ORIGINAL PAPER

Cold storage and cryopreservation of hairy root culturesof medicinal plant Eruca sativa Mill., Astragalusmembranaceus and Gentiana macrophylla Pall.

Sheng-Hui Xue Æ Xin-Juan Luo Æ Zhen-Hua Wu ÆHui-Li Zhang Æ Xin-Yu Wang

Received: 21 May 2007 / Accepted: 5 December 2007 / Published online: 18 December 2007

� Springer Science+Business Media B.V. 2007

Abstract To explore the possibility of an effec-

tively long-term preservation of the germplasm of the

HR lines of medicinal plant Astragalus membranac-

eus, Gentiana macrophylla Pall., and Eruca sativa

Mill., both cold storage and cryopreservation

approaches were attempted and compared. After

5-month cold storage on half strength Murashige

and Skoog (1962) (1/2 MS) agar medium (AM), up to

82.9, 75.7, and 100% of the A. membranaceus,

G. macrophylla and E. sativa hairy roots (HRs)

recovered growth, respectively. The survival rates

of A. membranaceus and G. macrophylla HRs sig-

nificantly decreased, whereas that of E. sativa HR

was unchanged with the addition of increased levels

of exogenous abscisic acid (ABA) during cold

storage. Using the encapsulation–vitrification (EV)

method for cryopreservation, the G. macrophylla

HRs died, whereas up to 6 and 73% of the A. mem-

branaceus and E. sativa HRs survived, respectively.

The HR lines evaluated with both methods showed no

significant differences in morphology and growth rate

compared with controls that were not subjected to

preservation methods. These results suggest that cold

storage is a more suitable alternative for the HR lines

of the three studied plant species and that specificity

of plant species have profound effects on the

effectiveness of preservation.

Keywords Astragalus membranaceus �Cryopreservation � Cold storage �Encapsulation–vitrification � Eruca sativa Mill. �Gentiana macrophylla Pall. � Hairy root

Abbreviations

ABA Abscisic acid

AM Agar medium

DMSO Dimethyl sulphoxide

EV Encapsulation–vitrifcation

ED Encapsulation–dehydration

FW Fresh weight

HRs Hairy roots

LN Liquid nitrogen

LM Liquid medium

LS Loading solution

MS Murashinge and Skoog (1962) medium

PVS Plant vitrification solution

Introduction

Hairy roots (HRs), produced from Agrobacterium

rihizogenes infected plant tissue, have been accepted

as a convenient and promising system for producing

interesting secondary metabolites and proteins.

Because HRs usually originate from a single plant

S.-H. Xue � X.-J. Luo � Z.-H. Wu � H.-L. Zhang �X.-Y. Wang (&)

Institute of Cell Biology, School of Life Sciences,

Lanzhou University, Lanzhou 730000, P.R. China

e-mail: wangxy@lzu.edu.cn

123

Plant Cell Tiss Organ Cult (2008) 92:251–260

DOI 10.1007/s11240-007-9329-x

cell infected by A. rhizogenes, they are considered

genetically stable in contrast to callus cultures.

Moreover, the HRs are characterized by stronger

growing potential, and their secondary metabolite

synthesis is not inhibited during the growth phase of

culture, compared with undifferentiated cells. There-

fore, the HRs usually produce secondary compounds

without the loss of concentration frequently observed

during callus or cell suspension cultures (Bourgaud

et al. 1997). To date, the HR lines of more than 100

plant species have been obtained (Georgiev et al.

2007), and many of these were found to exhibit

higher capacity of synthesizing the desired secondary

metabolite than the untransformed roots, combined

with good genetic and biochemical stability (Yoshik-

awa and Furuya 1987; Parr et al. 1988; Flem-

Bonhomme et al. 2004; Kumar et al. 2006).

Astragalus membranaceus, Gentiana macrophylla

Pall. and Eruca sativa Mill. are widely used as

traditional herbal medicines in China. Radix astragali

from the roots of A. membranaceus is rich in

calycosin, polysaccharides, isoflavonoids, c-amino-

butyric acid and various trace elements (Wagner

et al. 1997). Various medicinal properties have been

ascribed to compounds from R. astragali, which may

be utilized as immunostimulants, tonics, diuretics,

antidiabetics, analgesics, expectorants, and sedatives

(Sinclair 1998). The roots of G. macrophylla have

been successfully used to cure viral-induced, neuro-

pathic, respiratory, and cardiovascular diseases

(Zhang et al. 2003) due to the presence of gentiopi-

croside. Gentiopicroside has been found to play

roles in abirritation, defervescence, reducing blood

pressure, antibacterium, antiinflammation, and anti-

rheumatism (Zhang et al. 2003; Yu et al. 2004).

E. sativa contains glucosinolates. The hydrolysates of

glucosinolates, isothiocyanates, have been reported to

inhibit mitosis (Iori et al. 1999) and stimulate apop-

tosis in human tumor cells (Johnson 2002).

In order to explore the possibility of producing

interesting metabolites using HR cultures, we

recently obtained several HR lines from the above

three plant species by infecting their cotyledon or leaf

segments with A. rhizogenes R1000. The HR lines

grew well and showed the traits of the typical HRs.

Polymerase chain reaction (PCR) has demonstrated

that their genomes have been integrated with the rolB

or rolC genes of Ri T-DNA. Moreover, high perfor-

mance liquid chromatography (HPLC) has confirmed

that some of these lines can synthesize gentiopicro-

side or glucosinolates. However, the continuous

subculture is very time-consuming and expensive,

and permits the loss of stability of phenotype and

genotype of plant germplasm. Thus, it is necessary to

develop an effective method for long-term preserva-

tion of the useful HR lines.

In the past decades, cold storage and cryopreser-

vation were widely-explored as better alternatives

for germplasm preservation. Many approaches, e.g.,

slow cooling, vitrification, encapsulation–dehydration

(ED), and encapsulation–vitrification (EV) method,

were developed, and used with varying degrees of

success to preserve plants. The organs/tissues that

were successfully cold stored or cryopreserved mainly

included shoot tips (Pennycooke et al. 2000; Lam-

bardi et al. 2000; Xu et al. 2002), callus (An et al.

2003) and somatic embryo (Ford et al. 2000; Shiota

et al. 1999). However, only a few studies have

evaluated preservation for HRs (Benson and Hamill

1991; Teoh 1996; Yoshimatsu et al. 1996; Phunchin-

dawn et al. 1997; Hirata et al. 2002), and results

varied by plant species and methods used. The

purpose of the present work was to develop a protocol

for effective, long-term preservation of our HR lines

of A. membranaceus, G. macrophylla, and E. sativa.

Materials and methods

Plant material and culture condition

The HR cultures of Astragalus membranaceus,

Gentiana macrophylla Pall. and Eruca sativa Mill.

were established by transforming their cotyledon or

leaf explants with A. rhizogenes R1000, following

which the cultures were stored for *2 years via

regular subculture on hormone free 1/2 MS agar

medium (AM) with 0.088 M sucrose at 25�C in the

dark in 3 week intervals. The HR lines grew well and

had no visible morphological abnormalities.

Cold storage of the HR lines of A. membranaceus,

G. macrophylla, and E. sativa

Fresh HR tips *1-cm long were excised from the HR

cultures grown for 10 days on hormone free 1/2 MS

AM with 0.088 M sucrose, and transferred onto the

252 Plant Cell Tiss Organ Cult (2008) 92:251–260

123

same medium supplied with 0, 0.5 mg l-1 or,

1.0 mg l-1 ABA, respectively. After storage for

5 months at 4�C in the dark, they were retransferred

onto fresh hormone free 1/2 MS AM and cultured at

25�C in the dark. Two weeks later, their survival rates

(%), expressed as the ratio of survival HRs to total

cold-stored HRs, were evaluated. The HR cultures

stored for 5 months on the same medium at 25�C

were used as a control.

Cryopreservation of the HR lines

of A. membranaceus, G. macrophylla,

and E. sativa

The encapsulation–vitrification (EV) method (Hirai

and Sakai 1999) was employed, with minor modifi-

cations. Briefly, the HR tips excised from the HR

cultures grown for 10 days on hormone free 1/2 MS

AM were suspended in 2% (w/v) sodium alginate

solution supplied with 0.1, 0.3, or 0.5 M sucrose.

Then they were dropped in 50 mM CaCl2 solution

with 0.1, 0.3, or 0.5 M sucrose to be encapsulated

with a pipette. Afterwards, (i) the encapsulated HRs

were pre-cultured for 1 and 3 days at 25�C in 1/2 MS

liquid medium (LM) containing an identical level of

sucrose as that used during encapsulation, and then

vitrified for 60 min at 25�C with plant vitrification

solution 3 (PVS3) (Nishizawa et al. 1993), 50% (w/v)

glycerol and 50% (w/v) sucrose, with a change after

30 min (Protocol 1); (ii) the encapsulated HRs were

pre-cultured for 3 days in 1/2 MS LM with 0.3 M

sucrose at 25�C, treated for 20 min at 25�C in MS

LM containing 2 M glycol and 0.4 M sucrose

(loading solution, LS), and then vitrified for 60 min

at 25�C in PVS3 with a change after 30 min (Protocol

2); or (iii) the encapsulated HRs were pre-cultured for

3 days at 25�C in 1/2 MS LM with 0.3 M sucrose, LS

treated for 20 min at 25�C, and then vitrified for

0–120 min in PVS 3 or PVS2 (Protocol 3). PVS2

consisted of 30% glycerol (w/v), 15% (w/v) ethylene

glycol, 15% (w/v) dimethyl sulfoxide and 0.4 M

sucrose in MS medium (pH 5.8) (Sakai et al. 1990).

After vitrified, the encapsulated HRs were trans-

ferred into 2-ml-cryotubes and stored in liquid

nitrogen (LN). Three days later, they were thawed

in water bath at 40�C for 2 min, and then incubated

for 30 min in MS LM with 1.2 M sucrose in a 50-ml

conical flask 25�C with a change every 10 min.

Afterwards, they were transferred onto 1/2 MS AM

with 0.088 M sucrose and cultured at 25�C in the

dark. Two weeks later, their survival rates were

evaluated following the method above.

Analysis of growing potential of the HR cultures

after cold or cryopreservation

For appraising the effects of cold storage and

cryopreservation on the growing potential of the

HR lines, we inoculated the HR lines of the above

three plant species, which were subjected to cold

storage or cryopreservation and recovered growth,

onto 1/2 MS AM. After cultured at 25�C for 15 days,

their fresh weight (FW) was measured. Each bottle

was initially inoculated with five tips 1-cm long,

weighing *6 mg (E. sativa), 7 mg (G. macrophy-

lla), and 10 mg (A. membranaceus), respectively.

The growing potentials of the HR lines were

indicated by the increment of the HR lines, being

defined as the ratios of the final fresh weight (FW) to

the initial FW. The HR lines not subjected to cold

storage and cryopreservation were taken as control.

In addition, we inoculated the HR lines of

E. sativa and G. macrophylla, which were subjected

to cold storage or cryopreservation and recovered

growth, into 30 ml of 1/2 MS LM in 150-ml conical

flasks. Then they were shake-cultured at 25�C at

100 rpm and weighed every 5 days. Finally, their

growth curves were drawn. Each bottle was initially

inoculated with 30 HRs 3-cm long with branches,

weighing totally about 180 mg (E. sativa) and

310 mg (G. macrophylla), respectively.

All the media and solutions used were autoclaved

at 121�C for 20 min and all manipulations were

performed under strictly sterile conditions.

Statistical analysis

All the experiments above were repeated in triplicate,

each containing a minimum of three samples. The

final survival rates and increment of the HR lines

were represented by the means with standard devi-

ation (SD) of the three experiments. The differences

between various treatments were determined by

Duncan’s Multiple Range Test (P B 0.05) using

SPSS 10.0 software. The growth curves were drawn

with Excel software (Microsoft, Redmond, WA).

Plant Cell Tiss Organ Cult (2008) 92:251–260 253

123

Results

The feature of the HR lines of A. membranaceus,

G. macrophylla, and E. sativa

Figures 1a–c represent the HR cultures of A. mem-

branaceus, G. macrophylla and E. sativa grown on

1/2 MS AM at 25�C. The HR lines of the all three

plant species showed the features of typical HRs. But

the HRs of E. sativa (Fig. 1a) looked thinner and

more fragile than those of A. membranaceus (Fig. 1

b) and G. macrophylla (Fig. 1c). The HRs of E. sa-

tiva and A. membranaceus always appeared white

whether in the dark or light, whereas those of the

G. macrophylla gradually changed from white into

green when exposed to the light. Occasionally, small

callus and then plantlets formed spontaneously. In

addition, the HR lines of the three plant species grew

well in 1/2 MS LM (Fig. 1d–f).

Effects of cold storage on growth and survival

rates of the HR lines of E. sativa,

A. membranaceus, and G. macrophylla

Figures 1g–j represent the HR cultures of E. sativa,

A. membranaceus, and G. macrophylla cold storage

on 1/2 MS AM at 4�C, respectively. It can be seen

Fig. 1 Morphological

aspects of the HR cultures

of E. sativa (Es),A. membranaceus(Am), andG. macrophylla (Gm)grown under different

conditions. (a–c) The HRs

of Es, Am, and Gm on 1/2

MS agar medium (AM) at

25�C. (d–f) The HRs of Es,Am, and Gm on 1/2 MS

liquid medium (LM) at

25�C. (g, h) The HRs of Escold-stored for 5 months on

1/2MS AM with 0.5 and

1.0 mg l-1 ABA at 4�C. (i)The HRs of Gm stored for

5 months on 1/2MS AM

with 0.5 mg l-1 ABA at

4�C. (j) The HRs of Amstored for 5 months on 1/2

MS AM with 0.5 mg l-1

ABA at 4�C. (k) The HRs

newly came out from the

encapsulated Am HRs that

was cryopreserved. (l–n)The HRs came out from the

encapsulated Es HRs that

was cryopreserved. (o) The

HRs of Es that were

cryopreserved and allowed

to recover growth for

30 days. Bar equals 10 mm

(a–k), 2 mm (l, m) and

20 mm (n, o)

254 Plant Cell Tiss Organ Cult (2008) 92:251–260

123

that very limited growth was still maintained with

the HR lines of all the three plant species. The HRs

of E. sativa (Fig. 1g, h) appeared thin but dense, with

a white coloring, while those of A. membranaceus

(Fig. 1j) and G. macrophylla (Fig. 1i) appeared

strong rare and had white or beige coloring. In

addition, it was found that the HR cultures stored on

the medium without ABA universally grew better

than those on the medium with ABA and that the HR

system of E. sativa grew better than those of other

plants on the medium. These results were also

supported by the quantitative analysis of their fresh

weigh (FW) (Fig. 2).

Table 1 shows the survival rates of the HR lines of

the studied three plant species after 5 months in cold

storage. On the medium either with or without ABA,

all HRs of E. sativa recovered and showed high

survival (100%). However, the survival rates of the

HR lines of A. membranaceus and G. macrophylla

were only 82.9% and 75.7% at maximums, respec-

tively, being significantly less than that of E. sativa.

In addition, the survival rates of the HR lines of both

A. membranaceus and G. macrophylla significantly

decreased with increased ABA concentration. Also it

was found that the control HR lines of E. sativa and

A. membranaceus stored for 5 months at 25�C on

medium with or without ABA, all died. However, for

the HRs of G. macrophylla, the HRs grown on

medium with ABA all died, but the HRs on medium

without ABA exhibited a 100% survival rate.

Effects of sucrose concentrations and times

during pre-culture on cryopreservation of the HR

lines of E. sativa, A. membranaceus,

and G. macrophylla

After being pre-cultured in 1/2 MS LM with different

concentrations of sucrose at 25�C for 1 or 3 days

followed by PVS3-vitrification and LN storage (Pro-

tocol 1), all HR cultures of E. sativa recovered more or

less (from 6.6% to 67.8%) under any of the conditions

given in Table 2. The effect of Condition 5, 3-day pre-

culture in medium with 0.3 M sucrose, was optimal,

with a survival rate up to 67.8%. But for the HRs of

A. membranaceus, only those subjected to 3-day pre-

culture in medium with 0.3 M sucrose (Condition 5)

were able to survive, and had a very low survival rate

(about 6%). The HR line from G. macrophyll did not

survive under any of the given conditions in Table 2.

Effects of pre-culture and LS treatment

on cryopreservation of the HR lines of E. sativa,

A. membranaceus, and G. macrophylla

When pre-cultured for 3 days in 1/2 MS LM with

0.3 M sucrose, LS-treated for 20 min, PVS3-vitrified

for 60 min at 25�C, and then stored in LN (Protocol

2), 72.7% of the HR cultures of E. sativa survived

Fig. 2 The increment of fresh weight (FW) of the HR lines of

E. sativa, A. membranaceus, and G. macrophylla during cold

storage on MS agar medium with different concentrations of

ABA at 4�C. The increments are indicated as the final–initial

FW (mg)

Table 1 Effects of ABA concentrations and temperatures

during storage on survival rates of the HR lines of E. sativa,A. membranaceus and G. macrophylla

HR cultures ABA (mg l-1) Survival rates (% ± SD)*

4�C 25�C

E. sativa 0 100f 0a

0.5 100f 0a

1.0 100f 0a

G. macrophyll 0 75.7 ± 6.1e 100f

0.5 63.4 ± 4.7d 0a

1.0 18.4 ± 2.3b 0a

A. membranaceus 0 82.9 ± 4.0e 0a

0.5 46.5 ± 5.0c 0a

1.0 41.4 ± 2.0c 0a

* All the HRs was stored for 5 months at either 4 or 25�C.

Values represent the means with standard deviation (SD) of

three independent experiments. Each experiment consisted of

three repeats, each repeat contained 10 HRs at least. The

different letters following the values means a significant

difference between each two values (P B 0.05) according to

Duncan’s Multiple Range Test

Plant Cell Tiss Organ Cult (2008) 92:251–260 255

123

(Table 3), exhibiting a 5% higher survival rate than

samples where LS treatment was excluded (Number

1, Table 3). In contrast, the survival rate of A. mem-

branaceus HRs in the case of LS treatment present

(Number 2, Table 3) was significantly lower than that

in the case of LS treatment missing (Number 1,

Table 3), changing from 6.6% to 0%. All HR cultures

of G. macrophylla died, both with and without LS

treatment. In addition, we found that in the case of

pre-culture missing, the HR lines of all the three

plants completely died regardless of LS treatment

present or missing (Numbers 2 and 4, Table 3).

Effects of vitrification solutions and time

on cryopreservation of the HR lines

of A. membranaceus, E. sativa, and G. macrophylla

To examine the effects of different vitrification

solutions and times on cryopreservation of the HR

lines of the three plant species above, the

encapsulated HRs were vitrified with PVS3 and

PVS2 for various times after being pre-cultured in 1/2

MS LM with 0.3 M sucrose for 3 day at 25�C and

then LS treated for 20 min at 25�C, respectively

(Protocol 3). Finally, all HR cultures of G. macro-

phylla died, regardless of how long they were treated

with PVS3 or PVS2 (Table 4). The HR cultures of

A. membranaceus survived only when they were

vitrificated with PVS3 for 60 min and the survival

rate was only about 6%. All HRs of E. sativa vitrified

with PVS3 recovered. Their survival rates fluctuated

between 16% and 73%, and reached a peak at

60 min. The HRs of E. sativa vitrified with PVS2

recovered only when vitrified for 60 or 90 min, and

survival rates ranged from 12.3% to 25.0%, signif-

icantly less than the HRs vitrified with PVS3.

All the HR lines that were not subjected to PVS2

or PVS3 died, indicating that vitrification was

necessary for the cryopreservation of the HR lines

of the studied three plant species.

Table 2 Effects of sucrose concentrations and times during pre-culture on cryopreservation of E. sativa, A. membranaceus and

G. macrophylla HR culturesa

Condition Sucrose con. (mol/l) Pre-culture times (day) Survival rates (% ± SD)b

A. membranaceus E. sativa G. macrophylla

1 0.1 1 0a 6.6 ± 11.5ab 0a

2 0.3 1 0a 52.3 ± 4.1e 0a

3 0.5 1 0a 28.3 ± 2.8c 0a

4 0.1 3 0a 12.2 ± 10.7b 0a

5 0.3 3 6.6 ± 11.5ab 67.8 ± 1.9f 0a

6 0.5 3 0a 37.8 ± 3.8d 0a

a All the samples were vitrified in PVS3 solution and then storied in LN after pre-culturedb Values were expressed as the means with standard deviation (SD) of three experiments. Each experiment consisted of three repeats,

and each repeat contained 10 HRs at least. The different letters following values means a significant difference between each two

values (P B 0.05) according to Duncan’s Multiple Range Test

Table 3 Effects of pre-culture and LS treatment on cryopreservation of the HR lines of E. sativa, A. membranaceus, andG. macrophylla

Number Pre-

culture

LS

treatment

Survival rate (% ± SD)*

A. membranaceus E. sativa G. macrophylla

1 Yes No 6.6 ± 11.5a 67.8 ± 1.9b 0a

2 No Yes 0a 0a 0a

3 Yes Yes 0a 72.7 ± 2.2b 0a

4 No No 0a 0a 0a

* Values were expressed as means with standard deviation (SD) of three experiments. Each experiment contained three repeats and

each repeat contained 10 HR tips at least. The different letters following the values mean a significant difference between each two

values (P B 0.05) according to Duncan’s Multiple Range Test

256 Plant Cell Tiss Organ Cult (2008) 92:251–260

123

Recovery of growth of the HR lines

of A. membranaceus, E. sativa,

and G. macrophylla after cold storage

and cryopreservation

The HR lines undergoing cold storage began to

elongate 3 days after being transferred onto 1/2 MS

AM with 0.88 M sucrose at 25�C, respectively. The

HR lines undergoing cryopreservation began to elon-

gate 5 days after being transferred onto the same

medium. Quantitative analysis, based on solid

(Table 5) and liquid suspension (Fig. 3) culture,

revealed that their growth potentials (rate) had no

significant differences in comparison with the HR

cultures without undergoing cold storage or cryopres-

ervation. No significant differences were observed in

the growth rates between the HR cultures undergoing

cold storage and those undergoing cyropreservation

(Table 5). Further, no visible abnormalities were

observed in morphology (Fig. 1o).

Discussion

Cold storage

The present investigation indicates that up to 82.9,

75.7 and 100% of the HR cultures of A. membranac-

eus, G. macrophylla Pall., and E.sativa Mill. can

survive and continue to grow, following cold storage

for 5 months, as quickly and normally as cultures

where no preservation methods were utilized. The

optimal conditions determined experimentally indi-

cated the use of hormone free 1/2 MS AM at 4�C, at

which the HR lines of the three plants above were

preserved for at least 5 months.

To date, most studies have focused on shoot tips,

callus, mature embryos, and somatic embryos about

the cold storage of plant tissue or organ (Lai et al.

1997; Xu et al. 2002; Shiota et al. 1999; Peng et al.

1996). As for the preservation of plant’s HR system,

that of few plants was studied besides Panax

Table 4 Effects of vitrification solutions and time on cryopreservation of the HR lines of A. membranaceus, E. sativa, and

G. macrophylla

Time (min) Survival rate (% ± SD)*

A. membranaceus E. sativa G. macrophylla

PVS3 PVS2 PVS3 PVS2 PVS2 PVS3

0 0a 0a 0a 0a 0a 0a

10 0a 0a 16.1 ± 3.3cd 0a 0a 0a

30 0a 0a 54.0 ± 3.6e 0a 0a 0a

60 5.6 ± 9.8ab 0a 73.3 ± 2.8g 12.3 ± 10.7bc 0a 0a

90 0a 0a 41.0 ± 8.5f 25.0 ± 5d 0a 0a

120 0a 0a 17.8 ± 1.9cd 0a 0a 0a

* Values were expressed as means with standard deviation (SD) of three experiments. Each experiment consisted of three repeats and

each repeat contained 10 HRs at least. The different letters following the values mean a significant difference between each two

values (P B 0.05) according to Duncan’s Multiple Range Test

Table 5 Increments of fresh weight (FW) of the HR lines of A. membranaceus E. sativa and G. macrophylla undergoing cold and

cryopreservation 15 days after recovery growth on 1/2 MS agar medium

Storage methods A. membranaceus E. sativa G. macrophylla

Cryopreservation (5 days in LN) 25.2 ± 2.8b* 46.1 ± 2.5c –

Cold storage (5 months at 4�C) 28.7 ± 6.2b 48.3 ± 5.4c 6.3 ± 1.5a

Control (1 month at 25�C) 29.8 ± 5.7b 46.7 ± 7.4c 6.4 ± 1.7a

* Values represent the ratios of final to initial FW and were expressed as the means with standard deviation (SD) of three

experiments. Every experiment contains three repeats, each being inoculated five HR tips, about 6 mg (E. sativa ), 7 mg (

G. macrophylla), and 10 mg (A. membranaceus). The increment of the HR lines was defined as the ratios of the final fresh weight

(FW) to the initial FW. The different letters after the values mean a significant difference between each two values (P B 0.05)

according to Duncan’s Multiple Range Test

Plant Cell Tiss Organ Cult (2008) 92:251–260 257

123

(Yoshimatsu et al. 1996). The HR line of Panax was

reported to have survived for 4 months on hormone

free MS AM at 4�C, with 100% survival rate. It can

be seen the survival rate of E. sativa HRs is identical

to that of Panax HRs. The survival rates of the HR

lines of A. membranaceus and G. macrophylla,

82.9% and 75.7%, are not as high as that of E. sativa

and Panax, but are not excessively low. So it is

feasible to preserve the HR lines of the three plants

above using our present protocol.

Effect of ABA on cold storage

of A. membranaceus, G. macrophylla,

and E. sativa HRs

ABA is a hormone regulating plant seed germination

and development that responds to various environ-

ment stresses (Finkelstein et al. 2002). Previous work

has demonstrated that ABA enhances plant tolerance

to low temperature, drought, wounds, or pathogens,

when exogenously applied. The shoot of garlic was

successfully preserved on B5 medium with 0.3 mg l-

1 6-BA, 0.1 mg l-1 NAA and 10 mg l-1 ABA at 0�C

for a year (Xu et al. 2002). The carrot somatic

embryos undergoing 10-5 M ABA-treatment and

then desiccation were found to be able to survive

for 169 weeks at -25�C and at least 24 weeks at 5�C,

respectively (Shiota et al. 1999). Neither medium

with or without exogenously added ABA had any

effect on the survival rate of the HR lines of E. sativa

in the present work. For the HR lines of both

A. membranaceus and G. macrophylla, adding ABA

significantly reduced survival rates. This result is

different from the above author’s results, but not

inconsistent with other literature. For example, the

mature embryos of Citrus microcarpa were found to

survive on 1/2 MS medium with 5 mg l-1 chloroch-

oline chloride (CCC) at 25�C for a year without

subculture, with 100% survival rate. However, when

15 mg l-1 ABA was added, its survival rate dropped

sharply to 51.2% (Lai et al. 1997). These differences

can be explained by the disagreement of the plant

organ/tissues and/or media used by different authors.

It is possible that the effect of ABA differs with the

specificity of plant species, tissue/organ and/or

medium composition.

Cryopreservation

The approaches used to cryopreserve plant tissues

and organs mainly include slow cooling (SC),

vitrification (V), encapsulation–dehydration (ED),

and encapsulation–vitrification (EV) method, with

the latter two methods appearing to produce more

effective results. The EV method was employed in

the present work. The core of EV method is to first

encapsulate the plant tissue/organ and later vitrificate

with PVS before LN-storage. To improve the survival

rate of the HR lines, we added auxiliary treatments,

mainly including the pre-culture for different periods

of days in MS LM with sucrose of different

concentration, LS treatment and vitrification at dif-

ferent lengths of time (in min) with different PVS. It

was indicated that pre-culture was essential for the

preservation of E. sativa and A. membranaceus HRs,

but completely ineffective on that of G. macrophylla

HRs. LS treatment was valid for the preservation the

E. sativa HRs, whereas completely ineffective on the

HRs of other two plants.

Fig. 3 The growth curves of the HR lines of E. sativa (a) and

G. macrophylla (b) with and without undergoing cold storage

and cryopreservation in 1/2 MS liquid medium at 25�C. The

values were expressed as the fresh weight (mg) on different

days after culture. Each bottle was inoculated with 30 HRs 3-

cm long with branches, 180 mg (E. sativa ) and 310 mg

(G. macrophylla), respectively. Since none of G. macrophyllaHRs survived following cryopreservation under any given

condition, there is no growth curve of G. macrophyllaundergoing cryopreservation

258 Plant Cell Tiss Organ Cult (2008) 92:251–260

123

PVS2 is a vitrification solution most widely used in

cryopreservation of plant organs/tissues. In the pres-

ent experiment, however, it was found completely

ineffective on the preservation of G. macrophylla and

A. membranaceu HRs, but had a small positive effect

on the storage of E. sativa HRs. PVS3 was completely

ineffective on the preservation of G. macrophylla

HRs, slightly effective for A. membranaceus HRs and

significantly valid for E. sativa HRs (Table 4). The

optimal protocol for E. sativa HRs was 3-day pre-

culture in 1/2MS LM with 0.3 M sucrose at 25�C ?LS treatment for 20 min at 25�C ? PVS3 vitrification

for 60 min at 25�C, with 73% survival rate. The

optimal protocol for A. membranaceus HRs was 3-

day pre-culture in 1/2 MS LM with 0.3 M sucrose at

25�C ? PVS3 vitrification for 60 min at 25�C, with

6% survival rate. The HRs of G. macrophylla, did not

recover under any of the given conditions.

To date, the HRs of only Beta vulgaris, Nicotiana

rustica (Benson and Hamill 1991), Artemisia annua

(Teoh et al. 1996), Panax (Yoshimatsu et al. 1996),

Armoracia rusticana (Phunchindawan et al. 1997)

and Vinca minor (Hirata et al. 2002) have been

successfully cryopreserved, to our knowledge. It was

reported that 60% of Armoracia rusticana HRs was

recovered following cryopreservation with EV

method (Phunchindawan et al. 1997). This value is

smaller than in E. sativa HRs.

The relationship between plant HR’s preservation

and species

In the present work, the survival rate of the HRs of

E. sativa following cold-storage or cryopreservation

was always found to be significantly higher than that

of G. macrophylla and A. membranaceus HRs. The

HRs of G. macrophylla always exhibited lower

survival rates than that of the other two plants and

exhibited a 0% survival rate after cryopreservation.

Also, the response of the HR lines of the three plants

to LS treatment and PVS solution were different from

each other during the cryopreservation of the three

plants studied, E. sativa and A. membranaceus were

reported to be highly-resistant to drought (Tewari

et al. 1995; Sun et al. 2005; Guo et al. 1987), and

E. sativa. G. macrophylla is completely nonresistant

to drought. The plants exhibiting high drought-

resistance generally are capable of stronger osmotic

regulation. This suggests questions as to whether the

relatively high survival rates of E. sativa and

A. membranaceus HRs, following cryopreservation,

are linked with their stronger drought resistance.

In conclusion, the effect of cold and cryopreser-

vation of A. membranaceus, G. macrophylla, and

E. sativa HR lines were compared in the present

study. The HRs of the three plant species survived

after cold storage, with survival rate up to 82.9, 75.7

and 100%, respectively. But with EV cryopreserva-

tion method, only the E. sativa and A. membranaceus

HR lines remained alive, with 73 and 6% survive

rate. The HR lines of the three plant species with cold

storage or cryopreservation grew comparably to that

of controls without preservation in both solid and

liquid medium. These results suggest cold storage is a

better alternative for the long-term preservation of the

HR lines of the three plant species. In addition, cold

storage is simple, convenient, time-saving and low-

cost compared with EV cryopreservation. However, it

remains to be determined whether this approach

preserves stable germplasms of the HR lines with

respect to phenotype and molecular biology.

Acknowledgments We would like to show our thanks to

Ph.D. Michael Kirberger in the Department of Chemistry and

Max Oginsky in the Department of Biology, Georgia State

University for spending precious time to revise our paper.

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