Gene transfer into isolated and cultured tobacco zygotes by a specially designed device for electroporation
Gene transfer into isolated and cultured tobacco zygotes by a specially designed device for electroporation
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DOI：
10.1007/s002990000249
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发表时间：
2000-12
影响因子：
6.2
通讯作者：
Shisheng Li;H. Yang
中科院分区：
文献类型：
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作者：
Shisheng Li;H. Yang
建立了烟草合子的分离、培养和转化技术。受精卵通过显微切割或酶浸渍从受精胚囊中分离。与叶肉原生质体共培养的合子在培养3天后进行第一次分裂。显微分离法分离的受精卵第一次分裂频率（61.2%）高于酶法分离的受精卵（30.5%）。球形胚仅由显微切割的受精卵形成，在培养1-2周后的频率为8.7%。建立了一种高效的将DNA电穿孔到合子中的毫细胞装置。电穿孔的受精卵在体外分裂率为54.6%，并发育成原胚。引入的GFP基因构建体在约2.6%的电穿孔烟草合子中显示瞬时表达。
We have established a technique for isolating, culturing and transforming tobacco zygotes. Zygotes were isolated by microdissection or enzymatic maceration from fertilized embryo sacs. Viable zygotes cocultured with mesophyll protoplasts underwent first division after 3 days of culture. Zygotes isolated by microdissection underwent a higher frequency of first division (61.2%) than those isolated by enzymatic maceration (30.5%). Globular embryos were formed only from microdissected zygotes, at a frequency of 8.7% after 1–2 weeks in culture. An efficient millicell device for the electroporation of DNA into zygotes was established. The electroporated zygotes divided in vitro at a frequency of 54.6% and developed into proembryos. Introduced GFP gene constructs showed transient expression in about 2.6% of the electroporated tobacco zygotes.