柑桔抗盐突变体的原生质体培养与分离纯化研究

锦橙、桃叶橙抗盐胚性愈伤组织酶解后可得到大量的原生质体,FDA检查活性分别是92.5％和89.2％。抗盐原生质体在培养过程中首先逐渐再生细胞壁,覆盖整个细胞膜表面,然后恢复分裂。锦橙、桃叶橙抗盐原生质体经2个月培养,单个原生质体已分裂形成肉眼可见的细胞团,锦橙的细胞团平均达550×391μm,其中很多已分化形成球形胚状体,部分即将形成原生质体植株,桃叶橙原生质体的细胞团达389.2×280.0μm。

A PRELIMINARY REPORT ON PROTOPLAST CULTURE AND ISOLATION FOR SOLID MUTANTS FROM NaCl-RESISTANT CALLUS IN CITRUS

NaCl—Resistant mutants were obtained in two sweet orange eultivars "Jineheng" and "Taoyecheng" (Citrus sinensis Osbeek) by exposing their nucellar callus to mutagens ~(60)Co γ-ray(5-8Krad) or EMS(0.3%—0.5%) and screening with sodium chlorid. The mutant ealli were incubated in enzymatic solution with 0.3%-0.5% pectinase and cellulase at 26℃ for 12—16h, yielding numerous protoplasts with the viability being 92.5% in Jincheng and 89.2% in Taoyeeheng. The protoplasts were cultured in liquid MT added with 0.65mol/L sucrose or 0.14mol/L sucrose and 0.46 mol/L mannitol. One week after culture, 11.13% of the protoplasts in Jincheng and 4.40% in Taoyeeheng resumed division, forming clumps of 2—4 eells. One month later, the cell clumps reached a size of 73.3μm×53.0μm, and consisted of 20—40 cells. The frequency of divisionresuming protoplasts was 37.2% in Jincheng and 15.0% in Taoyeeheng. The individual protoplasts after two month culture, formed large number of visible cell clumps and developed many globular and heart-shaped embryos. The cell clumps formed 40 days after culture were transferred into MT medium with 0.8%-1.2% NaCl. and kept for 20 days. It was observed that a large portion of them could tolerate 0.8% NaCl and maintained the resistance. Above results suggest that by combining protoplast culture method with induced mutation technique, large number of solid (non—chimeral and stable) NaCl—resistant mutant plants can be obtained in Citrus.