An efficient protocol was developed for Agrobacterium tumefaciens-mediated transformation of tomato (Solanum lycopersicum) cultivars using cotyledon explants. The transformation frequency was assessed in response to several different factors, including seed germination medium, seedling age, pre-culture duration, pre-culture and cocultivation media, inoculation medium, medium pH, washing medium, and kanamycin concentration in initial selection medium. Cotyledons excised from 6-d-old seedlings germinated on half-strength Murashige and Skoog's (MS) basal medium containing 8.9 μM benzyladenine (BA) produced the most suitable explant material. Six days of explant pre-culture and 5 min inoculation with Agrobacterium culture in MS medium, containing 8.9 μM BA, 9.3 μM kinetin, and 0.4 mg l −1 thiamine at pH 5.0, significantly improved the transformation frequency. The addition of a tobacco feeder cell layer, however, did not lead to any significant improvement in the transformation rate. Kanamycin at 20 mg l −1 in the selection medium for the initial 10 d resulted in the highest transformation frequency. Combining the best conditions for each parameter resulted in an overall transformation efficiency of 44.3 %. Gene transfer was confirmed through PCR and Southern blot analyses. Mendelian inheritance ratios were found in 71.5 % of the independent transgenic lines from self-fertilized T 1 progeny. The optimized transformation procedure showed high transformation frequencies for all three tomato cultivars tested, namely, Kashi Vishesh (H-86), Hisar Anmol (H-24), and Kashi Amrit (DVRT-1), and is also expected to give reproducible results with other tomato cultivars.

An efficient protocol for rapid in vitro clonal propagation of spine gourd (Momordica dioica Roxb.) genotype RSR/DR15 (female) and DR/NKB-28 (male) was developed through enhanced axillary shoot proliferation from nodal segments. Maximum shoot proliferation of 6.2 shoots per explant with 100 % shoot regeneration frequency was obtained from the female genotype on Murashige and Skoog's (1962) medium supplemented with 0.9μM N6-benzyladenine (BA) and 200 mg l -1 casein hydrolysate (CH). While from the male genotype the optimum shoot regeneration frequency (86.6 %) and 6.4 shoots per explant was obtained on MS medium supplemented with 2.2μM BA. CH induced vigorous shoots, promoted callus formation, and proved inhibitory for shoot differentiation and shoot length, especially in explants from male genotype. Rooting was optimum on half-strength MS medium (male 92.8 %, female 74.6 %) containing 4.9μM indole-3-butyric acid (IBA). Plantlets were transferred to plastic cups containing a mixture of cocopit and perlite (1:1 ratio) and then to soil after 2-3 weeks. 84 % female and 81 % male regenerated plantlets survived and grew vigorously in the field. Genetic stability of the regenerated plants was assessed using random amplified polymorphic DNA (RAPD). The amplification products were monomorphic in the in vitro propagated plants and similar to those of mother plant. No polymorphism was detected revealing the genetic integrity of in vitro propagated plants. This micropropagation procedure could be useful for raising genetically uniform planting material of known sex for commercial cultivation or build-up of plant material of a specific sex-type.

To elucidate whether the transgenic crop alters the rhizospheric bacterial community structure, a 2-year study was performed with Cry1Ac gene-inserted brinjal crop (Bt) and their near isogenic non-transformed trait (non-Bt). The event of Bt crop (VRBT-8) was screened using an insect bioassay and enzyme-linked immunosorbent assay. Soil moisture, NH4 (+)-N, NO3 (-)-N, and PO4 (-)-P level had non-significant variation. Quantitative polymerase chain reaction revealed that abundance of bacterial 16S rRNA gene copies were lower in soils associated with Bt brinjal. Microbial biomass carbon (MBC) showed slight reduction in Bt brinjal soils. Higher MBC values in the non-Bt crop soil may be attributed to increased root activity and availability of readily metabolizable carbon compounds. The restriction fragment length polymorphism of PCR-amplified rRNA gene fragments detected 13 different bacterial groups with the exclusive presence of β-Proteobacteria, Chloroflexus, Planctomycetes, and Fusobacteria in non-Bt, and Cyanobacteria and Bacteroidetes in Bt soils, respectively, reflecting minor changes in the community structure. Despite the detection of Cry1Ac protein in the rhizospheric soil, the overall impact of Cry1Ac expressing Bt brinjal was less compared to that due to seasonal changes.

A b s t r a c t A r t i c l e I n f oColeoptilar nodal explants, which are independent of season, were used to develop high efficiency transformation method in maize. Conditions which affect Agrobacterium-mediated genetic transformation have been standardized by using these explants on hygromycin selection regime. Hyper virulent Agrobacterium strain EHA105 containing gateway vector pMDC99 with Ascorbate -Glutathione pathway coding genes harboring with hygromycin phosphotransferase (hpt II) and bar as plant selectable marker genes was used. The survival frequency of calli at three different stages viz., at the end of I sub-culture on normal medium and II selection as well as on regeneration media supplemented with hygromycin were taken into consideration for the assessment of optimal conditions after checking with PCR. Of the different parameters used with different conditions, one day pre-conditioning of explants, 0.8 optical density of bacterial culture in plain MS liquid infection medium having 5.8 pH along with 200 µM acetosyrengone, 15 minutes infection time for the explants after treating with 0.1% Macerozyme, application of vacuum infiltration for 10 minutes, three days of co-cultivation period were found to be optimal for getting high frequency of transformation through Agrobacterium-mediated genetic transformation in maize. Transformation frequency of 3.25% was obtained on average with these optimal conditions, for which the explants were co-cultivated with these conditions and inoculated on callus induction medium having hygromycin at 10 mg l -1 for selection. The selected calli were transferred onto regeneration medium supplemented with BAP and Kinetin, at a concentration of 1 mg l -1 each for plantlet development. Putative transformants were screened by using hpt II gene specific primers and confirmed after performing southern analysis.