xia Wang

Transgenic Populus tremula: a step-by-step protocol for its Agrobacterium-mediated transformation

In recent years, Populus species have acquired an important place in basic and applied research of woody plants. The practical role of Populus species in world forestry and their importance to research as a woody-plant model have led to increasing interest in tissue-culture and molecular techniques, as well as the development of transformation procedures for this genus. A simple technical procedure is described here step-by-step, for the first time, as a routine method for transforming Populus tremula using a disarmed Agrobacterium tumefaciens hypervirulent strain. The procedure begins with the inoculation of stem explants with bacterial suspension, followed by a short period of co-cultivation on a highly regenerative medium. Transformed shoots are selected on regeneration medium containing antibiotics and the presence of the inserted target genes is checked using a rapid and efficient PCR test. Selected shoots are transferred to a rooting medium, under the same selection pressure, and propagated via stem cuttings. Selected plants can be hardened and transferred to the green-house within 4 months of inoculation. The method has proven efficient for several gene constructs, selection on Kan or Hyg, and three different Agrobacterium strains.

Abiotic Resistance and Chaperones: Possible Physiological Role of SP1, a Stable and Stabilizing Protein from Populus

The molecular basis of abiotic stress tolerance in plants, especially drought, salinity and extreme temperatures, is rather limited. In view, the worldwide devastating problems of salinization and desertification, efforts are continuing to unravel some of the molecular controls and tolerance mechanisms. Discovery of new genes for abiotic stress tolerance, combined with controlled molecular breeding will have an important role in shaping agricultural plants in the post-genomic era (Wang et al., 200la).

Crystallization and preliminary X-ray crystallographic analysis of SP1, a novel chaperone-like protein.

SP1 (108 amino acids) is a boiling-stable stress-responsive protein. It has no significant sequence homology to other stress-related proteins or to small heat-shock proteins (sHsps). SP1 activity is ATP-independent, similar to other small heat-shock proteins. Based on these features, it is expected that the structure-function relationship of SP1 will be unique. In this work, the crystallization and preliminary crystallographic data of native SP1 and its selenomethionine derivative are described. Recombinant SP1 and its selenomethionine derivative were expressed in Escherichia coli and used for crystallization experiments. SP1 crystals were grown from 0.1 M HEPES pH 7.5, 20% PEG 3K, 0.2 M NaCl. One to four single crystals appeared in each droplet within a few Days and grew to dimensions of about 0.5 x 0.5 x 0.8 mm after about two weeks. Diffraction studies of these crystals at low temperature indicated that they belong to space group I422, with unit-cell parameters a = 89, b = 89, c = 187 A. Efforts to crystallize the selenomethionine derivative of SP1 are in progress.

Plant Tolerance to Water and Salt Stress: The Expression Pattern of a Water Stress Responsive Protein (BspA) in Transgenic Aspen Plants

Abiotic stress conditions, such as drought, salt, cold and heat, are major environmental factors affecting plant growth and productivity. Plants have developed several strategies to adapt or to tolerate stress conditions, one of which is the accumulation of compatible solutes helping to maintain an osmotic balance with environment (Rudulier et al 1984). Another important strategy is the expression of specific stress-responsive proteins which may function in plant tolerance (Dure 1993; Close 1997). A hydrophilic boiling stable protein (designated as BspA) has been identified by us as a major water stress and ABA-induced protein in aspen (Populus tremula L.) plants (Pelah et al 1995, 1997a). A differential expression pattern of BspA in poplar genotypes differing in their water stress tolerance, and a positive correlation between BspA accumulation and reduced ion leakage upon water stress was previously reported (Pelah et al 1997b), yet direct evidences for its precise mode of action are still lacking.

TOWARDS WATER STRESS-TOLERANT POPLAR AND PINE TREES: MOLECULAR BIOLOGY, TRANSFORMATION AND REGENERATION

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].

In Vitro Organogenesis, Transformation and Expression of Drought-Related Proteins in Forest Tree Cultures

A procedure for aseptic in vitro culture of Populus spp. and Pinus halepensis buds from adult trees was developed, and bud cultures have been used to elucidate the hormonal control of bud growth and development. Populus cultures served as well for establishing procedures for in vitro propagation. Axillary bud break was induced, and adventitious buds and roots were formed, resulting in acclimatized plants. Adventitious shoot formation in root cultures of Populus is being studied. Expiants of zygotic embryos, seedlings and rejuvenated mature trees of Pinus halepensis are examined for in vitro clonal propagation. Efficient procedures for massive induction of adventitious shoots from mature pine embryos were established, and good rooting was achieved. Efforts are being made to induce somatic embryogenesis. A novel 66 kD boiling-stable protein was highly expressed in Populus tremula shoot cultures and in callus, in response to gradual desiccation, cold and osmotic stress, and ABA application. Populus clones differing in their drought tolerance are characterized by different expression patterns of this protein. This protein shows a high degree of homology with similar proteins form other species. Similar boiling-stable proteins were differentially expressed in germinating pine seeds. Populus shoots and Pinus embryos were transformed using A rhizogenes. as monitored by root formation and GUS expression, and complete transgenic aspen plants were established.