Genetic transformation of sorgum [Sorgum bicolor (L). Moench] plants using Agrobacterium tumefaciens and the shoot apex /
Combining transformation by Agrobacterium and the shoot meristembased regeneration system is an alternative technique to improve sorghum (Sorghum bicolor L.) for which efficient and reliable methodology does not exist. Shoot apices isolated from germinated seedlings of sorghum cultivars, RTx430 and...
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| Format: | Thesis Book |
| Language: | English |
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[Place of publication not identified] :
[publisher not identified] ;
1995.
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| Online Access: | http://proxy.library.tamu.edu/login?url=http://proquest.umi.com/pqdweb?did=742161621&sid=1&Fmt=2&clientId=2945&RQT=309&VName=PQD |
| Summary: | Combining transformation by Agrobacterium and the shoot meristembased regeneration system is an alternative technique to improve sorghum (Sorghum bicolor L.) for which efficient and reliable methodology does not exist. Shoot apices isolated from germinated seedlings of sorghum cultivars, RTx430 and BTx3l97 were cocultivated with A. tumefaciens EHAL 01 carrying gus, NOSNPT //and bar genes under the control of CAMV 35S or rice actin 1 promoters. Inoculated shoots with CAMV 35S-gus were recovered rapidly to rooted plants (14%) within 3-5 weeks without selection pressure. The GUS activity in regenerated plants was undetectable in histochemical assays. The frequency of putative transformation, estimated from PCR amplification of introduced genes was 20%. Due to difficulties in the identification of actual transformants by the GUS assay and Southern analysis, proof of transformation relied on progeny tests. The Fl plants derived from a putative transformant indicated that both foreign genes (gus and nosnpt /@ were present via PCR analysis. Genomic restriction and Southern analyses confirmed that the introduced genes were stably incorporated into high molecular weight plant genomic DNA and transmitted into Fl and F2 generations. The possibility that the transferred genes under the control of the 35S promoter would not be expressed in transformed plants was considered. A new construct driven by rice actin 1 promoter on GUS expression was evaluated by histochemical and fluorometric assays. The promoter derived from Act 1 promoter produced significantly higher GUS activity levels compared to the CAMV 35S promoter. Recent experiments were conducted with two plasmid constructs containing either a 35S or Act 1 promoters driving the bar gene. The bar gene confers resistance to phosphinothricin, and transformed plants were selected in vitro with 0.5 mg/l PPT. Data from genomic Southern analyses and enzyme assays for primary plants proved that the introduced gene was stably integrated into plant genomic DNA and expressed. Fl plants from putative transformants were analyzed to confirm the sexual transmission of introduced gene and stable expression in progeny. |
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| Item Description: | Vita. "Major Subject: Plant Physiology and Plant Biotechnology". |
| Physical Description: | x, 107 leaves : illustrations ; 28 cm. Issued also on microfiche from University Microfilms Inc. |
| Bibliography: | Includes bibliographical references. |