花生毛状根诱导及其体外培养

目的]优化花生毛状根诱导条件,为体外培养毛状根生产白藜芦醇以及培养花生根结线虫奠定基础。方法]研究不同的发根农杆菌及其菌液浓度、外植体类型、侵染时间、共培养时间、乙酰丁香酮浓度对花生毛状根诱导的影响。结果]发根农杆菌ACCC10060的毛状根诱导率高于ATCC11325;幼叶外植体的毛状根诱导率高于子叶;最佳侵染菌液浓度为0.2～0.4;最佳共培养时间为2 d;最佳侵染时间为10～15 min;菌液中添加75μmol/L乙酰丁香酮(AS)可以提高毛状根诱导率。通过毛状根能在无激素的MS培养上自主生长以及毛状根冠瘿碱检测,确定发根农杆菌中Ri质粒中已整合到花生基因组中。毛状根体外培养的研究表明,液体培养效果好于固体培养,最佳液体培养基为无激素的MS培养基。结论]该研究建立的花生毛状根培养体系,将为花生转基因技术的完善及利用毛状根生产白藜芦醇和无菌的花生根结线虫提供试验基础。

Induction and in vitro Culture of Hairy Roots in Peanut (Arachis hypogaea L.)

Objective] To optimize the induction conditions of hairy roots in peanut,so as to lay foundation for producing resveratrol and the propagation of root-knot nematode.Method]Effects of different Agrobacterium rhizogenes strains,explants type,bacteria concentration,co-culture time,infection time and AS concertration on the induction of hairy roots in peanuts were studied.Result] Agrobacterium rhizogenes ACCC10060 had higher induction rate of hairy roots than strains ATCC11325.Leaflet had higher induction rate of hairy roots than cotyledon,the optimal bacteria concentration was 0.2-0.4,the optimal co-culture time was 2 d,the best infection time was 10-15 min,the dose of 75 μmol/L acetosyringone(AS) could improve induction rate of hairy roots.Hairy roots could grow independently on MS medium without hormone,and presence of opines from hairy roots of peanut confirmed that Ri plasmid of Agrobacterium rhizogenes was integrated into the genome of peanut.Hairy roots grew faster in liquid culture than in solid culture,and the optimal medium was MS liquid medium without hormone.Conclusion] Establishment of hairy roots culture system of peanut provided a foundation for the improvement of peanut transgenic technique,industrial production of resveratrol and propagation of sterile root-knot nematodes.