Breeding drought tolerant rice for shallow rainfed ecosystem of eastern India.

In shallow rainfed rice agro-ecosystems, drought stress can occur at any growth stage and can cause a significant yield reduction. During recent years, some rice varieties possessing tolerance of reproductive-stage drought stress have recently been developed. Tolerance of vegetative-stage drought stress is also required to improve rice productivity in drought-prone regions. In this study, we evaluated a set of rice breeding lines for their response to a range of different types of vegetative-stage drought stress in order to propose standardized phenotyping protocols for conducting vegetative-stage drought stress screening trials and also to identify genotypes combining tolerance of vegetative- and reproductive-stage drought stress. A soil water potential threshold of -20 kPa during the vegetative stage was identified as the target for effective selection under vegetative stage with grain yield reduction of about 50% compared to irrigated control trials. Genotypes identified as showing high yield under reproductive-stage drought stress were not necessarily the genotypes showing best performance under vegetative-stage drought stress. Genotypes IR72667-16-1-B-B-3, IR78908-126-B-2-B, and IR79970-B-47-1 showed tolerance of both vegetative-stage and reproductive-stage drought stress. For most, the genotypes that were best under vegetative stage drought or even vegetative stage + reproductive stage drought were different from the genotypes that were best under reproductive stage drought. Based on the cultivar superiority measure, IR69515-6-KKN-4-UBN-4-2-1-1-1 and IR78908-126-B-1-B were the stable genotypes (indicated by low Pi ) under both irrigated control and severe vegetative stress conditions, genotypes IR83614-203-B and IR78908-80-B-3-B were stable under irrigated control conditions and moderate stress, whereas IR72667-16-1-B-B-3 was stable under both moderate and severe vegetative-stage stress conditions.

Introduction

Drought is the most important factor limiting rice productivity in the rainfed rice agro-ecosystem (Huke and Huke, 1997, Pandey and Bhandari, 2009). Climate change is predicted to increase the frequency and severity of drought, which will likely result in increasingly serious constraints to rice production worldwide (Wassmann et al., 2009). Drought can occur at any stage of the rice crop in any year in rainfed areas. Modern rice varieties are highly sensitive to drought stress at seedling, vegetative, and reproductive stages and even mild drought stress can result in a significant yield reduction in rice (O'Toole, 1982, Torres and Henry, 2016). At seedling stage, drought affects crop establishment and seedling survival rates. At vegetative stage, drought reduces leaf formation and tillering, which subsequently reduces the development of panicles per plant, thus causing a yield loss; whereas, at reproductive stage, drought causes a reduction in the number of grains per panicle, increases grain sterility, and reduces grain weight (Pantuwan et al., 2002).

The reproductive stage is recognized as the most critical stage at which drought stress can cause a high yield reduction (Hsiao, 1973), and drought at the vegetative stage was earlier predicted to have a relatively small effect on grain yield in rice (Boonjung and Fukai, 1996). However, it should be noted that these conclusions are based on the effects of drought stress on the rice plant, rather than on which type of drought stress is most frequently occurring in farmers’ fields. Vegetative-stage drought has become a critical factor in reducing rice yield in shallow rainfed environments in recent years because of the late arrival of monsoon rains or long gaps between initial rains (Bunnag and Pongthai, 2013). In recent years, the frequency and intensity of vegetative-stage drought stress have increased in shallow rainfed areas of South and Southeast Asia, particularly in eastern India. Due to less initial rains, farmers fail to accumulate enough water in the field early in the season to prepare land and undertake transplanting. As a result, large areas in shallow rainfed ecosystem are left un-transplanted in years with less initial rainfall. Even when farmers are able to transplant, slow growth, less tillering, and in some cases death of early transplanted seedlings due to vegetative-stage drought stress cause heavy yield losses.

Increased incidence of vegetative-stage drought stress under climate change requires the development of rice varieties that combine tolerance of vegetative-stage and reproductive-stage drought stress in addition to having high yield potential under well-watered conditions. However, the responses to drought at the vegetative stage are different from the responses to drought at the reproductive stage (Kamoshita et al., 2008). Depending on the drought tolerance mechanism, traits that confer tolerance of reproductive-stage drought may not necessarily be effective under vegetative-stage drought stress. Recent research to improve rice yield under reproductive-stage drought stress while maintaining high yield potential (Verulkar et al., 2010, Mandal et al., 2010, Kumar et al., 2008, Kumar et al., 2014) has resulted in the identification of some QTLs that impart increased yield in the reproductive stage (Bernier et al., 2008, Dixit et al., 2015). These efforts have been successful through the development of standardized reproductive stage drought screening protocols that allowed for clear discrimination of drought-tolerant genotypes from drought-susceptible genotypes (Kumar et al., 2008). The timing and severity of drought (mild, moderate, or severe) in relation to the growth stage was appropriately determined so as to capture the genetic variation for reproductive-stage drought tolerance (Kumar et al., 2008).

However, for vegetative-stage drought stress screening, although some information is available on screening protocols (Verulkar and Verma, 2014), guidelines for the severity of drought stress to be imposed have not been very precisely defined. These guidelines are necessary to aid scientists in deciding on the appropriate timing of irrigation for vegetative-stage drought stress screening experiments. The lack of proper screening methods acts as a constraint and often delays the attainment of breeding objectives (De Datta and Seshu, 1982). Developing protocols for effective vegetative-stage drought stress screening in rice will facilitate the identification and development of varieties that are tolerant of vegetative-stage drought. Currently, very few varieties have been characterized to possess high yield potential as well as tolerance to drought at both stages. Selection for yield and yield-attributing traits at the vegetative and reproductive stages in standardized drought screens as well as high yield potential under well-watered conditions may allow breeders to select lines combining tolerance of vegetative- and reproductive-stage drought stress in high-yielding genetic backgrounds.

In the present study, a set of genotypes previously reported to be tolerant of drought at the reproductive stage (Verulkar et. al., 2010; Raman et al., 2012) and some additional genotypes identified to be tolerant to reproductive stage drought were evaluated for tolerance of vegetative-stage drought stress. The genotypes were developed by crossing high-yielding but drought-susceptible rice varieties with drought-tolerant donors and subjected to direct selection for grain yield under reproductive-stage drought and well-watered conditions. The lines were evaluated over three years under different levels of vegetative-stage drought stress with the aim of (i) devising a suitable dry-season vegetative-stage drought stress screening protocol by carefully monitoring soil moisture status over time, (ii) characterizing the degree of yield reduction under moderate and severe vegetative-stage drought stress, and (iii) identifying breeding lines that show tolerance of both vegetative- and reproductive-stage drought stress, in addition to having high yield potential under well-watered conditions.

Materials and methods

Field sites and experimental designs

Vegetative stress screening

Vegetative-stage stress screening trials at the National Rice Research Institute, Cuttack, Odisha, India (20°27′9″N, 85°56′25″E), were conducted during the dry season over three years, from 2008 to 2010. The experimental site had a sandy loam soil. The genotypes tested were advanced breeding lines of 100-120 days’ duration generated using crosses of popular high-yielding rice varieties with a diverse array of donors for drought tolerance developed at IRRI. A total of 210 lines were evaluated (72 in 2008, 75 in 2009, and 63 in 2010) along with popular-varieties IR64, IR36, and MTU1010 used as checks. An alpha-lattice design with three replications was used in all years for each trial. The vegetative stage was considered to begin following the seedling stage (1 month after direct seeding or 10 days after transplanting) and to end when panicle initiation was observed. Two separate vegetative-stage drought trials were planted in 2008 and 2009, which differed in severity due to differences in planting date, time of initiation of drought stress, and/or time of re-watering, and one vegetative-stage drought trial was planted in 2010. A separate irrigated control treatment was also planted in each year of the study in which the water level was maintained at a depth of ∼2 cm through flooding irrigation (Table 1).

Table 1

Trial-wise means and stress categorization of the 2008–2010 vegetative stage drought trials at Cuttack.

Trial	Year	Targeted treatment	Plot size	No. of lines tested	Trial mean yield (kg ha−1)	Heritability	% Yield reduction under stress	Assigned stress level
1	2008	Irrigated control	3.0 m2	72	2634	0.77		Well-watered
2	2008	Moderate	3.0 m2	72	773	0.85	71	Severe
3	2008	Severe	3.0 m2	72	99.6	0.82	96	–
4	2009	Irrigated control	3.0 m2	75	4554	0.55		Well-watered
5	2009	Moderate	3.0 m2	75	2138	0.81	54	Moderate
6	2009	Severe	3.0 m2	75	1035	0.80	77	Severe
7	2010	Irrigated control	3.0 m2	63	3950	0.80		Well-watered
8	2010	Moderate	3.0 m2	63	1630	0.86	59	Moderate

‘-‘ indicates that the trial was not considered for interpretation.

In 2008 and 2009, the trials were manually dry direct seeded into dry soil at a depth of 2–3 cm and a seeding rate of 60 kg ha−1 with 4–5 seeds per hill. This method gave uniform seedling emergence for all plots within 6-8 days. The seeds were dibbled in three rows of 2.5 m each at 20 cm x 15 cm spacing in 4-m-length rows. In 2010, 17-21-day-old seedlings (3–4 plants per hill) were transplanted at a spacing of 20 cm × 15 cm (Cuttack) in 4-m-length rows in puddled bunded fields. Inorganic NPK fertilizer was applied at the rate of 40-20–20 kg ha−1. P and K were applied as a single basal dose at the time of sowing, whereas N was applied in two equal splits, one at the 15-20-day-old seedling stage after the first weeding and the other at maximum tillering stage. Weeds were controlled by two hand weedings per season. Insect and pest control measures were applied when required.

Reproductive stress screening

A total of 215 of advanced breeding lines of 100–120 days’ maturity duration which included popular varieties as checks were tested from 2008 to 2010 at four sites chosen to represent a wide range of drought-prone shallow rainfed lowland rice production environments in India. At all sites, the trials were planted under irrigated control and reproductive-stage drought stress in alpha lattice designs with three replications. The general information on each trial is presented in (Table 2). List of lines evaluated under vegetative stage and reproductive stage drought stress is mentioned in Table 3.

Table 2

Details of reproductive stage drought experiments conducted during 2008–2010.

Location	Seeding date	Plot-size (sq.m)	No.of lines included	Trial mean (kg ha−1)	Targeted stress intensity	Assigned stress level
2008
NDUAT, Faizabad	22-June-08	3.2	75	3183	Irrigated Control	Non-stress
NDUAT, Faizabad	22-July-08	3.2	75	1639	rep. stress	Severe
NDUAT, Faizabad	22-June-08	3.2	75	1342	rainfed severe	Severe
CRURRS,Hazaribag	15-July-08	2.4	75	5194	Irrigated Control	Non-stress
CRURRS,Hazaribag	14-July-08	2.4	75	2234	rep. stress	Moderate
CRURRS,Hazaribag	14-July-08	2.4	75	1605	rainfed severe	Severe
IGKV, Raipur	28-June-08	3.2	75	3929	Irrigated Control	Non-stress
IGKV, Raipur	14-July-08	3.2	75	1009	rep. stress	Severe
IGKV, Raipur	17-June-08	3.2	75	2226	rainfed severe	Moderate
BAU, Ranchi	24-June-08	3.0	75	5268	Irrigated Control	Non-stress
BAU, Ranchi	25-June-08	3.0	75	4688	rep. stress	Non-stress
BAU, Ranchi	27-June-08	3.0	75	3883	rainfed severe	–
2009
NDUAT, Faizabad	20-June-09	4.0	63	3595	Irrigated Control	Non-stress
NDUAT, Faizabad	20-June-09	4.0	63	2499	rep. stress	Moderate
NDUAT, Faizabad	20-June-09	4.0	63	2239	Rainfed severe	Moderate
CRURRS,Hazaribag	03-July-09	3.2	63	4475	Irrigated Control	Non-stress
CRURRS,Hazaribag	03-July-09	3.2	63	2075	rep. stress	–
CRURRS,Hazaribag	03-July-09	3.2	63	2222	Rainfed severe	Moderate
IGKV, Raipur	30-June-09	2.4	63	5258	Irrigated Control	Non-stress
IGKV, Raipur	19-July-09	2.4	63	1895	rep. stress	Severe
IGKV, Raipur	30-June-09	2.4	63	2470	Rainfed severe	Moderate
BAU, Ranchi	27-June-09	2.4	63	3620	Irrigated Control	–
BAU, Ranchi	07-July-09	4.0	63	3170	rep. stress	–
BAU, Ranchi	17-July-09	4.0	63	1836	Rainfed severe	Severe
2010
NDUAT, Faizabad	21-June-2010	4.0	78	3925	Irrigated Control	Non-stress
NDUAT, Faizabad	20-June-2010	4.0	78	3575	rep. stress	–
NDUAT, Faizabad	20-July-2010	4.0	78	583	Rainfed severe	–
CRURRS,Hazaribag	14-July-2010	3.2	78	4326	Irrigated Control	Non-stress
CRURRS,Hazaribag	14-July-2010	2.4	78	3294	rep. stress	–
CRURRS,Hazaribag	14-July-2010	2.4	78	2628	Rainfed severe	–
IGKV, Raipur			78	5610	Irrigated Control	Non-stress
IGKV, Raipur			78	2708	rep. stress	Moderate
IGKV, Raipur			78	2943	Rainfed severe	–
BAU, Ranchi	25-June-2010	2.4	78	3405	Irrigated Control	Non-stress
BAU, Ranchi	01-July-2010	2.4	78	2459	rep. stress	Moderate
BAU, Ranchi	21-July-2010	2.4	78	1478	Rainfed severe	Severe

‘-‘ indicates that the trial was not considered for the combined analysis.

Table 3

Released varieties and breeding lines evaluated for two or more years in the vegetative (Cuttack) and reproductive stage drought trials.

SNO	Entry list (Veg. trials) SET 1	Parentage	Entry list (Rep. stage drought trials) SET 2
1	IR 36	IR1561-228-1-2/IR 1737//CR 94-13	IR 36
2	IR 64	IR5657-33-2-1/IR2061-465-1-5-5	IR 64
3	IR 70215-70-CPA-3-4-1-3	IR57519-PMI 5-B-2-2/IR57514-SRN 299-2-1-1//IR43524-55-1-3-2	IR 70215-70-CPA-3-4-1-3
4	IR 72667-16-1-B-B-3	WS 91/Abhaya//IR43070-UBN 511-2-1-1-1	IR 72667-16-1-B-B-3
5	IR 77298-14-1-2	IR64 (WH)/Aday Sel//3*IR64	IR 77298-14-1-2
6	IR 78875-131-B-1-4-1	IR55423-01 (NSIC RC 9)/IR64	IR 78875-131-B-1-4-1
7	IR 78878-53-2-2-2	IR55423-01 (NSIC RC 9)/IR72875-94-3-3-2	IR 78878-53-2-2-2
8	IR 78908-105-B-2-B	Vandana/IR64	IR 78908-105-B-2-B
9	IR 78908-126-B-2-B	Vandana/IR64	IR 78908-126-B-2-B
10	IR 78908-142-B-3-B	Vandana/IR64	IR 78908-142-B-3-B
11	IR 78908-193-B-3-B	Vandana/IR64	IR 78908-193-B-3-B
12	IR 78908-80-B-3-B	Vandana/IR64	IR 78908-80-B-3-B
13	IR 78937-B-4-B-B-B	IR47701-6-B-1/IR55435-05	IR 78937-B-4-B-B-B
14	IR 79899-B-179-2-3	CT 6510-24-1-2/IR72875-94-3-3-2	IR 79899-B-179-2-3
15	IR 79906-B-5-3-3	IR55419-04/IR55435-05	IR 79906-B-5-3-3
16	IR 79913-B-221-B-2	IR55419-04/Way Rarem	IR 79913-B-221-B-2
17	IR 79913-B-399-B-2	IR55419-04/Way Rarem	IR 79913-B-399-B-2
18	IR 79956-B-60-2-3	UPL RI 7/IR36	IR 79956-B-60-2-3
19	IR 79959-B-217-1-2	UPL RI 7/IR64	IR 79959-B-217-1-2
20	IR 79970-B-47-1	Vandana/UPL RI 7	IR 79970-B-47-1
21	IR 80013-B-141-4-1	IR55423-01 (NSIC RC 9)/CT 6510-24-1-2	IR 80013-B-141-4-1
22	IR 80416-B-152-4	IR70204-32-1-B-2/IR71676-90-2-2	IR 80416-B-152-4
23	IR 83614-203-B	IR78875-131-B-1–2/IR64	IR 83614-203-B
24	IR 83614-281-B	IR78875-131-B-1–2/IR64	IR 83614-281-B
25	IR 83614-315-B	IR78875-131-B-1–2/IR64	IR 83614-315-B
26	IR 83614-349-B	IR78875-131-B-1–2/IR64	IR 83614-349-B
27	IR 83614-394-B	IR78875-131-B-1–2/IR64	IR 83614-394-B
28	IR 83614-438-B	IR78875-131-B-1–2/IR64	IR 83614-438-B
29	IR 83614-46-B	IR78875-131-B-1–2/IR64	IR 83614-46-B
30	IR 83614-513-B	IR78875-131-B-1–2/IR64	IR 83614-513-B
31	IR 83614-564-B	IR78875-131-B-1–2/IR64	IR 83614-564-B
32	IR 83614-61-B	IR78875-131-B-1–2/IR64	IR 83614-61-B
33	IR 83614-673-B	IR78875-131-B-1–2/IR64	IR 83614-673-B
34	MTU 1010		MTU 1010
35	NDR 1119		NDR 1119
36	IR55419-04	IR12979-24-1 (BROWN)/UPL RI 5	IR 79975-B-83-4-3
37	IR69502-6-SRN-3-UBN-2-B-2-2-2	IR57514-PMI 5-B-1–2/IRRI 119//IR43524-55-1-3-2	IR 81039-B-173-U-3-3
38	IR69515-6-KKN-4-UBN-4-2-1-1-1	IR57514-SRN-299-3-2-4/IRRI 119//IR57519-PMI 5-B-2-2	IR 81044-B-112-U-4-2
39	IR70181-26-PMI-1-UBN-1-B-1-1-1	IR43524-55-1-3-2/IR57519-PMI 5-B-2-2//IR43487-11-2-3-2	IR 84887-B-153-CRA-25-1
40	IR70213-10-CPA-4-2-2-2	IR57519-PMI 5-B-2-2/IR52555-UBN 3-2-1//IR43524-55-1-3-2	IR 84894-B-143-CRA-17-1
41	IR74371-70-1-1	IR55419-4*2/Way Rarem	IR 84896-159-CRA-12-1
42	IR78875-123-B-4-B	IR55423-01 (NSIC RC 9)/IR64	IR 84899-B-182-CRA-12-1
43	IR78875-190-B-4-B	IR55423-01 (NSIC RC 9)/IR64	IR 84899-B-183-CRA-19-1
44	IR78875-66-B-3-B	IR55423-01 (NSIC RC 9)/IR64	IR 84900-B-149-CRA-2-1
45	IR78877-181-B-1–2	IR55423-01 (NSIC RC 9)/IR72	LALAT
46	IR78908-16-B-3-B	Vandana/IR64	R-RF-45
47	IR78908-201-B-4-B	Vandana/IR64	R-RF-65
48	IR79913-B-176-B-239-B-4	IR55419-04/Way Rarem
49	IR79966-B2-52-2	Vandana/IR55423-01 (NSIC RC 9)
50	IR80021-B-86-3-4	IR55423-01 (NSIC RC 9)/Way Rarem

Twenty-one-day-old seedlings were transplanted in puddled fields at 20 cm × 20 cm spacing in 4 rows of 4 m length with total plot size of 3.2 m2. Inorganic NPK fertilizer at the rate of 90–60–40 kg ha−1 was applied. P and K were applied as a single basal dose at transplanting, whereas N was applied in three equal splits, at transplanting and at 25 and 50–56 days after transplanting.

In stress trials, N was applied in two splits, at transplanting and at 25 days after transplanting. Weeds were controlled by two hand weeding.

Soil moisture monitoring and management of drought stress treatments

All vegetative drought stress trials were conducted in upper fields that did not retain standing water. Irrigation was initially applied at 3-4-day intervals and the soil in the stress plots was maintained saturated up to 25 days after germination/sowing (2008 and 2009) or 4 weeks after transplanting (2010). The stress treatment was then initiated by stopping irrigation. As the soil dried, gravimetric soil water content was monitored at 5-day intervals through soil sampling at the depth of 15–30 cm at three different locations in each replication. The fresh soil samples were weighed and then oven-dried and re-weighed to calculate the moisture content within 2–3 days of sampling ((fresh sample wt. − dry sample wt.)/dry sample wt. × 100). Soil water potential at a depth of 30 cm was recorded on alternate days through tensiometer tubes (Soil moisture Equipment Co.) placed randomly in five to six locations in the stress plots. In the stress treatments, piezometers constructed from perforated PVC pipes wrapped with coir rope or fabric (2-m length) were installed (two per stress experiment) after sowing/transplanting to monitor water table depth.

In each season, the drought stress treatment was maintained until the plants began to show stress symptoms such as severe leaf rolling and tip drying, which occurred at 15 and 53 days in 2008, at 17 and 56 days in 2009, and at 31, 7, 7, 7, and 7 days in 2010, respectively (Table 4). The stress treatments were re-watered by flooding the field with water when gravimetric soil moisture was about 12% and the soil water potential was about −15 kPa at a depth of 30 cm and correspondingly when the water table was deeper than 100 cm. The total rainfall recorded during the period of stress was 21.8 mm and 10.2 mm in 2008 and 2010 DS, respectively, with no rainfall in 2009 (Fig. 1). Once the vegetative-stage drought stress treatments finished, irrigation was resumed to allow the plants to recover from the stress. The recovered crop was grown under weekly irrigation until maturity.

Table 4

Irrigation schedule across the three dry seasons under moderate and severe vegetative-stage stress.

Year	Stress treatment	Method of establishment	Seeding date	Germination date	Transplanting date	Stress imposition date	DAG/DAP	Re-irrigation during stress treatment
								Date	Days after stress imposition
Cuttack
2008	Moderate	DSR	5.2.2008	10.2.08	–	10.3.2008	28 DAG	26.3.2008	15 (44 DAG)
2008	Severe	DSR	7.2.2008	12.2.08	–	10.3.2008	26 DAG	2.5.2008	53 (79 DAG)
2009	Moderate	DSR	16.1.2009	21.1.09	–	25.2.2009	35 DAG	14.3.2009	17 (52 DAG)
2009	Severe	DSR	16.1.2009	21.1.09	–	23.2.2009	33 DAG	21.4.2009	56 (82 DAG)
2010	Moderate	TPR	24.12.2009	1.1.10	28.1.2010	26.2.2010	29 DAP	29.3.2010	31 (60 DAS)
								5.4.2010	7 (67 DAS)
								12.4.2010	7 (74 DAS)
								19.4.2010	7 (81 DAS)
								26.4.2010	7 (88 DAS)

DAG: date after germination under dry direct-seeded trial, DAS: date after sowing nursery in transplanted trial, DSR: direct-seeded rice, TPR: transplanted puddled rice, DAG: days after germination, DAP: days after transplanting. Establishment time is given in DAG for Cuttack due to the delay in initial irrigation in some trials.

Fig. 1

Climate conditions during the 2008, 2009, and 2010 trials in Cuttack, Odisha, India.

For each drought-stress experiment, an irrigated control (non-stress) trial was planted at each of the locations. In the non-stress experiments, standing water was maintained at each site from transplanting to 10 days before maturity by maintaining the rainwater in the field or by providing supplementary irrigation through a pump as and when required. The reproductive-stage drought-stress experiments (stress, S) were irrigated like the non-stress experiments by keeping standing water up to 28 days after transplanting. Thereafter, the stress fields were drained to allow them to dry and stress to appear. The stress experiments were not provided with any supplemental irrigation after drainage even if the stress was very severe. This has been reflected in the difference in mean yield of stress trials at different sites (Table 2).

Traits measured

In all trials, the number of days to flowering (DTF) was recorded when panicles in 50% of the plants in each plot emerged and flowered. Plant height (PHT) was recorded on the main culm of five representative plants from ground level to the tip of the panicle before harvesting. Grain yield was measured from a whole-plot area of 3.0 m2, and was adjusted to 14% moisture content. Drought scores and recovery observations were taken following the 0–9 scoring method of the Standard Evaluation System (SES) (IRRI, 1996).

Data analysis

In every year of the vegetative as well as the reproductive stage drought trials, the set of genotypes not performing well under drought stress was discarded from the entry list of the subsequent trial and was replaced by new genotypes showing promising performances under drought. Across vegetative stress trials at Cuttack, the genotypes of interest were a subset of 50 genotypes grown in two or three years, of which 17 genotypes (set 1, Table 3) were common across all three years. Of the 215 genotypes tested in reproductive stage stress trials, 47 (set 2, Table 3) were tested in two or more years. 33 entries were common between set 1 and set 2. After harvest, each stress treatment was classified for the observed stress intensity as moderately stressed if the yield reduction compared with that of the irrigated control treatment was 30–65%, and severely drought-stressed if the yield reduction was more than 65%, according to Verulkar et al. (2010). The 2008 vegetative severe stress trial of Cuttack was not used included in the analysis since it showed a mean yield reduction of more than 85% compared with that of the irrigated control (Table 1), as such trials usually show low genetic variability, which prevents reliable differentiation between stress-tolerant and susceptible breeding lines.

Single trial analyses in both vegetative and reproductive drought stress trials were first conducted using a mixed model that considered genotype factor as a fixed effect and replicate and block within replicate effects as random effects. The model iswhere g is the effect of the i genotype, r the effect of the k replicate, b the effect of the l block within the k replicate, and ε is the error. The analysis was run using the MIXED procedure of SAS (Littell et al., 2006).

At each stress level a conventional combined analysis of variance across environments (site-year cross-classification) of the reproductive stage stress trials is based on the following model.where g is the effect of the i genotype, z is the effect of the j environment, zr is the effect of the k replicate of the of the j environment, zrbis the effect of the l block within the k replicate of the j environment, gzis the interaction of the i genotype in the j environment and εis the residual error. All effects except genotypes were set to random.

The cultivar superiority measure, P (Lin and Binns, 1988), based on the year-wise genotype predicted means (1), was used to assess stability. P is defined as the mean square of the distance between genotype i and the genotype with the maximum response in the individual treatment. Cultivars with the lowest P values are considered the most stable. For each stress level (irrigated control and vegetative-stage drought stress), P was obtained by the expressionwhere Pis the superiority of the ith cultivar at stress level S,  is the mean yield of the ith cultivar in the jth year at stress level S, is the maximum mean response obtained among cultivars in the jth year at stress level S, and n is the number of years at stress level S.

Results

Characterization of vegetative-stage drought stress severity

The severity of stress in each trial depended on the planting date in relation to seasonal rainfall, time of initiation of drought stress, rainfall during the stress treatment, soil properties affecting water retention and drainage, and time of re-watering. In the 2008 and 2009, in direct-seeded trials in Cuttack, gravimetric soil moisture (and soil water potential) declined to lower levels in the severe stress treatments (8–9% (−60 kPa)) than in the moderate stress treatments (16–12% (−25 kPa)) due to 38 days later irrigation of the severe stress treatment compared to the moderate stress treatment (Table 4). The water table declined progressively to below the depth of 60 cm for a major part of the stress period in both the moderate stress (80 cm) and severe stress (100 cm) treatments (Fig. 2). In the 2010 transplanted trial, the soil moisture declined to 12% and −14 kPa and the water table reached a depth of 112 cm at 92 DAS (31 days after initiation of the stress treatment; Fig. 2).

Fig. 2

Soil moisture levels in terms of soil water potential and gravimetric water content at depth of 30 cm, and water table depth during the 2008, 2009, and 2010 trials in Cuttack, Odisha, India.

Genotypic variation for grain yield under vegetative-stage drought stress

The individual trials were grouped into well-watered, moderately stressed, and severely stressed classes based on the mean yield reduction compared with that of the irrigated control. The percent yield reduction under drought compared with that of the irrigated control ranged from 96% in the 2008 severe stress treatment to 54% in the 2009 moderate stress treatment (Table 1). The trial-wise mean yields were highest in the 2009 irrigated control treatment (4554 kg ha−1) and lowest in the 2008 severe stress treatment (99.6 kg ha−1). The mean yields were relatively low in 2008 under all stress levels (Fig. 3). Significant differences in grain yield among lines were observed in all treatments in all years.

Fig. 3

Genotypic variation for grain yield in each treatment applied in Cuttack. Genotypes are listed in order of the mean yield across vegetative-stage drought stress trials.

Among lines that were tested in all three years across all stress levels, three distinct groups of genotypes showed indications of good performance (Table 5): (1) IR72667-16-1-B-B-3, IR78908-126-B-1-B, and IR79970-B-47-1 showed the highest yield across different conditions with yield of 2.5–5.1 t ha−1 under well-watered conditions and 0.9–2.3 t ha−1 under moderate or severe drought stress. Furthermore, genotype means under reproductive-stage drought stress of entries among the set of 50 tested across six sites during the same years (Verulkar et. al., 2010; Kumar et. al., 2012; Raman et al., 2012) showed that (1) IR72667-16-1-B-B-3, IR78908-126-B-2-B, and IR79970-B-47-1 did well under severe reproductive-stage stress conditions (1.5–1.7 t ha−1); (2) IR78908-80-B-3-B, IR83614-203-B, IR78908-193-B-3-B, IR83614-349-B, and IR83614-438-B showed high yield under moderate stress (1.8–2.9 t ha−1) and moderately high yield under irrigated control conditions (2.8–5.7 t ha−1); and (3) IR69515-6-KKN-4-UBN-4-2-1-1-1 and IR79966-B2-52-2 were the highest-yielding lines under severe vegetative stress. Of these, IR69515-6-KKN-4-UBN-4-2-1-1-1 also showed high yield under irrigated control conditions. Based on the cultivar superiority measure, stable genotypes (indicated by low P) under both irrigated control conditions and severe vegetative stress were IR69515-6-KKN-4-UBN-4-2-1-1-1 and IR78908-126-B-1-B (Table 6). Genotypes IR83614-203-B and IR78908-80-B-3-B showed low P values under irrigated control conditions and moderate stress, whereas IR72667-16-1-B-B-3 was stable under both moderate and severe vegetative-stage stress conditions (Table 6). Scatter plots (Fig. 4) show that under both moderate stress and severe stress the entries nearly maintained their performances across years with r=0.54** between the moderate stress trials and r=0.69** between severe stress trials respectively which indicates the uniformity in the evaluation of stress tolerance across years.

Table 5

Overall mean of genotypes across environments (location × year) under irrigated control and managed stress conditions under the vegetative stress experiments at Cuttack and reproductive stage stress experiments at four sites during 2008–2010.

ENO	GENOTYPE	Vegetative stress trials − Cuttack 2008–2010							Reproductive stress trials 2008–2010
		Non-stress			Moderate stress		Severe stress		Non-stressa	Moderate stressa	Severe stressa
		Control 2008	Control 2009	Control 2010	Moderate 2009	Moderate 2010	Moderate 2008	Severe 2009
1	IR 36 (CHECK)		3908	4753	2287	1849		1491	4067	2051	987
2	IR 64		4580	3920	2234	1646		686	4381	2219	1400
3	IR 70215-70-CPA-3-4-1-3	2349	5242	4141	1532	963	592	531	4594	2477	1657
4	IR 72667-16-1-B-B-3	3577	4994	3529	2345	2317	687	1987	4778	2642	1693
5	IR 77298-14-1-2	2836	3852	3099	1901	1448	727	687	4182	2125	1262
6	IR 78875-131-B-1-4	2429	4796		2194		633	833	4375	2508	1418
7	IR 78878-53-2-2-2		4561	3389	2012	1273		1064	4316	2452	1617
8	IR 78908-105-B-2-B	2692	4633	3936	1914	1993	718	958	4509	2594	1567
9	IR 78908-126-B-2-B	3678	5097	3937	2154	1719	914	1632	4507	2343	1721
10	IR 78908-142-B-3-B	1979	5063	3705	1946	1458	786	868	4455	2266	1498
11	IR 78908-193-B-3-B		4985	2834	2357	2407		1163	4505	2752	1733
12	IR 78908-80-B-3-B	3064	4454	4507	2322	1824	712	889	4318	2264	1654
13	IR 78937-B-4-B-B-B	3130	4604	3529	1825	942	545	473	5007	2296	1586
14	IR 79899-B-179-2-3	2479	4087	3330	2082	1500	422	452	4488	2323	1608
15	IR 79906-B-5-3-3	2431	4472	3288	1662	1348	589	595	4490	2501	1532
16	IR 79913-B-221-B-2	2300	4573	4076	1597	774	758	732	4257	2404	1635
17	IR 79913-B-399-B-2	2982	4513	3293	2276	1386	742	1058	4302	2527	1444
18	IR 79956-B-60-2-3	1802	4825	3500	2181	646	714	1200	4782	2609	1502
19	IR 79959-B-217-1-2	2371	4029	3898	2077	1205	392	638	4438	2434	1522
20	IR 79970-B-47-1	2495	4131	3831	2809	1543	923	2233	4161	2185	1543
21	IR 80013-B-141-4-1	2396	4559	4460	1668	732	527	817	4891	2573	1537
22	IR 80416-B-152-4		4289	4448	1720	1301		574	4533	2170	1471
23	IR 83614-203-B		5744	4227	2963	1998		1286	4483	2243	1814
24	IR 83614-281-B		4707	3467	1615	1291		678	4529	2382	1573
25	IR 83614-315-B		4650	3732	2140	1761		662	4338	2418	1758
26	IR 83614-349-B		4914	3627	2149	2012		589	4637	2442	1674
27	IR 83614-394-B		6005	3119	1987	1189		1436	4189	2369	1934
28	IR 83614-438-B		4439	3449	2226	1979		847	3814	2276	1581
29	IR 83614-46-B		5071	3771	1664	790		696	4507	2361	1631
30	IR 83614-513-B		4164	3126	2399	1794		1384	4636	2233	1772
31	IR 83614-564-B		3403	3087	2536	1651		1151	4529	2503	1548
32	IR 83614-643-B		5034	4101	1655	1893		503	4714	2300	1687
33	IR 83614-673-B		5003	4259	2888	1214		760	4557	2411	1887
34	MTU 1010 (CHECK)	2658	5456	4627	2075	1628	925	1344	5049	2572	1584
35	NDR 1119		4759	3678	1499	889		448	4484	2310	1734
LSD		901.818	1165.52	812.87	516.679	662.358	233.328	626.204	780.837	576.855	772.775

Indicates a combined analysis across all trials categorized for that stress level.

Table 6

Cultivar superiority measure (P) of genotypes tested across two to three years under irrigated control and vegetative-stage drought stress conditions in Cuttack. Cultivars with the lowest P values are considered the most stable.

No.	Entry	Years of testing	Pi (control)	Pi (mod. stress)	Pi (severe stress)
1	IR36 (check)	2	142	50
2	IR55419-04	2	196
3	IR64 (check)	2	147	65
4	IR69502-6-SRN-3-UBN-2-B-2-2-2	2	171		54
5	IR69515-6-KKN-4-UBN-4-2-1-1-1	2	23		44
6	IR70181-26-PMI-1-UBN-1-B-1-1-1	2	356		75
7	IR70213-10-CPA-4-2-2-2	2	131		89
8	IR70215-70-CPA-3–4-1-3	3	93	167	153
9	IR72667-16-1-B-B-3	3	110	27	30
10	IR74371-70-1-1	2	119
11	IR77298-14-1-2	3	220	97	131
12	IR78875-123-B-4-B	2	112		108
13	IR78875-131-B-1–4	2	80		118
14	IR78875-190-B-4-B	2	141		129
15	IR78875-66-B-3-B	2	95		152
16	IR78877-181-B-1–2	2	137		127
17	IR78908-105-B-2-B	3	113	62	103
18	IR78908-126-B-1-B	3	77	64	44
19	IR78908-142-B-3-B	3	146	94	110
20	IR78908-16-B-3-B	2	75		84
21	IR78908-201-B-4-B	2	190		84
22	IR78908-80-B-3-B	3	79	50	110
23	IR78937-B-4-B-B-B	3	131	147	162
24	IR79899-B-179-2-3	3	196	83	169
25	IR79906-B-5-3-3	3	180	121	145
26	IR79913-B-176-B-239-B-4	2	386		140
27	IR79913-B-221-B-2	3	123	181	125
28	IR79913-B-399-B-2	3	158	82	93
29	IR79956-B-60-2-3	3	181	159	81
30	IR79959-B-217-1-2	3	162	107	149
31	IR79966-B2-52-2	2	173		46
32	IR79970-B-47-1	3	155	54	15
33	IR80013-B-141-4-1	3	99	181	123
34	IR80021-B-86-3-4	2	33		115
35	MTU 1010	3	51	74	64
36	IR78878-53-2-2-2	2	207	105
37	IR78908-193-B-3-B	2	258	24
38	IR80416-B-152-4	2	125	121
39	IR83614-203-B	2	70	25
40	IR83614-281-B	2	187	130
41	IR83614-315-B	2	161	62
42	IR83614-349-B	2	156	48
43	IR83614-394-B	2	190	114
44	IR83614-438-B	2	209	46
45	IR83614-46-B	2	133	175
46	IR83614-513-B	2	274	49
47	IR83614-564-B	2	364	53
48	IR83614-61-B	2	103	85
49	IR83614-673-B	2	91	80
50	NDR 1119	2	160	177

Fig. 4

Scatter plots for grain yield between years each under moderate and severe stress levels.

Discussion

Monitoring severity of drought stress in vegetative stress trials at cuttack

If a variety’s potential for success is to be assessed realistically, the field growing conditions and crop management methods must simulate those in the target area where in the variety is expected to be grown (Mackill et al., 1996). Therefore, in studies related to drought tolerance phenotyping, the focus should be on careful choice of the testing environment, an effective irrigation management and timing, criteria and evaluation of yield reduction under water deficit conditions. In order to identify sources of drought tolerance, it is necessary to develop screening methods that are simple, reproducible, and predictive of cultivar performance in the target environment (Serraj et al., 2011).

Monitoring the gradual development of drought in the field, its severity, and its duration aid in categorizing cultivars for vegetative-stage drought tolerance. The aim of such screens is to reduce the mean yield of drought stress trials by 50–60% compared with that of irrigated control trials, which can amplify the genetic differences between the drought tolerant and the drought susceptible lines for effective selection to be employed (Fischer et al., 2012) and lines that consistently perform well under drought can be identified. In 2008 DS, with the imposition of stress on the 28th day after emergence (DAE), water table depth began to increase steadily. The moderate stress plots were re-watered 15 days after stress imposition (44 DAG) at which the water table depth reached 42 cms. Thereafter, irrigation was provided at intervals of four to ten days. The trial mean yield of the moderate stress trials was reduced by 71% as compared to the irrigated control (Table 1, Fig. 2). In the severe stress trial in 2008, the stress period was further extended. The severe stress plots were re-irrigated 53 days after stress imposition (79 DAG) when water-table depth stretched to 95 cms. Trial mean yield was reduced by 96% as compared to the irrigated control. The stress imposition steadily diminished the soil moisture levels in both moderate and severe stress trials (Fig. 2). Tensiometers were not installed in the stress plots during this season. In 2009 DS, irrigation was suspended on 35 DAG and 33 DAG in the moderate and severe stress trials respectively. With the inception of stress, the soil moisture retention curve indicated a progressive increase in SMT with a progressive decrease in SWC. The moderate stress plots were re-watered 17 days after the application of stress (52 DAG) when the SWC was about 15% and the corresponding SWT about −10 kPA. The water table depth at this stage was 70 cms. Thereafter the trial was irrigated at regular interval of about ten days. The trial mean yield of the moderate stress trials decreased by 54% as compared to control. The severe stress plots were re-watered 56 days after the stress imposition (82 DAG). The mean yield was reduced by 77% as compared to irrigated control. In 2010, the vegetative stage drought experiment was transplanted in place of direct seeded due to frequent initial rain. This provided opportunity to evaluate the performance of lines under two diverse system of cultivation (direct seeded and transplanted) likely to be practiced by farmers in different years depending upon the rainfall pattern in the season. In 2010 DS water deficit was imposed on the 29th day after transplanting (DAP). When the SWC was about 12% and the corresponding SWT about −14 kPA coinciding with a water table depth of 112 cms (60 DAP, 31 days after stress imposition), the stress plots were re-watered. Thereafter the stress plots were irrigated at regular intervals of seven days until harvest. The yield reduction due to induced water stress was about 59%. Therefore, if tensiometers are installed, it is suggested that the field should be irrigated when SWT = −10–15 kPA at a depth of 30 cm and SWC = 12–15% to get a stress severity appropriate to employ selection at vegetative stage.

Across the different severities of vegetative-stage drought stress trials in this study, the 2009 and 2010 moderate stress treatments in Cuttack were closest to the target screening criterion of reducing grain yield by 50% compared with that of the well-watered control. These results suggest that the field conditions and timing of re-watering were optimum in those trials compared with the 2008 severe stress treatment in Cuttack, which were re-watered later and less frequently and reduced yield by more than the target level. The advantage of achieving the targeted level of vegetative-stage drought stress is also evidenced by the identification of significant differences in grain yield among genotypes in the 2008 moderate, 2009 moderate and severe and 2010 moderate stress treatments in Cuttack,. Yield reduction under stress compared to control in these experiments ranged between 54 and 77 percent. We therefore recommend that a vegetative-stage drought stress treatment should be drained as early as possible after seedling establishment or transplanting (about 1 month after direct seeding or 25–30 days after transplanting) and re-watered minimally as needed to maintain the soil water potential above −20 kPa (ideally between −12 to −15 kPa) as monitored at a depth of 30 cm to achieve yield reduction between 50 and 77 percent under stress compared to control to clearly distinguish between drought tolerant and drought susceptible genotypes at vegetative stage drought. This soil water potential threshold is notably less negative than the recommended soil water potential threshold of −65 kPa for reproductive-stage drought stress screening, reflecting the different time frames and sensitivities of rice at the vegetative and reproductive stage due to lesser presence of roots at depth to access moisture from deeper soil. When the target-level stress is reached, the field should be irrigated to measure the recovery from vegetative-stage drought stress. The number of days required for the soil water potential to reach optimum kPa will be different in wet-season screens or in soils with different water retention properties. Strategies to maximize the degree of drought stress include timing of sowing to coincide with low-rainfall seasons, locating the experiment in an upper field, and installing drainage such as canals around the field. Rainfall, evaporative demand, and the soil characteristics of a site also influence the type of vegetative-stage drought stress applied. Apart from the test environment, factors that play a critical role in devising an appropriate irrigation protocol include stage of crop growth, physical characteristics of the soil, availability of water supply and monitoring soil-water relationships. In 2010, stress treatment was puddled transplanted that experienced little or no rainfall during the stress period. The soil at Cuttack dried to a water potential of −14 kPa over 31 days, (Fig. 2). The volumetric water potential at the end of the 2010 stress period when the water potential was −14 kPa can be estimated to be about 20% (12% gravimetric water content × bulk density of 1.7 g cm−3 as reported by Singh et al. (in review).

Environmental characterization and accurate measurements of plant and/or soil water status are critical in any experiment for which one is concerned about understanding the effects of differing water supply (Jones, 2007). Another important physical characteristic of fields used for vegetative-stage drought stress screening is the uniformity of the experimental field, since variation in the moisture-holding capacity of the soil can decrease the accuracy of the screening (Mackill et al., 1996) and complicate phenotyping. Furthermore, the dry season is useful for preliminary vegetative-stage screening of large numbers of genotypes as it is relatively easier to control soil moisture status (De Datta et al., 1988, Lafitte et al., 2006) and the chances of obtaining a reliable drought screen is expected to be higher as compared to the wet-season screening. However, responses among genotypes may differ from those under wet-season drought, which is typically the main cropping season (De Datta and Seshu, 1982). Nevertheless, the dry season may be useful for preliminary vegetative stage screening of large numbers of genotypes (Pantuwan et al., 2004). The selections from dry season screening can be tested for yield and other agronomic performances in succeeding wet seasons. The actual field drought reactions would be observed during a wet season when dry spells of varying degrees of severity occur (De Datta and Seshu, 1982). Farmers require cultivars that not only respond to favorable conditions but also produce an economically safe yield under drought conditions. Therefore, breeding lines need to be screened under both stress and non-stress conditions. Differences in climatic conditions between seasons can therefore be monitored and the performance of selections from dry-season screening can be validated in succeeding wet seasons if needed.

The evaluation of yield stability by the level of stress can be useful for targeting selection to regions with either a narrow range of stress severities or a high degree of variability in the level of stress across seasons. Previous studies have used P for assessing varietal stability and cultivar evaluation (Aremu et al., 2007, Silva et al., 2008, Bastos et al., 2009, Koppel and Ingver, 2010, Fikere et al., 2014). In this study, the identification of genotypes performing well under vegetative-stage drought stress was conducted in reference to their previous performance under reproductive-stage drought stress. Interestingly, some genotypes in this study that had previously been well-documented as high-yielding under reproductive-stage drought stress did not consistently stand out for grain yield under vegetative-stage drought stress. IR74371-70-1-1 and IR55419-04 were earlier identified to be reasonably good yielding and stable under both irrigated (4 and 5 t ha−1, respectively) and reproductive-stage drought stress (2.0–2.2 t ha−1; Verulkar et al., 2010, Kumar et al., 2012a, Kumar et al., 2012b, Raman et al., 2012), but showed only moderate performance under vegetative-stage drought (0.8–1 t ha−1). In comparison, IR72667-16-1-B-B-3, IR78908-126-B-1-B, and IR79970-B-47-1 performed well under both vegetative- and reproductive-stage drought stress, and were the most stable yielding under both moderate and severe vegetative-stage stress. These results indicate the involvement of the different plant traits responsible for resistance at the two stages and the distinct response of rice genotypes to vegetative-stage and reproductive-stage drought stress, but also suggest that some genotypes can show tolerance to both stages of drought stress.

Despite being highly susceptible even to moderate reproductive-stage drought stress, IR36 and IR64 showed good tolerance and yielded 1.6–2.2 t ha−1 under moderate vegetative-stage stress in this study. IR64 and IR36 are high-yielding lowland varieties popular in many rainfed areas of South Asia and Southeast Asia that have good yield potential under favorable conditions and preferred quality characteristics. Although developed for irrigated ecosystem, tolerance to mild to moderate vegetative stage stress could be possible reason for such varieties to become popular in rainfed drought prone ecosystems. However, due to susceptibility to reproductive stage drought stress, they need replacement (Atlin et al., 2008) and the varieties combining tolerance to vegetative and reproductive stage drought stress could help achieve providing appropriate varieties for cultivation to farmers of rained ecosystems. Since the selected lines from the present study showed yield similar to that of IR36 and IR64 under irrigated non-stress as well as possessed tolerance to both vegetative and reproductive stage drought tolerance, they may be appropriate candidate varieties targets as genotypes to replace IR36 and IR64. Among the lines found promising in the present study, IR 72667-16-1-B-B-3 has been released as a drought tolerant variety for rainfed lowland ecosystem in the Philippines.

Conclusions

For vegetative-stage drought screening in the dry season, maintaining the soil water potential above −20 kPa as monitored at 30-cm depth is recommended to be able to clearly distinguish between tolerant and susceptible genotypes at the time of harvest. This study identified some genotypes that were susceptible to drought in reproductive-stage drought stress but showed tolerance of vegetative-stage drought stress, indicating the distinct response of rice to drought stress at these two growth stages. Combining tolerance of vegetative-stage drought with tolerance of reproductive-stage drought could be accomplished by performing separate vegetative- and reproductive-stage drought stress screening trials, which in this study resulted in the identification of genotypes IR72667-16-1-B-B-3, IR78908-126-B-2-B, and IR79970-B-47-1 as tolerant of both stages of drought stress. Alternatively, tolerance of both vegetative-stage and reproductive-stage drought stress could be accomplished by crossing donor lines, one of which is tolerant of vegetative-stage drought stress and one of which is tolerant of reproductive-stage drought stress. The development of improved varieties with combined tolerance of drought stress at multiple growth stages will help farmers in rainfed rice-growing regions maintain stable yields across increasingly unpredictable climatic conditions