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(I .-..11;. .1. 11‘s o 11 ....Iq|f.u.cu.l.i(.. .(1: .3 7 11:1. 5. 1.. :tunry C .y...crr..0.1..{l..|r&oh..£h.11 g‘akuolqllb: r: CuJHth? 7 1.1 1 1. .3... to .1 .1l. . . . 1| .. \. A". 1 to; .. I}. 1 . . 1 1 $145314 P4522: bur-usuaiiul. 1... 10“. t. :5 .r Fh‘u :1 . ' 3' 'l . I III 7Jt€lq7él SETAT EUNIVERSITY L IBIRARIE'S\|IFi ”ERA“ lllllllllllTIM “33M “Hill; Ill Michigan State 3 1293 00572 University This is to certify that the dissertation entitled SOMATIC EMBRYOGENESIS IN ASPARAGUS OFFICINALIS L. presented by Amnon Levi has been accepted towards fulfillment of the requirements for Plant Breeding Ph. D . degree in and Genetics MIKK/a Major professor Date I "go -' Fig? MSU is an Affirmative Action/Equal Opportunin Institution PLACE N RETURN BOX to remove thle checkout from your record. TO AVOID FINES retun on or hetero dde due. ll DATE DUE DATE DUE DATE DUE l ll g— —--l —T__\. MSU le An Affirmative AdionlEquel Opportunity Institution SOMATIC EMBRYOGENESIS IN AfiEABAfiQfi QEEIQIHALIS L. BY Amnon Levi A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Horticulture 1989 1,:2 ‘i V l V9L/ ABSTRACT SOMATIC EMBRYOGENESIS IN ASEABAQQS QEEIQIEALIS L. by Amnon Levi The roles of explant source, auxin type and concentration, and carbohydrate source and level in asparagus somatic embryogensis were examined. Calli derived from in vitro established crowns (IVC) and lateral buds (LB) had a high embryogenic capacity; yielding 81-127(IVC) and 46-69 (LB) globular and bipolar mature embryos/g callus, on induction media (IM): Hurashige and Skoog salts and vitamins (MS) + 2,4-0 or NAA (0-0.1ppm) or kinetin at levels of 0.01- 0.1ppm. Calli derived from spear sections (SS) had a lower embryogenic capacity where only 9-23 embryoids/g callus formed on IN. The auxin 2,4-D at levels of 1-10ppm induced a high embryogenic frequency. However, 2,4-D also induced formation of abnormal embryos and a low rate of embryo conversion to plantlets on maturation medium (HM) of MS + NAA (0.05ppm) and kinetin (0.1ppm). On the other hand, NAA of the same levels induced a lower embryogenic frequency. However, NAA promoted normal embryo formation and a higher conversion rate than 2,4-D; up to 2(SS), 10.7(LB) and 13.7 plantlets/g callus (IVC) within six weeks on MM. Glucose (4-5%) in In containing 2,4-D (1.5ppm) significantly increased embryo formation while sucrose of the same levels had an intermediate effect and fructose reduced formation. However, in IN containing NAA (1.5ppm) the effect of glucose on embryo formation was not as pronounced, and there was no significant difference between glucose and sucrose, while fructose reduced formation. Fructose in MM enhanced the rate of embryo conversion into plantlets, and the combination sucrose in IN and fructose in MM at the level of 5% gave the highest rate (10.2 plantlets/g callus). Carbohydrate levels significantly affected conversion rate where levels of 2% gave the lowest while 5% yielded the highest rate; 3.4 and 7.4 plantlets/g callus, respectively. Embryogenic cell suspension cultures have been established and maintained over a 10 month period in liquid IH of MS + NAA (10-20ppm). Globular embryos were transferred from liquid IM to solidified MM containing glucose, fructose or sucrose (2-10%). Carbohydrate levels of 4-10% in HM enhanced the germination of somatic embryos to plantlets by 3.6-8.5 fold compared to 2%. Furthermore, transfer of the embryos from these levels to NM with a lower level (2%) enhanced the conversion of embryos‘ into plantlets by 2-4 fold. DEDICATION To my dear friend, Allison Fine and her children, Sheba, Ben and Nani iv ACKNOWLEDGMENTS My gratitude to Dr. K.C. Sink for his dedicated support and supervision. Thanks are also due to my co-workers R. Muleo, T. Ball, J. Smeenk, L. Dunbar, A. Guri, Miguel, and all my friends in the Department of Horticulture. Also a special thanks to Sherry Mulvaney, Gloria Blake and LuAnn. Gloden. Gratefully acknowledged are the valuable discussions and suggestions made by Drs. J. Hancock, 8. Sears, G. Hosfield, J. Saunders and F. Dennis. TABLES OF CONTENTS LIST OF TABLES O O O O O O O O O O O O LIST OF FIGURES O O O O O O O O O O O O O INTRODUflION O O O O O O O O O O O O O O LITERATURE REVIEW . . . . . . . . . . . . SECTION I SOMATIC EMBRYOGENSIS IN ASEABAQHS QEEIQIHALIS L. THE ROLE OF EXPLANT SOURCE AND GROWTH REGULATORS . . . Abstract . . . . Introduction . . Materials and Methods Results . . . . . Discussion . . . . Literature Cited . . SECTION II THE ANATOMY AND MORPHOLOGY OF ASPARAGUS QEEIQIHALIS L. SOMATIC EMBRYOS . . . . . . . . . . . . . Abstract . . . . Introduction . . Materials and Methods Results and Discussion Literature Cited . . O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O vi Page viii xi 50 51 52 54 57 74 80 83 84 85 86 88 98 INTERACTION OF SUGAR TYPE AND CONCENTRATION EMBRYOGENSIS IN AEBABAQQE QEEIQIEALIE Abstract Introduction Materials and Methods Results . Discussion . Literature Cited . PRODUCTION OF ASPARAGUS OFFICINALIS L. SECTION III SECTION IV THROUGH SUSPENSION CULTURE Abstract Introduction Materials and Methods Results . Discussion . Literature Cited . APPENDIX vii L. ON SOMATIC SOMATIC EMBRYOS 100 101 102 104 106 122 127 130 131 132 133 137 152 157 159 LIST OF TABLES Table Page SECTION I 1. AOV’s for callus increase, frequency of somatic embryos and mature plantlets on IM and MM for three asparagus explants . . . . . . . . . 65 2. SS-derived callus on IM and MM: Growth response and frequency of embryos and plantlets . . . . 66 3. LB-derived callus on IM and MM: Growth response and frequency of embryos and plantlets . . . . 68 4. IVC-derived callus on IM and MM: Growth response and frequency of embryos and plantlets . . . . 70 SECTION III 1. AOV’s for callus increase and frequency of somatic embryos as influenced by CHO type and concentration in IM and MM . . . . . . . . 114 2. The combined effect of CHO type and concentration in IM containing 2,4-D, and in MM on callus increase, frequency of globular (<3mm) and bipolar (4-7mm) embryos, and rate of conversion into plantlets . . . . . . . . . . . . . . 116 3. The combined effect of CHO type and concentration in IM containing NAA, and in MM on callus increase, frequency of globular (<3mm) and bipolar (4-7mm) embryos, and rate of conversion into plantlets . . . . . . . . . . . . . . 119 SECTION IV 1. Frequencies of single cells, organized cell clusters and globular embryos in liquid suspensions . . . . . . . . . . . . . 145 2. AOV’s of embryo frequencies as influenced by CHO type and concentration sequence . . . . . . 146 viii Number and type of somatic embryos and plantlets per gram callus as affected by CHO type and concentration in MM . . . . . . . . . . Root development and callus formation at the shoot base of plantlets on MM and survival rate of plants two weeks after transfer to planting medium . . . . . . . . . . . . . . . ix 147 149 Figure Figure Figure Figure APPENDIX Profile of embryo types in SS-derived callus induced on MS containing 2,4-D (0.5- 3ppm) for four weeks, followed by transfer to NAA (lppm) for six weeks . . . . . Combined effect of sorbitol and sucrose combination in MS + (ppm): NAA (0.3) and kinetin (0.8) on formation of shoots in SS- derived callus . . . . . . . . . . Effect of sugar type and concentration in IM containing 2,4-D (1.5ppm) on callus growth and formation of small globular embryoids. No. embryoids per gram callus . Effect of sugar type and concentration in TM containing NAA (1.5ppm) on callus growth and formation of small globular embryoids. No. embryoids per gram callus . . . . . Page 160 161 162 163 LIST OF FIGURES Figure Page SECTION I 1. SS-derived callus on IM and MM: Growth response and frequency of embryos and plantlets . . . . 67 2. LB-derived callus on IM and MM: Growth response and frequency of embryos and plantlets . . . . 69 3. IVC-derived callus on IM and MM: Growth response and frequency of embryos and plantlets . . . . 71 4. Frequencies of embryo types during 6 months on MM, following IM + 2,4-D or NAA and kinetin (0.1 - 1.5 ppm) O O O O O O O O O O O O O O O 7 2 5. Somatic embryos at different developmental stages on W O O O O O O O O O O O O O O O 73 SECTION II 1. A) Callus on induction medium showing early formation of embryogenic initial cells, B) Suspensor cells formed in embryogenic callus, C) Early globular stage in embryogenic callus, D) Early bipolar embryo, E) Bipolar embryo, F) Median longitudinal sections through an embryo in the late maturation stage, G) Shoot meristem forming in a mature embryo, H) Longitudinal section through shoot apex of mature embryo . . 94 2. A) Embryonic callus, B) Mature elongated somatic embryo, C) Zygotic embryos, D) Mature embryo with a well developed cotyledon, shoot primordia, and short radical, E) Typical germinating somatic embryos . . . . . . . . . . . . . . 96 SECTION III 1. The effect of sugar type and concentration in induction media containing 2,4-D (1.5ppm) on callus growth and formation of small globular embryoids . . . . . . . . . . . . . . 115 xi The combined effect of sugar concentration and type in TM containing 2,4-D, and in MM on callus increase, frequency of small (<3mm) and large (4- 7mm) embryos, and rate of conversion into plantlets . . . . . . . . . . . . . . The effect of sugar type and concentration in IM containing NAA on callus growth and formation of small globular embryoids . . . . . . . . . The combined effect of sugar type and concentration in IM containing NAA and in MM on callus increase, frequency embryos, and rate of conversion into plantlets . . . . . . . . Typical mature embryos and plantlets on MM containing sucrose, glucose, and fructose . . . SECTION IV Frequencies of embryos and plantlets as affected by sugar type and concentration sequence in MM . A) Embryo in early globular stage in liquid suspension. B) Bypolar embryo on MM. C) Mature embryos after two weeks on MM. D) Plantlets on MM. E) Crowns on MM . . . . . . . . . . xii 117 118 120 121 148 150 IHTBQDQSTIQN Asparagus officinglig L. is a monocot species that provides interesting challenges for breeding and physiological research. Asparagus has a low multiplication rate using traditional horticultural methods (Anderson and Ellison, 1965). These methods have restricted asparagus breeding programs and the selection and propagation of elite crowns (Yang and Clare, 1975). However, in recent years, efforts have been focused towards production of selected asparagus crowns by in vitro propagation (Murashige et a1. 1972: Hasegawa et al. 1973: Yang and Clare, 1973: Yang, 1977: Volokita et al. 1987). Despite many experiments, monocots have been found much more difficult to regenerate in culture than dicots (Vasil, 1987). However, successful regeneration and preliminary work on somatic embryogenesis has been reported in asparagus (Wilmer and Hellendorn, 1968: Steward and Mapes, 1971) and in other species of the Liliaceae family (Reuther, 1983). The goal of this work was to examine the roles of explant source, growth regulators and carbohydrate source and concentration on asparagus somatic embryogensis. Based on the information obtained in these experiments, preliminary studies on large scale production were 2 conducted. It is demonstrated here that somatic embryogensis . in asparagus can be obtained through different explant types and media sequences and that explant source, hormonal and nutritional factors play an important role. REVIEW OF LITERATURE The genus Asparagus belongs to the Liliagsas family and was first described by Lineaus (1775). Asparagus is comprised of about 150-300 herbaceous and woody perennial species spread throughout the temperate and tropical regions of the world (Baily, 1942: Lawrence, 1982). Asparagus gfifiginalis L. is the only species cultivated as an edible vegetable plant. It is an European-Sibirian continental plant and belongs to the northeast Mediterranean vegetation gene center (Reuther, 1984: Peirce, 1987). Asparagus Qfifiiginalis L. is considered one of the oldest garden vegetables. The ancient Greeks introduced it to their land from the eastern part of the Mediterranean. During the Imperial Roman era, it was a popular vegetable. It was probably the Roman Legions that subsequently introduced it to central Europe. This assumption is based on the fact that wild asparagus can be found in the vicinity of the ancient Roman Legion camps (Luzny, 1979). The name asparagus probably originated from the old Iranian word "Sparaga" which means shoot, rod, spray. The Greeks used the word "Asparagos". This was converted to the common name ”Asparagus" which has been used by Roman and other European nations (Luzny, 1979). 4 Native asparagus species are .described in several regions in Europe. Henderson (1890) described it as native to Great Britain, Russia, and Poland. It is suggested that the native forms of asparagus originated from cultivated types which then reverted to wild forms (Luzny, 1979). In Great Britain, asparagus grew in most vegetable gardens (Sturtevent, 1919). From there asparagus arrived in New England with the Puritans in the early seventeenth century. Here it escaped from cultivation and became adapted to sandy fields, roadsides and garden sites. By 1776 asparagus was growing in every colony along the Atlantic coast (Gleason and Cronquist, 1963). During the following century it became widely distributed in North America. Today, it grows everywhere in the United States except in areas of extreme heat. It is also grown as a high value crop in different parts of the world. California is the main producer in the United States, and Washington is second. In Michigan, the third largest producer, asparagus is grown on sandy soils not suitable for other vegetable crops. ' Properly maintained asparagus fields should remain productive for up to 20-25 years, producing annual yields of 3,000 pounds per acre (Ellison, 1986). However, despite the increase in asparagus production due to increased plantings, asparagus yields are significantly declining (Hartung, 1987). Presently asparagus fields are being removed after 8-15 years production due to decreased plant stands and concomitant decrease in spear size (Takatori and 5 Souther, 1978). The average yield for Michigan in 1974 was 1,600 pounds per acre: whereas, in 1981. it decreased to 900 pounds per acre. Toward 1987 it increased to 1,200 pounds per acre due to new planting (Michigan Crop Reporting Services, 1987). Most asparagus fields are planted with approximately 10,000 crowns per acre, but often fewer than half of the original crowns survive after the first five years. A 1978 survey of asparagus fields in Michigan showed that the average crown population was only 3,153 crowns per acre. This represent a 70% reduction in crown survival. This decline in yield and number of crowns is known as the ”Asparagus Decline Syndrome" and is not confined only to Michigan (Hartung, 1987). During the last four decades, asparagus production in New Jersey declined from 30,000 acres to less than 1000 acres (Herner and Vest, 1974). In California, the acreage of asparagus fields decreased from 44,000 acres in 1974 to 28,000 acres in 1978 (Takatori and Souther, 1978). Asparagus decline has also been reported in the Netherlands where it decreased from 500 ha in 1963 to 340 ha in 1970 (Vanbakel and Kerstens, 1970). In addition to the decreased longevity and productivity of established plantings, asparagus is re-established with difficulty in fields where asparagus was previously grown (Hanna, 1947: Hartungi 1987). The fusarium crown and root rot diseases are considered the main factors in the decline of asparagus fields, and no solution has yet been found (Hartung, 1987). Marxism. 11952111211211 W efficinalis is winter hardy and is considered a cool season crop with 24 to 29 C day, and 13 to 19C night temperatures favoring productivity and longevity (Peirce, 1987). As a perennial grass in the lilly family, asparagus establishes a crown which is an extensive underground mass of fleshy roots, fibrous roots, and stems (rhizomes). The rhizomes give rise to spears that develop into 4 to 6 ft tall ferns (Pierce, 1987). The fleshy or tuberous roots spread laterally to an 8-12 foot depth (Shelton, 1978). Each spring new fleshy roots arise from primary and secondary roots and also adventitously from rhizome tissue (Jones and Rosu, 1928). The fleshy roots give rise to the fibrous roots which function as absorbtive organs. The fibrous roots die in late fall at the end of the growing season and new ones develop the following spring. Each rhizome carries one or more lateral buds that give rise to new spear growth. The spears are triangular and consist of short internodes and lateral buds covered with peppery bracts. The bracts are the true leaves but do not have photosynthetic function. Instead, photosynthesis is active in the cladophylls (Pierce, 1987). Structurally, a cross section of a spear shows five anatomical regions: the epidermis, cortex, pericyclic fibers, ground parenchyma, and vascular bundles (Pierce, 1987). Elongation of spears proceeds first at the lowest internode and then within successive internodes resulting 7 in rapid spear growth. As the spears develop, cell walls in the pericycle and vascular bundles gradually become lignified, starting at the base of the spear (Sosa-Coronell, 1974). However, the fiber cell walls at the shoot tip region remain thin since no lignin deposition occurs there. Asparagus grfiisinalis L., is a dioecious species with a 1:1 sex ratio. Sometimes single plants will produce perfect flowers. This is a result of a rare mutation in the male plants which revert them to the andromoncious state. Andromoncious plants can be 10 to 20% of some asparagus populations (Wricke, 1979). The male flowers are slender, bell-shaped and greenish white in color. Each flower has an aborted ovary and well developed anther bearing orange pollen. Female flowers are smaller than those on male plants and contain vestigial, functionless anthers and a well developed ovary, style and three feathered stigma. The asparagus fruit is a red berry containing from one to six small seeds which are nearly round, flattened on one side with a hard black coat. The seeds are primarily endosperm tissue with a small elongated embryo. As the seed germinates, a radical appears first, followed by the primary stem. As subsequent shoots develop, the primary shoot senesces. There are also important differences in the growth of female versus male plants. Females produce fewer spears that are larger in diameter than those of male plants. On the other hand, male plants produce overall a greater number of 8 spears and show greater longevity than females. (Ellison and Scheer, 1959). Also, male plants do not produce seedling weeds which compete with the established crowns and may harbor diseases. For these reasons there are major efforts to produce all-male varieties (Ellison 1986). musicals Today, fusarium is considered the most limiting factor in asparagus production and resistance to it is listed as the most important goal in asparagus breeding. Over 26 vegetable varieties with resistance to fusarium wilt have been developed through plant breeding (Mace et a1. 1981). Grogan and Kimble (1959) suggested that resistant varieties are the most viable tool to arrest fusarium infection. In their selection process for developing resistant plants, they found only one asparagus plant line, which exhibited limited tolerance to fusarium. Takatori (1978) in California and Ellison (1986) in New Jersey made attempts to select for asparagus with resistance to fusarium, but to date no such cultivar has been found. Ellison (1986) also considered the possibility of using exotic germplasm in breeding for fusarium resistance. He collected numerous seed samples of Ar asurirslius in the wild i.e. in Crete, mainland Greece, Italy and Spain, and seed of wild Ar maririmus from Yugoslavia. Unfortunately, all of these accessions were found to be susceptible to fusarium. So far only the ornamental species Ar,springsrii and A; plumssus have been found to be highly resistant to fusarium, but 9 neither species will sexually hybridize with Au arrisiualis (Ellison 1986). Additional breeding goals are high yield and quality characterized by an increased number of spears of large diameter per plant, uniformity among the spears, low level of fiber, high resistance to diseases, and climatic adaptability. WW As a species with male and female plants, asparagus offers interesting breeding challenges. Lack of genetic markers is also a considerable barrier in asparagus breeding. Marker gene(s) to distinguish between male and female plants still have not been found. Such a marker would be of great value, since it would enable identification of male and female plants in early stages. So far, the inheritance of only a few morphological genes has been determined in asparagus. These include those involved in chlorophyll and carotene synthesis (Irizarry et al. 1965), and the purple stalk recessive gene which is a useful marker in identification of haploid seedlings (Bassett et al. 1971). Future mapping of the asparagus genome using isozymes and DNA techniques will make it possible to find more genetic markers useful in breeding. As a perennial grass, asparagus has a long life cycle. Asparagus plants start to yield after 4 to 5 years in the field, and the average harvesting period is about 10-15 10 years. Therefore, the development of new cultivars requires many years (Ellison, 1986). According to Ellison, Norton (1913) was the only one that was able to selecte for asparagus plants with resistance to disease. This may be because Norton consistently utilizing mass selection, screening large numbers of plants from different sources. It is likely that genetic diversity among the asparagus cultivars in North America is relatively low. Since asparagus was cultivated by the Greeks more than 2,000 years ago, and introduced by the Roman Legions to central Europe, the native forms of asparagus presently found there, may have originated from these cultivated types that reverted to wild forms (Luzny, 1979). Furthermore, it was brought to North America by the colonists from discreate regions in Europe (Gleason and Cronquist, 1963). In addition, essentially every strain of asparagus developed in the United States and Canada since 1930, has been a selection of Martha or Mary Washington (Ellison, 1986). Still, there is considerable variation in yield of individual plants of the same variety. Propagation of large numbers of clones from selected crowns will enable a better evaluation of such crowns at different field plots. Rigorous determination of the genetic diversity of asparagus would be assisted by comprehensive isozyme and DNA analyses. 11mm Asparagus has a low multiplication rate using conventional propagation methods. Only a limited number of 11 propagules can be obtained by division of a mature crown. Experiments in establishing rooted cuttings were of limited success (Anderson and Ellison, 1965). Yang and Clare (1973, 1975) offered an alternative method of propagating aerial crowns of asparagus stems in potted plants kept under high moisture. Following enlargement, shoots and roots are formed and are separated artifically to form new plants. However, this technique is slow and does not produce a large number of plants. So far, in vitro tissue culture techniques have proved to be the most promising for propagating asparagus lines (Hasegawa et a1. 1973: Yang and Clare, 1973: Chin, 1982: Reuther, 1984). Several studies have been devoted to developing practical in-vitro techniques for vegetative mass propagation of asparagus. Complete plantlets have been successfully obtained from: 1) shoot apexes excised from young lateral branches of field-grown ferns (Murashige et a1. 1972), 2) apices of terminal and lateral buds of field- grown spears (Chin, 1982: Volokita et a1. 1987), and 3) shoots and crowns produced in-vitro (Hasegawa et a1. 1973). Depending on the hormonal and nutrient composition of the medium, asparagus explants may form shoots, roots or callus. Rapid formation of shoots and roots, from shoot apices excised from buds, occurred on modified MS medium containing NAA (0.3ppm) and kinetin or ziP (0.1ppm) (Yang and Clore, 1973). Similar results were observed in other studies where auxin and cytokinin levels were varied from 0.01-0.3ppm for NAA, and from 0 - 0.1ppm for kinetin or ziP 12 (Chin, personal communication: Matsubara et a1. 1973: Reuther, 1984: Yang, 1977: Volokita et al. 1988). In addition, shoot tip explants (produced in-vitro) formed roots when induced with NAA (0.1ppm) followed by transfer to kinetin free MS medium (Reuther, 1984). A two step procedure including two hormonal levels promoted root formation (Volokita et al. 1988). In the first step buds from field grown spears or shoot segments produced in-vitro were placed on induction media of MS + NAA (0.3- 0.8ppm) and kinetin or ziP (0.1-0.3ppm). In the second step shoots and buds are divided and subcultured on MS with lower levels of NAA (0.03-0.08ppm) and kinetin or Zip (0.01- 0.03ppm), or hormone free MS medium. Within 2-3 weeks a large number of crowns formed roots. Cyclical splitting and subculturing of crowns produced a large number of rooted plants. Reculture of the plants on MS medium lacking NAA was conditional for successful transfer of the plants from in-vitro culture to the planting medium (Hasegawa et al. 1973). Histological examination showed that formation of roots is initiated adventitiously in a swollen mass of a callus-like tissue at the base of the shoot apex explant: whereas, spear formation resulted from growth of the axillary buds (Hasegawa et al. 1973). Experiments on callus formation and regeneration of adventitious shoots have been conducted using shoot tips (Reuther, 1977), apical and lateral buds of spears (Chin, 1982), spear segments (Takatori et al. 1968: Yakewa et al. 13 1971a, b), hypocotyls (Wilmer and Hellendoorn, 1968), and stem explants (Harada, 1973). Additionally, single cells of cladophylls (Jullien et al. 1979) were induced to form callus. Hypocotyl-derived callus formed a large number of dense globular embryo-like bodies when placed on Linsmaier and Skoog (1965) (LS) basal medium containing 4.5 uM 2,4-D and 1.5 uM kinetin followed by subculture to liquid medium of the same composition. Globular bodies (larger than 2mm) continued to dedifferentiate in suspension, and their number increased with decreased 2,4-D levels (Wilmer and Hellendoorn, 1968). Spear segments freed of lateral buds formed callus within 4-6 weeks on MS medium supplemented with NAA (0.5ppm) and 15% Coconut Water (Takatori et al. 1968). Replacement of 2,4-D by NAA (0.1-1ppm), in combination with BAP (0.1-1ppm) promoted root and shoot regeneration in calluses derived from spear segments (Yakuwa et al. 1971 a,b). Harada (1973) examined the effect of auxins, cytokinins and casein hydrolysate on formation of callus, roots and shoots from stem explants. NAA (0.1-1ppm) enhanced callus growth more than 2,4-D, while zeatin, BAP and kinetin had equal effects. Adventitious roots developed slightly more with NAA (1ppm) than with 2,4-D of the same level. Cytokinins had an inhibitory effect on rooting. This effect was highest with kinetin (0.01ppm), moderate with BAP, and lowest with zeatin. NAA (0.1ppm) in combination with zeatin (1ppm) promoted shoot regeneration from callus. BAP and kinetin were less effective when l4 replaced zeatin, and casein hydrolysate was stimulatory only for shoot development. Explanted shoot tips formed callus on MS medium supplemented with NAA (1ppm), and callus regenerated shoots on MS + BAP and IAA (Reuther, 1977). Shoot tip cultures often developed excessive callus which competed with the newly formed roots and frequently prevented formation of a vascular connection between the root and shoot. Incorporation of growth retardants such as ancymidol, which inhibits GA synthesis (Dennis et al. 1965), reduced the proliferation of excessive callus and promoted growth of more vigorous shoots and roots (Chin, 1982: Khunachak et al. 1987). MW Somatic embryogenesis is a developmental sequence linked to the totipotent ability of plant cells to express their genetic potential in a pathway similar to that of the zygotic embryo (Raghavan, 1986). Steward et al. (1958) were the first to demonstrate the totipotency of plant cells by culturing carrot callus in liquid medium where single cells become disassociated and continued to divide and differentiate to form intact plantlets upon transfer to solidified medium. Steward (1963) and Wetherell and Halperin (1963) reported that a suspension culture of carrot cells regenerated an enormous number of embryoids resembling the zygotic embryos. In the same year, Takeuchi (1963) described an embryogenic sequence of development from single cells originated from carrot root-derived callus. Similarly, 15 Vasil and Hildebrandt (1965) showed that single cell of a hybrid tobacco, nurtured in isolation from other cells in a defined medium, formed a completely organized plant. Backs-Husemann and Reinert (1970) also demonstrated that single cells from carrot callus cultures are evolved into embryoids: thus, reinforcing the conclusion that somatic embryos indeed have their origin in single totipotent cells. Morphological and anatomical observations show a close resemblance at the globular, heart and torpedo stages in carrot zygotic and somatic embryos (McWilliam et al. 1974) and in other plant species (Raghavan, 1986). Biochemical and physiological studies also point to the resemblance. At the biochemical level, somatic embryos of diverse plants contain fatty acids (Pence et al. 1981a), lipids (Janick et al. 1982), anthocyanins (Pence et al. 1981b), storage proteins (Crouch, 1982) and alkaloids (Schuchmann and Wellmann, 1983) that are characteristic of maturing zygotic embryos in vivo. Another example of the similarity between these cell types is the case of seeds and somatic embryos of grapes, where dormancy can be overcomed by cold treatment and is accompanied by a decrease in endogenous ABA content (Rajasekaran et al. 1982). These observations show that at least to a certain extent, somatic embryos can serve as a model for zygotic embryo development (Raghavan, 1986). Somatic. embryogenesis consists of an induction stage, early growth, and maturation and germination (conversion to plantlet) stages. In various plant species embryogenic 16 growth from somatic tissue occurs in two ways: direct and indirect embryogenesis. Direct embryogenesis occurs when certain cells are predetermined for embryogenic development, needing only permissive conditions to develop into an embryo (Sharpe et al. 1980, 1982). Direct somatic embryogenesis most often occurs on excised zygotic embryos (Steward et al. 1964) and somatic embryos (Rate, 1968), or on the plant arising from them, mostly from the hypocotyl region (Homes, 1986). Indirect somatic embryogenesis occurs from cells that have already been differentiated within the explant (i.e. cells of specific meristems or mature tissues). In this case, an induction treatment is needed to foster redetermination of the differentiated cells followed by development of the induced embryogenic cells (Ammirato, 1986). The earliest cell divisions in the embryogenically determined cells may occur in a few patterns without affecting the final embryogenic outcome (McWilliam et al. 1974). Embryogenically determined cells may differentiate singly or in groups to form embryos (Williams and Mashewaran, 1986). This capability has been demonstrated when enzyme maceration of embryonic callus gave rise to many single cells which regenerated embryoids in large numbers (Button and Botha, 1975). A In many plants the treatment where an auxin promotes cell dedifferentiation and development of densely cytoplasmic globular proembryos is considered as embryo l7 induction stage (Halperin, 1966: MCWilliam et a1. 1974: Street and Wethers, 1974: Ammirato, 1985). Following transfer to an auxin-free or reduced auxin medium, proembryos continue growth and differentiate forming mature embryos and plantlets. In different cases the process of callus growth and embryogenic induction have been separated into two stages. In alfalfa for example, kinetin-NAA medium fosters growth of unorganized callus. This callus remains unorganized when transferred to media with different levels of kinetin-NAA in combination. However, somatic embryogensis is induced when such callus is transferred to media supplemented with 2,4-D (Walker et al. 1979). Cells within the proembryos retain their embryogenic competence and rather than participating in the coordinated embryo. growth may themselves become embryogenic growth centers. Such patterns occur mainly in induction media supplemented with auxin where proembryos are formed, but do not continue to develop to a mature stage (Ramada and Harada, 1979). On induction medium supplemented with auxin, embryogenic cells continue to divide forming a larger proembryo, or budding off forming additional smaller, fused or separated proembryos (Ammirato, 1986). Such 'formation has been observed in different plant species including carrot (Street at al. 1970: McWilliam et al. 1974: Street and Withers, 1974), celery (Al-Abta and Collin, 1980), Arrspa ballaggnna (Konar et a1. 1972b). and Panimmaxim and Eemisstum purpursun (Karlsson and Vasil, 1896). Subculture of such l8 proembryo populations provides continuity of the process where new embryogenic cells are continuously formed (Ammirato, 1986). On maturation medium proembryos may develop in a sequence resembling that leading to maturation of zygotic embryos, i.e. the initiation and growth of cotyledonary primordia, the early stages of cell and tissue differentiation (usually changes in cell shape) and formation of shoot and root apices. However, in culture various events may occur prematurely, be delayed, absent or extended in time, leading to morphological and developmental abnormalities in mature somatic embryos (Halperin, 1966: Ammirato and Steward, 1971: Konar et al. 1972b: Ammirato, 1985). For instance, extended cell divisions during the proembryo stage may result in relatively large proembryos and mature embryos as compared to zygotic embryos. It can also result in cells forming new centers of embryonic growth leading to multiple or secondary embryos (Ammirato, 1986b). Somatic embryos may cluster or fuse to form twins, triple and multiple embryonic bodies that generate multiple shoots and roots during plantlet formation (Ammirato 1983b, 1986a, 1986b). Somatic embryos in different developmental stages may form additional, but smaller embryos at different loci along their axis such as at the radical hypocotyl junction or on the radical cap (Halperin 1966: Konar et a1. 1972: Ammirato 1985, 1986b). Developmental changes such as extensive cell divisions and/or premature cell enlargement 19 at the early heart-shaped stage may result in too many cell centers leading to formation of multiple cotyledons. In cases where cell division continues in the cotyledonary ring, even after the cotyledonary primordia are initiated, fused or fasicated cotyledons may be formed and develop poorly at a later stage. On the other hand, insufficient cell division or premature vacuolation and differentiation can result in aberrant or poor cotyledon formation and development. Here, somatic embryos may appear morphologically normal, but their shoot- apex may be affected and fail to develop shoots during germination (Ammirato, 1985, 1986b). Changes in the developmental sequence, i.e. cell divisions, enlargement and maturation can also result in aberrant or precocious formation of the shoot apex at the same time cotyledons are formed. Such abnormalities can arrest plant development (Ammirato, 1985). On the other hand, many somatic embryos that reach the early maturation stage 'may precociously germinate without the growth cessation associated with seed maturation. This early germination often leads to abnormal plantlet development (Ammirato, 1986b). Increased evidence links somatic embryogenesis to changes in the endogenous levels of auxin(s) (Fujimura and Komamine, 1979a: Sung, 1979: Kato, 1968: Raghavan, 1986). Exogenous auxins such as 2,4-D, NAA, IRA, and IAA play a key role in inducing somatic embryogenesis (Zaerr and Mapes, t 1985). Carrot cells showed sensitivity to auxin and 20 antiauxin, during embryo formation. In habituated callus of shamuti orange, which grows in the absence' of auxin and cytokinin, the addition of even a low concentration of IAA or NAA significantly reduced or inhibited embryogenesis. Conversely, the addition of auxin biosynthesis inhibitors greatly stimulated the embryogenic process (Kochba and Spiegel—Roy, 1977b). Experiments with gamma-irradiation, which is known to inactivate endogenous auxins in plants, showed that embryogenesis is stimulated when callus is irradiated prior to subculture (Kochba and Spiegel-Roy, 1977a). Such evidence suggests that the endogenous auxin level is important during early embryogenesis, whereas continual presence of auxin in the later stage is likely to inhibit embryoid formation (Halperin and Wetherell, 1964: Sung 1979). Successful embryoid formation occurred when calluses were transferred from an auxin-enriched medium to an auxin-free medium. For example, stem and embryo explants of several members of the carrot family (Umbsllirsras) had the best embryogenic response in cultures where the explant was nurtured in an auxin-enriched medium for callus induction followed by transfer of callus to an auxin-free medium (Steward et al. 1970: Ammirato, 1983a). In Maslsaya surgara, isolated mesophyll cells formed callus on medium supplemented with 2,4-D and kinetin. Omission of 2,4-D from the medium or its replacement with a weaker auxin such as IAA, stimulated formation of embryogenic callus (Lang and Kohlenbach, 1975). The functional connection between 21 reduction in auxin level(s) and embryogenesis has also been supported by experiments in which extensive washing of callus to remove 2,4-D before transfer to maturation medium enhanced somatic embryogenesis. This indicates that 2,4-D is sometimes a potent inhibitor of somatic embryogenesis (Ammirato, 1986). However, this is not the case for other auxins. For example, continuous treatment of carrot hypocotyl segments with IAA, NAA or IBA for a 5 week period favored somatic embryogenesis to the same extent as treatments where tissues were transferred to a medium lacking auxin following a 2 week period of exposure to the same auxins (Ramada and Harada 1979). Smatic embryogenesis occurred in several experiments, but mostly in low frequency when explants were cultured on auxin-enriched medium without transfer to hormone-free medium. Sharp et al. (1980) suggested that potential embryonic cells of the callus are mitotically quiescent in the presence of high auxin concentrations in the medium. A low frequency of somatic embryos may form on auxin-enriched medium. This may be due to a lack of auxin in the cellular vicinities of predetermined embryonic cells, thus enabling them to overcome mitotic arrest and form embryos. This also may explain why high embryogenic frequency occurs on the surface of fresh, white friable callus that emerges from older brownish tissues (Raghavan, 1986). Cytokinins have been incorporated in various' culture media to promote somatic embryogenesis (Zaerr and Mapes, 22 1985: Minocha 1987). Compounds with cytokinin activity which may exercise a considerable regulary role in somatic embryogenesis have been identified in the medium of carrot cell cultures (Salem et al. 1979). Cytokinins promote embryogenesis by enhancing division of proembryogenic cell masses. Sung et al. (1979) demonstrated a promotive role for cytokinins in somatic embryogenesis of wild-type carrots cells grown in 2,4-D enriched medium, and in 5- methyltryptophan-resistent carrot cell lines in which embryogenesis was induced by high levels of endogenous IAA. In another example (Fujimura and Komamine 1980a), the capacity of cell clusters to undergo embryogenesis was promoted by zeatin which enhanced cell division. Results leading to the same conclusion were also obtained when 21P or isopentenyladenosine were added to embryogenic cultures of Rippinslla anisun (Ernst and Oesterhelt, 1984). In celery cell suspensions kinetin enhanced the transition of embryo from the globular to torpedo shaped stage (Al-Abta and Collin, 1978). I Despite many experiments, there has been less success with somatic embryogenesis in monocots than in dicots. Although, somatic embryogenesis has been reported in Asparagus arriginalis (Wilmer and Hellendoorn, 1968: Steward and Mapes, 1971) and in other species of the Liliaceae such as pallsyilia rpmana (Lupi et al.1985), as well gasrsria ysrruspsa, and nausprrniairassara (Beyl and Sharma, 1983) and in species of taxonomically related families, such as 23 Iris (Reuther, 1977) and palms (Tisserat,1979: Reynolds and Murashige, 1979). Successful somatic embryogenesis has also been reported in various species of the graminsa (Vasil and Vasil, 1982). Frequently, the embryogenic cultures are initiated from excised immature zygotic embryos (Botti and Vasil, 1983: Vasil et al. 1985: Hakman and Fowke, 1987), but other plant tissues, most notably young leaves, have also been used (Lu and Vasil, 1981: Yeh and Chang, 1986: Szabados et al. 1987). Here, the slow regeneration of callus and the rapid loss of embryonic competence are two limiting characteristics (Raghavan, 1986). The development of monocot zygotic embryos proceeds through a well-defined pathway (Raghavan, 1972). The first division of the zygote cell gives rise to an apical cell and a basal cell. In the next round, the apical cell and the basal cell divide longitudinally and transversely, respectively. The basal cell gives rise to a suspensor complex of about 6-10 cells terminated by a large basal cell at the micropylar end and by the embryo proper at the chalazal end. Throgh active divisions the apical cell gives rise, to the spherical embryonal part, characterized by small, dense cytoplasmic cells. Compared to cells of the spherical embryo, those of the suspensor are more vacuolated and contain more endoplasmic reticulum, but appear to be depleted of ribosomes and stain less intensely for proteins and nucleic acids. This early developmental stage involves the same steps in monocots and dicots and is defined as the 24 globular or pre-embryo stage (Raghavan, 1976). However, there is disagreement as to whether to classify the early transverse and longitudinal cell divisions as the proembryo stage and the later formation of the spherical part as the globular stage (Raghavan, 1976, 1986). At this stage the globular zygote increases in size, mainly through active cell divisions, but retains its spherical shape. During this stage, the three primary meristems (protoderm, ground meristem and procambium) become visible. At the end of this stage, a ring of actively dividing cells appears at the apical end. Further cell divisions give rise to two portions: the lower part of the cotyledon, and to the upper part of the cotyledon and the hypocotyl, while the shoot tip is initiated at the junction of the hypocotyl and the cotyledon. A characteristic unique to the monocot embryo is the simultaneous growth of the cotyledon initial cells and the shoot apex. This may be explained by the ontogeny of monocots, in which the single cotyledon incorporated the primordia of both cotyledons and the original shoot apex found in the dicots, while a new functional shoot apex arises laterally from the subterminal tier of the proembryo. In the following stage, the process of cell differentiation in the cotyledon and hypocotyl are accompanied by considerable cell elongation resulting in formation of an elongated embryo axis. The cells derived from one half of the original basal cell form the root cap in the mature embryo, the other half of the original basal cell forms the 25 remaining part of the root cap and suspensor. By the time the embryo has matured, its length has increased to more than 2mm. This tremendous increase in size is supported by the uptake of raw materials from the endosperm and their utilization in the synthesis of complex substrates. As the embryo reaches maturity, the size of the individual cells is reduced to a size that is no bigger than a quarter of the early zygote cells. Mature embryos have a procambial system which differentiates throughout the hypocotyl and cotyledons, giving rise to xylem and phloem elements (Bisalputra and Esau, 1964). The mature embryo is surrounded by a mass of cellular endosperm. With the formation of a mature seed the embryo becomes quiescent with cessation in mitotic division, accompanied by a decrease in water content. The imbibition of water initiates germination which includes resumption of mitotic activity along with cell expansion and differentiation (Raghavan, 1986). As a carbon and energy source carbohydrates (CHO) are major and essential constituents of any tissue culture medium (Thorpe, 1982). Sucrose is the sugar of transport in most plants, and is considered the best sugar for supporting growth and differentiation of in vitro plant tissue cultures, including somatic embryogenesis (Thompson and Thorpe, 1987). The constituent hexoses of sucrose, i.e. glucose and fructose and several other monosaccharides such as galactose (Gross et al. 1981) and mannose (Wright and 26 Northcote, 1972), disaccharides like cellobiose and trehalose (Mathes et al., 1973), lactose (Hess et al., 1979) and melibiose (Nickell and Maretzki, 1970), and trisaccharides such as raffinose (Wright and Nortcote, 1972) also can support growth in plant tissue cultures (Maretzki et al., 1974: Opekarova and Kotyk, 1973) and embryogenesis (Verma and Dougall, 1977). Sugar alcohols such as myo-inositol, are required in small quantities for the culture of many plant species (Murashige and Skoog, 1962), but they generally do not serve as a sole carbon source. However, in a few studies sugar alcohols did support growth in vitro. Mannitol supported shoot initiation from black and white spruce epicotyl explants (Rumary, 1981) and sorbitol supported growth of malus tissue cultures (Chang and Taper, 1974). Sorbitol is considered important in the metabolism of apple and related species in vivo. The utilization of sorbitol and other cyclitols involves their conversion to the corresponding hexoses. Sorbitol dehydrogenase, which converts sorbitol to fructose, has been detected and characterized in apple callus tissue (Negm and Loescher, 1979). Carbohydrate metabolism in vitro has been studied in various tissue culture systems. An early study (Goris, 1954) showed that carrot callus readily interconverted glucose and fructose, and synthesized sucrose. Recent studies (Konemine et a1. 1978) confirmed that glucose and fructose are the most commonly metabolized hexoses, and to a 27 large extent initiation with different carbon sources will lead to their formation or their derived phosphates or nucleotides. The capacity of tissues to utilize different carbohydrates may vary with the species or explant (Hildebrandt and Riker, 1949) and with different cultivars (Chang and Taper, 1972). Furthermore, it may even vary with different organs of the same plant (Mathes et al. 1973). Although it has not been comprehensively studied, CHOs type may also influence the type of organ differentiated by the. primary explant (Kikuta and Okazwa, 1984). Mannitol, sorbitol and inositol supported shoot bud formation in potato tuber tissue, whereas, sucrose promoted root formation. Somatic embryogenesis may occur in two distinct stages as proembryogenic cell initiation on an auxin containing medium followed by embryo development on an auxin-free medium. It is possible that CHOs requirements and metabolism may differ in these stages. Few studies have examined the role of CHOs source in somatic embryogenesis. Galactose and galactose-containing saccharides were found to stimulate embryogenesis in habituated shamuti orange callus. Sucrose in combination with these sugars suppressed it (Kochba et al., 1978). Non-embryogenic calli lines responded to galactose at 7-10%. On the other hand, embryogenic lines responded to low galactose levels, (0.05%), which were unable to support callus growth. Here, the action of galactose or galactose containing sugars may involve 28 inhibition of auxin synthesis in .the habituated calli (Kochba et al., 1974). Such a phenomenon was also found in other systems (Thompson and Thorpe, 1987). In pausus sarpra maturation of somatic embryos on sucrose was slower than with other sugars (Verma and Dougall, 1977). In Inapprpma sagas low sucrose levels (1-2%), glucose and fructose (3- 5%), all stimulated faster growth than that of the zygotic embryo controls (Kononowicz and Janick, 1984). Embryogenic carrot cells on an auxin-containing medium are rich in starch (Street and Withers, 1974) which disappears during embryo formation on an auxin-free medium. It is not clear whether the metabolic changes are a direct result of the release of auxin from the medium or are associated with metabolic events involved in embryogenesis (Thorpe, 1980). Changes in starch content of organ-forming tissues have also been observed in several plant species (Brossard, 1977: Thorpe and Meier, 1974). In shoot-forming tobacco callus, maximum starch accumulation occurred just prior to meristemoid formation (Thorpe and Meier, 1972: Thorpe and Murashige, 1968). During meristemoid formation, starch was rapidly degraded (Thorpe and Meier, 1974). In meristematic tissue of cultured cotyledons of Einus ragiara (Patel and Thorpe, 1984), the decline in starch content was consistent with increased respiration and high succinate activity (Biondi and Thorpe, 1982). Higher respiration rates were also observed during shoot initiation from tobacco callus (Ross and Thorpe, 1973: Thorpe and Meier, 1972). Tobacco 29 callus showed increased activities of both glycolysis and the pentose phosphate pathways (Thorpe and Leishley, 1973) and increased levels of NADPH and NADP+ (Brown and Thorpe, 1980). Here, total adenosine phosphates increased during the early culture period and declined thereafter. However, the levels in shoot-forming tissue were always higher than in proliferating callus. Embryogenic cells have high mitotic activity (Dmitrieva and Mohamed, 1977: Warren and Flower, 1978), they are rich in mitochondria and have an intensified respiration rate (Street and withers, 1974). Dissolved 02 concentration influenced embryo development in carrot (Kessell et al., 1977). Below a critical level embryogenesis occurred, but above that level rhizogenesis was favored. Lowered 02 levels increased cyanide-sensitivity and cellular levels of ATP. Exogenous adenosine at higher 02 concentrations also raised the cellular levels of ATP. Both treatments enhanced, somatic embryo development (Kessell et al., 1977). These findings confirm the need for a higher energy requirement as well as for reducing power (NADPH and pentoses) for biosynthetic processes involved in organogenesis and embryogenesis (Brown and Thorpe, 1980). The typical tissue culture medium (e.g. Murashige and Skoog with 3% sucrose) has a water potential of -5 bars (Doley and Leyton, 1970). However, under in-vitro culture conditions, the cells are exposed to water potentials similar to those of field grown plants. In soil, water 30 potential may range from zero, after irrigation to -15 bars at drought (Thorpe, 1982). Osmotic potential is regulated in plant cells by both inorganic ions and organic molecules (including organic acids, sugars and sugar alcohols) (Zimmerman, 1978). It has been suggested (Wyn Jones et al. 1977) that the cytoplasmic inorganic ion concentration remains fairly constant, while osmotic adaptation of the cytoplasm is mainly achieved by the accumulation of non- toxic organic molecules (Wyn Jones et al. 1977). CHOs in plant culture medium are generally in excess of the tissue growth requirement (Thorpe, 1982). In some systems, relatively large quantities of CHOs may accumulate and increase the osmotic potential in the cell, unless they are rapidly removed by metabolic utilization (Thorpe, 1982). The osmotic potential of the medium has a prominent role in growth and differentiation. An increase in medium water potential from near zero to -1 bars significantly reduced the growth rate of callus derived from wound injury of Eraxina sxsslsipr (Doley and Leyton, 1970). At near zero water potential the callus had active surface growth and little internal differentiation. At water potentials greater than -1 bars, the callus had suberized surfaces and contained lignified xylem and scleroids. On medium with near zero water potential, callus appeared after 2-3 days, and at water potential of -6 to -10 bars it appeared after 6—9 days. Studies on the effect of mild and severe osmotic stress on tobacco tissue in culture (Klenovska, 1973) showed 31 that increasing the osmotic potential of the medium by 0.5 bars, with the addition of PEG 1000 at 1% w/v, reduced tobacco callus growth. The sucrose, fructose and glucose content of the tissue was also decreased. Water content of the callus was not affected but dry matter increased. More severe stress intensified these effects, and reduced the water content of the tissue. Reduction in the relative humidity of the air above the cultures from 90 to 32% increased the osmotic potential, dry weight, and sugar content of the tissue (Klenovska, 1976). An increase in medium water potential in soybean culture decreased cell size, but increased callus growth (Roberts et al. 1982). Small increases in osmotic stress (-2 bars) decreased growth rof sugar cane cells in culture, increased respiration, turnover of amino acids and sucrose content. At the same time it lowered the reduced sugar content and invertase activity. These effects were even greater in cells not preconditioned to the greater medium water potential (Thorpe and Meier, 1973). CHOs are utilized as the energy source for cell metabolism and for the formation of major cell components. The balance between these two functions varies with the stage of development (Maretzki et al. 1974). High sucrose levels promoted in vitro somatic embryogenesis (Ammirato and Steward, 1971), and development of zygotic embryos in vitro (Norstog, 1961). The younger the zygotic embryos, the higher the sugar level that was required for development in-vitro 32 (Yeung et al. 1981). Higher sucrose levels were also required for in-vitro rooting from cuttings of Pinus lampsrriana embryos (Greenwood and Cockerline, 1978), and for root formation from sugar cane cells (Maretzki and Hiraki, 1980). On the other hand, high sucrose levels reduced embryo growth, slowed their maturation and prevented precocious germination (Ammirato, 1983). Low sucrose levels, glucose and fructose stimulated somatic embryos of Inapprpma sagas to grow faster than the zygotic embryo control (Kononowicz, 1984). Increased sucrose levels reduced embryo growth. Instead, embryos produced storage lipids, anthocyanins and alkaloids characteristic of the maturing zygotic embryos. High sucrose levels affected fatty acid composition and anthocyanin content of somatic embryos comparable to the zygotic embryos (Pence et al. 1981a: Pence et al. 1981b). These findings indicated that sucrose at high levels suppressed precocious germination and regulated the embryo developmental maturation pattern (Pence et al. 1981b). Osmotic substitutes applied alone or in combination with sucrose could not duplicate the sucrose effect on regeneration of roots from cuttings of Pinus lampsrriana embryos. Isa-osmotic quantities of fructose, fructose plus glucose, or glucose alone were much less effective or totally ineffective in inducing maturation of Inapprpma sagas somatic embryos (Kononowicz and Jenick, 1984). These findings indicated that the effect of sucrose on embryo 33 development is not exclusively osmotic (Thompson and Thorpe, 1987). The optimum sucrose concentration for growth and formation of shoots in dark grown tobacco callus is 3% (Brown et al. 1979). Lower or higher sucrose levels reduced the number of shoots formed. However, the same number of shoots formed on medium with 2% sucrose when supplemented with mannitol to give the medium water potential equivalent to 3% sucrose. However, mannitol could not substitute to levels below 2% sucrose. Mannitol alone at any level could not serve as a sole energy source. Also, increased levels of bacto-agar in the medium did not replace CHOs including mannitol in promoting shoot formation. This indicated that in addition to a metabolic role, part of the CHO in the medium must be an osmoregulatory effect and that the osmoticum must enter the tissue (Brown et al. 1979). Studies with different tobacco callus lines indicated that the requirement for optimal sugar level in the medium differed for growth, greening and shoot formation (Berg and Umiel, 1977). Reduction in sucrose level while maintaining the medium water potential by adding manitol increased the number of shoots formed and improved their morphology. During shoot formation, tobacco callus had higher water, osmotic and pressure potentials when maintained by CHO and other metabolites (Maretzki and Hiraki, 1980). As in somatic embryogenesis, bud formation from callus occurred on an auxin-containing medium and further development of shoots 34 occurred, following transfer of buds to an auxin free medium for (Thorpe, 1977: 1980). Reduction in sucrose level in radiata pine from 3 to 2% (Aitken et al. 1981), and in alfalfa, from 3 to 1% (Stavarek et al. 1980) promoted shoot development. Similarly, reduction by half of the mineral content of the medium and lowered sucrose levels were optimal for rooting of conifer adventitious shoots (Thorpe, 1977). The osmotic potential has a distinct effect on membrane properties, and membrane proteins such as ATPase (Zimmermann, 1978). 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Physiol. 29:121-148. WI SOMATIC EMBRYOGENSIS IN W W L.: THE ROLE OF EXPLANTS AND GROWTH REGULATORS 50 53513591 The role of explants and growth regulators in somatic embryogensis in asparagus was examined. Calli derived from spear-cross sections (SS), lateral buds (LB) of spears, and callus taken from crowns established in, vitro (IVC) of asparagus male line (C-3) were subcultured twice at four weeks intervals on initiation media of Murashige and Skoog salts and vitamins medium (MS) + NAA (0.1-1ppm) and kinetin(0.1ppm). At the end of the second subculture calli were transferred to MS containing 2,4-D or NAA and kinetin alone and in combination at levels of 0-10ppm: designated as embryo induction media (IM). After four weeks, callus was visually and quantitatively evaluated for growth and formation of somatic embryos. The calli were transferred to maturation media_(MM) of MS + (ppm): NAA (0.05) and kinetin (0.1). After six weeks on MM, somatic embryogensis was evaluated again. Calli derived from LB and IVC were found to have a high embryogenic capacity: yielding 46-69 (LB) and 81-127(IVC) embryos (small globular and bipolar mature)/g callus on IM devoid of growth regulators or with auxin and/orkinetin at levels of 0.01-0.1ppm. Calli derived from SS ,had a lower embryogenic capacity where only 9-23 embryoids/g callus formed on such IM. The auxin 2,4-D at levels of 1-10ppm was found to induce a high embryogenic 51 52 frequency on IM: up to 491(SS), 450(LB) and 467(IVC) small globular embryos/g callus. However, 2,4-D also induced formation of abnormal embryos and a subsequent low rate of embryo conversion to plantlets on MM: up to 2(SS), 3.7(LB) and 7.7/g callus (IVC): 2, 5.1 and 6.8%, respectively. NAA at the same levels induced a lower embryogenic frequency: up to 95(SS), 306(LB), and 251(IVC) small globular embryos/g callus on IM. However, NAA promoted normal embryo formation and a higher conversion rate than 2,4-D: up to 2(SS), 10.7(LB) and 13.7/g callus (IVC): up to 5, 21 and 17.3%, respectively. There was no difference between kinetin levels (O-lppm) on callus growth and embryogensis. A high kinetin level (loppm) in TM was found inhibitory to embryogenesis. INTBQDQQTIQN Asparagus strisinalis L. is a dioecious crop species that offers interesting challenges for breeding and tissue culture research. Asparagus has a low multiplication rate using conventional propagation methods (Ellison, 1986), where only a few propagules can be obtained by crown division (Yang and Clore, 1973). This restricts asparagus breeding where the only method for determining yield potential is through individual plant records (Hanna, 1947). So far, in vitro tissue culture techniques have proved useful for propagation of asparagus lines (Reuther, 1983). However, the vast majority of tissue culture research in asparagus has concentrated on shoot proliferation and 53 plantlet division from cultured meristems and shoot tips (Murashige et al. 1972: Hasegawa et al. 1973: Chin 1982: Volokita et al. 1987). Somatic embryogenesis has been considered a potential system for propagating elite asparagus crowns (Reuther, 1983). Wilmer and Hellendoorn (1968) reported that hypocotyl-derived callus formed a large number of dense, globular embryo-like bodies when placed on Linsmaier and Skoog (1965) basal medium (LS) + 2,4-D (1.0ppm) and kinetin (0.3ppm). Globular embryoids continuously differentiated in liquid suspension of the same composition and gave rise to mature plantlets. Steward and Mapes (1971) reported the formation of somatic embryos from asparagus stem segments through a sequence of callus induction in White's liquid medium supplemented with coconut milk and NAA. At the following stage the NAA was replaced by 2,4-D. Embryos were induced in high salt medium supplemented with NAA,and root and shoot primordia were formed by transfer of embryos to medium supplemented with coconut milk and IAA. Reuther (1977) demonstrated that shoot tip- and shoot segment- derived callus formed on LS + NAA (1.0ppm) and kinetin (1.0ppm), gave rise to somatic embryos when transferred to LS + IAA (1.0ppm) and BA (0.1ppm) and subsequently to plantlets when transferred to LS or LS + IAA (0.5ppm). However, these reports did not consider the role of explant source and auxin type and concentration and did not quantify embryos and mature plantlets formed in culture. 54 The objectives of this study were to define an efficient scheme for producing somatic embryos of asparagus, concentrating on the role of explants and auxin type (2,4-D versus NAA). This research provides evidence that somatic embryogenesis in asparagus may occur in several pathways using different explants and media conditions, some of which were found more efficient than others. MATERIAL AND HEIHQDS Spear segments (SS), lateral buds (LB), and in vitro maintained crowns (IVC) of asparagus male line C-3 were assessed for the initiation of embryogenic callus. The SS and LB explants were obtained from greenhouse grown plants, sampling the upper 2-12 internodes, from the tip of 4-7 days after spears emerge. Spears were surface sterilized for 30 min in aqueous sodium hypochlorite (1.5% v:v) and 2 drops of Tween-20, and were thoroughly rinsed with 3 changes of sterile distilled water. The SS were transverse X-sections (3-5mm) end were free of lateral buds. The LB were free of leaflets. In order to initiate callus, SS were placed on Murashige and Skoog (1962) salts and vitamins medium (MS) solidified with 0.9% Difco agar and containing 3% sucrose and supplemented with 2,4-D and/or NAA and kinetin alone and in combination at levels of 0-10ppm. The cultures were kept in the dark at 27C. In 4-8 weeks, callus was separated from $8 and subcultured. Callus initiation from LB explants included two steps: 1) culture of LB for 4 weeks on MS + NAA (0.5ppm) and kinetin (0.1ppm), and 2) transfer of 55 LB to MS + NAA (0.1ppm) and kinetin (0.01ppm) where callus proliferated and was subcultured. IVC were maintained on MS + NAA and kinetin alone and in combination at levels of 0-1ppm under 16h/day of light 32uEm'2 sec"1 from cool white fluorescent bulbs, and gave rise spontaneously to calli. Such calli on MS + NAA (0.01, 0.1, 0.3 and lppm) and kinetin (0.01ppm) were separated from the IVC and subcultured. After two subcultures at 4 week intervals, calli derived from all explants were visually evaluated for growth and embryogenic capacity i.e. callus color, formation and frequency of spherical and bipolar embryos using a dissecting microscope. This information was used to determine the optimum medium for initiation of callus from each explant. For LB and IVC these were calli derived on MS + NAA (0.1ppm) and kinetin (0.01ppm), and for SS calli on MS + NAA (0.1-lppm) and kinetin (0.01ppm). These chosen calli were all transferred to MS + 2,4-D or NAA and kinetin alone and in combination at levels of 0-10ppm, designated embryo induction media (IM). Each callus piece placed on IM was 0.4-0.6 g and the experimental unit was 3 pieces per 100 x 15 mm Petri dish. After four weeks on IM, callus increase (%) was calculated from the weights as follows: (final fresh weight - initial fresh weight) x 100 / initial weight. Somatic embryogenesis on IM was evaluated by weighing callus pieces withdrawn at random and counting the number of small globular embryos (<3mm diam), and large elongated embryos (4-7mm diam) with the latter classified as bipolar embryos. 56 After four weeks on IM, calli selected at random were transferred to maturation media (MM) of MS + NAA (0.05ppm) and kinetin (0.1ppm). These cultures were incubated at 27C under 16h/day irradiance 32uEm’2 sec'1 from cool white fluorescent bulbs. After six weeks on MM, evaluation of somatic embryogenesis was performed by weighing callus pieces and counting globular, bipolar, abnormal embryos, and plantlets as described previously. Percent of embryos converting into plantlets was calculated as % plantlets = plantlets / (bipolar embryos + plantlets) x 100. The study was conducted as a separate experiment for each explant- derived calli source as a randomized complete blocks design (RCBD) 2 factor factorial with 10 auxin treatments (i.e. 5 levels each of 2,4-D and NAA) x 4 kinetin levels x 6 Petri dishes as replications. Such an experimental design was conducted in both IM and MM. Mean separation was performed using LSD (Steel and Torrie, 1980). In order to determine whether the abnormal formation of embryos observed in MM was due to a carry-over of auxin from IM to MM, the following experiment was conducted: IVC- derived calli of male line A-9 were placed on IM containing (ppm): 2,4-D (1.5) + kinetin (0.1), 2,4-D (0.3) + kinetin (0.1), or NAA (1.5) + kinetin (0.1), NAA (0.3) + kinetin (0.1), for 4 weeks. Calli were subsequently transferred to MM 'and subcultured every 4 weeks during a 6 month period. Prior to each subculture, callus pieces were withdrawn randomly and weight and the number of abnormal and normal 57 embryos and plantlets were counted. Analysis of variance (AOV) and means were calculated from 6-8 replications (100 x 15mm Petri dishes) per treatment. BESHLIE MMWM Callus was formed at the periphery of SS 4-8 weeks after placement on MS with 2,4-D or NAA and kinetin alone or in combination (0-10ppm). Segments from the upper part of the spear (4-12 internodes) formed callus more rapidly than those from the lower segments (14-22 internodes). Media of MS + 2,4-D (0.5-2ppm) and/or NAA (1-3ppm) + kinetin (0-1ppm) were optimal for callus initiation. On MS + 2,4-D (0.5- 2ppm) and kinetin (0-1ppm) the callus was compact, yellowish and mucilaginous with a high frequency of globular translucent embryoids. These embryoids consisted of small cells with a dense cytoplasm surrounded by large vacuolated cells: whereas, on MS + NAA (1-3ppm) and kinetin (0-1ppm) callus was yellowish and friable with sporadic globular embryoids surrounded by large vacuolated cells. Explants of SS placed on MS + low 2,4-D or NAA levels (0-0.2ppm) or on MS + kinetin alone (o-lppm) gave rise to sparse, non- vigorous callus. The interaction of auxin x kinetin was highly significant for both % callus growth and the number of globular embryos on IM (Table 1). 0n IM with kinetin (0- 1ppm), fresh weight of callus did not increase as 2,4-D levels increased from 0 to 0.1ppm. The fresh weight of 58 callus gradually increased at 0.1 to lppm and decreased 'at loppm. 0n IM with the same kinetin levels but with NAA, callus fresh weight did not increase much as NAA increased from 0 to 0.1ppm, sharply increased between 0.1 to lppm and decreased at loppm (Table 2, Fig.1A). Calli slightly increased as 2,4-D or NAA levels increased from 0 to 0.1 ppm: sharply increased at 0.1 to 1 ppm and decreased at 10 ppm (Table 2, Fig. 1A). The combinations of 2,4-D or NAA (1ppm) with kinetin (0-1ppm) gave the highest callus increase (341-395%) (Table 2, Fig. 1A). A high kinetin level (10 ppm) slightly increased callus growth mainly when media contained low NAA levels (0-0.1ppm) but in combination with 2,4-D (0.1-1 ppm) it reduced growth (Table 2, Fig. 1A). Kinetin alone (10ppm) mainly affected morphogenesis, where green hard callus with some shoot formation occurred. The absence of auxin or low levels of auxin (0, 0.01, and 0.1 ppm) in combination with all kinetin levels only induced a low frequency of small globular embryos (Table 2, Fig 13). High 2,4-D levels (1 and 10 ppm) in combination with kinetin (0-1 ppm) induced a 30-50 fold higher frequency: whereas, the highest kinetin (10 ppm) and 2,4-D (10ppm) combination inhibited their formation (Table 2, Fig. 13). High NAA levels (1-10ppm) also increased the frequency of globular embryos, but the effect was not as pronounced as that of 2,4-D: (Table 2, Fig. 1B). SS-derived callus had only a few large embryoids on IM (Table 2, Fig. 1B). 59 The interaction of auxin x kinetin in IM was significant in effecting the number _of bipolar embryos formed by SS-derived calli on MM (Table 1). Low levels of 2,4-D or NAA (0-0.1 ppm) with kinetin (0-10 ppm) in IM gave rise to only a few (2-11/g callus) bipolar embryos on MM (Table 2, Fig. 1C), while higher auxin levels (1 and 10ppm) significantly increased the frequency of such embryos: by 5- 40 fold (2,4-D) and 2-5 fold (NAA) (Table 2, Fig. 1C). However, 2,4-D also gave rise to a high number of abnormal embryo structures that resembled fused elongated roots, and only a few typical embryoids that gave rise to plantlets (Fig. 1C and D). In this experiment, SS-derived callus gave rise to a very low frequency of embryos with the capacity to convert into plantlets following treatment with the various IM (Fig. 1D). However, in a separate experiment (with male line A-9), SS-derived callus - gave rise to a higher conversion rate (11%) following transfer from MS + (ppm): 2,4-D (0.5-3) and kinetin (0.5) to MS + (ppm): NAA (1.0) and kinetin (0.3) (Appendix). WNW“ Within four weeks on the initiation medium of MS + (ppm): NAA (0.1) and kinetin (0.01), LB gave rise to a hard compact callus that simultaneously formed roots with a yellow friable callus and globular embryos. The friable callus portion was subcultured twice at four weeks intervals: it proliferated vigorously, forming new embryos while mature embryos developed into plantlets. At the end 60 of the second subculture stage, friable portions of the callus were transferred to IM. Auxin and kinetin concentrations alone had a significant effect on callus growth on IM, but the interaction was not significant (Table 1). The combination of 2,4-D or NAA at 1ppm with kinetin (0-1 and 0-10 ppm, respectively) gave the highest callus increases (431-471% and 439-514%, respectively) (Table 3, Fig. 2A). There was no difference between kinetin levels (0-1ppm) in effecting callus growth on IM with low auxin levels (0-0.1ppm) (Table 3, Fig. 2A). However, in combination with high auxin levels (1 and 10ppm), high kinetin level (10ppm) reduced callus growth (Table 3, Fig. 2A). The interaction of auxin x kinetin was highly significant in effecting the frequency of globular and bipolar embryos on IM (Table 1). There was no difference in the frequency of globular embryos on IM with low auxin levels (0-0.1 ppm) in combination with kinetin (0-1ppm): 28- 50 embryos/g callus, while high kinetin (10 ppm) reduced their frequency: 15-19/g callus (Table 3, Fig. 23). High auxin levels (1 and 10 ppm) in combination with kinetin (0- 1ppm), sharply increased the frequency of globular embryos: by 3.1-10 fold(NAA), and 5-15 fold(2,4-D), whereas, high kinetin (10ppm), particularly in combination with high auxin (1 and 10ppm) inhibited their formation (Table 3, Fig. 28). High kinetin (10ppm) had mainly a morphogenetic effect as it negated embryogenesis (Figs. 23, C and D) while inducing 61 greening of calli and some shoot formation. Calli derived from LB cultured on IM with auxin (0-0.1ppm) and kinetin (O- lppm) formed 17-21 bipolar embryos/g callus. High auxin (1 and 10ppm) and kinetin levels (10ppm) reduced the number of bipolar embryos but not significantly (5-13 embryos/g callus: Table 3, Fig. 23). High auxin levels xinhibited embryo growth, leading to a reduction in number of bipolar embryos while enhancing the formation of new globular embryos (Table 3, Fig. 23). The interaction of auxin x kinetin levels in IM was highly significant in effecting the frequency of bipolar embryos and their conversion rate into plantlets on MM (Table 1). Low auxin levels (0-0.1ppm) in combination with kinetin (0-1ppm) in IM induced only a low frequency of bipolar embryos on MM (between 17-27 per gram callus), while high levels of 2,4-D and NAA (1 and 10ppm) in combination with low kinetin levels (0-0.1 and 0-1ppm, respectively) sharply increased their frequency by 1.3-3.5 fold (Table 3, Fig. 2C). High kinetin (10ppm) sharply reduced the number of embryos and their conversion into plantlets on MM (Table 3, Fig. 20). Induction with various 2,4-D levels (0-10 ppm) or with low NAA levels (0-0.1) in combination with kinetin (O-lppm) gave rise to 1.7-4.7 plantlets/g callus (12.5- 22.3%) on MM, while induction with NAA at levels of 1 and 10ppm in combination with low kinetin levels (0-1 ppm), resulted in a higher number of plantlets: 8.3-10.7/g callus (14-21%) (Table 3, Fig. 2D). High 2,4-D levels (1 and 62 10ppm) induced the formation of abnormal embryos similar to those described for SS-derived callus. This resulted in a low conversion rate (1.3-3.7 plantlets/g callus: 2.3-5.7%) (Table 3, Fig. 20). mmmufllzderimsallus IVC-derived callus placed on MS + various levels of NAA and kinetin (0-1 ppm) occasionally formed a compact organized callus with shoot and root initiation, and simultaneously a yellowish friable embryogenic callus. The letter also consisted of embryos at various developmental stages together with a few plantlets. This callus was subcultured twice at four week intervals, and the fine friable portion of the calli was used for the experiment on IM. There was no interaction between auxin and kinetin with respect to callus growth on IM (Table 1). On IM the fresh weight of callus slightly increased as auxin levels increased from 0 to 0.1 ppm, sharply increased at 0.1 to 1 ppm, and decreased at 10 ppm. The combination of 2,4-D or NAA (1 ppm) and kinetin (0-1 ppm) gave rise to the highest callus increases (508-623 and 582-620%, respectively). High kinetin (10 ppm) reduced callus growth. This was particularly evident in combination with 2,4-D or NAA at levels of O-lppm (Table 4, Fig. 3A). The interaction auxin x kinetin was highly significant with respect to the frequency of globular and bipolar embryos on IM (Table 1). IVC-derived callus formed a 63 relatively large number of embryos in various developmental stages on IM with low auxin levels (0-0.1 ppm) and kinetin (0-1ppm) (18-37 globular and 59-94 bipolar embryos/g callus: Table 4, Fig. 33). However, higher auxin levels (1 and 10ppm) sharply increased the number of globular embryos, while decreasing the number of bipolar embryos, and high kinetin (10 ppm) inhibited embryo formation while (Table 4, Fig. 33) inducing greening and shoot formation. The interaction of auxin x kinetin in IM was significant with respect to the number of bipolar embryos and mature plantlets formed on MM (Table 1). Callus transferred from IM with low auxin levels (0-0.1ppm), gave rise to embryos in various developmental stages: 48-84/g callus (Table 4, Fig 3C). Higher levels of 2,4-D (1 and 10 ppm) or NAA (10 ppm) in combination with kinetin (0-1 ppm) increased the frequency of such embryos on MM: by 1.3-2.8 fold (2,4-D) and 1.2 fold (NAA) (Table 4, Fig. 3C). Low auxin levels (0-0.1ppm) in combination with kinetin (0-1 ppm) gave rise to 7.2-11.7 plantlets/g callus (9-17.7%) on MM. High 2,4-D (10ppm) reduced it to 1.7-2.7 plantlets/g callus (1.1-2.1%), while NAA (1 and 10ppm) slightly increased it to 9.8-13.7 plantlets/g callus (9-17.3%) (Table 4, Fig. 3D). Induction with high kinetin (10ppm), significantly reduced embryo formation and conversion rate to plantlets on MM (Table 4, Figs. 3C and D). Continuous subculture of embryos on MM at two-three weeks intervals, during a four to six month period, enhanced 64 the formation of new embryos and their conversion into plantlets. This was mainly in LB and. IVC-derived callus induced with NAA (10ppm): 4-13 plantlets/g callus in each subculture. 0n MM mostly typical embryos were formed. However, abnormal embryos in various frequencies were also observed (Fig. 4). Such embryos may have developed a cotyledon and radical, but had an undeveloped shoot meristem. 0n the other hand, some embryos had more than one shoot meristem (3-4) developing at the same time. In addition, fully developed embryos were partly fused or clustered with each other. Continual subculture of callus of line A-9 every 4 weeks on MM reduced the frequency of abnormal embryos while it slightly increased the number of embryos that converted into plantlets (Fig. 4). 65 Table 1. AOV for callus increase and frequency of somatic embryos and mature plantlets on IM and MM for three asparagus explants. Mixed callus IM MM Source DF Callus Globular Bipolar Bipolar Plantlets (%) (<3mm) (4-7mm) (4-7mm) R 5 NS NS NS NS NS A 9 *s as e as s K 3 e as as as NS A x K 27 * ** NS ** NS Marilee callus IM MM Source DF Callus Globular Bipolar Bipolar Plantlets (%) (<3mm) (4-7mm) (4-7mm) R 5 NS NS NS NS NS A 9 es s * es s: K 3 a *s as as as A x K 27 NS * *e as as mum callus IM MM Source DF Callus Globular Bipolar Bipolar Plantlets (%) (<3mm) (4-7mm) (4-7mm) R 5 NS NS NS NS NS A 9 ** ** es ** ** K 3 es *4 as as ea A x K 27 NS ** as e * **, *, NS, significant at the 1%, significant, respectively. R a Replications A - Auxin in IM K = Kinetin in IM levels, or not 66 Table 2. Responses of SS-derived callus on IM and MM. The effect of 2,4-D or NAA and kinetin on callus growth (%) and number of globular and bipolar embryos after 4 weeks on IM: and bipolar embryos and plantlets after 6 weeks on MM. IM MM 2,4-D Kinetin Callus GlObular Bipolar Bipolar *Plantlets (ppm) (ppm) (%) (<3mm) (4-7mm) (4-7mm) 0 O 109 5 8 7 ’0?3 0.1 86 6 3 7 0.3 1 190 8 9 9 0.5 10 178 8 11 7 0.5 0.01 0 136 5 7 3 0 0.1 133 7 4 8 0.5 1 206 6 10 8 0.3 10 146 7 8 8 0.2 0.1 0 295 13 6 2 0.2 0.1 243 8 6 5 0.3 1 244 14 8 8 0.2 10 109 15 8 9 0.3 1 0 392 284 4 82 1.8 0.1 341 312 2 77 1.5 1 366 314 7 50 0.8 10 276 49 6 29 0.8 10 0 264 491 3 147 1.2 0.1 261 479 2 180 2.0 1 269 489 4 161 1.3 10 221 180 8 67 1.8 NAA (PPM) 0 99 2 8 10 0.5 0.1 81 3 8 10 0.2 1 164 6 6 7 0.3 10 207 6 3 10 0.5 0.01 0 88 5 8 8 0.2 0.1 118 3 4 9 0.2 1 125 8 9 9 0.3 10 185 7 4 11 0.2 0.1 0 95 4 8 7 0.3 0.1 129 3 5 10 0.3 1 142 10 9 5 0.2 10 228 6 2 8 0.2 1 0 344 33 8 17 0.7 0.1 370 36 3 23 0.7 1 395 47 8 24 0.8 10 385 23 4 14 0.3 10 ' 0 256 95 3 27 1.5 0.1 239 84 2 35 1.3 1 239 88 6 37 1.3 10 294 65 2 22 0.8 L S D (1%) 101 41 6.4 26.5 1.4 67 A. ”mass of SS-Garlvad callus B. Globular (G) and Bipolar (B) Embryos on M on IM nu)n-nz-‘ u0v-‘ . a u I a O s 2 LOO-I... LSD-ll Fig. 1. Responses of SS-derived callus on IM and MM: A) % callus growth and 3) Number of globular embryos (<3mm) and bipolar embryos (4-7mm) after 4 weeks on IM, C) Number of bipolar embryos and D) plantlets after 6 weeks on MM. 68 Table 3. Responses of LB-derived callus on IM and MM. The effect of 2,4-D or NAA and KIN on: % callus growth, number of globular and bipolar embryos after 4 weeks on IM: and bipolar embryos and plantlets after 6 weeks on MM. IM' MM 2,4-D Kinetin Callus Globular BipoIar Bipolar Plantlets (99!!!) (PPM) ( 15) (<3mm) (4'71“!) (4'71“!) 0 O 206 39 21 22 3.6 0.1 208 48 19 20 3.6 1 217 30 17 23 3.3 10 201 16 10 4 0.3 0.01 0 191 48 21 20 4.1 0.1 186 50 19 22 4.5 l 217 28 18 23 3.0 10 189 19 10 4 0.3 0.1 0 286 47 20 24 4.0 0.1 242 49 20 23 4.2 1 321 31 17 23 3.7 10 220 18 10 4 0.3 1 0 431 265 13 68 3.7 0.1 414 241 13 68 1.8 l 471 230 10 32 1.7 10 352 29 7 6 0.3 10 0 325 433 6 52 2.3 0.1 277 448 7 41 1.3 1 242 450 5 48 '2.0 10 249 198 8 23 1.2 NAA (PPM) 0 0 242 41 20 28 3.5 0.1 276 38 19 25 4.5 l 185 39 19 17 3.3 10 191 17 8 6 0.5 0.01 0 229 48 20 24 3.5 0.1 251 45 18 25 4.5 l 212 35 20 19 3.2 10 225 17 9 6 0.7 0.1 0 217 47 19 27 4.2 0.1 216 48 18 26 4.7 1 220 31 16 21 3.7 10 266 15 9 7 0.7 1 0 504 133 20 41 9.2 0.1 574 168 13 45 8.3 1 472 170 10 51 10.5 10 439 37 7 4 0.5 10 0 343 294 13 58 8.5 0.1 310 280 9 60 9.2 1 266 306 7 71 10.7 10 217 75 5 27 3.5 LSD (1%) 112 49 15.4 9.3 2.3 69 A. summons-dementia smlelwamlelsmm on“ onlM U0<~oxa 10" a \‘ ‘\‘ s e r Leo-«(0), Is.a(a) D. Plantlets on W Iowan-p42)... Klnethbem) LOO-l3 Fig. 2. Responses of LB-derived callus on IM and M: A) % callus growth and 3) number of globular and bipolar embryos after 4 weeks on IM, C) Number of bipolar embryos and D) plantlets after 6 weeks on M. Table 4. 70 Responses of IVC-derived callus on IM and MM as cal % us growth, formation of globular and bipolar embryos after 4 weeks on IM. Number of bipolar embryos and plantlets after 6 weeks on MM. 2,4-D Kinetin (ppm) 6““— 10 NAA (PPM) 10 LSD (ppm) (%) (<3mm) (4-7mm) (4-7mm) "EF"" '259" “"30"’ "‘7§“' “‘63" IM ’MM Callus Globular Bipolar Bipolar Plantlets 0.1 207 30 83 71 1 306 18 63 48 10 195 9 22 15 0 289 31 83 70 0.1 211 31 91 67 1 323 26 65 53 10 180 10 16 12 0 312 35 87 75 0.1 297 33 94 84 1 353 26 66 70 10 209 10 17 13 0 508 317 15 118 0.1 531 372 15 119 1 623 288 19 p 86 10 414 49 12 42 0 319 449 8 165 0.1 374 467 6 142 1 356 400 6 143 10 294 158 4 108 0 276 28 89 71 0.1 314 36 83 75 1 294 30 74 73 10 223 8 23 21 0 266 32 91 69 0.1 319 33 89 84 1 322 21 59 68 10 232 7 16 18 0 297 37 78 79 0.1 364 f 34 90 84 1 318 20 74 75 10 247 7 16 15 0 582 114 44 78 0.1 601 133 37 86 1 620 110 37 81 10 447 23 8 26 0 367 219 22 99 0.1 390 251 21 96 1 406 226 20 107 10 334 117 8 52 (1%) 133 50 17.9 25.4 p \IQOQ \IQNIN \IQNH \INQU \INQU Hh‘ NNNH quq omoo omqw H440 H O O O O H HHH H H U! Pia-MOO HHUu HmI-HO bd‘OO HQ‘D‘D N (”\IQO UIWUO NOQN \IQNO (JUNO 71 A. i lnereasa IVC-Derived Calue B. Globular (G) and Bipolar (3) Embryos on M on IM less I M - _ 7 I I“. N . __ __ _ C Vase l I l E o . II I - g 5 NO . "'A a.I s ‘ we ' w\+\ O OO OO O. OO O. OO OI OI V u0