沙田柚杂种胚性愈伤组织的诱导及遗传鉴定

Induction and Genetic Identification of Embryogenic Calli from Hybrids of Shatian Pummelo

目的沙田柚(Citrusgrandis[L.]Osbeckcv.Shatian)是中国的特有品种,其愈伤组织难以诱导。为进一步进行细胞工程研究,本研究通过与其它品种杂交的方法获得杂种胚性愈伤组织。方法以沙田柚为母本,用体细胞杂种橘柚+无酸甜橙([C.reticulataBlanco×C.paradisiMacf.]cv.Nova+C.sinensis[L.]Osbeckcv.Succari)为父本进行杂交,90d后将幼胚取出,在MT+ME(麦芽提取物,500mg·L-1)和MT+GA3(1mg·L-1)两种固体培养基上培养。结果幼胚培养约5个月左右,在两种培养基上从胚状体和幼小植株的下胚轴处都长出了可继代保存的胚性愈伤组织,经细胞流式仪和SSR鉴定,该胚性愈伤是三倍体杂种愈伤,继代保存两年后,在MT+乳糖(50g·L-1)培养基上仍可以产生大量的胚状体,具有较强的胚状体发生能力。结论沙田柚与体细胞杂种杂交后,可以较容易地获得其杂种的胚性愈伤,对沙田柚种质资源的保存和利用具有重要价值。

[Objective] Shatian pummelo （Citrus grandis [L.] Osbeck cv. Shatian） is a peculiar variety in China and it is difficult to regenerate embryogenic callus. Other varieties were used to cross with it in order to gain the callus for further research on cell engineering. [Method] Shatian pummelo was used as the female parent and crossed with the somatic hybrid NS （Nova Tangelo ＋ Succari Sweet orange, [C. reticulata Blanco x C. paradisi Macf.] cv. Nova ＋ C. sinensis [L.] Osbeck cv. Succari）. About 90 days after pollination, the embryos obtained from crosses were cultured on the solid media of MT ＋ ME （malt extraction, 500 mg·L^-1） and MT ＋ GA3 （1 mg·L^-1）. [Result] The embryogenic callus was induced from the embryoids and plantlets＇ hypocotyls and can be subcultured. Flow cytometry and SSR analysis verified that the callus was from the triploid hybrid. The callus showed strong capability of embryogenesis and was able to produce a large number of embryoids on MT＋ Lactose （50 g·L^-1） medium after subcultured for two years. [Conclusion] It is comparatively easy to gain embryogenic callus when Shatian pummelo is crossed with somatic hybrid. The callus is valuable for the conservation and utilization of Shatian pummelo germplasm.