Crop Science 43:824-828 (2003)
© 2003 Crop Science Society of America
CROP BREEDING, GENETICS & CYTOLOGY
Genetic Variation and Relationships among Seedling Vigor Traits in Sorghum
N'Diaga Cissea and
Gebisa Ejeta*,b
a The National Center for Agricultural Research, Bambey, Senegal
b Dep. of Agronomy, Purdue Univ., 1150 Lilly Hall of Life Sci., West Lafayette, IN 47907-1150
* Corresponding author (gejeta{at}purdue.edu)
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ABSTRACT
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Seedling vigor in sorghum [Sorghum bicolor (L.) Moench] is important for improving stand establishment of the crop, particularly in arid regions and in areas where low soil temperatures prevail at planting time. This study was conducted to examine the genetic variation, heritability, and relationships among seedling vigor traits in a recombinant inbred (RI) sorghum population developed from two contrasting inbreds. One hundred RI lines and their parents [‘SRN39’, an African caudatum and ‘Shan Qui Red’ (SQR), a Chinese kaoliang] were evaluated for seedling vigor in experiments conducted in the field, in an incubator, and in a greenhouse. Data on 100-kernel weight, visual seedling vigor scores, percentage germination at 12 and 22°C, percentage emergence, seedling height, and shoot dry weight were collected. Genotypic differences were significant for many of these seedling vigor traits. Significant genetic and additive variances were observed for field vigor scores as well as for those traits measured in the more controlled environments. Heritability estimates for visual scores, percentage germination, emergence, and seedling height were high, while those for seedling dry weight were low to medium. Genetic correlation coefficients of visual seedling scores with the different estimates of vigor were significant except for 100-seed weight. Significant genetic interrelationships were revealed among traits measured in the greenhouse and incubator at 22°C. Visual scores taken in field experiments appeared effective in integrating germination, emergence at high temperatures, and shoot dry weight. The significant additive genetic variances obtained indicate that the superior seedling vigor observed in the kaoliang parent, SQR, can be effectively utilized for improving germination in cold temperature, as well as germination, emergence, seedling growth, and development at optimum temperature.
Abbreviations: RI, recombinant inbred • SDWT, seedling dry weight • SHT, seedling height • SQR, Shan Qui Red
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INTRODUCTION
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EARLY VIGOR is considered an essential component of crop plant development under most environmental conditions (Ludlow and Muchow, 1990). In arid environments, crop varieties with early seedling vigor and good stand establishment tend to maximize use of available soil water, resulting in increased dry matter accumulation and improved grain yield. In temperate environments, where low soil temperature and high moisture often prevail at time of planting, early sowing and use of minimum tillage accentuate germination and seedling growth problems (Keim and Gardner, 1984). Poor early vigor has been a limitation to the introduction of maize (Zea mays L.) hybrids based on Corn Belt germplasm in the European Atlantic coast because temperatures are too low in the spring for rapid growth (Revilla et al., 1999). Seedling tolerance to low temperature is enhanced by rapid germination, high percentage germination, and vigorous seedling growth (Keim and Gardner, 1984).
Plant characteristics that are responsible for differences in early seedling vigor among and within plant species have not been fully explored. Simple plant characteristics such as kernel weight, percentage germination, and seedling weight and height have been identified as good indicators of seedling vigor (Acevedo et al., 1991; Regan et al., 1992). Seedling vigor in sorghum has been assessed by direct measurement of seedling dry weight, which was highly correlated to leaf area, leaf number, and plant height (Maiti et al.,1981). Laboratory evaluations have been shown to be useful in estimating germination, and in establishing significant positive correlations between germination in the laboratory and emergence, as well as with seedling vigor in the field (Abdullahi and Vanderlip, 1972; Mendoza-Onofre et al., 1979; Brar and Stewart, 1994).
Our study was prompted by past field observations that certain Chinese kaoliang sorghum lines consistently showed early season seedling vigor superior to that of most sorghum lines and hybrids in our sorghum breeding nurseries. The objectives of this study, therefore, were to determine if these differences were genetic and to establish interrelationships among different estimates of seedling vigor in a RI population derived from the cross between SRN39, an African caudatum, and SQR, a Chinese kaoliang.
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MATERIALS AND METHODS
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Genetic Materials
Recombinant inbred lines were generated from a cross between SRN39, an African caudatum genotype, and SQR, a Chinese kaoliang line. These two parents differ in seedling growth habit with SQR possessing superior seedling vigor under both optimal and cool temperature conditions. One hundred random F2 plants of this cross were selfed and advanced to the F5 generation by the single seed descent method of plant breeding. Selfed seeds from each of these inbred plants were used to grow F5:6 progeny rows in Isabella, Puerto Rico, and several plants within each row were selfed and seed bulked to produce F5:7 seeds for a 2-yr field experiment. Selfed F5:8 seeds obtained in the summer of 1993 were used to evaluate seedling vigor differences among genotypes in the greenhouse and incubator experiments. Weight of 100 kernels from each RI line was recorded before samples were taken for use in the field, greenhouse, and incubator experiments.
Field Experiment
An experiment employing a randomized complete block design with five replications was conducted in 1993 and repeated in 1994 at the Purdue University Agronomy Research Center in West Lafayette, IN, to evaluate differences in seedling vigor among RI lines and their parents. The experiment included 100 RI lines and their parents. Fertilization and other standard cultural practices recommended for sorghum cultivation at this location were used. Single row plots, each 5 m long and with 75 cm between rows, were used. Each plot was drill-seeded on a Chambers silty clay loam soil (fine silty, mixed, mesic Typic Haplaquolls) and hand-thinned to a spacing of six plants per 30 cm. Thinning was accomplished after visual evaluation of seedling vigor was completed. The experiment was planted on 15 May in 1993 and 19 May in 1994 within the range of planting dates considered optimal for sorghum in Indiana. The average minimum and maximum soil temperatures at the 5-cm depth for the 30 d following planting were 15.5 and 27.1°C in 1993, and 12.2 and 25.5°C in 1994.
The entries were visually scored for seedling vigor on a scale of 1 (most vigorous) to 9 (least vigorous). The scores were assigned at the 2- to 3-leaf stage, when seedling vigor differences between entries were observed to be large. This stage corresponded to 29 d after planting for the 1993 experiment and 15 d after planting in 1994. The visual scoring system was a relative evaluation based on the range of variation for seedling size in the population under study. The estimates attempt to visually integrate percentage emergence, seedling height, and the length and width of individual leaves.
Greenhouse Experiment
An experiment was conducted on a sand bench during June–July 1995 to assess genotypic variation for emergence and seedling vigor among RI lines. The experiment included 97 RI lines and the two parents; three RI lines were not included because of inadequate amount of seeds. A randomized complete block design with three replications was employed. Seeds were surface sterilized with 0.6% sodium hypochlorite and rinsed several times with distilled water before seeding. Seeds were planted in single rows, each 30 cm long with 10 cm between rows. Fifty kernels per row were placed at the 2-cm depth and watered daily. Seedlings were grown in the greenhouse under daylight supplemented with fluorescent lamps that provided 295 µmol m-2 s-1 at seedling canopy level for 12 h per day. Temperature was not monitored, but 
20 to 25°C day and night temperatures were maintained. One week after planting, the number of emerged plants per row was recorded and percentage emergence calculated. At the same time, the first seedling height (SHT1) was measured, and each plot was thinned to 20 plants. Seedling height was taken again at 2 and 3 wk after planting and recorded as SHT2 and SHT3, respectively. These measurements were taken as sorghum seedling height from the soil surface to the tip of the uppermost leaf. At 2 and 3 wk after planting, 10 seedlings were harvested, dried at 120°C for 5 d, and shoot dry weights (SDWT1 and SDWT2) were recorded.
Laboratory Experiments
Two experiments with two replications each were conducted in an incubator to evaluate differences in germination among RI lines. The temperature in the incubator was set at 12°C for the first experiment and 22°C for the second; both were at 100% relative humidity. No lighting was provided as seeds were germinated in the dark. Each experiment included 100 RI lines and their two parents. Seed cleaning and sterilization procedures were as described above. Fifty seeds of each entry were placed on a filter paper in a 100- x 15-mm (diameter x height) Petri dish and moistened once every 2 d. One week after the beginning of each experiment, the seeds were placed at -70°C for 48 h to stop all physiological processes. The number of germinated seeds was subsequently recorded as determined by radicle protrusion through the seed coat, in accordance with the Association of Official Seed Analysts (1970) definition of germination.
Data Analysis
Analyses of variance were used to examine differences among RI lines for all traits measured in the field, greenhouse, and incubator experiments. The mean squares for family [MS(fam)] and for the experimental error [MS(error)] were obtained from the ANOVA and used to obtain the genotypic variance. The additive variance was calculated by adjusting for inbreeding using the formula 
2A = 2 - 
t - 1, where t is the generation of evaluation, so that at F7 the genotypic variance contains 31/16 of the additive variance in the F2 generation. Heritability estimates were calculated for the different variables measured in the field, greenhouse, and laboratory experiments. Broad-sense heritability (Hf) on a family mean basis was calculated as follows:
for field traits, and
for greenhouse and incubator experiments. Expected gain from selection was computed as gain per cycle (Gc) according to Fehr (1987):
where k = selection differential (at 10% intensity) expressed in standard units, 2A = additive genetic variance, and ph = square root of the phenotypic variance.
The ANOVA and covariance provided the sum of squares and cross products so that the genetic correlations could be obtained using the formula:
where rg = the genetic correlation between x and y, F(xy) = family covariance component between x and y, 2F
= family variance component for variable x, and 2F
= family variance component for variable y.
An ANOVA between traits measured in the field and those in the controlled environments was calculated assuming that the expected environmental covariance was zero, so that the mean cross product for family, MCP(fam) = 2F
. Standard errors for estimates of genetic correlations were calculated according to the procedure given in Mode and Robinson (1959). Significance at the 
= 0.05 and 0.01 levels for genetic correlations was declared if the coefficient exceeded its standard error by two and three times, respectively.
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RESULTS AND DISCUSSION
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Genetic Variation and Heritability for Vigor Traits
Highly significant differences for visual seedling vigor scores between parents and among RI lines were observed in the field experiments (Table 1). Differences in seedling vigor between the two parents were clear as reflected in their visual scores. Shan Qui Red had a mean of 1.8 in 1993 and 1.0 in 1994, while the corresponding scores for SRN39 were 7.9 and 7.0, respectively (Table 2). Considerable variation for field vigor among RI lines also was observed. In 1993, visual mean scores among RI lines ranged from 1.6 to 8.4, while in 1994 the values were between 1.0 and 9.0. Parental line performance for visual seedling vigor scores was consistent from year to year, while significant year x RI line interaction was observed for this trait (Table 1). The 2-yr field scores were highly correlated (r = 0.64, P = 0.0001), indicating that visual scoring was a reliable method for estimating seedling vigor.
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Table 1. Expected mean squares and ANOVA for seedling vigor scores, combined across years from field experiments in 1993 and 1994.*
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Table 2. Performance of parental and recombinant inbred (RI) lines for seedling vigor traits in field (visual scores), greenhouse (emergence, seedling height, and seedling weight), and laboratory (germination) experiments.
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Significant differences between parents and among RI lines were observed for all traits measured in the greenhouse and laboratory experiments (analysis not presented). Shan Qui Red had higher percentage emergence and its seedlings were taller and heavier than those of SRN39 (Table 2). Significant differences also were detected for 100-seed weight and percentage germination at 12° and 22°C. Higher temperature (22°C) increased germination percentage for the parents as well as for the inbred lines in the RI population. Similar results were obtained by Redona and Mackill (1996) on rice (Oryza sativa L.). Shan Qui Red had higher germination percentage than SRN39 under both low and high temperature conditions.
Significant additive genetic variances were found for visual seedling vigor scores recorded in the field experiments as well as for the different estimates of seedling vigor in the controlled environments (Table 3). Segregation of the F7 RI lines for seedling vigor fit a duplicate dominant inheritance model in 1993 (
2 = 0.053, 0.90 < P < 0.75) and a triplicate dominant model for the 1994 data (2 = 0.023, 0.90 < P < 0.75). The results of the 2-yr study indicated that seedling vigor estimated as visual scores is conditioned by two or three genes in the population. The qualitative nature of this trait in sorghum is consistent with observations on rice where seedling vigor was reported to be controlled by four or five genes (Anonymous, 1979). In maize, Moreno-Gonzalez (1988) reported that for early vigor, additive genetic variance was more important than dominance variance in dent x dent and dent x flint crosses. Heritability estimates for field scores, percentage germination, emergence, and seedling height at 1 and 2 wk were high, while those for seedling height at 3 wk and seedling dry weight were low to medium (Table 3). Comparable heritability estimates were reported in rice for shoot length (Redona and Mackill, 1996) and for visual seedling vigor evaluated in the field and germination at low temperature (Sthapit and Witcombe, 1998).
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Table 3. Genetic, additive, and environmental variances, broad-sense heritability (H) estimates, and predicted genetic gain (Gc) for seedling vigor traits.
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The observed significant additive genetic variances indicate that there is adequate variation to allow improvement for germination under low temperatures as well as for germination, emergence, and overall seedling growth and development at normal temperatures. Expected gain from selection was highest with field vigor scores, germination at 12°C, and percentage emergence (Table 3). It appears that the largest gain will be obtained by direct selection for seedling vigor in the field. The high heritability estimates for germination at 22°C, emergence, and early seedling height (SHT1 and SHT2) indicate that attempts to improve seedling vigor using these traits would be successful. But progress in breeding for improved seedling dry weight is expected to be slow, as suggested by the low to moderate heritability estimates (Table 3).
The germination percentage of SQR at 12°C was high (87%) compared with that of SRN39 (24%). The significant genetic variance and high heritability estimates in the population for percentage germination at the low temperature indicate that SQR may also be a good parental source for improving stand establishment of sorghum in environments where low soil temperatures prevail at planting time. This is corroborated by our repeated observation of the superior early seedling vigor of SQR and other Chinese sorghum lines in nurseries at West Lafayette, IN, where cool temperatures prevail in early spring. Shan Qui Red consistently shows rapid germination and high percentage emergence in both low and high temperatures. Domestication of the sorghum crop began in Africa (Harlan and de Wet, 1972) and the crop later moved over the Indian Ocean and the Arabian Peninsula to India, and eventually was carried over the Himalayas to China (Doggett, 1988). Because the kaoliangs are the only sorghum types that evolved in a temperate environment, we speculate that adaptation in temperate China may have given kaoliangs unique sets of genes for early season seedling vigor and cold tolerance. Carefully designed experiments and selection efforts need to be undertaken to substantiate our suggestion.
Relationships among Vigor Traits
Highly significant genetic correlations were detected between seedling height and dry weight as could be expected (Table 4). Genetic correlation coefficients between emergence and seedling height and weight were relatively small. Similar relationships between percentage emergence, seedling height, and seedling weight were reported in rice (Redona and Mackill, 1996).
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Table 4. Genetic correlation coefficients of field seedling vigor scores with emergence, seedling height, and seedling weight measured in the greenhouse.
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Significant associations were observed between germination at 22°C, percentage emergence, seedling height, shoot dry weight, and field vigor scores (Table 5). Significant genetic correlations also were observed between visual seedling vigor and seedling emergence, height, and weight (Table 4). The negative sign in the genetic correlations with vigor score results from the fact that a score of 1 was designated for the most vigorous phenotypes and a score of 9 represented the nonvigorous lines. Significant correlation between field vigor scores and plant height as well as kernel weight were reported in corn (Revilla et al., 1999), while vigor scores in rice were found to be significantly correlated with percentage germination, plumule vigor, and greening (Sthapit and Witcombe, 1998).
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Table 5. Genetic correlation coefficients of seedling vigor traits measured in the field, greenhouse, and the incubator.
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Germination at 12°C was significantly associated with early shoot dry weight measurement (SDW1), but not with any of the other estimates of seedling vigor recorded in the greenhouse or in the field (Table 5), suggesting that germination at low temperature, perhaps a valuable attribute for cold tolerance, is not necessarily an indicator of seedling vigor per se. Support for this idea is provided by the nonsignificant genetic correlation that was observed between percentage germination at 12 and at 22°C (Table 5). This result suggests that germination under cold temperature is controlled by a different set of genes than those controlling germination and emergence at normal temperatures. These findings support an early report (McConnell and Gardner, 1979) in maize, where a nonsignificant association between seedling vigor in the field and germination at 12°C was observed. The authors found poor correlation between emergence in the laboratory under cold temperature and field seedling vigor due to differences in conditions between the laboratory and the mild spring temperatures in the field during the 2 yr of their evaluation. However, when conditions of low temperature were more similar, germination in an incubator at 17°C and early field vigor were found significantly correlated in rice (Sthapit and Witcombe, 1998).
Kernel weight was not significantly associated with most seedling vigor traits except for percentage emergence (Table 5). This is in agreement with previous observations reported in sorghum (Vanderlip et al., 1973) and wheat, Triticum aestivum L. (Gan and Stobbe, 1996). Radford and Henzell (1990) and Mian and Nafziger (1992) also concluded that kernel weight had little effect on stand establishment of sorghum and wheat, respectively. However, Revilla et al. (1999) found maternal effects for kernel weight on estimates of early field vigor and plant weight in maize, recommending that the parent with larger kernel weight be used as the seed parent in hybrid seed production to improve early vigor.
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CONCLUSIONS
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Early germination, emergence, and stand establishment are important traits in sorghum and other field crops. Significant genetic variation for seedling vigor traits exists in sorghum. Chinese kaoliangs appear to be excellent sources of seedling vigor and stand establishment. In this study, we demonstrated that the vigorous seedling growth observed in the kaoliang line SQR is a heritable trait. Among all traits measured, field visual score had the highest heritability and showed the greatest potential response to selection. The parental line SQR may be a good source of both cold tolerance and overall seedling vigor even at higher temperatures. The significant genetic variances and heritability estimates for many of the vigor traits including germination at low temperature indicate that SQR can be used in improving seedling vigor and stand establishment of sorghum in both low and high temperature conditions. If the superior cold tolerance of kaoliangs is further substantiated, sorghum cultivars that withstand early season cold temperatures can readily be developed using Chinese lines as sources.
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ACKNOWLEDGMENTS
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We thank Terry Lemming for technical assistance with field experiments. This research was funded by USAID Grant DAN 254-G-00-002 through INTSORMIL, the International Sorghum and Millet Collaborative Research Support Program.
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NOTES
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Purdue Agric. Res. Program Journal Paper 16 292.
Received for publication June 9, 2000.
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ABSTRACT
INTRODUCTION
