影响大豆遗传转化主要因素及相关条件优化

Optimization of main factors and relevant conditions affecting soybean Agrobacterium-mediated transformation system

以浙春3号,华春6号,0428三个大豆基因型的胚尖、子叶节和下胚轴为受体材料,运用农杆菌介导法转化GUS基因,通过对GUS基因瞬时表达率的比较,探讨大豆遗传转化的主要影响因素(基因型、外植体等),并进一步对遗传转化条件进行优化。结果表明:以胚尖为受体时,3个基因型的GUS基因瞬时表达率都很低,在丛生芽的再生部位没有瞬时表达。以下胚轴为受体时,萌发5 d,共培养3 d后,浙春3号、华春6号、0428在丛生芽再生部位的GUS瞬时表达率达到最高,分别为43.92%,54.45%,58.47%。而以子叶节为受体时,按照常规方法(萌发5 d),浙春3号、华春6号、0428分别在共培养5,4,4 d时,其丛生芽再生部位的GUS瞬时表达率最高,分别为23.05%,13.79%,4.78%;利用浙春3号对子叶节法的萌发时间进行了进一步的优化,发现萌发时间为3 d时在丛生芽再生部位的GUS瞬时表达率、芽诱导率较萌发5 d都有较大幅度的提高。

In this study, we optimized main factors （genotypes, explants, etc） affecting soybean Agrobacterium-me-diated transformation system by comparing GUS transient expression rate with embryonic tip , cotyledonary node and hypocotyl of Zhechun No .3, Huachun No.6 and 0428.The results showed GUS transient expression rate of the three soybean genotypes was low in the embryonic tip transformation system and tufty shoots showed no expression .Using the hypocotyl transformation system , the maximum expression rate of Zhechun No .3, Huachun No.6 and 0428 in tufty shoots was 43.92%, 54.45%, and 58.47%, respectively, after 5 days of germination and 3 co-culture days. Using the cotyledonary node transformation system （ 5-day germination ） , the optimal co-culture time for Zhechun No.3, Huachun No.6 and 0428 were 5, 4, and 4 days respectively and the maximum expression rate of Zhechun No.3, Huachun No.6 and 0428 in tufty shoots was 23.05%, 13.79%, and 4.78%, respectively.The germina-tion time of cotyledonary node transformation system was further optimized using Zhechun No .3.The results showed that both gene expression and shoot induction increased dramatically in 3 germination days compared to 5 germination days.