HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Free Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in ISI Web of Science Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (6) Citing Articles via Google Scholar Google Scholar Articles by Klos, K. L.E. Articles by Brummer, E.C. Search for Related Content PubMed Articles by Klos, K. L.E. Articles by Brummer, E.C. Agricola Articles by Klos, K. L.E. Articles by Brummer, E.C.

CROP BREEDING, GENETICS & CYTOLOGY

Response of Six Alfalfa Populations to Selection under Laboratory Conditions for Germination and Seedling Vigor at Low Temperatures

Dep. of Agronomy, Iowa State Univ., Ames, IA 50011 USA

brummer{at}iastate.edu

	ABSTRACT

TOP NOTES ABSTRACT INTRODUCTION Materials and methods Results and discussion REFERENCES

 
Alfalfa (Medicago sativa L.) seeded in early spring often germinates and emerges under cold temperatures. We conducted this study to determine if phenotypic recurrent selection improved germination time and seedling growth under cool temperatures in the laboratory. Four selection methods were conducted in the laboratory for two cycles within six commercial alfalfa cultivars: 5454, Alfagraze, Amerigraze 401+Z, Innovator +Z, Magnum IV, and WL252HQ ranging in fall dormancy class from two to four. Cycles 0, 1, and 2 were evaluated in controlled environmental chambers. Two cycles of selection for rapid germination at 5°C decreased germination time (GT) by 29%. Response to selection was greatest in the first cycle. Two cycles of selection for high seedling vigor increased seedling height (SH) after 45 d at 10°C by 15%. In some cultivars, selection for high seedling vigor was effective at increasing SH in the laboratory and further increases may be expected. Selection for high seedling vigor increased GT, indicating an association between these traits under laboratory conditions. The mean realized heritability estimates were 0.49 for GT and 0.18 for SH. Selection for both early germination and high seedling vigor resulted in an average decrease in GT of 29% with no effect on SH after two cycles, but combined selection against both traits increased GT by 162% and decreased SH by 18%. Laboratory selection for decreased GT and increased SH under low temperatures can be successful, but the amount of improvement is population dependant.

Abbreviations: EG, early germination • HSV, high seedling vigor • LG, late germination • LSV, low seedling vigor • GT, germination time • SH, seedling height

	INTRODUCTION

TOP NOTES ABSTRACT INTRODUCTION Materials and methods Results and discussion REFERENCES

 
THE MOST COMMON SEASON for seeding alfalfa in the midwestern USA is late March through early May because moisture is abundant and forage can be harvested during the establishment year (Vough et al., 1995; Barnes and Sheaffer, 1995). However, soil temperatures during spring are commonly below 10°C and seeding into cold, wet soils can result in poor germination and weak stands (Vough et al., 1995). Alfalfa populations tolerant of cool temperatures during germination and the early stages of growth may be useful for establishing acceptable stands (Volenec and Nelson, 1995).

Germination of alfalfa, one of the most cold resistant forage legume species, can occur at 0 to 1°C (Arakeri and Schmid, 1949; Tysdal and Pieters, 1931; Coffman, 1923), but germination rate and percentage is optimum between 15 and 25°C, as determined on the basis of an evaluation of two intermediate dormancy alfalfa cultivars (Brar et al., 1991). Variation among cultivars for germination time (GT) and root growth response has been reported across a temperature range of 5 to 30°C (Klos, 1999). Among alfalfa cultivars we have studied, some fall dormant cultivars were found to germinate more slowly at 5°C than those with less dormancy; and germination rate had a weak negative correlation with seed mass (Klos, 1999). Differences among cultivars in initial radicle growth rate at 5 to 15°C were not associated with fall dormancy rating (Klos, 1999).

Correlations of field and laboratory measurements of germination responses at cold temperatures have been examined in several leguminous crop species. In alfalfa, rapid germination and radicle growth at suboptimal temperatures in the laboratory was positively correlated with better field emergence at one of two locations evaluated (Klos, 1999). In soybean [Glycine max (L.) Merr.] and common bean (Phaseolus vulgaris L.), the germination rate under low temperatures in the laboratory provided a good test for tolerance to cool field conditions (Dickson and Boettger, 1984; Saminy et al., 1987; Szyrmer and Szczepanska, 1981).

Progress using recurrent selection for improved germination at cold temperatures has been reported in maize, Zea mays L. (McConnell and Gardner, 1979), and cucumber, Cucumis sativus L. (Staub et al., 1988). The narrow-sense heritability of days to germination in cucumber was estimated to be between 0.15 and 0.20 when measured at 15°C using parent-progeny regression and 0.61 at 17°C using half-sib family means, a difference attributed to the evaluation temperature (Wehner, 1982, 1984).

The objectives of this study were to assess the progress realized from phenotypic recurrent selection in six alfalfa populations for GT and seedling growth at low temperatures under laboratory conditions, to evaluate changes in seed mass with selection for EG and SH, and to estimate the realized heritability of GT and SH in these cultivars.

	Materials and methods

TOP NOTES ABSTRACT INTRODUCTION Materials and methods Results and discussion REFERENCES

 
Selection Methods
The populations used for recurrent selection in this study represented commonly available cultivars in Fall Dormancy Groups 2 through 4 (Table 1) . Seed was obtained either from commercial sources or from Dr. T.A. Campbell, USDA-ARS. Two to three cycles of selection using the following four methods were conducted on each population.

High Seedling Vigor (HSV)
Seedling vigor was evaluated on the basis of seedling height (SH) after growth at 10°C. Six hundred scarified seeds were germinated in the greenhouse in 196 cavity trays (Model JT 196; Hummert Int'l., Earth City, MO) in equal parts vermiculite and topsoil, two seeds per cavity. Three to 4 d after planting, flats were randomly thinned to one plant per cavity and transferred to a growth chamber at 10°C with 20 hr light. Seedling height was recorded 45 d after planting, and the tallest 5% of the seedlings were selected . Selection between seedlings of equal height was made visually for greater leaf number and thicker stem diameter.

Early Germination and High Seedling Vigor (EG+HSV)
Three hundred scarified seeds were germinated at 5°C, as described for EG above. The earliest 30 seeds to germinate were transplanted into 196 cavity trays with one seedling per cavity. Flats were placed in a growth chamber at 10 C with 20 hr light for 45 d, at which time the tallest 15 plants were selected. Selection intensity was greater for GT than for SH.

Late Germination and Low Seedling Vigor (LG+LSV)
Procedures were identical to EG+HSV except that the last 30 seeds to germinate prior to 50 d after planting were transplanted to the growth chamber where the shortest 15 plants were selected after 45 d. Selection between seedlings of equal height was done visually for fewer leaves and thinner stem diameter.

Control Populations
Control (unselected) populations of Innovator +Z and 5454 were developed by intercrossing 15 randomly chosen plants of each cultivar in each cycle. No conscious selection was practiced.

Intercrossing
Selected plants were transplanted into 3.5 l pots in the greenhouse under 20 h light. Plants within each selected population were intercrossed by hand tripping without emasculation. Equal amounts of seed were bulked from each plant in the selected populations for use in the next cycle of selection. Sixty-one populations were created after 2 to 3 cycles of selection.

Evaluation
Prior to selection, average seed mass (mg) was measured on 100 seed bulk samples on all populations as the average of three samples. Laboratory and growth chamber evaluations were performed separately for SH and GT. Evaluation experiments included all C0, selected and control populations as entries. Experiments were arranged as randomized complete block designs with three replications and individual populations as the only treatment. Population effects were considered fixed.

Germination time was evaluated by the method established for EG selection in a randomized complete block design with three replications. Boxes were opened daily for 15 d and then every second day up to 50 d; the day of germination for each seed was recorded. The mean GT per box was calculated as the average number of days from planting to germination for all viable seeds.

Seedling height was evaluated in a randomized complete block design by planting 96 scarified seeds of each population into three row plots, with 4 cm between rows, in 1500 cm² flats filled with equal parts vermiculite and topsoil. Four populations were planted in each flat; border rows of `Vernal' were placed at each end. Flats were kept in the greenhouse for 3 d after planting to ensure even germination and then moved to a growth chamber at 10°C with 20 h light for 45 d. The first and last seedlings of each row were discarded and seedling height was recorded for all remaining seedlings, excluding the border rows. Three replications were planted, separated by time.

Statistical analysis was conducted using the GLM and CORR procedures in the SAS statistical software package (SAS Institute, Inc., 1996). Mean separations were determined using Fisher's protected LSD (p<0.05) (Steel and Torrie, 1980).

Realized Heritabilities
Realized heritabilities were calculated as the regression of the cumulative response on the cumulative selection differential:

where b_c is the unbiased estimator of realized heritability, and are the mean values of S_i and X_i, the cumulative selection differential and the population mean, respectively, for Cycle i (Hill, 1972). The estimates of the Cycles 0, 1, and 2 parental means used to calculate the selection differentials were obtained at different times but in the same controlled environmental chambers. The estimator of the unbiased sampling variance of b_c, V(b_c), was estimated as described by Hill (1972)(p. 772); the standard errors of the heritability estimates were calculated as the square root of V(b_c). Estimates of realized genetic correlation between GT and SH were calculated as:

where R is the total direct response and CR the total correlated response after two cycles selection for each trait (Falconer and Mackay, 1996, p. 317).

	Results and discussion

TOP NOTES ABSTRACT INTRODUCTION Materials and methods Results and discussion REFERENCES

 
Cultivars differed for seed mass and germination time at 5°C, but not for 45 d seedling height at 10°C (Table 1). Seed mass of treated seed lots was not evaluated. The percentage of normally germinating seeds, determined at 20°C by standard seed quality testing procedures (AOSCA, 1991), was approximately 80% and did not differ among the cultivars (data not shown). Alfagraze had the lowest seed mass and the longest mean germination time at 5°C (Table 1).

Control (Unselected) Populations
No differences in GT or SH were observed between C0 populations and unselected control populations of `5454' and `Innovator +Z' (Table 2) . The lack of change suggests that the changes in GT and SH that we observed in some selected populations were probably not caused by genetic drift or inbreeding depression. However, because only a single control population was developed and evaluated in only two of the cultivars, we cannot conclusively eliminate either of these causes in interpreting our results.

The average seed mass of EG-C1 and EG-C2 populations were 0.22 and 0.06 mg greater than C0, respectively, but similar responses were observed with all other selection methods (Table 3). The Innovator +Z Control-C1 and -C2 populations were 0.17 and 0.29 mg higher than C0, respectively. A similar comparison could not be made in 5454, which had been treated with fungicide before we received the seed. Because environmental factors are known to affect alfalfa seed size (Carleton and Cooper, 1972), the changes in seed mass we observed over cycles may be due to differences in the seed production environments experienced by the source (field grown) and selected (greenhouse grown) populations. No correlation between seed mass and GT in the laboratory at 5°C was observed over all populations (P < 0.05; data not shown).

HSV Selection
Two cycles of HSV selection increased SH by 15% (P < 0.05), averaged over populations (Table 3). HSV-C2 was not significantly (P < 0.05) different from HSV-C1 indicating that most response occurred in the first cycle, as was seen for EG selection. The response to HSV selection differed among cultivars (Table 2). In Innovator +Z, two cycles of HSV selection increased SH by 68% (P < 0.05) compared to its Control-C2 population; in this case, the response was greater in the second cycle than the first (Table 2). Linear contrasts among generations of HSV selection in Innovator +Z were significant (P < 0.05), indicating the potential for further SH increases. Amerigraze 401+Z HSV-C2 was 24% (P < 0.10) taller than C0 (Table 2). No other selected populations had increased SH (P < 0.10). Microclimate variation in the greenhouse and growth chamber during evaluation that was not accounted for by the experimental design affected SH measurements. This environmental variation together with variability for traits such as seed size hampered our ability to differentiate among populations for SH. Nevertheless, selection for increased SH at cool temperatures is effective in certain populations.

Two cycles of HSV selection increased GT by an average of 29% despite germinating seeds in the greenhouse prior to placement in a 10°C growth chamber for selection (Table 3). Cultivars differed in their GT response to selection for HSV, with increases (P < 0.05) observed after two cycles in 5454, Alfagraze, and Magnum IV (Table 2). In these populations, a large response occurred in C2 and significant (P < 0.05) linear contrasts among cycles indicated that GT would continue to increase with further HSV selection. In Amerigraze 401 +Z, Innovator +Z, and WL252HQ, however, changes in GT with HSV selection followed no clear trend (Table 2).

The seed mass of C1 and C2 populations selected for HSV was greater than C0 (Table 3), possibly due to the seed production environment. The average seed mass of HSV-C2 was lower than that of HSV-C1, suggesting that the increases in SH over cycles were not due to seed mass (Table 3). The greatest increase in SH with selection for HSV was observed in Amerigraze 401+Z, which also had the greatest seed mass of the cultivars measured (Table 1). However, seed mass was not correlated (P < 0.05) with SH in this study. Carleton and Cooper (1972) examined eight alfalfa clones and found no differences in 3 wk seedling dry matter even though the seed mass varied from 1.87 to 2.46 mg seed^-1. They suggested that unspecified environmental affects, rather than genetic affects, on alfalfa seed size were the more important source of seedling performance differences. Selection for seedling vigor may not increase seed mass, but selection based on uniformly sized seed may yield more consistent selection responses.

Realized Heritability
The average realized heritability of GT, calculated based on response to EG selection, was 0.49 in these populations (Table 4) . Realized heritability estimates for GT ranged from 0.67 in Alfagraze to 0.38 in Magnum IV (Table 4). The average realized heritability of SH, based on the response to HSV selection, was 0.18, though two of the estimates used in this average were not significantly different from zero (Table 4). Realized heritability estimates for SH range from 0.046 in 5454 to 0.40 in Innovator +Z (Table 4). Alfagraze had a selection differential approximately twice that of the other cultivars for GT but smaller than others for SH (Table 4). The standard errors for the estimates are rather large, as expected for short-term selection experiments which have few degrees of freedom among cycles (Hill, 1972).

Bidirectional Selection for Germination Time and Seedling Vigor
Averaged over populations, two cycles of EG+HSV selection decreased GT by 29% (P < 0.05) from C0, with the largest response observed in C1; SH was unaffected (Table 3). Alfagraze EG+HSV-C2 had a 90% earlier GT than C0 (P < 0.05) but did not differ from C1 (Table 2). The 5454 EG+HSV-C1 was 35% taller than the unselected control, but the response was lost with a second cycle of selection. No other GT or SH differences were observed among populations of the other cultivars (Table 2). The selection intensity with the EG+HSV method may not have been great enough to produce significant changes in SH after only two cycles, considering the low heritability observed for this trait. Alternatively, selection for GT and SH may be counteracting each other during EG+HSV selection, leading to little improvement for either trait. This explanation is supported by the high r²_A between SH and GT seen for some cultivars. Genetic drift caused by small sample size may have further affected SH and GT in selected populations.

In contrast to EG+HSV, selection for LG+LSV was much more successful, resulting in an average increase in GT of 162% after two cycles (Table 3). The germination time increased (P < 0.05) in all cultivars under selection, ranging from 145% in Innovator +Z to 234% in Amerigraze 401+Z (Table 2). In all cases, LG+LSV-C2 was later to germinate than C1 (Tables 2 and 3), and linear contrasts among generations indicated the potential for further increases in GT (P < 0.05). Seedling height decreased by 12% (P < 0.05) on average after two cycles of LG+LSV selection (Table 3), but selection was not effective in all cultivars (Table 2). 5454 LG+LSV-C2 was 26% shorter than C0 but not different (P < 0.05) from the Control-C2 population (Table 2). WL252HQ LG+LSV-C2 was 28% shorter (P < 0.10) than the C0. Differences in other source populations were not significant (P < 0.10).

The GT and SH responses were greater with LG+LSV selection than with EG+HSV selection (Table 3). Selection against these traits may have been more successful because cultivars have already been indirectly selected for high seedling vigor so further progress may be slow, while substantial genetic variation may exist for "unimproving" these traits. An asymmetrical response is commonly observed with divergent selection and may be due to differences in the selection differential, asymmetry in the effects of genetic and environmental variation, the presence of genes of large effect, genetic drift due to sampling error, or to the effects of indirect selection (Falconer and Mackay, 1996).

In summary, selection for both decreased germination time and improved seedling height under low temperatures appears to be possible in at least some alfalfa populations. A number of factors should be considered when similar types of selection programs are conducted: (i) stringent control of microclimatic variation in greenhouses and environmental chambers to improve the ability to identify superior plants, (ii) selection from uniformly sized seed, (iii) production of seed under common environmental conditions if possible to make evaluations more comparable, and (iv) consideration of population sizes selected to avoid genetic drift and inbreeding depression problems.SAS Institute Inc 1996

	NOTES

TOP NOTES ABSTRACT INTRODUCTION Materials and methods Results and discussion REFERENCES

 
Journal Paper No. J-18406 of the Iowa Agric. and Home Econ. Exp. Stn., Ames, IA 50011, Project No. 2569, supported by Hatch Act and State of Iowa Funds.

Received for publication May 24, 1999.

	REFERENCES

TOP NOTES ABSTRACT INTRODUCTION Materials and methods Results and discussion REFERENCES

 

• AOSCA. Rules for testing seeds. Assoc. of Official Seed Analysts. J. Seed Tech. 1991;12:54-57.
• Arakeri H.R., Schmid A.R. Cold resistance of various legumes and grasses in early stages of growth. Agron. J. 1949;41:182-185.[Free Full Text]
• Barnes, D.K., and C.C. Sheaffer. 1995. Alfalfa. In R.F Barnes, et al. (ed.) Forages. Vol. 1: An introduction to Grassland Agriculture. Iowa State Univ. Press, Ames, IA.
• Brar G.S., Gomez J.F., McMichael B.L., Matches A.G., Tailor H.M. Germination of twenty forage legumes as influenced by temperature. Agron. J. 1991;83:173-175.[Abstract/Free Full Text]
• Busbice T.H. Inbreeding in synthetic varieties. Crop Sci. 1969;9:601-604.[Abstract/Free Full Text]
• Carleton A.E., Cooper C.S. Seed size effects upon seedling vigor of three forage legumes. Crop Sci. 1972;12:183-186.[Abstract/Free Full Text]
• Coffman F.A. The minimum temperature of germination of seeds. Agron. J. 1923;15:257-270.[Free Full Text]
• Dickson M.H., Boettger M.A. Emergence, growth, and blossoming of bean (Phaseolus vulgaris) at suboptimal temperatures. J. Am. Soc. Hort. Sci. 1984;109:257-260.
• Falconer D.S., Mackay T.F.C. Introduction to quantitative genetics. 4 ed. Harlow, Essex, England: Longman, 1996.
• Hill R.R., Jr., Pedersen M.W., Elling L.J., Cleveland R.W., Graham J.H., Frosheiser F.I., Starling J.L. Comparison of expected genetic advance with selection on the basis of clone and polycross progeny-test performance in alfalfa. Crop Sci. 1971;11:88-91.
• Hill W.G. Estimation of realized heritabilities from selection experiments. II. Selection in one direction. Biometrics 1972;28:767-780.[ISI][Medline]
• Klos, K.L.E. 1999. Variation in alfalfa (Medicago sativa L.) for germination and seedling vigor at suboptimal temperatures; and laboratory and field responses to selection within six alfalfa populations. Ph.D. diss. (Diss. Abstr. 9924731). Iowa State Univ., Ames, IA.
• McConnell R.L., Gardner C.O. Selection for cold germination in two corn populations. Crop Sci. 1979;19:765-768.[Abstract/Free Full Text]
• Rumbaugh M.D., Caddel J.L., Rowe D.E. Breeding and quantitative genetics. In: Hanson A.A., et al. , ed. Alfalfa and alfalfa improvement. Madison, WI: ASA, CSSA, and SSSA, 1988:777-808.
• Saminy C., Tailor A.G., Kenny T.J. Relationship of germination and vigor tests to field emergence of snap beans (Phaseolus vulgaris L.). J. Seed Tech. 1987;11:23-34.
• SAS Institute Inc. SAS/STAT guide for personal computers, Version 6.12 edition. Cary, NC: SAS Institute Inc, 1996.
• Staub M.E., Lower R.L., Nienhuis J. Correlated responses to selection for low temperature germination in cucumber. HortScience 1988;23:745-746.
• Steel R.G.D., Torrie J.H. Principles and procedures of statistics: A biometrical approach, 2nd Ed New York, NY: McGraw-Hill, 1980.
• Szyrmer J., Szczepanska Krystyna Screening of soybean genotypes for cold-tolerance during germination. J. Pflanzenzucht. 1981;88:255-260.
• Tysdal H.M., Pieters A.J. Cold resistance of three species of lespedeza compared to that of alfalfa, red clover, and crown vetch. J. Agric. Res. 1931;43:177-182.
• Volenec, J.J., and C.J. Nelson. 1995. Forage crop management: Application of emerging technologies. In R.F Barnes, et al. (ed.) Forages. Vol. 2: The science of grassland agriculture. Iowa State Univ. Press, Ames, IA.
• Vough, L.R., A.M. Decker, and T.H. Tailor. 1995. Forage establishment and renovation. In R.F Barnes, et al. (ed.) Forages. Vol. 2: The science of grassland agriculture. Iowa State Univ. Press, Ames, IA.
• Wehner T.C. Genetic variation for low-temperature germination ability in cucumber. Cucurbit Genet. Coop. Rep. 1982;5:16-17.
• Wehner T.C. Estimates of heritabilities and variance components for low-temperature germination ability in cucumber. J. Am. Soc. Hort. Sci. 1984;109:664-667.

This article has been cited by other articles:

				M. D. Casler and D. J. Undersander Selection for Establishment Capacity in Reed Canarygrass Crop Sci., April 25, 2006; 46(3): 1277 - 1285. [Abstract] [Full Text] [PDF]

				K. L.E. Klos and E.C. Brummer Field Response to Selection in Alfalfa for Germination Rate and Seedling Vigor at Low Temperatures Crop Sci., September 1, 2000; 40(5): 1227 - 1232. [Abstract] [Full Text]

This Article Abstract Full Text (PDF) Free Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in ISI Web of Science Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (6) Citing Articles via Google Scholar Google Scholar Articles by Klos, K. L.E. Articles by Brummer, E.C. Search for Related Content PubMed Articles by Klos, K. L.E. Articles by Brummer, E.C. Agricola Articles by Klos, K. L.E. Articles by Brummer, E.C.