2012 Annual Report
1a.Objectives (from AD-416):
Develop an Agrobacterium-mediated transformation system for leafy spurge.
1b.Approach (from AD-416):
Initially, the Plant Transformation Research Center (PTCR) will use the healthy in vitro leafy spurge plants and the methodology supplied by the USDA for the propagation and the transformation experiments. The next steps will be the evaluation of explants regeneration capacity after co-cultivation with Agrobacterium on the different selection systems. Agrobacterium tumefacienens strains AGLO1, EAH105, GV3101 containing the binary plasmids pBI121, pCambia 1301, and pCambia 3300 will be used for the transformation experiments. The transformation vectors are under regulation of the CaMV 35S constitutive promoter, but the selectable marker gene in pBI121 is nptII for Kanamycin resistance, in pCambia 1301 is hpt for hygromycin resistance, and in pCambia 3300 is glufosinate that confers resistance to Basta herbicide. Confirmation of the transgenic tissue will be performed using genomic PCR with specific primers for the amplification of the corresponding selectable marker genes.
Initially, the UC Riverside Plant Transformation Research Center (UCR-PTCR) used the healthy in vitro leafy spurge plants and the methodology supplied by the USDA for propagation and transformation experiments and has successfully repeated subculture and in vitro propagation of two leafy spurge genotypes, SP6 and LS26. UCR-PTCR has then performed transformation experiments using different Agrobacterium strains and T-DNA binary vector combinations including Aglo1/pBI121, EHA105/pCambia3300, and GV3101/pCambia3300. All three vectors are under regulation of the CaMV 35S constitutive promoter, but the selectable marker gene in pBI121 is nptII for Kanamycin resistance, in pCambia 3300 is bar gene that confers resistance to Basta herbicide, and in pCambia 1301 is hpt for hygromycin resistance. After chemical selection, the regenerated plants were subjected to genomic DNA isolation and PCR analysis. At present, only EHA105/pCambia3300 combination showed promising results after PCR analysis, since among 20 regenerated SP6 plants, 9 showed positive results after PCR analysis (a PCR product of the transformed gene was identified) and among 27 regenerated LS26 plants, 6 showed positive results. Based on these results, Basta herbicide selection (EHA105/pCambia3300) appears to be more effective in preventing escapes in regenerated plants. However, final confirmation of these potential transgenic plants requires Southern Blot analysis.
An efficient DNA transformation system for leafy spurge can be applied to study gene functions based on the alteration of gene expressions and phenotypic changes in transgenic plants. The positive results for EHA105/pCambia3300 combination imply that Basta herbicide may be the method of choice for leafy spurge transformation.