Agrobacterium Mediated Transformation Of Bt Gene And Bar Gene On Upland Cotton (Gossypium Hirsutum L.)

Upland cotton (Gossypium hirsutum L.) is one of the most important economic crops, and one of the most phenomenal crops damaged by pests in China. Traditional breeding solutions for insect-resistant cotton are time-consuming and inefficient. Bt genes have been proved enjoying prowess against pests. By using transgenic technology, Bt genes transferred and expressed in cotton will enhance the resistance capacity against pests noticeably, which will result in the sharp decrease of the dose and practice frequency of pesticide in cotton field and then make huge economic achievements. Bar gene, a gene which detoxifies the ingredient of the herbicide Bialaphos PPT, serves as a mark gene in screening procedure for transformants, and also plays a role in new material creation for Bialaphos-resistant GM crops as a herbicide-resistant gene. Co-transformation of both Bt gene and Bar gene could bring us the cotton carrying resistance against insect and herbicide.By the way of Agrobacterium tumefaciens-mediated embryogenic callus transformation, Bar gene and each of the three novel Bt genes(CryIC, CryIIA, Cry9C) were transformed separately, into four upland cotton cultivars, EK10, E22, C201,YZ1. And the result is as follows:1. In this study, several key parameters have been modified and the transformation procedure is optimized. The optimal concentration of agrobacterium solution for transformation is 0.1OD; the existence of acetosyringone improves the efficiency of transformation, because adding 50mg/L As to the solid co-culture medium or liquid MGL medium demonstrates stronger performance; the optimal temperature for co-culture is 19℃; transformation by using embryogenic callus subcultured over extended period as material decreases the differentiation ability of somatic embryos, and the expression patterns differ according to source of embryogenic callus and plasmid construction.2. A highly efficient and stable system for selection and regeneration is established, and the period required is largely decreased. The optimal PPT concentrations are 20mg/L for Ek10, 5mg/L for E22, 10mg/L for C201, 10mg/L for YZ1, separately. The positive transformation frequency confirmed by PCR amplification varies from 60% to 100%, and the average value is 82.81% which prove the transformation plan based on Bar as selectable mark is effective. The insertion of DNA fragment is also confirmed by Southern hybridization.3. The antagonism against PPT for amino acids containing amide is observed during the experiments. For the antagonism against PPT, Asn and Gln present aratio of 1:1 approximately in function, which possibly shows that Asn possibly participates into the nitrogen cycle through the approach of Asn synthetase metabolism or other physiological routes, and takes the role of Gin to some extent. Regenerated plantlet, hypocotyl segment, cotyledon are more sensible than embryogenic callus, which maybe indicate that GS2, the chief form of GS in chloroplast, is more sensible for PPT than GS1, the dominating form of GS existing in callus. Additive lg/L Gin into the solid medium supporting the embryogenic callus of E22 increases the optimal screening concentration of PPT hugely, from 5mg/L to 50mg/L, which demonstrates that ratio of Gin versus PPT is approximately 1:22.22 in the combining competition with GS.4. Tests of pest feeding and herbicide toxicity were fulfilled. Some of the transgenic lines display outstanding potential in the characteristics of insect-resistance and herbicide-resistance.5. Graft is a favorable choice in solving the problem that regenerated cotton plantlet is hard to generate roots. Two graft skills (in-vivo grafting & in-vitro grafting) possess their own advantages and disadvantages, and in-vivo grafting is proved helpful in rooting initiation before transferring. The survivor rate for transferring will be increased obviously after acclimation.