Integration and expression of Bluetongue VP2 gene in somatic embryos of peanut through particle bombardment method.
After pre-culture and treatment of osmosis, zygotic embryos of peanut (Arachis hypogaea L.) were transformed via particle bombardment with a plasmid containing a Bluetongue VP2 gene (BTVP2) comprising neutralizing epitopes. Selection for Kanamycin resistant calluses and somatic embryos was initiated at 12th day post-bombardment on medium containing 25 mg/L Kanamycin. Under continuous selection, 12.38% Kanamycin resistant plantlets were regenerated from bombarded somatic embryos. The presence and integration of BTVP2 DNA in regenerated Kanamycin resistant plants were confirmed by southern hybridization assay using non-radioactive Digoxiginin BTVP2 probe. Beta-glucuronidase (GUS) enzyme activity was detected in transgenic somatic embryos but not from control, non-transformed embryos. The expression of the BTVP2 protein was confirmed through RT-PCR (reverse transcription polymerase chain reaction) using the RNA isolated from the transgenic callus employing BTVP2-specific primers. The production of transgenic peanut was mainly focused on evaluating a newly improved somatic embryogenesis regeneration system as well as the gene transfer method and to produce the Bluetongue outer coat protein that comprises the neutralizing epitopes.
Athmaram
T N
TN
Department of Microbiology and Biotechnology, Bangalore University, Jnanabharathi, Bangalore 560056, India.
Bali
Geetha
G
Devaiah
K M
KM
D010367:Arachis / Q000187:drug effects / Q000235:genetics* / Q000276:immunology; D015139:Blotting, Southern; D036022:Capsid Proteins / Q000096:biosynthesis / Q000235:genetics*; D018744:DNA, Plant / Q000032:analysis / Q000235:genetics; D000939:Epitopes / Q000235:genetics; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005966:Glucuronidase / Q000235:genetics; D007612:Kanamycin / Q000494:pharmacology; D030821:Plants, Genetically Modified; D010957:Plasmids / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis; D012367:RNA, Viral / Q000032:analysis; D011995:Recombination, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012639:Seeds / Q000235:genetics / Q000378:metabolism; D014170:Transformation, Genetic
D036022:Capsid Proteins; D018744:DNA, Plant; D000939:Epitopes; D012333:RNA, Messenger; D012367:RNA, Viral; C442520:VP2 protein, Bluetongue virus; D007612:Kanamycin; D005966:Glucuronidase