J Intl Cooper Agric Dev 2017; 15: 32–41

Journal ofInternational Cooperation for
Agricultural Development

Working Paper

Development of a New Cultivation
Technology for Cold Stress
Escape through Flowering Time
Manipulation by Water Managementin the Highlands of East Africa
Cornelius Mbathi Wainaina1, 2), Daigo Makihara3), Hiroaki Samejima3), Mayumi Kikuta3),Daniel Makori Menge3), John Munji Kimani3, 4) and Yoshiaki Inukai3)1) Department of Horticulture, Jomo Kenyatta University of Agriculture and Technology, Nairobi, Kenya
2) Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan
3) International Cooperation Center for Agricultural Education, Nagoya University, Nagoya, Japan
4) Kenya Agricultural and Livestock Research Organization, Mwea-Tebere Center, Kerugoya, Kenya
Received January 19, 2017

Accepted: February 10, 2017

Abstract. Cold stress is a major abiotic factor limiting rice production by reducing spikelet fertility. Under field conditions,cold stress varies with years and seasons in terms of its intensity, duration, and timing of occurrence. To reduce the risk ofcold damage because of its unpredictable nature, crop adaptation strategies, such as management practices, that complement
breeding are required. Flowering time (heading time) is a key trait that can be used to adapt rice to changing climatic conditions. Therefore, we screened a rice mutant line (T6-16) that has very shallow root system and exhibits delayed heading
time under moderate drought stress conditions. This mutant line was used in breeding and development of a new cultivation
technology for cold stress escape by introgression of its genetic segments into the background of a recurrent parent of New
Rice for Africa (NERICA), WAB56-104. The mutant-type F2 plants grown under moderate drought stress conditions showed
delayed heading by an average of 11 days. Three F2 plants that showed delay of heading by 11−17 days and maintained
over 95% grain weight under the stress conditions were identified. These F2 plants could be useful in breeding for cold
stress escape. These results suggest that the utilization of rice plants introgressed with the mutation gene and proper water
management practices for rice cultivation based on the predicted length of cold periods could escape the cold stress throughdelaying heading time.
Key words: Kenya, Rice, New cultivation technology, Cold stress escape, Flowering time manipulation

Introduction
Cold stress is a major abiotic factor affecting ricecultivation in high latitude and altitude areas1–3). In rice
growth stages, the booting stage, especially the early
Corresponding author: Yoshiaki Inukai, E-mail: inukaiy@agr.nagoyau.ac.jp

32 J Intl Cooper Agric Dev 2017pollen microspore stage that occurs approximately 10–12
days before flowering time (heading time), is the mostsensitive to cold injury4). Low temperature at the booting
stage reduces spikelet fertility and, in turn, reduces grain
yields1).
In cold-prone highlands of East Africa, such as Mwea
in Kenya (Fig. 1), cold stress exhibits yearly and seasonalvariations in terms of its intensity, duration and timing of

Fig. 1. Daily average air temperature in June to August in Mwea, Kenya between 2012 and 2015. Dotted arrow indicates minimum threshold temperature for normal rice growth and development.

occurrence5). In these locations, temperatures can reachbelow 18°C (i.e., during severe stressful years or seasons)
thereby increasing the risk of yield loss due to cold damage, resulting in yield losses of up to 100%. Due to theunpredictable nature of cold stress under field conditions,
breeding efforts for cold tolerance based on genetic improvement alone will not be enough to reduce the risk ofcold damage. Improved crop adaptation strategies, suchas new management practices, that would complement
breeding for stress tolerant varieties are needed. Thus,
exploration of key traits and management practices foradapting rice to variable low temperature environments
will play a major role in sustaining rice yields.
Heading time is mainly influenced by environmental
factors such as day length (photoperiod) and temperature,but is also affected by abiotic stresses such as cold, drought
and nutrient deficiency which can cause delay in heading
time of rice6–8). The shifting of heading time is a key
strategy that can be used to better adapt rice to changing
climatic conditions, and is a major goal for plant breeders9). Delaying heading time in rice (i.e., lengthening thevegetative growth during the cold period and switching tofloral transition when temperatures are optimum) is a potential option to adapt rice to cold-prone regions. Shiftingof the reproductive phase in rice may maintain the yieldpotential in cold-prone environments through preserving
spikelet fertility and grain filling.
In this study, we newly screened a rice mutant line, T6-16,
that has a shallow root system and exhibits delayed heading time when subjected to moderate drought stress. We
explore the possibility of using mutant traits for breeding
rice lines that can escape cold periods by delaying heading
time while sustaining yield or with minimal yield loss.

Materials and Methods
Plant materials
The plant materials (rice genotypes) used included a
newly screened rice mutant line, T6-16, WAB56-104, andan F2 population (115 individuals) derived from a crossbetween the T6-16 mutant and WAB56-104. The mutant
parental variety is Taichung 65 (TC65), a Taiwaneselowland rice variety. WAB56-104 is an upland improved
variety and parent of NERICA 1 to NERICA 1110).
Seed germination and transplanting
Pre-germinated seeds were sown in seedling trays in
mid-October, 2015. Each pre-germinated seed was sownin a cell measuring 5 cm × 5 cm × 5 cm and N fertilizer(ammonium sulfate) was applied 7 days after emergence
as a nutrient solution (5 g/20 L) to enhance rapid tiller production. At day 40, the seedlings were transplanted in thefield (on November 27, 2015) in single rows, 10 plants per
row at a spacing of 30 cm × 15 cm between rows or plants.
Each hill had 6 tillers by the transplanting time and was
split into two parts, one with the main tiller (main-stem
plant) was transplanted in an alternate wetting and drying(AWD) paddy field and the other (split-tiller plant) in acontinuously waterlogged (CWL) paddy field.
Field evaluation
The F2 population and the parent genotypes were evaluated in a paddy field at Kenya Agricultural and LivestockResearch Organization-Mwea Centre, Kenya (KALROMwea research farm (0°40'35"S, 37°18'06"E, and 1168 ma.s.l) from October 2015 to March 2016. The plant materials were evaluated under two water management practices:
J Intl Cooper Agric Dev 2017 33

AWD and CWL. The AWD field was kept flooded during
the first two weeks after transplanting and then re-irrigatedby surface irrigation up to 5 cm ponded water depth when
the soil water potential reached –30 kPa at 20 cm soil
depth. The threshold of –30 kPa was considered a moderate water stress condition11). Soil water potential in AWD
field was measured every day at two points in the paddy
field using tensiometers installed at 20-cm soil depth.
Fertilizer was applied as basal fertilizer at a rate of 25 kg
N/ha, 25 kg P205/ha, and 25 kg K2O/ha at transplanting
time. Top-dressing fertilizer was applied in two splits at a
rate of 25 kg N/ha (ammonium sulfate) at 21 and 45 days
after transplanting (DAT) (Total N rate = 75 kg N/ha). The
fields were regularly weeded, and other crop management
practices were carried out following recommended farmer
practices12).

hibited horizontal root distribution, while TC65 exhibited
vertical root distribution (Fig. 2a). The mutant distributed
62% of its roots in the 0–60° root growth angle region
whereas TC65 distributed only 36% of its roots in the sameroot growth angle region (Fig. 2b). These results reveal
a clear variation in root distribution patterns between the
mutant and the wild type.

Measurements
Heading time was determined as the date of 50% heading, during which the number of panicles per plant wascounted. The panicles were hand-threshed and the filled
spikelets were separated from the unfilled ones by floatation in water. These were then dried in the sun for a few
hours and weighed separately. The moisture content of thefilled spikelets was measured using a grain moisture tester(Riceter f, Kett Electric Laboratory, Tokyo). Grain weight
per plant, adjusted to 14% moisture content, was calculated using the weights of filled spikelets in each hill andthe measured moisture values. The total number of filled
and unfilled spikelets was counted. The percent spikelet
fertility was calculated by multiplying the number of
fertile spikelets by 100 then dividing by the total numberof spikelets. The number of spikelets per panicle was also
calculated.

Agronomic and yield traits
Heading time: WAB56-104 and the T6-16 mutant
significantly differed in heading time, with the heading
occurring at 58 DAT and 90 DAT, respectively, under the
AWD practice (Fig. 3b; Table 1). Under the CWL practice,
the heading time of WAB56-104 and the mutant was 58
and 75 DAT, respectively (Table 1). The heading time of
the wild-type F2 plants ranged from 45 to 87 DAT, with amean of 66 DAT under the AWD practice (Table 1; Fig.
4a), compared to 45 to 85 DAT, with a mean of 62 DAT
under the CWL practice (Table 1; Fig. 4a). The heading
time of the mutant-type F2 plants ranged from 51 to 102
DAT, with a mean of 79 DAT under the AWD practice(Table 1; Fig. 4b) compared to 46 to 92 DAT and a meanof 68 DAT under the CWL practice (Table 1; Fig. 4b). For
WAB56-104, the heading time was not affected by the
AWD practice, whereas the heading time of T6-16 mutant
was delayed by 15 days under the same practice (Table1). For the F2 plants, wild-type plants delayed heading by
an average of 4 days (0–9 days), whereas the mutant-type
plants delayed by 11 days (4–18 days) (Table 1). Over
86% of wild-type F2 plants reached heading time earlier
than 80 DAT (Fig. 4a and b) under both water management practices, which was similar to mutant-type F2 plants
under the CWL practice (Fig. 4b). When mutant-type F2
plants were grown under AWD practice, over 65% of theplants reached heading time after 80 DAT (Fig. 4a).
Spikelet fertility: Spikelet fertility of WAB56-104 andT6-16 mutant was 87.1% and 44.5%, respectively, underthe AWD practice (Table 1). Under the CWL practice,
spikelet fertility of WAB56-104 and T6-16 mutant was
90.4% and 75.6%, respectively (Table 1). Spikelet fertilityof wild-type F2 plants ranged from 0.9 to 82.2%, with amean of 38.6% under the AWD practice (Table 1; Fig. 4c),compared to 0.3 to 82.3%, with a mean of 40.6% under

Statistical analysis
To assess the differences in agronomic traits between
genotypes, analysis of variance (ANOVA) was performed
using GLM procedure in SAS program (SAS version
9.1, SAS Institute Inc., Cary, NC, USA, 2002). The data
for mean spikelet fertility was arcsine-transformed andthe means were reported after back-transformation. The
means were separated using the least significant difference(LSD) test at P < 0.05.

Results
Root distribution of T6-16 mutant line and parental
variety TC65
The differences in root distribution between T6-16 mutant line and the parental variety (TC65) were evaluated ina rootbox experiment using a pinboard13). The mutant ex34 J Intl Cooper Agric Dev 2017

Soil water potential and rainfall in the experiment site
The AWD paddy field experienced 4 wetting and drying
cycles during the growing season (Fig. 3b). Even though
the AWD practice was started at 14 DAT, the first cycle
was reached at 58 DAT owing to the high rainfall in December and January (Fig. 3a) that resulted in the pondingof water depth, high soil water moisture, and a rise in the
ground water table.

Fig. 2. Differences in root distribution between wild- and mutant-type plants and proportion ofroot distribution by root growth angle at 35 days after sowing. Root growth angle regions
are 0–30° (blue shaded), 30-60° (red shaded) and 60–90° (green shaded).

Fig. 3. Rainfall (a) and soil water potential at 20 cm (b) depth in the AWD paddy field in 2015–2016.
W and M indicate heading time of WAB56-104 and T6-16 mutant respectively, under AWD conditions; H indicate harvesting time.
J Intl Cooper Agric Dev 2017 35

Table 1. Performance of agronomic characteristics in F2 population and their parental varieties grown under AWD and CWL
conditions
Parents
WAB56-104
Trait

CWL

Heading time (DAT)
Spikelet fertility (%)
Panicle number
Spikelet number per panicle
Grain weight (g/plant)

58.0
58.0
(0 d) ns
90.4
87.1 (96.4%) **13.1
11.5 (87.8%) ns
154.2 108.3 (70.2%) ***46.6
29.2 (62.7%) **AWD

F2 population
T6-16 mutant

wild-typemutant-type

CWL AWDCWL AWDCWL AWD

75.0
75.6
19.8
78.1
31.0

62.0
40.6
17.3
99.1
16.4

68.0
40.4
15.0
81.7
14.4

90.0 (15 d) ***44.5 (58.9%) ***11.5 (58.1%) ***36.9 (47.2%) ***5.1 (16.5%) ***66.0(4 d) ns
38.6 (95.1%) ns
20.2 (116.8%) ns
96.7 (97.6%) ns
16.5 (100.6%) ns

79.0(11 d) *36.0 (89.1%) ns
16.0 (106.7%) ns
73.9 (90.5%) ns
13.4 (93.1%) ns*P < 0.05, **P < 0.01, *** P < 0.001. ns, not significant.
Values in parenthesis indicate heading delay in days (d) or percent proportion (%) of the trait in AWD relative to that under CWL.
AWD, Alternate wetting and drying; CWL, Continously waterlogged.

the CWL practice (Table 1; Fig. 4c). Spikelet fertility ofmutant-type F2 plants ranged from 2.1 to 81.1% (a mean of36%) under the AWD practice (Table 1; Fig. 4d) comparedto 1.3 to 90.6% (a mean of 40.4%) under CWL practice(Table 1; Fig. 4d). WAB56-104 maintained 96.3% of its
spikelet fertility under AWD practice, whereas that of theT6-16 mutant was greatly reduced (i.e., by 41.2%) (Table1). For F2 plants, the wild-type plants maintained 95.1%of their spikelet fertility whereas the mutant-type plants
maintained 89.1% of their spikelet fertility (Table 1).
However, the mean spikelet fertility in the F2 population
was low (< 50%) under both water management practices,suggesting that there were other causes of F2 sterility, suchas male sterility.
Panicle number: WAB56-104 and the T6-16 mutant
produced almost the same number of panicles under the
AWD practice (Table 1). Under the CWL practice, panicle
numbers of WAB56-104 and T6-16 mutant were 13.1 and
19.8, respectively (Table 1). Panicle numbers of wild-type
F2 plants ranged from 7 to 35 (a mean of 20.2) under the
AWD practice (Table 1; Fig. 4e), compared to 4 to 30 (amean of 17.3) under the CWL practice (Table 1; Fig. 4f).
Panicle numbers of mutant-type F2 plants ranged from 7 to30, with a mean of 16, under the AWD practice (Table 1;
Fig. 4e), compared to 8 to 23, with a mean of 15, under the
CWL practice (Table 1; Fig. 4f). WAB56-104 maintained
87.8% of its panicle numbers under the AWD practice,whereas in the mutant the number of panicles greatly
reduced (i.e., by 41.9%) under the same practice (Table1). For the F2 plants, the panicle numbers of both wildtype and mutant-type plants were not affected by moderatewater stress.
Spikelet numbers per panicle: Spikelet numbers per
panicle of WAB56-104 and T6-16 mutant were 108.3 and
36.9, respectively, under the AWD practice. Under the
CWL practice, spikelet numbers per panicle of WAB5636 J Intl Cooper Agric Dev 2017

104 and T6-16 mutant were 154.2 and 78.1, respectively(Table 1). Spikelet numbers per panicle of wild-type F2
plants ranged from 25.2 to 178.4, with a mean of 96.7,
under the AWD practice (Table 1; Fig. 4g) compared to30.7 to 183.1, with a mean of 99.1, under the CWL practice (Table 1; Fig. 4g). Spikelet numbers per panicle ofmutant-type F2 plants ranged from 29.5 to 134.2, with amean of 73.9, under the AWD practice (Table 1; Fig. 4h)compared to 45.2 to 133.1, with a mean of 81.7, under the
CWL practice (Table 1; Fig. 4h). Under the AWD practice,
WAB56-104 maintained 70.2% of its spikelet numbers per
panicle, whereas the T6-16 mutant had a reduced number(i.e., 52.8%) (Table 1). For the F2 plants, wild-type plants
maintained 97.6% of their spikelet numbers per panicle
whereas the mutant-type plants maintained 90.5% of their
spikelet numbers per panicle (Table 1).
Grain weights: Grain weight of WAB56-104 and T6-16
mutant was 29.2 g/plant and 5.1 g/plant, respectively,
under the AWD practice. Under the CWL practice, grain
weight of WAB56-104 and T6-16 mutant was 46.6 g/plantand 31 g/plant, respectively (Table 1). Grain weight ofwild-type F2 plants ranged from 0.7 to 35.4 g/plant, witha mean of 16.5 g/plant, under the AWD practice (Table 1;
Fig. 4i), compared to 0.1 to 41.2 g/plant, with a mean of
16.4 g/plant, under the CWL practice (Table 1; Fig. 4i).
Grain weight of mutant-type F2 plants ranged from 0.4 to
43.5 g/plant, with a mean of 13.4 g/plant, under the AWD
practice (Table 1; Fig. 4j), compared to 0.1 to 50.6 g/plant,with a mean of 14.4 g/plant, under the CWL practice(Table 1; Fig. 4j). Under the AWD practice, WAB56-104
maintained 62.7% of its grain weight, whereas the grain
weight of T6-16 mutant was greatly reduced, by 83.5%(Table 1). For the F2 plants, wild-type plants were not affected by the AWD practice whereas mutant-type plants
maintained 93.1% of their grain weights (Table 1) under
the same treatment.

Fig. 4. Distribution of heading time (a, b), spikelet fertility (c, d), panicle number (e, f), spikelet numbers per panicle(g, h) and grain weights (i, j) in F2 population under AWD and CWL conditions. Closed squares indicate
Wild-type F2 plants and stripped squares indicate mutant-type F2 plants.

J Intl Cooper Agric Dev 2017 37

Table 2. Trait mean values in selected mutant-type F2 plants
F2 plants

Water treatment

Heading time
(DAT)

Spikelet fertility(%)

Panicle number

Spikelet number
per panicle

Grain weight(g/plant)

1

CWL
AWD
Difference

75.0
86.0
11.0

72.1
61.2 (85%)
15.0

17.0
19.0 (112%)
–2.0

133.1
134.2 (101%)
–1.2

36.8
35.0 (95%)
1.8

2

CWL
AWD
Difference

71.0
82.0
11.0

66.9
68.5 (102%)
–2.4

10.0
23.0 (230%)
–13.0

103.4
98.5 (95%)
4.9

21.0
43.5 (207%)
–22.5

3

CWL
AWD
Difference

73.0
90.0
17.0

70.0
62.9 (90%)
10.0

15.0
30.0 (200%)
–15.0

54.1
61.4 (113%)
–7.3

20.8
39.9 (192%)–19.1

Negative value indicate not affected by moderate water stress.
Values in parenthesis indicate percent proportion of the trait in AWD relative to that under CWL.

Agronomic performance of selected F2 plants
Three F2 plants that showed good agronomic performance, in terms of delayed heading time and maintenanceof spikelet fertility, panicle numbers, spikelet number per
panicle, and grain weights, under water stress were identified (Table 2). Heading time delayed by 11-17 days. The
F2 lines maintained 85% or more of their spikelet fertility.
Their panicle numbers were not affected by moderate water stress. Similarly, the spikelet number per panicle wasalmost maintained under moderate water stress (relative
to continuously waterlogged conditions). The three F2lines also maintained 95% or higher grain weight undermoderate water stress. However, two of the three F2 plants
produced 2-fold higher grain weight, which was attributed
to the production of high number of panicles under moderate water stress (Table 2).

Discussion
Flowering in rice is delayed under environmental
stresses, such as under drought stress imposed at different
developmental stages 8, 14–21). In this study, we demonstrated the effectiveness of a mutation gene in causing delay
in heading time of rice under moderate water stress. Past
studies have reported the genes and pathways involved in
the control of flowering in rice based on the photoperiodic
response22–29). Under floral inductive period, exposure to
drought delays flowering through reduction in transcription of primary integrators of day length signals which
include EARLY HEADING DATE 1 (Ehd1), HEADINGDATE 1 (Hd1), Hd3a and RICE FLOWERING LOCUS T 1(RFT1) 30).
The parental cultivar of the mutant line (T6-16), Taichung 65, contains non-functional alleles of both Hd1 andEhd1, and flowers relatively late regardless of the natural
photoperiod26). In this study, the variation of delayed
38 J Intl Cooper Agric Dev 2017

heading time among the mutant-type F2 plants was very
wide under the AWD practice (4-18 days), indicating thata set of genes regulating heading time was very different
between the T6-16 mutant and WAB56-104. Therefore,
there is potential to identify optimal lines for the mutation
gene’s effect by selecting genotypes that have effective
genetic basis for delayed heading time. Indeed, heading
time of the three F2 plants was delayed by 11–17 days, andtheir spikelet fertility and grain weight were minimally or
not affected by moderate water stress. These three plants
have been selected as potential genotypes to be improved
and further tested for use as a strategy for cold stressadaptation to reduce yield losses. Work is underway toevaluate the selected plants’ descendants (F3 lines) during
the long-rains season (in June and July) when a cold spell
is likely to be experienced.
In this study, AWD was applied from 14 DAT as recommended by other researchers31). However, the first cycleof drying was reached late in the vegetative stage of theF2 plants (at 58 DAT) due to the frequent rainfall during
the 2015/16 short-rains season in Mwea, Kenya. Water
stress imposed at this stage resulted in heading delay bya mean of 11 days in mutant-type F2 plants. We speculatethat cumulative moderate water stress from early stagesof plant growth may result to a more prolonged delay inheading of rice with minimal yield losses. As such, there isneed to evaluate the effect of cumulative water stress fromearly vegetative stage to maturity. In addition, there is needto assess the effect of moderate water stress imposed at
specific growth stages on heading delay and yield performance using the advanced generations produced fromthis cross of mutant-type rice. This information would be
helpful for determining the best timing of water management practices as a cold stress adaptation strategy based on
weather forecasts.

Acknowledgements
This work was supported by the Japan Science andTechnology Agency (JST)/Japan International Cooperation Agency (JICA) and the Science and Technology
Research Partnership for Sustainable Development (SATREPS). CMW was also provided with a PhD scholarshipby the Ministry of Education, Culture, Sports, Science andTechnology (MEXT) of Japan.
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.
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Working Paper

東アフリカ高地を対象とした水管理による開花期の制御を通して低温ストレスを回避させうるイネ新栽培技術の開発
コーネリアス ムバティ ワイナイナ 1,2，槇原 大悟 3，鮫島 啓彰 3，菊田 真由実 3，ダニエル マコリ メンゲ 3，ジョン ムンジ キマニ 3,6，犬飼 義明 31) ジョモケニヤッタ農工大学 園芸学部
2) 名古屋大学 大学院生命農学研究科
3) 名古屋大学 農学国際教育協力研究センター
4) ケニア農業畜産研究機関 ムエア・テベレセンター
要約
低温ストレスは、種子稔性の低下を通してイネの収量を大きく低下させる。低温の程度や継続期間は年や季節によって異なるため、最も気温が低下する時期を回避して開花させうる新技術の開発が求められる。一般に、乾燥ストレスは出穂を遅延させる傾向があり、また根が浅く張る浅根性品種は軽微な乾燥ストレスに敏感に反応することが知られている。そこで本研究では、軽微な乾燥ストレス下で出穂期の遅延を示す突然変異体（T6-16）を選抜し、アフリカで有望視されているネリカ品種の反復親の 1 つである WAB56-104 と交配することで、本変異遺伝子の有用性を評価した。F2 個体群のうち、変異体型個体の出穂日は軽微な乾燥ストレス下で平均 11日間遅延する傾向を示した。特に 3 つの F2 個体では、11 〜 17 日間出穂日が遅延したが収量の低下は見られなかった。これらの結果は、浅根性に関わる遺伝子座の利用と軽微な灌水量の制限により、収量の減少を伴うことなく、最も厳しい低温期を回避して開花させうる新技術確立の可能性を示している。

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