Journal ofInternational Cooperation for
Agricultural Development

J Intl Cooper Agric Dev 2019; 17: 34–40

Field Report

Evaluation of a Newly Identified Mutation
Gene that Promotes Root Elongation for
Improvement of Drought Avoidance in
Rice
Tomomi Hasegawa1), Akihide Shibata1), Cornelius Mbathi Wainaina1, 2), Nonawin Lucob-Agustin1),
Daigo Makihara3), Hiroaki Samejima3, 4), Mayumi Kikuta3, 5), Daniel Makori Menge3, 6),John Munji Kimani6), Akira Yamauchi1), Yoshiaki Inukai3)

Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan
Department of Horticulture, Jomo Kenyatta University of Agriculture and Technology, Nairobi, Kenya
3)International Center for Research and Education in Agriculture, Nagoya University, Nagoya, Japan
4)
Graduate School of Agricultural Science, Kobe University, Kobe, Japan
5)
Applied Social System Institute of Asia, Nagoya University, Nagoya, Japan
6)
Kenya Agricultural and Livestock Research Organization, Mwea-Tebere Center, Kerugoya, Kenya
1)
2)

Received March 10, 2018 Accepted April 27, 2018
Abstract. Drought is one of the most serious abiotic stresses for rice cultivation, even under a water saving production
system such as the Alternate Wetting and Drying (AWD) system and upland irrigated fields. In this study, we evaluatedthe performance of larger root systems in a KM07 mutant under such water management conditions. This mutant line wasutilized for breeding and development of new rice germplasm adaptable to drought stress conditions in Mwea, Kenya, bycrossing it with the recurrent parent of New Rice for Africa (NERICA), WAB56-104. Three mutant-type F6 lines grown under
AWD and upland irrigated conditions showed different responses to drought stress. The mutant type’s greater root growth
was observed in lines 2 and 3 under AWD and upland irrigation conditions, respectively. These results suggest that the highly
developed root system derived from the KM07 mutant can avoid drought stress under field conditions in Kenya, and it should
be utilized as breeding material for drought stress avoidance, along with water management technology.
Key words: Kenya, rice, root system, drought, water saving technology

Introduction
Rice is the staple food for more than half of the world’s
population. Rice is one of the most important crops, notonly for its calories, but also as an economic commodity, particularly in many developing countries1). Riceconsumption among those countries is growing rapidly,
especially in sub-Saharan Africa (SSA) where it is thefastest growing staple food2). However, the productivity ofrice in SSA is less than half that of the world’s average,
and more than 60% of the total rice consumed there isimported, mainly from Asia3).
34 J Intl Cooper Agric Dev 2019

Drought caused by limited water resource is considereda major constraint to both upland and rainfed lowland
rice production in SSA4). Therefore, in order to address
this challenge and use the limited water resource more
efficiency, water management practices are necessary. A
water saving technology, known as ‘Alternate Wetting
and Drying’ (AWD), has been developed. In the AWD
system, rice is grown without continuously waterlogged(CWL) in the field, and irrigation water is applied toobtain flooded conditions after a certain number of days
have passed since the disappearance of ponded water5).
AWD was implemented after several studies showed that

it reduced irrigation water input by up to 38% 6, 7). Another
newly developed method is the aerobic or upland irrigatedmethod, where adapted rice varieties are grown as normal
field crops with or without supplementary irrigation inwell-drained, non-puddled, and non-saturated soils. Watersaving techniques, however, risk yield reduction because
plants may experience drought stress. By analyzing thedata from in total 31 field experiments, it is reported that
92% of the AWD treatments resulted in yield reductions
varying from just above 0% to 70% compared with flooded
control plots5). Therefore, in order to minimize drought
stress without decreasing yield under water saving condition, water input needs to be optimized and rice varieties
possessing drought tolerance or drought avoidance mechanisms should be selected. Adaptability of root systems todrought stress have been studied intensively under various
water management systems, including AWD, aerobic conditions, different soil moisture conditions, and fluctuating
soil moisture conditions8-16). For adaptation to these stressenvironments, one of the most important root attributes
is to maintain total root length under water deficient andrewatering conditions9, 17). These adaptations resulted inincreased water uptake and improved shoot dry matterproduction13, 18).
In this study, we identified a rice mutant line, KM07,
that has a highly-developed root system under hydroponic
conditions at the seedling stage. In order to evaluate thepossibility of using these mutant root traits for avoiding
drought stress under field conditions in Mwea, Kenya, weprepared F6 lines derived from the cross between KM07
mutant and WAB56-104, an upland improved variety andparent of NERICA 1 to NERICA 11, for improving riceproductivity in Africa.

Materials and Methods
Plant materials

Rice root mutant line KM07 and its parental variety,
Kimmaze (Oryza sativa L.) were utilized for pot experiments. Three F6 lines derived from the cross between the
KM07 mutant and WAB56-104 were evaluated in the field.
Each F6 line was classified into normal root phenotype(Normal-type) and mutant root phenotype (Mutant-type).
Pot experiment
Growth conditions

Kimmaze and the KM07 mutant were evaluated in
pot experiments conducted in a glasshouse at Nagoya
University, Nagoya, Japan (35°6’42”N, 137°4’57’E) in2017. Kimmaze and KM07 mutant seeds were sterilized bysoaking in water containing benomyl fungicide (2.5 g L–1)
in a growth chamber at 28°C under continuous light for 4d. Three pre-germinated seeds each of Kimmaze and theKM07 mutant were sown in a pot (20 cm in height and16 cm in diameter) filled with 4.0 kg of air-dried sandy
loam soil analyzed in our previous paper19). The soil was
mixed with fertilizer containing 120 mg nitrogen, 120 mg
phosphorus, and 120 mg potassium. The seedlings in eachpot were thinned to one at 10 days after sowing (DAS).
Kimmaze and the KM07 mutant were grown under CWL
conditions.
Root sampling and measurement

Roots were collected from three plants of each genotype
at 60 DAS. Roots were washed under running water toremove soil. Cleaned roots were spread on the acrylic
tray filled with water without overlapping. Digital images
were taken using an Epson scanner (ES-10000G) and thensaved as tiff files (6600 × 4879 pixels). Each root trait
was analyzed using WinRHIZO software v. 2013 (Regent
Instruments, Quebec, Canada).
Field experiment
Growth condition

Three F6 lines were evaluated in a field experimentconducted at Kenya Agriculture and Livestock Research
Organization-Mwea Centre, Kenya (KALRO-Mwea
Research Farm (0°40’35”S, 37°18’06”E) from April to
September 2017. The soil of the experimental field was
Nitisol analyzed in our previous paper20). Plant materials
were grown under CWL, AWD, and upland irrigatedfield conditions (Fig. 3b, c). For the upland irrigated field
condition, three seeds were directly sown per hill on April
12, 2017 and then the seedlings in each hill were thinned
to one at 10 DAS. Pre-germinated seeds were sown inseedling trays on April 12, 2017 for CWL and AWD treatment, and then seedlings were transplanted in the field on
May 3, 2017. As shown in Fig. 3a, seeds or seedlings weregrown in seven rows, seven plants per row at a spacing of
20 cm × 20 cm between plants. Each line and genotype
had three replicates. The AWD field was maintained in aflooded condition for 21 days after transplanting (DAT),and then re-irrigated up to 5 cm depth from the soil surface
whenever the water depth reached 20 cm from the soil
surface. Water depth in the AWD field was measured using
a water tube buried into the soil. The upland irrigated field
was kept wet but not flooded by applying water using asprinkler until harvest. Basal fertilizer was applied at a perhectare rate of 25 kg N, 25 kg P2O5, and 25 kg K2O at thetime of transplanting. Top-dressing fertilizer was applied
at a per-hectare rate of 25 kg N at 21, 45, and 75 DAT (42,66, and 96 DAS in the upland irrigated field, respectively).

J Intl Cooper Agric Dev 2019 35

Fig. 1. Root system profiles (a) and root traits (b) of parental variety (Kimmaze) and KM07 mutant plants at 60 days after
transplanting under continuously waterlogged (CWL) conditions. Bar = 10 cm.
*, significant at P < 0.05 between the two genotypes.

Root sampling and measurement

Roots were collected from four plants of each line andgenotype at 125 DAT. Before root sampling, shoot parts
were cut at 5 cm above the ground surface. A metal tube
(30 cm in height and 20 cm in diameter) was inserted
into the soil up to a depth of 20 cm and then pulled out tosample a core of soil and roots. The root samples from thecore were washed with running water to remove soil. Eachroot trait was measured by the same methods used in the
pot experiment.

Results
Root development of the KM07 mutant

The differences in root development between the KM07
mutant and Kimmaze were evaluated in the pot experimentat the vegetative stage. The roots of the KM07 mutant
were found to be distributed wider and deeper than thoseof Kimmaze (Fig. 1a). As shown in Fig. 1b, under CWL
conditions, total root length (TRL), root surface area (SA)and root volume (RV) in the mutant were 53%, 47% and
38% higher than those in the Kimmaze, respectively.
Water depth in AWD field and weather conditions
at the experimental site

In the AWD field, the cycles of wetting and drying wererepeated 19 times until harvesting (Fig. 2b). The air temperature reached below 15°C three times from the booting
stage to the maturity stage (Fig. 2a), and the cold damage
36 J Intl Cooper Agric Dev 2019

resulted in severe sterility in both genotypes (Fig. 3d).
Root phenotypic evaluation

Each root trait was analyzed using digital images in
WinRHIZO (Table 1). The rice root system typicallyconsists of two types of roots, nodal roots (NR) formed atlower stem nodes and lateral roots (LR) derived from thenodal roots. In this study, the root components, NR and
LR, were classified based on their diameter (D.). NR and
LR were included when their diameters were D.> 0.3 mm
and D.≤ 0.3 mm, respectively. LR was divided into threeranges, thin LR, middle LR and thick LR when diameters
were 0 mm <D.≤ 0.080 mm, 0.080 mm <D.≤ 0.15 mm
and 0.15 mm <D.≤ 0.30 mm, respectively21). Under CWL
condition, there was a tendency that Mutant-type plants
showed slightly higher root development than that ofNormal-type plants among all three F6 lines, however, in
all root traits there were no significant differences between
Normal-type and Mutant-type plants. Meanwhile, the effects of water treatment in AWD and upland irrigated onroot development were significant between the two genotypes. Under AWD conditions, only Mutant-type plants ofline 2 maintained significantly higher TRL, SA, RV, total
nodal root length (TNRL), total lateral root length (TLRL)
and thin TLRL compared with Normal-type plants. Under
upland irrigated conditions, only Mutant-type plants ofline 3 maintained significantly higher TRL, TLRL, andlateral root length (LRL) at each diameter compared with
Normal-type plants. As a result, Mutant-type plants of line

Fig. 2. Air temperature and rainfall in Mwea, Kenya (a) and ground water levels under alternate wetting and drying (AWD)
field (b) in 2017. Max, Ave, and Min represent maximum, average, and minimum temperature, respectively.

2 in AWD conditions and Mutant-type plants of line 3 inupland irrigated conditions had significantly higher developed root systems, but Mutant-type plants of line 1 did
not have significant higher root growth under either AWD
or upland irrigated conditions compared with Normal-type
plants of line 1 grown under both conditions.

Discussion
In this study, we evaluated the performance of rootsystems observed from the KM07 mutant under different
water management conditions, including CWL, AWD,
and upland irrigated conditions in Mwea, Kenya. Under
AWD and upland irrigated conditions, Mutant-type plants
developed larger root system with increased root lengthcompared with those in Normal-type plants, suggestingthat Mutant-type plants responded to the water deficit andrewatering more strongly, thereby maintaining a highly
developed root system under drought stress conditions.
Mutant-type plants exhibited greater root growth inlines 2 and 3 under AWD and upland irrigate condition,
respectively.
East Africa including Mwea, Kenya is located incold-prone highlands. In these location, cold stress, oneof the most important abiotic stresses for rice cultivation,
caused yield losses of up to 100%16). In this study, the airtemperature reached below 15°C several times from thebooting stage to the maturity stage, resulting in severe sterility in both Normal-type plants and Mutant-type plants.
Importantly, Mutant-type plants of lines 2 and 3 maintained
highly developed root systems even under a severe cold
stress environment. We previously reported a cold tolerant
gene locus that functions well in the cold-prone highlands
in Kenya22). In order to achieve stable yield under water
deficit as well as cold-prone conditions, we are now trying
to produce breeding lines that have both the cold tolerant
gene and the KM07 mutant gene.
Acknowledgement
This work was supported by the Japan Science andTechnology Agency (JST)/Japan International Cooperation
Agency (JICA) and the Science and Technology ResearchPartnership for Sustainable Development (SATREPS).
The authors are indebted to Mr. James Gichuki Kambo,
Ms. Christine Wambui Wangui, Mr. Francis Ngare Kambo,
Mr. Benson Mwangi Kairungu, Ms. Edith Micere Muriithi,
Mr. Arnold Kimanthi Njue, and Mr. Paul Nganga Njenga
for supporting the implementation of the experiments.
J Intl Cooper Agric Dev 2019 37

Fig. 3. Field map in continuously waterlogged (CWL),
alternate wetting and drying (AWD), and uplandirrigated field (a), growth conditions in CWL (b, left),in AWD (b, right) and in upland irrigated (c) andpanicle damaged by cold stress (d). □, Normal-type ofline 1; ■, Mutant-type of line 1; ○, Normal-type of line
2; ●, Mutant-type of line 2; ∆, Normal-type of line 3;▲, Mutant-type of line 3. Each symbol represents one
plant per hill.

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