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CHARACTERIZATION OF TRANSGENIC GROUNDNUT (Arachis hypogaea L.) PLANTS CARRYING COAT PROTEIN GENE OF TSV AND NUCLEOCAPSID GENE OF PBNV

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Title CHARACTERIZATION OF TRANSGENIC GROUNDNUT (Arachis hypogaea L.) PLANTS CARRYING COAT PROTEIN GENE OF TSV AND NUCLEOCAPSID GENE OF PBNV
 
Creator PATIL ABHINANDAN SURGONDA
 
Contributor RADHAKRISHNAN T.
 
Subject , groundnuts,
GENETICS AND PLANT BREEDING
 
Description Key words: transgenic groundnut, TSV, PBNV, coat protein, nucleocapsid
The present work has been carried out with the major objectives to characterize
marker free transgenic groundnut plants for integration, expression and
inheritance of coat protein gene of TSV and nucleocapsid gene of PBNV and to
evaluate the transgenic plants for resistance to TSV and PBNV under glasshouse
conditions. The experimental transgenic test plants of Arachis hypogaea, was
developed through Agrobacterium mediated transformation groundnut cv. Kadiri-
6/(K-6) at Biotechecnology Laboratory, ICAR-DGR, Junagadh.
PCR amplification of TSV-CP and PBNV-NC gene specific primers gave
amplification of approximately 717bp and 654bp, respectively. The presence of dual
transgene in five transgenic events (T3) was confirmed by Dot blot analysis and by
Southern hybridization. PCR analysis of genomic DNA of T1, T2 and T3 generation
plants showed segregation of gene in ratios 3:1 (T1) and 5:3 (T2). RT-PCR results
showed that transgenic plants expressed gene(s) at transcription level and genes
expression were analyzed at transcript level through real time PCR (qPCR).
Test plants were grown in glasshouse condition and mock-inoculated. Transgenics
were visualized with declining TSV expressions, concluded that the high expression of
TSV-CP protein retards and delays TSV disease development. In case of PBNV
challenge inoculation, transgenic plants with NC protein showed delayed (of 2-3
weeks) symptoms expressions and attenuation with PBNV concluded that the high
expression of PBNV-NC protein retards and delays the disease development by
PBNV. Interestingly, in the transgenics plants no axillary shoot proliferation was
observed.
The end result of DAC-ELISA indicated more virus titer values in wild type than
transgenic groundnut plants for TSV and PBNV. After challenge inoculum RT-PCR
analysis were carried out to determine the presence of virus inoculums load. Wild type
groundnut leaves showed larger phenotypic symptoms of micro-necrosis spots at
infection site than transgenic groundnut leaves when stained with lacto-phenol-trypan
blue.
Thus, these strategies can further be implemented through genetically engineered
viral disease resistance to other commercially important crops that are affected by
Ilarvirus and Tospoviruses. Also, these dual gene construct transgenic groundnut
plants were produced by the use of marker free genes that do not rely on antibiotic or
herbicide resistance
 
Date 2016-09-28T08:36:28Z
2016-09-28T08:36:28Z
2015-10
 
Type Thesis
 
Identifier http://krishikosh.egranth.ac.in/handle/1/79213
 
Language en
 
Format application/pdf