主栽小麦快速遗传转化方法

近日,Scientific Reports在线发表日本科学家Ryozo Imai团队题为“An in planta biolistic method for stable wheat transformation”研究论文,该研究以小麦成熟胚分生组织为受体材料,利用基因枪方法获得稳定转基因植株,该种方法并不需要愈伤诱导,再生等繁琐步骤。

小麦因为其具有较大的基因组,遗传背景复杂等原因导致遗传转化比较困难,使得小麦的分子生物学研究相对于滞后其他主要农作物。该研究团队以茎尖分生组织做为受体材料,利用基因枪将GFP基因的表达载体导入分省组织。12小时之后,可观察到GFP的瞬时表达并转移至Ms培养基上生长2-3周获得幼苗,经过对后代植株鉴定后证明通过该种方法可以获得稳定的转基因植株,该论文还对基因枪轰击的参数等进行了优化。该方法无疑解决小麦遗传转化一直以来受受体材料的限制的问题。

An in planta biolistic method for stable wheat transformation

Haruyasu Hamada1,2, Qianyan Linghu1, Yozo Nagira2, Ryuji Miki2, Naoaki Taoka2 & Ryozo Imai1,3

 

The currently favoured method for wheat (Triticum aestivum L.) transformation is inapplicable to many elite cultivars because it requires callus culture and regeneration. Here, we developed a simple, reproducible, in planta wheat transformation method using biolistic DNA delivery without callus culture or regeneration. Shoot apical meristems (SAMs) grown from dry imbibed seeds were exposed under a microscope and subjected to bombardment with different-sized gold particles coated with the GFP gene construct, introducing DNA into the L2 cell layer. Bombarded embryos were grown to mature, stably transformed T0 plants and integration of the GFP gene into the genome was determined at the fifth leaf. Use of 0.6-μm particles and 1350-psi pressure resulted in dramatically increased maximum ratios of transient GFP expression in SAMs and transgene integration in the fifth leaf. The transgene was integrated into the germ cells of 62% of transformants, and was therefore inherited in the next generation. We successfully transformed the model wheat cultivar ‘Fielder’, as well as the recalcitrant Japanese elite cultivar ‘Haruyokoi’. Our method could potentially be used to generate stable transgenic lines for a wide range of commercial wheat cultivars.

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