Department of Horticulture, Michigan State University, 48824, East Lansing, MI, USA.
Plant Cell Rep. 1995 Jul;14(10):652-6. doi: 10.1007/BF00232732.
Asymmetric somatic hybrid plants were obtained by a modified PEG/DMSO fusion procedure between protoplasts derived from suspension cells of an interspecific tomato hybrid, Lycopersicon esculentum x L. pennellii, and mesophyll protoplasts of Solanum melongena, eggplant. The tomato hybrid was previously transformed with Agrobacterium tumefaciens and contained the kanamycin-resistance marker gene. Prior to fusion, the donor protoplasts of the tomato hybrid were gamma irradiated at 9.0 krad. Thus, non-division of irradiated tomato hybrid protoplasts coupled with kanamycin sensitivity of eggplant enabled selection of somatic cell hybrids. Forty-nine calli selected post-fusion regenerated leaf-like structures in the presence of 50 mg/l kanamycin. However, only four of the 49 calli regenerated intact shoots which rooted in the presence of 50 mg/l kanamycin and were later transferred to the greenhouse. Analysis of phosphoglucoisomerase and peroxidase isozymes, and Southern hybridization with a nuclear-specific pea 45 S ribosomal RNA gene confirmed somatic hybrid status. Cytology revealed that the four hybrid plants had chromosome numbers of 45, 60, 42 and 57, respectively; they were all sterile.
通过改良的 PEG/DMSO 融合程序,将来自种间番茄杂种(Lycopersicon esculentum x L. pennellii)悬浮细胞原生质体和茄子(Solanum melongena)叶肉原生质体之间的不对称体细胞杂种获得。该番茄杂种先前已被根癌农杆菌转化,并含有卡那霉素抗性标记基因。在融合之前,番茄杂种供体原生质体被 9.0 krad 的γ射线辐照。因此,照射的番茄杂种原生质体不分裂,加上茄子对卡那霉素的敏感性,使体细胞杂种得以选择。在 50mg/L 卡那霉素存在下,融合后选择的 49 个愈伤组织再生出具有叶状结构的组织。然而,在 50mg/L 卡那霉素存在下,只有 49 个愈伤组织中的 4 个再生出完整的芽,并在温室中生根。磷酸葡萄糖异构酶和过氧化物酶同工酶的分析以及与核特异性豌豆 45S 核糖体 RNA 基因的 Southern 杂交证实了体细胞杂种的状态。细胞学研究表明,这 4 株杂种植物的染色体数分别为 45、60、42 和 57,它们都是不育的。