Abstract
Problem statement: The present investigation described a simple and reproducible protocol for transgenic cotton regeneration and characterization of chitinase (Chi II) gene expression against two different fungal pathogens in cotton. Approach: Transgenic cotton (Gossypium hirsutum cv. SVPR2) plants were produced by pCambia-bar-Chi II (13.8 kb) under the control of the CaMV 35S promoter, harbored in the strain LBA 4404 Agrobacterium tumefaciens by using shoot tip explants. Results: Finally, from the 10 experiments, 21.8% of transformation frequency was recorded. Segregation ratio of 3:1 was recorded in the T0 plant seeds. Polymerase chain reaction and southern blotting analysis were used to confirm the integration of Chi II transgene in the T0 plants genome of putative transgenics. Quantitiave and qualitative (SDS-PAGE) analyses were also carried out to confirm the expression of chitinase enzyme in T0 plants. Further, randomly selected transgenic plants (T0) were analyzed for disease tolerance by evaluating them with spores of Fusarium oxysporum and Alternaria macrospora. All the selected PCR positive plants showed enhanced disease resistance against Fusarium wilt. The plants selected randomly showed an enhanced survival rate compared with the control when they were grown in earthen pots inoculated with 1×105 spores 100-1 g of soil mixture. Another four randomly selected plantlets were sprayed with spores of Alternaria macrospora in order to test their tolerance to Alternaria leaf spot disease. After 20 days of culture, the number of lesions per leaf and the lesion length per leaf spot in non-transferred leaves increased. In the case of transgenic plantlets, lesion formation was completely absent. Conclusion: The disease resistance against Fusarium wilt and Alternaria leaf spot in cotton strains would serve as good breeding materials for producing fungal disease resistant cotton varieties.
Highlights
Cotton (Gossypium hirsutum L.) has been estimated that 180 million people depend on cotton fiber production
The data were analyzed for variance by Quantitative analysis of chitinase was determined by Duncan's Multiple Range Test (DMRT) using the SAS the leaf specific method[22]
Chi-1, SVPR2-Chi-15, SVPR2-Chi-31, SVPR2-Chi-45, bioassay of the T0 PCR and Southern-positive plants with Fusarium oxysporum and Alternaria macrospora showed that the infection level was significantly controlled in transgenic progenies having a higher expression of PR-proteins than in the non-transgenic control plants, indicating enhanced resistance to Fusarium wilt and Alternaria leaf spot diseases
Summary
Cotton (Gossypium hirsutum L.) has been estimated that 180 million people depend on cotton fiber production. Agrobacteriummediated transformation via somatic embryogenesis has been the most common method for transgenic cotton development It is a multi-step process involving laborintensive work over a 10-12 month period starting from co-cultivation of Agrobacterium culture with explants followed by production and maintenance of hundreds of calli derived from independent transformation events, induction of somatic embryos and development of somatic embryos into normal plantlets[10]. In this procedure, the transformation efficiencies are generally low due to the low frequency of embryogenesis and the difficulty in germination of transformed embryos[11]. In the present investigation, we targeted to produce the cotton plantlets with chitinase gene through Agrobacteriummediated transformation protocol by using shoot tip culture technique
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