Transformation of cotton plants through an alternate DNA delivery approach

The objective of this research was to introduce chitinase and beta-1,3-glucanase genes into cotton to improve resistance to fungal pathogens through the pollen tube pathway-mediated transformation (PTP). To confirm the success of this method, DNA of a plasmid pRTL2-GUS containing uidA (coding sequence for beta-glucuronidase) was introduced into cotton (Gossypium hirsutum L.) 'Paymaster HS26', 'Stovepipe', and 'CA3048'. Seeds formed from treated flowers were allowed to mature on the plants. Seedlings grown from seeds harvested from treated flowers were screened for the presence of uidA by PCR and GUS (beta-glucuronidase) expression by histochemical assay. The overall transformation frequency was 11.7%. Southern hybridizations of T1 progeny confirmed that the uidA transgene was integrated into the cotton genome. GUS expressions were detected in the plants of two generations. In T2 progeny, GUS segregation of uidA followed the expected 3:1Mendelian genetic ratio. Our results confirmed transformation of uidA via PTP.;Chitinases and beta-1,3-glucanases have been used in transgenic plants to improve resistance to pathogens. In this study, fertile, transgenic cotton (Gossypium hirsutum L.) plants expressing a bean chitinase and an Arabidopsis beta-1,3-glucanase were generated using pollen tube pathway-mediated transformation system. Ch5b from Phaseolus vulgaris L. and bg2 from Arabidopsis thaliana L. were cloned into plasmid pRTL2 driven by the CaMV 35S promoter. The transgenes ch5b and bg2 were successfully delivered into two breeding lines of cotton. Southern blot and slab blot analyses of DNA isolated from T2 progeny demonstrated that the transgenes were stably integrated into the genome of transgenic cotton plants and inherited by the offspring. Expression of the chitinase and beta-1,3-glucanase gene was detected in transgenic T2 cotton plants. Transgenic T 2 and T3 plants examined for resistance to the fungal pathogen Rhizoctonia solani Kuhn in a growth chamber exhibited less root rot and fewer hypocotyl lesions than non-transgenic control plants. Greenhouse screening tests indicated that the ability to resist the fungal pathogen R. solani increased in these plants. Our results suggest that the chitinase and beta-1,3-glucanase genes functioned in cotton and were expressed in fertile plants. The pollen tube pathway transformation strategy may be useful for the control of other fungal diseases of plants.