Generation of Transgenic <i>Medicago Sativa</i> Overexpressing "<i>Osmotin-Chitinase</i>" Gene Chimera

<i>Medicago</i> is widely used as a forage crop. It is often susceptible to various pathogenic infections and exhibits low growth in drought and extreme climatic conditions. In the current study, a strategy was developed for over-expressing an <i>“Osmotin-Chitinase</i>” gene chimera in transgenic <i>Medicago</i> that could potentially confer resistance to different biotic and abiotic stresses. Seed germination of several cultivars of <i>Medicago</i> (<i>M. sativa ssp. sativa, M. sativa ssp. falcata, M. sativa ssp. caerulea, M. truncatula</i>, and <i>M. Rugosa</i>) was tested to determine the cultivars with good germination rates. Among these, <i>M. sativa</i> ssp. <i>sativa</i> showed an average of 80% germination over a period of one week and was subsequently selected for regeneration and transformation experiments. Different explants (cotyledons, hypocotyls, petioles) were tested for regeneration. Among these, hypocotyl explants showed highest (46.17 %) percent regeneration. <i>Escherichia coli</i> harboring <i>Osmotin-Chitinase</i> (<i>OSM-CHI</i>) gene chimera cloned into binary vector pBTEX with <i>npt</i>II as a selection marker was mobilized in <i>Agrobacterium tumefaciens</i> strain EHA105 which was employed in the transformation of hypocotyl explants of <i>Medicago</i>. Transformed calli were grown on callus inducing medium containing kanamycin for screening. Further screening of the positive transgenics was performed using PCR. Southern hybridization was carried out for further confirmation of successful transformation. Transformed shoots will be grown on the root inducing medium for developing into plantlets which would then be transferred to the green house and later tested for their degree of resistance to various biotic and abiotic stresses.

Name thesis:degree_name

Master of Science

Discipline thesis:degree_discipline

Department of Biology

Year

2011

Author dc:creator

• Kancharla, Jahnavi Reddy

Contributors dc:contributor

• Dr. Shivendra V. Sahi (Director), Dr. Sigrid Jacobshagen, Dr. Linda Gonzales

• agrobacterium
• biotic stresses
• transgenics
• abiotic stresses
• Southern hybridization
• Agricultural Science
• Biology
• Plant Biology
• Plant Breeding and Genetics
• Plant Sciences

Repository record dc:identifier

https://digitalcommons.wku.edu/theses/246

OAI identifier oai:identifier

oai:digitalcommons.wku.edu:theses-1249