Genetic transformation of the cotton (Gossypium hirsutum L.) shoot apex by Agrobacterium tumefaciens /
A cotton transformation system using Agrobacterium-mediated shoot apices were isolated from two Texas genotypes MAR- CUBQHRPIS, and 'Tamcot Sphinx', and from 'Coker 312' as a control. Explants were cocultivated with 3 strains of Agrobacterium tumefaciens carrying plasmids with N...
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| Format: | Thesis Book |
| Language: | English |
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[Place of publication not identified] :
[publisher not identified] ;
1997.
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| Online Access: | http://proxy.library.tamu.edu/login?url=http://proquest.umi.com/pqdweb?did=736824931&sid=1&Fmt=2&clientId=2945&RQT=309&VName=PQD |
| Summary: | A cotton transformation system using Agrobacterium-mediated shoot apices were isolated from two Texas genotypes MAR- CUBQHRPIS, and 'Tamcot Sphinx', and from 'Coker 312' as a control. Explants were cocultivated with 3 strains of Agrobacterium tumefaciens carrying plasmids with Neomycin phosphotransferase (NPTII), Phosphinothricin acetyl transferase (Pat) and Hygromycin phosphotransferase (Hpt) genes. The NPTII gene was linked to a P-Glucuronidase GUS gene, and the Pat and Hpt genes carried the 8-endotoxin (CryIF) gene from Bacillus thuringiensis. The NPTII, Pat and Hpt genes confer resistance to kanamycin (Kn), to glufosinate-ammonium (PPT) and to hygromycin (Hyg), respectively. Experiments to determine the survival rates of shoot apices on media containing kanamycin, PPT and hygromycin, established that no shoots survived above I 00 mgL- 1 Kn, 2.5 mgL- 1 PPT and 25 mgL-1 Hyg. Several factors were identified as influencing the regeneration of putative transformed plants. Use of different strains of Agrobacterium and plasmid vectors showed variation in regeneration efficiencies of putative transformed plants. Putative transformed plants selected in vitro with 100 mgL-1 Kn resulted, in 0.8% shoot apices surviving and regenerating into plants in 2 months. For PPT, putative transformed plants were selected on a medium containing 2.5 mgL-1 PPT, resulted in 1.86% shoot apices surviving. Hyg selection at 25 mgL-1 resulted in 0.33% shoot apices surviving. It was expected that primary plants might be chimeric; therefore efforts were concentrated on screening a large number of progeny plants in the greenhouse by phenotypic evaluation. Only the progeny plants (T1) that showed resistance to the leaf application of kanamycin (2%), Ignite [ ] (0. I%) or hygromycin (1 000 mgL-1) were further evaluated by DNA blot analysis to confirm the presence of the selectable marker. To date, from 2208 progeny plants evaluated in the only four were positive for incorporation of the gene as indicated by DNA blot analysis probing for the GUS gene. From 1847 progeny plants evaluated, 29 were tolerant to PPT and from 598 progeny plants evaluated, 18 were tolerant to Hyg. DNA blot analysis revealed no introduced genes in these plants. |
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| Item Description: | Vita. "Major Subject: Plant Physiology". |
| Physical Description: | xii, 90 leaves : illustrations ; 28 cm. Issued also on microfiche from University Microfilms Inc. |
| Bibliography: | Includes bibliographical references: pages 75-89. |