扭果花旗杆组织培养与再生体系的建立

Establishment of tissue culture and regeneration system for Dontostemon elegans

【目的】以扭果花旗杆(Dontostemon elegans)根和下胚轴为外植体,建立扭果花旗杆组织培养体系,为后续遗传转化体系的建立提供技术基础。【方法】以MS培养基为基础培养基,利用萘乙酸(NAA)探究根和下胚轴直接诱导不定芽情况,利用2,4-二氯苯氧乙酸(2,4-D)、6-苄氨基嘌呤(6-BA)、NAA、苯基噻二唑基脲(TDZ)、吲哚丁酸(IBA)进行愈伤组织诱导及分化和不定芽生根试验,统计愈伤组织诱导及分化和不定芽生根情况,确定扭果花旗杆组培体系最佳培养基类型。【结果】(1)由根和下胚轴直接诱导不定芽的最适培养基分别为MS+0.3 mg/L NAA和MS+0.6 mg/L NAA,直接诱导不定芽的最佳外植体为根。(2)扭果花旗杆根和下胚轴都能诱导愈伤组织,其中根系诱导愈伤组织速度最快且状态最佳,其次为下胚轴。根系愈伤组织诱导的最佳培养基为MS+1.0 mg/L 2,4-D+0.2 mg/L 6-BA+0.1 mg/L NAA,诱导率可达100.00%;下胚轴愈伤组织诱导的最佳培养基为MS+0.3 mg/L 2,4-D+1.5 mg/L 6-BA+1.5 mg/L NAA,诱导率可达100.00%。(3)根系和下胚轴的最佳愈伤组织分化培养基均为MS+0.5 mg/L 6-BA+0.3 mg/L NAA,分化率分别为100.00%和86.66%。(4)最佳生根培养基为MS+0.5 mg/L IBA,生根率可达50%以上。(5)将组培苗移至土壤后,在光照培养室培养15 d,其存活率可达83.33%。【结论】建立的扭果花旗杆组培体系可获得与实生苗形态一致的组培苗,为扭果花旗杆遗传转化体系的建立奠定了技术基础。

【Objective】A tissue culture system of Dontostemon elegans was established with roots and hypocotyls as explant to provide basis for the establishment of subsequent genetic transformation system.【Method】Using MS medium as the basic medium,Naphthylacetic acid(NAA)was used to explore the direct induction of adventitious buds from roots and hypocotyls,and 2,4-dichlorophenoxyacetic acid(2,4-D),6-benzylaminopurine(6-BA),NAA,thidiazuron(TDZ)and indole butyric acid(IBA)were used for callus induction,differentiation and adventitious bud rooting experiments.The callus induction,differentiation and adventitious bud rooting were observed and counted,and the optimal medium type for the tissue culture system was determined.【Result】(1)The optimum medium for direct induction of adventitious buds from roots and hypocotyls was MS+0.3 mg/L NAA and MS+0.6 mg/L NAA,respectively.(2)Both root and hypocotyl of Dontostemon elegans induced callus,and root induced callus was the fastest and best,followed by hypocotyl.The best medium for callus induction was MS+1.0 mg/L 2,4-D+0.2 mg/L 6-BA+0.1 mg/L NAA with induction rate of 100.00%.The best medium for hypocotyl callus induction was MS+0.3 mg/L 2,4-D+1.5 mg/L 6-BA+1.5 mg/L NAA with induction rate of 100.00%.(3)The best callus differentiation medium for roots and hypocotyls was MS+0.5 mg/L 6-BA+0.3 mg/L NAA,and the differentiation rates were 100.00%and 86.66%,respectively.(4)The best rooting medium was MS+0.5 mg/L IBA with rooting rate of higher than 50%.(5)After tissue culture seedlings were transferred to soil and cultured in light culture room for 15 d,the survival rate was 83.33%.【Conclusion】The established tissue culture system obtained tissue culture seedlings with the same morphology as normal seedlings,which lays technical foundation for establishing the genetic transformation system of Dontostemon elegans.