Genetic engineering of Populus species to improve resistance to environmental stresses

An efficient Agrobacterium-mediated transformation protocol combined with direct shoot regeneration was developed for hybrid aspen, Populus canescens x P. grandidentata. In addition, the usefulness of hygromycin resistance as a selectable marker for transgenichybrid aspen plants was described. Factors affecting transformation efficiency including maturation of plant material, acetosyringone, pH, and inoculation duration were tested for their impact on the survival and health of explants. Cytokinins tidiazuron (TDZ) and 6-benzyl-adenine (BA) were tested to induce direct shoot regeneration from leaf explants.; The male sterility gene (DTA: Diphtheria toxin subunit A) and insect resistance genes (PI I: proteinase inhibitor I and Bt toxin, CRYIIIA) were introduced into hybrid aspen plants in order to reduce the risk of contamination of wild type species by transgenic plants and to reduce damage by insect feeding. Several binary vectors with plant expression cassettes of DTA, PI I and/or CRYIIIA were constructed to use in conjunction with Agrobacterium -mediated transformation methods. The presence of the DTA gene in transgenic hybrid aspen was confirmed by PCR and Southern analysis and absence of the DTA gene expression in vegetative tissues was confirmed by Northern analysis. Insect resistance genes (PI I and CRYIIIA) were introduced into transgenic plants of male hybrid aspen containing the DTA gene. Transgenic lines with PI I and/or CRYIIIA genes showed increased resistance to gypsy moth and cottonwood leaf beetle compared to untransformed control hybrid aspen.; The level of chloroplastic Cu/Zn SOD of two aspen clones, ozone-tolerant or ozone-sensitive, was altered by inserting the chloroplastic Cu/ZnSOD gene in sense or antisense orientation to study the role of chloroplastic Cu/Zn SOD in plant defense mechanisms during increased oxidative stress by ozone. The presence of sense or antisense genes in transgenic plants was confirmed by PCR, and the expression level at RNA or protein was measured before and after ozone fumigation. Transgenic aspen of ozone sensitive clone containing sense chloroplastic Cu/Zn SOD gene showed increased resistance to ozone visible injury compared to untransformed ozone sensitive aspen clone demonstrating that chloroplastic Cu/Zn SOD played an important role in reducing the sensitivity to ozone injury in trembling aspen.