3D-printed autoclavable plant holders to facilitate large-scale protein production in plants

Abstract

The Australian tobacco plant Nicotiana benthamiana is becoming increasingly popular as a platform for protein production and metabolic engineering. In this system, gene expression is achieved transiently by infiltrating N. benthamiana plants with suspensions of Agrobacterium tumefaciens carrying vectors with the target genes. To infiltrate larger numbers of plants, vacuum infiltration is the most efficient approach known, which is already used on industrial scale. Current laboratory-scale solutions for vacuum infiltration, however, either require expensive custom-tailored equipment or produce large amounts of biologically contaminated waste. To overcome these problems and lower the burden to establish vacuum infiltration in new laboratories, we present here 3D-printed plant holders for vacuum infiltration. We demonstrate that our plant holders are simple to use and enable a throughput of around 40 plants per hour. In addition, our 3D-printed plant holders are made from autoclavable material, which tolerate at least 12 autoclave cycles, helping to limit the production of contaminated waste and thus contributing to increased sustainability in research. In conclusion, our plant holders provide a simple, robust, safe and transparent platform for laboratory-scale vacuum infiltration that can be readily adopted by new laboratories interested in protein and metabolite production in Nicotiana benthamiana. Practical application Transient expression in Nicotiana benthamiana provides a popular and rapid system for producing proteins in a plant host. To infiltrate larger numbers of plants (typically >20), vacuum infiltration is the method of choice. However, no system has been described so far which is robust to use and can be used without expensive and complex equipment. Our autoclavable 3D-printed plant holders presented here will greatly reduce the efforts required to adopt the vacuum infiltration technique in new laboratories. They are easy to use and can be autoclaved at least 12 times, which contributes to waste reduction and sustainability in research laboratories. We anticipate that the 3D printing design provided here will drastically lower the bar for new groups to employ vacuum infiltration for producing proteins and metabolites in Nicotiana benthamiana.

Keywords: Nicotiana benthamiana; additive manufacturing; agroinfiltration; autoclavable 3D printing material; vacuum infiltration.

FIGURE 1

Plant holder design. (A) Shows the schematic drawing of the holder. Dimensions are provided in millimetres. All edges were rounded (fillet) with a 1 mm radius to avoid damage to the plants. (B) Shows the top view of an actual holder. (C) Shows a schematic representation of the vacuum infiltration cycle using the plant holder. (D) Shows the complete vacuum infiltration setup; by alternating between two desiccators, around 40 plants can be infiltrated per hour. (E) Successful vacuum infiltration of Nicotiana benthamiana as demonstrated by transient GFP expression. A representative plant is shown from multiple angles. Plants were infiltrated with Agrobacterium tumefaciens containing the pEAQ‐GFP‐HT plasmid [25]

FIGURE 2

Side‐by‐side comparison of operation times of 3D‐printed holders compared to two previously described vacuum infiltration systems [16, 18]. The violin plot shows the operation times in seconds for seven participants to complete the following actions, which simulate a real vacuum infiltration cycle: taking out three plants with their holding systems from the desiccator; disassembling the holding system; re‐assembling the holding system to three new plants. Each of the seven participants repeated the actions four times. Among the participants, four were experienced users (black triangles), and three were new to vacuum infiltration (gray circles). Below the violin plot, holding items as well as holding assemblies are shown

FIGURE 3

Plant holders described here can be autoclaved for disinfection. (A) Plant holders showed a gradual darkening over multiple autoclave cycles, but no other deterioration. (B) Autoclaving is a highly effective method to sterilise plant holders. All plant holders used for the sterilisation experiment had already been autoclaved four times, demonstrating that they tolerate multiple autoclave cycles. Abbreviation: CFU, colony forming units