Abstract:
Barley yellow dwarf viruses [BYDV; genus Luteovirus; family Luteoviridae]
are plant-infecting viruses that cause serious economic losses in cereals. Introduction
of virus-derived gene(s), through genetic engineering, is considered as a powerful tool
to confer resistance. The present study was designed to optimize Agrobacteriummediated
plant transformation protocol, with a sense coat protein (CP) gene of BYDV,
and to attain transgenic resistance. Briefly, BYDV-CP was amplified, cloned and
sequenced from a leaf sample showing leaf discoloration and dwarf stature – symptoms
typical of BYDV infection. The CP was sub-cloned into a binary plasmid vector
(pING71), in sense orientation. Agrobacterium tumefaciens was used to transform both
Nicotinana benthamiana and wheat cv. Bobwhite. Although literature suggests, N.
benthamiana genotype used in this study, however could not host aphids for the virus
transmission. Similarly, the transgenic Bobwhite could not be tested for resistance due
to limited study duration at McGill University, Canada. To come up with, a protocol
was optimized to transform an elite wheat variety (Sehar-2006) using different nutrient
media concentrations including salts and vitamins. The BYDV-CP transgenes were
selected on the antibiotic and confirmed through PCR and reverse transcription PCR.
The BYDV-CP transgenic wheat plants were challenged with the BYDV, using
viruliferous aphids. The transgenic wheat plants showed mild symptoms, when
compared with non-transgenic controls. Our results indicated that the transgenic wheat
(Sehar-2006), in general, was good in providing protection against BYDV, grown under
greenhouse conditions. The thesis may contribute significant knowledge to validate
protection using virus-derived gene and to optimize Agrobacterium-mediated wheat
transformation.
