Transgenic rice expressing a codon-modified synthetic CP4-EPSPS confers tolerance to broad-spectrum herbicide, glyphosate.

KEY MESSAGE: Highly tolerant herbicide-resistant transgenic rice was developed by expressing codon-modified synthetic CP4--EPSPS. The transformants could tolerate up to 1% commercial glyphosate and has the potential to be used for DSR (direct-seeded rice). Weed infestation is one of the major biotic stress factors that is responsible for yield loss in direct-seeded rice (DSR). Herbicide-resistant rice has potential to improve the efficiency of weed management under DSR. Hence, the popular indica rice cultivar IR64, was genetically modified using Agrobacterium-mediated transformation with a codon-optimized CP4-EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) gene, with N-terminal chloroplast targeting peptide from Petunia hybrida. Integration of the transgenes in the selected rice plants was confirmed by Southern hybridization and expression by Northern and herbicide tolerance assays. Transgenic plants showed EPSPS enzyme activity even at high concentrations of glyphosate, compared to untransformed control plants. T0, T1 and T2 lines were tested by herbicide bioassay and it was confirmed that the transgenic rice could tolerate up to 1% of commercial Roundup, which is five times more in dose used to kill weeds under field condition. All together, the transgenic rice plants developed in the present study could be used efficiently to overcome weed menace.

A comprehensive study of the hormetic influence of biosynthesized AgNPs on regenerating rice calli of indica cv. IR64.

Rice is one of the most widely cultivated crops worldwide; however, it is not amenable to genetic manipulations, owing to its poor response to tissue culture and regeneration in vitro. To improve its response to tissue culture, we evaluated the influence of biosynthesized silver nanoparticles on callus induction, regeneration and rhizogenesis in Indica rice cv. IR64. Silver nanoparticles were biosynthesized by using silver nitrate and Parthenium hysterophorus plant extract, and were characterized by UV-visible spectroscopy, Fourier-transform infrared spectroscopy, Transmission electron microscopy and X-ray diffraction. The biosynthesized silver nanoparticles (PHAgNPs), when supplemented in tissue culture medium, promoted callus induction frequency, callus regeneration and rhizogenesis at concentrations of 10 mg l -1, 5 mg l-1 and 10 mg l-1, respectively. Further examination of the endogenous hormonal levels in regenerating calli revealed that AgNPs enhanced regeneration by alleviating abscisic acid and ethylene levels in the plant tissue. The stimulatory influence eliciting the regeneration response was found to be optimal with the supplementation of 5 mg l-1 PHAgNPs in the regeneration medium; the malondialdehyde, proline and hydrogen peroxide levels were also lower than those in the control, thus suggesting improved antioxidant status. Our results indicated that biosynthesized PHAgNPs may have the potential to positively influence tissue culture of recalcitrant varieties.

Genotype-independent and enhanced in planta Agrobacterium tumefaciens-mediated genetic transformation of peanut [Arachis hypogaea (L.)].

Agrobacterium infection and regeneration of the putatively transformed plant from the explant remains arduous for some crop species like peanut. Henceforth, a competent and reproducible in planta genetic transformation protocol is established for peanut cv. CO7 by standardizing various factors such as pre-culture duration, acetosyringone concentration, duration of co-cultivation, sonication and vacuum infiltration. In the present investigation, Agrobacterium tumefaciens strain EHA105 harboring the binary vector pCAMBIA1301-bar was used for transformation. The two-stage selection was carried out using 4 and 250 mg l-1 BASTA® to completely eliminate the chimeric and non-transformed plants. The transgene integration into plant genome was evaluated by GUS histochemical assay, polymerase chain reaction (PCR), and Southern blot hybridization. Among the various combinations and concentrations analyzed, highest transformation efficiency was obtained when the 2-day pre-cultured explants were subjected to sonication for 6 min and vacuum infiltrated for 3 min in Agrobacterium suspension, and co-cultivated on MS medium supplemented with 150 µM acetosyringone for 3 days. The fidelity of the standardized in planta transformation method was assessed in five peanut cultivars and all the cultivars responded positively with a transformation efficiency ranging from minimum 31.3% (with cv. CO6) to maximum 38.6% (with cv. TMV7). The in planta transformation method optimized in this study could be beneficial to develop superior peanut cultivars with desirable genetic traits.