Non-destructive Determination of Shikimic Acid Concentration in Transgenic Maize Exhibiting Glyphosate Tolerance Using Chlorophyll Fluorescence and Hyperspectral Imaging

Abstract

The development of transgenic glyphosate-tolerant crops has revolutionized weed control in crops in many regions of the world. The early, non-destructive identification of superior plant phenotypes is an important stage in plant breeding programs. Here, glyphosate-tolerant transgenic maize and its parental wild-type control were studied at 2, 4, 6, and 8 days after glyphosate treatment. Visible and near-infrared hyperspectral imaging and chlorophyll fluorescence imaging techniques were applied to monitor the performance of plants. In our research, transgenic maize, which was highly tolerant to glyphosate, was phenotyped using these high-throughput non-destructive methods to validate low levels of shikimic acid accumulation and high photochemical efficiency of photosystem II as reflected by maximum quantum yield and non-photochemical quenching in response to glyphosate. For hyperspectral imaging analysis, the combination of spectroscopy and chemometric methods was used to predict shikimic acid concentration. Our results indicated that a partial least-squares regression model, built on optimal wavelengths, effectively predicted shikimic acid concentrations, with a coefficient of determination value of 0.79 for the calibration set, and 0.82 for the prediction set. Moreover, shikimic acid concentration estimates from hyperspectral images were visualized on the prediction maps by spectral features, which could help in developing a simple multispectral imaging instrument for non-destructive phenotyping. Specific physiological effects of glyphosate affected the photochemical processes of maize, which induced substantial changes in chlorophyll fluorescence characteristics. A new data-driven method, combining mean fluorescence parameters and featuring a screening approach, provided a satisfactory relationship between fluorescence parameters and shikimic acid content. The glyphosate-tolerant transgenic plants can be identified with the developed discrimination model established on important wavelengths or sensitive fluorescence parameters 6 days after glyphosate treatment. The overall results indicated that both hyperspectral imaging and chlorophyll fluorescence imaging techniques could provide useful tools for stress phenotyping in maize breeding programs and could enable the detection and evaluation of superior genotypes, such as glyphosate tolerance, with a non-destructive high-throughput technique.

Hyperspectral imaging combined with machine learning as a tool to obtain high‐throughput plant salt‐stress phenotyping

“…Recently, controlled environment HTP platforms (Klukas et al , ; Cabrera‐Bosquet et al , ; Chen et al , ), field‐based HTP platforms (Andrade‐Sanchez et al , ; Ballesteros et al , ; Sun et al , ) and an automated phenotyping robot (Salas Fernandez et al , ) were developed at the plant canopy or plot level to monitor large numbers of crop lines. These techniques for monitoring plant exposure to biotic and abiotic stresses are based on RGB imaging of plant biomass and morphological parameters (Chen et al , ), near‐infrared imaging of photoactive pigments, water content and biochemical components (Feng et al , ), infrared thermal imaging of canopy temperature (Biju et al , ) and fluorescence imaging of photosynthetic efficiency (Feng et al , ).…”

“…Among the current image‐based techniques used for plant phenotyping, hyperspectral imaging (HSI), which integrates imaging and spectroscopy, offers the greatest promise in quantitating complex morpho‐physiological traits in a fast and non‐destructive manner. For example, Feng et al () established shikimic acid prediction model to evaluate glyphosate effect on maize in a laboratory environment. Rumpf et al () developed early detection and classification model for foliar sugar beet diseases based on hyperspectral reflectance under greenhouse conditions.…”

Physiological and Morphological Responses of Jatobá Submitted to Weed Competition and Glyphosate Doses

“…Stress, as caused by glyphosate doses, can change leaf optical characteristics and that changes may result plant physiological changes, making it difficult interpretation by F 0 and F m variables (Baker, 2008). However, when plants are under any stress, the F v /F m ratio becomes a significant indicator of the inhibitory effect of PSII (Maxwell & Johnson, 2000;Baker, 2008;Feng et al, 2018). The Fv/Fm ratio ranging from 0.75 to 0.85 indicate that the plants are under some kind of stress (Bolhar-Nordenkampf et al, 1989).…”

“…Moreover, the stress ratio can be better analyzed by ETR together F v /F m ( Figure 2D). A decreased value of F v /F m indicated problems in ETR and subsequently damages to photosynthetic structures (Feng et al, 2018). Probably, the lower ETR in plants under glyphosate doses at 15 and 30 DAT were related to one of the glyphosate indirect effects in photosynthesis, which reduce the ATP synthesis and consequently incorporate less carbon (Malkin & Niyogi, 2000).…”

“…The light is absorbed mainly by chlorophyll that when excited it can dissipate the energy as fluorescence (Maxwell & Johnson, 2000;Baker, 2008). Plants under various types of stress alter the shape and the amount of this energy dissipation directly affecting photosynthesis (Baker, 2008;Olesen & Cedergreen, 2010;Yanniccari, Tambussi, Istilart, & Castro, 2012;Feng et al, 2018). Thus, this study was conducted to evaluate the effect of weed competition and intoxication by glyphosate in jatobá plants.…”