llllll l Ill .llllllllllllllll “425m 3 12901009 2926 This is to certify that the thesis entitled MORPHOLOGICAL AND CYTOLOGICAL ANALYSIS OF AN INTERSPECIFIC HYBRID EGGPLANT, SOLANUM MELONGENA L. X SOLANUM TORVUM SW. presented by Kenneth R. McCammon has been accepted towards fulfillment of the requirements for Master of Science degree in Horticulture Ami/Lac ' 4’" ”Wk—- Major professor Date2>u7 3/ //’[ l/ 0-7639 MS U is an Affirmative Action/Equal Opportunity Institution LES-mi??? Mm an atate Universitty ovaaouc mes: 25¢ per day per item .1 I (.‘i‘i‘fi _‘ RETURNING LIBRARY MATERIALS: J; * Place in book return to remove “3‘9"”, 1 charge from circulation records MORPHOLOGICAL AND CYTOGENETIC ANALYSIS OF AN INTERSPECIFIC HYBRID EGGPLANT, SOLANUM MELONGENA L. X SOLANUM TORVUM SW. by Kenneth R. McCammon A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Horticulture 1982 ABSTRACT MORPHOLOGICAL AND CYTOGENETIC ANALYSIS OF AN INTERSPECIFIC HYBRID EGGPLANT, SOLANUM MELONGENA L. X SOLANUM TORVUM SW. by Kenneth R. McCammon An interspecific hybrid between S. melongena L. cv. Mil- lionaire and s. torvum Sw. was produced by means of unilater- al sexual hybridization. S. torvum was used as the pollen parent. The cross was made in an effort to transfer resis- tance to Verticillium wilt (Verticillium dahliae) from S. torvum into the cultivated species. .The progenies were de- termined to be hybrids based on morphological observations. Attempts to self and backcross the hybrid to the parents were unsuccessful. Observations of the pollen from the F1 plants indicated low viability. Meiosis in the parents appeared normal. Cytological observations of hybrid PMCs showed gross abnormalities in all stages of meiosis. It appears that the sterility may be due to lack of homology and genic imbalances of the parental genomes. This results in abnormal pairing at metaphase I and altered chromosome distributions at anaphase I and subsequent stages, producing pollen with an abnormal complement of chromosomes. This thesis is dedicated to my grandmother, Mrs. B. O. LeBlanc, and to my parents, Mr. Frederick V. McCammon, Sr., and the late Joyce L. McCammon. ii ACKNOWLEDGEMENTS The author wishes to express his appreciation to Dr. Shigemi Honma for his encouragement in this research and helpful assistance in the preparation of the manuscript. Sincere graditude is also expressed to Dr. William Tai for his advice and guidance throughout the study, and for the use of his laboratory facilities, and to Dr. Hugh Price and Dr. Ardeshir Ghaderi for their comments and criticisms in reviewing this manuscript. Further appreciation is ex- tended to Dr. Ghaderi for filling in for Dr. Wayne Adams in his absence. Additional thanks is extended to Dr. Gary Bauchan for his advice and suggestions during the course of the study, for his aid in interpretation cf cytological observations, and for detailing the photographic techniques necessary for completion of this thesis. Final appreciation is expressed towards my wife, Marlise, and daughter, Mollie, for the sacrifices they made so that completion of this thesis could be realized. iii DEDICATION . ACKNOWLEDGEMENT. LIST OF TABLES . LIST OF FIGURES. INTRODUCTION LITERATURE REVIEW. TABLE OF MATERIALS AND METHODS. RESULTS. . . DISCUSSION . BIBLIOGRAPHY iv CONTENTS Page ii iii vi 12 28 32 Table Table Table Table Table Table Table Table Table LIST OF TABLES Comparison of certain morphological charac- teristics of the parents and F1 hybrid . . . Chromosome associations at diakinesis in S. melongena X‘S. torvum. . . . . . . . . . . . Chromosome associations at metaphase I in S. melongena X S. torvum. . . . . . . . . . . . Frequency of laggards and dipolar distribu- tions at anaphase I in S. melongena X S. tOMoeeoooeoeoooooeeooe Frequency of multipolar chromosome distribu- tions at anaphase I in S. melongena X S. tom 0 O O O O O O O O O O O O O O O O O 0 Frequency of chromosome distribution at metaphase II in S. melongena X S. torvum . . Frequency of quadripolar chromosome distribu- tion at anaphase II in S. melongena X‘S. t orvum O O O O O O O O O O O O O O C O O O 0 Frequency of multipolar chromosome distribu- tion at anaphase II in S. melongena X S. t 0mm 0 O O O O O O O O O O O O O O O O O O Microspore formation at the "tetrad" stage in S. melongena X S. torvum . . . . . . . . . . Page l3 18 21 22 23 23 25 26 27 Figure 1. Figure 2. Figure 3. LIST OF FIGURES Page Morphology of leaves and inflorescences of S. melongena, S. torvum and their Fl hybrideeooeoeoooooooooeoo15 Meiosis in S. melongena and S. torvum . . . l7 Meiosis of S. melongena X S. torvum hybrid. 20 vi INTRODUCTION Commercial production of eggplant (Solanum melongena L.) in Michigan is limited by a high incidence of 2233;; cillium wilt. This soil borne disease is caused by the fungus Verticillium dahliae Klebahn, (referred to as E. SSSgragggg,Reinke and Berth. in earlier literature), and results in severe wilting, stunted growth, vascular dis- coloration, reduced yields, and premature death. This pro- blem is compounded by the lack of genetic resistance within S. melon ena, and the absence of chemical or physical meth- ods for the control of Verticillium wilt. In an effort to solve this problem, Solanum torvum Sw., a wild species of eggplant, was crossed with S. melongena L., in order to transfer its resistance to 1. dahliae into commercial eggplant. An interspecific hybrid between the two species has been produced that is sterile. The purpose of this paper is two-fold: (l) to report on the morphologi- cal characteristics of the hybrid, and (2) to present a probable explanation of the sterility cytologically. LITERATURE REVIEW Burton and deZeeuw (1958) conducted a series of stu- dies to determine the importance of seed transmission of X. albo-atrum as a causative agent in Verticillium infection and determined that the disease was not seed transmitted. Wilhelm (1955) found the pathogen had a wide range of hosts, enabling it to survive in soil for long periods of time. Due to the lack of efficient means of soil treatment to eliminate the pathogen, Lockwood and Markarian (1961) at- tempted to breed cultivars of eggplant resistant to Verti- cillium, utilizing germplasm sources within S. melongena L., with limited success. According to Bhaduri (1951), the genus Solanum contains approximately 2000 species, the majority of which are of the non-tuberiferous type. They are mostly herbs and shrubs, though some may attain the size of small trees. Within the non-tuberiferous types are two types, with and without spines on the leaves and stems. Yamakawa, gt a} (1978) screened a number of species related to S. melongena for resistance to Verticillium and other diseases which attack eggplant. They found Solanum torvum Sw. to be resistant not only to Verticillium wilt, but Fusarium and Pseudomonas wilts as well. Both of these Solanum Species are spined. Rai (1959) reported that S. melongena L. originated in either India or tropical Africa, and is cultivated through- out the warmer regions of the globe. According to Rao (1972) and Deb (1979). S. torvum Sw. is indigenous to India and southeast Asia, but extends through Malaysia and the Phillipines, to Australia, Africa and tropical America. The general morphological features of S. melongena L. have been elucidated by Khan (1979), while S. torvum Sw. was described morphologically by Hossain (1972). A review of cytogenetic studies in the genus Solanum was conducted by Magoon, g£_§l (1961), in which they reported a chromosome number of n-12 for both species. Two previous attempts to hybridize these two species appear in the literature. The first, reported by Pearce (1975). involved the crossability of S. melongena L. with related species. He was able to produce healthy F1 progeny from a cross between S. melongena and S. torvum, however, they were sterile and exhibited few stainable pollen. Yamakawa, 33 g; (1978) also produced a hybrid between these species, but neither F2 nor backcross progenies were obtained. To the author's knowledge, the morphological characteristics of the hybrid or the cytologi- cal bases for their sterility have not been reported. Efforts to hybridize S. melongena and other related species appear in the literature. Tatebe (1936) made a cross between S. integrifgligm Poir. and S. melongena L., in which a hybrid was only produced when S. mglgnggna was used 4 as the pollen parent. The hybrid showed a closer resem- blance to S. integrifolium than to S. melongena, and was sterile. Although cytological observations suggested that meiosis was normal through the tetrad stage, shortly after liberation of the microspores from the tetrad, they were ob- served to disintegrate, resulting in inviable pollen pro- duction. Khan, 33 g; (1978) produced a semi-fertile hybrid with S. inte ifolium, using S. melongena L. var. Pusa Purple Long as the female parent. Meiosis appeared normal, though various meiotic abnormalities were noted in about 15% of the pollen mother cells (PMCs). They concluded that the two genomes had been differentiated by cryptic structural changes of the chromosomes. In a study involving the hybridization of S. melongena L. and S. cumingii Dunal, Campinpin, gg_§;_(l963) were able to produce a fertile hybrid. Cytological observations in- dicated that there was considerable similarity between the two parental genomes. In the F1, pairing was found to be normal, and the percentage of viable pollen was approximate- ly the same as that of the parents. In an effort to incorporate resistance to Verticillium wilt into S. melon ena, Nasrallah and Hcpp (1963) made crosses with S. giig and S. indicum. Reciprocal Fl's were produced, and all F1 plants were highly sterile. The hybrid S. melgngena,x S. gilg could be backcrossed to S. gilg. but not to S. melgngena. In this hybrid, pairing appeared to be 5 normal at metaphase I, however, irregular chromosomal dis- tributions as well as the formation of bridges were noted in anaphase I. Rajasekaran (1970) conducted cytogenetic analyses of the F1 hybrid S. indicum X S. melongena L. and its amphi- diploid. The cross was only successful when S. indicum was used as the pistillate parent. The hybrid exhibited normal meiosis, although it was semi-sterile, with 49% stainable pollen. They were able to produce a fertile amphidiploid and concluded that the sterility was due to small segmental differences existing in the chromosomes of the parental ge- nomes. Cytological studies were also conducted by Rajasekaran (1971), on the F1 hybrid S. xanthocggpum Schrad. and Wendl. X S. melongena L. and its colchicine derived amphidiploid. The hybrid was produced only by using S. xanthocarpum as the female parent. The resulting F1 was sterile, despite normal meiosis. Fertility was restored in the amphidiploid, sug- gesting cryptic structural hybridity. A similar study by Rajasekaran (1971) utilized S. 22? longena var. insanum Prain. A hybrid was produced only when S. xanthocarpum was used as the female parent. The result- ing hybrid was sterile, and efforts at selfing and back- crossing to either parent were unsuccessful. The most com- mon chromosomal association noted at metaphase I involved the formation of 10 bivalents and l quadrivalent. Only about one-third of the cells observed exhibited normal 6 pairing of 12 bivalents at metaphase I. Subsequent stages appeared normal. The pairing in the hybrid indicated a close affinity between the two parental genomes, and the differences appeared to be due to segmental interchange, as indicated by the formation of a quadrivalent at metaphase I, followed by normal anaphase I segregation. The breeding behavior of S. zuccagnianum Dun. with S. melongena L. was reported by Rajasekaran and Sivasubramanian (1971) using S. zuccagnianum as the female parent. As in previous studies, the reciprocal cross was unsuccessful. The hybrid was sterile, and backcrosses to the parents were unsuccessful. Examination of the pollen indicated sterility. Metaphase I associations of 10 bivalents and 1 quadrivalent were most commonly observed, with a small percentage of cells showing 12 bivalents. Subsequent stages generally appeared normal. The parental genomes appeared to possess some homo- logy, and the sterility was explained on the basis of seg- mental interchanges and small cryptic differences of the chromosomes. Solanum macrocarpon L. was used as the pollen parent and was crossed with two varieties of S. melongena L. in- digenous to India, by Wanjari (1975). While most PMCs show- ed regular bivalent formation at metaphase I, some univalents and multivalent formations were noted, with corresponding irregularities in subsequent stages. Sterility was postu— lated as due to cryptic structural differences between the genomes. 7 Schaff, SE S; (1980) also utilized S. macrocarpon in interspecific hybridizations with eleven genotypes of S. melongena. When S. melongena was used as the female parent, five of the eleven combinations of crosses set fruit. Using S. macrocarpon as the female, nine of the eleven combina- tions were successful. The fertility of the F1 progenies was variable, ranging from complete absence of flowers to the production of fruits with few seeds. Rao, g£_§; (1979) conducted a study to determine the inter-relationship between S. melongena L. and S. hispidum Pers. They utilized an Indian cultivar of S. melongena, var. Pusa Purple Long, as the female parent. The recipro- cal cross was unsuccessful. The resulting hybrid was high- ly sterile, with less than 2% pollen fertility. Hybrid meiosis was irregular. Few bivalents and large numbers of univalents were noted. The chromosomes appeared to be very lossely paired in the bivalents. Anaphase I exhibited er- ratic behavior of the univalents with varying numbers of laggards. Delayed separation of the bivalents, as well as bridges and fragments, were also noted in anaphase I. Micronuclei were observed in both telophase I and II, with most sporads containing multiple clumped and degenerating pollen grains. Based on the meiotic abnormalities observed, the pa- rental genomes were considered dissimilar. The high fre- quency of univalents, coupled with loose associations of the bivalents, suggest that the differences in the parental 8 genomes were due to structural changes within the chromo- somes which may have occured during the evolution and dif- ferentiation of the Species. Rao (1980) obtained similar results from the hybridiz- ation of S. melongena L. var. Baromishi X S. hispidum Pers. The resulting hybrid was sterile with 1.4% pollen viability. Hybrid meiosis was irregular, and anomalies were observed at every stage. He concluded that structural changes of the chromosomes, coupled with disharmonious interaction of the parental genes were responsible for the sterility of the hy- brid. Attavian, g; g; (1980) reported the crossability of several Solanum species and classified the crosses into three groups: A, species reciprocally crossable (S. 3;;2, Raddi X S. integrifolium Poir.; S. nodiflorum Jacq. X S. integrifolium), capable of producing fully fertile Fl plants, B, species unilaterally crossable (S. indicum L. X S. incanum L.; S. 5;;9_X S. nodiflorum), and C, species reciprocally noncrossable (S. gggg X S. indicum). Several varieties of S. melongena L. were used to investigate the possibility of gene transfer between species as a method of varietal im- provement, and they found that many species of Solanum are crossable inter EE- MATERIALS AND METHODS The parents utilized in the hybridization were S. melongena L. cv. Millionaire, (Takii Seed Company of Kyoto, Japan), and a selection of S. torvum Sw., supplied by Pro- fessor K. Yamakawa of the Vegetable and Ornamental Crops Research Station, Tsu-City, Japan. Morphological Siggy S. melongena L. is an erect, branched shrub, about one meter tall, with spines confined to the flower pedicel and calyx. The leaves are large, oblong-ovate, shallowly sin- uate-lobed, nearly glabrous above but densely tomentose be- neath (Khan, 1979). In S. meldngena L. cv. Millionaire, the flowers are large, solitary, or occasionally, in clusters of 2 to 3. The calyx is spiny, purple, deeply lobed and per- sistent. The corolla is about 3.7 cm in diameter. The fruit is a large elongate berry, an average of 20 cm long and 4.5 cm in diameter, dark purple in color. Hossain (1973) reports that S. torvum Sw. is usually 2 to 3 meters tall, a much branched and moderately spined shrub, with simply lobed to lobate-sinuate leaves. Its in- florescences represent a form of a cyme. The calyx is deep- ly five-lobed, white and deciduous. The fruit is a smooth, Spherical berry, 10 to 13 mm in diameter, green when young, yellowish green to yellow when ripe. 9 10 Plants of each species were grown in the greenhouse. After anthesis of the initial flowers, bud pollinations were made reciprocally by emasculating the female parents, fol- lowed by hand pollination. Repeated pollinations were made for each cross, and were successful only when S._melongena was used as the female parent. The fruit, upon harvest, yielded very few seeds. The seeds, when grown, produced both selfed and hybrid seedlings. The Fls were identified from the selfs by several morphological characters. The following hybrid and parent— al information were recorded: flower size and color, fruit Size, shape and color, leaf width, length and margin type, amount and location of spines, number of flowers per in- florescence, and the presence of stem anthocyanin. Data on flower size, fruit size, leaf width, leaf length, and flo- wers per inflorescence were obtained by averaging 10 mea- surements per character. All other data were based on visual observations. Cytological S33Qy_ In order to determine the proper stage for the col- lection of flower buds for cytological analysis, samples were collected from greenhouse grown plants of both parents and the hybrid, at two hour intervals, beginning at 9 AM and ending at 5 PM. Cytological observations indicated that buds collected between 9 and 11 AM and 3 and 5 PM, were most suited for analysis. However, buds collected at 10 PM yielded better cells for observing metaphase I chromosomes 11 for the hybrid. Buds were collected and fixed in a 1:3 acetic acid- absolute alcohol solution. After 24-48 hours, they were transferred to 70% alcohol and stored in a refrigerator un- til used. In preparation for analysis, buds were hydrolyzed in 1N H01 for 6 minutes. After hydrolysis, buds between 8 and 10 mm long, with anthers 4 to 5 mm long, were selected and the anthers removed and smeared in 1% acetocarmine. All observations were made on pollen mother cells. Photomicro- graphs were made to record the meiotic process for both parents. For the hybrid, data on meiotic abnormalities were re- corded at diakinesis, metaphase I and II, anaphase I and II, and the tetrad stage. Photomicrographs were made to record the anomalies at the various meiotic stages. The presence of micronuclei was determined by observing the tetrad stage. Pollen viability estimates were based upon the percent of pollen stainable with potassium iodide (IZKI). 1000 pollen grains were counted for the parents and the hybrid and those stained darkly were considered viable. For the study of hybrid mitosis, root tips were col- lected from actively growing roots and pretreated in 0.02% orthodichlorobenzene (ODB) for 45 minutes. After pretreat- ment they were fixed in a 1:3 acetic acid-absolute alcohol solution for 24 hours, hydrolyzed in IN HCl for 10 minutes, and smeared in 1% acetocarmine. RESULTS ' Morphological observations of the parents and the F1 hybrid are summarized in Table 1. Leaf length, leaf width and number of flowers per inflorescence were greater for S. torvum than for S. melongena, while flower diameter and fruit size were larger for S. melongena than for S. torvum. Due to sterility, the fruiting characteristics of the hy- brid are not available. The hybrid plant more closely resembled S. torvum in flower diameter, Spininess, leaf margin, inflorescence type and flowers per inflorescence. The lavender colored flowers resembled those of S. melongena. The leaves of the F1 ex- hibited the presence of anthocyanin as in S. melongena, but were spined and deeply lobed as in S. torvum (Figure 1). All other traits were intermediate between the parents. gytological Sgggy The parents and the F1 had 2n=24 chromosomes. No mi- totic irregularities were noted in the hybrid. Meiosis in the parents appeared normal, with 12 bivalents at metaphase I, followed by normal meiosis in subsequent stages (Figure 2). The pollen appeared well developed and exhibited a viability of 87.5% for S. melongena, and 96.9% for S. torvum. The size of the hybrid pollen varied greatly, viability l2 13 pcomohm we. H m.HN memo moonsmhoo moaoapma cam mo>moH .mempm peomnm mm. « m.we memo omenESHoo moaoapmm ocm mo>moH .mEmpm Summons OH. + H.H oHQEHm HmOHoma cam xmfimo cflcmhoonpc< monoomonoamcH\mHm3oam mama oodmomohoamcH soapmooq onamm I coonm magnum xnmn Moaoo waspm I 0H. « o.H am. « m.v npoag I HH. « N.H me. « o.oN spmcmg AEOV muflm padpm I pmfisnoaw mamchHo mamnm vague Hooco>mfi open; Hocco>mH poaoo nozoam ma. « a.m SH. « H.N ma. « >.m Agov empmsmflm umzoam nonoa manomc cocoa mamoon nonoanmpmscfim cflmnmz mama ow. H >.ma me. H ¢.ON mm. a m.oH cpofi3 we. « m.mH me. « m.om as. A e.eH cameos AEoV mufim Hmmg camp»: Hm .ammmmm .w snowmoaos .m QHSmfihopommmso .eapgh: as new mummpmm osp mo moflpmfipmpomhmso HousmOHonmnoE campmmo mo somHHdeoo .H mqm\J~ll\)\.nl\)1-‘NI‘—’I \O \N o Q Total 133 23 TABLE 5. Frequency of multipolar chromosome distri- butions at anaphase I in S. meloggena X S. torvum. Cells Observed Distribution No. % Tripolar 14-6-4 2 1.4 14-5-5 l 007 12-6-6 1 007 10-9-5 2 1.4 10-7-7 2 1.4 andripolar 1.1-6-6-1 l 007 7-7-6-4 l 0-7 Total 10 7.0 TABLE 6. Frequency of chromosome distribution at meta- phase II in S. melongena X S. torvum. Number of bivalents on Cells Observed metaphase plates No. % 12-12 3 20.0 13-11 4 26.7 14-10 4 26.7 15" 9 2 1303 12-10-2 2 13.3 'Total 15 100.0 24 (Figure 3F). The frequency of chromosome distribution observed at the second metaphase was determined from observing 15 PMCS (Table 6). Unequal distribution of chromosomes at metaphase II was probably due to the unequal distributions noted in anaphase I. The most frequent distribution types were 13-11 and 14-10, each occuring in 26.7% of the cells, followed by a normal 12-12 distribution in 20.0% of the cells. Quadripolar and multipolar distributions of the chromo- somes were noted when 57 PMCS were observed at anaphase II. Data on the frequency of quadripolar chromosome distribution at anaphase II are presented in Table 7. Segregation of 12 chromosomes to each of the poles was observed in 7.0% of the cells. Seven percent of the cells also showed distributions of 14-14-10-10 and 15-15-9-9, while 5.3% showed distribu- tions of 13-13-11-11 and 14-12-11-11. Multipolar distri- butions were observed in 26.25% of the cells (Table 8, Fig- ure 3G). Microspore formation at the tetrad stage is presented in Table 9. Examination of 69 sporads showed that the number of microspores formed from each microsporocyte was irregular, with 4.3% forming single cells, 18.8% dyads, and 18.8% triads (Figure 3H). Only 15.9% of the cells appeared to have normal tetrad formation. The remaining sporads contained 5 or 6 microspores (Figure BI). Micronuclei formation varied from 1 to 4, with 37.7% of the cells producing l micronuclei and 18.9% producing two. A maximum of 4 micronuclei per cell 25 TABLE 7. Frequency of quadripolar chromosome distri- bution at anaphase II in S. melongena X S. torvum. Cells Observed Distribution No. % 12-12-12-12 20-20- 4- 20-16- 9- 18-12-12- 17-17- 7- 17-12- 8- 16-16- 8- 16-15- 9- 16-14-11- 16-14- 9- 16-13-10- 16-12-12-10 15-15- 9- 9 15-12-11-10 14-14-10-10 14-13-12- 9 14-13-11-10 14-12-12-10 14-12-11-11 13-13-12-10 13-13-11-11 13-12-12-11 \OKDQmmxlxlmW-A NwHWNHH#H#HNNHNNHHHHH# U'IU'lU'IU'IU'I UH»-JVMfi~J\MD~JcrdanVQKHvPQ~J\%Q~JC) 01 coco-oeoooooo UTU'IKHW U'l memeHflHflwawaHHHHHQ 73.75 26 TABLE 8. Frequency of multipolar chromosome distri- butions at anaphase II in S. melongena X S. torvum. Cells Observed Distribution No. % Pentapolar 15-13- 9-7-2 1 1.75 14-11-10-8-5 1 1.75 14-11- 9-9-5 1 1.75 13-12-11-8-4 1 1.75 13-12- 8-8-7 1 1075 11-10-10-9-8 1 1.75 Hexapolar 19- 17- 4- 4- 2- 2 1 1.75 14- 12-8- 8- 3- 3 1 1.75 13- 13-7-6- 5- 4 1 1.75 13-11-9- 5- 5- 5 l 1.75 12-12-8-7 -5- 4 1 1.75 10-10-9- 8- 6- 5 1 1.75 9- 9- 9- 9- 6- 6 1 1.75 Heptipolar 11-11-8-5-5-5-3 1 1.75 Total 15 26.25 27 were observed in 2.9% of the cells. TABLE 9. Microspore formation at the "tetrad" stage in S. melongena X S. torvum. Cell Size Cells Observed Cells/"Tetrad" Large Small No. % l l " 3 403 2 2 - 13 18.8 3 1 2 2 2.9 3 2 1 11 15.9 4 2 2 4 5.8 4 3 1 1 1.5 4 4 - 11 1509 5 5 - l 105 5 3 2 2 2.9 5 4 1 14 20.3 6 2 4 2 2.9 6 4 2 5 7.3 Total 69 100.0 Determination of the presence of micronuclei was by visual observation, with no assurance that a "normal" appearing cell contained the proper chromosomal complement to insure fer- tility in the mature pollen. DISCUSSION The hybridity of the interspecific F1 from the cross of S. melongena X S. torvum was determined on the basis of mor- phological characteristics and from cytological observation of the meiotic abnormalities exhibited by the hybrid. The "loose" associations observed at diakinesis and metaphase I may be due to asynapsis, a failure of the chro- mosomes to pair at prophase, or desynapsis, a premature se- paration of bivalents prior to these stages. Univalents were observed in all of the cases. Short chromosomes have been reported to have a lower chiasma frequency than long chromosomes, and thus a tendency towards early separation (Kostoff, 1940). Short chromosomes are characteristic of many Solanum Species (Swaminathan, 23 El: 1954). The low chiasma frequency in the hybrid chromosomes may have con- tributed to the "loose" associations and occurence of uni- valents at diakinesis and metaphase I. Lack of homology between the two genomes may also be a cause of univalent formation. The degree of homology between the chromosomes of two species is generally con- sidered as an indication of the evolutionary relationship between them. However, pairing appears to be based upon a balance between chromosome development and the stage of mei- otic development. Rao, g3 El: (1979) suggest that these 28 29 timing relationships may be controlled in such a manner that slight changes in genetic constitution and genic balances may inhibit pairing of chromosomes which may otherwise pos- sess enough homology to pair. Bivalents resulting from allosyndetic pairing between chromosomes derived from the two parents, would suggest that the parental chromosomes possess homologous segments. How- ever, the occurence of 24 univalents in some of the PMCS, coupled with the “loose" associations of bivalents in the other cells, would suggest that the species may have differ- entiated by structural changes of the chromosomes. Based on observations at anaphase I, the aberrant be- havior of the chromosomes would probably not allow for the development of viable pollen grains. The abnormal segre- gations noted in anaphase I was a continuation of the fail- ure of the chromosomes to pair at metaphase I. When biva- lent formation is limited, the resulting univalents are scattered throughout the cell. AS the bivalents begin to divide, the univalents either move to the equatorial plate, or gather about the pole nearest their former position (Li, gE_§;, 1945). The univalents which fail to reach the equa- torial plate are seen to lag behind at anaphase I, and de- pending upon their proximity to the poles, are either in- cluded in the daughter nuclei, or are lost in the cytoplasm. These excluded chromosomes may, in part, be the micronuclei observed in the tetrads of the hybrid. The anomalies observed in anaphase I were present in 30 the subsequent stages of meiosis. Abnormal chromosome dis- tributions at anaphase II, and the occurence of multipolar meiosis at both anaphase I and II, may result in the pro- duction of gametes with unbalanced chromosome complements. According to Tai (1970), non-homology between genome-specif- ic spindle organizers can result in multipolar meiosis and each genome possesses a genome-Specific spindle organizer. During fertilization, the male spindle organizer enters the egg cell and either the spindle organizers fuse, or one de- generates in favor of the other. The behavior of the chromosomes may be due to an inter- action of chromosome homology and the homology between chro- mosomes and their Spindle organizers. Thus, if either the male or female Spindle organizer disintegrates, the affinity between the spindle organizer of one Species and the chromo- somes of another may be limited or non-existent. In such a case, chromosomes released from the control of their spindle organizer will move randomly within the cell, and may be ob- served as laggards. In this species hybrid, both male and female spindle organizers may be present, causing the sepa- ration of genomes into different groups by means of multi- polar meiosis. The abnormal microspore formation observed at the tetrad stage of the hybrid was most likely due to multipolar meio- sis occuring in the PMCS. Cytokinesis appears to result from an interaction of Spindle organizers, such that when- ever an organizer is present, cytokinesis occurs, cleaving 31 the cytoplasm and maintaining a constant ratio of one Spin- dle organizer per cell (Tai, 1970). AS the number of Spin- dle organizers increases, so does the number of cytokineses, resulting in the formation of more microcells. Cells con- taining more than four microspores would arise from this process. Cells with only one or two microspores were observed in the hybrid. Apparently in these cells, normal nuclear en- velope formation and cytokinesis were hindered by multipolar divisions and unpaired chromosomes, resulting in the forma- tion of one or two micrOSporeS. According to Machado (1978), this is a common occurence in interspecific hybrids. The formation of a restriction nucleus, which results from the inability to separate the chromosomes into individual nuclei, can result in a polyploid nucleus which would exhibit normal meiosis and give rise to a viable gamete. Although meiotic irregularities appear to be responsible for the sterility of this hybrid, it is very difficult to distinguish between genie and chromosomal sterility cytologi- cally (Stebbins, 1958). Thus, genic imbalances and lack of homology of the parental chromosomes may be acting jointly to produce sterility in this hybrid. l. 4. 10. BIBLIOGRAPHY Attavian, B. N., B. L. Pollack and G. Jelenkovic. 1980. Crossability of selected Solanum Species. Abstr. 341. J. Amer. Soc. Hort. 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