Engineering Drought Tolerance in Sugarcane (Saccharum officinarum L.)

Abstract

Water stress imparted by drought is the most prevalent abiotic stress that limits plant growth and productivity and it is more threatening for crops require a lot of water i.e. Sugarcane (Saccharum officinarum L.). The present study aimed to develop the drought tolerant transgenic sugarcane. Two commercial cultivars (CPF-245 and CPF-237) and three advanced lines (CSSG-668, S-2003US633, S-2003US114) of sugarcane grown in Punjab, Pakistan were evaluated for callus induction, embryogenic callus production and regeneration from apical discs. CSSG-668 was found to be the best genotype yielding maximum embryogenic callus production and regeneration whereas cv. CPF-245 exhibited the lowest callus induction and regeneration frequency. Selective agent (geneticin G-418) at five concentrations; 0, 20, 40, 60, and 80 mg/L was used to optimize selection conditions with non-transformed embryogenic calli. Variable effects of the selective agent were observed on different sugarcane genotypes for appropriate selection. The geneticin concentration at 60 mg/L was found to be the suitable dose to select the calli of genotypes CSSG-668, CPF-245 and S-2003US633. However, for S-2003US-114, 35 mg/L geneticin was found to be optimum. Similarly, 60 mg/L geneticin, was optimum dose to select regenerated plantlets of the cultivars CSSG-668 and CPF-245 while it was 40, 25 mg/L for the cultivars S-2003US-114 and S-2003US-633, respectively. Drought tolerant transgenic sugarcane lines were developed by exploiting the potential of vacuolar H+ pyro-phosphatase (AVP1 gene). The genetic transformation of sugarcane was done through Biolistic mediated transformation method. Seventy eight embryogenic calli plates of sugarcane cultivar CSSG-668 were bombarded and 120 putative transgenic plants resistant to selection marker (Geneticin) were obtained. Out of these, 34 plants showed the presence of both AVP1 and nptII (selection marker) genes. Southern results further confirmed the stable integration of transgene. One to four copies were present in sugarcane genome. Thirteen lines which showed better morphology were selected and transplanted in micro plots for drought tolerance screening. Two irrigation treatments well watered and limited water conditions were applied. Northern and Western analyses showed variable and higher expression of AVP1 gene in transgenic lines compared with non-transgenic line under stress condition. The effects of drought stress on agronomic parameters i.e., cane height, number of millable canes, number of internodes, cane diameter, brix % and physiological parameters i.e., relative water contents in the leaf, leaf water potential, osmotic potential, turgor potential, photosynthetic rate and transpiration rate were noted. Interaction among water stress treatment x sugarcane lines was statistically significant for all observed agronomic parameters and physiological parameters at P<0.05. Visually it was observed that transgenic sugarcane lines showed better growth and plant height when compared with non-transgenic line. Water stress significantly reduced the cane height, number of internodes, cane diameter, number of millable canes and Brix %. However transgenic sugarcane lines SP27, SP34, SP58 and SP10 showed better agronomic performance than respective non-transgenic line under stress condition. Physiological parameters such as relative water contents (RWC), leaf water potential (Ψw), osmotic potential (Ψo) and turgor potential (Ψp), photosynthetic rate, transpiration rate and stomatal conductance are the important traits for the identification of drought tolerant and sensitive lines. Significant variations were found in all tested genotypes for these traits. Transgenic sugarcane lines SP27, SP34 and SP58 showed better physiological performance water relation than non-transgenic line and other transgenic lines. This study provided an evidence regarding potential of AVP1 for drought tolerance.