甘蓝型油菜与菘蓝原生质体融合及植株再生体系的研究

以甘蓝型油菜(Brassica napus)和菘蓝(Isatis tinctoria)叶片为材料提取原生质体,采用PEG-高钙高pH法融合亲本原生质体,采用固液双层培养法诱导愈伤组织的形成.研究适宜原生质体分离的酶种类及浓度,并考察了激素种类和用量对愈伤组织诱导和分化的影响.结果表明,5 mg/L纤维素酶+3 mg/L离析酶适合甘蓝型油菜和菘蓝的酶解.各培养基中适宜的激素浓度分别为,诱导培养基PellB 1.0 mg/L6-BA+1.0 mg/L NAA+0.25 mg/L 2,4-D;增殖培养基PellC 2.0 mg/L 6-BA+0.1 mg/L NAA+0.1 mg/L2,4-D;分化培养基PellE 2.0 mg/L 6-BA +0.1 mg/L NAA+0.02 mg/L 2,4-D.获得了15株再生植株,杂种幼苗叶片均呈深绿色,叶表面覆有厚的蜡质,叶片肥厚,植株在成熟期时株高比甘蓝型油菜矮,开花较晚,雄蕊发育不完全,杂种体细胞染色体数目为48～66.

Study on the Protoplast Fusion and Plant Regeneration System between Brassica napus and Isatis tinctoria

Protoplast was isolated from leaves of Brassica napus and Isatis tinctoria and fused by PEG-high pH and high calcium method.Callus was then induced by solid-liquid double layer culture method.Effects of factors including enzyme type and concentration on protoplast isolation,type and dose of hormone on callus induction and differentiation were studied to improve the plant regeneration efficiency.The results suggested that the enzyme combination suitable for protoplast isolation was 5 mg/L cellulase+3 mg/L macerozyme.The concentration of hormone for protoplasts induction,proliferation and differentiation were 1.0 mg/L 6-BA+1.0 mg/L NAA+0.25 mg/L 2,4-D,2.0 mg/L 6-BA+0.1 mg/L NAA+0.1 mg/L 2,4-D and 2.0 mg/L 6-BA +0.1 mg/L NAA +0.02 mg/L 2,4-D.A total of 15 somatic hybrids were obtained.The leaves of the hybrids seedling was dark green and thick,covered with wax layer.The adult plants were shorter and flowered later than B.napus.Their stamens were ateleiosis;and the chromosomes number was 48～66 in somatic cells.