| Transgenic sorghum plants containing a codon-modified insecticidal Bacillus thuringiensic (Bt) crylAb gene were obtained through /Igrofeacrerium-mediated transformation, based on optimizations of regenerative and transformation system.The cultural medium was optimized by a statistical method and dissecting effect of single factor on in vitro culture. The optimal medium for direct regeneration of shoot apex was MS salts, B5 vitamins, KT 0.25mg/L, BAP 0.5mg/L, IAA 0.25mg/L, L-Asn 200mg/L, ascorbic acid lOmg/L, PVP (polyvinylpyrrolidone) lOOmg/L, sucrose 30g/L and agar lOg/L; for callus induction of seed (mature embryo) was modified MS salts, MS vitamins, 2,4-D 3mg/L, KT 0.05mg/L, L-Pro 50mg/L, casamino acid 500mg/L, PW 8mg/L, sucrose 30g/L and agar 8g/L; the medium for callus sub-culture contained modified MS salts, MS vitamins, 2,4-D 3mg/L, KT 0.5mg/L, L-Asn 200mg/L, L-Pro 50mg/L, PVP lOOmg/L, ascorbic acid lOmg/L, casamino acid 500mg/L, sucrose 30g/L and agar 8g/L; for callus induction of immature embryo was MS salts, MS vitamins, 2,4-D 2mg/L, KT 0.05mg/L, zeatin 0.05mg/L, L-Pro 700mg/L, L-Asn 150mg/L, casamino acid Ig/L, PW 8mg/L, ascorbic acid lOmg/L, NH4NO3 3.5g/L, KH2PO4 0.5g/L, sucrose 20g/L and agar 8g/L; for callus sub-culture was MS salts, MS vitamins, 2,4-D 2mg/L, zeatin 0.5mg/L, L-Pro 150mg/L, L-Asn 150mg/L, casamino acid 0.5g/L, PVP 8mg/L, ascorbic acid lOmg/L, Ntt^MOs 3.5g/L, KH2P04 0.5g/L, sucrose 20g/L and agar 8g/L; for callus induction of immature inflorescence is YI4 containing MS salts, MS vitamins, 2,4-D 2mg/L, zeatin 2.2mg/L, PVP 8mg/L, ascorbic acid lOmg/L, casamino acid 0.5g/L, sucrose 30g/L and agar 7.2g/L; for sub-culture was YI4 with KT 0.175mg/L.The regenerative capability of different explants and genotypes was comparatively investigated. It showed that the regenerative frequency of immature inflorescence-derived callus was higher than that of other types of explant-derived callus and shoot apex. The average regenerative frequencies of callus induced from immature inflorescence, immature embryo and seed (mature embryo), and shoot apex were 77.2%, 21.7%, 1.0% and 39.4%, respectively. The regenerative capability was different among the genotypes. Immature inflorescence, shoot apex and immature embryo were ideal explants suitable for further transformation study.The genetic transgenic system of sorghum mediated by Agrobacterium and factors were investigated using the transient expression of gus gene and the optimal conditions were determined as follows: for shoot apex, pre-culture 3 days, bacterial concentration ODeoo 0-5, infection time 10 min, and co-culture 3 days; for immature embryo and inflorescence-derived callus, pre-culture 3 days, bacterial concentration ODeoo 0.5 to 0.7, infection time 5 to 20 min, pH value of co-culture medium 5.2 to 5.6, co-culture temperature 22℃ to 25 ℃, and less than 3-day co-culture. To be higher transformation frequency, it was important to add AS to co-culture medium, pre-culture medium of Agrobacterium., and the bacterial cell suspension. To avoid the browning and death of explants after co-culture and improve the transformation frequency, it was necessary to add PVP 500mg/L to co-culture medium.The effects of antibiotics on bacterial growth and in vitro response were comprehensively investigated. Carbenicillin with 250-500mg/L was optional to remove the bacterial cells after co-cultivation in the process of Agrobacterium-mediated transformation. Sensitivity to kanamycin and hygromycin varied among the varieties and explants, and the higher sensitivity to hygromycin was observed than that to kanamycin. The preferable concentration of hygromycin for transformant screening is 25-50mg/L for shoot apex and immature embryo and 50-75mg/L for callus, respectively.The insecticidal Bacillus thuringiensis (Bt) crylAb gene were firstly hi the world inserted into the immature inflorescence-derived callus of sorghum varieties, i.e. 115, ICS21B and 5-27 through Agrobacterium-mediated transformation. After gradient selection with hygromycin, a... |
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