High Throughput Phenotyping Using NIR Sensor .pptx

High Throughput Phenotyping Using NIR Sensor For Abiotic Stress Tolerance

1. ICAR-Indian Agricultural Research Institute, New Delhi High Throughput Phenotyping Using NIR Sensor For Abiotic Stress Tolerance E-Publication Sudhir Kumar Division of Plant Physiology ICAR-Indian Agricultural Research Institute, New Delhi Akshay S. Sakhare , Plant Physiology Section ICAR-Indian Institute of Rice Research, Hyderabad

2. High Throughput Phenotyping Using NIR Sensor For Stress Tolerance Study Advanced phenotyping techniques use image processing with visible to near-infrared spectrum light sources to provide image datasets of the plant phenotype in a nondestructive manner ( Rahaman et al., 2015). The imaging techniques—including visible light imaging, hyperspectral imaging, infrared imaging, fluorescence imaging, and X-ray computed tomography, using a robust software system, generate unique, multilevel phenotyping data ( Sozzani et al. , 2014). With the current emphasis on precise phenotyping , imaging techniques that measure the interaction between light and plants, such as photons (transmitted, absorbed, or reflected), are contributing more to reaching the desired level of measurements related to quantitative phenotypic traits. ICAR-Indian Agricultural Research Institute, New Delhi

3. Employment of high dimensional phenotyping assays requires uniform experimental protocols with calibrated imaging sensors and precise analyses of raw data-processing methods. The imaging devices that are currently used for high-throughput phenotyping of crop plants are outlined as follows. 1. Visible Light (300–700 nm) Imaging 2. Infrared- and Thermal-Based Imaging 3. Fluorescence Imaging 4. Spectroscopy Imaging 5. Integrated Imaging Techniques ICAR-Indian Agricultural Research Institute, New Delhi

4. Importance Infrared thermal imaging allows visualization of infrared radiation emitted from the object using the Stefan-Boltzmann equation (R¼εσT4 ). This technology uses internal molecular movements of the objects that emit infrared radiation for imaging ( Kastberger and Stachl , 2003). Infrared imaging devices use two main wavelength ranges, near-infrared (NIR) (0.9–1.55μm) and far-infrared (FIR) (7.5–13.5μm). However, the sensitive spectral range of thermal cameras is 3–14μm (Li et al., 2014). In addition, by combining NIR imaging with visible imaging, that is, visible to short-wave infrared (VSWIR; 0.4–2.5μm), this method provides deeper insight into plant health under different stress conditions because it provides well-defined spectral features for pigments, leaf water content, and biochemicals such as lignin and cellulose (Yang et al ., 2013). Further, infrared thermography has also been used to study stomatal responses under salinity and drought by visualizing differences in canopy temperature ( Rahaman et al., 2015). Several earlier studies used NIR spectroscopy to indirectly assess crop growth and yield performance under stressed conditions. These infrared imaging technologies provide high spatial resolution images with precise measurements in large fields during varied climatic conditions at the same time (Li et al., 2014). ICAR-Indian Agricultural Research Institute, New Delhi

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6. Uses Thermal imaging cameras are sensitive to a spectral range of 3–14μm in the infrared region; within this wavelength, 3–5 and 7–14μm are the most commonly used wavelengths for imaging. The smaller wavelengths correspond to higher energy levels, and therefore, higher thermal sensitivity. Thermal imaging can measure leaf and canopy temperature to evaluate leaf water status. Gas exchange is measured by stomatal movement because plants are generally cooled by transpiration, and plant temperature increases when the stomata are closed. Canopy temperature differences between the canopy and the surrounding air can be used as a proxy for drought tolerance in dry environments. Thermal infrared imaging can be performed in the laboratory and the field to characterize tolerance to various stresses such as drought and salinity based on osmotic tolerance and Na+ exclusion. It can also be used to measure relative chlorophyll content, leaf color, and canopy temperature (Merlot et al. , 2002; Jones et al ., 2009; Munns et al ., 2010). Infrared thermal imaging systems have been used to measure stomatal behavior under various stress conditions, for example, to monitor salt tolerance in wheat genotypes ( Bayoumi et al ., 2014). ICAR-Indian Agricultural Research Institute, New Delhi

7. Use of NIR Sensor For Drought Stress Tolerance Tolerance to drought stress in plants is indicated by visible symptoms like, including leaf rolling, stay-green ability, stomatal closure, photochemical quenching, photo inhibition resistance, water use efficiency (WUE), osmotic adjustment, membrane stability, epicuticular wax content, mobilization of water-soluble carbohydrates and increased root length (Singh et al ., 2015a). These traits are often targeted for phenotyping under drought stress. Leaf rolling is the primary visible symptom—and one of the survival mechanisms against drought stress—reducing the transpiration rate and canopy temperature ( Joshi and Karan, 2013). ICAR-Indian Agricultural Research Institute, New Delhi

8. Use of NIR Sensor For Drought Stress Tolerance cont.. Drought-tolerant plants retain a higher relative water content (RWC) under water deficit conditions to sustain normal growth (Singh et al., 2015b). The impact of drought on photosynthesis has been differentiated into two groups; direct and indirect. The direct effect is measured as the increased restriction of CO 2 diffusion via stomata that limits CO 2 supply inside leaves and leads to a decline in CO 2 availability for Rubisco ( Martorell et al ., 2014). Indirect effects include alterations in the biochemistry and metabolism of the photosynthetic apparatus, membrane permeability, and the promotion of oxidative stress ( Aranda et al ., 2010; Wahid et al., 2014). Drought stress critically impedes wheat and grain legumes’ performance during flowering and grain-filling stages, primarily because of a reduced net photosynthesis rate, oxidative damage to chloroplasts, and stomatal closure, which finally leads to poor grain development ( Farooq et al ., 2014b, 2017). ICAR-Indian Agricultural Research Institute, New Delhi

9. Drought stress exerts osmotic pressure on plants, the response of which is to accumulate compatible solutes such as proline , betaine , and polyols ( mannitol , trehalose , etc.) in the cytosol to retain osmotic pressure that provides a driving gradient for the uptake of water and turgor maintenance ( Joshi et al ., 2016a). It is well established that proline plays an important role in the stabilization of cellular proteins and membranes under high osmotic concentrations. Secondary responses, such as oxidative stress, induce membrane damage during water stress and are characterized by membrane lipid peroxidation perceived by the accumulation of malondialdehyde (MDA) ( Farooq et al., 2010). Root length is another significant character that requires careful consideration, as roots are directly connected with soil and perceive the immediate effects of a reduction in soil water content ( Hochholdinger , 2016). Different next-generation phenotyping platforms with highly efficient software are being used to evaluate drought tolerance in different crops (Cobb et al., 2013); for example, PHENOPSIS ( Granier et al ., 2006) and WIWAM in Arabidopsis, and LemnaTec in barley ( Honsdorf et al ., 2014), maize ( Ge et al., 2016), tomatoes ( Petrozza et al., 2014), and wheat ( Feher-Juha´sz et al ., 2014). ICAR-Indian Agricultural Research Institute, New Delhi

10. 2 Salinity Tolerance- Salinity induces both ion toxicity and osmotic stress in crop plants by altering the physiological status and ionic homeostasis of cells ( Wungrampha et al., 2018). This cellular status is regulated by a plethora of genes ( Joshi et al., 2017). Crop plants are vulnerable to salinity, the extent of which depends on the plant growth stage and salt concentration ( Hossain et al., 2015). The mechanism of tolerance is cultivar specific. Salinity stress affects growth at different developmental stages and generally delays germination. During vegetative stages, it reduces leaf area, total chlorophyll content, biomass, and root length ( L€auchli and Grattan, 2007). In addition to phenotypic changes, depending on the severity and duration of the stress, salinity affects various physiological and metabolic processes by exerting osmotic and ionic toxicity (Gupta and Huang, 2014). Osmotic stress reduces the water absorption capacity of root systems and concurrently increases water loss from leaves ( Munns , 2005). However, the production of cellular osmolytes ( proline , glycine betaine , mannitol , etc.) alleviates the effect of osmotic stress ( Singla-Pareek et al., 2008). ICAR-Indian Agricultural Research Institute, New Delhi

11. Use of NIR Sensor For Salinity Stress Tolerance - Important physiological changes caused by osmotic stress include Membrane interruption, Nutrient imbalance, Impaired ability of ROS detoxification, Differences in antioxidant enzymes, Decreased photosynthetic activity, and r Educed stomatal aperture. Ion toxicity mainly occurs due to the higher accumulation of na + and cl ions in plant tissues exposed to high saline conditions. Higher uptake of Na+ and Cl - into cells results in a severe ionic imbalance and might cause considerable physiological changes. ICAR-Indian Agricultural Research Institute, New Delhi

12. Use of NIR Sensor For Salinity Stress Tolerance - High Na+ concentration inhibits K+ ion uptake, which is essential for growth and development, resulting in reduced productivity. Production of ROS, such as singlet oxygen, superoxide, hydroxyl radical, and hydrogen peroxide is a secondary response under salinity stress. Salinity induced ROS formation causes oxidative damage in various cellular components such as proteins, lipids, and DNA, thus interrupting vital cellular processes in plants. Plants develop various physiological and biochemical mechanisms to survive in soils with high salt concentration ( Joshi et al ., 2016b). Visible imaging-based phenotyping uses a standard evaluation system (SES) score, while RGB is used to measure total chlorophyll content ( Mishra et al ., 2016a,b). Next-generation phenotyping assays exploiting different light wavelengths are used to assess salt tolerance, such as PHENOPSIS ( Granier et al., 2006), WIWAM in rice ( Hairmansis et al., 2014), wheat, and barley (Harris et al., 2010; Humplı´k et al ., 2015a; Meng et al ., 2017). ICAR-Indian Agricultural Research Institute, New Delhi

13. Use of NIR Sensor For Temperature (Heat/Cold) Stress Tolerance-_ Temperature stress in plants occurs at either high or chilling/freezing temperatures. Phenotypic and biochemical features change in cultivated plant species in response to heat stress, for example, Poor Germination Ratio, Poor Seedling Emergence, Abnormal Seedling Development, Poor Seedling Vigor, Reduced Radicle And Plumule Growth, Inhibition Of Photosystem II ( Psii ) Activity, And ROS Production ( Jagadish Et Al., 2016). ICAR-Indian Agricultural Research Institute, New Delhi

14. Use of NIR Sensor For Temperature (Heat/Cold) Stress Tolerance-_ The response of cultivated species to temperature stress and the tolerance level of cultivars are analyzed on the basis of the affected traits. The vulnerability of any plant to heat stress is stage-specific, despite all crop species being susceptible to heat stress during their entire life cycle. However, the reproductive stage is considered the most sensitive stage for terminal heat stress. Even a few-degree rise in temperature during flowering can cause complete yield loss ( Ohama et al., 2017). Morphological changes due to high-temperature stress include scorching and sunburns of leaves, twigs, branches, and stems, senescence and abscission of leaves, inhibition of shoot and root growth, fruit discoloration, and permanent damage. High-temperature stress in sugarcane damaged leaf tips and margins, and caused leaf rolling, drying, and necrosis ( Hasanuzzaman et al ., 2013). ICAR-Indian Agricultural Research Institute, New Delhi

15. 3 Temperature (Heat/Cold) Stress Tolerance- In wheat, heat stress reduced tiller numbers and increased shoot length (Kumar et al. 2011). Biochemical changes due to high temperature stress include irreversible damage to photosynthetic pigments and Rubisco , enhanced rate of photorespiration, and inhibition of noncyclic electron transport, which enhances ROS accumulation in plant cells and limits CO 2 fixation ( Mathur et al ., 2014; Farooq et al ., 2016). Plants under abiotic stress tend to produce more ROS in chloroplasts and mitochondria, which severely damages DNA and causes lipid peroxidation in the cell membrane ( Kukavica and Veljovic-Jovanovic , 2004). Several studies have demonstrated that ROS detoxification mechanisms play a significant role in providing hightemperature stress tolerance in plants (Suzuki and Mittler , 2006). ICAR-Indian Agricultural Research Institute, New Delhi

16. 3 Temperature (Heat/Cold) Stress Tolerance- Thus, plant tolerance to environmental stress is closely correlated with their ability to scavenge and detoxify ROS ( Zandalinas et al., 2018). However, high temperatures reduce plant growth by inhibiting net assimilation rates in shoots, and thus total plant dry weight (Wahid et al ., 2007). Under elevated temperatures, programmed cell death (PCD) occurs in specific cells or tissues within minutes, or even seconds, due to the denaturation or aggregation of proteins. However, moderately high temperatures for extended periods cause gradual senescence in plants. In both conditions, leaf shedding, flower and fruit abortion, and even plant death have been observed ( Rodrı´guez et al., 2005). ICAR-Indian Agricultural Research Institute, New Delhi

17. 3 Temperature (Heat/Cold) Stress Tolerance- Low temperature affects plant growth and productivity and causes significant yield losses. In contrast to heat stress, chilling stress directly inhibits metabolic reactions and indirectly harms the osmotic imbalance, which reduces the expression of the full genetic potential of plants . Based on the temperature range, cold stress is defined as chilling stress biomembrane lipid composition, and the accumulation of small molecules ( Wani et al ., 2016). In contrast, tropical and subtropical plants are more sensitive to chilling stress and lack the cold acclimation mechanism. Thus, low-temperature resistance in these plants is a complex trait, involving various metabolic pathways and cell compartments (Barnes et al., 2016). Several studies have been conducted in various plant species to measure tolerance under cold stress or in combination with other stresses, such as drought and salinity. However, few details have been provided on the methodologies used to evaluate the stress response in plants. ICAR-Indian Agricultural Research Institute, New Delhi

18. 4. APPLICATION OF PHENOMICS IN IMPROVING ABIOTIC STRESS TOLERANCE IN PLANTS High-throughput phenotyping is a bottleneck in crop genetic improvement. The progress of developing high-yielding crop varieties adapted to an environment can be hindered by slow, and often subjective, manual phenotyping . It also requires destructive and laborious harvesting across many field seasons and environments. High-throughput and nondestructive crop evaluation in the field and controlled environments is missing in our breeding systems. Nevertheless, the need for developing plant phenomics approaches and infrastructure has been realized globally. ICAR-Indian Agricultural Research Institute, New Delhi

19. 1 Infrared Thermography (IRT) It is used to measure temperature differences by infrared wave emission. Using infrared imaging, Qiu et al. (2009) detected significant differences between leaf temperature, air temperature, and canopy temperature under drought and high temperature stress in melons, tomatoes, and lettuce. They further proposed that the transpiration transfer coefficient (hat) can be used to detect various environmental stresses in plants. Similarly, IRT has been used to evaluate osmotic stress in wheat and barley in response to salt stress ( Sirault et al., 2009), and plant responses to water stress in grapevines and rice ( Jones et al ., 2002). This approach allows high-throughput screening at the seedling stage that can be validated within the canopy in the field using the same tools and genotypes ( Furbank , 2009). More recently, Wedeking et al. (2017) used IRT to monitor leaf temperature and transpiration in Beta vulgaris plants subjected to progressive drought stress. ICAR-Indian Agricultural Research Institute, New Delhi

20. 2 Spectroscopic Techniques It can be used to study photosynthetic rates at leaf and canopy levels, as well as other biochemical activities. Kiirats et al. (2009) used a leaf spectrometer to investigate photosynthetic electron transport feedback regulation in Nicotiana sylvestris . Photosynthetic efficiency, its activity, and biochemical pathway have been monitored in pine (Busch et al., 2009) and barley ( Siebke and Ball, 2009), with large-scale use of reflectance spectroscopy. The Raman spectroscopic technique was developed by Altangerel et al. (2017) for high-throughput stress phenotyping and in vivo early stress detection. ICAR-Indian Agricultural Research Institute, New Delhi

21. 4.3 Fluorescence Imaging Spectral absorption and reflectance – It offer a noninvasive tool for investigating plant chemical composition and function, which is scalable from the cell to canopy level. Chlorophyll fluorescence is the most commonly used, and relatively affordable, tool at the leaf level. Jansen et al. (2009) combined chlorophyll fluorescence with 2D digital imaging of plant growth to monitor plant reactions under drought and chilling stress in Arabidopsis thaliana. Similarly, by preparing a 3D polygon model by combining the time series of chlorophyll, a fluorescence image taken from a high resolution scanning lidar , spatiotemporal changes of herbicide, that is, 3-(3,4 dichlorophenyl )-1,1-dimethylurea (DCMU) effects in whole melon plants was monitored three-dimensionally ( Konishi et al., 2009). Rungrat et al. (2016) reviewed the use of chlorophyll fluorescence to monitor the effect on photosynthetic activity in Arabidopsis thaliana under various abiotic stresses. ICAR-Indian Agricultural Research Institute, New Delhi

22. 4 Integrated Imaging Techniques PET is used for in vivo imaging and to study biochemical pathways, and ion assimilation and transport. Fatangare et al. (2015) characterized 2-deoxy-2-fluoro-D-glucose (FDG) metabolism in plants, which had been used to study plant defense ( Ferrieri et al ., 2012) and carbon allocation ( Fatangare et al., 2014). A study by Meldau et al. (2015) on carbon allocation in plants after herbivore attack expanded the scope of FDG to in vivo plants subjected to various biotic and abiotic stresses. Imaging techniques such as MRI ( Borisjuk et al., 2012) and X-ray-CT ( Dhondt et al., 2010) are used to obtain anatomical information. An MRI-PET co-registration system was used by Jahnke et al. (2009) to combine PET-obtained radioactivity information with MRI-obtained anatomical data. A bifunctional PET/CT was used to obtain 4D radiotracer dynamics combining CT-derived morphological data, and PET-derived corresponding radio signals ( Fatangare et al., 2014). ICAR-Indian Agricultural Research Institute, New Delhi

23. Case Study- I ICAR-Indian Agricultural Research Institute, New Delhi

24. Materials and methods Quantifying temperature response to salinity using thermal imaging Germplasm and growing conditions Seeds of barley ( Hordeum vulgare L.) cultivar Himalaya were surface sterilised with 1% hypochlorite and germinated in Petri dishes for 2 days. Germinated seeds were planted 1.5 cm deep, one per pot into square pots (6.5 cm width, 16 cm depth) containing a 50 : 50 mix of coarse river sand : perlite . Seedlings were watered with half-strength modified Hoagland solution (P reduced from 1mM to 100 mM ) according to Munns and James (2003). At 8 days after mergence, 25mM NaCl was added to the irrigation solution twice daily to obtain required salinity levels of 0, 50, 100, 150 and 200 mM . Seedlings were randomly allocated to the different salinity levels, four seedlings for each salinity level. Seedlings were grown in a controlled environment chamber with 10 h photoperiod, a photosynthetic photon-flux density of 650 mmolm–2 s–1 from a mix of incandescent metal halide and fluorescent light bulbs and temperatures of 24C during the day and 18C during the night. Relative humidity in the chamber was maintained at ~50%. ICAR-Indian Agricultural Research Institute, New Delhi

25. Materials and methods Image acquisition system Thermal images of seedlings were acquired between 1100 and 1400 hours, 3 days after imposing the salt treatment, in the controlled environment chamber using a ThermaCAM SC660 IR camera (FLIR Systems Inc., Boston, MA, USA). Additionally, a time-series analysis over two hours was also acquired to study variation in leaf temperature across time (see Accessory Publication to this paper). Thermal data from the seedlings were acquired at 20-s intervals. The SC660 IR camera uses a focal plane array, uncooled microbolometer with 640480-detector elements, a spectral range of 7.5–13 mm, a thermal resolution of 0.045C and an accuracy of 1%. A 24 lens was mounted on the camera and emissivity in this study was deemed to be 0.95 (Tanner 1963). The IR camera was placed in the controlled environment chamber 2 hours before the measurement series to allow the optics to reach thermal equilibrium with air temperature. The IR camera was positioned at a distance of 0.8 m, perpendicular to the seedlings. In each image, two salt-treated seedlings were juxtaposed with a (non-salt) control seedling to assess the difference in leaf temperature due to salinity, rather than determining absolute leaf temperature . ICAR-Indian Agricultural Research Institute, New Delhi

26. Materials and methods Stomatal conductance To determine environmental conditions that would maximise transpiration rate under controlled environment chamber conditions, a LI-6400 gas -exchange system (Li-Cor, Lincoln, NE, USA) was used to examine the influence of vapourpressure deficit of the air (VPD) on both conductance and transpiration rate. As a result, the relative humidity in the controlled environment chamber was set at 50% to give a VPD of 1.5 kPa . Abaxial stomatal conductance measurements were obtained using an AP4 cycling porometer (Delta-T Devices Ltd, Burwell, UK), three days after the final salt concentrations were reached. Abaxial stomatal conductance had previously been shown to be more sensitive to salinity than adaxial stomatal conductance of wheat grown in controlled environment chambers (James et al. 2008). Stomatal conductance measurements were made from the mid-portion of the most recently expanded leaf (leaf 2) immediately following IR measurements. Development of IR measurement protocol Images and data acquisition Images were stored directly onto a hard-drive as 14-bit resolution files using ThermaCAM Researcher Pro 2.9 (FLIR Systems Inc.). IR images were converted into matrices of raw temperature data in C ( T480,640(R)) using ThermaCAM Researcher Pro for the development of the segmentation algorithm. ICAR-Indian Agricultural Research Institute, New Delhi

27. Materials and methods Segmentation algorithm Temperature matrices T480,640(R) were transformed into 8-bit resolution, grey-level images for viewing in MATLAB release 2009a (The MathWorks , Natick, MA, USA), using a custom-built function ‘tmp2img.m’ (Fig. 2 a). The function scales and normalises the temperature data using the following transformation: ICAR-Indian Agricultural Research Institute, New Delhi

28. Materials and methods ICAR-Indian Agricultural Research Institute, New Delhi

29. Materials and methods Seedling temperature The binary matrix B was then used as a mask to derive the temperature of the two seedlings in the thermograph only. By multiplying arrays T480,640(R) and B480,640(R) according to array arithmetic rules – element by element multiplication, temperature values for the seedlings were derived. A structure identifying the location and the number of pixels for each labelled seedling was defined in Matlab , allowing automatic calculation of the average temperature for each labelled seedling (Fig. 2 d). The difference between the control and salt-treated seedling was then computed. Each seedling was represented by a minimum of 3000 independent, thermally calibrated data points. IR thermography screen validation : evaluation of durum wheat genotypes at 150mM NaCl Germplasm Seventeen durum wheat ( Triticum turgidum L. ssp. Durum Desf .) genotypes and one barley cultivar Franklin, varying for osmotic stress tolerance were chosen based on the published work by James et al. (2008). The durum wheats included cultivars and landraces from a range of international locations, and three Australian durum cultivars, Tamaroi , Wollaroi and Bellaroi . ICAR-Indian Agricultural Research Institute, New Delhi

30. Materials and methods Growth conditions and experimental design Sterilised and germinated seeds, six per genotype, were planted into pots containing a 50 : 50 mix of coarse riversand : perlite in square pots as described previously. At 8 days after emergence, two seedlings per genotype were randomly chosen and deemed ‘control’ seedlings and were watered with modified Hoagland solution. The remaining four seedlings were watered with NaCl solutions incrementing by 25mM twice daily over 3 days to the required salinity level of 150mM NaCl . Supplemental Ca2+ (CaCl2) was added to maintain a Na+ : Ca2+ ratio of 15 : 1. Seedlings were randomised according to a split-plot design with four replicates. Plants were grown in a glasshouse under natural light conditions (~1200–1500 mmolm–2 s–1) and transferred into a controlled environment chamber 1 day before the first series of IR measurements to acclimatise . Conditions in the controlled environment chamber were as described above. ICAR-Indian Agricultural Research Institute, New Delhi

31. Results Relationship between stomatal conductance and a change in leaf temperature due to different salinity levels The relationship between stomatal conductance and a change in leaf temperature of Himalaya barley grown in a range of NaCl concentrations increasing from 0 to 200mM NaCl , is shown in Fig. The relationship between conductance and leaf temperature was curvilinear and mathematically described by a fourth-order polynomial function: ICAR-Indian Agricultural Research Institute, New Delhi

32. Validation of IR thermography of durum wheat genotypes at 150mM NaCl - ICAR-Indian Agricultural Research Institute, New Delhi

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