Influence of Pressure on Germination of Garden Cress, Leaf Mustard, and Radish Seeds at Various Temperatures

doi:10.4236/ajps.2011.23050

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American Journal of Plant Sciences, 2011, 2, 438-442

doi:10.4236/ajps.2011.23050 Published Online September 2011 (http://www.SciRP.org/journal/ajps)

Influence of Pressure on Germination of Garden

Cress, Leaf Mustard, and Radish Seeds at

Various Temperatures

Akio Shimizu, Jun Kumakura

Department of Environmental Engineering for Symbiosis, Faculty of Engineering, Soka University, Tokyo, Japan.

Email: [email protected]

Received June 16th, 2011; revised July 12th, 2011; accepted July 25th, 2011.

ABSTRACT

The effects of hydrostatic pressure (0.1 - 400 MPa) and tempera ture (4˚C, 25˚C, and 35˚C) on the germination o f three

types of seeds (garden cress, leaf mustard, and radish ) were studied. The normal germination ra te of the three types of

seeds was decreased at high hydrostatic pressure, and germination time tended to be delayed. Pressure and tempera-

ture had two types of effects on seed germination. Germination of garden cress and leaf mustard seeds was more resis-

tant to pressure at lower temperature. Conversely, germination of radish seeds was most pressure-sensitive at low tem-

perature, and germination drastically decreased with treatment at 50 MPa and 4˚C. Generally, pressure and temp era-

ture effects on protein structure and enzyme activity have been classified into two types, “hillside”-like (pressurization

decreases the stable temperature range) and “tongue”-like (stabilizing effect of moderate pressure against heat dena-

turation). Therefore, the type of temperature-pressure effects on germination of garden cress and leaf mustard seeds is

classified as “hillside”-like and that of radish seeds is classified as “tongue”-like, similarly to the generally observed

effects on protein denaturation.

Keywords: High Hydrost atic Pressure, Germination, Seed, Temperature

1. Introduction

Several methods have been proposed for reducing mi-

crobial counts [1-8], but none of them completely elimi-

nate pathogens inside seeds. High hydrostatic pressure

treatment has been studied as a new approach [9,10-15].

Pressure is an important physical parameter on a par with

temperature, but because pressure experiments are diffi-

cult to perform, the effectiveness of pressure treatment

remains unclear. Nevertheless, high hydrostatic pressure

treatment has several merits as compared to other meth-

ods: because pressure does not use disinfectants, it leaves

no residue on the product [9,10,14], and whereas heat is

distributed inhomogeneously and cannot easily reach the

central parts of the seed, pressure reaches the inside of

seeds homogeneously and instantaneously [16]. On the

other hand, application of high hydrostatic pressure to

seeds influences germination ability [9,14,15]. Therefore,

prior to evaluating the effect of pressure on reducing mi-

crobial counts, we need to examine the effects of high

hydrostatic pressure and temperature on germination

percentage and germination time. These parameters have

been studied widely for spores [17-22], but rarely for

seeds [9,14,15]. In particular, to the best of our knowl-

edge, the effect of high hydrostatic pressure on seed

germination at low temperature (4˚C, typical refrigerator

temperature) has not been investigated. Determination of

the effects of pressure and temperature on seed germina-

tion would be of great importance not only for collecting

fundamental data on the sanitization of seeds but also

from the viewpoint of vegetable physiology. In this study,

we evaluated the effects of various combinations of hy-

drostatic pressures (0.1 - 400 MPa) and temperatures

(4˚C, 25˚C, and 35˚C) on the germinability and germina-

tion time of three types of seeds: garden cress, leaf mus-

tard and radish.

2. Materials and Methods

2.1. Seeds

Garden cress (place of production: Denmark) and leaf

mustard (place of production: Japan) seeds were pro-

vided by Takii Seed (Kyoto, Japan), and radish (place of

production: USA) seeds were provided by Nakahara

Influence of Pressure on Germination of Garden Cress, Leaf Mustard, and Radish Seeds at Various Temperatures439

Seed (Fukuoka, Japan). Seeds were stored at room tem-

perature until high hydrostatic pressure treatment.

2.2. High Hydrostatic Pressure Treatment

Approximately 50 seeds were packed in a silicone tube 8

mm in diameter, 1 mm thick and 80 mm long filled with

ultrapure water, with both ends of the tube stopped with

Teflon rods (7 mm diameter, 15 mm long). The tube was

set in a high-pressure vessel, which was filled with water

as the pressure medium and subjected to 50, 100, 150,

200, 250, 300 and 400 MPa pressure for 10 min at 4˚C,

25˚C, and 35˚C with a hydrostatic pump (Kouatu System,

Japan). The compression rate was about 150 MPa/min,

and decompression was immediate. Control seeds were

not pressurized but were otherwise treated in the same

way as pressure-treated seeds. The temperature of the

high-pressure vessel was maintained at a preset tempera-

ture by circulating water. Experiments were replicate two

times.

2.3. Determination of Germination

Control and pressure treated seeds were spread on 90-

mm-diameter filter paper moistened with ultrapure water

in a plastic dish and germinated at room temperature for

1 week in the dark. Ultrapure water was sprinkled on the

dish every day. The germination percentage (the number

of germinated seeds/total number of seeds) was deter-

mined by counting the number of germinated seeds.

3. Results

All seeds treated with high hydrostatic pressure in this

experiment kept their shape better, similarly to untreated

seeds; however, the seed coat of some seeds was slightly

cracked. Figures 1-3 show the time dependence of gemi-

nation percentage for the three types of seeds after high

hydrostatic pressure treatment at 4˚C, 25˚C, and 35˚C.

Based on these figures, garden cress seeds were the

most pressure-resistant at all temperatures studied. As

shown in Figure 1, differences in germination percent-

age of garden cress seeds were observed at shorter ger-

mination times up to 2 days; the germination percentage

decreased with high hydrostatic pressure treatment. The

germination percentage of garden cress reached over

90% on the seventh day at the three different tempera-

tures, except for seeds treated at 400 MPa and 35˚C.

Therefore, the effect of pressure on the germinability of

garden cress appeared to be negligible up to 300 MPa at

4˚C - 35˚C.

As shown in Figure 2, the effects of pressure and tem-

perature on the germinability of leaf mustard seeds

showed a similar tendency to that of garden cress, but

leaf mustard seeds were more pressure-sensitive than

garden cress. A significant decrease in the germinability

of leaf mustard was observed at 400 MPa, and the ger-

mination percentages on the seventh day decreased with

increasing temperature, with values at 4˚C, 25˚C, and

35˚C of about 70%, 60%, and 50%, respectively.

Similarly to the garden cress and leaf mustard seeds,

over 90% of the untreated control radish seeds germi-

: 0.1 MPa, : 50 MPa, : 100 MPa, : 150 MPa, : 200 MPa, : 250

MPa,



: 300 MPa,



: 400 MPa.

Figure 1. Effects of high hydrostatic pressure (0.1 - 400

MPa) treatment at 4˚C (a), 25˚C (b), and 35˚C (c) for 15 min

on the germination of garden cress seeds.

: 0.1 MPa, : 50 MPa, : 100 MPa, : 150 MPa, : 200 MPa, : 250

MPa, : 300 MPa,



: 400 MPa.

Figure 2. Effects of high hydrostatic pressure (0.1 - 400

MPa) treatment at 4˚C (a), 25˚C (b), and 35˚C (c) for 15 min

on the germination of leaf mustard seeds.

Influence of Pressure on Germination of Garden Cress, Leaf Mustard, and Radish Seeds at Various Temperatures

440

nated after 1 day at all three temperatures; however, the

germinability of pressure-treated radish seeds (Figure 3)

showed significant differences compared to both garden

cress and leaf mustard seeds (Figures 1 and 2). Radish

seeds were the most pressure-sensitive of the three types

of seeds. At 4˚C, the germination percentage decreased

to about 25% at a pressure of only 50 MPa, and this low

germination percentage was maintained up to 400 MPa.

However, detailed analysis of the data revealed that the

germination percentage recovered slightly at the higher

pressures of 350 and 400 MPa. The same tendency has

been observed for the germination of sesame seeds 20˚C

[14]. Therefore, it is possible that the germination of rad-

ish seeds recovers at higher pressures, but future experi-

ments are needed to verify this finding, as the change

observed in the present study was relatively small. In

contrast, at both 25˚C and 35˚C, the germination per-

centage of radish seeds decreased gradually with in-

creasing pressure up to 400 MPa.

The time to 50% germination of pressure-treated gar-

den cress seeds, when treated up to 250 MPa, did not

significantly differ compared with untreated seeds, but

showed an approximately 1-day delay with high pressure

treatment at 300 and 400 MPa. Leaf mustard seeds

showed the same tendency as garden cress seeds, with an

approximately 3-day delay at 25˚C and a 5-day delay

when treated at 35˚C at 400 MPa. The 50% germination

time of radish was highly pressure-sensitive compared to

both garden cress and leaf mustard seeds, as shown in

Figures 1-3; it was delayed with increasing pressure up

: 0.1 MPa, : 50 MPa, : 100 MPa, : 150 MPa, : 200 MPa, : 250

MPa, : 300 MPa,



: 400 MPa.

Figure 3. Effects of high hydrostatic pressure (0.1 - 400

MPa) treatment at 4˚C (a), 25˚C (b), and 35˚C (c) for 15 min

on germination of radish seeds.

to 200 MPa at 25˚C and 35˚C. However, when radish

seeds were treated with more than 200 MPa at 25˚C and

35˚C, the germination percentage did not exceed 50%

even after 7 days. Also, germination percentage of radish

seeds treated more than 50 MPa at 4˚C did not exceed

50% even after 7 days.

4. Discussion

In this experiment, we examined the effects of pressure

and temperature on the germinability of garden cress,

leaf mustard, and radish seeds. The order of germination

percentage for the three types of seeds on the seventh day

after high hydrostatic pressure treatment at all three tem-

peratures was as follows:

Garden cress > Leaf mustard > Radish.

Our results at 25˚C showed good agreement with those

reported at 20˚C by Wuytack et al., [21] although there

were minor differences in various experimental condi-

tions between our experiments, as follows. 1) We pres-

surized seeds for 10 min, but Wuytack et al. pressurized

for 15 min; 2) we spread seeds on plates holding mois-

tened filter paper, whereas Wuytack et al. spread seeds

on 1.2% agar plates; 3) the origin of the seeds was dif-

ferent.

Figure 4 shows the germination percentage on the

seventh day (maximum germination percentage) when

seeds were treated at various pressures and temperatures.

High germination percentages of over 90% were main-

tained for garden cress and leaf mustard seeds when

Figure 4. Germination percentage on the seventh day after

treatment with high hydrostatic pressure. Each seed was

treated with high hydrostatic pressure (0.1 - 400 MPa) at 4˚C

(), 25˚C (), and 35˚C () for 15 min. (a) Garden cress; (b)

leaf mustard; (c) radish.

Influence of Pressure on Germination of Garden Cress, Leaf Mustard, and Radish Seeds at Various Temperatures441

treated up to 300 MPa at 4˚C, 25˚C and 35˚C. The ger-

mination percentage decreased linearly by 2% - 3% by

100 MPa up to 300 MPa. On the other hand, the germi-

nation percentage of radish seeds decreased to about 25%

when treated at 50 MPa and 4˚C, and this low germina-

tion percentage was maintained at pressures up to 400

MPa. At 25˚C and 35˚C, the germination percentages of

radish seeds decreased linearly with increasing pressure

up to 400 MPa. Therefore, best-fit lines to the germina-

tion percentage at 4˚C and that at 25˚C and 35˚C cross at

about 300 and 400 MPa (Figure 4). Therefore, at more

than 400 MPa, the germination percentage of radish

seeds was suppressed greatly and no significant differ-

ence due to temperature was observed, at least in the

range of 4˚C - 35˚C.

There have been many investigations of pressure and

temperature effects on protein structures and enzyme

activities [23-32]. Hawley calculated the Gibbs free en-

ergy difference (∆G) of the denatured and native state of

chymotrypsinogen (his measurements) [25] and ribonu-

clease A (experiments by Brandts) [26] and obtained a

second-order curve for the denaturation conditions (∆G =

0) [25]. The experimental data on chymotrypsinogen and

ribonuclease A also confirmed that the shape of the phase

diagram of the unfolding of these proteins, assuming that

the unfolding of the protein is a two-state transition be-

tween the native state and denatured state, is elliptical.

However, the phase diagrams determined by Hawley for

chymotrypsinogen and ribonuclease A differ markedly in

shape. Whereas the phase diagram of chymotrypsinogen

shows a stabilizing effect of moderate pressure against

heat denaturation, pressurization decreases the unfolding

temperature of ribonuclease A. Therefore, the phase dia-

grams of proteins can be classified into two types as a

first approximation. Smeller called the phase diagram of

chymotrypsinogen “tongue”-like and that of ribonuclease

A “hillside”-like [31] Furthermore, the importance of the

presence of water is clearly proven by the fact that the

data shown in the elliptic diagram can only be observed

for protein solutions, and if proteins are in a dry state,

they are very pressure stable [31]. Because we pressur-

ized seeds in water, the seeds were more unstable than if

pressurized in a dry state. Also, the effects of pressure

and temperature on the germination of garden cress and

leaf mustard observed in the present experiments were

similar to ribonuclease A type protein denaturation, be-

cause germination was inhibited at high pressure and

high temperature. On the other hand, the effects on the

germination of radish seeds were similar to chymotry-

psinogen type protein denaturation, because pressure

inhibition was remarkable at lower temperatures. From

these results over a wide temperature range, the effects of

pressure on seed germination can be classified into at

least two types, similarly to proteins. This relationship

seems reasonable, because it has been suggested that

various proteins and/or enzymes contribute to germina-

tion [32]. To clarify why each type of seed shows such a

tendency, it will be important to clarify what enzymes

and proteins are influenced by pressure treatment.

In conclusion, the most important results in the present

study observed over wide ranges of temperature (4˚C,

25˚C, and 35˚C) and pressure (0.1 - 400 MPa) was that

the effects of pressure and temperature on germinability

could be classified into two types. One type, represented

by garden cress and leaf mustard seeds, showed more

pressure-resistant germination at lower temperatures. The

other type, represented by radish, showed the highest

pressure sensitivity at low temperature (4˚C) and greatly

decreased germination with treatment at lower pressure

(50 MPa).

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