Kondeti Subramanyam

Ectopic expression of Arabidopsis RCI2A gene contributes to cold tolerance in tomato.

AbstractCold is a major stress that limits the quality and productivity of economically important crops such as tomato (Solanum lycopersicum L.). Generating a cold-stress–tolerant tomato by expressing cold-inducible genes would increase agricultural strategies. Rare cold-inducible 2a (RCI2A) is expressed in Arabidopsis, but its molecular function during cold stress is not fully understood. Here we ectopically expressed ArabidopsisRCI2A in transgenic tomato to evaluate tolerance to cold stress without altering agronomic traits. Biochemical and physiological study demonstrated that expression of RCI2A in transgenic tomato enhanced the activity of peroxidase and ascorbate peroxidase (APX) and reduced the accumulation of H2O2, alleviated lipid peroxidation, increased the accumulation of chlorophyll, reduced chilling-induced membrane damage, retained relative water content and enhanced cold tolerance. A motif search revealed that the motifs of photosystem II (PSII) phosphoproteins PsbJ and PsbH and reaction-center proteins PsbL and PsbK were common to cold-inducible RCI2A and peroxidase proteins RCI3A, tomato peroxidase (TPX1), TPX2, tomato ascorbate peroxidase (APX1), and horseradish peroxidase (HRP-c). In addition to membrane protection, RCI2A may cross talk with PSII-associated proteins or peroxidase family enzymes in response to cold stress. Our findings may strengthen the understanding of the molecular function of RCI2A in cold-stress tolerance. RCI2A could be used to improve abiotic stress tolerance in agronomic crops.

Transfer and Targeted Overexpression of γ-Tocopherol Methyltransferase (γ-TMT) Gene Using Seed-Specific Promoter Improves Tocopherol Composition in Indian Soybean Cultivars

Soybean oil contains high levels of tocopherols which are an important source of vitamin E in human diet. The conversion of γ- to α-tocopherol catalyzed by γ-tocopherol methyltransferase (γ-TMT) is found to be the rate limiting factor in soybean which influences the tocopherol composition. Using Agrobacterium-mediated transformation, we overexpressed the γ-TMT gene of Perilla frutescens under the control of the seed-specific promoter vicillin in cultivar Pusa 16. Transgene integration and expression was confirmed in five independently transformed GUS positive soybean plants by polymerase chain reaction (PCR), Southern hybridization, and reverse transcriptase-PCR (RT-PCR). High-performance liquid chromatography (HPLC) analysis showed that overexpression of Pf-γ-TMT resulted in efficient conversion of γ-tocopherol to α-tocopherol and concomitant increase in seed α-tocopherol content in RT-PCR positive plants. The protocol was successfully applied to three more cultivars PK 416, Gujarat soybean 1, and VL soya 1 in which seeds of transformed plants showed elevated level of α-tocopherol than wild-type seeds.

RNAi-mediated resistance to yellow mosaic viruses in soybean targeting coat protein gene

In this genomic era, soybean has entrenched genomic database which offer an extensive scope for improvement through genetic manipulation, although demand for transgenics soybean with better production and enhanced quality has been handicapped due to Mungbean yellow mosaic India virus (MYMIV) belonging to the genus Begomovirus. MYMIV is a causative agent of yellow mosaic disease that has been emerged as a threat to the cultivation of bean family in India. In this study, transgenic soybean plants were generated using the intron-spliced hairpin construct encoding the coat protein sequence of MYMIV in the control of 35S promoter and ocs terminator. Integration of coat protein gene in independently transformed plants was confirmed by PCR and Southern hybridization where one transgenic line of coat protein-event A, two transgenic lines of coat protein-event B, and two transgenic lines from the coat protein-event C showed gene hybridization. Inoculation was performed on T1 seedlings of transgenic and non-transgenic plants where the viral replicative DNA level was assessed for ten plants and a quality concentration of viral replicative form was seen in the transgenic lines. Northern blot analysis detects siRNA in the transgenic line 2 of event A, line 5 and 6 of event B, as well as line 9 and 10 from event C inoculated with viruliferous whiteflies and a high level of siRNA (21–22 nt) was observed in the transgenic line 2 and line 10 which corroborated by the non-detectable level of viral replicative DNA and low concentration of viral transcript for replication as estimated in qRT-PCR. Results obtained in this study confirmed the transgene construct can be used to develop resistance against begomoviruses in soybean and other crops, as it targets the most conserved domain governing whitefly transmission.