多年生黑麦草再生体系的建立

Establishment of tissue regeneration system of Lolium perenne

关键词:  多年生黑麦草  组织培养  外植体  诱导  再生

Key words:  Lolium perenne  tissue culture  explants  induction  regeneration

摘要:

以多年生黑麦草‘高帽2号'(Lolium perenne ‘Top Hat 2')颖果为外植体,分别研究了胚端半粒颖果、纵切的胚端半粒颖果、胚端颖果小块、颖果小块这四种外植体处理方式与氯化汞、次氯酸钠两种表面灭菌方法对其愈伤组诱导的影响; 利用两因素(2,4-D和6-BA)随机区组试验设计研究了不同植物生长调节物质及其浓度配比对外植体愈伤组织诱导的影响; 利用三因素(NAA,6-BA和ZT)四水平正交试验设计研究了不同植物生长调节物质及其浓度配比对不定芽诱导的影响; 研究了0.10 mg·L^-1的NAA和IBA分别对不定根诱导的影响。结果表明:相同条件下,以饱满胚端半粒颖果为外植体,75%酒精表面灭菌1 min后,再用含1 mL·L^-1 Tween 20的次氯酸钠溶液(有效氯含量>10%)处理30 min,愈伤组织诱导率最高; 适宜的愈伤组织诱导培养基为MS+5 mg·L^-1 2,4-D+0.03 mg·L^-1 6-BA(pH=5.8),诱导率达68.7%,获得的高质量愈伤组织; 不定芽诱导培养基为MS+0.5 mg·L^-1 NAA、0.5 mg· L^-1 6-BA +0.9 mg·L^-1 ZT,不定芽诱导率最高,为56%; 不定根诱导培养基为1/2MS+IBA培养基时,根系粗壮,诱导生根率为90%。该研究建立了多年生黑麦草高效组培再生体系,为高效基因工程育种的开展奠定了良好基础。

Abstract:

The caryopsis of perennial ryegrass(Lolium perenne)cultivar ‘Top Hat 2' was used as explants. Firstly, this paper focused on the effect of four ways of explants treatments(embryo end half a caryopsis, slitting embryo end half a caryopsis, small pieces of embryo end for caryopsis, small pieces of caryopsis)and two ways of surface sterilization methods(disinfection with sodium hypochlorite solution, disinfection with mercuric chloride)on callus induction; Secondly, we studied the effects of different plant growth regulators(2, 4-D and 6-BA)and their concentrations on callus induction by randomized block design of two factors; Thirdly, we studied the effects of different plant growth regulators(NAA, 6-BA and ZT)and their concentrations on adventitious bud differentiation by orthogonal test of three factors and four levels; At last, we separately studied the effects of 0.10 mg·L^-1NAA and IBA on adventitious root induction. The results were as follows: Under the same conditions, explants of full end half a caryopsis embryos were surface sterilized with 75% alcohol for 1 min, then treated with sodium hypochlorite solution 1 mL·L^-1 Tween 20(the chlorine content>10%)for 30 min, the rate of callus induction were better than the other's; The explants inoculated on MS medium with 5 mg·L^-1 2,4-D and 0.03 mg·L^-1 6-BA(pH=5.8), provided us a relatively high induction rate of 68.7% and high quality callus; The most high differentiation rate was 56% when adventitious buds were inoculated in MS with 0.5 mg·L^-1NAA, 0.5 mg·L^-1 6-BA and 0.9 mg·L^-1 ZT. The best rooting medium was 1/2MS+IBA with nothing plant growth regulator obtaining stout root, then the regeneration rate was 90%. This paper has established efficient tissue culture regeneration system for perennial ryegrass and laid a good foundation for conducting highly efficient genetic engineering.