《植物生理学报》 2012, 48(10): 1015-1020

蓝猪耳离体受精初试

于金金¹, 成诚¹, 王雅英², 张亚楠², 田惠桥^1,*

1厦门大学生命科学学院, 厦门361005; 2厦门医学高等专科学校, 厦门市中医生物工程重点实验室, 厦门361008

通信作者：田惠桥；E-mail: hqtian@xmu.edu.cn；Tel: 0592-2186486

摘　要：

用体内-体外方法分离了蓝猪耳精细胞。用酶解和解剖方法分离了其成熟卵细胞。分离的精、卵细胞用电融合介导 尝试了体外诱导融合。在合适的渗透压(6%甘露醇)和合适的氯化钙(0.04% CaCl2•2H2O)溶液中, 用交流电场为30~35 V,10~25 s使精、卵细胞排队; 用直流电场400~600 V, 45~50 µs的脉冲穿孔条件可诱导30%的精、卵细胞融合和70%以上的卵 细胞之间的融合。尝试了人工合子的单细胞培养但未获成功。诱导蓝猪耳精、卵细胞融合的条件与玉米和水稻不同。

关键词：蓝猪耳; 精细胞; 卵细胞; 电融合; 离体受精

收稿：2012-08-30   修定：2012-10-07

资助：国家自然科学基金(30970275和31170289)和公益性行业(农业)科研专项经费(200903016)

The Primary Attempt of in Vitro Fertilization of Torenia fournieri

YU Jing-Jing¹, CHENG Cheng¹, WANG Ya-Ying², ZHANG Ya-Nan², TIAN Hui-Qiao^1,*

¹School of Life Sciences, Xiamen University, Xiamen 361005, China; ²Xiamen Key Laboratory of Biological Engineering for Traditional Chinese Medicine, Xiamen Medical College, Xiamen 361008, China

Corresponding author: TIAN Hui-Qiao; E-mail: hqtian@xmu.edu.cn; Tel: 0592-2186486

Abstract:

Sperm cells of Torenia fournieri were isolated from pollen tubes which were induced using an in vivo-in vitro method. Its egg cells were isolated by enzyme digesting followed by mechanical dissection. The electrical fusion method was applied to induce the fusion of the isolated sperm and egg cells. In a suitable solution containing 6% mannitol and 0.04% CaCl₂ ·2H₂O, 30% sperm-egg and over 70% egg-egg could be induced fusion using alternating current (30–35 V, 10–25 s) to align sperm-egg or egg-egg, following direct current (400–600 V, 45–50 µs) pulse to perforate the plasma member of both sperm and egg. The artificial zygotes were also tried to culture but no success. The conditions of inducing fusion of sperm cells and egg cells of Torenia fournieri are different with those of maize and rice.

Key words: Torenia fournieri; sperm cell; egg cell; electrical fusion; in vitro fertilization