Towards Water Stress-Tolerant Poplar and Pine Trees: Molecular Biology, Transformation and Regeneration

Abstract

Novel procedures for the improvement of current breeding, selection and forestation techniques can only be achieved by combining conventional breeding and selection procedures, rapid and efficient propagation, new planting and land use approaches, and novel biotechnological methodologies. An integrated biotechnological approach should be useful both for determining the physiological and molecular basis of tree tolerance, and for rapid selection and tree improvement. In the following, we present data from our studies on micropropagation, molecular analysis of water-stress tolerance, and transformation of Populus spp. and Pinus halepensis. Axillary bud break was induced in Populus stem and leaf cultures, and adventitious buds and roots were formed, resulting in acclimatized plants. Procedures for adventitious shoot formation in root cultures of aspen (Populus tremula) are currently under study. Expiants of mature zygotic embryos, seedlings, and cytokinin-treated mature trees ofP. halepensis are being studied for in vitro clonal propagation. Efficient procedures for the massive induction of first- and second-cycle adventitious shoots from embryos were established. Buds were also induced on pine seedling expiants and on detached needles from long-term embryo culture. Fascicular buds were induced in 3-year-old pine trees, and they were cultured in vitro for further regeneration. Somatic embryogenesis is also being studied. Drought-specific proteins/genes were studied in several Populus species and in P. halepensis. A novel 66 kDa boiling-stable protein was highly expressed in P. tremula shoots, as early as 1 h after gradual water loss and ABA application, as found by Pelah et al. [19]. Using Populus clones which differed in their drought tolerance, we found a good correlation between the expression of dehydrin-like proteins and sucrose synthase and the degree of drought tolerance and ion leakage [21]. cDNAs of the 66 kDa and related proteins are being employed for additional molecular characterization. Here we present additional evidence for the effect of cold and osmotic stresses on protein expression. The 66 kDa boiling-stable protein was highly expressed in aspen callus cultures subjected to osmotic stress in a medium containing 6 or 12% mannitol, simultaneously with a decrease in cellular water potential. The same protein accumulated in response to cold stress, upon culture of aspen callus and shoots at 4°C. A highly efficient transformation and regeneration procedure of aspen, which does not require a pre-selection stage on antibiotics, was established. P. halepensis embryos, seedlings and mature buds were also efficiently transformed, as monitored by root formation and/or GUS expression. These are fully described by Tzfira et al. [29, 31].