天蓝苜蓿花药愈伤组织诱导及再生体系的建立

Construction of a Highly Efficient Anther Culture Technology System of Medicago lupulina

为获得天蓝苜蓿单倍体再生植株,本研究以野生天蓝苜蓿花药为外植体,采用正交设计L_(16)(4^(4))筛选适宜天蓝苜蓿愈伤组织诱导培养基,比较不同基本培养基及生长调节剂组合筛选适宜的分化培养基,并用1/3MS、1/2MS和MS添加不同浓度的NAA研究不定生根。结果表明:天蓝苜蓿现蕾15~25 d的花药其愈伤组织诱导效果最好,高达60.5%。4℃低温预处理2~4 d有利于愈伤组织诱导,诱导率达77.2%。适宜花药愈伤组织诱导的培养基为NB+2,4-D 1.0 mg/L+NAA 0.5 mg/L+6-BA 0.5 mg/L+TDZ 1.0 mg/L,诱导率达78.5%。比较不同基本培养及生长调节剂的不同组合发现,NB培养基愈伤组织分化效果优于B_(5)和MS,NB+NAA0.5 mg/L+6-BA 2.0 mg/L适宜愈伤组织的分化,分化率为66.3%。不定芽在1/3MS+NAA 0.5 mg/L中培养,生根率最高,为86.55%。本研究建立了天蓝苜蓿花药培养再生体系,获得了单倍体植株,为天蓝苜蓿的育种实践及基因组学研究提供基础材料。

In order to obtain the haploid regeneration plant,the anther of wild Medicago lupulina was used as the explants.The appropriate medium was screened for induction of anther callus by orthogonal design L_(16)(4^(4)).In addition,compared with different basic medium and different growth regulator combination,the appropriate medium was screened for differentiation of anther callus.The rooting ability from buds was studied by adding different concentrations of NAA into 1/3 MS,1/2 MS and MS medium.The results showed that the highest induction rate of Medicago lupulina anther callus was in 15 to 25 days after budding,and the maximum value was 60.5%.4℃low temperature pretreatment for 2 to 4 d is conducive to callus formation,induction rate was 77.2%.The medium that was good for the induction of the callus of Medicago lupulina anther was made up of NB+2,4-D 1.0 mg/L+NAA0.5 mg/L+6-BA 0.5 mg/L+TDZ 1.0 mg/L,and the maximum induction rate was 78.5%.Compared with different basic medium and different growth regulator combination,NB medium was better than that of B_(5) and MS.The appropriate medium of callus differentiation was NB+NAA 0.5 mg/L+6-BA 2.0 mg/L,and differentiation rate was66.3%.The best culture medium for adventitious bud rooting was 1/3 MS with NAA 0.5 mg/L,and the maximum rooting rate was 86.55%.An anther culture regeneration system of Medicago lupulina was established successfully,and the regenerated haploid plants were obtained.It will also provide basic materials for breeding practice,genomics research of Medicago lupulina.