Tolerance of barley seed germination to cold- and drought-stress expressed

as seed vigour

O. Chloupek1, P . Hrstkova

1 and D. Jurecka2

1 Mendel University of Agriculture and Forestry, Zemedelska 1, CZ-613 00 Brno. E-mail: chloupek@mendelu.cz 2 CentralInstitute for Supervising and Testing in Agriculture, CZ-656 06 Brno, Czech Republic

With 3 tables and 2 figures

Received March 19, 2002/Accepted August 12, 2002Communicated by A. Graner

Abstract

In barley, high seed vigour is a precondition for rapid and homogenous

field emergence and good malting quality. Seed vigour was defined as

germination percentage under stress conditions (10�C, drought stress) 2 Bars) in 7–8 barley varieties grown in 7–8 locations in the Czech

Republic over 7 years. Three of the 7 years were not suitable for high

seed quality, probably because of unsuitable weather, as average seed

vigour reached only 61, 77 and 86%, respectively. In the remaining

4 years, the average vigour exceeded 94%. The impact of variety on

seed vigour was higher in the �bad years� and the impact of location

was higher in the other years. Varieties with higher vigour from all

locations in the �bad years� were identified. Lower vigour was related to

the high occurrence of fungi (indicated by ergosterol assays) and to

lower field emergence rates of seed samples. The results support the

possibility of selecting for improvement of barley seed vigour, which is

related to tolerance to various conditions during emergence and to

homogenous malting.

Key words: Hordeum vulgare — tolerance to drought and cold

stress — vigour — germination — emergence rate — varieties —provenance

Germination rate is an important character, not only for seedquality but also for malting quality, and is evaluated under

optimal conditions for germination, i.e. at laboratory temper-atures (20�C) and at optimal moisture. However, it differsduring germination in the soil and during malting procedures.

When field conditions are unfavourable, germination testresults are not correlated with field emergence (van de Venter2001), and so the term seed vigour was introduced. High

vigour is a measure of physiological seed quality, it is theprecondition for the good storage potential of seed, rapid andhomogenous field performance and for high yield under

different ecological conditions. Only healthy seed can reachhigh vigour.

Vigour can be evaluated using stress tests, biochemical testsand seedling growth and evaluation tests. Their reliability and

accuracy can be determined by comparisons of the relationshipbetween test results and field performance and/or seed storagepotential. Only a few are recommended by the International

Seed Testing Association (ISTA), as published by van deVenter (2001), mainly because of low reproducibility betweenlaboratories. In spite of that, vigour tests are frequently used

by the seed industry (TeKrony 2001).The vigour of barley caryopses was evaluated at low

temperature and under drought stress in some preliminaryexperiments. The relative contributions of varieties to the total

variation were higher for vigour than for germination. How-ever, the percentage of germination was not always related to

the percentage of vigour. Seed provenance generally caused thegreatest variation and its impact was greater on the germinationrate than on vigour (Chloupek et al. 1997). These experiments

have been extended and the results are presented in this paper.Ergosterol is the main sterol of fungal membranes. Its

content in living fungi is constant at approximately 5 mg/kg of

dry matter, and it does not occur in other organisms that decayliving matter. It can therefore be used as a biochemical indexfor living fungi (Gessner et al. 1997). The content of ergosterolin the caryopses of malting barley from harvests in 2000 was

evaluated in relation to seed vigour. Evaluation of 500 samplesfrom the whole Czech Republic showed that the visualoccurrence of fungi was abnormally high, probably because

of extremely wet and hot weather during ripening, and wasassociated with low germination rates (Psota 2000).The aim of this paper was to evaluate our approach to seed

vigour testing (Chloupek et al. 1997) in relation to variety,year and location. The relationship to field emergence rate wasalso studied.

Materials and Methods

Caryopses of 7–8 spring barley (Hordeum vulgare L.) varieties from

7–8 locations in the Czech Republic, harvested over 7 years of official

variety trials were studied. Experimental years, varieties evaluated and

their harvest location are given in Table 1. The barley caryopses came

from plots sown with seed treated with fungicides but only with basic

fertilization and without the application of any other pesticide. The

vigour was tested by germination at 10�C and drought stress at )2 Bars, measured after 4 and 7 days.

Drought stress was induced by polyethylenglykol (PEG 6000)

treatment. Each sample (each variety of each provenance) was tested in

four replications of 50 caryopses each. For analysis of variance the

values were transformed by y ¼ arcsin �p, where p is the proportion of

germinated caryopses.

As seed vigour is expressed as the ability to emerge quickly and

uniformly, the field emergence rate is also relevant. It was evaluated on

seed samples harvested in 1998, 1999 and 2000, the caryopses being

sown on two sites. The field emergence rate (FER) was therefore

evaluated in six environments. Each sample was sown in three

replications of 42 seeds at a depth of 6–8 cm, and the emergence was

assessed five times from the time when approximately 30% of the

seedlings emerged. Average values of the three middle terms were used

for evaluation. The percentage values were transformed for evaluation

of variability, as stated earlier.

Plant Breeding 122, 199—203 (2003)� 2003 Blackwell Verlag, BerlinISSN 0179-9541

U. S. Copyright Clearance Center Code Statement: 0179–9541/2003/2203–0199 $ 15.00/0 www.blackwell.de/synergy

The content of ergosterol was measured by liquid chromatography

(Gessner and Newell 1997) at the Department of Crop Science in the

University of Giessen, Germany. Only caryopses of eight varieties

from two locations with the highest and lowest seed vigours (Caslav

and Verovany) harvested in the year 2000 were analyzed.

ResultsImpact of year, variety and provenance on seed vigour

On average over the 7 experimental years the vigour amountedto 87%; high vigour, more than 90%, was found in 4 of the 7experimental years and low vigour, less than 90%, in the three

other years:

• Good years (vigour): 1992 (99%), 1995 (99%), 1999 (96%)and 2000 (94%)

• Bad years: 1993 (77%), 1997 (62%) and 1998 (86%)

The lower the seed vigour the lower was the relative influenceof provenance (r ¼ + 0.88, P < 0.01) and the higher the

influence of variety (r ¼ )0.93, P < 0.01) the higher was theinteraction between the varieties and the locations (r ¼ )0.75,P < 0.05) (Fig. 1). This means that the impact of variety onseed vigour was higher in the bad years and the impact of

provenance was higher in the good years.The analysis of variance only showed a significant effect of

varieties tested (P < 0.01) on vigour in 1993 and 1997

(Table 2). Its percentage of the whole variance ranged from7.7 to 32.7%, the average for all 7 years was 17.0%. In 1993(Table 1) a relatively high value was found in the variety

�Jubilant� (90%); the lowest was for �Forum� (61%). In thesecond group of varieties in 1997 the highest vigour (P < 0.01)was for �Kompakt� (81%), when an average of all varieties was

only 62%. In the third group, in 1998–2000, the significantlyhighest vigour levels were in �Krona� and �Akcent� (94% and93%, respectively), and the lowest was in �Kompakt� (88%).

Unlike variety, the provenance of samples influenced seedvigour significantly (P < 0.01) in all experiments. Its percen-tage of the whole variance in the experimental years rangedfrom 36 to 88%, with an overall average for all 7 years of 68%.

The values for vigour were only slightly related to the altitudeof growing locations (the correlation coefficient for all yearswas r ¼ )0.45; nonsignificant). In 1997 and 2000, the vigour

was also significantly influenced by the interaction of varietieswith locations (P < 0.01). Its percentage ranged from 3.4 to25.4, with an average of 15.3% over all seven experiments.

Remarkable results were detected when evaluating vigourdata from �bad years� (Table 1). In 1993 the difference betweenvarieties tested reached 29% (�Jubilant� 90%, �Forum� 61%),

and between locations reached 28% (Lednice 88%, Hradec60%). In 1997 it reached 26% (�Kompakt� 81%, �Akcent�55%), and between locations it reached 32% (Vysoka 74%,Lednice 42%). In 1998 it reached 13% (�Krona� 89%,

�Kompakt� 76%), and between locations it reached 18%(Sedlec 93%, Stankov 75%). This suggests that in the �badyears� the higher the influence of variety and provenance the

lower was the seed vigour. This means that, particularly insuch years, seed provenance and variety are of great import-ance.

Linear regression analysis showed (Fig. 2) that in 1993 thevigour of the variety �Jubilant� was better than the average ofall varieties. The variety �Orbit� responded to better environ-ments with better improvement of vigour than other varieties;

�Terno� in contrast had worse vigour. In 1997 �Kompakt� and�Sladko� had high and average vigour scores, respectively,without a response to different environments. �Amulet� wasrelatively sensitive to the environment. �Krona� showed overallbetter vigour in 1998 than the average of all varieties,�Olbram� had above average vigour without response to

Table 1: Mean values of seed vigour (%) for varieties and locations(harvest years 1992, 1993, 1995 and 1997–2000)

Varieties 1992 1993 1995 1997 1998 1999 2000

Akcent 100 83 99Forum 100 61 99Jubilant 99 90 99Orbit 99 81 100Rubın 99 72 99Sladko 99 80 99Terno 99 75 99Akcent 55 85 97 98Amulet 59 86 95 93Forum 58 88 96 95Kompakt 81 76 97 90Krona 59 89 97 96Nordus – 86 96 95Olbram 57 87 95 91Sladko 63 – – –Tolar 61 87 94 95

Locations (altitude) 1992 1993 1995 1997 1998 1999 2000

Caslav (290) 99 73 98 67 85 96 99Hradec (450) 100 60 100 56 79 98 99K. Udolı (647) 99 75 98 64 82 92 91Lednice (170) 100 88 100 42 90 – 97Sedlec (300) 99 84 99 62 93 93 98Stankov (370) 98 87 99 64 75 98 94Verovany (590) 100 79 99 64 90 97 82Vysoka (207) 99 73 100 74 90 97 93

Var

iati

on

Fig. 1: Percentage of factors involved in the variation (¼ 100%) in the7 experimental years

Table 2: Analysis of variance for varieties and locations in the years ofunfavourable for seed vigour

Source of variance df MS (1993) df MS1 (1997) MS (1998)

Varieties 6 1753.37** 7 1191.96** 251.60Locations 7 1946.24** 7 1524.35** 1108.05**Interaction 42 1688.71** 49 924.89** 323.18Error 168 251.14 192 235.03 275.24

** Significant at P ¼ 0.01.1 MS, mean square.

200 CHLOUPEK, HRSTKOV A and JURECKA

different environments, and �Kompakt� mostly had an overall

lower vigour. (Only varieties with differing responses to theenvironment are given in Fig. 2.)

Impact of year, variety and provenance on field emergence rate

The analysis of variance for FER (Table 3) showed significant

impact of the harvest year, followed by interaction between thelocation and the harvest year, and interaction among thevariety · the location · the year. The lowest FER (74%) wasfound in the harvest year 1998, in which the average vigour

was also the lowest (86%) of all 3 experimental years in which

FER was tested. Seed from some locations showed higherFER in some years than in others. The significant multipleinteraction indicates that different varieties have different

responses in different years.The highest FER value was for location Caslav (83%),

and the lowest was K. Udolı and Vysoka (75%). The variety

�Tolar� showed a significantly higher FER value (80%) thanthose of the other six varieties (76–78%). Seed vigour wasnot significantly related to the field emergence rate(r ¼ 0.50–0.54).

1993

r = 0.52

r = 0.69

r = –0.31

30

40

50

60

70

80

90

100

45 50 55 60 65 70 75 80

Average vigour of 7 varietiesfrom 8 locations

Vig

ou

r o

f p

arti

cula

r va

riet

y fr

om

8 lo

cati

on

s

Mean ´Jubilant´

´Orbit´ ´Terno´

Average vigour of 7 varietiesfrom 8 locations

Vig

ou

r o

f p

arti

cula

r va

riet

y fr

om

8 lo

cati

on

s

r = 0.14

r = 0.01

r = 0.81

1997

20

30

40

50

60

70

80

90

100

40 45 50 55 60 65 70 75

Mean ´Amulet´

´Kompakt´ ´Sladko´

Average vigour of 7 varietiesfrom 8 locations

Vig

ou

r o

f p

arti

cula

r va

riet

y fr

om

8 lo

cati

on

s

1998

50

60

70

80

90

100

70 75 80 85 90 95 100

Mean ´Olbram´

´Krona´ ´Kompakt´

Fig. 2: Regression lines for vigourof individual varieties in compar-ison with the average vigour of allvarieties from locations with differ-ent levels of the trait, but only inthe 3 years with generally lowlevels of the trait (1993, 1997 and1998)

Seed vigour of barley 201

Content of ergosterol and seed vigour

The content of ergosterol in caryopses from Caslav was onaverage 9.5 mg/kg of dry matter, and the vigour was 99%; forVerovany it was 11.5 mg and 82%, respectively. Highercontent of ergosterol (a symptom of higher occurrence of

living fungi) was therefore associated with lower vigour in theyear favourable for ear fungi. This was also evident from anonsignificant correlation between the content of ergosterol

and the vigour of eight variety samples from both locations(r ¼ )0.46).

Discussion

The values of germination under optimal conditions (20�C,drought stress 0 Bar) were not always related to the values ofvigour measured under stress conditions (10�C, ) 2 Bars).Low temperature is used in, for example, cold soil tests, which

combine the effects of suboptimal temperature and soil-bornepathogens. The growth of pathogens is encouraged by leakedsugars and amino acids, etc. from low vigour seeds (van deVenter 2001).

Little or no interaction of variety with assay temperature(5, 10, 15 and 20�C) was found for germination values (Briggsand Dunn 1999). Evaluation of vigour, including also drought

stress, seems to be more relevant to the conditions ofgermination in soil and for the malting procedure.Vigour in the poly factorial experiments was influenced by

variety, provenance and their interactions. The effect ofprovenance on vigour was not related to altitude, precipitationand temperature during the vegetative period at seed origin

locations, but it did include other factors connected withfarming practice (harvest time, machinery, postharvest treat-ment) to the occurrence of pathogens, etc.Severe stress during seed filling results in a larger proportion

of small seed and poor seed vigour (Dornbos and Mullen1991). However, in these experiments only caryopses over2.2 mm in sieve size were used.

Plant responses to stresses such as drought and low tempera-ture involve many physiological, biochemical and molecularchanges.Dehydrin-genes have been implicated as the key genetic

determinants of stress tolerance in anumber of species, includingbarley (Zhu et al. 2000); dehydrins accumulate during normal(nonstress) ripening in seeds and protect proteins duringdehydration or frost. Genetically similar lines of Vigna ungu-

iculata differed in emergence rate and also differed in dehydrins.It has been shown that better emergence was connected to35-kDa dehydrin occurrence (Ismail et al. 1997).

The data did not confirm that the seed with the highestvigour comes only from regions where barley is traditionallygrown for malting; good quality seed was also produced innontraditional areas. However, the malting varieties showed

higher vigour than those not suitable for malting (Chloupekand Hrstkova 2000).The differences in vigour caused by variety, provenance and

by their interaction could be influenced by fungal infection. Asmall proportion of infected caryopses diminished not onlytheir malting value but also their seed quality. Vigour therefore

seems to be a complex trait controlled by many genes, such asresistance genes to ear fungi, etc. Artificially reduced barleyseed vigour caused a reduction in competitive ability againstweeds (Rasmussen and Rasmussen 2000).

Vigour of barley caryopses proved to be geneticallycontrolled and had higher heritability in the years unsuitablefor high vigour. It can be used as a selection criterion. A

literature review concerning breeding for seed quality waspublished by Salisbury and Flood (1994). Perhaps it would beuseful to judge barley seed also by its provenance (origin), as

this is of relevance to seed and malting quality. By sowinghigh-vigour planting seed adequate emergence and coveragecan be ensured across a wide range of field conditions

(TeKrony and Egli 1991).The rate of barley seed germination under nonstress, cold-,

drought- and salt-stress conditions may be controlled by thesame genes (or physiological mechanisms), but additional

components may be involved that affect germination ratesunder specific stress conditions, as for tomato seed (Fooladand Lin 1999). Selection for rapid seed germination was

effective under cold stress and salt stress (Foolad et al. 1999).Seed vigour can be improved through breeding, while main-taining high yields because of the predominance of general

combining ability (GCA) effects in both seed vigour and seedyield (Cho and Scott 2000).In field trials, days to emergence were correlated with

germination resistance determined in the growth cabinet(Briggs and Dunn 2000). As high vigour and the emergencerate of barley grains are important not only for malting qualitybut also for agronomy, it could be useful to subject lines to

selection for this trait. Quantitative trait loci were found fordeep seeding tolerance in barley and corresponded with QTLfor abscisic acid and gibberellic acid responses (Takahashi

et al. 2001). Similar selection of alfalfa was successful becausea clone with significant effects of GCA for high germinationwas found and included as one of the parents in two synthetic

varieties. The populations showed higher germination rates(81.4%) and lower percentages of hard seeds (14.9%) incomparison with standard varieties (79.1 and 16.2%) in varietyregistration trials (Chloupek 1994).

The content of ergosterol in caryopses of wheat at harvestreached 3.0–5.6 mg/kg of dry matter and increased duringstorage to 8–15 mg/kg (Schnurer and Jonsson 1992). Similar

values were found in the present observation in the yearfavourable for field fungi after 1 month in storage (on average10.5 mg/kg).

Acknowledgements

The authors thank the Czech Government agencies for financial

support (projects of GACR 521/99/1576 and of the Ministry of

Education 432100001), Prof Dr h. c. Wilhelm Opitz von Boberfeld for

evaluation of ergosterol content and Dr Brian Forster for language

correction of the text.

Table 3: Analysis of variance for field emergence rate of eight varietiesharvested in seven locations and proved in 3 years on two plantbreeding stations in three replications

Source of variance df Mean squares

A. Varieties 7 202*B. Locations 6 1307**C. Harvest years 2 4873**A · B 42 112A · C 14 111B · C 12 1989**A · B · C 84 154**Residuum 840 95

Significant at * P ¼ 0.05 and ** P ¼ 0.01, respectively.

202 CHLOUPEK, HRSTKOV A and JURECKA

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