Integration and expression of Bluetongue VP2 gene in somatic embryos of peanut through particle bombardment method.

Abstract

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.

• Authors: T N Athmaram, Geetha Bali, K M Devaiah
• Journal: Vaccine (Vaccine) Vol. 24 Issue 15 Pg. 2994-3000 (Apr 05 2006) ISSN: 0264-410X [Print] Netherlands
• PMID: 16513222 (Publication Type: Journal Article)

Chemical References

• Capsid Proteins
• DNA, Plant
• Epitopes
• RNA, Messenger
• RNA, Viral
• VP2 protein, Bluetongue virus
• Kanamycin
• Glucuronidase

Topics

• Arachis (drug effects, genetics, immunology)
• Blotting, Southern
• Capsid Proteins (biosynthesis, genetics)
• DNA, Plant (analysis, genetics)
• Epitopes (genetics)
• Gene Expression
• Gene Transfer Techniques
• Glucuronidase (genetics)
• Kanamycin (pharmacology)
• Plants, Genetically Modified
• Plasmids (genetics)
• RNA, Messenger (analysis)
• RNA, Viral (analysis)
• Recombination, Genetic
• Reverse Transcriptase Polymerase Chain Reaction
• Seeds (genetics, metabolism)
• Transformation, Genetic