Professional Documents
Culture Documents
Seed Germination Paara
Seed Germination Paara
Seed Germination Paara
Extraction
Yousry A. El-Kassaby, Ian Moss, Dave Kolotelo, and Michael Stoehr
Abstract: A method for mathematically describing cumulative seed germination using the four-parameter Hill
function is described. The function’s four parameters allowed both direct or indirect biological interpretation of
germination behavior and the impact of seed pretreatments on germination improvement. Three parameters, a,
b, and c, allowed direct assessment of germination capacity (%), the shape and steepness of the germination
course, and germination speed (time to reach 50% germination). The fourth parameter y0 permitted estimation
of the lag time (germination onset). The mathematical expression of cumulative seed germination of unstratified
and stratified seed made it possible to quantitatively estimate seedlot dormancy and the amount of germination
improvement caused by a specific seed pretreatment. This was accomplished by estimating the area between the
two cumulative germination curves through integration. The utility of the proposed approach was demonstrated
T
HE GERMINATION PERFORMANCE of a seedlot can be better understand germination behavior and the effect of
characterized by three parameters; time of germina- seed pretreatments on germination enhancement. Many
tion onset (lag), germination speed (rate), and extent curve-fitting routines have been used and their suitabilities
or capacity (cumulative germination percentage at the end for characterizing and managing seedlots have been criti-
of the testing period). The genetic makeup of a seedlot cally assessed (Tipton 1984; Brown and Mayer 1988a,
varies from a bulk collection (e.g., originating from an 1988b).
unknown number of parents) to a full-sib family (derived Curve-fitting methods using the Weibull, Gompertz, and
from controlled pollination) and strongly affects the vari- probit functions have been used to characterize the germi-
ability of its germination parameters (El-Kassaby et al. nation of conifer seeds, compare seed pretreatments, and
2002). Germination parameters are useful for estimating the measure seedlot differences (Bonner and Dell 1976; Rink et
conversion of seeds to seedlings and, thus, the suitability of al. 1979; Campbell and Sorensen 1979; Bramlett et al. 1983;
a seedlot for commercial seedling production. Germination Dell et al. 1983; Leadem 1986, Stoehr et al. 1998). How-
parameters are also useful in determining the type of seed ever, the parameters in these functions do not lend them-
pretreatment as well as nursery management practices selves to simple biological interpretation. The use of curve-
needed to attain a high level of germination (Kolotelo et al. fitting to interpret and understand germination patterns has
2001). been reviewed and endorsed by Scott et al. (1984).
In most cases, germination capacity (% germination) is In this article, we introduce the four-parameter Hill func-
the most important parameter in determining the suitability tion (4-PHF) as a curve-fitting method for describing conif-
of a seedlot for commercial use, but germination rate influ- erous seed germination. We also show that this function is
ences the uniformity of emergence in nurseries (Ching amenable to biological interpretation, thereby enhancing
1959; Thomson and El-Kassaby 1993; El-Kassaby 2000). our understanding of germination differences among geno-
Several attempts have been made to simplify the character- types, seedlots, and seed pretreatments.
ization of seed germination performance by distilling the
various germination parameters into a single index or value
(e.g., Czabator 1962). However, reducing multiple germi- The 4-PHF and Germination Parameters
nation parameters into one index provides an incomplete
picture of germination behavior. The cumulative germination count of a seedlot was mod-
When germination is properly fitted to a mathematical eled to fit the 4-PHF (Equation 1) using the curve-fitting
function, the parameters of that function can be used to routine in MATLAB (The MathWorks, Inc. 2005), with the
Yousry A. El-Kassaby, University of British Columbia, Forest Sciences, 2424 Main Mall, Vancouver, BC V6T 1Z4, Canada—Phone: (604) 822-1821; Fax:
(604) 822-9102; y.el-kassaby@ubc.ca. Ian Moss, ForesTree Dynamics Ltd.—forestree@shaw.ca. David Kolotelo, British Columbia Ministry of Forests and
Range, Tree Improvement Branch, Tree Seed Centre— dave.kolotelo@gov.bc.ca. Michael Stoehr, British Columbia Ministry of Forests and Range, Research
Branch—michael.stoehr@gov.bc.ca.
Acknowledgments: We extend our thanks to M. El-Sharkawi (University of Washington, Seattle, WA), M. Fayed and M. Yasein (University of Victoria,
Victoria, BC, Canada), and M. Senousy (University of British Columbia, Vancouver, BC, Canada) for assisting with the model development and introduction
to MATLAB. This work was funded, in part, by the Johnson’s Family Forest Biotechnology Fund, British Columbia Forest Genetics Council’s Applied Forest
Genetics and Biotechnology Grant, and The Natural Sciences and Engineering Research Council of Canada-Industry Research Chair Funds to Y.E.K.
Manuscript received March 14, 2007, accepted September 21, 2007 Copyright © 2008 by the Society of American Foresters
ax b 80 40
y ⫽ y0 ⫹ , (1)
c ⫹ xb
b
70 35
Germination %
1st Derivative
60 30
where y is the cumulative germination percentage at time x,
y0 is the intercept on the y axis (ⱕ0), a is the asymptote, or 50 25
maximum cumulative germination percentage, which is 40 20
equivalent to germination capacity, b is a mathematical
30 15
parameter controlling the shape and steepness of the germi-
nation curve (the larger the b parameter, the steeper the rise 20 10
toward the asymptote a, and the shorter the time between 10 5
germination onset and maximum germination), and c is the
0 0
“half-maximal activation level” measured in days (Keshet 0 2 4 6 8 10 12 14 16 18 20
冑
line represents the fitted curves.
⫺ y0 cb
lag ⫽ b , (2)
a ⫹ y0
periodic germination increment. The former represents the
where y0 is the intercept on the y axis. maximum total amount of germination divided by the total
Another way to characterize the speed of germination is time elapsed, whereas the latter are maximum rates of
to consider the duration (Dlag-50) between the time at ger- germination at a point or small interval in time that may be
mination onset (lag) and that at 50% germination (c). The alternatively referred to as instantaneous rates of germina-
shorter the Dlag-50, the more prompt and uniform the ger- tion. By definition the maximum instantaneous rate must
mination and the steeper the germination curve (i.e., higher exceed the maximum average rate of germination and so
b). Thus, Dlag-50 and b should be negatively correlated, and too, the maximum instantaneous rate must occur in advance
Dlag-50 is a good indictor of germination speed, uniformity, of the maximum average rate of germination (Table 1). Both
and vigor. Collectively, the Dlag-50, c, and lag parameters TMGR and PV are important to the production of seedlings.
provide a better indicator of germination speed than R50⬘ However, TMGR defines the inflection point of the cumu-
alone. This is because R50⬘ is a function of time only and lative germination curve or the point in time when the
could be the same for two seedlots with different germina- instantaneous rate of germination starts to decline. It also
tion behaviors (i.e., similar R50⬘ but different germination defines the scale of the cumulative germination curve as this
onset times and/or different germination capacities). is proportional to the maximum instantaneous rate of ger-
The instantaneous rate of germination was also estimated mination. In contrast, PV is no more important than any
from the partial derivative of Equation 1 and can be used to other point along the line in terms of uniquely defining the
measure the sensitivity of any specific variable as follows: shape and scale of a sigmoid curve. Thus, TMGR is bio-
⭸y abc b x b⫺1 logically more meaningful. The shorter the TMGR, the
s ⫽ ⫽ b , (3) more vigorous is the seedlot and the time to reach R50⬘ is
⭸x 共c ⫹ x b 兲 2
shorter. Therefore, the 4-PHF is suitable for extracting
where s is the daily rate of germination. This function was meaningful germination parameters and characterizing the
plotted against time, and the time required to reach maxi- behavior of germination at different times during the ger-
mum germination rate (time at maximum germination rate mination test. Furthermore, if germination is expressed by a
[TMGR]) was determined as mathematical function, then this function could be used to
冑
assess the effectiveness of seed pretreatments using the
cb (b ⫺ 1) integration method of Richter and Switzer (1982). Using
TMGR ⫽ b , (4)
b ⫹ 1 this method, dormancy is defined as the amount of ger-
mination improvement caused by a specific dormancy-
The location of TMGR on the germination curve is the point breaking treatment. The improvement is quantitatively
at which the instantaneous slope is at maximum (Figure 1). determined by the difference between the areas under the
This point is different from Czabator’s “peak value (PV)” germination curves before and after seed pretreatment
which is “the maximum quotient derived from all of the (Figure 2) and is defined as the dormancy index (DI), which
cumulative full-seed germination percent on any day di- is calculated as
vided by the number of days to reach this percent” (Czaba-
tor 1962). The difference between PV and TMGR is similar
to the difference between mean germination increment and
DI ⫽ 兰 tn
t0
dt
共 y 1 ⫺ y 2) , (5)
Unstratified Stratified
Cumulative Cumulative
Day Germination PV TMGR Germination PV TMGR
1 0 0.00 0.00 0 0.00 0.00
2 0 0.00 0.00 0 0.00 0.12
3 0 0.00 0.01 0 0.00 1.24
4 0 0.00 0.04 0 0.00 5.97
5 0 0.00 0.13 2 0.40 16.57
6 0 0.00 0.36 42 7.00 26.08
7 0 0.00 0.84 61 8.71 21.14
8 0 0.00 1.67 70 8.75 12.30
9 3 0.33 2.84 81 9.00 6.18
10 5 0.50 4.07 84 8.40 3.03
Statistical Analyses
where DI is the dormancy index (surface area), y1 and y2 are
the 4-PHF for the seedlot after and before the dormancy Germination parameters (a, b, c, TMGR, and lag) for
breaking treatment, respectively, and t0 and tn are the time each replication within each family were generated by fit-
in days at the beginning and end of the germination test, ting the 4-PHF curve and then subjecting the parameters to
respectively. Thus, zero and negative values imply that the analyses of variance using the additive linear model,
treatment either has no effect on improving seedlot perfor- Y ijk ⫽ ⫹ T i ⫹ F j ⫹ TF ij ⫹ 共 ij 兲 k , (6)
mance, or is detrimental (see below).
where is the overall mean, Ti is the effect of the ith seed
pretreatment (stratification) (i ⫽ 1 to 2, fixed effect), Fj is
Parameters Demonstration
the effect of the jth family (i ⫽ 1 to 18, random effect), TFij
Germination parameters for 18 wind-pollinated seedlots is the effect of the interaction between ith family and seed
(families) from individual lodgepole pine (Pinus contorta ith pretreatment; and (ij)k is the residual error (k ⫽ 1 to 4).
var. latifolia) orchard parents were determined from stan- With the exception of germination capacity (which was
Table 3. ANOVA for the germination parameters of seedlots from 18 lodgepole pine families subjected to seed pretreatment (4 weeks stratification)
80
Germination %
4
Dlag-50
60
3
40
20 2
0 1
20
6
15
5
10
4
5 3
12
60
10 50
R'50
40
DI
8
30
6
20
4 10
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Family Family
Figure 3. Germination parameters for 18 stratified (E) and unstratified (F) lodgepole pine seedlots (families) (see text
for parameters explanation). Vertical lines represent 95% confidence intervals.
which is precisely why the parameter c is referred to as level of germination. Another correlation of interest was the
thehalf-maximum rate. Insofar as c decreases, so too will negative correlation between b and Dlag-50 (⫺0.71 and
the time to the point of inflection because the inflection ⫺0.82 for the stratified and unstratified seedlots, respec-
point will generally be somewhere in the vicinity of the 50% tively). This correlation confirms the role of germination
onset (promptness) and speed on compacting the germina-
70
tion duration, which in turn affects the curve shape and
steepness (Table 2). As expected, the faster the germination,
60
the earlier R50⬘ is reached, resulting in a shorter Dlag-50
50
period. Thus, correlations between the time of germination
onset (lag) and both TMGR and R50⬘ are expected to pro-
duce contrasting results for stratified (TMGR ⫽ 0.63, R50⬘
DI
40
⫽ 0.67) and unstratified (TMGR ⫽ 0.18, R50⬘ ⫽ 0.22) seed.
30
These correlations indicate that the stratification treatment
was effective in speeding the onset of germination as well as
20
the time needed to reach 50% germination (Table 2). This
result is further confirmed by the high correlation between
10
TMGR and Dlag-50 for both stratified (0.86) and unstratified
0 20 40 60 80 100 (0.89) seed (Table 2). The parameter b (shape and steep-
Stratified - unstratified germination % ness) of the stratified seed curve did not correlate with its
germination capacity (a), indicating that the steepness of the
Figure 4. Correlation between the difference between stratified and
unstratified germination percentages versus DI as determined by the
germination curve is not associated with higher germina-
Hill function for 18 lodgepole pine seedlots (families). tion. Finally as expected, b produced significant negative
80 80
Germination %
Germination %
60 60
40 40
20 20
0 0
US 1WK 2WK 3WK 4WK 5WK US 1WK 2WK 3WK 4WK 5WK
6 5
5
4
4
Lag (Days)
Lag (Days)
3
3
2
1 1
0 0
US 1WK 2WK 3WK 4WK 5WK US 1WK 2WK 3WK 4WK 5WK
5 5
4 4
D0-50 (Days)
D0-50 (Days)
3 3
2 2
1 1
0 0
US 1WK 2WK 3WK 4WK 5WK US 1WK 2WK 3WK 4WK 5WK
10 10
8 8
TMGR (Day)
TMGR (Day)
6 6
4 4
2 2
0 0
US 1WK 2WK 3WK 4WK 5WK US 1WK 2WK 3WK 4WK 5WK
45 45
40 40
35 35
30 30
25 25
DI
DI
20 20
15 15
10 10
5 5
0 0
1WK 2WK 3WK 4WK 5WK 1WK 2WK 3WK 4WK 5WK
Figure 5. Interior spruce (left) and lodgepole pine (right) germination parameters as affected by stratification duration.
Black and white columns represent interior spruce seedlots 60751 and 60425 and lodgepole pine 2001 and 2002 seed
orchard lots, respectively. US, unstratified.
correlations with early germination parameters such as differences (i.e., quantifying fixed effects violates analysis
TMGR and Dlag-50 (Table 2). of variance assumptions). The family effect and the seed
Variation in germination parameters was highly signifi- pretreatment ⫻ family interaction accounted for 23–59%
cant for the treatment ⫻ family interaction (Table 3). Thus, and 19 –52% of the variation, respectively (Table 3). These
graphical representations are best for interpreting treatment results are in line with previously published estimates for
and family effects (Figure 3). Seed pretreatment was highly the same species (Krakowski and El-Kassaby 2005). How-
significant for all germination parameters; however, the use ever, the germination parameters tested in the present study
of the mixed model did not allow us to estimate treatment are the product of curve fitting using the 4-PHF. Graphical