Name: Multipronged Phenotyping Approaches to Characterize Sugarcane Root Systems (Video) | JoVE Journal
Uploaded: 2022-08-17T13:00:00.000+00:00
Duration: 9 min 21 s
Description: Learn multipronged phenotyping approaches to characterize sugarcane root systems using field excavation, digitized imaging, and hydroponic analysis. This method advances breeding strategies for improved stress resistance and productivity.

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The authors acknowledge the infrastructure and support extended by the Director, ICAR-Sugarcane Breeding Institute, Coimbatore, for establishing root phenotyping facilities for sugarcane. Funding provided by the Science and Engineering Research Board, Department of Science and Technology, Government of India, in the form of Early Career Research Award to KV (ECR/2017/000738), is duly acknowledged. The authors acknowledge Brindha, Karpagam, Rajesh, Sivaraj, and Amburose for their assistance in generating data in a meticulous manner.

1. Field excavation by trench sampling
<ol>
	<li>Raise commercial sugarcane hybrids (hereafter called "varieties") in the field from two-budded setts planted at a row spacing of 120 cm, with 90 cm within rows, following the recommended package of practices (POP) to ensure good crop establishment and growth.</li>
	<li>At the end of the maturity phase of the crop, employ an excavator to dig a trench (1.5 m deep and 1.0 m wide) in the field. Through continuous water jetting, ensure that the soils from root zones are cleared without damaging the roots (Figure 1).</li>
	<li>When the adhering soil loosens, uproot the cane along with the root system and take it to the laboratory for manual measurement of the number of roots, root length, volume, and weight.</li>
</ol>
<img alt="Sugarcane root system analysis in soil trench, showing irrigation and root structure exposure." class="xfigimg" src="/files/ftp_upload/63596/63596fig01.jpg" data-altupdatedat="1762425657000" data-alt="Figure 1"> 
Figure 1: Trench sampling method for the field excavation of roots. (A) Sideways trench dug along the field, (B) water jetting, and (C) inner view showing the depth of the trench. <a href="https://www.jove.com/files/ftp_upload/63596/63596fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. Root core sampler to reduce sampling errors
<ol>
	<li>Use a cylindrical root core sampler of 61 cm height and 16 cm diameter weighing 8 kg, fabricated using mild steel (MS) material for sugarcane root sampling in the field. Provide a sharp edge at the bottom edge of the sampler to facilitate easy penetration while inserting it into the soil. Provide the top with collars of 3 cm diameter to lift the sampler (Figure 2).</li>
	<li>Raise sugarcane varieties in the field from two-budded setts planted at a row spacing of 120 cm, with 90 cm within rows, following the recommended POP to ensure good crop establishment and growth.</li>
	<li>At the maturity phase of the crop, fasten the top edge of the sampler to the primary shoot/cane and hammer continuously to reach the desired soil depth (45 cm). Lift the entire soil mass into the sampler and wash carefully under running water to separate the adhering roots.</li>
	<li>After thorough washing of the roots, record the volume, surface area, length, and weight by manual measurement, as well as by spreading the roots on transparent trays to scan and analyze the corresponding digitized images using the referenced software (see the Table of Materials).</li>
</ol>
<img alt="Soil water collection tube design diagram, 3D model, and installation in the field for research use." class="xfigimg" src="/files/ftp_upload/63596/63596fig02.jpg" data-altupdatedat="1762425657000" data-alt="Figure 2"> 
Figure 2: Root core sampler. (A) Dimensions, (B) top view, and (C) site of sampling. <a href="https://www.jove.com/files/ftp_upload/63596/63596fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Root phenotyping structure to facilitate sampling at different phenophases
<ol>
	<li>Construct a root phenotyping structure comprising three adjacent compartments of dimensions 4.5 m x 10.1 m for sampling of the sugarcane roots, with provisions for manually dismantling the side walls to reveal the underground root system (up to a depth of 80-100 cm) (Figure 3). Construct the side walls with precast slabs of dimensions 1.8 m in length, 30 cm in width, and 4 cm in thickness.</li>
	<li>Fill and compact the structure with field soil, leaving a headspace of ~20 cm, with adequate drainage holes to facilitate soil aeration.</li>
	<li>Sow the bud chips of germplasm clones comprising Saccharum officinarum L., Saccharum spontaneum L., Saccharum barberi Jesw., Saccharum sinense Roxb., and Saccharum robustum Brandes and Jeswiet ex Grassl. (hereafter called "Saccharum spp. clones") and allow them to germinate for 30 days in protrays comprising rooting media (red soil: farm yard manure: sand = 2:2:1). Transplant uniform and healthy settlings to the structure at a row spacing of 90 cm, with 60 cm within rows, following the recommended POP to ensure good crop establishment and growth.</li>
	<li>During the formative (60-120 days after planting [DAP]) and grand growth (120-150 DAP) phases, manually remove the side walls made of the precast slabs, followed by continuous spraying of the water jet to expose the roots.</li>
	<li>Uproot the entire root system and take it to the laboratory for manual measurement of the length, volume, and weight, and spread the roots on transparent trays to scan and analyze the corresponding digitized images in the referenced software (see the Table of Materials).</li>
	<li>Impose drought stress by withholding irrigation in one of the compartments, and plug the drainage holes in the second compartment to maintain soil saturation to simulate waterlogging stress. Irrigate the third compartment according to the recommended POP to maintain the field capacity and to serve as a control.</li>
</ol>
<img alt="Soil erosion control structure; includes design diagram and vegetated terracing for erosion prevention." class="xfigimg" src="/files/ftp_upload/63596/63596fig03.jpg" data-altupdatedat="1762425657000" data-alt="Figure 3"> 
Figure 3: Root phenotyping structure. (A) Dimensions, (B) overview of the three compartments, and (C) view of one compartment. <a href="https://www.jove.com/files/ftp_upload/63596/63596fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
4. Hydroponic culture of plants to study rhizosphere biology
<ol>
	<li>Fabricate an in-house hydroponic system in an environment-controlled glasshouse conducive for growing sugarcane to study the finer details of root biology. Add ~15 L of modified Hoagland's nutrient solution (Table 1) to glass tanks of dimensions 20 cm x 20 cm x 50 cm, with aeration provided by aquarium pumps (Figure 4).</li>
	<li>Sow the bud chips of sugarcane varieties and Saccharum spp. clones and allow them to germinate for 30 days in protrays comprising composted coir pith. Transplant uniform and healthy settlings to hydroponic tanks at the frequency of three settlings per tank, taking care to place the entire root in the nutrient solution. Cover the tanks with black cloth to ensure that roots are not exposed to light. Use a plastic mesh (20 cm x 20 cm) at the brim of the glass tanks to support the plants upright.</li>
	<li>At the end of the germination phase (60 days), collect the root exudates by immersing the roots of intact plants in 50 mL of trap solution (sterile double-distilled water) for 4 h (0800 h to 1200 h) during the time of peak photosynthetic activity. Filter the collected solution through Whatman filter paper, then pass it through glass columns filled with anion-exchange followed by cation-exchange resins12. Evaporate the eluted fractions to dryness and store at -20 °C until further processing.</li>
	<li>Analyze the processed root exudate samples by HPLC for the determination of organic acids12 and by spectrophotometry for the estimation of total phenolics13, proteins14, sugars15, and amino acids16 according to standard protocol.</li>
	<li>Monitor the root growth at weekly intervals to record the root tip pigmentation and root hair density. Assess the activity of the enzymes, peroxidase17 and superoxide dismutase18, and total phenolic content19 at the 3rd month according to standard protocol.</li>
	<li>Assess the response to root injury by inflicting a longitudinal slice in the primary root up to the root tip using a sterile surgical blade, and monitor the changes periodically.</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Chemical</td>
			<td>Final concentration</td>
		</tr>
		<tr>
			<td>Potassium nitrate</td>
			<td>0.608 g·L-1</td>
		</tr>
		<tr>
			<td>Calcium nitrate</td>
			<td>1.415 g·L-1</td>
		</tr>
		<tr>
			<td>Potassium dihydrogen phosphate</td>
			<td>0.164 g·L-1</td>
		</tr>
		<tr>
			<td>Magnesium sulphate</td>
			<td>0.560 g·L-1</td>
		</tr>
		<tr>
			<td>EDTA-ferric monosodium salt</td>
			<td>6.00 g·250L-1</td>
		</tr>
		<tr>
			<td>Boric acid</td>
			<td>1.43 g·250L-1</td>
		</tr>
		<tr>
			<td>Manganese chloride tetrahydrate</td>
			<td>0.91 g·250L-1</td>
		</tr>
		<tr>
			<td>Zinc sulphate</td>
			<td>0.11 g·250L-1</td>
		</tr>
		<tr>
			<td>Cupric suphate</td>
			<td>0.04 g·250L-1</td>
		</tr>
		<tr>
			<td>Molybdic acid</td>
			<td>0.01 g250L-1</td>
		</tr>
	</tbody>
</table>
Table 1: Composition of the modified nutrient solution for the hydroponic culture of sugarcane.
<img alt="Greenhouse experiment: hydroponic setup for plant growth study with visible root systems." class="xfigimg" src="/files/ftp_upload/63596/63596fig04.jpg" data-altupdatedat="1762425657000" data-alt="Figure 4"> 
Figure 4: Hydroponics setup. The setup (A) customized for growing sugarcane and (B) 5-month-old crop (black cloth removed for photography purpose only). This figure has been modified from Hari et al.9. <a href="https://www.jove.com/files/ftp_upload/63596/63596fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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<li>Sharma, S., Borah, P., Meena, M. K., Bindraban, P., Pandey, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evaluation+of+genotypic+variation+for+growth+of+rice+seedlings+under+optimized+hydroponics+medium%5BTitle%5D)+AND+%22Indian+Journal+of+Genetics+and+Plant+Breeding%22%5BJournal%5D)">Evaluation of genotypic variation for growth of rice seedlings under optimized hydroponics medium.</a> Indian Journal of Genetics and Plant Breeding. 78 (3), 292-301 (2018).</li>
<li>Soumya, P. R., Singh, D., Sharma, S., Singh, A. M., Pandey, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evaluation+of+diverse+wheat+%28Triticum+aestivum%29+and+triticale+%28%C3%97+Triticosecale%29+genotypes+for+low+phosphorus+stress+tolerance+in+soil+and+hydroponic+conditions%5BTitle%5D)+AND+%22Journal+of+Soil+Science%22%5BJournal%5D)">Evaluation of diverse wheat (Triticum aestivum) and triticale (× Triticosecale) genotypes for low phosphorus stress tolerance in soil and hydroponic conditions.</a> Journal of Soil Science. 21, 1236-1251 (2021).</li>
<li>Ganie, A. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Metabolite+profiling+and+network+analysis+reveal+coordinated+changes+in+low-N+tolerant+and+low-N+sensitive+maize+genotypes+under+nitrogen+deficiency+and+restoration+conditions%5BTitle%5D)+AND+%22Plants%22%5BJournal%5D)">Metabolite profiling and network analysis reveal coordinated changes in low-N tolerant and low-N sensitive maize genotypes under nitrogen deficiency and restoration conditions.</a> Plants. 9 (11), 1459(2020).</li>
<li>Venkatraman, T. S., Thomas, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Simple+contrivances+for+studying+root+development+in+agricultural+crops%5BTitle%5D)+AND+%22Agricultural+Journal+of+India%22%5BJournal%5D)">Simple contrivances for studying root development in agricultural crops.</a> Agricultural Journal of India. 18, 509-514 (1923).</li>
<li>Clark, R. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-throughput+two-dimensional+root+system+phenotyping+platform+facilitates+genetic+analysis+of+root+growth+and+development%5BTitle%5D)+AND+%22Plant%2C+Cell+and+Environment%22%5BJournal%5D)">High-throughput two-dimensional root system phenotyping platform facilitates genetic analysis of root growth and development.</a> Plant, Cell and Environment. 36 (2), 454-466 (2013).</li>
<li>Chopart, J. L., Rodrigues, S. R., Azevedo, M. C. B., Medina, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Estimating+sugarcane+root+length+density+through+root+mapping+and+orientation+modelling%5BTitle%5D)+AND+%22Plant+and+Soil%22%5BJournal%5D)">Estimating sugarcane root length density through root mapping and orientation modelling.</a> Plant and Soil. 313, 101-112 (2008).</li>
<li>Chopart, J. L., Azevedo, M. C. B., Le Mezo, L., Marion, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Functional+relationship+between+sugarcane+root+biomass+and+length+for+cropping+system+applications%5BTitle%5D)+AND+%22Sugar+Tech%22%5BJournal%5D)">Functional relationship between sugarcane root biomass and length for cropping system applications.</a> Sugar Tech. 12 (3-4), 317-321 (2010).</li>
<li>De Silva, A. L. C., De Costa, W. A. J. M., Bandara, D. M. U. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Growth+of+root+system+and+the+patterns+of+soil+moisture+utilization+in+sugarcane+under+rain-fed+and+irrigated+conditions+in+Sri+Lanka%5BTitle%5D)+AND+%22Sugar+Tech%22%5BJournal%5D)">Growth of root system and the patterns of soil moisture utilization in sugarcane under rain-fed and irrigated conditions in Sri Lanka.</a> Sugar Tech. 13 (3), 198-205 (2011).</li>
<li>Otto, R., Silva, A. P., Franco, H. C. J., Oliveira, E. C. A., Trivelin, P. C. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High+soil+penetration+resistance+reduces+sugarcane+root+system+development%5BTitle%5D)+AND+%22Soil+and+Tillage+Research%22%5BJournal%5D)">High soil penetration resistance reduces sugarcane root system development.</a> Soil and Tillage Research. 117, 201-210 (2011).</li>
</ol>

All authors declare that there are no conflicts of interest.

Root systems define the above-ground productivity of sugarcane, necessitating that all its facets be explored and understood thoroughly for the development of climate-resilient varieties. A team of scientists at ICAR-SBI comprising plant physiologists, a microbiologist, an agricultural engineer, a biochemist, and plant breeders employed multipronged approaches for sugarcane root phenotyping, including field excavation by trench sampling, the use of a root core sampler, raised platforms for root sampling, and raising plan...

Sugarcane (Saccharum spp.), an important food and bioenergy source, is a significant industrial crop suited for cultivation in many countries with tropical and subtropical climatic conditions. Owing to the C4 photosynthetic pathway for carbon assimilation, sugarcane is highly productive, efficiently using farm inputs such as water and fertilizers. Post-harvest processing of sugarcane yields economically valuable products such as sugar and jaggery, alongside its byproducts-molasses, ethanol, and energy. Sugarcane is produced by nearly 100 countries over an area of 25.97 Mha, which is approximately 1.5% of total arable land. India alone contributes to 16% of world sugarcane production (approximately 306 Mt), with an average productivity of 70 t&#183;ha&#8722;1 1. The major abiotic stresses drastically affecting sugarcane production include water deficits, water logging, temperature extremes, and soil properties such as nutrient deficiency, salinity, sodicity, and alkalinity. The biggest challenge for developing abiotic stress-tolerant crops is to pinpoint specific traits that confer a substantial yield advantage under stress conditions.
Several aspects of sugarcane physiology are poorly understood, including the root-shoot relationship, which drastically affects cane productivity. Sugarcane root is not as well studied as shoots, although the different types, such as sett roots, shoot roots, and adult roots, may be developmentally distinct with varying functions. Genotypic differences have been observed with regard to the number and length of sett roots emerging during germination2. Sett roots are implicated in the germination of sugarcane buds, ensuring early crop establishment, and are later replaced by shoot roots, which are robust, emerging from the base of the developing shoot3. Fine branches observed in the sett roots help in anchoring the young plants and aid in the absorption of water and nutrients until they are replaced by shoot roots. Similar to sett roots, shoot roots also arise from the root primordia present in the lower, unexpanded internodes of the cane.
As shoot roots persist in the plant for a longer duration, they are 4x-10x thicker than sett roots. Shoot roots constitute the sole root system of sugarcane, with an important role in further growth and development. The vigor of the shoot roots is positively associated with the overall vegetative vigor of the plant. The continuous development of roots resulting from the turnover of sett roots and shoot roots gives rise to the &#34;adult root system&#34; of sugarcane, which is ever-adapting to the prevailing environmental conditions. In general, a deeper, more prolific root system makes more water and nutrients available for the crop than does a shallower distribution of roots. Periodic dissections revealed that, when the soil moisture content was high, shallow root systems were observed, whereas a much deeper root system developed as the water table dropped2. The root system in sugarcane remains active even after harvesting of the crop, contributing to the growth of the ratoon crop until new shoot roots emerge from underground buds4. Root angle and the level of root branching are two important factors determining the volume of soil explored by plant roots. Root angle, a genotypic trait, may be altered through conventional breeding or molecular approaches to improve tolerance to biotic and abiotic stresses. On the contrary, the level of root branching is mostly influenced by the environment, necessitating periodic monitoring of root development and its response to localized soil conditions.
Anatomical features of sugarcane roots have been examined to ascertain differences with regard to genotype and environment. The anatomy of sett roots in sugarcane resembles that of mature roots in other grasses such as maize, wherein the cortex comprises well-differentiated cell layers in a regular pattern. The endodermis is suberized, followed by a single-layered pericycle. Metaxylem elements are the main conductors or water and nutrient ions, radially arranged and interspersed with groups of phloem, the latter comprising a sieve element with two companion cells. The large central mass of undifferentiated cells forms the root pith. Distinct anatomical features of sugarcane cultivars correspond to root hydraulic properties, thereby influencing water movement. Early studies on the differences in the root anatomical traits of sugarcane revealed that, under low moisture stress conditions, pronounced thickening of the cell wall was observed in the layers immediately inside of the endodermis, between the pith and the vascular region, and around the vessels5. Such thickened cells may be an adaptation to prevent the backward flow of sap and for mechanical strength during stress.
Some important traits implicated in the drought resistance of sugarcane include the relative thickness and number of exodermal layers, the ratio of cortex to stele, intercellular spaces in the cortex, and thickened root hair tips. The ratios of the area occupied by cortical cells to the area occupied by the stellar tissues of shoot roots are significantly different among sugarcane cultivars, with wide variability with respect to the area of the stele6. The hydraulic conductivity of sugarcane roots is related to the size and number of metaxylem elements in the shoot roots. Hydrophobic cell layers within the root are likely to define zones of apoplastic water movement. Suberized Casparian bands are found in the endodermis and in the hypodermis (termed as exodermis), which serve as hydrophobic barriers. The disintegration of cortical cells leads to the formation of lysigenous aerenchyma in older roots and in plants subjected to hypoxic conditions, irrespective of developmental age. The formation of aerenchyma during waterlogging stress is correlated with the maintenance of growth in resistant varieties7.
The morphology and anatomy of Erianthus arundinaceus [Retzius] Jeswiet (genera related to Saccharum spp.) roots are implicated in its strong tolerance to environmental stresses8. Erianthus arundinaceus roots exhibit nodal roots distributed at steep angles, with dense roots hairs to facilitate the uptake of water and nutrient ions from deeper soil zones. The deep-root system consists of many nodal roots growing with steep growth angles. The diameter of the nodal roots correlates with the size and number of large xylem vessels, the former varying widely from 0.5 mm to 5.0 mm. These nodal roots also form a rhizomatous sheath, with a hypodermis showing lignified sclerenchyma in the outer cortex (exodermis), lysigenous aerenchyma in the mid-portion of the cortex, and starch granules in the stele. In addition to architecture and morphological traits, root-exuded organic compounds play an important role in determining plant germination, establishment, and survival, with plausible allelopathic effects and/or affinity for microbial symbiosis.
Root enzymatic activity and the finer details of the morphology, including root cap pigmentation and rejuvenation potential upon injury, were documented in sugarcane varieties grown under hydroponic culture9. Root growth shows a highly plastic response to changes in the soil environment, both in terms of the form and size of the root system. The most efficient sugarcane variety would be one that has few or an optimal number of shoots, with a correspondingly lower number of roots, aiding better survival during stressful conditions. The systematic study of the root system should, thus, form an important component of any crop improvement program10. The majority of the experiments focusing on roots rely mostly on developmental aspects, while a focus on functional plasticity is often lacking11. Apart from the structural distribution, functional root plasticity plays a crucial role in survival under stress and would, therefore, support breeders in their efforts to include root system traits in the selection pipeline for abiotic stress tolerance and improve the robustness of sugarcane.
Considering its importance in sustaining growth and yield under stressful environments, it is essential to explore and utilize the inherent variability in root system traits of sugarcane. An emphasis on the selection of component traits and mechanisms imparting superior root systems is the way forward for better crop performance under changing climatic conditions. Phenotypic evaluation is a long and costly process; however, the integration of multipronged approaches would add tremendous value to its utility in crop improvement. In this manuscript, four different approaches for root phenotyping in sugarcane are described, each with its own set of merits and demerits, implying that a concerted effort is required to arrive at comprehensive and holistic results.

<table><tbody><tr><td>Aeration pump with pipeline accessories</td><td>Purchased from local sources</td><td>NA</td><td>Used for hydroponic culture of sugarcane</td></tr><tr><td>Boric acid</td><td>Sisco Research Laboratories, India</td><td>80266</td><td>Preparation of modified Hoagland&#039;s solution</td></tr><tr><td>Calcium nitrate</td><td>Central Drug House, India</td><td>27606</td><td>Preparation of modified Hoagland&#039;s solution</td></tr><tr><td>Composted coir pith</td><td>Purchased from local sources</td><td>NA</td><td>Used for germinating sugarcane setts</td></tr><tr><td>Cupric sulphate</td><td>Sisco Research Laboratories, India</td><td>38869</td><td>Preparation of modified Hoagland&#039;s solution</td></tr><tr><td>DEAE-cellulose</td><td>Sisco Research Laboratories, India</td><td>10529</td><td>anion exchange resin for processing of root exudates</td></tr><tr><td>EDTA-ferric monosodium salt</td><td>Sisco Research Laboratories, India</td><td>59389</td><td>Preparation of modified Hoagland&#039;s solution</td></tr><tr><td>Farm yard manure</td><td>Purchased from local sources</td><td>NA</td><td>Used for germinating sugarcane setts</td></tr><tr><td>Glass tanks</td><td>Fabricated in-house</td><td>NA</td><td>Used for hydroponic culture of sugarcane</td></tr><tr><td>HPLC</td><td>Agilent Technologies</td><td>1200 Infinity</td><td>Quantification of organic acids in root exudates</td></tr><tr><td>Magnesium sulphate</td><td>Sisco Research Laboratories, India</td><td>29117</td><td>Preparation of modified Hoagland&#039;s solution</td></tr><tr><td>Manganese chloride</td><td>Sisco Research Laboratories, India</td><td>75113</td><td>Preparation of modified Hoagland&#039;s solution</td></tr><tr><td>Molybdic acid</td><td>Sisco Research Laboratories, India</td><td>49664</td><td>Preparation of modified Hoagland&#039;s solution</td></tr><tr><td>Potassium dihydrogen phosphate</td><td>Central Drug House, India</td><td>29608</td><td>Preparation of modified Hoagland&#039;s solution</td></tr><tr><td>Potassium nitrate</td><td>Central Drug House, India</td><td>29638</td><td>Preparation of modified Hoagland&#039;s solution</td></tr><tr><td>Protrays</td><td>Fabricated in-house</td><td>NA</td><td>Used for germinating sugarcane setts</td></tr><tr><td>Red soil</td><td>Purchased from local sources</td><td>NA</td><td>Used for germinating sugarcane setts</td></tr><tr><td>Root core sampler</td><td>Fabricated in-house</td><td>NA</td><td>Used for in situ root sampling</td></tr><tr><td>Sand</td><td>Purchased from local sources</td><td>NA</td><td>Used for germinating sugarcane setts</td></tr><tr><td>Seralite-120</td><td>Sisco Research Laboratories, India</td><td>14891</td><td>cation exchange resin for processing of root exudates</td></tr><tr><td>Supporting frame</td><td>Purchased from local sources</td><td>NA</td><td>Used for hydroponic culture of sugarcane</td></tr><tr><td>Water motor pump</td><td>Purchased from local sources</td><td>NA</td><td>Used for hydroponic culture of sugarcane</td></tr><tr><td>Whatman filter paper grade 1</td><td>Universal Scientific</td><td>1001090</td><td>Processing of root exudates</td></tr><tr><td>WinRhizo PRO (software)</td><td>Regent Instruments Inc., Canada</td><td>STD4800</td><td>Two-dimensional root scanner with software for analysis of roots</td></tr><tr><td>Zinc sulphate</td><td>Sisco Research Laboratories, India</td><td>76455</td><td>Preparation of modified Hoagland&#039;s solution</td></tr></tbody></table>

multipronged phenotyping approaches to characterize sugarcane root systems

Multipronged Phenotyping Approaches to Characterize Sugarcane Root Systems

Roots are the primary conductors of water and nutrients and play a vital role in sustaining growth and yield under stressful environments. The study of plant roots poses methodological difficulties in in situ assessment and sampling, which is especially true for sugarcane (Saccharum spp.). Traditional methods during the 1920s documented the genotypic variation in sugarcane root systems, after which few studies were reported on sugarcane root traits per se until recently. In addition to morphology, rhizosphere characteristics, including allelopathic effects and/or affinity for microbial symbiosis, determine plant establishment and survival.
Ultimately, root systems define the above-ground productivity of sugarcane. With the impetus for climate-resilient varieties, it is becoming more relevant to explore and utilize the variability in root system traits of sugarcane. This paper describes multipronged approaches for sugarcane root phenotyping, including field excavation by trench sampling, the use of a root core sampler, raised platforms for root sampling, and raising plants under hydroponic culture, employed by a team of scientists at the Indian Council of Agricultural Research-Sugarcane Breeding Institute (ICAR-SBI).
Field excavation by trench sampling is imperative to assess the plant roots in their natural growing environment. The use of raised platforms simulating field conditions and a root core sampler are alternative approaches, with a considerable reduction in time, uniform sample size, and less loss of root material. Hydroponic plant culture allows the study of morphology, anatomical features, and rhizosphere biology, including the exudation of organic compounds and microbial interactions. Data generated from different experiments using diverse sampling methods add to the wealth of information on the root system traits of sugarcane.

Variation in the root morphological traits of sugarcane varieties 
Representative images of the root system in Co 62175, excavated from the field by trench sampling and grown in a hydroponic setup, are presented in Figure 5A,B. Long roots (~100 cm) were observed in the varieties Co 62175 and Co 99006, while Co 99006 recorded the highest root weight (127 g·clump−1). Root traits were recorded using a root core sampler, which revealed that Co 6217...

Characterizing root system traits is one of the areas of research that is still in its infancy, particularly in sugarcane. Integrating multiple approaches to precisely phenotype sugarcane roots leads to comprehensive and holistic results, enabling the utilization of identified traits and mechanisms for conventional and molecular breeding.

Roots are the primary conductors of water and nutrients and play a vital role in sustaining growth and yield under stressful environments. The study of ...

As a primary conductor of water and nutrients, roots are indispensable for growth and development. A thorough understanding of the root system is important for sugarcane as it takes a year to complete its lifecycle. The holistic approach here integrates the viable platforms for the phenotyping, and this will benefit in advancing the knowledge of sugarcane root systems and their responses to address the work conditions.This methodology is specific to the sugarcane crop as it has a unique root system as a long crop. However, with minor modifications, this method may be adapted to other crops. To begin, raise the commercial sugar cane hybrids or varieties in the field using two butted sets planted at a row spacing of 120 centimeters and 90 centimeters within the rows.Follow the recommended package of practices to ensure good crop establishment and growth. At the end of the maturity phase, employ an excavator to dig a trench of 1.5 meters deep and 1.0 meters wide in the field. Clear the soil from the root zones without damaging the roots with continuous water jetting.Once the adhering soil loosens due to jetting, uproot the cane along with the root system and take it to the laboratory for manual measurement of the growth parameters, namely the number of roots, root length, volume, and weight. Use a cylindrical root core sampler of 61 centimeters in height, 16 centimeters in diameter, weighing eight kilograms and fabricated using mild steel or MS material for sugarcane root sampling in the field. It is provided with a sharp edge at the bottom for easy penetration and has collars of three centimeters in diameter at the top to lift the sampler.At the maturity phase of the commercial sugar cane crop, fasten the top edge of the sampler to the primary chute or cane and hammer continuously to reach the desired soil depth of 45 centimeters. Then lift the entire soil mass into the sampler and wash it carefully under running water to separate the adhering roots. Measure the physical parameters of the plant, like plant height and leaf area in the laboratory.Then spread the roots on transparent trays for scanning and analyzing the corresponding digitized images using the software. Construct a root phenotyping structure comprising three adjacent compartments of 4.5 meters by 10.1 meters for sampling the sugar cane roots. Make provision for manually dismantling the sidewalls to reveal the underground root system up to a depth of 80 to 100 centimeters.Fill and compact the structure with field soil, leaving a head space of around 20 centimeters and having adequate drainage holes to facilitate soil aeration. Salve the bud chips of germplasm clones comprising saccharum officinarum, saccharum spontaneum, saccharum barberri, saccharum sinense, and saccharum robustum. Allow them to germinate for 30 days in per trays, comprising rooting media.After 30 days of germination transplant the uniform and healthy settlings to the structure in the rows with a spacing of 90 centimeters and 60 centimeters distance within rows. Follow the recommended POP to ensure good crop establishment and growth. During the formative phase of 60 to 120 days after planting and the grand growth phase of 120 to 150 days after planting, manually remove the sidewalls made of the precast slabs before continuously spraying the water jet to expose the roots.Uproot the entire root system and transfer it to the laboratory to measure the physical parameters. Then spread the roots on transparent trays for scanning and analyzing the corresponding digitized images in software. To study the rhizosphere biology and the finer details of root biology, fabricate an in-house hydroponic system in an environment controlled glass house conducive to sugar cane growth.In the 20 by 20 by 50 centimeters dimensions tanks with aquarium pumps for aeration, add around 15 liters of modified Hoagland's Nutrient Solution. Sell the bud chips of sugarcane varieties and saccharum species clones and allow them to germinate for 30 days in the per trays, comprising composted core pith before transplanting the uniform and healthy settlings to hydroponic tanks at the frequency of three settlings per tank. After 60 days when the germination phase ends, immerse the roots of intact plants in 50 milliliters of sterile double distilled water or trap solution for four hours, and collect the root exudates during the peak photosynthetic activity.Assess the activity of the enzymes, peroxidase, and superoxide dismutase and total phenolic content in the third month according to standard protocol. Finally, assess the response to root injury by inflicting a longitudinal slice in the primary root up to the root tip using a sterile surgical blade and monitor the changes periodically. In the root phenotyping, the longer roots of CO 62175 demonstrated a superior root system for cumulative root length, root surface area, root volume, and average root diameter.Saccharum species showed significant variations in morphological traits during the formative phase. Saccharum spontaneous had the highest cumulative root length per clump, while saccharum sinense showed the lowest. Saccharum spontaneum showed the highest root surface area, root dry weight, root volume, and the root to shoot ratio.Saccharum robustum showed the highest root volume and root diameter. Anatomical studies revealed that long root hair schlerenchymidis exodermis reduced cortical cell layers, increased cortical oerinchma, and increased steel area and xylem vessel number with large diameter were beneficial under drought conditions, whereas under water logging stress, increased root diameter and higher cortical cell area with increased aerenchymidis cells were observed. Saccharum robustum showed the highest total carboxylate exudation while the lowest exudation was recorded in saccharum spontaneum.In sugarcane varieties, a significant difference in the root hair density was observed with variation in shape and pigmentation of the root cap. The highest peroxidase activity and total phenolic content were present in C086032, and the lowest in C062175. During rejuvenation studies, Cox 671 showed the appearance of secondary roots in three days and Co-06022 took 10 days with symptoms of senescence on the third day.We need to exercise caution while interrupting the results obtained from individual protocol. An institute study of root system is ideal for sugar cane, but control platforms may be useful for specialist purposes. Understanding various aspects of root phenotype is essential to releasing the sugar cane varieties.Automating the techniques and integrating big data analytics or artificial intelligence to interpret the results would broaden our expertise in the field. Of the results, in characterizing root system traits under normal and periodic stress situations have paved the way for exploratory research areas like root plasticity, radiant nutrientisms, and miotic stresses.

multipronged-phenotyping-approaches-to-characterize-sugarcane-root

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JoVE Journal

Biology

This protocol describes a process for rapid collection of images of Arabidopsis seedlings responding to a gravity stimulus using commercially-available flatbed scanners.  The method allows for inexpensive, high-volume capture of high-resolution images amenable for downstream analysis algorithms.

Using Flatbed Scanners to Collect High-resolution Time-lapsed Images of the Arabidopsis Root Gravitropic Response

This article demonstrates how to culture Arabidopsis thaliana seedlings in a two-layer microfluidic platform that confines the main root and root hairs to a single optical plane. This platform can be used for real-time optical imaging of fine root morphology as well as for high-resolution imaging by other means.

Imaging the Root Hair Morphology of Arabidopsis Seedlings in a Two-layer Microfluidic Platform

An experimental protocol is presented for assessment of soil grown plant root systems with RGB and hyperspectral imaging. Combination of RGB image time series with chemometric information from hyperspectral scans optimizes insights into plant root dynamics.

RGB and Spectral Root Imaging for Plant Phenotyping and Physiological Research: Experimental Setup and Imaging Protocols

A software-based image analysis system provides an alternative method to study the morphology of stoloniferous and rhizomatous species. This protocol permits measurement of the length and diameter of stolons and rhizomes and can be applied to samples with a large amount of biomass and to a wide variety of species.

Measuring Stolons and Rhizomes of Turfgrasses Using a Digital Image Analysis System

We provide a novel method to improve the X-ray absorption contrast of maize tissue suitable for ordinary microcomputed tomography scanning. Based on CT images, we introduce a set of image-processing workflows for different maize materials to effectively extract microscopic phenotypes of vascular bundles of maize.

Micron-scale Phenotyping Techniques of Maize Vascular Bundles Based on X-ray Microcomputed Tomography

Visualizing and measuring root growth in situ is extremely challenging. We present a customizable rhizobox method to track root development and proliferation over time in response to nutrient enrichment. This method is used to analyze maize genotypic differences in root plasticity in response to an organic nitrogen source.

An Optimized Rhizobox Protocol to Visualize Root Growth and Responsiveness to Localized Nutrients

Parasponia andersonii is a fast-growing tropical tree that belongs to the Cannabis family (Cannabaceae) and can form nitrogen-fixing root nodules in association with the rhizobium. Here, we describe a detailed protocol for reverse genetic analyses in P. andersonii based on Agrobacterium tumefaciens-mediated stable transformation and CRISPR/Cas9-based genome editing.

Transforming, Genome Editing and Phenotyping the Nitrogen-fixing Tropical Cannabaceae Tree Parasponia andersonii

The protocol here describes the methods for the assessment of the arbuscular mycorrhizal colonization patterns and strategy in roots for two species: Zea mays and Festuca rubra. The use of the MycoPatt method permits the calculation of parameters, the conversion of mycorrhizal structures into digital data, and the mapping of their real position in roots.

Mycorrhizal Maps as a Tool to Explore Colonization Patterns and Fungal Strategies in the Roots of Festuca rubra and Zea mays

We use simple laboratory tools to examine the root system architecture (RSA) of Arabidopsis and Medicago. The plantlets are grown hydroponically over mesh and spread using an art brush to reveal the RSA. Images are taken using scanning or a high-resolution camera, then analyzed with ImageJ to map traits.

A Simple Protocol for Mapping the Plant Root System Architecture Traits

We designed an image-based phenotyping protocol to determine the morphological and physiological responses to single and combined heat, drought, and waterlogging treatments. This approach enabled the identification of early, late, and recovery responses at a whole plant level, particularly above-ground parts, and highlighted the necessity of using multiple imaging sensors.

High Throughput Image-Based Phenotyping for Determining Morphological and Physiological Responses to Single and Combined Stresses in Potato

Learn multipronged phenotyping approaches to characterize sugarcane root systems using field excavation, digitized imaging, and hydroponic analysis. This method advances breeding strategies for improved stress resistance and productivity.

Multipronged Phenotyping Approaches to Characterize Sugarcane Root Systems

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