Engineering salt and drought tolerance in cotton (Gossypium hirsutum L.)

Abstract

In the modern era of agricultural crop production, commercial applications of crop inputs and industrial activities have increased salt concentrations in our soil and water resources making them toxic for growing crops. During abiotic stresses, plants employ several strategies, among which salt pumping and compartmentalization into intracellular organelles (lumen of vacuole) is a major strategy. The present study involved the development of transgenic cotton cv. coker-312 tolerant to salt and drought stress. In the first part of the study, silicon carbide whisker mediated cotton calli transformation method was established using GUS and AVP1 gene. Fertile transgenic plants were produced from embryogenic calli of four events. PCR and southern hybridization of kanamycin resistant putative AVP1 embryogenic calli showed the presence and stable integration of transgenes in 1-4 copies in the genome. Results of the salt tolerance study of T1 AVP1 transgenic cotton showed significantly greater salt tolerance at 200mM NaCl than non-transformed control plants. Moreover, transgenes segregate in a Mendelian fashion indicated the authenticity of the method. In the second part of the study, AtNHX1 gene was cloned under the control of double CaMV 35S promoter and terminator in plant transformation vector pGA482. Agrobacterium tumefaciens harboring AVP1 and AtNHX1 genes were used to inoculate coker-312 hypocotyls. Putative transgenic kanamycin resistant calli of different events were produced and putative regenerated transgenic plants were obtained. The Overall transformation efficiency was 20-25 %. Regenerated T0 plants were grown to maturity in the containment for getting selfed T0 seeds and subsequently T1 and T2 seeds. PCR and Southern analyses showed the presence and genomic integration of 1-4 copies of the AVP1 and AtNHX1 genes. Northern analyses/cDNA analyses showed transcripts of transgenes in the transgenic plants. Transgenic plants (T2) having AVP1and AtNHX1 genes verified by PCR were exposed to salt studies executed with AVP1 transgenic in both hydroponic as well as in pots while plants having AtNHX1 gene were exposed only in pots. After 4-6 weeks of germination, NaCl was applied in solutions up to 200 mM. Transgenic plants showed significantly higher accumulation of Na+, K+, total free amino acids, proline, total soluble sugars and higher nitrate reductase activities than non-transformed control plants. A water stress study was performed on AVP1 T2 transgenic cotton after two weeks and 8-10 weeks of germination in pots. Stress was applied by withholding water for upto 10 days. Results revealed that transgenic AVP1 plants have significantly high water contents and low water potentials with less square shedding, retain high number of bolls than non-transformed control plants. Photosynthesis rate was significantly higher in transgenic (AVP1/AtNHX1) cotton progenies than non-transformed control plants. Transgenic cotton also showed significantly better growth and yield when compared with non-transformed plants. Fiber analyses by scanning electron microscopy and HV1 indicated that fiber of transgenic plant were healthy with uniform twisting in contrast to weak and shriveled fiber of the non-transformed control plants. Transgenic plants had high fiber strength, length and low mic value than non-transformed control cotton plants respectively. Transgenic germplasm developed in present study might be useful for its application in cotton breeding program aiming salt/drought tolerance in cotton cultivars.