Effect of ethanol and methanol

pre-treatment on the germination

of Vigna radiata seeds
Seeds of Vigna radiata (moong dal) were pre-soaked in 75% by volume ethanol and methanol for 3, 6, 9
and 12 minutes to study their effects on germination of these seeds. The seeds were allowed to germinate
for one week on cotton medium. The number of germinated seeds in each dish, the shoot and root lengths
of each germinated seed were measured for analysis to verify if the experiment had any effect on
germination.

1. The process of seed germination

Seed germination occurs through the following steps:
a) Imbibition: The dry seeds absorb water and the cellular constituents are rehydrated. This causes
swelling of the seed in great force and it ruptures the seed to allow the release of the radicle.
Imbibition causes the rehydration of macromolecules, polysaccharides and proteins.
b) Respiration: Post imbibition, metabolic activity starts in the seed and this initiates respiration.
c) Mobilization of reserves during seed germination: The seed undergoes expansion and division, and
the reserves move from the endosperm to the embryo via a scutellum. The outer layer of special
cells in the endosperm produce hydrolysing enzyms.
d) The embryo becomes heavily metabolic and the cells grow in size, and hence the seedling is formed.

2. Materials and methods

75% ethanol and methanol solutions were prepared and 135 seeds of Vigna radiata(moong dal) were used.
The seeds were allowed to grown on a cotton base over a petri dish.
15 seeds were used as control for the experiment, and were kept for germination without any treatment.
Equal volumes of 75% solutions of ethanol and methanol were prepared. The effect of the time of soaking
was to be found in the course of the experiment and the chosen times for pre-treatment were 3,6,9 and 12
minutes. 15 seeds were soaked for each of these times. The pre-treated seeds were collected and spread on
the cotton base. The seeds were kept undisturbed for a week with periodic watering.
The root lengths and shoot lengths were measured, along with the number of germinations per petri-dish.

3. Inferential Statistical methods

The goals of these methods are i) Estimation of population parameters from statistically obtained
parameters. ii) Hypotheses testing to check if statistically obtained data is close to the population
parameters and to obtain probabilities of getting such results.
 3.1 Central limit theorem
A sampling distribution is a distribution of all possible values of the statistic. The statistic is computed from
random samples from the whole population. The distribution of x’ will be normal if n>30.
Let the mean of x’ be X’.
X’~N(µ,σ 2/n)

Figure 1 Distribution of samplings

We know with 96% certainty that our sample X’ lies within ±2 σ/√n
σ/√n is called the Standard Error about Mean (SEM).
We have a point estimate X’ for the population mean µ, but we need to obtain a range to have a reasonable
chance of µ belonging in the range. From Central Limit Theory, we know that we can think of X’ as a
sample from N(µ, σ2/n). Therefore, 96% of samples should have X’ within 2 SEMs (2 σ/√ n) of µ. Then for
96% of random samples of size n from the population, µ must then be within 2 SEMs of X.
96% Confidence interval= X’ + 2SEM
3.2 Null hypothesis
The null hypothesis (A0) suggests that there is no change from status quo and there is no correlation
between the tested variables in the experiment. It takes a conservative position and dictates that there is no
change from the baseline or between groups.
The alternative hypothesis (Aa), most often the aim of the experiment, suggest there is a relation/change.
The null hypothesis suggests that all means are equal.
µ0= µ1= µ2
No experiment can give 100% certain results and statistical inference is used to arrive at a conclusion as to
which hypothesis is true. In this case, there is always a chance of error. This is portrayed by the matrix
below:

Scientist’s conclusion
µa is true µ0 is true
µa is No error Type II error
Reality true
µ0 is Type I error No error
true
Type I error is generally more dangerous than Type II, as one can intuitively infer. This could be
strengthened by drawing an analogy to verdicts by the Court of Law to execute an innocent when compared
to acquitting a guilty.
Let α,β be the probabilities of Type I and II errors respectively. These are in a reciprocal relationship and
hence both cannot be minimized simultaneously. Thus, the maximum allowed value for α is kept at 5% and
β at 20%.
Let p denote the probability of obtaining a result equal or more ‘extreme’ than what is observed, assuming
the null hypothesis is true. The threshold value for p is generally taken to be 0.05. An experimental value
higher than this would have strong evidence towards the null hypothesis and hence has to be adopted. The
value of p keeps the value of α below 0.05 too.
3.3 Analysis Of Variance (ANOVA)
Developed by RA Fischer, ANOVA is used to determine if there is any significant difference between
means of groups of data. In one-way ANOVA, these groups are meant to vary under a single factor.
ANOVA assumes the following:
i) Normal distribution of the data
ii) Constant variance
iii) Simple independent groups of data
When there is a significant difference, ANOVA leads to the rejection of the null hypothesis.
The ANOVA test is accompanied by a Tukey Q test. An ANOVA test can tell you if your results
are significant overall, but it won’t tell you exactly where those differences lie. This is accomplished by Tukey Q test.
After ANOVA, Tukey’s test could be used to find out how and if two groups are different. To test all pairwise
comparisons among means using the Tukey Honesty Significant Difference (HSD), we calculate HSD for each pair
of means using the following formula:
4. Observations

a) Number of seeds germinated

Control: 15

Time Ethanol Methanol

3 15 11
6 15 13
9 14 12
12 14 14
b) Methanol readings

Root length(mm) Shoot length(mm)

Control M3 M6 M9 M12 Control M3 M6 M9 M12
167 159 26 106 146 43 29 16 20 13
172 171 45 138 124 37 32 11 27 20
128 132 49 87 55 21 34 16 12 16
170 112 55 100 132 14 21 17 12 27
160 144 64 149 111 43 35 21 29 24
90 169 56 44 60 32 22 15 21 18
173 0 10 0 5 28 19 9 10 30
188 0 40 102 31 31 20 6 10 36
96 111 38 95 61 41 28 15 26 6
152 0 0 0 6 13 15 25 20 11
149 167 15 106 34 26 23 10 25 3
87 0 0 0 0 30 0 20 20 9
191 197 119 109 143 31 0 9 0 0
122 69 41 78 116 28 0 0 0 0
40 132 31 135 45 26 0 0 0 0
139 104.2 39.26667 83.26667 71.26667 29.6 18.53333 12.66667 15.46667 14.2
Root to shoot ratios

CONTROL M3 M6 M9 M12
0.257485 0.18239 0.615385 0.188679 0.089041
0.217647 0.285714 0.2 0.27 0.151515
0.109948 0.172589 0.134454 0.110092 0.111888
0.114754 0.304348 0.414634 0.153846 0.232759
0.25 0.204678 0.466667 0.210145 0.193548
0.2 0.152778 0.234375 0.14094 0.162162
0.291667 0.171171 0.236842 0.105263 0.491803
0.344444 0.118343 0.107143 0.227273 0.6
0.320313 0.212121 0.306122 0.298851 0.109091
0.325 0.113636 0.806452 0.148148 0.244444
0.174497 0.137725 0.666667 0.235849 0.088235
 c) Ethanol readings

Shoot length(mm) Root length(mm)

Control E3 E6 E9 E12 Control E3 E6 E9 E12
167 151 150 176 84 43 30 15 24 11
170 182 172 137 112 37 40 21 17 17
191 172 171 150 49 21 31 22 24 12
122 200 185 173 132 14 10 21 14 11
172 220 138 92 63 43 27 33 15 8
160 144 131 197 67 32 24 22 21 20
96 75 161 27 37 28 31 21 23 14
90 153 162 147 96 31 29 17 21 14
128 166 39 74 8 41 33 9 27 14
40 95 109 143 3 13 32 23 16 14
149 138 90 100 11 26 20 28 23 14
188 122 93 132 27 30 36 21 22 9
173 184 93 103 31 31 26 29 17 12
152 144 181 166 0 28 22 11 20 0
87 15 25 0 0 26 23 12 0 0
139 144.0667 126.6667 121.1333 48 29.6 27.6 20.33333 18.93333 11.33333

Root-shot ratios

CONTROL E3 E6 E9 E12
0.257485 0.198675 0.1 0.136364 0.130952
0.217647 0.21978 0.122093 0.124088 0.151786
0.109948 0.180233 0.128655 0.16 0.244898
0.114754 0.05 0.113514 0.080925 0.083333
0.25 0.122727 0.23913 0.163043 0.126984
0.2 0.166667 0.167939 0.106599 0.298507
0.291667 0.413333 0.130435 0.851852 0.378378
0.344444 0.189542 0.104938 0.142857 0.145833
0.320313 0.198795 0.230769 0.364865 1.75
0.325 0.336842 0.211009 0.111888 4.666667
0.174497 0.144928 0.311111 0.23 1.272727
0.159574 0.295082 0.225806 0.166667 0.333333
0.179191 0.141304 0.311828 0.165049 0.387097
5. Results of ANOVA and Tukey’s Q test

A) Ethanol

Shoot one-way ANOVA

Shoot matrix for ANOVA and Tukey's test

Root one-way ANOVA

Root ANOVA and Tukey's test matrix

 One-way ANOVA for root-shoot ratio

B) Methanol

One-way ANOVA for shoot

ANOVA for shoot and Tukey's test

 One-way ANOVA for root

ANOVA for root and Tukey’s test

One-way ANOVA for root shoot ratio

Pairwise ANOVA and Tukey's tests for root-shoot ratio

6. Analysis and Inferences
The germinated seeds of Vigna radiata showed differences in root and shoot lengths. The experimental
observations were analysed post hoc using ANOVA and Tukey’s pairwise Q tests. The results of these
tests show the following:
1. Seeds pre-treated with Ethanol
For the shoot length, the value of p from ANOVA was 3.673E-6 which is a very low value, and we can
safely reject the null hypothesis. We now can be sure that pre-treatment with ethanol alters the shoot
length during germination.
The results of Tukey’s pairwise Q tests show only E12 seeds have pairwise p values less than 0.05 and Q
values show significant differences only for E12. Results of E3, E6 and E9 showed p > 0.05 against each
other and against control as well. This could suggest that pre-treatment with ethanol has a significant
effect on the shoot length only if they are soaked for a minimum of 12 minutes. The similar pairwise Q
values and the p > 0.05 values of pairwise comparisons between E3, E6 and E9 indicate that they have
very closely equal means and that changing the time by 3 minutes has not very drastic effects on the
shoot length till it is soaked for at least 12 minutes.
For root length, the p value was found to be 2.843E-9 which is also very small and we can reject the null
hypothesis in this case as well. This implies that pre-treatment with ethanol has an effect on the root
length too during germination of Vigna radiata seeds. Pairwise results show large Q values and p <0.05
for all comparisons except E3-E6 and E6-E9. This implies the pre-treatment with ethanol has more
effects on the root length when soaked for a longer time. The E3-E6 and E6-E9 pairs show larger p
values because the effects of 3-minute soaking and 6-minute soaking were close and irresolvable.
As we can see from the averages of our data, soaking seeds in ethanol decreases the shoot and the root
lengths and the average decreases with time of pre-treatment. The shoot-root ratio, however gives a p value
greater than 0.05, implying that the ratio doesn’t change. We have established that the shoot and root length
change on pre-treatment, therefore, we know that the change in root length and shoot length due to pre-
treatment must be proportional.
2. Seeds pre-treated with methanol
ANOVA results for shoot length result in a p value of 2.73E-5 which directly leads us to reject the null
hypothesis. We can infer from this that methanol pre-treatment has an effect on the shoot length of
germinating seeds. The pairwise comparisons show p < 0.05 values against only control with the exception
of the M3-M6 pair.
The Q values also suggest not very significant difference except between M3 and M6. This indicates that
the effect of pre-treating seeds with methanol does not depend on the time the seeds are soaked. Thus, the
effect of methanol on the shoot length is immediate on immersion.
For the root, the ANOVA results give us a p of 0.0001807. The null hypothesis is rejected in this case
too and we can readily infer that the pre-treatment has resulted in some effect on the root length of the
seeds during germination. Pairwise tests show a single row of p values less than 0.05, the row pertaining to
that of the control. They also show a significantly large Q value in pairs that consist of the control. Here,
with no exceptions, it is evident that although soaking affects the germination of the seed, the time of
soaking in methanol does not influence the root length and the effect of methanol is instantaneous.
The ANOVA results of root-shoot ratio of methanol treated seeds give a p value of 0.01226, which implies
the root-shoot ratio is affected by methanol pre-treatment, or in other words, methanol pre-treatment
affects the root and shoot length disproportionately. However, pairwise tests show only M3-M6 and
M6-M9 pairs have p < 0.05. Tukey’s Q also shows maximum values at these pairs.
Since the effects of methanol pre-treatment do not depend on time, the disproportionate change of root and
shoot lengths also does not depend on time, and these pairs could be anomalous.
Summary of the inferences

 The null hypotheses were rejected in all cases but the shoot-root ratio of seeds pre-treated in
ethanol.
 Pre-treatment with ethanol alters the shoot length and root length proportionately and the shoot
length is affected only when soaked at least for 12 minutes.
 The root length is altered when seeds are pre-soaked in ethanol and this effect is dependent on the
time of soaking.
 The methanol pre-treatment alters the shoot and root length without any time-dependence
indicating the instantaneous effect of soaking the seeds in methanol.
The experiment results also suggest that 3-minute intervals are very short and the effects are irresolvable.
A larger time interval will provide clearer and resolved results and the implications that pre-treatments
affect seed germination could be quantified.