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以红姜花(Hedychium coccineum)未成熟花丝和花药为试材,研究了红姜花胚性愈伤组织的诱导、细胞悬浮体系的建立以及原生质体的培养。结果表明:在MS+4mg/L 2,4-D+4mg/L NAA+1mg/L 6-BA+30g/L蔗糖+7g/L琼脂上经过120d培养诱导出了愈伤组织,愈伤组织在增殖培养基上经过继代筛选获得浅黄色、松散易碎的胚性愈伤组织。胚性愈伤组织通过3个月的悬浮培养,得到均质稳定的胚性细胞悬浮系。以胚性悬浮细胞(ECS)为起始材料分离原生质体,酶解试验表明,在原生质体分离过程中,用于原生质体分离的酶液需要保持一定的渗透压以保护原生质体不被破坏。当甘露醇的浓度为0.14mol/L时,原生质体产量最高,达到2.25×105个/mL PCV ECS(packed cell volume,PCV)。在看护培养系统中,原生质体经过7d的培养,细胞第一次分裂,经过14d培养,细胞分裂频率达到12.3%,28d时,细胞团形成频率达到4.2%。所形成的细胞团具有典型的胚性细胞特征。
The embryogenic callus induction, establishment of cell suspension system and protoplast culture of red ginger flower were studied with immature filaments and anther of Hedychium coccineum. The results showed that callus and callus were induced after 120 days culture on MS + 4mg / L 2,4-D + 4mg / L NAA + 1mg / L 6-BA + 30g / L sucrose + 7g / After subculture on proliferation medium, embryogenic callus, which is light yellow, loose and fragile, was obtained. Embryogenic callus by 3-month suspension culture to obtain a homogeneous and stable embryogenic cell suspension system. Protoplasts were isolated from embryogenic suspension cells (ECS). The enzymolysis experiments showed that during the process of protoplast isolation, the enzyme solution used for protoplast isolation needs to maintain a certain osmotic pressure to protect the protoplasts from damage. When mannitol concentration was 0.14mol / L, the protoplast yield was the highest, reaching 2.25 × 105 cells / mL PCV ECS (PCV). In the nursery culture system, the protoplasts were cultured for 7 days and the cells were divided for the first time. After 14 days of culture, the frequency of cell division reached 12.3%. At 28 days, the formation rate of the cells reached 4.2%. The resulting cell mass has the typical characteristics of embryogenic cells.