科学家利用WIWAM植物表型平台在知名期刊Front Plant Sci发表了题为“VvEPFL9-1 Knock-Out via CRISPR/Cas9 Reduces Stomatal Density in Grapevine”的文章。文章研究用到了比利时根特大学和SMO开发的植物表型平台。

WIWAM植物表型成像系统由比利时SMO公司与Ghent大学VIB研究所研制生产，整合了LED植物智能培养、自动化控制系统、叶绿素荧光成像测量分析、植物热成像分析、植物近红外成像分析、植物高光谱分析、植物多光谱分析、植物CT断层扫描分析、自动条码识别管理、RGB真彩3D成像等多项先进技术，以较优化的方式实现大量植物样品——从拟南芥、玉米到各种其它植物的生理生态与形态结构成像分析，用于高通量植物表型成像分析测量、植 物胁迫响应成像分析测量、植物生长分析测量、生态毒理学研究、性状识别及植物生理生态分析研究等。

SMO是欧洲先进的机械设备制造与设计工程公司，是一家将大规模自动化理念和工业级零件和设备整合入 植物成像系统的厂家，在机械自动化以及机器视觉成像领域拥有丰富的设计和实践经验，为欧洲先进客户提供机械设计 解决方案，SMO公司将机械领域的先进理念带入了植物表型机器人领域，所采用的配件均为工业界广泛认可的高品质 配件，耐受苛刻环境，另外表型设备领域的诸多自动化配件，均由SMO公司自主设计，因公司拥有较为强大的工程师 团队，基于工业领域的丰富经验，可针对不同客户需求，提供复杂表型成像系统的解决方案。目前 WIWAM植物表型平台分为WIWAM XY，WIWAM Line、WIWAM Conveyor、WIWAM Mobile、WIWAM Imaging Box等几个系列，同时还提供提供野外表型成像系统设计方案。

VvEPFL9-1通过CRISPR/Cas9敲除降低葡萄气孔密度

摘要

EPFL9也称为气孔素，是一种富含半胱氨酸的肽，可诱导维管植物形成气孔，与其他表皮模式因子（EPF1、EPF2）起拮抗作用。葡萄中有两个EPFL9基因，EPFL9-1和EPFL9-2，在成熟功能C端结构域的蛋白质水平上具有82%的同源性。在这项研究中，CRISPR/Cas9系统被用于对高度可转化基因型“Sugraone”中的VvEPFL9-1进行功能表征研究。通过根癌农杆菌（Agrobacterium tumefaciens）在胚性愈伤组织中进行基因转移，选择一组再生植株进行评价。对于许多株系，目标位点的编辑图谱显示了一系列突变，主要导致编码序列的移码或影响第二个半胱氨酸残基。气孔密度分析表明，与对照相比，编辑植株的气孔数量显著减少，首次证明了EPFL9在多年生果树中的作用。在不同环境条件下进行的实验中，对三个编辑好的品系的生长、光合作用、气孔导度和水分利用效率进行了评估。与对照相比，编辑品系的内在水分利用效率有所提高，表明在未来的环境干旱情景下，在降低气孔密度方面可能具有优势。我们的结果表明，在不断变化的气候条件下，控制气孔密度有可能优化葡萄的适应性。

关键词：葡萄；气候变化；基因组编辑；气孔；用水效率。

VvEPFL9-1 Knock-Out via CRISPR/Cas9 Reduces Stomatal Density in Grapevine

Abstract

Epidermal Patterning Factor Like 9 (EPFL9), also known as STOMAGEN, is a cysteine-rich peptide that induces stomata formation in vascular plants, acting antagonistically to other epidermal patterning factors (EPF1, EPF2). In grapevine there are two EPFL9 genes, EPFL9-1 and EPFL9-2 sharing 82% identity at protein level in the mature functional C-terminal domain. In this study, CRISPR/Cas9 system was applied to functionally characterize VvEPFL9-1 in 'Sugraone', a highly transformable genotype. A set of plants, regenerated after gene transfer in embryogenic calli via Agrobacterium tumefaciens, were selected for evaluation. For many lines, the editing profile in the target site displayed a range of mutations mainly causing frameshift in the coding sequence or affecting the second cysteine residue. The analysis of stomata density revealed that in edited plants the number of stomata was significantly reduced compared to control, demonstrating for the first time the role of EPFL9 in a perennial fruit crop. Three edited lines were then assessed for growth, photosynthesis, stomatal conductance, and water use efficiency in experiments carried out at different environmental conditions. Intrinsic water-use efficiency was improved in edited lines compared to control, indicating possible advantages in reducing stomatal density under future environmental drier scenarios. Our results show the potential of manipulating stomatal density for optimizing grapevine adaptation under changing climate conditions.

Keywords: Vitis vinifera; climate change; genome editing; stomata; water-use efficiency.

Experiment 3: Well-Watered (WW) Conditions in an Automated High-Throughput Phenotyping Platform

Biological replicates of the edited line S-epfl9KO6 (n=6) and ‘Sugraone’WT (n=4), maintained in greenhouse for 12months, with a height range of 60–70cm and a weight brought to 3,000g (in 5l pots) were used. Plants were moved inside the phenotyping

platform (WIWAM, Ghent, Belgium) at the Plant Phenotyping Facility of Fondazione Edmund Mach where temperature was set to 28/25°C, photoperiod to 16/8h and average PPFD to 300μmolm−2 s−1 at apical leaf level. Plants were automatically

watered every day at 6:00AM to target weight (3,000g) and pot weight was evaluated before and after watering for 12days.