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Quantification of Soil-surface Roots in Seedlings and Mature Rice Plants    

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Abstract

Soil-surface roots (SORs) in rice are primary roots that elongate over or near the soil surface. SORs help avoid excessive reduction of stress that occurs in paddy, such as in saline conditions. SORs may also be beneficial for rice growth in phosphorus-deficient paddy fields. Thus, SOR is a useful trait for crop adaptation to certain environmental stresses. To identify a promising genetic material showing SOR, we established methods for evaluating SOR under different growth conditions. We introduced procedures to evaluate the genetic diversity of SOR in various growth stages and conditions: the Cup method allowed us to quantify SOR at the seedling stage, and the Basket method, using a basket buried in a pot or field, is useful in quantifying SOR at the adult stage. These protocols are expected to contribute not only to the evaluation of the genetic diversity of SOR, but also the isolation of related genes in rice.

Keywords: Gene isolation, Genetic diversity, Rice, Root system architecture, Soil-surface roots, Quantitative trait locus

Background

A subset of Indonesian lowland rice belonging to the ecotype Bulu develops thick crown roots above the soil surface (soil-surface roots; SORs) from the seedling stage, although such development is not observed in other commonly cultivated rice worldwide (Ueno and Sato, 1989, 1992). Lafitte et al. (2001) described that the occurrence of SORs is caused by selection pressure within the Bulu ecotype for adapting to anaerobic environments. The ecological and physiological functions of SORs in rice have not yet been understood in detail.


Recently, we demonstrated that SORs contribute to the prevention of excessive stress reduction in saline paddy through the cloning and characterization of a quantitative trait locus (QTL) for SOIL SURFACE ROOTING 1(SOR1) called qSOR1 (Kitomi et al., 2020). We also reported that SOR is a useful root trait for phosphorous uptake in phosphorus-deficient conditions (Oo et al., 2021). Therefore, the SOR phenotype is a valuable breeding target for the development of rice cultivars adapted to such abiotic stresses.


Several previous studies have investigated the genetic diversity of root system architecture in various rice cultivars. Accordingly, various root phenotyping methods, such as soil-core, soil-block, and soil-monolith around rice plants have been developed to date (Pavlychenko, 1937; Abe and Morita, 1994; Uga et al., 2013; Teramoto et al., 2019). These primarily field-based methods are suitable for investigating root biomass and length, but not for quantifying SOR.


Because SORs grow on the ground surface, the common root sampling method in a soil block cannot quantify the degree of SOR occurrence relative to the total roots. Therefore, a phenotyping method specific for SOR quantification is required, to evaluate the genetic diversity of the SOR phenotype in cultivated rice.


We developed a Cup method to quantify the SOR in a large number of samples for QTL mapping (Uga et al., 2012). The Cup method has been effective in identifying an SOR QTL on rice chromosome 7, by using recombinant inbred lines derived from a cross between a representative rice cultivar with SOR (cv. Gemdjah Beton), and without SOR (Sasanishiki) (Uga et al., 2012). A novel mutant gene SOR1, involved in soil-surface rooting, was also identified on rice chromosome 4, using the Cup method (Hanzawa et al., 2013). Furthermore, the Cup method was useful for phenotyping the QTL cloning of SOR (Kitomi et al., 2020).


The Basket method for plants grown in pots was originally developed to quantify the root growth angle of wheat seedlings (Oyanagi et al., 1993). This method was modified, and used to quantify the root growth angle of adult rice plants in the field (Uga et al., 2009). We modified the Basket method to quantify the SORs of rice grown in pots or flooded paddy fields (Uga et al., 2011, 2012; Kitomi et al., 2020). Here, we describe the protocols to quantify SOR in rice.

Materials and Reagents

  1. Cup method to quantify SOR at the seedling stage

    1. A plastic cup measuring 3.7 cm diameter × 4 cm depth; Beaker PP (AS ONE Corporation, Osaka, Japan)

    2. Stainless-steel tray (32 cm length × 25 cm width × 5.3 cm depth, any manufacturer) with drainage holes

    3. Plastic tray (44.5 cm length × 32.5 cm width × 7 cm depth, any manufacturer) capable of holding the previously mentioned stainless-steel tray

    4. Soil (Cultured soil for raising the seedlings, any manufacturer)

    5. Water


  2. Basket method to quantify SOR in pot cultures

    1. Open stainless-steel mesh baskets (6 cm diameter × 3 cm depth, any manufacturer)

    2. Pots (11 cm diameter × 15 cm depth, with a 1-cm diameter drainage hole at the bottom, any manufacturer)

    3. Plastic container (60 cm length × 35 cm width × 20 cm depth, any manufacturer)

    4. Soil (Cultured soil for raising the seedlings, any manufacturer)

    5. 5.Water


  3. Basket method to quantify SOR in paddy fields

    1. Open stainless-steel mesh baskets (15 cm diameter × 7 cm depth, any manufacturer)

Equipment

  1. Electric drill with bits of various sizes (Figure 1A)

  2. Sieve with various mesh sizes (Figure 1B)

  3. Scissors

  4. Knife

  5. Watering pot

  6. Water bucket



    Figure 1. Equipment used to quantify soil-surface roots using the cup and basket methods.

    (A) Electric drill; (B) Mesh sieve.

Procedure

  1. Cup method to quantify SOR at the seedling stage

    1. Drill holes (2 mm diameter, but size can be adapted to the seedling) at the bottom of each cup, for water supply and drainage (Figure 2A).

    2. Drill holes (5 mm diameter) at the bottom of each stainless tray, for water supply and drainage (Figure 2B).



      Figure 2. Preparation for sowing the seeds using the cup method to quantify soil-surface roots at the seedling stage.

      (A) Cylindrical plastic cup; (B) Stainless steel tray; (C) Cups arranged in stainless steel tray.


    3. Fill the cups with culture soil (or soil passed through a 3 or 4 mm-sized mesh sieve; can be adapted to the desired mesh size), and then place them in a stainless steel tray (or lightproof tray) (Figure 2C). The type of soil will depend on the purpose of the experiment.

    4. Irrigate the soil with sufficient water using the watering pot (Figure 3A).

    5. Soak the sterilized seeds in water at 30°C, in an incubator for two days.

    6. Select germinated seeds with 1–2 mm emerged radicle or/and plumule through the husk, and place these seeds on the soil, in the center of the cups (Figure 3B).

    7. Fill gaps between the cups with soil, and then cover the cups with a layer of culture soil of approximately 1 cm (Figure 3C).

    8. After irrigating the soil inside it with a watering pot, the stainless-steel tray was placed in a plastic tray.

    9. Maintain the water level at a depth of 2 cm, with supply from below the tray, until the two-leaf-stage (Figure 3D).

    10. Grow the rice plants in a greenhouse at 20–30°C temperature, using natural day length (can be adapted to the individual growing conditions).



      Figure 3. Sowing seeds and young seedlings using the cup method to quantify soil-surface roots at the seedling stage.

      (A) Cups in stainless steel tray; (B) Germinated seeds on soil in cups so that the seed plumules are at the center of a cup; (C) Cups in stainless steel tray covered with layer of culture soil of approximately 1 cm; (D) A water level of 2 cm in the plastic tray.


    11. From the two-leaf-stage to the time of rooting assessment, check and maintain the water level at the soil-surface every day.

    12. At the fourth or fifth leaf stage, cut the aerial parts using scissors, and measure traits such as plant height, number of leaves, or dry weight, which can vary depending on the purpose of the experiment (Figure 4A and 4B).

    13. Immerse the stainless steel tray in a bucket of water to remove the soil on and around the cups gently, and carefully retrieve each cup from the stainless steel tray, without cutting the primary roots (Figure 4C).



      Figure 4. A suitable stage for the investigation of the cup method to quantify soil-surface roots at the seedling stage.

      (A) Rice plants grown in a stainless-steel tray; (B) Stainless-steel tray without aerial parts of rice plants; (C) Rice plants with culture soil removed around the cups. Bulu rice cultivar “Gemdjah Beton” (GB; three rows from left), qsor1-NIL (NIL; two rows in center), and Japanese rice cultivar “Sasanishiki” (SA; two rows from right). qsor1-NIL is a near-isogenic line with Sasanishiki genetic background and a loss-of-function allele of qSOR1 derived from GB.


    14. Carefully take each cup from the stainless tray, and then count the primary roots that had elongated past the edge of the cup as SOR (Figure 5A).

    15. Release the plant from the cups, and then wash away the soil attached to the roots (Figure 5B).

    16. Untangle the roots, and then count their total number for each plant.



      Figure 5. Measurement of soil-surface roots using the cup method at the seedling stage.

      (A) Cups removed from the tray; (B) Plants removed from the cups. Bulu rice cultivar “Gemdjah Beton” (GB; left), qsor1-NIL (NIL; center), and Japanese rice cultivar “Sasanishiki” (SA; right).


    17. Define the soil-surface root ratio for each plant as the number of SORs divided by the total number of primary roots.

    18. The time required for above assessment is approximately 15 min per one cup done by one person.

    19. Recommend more than three times of replicates. Number of replications can be customized based on the purpose and design of the experiment.


  2. Basket method to quantify SOR in pot cultures

    1. Drill holes (5–10 mm in diameter) at the bottom of each pot, for water supply and drainage (Figure 6A).

    2. Spread a mesh or paper filter at the bottom of the pot, to prevent soil erosion through the holes (Figure 6B).

    3. Bury the open stainless-steel mesh basket just under the soil surface in each soil-filled plastic pot (Figure 6C and 6D). Use the sizes of the basket and pot (or container) depending on the experimental design. In addition, use the type of soil depending on the purpose of the study.

    4. Impregnate the culture soil in each pot with a watering pot.

    5. Soak the sterilized seeds in water at 30°C, in an incubator for two days.

    6. Select germinated seeds with 1–2 mm emerged radicle or/and plumule through the husk, and sow one germinated seed at the center of each basket in the soil-filled pot (Figure 6E).

    7. Cover the soil-surface with a layer of soil of approximately 1 cm, and then supply water to the container (Figure 6F).



      Figure 6. Preparation of the baskets before and after sowing seeds using the basket method.

      (A) Pots with drain hole; (B) A mesh or paper filter at the bottom of the pot; (C) Soil-filled plastic pot and open stainless-steel mesh basket; (D) Basket buried just under the soil surface in the pot; (E) One germinated seed sown at the center of the soil-filled pot; (F) Pots covered with soil in container.


    8. Maintain the water level at a depth of 2–5 cm, by supplying water from the bottom of the pots until the two-leaf-stage (Figure 7A).

    9. Grow the plants in a greenhouse at 20–30°C in natural daylight. Adopt growing condition according to individual experimental design.

    10. From the second leaf stage onward, maintain the water level in each plastic pot at the soil surface level (Figure 7B).



      Figure 7. Water management in the basket method to quantify soil-surface roots in pot culture.

      (A) Water level until the second leaf stage; (B) Water level from the second leaf stage onward.


    11. At the seven- or eight-leaf stage, carefully remove each basket out from the pot in a water bucket, and then remove the soil attached to the roots (Figure 8A). The time of assessment is dependent on the experimental design.

    12. Cut the aerial parts using scissors (Figure 8B), and measure traits such as plant height, number of leaves, and dry weight (traits depending on the individual experimental design).

    13. Carefully remove the baskets out from the pots without cutting the primary roots. Cut the primary roots growing over the open sides of baskets, and then count the number of roots as soil-surface roots (Figure 8C).

    14. Cut the primary roots penetrating form the side and bottom of the basket, and then count the number of roots as the shallow and deep roots.



      Figure 8. Water management in the basket method to quantify soil-surface roots in pot culture.

      (A) Basket removed from the pot in a water bucket; (B) Soil attached to the roots washed from the basket; (C) Primary roots growing over the edge, as well as from the side, and bottom of the basket.


    15. Define the soil-surface root ratio for each plant as the number of SORs divided by the total number of primary roots.

    16. The time required for the above assessment is approximately 30 min per one basket done by one person. The time can be customized based on the growth stage of plant.

    17. Measure the root length, thickness, and dry weight depending on the experimental design.

    18. More than three replicates are recommended; these can be customized based on the purpose and design of the experiment.


  3. Basket method to quantify SOR in paddy field

    1. Drain the flooding water from the paddy field. Use the field conditions, such as upland fields, vary based on the experimental design.

    2. Before transplanting, cut the roots of the seedlings into 1–2 cm length using scissors (Figure 9A).

    3. Dig up the paddy-field soil with an open basket, and then transplant the seedlings in the center of the open baskets, so that the base level of the seedling was just under the edge level of the open basket (Figure 9B).

    4. Return the basket to a prior location (Figure 9C).

    5. Cover the baskets (open-side up) with around 3 cm layer of paddy-field soil (Figure 9D).



      Figure 9. Positioning of the basket in the soil and the planting of seedlings using the basket method.

      (A) The basket and transplanting point in paddy field; (B) Basket containing paddy soil; (C) Basket returned to a prior location; (D) Basket buried under the paddy soil.


    6. At the end of the vegetative stage, cut the paddy soil around the circumference of the rice plant using a knife (circle of approximately 30 cm diameter, at a distance of approximately 15 cm from the plant) (Figure 10A).

    7. Remove the baskets along with the soil from the paddy field (Figure 8B), and carefully remove the soil attached to the baskets in a water bucket, without cutting the primary roots (Figure 8C).



      Figure 10. Sampling and measurement of soil-surface roots using the basket method in paddy fields.

      (A) Cutting the soil around the rice plant using a knife; (B) Basket dug up from the paddy field; (C) Removal of soil attached to the basket in a water bucket; (D) Gemdjah Beton plant; (E) Sasanishiki plant.


    8. Cut the aerial parts using scissors, and measure the traits such as plant height, number of leaves, and dry weight (traits can be measured depending on individual experimental design).

    9. Using scissors, cut the primary roots growing over the open sides of the baskets and count them as soil-surface roots (Figure 9A).

    10. Cut the primary roots penetrating form the side and bottom of the baskets (Figure 9B and 9C) and then count the number of shallow and deep roots, respectively.



      Figure 11. Counting the number of shallow and deep roots using the basket method.

      (A) Cutting of primary roots growing over the open sides of the basket; (B) Cutting of primary roots protruding from the side of the basket; (C) Cutting of primary roots protruding from the bottom of the basket.


    11. Define the soil-surface root ratio for each plant as the SORs growing over the upper edge of the baskets divided by the total number of primary roots, including penetrated from the entire basket mesh.

    12. The time required for above assessment is approximately one hour per one basket done by one person. The time can be customized based on the growth stage of plant and the condition of field.

    13. Measure root thickness depending on the experimental design.

    14. More than nine replicates are recommended; these can be customized based on the purpose and design of the experiment.

Notes

The results obtained using these methods have been published in the following papers: Uga et al. (2012), Hanzawa et al. (2013), and Kitomi et al. (2020).

Acknowledgment

This study was supported by KAKENHI #21H04152 [Grant-in-Aid for Encouragement Research] from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. The authors thank Shusei Sato and Kinya Toriyama for providing their laboratory spaces and Kunio Ichijyo for technical support in the paddy field. We would like to thank Editage (www.editage.com) for English language editing.

Competing interests

The authors declare no competing interest.

References

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  2. Hanzawa, H., Sasaki, K., Nagai, S., Obara, M., Fukuta, Y., Uga, Y., Miyao, A., Hirochika, H., Higashitani, A., Maekawa, M., et al. (2013). Isolation of a novel mutant gene for soil-surface rooting in rice (Oryza sativa L.). Rice 6: 30.
  3. Kitomi, Y., Hanzawa, E., Kuya, N., Inoue, H., Hara, N., Kawai, S., Kanno, N., Endo, M., Sugimoto, K., Yamazaki, T., et al. (2020). Root angle modifications by the DRO1 homolog improve rice yields in saline paddy fields. PNAS 177: 21242-21250.
  4. Lafitte, H. R., Champoux, M. C., McLaren, G., and O’Toole, J. C. (2001). Rice root morphological traits are related to isozyme group and adaptation. Field Crops Res 71: 57-70.
  5. Oo, A. Z., Tsujimoto, Y., Mukai, M., Nishigaki, T., Takai, T., and Uga, Y. (2021). Synergy between a shallow root system with a DRO1 homologue and localized P application improves P uptake of lowland rice. Sci Rep 11: 9484.
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  7. Pavlychenko, T. K. (1937). Quantitative study of the entire root systems of weed and crop plants under field conditions. Ecology 18: 62-79.
  8. Teramoto, S., Kitomi, Y., Nishijima, R., Takayasu, S., Maruyama, N., and Uga, Y. (2019). Backhoe-assisted monolith method for plant root phenotyping under upland conditions. Breed Sci 69(3): 508-513.
  9. Ueno, K. and Sato, T. (1989). Aerial root formation in rice ecotype Bulu. Jpn J Trop Agr 33: 173-175.
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  11. Uga, Y., Ebana, K., Abe, J., Morita, S., Okuno, K., and Yano, M. (2009). Variation in root morphology and anatomy among accessions of cultivated rice (Oryza sativa L.) with different genetic backgrounds. Breed Sci 59: 87-93.
  12. Uga, Y., Hanzawa, E., Nagai, S., Sasaki, K., Yano, M., and Sato, T. (2012). Identification of qSOR1, a major rice QTL involved in soil surface rooting in paddy fields. Theor Appl Genet 124: 75-86.
  13. Uga, Y., Okuno, K., and Yano, M. (2011). Dro1, a major QTL involved in deep rooting of rice under upland field conditions. J Exp Bot 62: 2485-2494.
  14. Uga, Y., Sugimoto, K., Ogawa, S., Rane, J., Ishitani, M., Hara, N., Kitomi, Y., Inukai, Y., Ono, K., Kanno, N., et al. (2013). Control of root system architecture by DEEPER ROOTING 1 increases rice yield under drought conditions. Nat Genet 45: 1097-1102.
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Copyright: © 2022 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Hanzawa, E., Kitomi, Y., Uga, Y. and Sato, T. (2022). Quantification of Soil-surface Roots in Seedlings and Mature Rice Plants. Bio-protocol 12(9): e4409. DOI: 10.21769/BioProtoc.4409.
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