新疆大叶紫花苜蓿(Medicago sativa.L)子叶、下胚轴、根的植株再生

Regeneration Plants of Cotyledon, Hypocotyl and Root of Xinjiang Big Leaf Alfalfa(Medicago.sativa.L)

随着植物抗逆性研究和植物转基因技术的发展,通过异源目的基因转化培育耐盐碱苜蓿品种的研究已引起人们的关注,植物受体高频再生体系的建立是异源转化高效的基础。选取新疆大叶紫花苜蓿种子萌发5～7d无菌苗的子叶、下胚轴及根为外植体,诱导愈伤培养基为MS+2,4-D0.1～3.0mg/L(8种不同水平)或MS+2,4-D2.0mg/L+KT0.01～0.5mg/L(10种不同水平),诱导芽培养基为MS+6-BA0.5mg/L+NAA0.05mg/L,生根培养基为MS。结果表明,外植体在MS+2,4-D2.0mg/L+KT0.2mg/L培养基中能够产生状态较好可再分化的愈伤组织,子叶、下胚轴、根的平均出愈率分别为93.1%、100%、100%。愈伤组织在MS+6-BA0.5mg/L+NAA0.05mg/L培养基中培养40～80d中均可分化芽,子叶、下胚轴、根的芽平均分化率为50%、78%、50%,将2cm以上的芽转入MS培养基中诱导生根,14d后,生根的小植株炼苗移入花土中,成活率达90%以上。子叶、下胚轴、根在该体系中均能获得再生植株,根也是一种较好的植株再生材料,以根为外植体进行植株再生的研究报道还较少。

Following the development of plants resistance and transgenic techonology, breed of new resistant alfalfa by transforming heterogenous gene has being become more and more remarkable to pepole. So,establishing a high-frequence system of regeneration plants is the base of high efficiency transform for heterogenous gene. Cotyledon, hypocotyl and root of 5-7 day-old seedling of Xinjiang big leaf alfalfa were selected as explants. MS and 8 groups of 2,4-D with different concentrations from 0. lmg/L to 3mg/L were used to induce callus, then MS and 2,4-D2mg/L and 10 groups of KT with different concertrations from 0.01mg/L to 0.5mg/L were used to induce callus; the shoots were induced in the medium of MS with additional 6-BA 0.5mg/L and NAA 0.05mg/L; MS was used to produce roots. Results showed that： callus tissues had much better growing status and could be used to produce differentiation shoots in medium of MS with 2,4-D 2.0mg/L and KT 0.2mg/L, the averaging calli frequences were 93.1%, 100% and 100% for cotyledon, hypocotyl and root; After growing callus tissues which originated from cotyledon, 40-80 days in the medium of inducing regeneration shoot, hypocotyl and root produce regeneration shoots and its averaging regeneration shoots frequence was 50%, 78% and 50% respectively; when shoots grew to about 2cm height, it was transferred into medium of MS to root; afer 14 days, regeneration plants were transferred into flower-soil and its survivable rate was more than 90%. All of cotyledon, hypocotyl and root can obtain regeneration plants in this culturing system. Moreover root can act as a good material of explant to obtain regeneration plant while researches in this field are still poor.