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DEVELOPMENT AND CHARACTERISATION OF TRANSGENIC GROUNDNUT (Arachis hypogaea L.) USING PLANT DEFENSIN GENE

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Title DEVELOPMENT AND CHARACTERISATION OF TRANSGENIC GROUNDNUT (Arachis hypogaea L.) USING PLANT DEFENSIN GENE
 
Creator MADHU BALA
 
Contributor Radhakrishnan T.
 
Subject TRANSGENIC GROUNDNUT
GENETICS AND PLANT BREEDING
 
Description Biotic and abiotic stresses happen to be the major constraints in groundnut production. Among the economically important biotic stresses foliar diseases like early leaf spot, late leaf spot and rust and the soil borne diseases like collar rot, stem rot incur huge economic losses every year. The present study outlines the application of Agrobacterium mediated genetic transformation in order to obtain putative transgenics with plant defensin genes. The de-embryonated cotyledons from cultivar GG-20 were co-cultured with the Agrobacterium tumefaciens strain GV2260 harbouring the plasmid vector pRD-400 carrying Tfgd2 (Trigonella foenum-graecum defensin 2) and Rs-AFP2 (Raphanus sativus antifungal protein 2) linked with each other by linker peptide sequence, which was fourth internal from naturally occurring IbAMP polyprotein precursor from Impatiens balsamina was constructed by directional cloning of 1.2 kb cassette of synthetic gene at HindIII position of pRD-400 vector with the neomycin-phosphotransferase (nptII) gene as selectable marker.
A high percentage of 85% explants from the de-embryonated cotyledons regenerated to form multiple shoot buds which were subsequently sub-cultured on MS medium containing 100 mgL-1
kanamycin for ensuring high selection pressure for screening putative transformants. From the total regenerated shoots, 45.81% were completely resistant to kanamycin whereas, all the control plants bleached in selection medium. Hundred percent rooting was observed in rooting medium. The transgenic plants with well-developed roots were transferred to earthen pots for hardening and 58.5% of the plants survived in the pots in the glasshouse.
The integration of the transgene was assessed by the PCR amplification of the genomic DNA from the transformants with plant defensin gene and nptII gene specific primers. From all the plants survived in glasshouse the DNA was isolated and 14 plants showed amplification of 498 bp band and 750 bp band with gene specific marker and selectable marker, respectively, in PCR analysis. The transformation efficiency obtained was 1.76%. The transgenics produced were further confirmed by southern hybridization and RT-PCR. Results of genomic southern was not clear, so PCR-Southern and Dot blot analysis was carried out which showed the presence of gene but no clue regarding the copy number. Reverse Transcriptase PCR was carried out that showed the expression of transgene in the T0 plants.
T0 plants’ seeds were harvested and sown to raise T1. Germination was 79%. T1 plants were PCR tested and the defensin showed segregation in 3:1 ratio in two lines and in the remaining ten lines the analysis was not confirmative due to the smaller population size. Analysis was not carried out for the two lines with only three seeds. Seeds were collected from PCR positive plants and sown to obtain T2 generation.
T2 plants were obtained in pots in glasshouse after sowing of 65 PCR positive lines. Germination was 83%. T2 plants were PCR tested to check the presence of transgene and showed that out of 65 T1 transgenic lines 14 transgenic lines were homozygous while the 51 T1 transgenic lines were segregating and hence not stable. Results of genomic southern was not
clear, so PCR-Southern and dot blot analysis was carried out which showed the presence of gene but no clue regarding the copy number. Reverse-Transcriptase PCR was carried out that showed the expression of transgene in the T2 plants.
Quantitative (real-time) PCR was carried out in T2 transgenic plants to determine the relative expression of the transcript of defensin.
Morphological characterization of T2 generation transgenic plants was carried out showing significant difference from the wild type plant for the five characters studied viz., days to 50% flowering, plant height of main stem (cm), plant width or spread (cm), number of primary branches and number of secondary branches.
Fungal bioassay was carried out by detached leaf method for both T1 and T2 generation plants. Analysis was not carried out for the two characters studied viz., days to disease appearance and days to defoliation as there was no disease occurrence on some transgenic lines. In T1 generation transgenic plants showed significant difference within the transgenic lines and between the transgenic lines as compared to the wild type for the characters lesion number and lesion size. T2 generation transgenic plants showed significant difference over the wild type for the characters studied lesion number and lesion size.
Stem rot screening was carried out for transgenic seeds, wild type (GG-20) and CS-19 as negative and positive controls, respectively. In the transgenic lines, the seeds germinated in the presence of inoculum and showed development of quadrifoliate but the seeds also showed the attack of mycelia of the fungi. But there was no germination and development of plumule and quadrifoliate in wild type (GG-20) variety and the CS-19 genotype seeds.
So, from the above study it could be concluded that using the defensin gene, transgenic lines were developed which conferred resistance
against leaf spot disease. Stem rot screening can be further validated using large scale trials in sick plots with heavy dose of inoculum. For determining the copy number in transgenic lines, further analysis and standardization should be carried out. The transgenic lines developed can be used in future for development of variety directly or in breeding programmes for development of disease resistant transgenic lines.
 
Date 2016-09-21T14:56:09Z
2016-09-21T14:56:09Z
2012-01
 
Type Thesis
 
Identifier http://krishikosh.egranth.ac.in/handle/1/78092
 
Language en
 
Format application/pdf