Molecular Analysis and Effectiveness Assay of AV1 Gene in Transgenic Tobacco for Resistance to Begomovirus

Tri Joko Santoso, Muhammad Herman, Sri H. Hidayat, Hajrial Aswidinnoor, Sudarsono Sudarsono

Abstract


Genetic transformation
of tobacco plant using AV1 gene was conducted at
the previously experiment and generated transgenic tobacco
plants positively carrying the selectable marker nptII gene.
The objectives of this experiment were to (1) analyze the
presence of Begomovirus AV1 gene in T0 generation putative
transgenic tobacco plants using PCR technique with specific
primers and its correlation with resistance phenotype, (2)
analyze the integration and copy number of the transgene in
T0 generation putative transgenic tobacco plants and its
correlation with resistance response, (3) screen the T0
generation putative transgenic tobacco plants with the target
virus infection and to detect the presence of the virus in the
transgenic plant tissue using universal primers. PCR
detection of AV1 gene in tobacco transgenic was conducted
by using specific primer for Begomovirus AV1 gene.
Meanwhile, Southern Blot analysis was conducted by using
the AV1 gene probe. The effectiveness of AV1 gene in
tobacco transgenic was tested by inoculation of target virus
using whiteflies vector. Result of the experiments showed
that there was a positive correlation between the presence
of the AV1 transgene in T0 generation putative transgenic
tobacco plants and the resistant phenotype. Transgenic
plants with a single copy integration of the transgene
exhibited more resistant than the multiple copy one. and
non transgenic plant. The resistance as a result of AV1 gene
expression was indicated with no symptom in T0 generation
transgenic tobacco plants and the accumulation of the virus
in the transgenic plants tissue. Northern and Western
hybridization analysis need to be perfomed for investigating
the presence of mRNA or protein accumulation so that the
resistance mechanism of the AV1 gene could be explained
more detail.


Keywords


Tobacco (Nicotiana tabaccum); molecular analysis; PCR technique; Southern Blot analysis; Begomovirus AV1 gene; transgenic.

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DOI: http://dx.doi.org/10.21082/jbio.v8n2.2012.p45-53

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P-ISSN : 1907-1094
E-ISSN : 2549-1547


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