Plant regeneration via somatic embryogenesis, and transient gene expression in sweet potato protoplasts
Plant regeneration via somatic embryogenesis, and transient gene expression in sweet potato protoplasts
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DOI：
10.1007/s002990050462
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发表时间：
1998-06-01
影响因子：
6.2
通讯作者：
Bhagsari, AS
中科院分区：
文献类型：
--
作者：
Dhir, SK;Oglesby, J;Bhagsari, AS
本文报道了一种利用宝石红薯叶柄原生质体进行离体植株再生的方法。在含有9%甘露醇的洗涤液中，用1%纤维素酶- r10、2%巨菌酶- r10和0.3%果胶酶Y-23消化1- 2cm叶柄（1g鲜重）4-6小时后，原生质体产量为3.0-5.0 × 10(6)。10(5)个原生质体/ml的电镀密度是后续分裂的最佳选择。在液体或琼脂糖固化的KP8培养基中，添加2,4-二氯苯氧乙酸（2,4- d） （0.9 μ M）和玉米素（2.3 CIM），初始分裂频率为12-15%。24 +/- 2℃黑暗培养4周后形成100-200个细胞的菌落，逐渐加入低渗透新鲜液体KP8培养基提高菌落形成频率。在连续光照和新鲜培养基的定期稀释下，再经过4-6周形成原愈伤组织（直径1- 2mm）。将愈伤组织转移到含有2,4- d （11.3 μ M）和苄基氨基嘌呤（2.2 μ M）的培养基上，形成球状和心形胚，发育为子叶期胚。随后，在添加2%蔗糖和3.5 μ M赤霉素酸的基础培养基上获得胚转化成植株。再生苗成功移栽于土壤中。成熟植株表型正常。在相同的叶柄原生质体群体中，电穿孔诱导的gusA基因瞬间表达。
A method for regenerating plants from petiole protoplasts of the in vitro-raised sweet potato cultivar Jewel is described. Protoplast yields of 3.0-5.0x10(6) were obtained following 4-6 h digestion of 1- to 2-cm petioles (1 g fresh weight) with 1% Cellulase-R10, 2% Macerozyme-R10, and 0.3% Pectolyase Y-23 in a washing solution with 9% mannitol. A plating density of 10(5) protoplasts/ml was optimal for subsequent division. An initial division frequency of 12-15% was obtained in liquid or agarose-solidified KP8 culture medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) (0.9 mu M), and zeatin (2.3 CIM). Colonies consisting of 100-200 cells were formed after 4 weeks in the dark at 24 +/- 2 degrees C. The frequency of colony formation was improved by the gradual addition of fresh liquid KP8 medium of lower osmoticum. Proto-calli (1-2 mm in diameter) were formed after an additional 4-6 weeks under continuous illumination and regular dilution with fresh culture medium. Morphogenic callus formed globular and heart-shaped embryos that developed into cotyledon stage embryos, following transfer of calli onto medium containing 2,4-D (11.3 mu M) and benzylaminopurine (2.2 mu M). Subsequently, embryo conversion to plantlets was obtained on basal medium with 2% sucrose and 3.5 mu M gibberellic acid. Regenerated plantlets were successfully transplanted in soil. Mature plants appeared phenotypically normal. The same petiole protoplast populations showed transient expression of the gusA gene introduced using electroporation.