Plant regeneration from mesophyll protoplasts of a medicinal plant, Phellodendron amurense Rupr.
Plant regeneration from mesophyll protoplasts of a medicinal plant, Phellodendron amurense Rupr.
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DOI：
10.1079/ivp2006808
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发表时间：
2006-11-01
影响因子：
2.6
通讯作者：
Yoshizawa, N.
中科院分区：
文献类型：
--
作者：
Azad, M. A. K.;Yokota, S.;Yoshizawa, N.
药用植物黄柏原生质体到植株的再生体系。，已经发展起来。选取微繁殖芽或小苗的叶片作为原生质体分离的植物材料。叶片原生质体的产量和活力受酶配、处理时间和渗透力的影响较大。在1%的纤维素酶Onozuka R-10和1%的Driselase-20中消化6小时，获得最高的活力（86%），每克鲜重产7.1 × 10(5)个原生质体。采用0.2%结冷胶固化或液态MS （Murashige and Skoog, 1962）培养基，每毫升4 × 10(5)-6 × 10(5)个原生质体，培养基中含有0.6M甘露醇、2.0 μ M 6-苄基氨基嘌呤（BA）和4.0 μ M α -萘乙酸（NAA）、吲哚-3-丁酸（IBA）或2,4-二氯苯氧乙酸（2,4- d），最有利于原生质体的持续细胞分裂和集落形成。将原生质体衍生的菌落转移到含有2.0 μ M BA和4.0 μ M NAA或IBA的固化MS培养基上，形成绿色致密的愈伤组织。在MS培养基上分别添加2.0 μ M BA和1.0 μ M NAA或2.5 μ M IBA，诱导原生质体愈伤组织再生。在含有1.0亩mba的MS培养基上，茎部增殖伸长。离体苗在MS培养基上生根，培养基中分别添加0.5 ~ 4.0 μ M的IBA或NAA。将再生剂转移到卡努马土壤中，并在温室条件下成功建立。
A regeneration system from protoplast to plantlet for a medicinal plant species, Phellodendron amurense Rupr., has been developed. Leaves of micropropagated shoots or plantlets were selected as plant materials for protoplast isolation. The yield and viability of leaf protoplasts were greatly influenced by the enzyme combination, treatment time and osmoticum. The highest viability (86%) with a yield of 7.1 x 10(5) protoplasts per gram fresh weight was obtained with a 6-h digestion in 1% Cellulase Onozuka R-10 plus 1% Driselase-20. Sustained cell division and colony formation from the protoplasts were best supported at a plating density of 4 x 10(5)-6x 10(5) protoplasts per milliliter using a 0.2% gellan gum-solidified or liquid MS (Murashige and Skoog, 1962) medium containing 0.6M mannitol, 2.0 mu M 6-benzylaminopurine (BA) with 4.0 mu M alpha-naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA), or 2,4-dichlorophenoxyacetic acid (2,4-D). The protoplast-derived colonies formed green compact calluses when transferred to a solidified MS medium containing 2.0 mu M BA with 4.0 mu M NAA or IBA. Shoot regeneration from protoplast-derived calluses was induced on MS medium supplemented with 2.0 mu M BA and 1.0 mu M NAA or 2.5 mu M IBA. Shoot multiplication and elongation occurred on MS medium containing 1.0 mu M BA. In vitro-grown shoots were rooted on MS medium with either 0.5-4.0 mu M IBA or NAA. Regenerants were transferred to the Kanuma soil and successfully established under greenhouse conditions.