———M‘ --A—_ . HYBRiDlZATION IN VETCH Thain fiat {-110 Dagrso a! M. S. MICH~§GAN STATE UNIVERSETY Ricfi'mré L". Camper 1958 ... . T‘. - _' 7‘... __ — TH £515 HYBRIDIZATION IN VETCH By Richard L. Cooper A THESIS Submitted to the School of Graduate Studies of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements MASTER OF SCIENCE Department of Farm Crops 1956 I] ' / «1". 'l I: —~ - . -11 p {.7594 ACKNOWLEDGMENT The author wishes to express his sincere gratitude to Dr. Fred C. Elliott for his guidance in this research project and for his helpful advice in the preparation of the manuscript. The author is very grateful to his wife Norma and to his children for their loyalty and patience through the course of this study. I am especially grateful to my wife for the typing of this manuscript. M‘MfiHI-fi-fiw ii HYBRIDIZATION IN VETCH By Richard L. Cooper AN ABSTRACT Submitted to the School of Graduate Studies of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements MASTER OF SCIENCE Department of Farm Crops Year 1958 ‘\ $§‘ Approved _;Agvflgg; ”Ara Ajifi ABSTRACT Some 2h species of Vicia were assembled and grown in the greenhouse. Many interspecific crosses were made, with special emphasis on crosses between 1. villosa, E. dasycarpa, ‘1. sativa, X, pannonica and X, atropurpurea. In addition several intraspecific crosses were made between 130 entries of 1. sativa. No hybrid seed was obtained from crosses of the.five major species. Seed was obtained, though, in crosses of X. calcarata with X. sativa. X, calcarata is listed as a distinct species by Darlington and Wylie (9). However, the strong morphological similarity of X. calcarata and 1. sativa suggests a possible synonymy of these two species. Other successful crosses were between X, cornigera and X, sativa, and between three purple flowered species, X. onobrychoides, X. bengalensis, and X, atropurpurea. However, becaiée of the strong morphological similarities of X, cor- nigera and X. sativa, it is suggested that X. cornigera is a selection of X. sativa rather than a distinct species. Simi- larly, the high cross compatibility and morphological similar- ity of the purple flowered species suggests that X. onobgy- choides and X. bengalensis are selections of 1. atropurpurea. 1. cornigera, E. onobrychoides and X. bengalensis were not listed by Darlington and Wylie (9). iv Intraspecific crosses in 1. sativa were quite fertile, producing 58 different hybrid combinations. These F1 hybrids, together with their parents were planted in the East Lansing nursery in the summer of 1958. In several cases, the F1 hy- brid exhibited greater vigor and set more seed than either parent. Preliminary experiments in embryo culture were conducted in an effort to find nutrient media satisfactory for growing small immature embryos of vetch. Various basal media were used, supplemented with varying amounts of sucrose, vitamins and the amino acids, asparagine and glutamic acid. Coconut milk and casein hydrolysate media were also tried. Nearly mature E. villosa embryos were successfully grown on Rijven's basal medium supplemented.with asparagine, 200, 800, and 1600 mg/l, and sucrose 2 and 12 percent. Large X. atropurpurea embryos germinated and grew in fiijven's basal medium supplemented with asparagine and glutamic acid, each at 200 mg/l, and sucrose 2 and 12 percent. All attempts to grow smaller embryos were unsuccessful. Vetch seeds, either presoaked in water or dry, were treated with 0.2, O.h.and 0.8 percent aqueous solutions of colchicine in petri dishes. If presoaked, the seeds were treated for six and 24 hours duration, and if not presoaked, for ten and 24 hours. In a side experiment, 250 and 500 ppm of gibberellin were added to the water or the colchicine solutions. One plant, X. villosa, was obtained that exhibited te- traploid sectors. This plant was obtained from seed treated directly in 0.8 percent colchicine solution for 2h hours. Using Nygren's technique, some one hundred X. atropurpr urea plants were treated with nitrous oxide gas. The plants were put in a gas chamber under pressures of 50, 75, and 100 pounds for durations ranging from four hours to 16 hours. Plants were treated at 10, 13%, 17, 20% and 24 hours after pollination in an effort to bracket a time that would place a plant under treatment when the first mitotic division of the newly formed zyogote occured. No seed was set in treatments more severe than 75 pounds for ten hours. Plants survived treatments as high as 100 pounds for ten hours. If duration of the treatment exceeded ten hours the only seed produced was at 50 pounds pressure. No seed was produced in the 16 hour treatments. Time did not permit cytological examination of root tips from all seeds produced, but in a sampling from each treat- ment, no polyploid cells were observed. References 1. Nygren A. "Polyploids in Melandrium Produced by Nitrous Oxide." Hereditas, ul:287-290, I955. 2. Rijven, A. H. G. C. "Glutamine and Asparagine as Nitro- gen Sources for Growth of Plant Embryos In Vitro: a Com- parative Study of Twelve Species." AustFElIan Journal of Biological Sciences, 9:511-527, 1958. vi TABLE OF CONTENTS INTHODUCTION-OOO0.00.0.0...0.000....0..OOOOOOOOOOOOOOOO. kaIEw OF LITERATUREOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO vetCh Utilizationeeeeeeeeeeeoeeeeoeeeeeeeeeeeeoeee Embryo Culture.................................... POlyplOid Induction............................... PART I HYBRIDIZATION IN VETCH................................. Materials and mathOdSeeeeeeeeeeeeeeeeeeeeeeeoeeeee RGBUltSeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee Summary..0OOOOOOOOOOOOOOOOOOOOOO...OOOOOOOOOOOOOOO PART II PRELIMINARY EXPERIMENTS IN EMBRYO CULTURE.............. Materials and MOthOdBeeeeeeeeeeeeeeeeeeeeeeeeeeeee RGSUltSeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee Summary........................................... PART III POLYPLOIDY INDUCTION................................... COlChiCine TreatmentS............................. Materials and Methods........................ RBSUltseoeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee Summary...................................... Nitrous OXide Treatmentseeeeeeeeeeeeeeeeeeeeeeoeee Materials and Methods........................ Results...................................... Summary...................................... GENERAL SUMMARY and CONCLUSIONeeeeeeeeeeeeeeeeeeeeeeeee LITERATURE CITEDeeeeeeeeeeeeeeeeeaeeeeeeeeeeeeeeeeeeeeo vii Page 10 13 13 l l 23 23 37 up .C...". O I O a O o e I U I O O a O I P I w I I I a O I O I D I O I O O I C I O I 0 O O D O I I I o C I O Q 0 O I u D O I G O a c I ‘ . U U O O O I I Q U I QO'COIOOOQ .ICOOOIII. .....‘..II 9 TABLE 1. 2. 3. 4. 5. o. 7. 8. 9. 10. 11. Embryo Embryo Embryo Embryo Embryo Embryo Embryo Embryo Embryo Embryo Culture Culture Culture Culture Culture Culture Culture Culture Culture Culture LIST OF TABLES Experiment Experiment Experiment Experiment Experiment Experiment Experiment Experiment Experiment Experiment l..................... 2..................... 3..................... 4..................... 5..................... b..................... 7..................... 8..................... 9..................... IOOOOOOOOCOOOOOOOIOOOO Nitrous 0x1de Treatments........................ viii Page 29 29 30 31 32 32 33 34 35 36 51 FIGURE 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. lb. Vicia Vicia Vicia Vicia Vicia Vicia Vicia Vicia Vicia Vicia LIST OF FIGURES Villosa, hairy vetch.................... dasycarpa, woollypod vetch.............. sativa, leucosperma, sativa, common vetch.................... common vetcn....... pannonica, Hungarian vetch.............. atropurpurea, purple vetch.............. calcarata............................... cornigera............................... angustifolia, narrowleaf vetch.......... tetragperma, sparrow vetch.............. Lens esculenta, lentil........................ 12.12. cracca, COW vetch....................... Cultured embryos on agar medium............... Colchicine doubled 1. villosa................. Nitrous 0x1d6 gas chamber..................... Somatic chromosomes, X. atropurpurea.......... ix Page 17 17 18 18 19 19 20 20 21 21 22 22 39 1+5 49 1+9 O I 0 e e O I O O O I . o c Q a I 1 O O .00. O C O Q INTRODUCTION In 1953. two and one half to three million pounds of hairy vetch seed were produced in Michigan (22). Most of this seed was shipped to the Cotton Belt States in the South where vetch is important as a winter cover and green-manure crop. Four years later, in 1956, only a few thousand pounds of vetch seed were produced in Michigan. The major reason for this sharp decline in vetch seed production has been the introduction and spread of the vetch bruchid throughout the state of Michigan. . The bruchid is highly specific, attacking only two species of vetch, X. villosa and E, dasygarpa. The larvae of this insect feed in the developing seed of these vetch species, destroying the germination. There are many species of vetch resistant to bruchid infestation including the economically important vetches E. sativa, 1. pannonica, and X, atropurpurea. Therefore in the summer of 1957. some 2h different species of vetch were assembled from various plant introduc- tion stations and foreign countries. These plants were grown in the greenhouse and many different interspecific crosses were attempted, with special emphasis on crosses be- tween major economic species. However, through personal communication with other in- vestigators working with vetch it was indicated that a high degree of species incompatibility exists in the genus Xigig. Thus, an effort was made to find techniques which could be used for overcoming these species incompatibilities. Several investigators have been successful in overcom- ing interspecies barriers in various genera by the use of embryo culture. Therefore experiments were set up in effort to obtain a culture medium that would satisfactorily grow vetch embryos. Another successful tool of the plant breeder in over- coming species incompatibilities has been polyploidy. Ex- periments were run with colchicine and nitrous oxide in effort to develop techniques which would readily produce polyploid vetches. LITERATURE REVIEW 0f the 150 species of Xigig (vetch) distributed through- out the world, only a few are of economic importance in the United States. Vetches are limited in their use because of disease and insect susceptibility and lack of winter hardi- ness. Attempts to combine desirable Characteristics of dif- ferent species have been unsuccessful due to species incom- patibility. Though embryo culture and polyploid induction have been used successfully in overcoming interspecific bar- riers in other genera, these techniques have not been uti- lized in a breeding program in vetch. ‘The.yigig species of major importance in the United States are: Vicia villosa, hairy vetch; X. sativa, common vetch; E. atrqpurpurea, purple vetch; and X. pannonica, Hungarian vetch, as listed by McKee (21). According to henson and Schoth (13), diseases of major importance on vetch are: anthracnose, false anthracnose, black stem, grey mold leaf spot, and various root rots. most insect pests that attack other legumes also attack vetch. Of particular importance is the green pea aphid. The vetch bruchid is a serious pest in areas wnere seed of 1, villosa is produced. According to Nelson and Janes (22), the vetch bruchid was first discovered in New Jersey in 1930 and had spread over the entire country by 1956. I. Pinckney and Stitt (26), in 1941 reported that in species tests for susceptibility to bruchid injury, 2. villosa and ‘1. dasycarpa showed considerable damage while X. atropurp- urea showed only slight infestation. All other 14 species of vetch tested showed no damage, including X. sativa and X. pannonica, even though a few eggs were laid on the pods of these species. Weimer and Bissell (42), in l9h2 reported similar results in testing resistance to bruchid with the exception that they found no damage on X. atropurpurea. Through personal communication with other investiga- tors maintaining a vetch breeding program, it was indicated that all efforts to transfer bruchid resistance to X. villosa by interspecific crosses have been unsuccessful. Species barriers prevented the formation of hybrid seed. These species barriers are particularly strong in the genus Xigig, and only a few interspecific crosses have been reported in literature. Yamamoto (46), in 1955 reported a successful cross between.£. sativa and X. tetrasperma and later in 1956, he reported obtaining hybrid seed between 3. sativa and 1. angstifolia (1+7). Yitzchake (49) also obtained hybrid seed in crosses between.X. sativa and X. angustifolia. Progent (29), reported the development and use of Cerdagne vetch, a hybrid between X. villosa and !. cracca. Of interest, is an intergeneric cross between £332 esculenta and X. sativa, reported by Buchinger (b). (Fig. 1-12) Embryo Culture Several investigators have been able to overcome inter- specific barriers by use of embryo culture. hannig (12), in l90h is recognized as the first investigator to excise and culture immature embryos successfully, while Knudson, L. (17), in 1916 is credited with establishing much of the basis for culturing embryos and seeds in zitgg in his work study- ing the influence of maltose on growth of corn embryos. Laibach (18), in 1929 realized the possibility of ob- taining hybrids from interspecific crosses which otherwise produced shrivelled, inviable seeds by excising immature hy- brid embryos. By means of embryo culture he was able to nurse the hybrid embryos of Eingm austriacum and giggm perenne to maturity. In 1942, Skirm (37). emphasized the use of embryo culture methods as an aid to plant breeding. He successfully cultured interspecific hybrid embryos in Prunus and Lillium, which when not excised did not fully develop due to embryo starvation. Keim (16), in 1952 reported success in growing excised embryos of several species of Trifolium and £9323 in Randolph's agar medium. (31) By using embryo culture he was able to obtain interspecific hybrids in Trifolium. He also devel- oped an embryo culture technique wherein he was able to ex- cise and culture embryos with a contamination of less than 1 percent. Since Hannig's first embryo culture in 1904, where he used Tollen's nutrient medium, many new and more elabor- ate ones have been developed taking into consideration the latest findings on the nutritional requirements of embryos. Because of numerous media it was decided to limit the dis- cussion to the more pertinent work. Basal media used in the present experiments were those developed by Randolph (31), Rappaport (32), White (44), and Rijven (34). Based on reports by numerous investigators, these basal media were then supplemented with: trace elements, sucrose, vitamins, amino acids and different organic sub- stances. ‘ White (43), in 1951 demonstrated that excised tomato roots required iron, copper and molybdenum and probably zinc, manganese, boron and iodine. Nitsch (24) developed a trace element solution that he added to his nutrient media. Similarly, Rijven (34) included the trace elements in his medium. Other workers such as Rappaport (32) limited their media to only a few of the trace elements. In the earlier media, iron salts used for an iron source had a tendency to precipitate in prolonged cultures rendering the iron unavailable. Rappaport (32) replaced the relatively unstable iron sulphate in Randloph's culture by a more stable iron complex, iron citrate. The use of iron citrate has in general been adopted by most investiga- tors. Carbohydrates A carbohydrate source in the nutrient media serves two purposes, one as an energy scurce and another as an osmotic agent. Rappaport (32) in a review of literature on embryo culture, 1952, found that in general investigators obtained best results by the use of sucrose as the carbo- hydrate source in their nutrient media. However, embryos of some plant species seem to prefer fructose, dextrose or other sugars in the nutrient media. Tukey (40), in 1938 reported that the sugar requirement of excised embryos depended on the stage at which the embryos were excised. Rappaport found, that in Datura, very young embryos in the "heart" and "preheart" stages, grew best on media containing up to 8 percent sucrose. The older the em- bryos at time of excision, the lower the optimal sucrose con- centration becomes until it reaches a level of about 0.5 percent with "late torpedo" stage. Sanders (35), in 1950 found that growth in Datura gtgg: monium embryos with 4 percent sucrose was 42 times that ob- tained with 0.5 percent, whereas over the same range, growth of three other Datura species increased only 1.2 to 2.7 times. Thus, even species within the same genus may react differently to different sugar concentrations. Honma (15), in 1955, by changing the sucrose concentration in his nutrient medium from 4 percent down to the 0 percent level was able to obtain in- terspecific hybrids in Phaseolus. Previous attempts to grow these interspecific hybrids had been unsuccessful. Amino Acids According to Rappaport (32) in his survey of embryo culturing techniques, many investigations with plant tis- sue fragments, organs, and embryos have shown that they can assimilate inorganic nitrogen. In general, organic nitro- genous compounch have been assumed to be inadequate nitro- gen sources or even toxic when used in synthetic media. However, Sanders and Burkholder (35). in 1948 reported that the addition of a mixture of 20 amino acids resulted in no- table growth of young Datura embryos. Rijven (33). in 1952 in experiments with Capsella em- bryos, grown by a sitting drop technique, obtained stimula- tion in growth by the addition of glutamine and asparagine to his nutrient medium. In later experiments, 1956, he was able to detect beneficial effects in the growth of embryos from eight different orders of angiosperms by adding 400 mg/l each of glutamine and asparagine to the basal medium (34). Glutamine was beneficial in every species tested whereas asparagine varied in effects, being inhibitory in some spe- cies in the order Rhoeadales. In a legume, Medicago 232i- cularis, asparagine was found to be progressively more bene- ficial at concentrations up to 2000 mg/l. Glutamine was shown to enhance embryonic growth con- siderably more than asparagine in all cases. This was ascribed to the fact that glutamic acid and glutamine have important primary roles in nitrogen metabolism, whereas asparagine is of lesser importance. Vitamins With the development of embryo culture techniques, it soon became apparent that some accessory growth factors were necessary in addition to inorganic salts and a carbohydrate source. White (44) Was one of the first workers to add a vitamin solution to his nutrient media. This was later modi- fied by La Rue (19), by taking 2.5 times the concentration of the vitamins and adding calcium pantothenate. Bonner gt El. (2, 3, and 4), was able to demonstrate that nicotinic acid, ascorbic acid, and thiamine stimulated growth of excised pea embryos. Van Overbeek, Conklin and Blakeslee (41), in 1942 re- ported that the addition of an arbitrary mixture of glycine, thiamine, ascorbic acid, pantothenic acid, nicotinic acid, vitamin B6' adenine, and succinic acid proved effective in promoting growth of Capsella burgg pastoris embryos in the torpedo stage. They had previously failed to obtain growth at this early stage without the added vitamin solution. OrganicIMggig Van Overbeek gt El° (41), in 1942 using autoclaved coco- nut milk observed an unorganized growth of Datura embryos. 10 With unheated coconut milk however, they were able to obtain normal growth of very young embryos. Chang (8), in 1957 re- ported success in growing barley embryos, 0.43 mm in length, by the use of nine parts coconut milk to one part White's medium. Sanders and Burkholder (36), in 1950 succeeded in grow- ing very young Datura embryos by adding casein hydrolysate to their medium. Rappaport (32) reported similar success using casein hydrolysate. Polyploid Induction There are several techniques for inducing polyploidy in plants. Colchicine is by far the most frequently used of these techniques. Nitrous oxide treatment has the advantage in that the zygote can be treated during the first mitotic division. In this way a completely doubled plant can be obtained. A. Colchicine Blakeslee and Avery (1) were among the first investiga- tors to utilize colchicine in producing polyploids in plants. Working with Datura, they were able to obtain from 45 to 65 percent polyploids by soaking pregerminated seeds in colchi- cine solutions varying from 0.2 to 1.6 percent concentrations for a period of ten days. Later experiments, using 0.4 per- cent concentrations, for one, two and four days he obtained .as high as 100 percent polyploidy in some treatments. ll Brewbaker (5), working with Trifolium species was able to obtain many polyploids by the use of colchicine. His most successful method was the aqueous drop technique. Seeds were germinated in petri dishes and as soon as the coty- ledons separated, drops of 0.15 to 0.30 percent aqueous col- chicine solution were placed on the growing points at three hour intervals. Duration of the experiments ranged from eight to 24 hours. The seedlings were weaned in water im- mediately after the treatment was completed and allowed to recover in petri dishes three to five days before trans- planting to light, well fertilized soil in pots. Evans (11), obtained polyploids in red clover, white clover and alfalfa, by the application of a 2 percent aqueous solution of colchicine to seedling by the drop method. She noticed that in addition to the normal indicators of polyploidy such as larger stomata and pollen grains, that polyploid red clover appeared more pubescent. Examination under the microscope showed that there is no difference in the number of hairs, but the polyploid red clover has thicker and longer hairs than the diploid, making it appear more pubescent. Burton (7), 1957, reported that aqueous colchicine seed treatments resulted in from 10 to 100 percent tetraploid sec- tors in Bahiagrass. Effective concentrations were, 0.4 and 0.8 percent for six hours, and 0.2, 0.4, and 0.8 percent for (+8 hours e 12 There are a few reports in the literature where poly- ploidy has been obtained in vetch by the use of colchicine. Yamamoto (48) obtained 2. sativa tetraploids with colchicine solution of 0.025 to 0.050 percent concentrations. Hertzch (14) reported obtaining 4n to lOn polyploid.!. villosa by the use of colcnicine. Nordenskiold (25) reported improved self fertility in tetraploid l. sativa by crossing two different strains of tetraploid X. sativa. Nitrous Oxide Ostergren (27) reported success in doubling the chromo- some number in grepis capillaris by placing the plants in a gas chamber under ten atmospheres (147 pounds per square inch) of nitrous oxide for periods from four to six hours. Later, Nygren (26), copying Ostergren's technique, was able to obtain many polyploids in the Melandrium species. He used various pressures of nitrous oxide for variable dur- ations. Five atmospheres pressure for durations of four to seven hours produced the highest percent of polyploids. In some cases 100 percent of the seeds produced on treated plants were doubled. PART I 13 HYBRIDIZATION IN VETCH Materials and Methods Twenty four different species of vetch were assembled from: the various Regional Plant Introduction Stations; Swedish Seed Association, Svalof, Sweden; the Botanical Gar- den, Nancy, France; the University of Perugia, Perugia, Italy; and the Agronomic Institute, Versailles, France. Ten seeds of each species were germinated on moist fil- ter paper in petri dishes and then transferred to four inch pots in the greenhouse. As the seedlings grew, they were supported with thin bamboo stakes, four feet long. Because of the vigorous growth of some species, it was found necessary to stretch wires across the top of the greenhouse, about ten feet above the pots and suspend strings from the wires to the top of the stakes. Temperatures in the greenhouse were maintained near 60°F and the seedlings were put under continuous illumination in effort to speed up growth and flowering. All species flow- ered under the continuous illumination, with the exception of X. cracca which bloomed sparingly in the summer under nor- mal day length. 11+ Many interspecific crosses were attempted between the 24 species grown, with special emphasis placed on crossing X. villosa, X. dasycarpa, X. sativa, X. pgnnonica, and X. atropurpurea. ‘Also, numerous intraspecific crosses were made between entries of l. sativa. In species with the raceme type infloresence, emascula- tion could be easily performed by pulling off the entire corolla tube, which at the same time removed the anthers which adhere to the inside of the tube. In species with single or multiple flowers sessile to the main stem this technique could not be used. It was necessary to remove the petals separately to expose the anthers which could then be removed with forceps. Results No interspecific hybrids were obtained between the five mayor agricultural species in which special emphasis was placed in this crossing program. However, several crosses were made between strains of X. sativa and the species 1. cornigera (Fig. 8) and Z. calgarata (Fig. 7). From the initial planting, only a few crosses were made between these species. Therefore, in a second planting sever- al plants of X. calcarata, X. cornigera and.X. sativa were grown. Many crosses were then made between these species with special care to avoid any pollen contamination.' Seed was readily set in these crosses, using either parent as the female. 15 There were three purple flowered species obtained in the seed assembled, X. atropurpurea, X. bengalensis, and X. onobrychoides. These species appeared quite similar morpho- logically and when crosses were made in all combinations, it was found that they intercrossed quite freely. 0n the basis of these observations it is suggested that X. bengalensis and 1. onobrychoides are strains of X. atropurpurea. Of special interest in this crossing program was the marked response in pod stimulation obtained in X. atropurp- ‘2323 by the application of pollen from nearly every species to which it was crossed. Unfortunately, however, after ten to 15 days, the pods began to brown at the tips and eventual- ly withered and dropped off. Attempts to excise embryos from these pods before this browning began, were unsuccessful. Upon cutting away the ovary wall, the exposed ovules were noticed to have a slight- ly sunken spot in the center. When a thin slice was removed from the lateral surface of these ovules a hollow space was observed in the center, with no trace of a deveIOping embryo. A high degree of compatibility was found between strains of 1. sativa in intraspecific crosses. Fifty-eight different hybrids were obtained from the 130 strains of X. sativa. Twenty-eight of these F1 hybrids were planted in the East Lansing nursery and observed for disease and insect resistance, vigor and seed yield during the 1958 growing season. In sev- eral cases the F hybrids out yielded either parent in seed 1 production and showed more vigor. 16 Summary Because of the high compatibility between 1. calcarata and X. sativa, and their morphological similarity, it is suggested that a possible synonymy exists between these two species. 1. cornigera appears quite similar to X. sativa and the two species cross readily. Since in a survey of literature no reference could be found on X. cornigera, it is suggested that X. cornigera is a selection from X. sativa rather than a distinct species. Similarly, it is suggested that two purple flowered species, X. onobrychoides and X. bengalensis, which readily cross with X. atropurpgrea are selections from X. atropurpurea. Most strains of X. sativa, though highly self fertile, will cross readily with each other and in some cases hybrid vigor is exhibited. Fig. l. .'1 u- (h 0 h) 0 ( Vicia villosa, Hairy vetch 211-111 Vicia villosa, glabrescence, Smooth vetch Ell-l ' vetch (3 ‘1 5.4. n I M I: r) p U: M n in '1 (u m U 34, 0 O H 4 ’U 0 (J 1'? Fig. 3. Fig. 4.0 Vicia sativa, Common vetch 2n I 12—01" 11+ ViciaAsativa, leucosperma, Common vetch Zni‘lc or I4 l8 \/ I ' Fig. 5. Vicia pannonica, Hungarian vetch 2n:12 Fig. 6. Vicia atropurpurea, Purple vetch 2n:14 Fig. 7. Vicia calcarata 211-111. Fig. 8. Vicia cornigera 20 Fig. 9. Vicia angustifolia, Narrowleaf vetch 2n 3 12 Fig. 10. Vicia tetragperma, Sparrow vetch 211.11: H g1“ 21 Fig. 11. Lens esculenta, Lentil Zn. 114. Fig. 12. Vicia cracca, Cow vetch I 12, IE, 01' 28 PART II 23 PRELIMINARY EXPERIMENTS IN EMBRYO CULTURE Materials and Methods In general, the techniques developed by Keim (16) for culturing legume embryos were used, with modification where necessary. Four species of ligig were used in these experi- ments, X. villosa, X; atropurpurea, X. sativa, and 1. 232' o nonica. The material was grown in the greenhouse at 60 F in continuous light. Embryos were excised from ten to twenty- five days after pollination, varying in size from 0.5 mm in length to fully developed embryos. A stereoscopic binocular was used with magnification of 201. Care was taken to avoid contamination by cleaning the table top and spraying the room with 50 percent aqueous "ST 37" solution. Forceps and scalpel were used in the ex- cision process. These were sterilized by flaming and placed in "ST 37" each time before use. The entire pod was placed in the dissecting dish and while holding it with the forceps, the scapel was used to cut around the margin of the pod. Having done this, the pod was held firm with the scalpel and the top half of the pod peeled back with the forceps, exposing the ovules. The dissecting instruments were then again flamed and placed in "ST 37". Special care was taken to avoid tearing the ovule 24 loose from the ovary wall. It was much easier to hold the ovule firmly if it remained attached. Holding the ovule firmly with the forceps, the scalpel was used to cut a very thin slice through the lateral surface of the ovule wall ex- posing the embryo in a cavity of maternal tissue. The scal- pel was then carefully eased beneath the embryo and it was lifted out. Then, holding the nutrient bottle slightly in- clined downwards, the embryo was placed cotyledons down slightly into the agar medium. The instruments were then flamed and placed in "ST 37" until the procedure was repeated. It was found that a scalpel blunt on the and rather than point- ed was more effective for removing the embryo from the ovule. The glass containers used to hold the nutrient media were 2 dram vials, 15 by 75 mm, with cotton plugs. (Fig. 13) These proved to work satisfactorily until warmer weather caused excessive evaporation. Covering the top of the vials with aluminum foil aided somewhat in reducing evaporation losses. The bottles containing the excised embryos were placed on a table top at room temperature under continuous florescent illumination. Attempts to grow embryos at constant tempera- ture of 320 C and in the dark as suggested by Rappaport (32), were unsuccessful. At this higher temperature, the problem of evaporation was intensified. 25 An agar medium 0.7 percent, was used in all of these experiments. In general three levels (2, 4, and 8 percent) of sucrose were used in each experiment in an attempt to bracket the optimum sucrose concentration for embryos of varying stages of development. The two dram vials were filled approximately half full by using the ring stand, funnel, rubber hose, and pinch clamp apparatus suggested by Keim (16). In this way, the vials could be filled rapidly without the media coming in contact with the upper portion of the vial. With the use of cotton plugs this was of particular importance as the plugs would stick in the vial, glued by the dried media. As the vials were filled, they were replaced in the original box which made iteasy to handle and sterilize two to three hundred vials at one time. The media was sterilized for thirty min- utes at 15 pounds pressure at 2400 F. Since the vials were not pyrex, care was taken to raise and lower the temperature and pressure slowly to avoid breakage. Also, this avoided "bumping" of the media which might cause the media to come in contact with the cotton plugs or even pushing them out. It was the intent of the writer to use embryo culture only as a tool to grow hybrid vetch embryos, not to do an intensive research in the field of embryo culture. There- fore, in general a simple media was used to begin with and as each failed to give the desired result, more elaborate media were tried. Since already established media were used 26 in these experiments, they will be referred to by the investi- gators who developed them, rather than listing the entire con- stituents of the media. Modifications to the media however will be specified. Experimental Methods The following preliminary experiments in embryo cultur- ing were performed in effort to obtain a nutrient medium which could be used in the culturing of excised hybrid emb- ryos in the XEEEE species: Experiment 1 -- Because of the success by Keim in growing clover and trefoil embryos on Randolph's medium, this was the first medium tried for the culturing of ex- cised vetch embryos. Sucrose at the 2 percent level was used. (Table 1) Experiment 2 -- Failure to grow embryos on Randolph's simple inorganic media plus sucrese suggested that a more complex medium might be needed. Rijven's basal medium with a complete source of trace elements was selected. To this medium were added the amino acids, glutamic acid and aspara- gine as the only nitrogen source. A stock nitrate solution was added to a portion of the media as a control. Sucrose was used at the 2 percent level. (Table 2) Experiment 3 -- According to various investigators, the smaller the embryos at excision, the higher the sucrose concentration should be in the nutrient media. Therefore, 27 using the same medium as in experiment 2, the sucrose level was varied from 2 percent to 12 percent in hopes of obtain- ing optimum levels for embryos excised at various stages of development. (Table 3) Experiment 4 -- Since a degree of success was obtained in the growing of nearly mature embryos in the asparagine supplemented media, variations in the concentration of as- paragine and sucrose were used in an attempt to find a more optimum level for smaller embryos. (Table 4) Experiment 5 -- Having had only partial success with Rijven's medium, a new media developed by Rappaport was tried. It was modified by adding Rijven's minor element solution and the vitamins, thiamine, niacin, and ascorbic acid. Three levels of sucrose were used. (Table 5) Experiment 6 -- Because of the emphasis some investiga- tors place on the level of sucrose, Randoph's media was used with sucrose at 1, 2, 3, and 4 percent sucrose. (Table 6) Experiment 7 -- Since in previous experiments no suc- cess was obtained in growing small immature embryos, it was decided to try an organic medium. Van Overbeek's (41) coco- nut milk medium was tried, modifying it by using Rijven's basal medium and adding Van Overbeek's (41) extensive vita- min solution with the exception of adenine. (Table 7) Experiment 8 -- Attempts to transfer the milk from the coconut aseptically to the nutrient medium by use of a 28 hypodermic needle was found to be unsatisfactory. A high percent of contamination resulted. Therefore, it was de- cided to try the heat stable amino acid complex, casein hydrolysate which could be sterilized in the autoclave. The basal medium used was that of Rappaport's plus the addi- tion of Rijven's trace element solution. The vitamin solu- tion used by Sanders and Burkholder (36), in their casein hydrolysate media was also added. (Table 8) Experiment 9 -- Upon purchase of a Seitz filter, the coconut milk media was again tried. The basal medium used was White's media, as modified by La Rue (19). Asparagine was added to a portion of the basal medium and the sucrose concentration was varied. The coconut milk was used at nine parts coconut milk to one part media, as was found to be quite successful by Chang (8). Experiment 10 -- La Rue's modified White's solution was again used, supplemented with asparagine and glutamine, (not glutamic acid as in previous experiments). In addition IAA was added as suggested by La Rue (19). (Table 10) These embryo culturing experiments were began in Decem- ber 1957. Ideal conditions were not available for a transfer room nor the growing of the embryos but an open room proved fairly satisfactory during the winter months. In the spring, however, as the temperatures increased in the culture room, rapid evaporation of water from the agar medium necessiated the termination of these experiments. 29 H\ws mm .mozm H\ms mm .mamozvso H\wa com us some scams unsung one ofio< OHSepsaw H\ms oom .ecammamdmd H\ms oom .eaoa sagaasae KN .emoaosm msHm Edfieea Hmmmm m.co>nam N pdofiaaodwm N mqmda omoaosm RN Edens: Hmmmm n.2ddoocsm H pcoafiaodxm H mqmnam m psosusedxm m mamas 31 Fifi—$19.? oaoaosm ouoaosm 000h05m oeoaosw enoaodm OQOfiofim omoaosm onoaosw ouohosm enoaosm eeoaosm omomosm H\ma ooom enamsaaan< H\ms coca odawesemed H\ws com .odawenumn< H\wE co: .odawsasdm< sauce: Hanan a.so>nam d pmoaahomxm e mamas 32 A:.m.mv .mm.mm aaeddom hm mOhhnSo sod oomaoXo Ho npsoaw on» ma amaoaModen on on csonm coon ops: owes oanmoomw one .caemac .omHEmHmB# R: omososm Rm omoaoSm sesame Hamam m.eaaoecam Rm omosoSm RH omoaosm % o pdeaahodxm o mqmda fin omoaosm &: omOAoSm soapsHom caawpfi> 02 am omonosm soapsaom pdoaoam momma m.co>nam suave: Hsmsm n.9aodmdaum Rm onoaosm H\ma 0.0m .Uaom canaooud H\waeo.o .aaaaaz emosesm H\wawo.o .Hom ocaswanev a: Rm onesesm m acosaaomxm m mqmda 33 °\ N eeoaosm envosm ouonesm ouoaosm emosodm ouchosm oaoaodm omonodm ouoaonm omonesm omonosm emoaeom soaaaaom cassaa> oz moanfiHom Gdampd> s.xeonse>o ne> aoaasaom sagaea> oz soaesaom assasa> u.xeonho>o nm> sass escoooo oz sauces Hsmsm a.uo>nam ssdcoa spawn com on pass a saws asaoaoo w pdosaseanm h mdm m.aovAomxasm was uaoocsms RN omoaosm QOAasAom udoSoAm comma m.do>nAm asAcoE Assam n.9nomsdmmm RNA omoaoum . sea oom .qoausaom mm ouoaosm scammeudhaa .osAopmho .cAomso RN eaoaosm RNA omomosm and 00A .sOApsAom ocsndopdhha .ecAopnho .cAoaso#* Rm onoaosm RN eeoaesm m pdosAaoaKm m mqmda 35 .opwsonpoacam sdAeAso no A mN.o esAd wdoAp Ianpdoodoe GAEspAp one GA endomodA Nm.N a use .NAooo no A mN wsAa aOApsAou asosvo some» n.noupAz no GOAvoos on» hp hAnAsa ceAMAoofl new asAoefl n.09Ansv am am Rm mm mm omoaosm omoaosm OMOHOSW omososm onenesm ouonosm e-ososm onoaezm saws ascoaoo oz ocAmeAstd oz ssAooa used A on upped o .mAAS usdoooo upsoaoAm comma m.moapAz .aoapsAom a.oe«s3 assesses u.osm sac 3AAS pzcoooo oz A\ws co: .onAwaanu¢ EdAooa sham A on mused o .AAAE assoeoo o adoSAnoQHm o mamas 36 .0 QAQmB moms mN omoaosm onenesm cachesm ouoaosm onohosm omoaosm omoaodm ouoaosm omonosm omonosm cachesm omososm «mA 02 A\ws co: .ocaaspsae A\wa ooom .edAwshsmmd H\maa .aaH <4H oz A\ma OQJ .edAampsAw A\ms A .eaa mdA oz A\ms ooom .osAmsamdud H\ms A .aeH GOApsAom pdoaoAm comma u.nompAz .soaasaom n.0aAs; easeseos a..sm was 0A pcoaAaoQKm 0A mqmda 37 Results Of the 500 plus vetch embryos excised and cultured in these preliminary experiments, only six embryos, which were nearly mature at excision, showed indication of growth. These six embryos germinated seven to 12 days after excision and subsequently developed into normal seedlings. No indica- tion of pregerminal growth was observed in either small or large embryos and no abnormal cell proliferation appeared. 1. One 3. villosa embryo germinated seven days after culturing on Rijven's basal medium supplemented with 200 mg/l of asparagine and 2 percent sucrose (Table 2). 2. One X. atropurpurea embryo germinated ten days after culturing on Rijven's basal medium supplemented with 200 mg/l of asparagine, 200 mg/l of glutamic acid, and 2 percent sucrose (Table 2). 3. One 1. villosa embryo germinated eight days after culturing on Rijven's basal medium supplemented with 200 mg/l of asparagine and 12 percent sucrose (Table 3). 4. One X. atropurpurea embryo germinated 12 days after culturing on Rijven's basal medium supplemented with 200 mg/l of asparagine, 200 mg/l of glutamic acid, and 12 percent su- crose (Table 3). 5. One 1. villosa embryo germinated seven days after culturing on Rijven's basal medium supplemented with 800 mg/l of asparagine and 2 percent sucrose (Table 4). 6. One 1. villosa embryo germinated ten days after culturing on Rijven's basal medium supplemented with 1600 mg/l of asparagine and 2 percent sucrose. (Table 4) 38 Fig. 13. Representative seedling growth from embryos cultured 20 days on Rijven's basal medium supplemented with amino acids. 40 SUMMARY 1. Preliminary experiments were run in effort to find a nutrient medium satisfactory for the culture of immature vetch embryos. 2. Six nearly mature embryos, excised and cultured in various media, germinated and developed into normal seed- # lings. No apparent growth was observed in any smaller imma- ture embryos. 3. Each of the six embryos developing into normal seedlings were grown in a nutrient media consisting of Rij- ven's basal medium supplemented with sucrose and asparagine at various concentrations. 4. There appeared to be a species differentiation in nutrient requirements in that X. atropurpurea embryos required in addition to asparagine, a glutamic acid supplement. No X. villosa embryos grew in media supplemented with glutamic acid. 5. Concentration of asparagine and sucrose did not seem to be critical for embryos at the nearly mature stage of development. 6. No apparent benefit was observed from the addition of vitamins, IAA, and the organic supplements, coconut milk and casein hydrolysate. 7. Though emphasis was placed on the culturing of immature vetch embryos, this was not accomplished. How- ever it is hoped that the information presented here will help give direction to further work in the embryo cultur- ing of the Vicia species. PART III POLYPLOIDY INDUCTION Because of failure to obtain many of the desired inter- species crosses at the diploid level attempts were made to develop a technique for obtaining polyploids in vetch, with the hope that species barriers could be broken down at a higher level of ploidy. Two techniques were employed to obtain polyploidy. The first technique involved colchicine solutions in seed treat- ments and the second involved nitrous oxide gas under pres- sure. Each technique will be discussed separately. Colchicine Treatments Materials and Methods This experiment actually consists of two sub-experiments. In the first experiment, four species of vetch were used, 1. atropurpurea, X. villosa, X. sativa and X. pannonica. Col- chicine solutions varying in concentrations from 0.2, 0.4, to 0.8 percent were prepared. The seeds were divided into two lots of ten seeds each for each species. One lot in each species was placed directly into the various colchicine solutions for intervals of ten to 24 hours, whereas the other half was first soaked 48 hours in distilled water and then transferred to the colchicine for six and 24 hours. 43 In the second experiment, only X. atropurpurea and X. villosa were used. Since it has been suggested that colchi- cine is most effective when cells are rapidly dividing, it seemed feasible that possibly the stimulating effect of gib- berellin on germination and growth could be utilized to ad- vantage. The same colchicine concentrations were used as in the previous experiment with the addition of gibberellin at 250 and 500 ppm. According to Wittwer and Bukovac(45), peas soaked over night in petri dishes with 250 ppm of gib- berellin showed hastened germination and rapid emergence. One lot of seed for each species was soaked over night in gibberellin and then treated with colcnicine six and 24 hours. The other lot was placed directlyninto a mixture of gibberellin and colchicine for intervals of ten to 24 hours. In both experiments, as each treatment was completed, the seeds were planted in rows in flats of sand in the green- house. Results The soaking of seeds in colchicine following germina- tion in water was a more severe treatment than when the dry seeds were put directly into the colchicine. Many plants were stunted particularly at the higher concentrations of colchicine and 24 hours of exposure. Plants suspected of polyploidy on the basis of stunted and abnormal growth were transplanted from the flats to four inch pots of sterile soil. 0f the 15 plants transplanted, only six survived. There ,c apparently was considerable root injury in these seedlings and they died before a root system became established. 0f the six surviving plants, only one showed apparent tetra- ploid sectors based on larger flowers. This plant arose from a treatment where the dry seed was placed directly into 0.8 percent aqueous colchicine solution and soaked for 24 hours. Another characteristic later observed on this plant and which proved to be quite accurate for detecting tetra- ploid sectors from diploid sectors was a more noticeable pubesence on the leaves and stems. Examination under the binoculars showed that this increased pubesence was not due to more hairs, but rather longer and thicker hairs. This same characteristic was discovered in tetraploid red clover by Evans (11). Final identification was made by cytolgical examination of pollen mother cells which showed the doubled chromosome number of 28. In the treatments to which gibberellin was added, a slight speed up in germination and growth of the seedlings was observed, but there was no indication that it enhanced the effect of colchicine. Summary Since several seeds germinated and produced normal seedlings after treatment in 0.8 percent colchicine solution for 24 hours, and the only polyploid plant produced was from -this same treatment, it is suggested that higher concentra- tions of colchicine might be used more effectively. Fig. 14. Comparison of diploid and tetraploid sectors produced on a colchicine treated X. villosa plant. 1+5 V V;//°50.J «vdxjeh EL 1110?? 1” 9w Clio/ck I Cl.“ 9 0'3 7o 19hr: 46 Root injury observed in plants from treated seed in- dicates that possibly seedling treatment with colchicine, in which the roots do not come in contact with the colchi- cine might be more successful. Nitrous Oxide Treatments Materials and Methods Since it appeared that the colchicine treatments were not too effective, it was decided to try the technique devel- oped and used by Ostergren and later used by Nygren, wherein they used nitrous oxide to obtain polyploids. Use was made of a gas chamber designed and set up by Dr. Elliott (10) for chromosome doubling work with other crops. (Fig. 15) A major advantage of nitrous oxide doubling over col- chicine is that the zygote can be treated at the first mito- tic division, producing a completely doubled embryo with no chimeras. Often, doubled sectors of colchicine treated plants are crowded out by the more vigorous diploid sectors. For nitrous oxide to be most effective, the zygote should be treated at its first mitotic division. Thus it was necessary to select a vetch species in which pollination could be easily controlled. Fortunately, in previous experi- ments it was found that X. atropurpurea could be easily emas- culated and would readily set seed following hand pollina- tion. Hence, this species was used in these experiments. 47 In order to bracket the right length of time from pollination to the first zygotic division, the plants were treated at 10, 13%, 17, 20%, and 24 hours after pollination. Three different pressures were used, 50 pounds, 75 pounds, and 100 pounds. The duration of the treatments ranged from four hours up to 16 hours. Some 130 plants of 1. atropurpurea were grown to assure plentiful material. At flowering, a pollination schedual was set up so that one plant was pollinated every three and one half hours over a seventeen and one half hour period. The plants were then placed under treatment ten hours after the last pollination. In this way five plants covering the range desired for "hours from pollination," could be treated at one time. Usually five to ten florets were pollinated per plant. In order to treat five plants at once, it was necessary to remove the plants from the pots. This was done carefully so as to disturb the roots as little as possible. It was found that if the pots were watered prior to removal of the plants, the soil held together better and also the moisture was beneficial to the plants during the treatment. The plants still staked up with four foot stakes were placed in a large plastic bag, long enough so the plants could be easily lowered into the gas chamber and easily pulled out when the treatments were completed. 48 After completion of a treatment, the pressure was slowly decreased, over a period of ten to 15 minutes, by gradually draining out the nitrous oxide. Before opening the gas chamber, it was first flushed out with nitrogen. More than one hundred plants were treated by this method, some of the treatments being repeated twice. In preparation for mitotic chromosome counting of treat- ed seedlings preliminary experiments were run with seminal root tips of X. atropurpurea seedlings. Root tips were first treated in oxyquinoline (20), then transferred to Newcomer's fixing solution (23), and finally stained with the propionic carmine smear technique (38). The best stage for excision was found to be when the roots had emerged from five to ten mm beyond the seed coat. Earlier excision failed to show any actively dividing cells. Also, the time of day that excision was made, seemed to be critical, with from 11:00 A.m. to 12: A.M. consistently being the best time. The length of time left in the oxy- quinoline seemed to be somewhat critica1.with about five or six hours being the optimum time. However some very shrunken Chromosomes were observed in material left in oxyquinoline for 24 hours. Root tips were left in Newcomer's solution for at least 48 hours before removal for staining. Fig. 15. Gas chamber set up used for treating plants with nitrous oxide. Designed by Dr. F. 0. Elliott (10). Fig. 16. Somatic chromosomes of root tips treated at 6° C for six hours in 0.002 M oxyquinoline, 48 hours in Newcomer's solution and then fixed in propionic carmine. X. atropurpurea 2n = 14 1500 X 49 50 Results As shown in Table 11, no seed was produced when the treatment was more severe than 75 pounds for 10 hours. Plants, however, did survive treatments of 100 pounds for 10 hours. When the duration of the treatment exceeded 16 hours, no seed was produced under any level of pressure and at 13 hours, the only seed produced was on plants under 50 pounds pressure. Time did not permit chromosome determination of all seed, but a sample of at least two seeds per treatment was germin- ated and their root tips excised for chromosome counting. Using the preparation and staining technique developed in preliminary experiments, approximately 75 percent of the root tips examined were at the right stage of division for chro- mosome counting. From this sampling of seed, no polyploid cells were observed. Summary The application of nitrous oxide treatments are rela- tively easy and many seeds are produced in some treatments, each seed being a potential polyploid. Thus it can readily be seen from these experiments, that if any effective treat- ment were found, numerous polyploids of vetch could be pro- duced without difficulty. In a sampling of seed from each treatment, no polyploids were found. However, it is hoped that from the 111 seeds produced on the treated plants, some polyploid seedlings will be obtained. Table 11 Nitrous Oxide Treatments Pounds Hours after No.of No. of Pressure Hours Pollination Plants Seeds 50 4 10 is 20% 24 50 7 10 13% 17 20% 24 so 10 10 13% 17 20% 24 so 13 10 ' Us 17 20a 24 50 16 10 13a 17 20a 24 75 u 101 13s 17 20% 24 7S 7 10 13% 17 20% 24 1,14 new hux~adnn t: p.- 00000 FU‘lOUIO OOOOO OOOOH OOOOO ODNOOO mmmmm HHHHH HPHHH HHHHH HPHHH mmmmm ‘ “t“ and 301* FHH Vb FHH $8 Pounds Pressure Hours Hours after Pollination No. of Plants No. of Seeds 52 75 75 75 100 100 100 100 100 10 13 16 10 13 16 10 13% 17 20% 24 10 13s 17 20a 2'4 10 13% 17. 20% 24 10 13% 17 20% 24 10 13% 1? 20$- 24 10 13% 17 20% 21+ 10 13s 17 20s- 24 10 . 13:3: 17 20$~ 24 ** HHHHH Nmmmm mmmmm HHHHH HHHHH NNNNN 00000 00000 00000 00000 00000 00000 00000 OHO¢N * Plant killed. 53 GENERAL SUMMARY AND CONCLUSIONS A strong incompatibility exists between species of Vicia. From numerous interspecific crosses made between 24 different species, the following crosses were successful; X. sativa X X. calcarata X. sativa X E. cornigera X. atropurpurea X X. onobrychoides E. atropurpurea X ‘1. bengalensis X. onobgychoides X X. bengalensis These crosses set seed readily, with reciprocal crosses being equally fertile. However, because of the high degree of compatibility and morphological similarity between species, it is suggested that synonymy may exist between.X. sativa, X. calcarata and X. cornigera, and similarly between.!. atro- purpurea, X. onobrychoides and E. bengalensis. Intraspecific crosses within 2. sativa exhibited high compatibility. Fifty-eight different hybrids were obtained from 130 entries of X. sativa. The F1 hybrids, together with both parents were planted in the East Lansing nursery in 1958. In several cases the F exhibited greater vigor and set more 1 seed than either parent. In E. atropurpurea, a strong pod stimulation was observ- ed from interspecific crosses. Attempts to excise embryos from the poorly developed seeds in theaapods were unsuccessful. 54 Failure to obtain hybrid embryos indicates that the barr- ier to interspecific hybridization is effective at ferti- lization or shortly after in these particular crosses. Attempts to develop a nutrient medium for the culture of vetch embryos was only partially successful. Nearly mature V. villosa and V. atropurpurea embryos were success- fully cultured on Rijven's basal medium supplemented with the amino acids asparagine and or glutamic acid. It is hoped that this Observed stimulation in growth of vetch em- bryos by asparagine and glutamic acid may help lead to the development of a nutrient medium satisfactory for the growth of small vetch embryos. Such a nutrient medium would be a valuable tool in effort to overcome interspecific barriers in vetch. Seed treatment of vetch with colcnicine produced poly- ploids only at the most severe treatment, 0.8 percent col- chicine for 24 hours. This indicates that seed treatment with higher concentrations of colchicine for longer durations may be more effective in producing polyploids. Many seeds were produced from the nitrous oxide treat- ments. However, in a sampling from each treatment, no doubled seed was observed. If a critical treatment is found that will produce polyploidy in vetch, it seems likely that numerous polyploid vetches could be readily produced by this treatment. 55 It is hoped that these initial steps, in the develop- ment of a nutrient medium and polyploid induction techniques for vetch, may be of value to other investigators for fur- ther development and eventual use to overcome species incom- patibilities in Vicia. 10. ll. 56 LITERATURE CITED Blakeslee, A. F. and A. G. Avery. "Methods of Inducing Doubling of Chromosomes in Plants." i. Heredity, 28:393-311. 1955. Bonner, J. "Nicotinic Acid and the Growth of Isolated Pea Embryos." Plant Physiology, 13:865-868, 1938. . and G. Axtmen. "The Growth of Plant Embryos In Vitro. Preliminary Experiments on the Role of Acces- sory Substances." Proceedings National Academy Science, 23: 53'4579 1937. . and D. Bonner. "Ascorbic Acid and the Growth of PIant Embryos." Proceedings National Academy Science, 24:70-75: 1938. Brewbaker, J. L. "Colchicine Induction of Tetraploids in Trifolium Species." Agronomy Journal, 44:592-594, 1952. Buchinger, A. "Osmotic Analysis of a Lentil-Vetch Hybrid and Its Parents." Biological Abstracts, 4:13993, 1930. Burton, G. W. "The Induction and Some Effects of Auto- tetraploidy in Pensacola Bahiagrass." Agronomy Abstracts, P- 53: 19570 Chang, Won Chong. "Comparative Growth of Barley Embryos In Vitro and In Vivo." M. S. Thesis, Department of Botany Efid PIant Pathalogy, Michigan State University, East Lansing, Michigan, 1957. Darlington, C. D. and A. P. Wylie. "Chromosome Atlas of Flowering Plants." 2d Edition, George Allen and Unwin, Lts., London, 1945. Elliott, F. 0., Associate Professor, Department of Farm Crops, Michigan State University, East Lansing, Michigan. Personal communication. Evans, A. M. "The Production and Identification of Poly- ploids in Red Clover, White Glover and Lucerne." New ‘Phytologist, 54:149-162, 1955. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 57 Hannig, E. "Zur Physiologic pflanzlicher Embryonen." as cited by Keim, W. F." InterSpecific Hybridization in Trifolium Utilizing Embryo Culture Techniques." Agron- omy Journal, 45:601-606, 1953. Henson, P. R. and H. A. Schoth. "Vetch Culture and Uses." U.S.D.A., Farmers' Bulletin No. 1740, 1955. Hertzch, W. "Observations on Polyploidy V. villosa." Plant Breeding Abstract, 22:476, 1952. Original not seen. Homna, S. "A Technique for Artificial Culturing of Bean Embryos." Pgoceedings American Society Horticultur- al Science, 65:405-498, 1955. Keim, W. F. "An Embryo Culture Technique for Forage Legumes." Agronomy Journal 45:509-510, 1953. Knudson, L. "Influence of Certain Carbohydrates on Green Plants." Cornell University Agricultural Experi- ment Station Memoir, 9:1-75, 1916. Laibach. F. "Ectogenesis in Plants. Methods and Genetic Possibilities of Propagating Embryos Otherwise Dying in the Seed." Journal of Heredity, 33:211-215, 1942. La Rue, C. D. Formerly University of Michigan, Ann Arbor, Michigan. Unpublished manuscript. Markarian, D. and J. S. Schaeffer. "A Squash Technique for Root Tips of Polyploids in Bromus." stain Technolgg , 32:3: 19570 McKee, R. as published in Forages, Hughes, Heath and Metcalfe. Iowa State College Press, Chap. 18, 1952. Nelson, L. V. and R. L. Janes. "Vetch Seed Production." Farm Crops Extension memeo, File No. 22.43, 1956. Mich- igan State University, East Lansing, Michigan. Newcomer, E. H. "A New Cytolo ical and Histologic Fixing Fluid." Sciencez 118:1 1, 1953. Nitsch, P. R. "Growth and Development In Vitro of Excised Embryos." American Journal Botany 38:555-575, 1951. Nordenskiold, H. "An Investigation Into Two Tetraploid Strains of Vetcn (Vicia sativa) and Their Hybrid Pro- ducts." Plant breeding Abstracts, 24:1191, 1954. Original not seen. ‘ 26. 27. 28. 29. 30. 31- 32. 33- 34. 35. 36. 37. 38. 39. Nygren, A. "Polyploids in Melandrium Produced by Nitrous Oxide." Hereditas, 4I3287-29O, 1955. Ostergren, G. "Polyploids and Aneuploids of Cre is capillaris produced by Treatment With Nitrous OxIde." Genetics 27:54-64. 1954. Pinckey, H. S. and R. E. Stitt. "Tests of Species and Varieties of Vetch for Resistance to the Vetch Bruchid." Biological Abstracts, 16:8192, 1942. (Original not seen Progent, A. "Cerdagne Vetch, an Annual Fodder to be Developed in South Western France." Biological Abstracts, 24311710, 1950. Original not seen. Rajan, S. S. "A Note on the Chromosome Number on Some Plants." Plant Breeding Abstracts, 24:959, 1954. Original not seen. Randolph, L. F. "Embryo Culture of Iris Seed." Amer- ican Iris Society Bulletin, 97:33—45, 1945. Rappaport, J. "In Vitro Culture of Plant Embryos and Factors ControllIHg TEeIr Growth." Botanical Review, 20:201-225, 1954. Rijven, A. H. G. C. "In Vitro Studies on the Embryo of Ca sella bursa pastoris. Acta Botanica Neerlandica, 1:157, I952. . "Glutamine and Asparagine as Nitrogen Sourcesfifhr Growth of Plant Embryos In Vitro: A Compara- tive Study of Twelve Species." Australian Journal of Biological Sciences, 9:511-527, 1956 Sanders, M. "Development of Self and Hybrid Datura Embryos in Artificial Culture." American JournaI Botany. 37:6-11, 1950. +_ . and P. R. Burkholder. "Influence of Amino Acids on Ufowth of Datura Embryos in Culture." Pro- ceedings National Academy Science, 34:516-526, 1948. Skirm, G. W. "Embryo Culturing as an Aid to Plant Breeding." Journal Heredity, 33:211. 1942. Smith, L. "The Acetocarmine Smear Technic." Stain Technology, 22:17-31, 1947. Stebbins, G. L. "Hybrid Inviability, Weakness, and Sterility." Advances in Genetics, 9:147-203, 1958 40. 46. 47. 48. 1+9. 59 Tukey, H. 5. "Growth Patterns of Plants Developed From Immature Embryos in Artificial Culture." Botanical Gazette, 99:630-665, 1938. Van Overbeek, J., M. Conklin, and A. F. BlakeSlee. "Cultivation In Vitro of Small Datura Embryos." American Journal Botany, 29:472-477, 1942. Weimer, J. L. and T. L. Bissell. "The Vetch Bruchid in Georgia." Journal Economic Entomolggy, 35:794, 1942. White, P. "Nutritional Requirements of Isolated Plant Tissues and Organs." Annual Review Plant Physiology, 2:231-244, 1951. . "A Handbook of Plant Tissue Cultures." The RonaId Press Company, NewYork, 1943. Wittwer,.S. H. and bukovac, M. J. "Gibberellin and Higher Plants." _ggarterly Bulletin Michigan Agr. Expt. Sta. Michigan State University, East Lansing, Michigan, 40:215-224. 1957 Yamamoto, K. "On the F of Vicia sativa L. X V. tetrasperma, Moench." Plant Breeding Abstracts 23:2106, 1955. Original not seen. . "On F of Vicia sativa x Vicia an usti- ToIIa." Plant Bree ingABstracts, 26:2374, 1958. OrIgInal not seen. . "Polyploid Vetch Induced by Colchicine Treatment?"Ir Plant Breeding Abstracts, 25:2147, 1955. Original not seen. Yitzchake, D. "Recognizing Cultivated Vetch in Israel." Plant Breeding Abstracts 25:1678, 1953. Original not seene 21.} \. ms. “are, a!“