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. ␤-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.

Somatic Embryogenesis: Fundamental Aspects and Applications

“…Athmaram et al (2006) described a protocol for the genetic transformation of peanut or groundnut (Arachis hypogaea) using immature zygotic embryos bombarded with tungsten microparticles coated with a binary plasmid carrying BTVP2 gene (pCAMBIABTVP2) encoding the major antigenic determinants of Bluetongue (BT) disease caused by a double-stranded RNA virus (Orbivirus genus), which affects primarily sheeps, but also goats, cattle, and wild ruminants. Transgene integration into the plastid genome was demonstrated by PCR.…”

Progress in genetic engineering of peanut (Arachis hypogaea L.)—A review

“…The expression and inheritance study for RPVH demonstrated stable integration in T 1 generation plants, which have been used for the oral administration of cattle for vaccination resulting into hemagglutinin-specific antibody production (Khandelwal et al, 2003b). Further, BTV-VP2 (bluetongue VP2 gene) has also been expressed in peanut for resistant to bluetongue virus (BTV) which is a causal agent of bluetongue of disease of sheep (Athmaram et al, 2006). Recently, HN gene expressing hemagglutininneuraminidase protein of Peste des petits ruminant virus (PPRV) in peanut plants has also been tested for oral administration in vaccination of sheep (Khandelwala et al, 2011).…”

The Uses of Somatic Embryogenesis for Genetic Transformation

“…Southern blot analysis revealed that transgenic plants regenerated after two subcultures of cell lines on selective medium with 350 mg L −1 spectinomycin showed heteroplasmy (presence of wild-type and transformed chloroplast genome), whereas those recovered after eight to ten subcultures of the cell lines were homoplasmic. Athmaram et al (2006) described a protocol for the genetic transformation of peanut or groundnut (Arachis hypogaea) using immature zygotic embryos bombarded with tungsten microparticles coated with a binary plasmid carrying BTVP2 gene (pCAMBIABTVP2) encoding the major antigenic determinants of Bluetongue (BT) disease caused by a double-stranded RNA virus (Orbivirus genus), which affects primarily sheeps, but also goats, cattle, and wild ruminants. Pods of the cv.…”