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Hyperhydricity (HH) is one of the most important physiological disorders that negatively affects various plant tissue culture techniques. The objective of this study was to characterize optical features to allow an automated detection of HH. For this purpose, HH was induced in two plant species, apple and Arabidopsis thaliana, and the severity was quantified based on visual scoring and determination of apoplastic liquid volume. The comparison between the HH score and the apoplastic liquid volume revealed a significant correlation, but different response dynamics. Corresponding leaf reflectance spectra were collected and different approaches of spectral analyses were evaluated for their ability to identify HH-specific wavelengths. Statistical analysis of raw spectra showed significantly lower reflection of hyperhydric leaves in the VIS, NIR and SWIR region. Application of the continuum removal hull method to raw spectra identified HH-specific absorption features over time and major absorption peaks at 980 nm, 1150 nm, 1400 nm, 1520 nm, 1780 nm and 1930 nm for the various conducted experiments. Machine learning (ML) model spot checking specified the support vector machine to be most suited for classification of hyperhydric explants, with a test accuracy of 85% outperforming traditional classification via vegetation index with 63% test accuracy and the other ML models tested. Investigations on the predictor importance revealed 1950 nm, 1445 nm in SWIR region and 415 nm in the VIS region to be most important for classification. The validity of the developed spectral classifier was tested on an available hyperspectral image acquisition in the SWIR-region.
Adventitious root (AR) formation is the basis of vegetative propagation in rose, be it via stem cuttings or via stenting. During this process, wounding plays a pivotal role since cell reprogramming takes place at the tissue adjacent to the wound. We investigated the effects of wounding on AR formation on leafy single-node stem cuttings of the rose rootstock R. canina ‘Pfänder’ (codes R02-3 and R02-6) and the cut rose cultivar Rosa ‘Tan09283’ (Registration name ‘Beluga’). Laser wounding treatments were based on the assisted removal of tissue layers located in the bark. The positioning of wounding was studied based on two marking directions: along the cutting base (strip pattern) and around the cutting base (ring pattern). Additionally, the effects of external supply of indole-butyric acid (IBA 1 mg L-1) on rootingwere analyzed. Results showedthat inorder toremovespecific tissue layers, the calculation of the laser energy density (J cm-2) in terms of cutting diameter was necessary. Interestingly, the application of energy densities from 2.5 J cm-2 up to approximately 8.5 J cm-2 were sufficient to expose the tissue layers of epidermis up to regions of phloem. Regarding AR formation for R. canina ‘Pfänder’, characterized by a low rooting response, an increase in the rooting percentage was registered when the laser treatment eliminated the tissue up to phloem proximities. Analysis of the nodal position showed that bud location was a preferential place for AR formation independently of wounding treatment. In case of Rosa ‘Tan09283’, laser treatments did not reduce its high rooting capacity, but an apparent reduction in rooting quality due to an investment in tissue healing was observed when wounding reached deeper layers such as parenchyma and sclerenchyma. Results also showeda strongARformation directly fromwounded regions in case of Rosa ‘Tan09283’ specifically when the woundwas located below the axillary bud. In conclusion, wounding by assisted-elimination of layers by laser can induce positive effects on AR formation of single-node stemcuttings of the rose if energy applied is able to expose phloemproximities,a longitudinalorientation, and relative position to the axillary bud are considered.
Recording of Low-Oxygen Stress Response Using Chlorophyll Fluorescence Kinetics in Apple Fruit
(2023)
Long-term storage of apples (Malus x domestica, Borkh.) is increasingly taking place under Dynamic Controlled Atmosphere (DCA). The oxygen level is lowered to ≤ 1 kPa O2 and the apples are stored just above the Lower Oxygen Limit (LOL). Low oxygen stress during controlled atmosphere storage can lead to fermentation in apples if oxygen levels are too low. Chlorophyll fluorescence can be used to detect low-oxygen stress at an early stage during storage. The currently available non-imaging fluorescence systems often use the minimal fluorescence (Fo) parameter. In contrast, the use of chlorophyll fluorescence kinetics is insufficiently described. Therefore, this study aimed to gain more knowledge about the response of chlorophyll fluorescence kinetics to low oxygen stress in apples using a fluorescence imaging system. The results show that the kinetic fluorescence curves differ under aerobic and fermentation conditions. The fermentative conditions initiated a decrease in fluorescence intensity upon application of the saturation pulses during exposure to actinic light. This result was made at 18 °C and 2 °C ambient temperatures. Interestingly, the kinetic curve changed at 2 °C before fermentation products accumulated in the apples. Non-photochemical quenching (NPQ) decreased under fermentation conditions in the dark phase after relaxation. Upon entering the dark relaxation phase after Kautsky induction, ɸPSII began to increase. Under atmospheric oxygen conditions, ɸPSII reached values of 0.81 to 0.76, while under fermentation, ɸPSII values ranged from 0.57 to 0.44.
Background
The current development of sensor technologies towards ever more cost-effective and powerful systems is steadily increasing the application of low-cost sensors in different horticultural sectors. In plant in vitro culture, as a fundamental technique for plant breeding and plant propagation, the majority of evaluation methods to describe the performance of these cultures are based on destructive approaches, limiting data to unique endpoint measurements. Therefore, a non-destructive phenotyping system capable of automated, continuous and objective quantification of in vitro plant traits is desirable.
Results
An automated low-cost multi-sensor system acquiring phenotypic data of plant in vitro cultures was developed and evaluated. Unique hardware and software components were selected to construct a xyz-scanning system with an adequate accuracy for consistent data acquisition. Relevant plant growth predictors, such as projected area of explants and average canopy height were determined employing multi-sensory imaging and various developmental processes could be monitored and documented. The validation of the RGB image segmentation pipeline using a random forest classifier revealed very strong correlation with manual pixel annotation. Depth imaging by a laser distance sensor of plant in vitro cultures enabled the description of the dynamic behavior of the average canopy height, the maximum plant height, but also the culture media height and volume. Projected plant area in depth data by RANSAC (random sample consensus) segmentation approach well matched the projected plant area by RGB image processing pipeline. In addition, a successful proof of concept for in situ spectral fluorescence monitoring was achieved and challenges of thermal imaging were documented. Potential use cases for the digital quantification of key performance parameters in research and commercial application are discussed.
Conclusion
The technical realization of “Phenomenon” allows phenotyping of plant in vitro cultures under highly challenging conditions and enables multi-sensory monitoring through closed vessels, ensuring the aseptic status of the cultures. Automated sensor application in plant tissue culture promises great potential for a non-destructive growth analysis enhancing commercial propagation as well as enabling research with novel digital parameters recorded over time.
Currently, only non-imaging chlorophyll fluorescence measurements are used to identify the Lower Oxygen Limit (LOL) in Dynamic Controlled Atmosphere - Chlorophyll Fluorescence (DCA-CF) storage. The disadvantage of non-imaging fluorescence is that no statement can be made about the spatial heterogeneity of the sample. In contrast, chlorophyll fluorescence imaging can detect spatial heterogeneity of photosynthetic activity and has been established in research for some decades because the information benefit is higher. In this study, the chlorophyll fluorescence (Fo, Fm, Fv, Fv/Fm) of apples (Malus x domestica, BORKH.) was measured with a fluorescence imaging system in situ during storage. Intact apples of ‘Braeburn’ and ‘Golden Delicious’ were stored under low-oxygen stress conditions (< 1 kPa). The metabolic shift from aerobic to fermentative metabolism was made visible with the chlorophyll fluorescence imaging and was spatially localized on the sample. Furthermore, a method was developed to identify the LOL based on the chlorophyll fluorescence imaging combined with the histogram division method. This method considers the heterogeneity of the fluorescence and bundles the measured Fo data as histograms. Our results showed that the fluorescence imaging combined with the histogram division method can be a powerful tool for identifying the LOL.