紫花苜蓿毛状根高效转化体系的建立

Developing a Hairy Root Transformation System for Alfalfa

紫花苜蓿是我国草食畜牧业首选的优质多年生牧草,为摆脱种子的进口依赖亟须加快苜蓿基因功能研究和生物育种进程,而建立高效、周期短的遗传转化体系是前提。目前常用的苜蓿叶盘转化法转化周期长、转化难度大,不适用于初期的基因功能鉴定和CRISPR/Cas编辑效率评价等研究。因此,本研究以“无棣苜蓿”的胚根为外植体材料,甘油醛-3-磷酸脱氢酶基因(MSgene72216)为Marker基因,将其构建的过表达载体pCAMBIA3301转入发根农杆菌(Agrobacterium rhizogenes)Ar. 1193菌株中诱导毛状根,经过共培养、二次切根、选择培养等过程获得毛状根转化植株后,通过PCR进行毛状根转化效率检测。结果表明:该方法可在2~3周获得用于鉴定的毛状根植株,大幅缩短了转化周期,其生根率可达100.00%,毛状根遗传转化率可达98.33%。该体系的建立对高通量开展基因功能研究提供了省时省力高效的转化方法,为推动紫花苜蓿基因功能研究和生物育种奠定了基础。

Alfalfa(Medicago sativa L.)is highly preferred perennial forage for herbivores in China.To reduce reliance on seed imports,accelerating research gene function and biological breeding of alfalfa is crucial.However,establishing efficient genetic transformation systems with rapid turnaround times is a prerequisite for such advancements.Existing leaf disc transformation methods are not ideal for initial gene function identification and evaluation of CRISPR/Cas editing efficiency due to their complexity and extended timelines.To address this limitation,this study used taproots from“Wudi”cultivar served as explants,and the glyceraldehyde-3-phosphate dehydrogenase gene(MSgene72216)acted as marker gene.The overexpression vector pCAMBIA3301 was transformed into Agrobacterium rhizogenes strain Ar.1193 to induce hairy root formation.Following co-culture,secondary root cuttings,and selection,hairy root-derived transgenic plants were obtained.PCR analysis confirmed the transformation efficiency.The results showed that this method enabled the generation of hairy root plants suitable for identification within 2-3 weeks,significantly shortening the transformation cycle.The system achieved a 100.00%rooting rate and a 98.33%transformation rate.This novel hairy root transformation system offers a time-saving and labor-saving approach for high-throughput gene function research in alfalfa.It has the potential to lay the groundwork for advancing research on gene function and biological breeding within this important forage.