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Transgenic plant production in monocotyledonous species has primarily relied on embryogenic callus induction from both immature and mature embryos as the pathway for plant regeneration. We have efficiently regenerated fertile transgenic wheat plants through organogenesis after Agrobacterium-mediated direct transformation of mechanically isolated mature embryos from field-grown seed. Centrifugation of the mature embryos in the presence of Agrobacterium was found to be essential for efficient T-DNA delivery to the relevant regenerable cells. The inoculated mature embryos formed multiple buds/shoots on high-cytokinin medium, which directly regenerated into transgenic shoots on hormone-free medium containing glyphosate for selection. Rooted transgenic plantlets were obtained within 10-12 weeks after inoculation. Further optimization of this transformation protocol resulted in significant reduction of chimeric plants to below 5%, as indicated by leaf GUS staining and T1 transgene segregation analysis. Direct transformation of wheat mature embryos has substantial advantages over traditional immature embryo-based transformation systems, including long-term storability of the mature dry explants, scalability, and greatly improved flexibility and consistency in transformation experiments.
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BACKGROUND: Mass propagation of date palm has attracted the interest of commercial producers. However, this technique still faces many obstacles that hinder production. This study investigated the effect of chitosan (CHT) at various concentrations for the possibility to apply it in combination with thidiazuron (TDZ) on the growth and development of tissue cultures of Barhee cultivar. RESULTS: The results showed that CHT and TDZ on in vitro proliferation of Barhee date palm cultivar were significant. The highest response rate and the number of shoots per jar were found in MS media supplemented with 15 mgL-1 CHT and 0.5 mgL-1 TDZ combination. Furthermore, we found that the combined application between 20 mg L-1 CHT+ 1.0 mg L-1 TDZ resulted in the highest shoots content of endogenous IAA, compared with other treatments. At the same time, the data revealed that the maximum cytokinins (CKs) content of shoots occurred in a medium supplemented with 15 mg L-1 CHT and 0.5 mg L-1 TDZ. The genetic stability of the discussed micropropagation protocol was confirmed in this study by DNA-based technique RAPD (random amplified polymorphic DNA). The results may indicate that the micropropagation protocol developed in this research paper was appropriate and applicable for producing genetically stable date palm cv Barhee plants. CONCLUSION: Applying the strategy of culture treatment with (CHT) and (TDZ) can be valuable for improving the propagation of date palm cv Barhee in vitro.
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Carpesium divaricatum Sieb. & Zucc. is a plant species rich in terpenoids of anti-inflammatory and cytotoxic activity, especially germacranolides of potential medicinal value. The present study describes in vitro multiplication of C. divaricatum, analysis of active constituents in the multiple shoots, and assessment of cytotoxic activities of extracts prepared from in vitro- and field-grown plants. The plant extracts were evaluated for cytotoxicity using two melanoma cell lines (HTB140 and A375); human keratinocytes (HaCaT); two colon cancer cell lines (Caco2 and HT29); human hepatocellular carcinoma cells (HepG2); two lines of prostate cancer cells (DU145 and PC3) and prostate epithelial cells (PNT2). Chemical compositions of the assayed extracts were analyzed by HPLC/DAD, in reference to isolated compounds. Maximum of 4.07 ± 1.61 shoots regenerated from a nodal explant of C. divaricatum, cultivated in a liquid MS medium supplemented with thidiazuron (1 µM). In vitro grown shoots and plantlets of C. divaricatum accumulated terpenoids that are known as active constituents of the intact plant. Cytotoxic activity of the extracts prepared from the in vitro cultured plants was like that demonstrated by the extracts prepared from field-grown plants and seemed to be more selective than cytotoxicities of the individual germacranolides.
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Cnidium officinale is a valuable medicinal plant cultivated in Asia for its rhizomes. This study reports the in vitro regeneration of Cnidium officinale plants and the induction of rhizomes from microshoots. The rhizomatous buds of Cnidium officinale induced multiple shoots on Murashige and Skoog (MS) medium supplemented with 0.5 mg L-1 BA, which led to the regeneration of plants within four weeks of culture. After four weeks of culture, the plants were assessed for fresh weight, the number of leaves, the number of roots, and the length of roots to compare the performance of the different clones. The clones with good growth characteristics were selected with the aid of a flow cytometric analysis of 2C nuclear DNA content. The plants bearing high DNA values showed better growth characteristics. Various factors, namely, sucrose concentration (30, 50, 70, and 90 g L-1), ABA (0, 0.5, 1.0, and 2.0 mg L-1), the synergistic effects of BA (1.0 mg L-1) + NAA (0.5 mg L-1) and BA (1.0 mg L-1) + NAA (0.5 mg L-1) + ABA (1.0 mg L-1) with or without activated charcoal (1 g L-1), and light and dark incubation were tested on rhizome formation from microshoots. The results of the above experiments suggest that MS medium supplemented with 50 g L-1 sucrose, 1.0 mg L-1 ABA, and 1 g L-1 AC is good for the induction of rhizomes from the shoots of Cnidium officinale. Plantlets with rhizomes were successfully transferred to pots, and they showed 100% survival.
Subject(s)
Cnidium , Plant Growth Regulators , Plant Shoots/genetics , Plant Growth Regulators/pharmacology , Charcoal/pharmacology , Clone Cells , Sucrose/pharmacologyABSTRACT
BACKGROUND: Gerbera jamesonii Bolus ex Hooker f. (African daisy) is listed among the top five most important ornamental plants in the global floricultural industry. To satisfy its demand, the floriculture industry relies on reproducible and effective propagation protocol while retaining the genetic uniformity of G. jamesonii. The present study, for the first time, reports the potential of picloram for enhanced induction of organogenic calli from leaves of G. jamesonii and its high-frequency indirect regeneration. RESULTS: The fastest induction of calli with maximum fresh and dry weight was recorded in the Murashige and Skoog (MS) semisolid medium supplemented with 1 mg/l picloram. In addition, callus induction was observed in 2,4-dichlorophenoxy acetic acid- and α-napthaleneaceticacid-supplemented media but with delayed response and reduced fresh and dry weight. The proliferated calli were transferred to shoot induction media containing MS salt and 0.5-1 mg/l N6-benzylaminopurine, kinetin, or thidiazuron. A mean number of ~6 shoots per callus were developed after 5 days of culture in the MS medium supplemented with 1 mg/l kinetin, with a mean length of 5.2 cm. Successful rooting of shoots was achieved in the MS medium fortified with 1.5 mg/l indole-3-acetic acid, wherein the earliest root initiation (~5 days), as well as the maximum number (~9) and length (~4.8 cm) of roots, were recorded. Complete plantlets were primarily acclimatized in sand before being transferred to a mixed substrate (of soil, sand, tea leaf waste, and cow urine) that secured >90% survival and further growth of the plantlets. Eventually, clonal fidelity of the in vitro regenerants assessed via inter-simple sequence repeats (ISSR) primers exhibited a monomorphic banding patterns that suggested genetic integrity within the plantlets as well as with their mother plant. CONCLUSIONS: The results of the present study should be of interest for commercial propagation and mutagenesis- as well as genetic transformation-related research.
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Bacopa monnieri (Linn.) Wettst. commonly known as waterhyssop, Brahmi plant, traditionally used for memory enhancement, nerve tonic, epilepsy, central nervous system (CNS), antidepressant, anxiety, blood pressure and antioxidant activities. Due to pharmaceutical demands its lost natural habitat. At this juncture we describe a resourceful protocol for micropropagation of water hyssop plant. Surface sterilized leaf and nodal explants were inoculated on basal MS semi-solid medium added with PGRs; auxins, cytokinins. Highest calli formation from leaf explants was obtained on NAA (2.5 mg-1) and showed (94.22%) accompanied via 2,4-D showed (2.5 mg-1; 82.43%), maximum calli formation in nodal explants was obtained on 2,4-D showed (2.5 mg-1; 71.14%) followed by NAA (2.5 mg-1) showed (62.15%), in internodes explants uppermost calli formation was obtained from 2,4-D showed (2.5 mg-1; 65.21%) followed by NAA (2.5 mg-1) showed (52.14%). The maximum somatic embryogenic callus, calli induction and formation (84%) was observed on 2,4-D + KIN (2.0 + 1.5 mg-1) amended solid medium. Uppermost shoot formation was observed in combination of IAA + BAP (1.0 + 1.0 mg-1) showed (78.54%) shoot formation followed by IBA (2.0 mg-1) alone showed (75.37%). The maximum shoot elongation was noticed from NAA + BAP (3.0 + 3.0 mg-1) with 21.21 cm followed by NAA (2.0 mg-1) showed (15.22 cm) although, chief root formation was obtained from IBA (2.0 mg-1) with 83.75% root formation along higher number of roots (47.43%) per shoot. Followed by IAA (2.0 mg-1) showed root induction (73.43%) and no of roots (38.54%) per shoot. In hardening under pot condition plants survivability (100%) was observed under glass house conditions, the present in vitro PTC techniques is extremely significant to gratifying its natural conservation.
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Rauvolfia serpentina (L.) Benth. ex Kurz., popularly known as Indian Snakeroot plant, belonging to Apocynaceae family, holds immense medicinal importance, owing to its rich source of multiple secondary metabolites such as ajmaline, ajmalicine, reserpine, and serpentine. To meet the constant demands for the key secondary metabolite (reserpine) by majority of the pharmaceutical industries, the present study assessed the effects of direct and indirect regeneration system on amelioration of reserpine accumulation in shoots of R. serpentina. In vitro multiple shoot cultures were established using shoot tip explants. Best results for shoot initiation, multiplication, and biomass production were obtained in case of Murashige and Skoog medium, supplemented with 1 mg/l N 6-benzyladenine. The multiple shoots were then sub-cultured on cytokinin-auxin combination media for further proliferation. Highest shoot and leaf multiplication rates and the most enhanced biomass were obtained in case of 1-1.5 mg/l Kinetin + 0.2 mg/l α-naphthalene acetic acid (NAA). Callus induction and its subsequent proliferation was obtained using 1.5 mg/l 2,4-dichlorophenoxyacetic acid. The best indirect shoot regeneration with highest shoot and leaf proliferation from calli was observed in case of 1 mg/l thidiazuron + 0.2 mg/l NAA. Reserpine content estimation via HPTLC from in vitro shoots (direct regeneration) and calli (indirect regeneration) were recorded to undergo an almost three-fold and two-fold increment (respectively) in comparison to that of the mother plant. Thus, in vitro direct regeneration system proved to be more effective and efficient in ameliorating the reserpine content.
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An optimised method for enhanced in vitro shoot multiplication of Rumex vesicarius (Polygonaceae)-a branched succulent herb-was achieved. The in vitro seed pre-treatment with 4% urea was able to show 95% seed germination on MS medium within 2 weeks of culturing. In vitro shoot bud induction from shoot tip explants was best in the presence of 2 mg L-1 kinetin on Murashige and Skoog medium (MS medium), wherein 8.3 shoots/explants were observed. Shoot elongation was found to be high (3.75 ± 0.5 cm) in 2 mg L-1 BA comprising medium. The transfer of micro-shoots on to MS medium comprising 1.5 mg L-1 IBA and 1% activated charcoal (w/v) supported efficiently in vitro rooting (2.5-4.0 cm) in 3-4 weeks. Upon hardening, 70% rooted plants survived under greenhouse conditions. Though friable callus was produced on MS medium-containing 2 mg L-1 BA followed by 3 mg L-1 BA, no organogenesis was noticed. The ascorbic acid content of 78.62 ± 0.25 mg 100 g-1 FW was recorded in callus cultures grown on medium supplemented with 2 mg L-1 BA, and it is 1.74-fold more compared to normal ex vitro leaves of the same age. In vitro raised plant leaf showed 1.98-fold more ascorbic acid (89.42 ± 0.18 mg 100 g-1 FW) to that of ex vitro leaves. The total phenolic content was found to be 60 mg in callus as compared to 610 mg (per 100 g GAE FW) of ex vitro leaves. The major phenolic compounds quantified were synergic, chlorogenic, ferulic, and generic acids, respectively. This optimised protocol will facilitate to pursue scale-up studies for in vitro ascorbic acid production and also to further investigate the kinetics of biosynthetic pathway genes involved.
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An efficacious, reproducible direct in vitro regeneration system has been developed from leaf base segments (LBs) of six high yielding genotypes of foxtail millet (Setaria italica (L.) Beauv.). LBs excised from 4-day-old seedling were inoculated on Murashige and Skoog (MS) medium supplemented with different types and concentrations of cytokinins. The shoots induced per explant significantly increased with the supplementation of BAP to auxin containing medium. The results showed that a maximum shoot induction, 58.8% was obtained on MS medium incorporated with 8.9 µM BAP and 2.7 µM NAA in 'CO5' genotype. Further, the highest frequency of multiple shoots was produced on MS(I) medium containing 8.9 µM BAP, 2.7 µM NAA, 700 mg L-1 proline, 0.5 mg L-1 cysteine, 2.0 mg L-1 glycine and 150 mg L-1 arginine. MS(I) medium additionally fortified with 5.0 g L-1 activated charcoal (AC) was found to achieve the best precocious plant regeneration. Elongated shoots were rooted on half-strength MS medium amended with 2.9 µM IAA and achieved maximum root number (8.7) within 10 days. Rooted plantlets were acclimated in soil with 92% survival rate. Molecular marker analysis of in vitro regenerated and field grown plants revealed no somaclonal variations. Briefly, amino acids and activated charcoal could significantly enhance the foxtail millet direct multiple shoot proliferation and plant regeneration. Here we report, a short-term, genotype independent, direct plant regeneration protocol for future genetic transformation studies in foxtail millet genotypes.
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The objective of the present study is to determine the effect of light source on enhancement of shoot multiplication, phytochemicals, as well as, antioxidant enzyme activities of in vitro cultures of date palm cv. Alshakr. In vitro-grown buds were cultured on Murashige and Skoog (MS) medium and incubated under a conventional white fluorescent light (control), and combinations of red + blue light emitting diode (18:2) (CRB-LED). Results revealed that the treatment of CRB-LED showed a significant increase in the number of shoots compared with the white florescent light. Total soluble carbohydrate "TSCH" (7.10 mg g(-1) DW.), starch (1.63 mg g(-1) DW.) and free amino acids (2.90 mg g(-1) DW.) were significantly higher in CRB-LED (p < 0.05). Additionally, CRB-LED induced a higher peroxidase activity (25.50 U ml(-1)) compared with the white fluorescent light treatment (19.74 U ml(-1)) as control treatment. Potassium, magnesium and sodium contents in (3.62, 13.99 and 2.76 mg g(-1) DW.) were increased in in vitro shoots under CRB-LED treatment in comparison with fluorescent light (p < 0.05). Protein profile showed the appearance of newly bands with the molecular weight of 38 and 60 kDa at the treatment CRB-LED compared with control treatment. Our results demonstrate the positive effects of CRB-LED light during the course of date palm tissue cultures.
Subject(s)
Peroxidase/analysis , Phoeniceae/growth & development , Phytochemicals/analysis , Tissue Culture Techniques/methods , Gene Expression Regulation, Developmental , Gene Expression Regulation, Plant , In Vitro Techniques , Light , Phoeniceae/metabolism , Plant Proteins/analysis , Plant Shoots/growth & developmentABSTRACT
Methods were developed in the present investigation for cloning and large scale plant production of Passiflora foetida L. germplasm selected from the East-Coast region of South India. Nodal shoot segments were used as explants. The explants were dressed and surface sterilized with 0.1% (w/v) HgCl2. Multiple shoots were induced (6.13 ± 0.22 shoots per explant) by proliferation of nodal shoot meristems on Murashige and Skoog (MS) semi-solid medium + 2.0 mg l-1 6-benzylaminopurine (BAP). The shoots of P. foetida were further multiplied (16.45 ± 0.44 shoots per explant) on MS medium + 0.5 mg l-1 each of BAP and Kinetin (Kin). The in vitro generated shoots were rooted on half-strength MS medium containing 2.5 mg l-1 indole-3 butyric acid (IBA). By this method 67% shoots were rooted. About 97% shoots were rooted ex vitro (8.33 ± 0.29 roots per shoot) when the cut ends of the shoots were treated with 300 mg l-1 IBA for 5 min. The in vitro rooted plants were hardened and acclimatized in the greenhouse and successfully (100%) transplanted to the field.
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An Agrobacterium tumefaciens-mediated corn transformation method based on multiple shoot tissue cultures was developed, which is effective with a variety of corn inbred lines and standard binary vectors. Six factors that affected the success of corn transformation were tested, including A. tumefaciens strain, corn genotype, tissue culture growth stage, medium composition, co-culture temperature and surfactant treatment. Agropine-type bacteria (EHA 101 and AGL 1) were eightfold more effective than octopine-type strain for corn multi-shoot tissues transformation. The average frequency of Glucuronidase (GUS)-positive explants obtained from 14 corn genotypes ranged from 36% to 76%. L-proline (0.7 g L-1) in the co-culture medium apparently improved the frequency of transformation. The newly initiated multi-shoot tissues were most responsive to Agrobacterium infection. A positive correlation was found between multi-shoot tissue susceptibility to Agrobacterium and the proportion of cells in G1 phase. Transformants were identified by reverse transcription Polymerase Chain Reaction (PCR) and by southern blot hybridization assays. The frequency of transformants was approximately 2% based on the number of multi-shoot explants co-cultivated with Agrobacterium.
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Experiments were conducted to develop methodology for in vitro propagation and rapid multiplication of Viola serpens Wall. using petiole explant. The MS medium supplemented with 2, 4-D (6.78 µm) was found most suitable for callus induction in petiole explant. The best growth response and higher rate of shoot regeneration from petiole callus was observed on MS medium containing BAP (11.10 µm) as the average number of shoots could be increased to 36.4 on fourth successive subculturing. Higher rooting response with larger number of roots were observed in shoots inoculated on the half-strength MS medium supplemented with IBA (19.68 µm).
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An in vitro propagation technique based on axillary bud proliferation was developed for the first time to mature annatto (Bixa orellana L.) tree. Nodal segments cultured on Murashige and Skoog (MS) medium supplemented with 1.0 µM benzyl adenine (BA) and tender coconut water (10 %) showed significantly high (P < 0.05) explant response (67.0 %), development of elongated shoots (3.36), shoot buds (8.9) and shoot elongation (3.53 cm). Cytokinins like zeatin, isopentenyl adenine (2-iP), kinetin, or thidiazuron (TDZ) were inferior to BA to induce multiple shoots. Seasonal variations significantly affected the in vitro response of nodal explants. In vitro rooting experiments have showed 55.6 % rooting on MS medium containing 15 µM indole-3-butyric acid (IBA). Alternatively, in vitro raised shoots were rooted (61.1 %) ex vitro, by 10 mM indole-3-butyric acid (IBA) for 30 s. The results of the RAPD marker system revealed the genetic stability among the micropropagated plants. The present protocol in brief, can be used for the clonal propagation of the superior genotype and preservation of germplasm.
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A successful report on the in vitro propagation of Homalomena aromatica via rhizome axillary bud multiplication is presented. Rhizome bud explants were cultured on Murashige and Skoog medium supplemented with various concentrations of cytokinins to induce multiple shoot formation for micropropagation. The highest number of shoots was achieved in MS medium supplemented with 2.0 mg l(-1) 6-benzylaminopurine. The regenerated shoots rooted most efficiently on half-strength MS medium supplemented with 0.5 mg l(-1) α-naphthalene acetic acid. The regenerated plantlets showed no morphological differences from the parent plant. This protocol takes approximately 6 months to reach the acclimatization stage from the initiation stage and facilitates commercial and rapid propagation of H. aromatica.
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Cotton (Gossypium hirsutum L.) is one of the most commercially important fiber crops in the world. Compared with other crops, cotton represents a recalcitrant species for regeneration protocols. The development of efficient and rapid regeneration protocol for elite Indian cotton variety could help improve the quality characteristics and biotic or abiotic stress tolerance. Here we report a novel regeneration protocol in Indian cotton cultivar Narashima. The maximum number of multiple shoots obtained was 16 per explants, performance which has never been achieved in any prior reports. The embryo apex explants were isolated from 2 d old in vitro growing seedlings. Explants were cultured on MS medium containing different plant growth regulator combinations in order to induce multiple shoots. Among the tested combinations, the 2 mg/l of 6-benzylaminopurine (BAP) and 2 mg/l kinetin (KIN) proved to be most suited for achieving the maximum number of multiple shoots. The elongation of multiple shoots was obtained in media supplemented with gibberellic acid (GA3). The regenerated plants were successfully hardened in earthen pots after adequate acclimatization. This method avoids callus tissue, the stage of regeneration which may lead to somaclonal variation. The important feature of the presented method is shortening of regeneration time, as well as the induction of a high number of multiple shoots per explants. The present protocol may provide an efficient and rapid regeneration tool for obtaining more stable transformants from embryo apex explants of Indian cotton cultivar Narashima.
Subject(s)
Gossypium/growth & development , Plant Growth Regulators/pharmacology , Plant Shoots/growth & development , Regeneration , Seeds/growth & development , Benzyl Compounds , Culture Media , Gibberellins/pharmacology , Gossypium/drug effects , Kinetin/pharmacology , Meristem , Plant Shoots/drug effects , Plant Somatic Embryogenesis Techniques/methods , Purines , SeedlingsABSTRACT
An efficient, rapid and improved in vitro plant regeneration protocol has been established for Withania somnifera L. using shoot tip and nodal explants, excised from 15 days old aseptic seedlings. A range of cytokinins were investigated for multiple shoot regeneration. Of the three cytokinins, 6-benzyladenine (BA), Kinetin (Kin) and 2-isopentenyl adenine (2-iP) evaluated as supplement to Murashige and Skoog (MS) medium, BA at an optimal concentration of 2.5 µM was most effective in proliferating apical and axillary buds. The highest regeneration frequency (95 %) and number of shoots (36.1 ± 0.33) were obtained on MS medium fortified with BA (2.5 µM) and NAA (0.5 µM) from nodal segments. High frequency of rooting (100 %) was obtained in in vitro raised shoots when transferred to half-strength MS medium supplemented with NAA (0.5 µM). Histological sections revealed that additional shoot bud primordia were differentiated within the explants just underneath the suberized cells which appeared to be arrested in their development. The presence of additional bud primordia within the explants is thereby helpful to maximize the potential of this system. The regenerated plantlets with well developed shoots and roots were hardened successfully, established in earthen pots containing garden soil and maintained in greenhouse with 95 % survival rate.
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Objective: This is the first attempt for an efficient plant regeneration protocol through in vitro direct organogenesis for a valuable medicinal plant, Dipteracanthus prostratus using nodal segment. Methods: Multiple shoots were induced from nodal explants cultured on Murashige and Skoog medium supplemented with kinetin (KIN), 6-benzylaminopurine (BAP) and 1-Naphthaleneacetic acid (NAA). Results: Maximum shoot responses (80%) were obtained with kinetin at 1.0 mg-1. The rate of shoot multiplication was maintained in subsequent subculture on similar fresh culture medium. The highest shoot length (3.96cm) was obtained with seventy three percentages of shoots at 0.2 mg-1 NAA along with 1.0 mg-1 kinetin. Maximum length of root (3.63cm) was formed at 0.5 mg-1 IBA with significant responses (80%). Rooted plantlets were then transferred to perforated plastic cups and grown in the green house at 80% survival rate. Conclusions: The highest survival rate was noticed and this plant developmental protocol could be used for large- scale regeneration of D. prostratus.
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Efficient methods were developed for both in vitro seed germination and micropropagation of an economically important dye yielding multipurpose tree, Bixa orellana L. Mature seeds were inoculated onto Murashige and Skoog (MS) medium supplemented with different concentrations of gibberellic acid (GA3). Highest frequency of germination (93.3 %) was recorded on medium supplemented with 3 µM GA3 against 13.33 % in control. Nodal explants cultured on MS medium fortified with 5 µM isopentanyl adenine (2-iP) produced maximum explants response (93.3 %) and highest number of shoots (35.71). Addition of relatively higher concentration (15 µM) of benzyl adenine (BA) resulted in the production of significantly (P < 0.05) reduced number of shoots (12.66). Sucrose at 87.6 mM was found to be the best carbohydrate source for multiple shoot induction compared to glucose and fructose. Regenerated shoots (3-4 cm) were rooted (95.5 %) on agar gelled MS medium supplemented with 10 µM indole-3-butyric acid (IBA). In vitro developed plantlets with well-developed roots were potted and acclimatized initially in the growth chamber and then moved to a green house with 83.3 % survival. The present protocol avoids the use of auxins in shoot multiplication medium, which will lower the cost, avoid callus formation and thus reduces the possibility of somaclonal variation in the regenerated plants. The method is efficient to produce over 32,000 hardened plants within a 10-month culture period starting from a single nodal explant.
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Ficus religiosa (Pipal) is a long-lived valuable multipurpose forest tree. The tree is exploited because of its religious, ornamental and medicinal value and the regeneration rate in natural habitat is low. An in vitro propagation protocol has been developed from nodal segments obtained from a 45-50-year old tree. The highest bud break frequency (100 %) followed by maximum number of multiple shoots (13.9) as well as length (2.47 cm) were obtained on Woody Plant medium (WPM) supplemented with 1.0 mg/l BAP along with 0.5 mg/l IAA. Two modifications in this medium resulted in enhanced shoot regeneration-one with 200 mg/l glutamine + 150 mg/l ADS (called as MM-1) giving 32.5 shoots per nodal explant while another modification-with 200 mg/l glutamine + 150 mg/l ADS + 100 mg/l phloroglucinol (called as MM-2) giving 35.65 shoots per explant. These two media were used for sub-culturing of shoots for 4 months. The rate of shoot multiplication was same during the first three sub-cultures on MM-1 and the shoots regenerated were healthy, afterwards shoot multiplication declined. While on MM-2, shoot multiplication declined after first sub-culture and shoots underwent the problem of early leaf fall. Rooting was best induced in micro-shoots excised from proliferated shoot cultures on semi-solid as well as liquid WPM modified with 2.0 mg/l IBA and 0.5 mg/l IAA. The in vitro-raised plantlets were potted and acclimatized under culture room conditions for 25-30 days before transfer to soil conditions, where the established plants showed more than 90 % survival.