Detection of T-DNA sequence in transgenic PRSV-resistant 'Davao Solo' papaya
2007
Galvez, H.F. | Bercansil, M.C.M.L. | Tengson, E.J.U. | Collee, M.G. | Hautea, D.M., Philippines Univ. Los Banos, College, Laguna (Philippines). Crop Science Cluster. Inst. of Plant Breeding
The Philippine 'Davao Solo' papaya is bioengineered to acquire resistance against the devastating Papaya Ringspot Virus (PRSV) disease. The coat protein-(CP) gene of Philippine PRSV (Bulacan isolate) was introduced into the genome of 'Davao Solo' papaya using the disarmed T-DNA of Agrobacterium as the carrier in transformation. Up to forth generation (T3) at least two lines are selected to have shown generational stability for the presence of CP transgene and expression of resistance against PRSV. Genetic analysis has confirmed the Mendelian inheritance of the transgene as detected by polymerase chain reaction (PCR) and based on resistance expression. Transgene detection in PRSV-resistant papayas was done by PCR analysis of leaf DNA samples. Using CP-LB primer pair. PRSV-resistant T3 samples amplified a strong 650 bp PCR product at 60 deg C annealing temperature. The amplicon was not present from DNA samples of ten untransformed 'Davao Solo' papaya plants. CP-LB primer pair targets the detection of downstream sequence of CP transgene up to the left border (LB) sequence of the integrated T-DNA. The detection of the 650 bp amplicon was verified in at least three PCR analyses, prick PCR and PCR coupled with Southern Blot. Southern blot of the PCR product with pMON plasmid as hybridization probe verified the integrity of the fragment as the expected T-DNA sequence. The fragment is currently being cloned from representative T3 papaya plants for nucleotide sequencing and to design primers for use in later large-scale PCR screening of transgenic 'Davao Solo' papaya plants.
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