玉米自交系愈伤组织诱导及植株再生体系的建立

摘要

玉米的组织培养比较困难,其再生体系还不完善,建立骨干自交系稳定、高效的遗传转化体系是玉米转基因技术的必要前提。以玉米常用自交系齐319、478、502、黄C和7922不同大小（1.0、1.5和2.0 mm）的幼胚为外植体,探讨不同基因型、胚大小和植物生长调节剂浓度对幼胚再生的影响,从而建立玉米高频再生体系。结果表明：以1.5 mm大小的齐319幼胚为外植体,愈伤诱导率最高,达到了86.2%;1.5 mg/L的24-D有利于胚性愈伤的形成和胚性的保持;两步法分化培养可以提高愈伤组织分化、再生成苗。确定了玉米自交系齐319较适合的培养程序为：将1.5 mm大小的幼胚盾片向上接于诱导培养基（N6＋24-D 1.5 mg/L＋L-脯氨酸0.7 g/L＋蔗糖30 g/L＋琼脂粉8 g/L＋硝酸银5μmol/L）,在25℃下暗培养20 d;挑选胚性愈伤组织在温度25℃、暗培养条件下进行继代培养,继代培养基与诱导培养基相同,每14 d继代1次;将愈伤组织继代42 d后转移至分化培养基1（MS＋肌醇100 mg/L＋蔗糖60 g/L＋gelrite 3 g/L）,继续暗培养10 d左右,待愈伤组织形成象牙白色的块状物且有绿点出现时,将其转移到分化培养基2（MS＋肌醇100 mg/L＋蔗糖30 g/L＋gelrite3 g/L）进行光培养,2～3 d即可分化出苗;待幼苗长到4～5 cm高时,移至生根培养基（1/2 MS＋IBA0.8 mg/L＋蔗糖30 g/L）,14～21 d幼苗长出大量的根系,形成完整的植株。%Tissue culture of maize is relatively difficult,and establishing a stable and efficient transformation system is the premise of maize transgene.Using elite inbred lines（Qi 319,478,502,Huang C and 7922）as explants,the effects of different genotypes,embryo sizes and plant growth regulator concentrations on callus induction and regeneration was studied.The results showed that the frequency of callus induction of 1.5 mm Qi 319 embryos was highest,which reached 86.2%;2,4-D 1.5 mg/L solution was favorable to callus induction and maintaining the growth of callus;differentiation in two steps could improve plant regeneration.The proper procedure of Qi 319 tissue culture was determined：putting the immature embryos scutella side up on the induction medium（N6＋2,4-D 1.5 mg/L＋L-proline 0.7 g/L＋sucrose 30 g/L＋agar powder 8 g/L＋AgNO3 5 μmol/L）;incubating at 25 ℃（dark） for 20 d;Selecting the callus to subculture medium（same with induction medium）,once for every 14 d;42 d later,transferring all callus to differentiation medium 1（MS＋ inositol 100 mg/L＋sucrose 60 g/L＋gelrite 3 g/L） and incubating for about 10 d（dark）,till some green spots appeared on the callus;transferring all callus to differentiation medium 2（MS＋inositol 100 mg/L＋sucrose 30 g/L＋gelrite 3 g/L） in light for germination,and somatic embryos would sprout leaves within 2-3 d;when seedlings reach 4-5 cm,transferring them to root medium（1/2 MS＋IBA 0.8 mg/L＋sucrose 30 g/L） and incubating for 14-21 d;when lots of fresh root generated,the seedlings would be ready to transplant.