pg_0001

Botanical Studies (2008) 49: 243-251.

Corresponding author:

E-mail: liyi2001@gmail.com.

INTRODUCTION

Today facing with the great loss of plant biodiversity

around the world, one way to protect is to store the plant

seeds in gene bank. The effective preservation of seeds de-

pends on their moisture content and the store temperature

(Hsu et al., 2000; Tsou and Mori, 2002), but in developing

countries where the costs of cold storage are prohibitive

(Zheng and Jing, 1998). Low moisture content conserva-

tion (it also called ultra-dry seed storage) through long-

term storage of seed is possible for a significant proportion

of higher plants. Where feasible, long-term seed storage

serves as a safe and relatively inexpensive method of plant

genetic resources conservation (Hong and Ellis, 1996).

Ultra-dry seed storage is a technique for decreasing the

seed moisture content to less than 5% and stored at ambi-

ent temperatures, it can reduce the cost for constructing

and maintaining the genebank and has brought worldwide

attention because of its potential economic effect and

promising application in germplasm conservation. A lot of

studies have been confirmed that ultra-dry seed storage not

only can be used to maintain the quality of seeds but also

improve the storability of seeds (Wang et al., 2003). Posi-

tive results of ultra-dry storage to improve storability have

been reported (Eills et al., 1989, 1990a, 1992, 1993, 1994,

1995; Cheng et al., 1991; Zheng and Jing, 1998; Wang et

al., 1999; Zhu et al., 2001; Huang et al., 2002; Wang et al.,

2005; Li et al., 2007).

Seeds during long-term storage at last lost their ability

to germinate. There are some papers have identified lipid

peroxidation, enzyme inactivation or protein degradation,

disruption of cellular membranes, and damage to genetic

integrity as major cause (Priestly, 1986; Smith and Berjak,

1995; Walters, 1998; McDonald, 1999; Narayana Murthy

et al., 2003). Under accelerated aging conditions such

as high temperature and high seed water moisture lead to

biochemical deterioration during seed aging (McDonald,

1999). In these cases, lipid peroxidation and the loss of

membrane phospholipids are major cause of seed aging

under accelerated aging conditions; the consequence of

A report on ultra-dry storage experiment of Zygophyllum

xanthoxylon seeds

Yi LI

* , Jianjun QU

, Xiaoming YANG

, and Lizhe AN

Dunhuang Gobi and Desert Ecology and Environment Research Station, Cold and Arid Region Environmental and

Engineering Research Institute, Chinese Academy of Sciences, Lanzhou Gansu, 730000, P.R. China

Gansu Academy of Agricultural Sciences, Lanzhou Gansu, 730070, P.R. China

School of Life Science, Lanzhou University, Lanzhou Gansu, 730000, P.R. China

(Received September 21, 2007; Accepted March 11, 2008)

ABSTRACT.

This research aimed to determine whether ultra-dry storage improves the longevity of

Zygophyllum xanthoxylon seeds. Moisture content of Z. xanthoxylon seeds was dried to 4.81%, 3.81%, 2.41%

and 1.99% (w.b.) in a desiccating container with silica gel, and stored at 45°C, 25°C and 15°C for 24 months.

The data from 24 months showed that the optimum moisture content for storage varies with temperature.

Our results found that optimum moisture can not be considered independently of temperature. After ultra-

drying the seeds were accelerated aged (50°C, 1 month), some physiological indices were tested. The results

indicated that Dehydrogenase, POD, SOD and CAT activities of the ultra-dry seeds were higher than those

of the control seeds, while volatile aldehydes and malondialdehyde were lower than the control group. The

results indicate that moisture content of seed was a key index for storage at ambient temperature (25°C) and

3.81% seem to be the best moisture content for ultra-dry seeds in our research. RAPD markers were also used

to evaluate the genetic fidelity of seeds, all RAPD profiles from ultra-dry seeds were monomorphic and similar

to non-ultra-dry seeds, we conclude that variation is almost absent in ultra-dry storage. From these results, we

suggest that seed moisture content less than 5% enhances longevity and ultra-dry could be an economical way

for conservation of the plant genetic resource.

Keywords: Moisture content; Physiological indices; RAPD; Seed storage; Ultra-dry; Zygophyllum

xanthoxylon.

PHYSIOLOGY

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244

Botanical Studies, Vol. 49, 2008

formation of an increase amount of free oxygen radicals

(Goel and Sheoran, 2003). Several protective mechanisms

including free radical and peroxide scavenging enzymes,

for example superoxide dismutase (SOD), peroxidase

(POD) and catalase (CAT) have been evolved within seeds

(McDonald, 1999). SOD is a key enzyme in the regula-

tion of the amount of superoxide radicals and peroxides.

Hydrogen peroxide can react in the Haber-Weiss reaction

forming hydroxyl radicals (Bowler et al. 1992) that cause

lipid peroxidation. CAT and POD are implicated in the

removal of H

(Goel and Sheoran, 2003). The removal

of H

through a series of reactions is known as an ascor-

bate-glutathione cycle in which ascorbate and glutathione

participate in a cyclic transfer of reducing equivalents

resulting in the reduction of H

to H

O using electrons

derived form nicotinamide adenine dinucleotide phosphate

(NADPH) (Goel and Sheoran, 2003).

Zygophyllum xanthoxylon is a dominant plant in the

stabilized sand fields in the northern desert of China. They

appear to be suitable for desert and have a reputation for

high tolerance to water deficiency (Zhao and Zhu, 2003;

Zhou et al., 2006). Although little is known about their

uses, they have great potential to provide different services

such as traditional medicine, halting desert encroachment

and stabilizing sand dunes (Zhou et al., 2006). The avail-

able data about this species are its botany characteristics,

cultivation method, brief descriptions of its habitat condi-

tion, and the range of its geographical distribution (Zeng et

al., 2004; Zhou et al., 2006). However, reports on the pro-

tection germplasm resource and biochemical basis of seed

storage in Z. xanthoxylon are very few. Hence, our aim

was to investigate seed germination ability and viability in

Z. xanthoxylon seeds after ultra-drying and to explore the

physiology mechanism of ultra-dry storage.

MATERIALS AND METHODS

Plant material

Seeds of Zygophyllum xanthoxylon were harvested from

at least 10 plant species in October 2004 at Minqin Desert

Plant Botanical Garden (38°34’ N, 102°58’ E) Gansu

Province, China and represented the equilibrium moisture

contents for seeds in open storage in the Lanzhou area in

the summer (25°C, 75% RH), there an initial germination

percentage of 90.9% and moisture content (MC) of

11.43% were determined.

Seed ultra-drying treatment and pre-humidifica-

tion

Seeds were packed in plastic net bags, the ratio of the

seeds to silica gel was 1:5 (w/w). Seed bags were buried

into silica gel in a desiccator at normal atmospheric

temperature (25°C) for 15 d to reduce the moisture content

of seeds to 4.81%, 3.81%, 2.41% and 1.99%. The ultra-

dried seeds were kept in sealed aluminum foil packages

for experiment.

The rapid uptake of water by dry seeds can result in

imbibition injury (Powell and Matthews, 1978). Seeds

are more likely to be damaged the lower their initial

moisture content (Ellis et al., 1990b) at which they imbibe

water. Imbibition injury can be avoided by conditioning

(humidifying) the seeds in a moist atmosphere (close to

100% RH) in order to raise seed moisture contents before

the seeds are set to germinate in contact with liquid water

(Ellis et al., 1985). In our research to avoid the imbibition

injury, the ultra-dried seeds were hydrated for 48 h in a

sealed desiccator containing saturated CaCl

solutions

(RH is 35%) and then they were hydrated for 48 h in a

sealed desiccator containing saturated NH

Cl solutions

(RH is 75%) at normal atmospheric temperature (25-30°C)

(Huang et al., 2002) before the germination assessment

and the following experiment.

Measurement of seed moisture content (MC),

germination percentage (GP), germination index

(GI) and vigor index (VI)

According to International Rules for Seed Testing

(ISTA, 1993). Moisture content (MC) of three samples

of 100 seeds was determined gravimetrically by the oven

method (8 h at 110°C ± 1°C) and could be expressed on

the wet basis (%, w.b.). The seed surfaces were sterilized

using 10% Na-hypochlorite the before the germination

process. Seeds were tested for germination on top of

three piece of filter paper moistened with 4 cm

distilled-

deionized water in 6-cm-diameter Petri dishes at 20°C

±1°C. Four replicates of 50 seeds were used for each

treatment. Emergence of the radicle was the criterion used

to assess germination. Germination was counted for 7

days. Seeds vigor index (VI) was determined according to

the following equation: VI =G

×S

, G

=.(G

), where G

is germination index, S

is radicle mean length x days after

germination and G

is germination percentage after t days,

is days of germination.

Ultra-dry storage experiments and accelerated

aging

To investigate the storage longevity of the ultra-dry

seeds over 24 months at three different temperatures,

the seed lot was split into three sub-samples. Seeds were

stored at 45, 25 and 15°C, respectively. Each treatment

combination of MC, temperature and storage duration was

represented by a sample in one sachet, which was used to

determine the percentage of germinated seeds.

The ultra-dried seeds and non-ultra-dried seeds (control)

were accelerated aged at 50°C for 1 month in an oven

(Wang et al., 2005). After accelerated aging, the seeds

were put into nylon bags.

Seed conductivity test

Seed conductivity tests were performed by soaking 100

seeds (uniform in size and without visual injury) in 300

ml of deionized distilled water at 25°C for 12 h (Zheng

et al., 1988). The conductivity of the soaking water was

measured by conductivity meter (model DDS SJ-308A,

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LI et al. — A report on ultra-dry storage experiment of

Zygophyllum xanthoxylon

245

made in Shanghai). Leakage rate was expressed in l/μs.cm.

Seed volatile aldehydes and malondialdehyde

test

5 g seeds were soak in distill water at 20°C for 12 h.

Seeds were homogenized in cold 50 mmol/L phosphate

buffer (pH=7.0). The homogenate was centrifuged at

15000×g under 4°C for 20 min and the supernatant was

collected (Zhu et al., 2001). The content of volatile alde-

hydes was determined by the method of Wilson and Mc-

donald (1986). The malondialdehyde (MDA) content was

assayed according to Bailly et al. (1996).

Enzyme extraction and assays

For enzyme extraction, 1.0 g seeds were soaked in

distilled water at 25°C for 12 h and then homogenized

on ice with 50 mmol/L phosphate buffer (pH=7.0). The

homogenate was centrifuged at 15,000 g for 20 min and

the supernatant was used for enzyme assay (Zhu et al.,

2001).

Dehydrogenase activity was determined by triphenyl

tetrazolium chloride (TTC) method (Kun and Abood,

1949).

Superoxide dismutase (SOD) activity was determined

by measurement of inhibition of photochemical reduction

of nitro blue tetrazolium (NBT) at 560 nm (Giannoplitis

and Ries, 1977). The 3 mL reaction mixture contained

50 mmol/L phosphate buffer (pH=7.8), 0.1 mmol/L

ethylenediaminetetra-acetic-acid (EDTA), 13 mmol/L

methionine, 75 μmol/L NBT, 16.7 μmol/L riboflavin and

enzyme extract. Riboflavin was added at last and the re-

action was initiated by placing the tubes under two 9W

fluorescent lamps. The reaction was terminated after 15

min by removal from the light scource. An illuminated

blank without protein gave the maximum reduction of

NBT, therefore, the maximum absorbance at 560 nm. SOD

activity is present as absorbance of sample divided by ab-

sorbance of blank, giving the percentage of inhibition. 1

unit of SOD is define as the amount required to inhibit the

photoreduction of NBT by 50%. The activity of SOD was

expressed as unit/mg protein.

POD activity was determined by measurement of Ka-

lpana and Madhava Rao (1995). The reaction mixture

contained 0.1 mL enzyme extract, 2 mL 0.1 mol sodium-

acetate buffer (pH=4.5) and 0.5 mL o-dianisidine solution

(0.2% in methanol, freshly prepared). The reaction was

initiated with the addition of 0.1 mL of 0.2 mol H

. The

change of absorbance was recorded at 470 nm at an inter-

val of 15 s for 2 min. One unit of POD was defined as 0.1

470

min

-1

CAT activity was estimated by the method of Goel and

Sheoran (2003). The reaction mixture contained 0.6 mL

enzyme extract, 0.1 mL of 10 mmol H

and 2 mL 30

mmol phosphate buffer (pH=7.0). The absorbance was

recorded at 240 nm immediately after addition of enzyme

extract at an interval of 15 s for 2 min. The blank was

without enzyme extract. One unit of CAT was defined as

0.1

240

min

-1

RAPD marker

DNA of seeds derived from radicel and the method de-

scribed by Hanania et al. (2004).

For PCR amplification, eight arbitrary 10-base primers

were selected for PCR amplification. Amplification

reactions were performed with 25 dm

of 10×assay buffer,

2.0 of 1.25 mM each of dNTP’s, 15 ng of the primer, 1×

Taq polymerase buffer, 0.5 units of Taq DNA polymerase

(TaKaRa), 2.5 mM MgCl

, and 30 ng of genomic DNA.

DNA amplification was performed in a Perkin Elmer

Cetus 480 DNA Thermal Cycler programmed for 45

cycles as follows: 1st cycle of 3.5 min at 92°C, 1 min at

35°C, 2 min at 72°C; followed by 44 cycles each of 1 min

at 92°C, 1 min at 35°C, 2 min at 72°C followed by one

final extension cycle of 7 min at 72°C. The amplification

products were separated by electrophoresis in 1.2% (w/v)

agarose gels with 0.5×TBE buffer, stained with 0.2 mg

-3

ethidium bromide. A 1 kb DNA ladder was used as

molecular standards and the bands were visualized and

analyzed by JD-801 Gel Electrophoresis Image analytic

system (Jiangsu, China). All the reactions were repeated at

least twice.

Statistical analysis

Student’s t-test was used to statistically test the

difference between two means. For comparison of means

multiple treatments, Tukey’s test was used. To fit the

normality, the percentage values were arcsine transformed

prior to statistical analysis. Significance lecel was at

P=0.05.

RESULTS

Germination of seeds at different temperature

stored for 24 months

Seeds stored at 45°C, the lower MC that was

maintained in seeds, the better was germination (Figure

1). Seeds at 1.99% MC retained higher viability after 24

months, whereas seed viability was reduced gradually if

moisture content was maintained at original MC or higher

than 1.99%. These results showed that Z. xanthoxylon

seed stored at high temperature (45°C) the MC had a

significant effect on viability, but after ultra-dry treatment,

the seed storability could be improved. Results suggested

that seeds stored at 45°C the optimum MC was 1.99%.

When the storage temperature was 25°C, MC of 11.43%,

4.81% and 1.99% reduced the germination percent greatly,

but for MC 3.81% and 2.41%, only a little decreased.

This suggested that the storability could be improved by

ultra-dry condition. Stored at 25°C the optimum MC was

3.81%-2.41% (Figure 2). Storing seeds at 15°C, however,

could extend seed storage life and good viability for seeds

with all MC retained for at least 24 months, except for the

control (MC 11.43%) (Figure 3).

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Botanical Studies, Vol. 49, 2008

Seed vigor

After accelerated aging, the ultra-dry seeds still kept

higher vigor levels in comparison with the control group.

Table 1 shows that the Z. xanthoxylon seeds are tolerant to

dehydration. They were highly tolerant to aging with low

moisture content (MC) (MC less than 5%). After 1 month

of accelerated aging, the germination percentage (GP) and

VI of the control (MC 11.43%) seeds decreased greatly,

meanwhile those of the ultra-dried seeds (MC 3.81% and

MC 2.41%) remained at a high level. The effect of ultra-

dry storage for Z. xanthoxylon seeds with a MC of 3.81%

was almost the same as that of low temperature storage

(-18°C). However, for the seeds with a MC of 2.41%, the

vigor began to decline. From Table 1 the electrical con-

ductivity of ultra-dry seeds (MC 3.81%) was not signifi-

cantly different from that of MC 11.43% stored at -18°C.

This suggests that the integrity of the membrane system in

ultra-dry seeds can be maintained. These results suggest

that ultra-drying of seeds within certain MC limits has no

negative effects on Z. xanthoxylon seed vigor, however the

Z. xanthoxylon seeds can not be dried too severely.

PhysiologIcal indices

We have found that after accelerated aging, volatile

aldehydes and malondialdehyde (MDA) contents of

ultra-dry seeds were lower than those of the control (MC

11.43%). It indicates that the deterioration of ultra-dry

seeds was less than the control. Perhaps the ultra-dry seeds

had an efficient antioxidant defense system that made the

degree of lipid oxidation and lipid peroxidation lower

(Figure 4). After accelerated aging, the activities of de-

hydrogenase (Figure 5) was higher than those of the non-

ultra-dry seeds (MC 11.43%). This result is consistent with

changes of germination and vigor (Table 1).

Figure 1. GP (%), GI and VI of Z. xanthoxylon seed stored at

45°C for 24 months.

Figure 2. GP (%), GI and VI of Z. xanthoxylon seed stored at

25°C for 24 months.

Table 1. Germinating ability, vigor and electrical conductivity of ultra-dry seeds after accelerated aging for 1 month.

Treatment

MC (%) GP (%) Mean radicle length (CM) VI Electrical Conductivity (μS·cm)

-18°C storage

11.43 90.93 a

6.51±0.36 a

32.88 a

38.39 a

50°C aging

11.43 30.12 c

0.14±0.02 c

1.56 c

75.43 c

50°C aging

3.81 90.55 a

6.57±0.31 a

33.00 a

38.01 a

50°C aging

2.41 68.88 b

5.35±0.24 b

14.15 b

52.11 b

The values in a column with the same alphabetical letter are not significantly differen. All values are means ± SD of three replicates.

pg_0005

LI et al. — A report on ultra-dry storage experiment of

Zygophyllum xanthoxylon

247

Monitoring of genetic fidelity by RAPD

In order to confirm genetic fidelity (at molecular level)

of seeds after ultra-dry treatment, the seeds were screened

with the 8 random RAPD primers, one primer that

produced distinct amplification profiles. The representative

profile of the ultra-dry seeds and the control (non-ultra-

dry) with primer is shown in Figure 7. It was obvious that

the ultra-dry seeds showed identical RAPD profiles (i.e. no

polymorphism was observed). These results confirmed the

genetic fidelity of the ultra-dry seeds.

DISCUSSION

At low temperature (15°C) the aging of seeds is quite

slow (Figure 3). In our experiment GP remained at

very high even after 24 months of storage. We showed

that optimum MC was observed for three temperatures

study and the critical value decreased with temperature

increased (Figures 1-2). This means Z. xanthoxylon seeds

can be stored over a wide range of temperatures at the

relatively low MC (MC <5%), their longevity begins to

vary greatly as seed MC is increased (Figures 1-3). Since

maintaining seed viability during long-term storage is of

the utmost importance, it appears that storage at this low

MC (MC <5%) and at ambient temperature, is absolutely

necessary. So, it is very important to be able to store seeds

without use of low temperature if seed longevity and vigor

can be maintained. But reports using numerous species

(Vertucci et al., 1994; Ellis et al., 1995; Chai et al., 1998;

Hu et al., 1998a, b; Kong and Zhang, 1998; Shen and Qi,

Figure 3. GP (%), GI and VI of Z. xanthoxylon seed stored at

15°C for 24 months.

Figure

4. Volatile aldehydes and malondialdehyde contents. All

values are means ± SD of three replicates.

Figure 5. Effects of ultra-dry storage on dehydrogenase of Z.

xanthoxylon seed. All values are means ± SD of three replicates.

Figure

6. Changes of the activities of CAT, SOD and POD after

accelerated aging at 50oC for 1 month. All values are means ±

SD of three replicates.

pg_0006

248

Botanical Studies, Vol. 49, 2008

1998) have demonstrated that the seeds aged more rapidly

under extremely dry conditions. So we can conclude that

drying to extremely low MC may shorten seed longevity.

Our data confirmed this viewpoint (Figure 2), at ambient

temperature (25°C) the optimum MC of Z. xanthoxylon

seeds is 3.81% and 2.41%, not 1.99%. However, for ex

situ genetic resource conservation, which is typically

considered as more than 10 years, further storage testing

for this species will be required (our trials only extended 2

years).

In our research, the seeds of Z. xanthoxylon were dried

to a moisture content of less than 5%, the viability and

vigor were not statistically significantly influenced; on

the contrary, the aging-resistant capability was greatly en-

hanced (Figures 4-6). After accelerated aging, the results

showed that the 3.81% moisture content was more ap-

propriate for Z. xanthoxylon seed ultra-dry storage. GP, VI

and mean radicle length were kept higher than non-ultra-

dry seeds, implying that no biochemical and biophysical

reaction might have occurred to injure the seed cells under

the conditions of low MC (Table 1). The conductivity test

was based on the assumption that a disintegration of cell

membranes in the seed takes place during seed deteriora-

tion. Seed deterioration can be explained by the findings

that the aging of seeds leads to lipid peroxidation that sub-

sequently causes membrane perturbation (Ponquett et al.,

1992; Chang and Sung, 1998; Goel and Sheoran 2003).

Such changes in the membrane of aged seeds lead to

electrolyte leakage. So tolerance of desiccation can be ex-

pressed by the electrolyte leakage rate (Berjak et al., 1993;

Leprince et al., 1995). In this study, the electrolyte leakage

rate of Z. xanthoxylon increased after aging (Table 1, MC

11.43%), indicating a loss in membrane integrity. How-

ever, the electrical conductivity of ultra-dry seeds (MC

3.81%) significantly decreased compared with the control

(MC 11.43%), indicating that ultra-drying could improve

the membrane function during Z. xanthoxylon seed desic-

cation. After high temperature aging, the ultra-dry seeds

showed strong storability. Compared with the control

group (seeds stored at -18°C), the electrical conductiv-

ity remained stable, which means that the integrity of the

membrane system in the ultra-dry seeds was maintained

during storage.

The imbibition injure occurred in a wide range of crops

when dried seeds were dipped directly in the water. So the

imbibition injure is inevitable to ultra-dried seeds (Zheng,

1994). Even if there is imbibition injure, the pre-humiti-

fication of ultra-dried seeds can be repaired. So we could

suppose that the water depletion induces the structural

changing of a seed’s cell membrane, which make ultra-

dried seeds lose vigor. The effects of pre-humidification

were correlated to the recovery of the membrane and

enzyme, which improve the aging-resistant capability of

ultra-dried seeds. The results of this experiment show that

in the ultra-dried seeds, high activities of dehydrogenase,

CAT, SOD and POD were kept. Free radical-induced dam-

age plays a key role in seed deterioration during aging

(Pinhero et al., 1998). Seed deterioration has been suspect-

ed to be associated with an accumulation of active forms

of oxygen, for example superoxide radical (O

), hydrogen

peroxide (H

) and hydroxyl radical (

OH). The chief

toxicity of O

and H

is thought to reside in their ability

to initiate cascade reactions that result in the production

of the hydroxyl radical and other destructive species, such

as lipid peroxides (Noctor and Foyer, 1998). Efficient de-

struction of O

and H

requires the action of several an-

tioxidant enzymes acting in synchrony (Song et al., 2004).

Antioxidant defense systems in plants include free radical

and peroxide-scavenging enzymes. Superoxide produced

in the different compartments of plant cells is rapidly con-

verted to H

by the action of SOD. CAT converts part of

to water and O

. SOD activity is involved in the reg-

ulation of intracellular concentration of superoxide radical

and H

. During our research, the activities of SOD and

CAT significantly increased after accelerated aging (Fig-

ure 6). These results show that the changes of activities of

antioxidant enzymes are closely related to desiccation tol-

erance and the ultra-drying does not destroy the enzymes.

The ultra-drying treatment can prolong the seed storage

life by increasing SOD activity.

Lipid peroxidation mediated by free radical and perox-

ides is one of the probable reasons for seed viability loss

during storage (Sung, 1996; Goel and Sheoran, 2003).

Loss of viability and vigor were associated with increased

peroxidation in rapidly aged seeds. The volatile aldehydes

and MDA is the products of lipid oxidation and peroxida-

tion. So, the contents of these products in seeds can tell

its deterioration degree. So determination of the volatile

aldehydes and MDA is a convenient method of quantify-

ing the extent of lipid peroxidation. In our research, the

contents of the volatile aldehydes and MDA decreased af-

ter accelerated aging and were correlated with the increase

in the activities of SOD, POD and CAT (Figures 4-6). The

results of this experiment show that the lipid peroxidation

was greatly suppressed under the ultra-dried condition.

This implied that the enzyme systems were not destroyed

and high activities of antioxidant enzymes were kept in

ultra-dry seeds.

Figure 7. RAPD bands of Z. xanthoxylon seeds. M~DNA Mark-

er; 1~MC 11.43%; 2~MC 4.81%; 3~MC 3.81%; 4~MC 2.41%;

5~MC 1.99%.

pg_0007

LI et al. — A report on ultra-dry storage experiment of

Zygophyllum xanthoxylon

249

Somaclonal variation can either bring changes at the

DNA level or it may induce changes in chromosome num-

bers. In general, morphological markers, chromosome

analysis, isoenzyme or DNA markers may be used to detect

somaclonal variation. As found in the present study, vari-

ous investigators have observed the absence of variations.

Cheng et al. (1997) and Meng et al. (2003) used RAPD

markers to evaluate genetic stability of peanut seeds and

three vegetable seeds, respectively. In a similar way, an-

other author using RFLP to observe somaclonal variations

in various crops (Hu, 2005). In our study, RAPD was cho-

sen to detect somaclonal variation. No genetic instabilities

were detected between the ultra-dry seeds and the control

seeds (non-ultra-dry seeds). We conclude that somaclonal

variation is almost absent in our ultra-dry seeds, the simi-

larity in RAPD banding may suggest genetic fidelity, and

therefore we believe that the methodology used to dry the

seeds did not induce major genetic changes. Although in

the present experiment the analysis of somaclonal varia-

tion was based on RAPD markers, it will be required to

obtain consistently seedlings and to analyze relative dif-

ferences in seedling characteristics. In future, it would be

surely useful to extent the spectrum of DNA markers for

genetic fidelity studies in ultra-dry storage.

The above conclusions have confirmed that the ultra-

drying technique has not only caused less injury to the

seeds but strongly enhanced the aging-resistant capability

and storability of Z. xanthoxylon seeds. This technique will

be potentially useful for the preservation of Z. xanthoxylon

germplasm.

Acknowledgements. This project was supported by

National Natural Science Foundation of China (90302010).

The authors wish to thank anonymous referees for

suggestive comments and modification.

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