ROOT PHENOTYPING METHODS
Phenotyping presents many challenges in front of plant breeders for enhancing stress tolerance in crop plants as screening roots for complex genetic traits is extremely difficult under field conditions (Sharma et al 2016). Root phenotyping methods involves combination of imaging and image processing with automation. Image-based phenotyping is a non-destructive method involving optical analysis of plant traits (Furbank et al 2011; Fiorani and Schurr 2013), and it mainly involves characterization of biochemical, physiological and anatomical properties of roots (Guo and Zhu 2014).
Minirhizotrons involves non-destructive analysis in which a clear tube is positioned in ground and root growth adjoining the tube is detected and image is captured using camera which is installed down the tube (Smit et al., 2000). However, it has certain limitations, as tube and soil interface provides an unnatural environment for the growth of roots, which results in false assessments of characteristics of growth in plants (Wasson et al., 2012). Electrical resistance tomography (ERT) is another approach used for analyzing physical properties of soil (Basso et al., 2010) and how soil water is being exploited by roots in different crops (Srayeddin and Doussan, 2009) both are the measures to detect root behavioral patterns indirectly. Various software such as WinRhizo, Image J, EZ-Rhizo, Root system analyzer, Smart Root, Optimas analysis, Root trace, and Root Nav are popular for image-based analysis of root phenotyping (Sharma et al., 2016). For measuring growth kinetics and branching angles Smart Root software can be used. DART is another software that can be used for studying root structure. It is a manual software coded in JAVA (Bot et al., 2010) and produces flexible datasets of individual parameters of root. Manual softwares like WinRHIZOTM, DART etc., helps to study root lifespan by monitoring color of roots manually (Li et al., 2022). X-ray computed tomography (CT) can be used for assessment of roots within soil profile and for visualizing 3D configuration of roots which helps to decide which character of roots is useful under drought conditions for obtaining high crop yield (Wasaya et al. 2018). The 3D magnetic resonance imaging (MRI) is a non-destructive method that is being practised commonly as a 3D root phenotyping process (Metzner et al., 2014). Image quality of MRI is known to be influenced by substrate type and water content (Rogers & Bottomley 1962). Daniel et al. (2022) has recently used this method for phenotyping roots of 288 wheat seedlings using a new pipeline which is designated as medium-throughput phenotyping. Electrical capacitance (EC) is another 3D root phenotyping method used in the field, which analyzes alternating current of low frequency (less than 1kHz) between the soil and base of stem and then evaluate the dielectric properties for re-establishing root architecture (Dalton, 1995). This method has been used for phenotyping roots of different crops, including maize (Imre et al., 2018), soybean (Cseresnyés et al., 2017) and wheat (Cseresnyés et al., 2021). Ground-penetrating radar (GPR) is another non-destructive geophysical device which uses electromagnetic waves for detecting surfaces which is based on physical and dielectric properties of materials. It is used for analyzing features of RSA and estimation of its biomass (Lorenzo et al 2010). When compared to soil, roots have distinct dielectric permittivity thereby, GPR is useful to detect number, diameter and depth of coarse roots by measuring the reflected signals produced by hyperbolic reflections on radargrams (Wu et al. 2014; Lombardi et al., 2021). For example, GPR was used to measure coarse roots in plants, including, cassava (Delgado et al., 2017), citrus (Zhang et al., 2019) and willow (Li et al., 2015).
For observing roots in controlled environment, various soil-less growth media are being employed, such as gels, paper rolls, and aerated aqueous solutions. Hydroponics techniques are practiced commonly for growing plants using transparent plexiglass nail board sandwiches packed with glass beads of about 1.5 mm size for maintaining proper flow of nutrients (Zhu et al 2005). These systems help to measure total root length, root traits, root branching angles through imaging technique or manually.