Agrobacterium-mediated transformation of the maize shoot apex and the expression and inheritance of transgenes /
The use of Agrobacterium-mediated transformation and the shoot apex based regeneration system is a rapid technique to improve maize (Zea mays L.) for which a reproducible methodology has been endlessly sought. Shoot apices isolated from germinated seedlings of maize genotypes Embu Pool A and Embu P...
| Main Author: | |
|---|---|
| Format: | Thesis Book |
| Language: | English |
| Published: |
[Place of publication not identified] :
[publisher not identified] ;
1996.
|
| Subjects: | |
| Online Access: | http://proxy.library.tamu.edu/login?url=http://proquest.umi.com/pqdweb?did=739667551&sid=1&Fmt=2&clientId=2945&RQT=309&VName=PQD |
| Summary: | The use of Agrobacterium-mediated transformation and the shoot apex based regeneration system is a rapid technique to improve maize (Zea mays L.) for which a reproducible methodology has been endlessly sought. Shoot apices isolated from germinated seedlings of maize genotypes Embu Pool A and Embu Pool B were cocultivated with four strains of Agrobacterium tumefaciens, all carrying the bar/pat gene together with one carrying a potato proteinase inhibitor 11 gene (pin2) and three carrying Bacillus thuringiensis CrylF gene. The bar/pat genes confer resistance to the herbicide glufosinate-ammonium (PPT). Experiments to determine the survival rates of isolated shoot apices on media containing PPT, established that no shoots survived above 2. 0 mg L-' PPT. Several factors were identified as influencing the regeneration of putative transformed plants. Results demonstrated that wounding of shoot apices highly reduced their regeneration capability even though the wounding treatment of meristematic cells was supposed to promote transformation. Use of different strains of Agrobacterium and plasmid vectors did not show any variation in regeneration efficiencies of putative transformed plants. Differences were observed in the number of plants regenerated between selected and unselected treatment. Results also showed that pre-induction of Agrobacterium with either acetosyringone or with potato shoot extract did not improve the number of regenerated plants. Putative transformed plants were selected in vitro in medium containing 1.0 mg L-' PPT and 3.6% (40 out of 1124 shoot apices) survived and regenerated into plants in 3-5 wk. These plants were expected to show the bar/pat gene expression after herbicide treatment. Most plants died after treatment although a few showed different responses and they were regarded as chimeric. Southern analyses revealed no introduced genes in the samples from primary regenerants. Due to the difficulties in the identification of primary transformants by herbicide application and Southern analyses, proof of transformation relied on progeny tests. Data from herbicide treatment of progeny of the PPT selected primary plants indicated that bar/pat genes were not expressed in the pro analyses failed to confirm that the introduced genes weregeny. Southern integrated into the maize genomic DNA. |
|---|---|
| Item Description: | Vita. "Major Subject: Plant Breeding". |
| Physical Description: | xi, 79 leaves : illustrations ; 28 cm. Issued also on microfiche from University Microfilms Inc. |
| Bibliography: | Includes bibliographical references: pages 67-78. |