Transformation and regeneration of Texas cotton

Abstract

An in vitro shoot apex grafting technique was developed to increase recovery of intact plants from shoots regenerating in culture. Induction of root organogenesis in regenerating cotton shoots is genotype-dependent and often unreliable. The resulting loss of regeneration potential due to failure to form roots can vary from 30-80% according to genotype and represents a significant bottleneck in the overall recovery of plants from culture. If the non-rooting shoots are transgenic, the loss in regenerated plant material can be substantial. In vitro grafting of shoots to seedling rootstock proved to be a simple and reliable method that allowed recovery of non-rooting shoots from culture. Following shoot apex grafting, the survival and recovery of plants averaged 90-100% under the final optimized conditions. Success of any given graft was directly related to scion size and age of the seedling rootstock; a scion of approximately 0.8-1.0 cm and a rootstock of 14-35 days was optimal. The method appeared to be genotype-independent, and varietal differences between rootstock and scion did not effect the rate of plant recovery from culture. Five TAES cotton (Gossypium hirsutum L.) cultivars were transformed using two vectors, pXBOP and pJMA4, which contained genes associated with pathogen resistance (PR genes): osmotin (PR-5) and a basic PR-1 in pXBOP, and lox1 in pJMA4. Plants were transformed using the shoot apex/Agrobacterium method (US Pat. # 5,164,310; Smith et al., 1992) developed earlier for cotton (Gould et al., 1991a; 1991b). After kanamycin selection, shoots (T₀) were grafted onto in vitro grown seedling rootstocks (Luo and Gould, 1999). Grafted/regenerated plants were normal, fertile, and produced normal progeny. The transferred genes were detected in the DNA of regenerated plants (T₀) and in progeny (T₁) using PCR amplification and Southern DNA analyses. Gene expression was detected by RT-PCR in the progeny.