Effects of N-1-naphthylphthalamic acid on the growth and bud formation of tobacco callus grown in vitro

The effect of N-1 -naphthylphthalamic acid (NPA), indole-3-aceticacid (IAA) and kinetin on callus growth and bud formation wasstudied mainly by a tobacco callus culture method. Callus producedfrom Nicotiana tabacum var. Wisconsin 38 was used as the testplant material. Callus growth on nutrient agar containing 2mg/liter of IAA was promoted by NPA added at a concentrationof 0.5 mg/liter with 0.4 mg/liter of kinetin or by NPA addedat 5 mg/liter in the absence of kinetin. At a high concentrationof 50 mg/liter, however, NPA inhibited growth on the mediumcontaining 2 mg/liter IAA and no kinetin. Kinetin reduced thisNPA inhibition. In the presence of 0.4 mg/liter kinetin and2 mg/liter IAA, when the concentration of NPA was 50 mg/liter,buds were initiated after calluses were grown on the test mediumfor 7 weeks in dim light, but no buds formed when NPA was omittedfrom the above medium. The control of callus growth and bud initiation is based onthe active ratio of auxin (IAA) to cytokinin (kinetin) in themedium and NPA added to the medium can promote or inhibit callusgrowth and induce bud formation. Therefore, it is proposed thatNPA can itself reduce auxin activity or enhance cytokinin activityand hence change the active ratio of the two regulators. NPAmay enhance the activity of cytokinin (here supplied as kinetin)but cannot substitute for it. ¹Present address: Department of Biology, Wisconsin State University,Oshkosh, Wisconsin 54901, U. S. A. (Received March 10, 1969; )     

ATTEMPTS TO OBTAIN BACTERIA-FREE PLANTS OF PSYCHOTRIA PUNCTATA (RUBIACEAE): GROWTH AND ROOT FORMATION IN CALLUS CULTURES

Attempts were made to obtain bacteria-free plants of Psychotria punctata from tissue cultures. Stem explants and callus derived from them were induced to form roots but failed to form buds on Linsmaier and Skoog medium and 96 chemical modifications of it, including most of those known to induce bud formation in other species. Roots formed with ample IAA (2 mg/liter or more) and a low kinetin concentration (0.25 or 0.50 mg/liter). Adenine inhibited root formation in these media, but tyrosine did not. Tyrosine did lower the percentage of calluses commencing growth. When enzyme-hydrolyzed lactalbumin (1.3 g/liter), kinetin (0.5 mg/liter) and IAA (5 mg/liter) were added to Linsmaier and Skoog medium modified by decreasing inorganic nitrogen and increasing inorganic phosphate, callus grew at the fastest rate observed (increasing threefold in fresh weight in three weeks) and formed numerous roots. This was adopted as the stock callus medium. Casein hydrolysates also stimulated growth but less so than lactalbumin hydrolysate. When lactalbumin hydrolysate or a casein hydrolysate lacking tryptophan was supplied, growth occurred without added auxin if sufficient cytokinin was added. Cytokinin was required at unusually high concentration and was tolerated at still higher concentration. Formation, elongation, and branching of roots persisted on a saturated solution of BA which inhibited callus growth about 70 % and delayed callus senescence. Light caused earlier callus senescence after growth had ceased but did not affect callus growth or root formation. Light-induced senescence was prevented by a high cytokinin concentration.     

CONTROL OF IN VITRO SEPAL DEVELOPMENT OF EXCISED FLORAL BUDS OF AQUILEGIA (RANUNCULACEAE)

Excised floral buds of Aquilegia formosa Fisch. were grown on a coconut-milk medium containing the minerals and vitamins of Murashige and Skoog, sucrose, and kinetin. The plant growth regulators indoleacetic acid (IAA, 0.5 mg/liter) and gibberellic acid (GA, 2.0 mg/liter) were added singly and in combinations; both were omitted from the control medium. The addition of GA to the basal medium was required to support sepal development on flowers at all phases of development. The formation of stomatal complexes in the epidermis of the sepals occurred only in the presence of GA. Sepals grown in the presence of GA also contained trichoblasts and developing trichomes, while none were formed in the absence of GA. The role of IAA in the development of these idioblasts was not clear but it appeared to have no effect. The hormones GA and IAA had different effects on the growth of the sepals. In the presence of GA the sepals increase in length until comparable with sepals grown in vivo. However, the sepals remained small when GA was omitted from the medium. Upon closer examination of this effect, it was determined that there was a direct proportionality between an increase in cell number in the epidermis and an increase in sepal length. The role of the two hormones in increasing epidermal cell length in sepals was distinct and separate. Exogenous IAA had no effect upon cell division but was required for cell elongation, while GA was required for cell division and had no effect on cell elongation. The GA effect in promoting cell division in the sepals was substantiated by use of autoradiography. If the buds were grown on media with GA, twice as many epidermal cells along the central file incorporated significant amounts of tritiated thymidine. The cell cycle of the epidermal cells of the GA-treated sepals was ca. 8.7 hr in duration and ca. 13.0 hr if GA was deleted from the medium.     

DEVELOPMENTAL POTENTIAL OF EXCISED PRIMORDIAL AND EXPANDING LEAVES OF COLEUS BLUMEI BENTH.

Coleus blumei Benth. primordial leaves 1 through 4 and expanding leaves 5 to 8 were isolated and cultured to examine the effects of auxin and kinetin on development. Without the plant growth regulators in the medium, expanding leaves 7 and 8 developed as leaves; younger leaf primordia did not develop. With 0.01 to 5.0 mg/1 IAA, 2–7% of the youngest pair of primordial leaves (1 and 2) developed as roots. Small leaf blade development occurred on IAA at 0.5 to 5.0 mg/1 with 10–12% of the explants, and shoots developed from 2% of the youngest primordia explants at 3 mg/1 IAA. With 2–28% of the second set of primordial leaves (3 and 4), a leaf with a root developed with 0.01 to 5.0 mg/1 IAA. At 3.0 mg/1 IAA, in addition to leaf formation, 2% of the explants formed a rosette of leaves and 1% formed a shoot. With the highest level of IAA (5 mg/1), 2% of the explants formed a root. Expanding leaves 5 through 8 developed mostly into leaves without petioles on IAA and kinetin. Plant development occurred from 2% of the youngest primordial leaves on 0.03 mg/1 kinetin; otherwise, these primordia on 0.003 to 2 mg/1 kinetin developed into abnormal leaves. Primordia 3 and 4 developed into normal appearing leaves at levels of kinetin between 0.03 and 2 mg/1. At lower levels the leaves were abnormal.     

Responses of tobacco pith nucleic to growth substances

Summary A cytological study was made of excised segments of pith of tobacco (ev. Wisconsin 38) grown in vitro on media containing various growth substances. Explants were cultured on a basal medium and on media containing kinetin, indoleacetic acid, gibberellic acid, and combinations of these three growth substances. Controls consisted of pith segments chemically fixed immediately after excision. The nuclei of explants on the basal medium and those on media containing the growth substances showed a response as early as 1 day after the beginning of the experiment. Nucleoli were indicators of initial stimulation in that they became swollen and displayed internal vacuolation, a known characteristic of active cells. These fatures were more pronounced by the second day for cultures grown on media containing growth regulators. Nucleoli were centers of nucleohistone staining as shown by the ammoniacal silver method. Continued and sustained growth of explants was possible only on media containing combinations of IAA and kinetin, or a combination of kinetin, IAA and GA. The greatest initial nucleolar stimulation occurred with IAA alone, even though subsequent growth did not take place. There was no nucleolar response of explants grown on abscisic acid alone.Abbreviations ABA abscisic acid - GA gibberellic acid - IAA indole-3-acetic acid The senior author is most grateful for support by fellowships from the Fulbright Commission and the John Simon Guggenheim Memorial Foundation. The authors wish to thank Mrs. Colette Nitsch, Miss Simone Hamon and Miss Linda Robinson for their valuable technical assistance.     

Development of Asparagus plumosus Shoot Tips Grown in vitro

Lateral shoot tips from young Asparagus setaceus (Kunth) Jessop (syn. A. plumosus Baker) shoots were grown on a modified Murashige and Skoog medium. Tips from 5 to 20 mm lateral shoots had significantly better growth and development than tips from lateral shoots (2 mm) still covered by leaf-scale. The optimum temperature for growth and development of the explants was 17 to 24°C. The initial growth was fast at 24°C but stopped after about 4 weeks. At 17°C the growth was slow but in return the cultures continued to grow. Kinetin was necessary for growth. Without any kinetin all cultures died. Optimum growth was found with 2 mg/l kinetin. There was no growth at all with IAA alone. A low IAA concentration had no effect, but at high concentrations IAA inhibited the kinetin induced growth.     

IN VITRO CULTURE OF FLORAL BUDS OF AQUILEGIA

Tepfer, S. S., R. I. Greyson, W. R. Craig, and J. L. Hindman. (U. Oregon, Eugene.) In vitro culture of floral buds of Aquilegia. Amer. Jour. Bot. 50(10): 1035–1045. Illus. 1963.—Floral buds at various stages of development, from early stages before sepal initiation to late stages with young carpel primordia present, were grown in culture on various agar media. A basic medium containing White's minerals, Nitsch's trace elements, coconut milk, sucrose, and assorted water-soluble vitamins was developed for growth of the buds. The addition of indoleacetic acid, gibberellic acid, and kinetin to the basic medium extended the developmental limits of buds at nearly all stages and decidedly improved the continued development of carpels. On this medium buds produce all of their organ primordia, growing from early stages to about the size of flowers at anthesis, but will not develop unless the sepals are removed. This inhibiting effect of sepals is not understood at this time. Stamen development is consistently poor beyond the point of differentiation of anther and filament, even with the addition of hormones. The development of buds in culture is illustrated and compared with development in intact plants. With further improvement of the medium, it is hoped that these buds may be used for experiments testing theories of floral morphogenesis.     

Morphogenic Potentialities of Flower Buds of Kalanchoe pinnata Pers. grown in vitro

Excised flower buds of Kalanchoe pinnata Pers. representingtwo early stages of development (designated sets I and II),were cultured on modified White's medium (WB). They failed toattain full development on WB or on WB containing any of thefollowing supplements: indole-3yl-acetic acid (IAA), 2,4-dichlorophenoxyaceticacid (2,4-D), naphthaleneacetic acid (NAA), kinetin, or coconutmilk (CM). A slight stimulation of the growth of corolla wascaused by kinetin (10 ppm). IAA (I ppm) and NAA (I ppm) inducedrooting from the cut end of the pedicel and from the proliferatedtorus tissue situated between the sepals and petals. 2,4-D (Ippm) either singly or in concert with CM (10 per cent) stimulatedthe formation of shoot buds and root growth. Addition of kinetin(I and 10 ppm) to WB favoured shoot formation, but suppressedrooting. Flower buds of set II developed shoot buds more readilythan those of set I. Thus, the primordia floral organs presentin the immature buds lose their ability for normal morphogenesisunder culture conditions. Buds destined to form flowers canbe made to revert to vegetative growth.     

GROWTH AND DIFFERENTIATION OF JUICE VESICLES OF ORANGE GROWN IN VITRO

Juice vesicles of Valencia orange were grown on agar bases containing different concentrations of kinetin plus mineral and organic constituents, or in comparable liquid solution (shake cultures), maintained at approximately 26 C. Shake cultures enlarge most rapidly, but both maintain similar patterns of anatomical development. Typically, marginal parenchymatous cells of the sac become meristematic and develop, largely by periclinal division, generalized cambial meristems that enlarge the callus by the addition of linear rows of cells. Chloroplasts and wound tracheids with bordered pits mature within 30 days, but to date no further differentiation has been noted. Surface cells frequently enlarge, detach, and grow into branched hyphal aggregations of cells. Media containing 1.0 mg/liter kinetin causes greater enlargement of callus than media with 0.02 mg/liter kinetin.     

Tissue culture inHaworthia

Effects of three auxins and kinetin on growth of the calluses of two species ofHaworthia, H. aristala andH. setata, were investigated. Stock calluses derived from the flower buds of these species were maintained for two years on RM-1964 agar medium containing 5 mg/l NAA. Small pieces of the stock calluses were transferred to the basal medium containing either auxin, IAA, NAA or 2,4-D in six different concentrations (0.1–50 mg/l) combined with three concentrations (0–2 mg/l) of kinetin; in total, 54 kinds of media were used. Fresh weight of the calluses was measured 0 to 30 days from transfer and transformed to the natural logarithm. The linearity of their growth curves against the culture period was tested. The growth curves of theH. aristata calluses grown in dark and under continuous light and that of theH. setata callus grown in dark gave similar regression coefficients of 0.07 to 0.11, indicating that the doubling time of the callus mass was about 6.3 to 10.1 days. After 42 to 50 days from inoculation, the fresh weight of each individual callus was recorded, and the data were statistically analyzed. All auxins at the concentration of 50 mg/l significantly inhibited callus growth. Kinetin did not affect growth of theH. aristata callus in dark, while its effect on theH. setata callus was detected under light. Interaction of kinetin was found with IAA and 2,4-D inH. aristata and with IAA and NAA inH. setata. REsponses of theH. aristata callus to auxins and kinetin, when grown in dark, were different in several points from those of theH. setata callus grown under light. The best callus growth was observed in the following media; 0.2 mg/l kinetin supplemented with 1 mg/l IAA, or 0.5 mg/l 2,4-D, and 2 mg/l kinetin with 0.5 mg/l NAA inH. aristata, and 0.2 mg/l kinetin supplemented with 1 mg/l IAA, 5 mg/l NAA or 0.1 mg/l 2,4-D inH. setata. Contribution from the Laboratory of Genetics, Faculty of Agriculture, Kyoto University, Japan, No. 413.     

In vitro flowering and pod formation from cotyledons of groundnut (Arachis hypogaea L.)

Summary The response of groundnut cotyledons to the presence of various growth regulators in concentrations from 0.1 to 5 mg/l has been studied in detail using several genotypes of groundnut on two different media. Cotyledons with embryo axis, cultured on Blaydes' medium with cytokinins, produced shoots, in the axils of which 2–7 flower buds could be seen. The frequency of flower bud induction in general increased with increasing concentrations of cytokinins, the optimal levels being 3 mg/l of KN or 4 mg/l of BAP. Cotyledons without embryo axis, cultured on Blaydes' medium with BAP (0.5 mg/l), produced a cluster of flower buds directly, ranging in number from 8–28, without any vegetative growth. Excised embryo axes cultured on the same medium gave plantlets without flower buds. The growth regulators IAA, NAA, GA₃ and ABA failed to induce flower buds in independent treatments. However, lower concentrations of IAA and NAA in combination with cytokinins exerted a positive influence on flowering. The blooming of the flower buds was facilitated on media supplemented with low concentrations of cytokinins. Six percent of the induced flowers resulted in gynophore development and ultimately formed pods when cultured under complete dark conditions in modified MS medium supplemented with kinetin.     

NUTRITIONAL AND MORPHOGENETIC INVESTIGATIONS ON CALLUS CULTURES OF NEOMAMMILLARIA PROFILERA MILLER (CACTACEAE)

Neomammillaria prolifera (Cactaceae), when grown on Murashige and Skoog's medium supplemented with fresh coconut milk, showed very little growth. Various concentrations and combinations of growth regulators which did not cause callusing had no apparent effect on the normal growth rates of intact plants. Healthy green calli obtained on a 2,4-D and kinetin-containing medium exhibited extremely fast growth and very specific growth requirements. Relatively high amounts of 2,4-D (10–20 mg/liter), kinetin (1–2 mg/liter), and coconut milk (20–60%) were required at all times for continued proliferation of callus on subculturing. Moreover, the callus was very tolerant to extremely high concentrations of other growth regulators (IAA, NAA, IBA, and GA up to 100 mg/liter) in the presence of 2,4-D and coconut milk. These substances could not replace 2,4-D for callusing or continued growth of callus. It was not possible to establish root cultures or to induce callusing of roots. Attempts to induce differentiation in callus were unsuccessful, except for sporadic root initiation in some cultures. A comparison of these results with similar studies on other succulents demonstrates some basic physiological similarities among this group of plants.     

HORMONAL REQUIREMENTS OF EXCISED DIANTHUS CARYOPHYLLUS L. SHOOT APICAL MERISTEM IN VITRO

Whereas a medium containing kinetin alone enabled a few Dianthus caryophyllus L. apical meristem dome explants to develop into rooted plants, the highest frequency of plants was obtained in one containing supplements of both IAA and kinetin. In an unsupplemented medium, continued development required that explants have 2 pairs of primordial and a pair of expanding leaves. Kinetin alone caused production of many new leaves, but the development was significantly less than when it was furnished in combination with IAA. IAA given alone caused meristem explants to develop primarily callus, roots, and a few leaves. Gibberellin and abscisic acid were without promotive effects on leaf and shoot formation. A balance of hormonal substances, synthesized in young leaf structures and relocated to the meristem, is proposed as the fundamental mechanism that regulates new leaf initiation in the shoot apex.     

Plant regeneration from calli of melon (Cucumis melo L., cv. ‘Amarillo Oro’)

Callus cultures from cotyledon and hypocotyl explants of a Spanish cultivar of melon (Amarillo Oro) have been tested for their growth and morphogenic capacity on a series of media with different concentrations of indole-3-acetic acid (IAA) and 6-furfurylaminopurine (kinetin). Melon tissues were able to undergo morphogenesis both via organogenesis and embryogenesis, depending on culture conditions and explant source. Shoot buds were obtained at high rates in cotyledon explants. In response to 1.5 mg/l IAA and 6.0 mg/l kinetin, more than 90% of the calli produced well-developed shoots. Hypocotyls failed to form shoots but formed somatic embryos on auxin containing media while cotyledon explants usually gave abundant shoots but only rarely formed embryos. It was possible to maintain organogenic callus lines for at least 12 months under defined conditions. Plants were recovered from adventitious shoots produced both in cotyledon-derived calli and from organogenic cell lines.     

THE EFFECTS OF HORMONES UPON THE DEVELOPMENT OF EXCISED FLORAL BUDS OF AQUILEGIA

Young excised floral buds of Aquilegia were grown on defined medium containing kinetin, indoleacetic acid (IAA), or gibberellic acid (GA₃). Only when 10⁻⁶ or 10⁻⁷ m kinetin was added to the basal medium was there a significant increase in the number of initiated whorls of primordia. Buds on the basal medium or on medium with IAA or GA₃ failed to initiate carpels. On medium with 10⁻⁶ or 10⁻⁷ m kinetin, buds successfully initiated a normal whorl of five carpels. A high level of inorganic nitrogen was also required for the initiation of carpels. With 10⁻⁵ m kinetin, individual buds initiated from 6–18 carpels. Staminodial primordia of these buds were replaced with carpels, or the floral apex enlarged to accommodate a single whorl of many carpels. Kinetin did not support the further differentiation of the floral organs. Sepals, petals, and carpels did differentiate on medium with GA₃, but stamens aborted. However, on medium with GA₃ and kinetin, stamen primordia differentiated into short filaments and anthers. Further unknown growth factors appear to be required for the complete differentiation of floral primordia into mature organs.     

Micropropagation of globe artichoke (Cynara scolymus L.) from underground dormant buds (“ovoli”)

Summary Side shoots excised from underground dormant buds ofCynara scolymus L. were used as primary explants to establishin vitro cultures. A 3×3 factorial experiment with all possible combinations of three concentrations (0.5, 1.0, 2.0 mg/liter or 2.22, 4.44, 8.88 μM) ofN ⁶-benzyladenine (BA) and three concentrations (0, 0.1, 0.2 mg/liter or 0, 0.54, 1.07 μM) of 1-naphthaleneacetic acid (NAA) was used to determine the optimum growth regulator combination for shoot multiplication. The highest rate of axillary shoots was induced on Murashige and Skoog agar medium supplemented with 0 mg NAA/liter and 1.0 mg BA/liter (4.44 μM). Other cytokinins tested (kinetin, zeatin, and 2-isopentenyl-adenine were less effective than BA in inducing axillary shoot growth. Up to 60% of elongated microshoots rooted after 5 weeks on 1/2 MS agar medium supplemented with 2 mg/liter (11.42 μM) indole-3-acetic acid (IAA). Seventy percent of rooted plantlets were transferred successfully into soil. Plants are under evaluation for their genetic uniformity and clonal fidelity.     

In vitro propagation of shoot buds of Carica papaya L. (caricaceae) var. Honey Dew

Shoot buds from the saplings and the fruit bearing plants of Carica papaya L.. var. Honey Dew (papaya) initially treated with Gentamycin were cultured in modified MS media, each with a different hormonal combination, for the establishment of cultures and multiplication and rooting of plants. About 43% of explants from fruit bearing plants and 69% of those from saplings remained free of contamination and retained regeneration capacity when treated in 500 mg/l Gentamycin. For the establishment of the explants a medium containing 1 mg/l GA₃ and 2 mg/l kinetin was necessary. When established buds were transferred to medium containing 1 mg/l NAA and 3 mg/l kinetin, calli were initiated at cut ends of shoot buds; multiplication started on transfer to NAA (0.1 mg/l) and BAP (0.5 mg/l) medium. Cultures have been maintained for the last twenty months without any loss in multiplication rate. Rooting was induced in medium with reduced salt concentration containing 2 mg/l IBA. Shoot elongation was induced after prolonged culture in the same rooting medium.Abbreviations MS Murashige and Skoog, 1962 - SH Schenk and Hildebrandt, 1972 - GA₃ Gibberellic acid - Kn Kinetin - NAA Napthaleneacetic acid - BAP 6 -Benzylaminopurine - IBA Indole-3-butyric acid - IAA Indole-3-acetic acid     

Somatic embryogenesis in asparagus: the role of explants and growth regulators

Summary The explant used to initiate embryogenic callus and the growth regulators used in subsequent induction (IM) and embryo development media (EDM) both influenced rate of somatic embryo development and conversion to plantlets in asparagus. Embryogenic callus derived from spear-cross sections (SS), in vitro crowns (IVC) and lateral buds (LB) was cultured on IM of MS salts and vitamins with 2, 4-D or NAA at 0, 0.01, 0.1, 1.0 or 10 mg/l and kinetin at 0, 0.1, 1.0 or 10 mg/l. The auxin 2,4-D at 1–10 mg/l, in combination with kinetin at 0–1 mg/l, in IM induced the highest frequency of embryos after four weeks; callus derived from SS, IVC and LB had means of 394, 382, and 344 small globular embryos, and 4, 11 and 9 bipolar embryos per gram of callus, respectively. After 6 weeks on EDM, 128, 116 and 51 bipolar embryos (4–7 mm in length) occurred per gram callus and 4.5, 1.4 and 2.1 embryos converted for IVC, SS and LB, respectively. NAA at 1–10 mg/l, in combinations with kinetin 0–1 mg/l, yielded means of 64, 175 and 225 small globular embryos per gram callus on IM for SS, IVC and LB, respectively. NAA promoted a higher rate of embryo development: means of 27, 54 and 91 bipolar embryos per gram callus for SS, LB and IVC, respectively, on EDM. There were 0.5, 9.4 and 11.9 plantlets from these respective callus sources. There was no difference between kinetin levels of 0–1 mg/l on callus growth and embryogenesis, whereas, 10 mg/l in IM was inhibitory.Abbreviations 2,4-D 2,4 dichlorophenoxyacetic acid - EDM embryo development medium - IAA indole-3-acetic acid - IM induction media - IVC in vitro crowns - LB lateral bud - LS Linsmaier and Skoog (1965) - MS Murashige and Skoog (1962) - NAA naphthaleneacetic acid - SS spear-cross section     

Effects of Nitrogen, Sucrose, IAA and Kinetin on Explants of Beta vulgaris Grown in vitro

Hypocotyl explants of Beta vulgaris L. were grown on defined agar media with different combinations of IAA and kinetin at varying concentrations of nitrogen or sucrose. The cultures were kept in light (18 h a day) at 27°C for 5 weeks. Root initiation and callus growth were recorded and the callus tissue was analysed for N and K. Root formation was found to increase with increasing nitrogen concentration (from 5 mM to 23.3 mM) in the medium at 10.0 mg/1 of IAA, whereas no stimulation was found at 0.1 mg/1 of IAA. When raising the sucrose level from 20 g/1 to 100 mg/1 at 10.0 mg/1 of IAA and 1.0 mg/1 of kinetin, root initiation was also stimulated. At a lower kinetin and auxin level, however, no increase was recorded. Callus growth was affected by changes in the nitrogen or sucrose concentration of the culture media. The nitrogen content of the callus tissue increased with rising nitrogen concentration of the media. When raising the sucrose level instead of the nitrogen level, the nitrogen content of the tissue decreased.     

Somatic embryogenesis from leaf derived callus of Tylophora indica (Burm. f.) Merrill

Mature leaf explant derived callus of Tylophora indica (Burm. f.) Merrill yielded somatic embryos on MS medium supplied with BA(1-2 mg/L) or kinetin(1-5 mg/L) or kinetin/BA (1-2 mg/L) used along with IAA(0.1-1 mg/L). Maximum somatic embryos (30) could be recovered from 100 mg of embryogenic callus within 60 days at an optimum concentration of 2 mg/L of BA which was also best suited for providing the maximum conversion rate (90%) of embryoids to plantlets. Kinetin (1-5 mg/L), used as the sole growth hormone, induced the development of embryoids showing either shoot or root primordia in 30% of the cultures. However, embryoids with shoot primordia developed roots upon transfer to medium containing IAA(0.1 mg/L) and kinetin(2 mg/L). Embryoids from all cultures germinated in the initiation medium and were transplanted to sterile vermiculite for hardening. After two weeks of hardening, the plantlets were transferred to the green house where they grew and established well showing a high rate of survival (90%).