摘要
以小麦品种济南１７７、３８４、４７１的幼胚和济南１７７的胚性愈伤组织为材料，质粒为ｐＰＰＩ２［含大麦黄矮病毒外壳蛋白（ＣＰ）基因和ＧＵＳ（β－葡萄糖苷酸酶）基因］；ｐＰＰＩ５（含ＣＰ基因）＋ｐＥｍｕＧＮ（含ＧＵＳ基因），用高速基因枪轰击钨粉质粒进入幼胚和胚性愈伤组织细胞．ＧＵＳ基因的瞬间表达平均频率幼胚约为４２％，愈伤组织为１８．５％．转化处理后用ＰＣＲ扩增技术检测植株中ＣＰ基因是否存在并稳定遗传．检测结果表明，来源于幼胚的Ｔ０代转化频率为２～９％（１９９３～１９９４年），并获得Ｔ１、Ｔ２和Ｔ３代稳定表达的转化植株．用愈伤组织转化，其转化频率３月龄者为０．４％；２年龄者为零．潮霉素（Ｈｍ）用于对转化幼胚和愈伤组织的筛选，低浓度时不能抑制非转化细胞的生长，高浓度则使细胞受伤害。
Transformation via bombardment of immature embryos Wheat influorescences about 12 days after pollination were surface sterilized with 70% alcohol for 5 sec. Transparent immature embryos were excised and inoculated with the scutellum of embryos exposed onto a modified MS medium containing 2 mg/L 2,4-D. They were incubated at 25℃ in the dark for 3￣4 days until small proliferat ing tissue was visible at the edges of the embryos. At this time,the embryos were bombarded by a high velocity gene gun apparatus, type JQ-700 (Manufactured by Institute of Biophysics, Academy of Sciences,P R C).Before bombardment,CaCl2 and spermidine were employed to coat the tungsten particles of 1. 09 μm diameter. The coated particles were dropped on the small hole of the stopping plate,which was 5. 5 cm above the target embryos plated on the agar medium in a dish. The velocity of the bullet was about 440 m/s. Under the air pressure of 0. 085￣0. 092 MPa.Two kinds of plasmids were used in the experiment: (1)pPPI2 and (2)pPPI5+pEmuGn (1: 1) (co-transformation). (Fig. 1)24 hs after bombardment, 100 embryos were randomly sampled to assay the GUS activity by incubation with X--Glue buffer. There was no remarkable difference in activity of GUS gene expression between pPPI2 and pPPI5+pEmuGN.7￣ 10 days following bombardment,the embryos were transferred to culture medium without hormone to induce differentiation. When 2￣ 3 leaflets were formed, the seedlings were removed to a medium containing MET for strengthening and rooting.They were then planted in the plastic pots with loamy soil and placed in green house.In 1993 a total of 600 embryos of wheat cv. Jinan 177 were bombarded and 108plants survived in soil. Among them 13 were proved transgenic upon PCR assay as described (Cheng et al. 1994), 11 were fertiled and set seeds. 48 seeds were taken randomly and sown in soil. At 3--leaf stage of the T1 seedlings,their DNA were extracted and assayed and 9 of them were proved PCR positive.In 1994,in addition to cv. Jinan 177,cv. 384 and cv. 471
出处
《山东大学学报（自然科学版）》
CSCD
1996年第1期94-101,共8页
Journal of Shandong University(Natural Science Edition)
基金
国家863高技术项目
关键词
小麦
微弹轰击
BYDV
CP基因
转基因可育植株
Wheat (Triticum aestivum)
Microprojectile bombardment
BYDV CP gene
Fertile transgenic wheat plants
