Plants with modified growth
a technology of plant growth and growth, applied in the field of plants with modified growth, can solve problems such as the inability to collect molecular cell cycle data in plant systems
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example 1
[0142] Construction of the Chimeric Genes
[0143] 1.1 Construction of the CaMV35S-AthcycD2 Chimeric Gene and inclusion in a T-DNA Vector.
[0144] A 1298 bp NcoI-SacI fragment comprising the DNA encoding CYCD2 from A. thaliana (having the nucleotide sequence of EMBL Accesion N.degree. X83370 from nucleotide position 194 to nuceotide position 1332) was treated with Klenow polymerase to render the protruding termini blunt, and ligated to Smal linearized pART7 (Gleave, 1992), yielding plasmid pCEC1. In this way, a chimeric gene flanked by NotI sites was constructed, wherein the DNA encoding the CYCD2 was operably linked to a CaMV35S promoter of the CabbB-J1 isolate (Harpster et a., 1988) and a 3'ocs region (MacDonald et al., 1991). The chimeric gene was then inserted between the T-DNA border of a T-DNA vector, comprising also a selectable chimeric marker gene.
[0145] To this end, the chimeric cycD2 gene was excised from pCEC1, using NotI, and ligated to NotI linearized pART27 (Gleave, 1992) ...
example 2
[0151] Agrobacterium-mediated Transformation of Tobacco Plants with the T-DNA Vectors of Example 1.
[0152] T-DNA vectors pCEC5 and pCRK9 were introduced in Agrobacterium tumefaciens LBA4404 (Klapwijk et al., 1980) by electroporation as described by Walkerpeach and Velten.(1995) and transformants were selected using spectinomycin and tetracycline respectively.
[0153] T-DNA vectors pCEC5b and pCRK9b are introduced in A. tumefaciens C58C1 Rif.sup.R by triparental mating (Diffa et al., 1980).
[0154] The resulting Agrobacterium strains were used to transform Nicotiana tabacum var Xanthi, applying the leaf disc transformation method as described in An et al. (1985).
[0155] Eight tobacco plants transformed with pCRK9 (designated 1 K9, 2K9, 3K9, 4K9, 8K9, 10K9, 17K9, 19K9 and 28K9) were generated and eleven tobacco plants transformed with pCEC5 (designated C8 lines 1 to 3 and 5 to 12).
[0156] Plants transformed by pCRK9 T-DNA were analyzed for the copy number of the inserted transgenes by Southe...
example 3
[0161] Phenotypic Analysis of the Transformed Tobacco Plants.
[0162] 3.1. Tobacco Plants Comprising the CaMV35S-AthCycD2 Chimeric Gene.
[0163] Seeds from primary transformants (T0 plants) were surface sterilized in 10% bleach for 15 minutes and thoroughly washed in sterile water. The surface-sterilized seeds were germinated on GM medium containing kanamycin to a final concentration of 100 .mu.g / ml. Seeds on plates were placed for 5 days at 4.degree. C. (vernalization) and then moved to 23.degree. C. in a growth chamber. All time points refer to the day of placing in the growth chamber. Eighteen days after moving to the growth chamber (ie after 23 days in total), the kanamycine-resistant seedlings were transplanted into seed trays containing soil, and grown under 18 hr photoperiod in a growth room. After a further 10 days these plants were transferred to 3 inch plant pots and after an additional 15 days to 8 inch plant pots where they remained for the rest of the experiment. The 3 inch...
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