Variation in rice root traits assessed by phenotyping under drip irrigation

Roots are the key elements in water saving rice cultivation. So, Background: the response of rice roots are to be phenotyped under varied drip irrigation treatments. This study describes an investigation on rice root Methods: phenotyping under drip irrigation treatments in split-split plot design. Two lateral spacing levels (0.8 and 1.2m), two depths of irrigation (5-10 and 15-20 cm) by solar powered and well operated irrigation were tested using TNRH 180, JKRH 3333 and ADT(R)45 rice genotypes during the summer season (2013 & 2014) in Coimbatore, India. Conventional aerobic irrigation was considered as control. An increased root length, root density (length Results and Discussion: and weight), root Adinosine Tri Phosphotase enzyme activity, root volume and filled grain percentage were favored in aerobic rice under the conditions of 0.8m lateral distance with 5-10cm depth of sub surface drip irrigation (SDI). Improved root characteristics were observed in JKRH 3333 rice hybrid, and root density and thickness favored the filled grains and yield increment in rice by drip irrigation. The 0.8m lateral distance laid out at 5-10cm depth SDI proliferated more roots at subsurface soil layer with significant yield increment in rice. T. Parthasarathi ( ) Corresponding author: parthasa@post.bgu.ac.il Competing interests: No competing interests disclosed. Parthasarathi T, Vanitha K, Mohandass S How to cite this article: et al. Variation in rice root traits assessed by phenotyping under drip 2017,  :125 (doi: ) irrigation [version 1; referees: 1 not approved] F1000Research 6 10.12688/f1000research.9938.1 © 2017 Parthasarathi T  . This is an open access article distributed under the terms of the , Copyright: et al Creative Commons Attribution Licence which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Data associated with the article are available under the terms of the (CC0 1.0 Public domain dedication). Creative Commons Zero "No rights reserved" data waiver The project was funded by Netafim Irrigation Ltd., Israel. Grant information: The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. 10 Feb 2017,  :125 (doi: ) First published: 6 10.12688/f1000research.9938.1 1 2 3 4

The project was funded by Netafim Irrigation Ltd., Israel.

Grant information:
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.The growth of the root system in rice is restricted under aerobic conditions, which is the reason for poor yield (Kato et al., 2010).
In aerobic soil conditions, the soil's interaction with rice is primarily focused on the root system.Therefore, the root system is the first barrier to face stress found in aerobic soil conditions.Water management of rice crops is a sensitive tool in aerobic rice cultivation practice, which has been demonstrated by alteration in rice root anatomy (Mostajeran & Rahimi-Eichi, 2008).
A deeper root system of rice eases water stress and improves the uptake of nutrients and water in deep soil layers (Lilley et al., 1994).Rice cultivars with deep rooting and higher root density are much more favorable to aerobic conditions than lowland conditions (Matsuo et al., 2010).
Regulation of root traits to aerobic conditions could be attained by phenotyping for root traits and rhizosphere management by application of water and nutrients to the root zone (Zhang et al., 2010).Drip irrigation stimulates fibrous root production with specific changes in root system architecture (Raj et al., 2013).
This study focuses on phenotyping of root traits, such as root length, density, and distribution, under various drip treatments, related to the root response of rice genotypes.Consequently, the phenotyping of root traits and grain yield under drip environment was analyzed by the present study.

Experimental set up
A root phenotyping experiment was conducted during the summer season of 2013 and 2014, using JKRH 3333, TNRH 180 and ADT(R)45 as the test rice varieties at Tamil Nadu Agricultural University (Coimbatore, Tamil Nadu, India).Seeds were manually sown in the field at 20×10cm spacing.The open pan evaporation (PE) values (125% pan evaporation) were used to calibrate irrigation scheduling, and drip irrigation was supplied via pipe at 40mm outside diameter (OD) by 7.5HP motor with a pressure of 1.5kg/cm -2 from a bore well.Solar powered and well-operated drip irrigation sources, 0.8 and 1.2m lateral distances, 5-10 and 15-20cm depth sub-surface drip (SDI) were the treatments adopted at field level.The conventional aerobic practice was scheduled at 1.25 irrigation water (IW)/cumulative pan evaporation (CPE) ratio to 3.0cm.It was named as conventional aerobic rice.The recommended dose of 150:50:50 kg:ha -1 : NPK water soluble fertilizers were used to fertigate the crops using the Venturi flume at weekly intervals.Further information on genotypes, experimental set up and fertigation schedule are given in Supplementary File 1.

Measurements
Root length was estimated during the flowering phase (80 days after sowing) from core samples (Kato et al., 2006).Rice roots were removed carefully from the soil by root auger without damaging the roots.After the samples were oven-dried at 80°C for 72h, root lengths and weights were measured.Root length (m hill -1 ) = sample root length (cm) × total root weight (g)/sample root weight (g).Root dry weight was expressed as g/hill.The specific root length was estimated as a ratio by root length to root dry weight.Four soil cores (50mm diameter, 35cm depth) per plot were taken next to the plant and between the rows (20cm) with a soil sampler.Cores of soil were separated into 0-15 and 15-35cm, then washed using water and sieved by 0.5mm mesh sieve.The root length (RLD), as well as root mass density (RMD), was determined using the formula of Pantuwan et al. (1997), and the values were expressed as cm/cm 3 and mg/cm 3 of the soil, for RLD and RMD respectively.Root volume was recorded using the water displacement technique (Bridgit & Potty, 2002) and expressed as cm 3 /hill.
Core sampled roots were washed thoroughly and dehydrated using 80, 90 and 100% alcohol.Dehydrated roots were embedded in slides using paraffin.Slides were kept for imaging the root system using camera (Sony 12.1 megapixel) mounted Leica D1000 microscope at 10X magnification (Guo et al., 2008).
Adenosine triphosphatase (ATPase) activity of the root was assayed according to Wayne (1955) at 32°C using ATP (sodium salt) as a substrate, and the reactions were terminated by the addition of 2.0 mL cold 10% trichloroacetic acid.The ATPase enzyme activity was expressed as µg Pi g -1 h -1 .
Harvesting of the crop (grain) was performed from the net plot level (2.4×7.0m) at 120 days after sowing.The yield of rice grain was calculated to hectares at 14% moisture level and expressed as kg ha -1 .The filled grain percentage (%) was calculated by the ratio of total filled grain with total spikelet numbers in panicles.
The dry weight of grain and total grain dry weight per hill ratio was used to measure the harvest index (HI; %) at harvest stage of the crop (Yoshida et al., 1971)

Results
Total root length (TRL) is the size of the total root system, which is the major determinant for water and nutrient uptake.The drip irrigation system used in the present study in aerobic rice showed significant variation among root traits.Regarding the TRL for the different rice genotypes, a longer length was observed in with JKRH 3333 (6.2m/hill), followed by TNRH 180 (50.7m/hill) and ADT(R) 45 (38.6m/hill) (Table 1).
Among the genotypes, a significantly higher root volume was observed in JKRH 3333 (66cm 3 /hill), followed by TNRH 180 (61.7cm 3 /hill) and ADT(R)45 (53.2cm 3 /hill).From the main plot treatment, the solar drip irrigation recorded an increased root volume of 43.4% compared with the well-operated drip irrigation treatment.
JKRH 3333 genotype was statistically superior among all the genotypes in grain yield.The grain yield was observed to be significantly higher in the solar operated drip irrigation treatment (4817kg/ha) compared with well-operated drip irrigation (4313kg/ha) (Table 2).Among the performance of genotypes under drip irrigated aerobic rice, JKRH 3333 was statistical superior in mean grain yield (4831kg/ha) followed by TNRH 180 (4639kg/ha) and ADT(R)45 (4224kg/ha).

Discussion
Roots are the main component in the absorption of water and minerals, which are essential in plant physiological processes.Fageria ( 2007) observed that root length followed a significant quadratic response with the advancement of plant age from 19 to 120 days after sowing, and shows a linear increase in root length during flowering.
Favored root length under SDI at 5-10cm treatment is due to deep rooting of rice to combat water limited conditions.Genotypic variation in TNRH 180 revealed deep rooting to reduce the limited water application effect.The increased root growth and development of the root system help the rice to explore the wider area of soil and the deeper soil layers for water and nutrients.These results were corroborated with Henry et al. (2011) in rice under drought.
The genotype JKRH 3333 registered an increased root length and specific root length of 34.9% and 3.9% over conventional aerobic rice (Figure 2).Specific root length was an indicator for environmental changes.The genetic potential of this rice genotype for maintenance of increased root length favors lateral root branching (Figure 3).This effect was in accordance with Kato & Okami (2011) in rice.
Root volume of plants covers huge soil volumes and water uptake from the soil in water-limited conditions (Kanbar, 2004).Altered root volumes were observed in the present study under SDI with a 5-10cm drip laid out at 0.8m LD, due to greater assimilation allocation in rice roots by drip irrigation.Similar results were observed by Parthasarathi et al. (2012) under drip irrigation.
The root length density (RLD) is the length of roots per unit volume of soil, is an important parameter required to understand plant performance.In the present experiment, the SDI at 5-10cm depth using JKRH 3333 increased the RLD and RMD, due to the root zone of rice exposed to frequent wetting and slight drying and nutrient accessibility.The dry weight of roots was 36.8% superior in JKRH 3333 hybrid under drip irrigation.Similar variation obtained in rice was observed by Vanitha (2011) and could support the present results.This unique response of root length and mass density under drip irrigation to improve nutrient and water accessibility was due to more root proliferation at topsoil.Comparing the root images of genotypes (Figure 3) revealed that, even though the appearance of white roots was common, an increase in root numbers and density was higher in drip irrigation.
Light energy absorbed by chlorophyll is converted into stable chemical energy and drives ATP formation via ATPase in the plastids of roots.ATPase is widely present in plant tissues and involved in the active transport of ions across membranes of the cell (Martínez-Ballesta et al., 2003).In the present study, higher levels of ATPases were observed in SDI + 0.8m LD at 5-10cm lateral depth with the JKRH 3333 hybrid.
The grain filling percentage is an important contributory factor to grain yield.The SDI laid out at 5-10cm depth with 0.8m LD treatment registered more grain production and filling percentage.Among the genotypes, the hybrid (JKRH 3333) excelled the variety in filled grain percentage by 15.4% (Figure 4).The increase in the water supply to the spikelets might reduce the floret abortion during flowering, and may be the reason behind higher filled grains in SDI.These results are indirectly supported by Kato et al. (2008) in aerobic rice.Harvest index (HI) reflects the proportion of assimilate distribution between economic and total biomass (Donald & Hamblin, 1976).Among the genotypes, a higher in HI was recorded in JKRH 3333 with a 1.6 and 4.5% increment over the TNRH 180 and ADT(R)45 genotypes, respectively (Figure 3).This might be attributed to the fact that producing a larger sink size and efficient transport of assimilates from leaves and stems ('source') into developing spikelets ('sinks'), thus resulting in the increased grain yield (Guan et al., 2010).
The higher grain yield of JKRH 3333 recorded a 21.4% increase in drip over conventional aerobic rice cultivation.Comparing the depth of SDI treatment at a 5-10cm soil depth achieved a 18.9 and 13.0% increased yield over 15-20cm soil depth and conventional aerobic irrigation method, respectively.The SDI system maintained equal soil wetting, reduced the evaporation with direct point application of water in root, which improves the grain yield of rice.A previous study supports this argument (Douh et al., 2013).

Conclusions
This drip-irrigated aerobic rice study concluded that there is an increase in grain yield along with increased root parameters.
Based on the data of lateral spacing, discharge variations and the root characters of rice under drip significantly showed that there was characteristic flexibility in the roots of the rice plant.The root length, root density, root hairs and root ATPase activity exhibited a significant association with filled grain percentage and grain yield.The genotype JKRH 3333 showed 14.3% increased grain yield with favorable root density and root dry weight over ADT(R)45.It could be recommended that 0.8m lateral distance laid out at 5-10cm depth SDI may proliferated more roots at subsurface soil layer with a yield increment in rice.
I accept your comments on including the interaction effect of treatments.My co-authors also accepted your view.In the version one, interactive effects of treatments are missing.
As per your comments, I have included the main, sub and sub-sub plot, interaction treatment results in the paper.I have mentioned the replications, experimental design, plot dimension in the paper.Also, please view the supplementary file for additional details such as experiment layout, drip layout.
Thank you for your peer review to made the manuscript better.
Please review the revised version (Version 2) and give your comments.
Thanking you.

Kind regards Parthasarathi
No competing interests were disclosed.Competing Interests: to rice grown under non-water stagnation.

Figure 2 .
Figure 2. Responses of root dry weight (black bar) and specific root length (gray bar) of rice.

Figure 3 .
Figure 3. Imaging of root system of rice genotypes under microscope.

Figure 4 .
Figure 4. Effect of filled grain percentage (black bar) and harvest index (gray bar) of rice.

Table 1 . Phenotyping of root length (g hill -1 ) and volume (cc hill -1 ) of rice
* Figure 1.Response of root length (black bar) and mass density (gray bar) of rice.