农杆菌介导美味猕猴桃基因转化影响因素研究

[目的]建立美味猕猴桃叶片外植体转化体系,为猕猴桃遗传转化研究奠定良好基础.[方法]以美味猕猴桃组培苗叶片为材料,研究不同浓度乙酰香酮（AS）对外植体β-葡萄糖苷酸酶基因（GUS）瞬时表达阳性率的影响,及暗培养时间与抗褐化剂二硫苏糖醇（DTT）、聚乙烯呲咯烷酮（PVP）对外植体褐化、抗性芽诱导的影响.[结果]仅在侵染菌液中添加AS时,100、150、200 μmol/L AS 3个处理的外植体GUS阳性率均极显著高于对照,分别为14.6％、16.7％、10.7％;而当农杆菌侵染液与共培养基中同时添加100～150μmol/L AS,可使GUS瞬时表达阳性率提高至37.9％.选择培养初期经过2～12 d暗培养后,外植体褐化率不断下降,抗性芽诱导率不同程度提高,其中以暗培养6、8d处理的褐化率相对较低,分别为33.2％和30.6％,且相应的抗性芽诱导率最高,分别为14.2％和13.1％,极显著高于其他处理与对照.在分化培养基中添加1.0～2.0 g/L的DTT并暗培养6d的效果优于对应浓度的PVP;其中1.5 g/L DTT并暗培养6d,可使外植体褐化率降至13.4％,卡那霉素抗性芽诱导率提高至24.3％.卡那霉素抗性植株叶片GUS染色与PCR扩增结果显示,ShivaA基因已转化至美味猕猴桃秦美.[结论]AS不同使用方式及其浓度对叶片GUS瞬时阳性表达率的提高具有明显影响;选择培养初期进行暗培养6～8 d或暗培养结合添加1.0～2.0 g/L DTT抗褐化剂,均可降低美味猕猴桃叶片转化芽再生过程中的褐化,提高转化效率.

Influencing factors on Agrobacterium-mediated gene transformation of Actinidia delicious cv Qinmei

[Objective]Transformation system of leaves from Actinidia delicious cv Qinmei plantlets was established to provide useful references for kiwi gene transformation. [Method]Young leaves of plantlets were used as explants to investigate effects of different concentrations of AS on transient expression positive rate of GUS and effects of dark culture duration and anti-browning agent of DTF and PVP on explant browning and resistant bud induction. [Result]GUS transient expression rate of explants under 100,150 and 200 μmol/L AS was significantly higher than that of the control, which was 14.6%, 16.7% and 10.7%, respectively. GUS transient positive expression rate increased to 37.9% adding 100-150 μmol/L AS in Agrobacterium infection fluid and co-cultivation medium. After 2-12 d dark culture in initial culture period, explant browning rate continu- ously dropped and resistant bud induction rate improved to some extent. On 6 and 8 d, browning rate was relatively low （33.2% by 30.6%） and resistant bud induction rate was the highest （14.2% and 13.1%）, which was extremely signifi- cantly higher than those of other treatments and the control. The 6 d dark culture in differential medium with 1.0-2.0 g/L DTT yielded better effects than PVP of the same concentration. The 6 d dark culture in 1.5 g/L DTT brought explant browning to 13.4% and increased kanamycin resistant bud induction rate to 24.3%. GUS staining of kanamycin resistant leaf and PCR am- plification showed that Shiva A gene already transformed to Actinidia delicious cv Qinmei. [Conclusion]Different application methods and concentrations of AS significantly improved leaf GUS transient positive expression rate. In initial culture period, 6-8 d dark culture or 1.0-2.0 g/L DTT both reduced browning during bud regeneration and improved conversion efficiency.