Department of Biological Sciences, The University of Newcastle, 2308, NSW, Australia.
Plant Cell Rep. 1995 Mar;14(6):349-53. doi: 10.1007/BF00238595.
Medicago truncatula (barrel medic) is an annual legume of agricultural and biological interest. In this report regeneration from isolated mesophyll protoplasts is described. A specifically developed, highly regenerable seed line is essential for regeneration. Other critical requirements for regeneration are the starting plant material, the use of agarose droplets incubated in a shallow layer of liquid medium, and protoplast density. Plants are grown in controlled environment conditions. Protoplasts are purified using a Percoll-based flotation procedure, then embedded in 100 μl agarose droplets containing a basal medium plus 25 μM NAA and 4 μM BAP (the same medium as in the surrounding shallow liquid layer) to induce protoplast division. A protoplast density of 6-8×10(5) ml(-1) is required for maximum colony formation. M. truncatula plants previously transformed for kanamycin resistance yielded embryogenic callus and also regenerated plants. Protoplasts from other annual Medicago (M.intertexta and M.scutellata) species readily form calli by the procedure we have described.
蒺藜苜蓿（桶状苜蓿）是一种具有农业和生物价值的一年生豆科植物。本报告描述了从分离的叶肉原生质体再生植株的过程。专门开发的、高再生的种子系对于再生是必不可少的。再生的其他关键要求是起始植物材料、使用在浅层液体培养基中孵育的琼脂糖液滴，以及原生质体密度。植物在受控环境条件下生长。使用基于聚蔗糖的浮选程序来纯化原生质体，然后将其嵌入包含基础培养基加 25 μM NAA 和 4 μM BAP 的 100 μl 琼脂糖液滴中（与周围浅层液体层中的培养基相同），以诱导原生质体分裂。对于最大集落形成，需要 6-8×10(5) ml(-1) 的原生质体密度。先前经过卡那霉素抗性转化的蒺藜苜蓿植物产生了胚性愈伤组织，并且也再生了植株。按照我们所描述的程序，其他一年生 Medicago（M.intertexta 和 M.scutellata）物种的原生质体很容易形成愈伤组织。
