摘要
采用根癌农杆菌直接注射子房,对8个棉花栽培品种进行活体方式的遗传转化。为了确立注射细菌的适宜时期,先采用含考马斯亮蓝染料的菌液对授粉前3d(-3 DPA,days post-anthesis)至授粉后2d(2DPA)的棉花子房进行注射,观察到细菌(染料)可以在-2 DPA至授粉后24 h之间进入子房。为了避免注射对花柱的损伤并影响授粉,选取授粉后24h已完成受精的子房,利用微量注射器经花柱将10μL约含1000个细菌细胞的农杆菌菌液注入子房。对8个棉花品种转化结果表明,该方法可以有效转化棉花。种子成熟后田间播种,经卡那霉素抗性筛选及PCR(Polymerase chain reaction)分子检测,转基因效率平均为2.78%,转化效率与受体的基因型无明显的相关性,相对组织培养的农杆菌转化法和DNA子房注射法有一定的优势。
In this study, 8 cultivars of Gossypium hirsutum were transformed by in planta agrobacterium injection, the bacteria are injected into the ovary after fertilization. In order to establish an optimized development stage of the ovary for efficient injection, the Coomassie brilliant blue stained agrobacterium are injected into ovary in several development stage of flower beforehand. We take the flower from -3 days post-anthesis （-3 DPA） to 2 days post-anthesis （2 DPA） for the test. It showed the bacterium can enter the ovules during -2 DPA and 24 h after pollination by observation of the stain detained in the ovary. Considering that the injection process might damage to the stigma and pistil thus to block its pollination, we determine to use fertilized flowers at 1 DPA for the transformation injection. Some 10 μL of agrobacterium of almost 1000 cells contented was injected into the ovary through stigma and pistil using micro-liter syringe. The transformation for the eight varieties showed that this method has a reasonable transformation effective for cotton transformation without significant difference in the ratio. The average transgenic efficiency was 2.78% by tested with kanamycin resistance screening in field and PCR amplification of the target molecule validating. It is concluded that in planta transgenic method is efficient and without a significant genotype correlation. It has comparable advantages to other transformation method currently applied in cotton transformation such as agrobacterium-mediated tissue culture dependent method or DNA plasmid direct ovary injection transformation.
出处
《棉花学报》
CSCD
北大核心
2011年第4期311-316,共6页
Cotton Science
基金
湖南省科技计划项目(603002003)
关键词
棉花
农杆菌
子房注射
转化率
Gossypium hirsutum
agrobacterium
ovary-injection
in planta transformation
