In lentil, flowering is known to be very sensitive to changes in external environment especially in temperature and photoperiod. Therefore, heat stress at reproductive stage causes heavy loss in grain yield of lentil (Summerfield et al., 1985). Thus, heat tolerant cultivars can provide not only an opportunity of horizontal expansion of lentil cultivation in rice-fallow lands but also can help to increase lentil productivity by minimizing the yield losses occurring due to forced maturity. It can be visualized that the increases in temperature will have more adverse effects on cool-season crops (e.g. lentil) than the rainy-season crops (Kumar, 2006). Therefore, identification of heat tolerant genotypes in available germplasm and their utilization can help to tackle situation of terminal heat stress through the development of heat tolerant cultivars. Earlier, efforts have been made in other cool-season legume like chickpea to identify the heat tolerant genotypes under field conditions (Dua, 2001; Krishnamurthy et al., 2011).

 

Yet, it is still not clear how heat affects the growth and development of lentil and whether that can explain part of the differences in seed yield under heat stress. Therefore, it is an urgent need to identify the traits that can be used effectively in field conditions for screening germplasm and breeding materials at reproductive stage. Keeping this in view, the present study aimed (i) to establish an effective screening technique under the field condition by identifying the morphological traits related to heat tolerance and yield and (ii) to validate identified heat tolerant genotypes using laboratory test.

 

The procedure used to screen the heat tolerant genotypes is presented in Figure 1. In the present study, out of 334 accessions, 174 accessions flowered early and matured within 80-85 days after sowing. These accessions escaped the heat stress and thus were excluded for further analysis. Another 64 accessions which did not flower or flowered rarely were considered as highly sensitive to heat. The remaining 96 accessions whose flowering and podding stage coincided with high temperature and still flowered were observed for the number of filled pods/plant, number of unfilled pods/plant, and number of filled and unfilled pods on terminal branch of individual plants. These accessions flowered in 56 to 85 days (between March 15 and April 30, 2012) when the maximum day temperature varied between 30.6 and 43 ºC (Figure 2). As a result, development of pods at maximum day temperature (>35 ºC) was used as a criterion to classify accessions as tolerant or sensitive to heat. Thirty seven (37) accessions, which podded normally and showed pod formation on terminal branch, were classified as heat tolerant while remaining 59 accessions that flowered but podded rarely were classified as sensitive to higher temperature. The mean, range and standard error of mean (s.e.m) over two years of these 37 accessions were calculated, which is presented in Table 1. Among 37 accessions, number of filled pods/plant ranged from 3.3 to 51.2 with an average of 23.6 while unfilled pods ranged from 5.5 to 45.0 with an average of 24.2. On terminal branch, filled pods were varied from 1.5 to 10.0 with an average of 3.3 and unfilled pods were varied from 0.0 to 3.8 with an average of 1.3. The 100-seed weight in these 37 accessions ranged from 1.1 to 2.5 g with an average of 1.3 g. The combined analysis of variance over the years indicated significant genotypic variability for filled and unfilled pods/plant, filled pods on terminal branch and also for 100-seed weight (100-SW). Heritability estimates ranged from 8.94 to 58.13% (Table 2). Highest heritability (58.13%) was observed for filled pods/plant on terminal branch and it was lowest (8.94%) for unfilled pods/plant.

 

The results indicated significant year effects on filled and unfilled pods/plant and hence wide variation in s.e.m over years. It was ranged from 0.2 to 37.8 for filled pods/plant and 0.0 to 16.5 for unfilled pods. Therefore, stable genotypes were identified with lower s. e. m. over the years along with highest pod formation per plant and on terminal branch.

 

In the present study, eight genotypes FLIP2009-55L (43.7) IG2507 (43.7), IG3327 (43.3), IG 3330 (45.5), IG 3546 (47.2), IG 3745 (51.2), IG 4258 (47.7) and IG 5146 (47.7) had significantly large number of filled pods per plant at higher temperature (>35 ºC). Also, genotypes, namely, IG2507 (7.0), IG 3984 (8.0), FLIP2009-55L (8.3), and IG4258 (10.0) had significantly more number of filled pods on terminal branch. However, significantly large numbers of unfilled pods were observed in IG3330 (37.0), IG 3364 (45.0), IG3546 (28.5), IG 4242 (29.5) and   IG 4258 (30.5). Based on these observations, only three genotypes, FLIP2009-55L, IG2507 and IG4258 were identified as heat tolerance genotypes because these three genotypes had significantly more number of filled pods per plant as well as on terminal branch of each individual plant. These genotypes also had significantly less number of unfilled pods/plant except on IG4258 (Table 3). In order to see the impact of late-sown conditions on seed size, data were recorded on 100-SW under late- and normal-sown conditions. The percent reduction in seed size observed under late-sown condition is presented in Figure 3. It ranged from 2.4 % to 67.2%. This was lowest in IG 4242 (2.4%) and highest in ILL 6002 (67.2%). However, three heat tolerant genotypes FLIP2009-55L, IG2507 and IG4258 had reduction in seed size 5.7%, 26.2% and 28.3%, respectively (Table 2). The pollen viability was used as physiological trait and tested in laboratory by collecting pollens at higher temperature (>35 ºC). The pollen viability of FLIP2009-55L, IG2507 and IG4258 was 63.2%, 72.4% and 60.9%, respectively (Table 2). Pollen viability showed significant positive correlation with filled pods/plant (r=0.79).

In India, temperature fluctuation during the grain filling period causes drastic yield losses in cool-season legumes. In chickpea, grain yields is estimated to reduce by 53-301 kg/ha if mean temperature rises 1 ºC (Kalra et al., 2008). Photoperiod and temperature are the major factors affecting flowering initiation in crop plants. Pulses are particularly sensitive to heat at flowering and pod development stages. If the crop encounters a few days of exposure to high temperatures (30-35°C) at these stages, heavy yield losses are reported due to flower drop and pod abortion (Summerfield et al., 1985; Sarker et al., 1999; Roberts et al., 1986; Gopesh et al., 2013). However, this sensitivity varies from genotype to genotype. The temperature during the reproductive stage in both years of experimentation was above the threshold level (>30°C; Figure 2), which suggested suitable conditions for identification of heat tolerant genotypes in lentil. Similar environmental conditions have also been used earlier to screen heat tolerant genotypes in chickpea (Krishnamurthy et al., 2011). Based on flowering and podding under higher temperature, in the present study, lentil genotypes were clearly categorized into three main groups, (i) early flowering, (ii) no flowering or rarely flowering and (iii) normal flowering and pod setting. In the present study 174 genotypes flowered early and matured within 80-85 days after sowing. Because these genotypes escaped from high temperature conditions and hence excluded from screening studies conducted further in next year. Earlier studies showed that heat stress delays flowering and accelerates maturity (Krishnamurthy et al., 2011) and hence probably due to this, 64 accessions in the present study did not flower or flowered rarely. The degree of tolerance was studied among 37 genotypes, which had filled and unfilled pods on individual plant as well as on terminal branch. The combined analysis of variance showed significant genetic variability for heat tolerance among these genotypes for filled pods/plant, unfilled pods/plant, filled and unfilled pods on terminal branch, 50% flowering and 100-SW. Similarly, genetic variation for heat tolerance among chickpea genotypes was reported (Krishnamurthy et al., 2011).

 

Heritability determines the proportion of parental characters that is inherited to their off-springs and hence it is an important parameter to study the inheritance of quantitative characters (Allard, 1960). A trait with high heritability suggests maximum genetic gain in response of selection and can be used reliably for screening the tolerant genotypes under heat stress conditions. In the present investigation, we observed high heritability for filled pods/plant and filled pods on terminal branch. Therefore, these traits could be useful to select tolerant genotypes at higher temperature. Lucas et al. (2012) also used number of pods per peduncle for identification of heat tolerance in recombinant inbred lines population of cowpea. In the present study, three genotypes, IG 3745, IG 4258 and IG 5146 were identified as heat tolerant because these accessions had significantly more pods per plant (>43 pods/plant) at higher temperature. On the basis of the number of pods on the terminal branch at higher temperature, FLIP2009-55L, IG2507 and IG4258 showed more number of pods on terminal branch and thus highly heat tolerant. Though another genotype (IG-3984) also showed more pod formation on terminal branch, it was poor in total number of effective pods per plant. We observed significantly higher unfilled pods for some genotypes, indicating the impacts of high temperature on pod formation. In legumes high temperature during anthesis reduces seed set due to impaired pollen tube growth and fertilization (Gross and Kigel, 1994).

 

The present study showed instability in performance of filled pods/plant over the years in most of the genotypes. The combined analysis of variation for filled pods/plant reflected that a large proportion of total phenotypic variance was due to environmental factors. In pulses, environment and genotype × environment interactions contribute >70% of total phenotypic variance as reported earlier (Kumar and Ali, 2006). However, two genotypes (i.e. FLIP2009-55L and IG2507) that classified as highly tolerant to heat showed stable performance over the years as reflected by low s.e.m. These genotypes can be used in lentil breeding program for developing improved cultivars having tolerance to terminal heat.

 

In the present investigation, impacts of high temperature were also observed on seed size, which varied from 2.4% to 67.2% over the normal sown conditions. However heat tolerant genotypes showed 5.7% to 28.3% reduction in seed size which is comparatively lower than other genotypes due to efficient accumulation of photosynthesis in seeds during grain filling at higher temperature. Though a few days of exposure to high temperatures (30–35 °C) during seed filling accelerates senescence, diminish seed set and seed weight, and reduce yield in pluses (Siddique et al., 1999), different genotypes within a species have different capabilities in coping with the heat stress (Wahid et al., 2007). 

 

High temperature leads pollen sterility (Saini et al., 1984) and hence seed yield depends on the temperature during pollen development (Ploeg Van der and Heuvelink, 2005). In the present study, high pollen viability (60-70%) was observed for two heat tolerant genotypes (FLIP2009-55L and IG2507) and showed highly positive correlation with number of pods per plant. Our results suggest that pollen viability test could be used in laboratory for identification of heat tolerant genotypes in lentil. The impact of heat stress on pollen viability has already been demonstrated in several legume crops including chickpea, common bean, groundnut, and soybean (Prasad et al., 1999; Porch and Jahn, 2001; Devasirvatham et al., 2012; Djanaguirama et al., 2013).

 

The present investigation shows that heat stress significantly affects number of flowers, pods, and seeds. Therefore, filled and unfilled pods on a single plant basis and on the terminal branch are important traits for phenotyping heat tolerance under field conditions. Further, the pollen viability is a useful trait for identification of heat tolerant genotype in lentil. Our results clearly demonstrated that significant genetic variability exits for these morphological traits in cultivated gene-pool of lentil. These genotypes can be considered as potential genetic resources to be used in lentil breeding program for the development of heat tolerant cultivars.

 

The present study included 334 lentil genotypes representing local and exotic germplasm originating from drought-prone areas, elite breeding lines from national and international programs and improved cultivars released in India. Breeding lines used in this study were developed at the Indian Institute of Pulses Research (IIPR), Kanpur, India. These lines are derived from crosses involving parents adapted to terminal heat-prone environments. These accessions were evaluated in 2011-12 and 160 accessions (out of above 334 accessions) that faced high temperature (>35 ºC) during reproductive stage were again screened for heat tolerance in 2012-13 (Table 4).

Field experiments involving a set of 334 and 160 accessions were conducted on 15 January 2011-12 and 2012-13, respectively at Main Research Farm of Indian Institute of Pulses Research, Kanpur (268270N, 808140E; 152.4 m above sea level). These experimental materials were evaluated both years in augmented design. Planting of these accessions was delayed 60-70 days from normal planting of lentil during the winter season in order to synchronize high temperature at reproductive stage. Planting was done on ridges and the plot size was a single row of 3 m length with 30 cm spacing between rows and 5 cm between plants within the rows. To avoid possible drought effects, sufficient moisture in soil was maintained by applying regular irrigation. Other standard agronomic practices were also followed in order to raise a good crop.

 

Data was collected weekly on temperatures [(ºC), maximum and minimum] and precipitation (mm) during the growth period of crop from the agrometeorological observatory of the Indian Institute Pulses, Research, Kanpur.

 

Delayed sowing exposed plants to higher temperature at reproductive phase under irrigated conditions in field. Ten plants were selected randomly from each single row plot in order to take observation on individual plant. Observations were recorded visually on formation of flowers and quantitatively on filled and unfilled pods per plant. Moreover the top 7-8 cm terminal branch of each individual plant was quantitatively recorded for number of filled and unfilled pods under high temperature (>35^oC). The post-harvest data was recorded on 100-seed weight (g) for each genotype. Based on these traits, present germplasm accessions were characterized into (i) sensitive (i.e. plants with flowers but no or rare pods), (ii) highly sensitive (i.e. plants with no or rare flowers and pods), (iii) tolerant (i.e. plants with filled pods but rare/no pods on terminal branch) and (iv) highly tolerant (i.e. individual plans and terminal branch with normal podding) categories (Figure 4).  

Pollen viability of the fresh pollens was studied in those accessions which showed tolerance on the basis of morphological characters. It was determined by acetocarmine technique (Robert, 1977) and those pollen grains stained deeply and looking normal were counted as viable and weakly stained were counted as non-viable (Pearsonand Harney, 1984). For each genotype, 2000 pollen grains were recorded by counting 200 pollen grains per slide. The pollen viability (%) was calculated using the following formula.

Pollen viability (%) = (Number of stained pollen / Total number pollen counted) × 100

 

Analyses of variance (ANOVA) were carried out for year-wise data on various traits using the statistical analysis tool of GENSTAT 14th edition (Payne et al., 2011). A combined analysis of variation over years was carried out for partitioning the phenotypic variance into year, genotype, and error variances. Phenotypic correlations were calculated using the mean values over years (SAS Institute, 2007). The critical difference (CD) at 5% significance level was calculated by using following formula.

CD= √2 MSe/no. of years × t_5% for error degree of freedom

Where, MSe is the error means of square.

 

Thanks are due to DAC, Government of India, New Delhi and to CGIAR program on Grain legumes for providing financial support to carry out this study.

 

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