Developing a Hairy Root Transformation System for Alfalfa
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 pre-requisite 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%transfor-mation 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.
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