马铃薯'新疆2号'遗传转化体系的建立OA

[Objective]To establish an efficient regeneration and genetic transformation system for potatoes(Solanum tuberosum L.)variety'Xinjiang No.2',and to provide technical support for virus-resistant breeding.[Method]Using'Xinjiang No.2'potato as the experimental material,Agrobacterium-mediated transformation was employed to introduce the plant interference vector plasmid pCAMBIA2300-CP-RNAi into potato stem explants.The effects of pre-culture duration,plant hormone combinations,and types and concentrations of antibiotics on callus induction and shoot differentiation were investigated.By real-time quantitative PCR(RT-qPCR)technology,with the Actin gene as an internal reference,the mRNA expression of the CP gene was detected.The actual content of PVY virus in plants was quantitatively measured using enzyme-linked immunosorbent assay(ELISA)to validate the RNAi silencing effect and virus suppression.[Result]The optimal hormone and antibiotic combination for callus induction was 2.0 mg/L 6-BA+0.8 mg/L 2,4-D+50 mg/L Kan+200 mg/L TMT when pre-culture and co-culture durations were both 2 days,achieving an induction rate of 85%.The callus showed a compact morphology,fresh green color,and a dumbbell-shaped swelling at both ends of the stem segments.For shoot differentiation,the optimal hormone and antibiotic ratio was 2 mg/L 6-BA+2 mg/L ZT+0.5 mg/L GA3+50 mg/L Kan+200 mg/L TMT,resulting in the highest germination and transformation rates,with well-developed seedlings.Transgenic seedlings regenerated into plants on 1/2 MS medium supplemented with 0.6 mg/L IAA+75 mg/L Kan+200 mg/L TMT,demonstrating robust root systems.Compared to non-transformed control plants,the relative expression of the CP gene in transgenic plants was significantly downregulated,and PVY virus content was markedly reduced,confirming that the constructed RNAi vector successfully triggered specific gene silencing and effectively inhibited virus accumulation in plants.[Conclusion]This study successfully establishes a stable and efficient genetic transformation system for the potato variety'Xinjiang No.2'.The constructed RNAi interference vector effectively suppresses Y virus accumulation in plants.