拟青山海关杨高效遗传转化系统的建立

Establishment of A High Efficient System for Genetic Transformation of Populus pseudo-cathayana ×P. deltodides Barry CV. ‘Shan Hai Guan’

为建立拟青山海关杨的再生和高效遗传转化系统,以该杨树叶片为外植体,研究了适合叶片再生的最适培养基;并利用GUS瞬时表达法,研究了菌种、预培养时间、侵染菌液浓度、侵染时间和乙酰丁香酮(AS)浓度5个因素对拟青山海关杨叶片遗传转化的影响。结果表明,拟青山海关杨叶片最适不定芽诱导培养基为MS添加0.5mg/L的6-BA和0.1mg/L的NAA,叶片再生频率为95%,平均不定芽数为10.07个;该杨树的高效遗传转化体系为:叶片无预培养,菌种使用GV3101,侵染液OD600为0.4～0.6,侵染10min,共培养培养基中添加乙酰丁香酮200μmol/L,在此条件下,GUS荧光定量分析结果显示叶片的GUS活性达到最大值。

In order to establish the system of regeneration and genetic transformation for Populus pseudocathayana ×P. deltodides Barry CV. ‘Shah Hal Guan＇, the optimal medium for the leaf-explant regeneration in vitro was studied; furthermore, the five factors related to genetic transformation were evaluated via GUS transient expression, including Agrobacterium strains, pre-cuhure time, the concentration of Agrobacterium, inoculation time and the concentration of acetosyringone （AS）. The results indicated that the optimal medium for adventitious bud regeneration was MS medium supplemented with 0.5mg/L 6-BA and 0.1mg/L NAA, on which the rate of regeneration reached 95% and the number of average adventitious buds were 10.07; a high efficient system for genetic transformation of Populus pseudo-cathayana ×P. deltodides Barry CV. ‘Shah Hai Guan＇ were： no pre-cuhure for leaf-explants, GV3101 strain, the bacterial concentration at OD600=0.4- 0.6,10 min for inoculation and supplemented of 200μmol/L AS in co-culture medium; Under this condition , the transient GUS expression reached to its maximum by fluorescence quantification method.