Transformation of soybean [Glycine max (L.) Merrill] using proliferative embryogenic tissue maintained on semi-solid medium

被引：45

作者：

Santarém E.R. ^{[1
,2
]}

Finer J.J. ^{[1
]}

机构：

[1] Dept. of Hort. and Crop Science, OARDC, Ohio State University, Wooster

[2] Universidade de Cruz Alta, Lab. de Cultura de Tecidos Vegetais, Campus Universitario, Cruz Alta-RS 98025-810

来源：

In Vitro Cellular & Developmental Biology - Plant | 1999年 / 35卷 / 6期

关键词：

D20; medium; Embryogenesis; Hygromycin resistance; Particle bombardment;

D O I：

10.1007/s11627-999-0067-0

中图分类号：

学科分类号：

摘要：

Transgenic soybean can be efficiently produced by particle bombardment of embryogenic suspension culture material. Unfortunately, the time required to obtain a transformation-competent soybean suspension culture line is often lengthy and can result in reduced fertility of regenerated plants. In addition, establishment and maintenance of embryogenic suspension cultures can be very difficult. The objective of this work was to minimize the time required to obtain transformation-competent embryogenic tissue and optimize DNA delivery into that tissue. Somatic embryos were induced from immature cotyledons of soybean [Glycine max (L.) Merrill cv 'Jack'] by placement of cotyledons, adaxial side up, on a MS-based induction medium containing 40 mg (181 μM) 2,4-dichlorophenoxyacetic acid (2,4-D) per 1 and 6% sucrose. Embryogenic tissues, which formed from the surface of the cotyledons within 2-4 wk, were transferred to an embryo proliferation medium containing 20 mg (90 μM) 2,4-D per 1 and 3% sucrose. After 4 wk, proliferative embryogenic tissue could be used for transformation via particle bombardment. Desiccation of target tissue, period of subculture prior to bombardment, and the number of bombardments per target tissue were evaluated for enhancement of transient β-glucuronidase (GUS) expression. The highest number of blue loci was observed when the target tissue was desiccated for 10 min in an uncovered Petri plate containing proliferation medium, subcultured on the same day of bombardment, and bombarded three times on a single day. For stable transformation, selection was started 20 d after bombardment using 9 mg hygromycin per 1 for 4 wk, and 18 mg per 1 thereafter. Stably transformed clones were obtained from tissue bombarded once and twice on a single day. GUS assays and Southern hybridization analysis of DNA from putative clones confirmed stable integration of the introduced genes. Fertile transgenic plants were obtained in 11-12 mo following culture initiation.

引用

页码：451 / 455

页数：4

共 28 条

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