Document Type

Thesis - University Access Only

Award Date

2003

Degree Name

Master of Science (MS)

Department / School

Biology and Microbiology

First Advisor

Thomas Cheesbrough

Abstract

Cholinephosphotransferase (CPf) is an important enzyme reversibly catalyzing a reaction, in which diacylglycerol (DAG) is converted to phosphatidycholine (PtdCho). Monounsaturated-PtdCho is in tum the substrate for polyunsaturated-PtdCho. Reversal of the CPf reaction with polyunsaturated-PtdCho results in increase of polyunsatu-ated fatty acids in soybean seeds. DAG is mainly the substrate for triacylglycerol (TAG) synthesis. Therefore, the down regulation of CPr catalysis is expected to increase monounsaturated fatty acids content and improve the quality of soybean oil. This pu-pose may be achieved by the insertion of a CPr antisense gene. To transfer the CPf antisense gene into soybean, plasmid pCB-GFl was constructed previously. Plasmid pCB-GFl, 6293bp, harboring a CPT antisense gene and a marker gene, bialaphos resistance gere (Bar), located between the T-DNA borders. This plasmid was designed for Agrobacterium-mediated transformation, which is a biological regulation process. The identification of some factors influencing Agrobacterium-mediated transformation has facilitated the development of plant transformation. However, Agrobacterium inoculum density, acetosyringone (AS), and light condition for soybean optimum transfonnation are still unknown. Therefore, the objective of this project is to investigate the factors influencing Agrobacterium-mediated transformation of CPf antisense gene in soybean. After the plasmids were transferred into Agrobacterium tumefaciens EHA105, six experiments were constructed, and the factors including bacterium inoculum density, AS concentration, light intensity, photoperiod, transferred genes, auxins, and antibiotics were investigated using a standard protocol for Agrobacterium-mediated soybean transformation of cotyledonary nodes. Presumed transgenic shoots were selected by herbicide (glufosina te) during shoot initiation and elongation. Our results indicated that AS concentration, light (photoperiod), and genes transferred affected transfonnation efficiency significantly. The optimal efficiency of transformation may be achieved under the following conditions: Agrobacterium inoculation density of O~oo=l.0, AS concentration of 50 ?M, photoperiod of 16 hours with light intensity of 25? mol ni1 s-? during co-cultivation. This study also suggested that it is necessary to supplement exogenous auxins indole-3-butyric acid (IBA) in rooting medium when antibiotics are used.

Library of Congress Subject Headings

Soybean -- Genetic engineering
Plant genetic transformation
Agrobacterium
Phophotransferases

Publisher

South Dakota State University

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Rights Statement

In Copyright