Seed Germination and Stress Response of Mung Bean
Seed Germination and Stress Response of Mung Bean
Seed Germination and Stress Response of Mung Bean
) on Various Abiotic
Factors
Custodio, SD.1 De Vera, S.1 Guillermo, KS.1 Maramba, CN.1 Salgado, CA.1
1
Department of Biology, College of Science, University of the Philippines Baguio
February 18, 2015
ABSTRACT
Seeds undergo series of events during germination, wherein a radicle
emerges through the seed coat. This experiment was conducted to understand the
phenomenon on seed germination and seedling growth. Mung Bean, scientifically
known as Vignaradiata L., was used as a test organism. It was exposed to
different stresses pertaining to temperature, pH, osmotic concentration, light, and
hormones. The effect on the rate of seed germination and growth of hypocotylroot length of the germinated seeds were observed and recorded. Data gathered
were analyzed using ONE-WAY ANOVA. On the one hand, based on the results
obtained, seed germination did not occur under low temperature. On the other
hand, increase seedling growth is high at increasing temperature, non-salty
conditions, and with the presence of hormone Gibberellin. However, seedling
growth varies in any pH and light conditions, yet, according to research, higher
rate of growth is more preferable at neutral pH and with the presence of light.
Therefore, based on the stress responses observed in the experiment, it is
indicative that seed germination and seedling growth are possible at different
conditions they have been exposed at. But under optimum conditions, they could
grow as a healthy plant.
INTRODUCTION
In this experiment, it is necessary to comprehend how seed and seedling, germinate and
grow, respectively. To observe the effects of different factors, such as temperature, pH, varying
osmotic concentration, light, and hormones, on the developmental process.
Seeds are embryonic plant in resting condition or seed dormancy. These remain dormant
or inactive until conditions are right for germination. The resumption of growth of this
embryonic plant is known to be the germination stage. Mung Bean (Vignaradiata L) belonging to
the Legume Family, like other plant species, has to meet its optimum conditions, such as having
enough sunlight and plenty of water, in order to germinate and grow as a healthy plant. (Leubner,
2000).
Seed germination is a complex physiological process triggered by imbibitions, where the
uptake of water by dry seed occur, after possible dormancy mechanisms have been released by a
prompt. Germination depends on both external and internal conditions of the growing plant. It
starts when a seed is provided with appropriate water and temperature. Seeds expand as they
imbibe water, and their enzymes and food supplies become hydrated. Hydrated enzymes become
active, thus the seed increases its metabolic activities to produce energy for the growth process.
Moreover, the size of the cell is in proportion with the increase in pressure inside caused by the
water. Under favorable conditions, rapid expansion on embryos growth culminates in the rupture
of the covering layers and emergence of the radicle. The emergence of the radicle is considered
as the completion of germination where the protrusion of the radicle tip is visible. According to
seed physiologists, this transition point is also characterized by the loss of desiccation tolerance
and is a molecular checkpoint
a developmental molecular switch from the germination
program to the seedling program. (Leubner, 2000).
Set-up
Effect of
Table 1. Raw data of the seed germination and seedling growth length.
Percent
Condition Germinati
Hypocotyls-Root Length (cm)
on
Room
90%
7.8 5.5 6.8 4.6 4.3 7.1 5.9 5.3
5.1
4.3
Temperatu
re
Temperatur
e
Effect of
pH
Effect of
Varying
Osmotic
Concentrati
on
Effect of
Light
Effect of
Temperatur
e
Oven
Refrigerat
or
pH 7
(distilled
water)
pH 3
pH 11
0.5 g
NaCl/200
ml
solution
2 g NaCl/
150 ml
solution
5 g NaCl/
150 ml
solution
100%
10.
7
13.
1
9.2
12.
7
13.
5
8.2
7.3
7.3
0%
90%
No seed germinated
7.2
6.7
7.0
6.9
6.5
7.3
7.2
6.9
6.4
7.1
100%
70%
5.5
6.8
6.0
6.7
6.3
5.5
4.8
5.3
6.4
4.8
4.0
5.1
3.9
5.9
3.5
6.2
3.8
4.7
5.8
5.5
100 %
4.0
0
3.9
0
4.4
0
4.7
0
5.1
0
5.0
0
4.6
0
4.1
0
5.3
0
4.8
0
40 %
1.4
0
1.1
0
1.3
0
1.2
0
1.2
0
1.2
0
1.2
0
1.1
0
1.0
0
1.2
0
100 %
1.2
0
1.3
0
1.1
0
1.1
0
1.1
0
1.2
0
1.1
0
1.2
0
1.1
0
1.2
0
5.8
0
6.8
0
7.6
4.5
0
5.4
0
8.2
5.3
0
6.0
0
7.9
4.7
0
5.8
0
8.3
5.2
0
6.2
0
8.2
4.8
0
5.5
0
7.6
5.5
0
6.6
0
7.7
5.6
0
5.6
0
8.4
5.1
0
5.5
0
8.1
Light
100 %
Dark
100%
Water
Indole
Acetic
Acid
Gibberelli
c Acid
100%
4.5
0
6.6
0
8.0
90%
2.8
2.8
3.6
3.4
3.2
3.1
2.6
3.5
3.2
3.6
100%
7.5
0
7.2
0
6.8
0
7.8
0
7.1
0
6.9
0
7.5
0
6.8
0
7.4
0
7.2
0
Mean
Std.
Deviation
LENGTH
Std.
95% Confidence Interval
Error
for Mean
Minimu
m
Maximu
m
room
oven
ref
Total
10
10
10
30
5.6700
9.6800
1.1200
5.4900
1.21568
2.59991
.13984
3.90034
Lower
Bound
4.8004
7.8201
1.0200
4.0336
.38443
.82217
.04422
.71210
Upper
Bound
6.5396
11.5399
1.2200
6.9464
4.30
7.00
.80
.80
7.80
13.50
1.30
13.50
Table 3. The table that shows the output of the ANOVA analysis of the mung bean seedling
length subjected to varying temperatures
ANOVA
LENGTH
Between
Groups
Within Groups
Total
Sum of
Squares
df
Mean
Square
Sig.
366.854
183.427
66.644
.000
74.313
441.167
27
29
2.752
We can see that the significance level is below 0.05 (table 3). Therefore, there is a
statistically significant difference in the mean length of the mung beans subjected to varying
temperatures (see appendix to see post-hoc results).
pH
Next, the effects of pH to the mung beans were observed in the experiment. All of the
mung beans germinated at the three varying pH setting after five days. The mung bean seedlings
added with water which is neutral had lengths ranging from 6.4cm to 7.3cm. The mung bean
seedlings added with a solution at pH 11 which is basic had lengths raging from 4.7cm to 6.8cm.
The mung bean seedlings added with a solution at pH 3 which is acidic had lengths ranging from
3.5cm to 6.3cm (Figure 3).
Neutral pH is the most preferable pH for seed to germinate. Some enzymes may be
inactivated by the very acidic environment. Sometimes, the presence of H + ions only has a
negative effect on plant development (Chodura, 2004).
In the case of some plants their growth in acid soil is possible but seed germination must
take place in less acidified environment because of the need for maintenance of the appropriate
pH of the soil solution for amylolytic enzymes initiating germination (Lee, 1998). However, acid
soil pH stimulates initial development phases of some species. These species include plants with
thick seed coats.
The effect of acid pH may be direct, manifesting itself in dissolution of the seed coat or
indirect which involves the stimulation of conditions for development of some species of fungi
whose action causes perforation of the seed coat (Vleeshouwers et al., 1995)
A study conducted by Bukvic et al. (2007) on the germination of Pisum sativum showed a
higher seed germination affected by lowering pH to 5.0, yet a further development of seedlings
was better at a higher pH.
The measured lengths of the mung bean seedlings were also subjected to statistical
analysis (table 4 and 5) to see if there are significant differences among the different pH levels.
Table 4. The mean, standard deviation and 95% confidence intervals for the dependent variable
(Length) for each separate group (water, pH11, pH3), as well as when all groups are combined.
Descriptives
water
ph11
ph3
Total
N
10
10
10
30
Mean
6.9200
5.6500
5.0000
5.8567
Std.
Deviation
.30478
.73673
1.12940
1.11840
LENGTH
95% Confidence Interval
for Mean
Std.
Lower
Upper
Error
Bound
Bound
.09638
6.7020
7.1380
.23298
5.1230
6.1770
.35715
4.1921
5.8079
.20419
5.4390
6.2743
Minimu
m
6.40
4.70
3.50
3.50
Maximu
m
7.30
6.80
6.40
7.30
Table 5. The table that shows the output of the ANOVA analysis of the mung bean seedling
length subjected to pH levels
ANOVA
LENGTH
Between
Groups
Within Groups
Total
Sum of
Squares
df
Mean
Square
Sig.
19.073
9.536
14.969
.000
17.201
36.274
27
29
.637
We can see that the significance level is below 0.05 (table 5). Therefore, there is a
statistically significant difference in the mean length of the mung beans subjected to different pH
level (see appendix to see post-hoc results).
Osmotic Concentration
intake of the seed in salty conditions, osmotically and by the ion toxicity with accumulation of
Na and Cl ions highly around the seeds, prevents the seed germination.
In the experiment, low solute concentration showed the highest hypocotyl-root axis
length and the high solute concentration showed low or none hypocotyls-root axis length. The
higher the salt concentration, the lower is the hypocotyls-root axis length. Higher solute inside
the seed, then there is lower solute concentration in the outside environment. As the solute moves
out, water goes inside the seed.
Strong delay of germination was observed mainly at the higher level of salt
concentration. A study by Jamil et al. (2005) reported that germination of Brassica species
( cabbage, cauliflower, canola) decreased as the salinity concentration increased.
According to the study conducted by Rahman et al(2009), ascending salt concentrations
not only prevent the germination of the seeds but also extend the germination time by delaying
the start of germination. High salt concentration decreases germination percentage. Salt tolerance
studies which have done during germination time are important for determining the plants salt
tolerance in the early and late growing phases (Zapata et al., 2003).
Seeds require higher amount of water uptake during the germination under the salt stress
due to the accumulation of the soluble solutes around the seeds which increases the osmotic
pressure. This causes excessive uptake of the ions which results in toxicity in plants (Jones,
1986). Moreover, water potential gradient between the external environment and the seeds also
inhibits the primary root emergence (Eneas Filho et al., 1995).
The measured lengths of the mung bean seedlings were also subjected to statistical
analysis (table 6 and 7) to see if there are significant differences among the different osmotic
concentrations.
Table 6. The mean, standard deviation and 95% confidence intervals for the dependent variable
(Length) for each separate group (0.5g NaCl/ 200 ml solution, 2 g NaCl/ 150 ml solution, 5g
NaCl/ 150 ml solution ), as well as when all groups are combined
Descriptives
LENGTH
0.5g
2g
5g
Total
N
10
10
10
30
Mean
4.5900
1.1900
1.1600
2.3133
Std.
Deviation
.48178
.11005
.06992
1.66085
Std.
Error
.15235
.03480
.02211
.30323
Minimu
m
3.90
1.00
1.10
1.00
Table 7. The table that shows the output of the ANOVA analysis of the mung bean seedling
length subjected to varying temperatures
Maximu
m
5.30
1.40
1.30
5.30
ANOVA
LENGTH
Between
Groups
Within Groups
Total
Sum of
Squares
df
Mean
Square
Sig.
77.753
38.876
468.181
.000
2.242
79.995
27
29
.083
We can see that the significance level is below 0.05 (table 7). Therefore, there is a
statistically significant difference in the mean length of the mung beans subjected to varying
osmotic concentrations (see appendix to see post-hoc results).
Light
light
dark
Total
N
10
10
20
Mean
5.1000
6.0000
5.5500
Std.
Deviation
.46188
.52281
.66610
LENGTH
95% Confidence Interval
for Mean
Std.
Lower
Upper
Error
Bound
Bound
.14606
4.7696
5.4304
.16533
5.6260
6.3740
.14894
5.2383
5.8617
Minimu
m
4.50
5.40
4.50
Maximu
m
5.80
6.80
6.80
Table 9. The table that shows the output of the ANOVA analysis of the mung bean seedling
length subjected to varying temperatures
ANOVA
LENGTH
Between
Groups
Within Groups
Total
Sum of
Squares
df
Mean
Square
Sig.
4.050
4.050
16.644
.001
4.380
8.430
18
19
.243
We can see that the significance level is below 0.05 (table 9). Therefore, there is a
statistically significant difference in the mean length of the mung beans subjected different light
availability.
Hormones
Figure 2. Lengths of the mung bean seedlings added with different hormones
Lastly, the effects of hormones to the mung beans were observed in the experiment. All of
the mung beans germinated at the three different hormone setting after five days. The mung bean
seedlings added with Gibberellic Acid had lengths ranging from 6.8cm to 7.5cm. The mung bean
seedlings added with Indole Acetic Acid had lengths raging from 2.6cm to 3.6cm. The mung
bean seedlings added with water had lengths ranging from 2.6cm to 3.6cm (Figure 6).
The evidence for hormone participation comes from the association of hormone
concentration with specific development stages, effects of applied hormones and the relationship
of hormones to metabolic activities. The applications of gibberellins increases the seed
germination percentage by adding the fact that gibberellins also increases the amino acid content
in embryo and cause release of hydrolytic enzyme required for digestion of endospermic starch
when seeds renew growth at germination (Chauhan, J. S. et al., 2009).
The measured lengths of the mung bean seedlings were also subjected to statistical
analysis (table 10 and 11) to see if there are significant differences among the different
hormones.
Table 10. The mean, standard deviation and 95% confidence intervals for the dependent
variable (Length) for each separate group (GA, IAA, water), as well as when all groups are
combined
Descriptives
Mean
Std.
Deviation
LENGTH
95% Confidence Interval
for Mean
Std.
Lower
Upper
Error
Bound
Bound
Minimu
m
Maximu
m
GA
IAA
water
Total
10
10
10
30
7.2200
3.1800
8.0000
6.1333
.33267
.35528
.29059
2.17166
.10520
.11235
.09189
.39649
6.9820
2.9258
7.7921
5.3224
7.4580
3.4342
8.2079
6.9442
6.80
2.60
7.60
2.60
7.80
3.60
8.40
8.40
Table 11. The table that shows the output of the ANOVA analysis of the mung bean seedling
length subjected to varying temperatures
ANOVA
LENGTH
Between
Groups
Within Groups
Total
Sum of
Squares
df
Mean
Square
Sig.
133.875
66.937
624.934
.000
2.892
136.767
27
29
.107
We can see that the significance level is below 0.05 (table 11). Therefore, there is a
statistically significant difference in the mean length of the mung beans subjected to different
hormones (see appendix to see post-hoc results).
The experiment shows the different evidences about the contributing factors which
governs the overall development of plant. Plant growth is not only regulated by their genes but
also regulated by the growth hormones, nutrient and environmental factors.
LITERATURE CITED
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APPENDIX
EFFECT OF TEMPERATURE
LENGTH
Tukey HSD
Subset for alpha = .05
TEM
P
N
1
2
3
ref
10
1.1200
room 10
5.6700
oven 10
9.6800
Sig.
1.000
1.000
1.000
Means for groups in homogeneous subsets are displayed.
a Uses Harmonic Mean Sample Size = 10.00
EFFECT OF PH
Table 13. Multiple Comparisons at the effects of pH
Dependent Variable: LENGTH
Tukey HSD
95% Confidence Interval
Mean
(J)
Difference Std.
Lower
Upper
PH
(I-J)
Error
Sig.
Bound
Bound
ph11 1.2700(*) .35695
.004
.3850
2.1550
ph3
1.9200(*) .35695
.000
1.0350
2.8050
ph11 water -1.2700(*) .35695
.004
-2.1550
-.3850
ph3
.6500
.35695
.182
-.2350
1.5350
ph3
water -1.9200(*) .35695
.000
-2.8050
-1.0350
ph11 -.6500
.35695
.182
-1.5350
.2350
* The mean difference is significant at the .05 level.
(I)
PH
water
LENGTH
Tukey HSD
Subset for alpha = .
05
PH
N
1
2
ph3
10
5.0000
ph11 10
5.6500
water 10
6.9200
Sig.
.182
1.000
Means for groups in homogeneous subsets are displayed.
a Uses Harmonic Mean Sample Size = 10.000.
EFFECT OF OSMOTIC CONCENTRATION
Table 14. Multiple Comparisons at the effects of osmotic concentration
Dependent Variable: LENGTH
Tukey HSD
Mean
(I)
(J)
Difference Std.
OSMOTIC OSMOTIC (I-J)
Error
Sig.
0.5g
2g
3.4000(*) .12887
.000
5g
3.4300(*) .12887
.000
2g
0.5g
-3.4000(*) .12887
.000
5g
.0300
.12887
.971
5g
0.5g
-3.4300(*) .12887
.000
2g
-.0300
.12887
.971
* The mean difference is significant at the .05 level.
LENGTH
Tukey HSD
Subset for alpha = .
05
OSMOT
IC
N
1
2
5g
10
1.1600
2g
10
1.1900
0.5g
10
4.5900
Sig.
.971
1.000
Means for groups in homogeneous subsets are displayed.
a Uses Harmonic Mean Sample Size = 10.000.
EFFECTS OF HORMONES
Table 15. Multiple Comparisons at the Effects of Hormones
Dependent Variable: LENGTH
Tukey HSD
Mean
(I)
(J)
Difference Std.
HORMONE HORMONE (I-J)
Error
GA
IAA
4.0400(*) .14636
water
-.7800(*) .14636
IAA
GA
-4.0400(*) .14636
water
-4.8200(*) .14636
Water
GA
.7800(*)
.14636
IAA
4.8200(*) .14636
* The mean difference is significant at the .05 level.
Sig.
.000
.000
.000
.000
.000
.000
LENGTH
Tukey HSD
Subset for alpha = .05
HORMO
NE
N
1
2
3
IAA
10
3.1800
GA
10
7.2200
water
10
8.0000
Sig.
1.000
1.000
1.000
Means for groups in homogeneous subsets are displayed.
a Uses Harmonic Mean Sample Size = 10.000.