Establishment Of Cotyledonary Nodes Regeneration System Of Soybean And The Transformation Of Agrobacterium-mediated Antimicrobial Peptide Gene

Soybeans are important oil crops and food crops. They are not only the source of the world of cooking oil and vegetable protein, but also the main source of industrial raw material and the important feed for live-stock and domestic bird. Antimicrobial peptide is a kind of high efficient and broad spectrum of active peptides. It has resistance ability against bacteria, fungi, viruses and protozoa. It is foundation of soybean germplasm resources for cultivating high-quality resistance that antimicrobial peptides are transplanted in soy gene.It is prerequisite that establishment of efficient stable soybean regeneration system for soybean genetic transformation. Liao dou 17, 18 and 23 are test materials in this study. They are induced in adventitious bud, and are compared inductivity of adventitious bud as explant with not keeping cotyledon, keeping half of cotyledon and the top of embryo. Fast and effective cotyledons section regeneration system are built. After soybean seed is disinfected by chlorine fumigation, vaccination on germination medium and training 5 days. Explants are inducted to adventitious bud in induction media (1/2MSB + 4.0 mg/L 6 - BA + 0.05 mg/L IBA + 3% sucrose + 0.7% AGAR). When adventitious bud grows out, it will be transfered into adventitious bud induction media for adventitious bud induction media(1/2MSB + 0.5 mg/L 6 - BA + 0.05 mg/L IBA + 3% sucrose + 0.7% AGAR). When adventitious bud grows into 2 ~ 3cm, it will be transfered into adventitious roots induction media(1/2MSB + 1.0 mg/L IBA + 2% sucrose + 0.7% AGAR) . When the root grow into 3 ~ 5cm, it will be transfered for training to become mature plants in nutritional soil, sphagnum and vermiculite (1:2:2).Liao Dou 17 are test materials, and antibacterial peptide genes are induced in soybean genome by agrobacterium-mediated technique. After analysis optimal degerming concentration of cephalosporins is 300mg/L; Screening concentration of kanamycin is 60 mg/L; Secondary screening without kanamycin is during adventitious roots growing. After PCR detection and green fluorescent protein detection, antibacterial peptides gene has been proved into Liao Dou 17 plants preliminarily and obtained expression, conversion rate is 10.3%. 2 T1 plants are acquired, and one plant performance is positive in PCR analysis result.