大叶紫花苜蓿愈伤组织原生质体再生植株

PLANT REGENERATION FROM CALLUS PROTOPLASTS OF MEDICAGO SATIVA SSP.SATIVA

大叶紫花苜蓿下胚轴诱导的愈伤组织在继代培养基上生长快速，易于分散。继代第１２ｄ的愈伤组织原生质体的得率为６．５×１０７／ｇ鲜重。原生质体培养基为ＳＨ基本培养基，含有１．０ｍｇ／Ｌ２，４－０、０．５ｍｇ／ＬＢＡ、２．０ｇ／ＬＣＨ、２％蔗糖、６％葡萄糖、５ｍｍｏｌ／ＬＭＥＳ，培养密度为１．０×１０５／ｍＬ。培养至第１２ｄ时的原生质体再生细胞植板率为３．７％。由原生质体形成的小愈伤组织在含２．０ｍｇ／Ｌ２，４－Ｄ的ＭＳ固体培养基上大量增殖。增殖的愈伤组织转移至２．０ｍｇ／Ｌ２－ｉｐ＋０．１ｍｇ／ＬＮＡＡ的Ｂ５培养基上，形成体细胞胚并发育成完整植株。

Calli derived from hypocotyledon of Medicago sativa ssp. sativa grew rapidly and dispersed easily on subculture media, Protoplasts isolated from calli of l2 days after subculture at a high yield of 6. 5×l0￣7/g fresh weigh. Protoplasts were cultured on modified SH medium containing l.0mgl/L 2,4-D,0.5mg/L BA,2.0g/L CH,2%sucrose,6% glucose, 5 mmol/L 2-(N-morpholino)-ethanesulfonic acid with plating density of 1.0×10￣5/mL. Protoplast plating efficiency was 3.7%after 12 days in culture. Microcalli derived from protoplasts multiplicated on solid MS medium containing 2.0mg/L 2,4-D. Somatic embryos and plant regeneration were obtained when these calli were transferred to B5 medium containing 2.0mg/L 2-ip and 0.1mg/L NAA.