Digitally Quantifying Growth and Verdancy of Lolium Plants In Vitro

Abstract

The image analysis of plants provides an opportunity to measure changes in growth and physiology quantitatively, and non-destructively, over time providing significant advantages over traditional methods of assessment which often rely on qualitative and subjective measures to distinguish between different treatments or genotypes in an experiment. Image analysis techniques are commonly deployed for the analysis of plants in the field or glasshouse, but few studies have demonstrated the use of image analysis to phenotype plants grown under aseptic conditions in culture media. Lolium × hybridum Hausskn 'Shogun' plants were germinated in vitro and cultured on media containing combinations of thidiazuron [1-phenyl-3-(1,2,3-thiadiazol-5-yl) urea] (TDZ), N6-benzylaminopurine (BA) and gibberellic acid (GA3) or on phytohormone-free control media. RGB images were taken of the plants throughout the experiment and morphological image analysis techniques were used to quantify changes in plant development. A novel approach to quantitatively measure 'greenness' in plants using the CIELAB colour model (L*a*b) colour space from RGB images was developed. This methodology could be utilised to develop improved in vitro growth protocols for Lolium and grass species with similar morphology.

Keywords: fescue; grasses; image analysis; plant growth regulators; ryegrass; tissue culture.

Figure 1

Mean greenness values for ryegrass plants growing in vitro at Days 30, 57 and 86. Error bars represent standard error to a 95% confidence interval.

Figure 2

(A) Mean area values for ryegrass plants growing in vitro at Days 30, 57 and 86; (B) mean convex hull area for ryegrass plants growing in vitro at Days 30, 57 and 86; (C) mean perimeter values for ryegrass plants growing in vitro at Days 30, 57 and 86; (D) mean solidity values for ryegrass plants growing in vitro at Days 30, 57 and 86. Error bars represent standard error to a 95% confidence interval.

Figure 3

Representative images of Lolium plants growing in vitro at Day 86 of the experiment: (A) Control; (B) BA; (C) GA₃; (D) GA₃ + BA; (E) GA₃ + TDZ; (F) TDZ.

Figure 4

Schematic diagram of imaging station used to collect images of tissue culture vessels for this experiment.

Figure 5

A summary of the steps in the image analysis pipeline used to identify ryegrass plants in vitro: (A) original RGB image; (B) conversion of the RGB image into CMYK with the Y-channel selected; (C) binarisation of the Y-channel image; (D) conversion of the RGB image into L*a*b with the *a channel selected; (E) binarisation of the *a-channel; (F) logical-OR operation to combine Y-channel and *a-channel binary masks; (G) morphological analysis using PlantCV; (H) measurement of foreground area above and below the medium level.

Figure 6

(A) The CIELAB colour space diagram. The L*a*b, colour system represents quantitative relationship of colours on three axes: L value indicates lightness, and a and b are chromaticity coordinates. (B) The mean values of a (where a < 0) and b (where b > 0) can be used to generate a (b, a) co-ordinate. The angle created between (b, a) and (0, 0) can be used to create an index of “greenness” by rescaling it from 0 (θ = 0°) to 100 (θ = 90°), where θ increases with increasing verdancy.