Thiadiazuron Research Articles

Ex situ Conservation Using In vitro Methods of an Endangered Plant Sterculia urens Roxb.: A High Volume Trade Plant for Gum Karaya

Sterculia urens is a vanishing non timber tree species of India, known for its gum exudates having high worldwide trade value and hence needs to be protected for its sustainable supply in nature. In present research, an efficient regeneration protocol is standardized using cotyledonary node of Sterculia urens (Gum Karaya) seedlings. In addition, appearance of different anomalies such as high callogenesis, yellowing of leaves, translucent shoots, shoot etiolation, reduced leaf number, and leaf abscission was observed during the study. Thus based on the assumption that these abnormalities can be a result of ethylene accretion, effect of silver nitrate (AgNO3; ethylene inhibitor) to overcome such abnormalities and enhancement of shoot quality was studied. Among different media compositions tried, full strength Murashige & Skoog (MS) medium supplemented with 0.33 mg/L thiadiazuron (TDZ), was found to be optimum resulting highest shoot multiplication (19.62 ± 0.27) after five weeks incubation with 81.9% regeneration response recorded. After mass regeneration shoots were transferred to full strength MS medium fortified with 0.2 mg/L N6-Benzyladenine (BAP), 1.5 mg/L 6-furfurylaminopurine (Kn), and 2.0 mg/L gibberellic acid (GA3) in combination, which resulted optimum shoot elongation (5.06 ± 0.41 cm) within 2 weeks. Treatments, AgNO3 (1.0 mg/L) with Kn (1.0 mg/L) in MS medium gave adequate shoot quality having dark green & shiny leaves, strengthened stem (dark green), reduced inter-nodal length (de-etiolation) and reduced callus formation. The optimum rhizogenesis (5.67 ± 0.58) was obtained on half strength MS fortified with IBA (2.0 mg/L) when shoots were given pulse treatment using IBA (200 mg/L) for 3-4 min. Further, process of biotization at hardening stage using Glomus sp. enhanced the plant survival rate (80%) at acclimatization stage. Mycorrhizae treated plants were healthy dark green in color having improved root system and less susceptibility to infections in comparison to untreated plants. Tissue culture raised plants revealed 95% genetic homogeneity through Jaccard’s silmilarity coefficient using RAPD markers. The established protocol can significantly be used to generate quality plantlets in elevated amounts to overcome declining abundance of S. urens in natural environment.

Micropropagation protocol for Stevia rebaudiana through axillary shoot proliferation

The present investigation was carried out at Anand Agricultural University, Gujarat during the year 2016 to develop a micropropagation protocol for mass multiplication of medicinally rich stevia plants. Axenic culture was established by sequential application of 200 mg/l Kanamycin, 200 mg/l Carbendazim-50% and 1000 mg/l HgCl2. Out of two different basal media, viz. Gamborg (B5) and Murashige and Skoog (MS) used, full strength MS gave highest (98%) culture establishment. Out of three different basal media, viz. Gamborg’s (B5), Linsmair and Skoog (LS), Murashige and Skoog (MS) and four different cytokinins, viz. 6-benzylaminopurine (BAP), Kinetin (Kn), Thiadiazuron (TDZ), Zeatin (Zn) tested for multiple shoot induction, full strength MS medium and 2 mg/l BAP, respectively, promoted maximum shoot formation. In the experiment to study the effect of varying strength of MS media and various plant growth regulators, viz. Indole-3-acetic acid (IAA), Indole-3-butyric acid (IBA), Naphthalene acetic acid (NAA) on in vitro rooting, ½ strength MS and 1 mg/l NAA, respectively, gave superior response. Among the 26 different combinations of potting mixtures used for primary acclimatization of in vitro rooted plants, cocopeat based substrates yielded highest survival rate (75.06%). Secondary acclimatization was carried out in the soil bags in the poly house. Thus, a reproducible and reliable micropropagation protocol for mass multiplication of Stevia rebaudiana (Bertoni) Bertoni using nodal segments has been developed.

Effect of Plant Growth Regulators on Coloured Callus Formation and Accumulation of Azadirachtin, an Essential Biopesticide in Azadirachta indica.

For centuries, Azadirachta indica or neem has been utilized as a primary source of medicine due to its antimicrobial, larvacidal, antimalarial and antifungal properties. Recently, its potential as an effective biopesticide has garnered attention, especially towards efficient and continuous production of its bioactive compounds. The present study investigated the effect of the plant growth regulators (PGRs) thiadiazuron (TDZ) and 2,4-dichlorophenoxyacetic acid (2,4-D) on the induction of colored callus formation and subsequent accumulation of azadirachtin (AZA) in A. indica. An efficient protocol was established for micropropagation and colored callus production of this species, followed by quantification of AZA (a mixture of azadirachtin A and B) and its safety assessment. For induction of the callus, leaf and petiole explants obtained from a young growing neem plant were excised and cultured on Murashige and Skoog (MS) medium supplemented with TDZ (0.2–0.6 mg L−1) and 2,4-D (0.2–0.6 mg L−1), either applied singly or in combination. Callus was successfully induced from both explant types at different rates, where media with 0.6 mg L−1 of TDZ resulted in the highest fresh weight (3.38 ± 0.08 g). In general, media with a single hormone (particularly TDZ) was more effective in producing a high mass of callus compared to combined PGRs. A culture duration of six weeks resulted in the production of green, brown and cream colored callus. The highest callus weight and accumulation of AZA was recorded in green callus (214.53 ± 33.63 mg g−1 dry weight (DW)) induced using TDZ. On the other hand, small amounts of AZA were detected in both brown and cream callus. Further experimentation indicated that the green callus with the highest AZA was found to be non-toxic (LC50 at 4606 µg mL−1) to the zebrafish animal model. These results suggested that the addition of different PGRs during in vitro culture could prominently affect callus and secondary metabolite production and can further be manipulated as a sustainable method for the production of a natural and environmentally friendly pesticide.

English

Regeneration of the coconut (Cocos nucifera L) through indirect somatic embryogenesis using 9 to 12 months embryos explants was established in Y3 medium supplemented with 100 to 250 µM 2,4 -dichlorophenoxyacetic acid (2, 4-D) alone and in combination with gibberellic acid (GA3), 6-benzylaminopurine (BAP) and thiadiazuron (TDZ). The highest callus induction (100%) was observed in medium containing 150 and 250 µM 2, 4-D + 5 µM BAP while the least was in 2, 4-D alone (16.7%) and in combination with 0.35 µM GA3 (0). The highest embryogenic calli were observed in medium with 125 µM 2, 4-D + 5 µM BAP (100%) and the least was in medium with 2, 4-D alone (16.7) and in combination with 0.35 µM GA3 (0). Multiple shoot induction was observed in medium supplemented with 10 µM kinetin, 10 µM BAP, 0.5 µM GA3, and 200 µM NAA. Maximum shoot elongation (1.667 cm) was in medium containing 10 µM BAP and the least (0.811 cm) was in medium with 5 µM BAP. Rooting was done in Y3 medium containing three levels of indole-3-butyric acid (IBA). The highest response was observed in Y3 medium supplemented with 5 µM IBA+ 0.5 µM GA3 both with respect to the number of roots 8.33 and root length 5.10 cm while the least was with 10 µM IBA+ 0.5 µM GA3 with regard to the number of roots 3.33 and root length 2.83 cm. Acclimatization was achieved in media prepared with soil: sand: manure ratio (3:1:1) with 25% survival rate and vermiculate medium with 8.3% survival rate. Hence, in vitro regeneration of coconut through somatic embryogenesis is a viable alternative for mass propagation.   Key words: Cocos nucifera L., somatic embryogenesis, zygotic embryos.

In vitro propagation and reintroduction of golden paintbrush (Castilleja levisecta), a critically imperilled plant species

Golden paintbrush (Castilleja levisecta Greenm.) is a hemiparasitic herbaceous perennial native to the Pacific Northwest of North America and is considered critically imperilled with only 11 populations remaining in the wild. The main objective of this study was to develop ex situ and in situ conservation through micropropagation and field plantings. In vitro cultures were initiated using nodal explants from two plants raised from seeds collected from a natural population. Shoots were then multiplied on Murashige and Skoog basal medium with 2.0 μmol L−1 6-benzylaminopurine (BA), 3.0 μmol L−1 kinetin (Kn), 2.2 g L−1 phytagel, and 3% sucrose. Explant position on source plants, culture vessel design, and application of different plant growth regulator levels for BA, Kn, and thiadiazuron (TDZ) were tested to optimize micropropagation protocols. Clones from the plants showed differences in plant height and number of nodes in response to various BA and TDZ concentrations. In vitro shoots were successfully rooted under ex vitro conditions using commercial rooting powder (0.8% indole-3-butyric acid) with an average of ∼17 roots per shoot and acclimatized in the greenhouse with 100% survival rate. Two-month-old plants were transferred to a Parks Canada restoration site at Fort Rodd Hill, Victoria, BC, with 7.5% survival. The use of micropropagation in combination with reintroduction efforts offers an excellent opportunity for conserving endangered plant biodiversity in vitro and facilitating in situ conservation efforts by providing plants for reintroduction.

High incidence regeneration system in Ceratonia siliqua L. articulated with SEM and biochemical analysis during developmental stages

The article describes refined regeneration process in Ceratonia siliqua using different plant growth regulators along with antioxidant, SEM and IRGA analysis to understand the developmental behaviour of the plantlets. The present study describes a simplified seed germination process in Ceratonia siliqua L. and gives a comparative analysis of hormonal supply for enhanced adventitious shoot regeneration through aseptic seedling-derived cotyledonary nodes. The axillary bud induction and multiplication was greatly influenced by the concentration and type of cytokinin viz. 6-benzylaminopurine (BA), meta-Topolin (mT), Kinetin (Kn) and Thiadiazuron (TDZ). Adenine-based cytokinins, BA and mT, augmented in Murashige and Skoog medium, were proved to be more responsive. The medium containing 10 µM BA produced a maximum of 9.1 ± 3.0 shoots/ explant. A combination of optimal concentration of BA with NAA (0.5 µM) however, resulted into a significant production of 28.10 ± 0.14 shoots per CN. Half-strength MS + IBA (10 µM) in the presence of light ensured more number of root formation while half-strength MS + IBA (5.0 µM) and stimulation in the dark for 1 week ensured better root growth (4.98 ± 0.08 cm). Plantlets were successfully acclimatized in soilrite™, showing 65% survival. The work is supported with the studies based on antioxidant enzyme activity as well as on net photosynthetic rate and its related attributes in comprehension with SEM analysis of leaves showing stomatal micromorphology during three stages: in vitro, during acclimatization and in net house conditions. The studies during acclimatization reflect better understanding of the stabilization of micropropagated plantlets to the environmental conditions.

English

Jatropha curcas L. is peculiar treasured tree species for its uses and considerable economic potential as a biofuel plant. Propagation using seeds is limited by low viability and germination hence unable to provide high quality planting material for sustainable use. To achieve mass production of J. curcas, regeneration from leaf discs and petioles using different treatments of plant growth regulators (PGRs) including 6- benzyl aminopurine (BAP), Kinetin (KIN), Indole -3-acetic acid (IAA) and Thiadiazuron (TDZ) was explored. Maximum callus formation efficiency (85.00%) and the shoot proliferation per explants (8.25) was observed on MS medium supplemented with 1.5 mg/L BAP, 0.6 mg/L KIN, 0.3 mg/L IAA and 0.1 mg/L TDZ. Rooting was induced from elongated shoots cultured on half strength Murashige and Skoog (MS) medium fortified with different regimes of indole-3-butyric acid (IBA) and Naphthalene acetic acid (NAA). MS supplemented with 3.5 mg/L IBA and 3.5 mg/L NAA gave optimum root formation of 2.5 cm on JCO4, JN1, JE4 and JRV1 accessions. The rooted plants were established in forest soil, sand and manure mixed in the ratio of 2: 1:1 in the green house with 20% survival rate. This successful in vitro regeneration is vital for seedling system for mass production. Key words: Plant growth regulators, callus, shoot induction, root formation.

English

The response of eleven Indian cultivars of pigeon pea for in vitro multiple shoot bud induction and regeneration from plumule junction explants under variable concentration of 6-benzyl amino purine (BAP), kinetin and thiadiazuron (TDZ) was assessed in the present study. The cultivar IPA-3088 showed best response with a maximum of 20 buds per explants in Murashige and Skoog (MS) media supplemented with 0.05 mgL -1 TDZ. Among the hormones tested, lower concentration of TDZ gave the best response for these cultivars though higher concentration of BAP was also effective in multiple shoot bud induction and regeneration from plumule junction explants. The elongation of multiple shoot buds was achieved in the same medium and the nature of the regenerants in most of the cases was shoots, though in few cases shoot buds and shoot primoridia were also observed. Rooting of plumule junction derived shootlets was found to be better in the presence of NAA as compared to IAA and IBA for most of the cultivars producing maximum number of primary roots. The best responding cultivar IPA 3088 showed efficient rooting in the presence of 0.2 mgL -1 of NAA. The regenerated plantlets were acclimatized in soil with percentage of acclimatization varying from 40-80% for different cultivars. Keywords: Pigeon pea, cultivars, multiple shoot bud induction, organogenesis, acclimatization, elongation, Cajanus cajan (L) Mill sp. African Journal of Biotechnology , Vol 13(41) 4061-4069

High efficiency direct shoot organogenesis from leaf segments of Aerva lanata (L.) Juss. ex Schult by using thidiazuron.

An efficient protocol for direct shoot organogenesis has been developed for the medicinal plant Aerva lanata (L.) Juss. ex Schult. Regeneration was achieved from leaf segments of 20 days old in vitro plantlets raised on Murashige and Skoog (MS) medium containing 0.25–2.0 mg L−1 thiadiazuron (TDZ), 3% sucrose, and 0.8% agar. After 21 days of culture incubation, maximum number of shoot organogenesis (23.6 ± 0.16) was obtained on medium containing 1.0 mg L−1 TDZ. The shoots were able to produce in vitro flowers on medium containing 1.0 mg L−1 TDZ in combination with 0.25–0.5 mg L−1 α-naphthaleneacetic acid (NAA). Histological observation showed that the epidermal cells of the leaf explants exhibited continuous cell division led to formation of numerous dome shaped meristematic protrusions and subsequently developed into adventitious shoots. Upon transfer of shootlets to half strength MS medium containing 1.0 mg L−1 indole-3-butyric acid (IBA), around 86% of the regenerated shoots formed roots and plantlets. Rooted plants were hardened and successfully established in the soil at the survival rate of 92%. The regeneration protocol developed in this study provides an important method of micropropagation of this plant. Furthermore, this protocol may be used for a large scale production of its medicinally active compounds and genetic transformations for further improvement.

In vitro plant regeneration of Albizia lebbeck (L.) from seed explants

Objectives: An efficient and reproducible regeneration protocol for rapid multiplication of Albizia lebbeck (L.) was developed by using intact seed explants.Methods: Murashige and Skoog's (MS) medium supplemented with different hormones (BA, Kn, GA3 and TDZ) was used for the induction of multiple shoots from the seed explants. Ex-vitro rooting was performed by using pulse treatment method in auxins (IBA and NAA) and the complete plantlets were transferred to the field.Results: High frequency direct shoot induction was found in aseptic seed cultures of A. lebbeck on Murashige and Skoog medium supplemented with 5.0 µM TDZ (Thiadiazuron). Seeds were germinated after 7 days of culture and induced maximum 8 shoots from the region adjacent to the apex of the primary shoot of the seedling upto 25 days of incubation. Proliferating shoot cultures with increased shoot length was established by sub-culture of excised sprouting epicotyls on MS medium supplied with reduced concentrations of TDZ. Maximum shoot regeneration frequency (76 %) with highest number of shoots (21) and shoot length (5.1 cm) per sprouting epicotyl was observed in the MS medium supplemented with 0.5 µM TDZ after 8 weeks of culture. Different concentrations of Indole-3-butyric acid (IBA) and α-naphthalene acetic acid (NAA) were tested to determine the optimal conditions for ex-vitro rooting of the microshoots. The best treatment for maximum ex-vitro root induction frequency (81 %) was accomplished with IBA (250 µM) pulse treatment given to the basal end of the microshoots for 30 min followed by their transfer in plastic cups containing soilrite and eventually established in normal garden soil + soilrite (1:1) with 78 % survival rate. In addition, histological study was undertaken to gain a better understanding of the regenerated shoots from the epicotyl region.Conclusion: The findings will be fruitful in getting a time saving and cost effective protocol for the in vitro propagation of Albizia lebbeck.Keywords: Woody legume; TDZ; multiplication; epicotyl segment; shoot stumps.Abbreviations used: GA3 (Gibberellic acid); BA (6-Benzyladenine); Kn (Kinetin); TDZ (Thidiazuron); MS (Murashige and Skoog Medium); IBA (Indole-3-butyric acid); NAA (α-Naphthalene acetic acid).

Influence of polyamines on in vitro organogenesis in bitter melon (Momordica charantia L.)

An efficient protocol for in vitro organogenesis was achieved from callus derived petiole explants of bitter melon. The effects of polyamines (putrescine, spermidine and spermine) along with naphthaleneacetic acid (NAA) and thiadiazuron (TDZ) for callus induction and multiple shoot induction were investigated. Murashige and Skoog (MS) plus Gamborg et al. (B5) medium containing a combination of 3.0 μM NAA, 1.0 μM TDZ and 1.0 μM putrecine induced (95.0%) callus induction. Regeneration of adventitious shoots from callus (53 shoots per explant) was achieved on MS medium containing 3.0 μM TDZ with 1.0 μM NAA and 1.0 μM spermidine. The regenerated shoots were elongated on the same medium. Elongated shoots were transferred to the MS medium fortified with 2.0 μM indole 3-butyric acid (IBA) and 1.0 μM putrescine for root induction. Rooted plants were hardened and successfully established in soil with a 95% survival rate. This protocol yielded an average of 53 plants per petiole explant with a culture period of 85 days. Key words: Adventitious shoots, growth regulators, spermidine, putrescine, organogenic callus, hardening.

Direct somatic embryogenesis and plantlet regeneration in oil palm

Direct embryogenesis without an intervening callus phase from cotyledonary nodes of germinated immature zygotic embryos of hybrids viz. DG1 and DG21 of oil palm (Elaeis guineensis) is reported here. Direct embryogenesis was achieved when the cotyledonary nodes of germinated immature zygotic embryos were cultured in dark for 8 weeks on Eeuwens media (Y3) supplemented with 40 μM 2,4-Dichlorophenoxyacetic acid (2,4-D), 40 μM α-Naphthaleneacetic acid (NAA), 10 μM 2,4,5-Trichorophenoxyacetic acid (2,4,5-T), 10 μM Thiadiazuron (TDZ), 10 μM 6-Benzyladenine (BA). The globular embryos with clear suspensor region appeared directly on the explants and multiplied. On subculture to fresh media, the other stages such as torpedo and heart shaped embryos were seen. On transfer to light in Y3 media containing BA (2 μM) and ABA (1 μM) they matured into complete plantlets. In 2% of the cultures secondary embryogenesis also was seen. Along with several other advantages of direct somatic embryogenesis this protocol opens up the prospect of genetic transformation in this important commercial crop.

Adventitious shoot regeneration from cotyledonary explants of rapid-cycling fast plants of Brassica rapa L

Rapid-cycling fast plants (Brassica rapa; RCBr) is also known as Wisconsin Fast Plant and is widely used in K-12 and undergraduate studies. RCBr has a short generation time (seed-to-seed in 30–60 days), which allows for the completion of experiments in a semester. Previous studies have shown that cotyledonary explants with attached petioles are capable of generating shoots. However, there is no published adventitious shoot regeneration protocol to date. Sterile cotyledonary explants were excised; all edges and petioles were removed. Five-day-old cotyledonary explants produced shoots on a Murashige and Skoog medium containing 1.5 mg/L thiadiazuron (TDZ) and 0.5 mg/L 1-naphthaleneacetic acid (NAA) (FPM I) at a mean rate of 8.8%. This rate increased to 14.8% in explants placed on FPM I medium supplemented with 5.0 mg/L silver nitrate (AgNO3) (SRM 2). The rate increased to 32.5% when 5-day-old explants, excised from the part of the cotyledon nearest to the petiole, were placed adaxial side up on SRM 2 medium. The shoot regeneration rate increased to 44.5% using 4-day-old cotyledonary explants. A shoot regeneration rate of 23% was observed among 9-day-old leaf explants. Shoots from cotyledonary explants were elongated on basal medium with 0.5 mg/L NAA, rooted on basal medium, and later acclimatized. This is the first report of shoot regeneration from cotyledonary explants of rapid-cycling Brassica rapa without pre-existing meristematic tissues.

AN EFFICIENT REGENERATION SYSTEM FOR FOUR O'CLOCKS (MIRABILIS JALAPA)

As a model system for studying ethylene-independent floral senescence we have selected Four o’clocks, Mirabilis jalapa. As a first step to developing a transformation/regeneration (T/R) system, we have investigated ways to obtain rapid and efficient regeneration of Mirabilis jalapa plants. After testing a wide range of explants, media, and culture conditions, we determined that we could obtain high rates of meristem formation using cotyledons excised from mature embryos. The cotyledons and their subtending petioles were isolated from mature seeds and cultured in the dark for one week on Murashige and Skoog (MS) medium containing 1mg.L -1 IAA and 1mg.L -1 thiadiazuron (TDZ). The cotyledons were then transferred to regeneration media and cultured in the light. The regeneration medium contained MS salts and vitamins and 2 mg.L -1 TDZ. After four weeks, shoot meristems appeared on more than 80% of the petioles. Most of the meristems could develop to mature shoots, and then to intact plants.

Microprojectile-mediated genetic transformation and regeneration of Chinese elm

A protocol for the genetic transformation and regeneration of Chinese elm ( Ulmus parvifolia Ja cq.) is reported. Cell suspension cultures were initiated from hypocotyls and were maintained on Murashige and Skoog (MS) basal semi-solid medium supplemented with varying concentrations of thiadiazuron (TDZ) and 1-naphthaleneacetic acid (NAA). Two week- old cell suspension cultures were bombarded with gold particles coated with plasmid harboring the Basta®resistance and â -glucuronidase genes. Elm cultures produced green calli within a month on selection media containing 20 mg L-1phosphinothrycin. These c alli were transferred to the same medium without the herbicide and were regenerated into shoots within 2 mo. The expression of uidA reporter gene in transformed elms was detected by histochemical analysis for â-glucuronidase activity. The presence of the Basta®resistance insert was demonstrated by polymerase chain reaction analysis. Key words:

Somatic embryogenesis and plant recovery from callus of ‘Nabali’ Olive ( Olea europea L .)

Regeneration via somatic embryogenesis from callus was studied in ‘Nabali’ olive ( Olea europea L.). Among different explant sources (leaf blades, leaf petioles, hypocotyls of germinated seeds and roots of germinated seeds), roots gave the highest (46%) callus induction. Somatic embryogenesis was induced from root callus on embryogenesis induction medium (EIM) containing 5.0 μM 2,4-D, 0.5 μM kinetin and 5.0 μM NAA in darkness. Embryo regeneration was studied by transferring the callus from EIM to embryogenesis expression medium (EEM) containing different concentrations (0.0, 5.0, 10.0 and 15.0 μM) of 2-isopentenyladenine (2iP), BA, thiadiazuron (TDZ), zeatin or kinetin. Among the tested concentrations, 2iP at 10.0 μM outperformed the other growth regulators. 2,4-D at 5.0 μM in the EIM was satisfactory for embryogenesis induction. Sucrose at 0.2 M evoked higher embryogenesis than any other concentration of fructose and glucose in EIM, while sorbitol and mannitol at 0.1, 0.2 or 0.3 M reduced embryogenesis significantly and inhibited it totally at 0.4 M. Somatic embryos were rooted by transferring them to hormone-free medium (HFM). About 85% of embryos converted to rooted plantlets, 5% showed secondary embryogenesis and 10% were not developed and died. Rooted plantlets gave 95% survival when acclimatized ex vitro. Acclimatized plantlets developed into whole plants in the greenhouse and they were phenotypically similar.

Differential cytokinin effects on the stimulation of in vitro shoot proliferation from meristematic explants of castor (Ricinus communis L.).

A highly efficient and reproducible method of in vitro propagation using meristematic explants has been developed for castor. Embryo axes and shoot tips were cultured on Murashige and Skoog (MS) medium supplemented with 0.5-10.0 mg/l of adenine, N6-benzyladenine (BA), kinetin (Kn), thiadiazuron (TDZ) and zeatin. TDZ (1.0-10.0 mg/l) gave the maximum number of shoots (37.8-40.0) from embryo axes, while BA (2.0 mg/l) was found superior to other cytokinins for obtaining the highest number of shoots (46.7) from the shoot apex. Adenine and Kn at all of the tested concentrations resulted in low proliferation rates from embryo axes. The carryover effect of the cytokinins was tested by subculturing proliferating shoot cultures from various media onto the medium fortified with 0.5 mg/l BA. There was no significant influence of the cytokinins on subsequent proliferation from the two explant types except for TDZ with embryo axes. The number of shoots from TDZ-habituated embryo axes ranged between 36.0 and 81.7, while it varied from 5.7 to 22.0 and 3.7 to 28.3 in axillary buds and embryo axes, respectively, on the other media. For elongation of shoots, gibberellic acid (GA3) (0.1-1.0 mg/l) was added to the medium supplemented with 0.2-0.5 mg/l BA. Incorporation of GA3 (0.1 mg/l) significantly enhanced the frequency of elongated shoots but drastically reduced the multiplication ability. Hence, proliferating shoot clusters were periodically transferred to the medium supplemented with 0.5 and 0.2 mg/l BA for further multiplication and elongation. Well-developed shoots were rooted on half-strength MS medium supplemented with 1.0 mg/l indole-3-butyric acid. The rooted plantlets were acclimatized with more than 60% success.