Multiple shoot induction and callus regeneration in Sarcostemma brevistigma Wight & Arnott, a rare medicinal plant

An efficient micropropagation protocol based on multiple shoot induction and callus regeneration has been standardized in Sarcostemma brevistigma, a rare medicinal plant. The nodal cuttings were cultured on MS medium supplemented with BA (0.5–8 μM) or Kn (0.5–8 μM) alone or in combination with NAA (0.5–1.5 μM). Maximum multiple shoot induction was observed on MS medium supplemented with 4 μM BA. On this medium, 100% cultures responded with an average number of 11.3 shoots per explant. However, the average shoot length was limited to only 0.9 cm on this medium. The addition of 1 μM NAA along with 4 μM BA gave rise to an average number of 10.9 shoots with an average shoot length of 1.8 cm. Luxuriantly growing callus was obtained on MS medium supplemented with BA (5 μM) and 2,4-D (2 μM). The callus was subcultured on MS medium supplemented with BA (2–15 μM) or Kn (2–15 μM) alone or in combination with NAA (0.5–2 μM) for shoot organogenesis. Optimum callus regeneration was obtained on MS medium supplemented with 10 μM BA and 1 μM NAA. On this medium, 100% cultures responded with an average number of 13.4 shoots per culture. The shoots obtained via multiple shoot induction and organogenesis were rooted on half-strength MS medium supplemented with NAA (1–7 μM) or IBA (1–7 μM). IBA was better than NAA in terms of both the percentage of cultures that responded and the average number of roots per explant. The rooted shoots were successfully transplanted to soil with 86% success. This standardized protocol will help to conserve this rare medicinal plant.     

High frequency adventitious shoot induction and plant regeneration from leaves of statice

An efficient regeneration system for large-scale propagation of statice (Limonium altaica cv. Emille) was developed using leaves from mature plants. Leaf segments (5×5 mm sections) were cultured on Murashige and Skoog's medium supplemented with N⁶-benzyladenine (BA) and thidiazuron (TDZ) individually and in combination with indole-3-acetic acid (IAA) and α-naphthaleneacetic acid (NAA). Prolific direct adventitious shoot regeneration occurred on most of the media. The best response in terms of frequency of shoot regeneration (99.5%) and number of shoots per explant (112 shoots per explant) was observed on medium supplemented with 2.85 μM IAA and 1.14 μM TDZ. The shoots rooted easily on half strength MS medium and MS medium with indole-3-butyric acid. In vitro propagated plants could be transferred to soil with survival rates of more than 95%. This revised version was published online in June 2006 with corrections to the Cover Date.     

Indirect organogenesis and plant regeneration in Helicteres isora L., an important medicinal plant

Abstract    Helicteres isora is a medicinal plant effective against asthma, diabetes, hypolipidemia, HIV, polio besides a good source of diosgenin. Seed dormancy and low natural fruit production rate make this plant a perfect candidate for developing an in vitro regeneration method. However, to date, no such work has been procured in this plant. An efficient method for plant regeneration via shoot organogenesis from callus cultures has been developed using nodal explants in H. isora. Murashige and Skoog (MS) media counting 2,4-Dichlorophenoxyacetic acid (2,4-D, 2.26 to 13.57 μM), Indole-3-acetic acid (IAA, 2.85 to 17.13 μM), Indole-3-butyric acid (IBA, 2.46 to 14.70 μM), 6-Benzylaminopurine (BA, 2.22 to 13.32 μM) and Kinetin (Kin, 2.32 to 13.92 μM) either singly or in the following combinations (IAA + BA; IAA + Kin, and BA + Kin) produced granular callus except BA + Kin which resulted in compact, hard, greenish-white (CHGW) callus. The optimum CHGW callus (2.62 g fresh weight/ explant) was produced on MS media with 13.32 μM BA + 2.32 μM Kin with over 93% callus induction frequency. Optimum shoot organogenesis (67% frequency) was achieved in CHGW callus with lower level of BA (2.22 μM) and Kin (2.32 μM) and produced 3.2 shoots/0.5 g callus within 35 d of culture. Microshoots were rooted successfully (62% frequency) after 35 d of culture on 1/2MS containing 4.90 μM IBA and hardened off. Antioxidant enzymes such as catalase, peroxidase, polyphenol oxidase, and biochemical parameters viz. hydrogen peroxide, reducing and nonreducing sugars, starch, proteins, phenols, and proline contents were studied in regenerating and nonregenerating CHGW calluses to establish a correlation between these parameters and shoot morphogenesis. All the enzyme activities and biochemical parameters were found more in regenerating callus than in nonregenerating except phenols.     

Direct shoot organogenesis and plant regeneration in safflower

Summary  Adventitious shoot buds were induced directly on the adaxial surface of the cotyledons of eight safflower cultivars after 14 d of culture initiation on Murashige and Skoog's (MS) medium supplemented with various levels of 6-benzylaminopurine (BA). Maximum shoot organogenesis of 54.4% with 10.2 shoots per responding cotyledon was obtained with 8.87 μM BA in the cv. S-144. Regenerated shoots were classified into three groups on the basis of their morphological features and were found to be correlated with the levels of BA. The highest number of normal shoots was obtained from 2.2 μM BA treatment. The regenerated shoots of group I (normal shoots) were rooted on half-strength MS medium supplemented with 5.3 μM α-naphthaleneacetic acid, 3% sucrose and 0.8% bacto-agar. Rooted plantlets were successfully transferred to soil and appeared morphologically normal. Histological studies revealed that shoot buds originated adventitiously from subepidermal cells.     

Ethylene and shoot regeneration: hookless1 modulates de novo shoot organogenesis in Arabidopsis thaliana

We have investigated the role of ethylene in shoot regeneration from cotyledon explants of Arabidopsis thaliana. We examined the ethylene sensitivity of five ecotypes representing both poor and prolific shoot regenerators and identified Dijon-G, a poor regenerator, as an ecotype with dramatically enhanced ethylene sensitivity. However, inhibiting ethylene action with silver nitrate generally reduced shoot organogenesis in ecotypes capable of regeneration. In ecotype Col-0, we found that ethylene-insensitive mutants (etr1-1, ein2-1, ein4, ein7) exhibited reduced shoot regeneration rates, whereas constitutive ethylene response mutants (ctr1-1, ctr1-12) increased the proportion of explants producing shoots. Our experiments with ethylene over-production mutants (eto1, eto2 and eto3) indicate that the ethylene biosynthesis inhibitor gene, ETO1, can act as an inhibitor of shoot regeneration. Pharmacological elevation of ethylene levels was also found to significantly increase the proportion of explants regenerating shoots. We determined that the hookless1 (hls1-1) mutant, a suppressor of the ethylene response phenotypes of ctr1 and eto1 mutants, is capable of dramatically enhancing shoot organogenesis. The effects of ACC and loss of HLS1 function on shoot organogenesis were found to be largely additive.     

Direct shoot organogenesis and plant regeneration in Fortunella crassifolia

An efficient in vitro regeneration system in kumquats (Fortunella crassifolia Swingle) was established. Explant types and orientations, concentrations and combinations of plant growth regulators were evaluated for their influences on efficiency of plant regeneration. It was found that the optimum explant and its orientation was epicotyl planted vertically with upper part upward, and a shoot regeneration frequency of 1.48 shoots per explant was obtained on Murashige and Skoog (1962; MS) medium supplemented with 22.19 μM 6-benzyladenine (BA). A rooting percentage as high as 74 % was obtained on 1/2 MS supplemented with 0.54 μM 1-naphthaleneacetic acid (NAA), 9.29 μM kinetin (KN), and 0.5 g dm⁻³ activated charcoal.     

In vitro regeneration through organogenesis and somatic embryogenesis in pigeon pea [Cajanus cajan (L.) Millsp.] cv. JKR105

In vitro regeneration of pigeon pea through organogenesis and somatic embryogenesis was demonstrated with pigeon pea cv. JKR105. Embryonic axes explants of pigeon pea showed greater regeneration of shoot buds on 2.5 mg L⁻¹ 6-benzylaminopurine (BAP) in the medium, followed by further elongation at lower concentrations. Rooting of shoots was observed on half-strength Murashige and Skoog (MS) medium with 2 % sucrose and 0.5 mg L⁻¹ 3-indolebutyric acid (IBA). On the other hand, the regeneration of globular embryos from cotyledon explant was faster and greater with thidiazuron (TDZ) than BAP with sucrose as carbohydrate source. These globular embryos were maturated on MS medium with abscisic acid (ABA) and finally germinated on half-strength MS medium at lower concentrations of BAP. Comparison of regeneration pathways in pigeon pea cv. JKR105 showed that the turnover of successful establishment of plants achieved through organogenesis was more compared to somatic embryogenesis, despite the production of more embryos than shoot buds.     

High frequency shoot regeneration in Agave sisalana

Callus cultures of Agave sisalana were initiated from rhizome, and stem explants on MS, SH, Gamborg and White's medium supplemented with different concentrations of BA, kinetin, NAA, IAA and 2,4-D either in combination or singly. Optimum numbers of shoots were obtained from stem and rhizome explants either directly or from callus. The capacity for the shoot regeneration remained constant in the callus for more than 32 months. Regenerated shoots rooted readily within 21-35 days on fine sand with half the strength of inorganic salts of MS medium. 100% of the rooted plants successfully adapted to field conditions and grown in the soil. Regenerated plants were morphologically similar to the field grown mother plants. This revised version was published online in June 2006 with corrections to the Cover Date.     

Plantlet regeneration through indirect shoot organogenesis and somatic embryogenesis in Justicia gendarussa Burm. f., a medicinal plant

A protocol for the regeneration of a large number of plantlets via indirect shoot organogenesis and somatic embryogenesis has been developed from the stem and leaf explants of Justicia gendarussa Burm. f. The callus was efficiently induced from the explants using Murashige and Skoog (MS) medium supplemented with α-Naphthalene acetic acid (NAA) + Benzyl amino purine (BAP) (1.0?+?0.1 mg/l). The highest number of plantlets through indirect shoot organogenesis was obtained when the callus was subcultured to MS medium with BAP + NAA (0.1?+?1.0 mg/l). The maximum number of plantlets via somatic embryos was obtained in the medium with BAP + NAA (1.0?+?0.1 mg/l) for stem derived calli and Kinetin (Kn) + NAA (2.0?+?0.1 mg/l) for leaf derived calli. The in vitro developed shoots were rooted well in half strength MS medium supplemented with 0.5 mg/l of Indole-3-acetic acid (IAA). The in vitro regenerated plantlets were hardened using a mixture of sterile sand:soil:manure (1:1:1). The present study is the first report on the regeneration of plants through somatic embryogenesis from stem and leaf derived calli of J. gendarussa.     

Effects of plant growth regulators on high frequency shoot multiplication and callus regeneration of an important Indian medicinal plant, nirgundi (Vitex negundo L.)

Summary A rapid shoot multiplication protocol was established for an important medicinal plant, Vitex negundo L., belonging to the family Verbenaceae, using Murashige and Skoog medium, achieved by shoot multiplication as well as callus regeneration. Shoot multiplication was induced by different concentrations of 1-phenyl-3-(1,2,3-thiadiazol-5-yl)-urea (TDZ), Benzyladenine and 6-furfuryl amino purine separately along with 10% (v/v) coconut water. Green organogenetic callus was obtained by the combined effect of 0.5–2.15 μM TDZ and 1.7 μM indole-3-acetic acid (IAA) along with 1% polyvinylpyrrolidone (PVP), and produced the maximum number of shoots when subcultured onto medium containing 2.7 μM TDZ alone. Elongation of in vitro shoots was observed in MS medium containing 2.4 μM gibberellic acid and rooting was induced by the combined effect of 1.71 μM IAA and 1.62 μM α-naphthalene acetic acid.     

High frequency plant regeneration and accumulation of tropane alkaloids in regenerated plants of Scopolia parviflora

An efficient in vitro plant regeneration system was established from callus culture of Scopolia parviflora. Callus was induced from adventitious roots on B5 medium with 0.45–9.04 μM 2,4-dichlorophenoxyacetic acid (2,4-D). In vitro plantlet regeneration was achieved on B5 medium supplemented with 44.38 μM benzyladenine (BA), 3% sucrose, and 0.38% gelrite. Plantlets were transplanted to artificial soil and grown to maturity successfully in a greenhouse. The tropane alkaloid contents in regenerated plants were analyzed using high-performance liquid chromatography (HPLC), and were found to be higher than those of adventitious roots, native growing plants, and acclimated plants. Regenerated plants from organogenic callus cultures produced a greater amount of tropane alkaloids.     

An efficient in vitro plant regeneration system for the medicinal plant Teucrium stocksianum Boiss.

An efficient and rapid plant regeneration system via direct organogenesis was established for Teucrium stocksianum Boiss. (Lamiaceae), an endangered and valuable medicinal plant. Hypocotyl explants excised from seedlings germinated in vitro were cultured on Murashige and Skoog (MS) medium supplemented with different concentrations of kinetin and indoleacetic acid (IAA) to induce shoot formation. Differentiation of multiple shoots was initiated within 3 weeks of culture. Optimal regeneration was achieved on medium containing 3 mg/l kinetin and 0.5 mg/l IAA. This particular medium composition significantly improved the production of multiple shoots directly from hypocotyl explants compared to other combinations of plant growth regulators. Root induction was achieved on half-strength MS medium containing indole-3-butyric acid. Rooted plantlets were successfully acclimatized, with a survival rate of 75–80%. The protocol developed in this study could be used for long-term in vitro conservation and mass propagation of this species.     

Rapid clonal multiplication through in vitro axillary shoot proliferation of Aegle marmelos (L.) Corr., a medicinal tree

Rapid clonal multiplication of Aegle marmelos (L.) Corr. (Rutaceae), a medicinal tree, was achieved by enhanced axillary bud proliferation in young single-node segments of a 25-year-old tree cultured in Murashige and Skoog (MS) nutrient medium. Bud break was dependent on cytokinin supply, but the synergistic combination of 2.5 mg l^–1 6-benzylaminopurine (BAP) and 1.0 mg l^–1 indole-3-acetic acid (IAA) induced the formation of 12.1 shoots of up to 5.2 cm length in 48% of the explants after 7 weeks of culture. Explants of in-vitro-grown shoots – node, whole leaf, shoot tip and internode – were subcultured in the presence of 0.05–2.5 mg l^–1 BAP to produce 11.3, 18.4, 5.3 and 3.2 shoots and shoot buds at a 100%, 70%, 95% and 40% rate respectively, in 7 weeks. Different shoot nodes and leaves were equally regenerative and adventitious organogenesis in the latter was confined to cut petiolar ends. Nodal explants responded most favourably at low BAP (0.05–0.1 mg l^–1) and produced uniform (3.8–5.3 cm) shoots facilitating their simultaneous harvest for rooting. Repeated subculturing through five cycles of nodes and leaves of shoot cultures enabled continuous production of healthy callus-free shoots without any sign of decline. Shoot cuttings (3.0–5.2 cm) were best rooted in half-strength MS medium with 0.5 mg l^–1 IAA (70%) or 10.0 mg l^–1 indole-3-butyric acid (90%). Eighty-eight percent of the rooted plants were established in polybags after hardening. Received: 4 April 1996 / Revision received: 8 September 1997 / Accepted: 20 September 1997     

High frequency plant regeneration of Cymbopogon martinii (Roxb.) Wats by somatic embryogenesis and organogenesis

Embryogenic callus cultures were obtained upon repeated sub-culture of non-embryogenic callus from nodal segments of Cymbopogon martinii (Roxb.) Wats. Murashige and Skoog's medium supplemented with 1mg/l 2,4-dichlorophenoxyacetic acid and 0.5 mg/l kinetin and Linsmaier and Skoog's medium supplemented with 2mg/l 2,4-dichlorophenoxyacetic acid and 0.4 mg/l kinetin were used as maintenance media for non-embryogenic and embryogenic cultures, respectively. Plant regeneration occurred through organogenesis in MS basal media containing 2 mg/l kinetin, 1 mg/l 6-benzylaminopurine, 0.2 mg/l biotin, 0.2 mg/l Ca-pantothonate and 0.1 mg/l napthalene acetic acid. Embryogenesis was induced in LS medium supplemented with 1 mg/l kinetin, 0.5 mg/l 6-benzylaminopurine and 0.1 mg/l 3-indole acetic acid. Plant regeneration at high frequency was recorded both through organogenesis and embryogenesis in different passages of long term callus cultures.Abbreviation MS Murashige and Skoog medium - LS Linsmair and Skoog medium - BAP 6-benzylaminopurine - kin kinetin - 2,4-D 2,4-Dichlorophenoxyacetic acid - IAA Indole-3-acetic acid - CH Casein hydrolysate - CaP calcium pantothonate - NAA napthalene acetic acid     

Somatic embryogenesis and in vitro plant regeneration of Bacopa monnieri (Linn.) Wettst., a potential medicinal water hyssop plant

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.     

High-frequency plant regeneration via somatic embryogenesis and organogenesis and in vitro flowering of regenerated plantlets in Panax ginseng

 The morphogenesis ability of light yellowish globular callus derived from cotyledons of mature zygotic embryos of Panax ginseng was investigated. The optimal media for somatic embryogenesis and shoot organogenesis were MS medium containing 0.5 mg l^–1 2,4-dichlorophenoxyacetic acid, 0.1 mg l^–1 6-benzyladenine (BA), and 500 mg l^–1 lactoalbumin hydrolysate, and SH medium supplemented with 0.5 mg l^–1 α-naphthaleneacetic acid, 0.1 mg l^–1 BA, and 500 mg l^–1casein hydrolysate. The influences of glucose, mannose, fructose, and sorbose in the media on somatic embryogenesis and shoot organogenesis were revealed as differences in the numbers of somatic embryos and adventitious shoots per gram of morphogenic callus. The best regeneration of somatic embryos was obtained on medium containing glucose, with a mean of 8.7 somatic embryos per gram of callus. The best regeneration of shoots was observed on medium containing fructose, with an average of 12.2 adventitious shoots per gram of callus. Of the somatic embryos 95% were converted into regenerated plantlets, and 100% of adventitious shoots rooted to form regenerated plantlets. Regenerated plants were successfully established in soil. Flowering was observed in 5.7% of the regenerated plants derived from shoot organogenesis and in 1.4% of the regenerated plants derived from somatic embryogenesis. Received: 1 December 1998 / Revision received: 13 September 1999 / Accepted: 20 September 1999     

An efficient in vitro shoot regeneration from immature inflorescence and ex vitro rooting of Arnebia hispidissima (Lehm). DC. - A red dye (Alkannin) yielding plant

Arnebia hispidissima, which belongs to the family Boraginaceae, is an important medicinal and dye yielding plant. The alkannin, a red dye, are root-specific secondary metabolites of A. hispidissima. Shoots were regenerated from callus derived from immature inflorescence explants obtained from field grown plants. MS medium containing 4.52 μM 2, 4-D and 3.33 μM BAP was found to be most effective for the proliferation of callus, induced on medium containing 4.52 μM 2, 4-D. Maximum number (43.1 ± 0.25) with average length (5.2 ± 0.23) of shoots regenerated when callus was transferred to MS medium supplemented with 1.11 μM BAP, 1.16 μM Kin and 0.57 μM IAA. About 75.5 % of in vitro regenerated shoots were rooted on half-strength MS medium supplemented with 9.84 μM of IBA and 200 mg l⁻¹ of activated charcoal. In comparison to in vitro, higher percent (90.2 %) of shoots were rooted under ex vitro conditions when treated with IBA (0.98 mM) for 5 min. Plantlets rooted in vitro as well as ex vitro were acclimatized successfully under the green house conditions. Ex vitro rooted plants exhibited higher survival percentage (75 %) as compared to in vitro rooted plantlets (60 %). Present study may be applicable in the large-scale root-specific red dye (alkannin) production via root induction under ex vitro condition.     

In vitro shoot and root organogenesis, plant regeneration and production of tropane alkaloids in some species of Schizanthus

A rapid in vitro propagation system leading to formation of shoots from callus, roots, and plantlets was developed for Schizanthus hookeri Gill. (Solanaceae), an endemic Chilean plant. The genus Schizanthus is of particular interest due to the presence of several tropane alkaloids. So far, in vitro propagation of species of this genus has not been reported. Propagation of S. hookeri consisted of two phases, the first one for callus initiation and shoot formation and the second for rhizogenesis and plantlet regeneration. From a single callus that rapidly increased in cell biomass (from approximately 50 mg to approximately 460 mg/culture tube [25 x 130 mm] in 60 days) in the presence of 2.69 microM NAA and 2.22 microM BA, more than 10 shoots/callus explant were formed. From the latter, approx. twenty plantlets formed after 90-110 days shoot subculture in medium devoid of growth regulators that favored root formation. Ten alkaloids ranging from simple pyrrolidine derivatives to tropane esters derived from angelic, tiglic, senecioic or methylmesaconic acids were obtained from in vitro regenerated plantlets. One of them, 3alpha-methylmesaconyloxytropane, was not previously described. The same growth conditions, as well as other growth regulator levels tested, were required to induce callus and root formation in S. grahamii Gill. Root organogenesis occurred despite a high level of BA vs. NAA used, (i.e., 4.44 microM BA and 0.54 microM NAA); however no shoot formation was achieved. In the case of S. tricolor Grau et Gronbach, only callus formation was obtained in the presence of various growth regulators.     

Rapid in vitro propagation of Saussurea lappa, an endangered medicinal plant, through multiple shoot cultures

Summary Rapid micropropagation of Saussurea lappa C. B. Clarke, an endangered medicinal plant endemic in the valley of Kashmir and western Himalayas of northern India, was achieved by culturing shoot tips (0.5–1 cm) of 2-wk-old seedlings on Murashige and Skoog’s medium (MS) supplemented with thidiazuron (TDZ, 0.45 μM). Although callus-free multiple shoots were obtained both on N⁶-benzyladenine- (BA) and TDZ-containing media, TDZ was most effective (90%) in inducing multiple shoots. Shoot tips containing proliferative buds were divided into equal halves and subcultured on MS liquid medium containing 0.225 μM TDZ for further multiplication and elongation. Multiplication of induced shoot buds was more effective when cultured in liquid medium than on agar-solidified medium. Shoots (8–10 cm) developed were rooted in MS medium containing naphthaleneacetic acid (NAA, 1.07 μM). Micropropagated plantlets were successfully transferred to soil after hardening.     

High frequency shoot regeneration and Agrobacterium-mediated DNA transfer in Canola (Brassica napus)

Five different varieties of Brassica napus (Cyclone, Dunkled, Oscar, Rainbow and KS75) were tested for their regeneration response. Cyclone showed a very high frequency of regeneration (92%). The use of silver nitrate was a pre-requisite for efficient shoot regeneration. Hypocotyls were selected as the starting material for transformation experiments on the basis of high transient GUS expression. Explants were co-cultivated with Agrobacterium strain EHA101 harboring a binary vector pIG121Hm containing neomycin phosphotransferase II (NPTII) gene, conferring resistance to kanamycin, hygromycin phosphotransferase (HPT) gene, conferring resistance to hygromycin as selectable markers and -glucuronidase (GUS) gene as a reporter. Acetosyringone promoted the transformation but was not an absolute requirement. A pre-selection period of 7 days after co-cultivation was essential for successful transformation. Kanamycin was efficient selective agent for selection and maximum transformation efficiency was 24%. GUS activity was evident in leaf tissues. All the transgenic plants have an expected band of 0.43 kb fragment by PCR analysis confirming the presence of foreign DNA into plant genome.