Right place, right time: Targeted auxin delivery to improve tree cutting propagation

政府制定了一个目标，到2025年，每年在英格兰种植1万公顷的树木。为了解决树木繁殖知识的不足，已经提供了新的资金。我们申请并成功获得了为期6个月的概念验证资金，预计将成为未来3年的资金，以达到政府的目标。然而，在大规模生产之前，有些物种需要更详细的研究来了解潜在的生物学，这就是本博士提案所要解决的问题。树木扦插繁殖依赖于茎基部的生根形成，但不同树种间的生根成功率存在显著差异。这种变异依赖于信号网络，包括植物激素，如生长素，这是许多生根粉的核心成分。然而，生长素处理并不总是能改善难根物种的生根，部分原因是生长素降解增加。在这个项目中，学生将确定之前由主要导师发现的生长素反应和降解是否可以应用于山茱萸物种的工业相关扦插（可食用和生物多样性价值），同时开发一种靶向递送生长素的新方法。通过在正确的时间向正确的细胞中添加生长素，我们可以克服与生长素含量低相关的生根障碍。目的1：确定以前在模型系统中发现的生长素反应是否也适用于山角扦插。在Rasmussen等人2015年的基础上，生长素水平和响应性将在从Whetman Plants International收集的较难繁殖的山角扦插中进行测量。嫁接将用于确认信号是源于茎还是源于根。将在生根区测量生长素、细胞分裂素、独角子内酯和茉莉酸的水平，以与我们的豌豆模型进行比较。激光消融层析成像将用于三维跟踪根的形成和萌发过程。目标2：开发新的生长素递送药物载体，在切割后的关键时刻操纵局部生长素。学生将合成纳米载体，以空间和时间靶向的方式递送生长素和生长素代谢物。这将涉及生产（生物）基于聚合物的胶囊（例如，PLGA， PCL，几丁质，壳聚糖）将生长素捕获在里面。这些将被喂到豌豆DR5:GUS系的切割端，这将提供一个标记来证明生长素的释放。然后将它们放入商业Cornus sp.插枝中，以测试对根形成的影响，并与其他方案进行比较，包括棉花喂养、微注射、微透析输送和羊毛脂混合。生长素IBA、2-4-D和NAA对IAA的移动性不同，具有较强的稳定性，将在添加和不添加NPA（生长素运输抑制剂）的情况下，与三种最有效的稳定生长素的多酚（阿威酸、咖啡酸和香兰素）一起施用于扦插。对于这些化合物中的每一种，PsDR5:GUS将用于观察山茱萸中与生根反应平行的生长素反应的变化。交付成果：有效的树木繁殖方案，使行业合作伙伴受益，同时满足政府的植树目标。靶向释放分子的新技术（无需基因改造）对如何使用传统生理学方法来操纵内部生长素水平以及不定根发育随年龄下降的原因的新见解里程碑：第一年：综述论文（与第一年的综述要求一致）。第二年：研究论文1：生长素对不定根的调控：模式植物和木本植物的比较（例如：New phyologist /Journal of Experimental Botany）。第3年：研究论文2：植物发育中生长素和生长素降解阻滞剂的靶向释放（Nature/Science）

The Government has set a target to plant 10,000 hectares of trees per year in England by 2025. To address the shortfall in tree propagation knowledge new funding has been made available. We applied and have successfully obtained 6-month proof-of-concept funding, which is expected to become a further 3 years funding to upscale to meet government targets. However, there are species that require more detailed research to understand the underlying biology before large scale production will be possible which is what this PhD proposal addresses. Tree cutting propagation depends on root formation from stem bases, however there is significant variation in rooting success between species. This variation is dependent on signalling networks including plant hormones such as auxin which is the core ingredient of many rooting powders. However auxin treatments do not always improve rooting on difficult-to-root species, partly due to increases in auxin degradation. In this project the student will identify whether auxin responses and degradation previously discovered by the primary supervisor can be applied to industry-relevant cuttings of Cornus species (edible and biodiversity value) while developing a novel method for targeted delivery of auxin. By adding auxin to the right cells at the right time for root initiation we can overcome low-auxin-related blocks to rooting. Objective 1: Identify if auxin responses previously discovered in model systems also apply to Cornus cuttings. Building on Rasmussen et al 2015, auxin levels and responsiveness will be measured in harder to propagate Cornus cuttings collected from Whetman Plants International. Grafting will be used to confirm if the signal is shoot or root-derived. Auxin, cytokinins, strigolactones and jasmonic acid levels will be measured in the rooting zone to compare to our pea model. Laser Ablation Tomography will be used to track root initiation and emergence processes in 3D. Objective 2: Develop pharmaceutical carriers for novel auxin delivery to manipulate local auxin at key times post-cutting The student will synthesise nanocarriers to deliver auxin and auxin metabolites in a spatially and temporally targeted manner. This will involve producing (bio)polymer-based capsules (e.g., PLGA, PCL, chitin, chitosan) trapping Auxin inside. These will be fed to the cut end of DR5:GUS lines of Pea which will provide a marker to demonstrate auxin release. They will then be fed to commercial Cornus sp. cuttings to test the impact on root formation compared to alternative protocols including cotton feeding, microinjection, microdialysis delivery and mixed in lanolin. Objective 3: Interference with auxin movement and metabolism to improve rooting The auxins IBA, 2-4-D and NAA are more stable with different mobility to IAA and will be applied with and without NPA (auxin transport inhibitor) to cuttings along with the three most effective polyphenols for stabilising auxin (ferulic acid, caffeic acid and vanillin). For each of these compounds PsDR5:GUS will be used to observe changes in auxin response parallel to rooting response in Cornus sp. Deliverables: Protocols for effective tree propagation to profit the industry partner while meeting government tree-planting targets.New technology for targeted release of molecules (no genetic modification required) Novel insights into how traditional physiology methods can be used to manipulate internal auxin levels and the cause behind the adventitious root development decline with age Milestones: Year 1: Review paper (aligned with first year review requirements). Year 2: Research paper 1: Auxin regulation of adventitious rooting: a comparison between a model and woody species (New Phytologist/Journal of Experimental Botany for example). Year 3: Research paper 2: Targeted release of auxin and auxin degradation blockers in plant development (Nature/Science)