Abstract
Potato late blight is a pandemic disease caused by the highly virulent (Phytophthora infestans) fungus. The regeneration capacity was done among three tested potato cultivars (Spunta, Diamont and Desiree). Two different types of explants (inter-node and leaf) were cultured for calli induction and plant regeneration .The highest value of leaf explants was for Desiree cultivar (80%), which induced from developed callus in the 2,4-D medium. The best value of maximum shoot regeneration was also for Desiree cultivar in the BA, IAA and GA3 media from leaf which proved to be more effective. Then cloning of glucanase gene in pRI plasmid which carrying kanamycin resistance (nptII) gene was perfomed and followed by transformation in Agrobacterium tumefaciens LBA4404 strain which used for plant transfection. Nucleotide and amino acid sequences of transformed Agrobacterium were analyzed. The putative transgenic plantlets genomes and cDNA of the isolated RNA for glucanase gene.
Highlights
Potato late blight is a pandemic disease caused by the highly virulent (Phytophthora infestans) fungus
The major goal of the present study are: 1) to produce transgenic potato cultivars resistant to late blight, 2) Introduce glucanase gene into potato cultivars through genetic engineering techniques by Agrobacterium mediated transformation and 3) Confirming the integration of the introduced genes in the genomic DNA of the putatively transformed potato plants using molecular analysis on DNA level (Polymerase chain reaction, polymerase chain reaction (PCR)) and mRNA level (Reverse transcription PCR, RT- PCR)
The present protocol described the methodology for transformation of potato using LBA4404 A. tumefaciens strain which carrying a disarmed pGV2260 Ti plasmid in a pRI201-AN binary vector with a selectable marker gene that conferred resistance against the kanamycin antibiotic to the transformed cells
Summary
Potato late blight is a pandemic disease caused by the highly virulent (Phytophthora infestans) fungus. The regeneration capacity was done among three tested potato cultivars (Spunta, Diamont and Desiree). Two different types of explants (inter-node and leaf) were cultured for calli induction and plant regeneration .The highest value of leaf explants was for Desiree cultivar (80%), which induced from developed callus in the 2,4-D medium. The best value of maximum shoot regeneration was for Desiree cultivar in the BA, IAA and GA3 media from leaf which proved to be more effective. Cloning of glucanase gene in pRI plasmid which carrying kanamycin resistance (nptII) gene was perfomed and followed by transformation in Agrobacterium tumefaciens LBA4404 strain which used for plant transfection. Nucleotide and amino acid sequences of transformed Agrobacterium were analyzed.
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