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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 calli were cultured in 1/2 of MS medium supplemented with different concentrations of TDZ using tissue culture method. The regenerated plants were obtained. The regeneration rate of 92.5% adventitious buds was obtained on the medium supplemented with 0.03 mg · L-1TDZ, and the regenerated shoots developed well. The result of selecting antibiotic screening test showed that callus of Toxoplasma gondii was sensitive to hygromycin, and the addition of hygromycin at the concentration of 5 ~ 35 mg · L-1 in the culture medium had a great effect on callus differentiation, When the concentration of hygromycin was 5 mg · L-1, all the explants were browned and died within 4 weeks. When the concentration of kanamycin was 0-100 mg · L-1, kanamycin The differentiation of callus had a certain degree of effect. When the concentration of kanamycin was 80 mg · L-1, the callus basically did not differentiate. Thus, kanamycin was selected as the suitable selection antibiotic for the transformation of Melastoma. The optimum selection pressure was 80 mg · L -1. The results of antibacterial antibiotic screening showed that both 200 mg · L-1 cefotaxime and 200 mg · L-1 carbenicillin could effectively inhibit the growth of Agrobacterium tumefaciens strain LBA4404, Bud differentiation has little effect, can be identified as suitable antibacterial antibiotics. Agrobacterium-mediated transformation of Jincang Melastomataceae calli callus genetic transformation obtained kanamycin-resistant plants 164, and initially identified pre-culture 1d, diluted 10 times the bacteria, infection 4min, co-culture for 2d Jinshan Spiraea optimal genetic transformation system for Jinshan Melaleaga laid the foundation for genetic engineering breeding.