PhenoWell®-A novel screening system for soil-grown plants

Abstract

As agricultural production is reaching its limits regarding outputs and land use, the need to further improve crop yield is greater than ever. The limited translatability from in vitro lab results into more natural growth conditions in soil remains problematic. Although considerable progress has been made in developing soil-growth assays to tackle this bottleneck, the majority of these assays use pots or whole trays, making them not only space- and resource-intensive, but also hampering the individual treatment of plants. Therefore, we developed a flexible and compact screening system named PhenoWell® in which individual seedlings are grown in wells filled with soil allowing single-plant treatments. The system makes use of an automated image-analysis pipeline that extracts multiple growth parameters from individual seedlings over time, including projected rosette area, relative growth rate, compactness, and stockiness. Macronutrient, hormone, salt, osmotic, and drought stress treatments were tested in the PhenoWell® system. The system is also optimized for maize with results that are consistent with Arabidopsis while different in amplitude. We conclude that the PhenoWell® system enables a high-throughput, precise, and uniform application of a small amount of solution to individually soil-grown plants, which increases the replicability and reduces variability and compound usage.

Keywords: Arabidopsis; abiotic stress; growth and development; high‐throughput screening assays; maize; nutrients; phytohormones; plant leaves; seedlings; soil.

FIGURE 1

Design of the PhenoWell® plates. (a) Technical drawing of the insert plate of the PhenoWell® system. (b) Drawing of the corresponding adapter plate. (c) 3D rendering of the PhenoWell® design showing the assembly of the insert and adapter plate. (d) Example of a treatment of seedlings with five concentrations of the artificial auxin 1‐Naphthaleneacetic acid.

FIGURE 2

Overview of the image‐analysis pipeline. (a) An example of an RGB image, cropped to a single PhenoWell® plate, taken during the experiment. (b) Mask file in which dots with the same color indicate the position of wells with the same treatment. (c) Binary image, generated after image segmentation, which is used to measure the different growth traits.

FIGURE 3

Determination of the optimal treatment regime for the PhenoWell® assay. (a) Changes in PRA of the early (5 & 7 DAS) and the later treatment (7 & 10 DAS) with 10 μM NAA over time. (b) Representative pictures at 14 DAS of control plants (left), a plant treated with 10 μM NAA at 5 & 7 DAS (middle) and at 7 & 10 DAS (right). (c, d), Means of compactness (c) and stockiness (d) measurements over time. Error bars indicate standard error of the mean (n = 4). DAS: Days after sowing; 5d_7d: Treatment at 5 and 7 DAS; 7d_10d: Treatment at 7 and 10 DAS.

FIGURE 4

Changes in projected rosette area (PRA) after the treatment with different phytohormones compared with the control treatment in the PhenoWell® system. (a) Abscisic acid (ABA). (b) 1‐Aminocyclo‐propane‐1‐carboxylic acid (ACC). (c) 6‐Benzylaminopurine (BAP). (d) Brassinolide (BL). (e) Gibberellic acid 3 (GA). (f) 1‐Naphthaleneacetic acid (NAA). Error bars indicate standard error of the mean (n = 4).

FIGURE 5

Effect of different phosphate concentrations on Arabidopsis seedling growth. (a–c), Changes in PRA (a), stockiness (b) and compactness (c) of plants exposed to different concentrations of KH₂PO₄ compared with control plants. Error bars indicate standard error of the mean (n = 4).

FIGURE 6

Effect of different osmotica and NaCl on plant growth in the PhenoWell® assay. (a, b), Changes in PRA of plants exposed to different concentrations of mannitol (a) and sorbitol (b) compared to control plants treated with water. (c) Representative pictures of plants treated with different NaCl concentrations. (d, e), Changes in PRA (d) and compactness (e) of plants exposed to different concentrations of NaCl. Error bars indicate standard error of the mean; (n = 4).

FIGURE 7

Effect of different drought stress regimes on plant growth. (a) PRA over time for different soil water humidity (SWH) levels. (b) Representative pictures of plants grown with different SWH levels. (c) Compactness for different SWH levels over time. (d) Stockiness for the different SWH levels over time. Error bars indicate standard error of the mean (n = 4).

FIGURE 8

Leaf 2 growth of the maize B104 line with GA or control treatments in the maize PhenoWell® system. (a) Changes in leaf 2 length over time of B104 plants with 100 μM GA or control treatment. (b) The representative picture of B104 plants grown with control (left) or 100 μM GA (right) treatment. Error bars indicate standard error of the mean (n = 2).