棉花(Gossypium hirsutum L.)原生质体培养的体细胞胚胎发生及植株再生

Somatic Embryogenesis and Plant Regeneration from Protoplast Culture of Cotton (Gossypium hirsutum L.)

以陆地棉(G.hirsutum L.)3118、晋棉4号和柯字棉312等3个品种为材料,取种子无菌苗的下胚轴诱导愈伤组织,建立胚性细胞悬浮培养系。以2％纤维素酶Ono-zuka RS和0.1％离析软化酶Y-23组成的混合酶液,从细胞悬浮培养物游离出原生质体。用含低融点琼脂糖的K_3培养基包埋原生质体,采用园柱及薄层漂浮平板培养方式。3个品种的原生质体培养都得到了愈伤组织,其中晋棉4号和柯字棉312品种的愈伤组织转到低激素水平或无激素的MS-2培养基上诱导产生胚状体,胚状体在无激素的MS-1培养基上形成了植株。

Until now protoplast culture of cotton remained at the level of small cell clusters and calli. Although EI-Shihy and Evans had briefly reported that regenerated plants from protoplasts of Gossypium barba-dense L. had been obtained, their thesis has not been found so far. The first instance of the regenerated plants from the protoplasts of G. hirsutum L. is reported here. The protoplasts were isolated from the hypocotyl-derived suspension cultures of G. hirsutum L. cultivars 3118, Jinmian 4 and Coker 312. The enzymes consisted of 2% ( W/V ) cellulase Onozuka RS and 0.1% pecto-lyase Y-23. The protoplasts were embedded with K3 medium containing agarose of low melting point and supplemented with 0.1-0.5 mg/1 2,4-D and 0.1-0.5 mg/1 KT, and cultured at a density of 2-3×l05/ml in droplets or thin layers. The protoplasts started to divide after 3-4 days of culture. Three weeks after culture, calli were observed in all of the three cultivars. Callus pellets formed from the protoplasts were slender, soft and white. The calli became yellow and compact after they were transferred in MS-1 medium plus 0.5 mg/1 2,4-D and 1 mg/1 KT. Then, when the calli were transferred to a hormone-free or lower concentration MS-2 medium,embryoids were obtained in Jinmian 4 and Coker 312. Regenerated plantlets occurred from the embryoids transferred in a hormone-free MS-1 medium. In a 5-month period, the whole process from protoplast culture to plant regeneration was completed. It is important to regulate the medium composition according to the color and status of cultures for successful protoplast culture. When the cell suspension cultures are turning white, slender and slower growth, the concentrations of salt ions and organic nitrogen and the level of hormones need to be enhanced.