Studies On Transformation Of CpTI And Bar Gene Into Peanut(Arachis Hypogaea L.cv.Shanyou 523) By Agrobacterium-mediated Genetic Transformation

The cowpea trypsin inhibitor (CpTI) gene and the bar gene discovered in recent years are found to be good used for insect-resistant and anti -herbicide respectively because of their broad spectrum and are used in gene improvement of many crops widely now. This report did a lot of research on transformation of CpTI and bar gene into peanut (Arachis hypogaea L. cv. Shanyou 523) by agrobacterium-mediated genetic transformation comprehensively, which provided a foundation for modern breeding technology applied in insect-resistance and anti-herbicide research ofpeanut.Factors influencing shoot induction from peanut explants, including explanttype, plant hormones, media components and transplantation method etc., were examined. A highly effective regeneration system from leaflets of peanut in vitro was developed and achieved regeneration plants successfully. The result showed: Middle sections of 9-10d germinated leaflets were the best materials from which the shoot formation rates were highest in shorter time. The plant hormones used were varied in different period of shoot formation. At the early shoot induction stage, lower concentrations of NAA were best used with BA at slightly higher levels, the highest shoot induction (79.1%) was attained in BA at 3mg/L and NAA at 0.8mg/L; 4 weeks later, the buds were subcultured in the secondary medium by just adding 3mg/L BA. At this time, we found B A alone was more favorable in promoting shoot elongation. And then adding NAA at low concentration in MS was used to give rise to roots. We also found that lower level of hormone could control effectively browninng and vitrifaction during the culture and G1 n (6mg/L) and AgNOj (2mg/L) supplemented in shoot induction media could improve the shoot information rates apparently (about90%) .The whole period of plant regeneration from leaflets of peanut could be divided as five steps: germination - shoot induction -shoot elongation-rooting- tranplant. Seeds were germinated on half-strength MS. Then we used MS supplemented with 3mg/L BA+ 0.8mg/L NAA+2mg/L AgN03+6mg/LGin for shoot induction of peanut (Arachis hypogaea L. cv. Shanyou 523) under 2500 Lux at 27 + 1℃. Buds were effectively induced 12-14 days later from middle sections of 9-10d sterile peanut leaflets. After 4 weeks, we transferred the multiple buds to secondary medium (MS + 3mg/L BA+2mg/L AgNOj) to promote their further growth. The induction rate reached 90.2%, with 9 multiple shoots from each explant. When grown to 3-4cm, the shoots were transcultured for rooting (MS 4-0.8mg/L NAA+2mg/L AgNO3). Once the roots occurred, the plantlets were planted in a mixture of vermiculite: sand (1:1) for further growth, the survive rate arriving 98%.However, factors which would effect the agrobacterium-mediated genetic transformation from the leaflets of peanut, such as the selective stress, the concentration of the agrobacterium, the time of infection and coculture were also studied. After precultured for 3d, leaflets were rinsed in the agrobacterium liquid( OD600=0.3) for 5 mins, and then were conculture with agrobacterium on shoot induction media. 2d later, we washed the explants 3 times with sterile water adding 500mg/L Cb and transferred them to MS containing 3mg/L BA+0.8mg/L NAA+2mg/L AgNO3+6mg/L Gln+SOOmg/L Cb to restrain the excess growth of the agrobacterium. And if the explants were cultured on media adding 0.25mg/L PPT as select stress, the percentage of buds was below 30% and their development was almost hold back and turned black gradually till died. While above 80% buds could be induced normally from the leaflets of peanut without PPT, the shoot induction frequency of leaflets arriving 30%. 20 regeneration shoots were made PCR analysis, the result proved that 8 tested material showed 31 Obp DNA segment, which were thought to be CpTI gene and its transformation rate was 40.0%.