Improvement of potato (Solanum tuberosum L.) for abiotic stress tolerance through genetic engineering

Abstract

Input applications of agricultural and industrial activities have increased salt levels in our soils. In the modern era of agricultural production of crops, shortage of water resources makes them inaccessible for growing crops. Plant species have different mechanisms that deal with the salt tolerance; but the capability to sustain low cytosolic Na+ is supposed to be one of the vital factors of plant salt tolerance. Removal of Na+ from the cytoplasm of the cells and/or the maintenance of the low cytosolic Na+ concentrations is carried out either by pumping Na+ out of cells (plasma membrane antiporter) or into the vacuoles (vacuolar antiporter) under high salinity conditions. This process is brought about by the operation of plasma membrane-bound H+-pumps responsible for energizing Na+/H+ antiporters. In addition to this, engineering of the regulatory machinery involving transcription factors has emerged as a new tool now for controlling the expression of many stress-responsive genes. Development and use of transgenic plants with enhanced capability of salt tolerance by over-expression of genes may help to meet the future challenges of abiotic stresses. The effect of plant growth regulators in different combinations on in vitro regeneration of currently grown potato cultivars (cvs). ‘Kuroda’, ‘Cardinal’ and ‘Desiree’ were determined. Overall, the callus production and in vitro regeneration efficiency was maximum in Cardinal and Kuroda. Media combination and cultivars having potential for good callus induction and regeneration were selected for transformation. The novel synthetic HSR1 gene was cloned under double CaMV35S promoter in the pGreen0029 plant expression vector. HSR1 and AVP1 genes were transformed in potato through Agrobacterium-mediated transformation method. Putative transgenic calli and regenerated shoots were obtained in the presence of kanamycin (50 mg/ml) as plant tissue selection agent. Varying transformation efficiencies (30 and 25 %) were observed in different batches for Kuroda and Cardinal, respectively. A total of 57 transgenic plants were obtained from independent events and were successfully established in pots containing sterilized sand. 2 Transgenic plants were confirmed by PCR and Southern hybridization. Variable numbers (1-4) of integration sites for the transgenes were observed in the genomic DNA of transgenic potato plants when AVP1/HSR1 specific probes were used for Southern analysis. In order to check abiotic stress tolerance potential, transgenic plants were subjected to in vitro screening in response to different levels of stress inducing agents like NaCl and PEG (6000). Agronomic parameters (shoot length, root length, leaf area index, shoot fresh weight, shoot dry weight, root fresh weight, root dry weight and relative water contents) were recorded that shows the transgenic potato lines performed better under stress conditions compared to the control plants. Transgenic potato plants containing AVP1 and synthetic HSR1 genes were analyzed for salt stress tolerance. Significantly (P ≤ 0.05) higher photosynthetic rates, stomatal, sub-stomatal conductance and transpiration rate were observed in transgenic plants harboring HSR1 and AVP1 genes compared to control plants. Higher Membrane Stability Index was noted in transgenic plants than non-transformed plants. Transgenic plants showed higher accumulation of Na+, K+ and a higher Na+/K+ ratio than non-transformed plants. Salt analyses showed high accumulation of total free amino acids, proline contents, and total soluble sugars indicating salt tolerance manifested by transgenic AVP1 and HSR1 plants. To avoid the toxic level of ROS and protect the cells from oxidative injury, accumulation of complex antioxidant enzymes such as superoxide dismutase (SOD) (EC 1.15.1.1), peroxidase (POD) (EC 1.11.1.7), ascorbic peroxidase (APX) (EC 1.11.1.11) and catalase (CAT) (EC 1.11.1.6) were determined in stressed leaves which showed a significantly higher accumulation in transgenic potato plants compared to controls. Tunnel experiment was performed for yield components to check the potential of transgenic plants under various levels (100, 75 and 50%) of drought stress. Significantly (P ≤ 0.05) higher biomass, number of tubers/plant and weight of tubers/plant was observed at 75 and 50% of drought stress. This study provides an efficient protocol for regeneration efficiency of potato cultivar Kuroda, Cardinal and Desiree using internodal explants. The results suggest that transgenic plants expressing higher levels of AVP1 and HSR1 transcripts in potato are able to withstand salt and drought stress regimes.