i ) . Not a l l the treatments were examined as,extensively 42 as i n other experiments because of the large number of treatments and the apparent lack of major.differences between them. Anther color development and enlargement were also similar to experiment I V ( i ) . V CHERRY V ( i ) . (Liquid coconut milk) This was one of,the most'encouraging experiments i n the investigations because doubled cel l e d pollen grains and possible a t r i c e l l u l a r pollen grain were observed (Fig 4 and 5). Abnormal germination (Fig. 6) was extensive and hypertrophied germ tubes were also seen (Fig. 8). After an i n i t i a l s l i g h t enlargement of the anthers (Fig. 3) they remained the same size for the duration of the experiment. In the anthers which remained healthy the o r i g i n a l yellow color became a d u l l straw color. V ( i i ) . (Three flower stages;IAA, vitamins and coconut milk alone and i n combination) No m u l t i c e l l u l a r pollen grains were"observed i n this experiment. Abnormal germination and hypertrophied germ tubes similar to those i n experiment V(i) were not uncommon. Neither the stage of development of the anthers nor the medium affected the frequency of these types of pollen growth. The color development and enlargement of the anthers was similar to experiment V ( i ) . V ( i i i ) . (Four mineral solutions; IAA and vitamins and coconut milk alone and combined) Although t h i s experiment with germinated pollen was repeated '43 three times, the second and t h i r d time with a n t i b i o t i c s , i t was a f a i l u r e because of t o t a l .contamination. The technique used by Kameya- -and Hinataj;,' (1970) should make this experiment possible. V ( i v ) . (Five mineral solutions; l i g h t and dark) No encouraging results were obtained i n this experiment. A l l the anthers which were examined contained almost exclusively degenerate pollen. The anthers incubated at 17°C remained yellow for longer than the others. Also,.they did not enlarge as much and degenerated more' slowly than those i n the l i g h t regime. The results were,the same for a l l the media i n the experiment. V(v). ( s o l i d medium; coconut milk) Examination of the pollen in.this;experiment indicated degeneration of the majority of the grains. However, a few hypertrophied pollen.grains were observed. The anthers enlarged s l i g h t l y to about one-half the length of mature anthers. They turned pale brown soon after the beginning of incubation. Fragments of flower buds which were inadvertantly incubated along with the anthers, enlarged. Sepals developed a normal'green color. A few petals which were attached to the sepals also enlarged and developed t h e i r characteristic color. The anthers which were attached to these fragments remained a healthy yellow color after the unattached ones had become a d u l l brown but abnormalities which were found i n the latter were less common, i n anthers attached to flower bud fragments. 44 V(vi) . (Nitsch's medium with coconut milk alone or combined; k i n e t i n plus coconut milk alone or combined with Nitsch's medium) Antherssquashes two days after the beginning of incubation showed that many pollen grains had germinated. Later i n the incubation period a pollen grain was found which had enlarged to about eight times the diameter of normal pollen (Fig. 9). Abnormal germination and protrusions (Fig. 7) were also observed but m u l t i c e l l u l a r pollen grains were not. Anthers, which were yellow at the beginning of incubation, became straw colored by the end.' V ( v i i ) . (2,4-D, k i n e t i n and IAA alone or combined; NAA, k i n e t i n and IAA alone or combined) Anther squashes showed that most of the pollen i n this experiment had degenerated. No interesting growth was observed. The anthers enlarged during the f i r s t ten days of incubation at which time the size of the anthers s t a b i l i z e d . Their original»yellow color became progressively darker and eventually the anthers were a dark brownish yellow. Two treatments one containing NAA plus k i n e t i n and.the other 2,4-D plus k i n e t i n , induced callus growth from flower bud tissue. There were numerous c a l l i i n both these treatments, In both 1 treatments the c a l l i were green. Those originating on the NAA treatment were much faster growing although the c a l l i from the 2,4-D treatment became large enough to subculture the NAA medium was considered the most satisfactory for callus growth so c a l l i were only subcultured onto this medium. V ( v i i i ) . (Nitsch's medium plus ei t h e r k i n e t i n or coconut milk; f i v e temperatures) -No p o l l e n growth was observed - i n t h i s -experiment although some p o l l e n appeared to remain i n a dormant state, degeneration was more.common. . C a l l i grew from anthers on the coconut milk medium incubated at 31^C. Although only f i v e of the tubes had c a l l i growing i n them there was always' more than one c a l l u s per test tube (usually about f i v e ) . A l l the c a l l i . i n the tube appeared to have begun.growing at about the,same time as they were nearly the same s i z e ( F i g . 11a). Although the c a l l i c l e a r l y originated i n s i d e the^anther ( F i g . l i b ) they are. thought to have grown from connective tissue rather than p o l l e n . Chromosome counts indicated d i p l o i d rather than haploid chromosome numbers ( F i g . 12) This alongJwith-the conspicuous lack of m u l t i c e l l u l a r p o l l e n or other p o l l e n abnormalities makes the o r i g i n of the c a l l i from pol l e n u n l i k e l y . Anthers on the coconut milk medium i n the 31°C treatment were straw colored when the c a l l i emerged. The anthers did not open as i f they were dehiscing. Those on the k i n e t i n medium became a darker brown. In a l l the other treatments the anthers on both media developed, v a r i a b l y within a tube. Some remained yellow and others became brown. V ( i x ) . (Five d i f f e r e n t pH's) Microscopic examination indicated no major differences i n p o l l e n growth between media d i f f e r i n g only i n pH. A l l the anthers i n a tube were from one flower and were homogeneous.in color and degree of enlargement throughout the incubation period. There was considerable 46 v a r i a t i o n between test tubes i n the same treatment. Response to,pH was indicated by the color and enlargement of the anthers and the tendency to form enlarged.filaments. Large anthers were the only ones' which developed swollen filaments: - Small anther size was usually associated with dark color. The only consistent effect of pH was on anther s i z e . Anthers f a i l e d t oenlarge at pH 7.5. The anthers i n the pH'7.5 treatments were dark brown; the anthers i n the other treatments were a brownish straw color. There was no other obvious relationship between pH and anther -morphology. V(x). (Eight nitrogen sources) Anther color and degree of enlargement. and . the;,;tendency ; to induce callus growth were used as indicators of the s u i t a b i l i t y of the medium i n this experiment because very l i t t l e pollen growth occurred. Anther color and the degree of enlargement seemed d i r e c t l y related to each other; anthers which enlarged only s l i g h t l y became dark brown early i n the incubation period and anthers which became considerably.; enlarged remained straw colored u n t i l the end of i t . Small brown anthers were associated with urea,except when urea was i n combination with ammonium. In other treatments anthers became.enlarged and straw colored. There were no other differences among treatments but less d i s t i n c t associations may have beennobscured by the variations among tubes i n a treatment. There were differences i n the tendency of the treatments to induce the formation of c a l l i . The n i t r a t e alone medium was the only one i n which c a l l i were induced i n every tube and; the most p r o l i f i c callus growth was from this treatment. The medium containing peptone' but not bovine serum albumin also induced callus growth but not to as 47 •. great an extent as did the n i t r a t e medium. A l l the c a l l i i n this experiment originated from .either the anther filaments or flower bud tissue.' C a l l i originating on.either of these media were fast enough growing to sub-culture . V ( x i ) . (IAA or NAA plusrvaring concentrations of coconut milk) In t his experiment p r o l i f i c callus growth was induced i n some treatments but no abnormal pollen growth was observed. Nearly a l l the anthers became straw colored by about a week after inoculation and retained this color u n t i l incubation was terminated. About.three weeks after the beginning of inoculation small c a l l i were observed i n many tubes. In the NAA plus 10% coconut milk treatment there was at.least:one and often several c a l l i i n about 90% of the tubes. Although considerable callus growth was i n i t i a t e d i n the NAA plus 20%. coconut milk treatment i t was only about one-half as extensive as on the other NAA treatment. Nearly a l l the c a l l i induced i n . the experiment appeared." to : . originate within the anther i n a similar manner to those'in experiment V ( v i i i ) . V ( x i i ) . (Mannitol,.mannose and sucrose combined at.various concentrations) No abnormal pollen growth was observed i n this experiment. C a l l i were induced to grow i n some of the treatments and :there were d i s t i n c t differences i n the extent of enlargement and the color of the anthers. In treatments of mannitol alone.at concentrations of 1, 2, or 3% callus growth was induced!:.- i n the 1% treatment three c a l l i were produced i n the 2%, two and in.the 3%,..three. A l l of these c a l l i were.first observed 48 about three weeks after inoculation and they grew slowly i f at a l l after a week. The c a l l i apppeared to originate from the flower bud ; . fragments. A l l the anthers i n th i s experiment became very enlarged and straw colored by the t h i r d week of incubation. There was no further-enlargement after this time but they did become progressively darker. In - a l l the treatments i n which sucrose was included the anthers enlarged only s l i g h t l y and became straw colored soon-after inoculation. After a week of incubation the color and size, of the anthers s t a b i l i z e d , , remaining the same for the duration of the experiment. V ( x i i i ) . (Five v a r i e t i e s ) • Although:.-no abnormal pollen growth was observed i n this experiment c a l l i did grow from.tissue of four of the f i v e c u l t i v a r s . There were c a l l i i n seven out of the twenty tubes of S t e l l a , eight of Royal Ann, eight of Lambert, thirteen of 5G-9-11 and none of 5G-29-8. A l l the c a l l i originated from either anther filaments or flower bud fragments. In a l l of the treatments i n this experiment the anthers enlarged only s l i g h t l y . They had become straw colored by two,weeks after inoculation. I t was also about this time that the f i r s t c a l l i were observed. .49. VI PEACH • V I ( i ) . ( S o l i d medium; coconut milk) There were no marked differences i n the responses of anther or p o l l e n to d i f f e r e n t treatments i n t h i s experiment. Examination revealed some hypertrophied p o l l e n ( F i g . 10) and some which had grown to the s i z e of mature p o l l e n . The enlarged p o l l e n stained darkly with aceto-carmine. The anthers enlarged s l i g h t l y and by the end of the experiment they had become a very dark brown. .By the end of the incubation period some of the anthers had s p l i t open i n anmanner s i m i l a r to dehiscing anthers. However, probably no p o l l e n l e f t the locule of the anther because of.the high humidity i n s i d e the tube. V I ( i i ) . (Nitsch's medium or coconut milk alone or combined; k i n e t i n plus coconut milk alone.or combined with Nitsch's medium) Although a few hypertrophied p o l l e n grains were seen in.KthiS; experiment most of the p o l l e n had degenerated. There were no d i s t i n c t differences between treatments. The anthers began.turning a dark color soon a f t e r the beginning of incubation and became progressively.darker u n t i l by the end of the experiment they were a slark purple. V l ( i i i ) . (2,4-D, k i n e t i n and IAA, alone or combined; NAA, k i n e t i n and IAA alone or combined) Degenerate p o l l e n interspersed with a few hypertrophied p o l l e n grains was c h a r a c t e r i s t i c of the treatments i n t h i s experiment; The anthers became s l i g h t l y enlarged but once color development.was w e l l under way anther enlargement stopped. The coler continued to i n t e n s i f y u n t i l at the end of the experiment they were a dark purple. 50 Several c a l l i grew from the 2,4-D plus k i n e t i n treatment'but they ;only grew slowly and d i d i i not become larger than 5 mm i n diameter. The c a l l i , f i r s t observed about,a month a f t e r i n o c u l a t i o n , were very f r i a b l e , pale or translucent and composed," of large c e l l s which could be seen under twenty power magnification.; Some,of the c a l l u s c e l l s became br i g h t purple when growth stopped.. These c e l l s were assumed to contain anthocyanins. They were scattered randomly on the surface.of the c a l l u s and were not usually adjacent to each other. V I ( i v ) . (Nitsch's medium plus ei t h e r k i n e t i n or coconut milk; Five temperatures) 1 Differences between, color of anthers was more apparent i n this.experiment than i n others. In a l l but the 31°C treatments the v a r i a t i o n w i t h i n tubes was greater than i n other experiments. Nearly a l l the p o l l e n degenerated except i n the 31°C treatments i n which hypertrophied c e l l s were found. Degeneration proceededmore•slowly at the lower temperatures. One c a l l u s s i m i l a r but smaller than that i n experiment V I ( i i i ) grew from an anther filament on the- coconut milk medium i n the 17°C treatment. The anthers i n the-31° C treatments became s l i g h t l y enlarged but at the other temperatures enlargement.was less prominent. By the end of incubation thea3Qi°C treatments had become' brown. In a l l the other treatments there was a mixture of yellow and brown anthers i n each.tube,.the r a t i o being about the same at a l l these temperatures. No differences were'observed between the coconut milk and k i n e t i n media at any temperature. 5 i . VI(v). (Five d i f f e r e n t pH's) No abnormal p o l l e n growth other than the usual number of hypertrophied p o l l e n grains was observed i n t h i s experiment. Most of the p o l l e n degenerated. The d i f f e r e n t treatments influenced s l i g h t l y anther color and s i z e . On the k i n e t i n medium adjusted to pH 7.5 the anthers became l i g h t brown. This was i n contrast to the pale brown.of the anthers i n a l l other treatments on ei t h e r media. Anthers on the coconut milk medium adjusted to pH'7.5 were smaller than the medium sized ones i n a l l the other treatments. Although no c a l l u s growth was i n i t i a t e d , anther, filaments on the coconut milk medium at.both pH 6.0 and 5.0 became 'swollen and white i n c o l o r . V I ( v i ) . (Eight nitrogen sources) In t h i s experiment a medium which supported the growth of peach c a l l u s was. found. The c a l l i grew from anther filaments and flower bud fragments.- Examination of po l l e n showed that no abnormal growth had . occurred other than a few hypertrophied p o l l e n grains. In the l a t t e r respect the treatments did not d i f f e r . When n i t r a t e was i n combination .. . with ei t h e r urea or ammonium the anthers became more enlarged than i n . other treatments: Ammonium i n combination with n i t r a t e caused the anthers to develop a dark brown co l o r . When ammonium was combined with urea only the midribbdeveloped the dark brown color while the rest of the'.-anther'. • remained a pale yellow. When ammonium alone was used as the nitrogen source the anther became a pale yellow. Anthers i n a l l other treatments were pale,brown. Callus growth was i n i t i a t e d on both the n i t r a t e alone and on the peptone medium. Six c a l l i grew, on the peptone medium and three on the n i t r a t e . Only those from the peptone grew quickly when subcultured. These subcultured c a l l i were cream.colored and f r i a b l e . In the subculture medium the concentration of peptone was increased from 0.1 gm/1 to 1.0 gm/1 and the pH was changed from 5.5 to 6.5. These changes markedly incresed the rate of growth of the c a l l u s . The anther filaments usually became swollen before callus growth was i n i t i a t e d from them. Only the n i t r a t e and ammonium medium i n i t i a t e d callus growth from the swollen fllamerits' However, anther filaments on ammonium plus n i t r a t e and the bovine serum albumin media,did become swollen. V I ( v i i ) . (Mannitol, mannose and,sucrose alone and combined at various combinations) Different carbon sources had d i s t i n c t effects upon antherss color and enlargement but there were no differences i n the development.of pollen. Most pollen degenerated although some hypertrophied pollen grains were observed. In treatments containing mannose very l i t t l e enlargement of the anthers occurred and their color remained a pale yellow.. No callus growth was i n i t i a t e d on any of the media containing mannose. In the two treatments i n which mannitolwwas combined with sucrose the anthers became quite enlarged and brown,in color, dark brown i f the medium contained 2% mannitol and 2% sucrose and l i g h t brown when both,were 1%. In the three treatments containing mannitol alone'the anthers became greatly enlarged. Those i n the 3% mannitol treatment were the largest (approximately 5 mm)' and those i n the 2% and 1% treatment'slightly smaller although s t i l l r e l a t i v e l y large (approximately 3 mm). The color of the anthers on.the 2 and 3% treatments was pale yellow but those on the 1% treatment developed a l i g h t brown color. Three c a l l i grew from the 3% medium and one each from the 2% and 1% media. A l l the c a l l i originated from 53 anther filaments. They were.slow growing and became brown!about 1 two weeks'after growth was i n i t i a t e d : A l l the c a l l i died a f t e r reaching a maximum diameter ofifabout 4 mm. - The anthers i n the mannitol and sucrose plus mannitol treatments s p l i t open l o n g i t u d i n a l l y as i f they were dehiscing. V l ( v i i i ) . These adjustments to the medium caused' the subcultured c a l l u s to grow much more vigorously than didx the parent c a l l i . A f t e r the transfer c a l l i grown i n the dark changed i n color from dark brown or black to pale brown and became more f r i a b l e and.smooth i n texture. C a l l i subcultured i n the l i g h t became green and.retained the rough texture of the parent c a l l i . Compared to the l i g h t - g r o w n . c a l l i the dark-grown had a f a s t e r growth rate; DISCUSSION SPECIAL DIFFICULTIES WITH CHERRY AND PEACH At the onset this, project two characteristics of cherry and( J, peach'were expected to make the successful adaption of the anther culture method to these .crops elusive r e l a t i v e to other crops. Callus can be grown'from many woody angiosperms but to date plants have only been regenerated from coffee (Staritsky, 1970) . I f haploid c a l l i grew i t . was expected that this c h a r a c t e r i s t i c of.most woody species would - , make the regeneration, of plantlets from peach and cherry d i f f i c u l t . Secondly, outbreeding species are known to possess many degenerative • genes which manifest themselves i n haploids and homozygous diploids derived from haploids. Nitsch (1969) has demonstrated a.reduced frequency of haploid production i n obligate outbreeding species of tobacco compared to inbreeding ones. The-reduction''Was probably caused by degenerative genes. Because cherry i s normally outbreeding ( although.seyeralrmutants which^are s e l f fertile-have been obtained) i t was expected that degenerative genes would reduce the chance of success p a r t i c u l a r l y i f conditions present precluded a l l but a small percentage of anthers, with the potential to produce haploid embryoids or c a l l u s , from developing. Most "peach, v a r i e t i e s are both cross.and self-compatible but, as with cherry, peach i s very heterozygous. Many degenerative genes are also present.in peach,.the evidence for this being the extremely reduced vigor of several naturally occurring haploids (G.M.Weaver, personal communication). I t -i s not known i f this second factor would reduce the chance of haploid Table II The requirements for androgenesis REFERENCE • SPECIES MEDIUM LIGHT GROWTH FACTORS TEMP. MATURITY NUCLEI PERIOD YEILD Guha and Maheshwari, 1967 1 Datura innoxia Datura stramonium Nitsch or H e l l e r 15% coconut milk or plum i u i c e or k i n e t i n mature ' 2 60 days 100% Nitsch, 1969 Nicotiana a l a t a N. glutinosa N. r u s t i c a N. s y l v e s t r i s N. tabacum Nitsch l i g h t : dark IAA i_ k i n e t i n 24: 30°C immature \ 2 25 days 67% •Nitzsche, 1970 Lolium multiflorum X Festuca arundinacea i - — — — mature 3 — .002% N i i z e k i and Oono , Oryza s a t i v a Blaydes dark IAA plus 2,4-D plus cinetin 28°C immature 3 28-56 days 0.57% Kameya and Hinata, 1968 a) Pollen cultures Brassica olreaceae Brassica olereae. X B. alboglabra b) Anther culture Brassica olereae Brassica olereae '1 X B. alboglabra Nitsch Nitsch diffuse l i g h t dark L0% coconut milk 2,4-D or 2,4-D plus • k i n e t i n : or , 10% coconut milk 20°C • 20°C mature mature 3 3 -28 days 49 days 1.6-8.7% Nakata and Tanaka, 1968 (Nicotiana tabacum RM-1964, Linsmairer c\r\4 Skoog, White l i g h t IAA and k i n e t i n — immature 95 days 6.0% Cn-On 1 56.. growing from."pollen, although i t would probably cause a low frequency of . embryoids developing d i r e c t l y . i REQUIREMENTS FOR ANDROGENESIS A comparison of the requirements and the results from species i n which androgenesis has been successful i s summarized i n Table I I . The basal medium which i s used i s probably not absolutely c r i t i c a l . In Datura innoxia, the Datura species studied i n most d e t a i l , either Nitsch's or Heller's medium were e f f e c t i v e . In tobacco Nakata and Tanaka (1968) used a different ..medium from that used by. Nitsch. Nitsch (1969) t r i e d several basal media and a l l worked equally w e l l . • No experiments i n which a variety of basal media was used have been reported for other species. However, Nitsch's medium was used for at least three of the f i v e species e x i b i t i n g androgenesis and thus appears to be generally suitable and i t s use i n further attempts at androgenesis appears j u s t i f i e d at this time. I t i s not possible to generalize to such an extent about growth regulator requirements. In both Brassica and Datura, haploid tissue grows when the basal medium plus coconut milk i s used. In Datura both IAA and 2,4-D are i n h i b i t o r y . With tobacco (Nitsch, 1969) thergrowth regulator content of the medium i s not c r i t i c a l but.the basal medium plus IAA i s most suitable. In r i c e , IAA, 2,4-D and k i n e t i n all.appear to be required. In experiments with suspension cultures of Brassica pollen only coconut milk i s ef f e c t i v e . This i s i n contrast to Datura where k i n e t i n , p a r t i a l l y , and plum juice completely, replace the requirement ' for coconut milk. I t i s interesting that when the anther culture method: i s used with Brassica rather than the pollen suspension method the coconut milk requirement can. be ..replaced by 2,4-D plus k i n e t i n . In the successful experiments with Datura and Brassica, mature pollen was used and i n both'species the growth regulator requirement could be s a t i s f i e d with coconut milk. In addition, i n cherry experiment V ( i ) , mature pollen and. a. medium consisting e n t i r e l y of coconut milk was used and some m u l t i c e l l u l a r pollen were observed. There i s l i t t l e mention of temperature of l i g h t requirements for androgenesis i n the reports sumarized.in Table I I . S i m i l a r i l y , no experiments i n which either l i g h t i n g conditions'or temperature were varied have been reported to date; In tobacco, the developmental stage of the pollen at the time of incubation i s a very important factor i n successful anther culture (Nitsch, 1969). Immature pollen placed on the medium just before mitosis occurs has been found to be the best. In Datura,, mature anthers placed on the medium just before anthesis^were the most suitable (Guha and Maheshwari, 1967). Other reports although they mention the maturity of a.nthers at the time of incubation do not discuss experiments i n which anthers of varing maturity wereeused. Successful use of either mature or immature anthers i s possibly correlated with the number of nuclei i n the pollen at anthesisi • Species with three nuclei i n mature pollen grains require mature anthers at the time of incubation. Conversely species with two require immature pollen for successful androgenesis. This association i s true for tobacco, the F hybrid of Lolium X Festuca and Brassica but i s not true for r i c e or Datura. With so few examples to draw from i t may be•immature to suggest • 5 » associations such as those mentioned above.but i t i s worth while to be. aware of thei r possible existence. I t may be he l p f u l i n reducing the number of variables which are necessarily compounded upon,each other when determining the requirements for androgenesis; they would be he l p f u l i n determining which experiments to do in.preliminary studies and.they could be used to survey quickly, a large number of species which for one. reason or another could not be investigated i n d e t a i l . The requirements for both haploid and d i p l o i d callus i n i t i a t i o n and growth and regeneration of plantlets from c a l l i i s known f o r , Brassica and tobacco. A comparison of the haploid and.diploid requirements i s useful because many species of plants have been regenerated from d i p l o i d c a l l i and the requirements for this may be useful i n elucidating the requirements for androgenesis i n these species. I f haploid and di p l o i d callus i n i t i a t i o n occurs concomitantly a great deal of ef f o r t could be avoided i f i t were possible to select for haploid c a l l i growth . only. As w i l l be shown this may•be possible by making changes i n the growth regulator content of the medium. Diploid callus growth.was i n i t i a t e d from r i c e seedlings on Linsmaier and Skoog's medium containing 2,4-D without k i n e t i n . C a l l i appeared ten days after inoculation. After s i x t y days of incubation the c a l l i were subcultured on a medium containing a cytokinin but no auxin. Incubation was i n the l i g h t at 25°C and regeneration of plantlets occurred after t h i r t y to si x t y days (Nishi, Yamada and Takahashi, 1968). Haploid r i c e callus was.initiated on Blaydes' medium containing IAA,"2,4-D and k i n e t i n . C a l l i emerged t h i r t y to sixty.days after incubation at 28°C i n the dark: D i f f e r e n t i a t i o n of roots and shoots occurred only when the c a l l i were subcultured on a.medium containing both IAA and k i n e t i n 59. and incubation was i n the l i g h t . P l a n t l e t s were v i s i b l e s i x t y days after subculturing ( N i i z e k i and Oono}1968). These authors also observed that d i p l o i d ' c a l l u s was faster growing than haploid c a l l u s . Diploid Brassica callus was i n i t i a t e d , on Murashige and Skoog's medium containing 2,4-D plus k i n e t i n . After ten to t h i r t y days of . , incubation i n natural daylight at 20 - 24°C,,greyish yellow callus was produced; When the c a l l i were subcultured on a medium without 2,4-D roots:appeared i n three to ten days followed by leaves and shoots . ten to t h i r t y days l a t e r (Lustinec and Horvak, 1970). Haploid Brassica''callus i n i t i a t e d growth on Nitsch's medium containing :,,-th_; vsame growth, regulators as. was used with d i p l o i d Brassica. The regeneration of plants from callus had a different requirement: NAA plus k i n e t i n or 10% coconut milk. I t i s curious that i t was not possible to induce d i f f e r e n t i a t i o n i n the c e l l clusters obtained from suspension cultures,of Brassica pollen (Kameya and Hinata, 1970). Growth regulators do not have.a profound effect on the growth of haploid embryoids from tobacco pollen. Some work has been done to compare the requirements for the regeneration of plants from haploid and d i p l o i d c a l l u s . The requirements appear to d i f f e r . Benz(a)anthracene, a carcinogen, replaced the requirement for auxin plus k i n e t i n for the regeneration of tobacco plants from haploid but not di p l o i d callus (Kochhar et a l . 1970). Nitsch (1969) has doubled the chromosome number of haploid callus and i s able to regenerate plants from this callus by removing a l l growth regulators from the medium. There appear to be different requirements for d i p l o i d and haploid callus i n i t i a t i o n i n r i c e , differences i n growth regulators required for the induction of plantlets from d i p l o i d and haploid r i c e and Brassica • 60. ,; and a different response of growth regulators'of both haploid, double haploid and d i p l o i d callus of tobacco. DIFFICULTIES IN DETERMINING THE REQUIREMENTS FOR PEACH AND CHERRY Ina an investigation such as the present one'it would be expected that the experiments would be'in a: logical;.order one leading •tov-> the next. Hopefully such a progression would develop convergently to a f i n a l experiment which yielded a p r o l i f e r a t i o n of tissue originating from pollen. In the present study the experiments were considerably more divergent than convergent.- There are several factors contributing to the explaination of why one,experiment did not delineate the next. I t was impossible to grow either peach or cherry plants so that flowers were continuously available. Trees of both species are several years old when they f i r s t flower. A 1000 hour dormant c h i l l i n g period i s required before flowering proceeds, and after flowering approximately two months pass before new flowers are d i f f e r e n t i a t e d . In order to have an adequate supply of flowers the year round a large number of trees must alternate between the coldroom and the greenhouse. An alternative to using flowers from whole plants i s to store dormant pruningsand to force flowering as required. A possible disadvantage of this i s the increased frequency,of meiotic abnormalities reported by.Whelan et a l . (1968). They reported that while 2.5% of the tetrads collected from.the orchard just- after meiosis were abnormal, storage at thirteen, weeks at from -3 to -5°C followed by forcing at 15°C increased the frequency of abnormal tetrads tO 42%-. The percentage of s t e r i l e pollen varies from c u l t i v a r to c u l t i v a r (whelan et a l . 1968) but i t i s not known i f storage at 61.. 3°C would effect the frequency meiotic abnormalities i n the same way as did storage at -3 to -5°C. Although meiotic abnormalities cannot be completely discounted as a serious disadvantage, mostly s t e r i l e pollen i n the anthers of the hybrid Lol'ium multiflorum X Festuca arundinacea didinot preclude successful anther culture (Nitzsche, 1970). In this study the only cold temperature available for storage of potted cherry trees and cherry and.peach prunings was 3°C. Storage at this temperature was found to be an unexpected handicap. In both the prunings and the trees a browrincolor developed i n the stem cambium after three months -storage; indicating degeneration of the vascular system. This damage prevented forced flowers from developing f u l l y thus the use of mature pollen was not possible except when i t could be gathered from orchard trees. The flower buds on the damaged shoots ' appeared healthy. At 3°C the flower buds and anthers gradually enlarged. This also was a.handicap because the development of the anthers and pollen eliminated the p o s s i b i l i t y of using very immature pollen. After nine months i n storage the flower buds also became brown and could not be used. However i t was possible during the winter months to c o l l e c t immature, flowers from orchard trees. The stage of development of the pollen used i n an experiment therefore was largely dictated by how long the prunings had been i n storage when the experiment was begun. In most cases there was at least a month between the i n i t i a t i o n of an experiment and.the time when results became apparent. Experiments designed on the basis of previous results were thus delayed at least a month. The lack of d i f f e r e n t i a l responses among treatments also made i t -d i f f i c u l t to base the design of experiments on previous ones. These--factors a l l contributed to . the divergent rather than convergent nature of - . 62 many experiments. The r e s u l t s of experiments, were analysed by changes i n four parameters: anther color, anther enlargement,.callus i n i t i a t i o n and.growth and p o l l e n abnormalities. Apart from the occurence of m u l t i c e l l u l a r p o l l e n there i s no guarantee that these parameters t r u l y i n d i c a t e a r e l a t i o n s h i p between the medium and it-;propensity f or the production of haploid t i s s u e from p r o l i f e r a t i n g p o l l e n , Following i s a d e s c r i p t i o n of how these parameters were7used i n analysing the r e s u l t s and the j u s t i f i c a t i o n for doing so. Because pale brown.anthers which were incubated i n the l i g h t opened as i f they were dehiscing pale brown was judged to be the optimum color for anthers incubated i n the l i g h t . However, darkly colored anthers incubated i n teh dark sometimes did the same thing. The accumulation of purple pigment, presumably anthocyanins, i n some peach anthers was. considered an i n d i c a t i o n of senescence because anthocyanins tend to accumulate under conditions of s t r e s s . Because the accumulation was towards the end of the incubation period, when senescence would be expected to occur and because during the f i r s t stages of accumulation i n d i v i d u a l c e l l s became purple while others remained a normal yellow c o l o r , senescence was in d i c a t e d . Dark brown c o l o r a t i o n of'anthers was considered to r e s u l t from a t y p i c a l browning re a c t i o n c h a r a c t e r i s t i c of degenerating plant t i s s u e s . Anthers which were t h i s color as w e l l as those containing anthocyanins were considered to be senescing. S l i g h t enlargement was considered more desirable than very l i t t l e or extensive enlargement because those anthers which contained m u l t i c e l l u l a r p o l l e n and those from which c a l l u s emerged were only s l i g h t l y 63 enlarged and because there i s no mention of anther enlargement i n other reports. Any type of pollen development was considered encouraging especially i f there were a d i v e r s i t y of types observed together. Germination i n experiments i n which mature pollen' was used was not considered p a r t i c u l a r l y s i g n i f i c a n t . Pollen enlargement and hypertrophy and germination andfprotrusions from pollen occurring i n experiments i n which immature pollen was used was considered s i g n i f i c a n t . Of course m u l t i c e l l u l a r pollen provided the most positive and encouraging sign. Throughput the i n v e s t i g a t i o n ' i t was often d i f f i c u l t to decide i f anthers were dead of merely i n a quiescent state. In practise the difference between the two was academic because no anthers were observed i n which any development occurred after the anther had entered the "quiescent" state. DEVELOPMENTAL CHARACTERISTICS ASSOCIATED WITH ANDROGENESIS References made to developmental characteristics i n the.reports of successful androgenesis are incomplete. Datura anthers (Guha and Maheshwari 1967) and tobacco anthers (Nitsch and Nitsch, . 1969) open as i f dehescing before the embryoids emerge. Both r i c e ( N i i z e k i and Oono, .1968) and tobacco anthers turn black before callus or embryoids emerge. This i s i n contrast to Brassica anthers which remain yellow (Kameya and Hinata, 1970) . Guha and Maheshwari (1967) report callus growth from stem and leaf fragments on media where embryoids were produced i n lower frequencies than i n the most suitable media. Callus sometimes grew from connective t i s s e on media where ho embryoids were produced. ' 64-Four reports of successful anther culture include.descriptions of abnormal pollen development. Guha&and Maheshwari<..'•" (]967) noted a general increase i n the size of pollen which had been cultured on a suitable medium for androgenesis when compared to normal pollen or that cultured on.the basal medium without organic addenda. In addition to this there was some germination of pollen and 50 - 60% of the pollen became m u l t i c e l l u l a r . In Brassica (Kameya and Hinata; 1970) some pollen germinated in- the l i q u i d suspension cultures. Other types of pollen growth were not mentioned although undoubtedly some m u l t i c e l l u l a r pollen was present. In anther cultures of the F^ hybrid of Lolium X Festuca (Nitzsche, 1970) nintey-two anthers were examined, twenty-four m u l t i c e l l u l a r pollen grains were found as well as an unspecified number of embrysac-like pollen grains (for a description of embryosac-like pollen grains see Stowe, 1930). In experiments with ..tobacco (Sunderland, 1970) a small portion of pollen germinated. Some pollen underwent rapid enlargement bursting the excine i n the process. These giant c e l l s accumulated starch grains (the c e l l i n F i g . 9 observed i n experiment V(v) i s probably of this type). Tobacco i s the only species i n which the development of pollen into m u l t i c e l l u l a r masses has been examined i n d e t a i l (Sunderland,1970). The generative c e l l remains intact but does not d i v i d e , . i t i s the vegetative c e l l which divides; a l l the c e l l s i n the haploid tissue except the generative c e l l being derived from i t . Nitsch (1969) feels that there i s a p o s s i b i l i t y that some of the embryoids may develop from p r o l i f e r a t i o n of germ tubes (Fig. 8). Nakata and Tanaka • (1968) suggest that i n tobacco the pollen either degenerates 1 or develops;'in one of three ways, namely a) germination 65 .-b) formation of large starch f i l l e d c e l l s including hypertrophied c e l l s which i n turn.give r i s e to giant pollen grains or: c) formation of m u l t i c e l l u l a r pollen which pr o l i f e r a t e s tomfofm undifferentiated masses of c e l l s which eventually organize, to become the embryoid; In t h e i r study-the pollen was to maturity the higher was the frequency of pollen germination germination. The incompleteness of t h i s review presumably indicates either no differences between treatments with regard to anther color, anther • enlargement, callus growth from flower fragments and pollen abnormalities or that the differences were not correlated with the capacity of the medium for the production of haploid . c a l l i or embryoids. In any case i t i s not possible to be certain of the importance of these parameters i n r e l a t i o n to successful androgenesis. More'detailed descriptions of them would have been very hel p f u l i n the present work and future accounts should give more attention to these d e t a i l s than has been given up u n t i l now. DISCUSSION OF RESULTS In the tobacco experiment I ( i ) the largest percentage of anthers producing embryoids (Table I), was less than the average of 45% reported by Nitsch and Nitsch (1969). Part of this reduction was because of the lower than optimal concentration of iron which was used This could reduce the nu number of anthers producing embryoids because iron has been shown by Nitsch (1969) to be a most important mineral i n the medium. Even with this r e latively'low percentage of anthers producing embryoids i t i s s t i l l e a s i l y possible to obtain large "numbers of haploid plants. 66 I f the apparent increased percentage due' to the increased Mg concentration i s r e a l i t may be.because i t i s substituting for iron-or perhaps because when the parent plants were harvested they showed symptoms sim i l a r to Mg deficiency. I t .was not immediately obvious i f the f a i l u r e of the anthers stored i n liquid'nitrogen was because of damage to the anther or both the anther and the pollen. Because pollen can be stored i n . l i q u i d nitrogen.and remain v i a b l e , damage to the anthers seem most l i k e l y . I f so this would indicate an involvement of anther metabolism i n the development of the embryoids. Nitsch (1969)" has attempted to grow embryoids from individual.pollen grains but was unsuccessful. This may have been because of technical short commings (Nitseh, .1969) a view supported by the report of the developmentitof haploid c e l l clusters from Brassica pollen suspensions (Kameya and,Hinata^ 1970). In some experiments the v a r i a t i o n of anthers withinaa^tube was much less than between tubes of the same treatment. Because a l l the anthers i n a tube were from the same flower, v a r i a t i o n between tubes i n the same treatment may have been because of small differences i n the • stage of development of the anthers or the handling of the anthers when they were removed .from the flower bud.. I t i s possible that these small differences caused the accumulation of one or more i n h i b i t i n g substances i n some tubes or that one or more stimulating substances accumulated i n the tubes i n which anthers grew large, remained yellow : and i n the tubes i n which c a l l i grew. Similar small differences, of which flower maturity appears to be the most important, also caused d i s t i n c t differences between similar treatments i n different experiments. This best example of t h i s are 67 experiments V ( y i i ) ans V(xi) both.of which included the treatment Nitsch's medium plus IAA and coconut'milk*. In experiment V ( v i i i ) many c a l l i grew .from.inside of anthers but in"experiment V(xi) very few did. Recent reports (Kameya and Hinata; 1970, Nitzsche, 1970 and. Niizeki.and Oono, 1968) have shown that the anther culture method can • be used for the production of haploid plants i n families other than Solanaceae. In the present. investigation of the anther culture of cherry.:; and peach i t has been shown that a number of types of pollen growth including m u l t i c e l l u l a r pollen can"occur when l i q u i d media and coconut milk are used,incubation i s i n the dark and mature pollen i s used'. THE DIRECTION OF FURTHER INVESTIGATIONS I t was unfortunate that l i m i t a t i o n s on the a v a i l a b i l i t y of mature anthers r e s t r i c t e d the number of experiments i n which mature pollen could be used. Further experiments with mature pollen would appear to be the d i r e c t i o n of the most promising future investigations;; These could include the use of l i q u i d media and pollen suspension cultures as w e l l further studies with incubation at low temperatures. This would seem.to be the most promising dir e c t i o n for .further studies because the conditions i n which m u l t i c e l l u l a r pollen were found as w e l l as a greater frequency and d i v e r s i t y of pollen development, were i n l i q u i d media-used in:experiment V(i) . In experiment V(vi) a medium consisting of s o l i d i f i e d coconut milk was used and incubation was i n the dark but few abnormal pollen grains were found. I f the advantage of the l i q u i d media i s closer contact between the pollen and media; pollen suspension culture's would be a l o g i c a l next step p a r t i c u l a r l y so because pollen suspension cultures of Brassica pollen p r o l i f e r a t e to form c e l l clusters 68, (Kameya and Hinata, 1970).' Androgenesis i n Brassica occurs at a temperature of 20°C. Another suggestion, that incubation at low temperature should be investigated more thoroughly comes from the somewhat analogus problem of growing, rust fungi, a x e n i c a l l y from germinating urediospores. The optimum growth'-of tissues derived from the urediospores i s when i s o l a t e d sporessare cultured on l i q u i d media at 17°C i n the dark (A.Bose personal communication); SUMMARY 1. The recently reported procedure'for the production of haploid plants from tobacco p o l l e n cultured i n v i t r o was v e r i f i e d . I t was found that anthers which had been stored i n l i q u i d nitrogen loss t h e i r , androgenic capacity. 2. At l e a s t f o r the stage of development of the pol l e n which was used, therrequirements f o r androgenesis of apple, cranberry, rose, tcherry and peach are not the same as those for tobacco. 3. Culturing cherry p o l l e n i n v i t r o can r e s u l t i n high frequencies and a number of types of abnormal p o l l e n growth which have been shown to be associated with androgenesis i n tobacco and other species,, in c l u d i n g m u l t i c e l l u l a r p o l l e n . 4. Cherry c a l l i , probably o r i g i n a t i n g from the connective ti s s u e of the anther, grow when immature anthers are incubated on Nitsch's medium plus NAA and coconut milk and are incubated i n the dark at 31°C. 5. Mature cherry anthers cultured on a l i q u i d medium consisting e n t i r e l y of coconut milk and incubated at 31°C i n the dark were found to contain some m u l t i c e l l u l a r p o l l e n . Further in v e s t i g a t i o n s with mature p o l l e n and l i q u i d media are suggested. 6. A comparison i s made ofmthe requirements f o r the induction and regeneration of p l a n t l e t s from haploid versus d i p l o i d c a l l u s . In the species f o r which the requirements are know there appear to be di f f e r e n c e s . 7. A comparison of the requirements for androgenesis i n a l l the species f o r which they are known pointed out associations between several of the components. 70 LITERATURE CITED • Blakeslee, A., J.Belling, M.E.Farnham, A.D.Berger. 1922. A haploid mutant in Datura stramonium. Science 55:646-647. Blaydes; D.F. 1966. Interaction of kinetin and various inhibitors in the growth of soybean.tissue. Physiol. Plant. 19: 748-753. Bonga, J.Mv. and';D.P;Fowler. 1970. Growth and differentiation in gametophytes of Pinus resinosa cultured in vitr o . Can. J. Bot. 48: 2205-2207. Bourgin, J.P. and J.P.Nitsch. 1967. Obention de Nicotiana haploides.a partir d' examines cultivees in vitro j- Annls. Physiol Veg. 9: 337-382. Brown CL. and R.H.Lawrence. 1968. Culture of pine callus on a,defined medium. Forest Sci. 14: 62-64. Carlson P.S. 1970. Induction and isolation of auxotrophic mutants in somatic c e l l cultures of Nicotiana tabacum. Science. 168: 487-489. Dodds K.S. 1955. Hybrid vigor in plant breeding. Roy. Soc. Lond. Proc. 144: 185-192. Gamborg, O.L. and D.E.Eveleigh. 1968. Culture methods and detection of glucanases in suspension cultures of whear and barley. Can. J. Biochem. 46: 417-423. Guha, S. and S.C.Maheshwari. 1964. In.vitro production of embryos from anthers of Datura. Nature. 204: 497. Guha, S. and S.C.Maheshwari, 1966. Cell division and differentiation of embryos i n the pollen grains of Datura. Nature. 212: 97-98. Guha, S; and S.C.Maheshwari. 1967. Development of embryoids from pollen grains of Datura in vitr o . Panchanan Maheshwari Mem.Vol. Phytomorphology. 17: 454-461. Hildebrant, A.D. 1962. Tissue and single c e l l cultures of higher plants as a basic experimental method, pp. 383-421. In: Modern methods of plant analysis; H.F.Linskens and M.V.Tracey eds., Springer-Verlag, Berlin. Kameya, T. and K.Hinata. 1970. Induction of haploid plants from pollen grains of Brassica. Japan. J. Breeding 20: 82-87. Kasha,.K.J. and K.N.Kao. 1970. High frequency of haploid production in Hordeum vulgare L. Nature 225: 874-875. Katayama, Y. and M.Tanaka. 1969. Studies on haploidy in relation to plant breeding V. Further proposal of haploid method of plant breeding. Seiken Zih© 21: 37-44: 71 -. Kihara, G. and K.Tsunewaki. 1962. The use of a l i e n cytoplasm as a new method of producing haploids. Japan. J . Genet. 37: 310-313. Kimber, G. and R.Riley. 1963. Haploid angiosperms. Bot. Rryiew 38: 310-313. Kochhar, T.S., P.R.Bhalla and P.S;Sabharwal. 1970. In .vitro induction of vegetative buds by Benz(a)anthracene i n tobacco c a l l u s . Planta-94: 246-249. Konar, R.N. 1963a. A haploid tissue from the pollen of Ephedra f o l i a t a Boiss. Naturwissenschaften 54: 203. Konar, R.N. 1963b. Studies on submerged callus culture of Pinus  gerardiana Wall; Phytomorphology 13: 165=169. LaRue, C.D. 1948. Regeneration in.the megagametophyte of Zamia floridana. B u l l . Torrey Bot. Club 75: 597-603. Linsmairer, E.M. and K.Skoog. 1965. Organic growth factor requirements of tobacco tissue culture. Physiol. Plantarum 18: 100-127. Lustinec, J . and J.Horvak. 1970. Reduced regeneration of plants-in tissue cultures of Brassica oleracea. Experientia 26: 919-920; Melchers,G. and G.Labib. 1970. Die 1Bedeutung haploider hoherer Pflanzen fur Pflanzenphysiologie und Pflanzehzuchtung. Ber. Dtsch. Bot. Ges. 83: 129-150. Murashige, T. and.F;Skoog. 1962. A revised medium for rapid growth and bio-asseys with tobacco tissue cultures. Physiologia Plantarum 15: 473-497. t Nakata; K. and M.Tanaka. 1968. D i f f e r e n t i a t i o n of embryoids from developing germ c e l l s i n anther culture of tobacco; Japan. J . Genetics 43: 65-71. Nei,.M. 1963. The ef f i c i e n c y of the haploid method of plant breeding. Heredity 18: 95-100. • ** Nemec, B. 1898. Uber den pollen der. petaloid antheren von Hyacinthus  o r i e n t a l i s L. Rozpravy Cesks Akad. Prag I I . 7(17). N i i z e k i , H. and K.Oono. 1968. Induction of haploid r i c e plant from anther culture. Proc. Japan. Acad. 44: 554-557. N i s h i , T., Y.Yamada and E.Takahashi. 1968. Organ r e d i f f e r n t i a t i o n and plant restoration i n r i c e c a l l u s . Nature 219: 508-509.' Nitsch, J.P. and C.Nitsch. 1969. Haploid plants from pollen grains. Science 163: 85-87. Nitsch,.J.P. 1969. Experimental androgenesis i n Nicotiana. Phytomorphology 19: 389-404. 72 . Nitzsche, W. 1970. Herstellung haploider Pflanzen aus Festuca-Lolium Bastarden. Naturwissenchaften 57:• 199-200. Magoon, M.L. and K.R.Khanna. 1963. Haploids. Carologia 16: 191-235 S t a r i t s k i , G. 1970. Embryoid formation i n callus cultures of coffee. Acta. Bot. Neerl. 19: 509-514. Stowe, E. 1930. Experimental studies on the formation of embryosac-like giant pollen grains of Hyacinthus b r i e n t a l i s . Cytologia 5: 88-108. Sunderland, N: 1970. Pollen plants and there significance. New Sc i e n t i s t 47 no. 710, pp. 142-144. > Tanaka, M. and K.Nakata. 1969. Tobacco plant obtained from anther culture and the experiments to get.diploid seed from the haploids.' Japan. J . Genetics 44: 47-54. Tulecke, W. 1953. Atissue derived from the pollen of Ginkgo biloba. Science 117: 599-600 Tulecke, W. 1959. The pollen cultures of LaRue: a tissue from the pollen of Taxus. B u l l . Torrey Bot. Club 86: 283-289. Tulecke, W. 1960. Arginine-requiring s t a i n of tissue obtained from Ginkgo pollen. Plant Physiology 35: 19-24. Tulecke, W, and N.Sehgal. 1963. C e l l p r o l i f e r a t i o n from the pollen -v, of Torreya nucifera. Contrib. Boyce Thompson Inst. 22: 153-163. Urgent, D. 1970. The potato. Science 170: 1161-1166. Whellan, E.D.R;, C.A.Hornby and G.W.-Eaton. 1968. Meiosis i n Prunus avium L. I I . The envoirnmental effects of bud forcing and storage on meiosis i n cv. Lambert. Can. J . Genet. Gy.tiol. 10: 819-824. White, P.R. 1943. A handbook of plant tissue culture. The Ronald Press Co. New York. Wittmann, W. 1965. Aceto-iron-haemetoxylin-chloral hydrate for chromosomes staining. Stain Tech. 10: 161-163. Wolter, K.E. and F.Skoog. 1966.'Nutritional requirements of Fraxinus callus cuttures. Amer. J. Bot. 53: 263- 270. 73 APPENDIX•I NITSCH'"S MEDIUM . (Nitsdh 196?) Ingredient mg/1; KN03 950 NH3NO, 720 4 3. MgS04 185 CaCl 2 166 KH2P04 68 MnS0..4H.0 25 4 2 H 3B0 3 10 ZnSO..7H„0 10 4 2 Na.MoO..2Ho0 0.25 2 4 2 CuS0..5H„0 0.025 4 2 Myo-inositol 100 Nicotinic acid 5 Glycine 2 . . Pyridoxin HC1 0.5 Thiamin HC1 0.5 Folic acid 0.5 Biotin • .0/05 -Sucrose 20,000. ' - • . Irons 5 ml of stock solution pH 5.5 § Stock solution made by dissolving in'.one'liter of water 5.57g of .FeSP4.7H-20 and 7.45g of Na EDTA. 74 APPENDIX I I PRL-4 MEDIUM (Gamborg and Eveleigh, 1968) Ingredient mg/1 NaELPO. .Ho0 90 2 4 2 NaJHPO. 30 2 4 • KC1 300 (NH.)oS0. 200 4 2 4 MgS04. 7H20" 250 KN03 1000 CaCl 2.2H 20 150 K l 0.75 I r o n 1 28 2 Micronutrients 1.0 ml 3 Vitamins 10.0 ml Sucrose , • 20.0 gm -N-Z Amine type A 2.0 mg/1 2,4-D 2.0 mg/1 pH (f i n a l ) 6.2 1 Sequestrene 330 Fe 2, Stock solution dissolved i n 100 ml water: i gm MnSO^.H^, 300 mg H 3_0 3, 300 mg ZnS04.7H20, 25 mg Na2Mo04.H20, 25 mg CuS04, 25 mg CoC1.6H20. 3 Stock solution dissolved i n 100 ml water: 10 mg n i c o t i n i c acid, 100 mg thiamin, 10 mg pyridoxin, 1 gm myo-inositol. 75 APPENDIX I I I M.S. MEDIUM ( Murashige and Skoog, 1962) Ingredient mg/1 NH.N0o 1650 4 3 KN03 1900 H 3B0 3 6,2 KELPO, 170 2 4 -KI 0.83 Na_MoO;.H.O 0.25 2 4 2 CoCl 2.6H 20 0.025 CaC1.2H20 440 MgS04.7H20 370 MnS0..4H„0 22.3 4 2 ZnS0..7H.O 8.-6 4 2 CuS0..5H-0 0.025 4 2 Na 2 EDTA 37.35 FeS0..7H.O 27.85 4 ' 2 Thiamin HC1 0.1 Ni c o t i n i c acid 0.5 Pyridoxin HC1 0.5 Glycine 2.0 Sucrose 30 gm/1 Myo-inositol 100 mg/1 Indole-3-acetic acid 10 mg/1 Kinetin 0.04 mg/1 Agar 10 gm/1 pH (fi n a l ) 5.7 - 5.9 APPENDIX IV WOLTER'S MEDIUM (Wolter and Skoog, 1966) Ingredient mg/1 MgS0 4 764 Na.SO. •= 425 ' 2 4 C a ( N 0 3 ) 2 425 KN0 3 170 KC1 140 NaH oP0. 35 2 4 MnSO. 9.0 4 ZnSO, 3.2 76 H 3B0 3 3.2 K l 1.6 FeNa 2 EDTA 5.5 NH.NO„ 50 4 3 M y o - i n o s i t o l 10 P y r i d o x i n HC1 0.1 2,4-D 0.04 K i n e t i n ; T .1.0 Sucrose 20,000 Agar 10,000 pH ( f i n a l ) 5.6 - 5.8 

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