利用未成熟种子建立野生阿宽蕉胚性细胞悬浮系和植株再生的研究

以纵切的野生阿宽蕉60d龄未成熟种子为外植体,在MI1愈伤组织诱导培养基(MS大量和微量元素及铁盐+MorelandWetmore维生素+0.1mmol/LKH2PO4+87mmol/L蔗糖+4.5μmol/L2,4-D+1g/LGelrite)中培养60d后开始出现浅黄色松散的胚性愈伤组织,150d后愈伤组织诱导率为(15.11±1.95)%。该类愈伤组织被转移到含18μmol/L2,4-D的MI2培养基中继代60d后,可获得状态均一的理想的胚性愈伤组织。胚性愈伤组织悬浮培养后,通过2个月的筛选和继代培养,可得到均质的胚性细胞悬浮系。理想的胚性愈伤组织在体细胞胚诱导培养基中培养10d后可见到白色半透明体细胞胚的发生,体细胞胚诱导率为7.70×105个/gFW。成熟体细胞胚的萌发率为(33.33±3.37)%,植株再生率为(20.83±1.68)%。

Establishment of embryogenic cell suspensions and regeneration of Musa itinerans from immature seeds

Establishment of a stable embryogenic cell suspension (ECS) is a prerequisite for the biotechnological breeding methods. In this study a system for embryogenic cell suspension was established from 60 days old immature seeds of Musa itinerans Cheesm, an important wild germplasm in China. After culture for 60 days on callus induction medium, which consisted of MS salts and Morel and Wetmore vitamins supplemented with 4.5 μmol/L 2,4-dichlorophenoxyacetic acid (2,4-D), 0.1 mmol/L KH2PO4, 87 mmol/L sucrose, and solidified with 1 g/L Gelrite, yellow, friable embryogenic calli were induced from the explants, and (15.11±1.95)% of explants induced embryogenic calli after culture of 150 days. This kind of embryogenic calli were transferred to callus subculture medium, contained 18 μmol/L 2,4-D, to culture for 60 days, and ideal, homogeneous embryogenic calli were obtained. Suspension cultures were initiated from these embryogenic calli in liquid medium supplemented with 4.5 μmol/L 2,4-D. After selection of the cultures using a stainless steel metallic strainer with a 154-μm pore size at 15-day intervals for 2 months, homogeneous and yellow embryogenic cell suspensions, composed of single cells and small cell aggregates, were established. Planting of embryogenic callus on semi-solid medium of somatic embryo induction and development (MSD) resulted in approximately 7.70×105 somatic embryos/g fresh weight embryogenic calli. MSD contained SH macronutrients, micro-nutrients, Fe-EDTA and MS vitamins supplemented with 4.5 μmol/L biotin, 680 μmol/L glutamine, 2 mmol/L proline, 100 mg/L malt extract, 1.1 μmol/L NAA, 0.2 μmol/L zeatin, 0.5 μmol/L kinetin, 0.7 μmol/L N6-(2-isopentenyl) adenine, 29 mmol/L lactose, 130 mmol/L sucrose and solidified with 2 g/L gelrite. After 2 months of maturity on MSD, (33.33±3.37)% of somatic embryos were observed to germinate on germination media (MG), consisted of MS salt, Morel and Wetmore vitamins, 0.2 μmol/L 6-BA, 1.1 μmol/L IAA, 87 μmol/L sucrose and solidified with 2 g/L gelrite, and (20.83±1.68)% of somatic embryos could develop into normal plantlets on rooting media contained the same composition as that of MG but without auxin and cytokinin.