地被菊间接体细胞胚发生途径的转基因受体体系的研究
Studies on Transgen ic Acceptor System of Ground-CoverChrysanthemum Via Indirect Soma tic Embryogenesis
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摘要: 采用三步培养法建立了地被菊花品种‘玉人面’间接体细胞胚发生途径的转基因受体体系。试验以地被菊花品种‘玉人面’茎段(节间)为外植体,研究了生长调节剂、光照强度等因子对其间接体细胞胚诱导和分化的影响,同时进行了抗生素敏感性试验。结果表明:胚性愈伤组织诱导培养基为MS + KT 2. 0 mg·L-1 + 2, 4-D 2. 0 mg·L-1 +NAA 0. 5 mg·L-1 ,诱导15 d后,将获得的黄绿色致密颗粒状胚性愈伤组织转入胚性愈伤组织分化培养基MS + KT2. 0 mg·L-1 + 2, 4-D 1. 0 mg·L-1 +NAA 0. 5 mg·L-1中,诱导15 d后再转入MS + KT 2. 0 mg·L-1 +NAA 0. 5 mg·L-1的分化培养基中培养20 d,光照强度为1 000～2 000 lx,胚性愈伤组织诱导率、分化率分别为95. 3%、92. 7% ,平均每个外植体分化的芽数为17. 8个,获得的再生芽的稳定性为99. 5%。抗生素敏感性试验表明:地被菊花品种‘玉人面’间接体细胞胚发生途径的转基因受体体系中,卡那霉素选择压为20 mg·L-1 ;头孢霉素在胚性愈伤组织诱导时的选择压为300 mg·L-1 ,在胚性愈伤组织分化培养时选择压为100 mg·L-1。Abstract: In this paper transgenic acceptor systems of chrysanthemum cv.‘Yurenmian’via indirect somatic embryogenesiswere established by three steps cultivation. The authors studied the effects of hormones, illumination intensity on indirect somatic embryogenesis through stem segments ( internodal segments) as explants. Results showed that: embryogeniccalli induction medium wasMS +KT 2. 0 mg· L-1 + 2, 4-D 2. 0 mg·L-1 +NAA 0. 5 mg·L-1 , 15 days later, yellowgreenish, compact nodular calliwere transferred embryogenic calli differentiationmediumMS plus KT 2. 0mg·L-1 , 2, 42D 1. 0 mg·L-1 and NAA 0. 5mg·L-1 for 15 days, andwere transferred differentiationmediumMS plus KT 2. 0mg·L-1and NAA 0. 5 mg·L-1 for 20 days. Illumination intensity was 1 000～2 000 lx. The highest rate of embryogenic callireached 95. 3%, the highest rate of embryogenic calli differentiation reached 92. 7%, average number of shoots per stemsegment explantwas 17. 8, and stability of regeneration shoots reached 99. 5%. Experiments of the sensitivity of antibiotics for transgenic accep tor systems of chrysanthemum cv.‘Yurenmian’via indirect somatic embryogenesis showed: the selection concentrations of kanamycin was 10 mg·L-1 , the selection concentrations of cefotaxime was 300 mg·L-1while embryogenic calliwere induced and 100 mg·L-1 while embryogenic calli differentiation.
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