The video explains the use of a 3D-Printed Direct Plant Infusion (DPI) device to apply compounds to plants effectively. It details the assembly, filling, and maintenance process of the DPI, highlighting experimental results with CFDA dye, streptomycin, and Imidacloprid, demonstrating their effects on plant health and pest management.

applying compounds to citrus plants using a novel 3d-printed direct plant infusion (dpi) device

使用直接输注装置在植物中递送分子

The management of plants in commercial and landscape settings often requires the use of chemical compounds to optimize the plant growth and health. How these molecules are delivered depends on the type of molecule, the function of the molecule, the type of plant, and the management system in place. Foliar and soil applications are the easiest delivery strategies, but limitations in the uptake of some molecules necessitate direct delivery. An example of these molecules is therapeutic molecules that function best when they move systemically within the plant but cannot be effectively delivered by simple topical applications1. This is the case with Huanglongbing (HLB), also called citrus greening disease. HLB is a disease associated with a phloem-limited bacterium, Candidatus Liberibacter asiaticus (CLas), which cannot be cultured outside of the plant, or its insect vector, Diaphorina citri Kuwayama (D. citri)2.
If the putative therapeutic molecules are gene products, they can be tested by creating transgenic plants expressing these compounds. However, transgenic plant production can be time- and resource-intensive, is highly genotype-dependent, and can be inhibited by gene silencing3. In addition, even if these transgenics show promising results, regulatory and public perception constraints reduce the likelihood of their commercial acceptance4,5. The exogenous application of compounds, however, simplifies the testing of biological and synthetic molecules because it does not require the production of stable or transiently expressing transgenic plants, which reduces the time and resources for testing a molecule&#39;s effects. A method for the effective and efficient systemic plant delivery of exogenous compounds could be used for a wide variety of research and screening purposes.
One of these applications is the analysis of systemic molecule movement within a plant&#39;s vascular system, which can be done using trackable markers, whether they are fluorescent, visible, or unique chemical isotopes6,7,8,9. One commonly used fluorescent marker is 5,6-carboxyfluorescein-diacetate (CFDA), which is a membrane-permeable dye that is degraded by intracellular esterases into 5,6-carboxyfluorescein (CF) and, subsequently, becomes fluorescent and membrane-impermeable10. CFDA has been extensively used to monitor phloem transport, sink and source relationships, and vasculature patterning in plant tissue11,12.
In addition to these markers, certain compounds may directly alter the plant&#39;s physiology to increase the productivity or kill the plant in the case of herbicides. Both insecticides and antimicrobial compounds are a means of increasing plant productivity, especially in the presence of HLB. An example of an antimicrobial molecule that is used to control CLas is streptomycin. Streptomycin is an aminoglycoside antibiotic that was originally isolated from Streptomyces griseus and has been shown to inhibit bacterial growth through the inhibition of protein biosynthesis13. In terms of insecticides, the main target for HLB research is D. citri, which transmits CLas from tree to tree14. For this purpose, neonicotinoids, such as imidacloprid, are commonly utilized, as they are the gold standard for controlling insect pests15. All these varied uses are important aspects of current plant management strategies, and the development of novel products is dependent on efficient screening assays.
One method that is used for the introduction of compounds into woody plants is direct injection into the trunk. A variety of systems have been designed that vary in their needs for predrilled injection sites, and these systems utilize either pressure-based injection or passive flow16. Although pressure-based systems allow for the quick introduction of a given compound, the potential physical damage caused by forcing liquid through blocked or embolized vasculature needs to be considered17. Although the foliar or drench application of compounds is less time-intensive to implement, direct plant injection reduces waste of the compound of interest due to losses to the air or soil and can also lengthen the time that compounds are in an active state by reducing the exposure to the outside environment18. Both these aspects are important for preserving expensive reagents and ensuring consistency among replicates in research settings.
This study describes the design, construction, and use of an innovative direct plant infusion (DPI) device, which can be used to assess how compounds of interest affect a host plant. A standard 3D printer was used to manufacture both the device itself and several components associated with its construction. This in-house construction method allows researchers to modify the device and device components based on their specific experimental needs and reduces the reliance on commercially available plant injection devices. The device setup is simple and efficient, and all the auxiliary components are readily available and inexpensive. Although the system was designed for use with a variety of plant species, the examples presented here pertain to potted citrus plants. Additionally, this study demonstrates that this device is capable of efficiently delivering multiple types of compounds systemically to young citrus plants without causing lethality. The compounds tested included CFDA, which was used to assess the compound distribution in the plant, and streptomycin and imidacloprid, which were used to verify that the antimicrobial and insecticidal effects of these compounds were observed when delivered via DPI.

作者聚焦：用于植物分子递送的直接输注装置  

用于植物分子输送的直接输液装置

Watch this Scientific Journal Video about Applying Compounds to Citrus Plants Using a Novel 3D-Printed Direct Plant Infusion DPI Device at JoVE.com

Applying Compounds to Citrus Plants Using a Novel 3D-Printed Direct Plant Infusion (DPI) Device  

使用新型3D打印直接植物输液（DPI）设备将化合物应用于柑橘类植物

要使用新型3D打印直接植物输液或DPI设备将化合物应用于植物，请确保该设备正确安装到植物上。组装正确后，塑溶胶环应与DPI设备齐平，喷口应与树上钻的孔对齐。使用注射器或移液器将感兴趣的化合物溶液填充DPI设备。使用注射器穿透DPI设备对面的硅胶带和塑料溶胶环，从内部通道抽出空气。将任何提取的化合物置回DPI设备。在注射器形成的孔上再添加一小块硅胶带，以加强该区域并防止撕裂。检查设备在连接点处是否有可见泄漏，并检查设备储液罐的液位是否稳定。用蜡密封膜覆盖DPI装置的开口端，并将其向下拉以形成密封，以减少实验化合物的蒸发。用注射器尖端在蜡膜上戳一个孔以防止真空的发展，然后重新填充设备。每天检查设备以确保组件正确对齐，并使用注射器加满液体以避免储液槽干涸运行，直到输送所需的量。当使用DPI设备将两毫升两毫摩尔CFDA染料引入植物时，在处理植物的脉管系统中检测到荧光信号，但在用20%DMSO水处理的受控植物中不存在荧光信号。在治疗后24小时内观察到该信号，均匀地在整个解剖的植物组织类型中。与对照组相比，对亚洲自由念珠菌或CLass阳性植物进行链霉素治疗显示，治疗后平均细菌滴度降低至28天。处理后的植物在所有组合时间点上均显示出较低的平均CLas滴度。与对照组相比，链霉素处理的植物在60天后显示新的健康潮红生长偶尔增加。与相应的卵数相比，在最高吡虫啉浓度下对植物进行吡虫啉处理显着减少了若虫的出现。这在若虫蜜露产量的减少中表现得很明显。

applying-compounds-to-citrus-plants-using-novel-3d-printed-direct

Research

JoVE Journal

Biology

Applying Compounds to Citrus Plants Using a Novel 3D-Printed Direct Plant Infusion (DPI) Device

297 次观看. 要使用新型3D打印直接植物输液或DPI设备将化合物应用于植物，请确保该设备正确安装到植物上。组装正确后，塑溶胶环应与DPI设备齐平，喷口应与树上钻的孔对齐。使用注射器或移液器将感兴趣的化合物溶液填充DPI设备。使用注射器穿透DPI设备对面的硅胶带和塑料溶胶环，从内部通道抽出空气。将任何提取的化合物置回DPI设备。在注射器形成的孔上再添加一小块硅胶?...

视频: Applying Compounds to Citrus Plants Using a Novel 3D-Printed Direct Plant Infusion (DPI) Device  

Direct Infusion Device for Molecule Delivery in Plants

Testing the function of therapeutic compounds in plants is an important component of agricultural research. Foliar and soil-drench methods are routine but have drawbacks, including variable uptake and the environmental breakdown of tested molecules. Trunk injection of trees is well-established, but most methods for this require expensive, proprietary equipment. To screen various treatments for Huanglongbing, a simple, low-cost method to deliver these compounds to the vascular tissue of small greenhouse-grown citrus trees infected with the phloem-limited bacterium Candidatus Liberibacter asiaticus (CLas) or infested with the phloem-feeding CLas insect vector Diaphorina citri Kuwayama (D. citri) is needed.
To meet these screening requirements, a direct plant infusion (DPI) device was designed that connects to the plant&#39;s trunk. The device is made using a nylon-based 3D-printing system and easily obtainable auxiliary components. The compound uptake efficacy of this device was tested in citrus plants using the fluorescent marker 5,6-carboxyfluorescein-diacetate. Uniform compound distribution of the marker throughout the plants was routinely observed.
Furthermore, this device was used to deliver antimicrobial and insecticidal molecules to determine their effects on CLas and D. citri respectively. The aminoglycoside antibiotic streptomycin was delivered into CLas-infected citrus plants using the device, which resulted in a reduction in the CLas titer from 2 weeks to 4 weeks post treatment. Delivering the neonicotinoid insecticide imidacloprid into D. citri-infested citrus plants resulted in a significant increase in psyllid mortality after 7 days. These results suggest that this DPI device represents a useful system for delivering molecules into plants for&#160;testing and facilitate research and screening purposes.

This manuscript describes a novel direct plant infusion device for screening the effectiveness of molecules against the bacteria (Candidatus Liberibacter asiaticus) or its insect vector (Diaphorina citri, Kuwayama) that, in combination, are associated with Huanglongbing citrus disease.

Testing the function of therapeutic compounds in plants is an important component of agricultural research. Foliar and soil-drench methods are routine but ...