Screening of Pearl Millet F1 Hybrids for Heat Tolerance at Early Seedling Stage

Ten pearl millet genotypes selected on the basis of response to supra-optimal temperature tolerance were crossed in a half-diallel mating system. The 45 F 1 hybrids produced were tested along with parents for heat tolerance and related traits at seedling stage. Field screening and laboratory screening techniques were simultaneously used for the evaluation of F 1 hybrids and their parents. Heat tolerance was measured as seedling thermotolerance index (STI) and seed to seedling thermotolerance index (SSTI) under field conditions, but membrane thermostability (MTS) in the laboratory. The hybrid H77/29-2 × CVJ-2-5-3-1-3 showed highest STI value followed by H77/833-2 × 96AC-93. The genotype H77/833-2 × 96AC-93 had the highest worth for SSTI. These three indices were highly correlated among themselves. STI values were invariably high, whereas SSTI has lower values, as it also covers the effect of under soil mortality (USM). It was seen that the heat tolerance indices STI and SSTI were not showing any perceptible pooled correlation with developmental traits except germination and emergence rate. Based on our results, it could be suggested that membrane thermostability (MTS) may be used for screening large number of genotypes. Field based indices STI and SSTI may be used for evaluation of hybrids and varieties before they are released.


Introduction
Pearl millet [Pennisetum glaucum (L.) R. Br.] is a multipurpose cereal grown for grain, stover, and green fodder.It is the most important staple crop in the semiarid and arid regions of Asia and sub-Saharan Africa.It shall continue to play a prominent role in the integrated agricultural and livestock economy of the country particularly in rainfed areas due to its drought hardiness.Pearl millet provides an excellent nutritious food because of high biological value, more protein (11.6%), fat (5%), and mineral content (2.3%).It also contains adequate amount of essential amino acids such as tryptophan, threonine, arginine, and lysine.Pearl millet is also high in lipids and calcium and low in crude fibre.
Adequate crop stand establishment problems are severe constraints to get good production of pearl millet.Failure to obtain adequate plant populations is often associated with high temperatures occurring during the germination and seedling establishment period of pearl millet.The temperature is one of the key climatic factors and has profound effect on the growth and development of the pearl millet.The soil temperatures in farmers' field in India and Africa commonly exceed 45 ∘ C and the temperatures as high as 60 ∘ C have occasionally been measured [1][2][3].The supraoptimal temperatures are known to be the most important cause of poor crop stands in farmer's field.This can only be managed through developing hybrid varieties which can tolerate high temperature during germination and early seedling stages.Genetic variability for adaptation to high temperature exists in crop plants.Screening of F 1 crosses and identification of the superior cross combinations for seedling heat tolerance is essential for effective manipulation through hybrid breeding.The objective of this study was to understand the response of different cross combinations for heat tolerance during 2 Advances in Agriculture T o l e r a n t I n b r e d Bred at CCSHAU, Hisar, and has profuse tillering, narrow leaves, and typical orange brown anthers identify some promising F 1 hybrids improvement for this trait particularly for commercial cultivation.

Plant Material.
A field screening technique, that is, thermotolerance index [4], was applied to screen the pearl millet F 1 hybrids for heat tolerance at seedling stage.This technique could be used in the hottest months (May-June) only with no rainfall.Since the technique was applicable to the very young seedlings of pearl millet, it was also of concern to test the association of seedling thermotolerance to other phenotypic characteristics of seedlings.The membrane thermostability (based on quantification of electrolyte leakage) was also conducted for screening the pearl millet F 1 hybrids at seedling stage.Ten pearl millet genotypes, namely, H77/833-2, H77/29-2, G73-107, 77/245, CVJ-2-5-3-1-3, 1305, 77/371 × BSECT CP-1, 96AC-93, Togo-II, and 99HS-18, were selected from the germplasm on the basis of different response to supraoptimal temperature tolerance (Table 1) in separate experiment [5].These were crossed in a half-diallel mating system (excluding reciprocals).The 45 F 1 hybrids produced were tested along with parents for heat tolerance and related traits at seedling stage.

Experimental Site and Conditions of Growth.
The experiment was conducted at the research area of Department of Genetics and Plant Breeding, Chaudhary Charan Singh Haryana Agricultural University, Hisar (Lat.: 29 ∘ 10  N, Long.: 75 ∘ 46  E, and 215.2 m above mean sea level), located in subtropical region of Haryana, India.The 45 F 1 hybrids produced were tested along with parents for heat tolerance and related traits in three replicated plots under supraoptimal temperature exposure at seedling stage in two stress environments (environment-1 and environment-2) created through different dates of sowing on May 21 and June 13 (the hottest period in North India), respectively, so as to conduct the experiment under different range of temperatures with no rainfall.A presowing medium light irrigation was done through flooding and the field was precisely levelled before sowing.After sowing no further irrigation was given to the stressed environments (environment-1 and environment-2).One nonstress environment (Table 2) was created through sowing on July 17 (normal sowing period during monsoon).Each genotype was grown in 3 rows each of 3 m length spaced 30 cm apart with 10 cm plant to plant spacing.The seeds were sown manually.The soil of experimental field was sandy loam in texture.The water holding capacity, field capacity, and permanent wilting point of the experimental field were 40, 17, and 6 percent, respectively [6].Irrigation was withheld during the course of experiment; however, drought stress-free conditions were ensured through monitoring at regular interval (on alternate days) by measuring the moisture status of the soil by gravimetric method.The soil samples of about 50 g were taken up to 20 cm depth from the experimental plot.The moisture content on dry weight basis may be calculated [7] using the following formula: ( The heat stress period was terminated by irrigating the experimental field before reaching the soil moisture level at permanent wilting point.A condition of no drought was maintained in order to determine the exclusive effect of temperature at seedling stage.This was maintained by measuring the moisture status of the soil by gravimetric method on alternate days.The soil surface temperature was measured by a soil thermometer at soil surface; temperature at 5 cm above the soil surface and air temperature at 1 m above the soil surface were recorded between 2:00 p.m. and 2:30 p.m. daily (Figure 1).
Emphasis of observation is centred on germination, survival, and mortality of the seedlings.

Plant Characteristics/Screening
Techniques.The emphasis was given to the observations of germination, survival, and mortality of the seedlings.The seedlings were inspected every day in the morning.The seedlings which had died due to Advances in Agriculture heat stress were noted in each genotype on daily basis.Observations continued till the surviving seedlings were established (15 DAE) and there was no further mortality.Moreover, the soil moisture was depleting near to the permanent wilting point and drought stress could also come into play along with heat stress to further continue the experiment.Since experiment proceeded without any stress after the establishment of seedling [4], the pearl millet plants show great resilience and recovery.The main impact of supraoptimal temperature at germination and seedling stage was observed on the seedling survival and resulted in poor plant stand/population of pearl millet.Poor crop stand reduces the productivity (fodder and grain) drastically, whereas after seedling establishment no impact of heat stress at seedling stage was observed on later growth stages.

Thermotolerance Characteristics.
For heat tolerance characteristics 3 indices, namely, seedling thermotolerance index (STI) [4], seed to seedling thermotolerance index (SSTI) [8], and membrane thermostability (MTS), were applied to evaluate the hybrids and parents.STI was calculated as the ratio of number of seedlings survived to the number of seedling emergences, expressed in percentage.Seed to seedling thermotolerance index (SSTI) was calculated as the ratio of seedling survival to the number of seedlings expected to emerge, expressed in percentage [8].The SSTI is an extension of STI by taking expected germination into account.It was necessary to correct the effect of under soil mortality (USM).The loss of germination due to heat (i.e., under soil mortality) can be measured only after a standard germination test.Therefore, the germination of seed from the same lot under monsoon environment (main pearl millet growing season, where no under soil mortality occurred due to heat stress) was taken as expected germination for calculation of SSTI.

Membrane
Thermostability.These genotypes were also screened through laboratory technique (membrane thermostability) [9,10].The young leaves of 21-day-old seedlings of each genotype in three replication plots were used for membrane thermostability test.A middle portion of 10 cm from each leaf was taken and the midrib was removed and two equal halves of the 10 cm each were made.Each half was taken to provide replications.Each leaf portion was cut into pieces and thoroughly washed and rinsed with distilled water two times to remove the electrolytes adhering to the plant tissue, as well as electrolyte released from the cutting of plant tissue, and put them into test tubes.After final rinsing, tubes were drained, maintaining sufficient water to prevent desiccation of plant material during heat treatment.These test tubes were covered with aluminium foil and incubated in water bath at 55 ∘ C for 15 minutes for giving heat treatment.After the heat treatment period, 10 mL of distilled water was added to the test tubes.Then all the test tubes were held at 10 ∘ C overnight to allow diffusion of electrolytes from plant material.Test tubes were brought out of refrigerator and shaken well to mix the contents.An initial electrical conductivity (EC) of both replications of each treatment ( 1 ) was determined with electrical conductivity meter.Then these test tubes were autoclaved at 15 psi for 2 minutes for complete electrolyte leakage from the leaf samples.These test tubes were then cooled at room temperature and contents mixed and final EC was taken for all the treatments ( 2 ).Membrane thermostability (MTS) was calculated according to Ibrahim and Quick [9] and Yadav et al. [10] as follows: where  1 is conductivity reading after heat treatment and  2 is conductivity reading after autoclaving.
Completely randomized design (CRD) for membrane thermostability was conducted to detect variations among the genotypes.

Morphological Characteristics.
The seedling growth characteristics, namely, germination (%), emergence rate (ER), number of leaves/seedling (two-week stage), seedling height (two-week stage), seedling fresh weight (g) (fourweek stage), and seedling dry weight (g) (four-week stage), were recorded.The data for seedling height and number of leaves per seedling was recorded on 10 random seedlings per plot.Emergence rate (an index for speed of germination) can be used as a tool in pearl millet breeding programs for evaluation of seedling vigour.Speed of germination is one of the oldest concepts of seedling vigour.The emergence rate (ER) was calculated by dividing the number of normal seedlings obtained at each counting by the number of days seeds had been in the seed bed (DAS).The values obtained at each count were then summed at the end of the germination test to obtain the emergence rate [11]: After termination of the heat stress on the seedlings by irrigating the field, the experiment was continued to study the effect of seedling heat stress on maturity traits on the survived plants.The maturity traits observed were panicle emergence, effective tillers/plant, plant height, ear length, ear weight/plant, dry fodder yield/plant, grain yield/plant, and total biological yield/plant.

Statistical Analysis.
Analysis of variance for randomized block design (RBD) for field traits, that is, germination, emergence rate, number of leaves/seedling, seedling height, seedling fresh weight, and seedling dry weight, and completely randomized design (CRD) for membrane thermostability test was conducted under each environment on mean basis [12].Analyses were done using the software OPSTAT developed at CCS HAU, Hisar.Correlations among the various characters studied were calculated to establish the nature and magnitude of associations among the traits [8,13] with the software SPSS (SPSS Inc.).Multivariate clustering was done based on paired group cluster method using similarity coefficient under Past 1.40 software [14].Multivariate analysis based on the Euclidean distance matrix was estimated among genotypes to distinguish genotypes from each other.The grouping of genotypes was generated using paired group cluster method.

Results and Discussion
The excessive heat (soil temperature up to 63.0 ∘ C in E 2 ) about 18 ∘ C higher than the normal atmospheric temperature (Figures 1(a) and 1(b)) was responsible for mortality of seedlings.Many seedlings were found desiccated at the point of contact besides the desiccation at the tips.The temperature around the tip level (5 cm above the soil surface) also reached up to 50 ∘ C (Figure 1(c)).It could therefore be concluded that the seedlings had to survive in a really high temperature.The moisture level remained above the permanent wilting point (Figure 1(d)); therefore, drought effect was not in the play.
The heat tolerance was measured as STI (Table 3) through the field screening technique of Peacock et al. [4].Another index known as SSTI was also calculated as an improvement and extension.The analysis of variance (RBD) for 8 characters in 3 environments showed significant genotypic differences.No variability was observed for STI and SSTI in E 3 only.In this environment no seedling mortality was observed and the germination was taken as standard.The values for STI and SSTI were therefore equal and the highest for all the genotypes in this environment and hence no variation was observed.
The hybrids were evaluated on the basis of mean performance for various characters in each environment (Table 3).On the mean basis, STI value ranged from 53.32 to 82.24 with an average of 70.72.The hybrid H77/29-2 × CVJ-2-5-3-1-3 was the best scorer for heat tolerance followed by H77/833-2 × 96AC-93 and (77/371 × BSECT CP-1) × Togo-II.The hybrid H77/833-2 × 1305 was found susceptible to heat stress.Seed to seedling thermotolerance index (SSTI) which is expected to be more rigorous index deviated the values from STI according to variation in germination under normal environment (E 3 ) as the SSTI also covers/includes the under surface mortality (USM) during germination.The reductions in values were not strictly proportional to the STI.The reduction is dependent on thermosensitivity of germination.We also came across such hybrids which showed poor germination and high STI.SSTI values ranged from 46.05 to 72.87 with an average of 60.90.The genotype H77/833-2 × 96AC-93 had the highest worth for SSTI on mean basis followed by G73-107 × (77/371 × BSECT CP-1) and H77/833-2 × (77/371 × BSECT CP-1) (Table 3).The analysis of variance for ten different characters in three different environments (two stressed and one normal) was carried out and the results showed significant genotypic differences for all the characters (Table 4).
A caution is needed for evaluating such genotypes under STI.The SSTI gave a highly reduced value putting these crosses in the susceptible list.It may be concluded therefore that SSTI is more dependable and desirable tool for screening and evaluating the genotypes for heat tolerance.The germination was lower in E 2 than in E 1 .This period has the highest range of temperature with a high average.Genotype-wise, the highest germination (62.09) was recorded for the genotype H77/29-2 × (77/371 × BSECT CP-1) on mean basis, which gives a preliminary indication of resistance to heat, while the genotype 1 × 6 had the lowest germination (35.33).The hybrid H77/833-2 × G73-107 was adjudged as the fastest emergence of seedlings.The genotype (77/371 × BSECT CP-1) × 96AC-93 had the highest fresh weight/seedling.The genotype H77/29-2 × Togo-II had the highest seedling dry weight.E 1 had lower dry weight/seedling than E 2 indicating the more heat stress during the E 1 .

Correlations.
The pooled correlations among the characters were calculated (Table 6) around heat tolerance indices (STI, SSTI, and membrane thermostability) with a view to find whether there was any association of these characters with seedling as well as maturity traits.It was observed that STI and SSTI recorded the highest significant correlation among themselves as they are related indices for heat tolerance.Membrane thermostability also showed high significant correlation with STI as well as SSTI.STI had a significant positive association with germination of seed only among seedling and maturity traits studied.SSTI, in addition, was also positively correlated with germination and emergence rate and negatively associated with number of effective tillers/plant.Membrane thermostability also showed significant positive correlation with seed germination among seedling and maturity traits studied.While evaluating the diallel progenies it was also seen that the heat tolerance indices, STI, SSTI, and membrane thermostability, were not showing any perceptible correlation with the rest of the developmental traits (seedling and maturity traits).The stress period was terminated by irrigating the field experiment after 15 days from date of sowing when the seedlings get established and further mortality stopped due to heat.Moreover, the soil

Sr.
No.   moisture was depleting near to the permanent wilting point and drought stress could also come into play along with heat stress to further continue the experiment.Since experiment proceeded without any stress after the establishment of seedling [4], the pearl millet plants show great resilience and recovery.The main impact of supraoptimal temperature at germination and seedling stage was observed on the seedling survival and resulted in poor plant stand/population of pearl millet.Poor crop stand reduces the productivity (fodder and grain) drastically, whereas after seedling establishment no impact of supraoptimal temperature at seedling stage was observed on later growth stages.Hence, the supraoptimal temperature at early seedling stage in pearl millet could have no impact on its later growth and development stages.The

Figure 1 :
Figure 1: (a) Soil surface temperature, (b) air temperature, (c) temperature 5 cm above the soil surface, and (d) soil moisture status in different environments.
Soil moisture (%) = (Weight of wet soil − Weight of dry soil) Weight of dry soil × 100.

Figure 2 :
Figure 2: Dendrogram representing similarity among pearl millet genotypes (parents and hybrids) based on PAST cluster analysis of Euclidean distance based on phenotypic traits.

Table 1 :
Description of pearl millet genotypes used as parents for hybridization.

Table 3 :
Performance of the parents and the crosses for different characters.

Table 4 :
Analysis of variance for 10 × 10 half-diallel for different characters in three environments.
*Values are transformed values.

Table 6 :
Pooled correlation coefficient among various characters in three environments.