i§§ HHHHIHHW .W_-a._‘~——— ' "" W ' mu- :- \\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\3\3l3\\\\ ‘3 1.x THESIS. ' 3 1293 10408 1 _ 3 . A __ 1 {- mew ; 6167 £3 51qu * :f- ijT'i-Qi‘s 0‘ If gvfiggkd‘ J” ’l "A - )'-"‘-~":I'ré (59% L ' if 3;“! (M2? I i. wm“: {33¢ Zn33 3L3] i j a"? L . tum, 1‘ This is to certify that the dissertation entitled STUDIES IN ALLIUM CEPA I. DEVELOPMENT OF AN ANNUAL GENERATION CYCLE II. PROTOCOL FOR PROTOPLAST' ISOLATION AND CULTURE presented by Eric Ayeh has been accepted towards fulfillment of the requirements for Ph . D . degree in Horticulture and Genetics Dr. James F. Hancock Major professor Date August 9, 1982 MS U is an Affirmative Action/Equal Opportunity Institution 0-12771 MSU LIBRARIES RETURNING MATERIALS: Place in book drop to remove this checkout from your record. FINES will be charged if book is returned after the date stamped below. STUDIES IN ALLIUM CEPA I. DEVELOPMENT OF AN ANNUAL GENERATION CYCLE II. PROTOCOL FOR PROTOPLAST ISOLATION AND CULTURE By Eric Ayeh A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Horticulture and Genetics I982 ABSTRACT STUDIES IN ALLIUM CEPA I. DEVELOPMENT OF AN ANNUAL GENERATION CYCLE II. PROTOCOL FDR PROTOPLAST ISOLATION AND CULTURE By Eric Ayeh In an effort to decrease the time required to produce long- day hybrid onion cultivars, an annual seed-to—seed generation cycle was devel0ped. A five x two x five factorial experiment was used in a randomized block design with 5 genotypes, 2 plant ages, and 5 vernali- zation periods. Significant differences were found among genotypes, vernalization periods, and the interaction genotype x plant age. Long- day genotypes bloomed earliest when young plants (86 days) were vernali- zed and short-day genotypes bloomed earliest when older plants (l54 days) were subjected to cold treatment. The fastest seed to seed generation cycle observed was l0 months. A second study was conducted to determine a protocol for onion protoplast isolation and culture. Two weeks and 3~month-old seedlings representing physiologically active cells and storage bulbs (dormant state of growth) were exposed to vernalizing and nonvernalizing tem— peratures. Protoplasts were isolated from leaf, flower scape, bulb and stem disc tissues and cultured in variable media. AProtdplast yields and viability were significantly greater when derived from _ Eric Ayeh physiologically active tissues exposed to vernalization temperature regardless of enzyme or asmoticum types. Similarly, streaming and division were only observed in protoplasts derived from vernalized tissues. Zeatin was required for cell wall regeneration in mesophyll and flower scape protoplasts, but bulb protoplasts regenerated cell walls in the absence of zeatin. The results suggest that protOplasts derived from cold stimulated plants mimic cold temperature effect on cells in vivo. r... ACKNOWLEDGMENTS My appreciation and thanks to Dr. Jon Forbes, my major advisor, for his guidance and encouragement during this study. Thanks also to the other guidance committee members: espe- cially Dr. Hugh Price for his advice and interest in my work. Drs. David Reicosky, Clifford Pollard, and James Hancock for their review of this manuscript. Sincere appreciation is also expressed to Dr. Kenneth Sink for the use of his research facilities and to his advice and review of this manuscript. Others must also be thanked: The field study of the Department of Horticulture and fellow students for their support during my study. Warmest appreciation to my parents for their patience and support throughout my education. ii TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES INTRODUCTION LITERATURE REVIEW Morphology and Development . Effects of Temperature on Bulbing and Flowering Somatic Cell Culture in Monocotyledons . Chapter I. DEVELOPMENT OF AN ANNUAL GENERATION CYCLE IN A, CEPA . Introduction Materials and Methods Greenhouse Treatment . . Cold Temperature Treatment . Results and Discussion . ONION PROTOPLAST ISOLATION AND CULTURE Introduction Materials and Methods Harvesting of Explants . Surface Sterilization of Explants Protoplast Isolation . Preparation of Protoplasts . Protoplast Count and Staining Estimation of Percent Viability and Protoplast Survival . . . Culture Media Enzyme Sources . Results and Discussion . Isolation of Mesophyll and Flower Scape .Protoplasts. Isolation of Bulb and Stem Disc Protoplasts iii Page vi —_J 0301-5 4}- Chapter Page Culture of ProtOplast . . . . . . . . . . . 36 Leaf and Flower Scape . . . . . . . . . . 36 Bulb and Stem Disc . . . . . . . . . . . . 44 Biennial Periodicity and Protoplast Culture . . . . 44 LITERATURE CITED . . . . . . . . . . . . . . 48 iv Table l0. ll. 12. I3. LIST OF TABLES Tissue Culture in the Genus Allium: State of the Art . Interaction of Vernalization Period, Genotype, and Seedling Age on the Induction of Flowering in Alluim cepa‘L. Analysis of Variance of the Effect of Vernalization Period, Genotype and Seedling Age on Flowering in Allium cepa L. . . . . . . . . . . Genotype and Age Interaction with Long Day (LD) and Short Day (SD) Onion Cultivars . . . . Independent Comparisons for Vernalization Periods in Allium cepa L, Enzyme Solutions Used in Onion Protoplast Isolation Onion Protoplast Culture Media Viability and Yield Estimates of Enzyme Extracted Protoplasts from Leaf Tissues using KCL as the Osmoticum . . Viability and Yield Estimates of Enzyme Extracted Protoplast from Leaf Tissues Using Mannitol as the Osmoticum . . . . . Yields of Mesophyll Protoplasts Extracted from Tem- perature Pre-Treated Plants . . . . Analysis of Variance of Temperature and Osmoticum Influences on Mesophyll Protoplast Yields . Protoplast Yields from Bulb and Stem-discs Maintained Under Differing Conditions . Survival and Cytoplasmic Streaming in Protoplasts Derived from Different Sources and Conditions Page 8 l2 l3 l4 IS 25 26 32 33 34 35 37 38 Figure IO. ll. LIST OF FIGURES The mean and cumulative values of flowering plants against vernalization period (7°C) Relative length of phases for an annual generation cycle in onion: l8 hr photoperiod Histogram depicting yields of mesophyll protoplasts extracted in enzyme solution E3 (Table 6) . . . MeSOphyll protoplast of onion . Fluorescence of regenerated cell walls of onion meSOphyll protOplasts treated with Calcufluor White Onion mesophyll protoplasts showing enlargement and elongation prior to streaming . . . Flower scape protoplast showing cytoplasmic channeling and streaming . . . . . . . Onion mesophyll protoplast undergoing apparent first and second division . . . . . . . . Onion flower scapes showing hollow and nonhollow cavities . . . . Onion flower scape protoplasts derived from plants maintained at l0°C . . . . . . Mesophyll protOplast undergoing apparent budding vi Page l6 l9 3T 40 4O 4O 42 42 45 45 45 INTRODUCTION Male sterility has been described in many economic plant species. In both natural and cultivated plant populations, male sterility serves as a dependable outbreeding mechanism. Cytoplasmic male sterility (CMS) has been found and utilized in the production of hybrid seeds in: sugar beet (Lichter, l978), sorghum (Shertz et al., l978), corn (Duvick, l965), carrot (Thompson, l978), onion (Jones and Mann, l965), radish (Ogura, l968), and rice (Pearson, l98l). CMS has the advantage of being maternally inherited and large amounts of sterile progenies, can be produced depending on the presence or absence of nuclear gene restorer(s). Male sterility can also be used to eliminate detassling (i.e., in corn) and emasculation (i.e., in onion, carrots, etc.) in hybrid seed production. The cultivated onion is self-compatible, but is predominately an outbreeder that is subject to inbreeding depression. Its out— breeding mechanisms protoandry flowering (pollen matures before stigma becomes receptive) and insect pollination (Pearson, T981) make the use of CMS in hybrid seed production feasible, but hybrid seed yields are decreased as a consequence of backcrossing. Additionally, the biennial generation cycle of the crop makes time consuming the conventional breeding methods of simultaneously transforming CMS and developing inbred parent lines. The characteristic pollen sterility of CMS in the onion is the result of microspore degeneration after meiosis, phenotypically expressed as brown anthers (Monsmith, 1928). It is under the con- trol of a recessive nuclear gene and a cytoplasmic factor (Jones, l943). Since bulbs are the most economically valued part of the onion plant, sterility of the F1 plants is of little importance. With conventional breeding, a minimum of six backcross generations (l2 yrs.) to the recurrent, CMS recipient parent is needed to insure successful incorporation. This is a relatively long period of time and shortening the generation cycle for a more rapid backcrossing scheme is desirable. The somatic transfer of CMS via protoplast fusion has been achieved in two plants. Zelcer (l978) and co-workers transferred CMS from Nicotiana tabacum into the nuclear genome background of N, sylvestris by making the N. tabacum nucleus nonfunctional through x-irradiation. Izhar and Power (l979) used a selective media for the recipient Petunia axillaris to transfer CMS from E, hybrida. They used a media which selected against the genome of hybrida and nuclear hybrids between the two species. Successes via cell fusion transfer of plant cell genome or genes in other crop plants awaits refinement in cultural techniques to the point where functional plants can be regenerated from protoplasts and appropriate makers are available for selection in culture. The examples with Nicotiana and Petunia demonstrate two different techniques which can transfer CMS in diploid organisms while keeping intact the recipient nuclear genome. Use of enucleated protoplasts in creating cybrids provides a third alternative to com- bine specific cytOplasmic and nuclear genomes in plant cells. The successful use of high speed centrifugation and density gradients to isolate subprotOplast from cultured cells in Hordeum and Zea mays (Lorz et al., l980) and the production of enucleated protoplast from the epidermis of onion bulbs (Bradley, 1978) suggest that CMS can be transferred via protoplast fusion in onion. However, the use of these techniques in onion has been limited since regeneration to plants from protoplasts has not yet been achieved. The objectives of this research were to (l) develop an annual seed-to-seed cycle for greenhouse seed production, and (2) develop a protocol for onion protoplast isolation and culture as a prerequisite for the eventual transfer pf CMS using protOplast fusion. LITERATURE REVIEW The ontogeny of organ formation and devel0pment in Alligm varies among different species, but in fl._gepg ontogeny also depends on the environmental conditions under which the plant is grown. The most important traits of the cr0p involve the bulbing response which is controlled by daylength and high temperatures, and the bolting response which is regulated by low temperatures (Jones and Mann, l963; Health and Holdsworth, l948; Heath, l944, l945; Hoffman, l933). Seeds from the same packet, planted under different environmental conditions of temperature and daylength, may provide scallions, sets, large bulbs or seeds. To understand these differences and their possible effects on the annual breeding cycle and onion protoplast culture, a review on onion plant development is provided. Morphology and Development After the seedling is established, the young onion plant continues to produce new foliage and adventitious roots, and the stem slowly elongates and broadens. Early in development the entire leaf is solid and meristematic; at a later date only the sheath and base of the blade isneristematic; and still later only the base of the sheath has the capacity to grow since the rest of the leaf becomes hollow (Hoffman, T933; Jones and Mann, 1963). Before this cavity is formed, all the inner cells of the leaf contain functional nuclei, 4 although the volume of the intracellular space is probably greater than that occupied by the cells. The mature leaf consists of this central cavity, surrounded by eight to ten layers of parenchyma with large intracellular spaces, the vascular bundles, the palisade layers and the epidermis (Hoffman, l933). The shoot apex, found at the center and upper side of the broadstem (stem-disc) is protected at the bottom of the youngest tubular leaf. Each leaf surrounds the next younger leaf. The inflorescence axis or flower scape always arises at the apex of the stem. It follows the same growth pattern of a leaf with the scape becoming hollow at a later date. This pattern of growth, described for the common onion, is essentially the same in all cultivated Species of Allium. Effects of Temperature on Bulbing_and Flowering With inductive daylength, the onset of bulbing is character— ized by the swelling of one or more of the innermost enlarged leaves and three or more of the outermost, nonemerged leaf initials. The swelling results from an increase in cell size and the develOpment of intracellular spaces, without cell division (Heath, l945). For a given daylength, bulbing is accelerated by high temperature and is greatly delayed or prevented by low temperature. Several workers have shown that the emergence of leaves normally ceases immediately or soon after bulb induction depending on temperature (Heath, l943a, l943; Heath and Holdworth, l943a, Aoba, l954). Thus, the entire process of bulbing and maturation does not include cell division as long as temperatures are kept at levels noninductive for flowering. Cold temperatures revert the process taf bulbing by inducing or maintaining cell-division at the shoot apex and at the young leaf initials (the same cells required for enlargement in bulbing). Photo- period and noninductive temperatures for flowering may be required in concert Uainduce bulbing. The existence of a critical bulb or plant size and a minimum requirement of cold temperature exposure for flower induction has been documented by several workers (Jones and Emsweller, 1939; Heath, 1944, 1945; Mather and Heath, 1944; Woodbury, 1950; Shishido and Saito, 1976; and Hesse et al., 1979). An effective vernalization tempera- ture range of 5° to 12°C for flower induction (Demille and Vest, 1975) and a lack of effect of photoperiod on flower initiation or emergence has also been reported (Heath, 1945; Shishido and Saito, 1975). The plant or bulb size plays a major role in triggering cold temperature responses for flowering. A critical mother bulb size (Jones and Emsweller, 1939) and plant (Shishido and Saito, 1976) have both been recognized as factors which affect flower induction in the cultivated onion. Gregory (1936) refers to this critical size as "the ripeness to flower.“ Somatic Cell Culture in Monocgtyledons The techniques of culturing i__vitro single somatic cells, protoplasts or micrOSpores under defined conditions, and proceeding via somatic embryogenesis or organ regeneration to a genetically stable plant has been demonstrated with a few species, mostly herba- ceous dicots (Potrykus, 1979). Several workers have found that monocotyledons often do not respond well to the wide range of con- ditions that successfully induce division of somatic cells in herba- ceous dicotyledons (Carter et al., 1967; Krikoran et al., 1969; Hussey, 1975; Dudits, 1976; Koblitz, 1976; Thomas et al., 1978; Potrykus, 1979, 1980). However, within the last decade and a half, an increasing number of monocots have been regenerated via tissue or cell culture. Plant regeneration from mesocotyl derived callus has been obtained in all cereals (Potrykus, 1980). Propagation by tissue culture of members of the Liliaceae, Irideacea, and Amarylli— daceae has proven highly successful (Hussey, 1975, 1979). Plant regeneration from basal disc and root derived callus of 5. £222 and from the leaf, stem and apical meristem of 5. sativum has also been successful (Table l). Callus has been derived from protoplasts in such cereals as Ogyza sativa (Deka et al., 1976; Tsai et al., 1978). Triticum gp, (Dudits et al., 1976), leg mgyg (Potrykus, 1977, 1979), Hordeum vulgare (Koblitz, 1976). Sorghum bicolor (Brar et al., 1980) and saccharum gp. (Maretzki et al., 1972), but attempts to regenerate plants from protoplasts in any of the aforementioned species has not been successful. So far only four monocots have not been recalcitrant in culture; successes have only been obtained in Bromus inermis (Kao et al., 1973) Asparagus officinalis (Ha and Mackenzie, 1973), Pennisetum americanum (Vasil et al., 1980) and Manihot esculenta (Shanin et al., 1980). Protoplast studies in the genus Allium have been limited to morphological and physiological studies of the cell, involving protoplasts isolated from the root and bulb epidermal layer of A. cepa (Table l). TABLE 1. Tissue Culture in the Genus Allium: State of the Art. _.__. .- *~ ~ w- References Allium porrum (Leek) Basal disc + Callus Allium cepa (Onion) Basal disc + Callus Callus + Root Callus + Shoot Root + Callus Callus + Shoot Callus » ProtOplast Bulb + ProtOplast Epidermal layer + Protoplast Flower head + Shoot Embryo 4 Callus Allium sativum (garlic) Leaf » Callus Callus » Shoot Callus 4' Plant Apical meristem 4 Callus + plant Stem + Callus Callus 4 Shoot Callus + Embroids Shoot buds + plant A. fistolosum Shoot tips + plant Dunstan and Short, 1979 Dunstan and Short, 1977 Fridborg, 1971 Dunstan and Short, 1978 Klein and Edsall, 1968 Selby and Collin, 1975 Dunstan and Short, 1978 Bawa and Torrey, 1971 Roland/Prat/Pilet, 1971 Vreugdenhill, 1957 Hepler/Zeigler, 1976 Schnaubl/Borman/Zeigler, 1978 Bradley, 1978 Dunstan/Short, 1978 Guha/Johri, 1966* Havranek/Novak, 1972, 1974 Mostafa/El-Nil, 1976 Novak, 1979 Kehr/Schaeffer, 1976 Mostafa/El-Nil, 1976 Mostafa/El-Nil, 1976 Mostafa/El-Nil, 1976 Bhojwani, 1980 Fujieda/Ando/Fujita, 1977 *Including 13 yitro development of ovary and ovules. CHAPTER I DEVELOPMENT OF AN ANNUAL GENERATION CYCLE IN A. CEPA Introduction External environmental factors (i.e., temperature and day- length) play an important role in the develOpment of the onion. The literature on E- ggpg indicates that there is a critical plant or bulb size and vernalization regime which affects flower induction. A vernalization range of 5 to 12°C is optimum for flowering in all cultivars, while the critical bulb size varies between genotypes—- a minimum plant diameter of 3-6 mm across cultivars was observed by Shishido et a1. (1976). Daylength does not appear to control flower initiation directly (Heath, 1945), but it seems to modify the effect of temperature on flowering (Shishido et al., 1975). The objective of this study was to determine how plant age, genotype and vernalization period affect the generation cycle of onion under Michigan greenhouse conditions. Materials and Methods The experimental design was a 5 x 2 x 5 factorial in a ran- domized complete block. The variables were 5 genotypes, 2 plant ages, and 5 vernalization periods. Five plants per experimental unit 10 were used in each of two blocks, yielding a total of 250 plants per block. Onion inbreds MSU 6693A, MSU 23998, and their hybrid progeny MSU (6693 x 2399) were utilized as examples of longday (LD) onion. Dessert Seed Company inbred 986 and the hybrid cultivar 'Dessex' were included as short day (SD) onions. Greenhouse Treatment Seeds of all 5 genotypes were sown in January in separate flats filled with Houghton Muck soil and kept moist. Soon after germination (35 days), seedlings were transplanted into 10 x 10 cm clay pots and were placed in 2 greenhouses under conditions of 26 i 2°C day/20 : 2°C night. They were irrigated 4 times per week and fertilized twice a week with a 20-20-20 mix of 200 ppm of nitrogen per application. Greenhouse lighting was supplemented with fluores- cent lamps to maintain an 18 hr. photoperiod. Sixty-eight days after the first sowing date, an additional set of seeds from all 5 genotypes were sown into similar flats and treated as above. The 50 treatment combinations were randomized in each greenhouse. Cold Temperature Treatment When the individuals sown on different dates were 86 and 154 days old, they were transferred into a cold room maintained at 7°C with an 18 hr. photoperiod. Watering was reduced to one application per week and fertilization to one application of 20—20-20 fertilizer every other week. Groups of 100 plants were returned to the green— house at 8, 10, 12, 14, and 16 week intervals. Upon return to the ll greenhouse, plants were tagged on the first day of umbel (flower head) appearance. Results and Discussion Overall, LD genotypes yielded the highest number of umbels, with the young (86 day) plants being the most responsive. Although all vernalization regimes were effective in inducing flowering, the longer vernalization periods yielded greater amounts of flower heads (Tables 2 and 5); the 16 week treatment produced significantly more flowers than 14 weeks, although there was no significant difference between 8 and 10 weeks. The significant effects of longer vernaliza- tion periods on flower head formation (Table 5) and the cumulative response of increased vernalization period (Table 2) suggest that longer cold temperature exposure periods are a significant factor in flower initiation. There was also a significant genotype x age interaction (Table 3). Both of the older (154 day) SD genotypes yielded more flower heads than the younger ones, but in the LD genotypes, 86-day— old plants produced twice as many flower heads as older ones (Table 4, Figure 1a). LD genotypes MSU 23998 and MSU (6693 x 2399) showed sig- nificant differences, but not MSU 6693A. Considerable variability was found in the age necessary for flowering in long— and short-day onion genotypes. Long—day plants flowered after 86 days of vegetative growth, whereas short-day plants required longer periods of time. Umbels appeared within 30 days upon return of the plants to the greenhouse following vernalization. Thus, 12 .xmv “cosm11om .xwv m:o_-1o4 mmcwpysn Low xcmmmmum: vOWLwaowoga P Fm— CW LO I {I'll Ill 1' I... null ll ‘ All I III, I! 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U0 .QEmH mULDOm 58L: so c. na:_aeocwm owamc_30uxu a u_ oczufiau ummPQOuoca .mc: we Loucc _c>_>c:m --.goecccmzu. _ .. _ ..- : .mcopu_v:ou new mooL30m “cococc_o EoLc cm>_cco mamopachcg :_ 7:_Ecccum o_Emc_a0uxu new _m>w>czm .m_ mgmqe j at aft 5111 no de ad CE 39 flower scape protoplasts. Protoplasts were incubated in the dark at three temperature levels and pr0t0p1ast survival was estimated after 48 hrs. Leaf and flower scape protoplasts cultured in BDS supplemented with only 2,4-D and 6BAP remained alive for 7 days with no apparent cell wall formation. Cell wall formation could be detected by Calcufluor staining after 4 days in medium with zeatin added (Figure 5). Approximately 60% of the pr0t0plasts regenerated cell walls during a 21—day culture period. Leaf and flower scape protoplasts cultured in Medium I showed elongation and enlargement of the protoplast by the 5th day in cul— ture (Figure 6). The resultant oblong-shaped pr0t0p1asts also showed cytOplasmic streaming characterized by an enlargement of the nucleus, and a more pronounced cytoplasm with diffused and dispersed cytoplasmic inclusions (Figure 7). The cytoplasms which were streaming were heavily channeled and chloroplasts could be seen in motion under the light microsc0pe. Anywhere from 10 t0 approxi- mately 90 percent of pr0t0p1asts were recorded streaming after 7 days in culture. When treated in conjunction with 6BAP at 0.3 mg/l, both 2,4—0 and zeatin at concentrations of 0.3 or 0.5 mg/l increased the number of pr0t0p1asts undergoing streaming. First division depicted by cytokinesis was also noticed in a few instances after ten days in culture (Figure 8). Figure 4. Figure 5. Figure 6. 4O Mesophyll protoplast of onion. Fluorescence of regenerated cell walls of onion mesophyll protoplasts treated with Calcufluor White. Onion mesophyll protoplasts showing enlargement and elongation pri0r to streaming. 41 Figure 7. Figure 8. 42 Flower scape protoplast showing cytoplasmic channeling and streaming. Onion mesophyll pr0t0plast undergoing apparent first and second division. 43 W1 44 Bulb and Stem Disc While the hormone zeatin was necessary for cell wall forma- tion in leaf and flower scape protoplasts, bulb pr0t0p1asts regen— erated a cell wall after a week in culture in the absence of zeatin. Bulb pr0t0p1asts survived up to 12 weeks in culture with no obvious signs of protoplast structural changes. Stem disc pr0t0p1asts sur— vived up to 25 days in culture without apparent regeneration of cell wall. Biennial Periodicigy and Protgplast Culture The yield and culturability of mes0phyll protoplasts derived from 10°C treated plants, was superior to those pretreated at 26°C. Besides bolting, the response of the plant to cold temperature was also manifested in leaf explants from plants ”ripe to flower" and from flower scape tissue during their development at 10°C. This response was characterized by nonhollow leaf and scape cavities (Figure 9). Apparently, the leaf meristematic region mentioned by Hoffman (1933) continued to divide once triggered when it is main- tained under vernalization temperatures. In nutrient media solution, meristematic protoplasts float to the t0p of the tube after centri— fugation for 15 minutes at 35 x g and can be separated from the pre- cipitating me50phyll pr0t0p1asts or the denser parenchyma proto- plast of the scape (Figure 10). Me50phyll pr0t0p1ast from ”nonripe to flower” but vernalized seedlings (2 weeks-old seedlings) had the lowest yields at all 10°C Figure 9. Figure 10. 1 Figure 11. l l 45 Onion flower scapes showing hollow and nonhollow cavities. 9-1 and 9-3. Onion flower scape showing cavities filled with cells. These scapes developed at 10°C. 9-2. Onion flower scape showing hollow cavity. This scape developed at 26°C Onion flower scape protoplasts derived from plants maintained at 10°C. Note the large meristematic and small parenchyma protoplasts. Mesophyll protoplast undergoing apparent budding. 46 47 pretreated plants (Tables 8 and 9). None showed streaming or division in any of the four culture media tested. The characteristic biennial periodicity known in the culti- vated onion has been shown to be under the control of temperature. Inductive temperatures of 5°-12°C induce flowering and prevent bulb- ing (Jones and Emsweller, 1939; Heath, 1943, 1944, 1945; Aoba, 1954). This cold temperature effect, requiring a critical plant or bulb size, is responsible for a qualitative change at the cellular level and not the induction of ripeness to flower (Gregory, 1936; Shishido and Saito, 1976). The onset of or cessation of cell division in inducing flowering and bulbing respectively is a direct product of temperature in enhancing flower and bulb organs formation, the same organs defining biennial periodicity in the onion crop. The data generated in this study reveal that vernalization temperature also influences the onion cells' behavior ig_gi£:g. Only protoplasts from plants responsive to vernalization stimulus showed cytoplasmic stream- ing and division in culture. The high pr0t0p1ast yields and via- bility of vernalized plants and the continuous and vigorous cell division within the cavities 13f vernalized leaf tissues and flower scapes are direct evidence of qualitative changes at the cellular level, induced by temperature, and expressed in culture. LITERATURE CITED 48 Ab Ac Be LITERATURE CITED Abo El-Nil, M. M. 1977. Organogenesis and embryogenesis in cul- tures of garlic. Allium satirum. Plant Science Letters 9:259. Aoba, B. 1954. On bulb formation and dormancy in onion II. On the process of bulb formation and development of scales. J. Hort. As§. Japan 23:249. Bawa, S. B. and Torrey, I. G. 1971. ”Budding” and nuclear division in cultured protoplasts of corn, convolvulus and onion. Bot. 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