农杆菌介导的小麦幼胚遗传转化及非基因型依赖性评价OA

Agrobacterium-mediated transformation is an important approach for genetic modification of wheat,but it still suffers from several unresolved critical technical bottlenecks,including low trans-formation efficiency,strong genotype dependence,severe browning of calli,and low regeneration effi-ciency.In this study,immature embryos of the model cultivar Fielder were used as explants to sys-tematically investigate the effects of embryo age,acetosyringone(AS)concentration and Agrobacterium infection duration,phosphinothricin(PPT)selection pressure,and exogenous hormone(abscisic acid,ABA;kinetin,KT)ratios on callus induction,regeneration and transformation efficiency.Using the optimized protocol,applicability was evaluated in the cultivars Yangmai 20,Zhengmai 7698,and Kenong 199.The results showed that embryos harvested 12 days after anthesis exhibited the lowest browning rate(4.40％)and were most suitable as recipients for transformation.When the Agrobacte-rium suspension was adjusted to OD600=0.5,supplementation with 200 μmol·L-1 AS and a 10 min infection produced the highest induction rate of resistant green callus spots(34.29％).Addition of 0.5 mg·L-1 ABA to the induction medium and 1.5 mg·L-1 KT to the regeneration medium signifi-cantly enhanced embryogenic callus formation and plant regeneration.The optimal PPT selection con-centration was determined to be 10 mg·L-1.Under the optimized conditions,transformation effi-ciency of Fielder increased significantly from 8.16％before optimization to 14.83％;transformation efficiencies of Yangmai 20,Zhengmai 7698,and Kenong 199 reached 9.03％,7.32％and 8.70％,re-spectively,approaching or exceeding the pre-optimization level of Fielder.In summary,the optimized system established in this study improves wheat genetic transformation efficiency and is broadly appli-cable across different genetic backgrounds,providing a technical platform for gene functional analysis and molecular breeding in wheat.