Transformation of aspen hybrids with RolB gene for root induction in hardwood cuttings

An efficient regeneration system was developed for three elite aspen hybrid clones (Populus tremuloides x P. tremula, P. tremuloides x P. davidiana, and P. x canescens x P. grandidentata). Leaf explants from in vitro shoots generally formed more callus on modified MS medium (MSA) and woody plant medium (WPM) supplemented with 2.0 mg/l zeatin + 1.0 mg/l naphthaleneacetic acid (NAA) than on media with other combinations of growth regulators. Healthier shoots were regenerated from callus tissues on WPM medium supplemented with 2.0 mg/l zeatin than on MSA medium and other combinations of growth regulators. The regenerated shoots were easily rooted in vitro and established in the greenhouse. Clone P. x canescens x P. grandidentata exhibited the greatest callus and shoot production efficiency among the three clones. An Agrobacterium-mediated transformation protocol was developed for aspen hybrid P. x canescens x P. grandidentata. Genetically transformed plants were regenerated after co-cultivation of in vitro leaf explants with A. tumefaciens strain EHA 105 that harbored a binary vector (pBI121) carrying nptII under nopline synthase promoter and rolB/uidA genes under the CaMV 35S or heat shock promoter. The optimum conditions for transformation included use of the above-described regeneration system, co-cultivation of explants with agrobacteria at 1.0 absorbance at OD₆₀₀, and addition of acetosyringone (AS) to the co-cultivation medium and/or to the callus induction medium. Polymerase chain reaction (PCR) and Southern blot analyses confirmed that the foreign genes were integrated into the aspen genome. Tissue-specific expression of GUS gene was evaluated. When CaMV 35S was used as a promoter, the GUS gene was expressed in all parts of mature stem except pith, with the strongest activity in the phloem. The heat shock promoter gave rise to a very strong expression, mainly in parenchyma of young stem and only in the epidermis and phloem of mature stems. In leaves, the GUS activity was primarily located in the veins and mesophyll, and no difference was found between CaMV 35S and heat shock promoter. In root tips, the GUS gene driven by both CaMV 35S and heat shock promoter was expressed in the columella, vascular, and apical meristem with a very strong expression in the apical meristem. A rooting experiment using in vitro cuttings showed that transformation of the rolB gene did not increase the rooting ability, probably because cuttings from all transgenic plants were mixed. With hardwood cuttings, transformed plants rooted significantly higher than non-transformed plants. Heat shock- rolB-transformed plants gave higher rooting percentage than CaMV 35S-rolB transformed plants. Heat treatment of heat shock- rolB transformed hardwood cuttings rooted significantly higher than those not receiving the heat treatment when cuttings were exposed to 100 or 1000 ppm IBA. Although more roots were induced when cuttings were exposed to 100 or 1000 ppm IBA, the insignificance of rooting percentages among three IBA concentrations suggest that the rolB-transformed cuttings have higher cellular sensitivity to auxin, in this case, primarily to the endogenous auxin. This research demonstrated the potential of using genetic engineering with the rolB gene to improve rooting of hardwood cuttings in difficult-to-root woody plants.