番木瓜成熟种子的体细胞胚简易诱导和植株再生

A Simple Protocol of Plant Regeneration of Carica papaya L. Through Somatic Embryogenesis from Mature Seeds

目前中国番木瓜产业受到环斑花叶病等病害的严重威胁,利用生物技术手段创制新种质、培育高产优质的抗病新品种成为番木瓜产业发展的亟需。本研究以‘一尺瓜’品种的140~150 d龄果实成熟种子为外植体,研究了其体细胞胚诱导和植株再生技术。结果发现了一种非常简单有效的外植体杀菌消毒方法,即用自来水将果实外表清洗干净,然后用70%酒精对果实进行表面消毒即可。该品种成熟种子理想的胚性愈伤组织诱导培养基组成为:1/2 MS(Murashige and Skoog,1962)+2 mg/L 2,4-D(2,4 dichlorophenoxyacetic acid)+400 mg/L谷氨酰胺+60 g/L蔗糖+7 g/L琼脂粉,胚性愈伤组织诱导率可达45%。将胚性愈伤组织置于含1 mg/L2,4-D的增殖培养基继代培养2个周期后,可获得大量均质的表面含有白色体细胞胚的胚性愈伤组织。胚性愈伤组织团在不含任何激素的培养基上可完成体细胞胚的诱导、成熟、萌发和生根过程。挑取具有芽和根的体细胞胚转移到再生培养基培养30 d后可获得具有正常叶片和根系的完整植株,其发育为正常植株的百分率为52.5%。本研究为番木瓜生物技术育种建立一套简单、高效的体细胞胚再生技术体系提供技术支撑。

At present, the papaya industry in China was seriously threatened by the diseases such as ring Mosaic virus, and it was urgent to use biotechnology to create new germplasms and breed high yield and high quality anti-disease new varieties. In this experiment, the seeds of the mature seeds from 140 to 150 days ＇Yi Chigua＇cultivar（Carica papaya L.） were cultivated as explants, and studied the induction of somatic embryo and plant regeneration. During the study, a very simple and effective sterilization method for explants was found. The surface of the fruit was cleaned with tap water, and then the fruit was disinfected with 70% alcohol. The optimum medium for friable embryogenic calluses induction of mature seeds was half-strength MS（Murashige and Skoog, 1962） basal salts and vitamins, 400 mg/L of glutamine and 60 g/L sucrose supplemented with 2.0 mg/L2,4-D（2,4 dichlorophenoxyacetic acid）, and the induction rate reached to 45%. After embryogenic callus was subcultured for 2 cycles on the embryogenic calluses induction medium, in which the 2,4-D concentration decreased to 1 mg/L, a large number of homogeneous embryogenic calluses with whitesomatic embryos in thesurfacewere obtained. Embryonic callus can complete somatic embryo induction, maturation, germination and rooting in medium without any hormones. These somatic embryos with shoot and root were transferred to the plant regeneration medium（half-strength MS basal salts and vitamins, 1 mg/L IBA and 5 g/L activated carbon）, after30 days, com-plete plants with normal leaves and roots were obtained, and the percentage of normal plants was52.5%. To establish a simple and efficient technology system of plant regeneration through somatic embryogenesis can provide technical support for papaya biotechnology breeding.