籼稻品种93-11遗传转化中抗生素适宜剂量的筛选

为优化农杆菌介导转化体系,研究了两种常用抑菌剂头孢霉素(cef)和羧苄青霉素(cb)的抑菌效果和对籼稻品种93-11成熟胚培养的影响,并进一步研究选择剂潮霉素(hyg)对愈伤诱导、分化和种子萌发生根的影响,确定其作为选择剂的有效浓度.对cb和cef共设置12种质量浓度处理,各处理都能完全抑制农杆菌的生长,同时对愈伤的诱导和生长没有不良影响,但是有些处理使愈伤的分化率下降.150、250 mg.L-1cb的单独作用,400、500 mg.L-1cef的单独作用,以及cb为100mg.L-1时,300、400、500 mg.L-1cef的3种互作处理都不影响愈伤的分化率;但cef高达600 mg.L-1时明显降低愈伤的分化率,达极显著水平,使分化率降到7%;cb为50 mg.L-1时,350、450、550 mg.L-1cef的3种互作处理都极显著地降低愈伤的分化率,表明高浓度的cef以及添加小量cb的处理与cef之间的互作对分化率的影响十分显著.两种抑菌剂单独使用(400-500 mg.L-1cef,150-250 mg.L-1cb)即有效抑制转化和培养过程中农杆菌的繁殖,同时不影响愈伤的生长和分化.对hyg的研究表明,愈伤诱导和分化对hyg最为敏感,5 mg.L-1的剂量足以使出愈率为0,使愈伤的分化率为0;hyg对愈伤的致死剂量为40 mg.L-1;hyg对种子萌发生根的致死剂量为25 mg.L-1.因此,在愈伤分化阶段不宜加入选择剂;在抗性愈伤组织筛选阶段加入hyg的适宜质量浓度要大于40 mg.L-1;利用愈伤诱导和种子萌发生根对hyg的敏感性,可用于转基因种子的筛选,筛选质量浓度分别要大于5和25 mg.L-1.

Screening of suitable antibiotic concentration for the genetic transformation of indica rice cultivar 93-11

To optimize the transformation system mediated by Agrobacterium,this paper deals with the effects of ceftaxime(cef) and carbenicillin(cb) on Agrobacterium and rice 93-11,and the effects of hygromycin(hyg) on callus inducing,differentiation and seed germination.12 concentration treatments were designed for cb and cef.Each concentration treatment could inhibit the growth of Agrobacterium and had no bad effects on callus inducing and growth,but some of them lowered calli differentiation rate.Single treatments of 150 and 250 mg·L-1 cb,400 and 500 mg·L-1 cef,and 3 mixed treatments of 100 mg·L-1 cb and 300,400,500 mg·L-1 cef did not affect differentiation rate.When cef was high to 600 mg·L-1,it obviously lowered the differentiation rate to 7% extremely significantly.3 mixed treatments of 50 mg·L-1 cb and 350,450,550 mg·L-1 cef lowered the differentiation rate extremely significantly.This illustrated that single treatment of high concentration of cef and mixed treatments of cef and lower concertration of cb could obviously affect the differentiation rate.So,single treatment of 400-500 mg·L-1 cef or 150-250 mg·L-1 cb was effective in inhibiting Agrobacterium for transformation and culture.The studying results for hyg showed that callus inducing and differentiation was very sensitive to hyg.At the low concentration of 5 mg·L-1,the rates of callus inducing and differentiation were all 0.Lethal dose of hyg to callus was 40 mg·L-1,and that to seed germination and rhizogenesis was 25 mg·L-1.Therefore,it is not suitable to add selection substance in the phase of calli differentiation;hyg higher than 40 mg·L-1 is suitable in the phase of resistant calli growth;the sensitivity of calli inducing,seed germination and rhizogenesis to hyg can be used to identify transgenic seed,and the corresponding mass concentration is higher than 5 and 25 mg·L-1 respectively.