Table 1.
Media used in the present investigation.
Fig 1.
Schematic representation of genes between T-DNA right border (RB) and left border (LB) of the gene construct pCAMBIA 2301 used for Agrobacterium-mediated transformation of W. somnifera.
The nptII gene is presented for kanamycin resistance under the control of the CaMV 35S promoter (left) and the gusA gene is used as a scorable marker under the control of the CaMV35S promoter (left).
Fig 2.
Effect of various antibiotics concentrations on the regeneration ability of nodal explants of W. somnifera.
Data were obtained after 4 weeks for the percentage of response of nodal explants. Each value represents the mean of three independent experiments with 25 explants per treatment.
Table 2.
Effect of kanamycin on sensitivity of nodal explants to regeneration and elongated shoots of W. somnifera on root induction.
Fig 3.
Transient GUS expression in regeneration site of nodal explants of W. somnifera under different parameters tested.
Effect of culture OD600 at 0.1 (a), 0.2 (b), 0.5 (c) and 1.0 (d); effect of AS concentrations-e. 50 μM, f. 75 μM, g. 100 μM, h. 125 μM, i. 150 μM, and j. 175 μM; effect of sonication treatment-k. 5 sec, l. 10 sec, m. 15 sec, n. 20 sec, o. 25 sec; effect of vacuum infiltration-p. 5 min, q. 10 min, r. 15 min, s. 20 min; t. combined effect of sonication (10 sec) and vacuum infiltration (10 min); effect of L-cysteine concentrations- u. 100 mg/l, v. 200 mg/l, w. 300 mg/l, x. 400 mg/l, y. 500 mg/l; effect of STS concentrations- z. 50 mg/l, a1. 75 mg/l, b1. 100 mg/l, c1. 125 mg/l, d1. 150 mg/l, e1. 175 mg/l; effect of DTT concentrations—f1. 25 mg/l, g1. 50 mg/l, h1. 75 mg/l, i1. 100 mg/l, j1. 125 mg/l; k1. Combined effect of L-cysteine (100 mg/l), STS (125 mg/l) and DTT (75 mg/l); l1. Control explant. The GUS expression was performed on 6 day pre-cultured nodal explants after 3 days co-cultivation
Table 3.
Factors that affect the transient expression of gusA gene in nodal explants of W. somnifera infected with A. tumefaciens strain LBA4404 containing vector pCAMBIA2301.
Fig 4.
Regeneration of putative transgenic plants of W. somnifera via Agrobacterium-mediated transformation of nodal explants.
a. 6 day-old nodal explants pre-cultured on WsSIM; b and c, multiple shoot induction from nodal explants after 15 days of culture on WsSSM-I after inoculation with A. tumefaciens LBA4404; d and e, multiple shoot proliferation from nodal explants on WsSSM-II after 15 days of culture; f, elongated transgenic shoot cultured on WsSSM-III after 15 days; g, rooting of elongated putative transgenic plantlet on WsRSM; h, T0 transgenic plants growing in the controlled environmental growth chamber; I, flowering of T0 transgenic plant in the greenhouse; j, T1 transgenic plants growing in the controlled environmental growth chamber
Table 4.
Transformation frequency of shoots regenerated from nodal explants of W. somnifera in WsSIM+L-Cys+STS+DTT+AS under three selection pressure for recovery of transgenic shoots.
Fig 5.
Stable gusA gene expressions in regeneration of transgenic plants of W. somnifera via Agrobacterium-mediated transformation using pCAMBIA 2301.
a. Transformed cluster of multiple shoots from nodal explants reared on WsSSM-II after 30 days of culture, b. Elongated transgenic shoot cultured on WsSSM-III after 45 days of culture, c. Transformed full plantlet, d and e. Transformed T1 progeny seed and seedlings, f-h. Respective control plants and seedlings.
Table 5.
Stable transformation frequency of rooted-elongated shoots of W. somnifera in WsRSM under three selection pressure for recovery of transgenic plants.
Fig 6.
Molecular confirmation of T0 and T1 transgenic W. somnifera plants by PCR and Southern blot analysis.
a. PCR amplification of the 515 bp fragment of the gusA gene in T0 transgenic plants. Lane 1–100 bp ladder, lane 2-Plasmid DNA (pCAMIA 2301; positive control), lanes 3, 4, 6, 7, 8- putatively transformed plant DNA, lane 5-non-transformed plant DNA (negative control); b, Southern hybridization analysis to study the T-DNA integration (5391 bp) in T0 plants; lane 1 DNA molecular weight marker (λDNA/EcoRI), lane 2-plasmid DNA digested with EcoRI (positive control), lanes 3–6 DNA isolated from putative transformed plants, lane 7-non-transformed plant DNA (negative control); c, PCR amplification of the 515 bp fragment of the gusA gene in T1 transgenic plants. lane 1–100 bp ladder, lane 2-plasmid DNA (pCAMIA 2301; positive control), lanes 3-8- putatively transformed plant DNA, lane 9-non-transformed plant DNA (negative control); d, Southern blot analysis of putatively transformed T1 plants. lane 1, pCAMBIA 2301 plasmid (positive control); lanes 2–6, putatively transformed W. somnifera genomic DNA digested with EcoRI and probed with a 515-bp PCR-amplified product of the gusA gene.
Fig 7.
Quantities of withanolide A, withanolide B, withaferin A and withanone contents in T0 (FT1-FT5) and T1 (T1–T5) transgenic lines of W. somnifera and its control plant.
Data represents mean ± standard error of three replicates.