Genetic Transformation Of Cotton With Rice Chitinase Gene Through Shoot Tip Culture Confers Resistance To Two Fungal Pathogens

摘要

The present investigation was aimed to standardize the simple and reproducible protocol for transgenic cotton (Gossypium hirsutum L. cv. SYPR 2) regeneration through shoot tip culture by using rice chitinase gene. Transgenic cotton plants were transformed by the Construct pCambia-bar-Chi Ⅱ (13.8 kb) harbored in strain LBA 4404 Agrobacterium tumefaciens infection. T-DNA containing a rice chitinase gene under the control of the CaMV 35 S promoter and the bar gene under control of the ubiquitin promoter as a selectable marker was used. Multiple shoot induction medium containing 5 mg/L Phosphinothricin (PPT) was identified as suitable selection agent for the isolation of transgenic shoots. Finally, from the total of 10 experiments conducted, 12 plantlets were harvested in each experiment with 30.8 transformation frequency. These plantlets were considered as putative transgenic plants. The morphological features of the transgenic plants did not differ from those of non-transgenic plants. Polymerase chain reaction was used to confirm the integration of Chi Ⅱ and npt Ⅱ transgenes in the T0 plants genome. Integration of Chi gene into the genome of putative transgenic was confirmed by southern blot analysis. Further, randomly selected transgenic plants were analyzed for disease tolerance by challenging them with spores of Fusarium oxysporum and Alternaria macrospora. Nine plants were selected as Fusarium oxysporum tolerant from a total of 10 plants tested. The selected plants showed an enhanced survival rate compared with the control when they were grown in earthen pots inoculated with 1×105 spores/100 g of soil mixture of Fusarium oxysporum. Another four randomly selected plantlets were sprayed with spores of Altemaria macrospora in order to test their tolerance to Altemaria leaf spot disease. After 20 days of culture, the number of lesions per leaf significantly increased from 8.5 to 34.9 in the non-transformed plantlets and the lesion length was also increased from 2.2 mm to 5.8 mm per leaf spot in these plants. In the case of transgenic plantlets, lesion formation was completely absent. Evan after 20 days of maintenance, no lesions was recorded. The disease resistance against Fusarium wilt and Altemaria leaf spot in cotton strains would serve as good breeding materials for fungal disease resistance cotton production.