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

英国政府制定了到2025年每年在英格兰植树1万公顷的目标。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，预计这将成为未来3年的资金，以满足政府的目标。然而，在大规模生产成为可能之前，有些物种需要更详细的研究来了解潜在的生物学基础，这就是本博士提案所针对的问题。插穗繁殖依赖于树干根基的生根，但不同树种间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，它是许多生根粉的核心成分。然而，生长素处理并不总是改善难以生根的物种的生根，部分原因是生长素降解增加。在这个项目中，学生将确定初级主管以前发现的生长素反应和降解是否可以应用于与行业相关的山茱萸物种的插条(可食用和生物多样性价值)，同时开发一种定向输送生长素的新方法。通过在合适的生根时间向合适的细胞添加生长素，我们可以克服与生长素低相关的生根障碍。目标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茱萸插穗。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收集的难以繁殖的山茱萸插条中衡量。嫁接将被用来确认信号是来自枝条还是来自根。生长素、细胞分裂素、内酯和茉莉酸水平将在生根区进行测量，以与我们的豌豆模型进行比较。激光消融断层扫描将被用来在3D中跟踪牙根的萌发和萌发过程。目的：研制新型生长素递送药物载体，用于在切割后的关键时间操纵局部生长素。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)聚合物胶囊(例如，PLGA、PCL、甲壳素、壳聚糖)，将生长素困在体内。这些将被提供给豌豆DR5：GUS系的切端，这将提供一个标记来证明生长素的释放。然后，它们将被喂给商业山茱萸。通过插条试验，与喂棉、微量注射、微渗析和混合羊毛脂等替代方案进行比较，以测试其对生根的影响。目的：干扰生长素的运动和代谢以促进生根生长素IBA、2-4-D和NAA更稳定，对IAA的迁移率不同，可与三种最有效的稳定生长素的多酚(阿魏酸、咖啡酸和香草醛)一起应用于插条上。对于这些化合物中的每一种，PsDR5：GUS将被用来观察与山茱萸生根反应平行的生长素反应的变化。交付成果：有效的树木繁殖协议，在满足政府植树目标的同时，为行业合作伙伴带来利润。定向释放分子的新技术(无需基因修改)。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里程碑下降的原因的新见解：第一年：综述论文(与第一年审查要求一致)。第2年：研究论文1：不定根的生长素调节：模式植物和木本植物之间的比较(例如，新植物学家/实验植物学杂志)。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(自然/科学)

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)