Development of stable transgenic maize plants tolerant for drought by manipulating ABA signaling through Agrobacterium-mediated transformation

Abstract

Abstract Background In crop plants, to cope up with the demand of food for rising population, revolutionary crop improvement programmes are being implemented for higher and higher yields. Abiotic stress, especially at flowering stage, causes drastic effect on yield in plants. Deforestation and urbanization made the water table very low and changed the climate which led to untimely and unforeseen rains which affect the yield of a crop through stress, both by lack of water as well as water logging (abiotic stress). Development of tolerant plants through breeding is a time-consuming programme and does not perform well in normal conditions. Development of stress-tolerant plants through transgenic technology is the better solution. Maize is a major crop used as food and fodder and has the commercial value in ethanol production. Hence, the genes viz., nced (9-cis-epoxycarotenoid dioxygenase) and rpk (receptor-like protein kinase), which play the key roles in the abscisic acid pathway and upstream component in ABA signaling have been transferred into maize plants through Agrobacterium-mediated transformation by optimizing several parameters to obtain maximum frequency of transformation. Results Cultures raised from immature embryos of 2-mm size isolated from maize cobs, 12–15 days after pollination, were used for transformation. rpk and nced genes under the control of leaP and salT promoters respectively, cloned using gateway technology, have been introduced into elite maize inbred lines. Maximum frequency of transformation was observed with the callus infected after 20 days of inoculation by using 100 μM acetosyringone, 10 min infection time, and 2 days incubation period after co-cultivation resulted in maximum frequency of transformation (6%) in the NM5884 inbred line. Integration of the genes has been confirmed with molecular characterization by performing PCRs with marker as well as gene-specific primers and through southern hybridization. Physiological and biochemical characterization was done in vitro (artificial stress) and in vivo (pot experiments). Conclusions Changes in the parameters which affect the transformation frequency yielded maximum frequency of transformation with 20-day-old callus in the NM5884 inbred line. Introducing two or more genes using gateway technology is useful for developing stable transgenic plants with desired characters, abiotic stress tolerance in this study.