Agrobacterium Mediated Transformation of Cp Genome,Phosphotransferase,Chlormphenicol,Hybridization

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Transplastomic Plants and Chloroplast Engineering Transplastomic Plants and Chloroplast Engineering Introduction Engineering of Chrloroplast Genome in Chlamydomonas Transformation of Chloroplast (Cp) Genome in Higher Plants Advantages of Chloroplast Transformation Difficulties in Producing Transplastomic Plants Methods of Tranformation of Cp Genome Agrobacterium Mediated Transformation of Cp Genome Stable Transformation of Cp Genome Using Particle Gun Method Other Possible Methods for Introduction of DNA into Plastids DNA Uptake by Chloroplasts in vivo

Home >> Plant Biotechnology and Genomics >> Transplastomic Plants and Chloroplast Engineering >>Agrobacterium Mediated Transformation of Cp Genome

Agrobacterium mediated transformation of Cp genome. A Ti plasmid vector was constructed using a chimeric gene construct consisting of the following: (i) promoter region of nopaline synthase gene (Pnos); (ii) coding sequence of chloramphenicol acetyltransferase (CAT) gene (cat); (iii) complete gene for nopaline synthase (nos); (iv) gene for amino glycoside phosphotransferase type Ii or APH Ii (aphII) and (v) the gene for lactamase (bla). The structure of the T-DNA construct carrying the above sequences.

The gene construct was introduced into tobacco leaf protoplasts by co cultivation with A. tumefaciens containing the hybrid Ti plasmid. Calli were derived from co cultivation protoplasts on a medium containing chloramphenicol, so that chloramphenicol resistant (Cm^R) calli were selected. Of these Cm^R calli, only 8% had nopaline, suggesting that nos gene was lost or inactivated in majority of cases. Of 20 plantlets regenerated from Cm^R nos^- calli, only eight (40%) were able to root on medium containing chloramphenicol. One of these plants exhibited the presence of CAT activity and APH II activity in chloroplast fraction obtained after cell fractionation. In reciprocal crosses with wild plants, it was found that cat gene was transmitted through the ovule and not through the pollen (maternal inheritance), confirming location of cat gene in the Cp genome.

The presence of cat gene in chloroplast genome was further confirmed, when Southern blots derived from XhoI restriction digests of nuclear, mitochondrial and chloroplast DNAs were mitochondrial and chloroplast DNAs were hybridized with a probe having cat gene (only CpDNA showed hybridization). Evidence was also available showing integration of cat and aphII genes with the chloroplast DNA.

Agobacterium Mediated Transformation of Cp Genome

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