金山绣线菊遗传转化体系的建立

Establishment of Genetic Transformation System for Spiraea×bumalda 'Golden Mound'

以金山绣线菊愈伤组织为受体材料,采用组织培养的方法,在附加不同浓度TDZ的1/2MS培养基上诱导培养,获得再生植株。在附加0.03mg·L-1TDZ的培养基上获得了92.5%不定芽的再生率,且再生芽发育良好。选择抗生素筛选试验的结果表明:金山绣线菊愈伤组织对潮霉素较为敏感,在培养基中添加浓度为5～35mg·L-1的潮霉素均对愈伤组织分化影响较大,潮霉素浓度为5mg·L-1时,4周时间可使外植体全部褐化死亡;在培养基中添加0～100mg·L-1的卡那霉素,不同浓度卡那霉素均对愈伤组织分化产生一定程度的影响,当卡那霉素浓度为80mg·L-1时,愈伤组织基本不发生分化。由此确定卡那霉素为绣线菊遗传转化中适用的选择抗生素,最适选择压为80mg·L-1。抑菌抗生素的筛选试验结果表明:200mg·L-1的头孢霉素和200mg·L-1的羧苄霉素都能有效抑制农杆菌菌株LBA4404的生长,却对金山绣线菊愈伤组织的芽分化影响不大,可确定为适宜的抑菌抗生素。利用农杆菌介导法对金山绣线菊愈伤组织进行遗传转化,得到卡那霉素抗性植株164株,并初步确定预培养1d、菌液稀释10倍、侵染4min、共培养2d为金山绣线菊最优遗传转化体系,为金山绣线菊的基因工程育种奠定基础。

The shoots were induced using the callus that regenerated from stem explants of the cultivar, Spiraea xbumalda ‘Golden Mound＇, the regeneration rate reached 92.5% after further regeneration culture on 1/2MS medium containing 0.03 mg.L^-1TDZ, and the regenerative plants grew well. Selective antibiotic screening test results indicated that the callus of Spiraea×bumalda ＇Golden Mound＇ were more sensitive to hygromycin than kanamycin, the callus were badly influenced on the medium supplemented with 5-35 mg.L^-1 hygromycin. When the concentration of hygromycin was 5 mg.L^-1, all the explants died in 4 weeks. Adding 0-100 mg.L^-1 kanamycin in the medium could influence the differentiation of the callus in some degree, and there was little bud differentiation occurred on the media adding with kanamycin at 80 mg.L^-1. Therefore the optimal antibiotic was kanamycin with an optimum concentration of 80 mg L^-1. Bacteriostatic antibiotic screening test results showed that cefotaxime and carbenicillin with a concentration of 200 mg L^-1 could inhibit the development of the Agrobacterium strain LBA4404 and affected the bud differentiation slightly. Cefotaxime and carbenicillin were considered to be the appropriate antimicrobial antibiotic. 164 transformants were obtained from the callus of Spiraea×bumalda ‘Golden Mound＇ after kanamycin resistance assays by Agrobacterium-mediated method. The optimal genetic transformation was： the time of precondition were 3 days, the dilution multiple were 10 times, the time of infection was 4 min, and the time of co-cultivation were 2 days. This study could provide a basis for gene engineering of Spiraea × bumalda ‘Golden Mound＇.