Studies On In Vitro Regeneration And Genetic Transformation In Pepper (Capsicum Annuum L.)

Transgenic technology has been widely used in modern genetics and breeding, which has madegreat achivements. Some GMO crops have been cultured commercially in a wide range, such ascotton, maize, soybean and so on. Capsicum annuum L. is a recalcitrant species for in vitroregeneration, it’s hard to induce normal regenerate buds, and the buds are difficult to elongate, inaddition, the transgenic rate is low. These circumstances combinedly hindered the application oftransgenic technology in the field of pepper genetic modification.In order to improve the efficiency of pepper regeneration and lay foundation for the applicationof transgenic technology with marker-free T-DNA in pepper, studies on in vitro regeneration andgenetic transformation have been carried out. The major contents and results in this study are listed asbelow:1. Three sweet pepper inbred lines （0510,0516,77013） and two hot pepper inbred lines （0818,0819） were used as material, and the cotyledons and different types of hypocotyls were used asexplant for regeneration. The effect of6-BA, IAA and AgNO3on regeneration were also studied.It showed that: the half-seeds consisting of the radicles and part of the hypocotyls cultured onMS medium without plant growth regulators （PGRs） gave the best response to in vitro regeneration,and the regenerate rate was17.33%; as to genotype, the regenerate rates of two hot inbred lines were10.00%and8.19%respectively, significantly higher than those of sweet ones; differentconcentrations of6-BA, IAA and AgNO3in the medium were tried for cotyledon regeneration, and5mg·L^-1,0.8mg·L^-1and5mg·L^-1respectively gave the highest regenerate rate1.25%.2. Half-seeds consisting of the radicles and part of the hypocotyls from two hot pepper inbredlines （0818,0819） were used as explant and infected by Agrobacterium tumefaciens harboringmarker-free T-DNA plasmid for genetic transformation. The effect of Agrobacterium tumefacienstype, infect time and acetosyringone （AS） on genetic transformation of pepper were studied.It showed that: the regenerate rate infected by C58was2.57%, higher than those of EHA105and Agl-0（1.67%and0）; the regenerate rate infected for10min was5.71%, higher than those of15min and20min （2.39%and0.76%）; the regenerate rate with200μmol·L^-1AS was5.71%,significantly higher than that of without AS.The optimized transformation system based on regeneration of pepper is as follows: half-seedsconsisting of the radicles and part of the hypocotyls from two pepper inbred lines （0818and0819）are used as explant, and infected by Agrobacterium tumefaciens of C58harboring marker-freeT-DNA plasmid for10min, and200μmol·L^-1AS added to the medium during infection, the explantare cultured on MS medium without PGRs.