Adventitious shoots formed on the proximal cut edges of different cotyledonary explants of watermelon [Citrullus lanatus (Thunb.) Matsum. & Nakai; cvs. Sweet Gem and Gold Medal] cultured on Murashige and Skoog's (MS) medium with 1 mgl(-1) 6-benzyladenine (BA). Light (16-h photoperiod, about 7 Wm(-2) cool-white fluorescent lamps) was essential for shoot formation. To obtain transformed plants, cotyledonary explants of 'Sweet Gem' were cocultured with Agrobacterium tumefaciens LBA4404, a disarmed strain harboring a binary vector pBI121 carrying the CaMV 35S promoter-β-glucuronidase (GUS) gene fusion used as a reporter gene and NOS promoter-neomycin phosphotransferase gene as a positive selection marker, for 48 h on MS medium with 1 mgl(-1) BA and 200 μM β-hydroxyacetosyringone. After 48 h of culture, explants were transferred to medium with 1 mgl(-1) BA 250 mgl(-1) carbenicillin, and 100 mgl(-1) kanamycin and cultured in the light. Adventitious shoots formed on the explants after 4 weeks of culture. When subjected to GUS histochemical assay, young leaves obtained from the shoots showed a positive response at a frequency of up to 16%. Preculturing cotyledonary explants on MS medium with 1 mgl(-1) BA for 5 d enhanced the competence of the cells to be transformed by Agrobacterium. Southern blot analysis confirmed that the GUS gene was incorporated into the genomic DNA of the GUS-positive regenerants. The transformed plants were grown to maturity.
Fourier transform infrared spectroscopy (FTIR) provides biochemical profiles containing overlapping signals from a majority of the compounds that are present when whole cells are analyzed. Leaf samples of seven higher plant species and varieties were subjected to FTIR to determine whether plants can be discriminated phylogenetically on the basis of biochemical profiles. A hierarchical dendrogram based on principal component analysis (PCA) of FTIR data showed relationships between plants that were in agreement with known plant taxonomy. Genetic programming (GP) analysis determined the top three to five biomarkers from FTIR data that discriminated plants at each hierarchical level of the dendrogram. Most biomarkers determined by GP analysis at each hierarchical level were specific to the carbohydrate fingerprint region (1,200-800 cm(-1)) of the FTIR spectrum. Our results indicate that differences in cell-wall composition and structure can provide the basis for chemotaxonomy of flowering plants.
Hypocotyl explants of Catharanthus roseus produced hairy roots when cultured on Murashige and Skoog (MS) basal medium after infection by Agrobacterium rhizogenes. Explants gave rise to adventitious shoots at a frequency of up to 80% when cultured on MS medium supplemented with 31.1 microM 6-benzyladenine and 5.4 microM alpha-naphthaleneacetic acid. There was a significant difference in the frequency of adventitious shoot formation for each hairy-root line derived from a different cultivar. Plants derived from hairy roots exhibited prolific rooting and had shortened internodes. Approximately half of the plants had wrinkled leaves and an abundant root mass with extensive lateral branching, but otherwise appeared morphologically normal. Plants with hairy roots that were derived from the cultivar Cooler Apricot developed flowers with petals that were white in the proximal region, whereas the wild-type flower petals are red. PCR and Southern blot analyses revealed that plants derived from hairy roots retained the Ri TL-DNA.
Genetic transformation is an important technique for functional genomics study and genetic improvement of plants. Until now, Agrobacterium-mediated transformation methods using cotyledon as explants has been the major approach for cucumber, and its frequency has been up to 23%. For example, significantly enhancement of the transformation efficiency of this plant species was achieved from the cotyledon explants of the cultivar Poinsett 76 infected by Agrobacterium strains EHA105 with efficient positive selection system in lots of experiments. This review is to summarize some key factors influencing cucumber regeneration and genetic transformation, including target genes, selection systems and the ways of transgene introduction, and then to put forward some strategies for the increasing of cucumber transformation efficiency. In the future, it is high possible for cucumber to be potential bioreactor to produce vaccine and biomaterials for human beings.
Agrobacterium tumefaciens-mediated transformation was used to produce transgenic watermelon. Cotyledonary explants of Citrullus lanatus Thumb (cv. Daesan) were co-cultivated with Agrobacterium strains (LBA4404, GV3101, EHA101) containing pPTN289 carrying with bar gene and pPTN290 carrying with nptII gene, respectively. There was a significant difference in the transformation frequency between bacteria strains and selective markers. The EHA101/pPTN289 showed higher transformation frequency (1.16 %) than GV3101/pPTN289 (0.33 %) and LBA4404/pPTN289 or /pPTN290 (0 %). The shoots obtained (633 and 57 lines) showed some resistance to glufosinate and paromomycin, respectively. Of them, the β-glucuronidase positive response and PCR products amplified by bar and nptII specific primers showed at least 21 plants resistant to glufosinate and at least 6 plants to paromomycin. Southern blot analysis revealed that the bar gene integrated into genome of transgenic watermelon. Acclimated transgenic watermelons were successfully transplanted in the greenhouse and showed no phenotypic variation.
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