Growth pouches For Soil-Free Root Phenotyping
Growth pouch systems are suitable for screening root system architectures (RSA) of large numbers of seedlings in a rapid and cost-effective manner - allowing their inclusion into high-throughput root phenotyping platforms. Such root phenotyping systems allow the lengths, branching and angles of different root types to be measured, which form the basis of models to generate RSAs of crop genotypes and estimate the consequences for soil exploration and resource uptake. While soil-free techniques such as germination pouches provide great contrast between root and substrate, allowing for accurate extraction of traits by imaging, the root systems of plants grown in artificial media can deviate from those grown in soil (such as in rhizoboxes). However, pouch systems using plants grown vertically on germination paper have been successfully used in seedling screens for many years and species - including bean, maize, wheat, oilseed rape, and pearl millet. The measurement of even "simple" root traits such as seminal, nodal root numbers and branching angles has shown to hold predictive value for breeding crops with greater yields and stress tolerance. Researchers have used germination pouch set-ups of many different configurations, below we showcase two contrasting examples, but are open to manufacture custom-made growth pouches meeting your aims. For short and effcient (pre-)screens of small plants, e.g. for treatment testing and bioassays, ready-to-use germination pouch systems may be suitable.
General application of growth pouches For Root phenotyping
How are growth pouches generally used? While the exact procedure differs depending on the pouch system and species, in brief, seeds are placed in a trough formed by the germination paper, usually thick creped paper-types are used, or seeds are fixated inbetween papers and acrylic plates, and then water or a liquid fertilizer solution is added. Most seeds can be grown in germination pouch systems. In general it is advisable to moisten the paper well before and after placing the seeds. Seeds of some species should be pre-germinated / soaked before beeing placed. Anyhow, eventually, the paper pouch is closed within a plastic sheet/envelope (type "one" below) or acylic cover (e.g. for corn, type "two" below) and the growth pouch is placed in an upright position - the protruding base of the germination paper often taps into a bottom reservoir of water or nutrient solution to re-moist. Growth pouch system dimensions should be chosen according to the seed size and the expected root growth during the experimental period. Finally, all types of pouches must be covered by dark plastic foil or placed in darkend boxes etc. to prevent light reaching the root systems. It is important to work clean (i.e. towards sterile conditions) when preparing the seeds and pouches - as the system is, in the absence of natural antagonists in soil, prone to fungal and bacterial contamination. To minimize fungal growth, it may be helpful to surface sterilze seeds and acylic plates , the germination paper is generally not reusable.
Type 1: Cost-Efficient Germination Pouch "Positioning System"
In this simple, yet efficient growth pouch system (Adeleke et al. 2020) a central acrylic plate is used to hold two germination paper in plastic envelopes (one on either site of the plate). Notches at the top of the acylic plate allow to hang the pouch system into containers/boxes with nutrient solution, allowing the germination paper to re-wet itself by capillary forces and to prevent light from reaching the root systems. When using closed plastic envelopes (at the bottom, care has to be taken not to overly irrigate the system - resulting in fast root decay. Ready-to-use germination pouch systems are available for growing and treating seedlings over short eperimental periods by commercial suppliers.
Type 2: Germination Pouch System with Acrylic Cover Plates and Spacers
Alternatively to the minimalistic root phenotxping pouch system illustrated above, some researchers proposed growth "pouch" systems with acrylic plates and spacers to enclose the growing paper and developing root system. Those systems resemble rather rhizoboxes lined with germination papers than classical pouches. Here, a system frequently used for corn root phenotyping is exemplified, using three to four layers of germination paper and 10 mm spacers - providing sufficient growing space and good mechanical protections for the developing root systems. Ultimately also such a system needs to be shielded from light by dark foil or more rigid structures (e.g. covering sleeves). They can be developed into high throughput platforms by adding well planned containers for light shielding and remoistening, allowing eventually even for automation of imaging by robots. An potentially enhanced version of this rot screening system, providing more resistance to the growing root systems by EPDM layers, are root phenoboxes.
Custom Growing Pouch Systems By Vienna Scientific
Vienna Scientific Instrument develops and builds growing pouch / rhizoslide systems for high throughput root phenotyping platforms - we currently do not sell the growth paper itself (see below for properties, suppliers). We hold ample experience on root phenotyping technology and can advice on cost-effective systems suitable for your target plant species and the scope of your projects. We can also develop the appropriate storage solutions for your growth pouch-based, high troughput phenotyping platform. Refer to our root phenobox system if you aim for enhanced, rather soil-like resistance during root system establishment. Get in contact to discuss your (high throughput) root phenotyping goals!
Germination PAper properties
In brief, germination paper for growing pouches should possess a creped surface. The paper should have thus an open, porous formation and be inert, i.e. in particular free from impurities or toxic substances that may affect seed germination and root growth. Papers should provide a high, uniform water retention, remain mechanically stable under wetted conditions, and should not allow for ingrowth of roots or root hairs. In addition, the germination paper should also be free of fungi or bacteria which might interfer with seedling growth and evaluation of results. Seeds may be surface sterized to reduce the risk of microbial contamination. While a range of companies offer creped germination papers, a classical provider of such papers for root observation tasks is the Anchor Paper Company, MN, USA.
Selected Readings on Growth Pouches for Root Phenotyping Tasks
- Adeleke E, Millas R, McNeal W, Faris J, Taheri A. 2020. Variation Analysis of Root System Development in Wheat Seedlings Using Root Phenotyping System. Agronomy, 10(2):206.
- Hund, A., Trachsel, S., & Stamp, P. 2009. Growth of axile and lateral roots of maize: I development of a phenotying platform. Plant and Soil, 325, 335-349.
- Jing, X., Cai, C., Fan, S., Wang, L., & Zeng, X. 2019. Spatial and temporal calcium signaling and its physiological effects in Moso Bamboo under drought stress. Forests, 10(3), 224.
- Le Marié, C., Kirchgessner, N., Marschall, D., Walter, A., & Hund, A. 2014. Rhizoslides: paper-based growth system for non-destructive, high throughput phenotyping of root development by means of image analysis. Plant methods, 10(1), 1-16.
- Shorinola, O., Kaye, R., Golan, G., Peleg, Z., Kepinski, S., & Uauy, C. 2019. Genetic screening for mutants with altered seminal root numbers in hexaploid wheat using a high-throughput root phenotyping platform. G3: Genes, Genomes, Genetics, 9(9), 2799-2809.
- Waller, S., Wilder, S. L., Schueller, M. J., Housh, A. B., & Ferrieri, R. A. 2020. Quantifying plant-borne carbon assimilation by root-associating bacteria. Microorganisms, 8(5), 700.