Direct somatic embryogenesis through thin cell layer culture in Panax ginseng

Direct somatic embryos were differentiated on cotyledon transverse Thin Cell Layers (tTCLs) of Panax ginseng after 9 weeks in the Murashige and Skoog basal (MS) medium containing 2,4-d (5M). When MS medium containing 2,4-d (5M) was used for seedling pretreatment and for tTCLs culture, somatic embryos were observed 2 weeks earlier, i.e. after 7 weeks of culture. On the tTCLs from seedlings pretreated with 2,4-d (5M) combined with benzyladenine and zeatin at 0.1 M (BZ), somatic embryos were observed after 6 weeks of culture and the percentage of embryogenesis was higher (62%) than when 2,4-d was used alone for pretreatment (40%). Similar results were also obtained from pretreatment with combinations of 2,4-d (5M) and thidiazuron (TDZ) (0.01, 0.1M). When a combination of 2,4-d (5M) and BZ (0.1M) was used both for seedling pretreatment and for tTCLs culture, both somatic embryos and shoots were observed after only 3 weeks. As the concentration of BZ increased, the percentage of somatic embryogenesis decreased but the percentage of organogenesis increased. Similar responses were obtained with a combination of 2,4-d (5M) and TDZ (0.01M). On the medium containing both NAA (0.3M) and BZ (1M), globular- and heart- stage embryos developed after 4 weeks of culture into cotyledonary-staged embryos which remained dormant after a short elongation of the embryo axis. The importance of seedling pretreatment by growth substances in enhancing somatic embryogenesis is reported.Abbreviations BA 6-benzyladenine - BZ combination of BA and zeatin - 2,4-d 2,4-dichlorophenoxyacetic acid - MS medium Murashige and Skoog basal medium - NAA a-naphthaleneacetic acid - TDZ thidiazuron - tTCLs transverse thin cell layers - TCL longitudinal thin cell layer     

Adventitious bud regeneration from leaf expiants of the shrubby ornamental honeysuckle,Lonicera nitida Wils. cv. ‘Maigrün’: effects of thidiazuron and 2,3,5-triiodobenzoic acid

Different combinations of auxins and cytokinins were employed to assess the regeneration capacity from in vitro leaf explants of Lonicera nitida Wils. cv Maïgrün. A high frequency of rhizogenesis was noticed, with 2.3 M thidiazuron plus 2.9 M indole-3-acetic acid as the only hormonal combination to support caulogenic responses. Increasing thidiazuron concentration and/or suppressing auxin did not improve caulogenesis. Combining thidiazuron with 2,3,5-triiodobenzoic acid produced a dramatic increase in the percentage of caulogenic explants. A maximum of 74% of adventitious bud forming explants was obtained with 2.3 M thidiazuron plus 20 M 2,3,5-triiodobenzoic acid. Buds were often in a rosette form and were vitreous, so that shoot elongation was difficult to obtain. The effect of the duration of the 2,3,5-triiodobenzoic acid treatment on shoot elongation was investigated.Abbreviations BAP benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - GA3 gibberellic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - 2 IP 2-isopentenyladenine - MS Murashige and Skoog - NAA -naphthylacetic acid - TDZ N-phenyl-N-1,2,3-thidiazol-5-ylurea (thidiazuron) - TIBA 2,3,5-triiodobenzoic acid - Z zeatin     

Establishment and characterization of Rubus tissue culture systems for in vitro bioassays against phytotoxins from Rubus fungal pathogens

The Rubus species R. parviflorus, R. spectabilis and R. strigosus interfere with conifer seedling establishment on forest regeneration sites in Canada and the United States. As a first step towards microbial metabolite-based control, callus and cell suspension cultures of the Rubus species were developed as a bioassay system to detect phytotoxic compounds that may have relevance in a vegetation control context. Rapidly growing friable callus and suspension cultures were obtained from leaf disks of the three weedy Rubus species using similar culture media conditions (modified Murashige and Skoog) but required different plant growth regulators (R. parviflorus, 4.5 M 2,4-D; R. spectabilis, 26.9 M NAA/0.5 M zeatin; and R. strigosus, 12.4 M picloram). Cell growth and health attributes including callus circumference, degree of browning and suspension culture cell viability as measured by the TTC vital stain assay were developed and were rapid and convenient to use. We have established Rubus tissue culture systems that will make it possible for large scale screening of phytotoxic metabolites.     

Plantlet regeneration from mesophyll protoplasts ofBetula platyphylla var.japonica

Summary InBetula platyphylla var.japonica, colonies were induced efficiently from mesophyll protoplasts cultured in half strength MS (1/2MS) liquid medium containing 0.6 M mannitol, 0.09M sucrose and 1 M 4-PU and 1 M NAA at a cell density of 5 × 10⁴/ml. The colonies grew actively and developed into callus after 3 months of culture.Roots differentiated from the protoplast-derived white calluses cultured on the 1 /2MS solid media supplemented with 0.1–1 M 4-PU and 1 M NAA, and 10 M zeatin with no supplementation of NAA. Furthermore, the protoplast-derived green callus differentiated shoots with 1/2MS solid medium containing 1 M 4-PU or 10 M zeatin with no supplementation of NAA. When shoots obtained were cultured on the cytokinin-free MS solid medium with 2.5 M IBA and 0.1 M NAA, they rooted and developed into plantlets after one month of culture.The phenylurea-type cytokinin, 4-PU, was effective for plantlet regeneration from the mesophyll protoplasts ofB. platyphylla var.japonica. This suggests that there is potential for the use of 4-PU in the culture of protoplasts in many forest tree species.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - FDA fluorescein diacetate - IBA indole-3-butyric acid - 2ip N ⁶-(2-isopentenyl)-adenine - NAA 1-naphthaleneacetic acid - 4-PU N-(2-chloro-4-pyridyl)-N–phenylurea - TDZ thidiazuron     

Somatic embryogenesis in callus cultures of Rosa hybrida L. cv. Landora

Callus was initiated from immature leaf and stem segments of rose (Rosa hybrida cv. Landora) and subcultured every four weeks on a basal medium of half-strength Murashige & Skoog (1962) salts plus 30 g l^-1 sucrose (1/2 MS) and supplemented with 2.2 M BA, 5.4 M NAA and 2.2–9.0 M 2,4-D. Embryogenic callus and subsequently somatic embryos were obtained from 8-week-old callus culture on 1/2 MS+2.2 M BA+0.05 M NAA+0.3 M GA₃+200–800 mg l^-1 L-proline. Long-term cultures were established and maintained for up to 16 months by repeated subculture of embryogenic callus on L-proline deficient medium. About 12% of cotyledonary stage embryos taken from cultures cold-stored at 8±1°C for 4 days germinated on 1/2 MS+2.2 M BA+0.3 M GA₃+24.7 M adenine sulphate.Abbreviations BA benzyladenine - NAA -naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid     

Axillary shoot induction and plant regeneration in Plantago ovata Forssk

Axillary shoot induction and plant regeneration were obtained in Plantago ovata. The optimum medium for inducing axillary shoots was Murashige & Skoog (MS) medium [5] supplemented with 4.6 M kinetin and 0.05 M NAA. Rooting of shoots was best on half-strength MS medium containing 5.0 M IBA and 0.05 M kinetin. The regenerated plants were similar to the control plants in karyotypic and phenotypic details.     

Plant regeneration from cotyledons of Prunus persica,Prunus domestica,and Prunus cerasus

Shoots were regenerated from the proximal region of immature cotyledons (with the embryonic axis removed) of Prunus persica (peach) and from the same area in mature cotyledons of P. domestica (plum) and P. cerasus (sour cherry) on MS medium containing (in mgl^-1) thiamine-HCl, 0.4; nicotinic acid, 0.5; pyridoxine-HCl, 0.5; sucrose, 25 000; and 0.7% agar. The medium was supplemented with 0.0–2.5 M indole-butyric acid and 5–12.5 M thidiazuron. Cultures were incubated at 24 °C under 16h photoperiod. Shoots regenerated adventitiously over a broad range of thidiazuron concentrations and 2.5 M indole-butyric acid in 35 days. The presence of the embryonic axis inhibited the development of shoots. Regenerated shoots of peach and plum were rooted on half-strength MS inorganic semi-solid medium with 2.5–5.0 M indole-butyric acid. Rooted plants were acclimatized and transferred to the greenhouse.Abbreviations BAP 6-benzylaminopurine - IBA indole-butyric acid - TDZ N-phenyl-N-1,2,3-thidiazol-5-ylurea - IAA indole-acetic acid - NAA -naphthalene-acetic acid     

Filterable marine bacteria found in the deep sea: Distribution,taxonomy, and response to starvation

A significant number of viable colony-forming bacteria were recovered from deep-ocean bottom water samples passed through a 0.45m filter. However, these bacteria small enough to pass through a 0.45m membrane filter and termed filterable bacteria were less abundant in open-ocean surface water and coastal water samples. The reduced size of bacterial cells present in deep-ocean bottom water samples was documented by scanning electron microscopy. The concentration of ATP in the water samples was found to be correlated with results of direct counts of bacteria.Numerical taxonomy of bacterial strains isolated from water samples collected at two stations in the deep sea yielded taxonomic clusters grouped according to sample and size fraction. The generic composition of bacterial populations of bottom water filtrates was compared with that of bacteria retained by 0.45 m filters. Strains ofAlcaligenes, Flavobacterium, Pseudomonas, andVibrio spp. were identified among those retained by, as well as passing through, 0.45m filters.Two marine isolates obtained from the filtrate of a deep-ocean water sample were incubated for 9 weeks in nutrient-free artificial seawater, during which the cells became rounded and reduced in size. After the 9-week incubation period, more than 10% of the viable cells of both cultures were able to pass through a 0.4m filter. The viable count at 9 weeks wasca. 10% of that of the initial population, although from direct counts the total population number remained relatively constant throughout the incubation period. From the observed reduction in cell size and increased starvation resistance of cells held under low nutrient conditions, it is concluded that a significant relationship exists between decreased cell size and increased survival of marine bacteria in the deep sea.This investigation was supported by Grant No. OCE 76-82655 from the National Science Foundation.     

In vitro regeneration of silktree (Albizzia julibrissin) from excised roots

Root segments (1 cm long) were excised from 15–20 day old seedlings of silktree (Albizzia julibrissin) grown on B5 medium. About 50% of the control (no growth regulators added) root explants formed shoot buds within 15 days after placement on the culture medium. After 30 days, there were about 4 shoots per control explant. Addition of low levels of various auxins (0.5 M) did not influence the formation of shoot buds from the explants. Higher concentrations (5M), however, decreased shoot regeneration. Kinetin and 2iP did not influence shoot regeneration at the concentrations tested (1 & 10 M). Addition of benzyladenine, Zeatin, or thidiazuron to the culture medium increased both the percentage of explants that formed shoots and the number of shoots per explant. Thidiazuron was highly effective in stimulating shoot formation at low concentrations (<1 M). At 0.05 M thidiazuron, 95% of the explants produced shoots and about 10 shoots were formed per explant. Compared to TDZ, higher concentrations (10 M) of benzyladenine and Zeatin were required to enhance shoot formation. Upon excision and transfer to B5 medium, regenerated shoots developed into normal rooted plantlets.Abbreviations BA Benzyladenine - IAA Indoleacetic acid - IBA Indolebutyric acid - NAA Naphthaleneacetic acid - TDZ Thidiazuron - 2ip Isopentenyladenine     

Long-term effects of thidiazuron are intermediate between benzyladenine,kinetin or isopentenyladenine in Miscanthus sinensis

The thidiazolylurea derivative thidiazuron has been reported to be considerably more effective than benzyladenine in promotion of in vitro shoot formation in a number of dicotyledonous species. In the present study, axillary shoots of Miscanthus sinensis (Thunb.) Anderss. Giganteus that had been subcultured four times on modified Murashige & Skoog medium with 20M benzyladenine were transferred to media with benzyladenine, kinetin, isopentenyladenine or thidiazuron at concentrations of 0.01, 0.1, 1, 10, 30 or 100M and grown over four subcultures. Shoot and root formation stabilized after the first subculture and results from the three subsequent subcultures are presented. The common effects of cytokinins, i.e., promotion of axillary bud growth, inhibition of root formation, reduced stem growth and delay of senescence, were observed for all four cytokinins. In a descending order regarding shoot formation, the four cytokinins at the optimum concentration could be ranked as follows: benzyladenine, thidiazuron, kinetin and isopentenyladenine. Benzyladenine and thidiazuron had optimum effects at the same concentration with regard to axillary shoot formation but thidiazuron induced a significantly lower number of shoots than benzyladenine. The number of roots, shoot size and percentage of chlorotic shoots were also the same for benzyladenine and thidiazuron. When transferring shoots from benzyladenine or thidiazuron medium to rooting medium, shoots previously grown on thidiazuron became taller and formed fewer roots than shoots previously grown on benzyladenine.Abbreviations BA benzyladenine - 2iP isopentenyladenine - KIN 6-(furfurylamino)-purine (kinetin) - MS Murashige & Skoog medium - NAA naphthaleneacetic acid - THI N-phenyl-N(1,2,3-thidiazol-5-yl)-urea (thidiazuron)     

Factors influencing shoot regeneration from cotyledonary explants ofCucumis melo

Cotyledonary explants of 4-day-oldCucumis melo cv. Hale's Best Jumbo in vitro seedlings showed maximum initiation of shoot buds when cultured onto a revised Murashige & Skoog medium supplemented with 5 M indole-3-acetic acid and 5 M benzylaminopurine and cultured at 25–29°C under low light intensity (5–30 mol m^-2 s^-1). Subculture of the shoot buds onto the same medium without auxin and supplemented with 3 M benzylaminopurine caused the development of shoots from 30% of the buds. The presence of abscisic acid significantly increased the number of explants producing shoot buds. Bud initiation was affected by genotype, seedling age, light intensity, and temperature. Addition of gibberellic acid, thidiazuron or silver nitrate to regeneration medium did not improve either bud initiation or shoot regeneration.     

Agrobacterium-mediated transformation of hybrid poplar suspension cultures and regeneration of transformed plants

A method for Agrobacterium-mediated transformation of hybrid poplar (Populus alba x P. grandidentata cv. Crandon) suspension cultures and regeneration of transformed plants is described. Transformants were recovered when suspension cultures were inoculated with Agrobacterium tumefaciens at a density of 10⁷ colony-forming units ml^-1, cocultivated for 48 h, and plated to cellulose acetate filters on Woody Plant Medium containing 4.5 M 2,4-dichlorophenoxyacetic acid and 250 mg l^-1 cefotaxime. Levels of cefotaxime greater than 250 mg l^-1 were unnecessary for control of residual bacteria and inhibited callus growth. Transgenic plants were regenerated by culturing the transformed callus on media containing 0.11 to 27 M thidiazuron. In contrast to thidiazuron, N⁶-benzyladenine had a negative effect on shoot regeneration; the callus became necrotic when we attempted to induce shoots with concentrations of 1.1 to 8.9 M, and growth was inhibited when concentrations of 0.11 or 0.22 M were used to regenerate callus from suspension cultures. Following cocultivation of poplar suspension cultures, we recovered transgenic plants containing the maize transposon Ac, and callus containing an insect toxin gene from Bacillus thuringiensis.Abbreviations BA N⁶-benzyladenine - CIM callus initiation medium - CaMV cauliflower mosaic virus - cfu's colony-forming units - HPT hygromycin phosphotransferase - MS Murashige and Skoog medium (Murashige & Skoog 1962) - NPT-II neomycin phosphotransferase-II - PAR photosynthetically active radiation - PCR polymerase-chain-reaction - TDZ thidiazuron - WPM Woody Plant Medium (Lloyd & McCown 1980) - 2,4-d 2,4-dichlorophenoxyacetic acid     

Adventitious shoot regeneration from in vitro-cultured leaves of Rubus genotypes

Regeneration of adventitious shoots from leaves of several in vitro-cultured Rubus genotypes was affected by media components and incubation conditions. Leaves cultured at 20°C with a photosynthetic photon flux (PPF) of 40 mol m^-2 s^-1 had a higher regeneration frequency and more shoots per regenerating leaf than ones cultured at 25°C with a PPF of either 40 or 80 mol m^-2 s^-1. Thidiazuron (TDZ) was significantly more effective than benzyladenine. Medium containing 1 M TDZ had the highest percentage regeneration for leaves of Autumn Bliss, Canby, Summit and Sentry red raspberries, whereas leaves of MD-ETCE-1 blackberry hybrid responded more to 10 M TDZ. Higher regeneration frequencies were obtained with 0.5 M indolebutyric acid (IBA) than with 1 M, but no significant difference was observed between 0.5 M and no IBA in other experiments. More shoots per regenerating leaf formed on Murashige and Skoog (MS) and N6 media, than on half-strength MS, Anderson, or Woody Plant media for all genotypes tested. The youngest two expanding leaves near the shoot apex were the most regenerative and yielded the highest number of shoots per regenerating leaf.Abbreviations AND Anderson (1980) - BA benzyladenine - IBA indolebutyric acid - MS Murashige & Skoog (1962) - N6 Chu et al. (1975) - NAA naphthaleneacetic acid - PPF photosynthetic photon flux - TDZ thidiazuron (N-phenyl-N'-1, 2, 3-thiadiazol-5-ylurea) - WPM Woody Plant Medium [Lloyd & McCown (1980)]     

Callus induction and plant regeneration from mature zygotic embryos of a tetraploid Alstroemeria (A. pelegrina × A. psittacina)

Summary A simple procedure was developed to induce callus growth and whole plant regeneration for a tetraploid cultivar of Alstroemeria. The callus, induced from mature zygotic embryos cultured on a medium supplemented with 20 M kinetin with 10 or 20 M NAA, could be maintained for one year without any loss of regeneration potential. Maximum frequency of regeneration (40%) was obtained with calli maintained on the medium containing 20 M kinetin and 20 M NAA. Whole plant regeneration occurred via somatic embryogenesis in the absence of growth regulators and the plantlets grew to maturity and flowered in the greenhouse conditions.Abbreviations BAP N⁶-benzylaminopurine - MS Murashige and Skoog (1962) medium - MSO Basal medium devoid of any plant growth regulator - NAA -Naphthaleneacetic acid - TDZ N-phenyl-N 1,2,3,-thiadiazol-5-ylurea (thidiazuron) - IAA Indole-3-acetic acid - 2,4-D 2,4-Dichlorophenoxyacetic acid     

In vitro propagation of Anacardium occidentale L.

Multiple buds have been induced from cotyledonary nodes of cashew, Anacardium occidentale L. Maximum number of buds were initiated on Murashige & Skoog's (MS) medium supplemented with 117 mM sucrose, 14.6 mM maltose and 22.2 M BA. The buds were harvested at each subculture after elongation on MS medium supplemented with 100 ml l^-1 coconut water, 14.6 mM maltose, increased sucrose (146 mM) and decreased BA (4.4 M). Excised microshoots were rooted in vitro on MS medium in the presence of 117 mM sucrose, 2.9 M IAA and 4.9 M IBA. Plantlets have been successfully transferred to soil and have been established in the field.     

Somatic Embryogenesis and Plant Regeneration in Pigeonpea

Somatic embryogenesis in pigeonpea [Cajanus cajan (L.) Millsp.] has been achieved using cotyledon segments of mature seeds as explants. A large number of globular somatic embryos were induced directly from cotyledons of genotypes T-15-15, GAUT-82-90 and GAUT-82-99 when cultured on EC6 basal medium supplemented with 2.22, 4.44, 13.32 or 22.2 M N⁶-benzylaminopurine (BAP) and 0.45, 1.36, 2.27, 4.54 and 13.62 M thidiazuron. Somatic embryos developed into cotyledonary stage when the globular embryos were transferred to Murashige and Skoog's (MS) basal medium containing 2.89 – 14.43 M gibberellic acid. Maturation of somatic embryos was achieved on half strength MS medium with 0.38 M abscisic acid. The mature somatic embryos were germinated on MS medium supplemented with 0.44 M BAP and the plantlets were hardened and transferred to soil.     

Clonal propagation of Camptotheca acuminata through shoot bud culture

The chinese tree Camptotheca acuminata produces the anti-cancer and anti-retroviral drug camptothecin. Methods were developed for the clonal propagation of this important medicinal plant through shoot bud culture. Shoot buds were excised from 25 to 30 day old seedlings, presoaked for 48 h in three different liquid media containing either BA (2.22–17.4 M), kinetin (2.32–18.58 M), or thidiazuron (0.1–10 M) and were subsequently cultured on semi-solid medium of the same composition. Multiple shoots only developed from the 6-benzyladenine presoaked explants with the maximum number of shoots initiated from buds presoaked in and grown on B5 medium containing 17.4 M 6-benzyladenine. Individual shoots were removed from clusters and rooted on B5 supplemented with indole-3-butyric acid (4.9–19.6 M). The lowest concentration of indole-3-butyric acid (4.9 M) gave the highest percentage of rooting (82%) and the shortest root initiation period (18 d). Over 90% of the in vitro rooted plantlets survived transfer to soil.Abbreviations BA 6-benzyladenine - B5 Gamborg's B5 medium (Gamborg et al., 1968) - CPT camptothecin - 2,4-d 2,4-dichlorophenoxyacetic acid - IBA indole-3-butyric acid - kinetin 6-furfurylaminopurine - LS Linsmaier & Skoog medium (Linsmaier & Skoog, 1965) - MS Murashige & Skoog (Murashige & Skoog, 1962) - NAA I-naphthaleneacetic acid - PGR plant growth regulator - TDZ thidiazuron - WPM woody plant medium (Lloyd & McCown, 1981)     

In vitro plant regeneration via somatic embryogenesis from root culture of some rhizomatous irises

A method for plant regeneration of Iris via somatic embryogenesis is described. Root and leaf pieces from in vitro-grown plants of several genotypes of rhizomatous Iris sp. were cultured in vitro. Callus induction occurred only on root cultures incubated under low light intensity (35 mol m^-2 s^-1) on two induction media containing 2,4-D (4.5 or 22.5 M), NAA (5.4 M) and kinetin (0.5 M). Somatic embryos developed after transfer of callus onto four regeneration media containing 9 or 22 M BA, or 5 M kinetin and 2 M TIBA or 9 M BA and 4 M TIBA. Plantlets could be obtained from these somatic embryos. Genotypic differences were found both in callus induction and somatic embryo formation, with I. pseudacorus responding better than I. versicolor or I. setosa. Cytological analysis performed on root tips of 80 regenerated plants revealed that two of the I. pseudacorus regenerants were tetraploid.Abbreviations 2,4-D dichlorophenoxy acetic acid - NAA naphthaleneacetic acid - BA 6-benzyladenine - TIBA 2,3,5-triiodobenzoic acid - IBA indolebutyric acid     

Somatic embryogenesis and plant regeneration from immature embryos of saw palmetto,an important landscape and medicinal plant

Somatic embryogenesis and plant regeneration from immature zygotic embryos was achieved for saw palmetto (Serenoa repens (Bartr.) Small). Embryos, isolated from immature fruit of native-grown plants, were cultured on Murashige and Skoog medium plus 0.15% (w/v) activated charcoal and supplemented with 452 M 2,4-dichlorophenoxyacetic acid (2,4-D) and 14.7 M N⁶-(2-isopentenyl)adenine (2iP). Clusters of somatic embryos developed from all immature zygotic embryos 5 weeks after culture initiation. After 12 weeks, explants were transferred to the same medium with the amount of 2,4-D reduced to 90.4 M which resulted in somatic embryo proliferation. Somatic embryos were then transferred to the basal medium containing 0.9, 9 M thidiazuron (TDZ), or no growth regulator for conversion into plantlets. The 9 M TDZ treatment was ineffective for plant regeneration. However, 12% of the embryos subcultured on 0.9 M TDZ were able to produce complete plantlets. Shoot production was obtained from 35% of the embryos subcultured in the absence of growth regulators. Rooting (100%) was achieved when these shoots were transferred onto medium containing 22.2 M -naphthaleneacetic acid (NAA).     

In vitro regeneration of chickpea (Cicer arietinum L.): Stimulation of direct organogenesis and somatic embryogenesis by thidiazuron

In vitro regeneration in chickpea (Cicer arietinum L.) was achieved by direct culture of mature seeds on Murashige and Skoog (MS) medium supplemented with either N-phenyl-N(-1,2,3-thidiazol-5-yl) urea (thidiazuron, TDZ) or N⁶-benzylaminopurine (BAP). Multiple shoots formed de novo without an intermediary callus phase at the cotyledonary notch region of the seedlings within 2 to 3 weeks of culture initiation. TDZ was found to be more effective compared to BAP as an inductive signal of regeneration. The former induced multiple shoot formation at all the concentrations tested (1 M to 100 M), although, maximum morphogenic response was observed at 10 M concentration. Addition of naphthaleneacetic acid (NAA) alone or in combination with BAP to the MS medium failed to invoke a similar response. When the TDZ supplemented medium was amended with L-proline, the resultant regenerants were mostly somatic embryos. Histological investigations confirmed the switch in the regeneration pathway from directly formed adventitious shoots to embryogenesis. For obtaining plantlets, adventitious shoots were rooted on MS medium supplemented with 2.5 M NAA; somatic embryos were germinated and established on MS medium. Normal plants were regenerated from both adventitious shoots and somatic embryos and transferred to soil.Abbreviations BAP 6-benzylaminopurine - MS Murashige and Skoog [14] basal medium - NAA naphthaleneacetic acid - TDZ thidiazuron [N-phenyl-N(-1,2,3,-thidiazol-5-yl)-urea]