影响农杆菌介导旱稻转化效率主要因素的研究

Optimization of the Agrobacterium-Mediated Transformation System on Upland Rice

针对旱稻在基因工程中转化效率低这一难题,以4个旱稻品种为对象,对农杆菌介导的遗传转化的各个步骤进行了优化试验,结果表明NB(N6 macro elements;B5 microelements and vitamins;proline 500mg L-1,L-Glutamine 500mg L-1,casamino acid 300mg L-1,sucrose 30 gL-1,2,4-D2mg L-1,agar8g L-1,pH5.8)培养基适合于旱稻愈伤组织诱导;AAM-AS(Na2HPO4.2H2O339.2mg L-1;KCl2.95g L-1;MgSO4·7H2O500mg L-1;CaCl2114.4mg L-1;MnSO4·4H2O10mg L-1;H3BO33mg L-1;ZnSO4·7H2O2mg L-1;KI0.75mg L-1;NaMO40.25mg L-1;CuSO4.5H2O0.0387mg L-1;CoCl2.6H2O0.025mg L-1;VB10.5mg L-1;VB60.5mg L-1;烟酸0.5mg L-1;肌醇100mg L-1;甘氨酸7.5mg L-1;精氨酸174mg L-1;谷氨酰铵876mg L-1;casamino acid 500mg L-1;蔗糖68.5g L-1;葡萄糖35g L-1;AS200μmol L-1pH5.2)是农杆菌的最佳重悬液;对NPT(new plant type,NPT)这种侵染后易水渍化品种,滤纸法共培养可将其抗性愈伤发生率提高15倍以上;筛选及分化前对抗性愈伤组织进行2d的干燥培养可显著加快愈伤组织分化进程;在分化培养基中加入AgNO3有助于提高分化率;而短期高浓度的CuSO4处理可显著提高分化率。利用此体系获得了一大批PCR鉴定阳性的转基因植株;Southern blot检测证明外源基因大多数以单拷贝形式整合在转基因植株中。

Agrobacterium-mediated transformation has been used very successfixlly in some monocot plant such as rice （Oryza sativa L. ） and maize （Zea mays L.）, by which the transgenic plants obtain stronger resistance or better characters. But the efficiency of Agrobacter/um-mediated transformation system is still not so high as our expectation. Upland rice is a variety of rice （ Oryza sativa L. ）, which has many good characters, such as drought resistance, and tolerance to sterile soil. Upland rice has been praised as one of the new source of food supplies in the 21st century in China. However, studies on the Agrobacterium mediated transformation system of upland rice are less compared with these of rice, and many problems that affected the transformation efficiency should be solved. Therefore, setting up an effective transformation system of upland rice is still a challenge to investigators. Several important factors affecting upland rice transformation mediated by Agrobacterium were investigated with four upland rice cultivars. In this paper callus induced from mature embryos of upland rice seeds were used mediated as explants to study the factors affecting the transformation rate. Callus were collected in a sterile trigonal bottle, Agrobacter/um suspension was then added to the bottle, and vortexed for 30 s, followed by a 20 rain of soaking in the bacteria solution. 3he callus were then transfered on co-cultivation medium（C-C1 or C-C2） in Petri plates and cultured in the dark for three days. After washed with sterile water, the explants were then put onto SX medium containing hygromycin for selection. After 6-8 weeks, surviving callus were transferred to differentiation medium. 3hen, the induced seedlings in 4-5 centimeters long were transferred onto root-inducing medium. Finally, they were transplanted into soil when the roots had formed, and identified by PCR and Southern blot. The transformation rate was affected by five factors in this process. NB medium was suitable for callus induction and subculture. Re-suspending the strain by AAM-AS medium before transformation could improve the resistant callus rate significantly. After infected by the strain, the callus were put on the two layers of sterile filter papers （d = 12 cm） absorbed 4 mL C-C2 medium for co-cultivation, instead of being on agar medium, which could improve the resistant callus rate of NPTfmm 2% to 31.7%. Before selection and regeneration, desiccation for two days on sterilized filter papers could increase the resistant callus rate. Adding 10 mg L^- 1 igiO3 or 8 mg L^-1 CLISO4 into the differentiation medium could improve the regeneration rote, especially for the callus subeulturd for two months or longer, many putative transgenic plants identified by PCR were acquired by using this transformation procedure, and Southern blot analysis showed the target gene integrated in most transgenic plants just by single copy.