Expl Agric.

: page 1 of 8 
C Cambridge University Press 2017

doi:10.1017/S0014479717000114

P R E H A RV E S T S P RO U T I N G A S S E S S M E N T I N W H E AT
G E N OT Y P E S I N F LU E N C E D B Y T E M P E R AT U R E A N D
D E G R E E DAYS

By L. A. OKUYAMA†§, N. S. FONSECA JUNIOR†, P. H. CARAMORI†

and M. M. KOHLI‡
†Instituto Agronômico do Paraná (IAPAR), Caixa Postal 10030, CEP 86057–970 Londrina,
Paraná, Brazil and ‡Capeco/Inbio, Asuncion 1512, Paraguay

(Accepted 8 February 2017)

SUMMARY
The environmental conditions and test duration in rainfall simulators have been major constraints to find
reliable differences in preharvest sprouting (PHS) among wheat cultivars. This study aimed to elucidate
the temperature conditions and degree-days (DD) that enable higher discrimination of genotypes in a
PHS test. Thirteen genotypes with different degrees of PHS (BR 18, BRS 220, CD 104, CD 105, CD
108, CD 114, CD 116, Frontana, IPR 085, IPR 128, IPR 136, OR1 and Safira) were evaluated in a
rainfall simulator, during four years, conducting two trials per year. The tests were carried on 20 spikes
per cultivar, kept in a vertical position on Styrofoam plates, spaced 10 cm between rows and 5 cm within
the row. The air temperature data were recorded on a thermograph and the DD were calculated from the
mean daily air temperature assuming a base temperature of 4 °C. The Falling Number (FN) parameter
and germination percentage (GP) data were averaged for each test. The temperatures (minimum,
mean and maximum) and the DD were regressed and correlated with FN and GP. Any increase in the
minimum and mean air temperature and accumulated DD decreased FN values and increased GP. Higher
genotypic discrimination was achieved at an average air temperature of 21.5 °C and 35 DD. These values
can be used as reference to set simulated rain testing condition for screening genotype for PHS based on
FN or pericarp rupture of grains.

I N T RO D U C T I O N

Preharvest sprouting (PHS), grain germination before the harvest, is a serious problem
in many wheat-growing areas of the world. Periods of rainfall and high humidity
after grain maturity but before the harvest can induce PHS. Germination initiates
when grains absorb moisture. Visible symptoms of PHS can vary from grain swelling,
germ discoloration, seed coat splitting to root and shoot emergence. The weather
conditions during ripening and preharvest are important factors affecting sprouting
(Paterson et al., 1989) and FN (Mares and Mrya, 2008). The grain ripening under
low temperature conditions induces higher level of dormancy than under higher
temperature conditions (Reddy et al., 1985). The grain and flour quality parameters
impacted by PHS are strongly related with climatic variables and soil water condition
(Guarienti et al., 2003).

§Corresponding author. Email: lauro2288@gmail.com

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 2 L . A . O K U YA M A et al.

There are several methods to evaluate PHS and these include artificial sprouting
of whole (intact) spikes, germination tests, natural weathering of crop in field
trials, artificial weathering trials, and indirect assessment of sprouting by measuring
Hagberg FN and molecular markers (DePauw et al., 2012). Among these, the artificial
weathering trials have been used to evaluate a large number of genotypes quickly
and to eliminate inferior ones (Bassoi et al., 2006; Franco et al., 2009; MacMaster and
Derera, 1976). The time period in the assessments of PHS in rain simulators varied
from 60 h (Franco et al., 2009; Gavazza et al., 2012) to 96 h (Shorter et al., 2005) and
as much as 120 h (Singh et al., 2014).
In addition, the heat units expressed in degree-days (DD) is a useful index to
study the timing of biological processes (McMaster and Wilhelm, 1997). For the PHS
studies conducted in a rain simulator, the values of temperature and accumulated
degree-days (ADD) that enable better characterization of genotypic differences are
not clear. In order to determine the test conditions that enable larger expression of
genetic variability for PHS, this study aimed to evaluate the effects of temperature
(minimum, mean and maximum) and DD on the FN and germination percentage of
wheat grain.

M AT E R I A L S A N D M E T H O D S

Field experiments were conducted at the Experimental Station of IAPAR (Instituto

Agronômico do Paraná), located at Londrina (23°23ʹS, 50°01ʹW, altitude 556 m),
in state of Paraná, Brazil. The soil is a Typic Haplorthox, characterized by a
deep profile, fine texture, and very deep water table. Fertility is constrained by
low organic matter and phosphorus content. The region has a subtropical humid
climate characterized by long hot summers and mild sunny winters. Experiments
reported in this study were conducted in four years with two planting dates, and
comprised of 13 spring wheat (Triticum aestivum L.) cultivars (BR 18, BRS 220, CD
104, CD 105, CD 108, CD 114, CD 116, Frontana, IPR 85, IPR 128, IPR 136,
OR1 and SAFIRA). Old Brazilian cultivar Frontana, reported as tolerant to PHS
(Czarnecki, 1986), was used as a check in the study. Field plots consisted of three
rows of two meters in length, spaced 30 cm apart and seeded at a rate of 350
seeds m−2 . Recommended agronomic management practices for wheat were used
during the crop season (IAPAR, 2003). At physiological maturity, around 80 uniform
spikes per cultivar were hand harvested (Hanft and Wych, 1982) and kept at room
temperature.
The PHS tests were performed in a greenhouse (length, width and height of 10, 4,
and 3 m, respectively), made of galvanized pipes and covered with a dark polyethylene
screen mesh with plastic transparent sheeting on the roof. Photosynthetically active
radiation (PAR) inside the greenhouse was measured with portable equipment
(LCpro, ADC Bioscientific Ltd., UK) and was determined to be 29% of the outside
environment.
Twenty spikes per cultivar, attached to a 4.0 cm peduncle, were inserted in rows
on polystyrene trays with 100 cm × 50 cm × 5 cm of length, width and height,

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 Preharvest sprouting assessment 3
Table 1. Accumulated degree-days (ADD), temperature, falling number and germination percentage of 13 wheat
genotypes under rain simulator with 48 h duration.

Temperature (o C) Falling number (s) Germination (%)

Year Blk∗ ADD Min Mean Max Min Mean Max CV Min Mean Max CV

2007 1 36.0 16.0 22.0 28.0 147.0 257.5 488.0 38.8 0 33.9 81 64.2
2007 2 43.5 19.0 25.8 32.5 63.0 185.8 282.0 34.8 16 52.3 93 41.3
2008 1 45.0 23.8 26.5 29.3 62.0 166.2 309.5 49.7 13 55.4 92 41.7
2008 2 44.0 23.5 26.0 28.5 62.0 132.0 217.0 44.9 33 65.4 90 28.5
2011 1 32.5 16.0 20.3 24.5 72.5 204.0 427.5 55.6 0 40.7 76 57.5
2011 2 32.0 11.0 20.0 29.0 185.0 352.0 481.0 26.4 1 21.5 60 100.4
2012 1 40.5 18.0 24.3 30.5 63.5 155.5 438.0 73.8 0 40.5 62 48.1
2012 2 45.0 21.0 26.5 32.0 62.0 163.6 250.5 41.5 13 51.9 91 43.5
Mean 39.8 18.5 23.9 29.3 89.6 202.1 361.7 45.7 9.5 45.2 80.6 53.2
∗ Test within each year.

respectively. Spikes were spaced at 10 cm between the rows and 5 cm within the row.
The trays were kept on a table at a height of 1.4 m above ground and 0.8 m below the
micro-sprinkler system. The sprinkler system consisted of four tubes distanced 80 cm
apart with sprinklers placed at 60 cm on the line. Each test started around 9:00 a.m.
and finished 48 h later. The nebulizer was controlled by a timer, set to turn on and off
each 30 min, which gave the equivalent to 280 mm of rainfall per day. The polystyrene
trays were moved manually, three times a day, to assure uniform misting. After 48 h
of misting, the spikes were removed from the greenhouse and sun-dried to stop the
germination process. All the spikes were threshed manually and grain germination
was observed in duplicate using 50 random seeds. A magnifying glass (10X) was used
to observe pericarp rupture as an indication of the initiation of germination (Bassoi
and Flintham, 2005; Nyachiro et al., 2002).
The FN (also known as Hagberg Number) is an internationally standardized
method to measure the α-amylase activity in the grain, which indirectly relates to
the degree of sprouting damage. It was determined in samples (7 g) of ground wheat,
which were mixed with 25 mL of distilled water in a FN tube with a stirrer and shaken
to form slurry. This slurry was heated in a boiling water bath at 100 °C and stirred
constantly. The time it took the stirrer to drop through the paste was recorded as the
FN value in a Perten FN 1500 (Perten Instruments), according to the 56–81 B method
of the American Association of Cereal Chemists (AACC, 2000).
The ADD sum of daily DD, used in the study was calculated as follow: DD =
Tmean – Tbase, where Tmean = (Tmax + Tmin)/2, Tmax and Tmin are daily
maximum and minimum air temperature, respectively, and Tbase is the base
temperature of 4 °C (Acevedo et al., 2002). The temperatures (recorded on a
thermograph R. Fuess, Germany) and ADD for the duration of the experiments are
presented in Table 1.
Regression analysis and Pearson correlation coefficients of FN and GP with
temperatures, ADD and coefficient of phenotypic variation were performed using
the softwares Microsoft Office Excel and SAS System.

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 4 L . A . O K U YA M A et al.
R E S U LT S A N D D I S C U S S I O N

There are several methods for testing PHS. The duration of these tests may
vary considerably according to the method and/or type of assessment. This study
considered the FN test as an appropriate measure to determine the damage caused
by PHS in wheat genotypes. In general, the FN value can vary from 62 s for heavily
sprouted grains, with excessive enzyme activity, to well over 500 s for grains from
warm and dry areas. The exact FN values for industrial quality segregation vary
between countries and/or markets depending on the end use purpose of the grain.
The minimum values of the FN required for the top quality and general bread wheat
class in Brazil are 250 and 220 s, respectively (Brazil, 2010).
The statistical analysis, correlation and regression, performed on a large data set
generated over the years in different experiments allowed us to determine that FN
and GP values were highly associated with Tmin, Tmean and ADD, but not Tmax.
The minimum temperature varied from 11.0 °C to 23.8 °C over the years under study
(Table 1). A regression and correlation analysis of Tmin with FN (R2 = 0.7802∗∗ ;
r = −0.8822∗∗ ) and GP (R2 = 0.9076∗∗ ; r = 0.9518∗∗ ) showed a highly significant
influence on the process leading to grain sprouting (Figure 1a). Any increase in the
Tmin led to a reduction in the FN and promoted the grain germination. On the
other hand, the relationship of Tmean with the sprouting was not seen as strong as in
the case of Tmin (Table 1). In spite of the dispersal of data points for both FN and GP
shown in the Figure 1b, the analysis revealed a moderate influence of Tmean on FN
(R2 = 0.6145∗ ; r = −0.7870∗ ) and GP (R2 = 0.7258∗ ; r = 0.8523∗∗ ) values. Therefore,
any increase of the mean temperature, caused by a related rise in Tmin or otherwise,
reduced FN values and increased the percentage of sprouted grains. A similar analysis
conducted on the effect of Tmax with FN and GP was non-significant (Figure 1c).
Our study demonstrates that the minimum and mean temperatures exert far more
impact on the genotype discrimination for sprouting than the maximum temperature.
Similar temperature influence was observed by Mares (1993), who asserted a higher
dormancy in cultivars exposed to temperature regime (day/night) of 26.5/8.5 °C
compared to those exposed to 35.6/17.5 °C.
With respect to the aggregate thermal influence expressed as ADD during the
nebulization tests, the values ranged from 32 to 45 (Table 1). A statistical analysis
of this parameter showed a dispersal of points similar to the mean temperature
and a significant relationship of ADD with FN (R2 = 0.6145∗ ; r = −0.7838∗ ) and
GP (R2 = 0.7258∗∗ ; r = 0.8515∗∗ ), as shown in Figure 2a. As mentioned earlier,
this observation confirms that any increase of DD results in a decline of FN value
leading to higher percentage of sprouting. Taking the reference FN values between
220 and 250 s, accepted by the industry, for selection of genotypes tolerant to PHS,
it was observed that these values correspond to a range of 35–38 ADD and 36–42
% of sprouted grains (Figure 2a). However, larger phenotypic coefficient of variation
observed for GP at lower ADD (R2 = 0.683∗ , r = −0.8267∗ , Figure 2b), indicates
better discrimination of sprouting ability among genotypes at lower ADD values. The
data presented in the Figure 2b also indicates lower coefficient of variation for GP,
confirming our inability to discriminate among genotypes at higher ADD.

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 Preharvest sprouting assessment 5

Figure 1. Falling Number (FN) and germination percentage (GP) of 13 wheat genotypes subjected to simulated
rainfall, related to: (a) minimum temperature; (b) mean temperature; (c) maximum temperature. Each symbol
represents the mean value of 13 genotypes.

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 6 L . A . O K U YA M A et al.

Figure 2. Accumulated degree-day (ADD) of 13 wheat genotypes subjected to simulated rainfall, related to: a) Falling
number (FN) and germination percentage (GP): (b) falling number coefficient of variation (FNCV) and germination
percentage phenotypic coefficient of variation. Each symbol represents the mean value of 13 genotypes.

Thus, for the selection of genotypes based on FN or GP, we suggest using an ADD
value close to 35 to select for FN values between 220 and 250 s. As shown in the
Table 1, the ADD value of 36 coincides with FN values between 147 and 488 s and
grain germination values between 0 and 81%, which allowed us to visualize a wide
range of variation in the performance of genotypes for PHS.
The duration of a test for sprouting resistance in a rain simulator depends on the
weather conditions (Bizova et al., 2011; DePauw et al., 2012) and the type of evaluation

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 Preharvest sprouting assessment 7

(Paterson et al., 1989). Test durations between 60 and 120 h have been used for visual
evaluations of genotypes (Franco et al., 2009; Gavazza et al., 2012; Shorter et al., 2005;
Singh et al., 2014). Converting these durations to DD, based on the temperature data
reported in each of these studies, it was observed that these researchers conducted
their tests at 54 ADD (Shorter et al. 2005), 59 ADD (Gavazza et al., 2012) and 70
ADD (Singh et al., 2014). For analytical evaluations, such as FN or grain rupture
(observed through the magnifying glass), no references were found associating these
data with the measurements of DD.
The research reported here is based on mist duration of 48 h, generating an ADD
between 31 and 45 (Table 1). Under these conditions, ADD value of 35 allowed most
consistent expression of genetic variability for sprouting. These results can serve as an
indicator to calculate the duration of sprouting tests based on the temperature regime
of an area or test site. As an example, at a mean temperature of 21.5 °C, test duration
of 48 h in a rain simulator may be best to discriminate genotypes for FN and/or
percentage of sprouting based on pericarp rupture. We believe that the results of this
large study provide an ideal set of experimental conditions, as well as determine the
test duration for sprouting under controlled conditions. In our evaluations of PHS, we
found desirable to finish the test early in the morning to enable sun or oven drying of
spikes on the same day.

C O N C LU S I O N S

In a rain simulator study to determine PHS resistance of 13 wheat genotypes,

the thermal sum of around 35-DD allowed the highest genotypic differentiation
based on FN values and/or grain rupture of pericarp, expressed as germination
percent. In addition, a mean temperature of around 21.5 °C and test duration of
48 h is considered ideal to evaluate genotypes for the above-mentioned parameters.
Our results show a significant relationship of FN and GP parameters with the
minimum and mean air temperatures and thermal index (ADD). Any increase in the
temperature regime and/or the thermal index led to decreases in FN and increases
in grain germination.

REFERENCES

AACC (2000). Approved Methods of the American Association of Cereal Chemists, 10th edn. St Paul, MN, USA: American
Association of Cereal Chemists.
Acevedo, E., Silva, P. and Silva, H. (2002). Wheat growth and physiology. In Bread Wheat: Improvement and
Production, Food and Agriculture Organization of the United Nations, Rome, Italy. (Eds B. C. Curtis, S. Rajaram, and
H. Gomez Macpherson), Available from: http://www.fao.org/docrep/006/Y4011E/y4011e06.htm (Accessed
May 25, 2016).
Bassoi, M. C., Flintham, J. and Riede, C. R. (2006). Analysis of preharvest sprouting in three Brazilian wheat
populations. Pesquisa Agropecuária Brasileira 41:583–590.
Bassoi, M. and Flintham, J. (2005). Relationship between grain colour and preharvest sprouting-resistance in wheat.
Pesquisa Agropecuária Brasileira 40:981–988.
Bizova, I., Hromadko, M. and Svec, K. (2011). Testing of pre-harvest sprouting of wheat and triticale at the breeding
station Uhretice, Selgen Corp., Czech Republic. Tagung der Vereinigung der Pflanzenzuchter 62:109–110.

Downloaded from https:/www.cambridge.org/core. UNIVERSIDADE ESTADUAL DE LONDRINA, on 14 Mar 2017 at 16:28:50, subject to the Cambridge Core terms
of use, available at https:/www.cambridge.org/core/terms. https://doi.org/10.1017/S0014479717000114
 8 L . A . O K U YA M A et al.
Brasil (2010). Ministério da Agricultura, Pecuária e Abastecimento. Instrução Normativa n° 38, de 30 de novembro
de 2010. Regulamento técnico do trigo. Diário Oficial da República Federativa do Brasil, Brasília, DF,
n 229, Seção 1. Available from: http://sistemasweb.agricultura.gov.br/sislegis/action/detalhaAto.do?method=
visualizarAtoPortalMapa&chave=358389789. (Accessed May 02, 2016).
Czarnecki, E. (1986). Breeding and selecting for preharvest sprouting resistance in red wheats. In Proceedings of 4th
International Symposium on Pre-Harvest Sprouting in Cereals, 45–53 (Ed D. J. Mares, ). Boulder: Westview Press.
DePauw, R. M., Knox, R. E., Singh, A. K., Fox, S. L, Humphreys, D. G. and Hucl, P. (2012). Developing
standardized methods for breeding preharvest sprouting resistant wheat, challenges and successes in Canadian
wheat. Euphytica 188:7–14.
Franco, F. A., Pinto, R. B., Scapim, C. A., Schuster, I., Predebon, C. T. and Marchioro, V. S. (2009). Tolerância à
germinação na espiga em cultivares de trigo colhido na maturação fisiológica. Ciência Rural 39:2396–2401.
Gavazza, M. I. A., Bassoi, M. C., Carvalho, T. C., Bespalhok Filho, J. C. and Panobianco, M. (2012). Methods for
assessment of pre-harvest sprouting in wheat cultivars. Pesquisa Agropecuária Brasileira 7:928–933
Guarienti, E. M., Ciacco, C. F., Cunha, G. R., Del Duca, L. J. A. and Camargo, C. M. O. (2003). Avaliação do efeito
de variáveis meteorológicas na qualidade industrial e no rendimento de grãos de trigo pelo emprego de análise de
componentes principais. Ciência e Tecnologia de Alimentos 23:500–510.
Hanft, J. M. and Wych, R. D. (1982). Visual indicators of physiological maturity in hard red spring wheat. Crop Science
22:584–587.
IAPAR (2003). Informações Técnicas Para as Culturas do Trigo e Triticale no Paraná, 202 Londrina: Instituto Agronômico do
Paraná (IAPAR), (Circular, 126)
MacMaster, G. J. and Derera, N. F. (1976). Methodology and sample preparation when screening for sprouting
damage in cereals. Cereal Research Communications 4:251–254.
Mares, D. J. (1993). Preharvest sprouting in wheat. I Influence of cultivar, rainfall and temperature during grain
ripening. Australian Journal of Agricultural Research 44:1259–1272.
Mares, D. J. and Mrva, K. (2008). Late-maturity alpha-amylase: Low falling number in wheat in the absence of
preharvest sprouting. Journal of Cereal Science 47:6–17.
McMaster, G. S. and Wilhelm, W. W. (1997). Growing degree-days: One equation, two interpretations. Agricultural and
Forest Meteorology 87:291–300.
Nyachiro, J. M., Clarke, F. R., DePauw, R. E., Knox, R. E. and Armstrong, K. C. (2002). Temperature effects on
seed germination and expression of seed dormancy in wheat. Euphytica 126:123–127.
Paterson, A. H., Sorrells, M. E. and Obendorf, R. L. (1989). Methods of evaluation for preharvest sprouting resistance
in wheat breeding programs. Canadian Journal of Plant Science 69:681–689.
Reddy, L. V., Metzger, R. J. and Ching, M. (1985). Effect of temperature on seed dormancy of wheat. Crop Science
25:455–259.
Shorter, S. C., Munro, C. A. and Hodgkinson, J. (2005). Predicting pre-harvest sprouting susceptibility in New
Zealand wheat cultivars. Euphytica 143:309–312.
Singh, A. K., Knox, R. E., Clarke, J. M., Clarke, F. R., Singh, A., DePauw, R. D. and Cuthbert, R. D. (2014).
Genetics of pre-harvest sprouting resistance in a cross of Canadian adapted durum wheat genotypes. Molecular
Breeding 33:919–929.

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of use, available at https:/www.cambridge.org/core/terms. https://doi.org/10.1017/S0014479717000114