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以红叶石楠(Photinia fraseri)的胚性愈伤组织为受体材料,利用基因枪法导入外源抗寒基因rd29A,以期建立基因枪转化红叶石楠的技术体系。结果表明,基因枪转化红叶石楠的最佳组合条件为:胚性愈伤组织在诱导培养基MS+6-BA 2.0 mg·L-1+NAA 5.0 mg·L-1+2,4-D 0.5 mg·L-1+琼脂5.0g·L-1+蔗糖20 g·L-1里,用0.3 mol·L-1甘露醇处理15~16 h;基因枪轰击时添加12.5 mol·L-1氯化钙50μL+0.1 mol·L-1亚精胺50μL,每枪含金粉300μg+质粒DNA3.0μg,轰击距离9 cm,轰击压力1 100 psi,轰击次数1次。经多次筛选获得了6株转基因植株,转化植株经PCR检测和Southern分析,证实了rd29A已经整合到红叶石楠基因组中。
The embryogenic callus of Photinia fraseri was used as the receptor material, and the foreign gene rd29A was introduced by gene gun, in order to establish the technical system of gene gun transformation of Photinia fraseri. The results showed that the optimal conditions for the gene gun transformation of Photinia fraseri were as follows: embryogenic callus was cultured in the medium of MS + 6-BA 2.0 mg · L-1 + NAA 5.0 mg · L-1 + 2,4-D 0.5 mg · L-1 + agar 5.0g · L-1 + sucrose 20 g · L-1, treated with 0.3 mol·L-1 mannitol for 15-16 h, and added 12.5 mol·L-1 chlorine 50 μL of calcium chloride and 0.1 μL of 0.1 mol·L-1 spermidine, 300 μg of gold powder per gun, 3.0 μg of plasmid DNA, a bombardment distance of 9 cm, a bombardment pressure of 1,100 psi and a bombardment frequency of 1 time. Six transgenic plants were obtained after multiple screening. The transformed plants were confirmed by PCR and Southern analysis, which confirmed that rd29A was integrated into the genome of Photinia fraseri.