广藿香原生质体制备、培养与融合技术优化研究

Optimization of protoplasts preparation, culture and fusion in Pogostemon cablin

关键词:  广藿香, 聚乙二醇, 原生质体制备, 原生质体融合, 新品种选育

Key words:  Pogostemon cablin, PEG, protoplast preparation, protoplast fusion, breeding

摘要:

为建立高效稳定的广藿香原生质体培养与融合技术体系,该研究以广藿香愈伤组织悬浮细胞为材料,研究了原生质体制备的酶解条件和培养方法、细胞密度、激素种类和浓度等因素对原生质体培养的影响,并通过测定融合产物直径确立融合细胞筛选范围,进一步研究聚乙二醇浓度、细胞密度、融合时间及融合液加入量等因素对原生质体融合的影响。结果表明:制备原生质体的适宜条件为pH5.8,酶解温度25 ℃; 原生质体培养以铵盐减半的MS₁培养基进行海藻酸钠包埋、激素选用0.2 mg·L^-1 NAA、2.0 mg·L^-1 6-BA,培养密度2.0×10⁵个·mL^-1、蔗糖添加量1.0%、酸水解酪蛋白500 mg·L^-1的条件下原生质体分裂频率、植板率均较高,且开始分裂时间和细胞团形成时间都较短; 双细胞融合产物筛选范围为69.33～87.35 μm; 以40% PEG 6000化学促融30 min、加入0.5倍体积的融合液、细胞密度2.0×10⁵个·mL^-1的条件进行原生质体融合,聚合率可达57.19%; 获得的融合产物经海藻酸钠包埋培育2个月后可观察到再生愈伤组织。

Abstract:

For establishing a high efficient and stable protoplast culture and fusion technology system, the protoplasts obtained from Pogostemon cablin's callus, were studied as the material for enzymatic hydrolysis conditions of protoplast preparation, and on this base, the effects of culture methods, cell density, hormone types and concentrations on the protoplast culture were studied. Screening range of fusion cells was determined by measuring the diameter of fusion products. The effects of PEG concentration, cell density, incubation time and amount of fusion fluid on the fusion rate of protoplasts were studied by single factor analysis. The results showed that the vitality of protoplast reached the highest value at 25 ℃ and the optimal pH for enzymatically hydrolyzing was 5.8. The improved medium MS₁, which consisted half of ammonium salt(NH₄NO₃ 825 mg·L^-1), added the hormones about 0.2 mg·L^-1 NAA and 2.0 mg·L^-1 6-BA was used to embed in sodium alginate. With the conditions of 2.0×10⁵ protoplasts·mL^-1 of the cell density, 1% sucrose content and 500 mg·L^-1 acid hydrolyzed casein, protoplasts got into division and formation of cell clusters earlier and had the higher division frequency and plating efficiency. The range of double cell fusion products was 69.33-87.35 μm; 40% PEG 6000 was selected to promote the chemical fusion. With the increase of fusion time, cell density and amount of fusion liquid, both polymerization rate and polycondensation rate increased. With 0.5 times volume of the fusion solution, 2.0 × 10⁵ protoplasts·mL^-1 of the cell density and 30 min of fusion time, the polymerization rate was up to 57.19%. After two months' embedding cultivation, the callus was observed.