以PMI为选择标记基因的雪柑遗传转化体系的优化

Optimization of Genetic Transformation System of 'Xuegan' Based on the PMI Selection System

[目的]以PMI为选择标记基因对雪柑的遗传转化体系进行优化。[方法]对农杆菌的菌液浓度、实生苗培养条件、甘露糖筛选压、2,4-D浓度、2,4-D预处理时间、农杆菌侵染时间、共培养时间进行梯度试验来研究转化后的再生率,并对筛选培养基中6-BA和NAA不同浓度组合进行试验来研究抗性大苗的筛选。[结果]经过暗培养20 d光培养10 d条件的实生苗外植体在农杆菌菌液OD600为0.6,1.0 mg/L 2,4-D预处理3 h,农杆菌侵染30 min,共培养时间4 d,筛选压为甘露糖20 g/L的情况下转化后的再生率大大提高,而在筛选培养基中加入2 mg/L 6-BA+0.5 mg/L NAA有助于抗性大苗的筛选。[结论]通过对影响转化的各种因素的优化试验建立了雪柑以PMI基因/甘露糖选择标记系统的高效遗传转化体系。

[Objective] To optimize the genetic transformation system of Citrus using PMI as a selective marker gene.[Method] Gradient experiments were carried out to study the regeneration frequency of ‘Xuegan’by the concentration of bacterial solution,culture condition of seedlings,screening pressure of mannose,concentration of 2,4-D,pretreatment time of 2,4-D,infection time of Agrobacterium and co-culture time. The selection of resistant plantlets with different concentrations of 6-BA and NAA in screening medium was studied. [Result] The seedling explants cultured under dark culture for 20 days and light culture for 10 days were pretreated with Agrobacterium solution OD600 for 0.6,1.0 mg/L,2,4-D for 3 hours,infected with Agrobacterium for 30 minutes,co-cultured for 4 days,and pressed with mannose for 20 g/L. The regeneration frequency of transformed seedlings was greatly increased,while 2 mg/L 6-BA+0.5 mg/L NAA added to the screening medium was helpful for screening resistant seedlings. [Conclusion] An efficient genetic transformation system of‘Xuegan’with PMI gene/mannose selective marker system was established by optimizing the factors affecting transformation.