Generation Of Transgenic Sugarcane (Saccharum Officinarum)Expressing TERF1and TSRF1and Characterization Of Its Tolerance To Drought And Salt

Sugarcane (Saccharum officinarum L.) is one of the most important field crops grown in the tropical and sub-tropical regions and more than70%world’s sugar is produced from sugarcane. Drought and salinity are the major abiotic stresses severely affect sugarcane growth and sucrose contents. Improvement of sugarcane through conventional breeding became limited due to its complex genome, high polyploidy level, heterozygosity and poor fertility. Genetic engineering might be a solution for improving tolerance level of sugarcane against these stresses. Thus, the present study was designed to transform sugarcane with TERF1and TSRF1gene through Agrobacterium. Embryogenic callus of sugarcane cultivars XintaitangR22and Guitang281were used for transformation with Agrobacterium strain LBA4404harboring pROK2vector containing TERF1and TSRF1gene driven by35SCaMV promoter.Highest transformation efficiency (78%) and PCR positive plants (4.6%) were obtained from the cultivar Guitang281when it was transformed with TERF1gene having inoculum density OD600at0.4and co-cultivated for4days on MS based medium. Under normal condition transgenic lines displayed similar growth status as compared with wild-type plants, in contrast only transgenic plants were able to withstand in water deficit as well as higher salt stress condition, showing tolerance against drought and salinity stresses. Physiological and biochemical assay revealed that TERF1and TSRF1overexpressing plants not only increased accumulation of proline, soluble sugars and glycine betaine than wild-type plants but also reduced MDA and H2O2content in response to drought and salt stress. The findings of this study demonstrated that TERF1and TSRFl conferred drought and salt tolerance to transgenic sugarcane through modulating the accumulation of osmo-protectant and antioxidant metabolism, indicating that these genes may have regulatory roles in response to abiotic stress in sugarcane.To understand proteome changes of transgenic sugarcane leaves overexpressing TERFI in response to drought, seven months old green house grown transgenic and WT sugarcane plants were exposed to polyethylene glycol (PEG8000;20%w/v) stress for21days. Total protein was extracted from leaves of OE and WT plants and separated by2-DE. Among differentially expressed proteins of OE plants compare with WT plants under drought stress,13protein spots in XintaitangR22and36protein spots in Guitang281were identified by MALDI TOF/TOF MS analysis. In XintaitangR22all proteins were up regulated and in Guitangtang34proteins were upregulated and two were downregulated in OE transgenic line compare with WT in response to drought stress. In XintaitangR22, nine identified proteins were involved in metabolism and energy function, one in protein synthesis and three functioned as transporter. In Guitang281, among identified proteins22were involved in metabolism and energy function, two in protein synthesis, one transcription function, one intracellular traffic function, one signal transduction function and nine functions for disease/defense. Among upregulated proteins in transgenic XintaitangR22, photosynthetic protein (RuBisCO large subunit), thioredoxin, ATP synthase protein, glycolysis and sugar metabolism protein (triose phosphate isomerase), malate dehydrogenase, and eukaryotic translation initiation factor5A proteins are vital. In transgenic Guitang281, peptidyl-prolyl cis-trans isomerase and penta tricopeptide repeat containing proteins were down regulated. Among upregulated proteins in transgenic Guitang281, metabolism protein (14-3-3like protein), photosynthetic protein (RuBisCO large subunit, PEP carboxylase), the ferredoxins (Fds), glyceraldehyde3-phosphate dehydrogenase (GAPDH), elongation factor Tu, several small heat shock proteins (17.4kDa,17.5kDa,17.6kDa,17.8kDa,18kDa,18.1kDa) and peroxidases (PHGPx, APx) proteins are vital. Analysis of protein properties implied that majority of the differentially abundance proteins associated with drought were acidic, stable and transmembrane proteins, enriched with hydrophobic amino acid alanine, valine and leucine. These results unravel the regulatory mechanism of TERF1for drought stress tolerance of transgenic sugarcane and provide new insight into adaptation to water deficit stress through altering the expression of specific proteins.