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Sep 2020

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Quantification of Soil-surface Roots in Seedlings and Mature Rice Plants
幼苗和成熟水稻植物土壤表面根的量化   

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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 (基因分离), Genetic diversity (遗传多样性), Rice (水稻), Root system architecture (根系统架构), Soil-surface roots (土壤表面根), Quantitative trait locus (数量性状基因座)

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

  1. Abe, J. and Morita, S. (1994). Growth direction of nodal roots in rice: its variation and contribution to root system formation. Plant Soil 165: 333-337.
  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.
  6. Oyanagi, A., Nakamoto, T., and Morita, S. (1993). The gravitropic response of roots and the shaping of the root system in cereal plants. Environ Exp Bot 33: 141-158.
  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.
  10. Ueno, K. and Sato, T. (1992). Varietal difference in growth directions of rice crown roots and relation to gravitropic response and diameter of crown roots. Jpn J Breed 42: 779-786.
  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.

简介

[摘要]水稻土壤表层根系(SORs)是在土壤表层之上或附近伸长的原生根。 SOR 有助于避免过度减少稻田中发生的压力,例如在盐碱条件下。 SORs也可能有利于缺磷条件下的水稻生长 稻田。因此,SOR 是作物适应某些环境胁迫的有用性状。为了确定显示 SOR 的有希望的遗传材料,我们建立了在不同生长条件下评估 SOR 的方法。我们引入了评估不同生长阶段和条件下 SOR 遗传多样性的程序:Cup 方法允许我们在幼苗阶段量化 SOR,而 Basket 方法使用埋在花盆或田地中的篮子,可用于量化 SOR在成人阶段。这些协议不仅有助于评估SOR的遗传多样性,而且有助于分离水稻中的相关基因。

[背景] 属于 Bulu 生态型的印度尼西亚低地水稻的一个子集从幼苗阶段开始在土壤表面(土壤表面根;SORs)上形成厚的冠根,尽管在世界范围内其他常见种植的水稻中没有观察到这种发育(Ueno 和 Sato,1989 , 1992) 。拉菲特等人。 (2001)描述了 SORs 的发生是由 Bulu 生态型内适应厌氧环境的选择压力引起的。水稻中 SOR 的生态和生理功能尚未得到详细了解。
最近,我们通过克隆和表征数量性状基因座 (QTL) 证明了 SOR 有助于防止盐田过度胁迫。 用于土壤表面生根 1(SOR1) ,称为 qSOR1( Kitomi等人,2020)。我们还报道了 SOR 是在缺磷条件下吸收磷的有用根性状( Oo 等。 , 2021) 。 因此,SOR 表型是开发适应这种非生物胁迫的水稻品种的有价值的育种目标。
各种水稻品种根系结构的遗传多样性。因此,各种根表型方法,如土壤核心、土壤块和土壤整体水稻植株周围 迄今已开发( Pavlychenko ,1937; Abe 和 Morita, 1994; Uga 等人,2013;寺本 等人,2019)。这些主要基于现场的方法适用于研究根生物量和长度,但不适用于量化 SOR。
因为 SOR 生长在地面上, 土壤块中的普通根采样方法无法量化 SOR 相对于总根的发生程度。因此,需要一种特定于 SOR 定量的表型分析方法,以评估栽培稻中 SOR 表型的遗传多样性。
我们开发了一种 Cup 方法来量化大量样本中的 SOR 以进行 QTL 映射( Uga 等人,2012)。 Cup 方法在鉴定水稻第 7 号染色体上的 SOR QTL 方面是有效的,它使用源自具有 SOR 的代表性水稻品种(cv. Gemdjah)杂交的重组自交系。 贝顿), 并且没有 SOR ( Sasanishiki ) ( Uga 等人,2012)。使用Cup法( Hanzawa 等人,2013)。 此外,Cup 法可用于SOR ( Kitomi ) 的 QTL 克隆表型分析。 等人,2020)。
盆栽植物的篮法最初是为了量化小麦幼苗( Oyanagi )的根系生长角度而开发的。 等人,1993)。对该方法进行了修改,用于量化田间成年水稻植株的根系生长角度 (乌加 等人,2009)。我们修改了 Basket 方法来量化盆栽或淹水稻田中水稻的 SOR( Uga 等人,2011 年,2012 年;北见 等人,2020)。在这里,我们描述了量化大米中 SOR 的协议。

关键字:基因分离, 遗传多样性, 水稻, 根系统架构, 土壤表面根, 数量性状基因座



材料和试剂


A. 苗期SOR量化的C up方法
1.一个 直径3.7厘米×深4厘米的塑料杯;烧杯 PP(AS ONE Corporation,大阪,日本)
2. 带排水孔的不锈钢托盘(长32cm×宽25cm×深5.3cm,任意厂家)
3. 塑料托盘(长44.5cm×宽32.5cm×深7cm,任意厂家),可装上述不锈钢托盘
4. 土壤(育苗培养土,任何厂家)
5. 水


B. 量化盆栽培养中 SOR 的篮法
1. 打开不锈钢网篮(直径6厘米×深度3厘米,任何制造商)
2. 花盆(直径11厘米×深15厘米,底部有直径1厘米的排水孔,任何厂家)
3. 塑料容器(60厘米长×35厘米宽×20厘米深,任何制造商)
4. 土壤(育苗培养土,任何厂家)
5. 水


C. 稻田SOR的篮法
1. 开放式不锈钢网篮(直径 15 厘米 × 深度 7 厘米,任何制造商)


设备


1. 各种尺寸s钻头的电钻(图 1A)
2. 用筛子_ 各种网格尺寸(图 1B)
3. 剪刀
4. 刀
5. 喷壶
6. 水桶




图 1. 使用杯子和篮子方法量化土壤表层根部的设备。 (一)电钻; (B) 网筛。


程序


A. 杯法在苗期量化 SOR
1. 在每个杯子的底部钻孔(直径 2 毫米,但大小可以适应幼苗),用于供水和排水(图2A )。
2. 钻孔(5 mm 直径)在每个不锈钢托盘的底部,用于供水和排水(图 2B)。




图2 。播种准备使用杯法在幼苗阶段量化土壤表面根。 
(A)圆柱形塑料杯; (B) 不锈钢托盘; (C) 在不锈钢托盘中排列的杯子。


3. 培养土(或通过3 或 4 毫米筛网的土壤;可调整到所需的目数)填充到杯子中,然后将它们放入不锈钢托盘(或不透光托盘)中(图2C ) .土壤的类型将取决于实验的目的。
4. 喷壶用足够的水灌溉土壤(图 3A) 。
5. 将灭菌的种子浸泡在 30 °C 的水中,在培养箱中浸泡两天。
6. 选择具有 1 – 2 毫米长的胚根或/和胚芽穿过外壳的已发芽种子,并将这些种子放在土壤上,在杯的中心(图3 B)。
7. 用泥土填充杯子之间的间隙,然后 c在杯子上 有一层培养土 大约 1 厘米(图3 C)。
8. 用喷壶浇灌里面的土壤后,将不锈钢托盘放入塑料托盘中。
9. 将水位保持在 2 厘米的深度,从托盘下方供应,直到两叶阶段(图3 D)。
10. 为 20 – 30°C 的温室中种植水稻植物,使用自然日长(可适应个体的生长条件)。




图3.使用杯法播种和幼苗在幼苗阶段量化土壤表层根系。 
不锈钢托盘中的杯子; (B) 将发芽的种子放在杯子中的土壤上,使种子胚芽位于杯子的中心; (C)不锈钢托盘中的杯子,上面覆盖着大约 1 厘米的培养土层; (D) 塑料托盘中 2 厘米的水位。 


11. 从两叶期到生根评估时,每天检查并保持土壤表层的水位。
12. 在第四或第五叶阶段,用剪刀剪断地上部分,并测量植物高度、叶数或干重等性状,这可能因实验目的而异(图4 A和 4B )。
13. 不锈钢托盘浸入一桶水中,轻轻去除杯子上和周围的土壤,然后小心地从不锈钢托盘中取出每个杯子,不要切割主根(图4 C)。




图4.一个适合研究杯法在幼苗阶段量化土壤表面根的阶段。 
种植在不锈钢盘中的水稻; (B) 不带水稻地上部分的不锈钢托盘; (C)在杯子周围去除培养土的水稻植株。布鲁水稻品种“ Gemdjah ” Beton ”(GB;左起三排)、qsor1-NIL(NIL;中间两排)和日本水稻品种“ Sasanishiki ”(SA;右起两排)。 qsor1-NIL 是具有Sasanishiki遗传背景的近等基因系和源自 GB的qSOR1功能丧失等位基因。


14. 小心地从不锈钢托盘中取出每个杯子,然后将伸长超过杯子边缘的主根数为 SOR(图5A )。
15. 杯子s中释放出来,然后洗掉附着在根部的土壤(图5B )。
16. 解开根部,然后计算每株植物的总数。






图5 。在苗期使用杯子法测量土壤表面根。 
(A) 从托盘中取出的杯子; (B)从杯子中取出的植物。 布鲁水稻品种“ Gemdjah ” Beton ”(GB;左)、qsor1-NIL(NIL;中)和日本水稻品种“ Sasanishiki ”(SA;右)。


17. 将每种植物的土壤表面根比定义为SOR的数量除以主根的总数。
18. 上述评估所需的时间约为1人每杯15分钟。
19. 建议重复三次以上。重复次数可以根据实验的目的和设计进行定制。


B. 在盆栽培养物中量化 SOR 的篮法
1. 花盆底部钻孔(直径5-10毫米),用于供水和排水(图6 A)。
2. 在花盆底部铺上网状或纸质过滤器,以防止水土流失通过孔(图 6B)。
3. 埋葬开放的不锈钢网篮 就在每个充满土壤的塑料盆的土壤表面下方(图6C 和 6D )。根据实验设计使用篮子和锅(或容器)的尺寸。此外,根据研究目的使用土壤类型。
4. 盆浸透每个盆中的培养土。
5. 将灭菌的种子浸泡在 30 °C 的水中,在培养箱中浸泡两天。
6. 选择1-2毫米的胚根或/和胚芽穿过外壳的已发芽种子,并在装土的盆中每个篮子的中心播种一粒已发芽的种子(图6E )。
7. 在土壤表面覆盖一层约 1 cm 的土壤,然后向容器供水 (图6F )。








图6 。使用篮法播种前后篮子的准备工作。
(A) 带排水孔的花盆; (B) 锅底的网状或纸质过滤器; (C) S充油塑料锅和开放式不锈钢网篮; (D) 篮子埋在花盆内的土壤表面之下; (E) 在填土盆中央播种一粒发芽种子; (F)在容器中用土壤覆盖的花盆。


8. 通过从花盆底部供水直到两叶阶段(图 7A) ,将水位保持在 2-5 厘米的深度。
9. 在 20-30°C 的温室中在自然光下种植植物。根据个体实验设计采用生长条件。
10. 从第二叶阶段开始,将每个塑料盆中的水位保持在土壤表面水平(图 7B) 。




图 7. 篮法中的水分管理,以量化盆栽中土壤表面的根。 
(A) 直到第二叶阶段的水位; (B) 从第二叶阶段开始的水位。


11. 在七叶或八叶阶段,小心地将每个篮子从水桶中的花盆中取出,然后去除附着在根部的土壤(图8A )。评估时间取决于实验设计。
12. 剪断地上部分(图8B ) ,并测量植物高度、叶片数和干重等性状(性状取决于个体实验设计)。
13. 小心 r将篮子从花盆中取出,不要切断主根。切割生长在篮子开口侧的主根,然后将根数计算为土壤表面根(图 8C) 。
14. 的侧面和底部切下穿透的初生根,分别计算根数为浅根和深根。




图8 。篮法中的水分管理量化盆栽土壤表层根。 
(A) 将篮子从水桶中的锅中取出; (B) 从篮子中洗出的附着在根部的土壤; (C) 主根生长在边缘以及篮子的侧面和底部。


15. 将每种植物的土壤表面根比定义为SOR的数量除以主根的总数。
16. 上述评估所需时间约为30分钟 每篮 一个人做的。时间可以根据植物的生长阶段定制。
17. 根据实验设计测量根长、厚度和干重。
18. 重复三次以上;这些 可以根据实验的目的和设计进行定制。


C. 稻田SOR量化的篮法
1. 稻田里的积水。使用田间条件,例如高地田地,因实验设计而异。
2. 移植前,用剪刀将幼苗的根部剪成1-2 厘米长(图9 A)。
3. 挖好水田土,然后将秧苗移栽到开篮的中央,使秧苗的底面刚好在开篮的边缘水平之下(图9B )。
4. 将篮子放回之前的位置(图9 C)。
5. 用大约 3 厘米的稻田土壤覆盖篮子(打开的一面朝上)(图9 D)。 




图9 。将篮子定位在土壤中并使用篮子方法种植幼苗。 
(A) 水田中的篮子和移栽点; (B) 装有稻田的篮子; (C) 篮子返回之前的位置; (D) 篮子埋在稻田土壤下。


6. 在营养阶段结束时,用刀(直径约30 厘米的圆圈,距离植物约15 厘米)切割水稻植株周围的水稻土(图10A )。
7. 连同土壤一起取出篮子 从稻田 (图 8B), 并小心地去除附着在水桶中的篮子上的土壤,不切割主根(图 8C)。 




图10 。 稻田篮法土壤表层根系取样与测量[J]. 
(A) 用刀切割水稻植株周围的土壤; (B) 从稻田挖出的篮子; (C)在水桶中清除附着在篮子上的土壤; (四) 金贾 贝顿厂; (E) Sasanishiki工厂。


8. 剪掉地上部分,然后测量 性状,如株高、叶数和干重(性状可根据个体实验设计进行测量)。
9. 使用剪刀,剪下生长在篮子开口侧的主根,并将其计为土壤表面根(图 9 A )。
10. 从篮子的侧面和底部切下穿透的初生根(图 9B和 9C ),然后分别计算浅根和深根的数量。




图11 。使用篮法计算浅根和深根的数量。 
(A) 切割生长在篮子开口两侧的初级根; (B)切割从侧面突出的主根 篮子的; (C)切割从突出的主根 篮子的底部。


11. 将每种植物的土壤表面根比定义为生长在篮子上边缘的 SOR 除以主根总数,包括从整个篮子网格中穿透的总根数。
12. 上述评估所需时间约为一人完成一篮子一小时。时间可根据植物生长阶段和田间情况定制。
13. 根据实验设计测量根厚度。
14. 建议重复九次以上;这些可以根据实验的目的和设计进行定制。


笔记


使用这些方法获得的结果已发表在以下论文中: Uga 等。 (2012),半泽 等。 (2013)和Kitomi 等。 (2020 年)。


致谢


本研究得到日本文部科学省 KAKENHI #21H04152 [Grant-in-Aid for Encouragement Research] 的支持。作者感谢 S husei Sato 和金雅 Toriyama提供他们的实验室空间和 Kunio Ichijyo为稻田提供技术支持。我们要感谢Edi ta ge ( www.editage.com ) 的英文编辑。


利益争夺


作者声明没有竞争利益。


参考


1. Abe, J. 和 Morita, S. (1994)。水稻节根的生长方向:其变化及其对根系形成的贡献。 植物土壤165:333-337。
2. Hanzawa , H., Sasaki, K., Nagai, S., Obara , M., Fukuta , Y., Uga , Y., Miyao , A., Hirochika , H., Higashitani , A., Maekawa, M.,等人。 (2013)。水稻土表生根新突变基因的分离( Oryza sativa L.)。 米饭 6:30 。
3. Kitomi , Y., Hanzawa , E., Kuya , N., Inoue, H., Hara, N., Kawai, S., Kanno , N., Endo, M., Sugimoto, K., Yamazaki, T.,等人。 (2020 年)。 DRO1同系物的根角修饰提高了盐田的水稻产量。 PNAS 177:21242-21250。
4. Lafitte, HR, Champoux , MC, McLaren, G. 和 O'Toole, JC (2001)。 水稻根系形态性状与同工酶群和适应有关。 田间作物水库71:57-70。
5. Oo ,AZ, Tsujimoto ,Y.,Mukai,M., Nishigaki ,T., Takai ,T.,和Uga ,Y.(2021 年)。具有DRO1同源物的浅根系与局部施磷之间的协同作用提高了低地水稻对磷的吸收。 科学代表11:9484。
6. Oyanagi , A.、Nakamoto, T. 和 Morita, S. (1993)。谷类植物根系的重力响应和根系的形成。 环境实验机器人33:141-158。
7. 帕夫利琴科,TK (1937)。在田间条件下对杂草和农作物的整个根系进行定量研究。 生态学18:62 - 79。
8. Teramoto , S.、 Kitomi , Y.、 Nishijima , R.、Takayasu, S.、Maruyama, N. 和Uga , Y. (2019)。反铲辅助整体法在高地条件下进行植物根系表型分析。 品种 Sci doi :101270/ jsbbs 。 19019 年。
9. Ueno, K. 和 Sato, T. (1989)。水稻生态型布鲁的气生根形成。 Jpn J Trop Ag r 33:173-175。
10. Ueno, K. 和 Sato, T. (1992)。水稻冠根生长方向的品种差异及其与向地反应和冠根直径的关系[J]. Jpn J Breed 42:779-786。
11. Ug a , Y.、 Ebana , K.、Abe, J.、Morita, S.、 Okuno , K. 和 Yano, M. (2009) 。不同遗传背景的栽培稻( Oryza sativa L.)种质根形态和解剖结构的差异。 品种科学 59:87-93。
12. Uga , Y.、 Hanzawa , E.、Nagai, S.、Sasaki, K.、Yano, M. 和 Sato, T. (2012)。水稻田土壤表层生根的主要水稻QTL qSOR1的鉴定。 Theor Appl Genet 124:75-86。
13. Uga , Y.、 Okuno , K. 和 Yano, M. (2011)。 Dro1是旱田条件下水稻深生根的主要QTL。 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.,等人。 (2013)。通过DEEPER ROOTING 1控制根系结构可提高干旱条件下的水稻产量。 Nat Genet 45:1097-1102。
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引用: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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