Crop management and physiology

Screening for low-temperature stress tolerance in boro rice

P. Satya and A. Saha, Department of Genetics and Plant Breeding, Uttar Banga Krishi Viswavidyalaya, Coochbehar 736165 (present address of first author: Crop Improvement Division, Central Research Institute on Jute and Allied Fibres, Nilganj, Barrackpore 700120), West Bengal, India, and N. K. Singh, Rajendra Agricultural University, Pusa, Bihar, India E-mail: pratiksatya123@yahoo.co.in

Keywords: boro rice, cold tolerance, low-temperature germination tolerance, screening method

Low temperature during germination and seedling growth is a common environmental stress for boro (winter-season) rice cultivated in diverse ecologies of eastern India, Bangladesh, and Nepal. Studies reveal that tolerance for lowtemperature stress during germination and subsequent growth is controlled by a complex interaction of multiple traits (Onishi et al 2004, Sthapit and Witcombe 1998). IRRIs Standard evaluation system for rice (SES) provides a qualitative evaluation (based on leaf yellowing) when screening for low-temperature stress tolerance/susceptibility at the seedling stage. Others have attempted screening for low-temperature tolerance based on percent germination or reduction in coleoptile length under stress conditions (Nilanjaya et al 2003, Cruz et al 2006). However, there is no standard method for screening for low-temperature tolerance in boro rice. Because of variability in temperature stress and cultivation practices, boro rice is sown from November to March in different regions. None of the methods developed so far is adequate enough to identify tolerant genotypes under these variable ecologies and growth stages. Here, we propose a method for tolerance screening at the germination and vegetative stages, combining responses during germination and seedling growth. Two experiments were conducted: one under field conditions and the other under controlled temperature conditions. In the field experiment, 17 diverse indica rice genotypes were evaluated at six different growth periods (Table 1) during the boro seasons of 2003-04 and 2004-05 using a randomized complete block design with three replications. In the controlled-temperature experiment, the genotypes were grown in a growth chamber at 28 C (control) and 15 C (low-temperature stress) to confirm the results obtained from the field tests. In the first experiment, six treatments were used (see Table 1). Under E1 and E2, higher mean germination was recorded for Bhog Jira 1 (97%) and Hei

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International Rice Research Notes (0117-4185)

Crop management and physiology

Bao (87%), whereas standard boro genotypes such as IR64 (56%) and BRRI dhan 28 (54%) had low germination during the first week. Under E4, 11 genotypes, including IR64, did not germinate in the first week, confirming that these genotypes are susceptible to low-temperature stress. The germination percentage of these genotypes increased during the third week when stress was less severe (Table 1). In such stress conditions, Bhog Jira 1 successfully tolerated cold stress; it had 82% germination. Moderate tolerance was shown by Hei Bao, Pusa 44, Ajaya, IET17900, and IET17901. Under moderate stress (E5 and E6), these genotypes exhibited higher germination and vigor than IR64 and BRRI dhan 28. We constructed germination indices representing the rate of germination in the first, second, and third week and found that genotype-by-environment (G E) interactions was highly significant for these indices, establishing them as a valuable marker. The mean square values for G E interactions for rate of germination during the first and second week after sowing were 11.72** and 43.63**, respectively. Several researchers have used percent germination as the screening criterion for cold tolerance (Nilanjaya et al 2003, Fujino et al 2004). However, our study indicates that, although percent germination is a valuable morphological marker for low-temperature stress tolerance, its reliability depends on the nature and time of the onset of the stress. This is evident from the fact that 11 genotypes showed more than 50% germination under E1, E2, E5, and E6, on the basis of which some of them may be considered moderately tolerant (Table 1). But some of these genotypes did not germinate under E4 in the first week. It is therefore essential to screen on the basis of seedling vigor, which is indicative of plant height as well as germination. As seedling vigor is calculated using percent germination multiplied by seedling height (Abdul-Baki and Anderson 1973), direct use of vigor as an index might be erroneous as genotypic variability in rice seedling height exists and taller genotypes may be classified as highly vigorous under stress. To standardize, we propose the use of reduction in seedling vigor by a two-way classification based on reduction in germination and seedling height. The results of field experiments indicated sufficient stress on germination as well as on seedling growth (Table 1), for which tolerant genotypes were less affected than susceptible genotypes.

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International Rice Research Notes (0117-4185)

Crop management and physiology

Table 1. Mean performance of six tolerant and 11 susceptible genotypes evaluated for their responses to cold stress at six different growth periods in the field.
Growth environment Extent of lowtemperature stress Moderate Low Severe Severe Moderate Low Mean air temperature during the 1st week after sowing (C) Maximum E1 E2 E3 E4 E5 E6 27.7 26.6 23.9 22.0 27.5 27.2 Minimum 12.8 15.5 9.3 8.1 12.4 14.0 November, 2nd week December, 1st week December, 2nd week December, 3rd week November, 1st week November, 4th week Sowing period Mean germination (%) 7 d after sowing Tolerant (6) 87.337.68 85.503.45 78.8912.08 45.6719.38 78.509.67 87.885.17 Susceptible (11) 58.0914.60 72.3614.25 44.7510.72 0.00 61.697.18 77.063.87 Mean germination (%) 21 d after sowing Tolerant (6) 93.784.72 95.172.56 87.836.65 83.1610.01 94.672.34 94.444.92 Susceptible (11) 83.486.84 84.158.22 64.817.90 60.2710.71 80.615.01 85.612.86 Mean seedling height (cm) at transplanting (45-leaf stage) Tolerant (6) 19.054.79 21.15.49 10.243.46 10.414.36 16.483.99 17.575.95 Susceptible (11) 17.652.32 20.131.87 8.731.12 8.570.85 15.781.25 16.432.95

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Crop management and physiology

Under controlled-temperature conditions, 100 mature seeds of each genotype were surface-sterilized with mercuric chloride (0.1%) and germinated in sterile petri plates at 28 C and in a growth chamber at 15 C. All treatments were replicated three times and observations on percent germination and seedling height after 21 d were recorded. Based on the field and growth chamber observations, two indices were developed for screening: Standardized germination reduction (X1) = [(% germination under control % germination under stress)/% germination under control] 100 Standardized seedling height reduction (X2) = [(seedling height under control seedling height under stress)/seedling height under control] 100 where control and stress refer to the value of the parameter at 28 C and 15 C, respectively. The significant correlation of X1 and X2 (0.76**) suggested that these two indices can be considered reliable parameters in evaluating genotypic differences under cold stress. A comparison of the ranking of genotypes, based on reduction in germination in the controlled growth chamber experiment and field evaluation, shows that the screening method under these two conditions exhibited consistent results for X1 and X2 (Table 2). This implies that screening methods based on X1 and X2 would provide similar results in both field and controlled conditions. Low-temperature stress tolerance mechanisms may be controlled by different genes at the germination and seedling stages as IET17901 and IET17295 exhibited tolerance for cold stress at the germination stage but not during seedling growth. However, other genotypes (Bhog Jira 1 and Hei Bao) exhibited tolerance for lowtemperature stress at both stages. Since breeding for boro rice should target simultaneous improvement in stress tolerance at germination as well as early vegetative growth stages, such materials can be used as donor lines for developing low-temperature-tolerant boro rice. The screening system proposed here would be helpful for rapid screening under field and controlled conditions to identify low-temperature-tolerant breeding materials in rice.

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International Rice Research Notes (0117-4185)

Crop management and physiology

Table 2. Relative ranking of genotypes based on reduction in germination and seedling height in controlled growth chamber experiment and field experiment, 21 d after sowing.
Genotype Description/parentage Standardized germination reduction Ranking in Ranking in field experiment controlled (comparison of E2 and E4) experiment 1 2 2 5 3 3 4 1 5 6 6 8 7 4 8 17 9 10 11 12 13 14 15 16 17 12 11 9 7 14 10 15 13 16 Standardized seedling height reduction Ranking in controlled Ranking in field experiment experiment (comparison of E2 and E4) 2 8 9 1 5 6 15 14 10 3 7 11 12 16 17 4 13 4 5 12 1 2 3 11 16 15 6 7 14 9 17 13 10 8

Bhog Jira 1 IET17900 IET17901 Hei Bao Ajay Pusa 44 IET17295 BR28 Pusa Basmati 1 Pusa Sugandh 4 IET17197 IET17294 Pusa 1302 IET17286 IET17196 Pusa 1412 IR64

Landrace Goram/MW10M//N22M Sneha/RR149-1129 Landrace Variety released in India Variety released in India SYE-1/Suraksha Variety released in Bangladesh Variety released in India Variety released in India Sattari/Jaya IR67469-1-2-M-1-1-1 P1176-91-1-3/KLM 28 IR64/K-3 CR1064-5/Dular P1121/Pusa Basmati 1 IRRI variety

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International Rice Research Notes (0117-4185)

Crop management and physiology

References
Abdul-Baki A, Anderson JD. 1973. Vigor determination in soybean seed by multiple criteria. Crop Sci. 13:630-633. Cruz RP, Milach SCK, Federizzi LC. 2006. Inheritance of rice cold tolerance at the germination stage. Genet. Mol. Biol. 29:314-320. Fujino K, Sekiguchi H, Sato T, Kinuchi H, Nonone Y, Takenuchi Y, Ando T, Lin SY, Yano M. 2004. Mapping of quantitative trait loci controlling low temperature germinability in rice (Oryza sativa L.). Theor. Appl. Genet. 108:794-799. Nilanjaya TR, Singh IRP, Singh NK. 2003. Evaluation of germination, cold tolerance, and seedling vigor of boro rice germplasm. Int. Rice Res. Notes 28:19-20. Onishi K, Oka NI, Adachi M, Sano Y. 2004. Responses to chilling temperature at the early stage of development in rice: geographical clines and genetic bases as revealed by QTL analysis. In: Toriyama K, Heong KL, Hardy B, editors. Rice is life: scientific perspectives for the 21st century. Los Baos (Philippines): International Rice Research Institute. p 460-464. Sthapit RB, Witcombe R. 1998. Inheritance of tolerance to chilling stress in rice during germination and plumule greening. Crop Sci. 38:660-665.

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International Rice Research Notes (0117-4185)