An efficient protocol was standardized for successful regeneration of Cissampelos pareira (L.) through indirect organogenesis. Nodal explants were cultured on MS fortified with 0.5 ± 1.0 mg/l BAP, Kn either single or in combination with NAA 0.5 mg/l. The combinations induced profuse, compact, light green to greenish coloured calli. Some differences in the morphology of callus such as change in the colour and texture was also observed with increasing the concentration of BAP 0.5 ± 2.0 mg/l + NAA 0.5 mg/l. Maximum callus induction was observed on 1.0 mg/l BAP and 0.5 mg/l NAA showed greenish, friable and granular lush colour. The calli were subcultured on fresh MS that contained BAP and Kn single or in combination with NAA (BAP 0.5 ± 2.0 mg/l, Kn 0.5 ± 2.0 mg/l, NAA 0.5 mg/l). The maximum regeneration frequency of shoot organogenesis was recorded on BAP (2.0 mg/l) + NAA (0.5 mg/l). Healthy microshoots were separated and transferred to the rooting medium. Here, MS augmented with IBA 1.0 mg/l showed maximum rooting. Well rooted plantlets were transferred to the field and maximum survival frequency was recorded when BAP (1.0 mg/l) + NAA (0.5 mg/l) for callus induction, for shooting BAP (2.0 mg/l) + NAA (0.5 mg/l) and for rooting IBA (1.0 mg/l) was used. The regenerated whole plants were subjected for hardening where the maximum survival frequency was found to be 80%. This reproducible protocol can be used for regeneration and genetic transformation studies.

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Background: Optimization of co-cultivation parameters during Agrobacterium-mediated transformation to Euphorbia tirucalli evaluated were bacterial density, infection period, acetosyringone (AS) concentration and co-cultivation temperature. Optimized parameters resulted in high transformation efficiency 3 fold increase at transient GUS expression .The optimized conditions were Agrobacterium tumefaciens growth phase of A600nm 0.17, infection period of 30 min, addition of acetosyringone (AS) in co-cultivation medium (100 µM) and cocultivation temperature of 25°C. Methods: Nodal explants were used for transformation. Bacterial culture was added to 50 ml of liquid YEP medium with kanamycin and rifampicin and grown until reaching the growth phase (A600nm). Bacterial density ranged from A600nm 0.17, 0.56, 0.8 and 1.2 OD were used in the present study. The co-cultivation medium made of solid MS medium consisted of NAA 2 mg L-1 and various concentrations of AS at 0, 50, 100, 200, 400, 600 and 800 µM. Histochemical analysis of gus gene expression was carried out. Result: Higher bacterial density resulted in more transformation efficiency, but also higher necrosis in the explants. Dilution of bacterial suspension reduced necrosis in explants and resulted in higher transformation. The transformation efficiency is 72% when the infection process was carried out with acetosyringone in co-cultivation medium (100 µM). Our studies proved that among the optimized conditions, cocultivation temperature and acetosyringone were the critical parameters during Agrobacterium mediated transformation.

Background: Optimization of co-cultivation parameters during Agrobacterium-mediated transformation to Oxalis corniculata evaluated were bacterial density, infection period, acetosyringone (AS) concentration and co-cultivation temperature. Five fold transformation efficiency is achieved in transient gus gene expression after optimizing various parameters. The optimized conditions were Agrobacterium tumefaciens growth phase of A600nm 0.17, infection period of 30 min, addition of acetosyringone (AS) in co-cultivation medium (400 μM) and cocultivation temperature of 22°C. Methods: Oxalis corniculata (L.) were used for transformation. Bacterial culture was added to 50 ml of liquid YEP medium with kanamycin and rifampicin and grown until reaching the growth phase (A600nm). Bacterial density ranged from A600nm 0.17, 0.56,0.80 and 1.34 OD were used in the present study. The co-cultivation medium made of solid MS medium consisted of BAP 2 mg L-1 and NAA 0.5 mg L-1 and various concentrations of AS at 0, 50, 100, 200, 400,600 and 800 μM. Histochemical analysis of gene expression was carried out. Result: Higher bacterial density resulted in more transformation efficiency, but also higher necrosis in the explants. Dilution of bacterial suspension reduced necrosis in explants and resulted in higher transformation. The transformation efficiency is 64% when the infection process was carried out with acetosyringone in co-cultivation medium (400 μM). Our studies proved that among the optimized conditions, cocultivation temperature and acetosyringone were the critical parameters during Agrobacterium mediated transformation.

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