Molecular analysis of activation tagged aspen-Populus variants II

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So far, only very few naturally available tree mutants are known, most of them showing alterations in tree habitus (e.g. dwarf, columnar, pendula mutants) and/or variations in leaf form/shape/color (e.g. lanceolate, brevifolia, and pale green leaf mutants. In two preceding proposals, the fundamentals for transposon (Ac from maize) based activation tagging in a tree species (Populus spec.) were developed. Proof-of-concept for using this system to generate poplar mutants was already provided. In this proposal, we are aiming to consider highly innovative aspects, namely (a) to investigate already available poplar variants obtained in the preceding project and characterize the genes involved in the mutation by over-expressing of the candidate genes in poplar, (b) to produce new mutants by employing a novel and efficient tagging strategy, and (c) to test different possibilities for the long-term storage of mutant plants and mutant tissue (incl. cryopreservation) of a tree species. Characterization of the already available hybrid aspen variants will be done as following: (a) all putative activation tagged variants still available will be further investigated molecularly as well physiologically, (b) for many of the variants, a candidate gene was identified putatively responsible for the variant phenotype: the gene of up to 5 most interesting variants will be cloned into a binary vector and over-expressed in hybrid poplar through genetic transformation. We will be able to prove whether the candidate gene is responsible for the variant phenotype latest in the third year of the project. Compiling all the information obtained, we will develop a vision of what to do with the variants and how to save all the lines produced to build up a collection of poplar variants. As a revised tagging strategy, we will establish a functional ATDs-tms-based transposon approach to produce plants with high probability of transposition (being putative variants). For this, first, the NAM concentration in the regeneration medium has to be optimized, and second the number of different transgenic lines will be enlarged to eliminate transposition-silenced lines at an early stage of the experimental procedure. For long-term storage of variants, at first regenerative callus cultures will be established from the mutant lines and be kept at low temperature. Also re-cutting the mutant plants during glasshouse culture will be tested. In cooperation with a French lab, cryopreservation of the regenerative callus cultures in liquid nitrogen will be tested. In the third year, the stability of mutant phenotypes will be tested.