Molecular analysis of activation tagged aspen-Populus variants II

到目前为止，只有很少的天然树木突变体是已知的，他们中的大多数表现出改变树的习性（如矮化，柱状，下垂突变体）和/或变化的叶形/形状/颜色（如披针形，短叶，和浅绿色叶突变体。在前面的两个提议中，基于转座子（来自玉米的Ac）的树木物种（杨属物种（Populus spec.））被开发出来。已经提供了使用该系统产生白杨突变体的概念验证。在这个提议中，我们的目标是考虑高度创新的方面，即（a）研究在先前项目中获得的已经可用的白杨变体，并通过在白杨中过表达候选基因来表征参与突变的基因，（B）通过采用新颖且有效的标记策略来产生新的突变体，和（c）测试长期储存突变植物和突变组织的不同可能性（包括，冷冻保存）的一个树种。已经可用的杂种白杨变体的表征将如下进行：（a）将进一步研究仍然可用的所有推定的激活标记的变体的分子以及生理学。（B）对于许多变体，鉴定了候选基因，其对变体表型负责：将多达5个最感兴趣的变体的基因克隆到双元载体中，并通过遗传转化在杂种白杨中过表达。我们将能够证明候选基因是否负责变异表型最迟在第三年的项目。汇编所有获得的信息，我们将制定一个愿景，该怎么做的变种，以及如何保存所有生产线，以建立一个集合的白杨变种。作为一个修改后的标签策略，我们将建立一个功能性的ATDs-tms转座子的方法来产生转座的概率高的植物（被推定的变体）。为此，首先，必须优化再生培养基中的NAM浓度，其次，将扩大不同转基因系的数量以在实验程序的早期阶段消除转座沉默系。对于变体的长期储存，首先将从突变株系建立再生愈伤组织培养物并保持在低温下。还将测试在温室培养期间重新切割突变体植物。与法国实验室合作，将对再生愈伤组织培养物在液氮中的冷冻保存进行测试。在第三年，将测试突变表型的稳定性。

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.