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ORIGINAL PAPER

The survival of in vitro shoot tips of Garcinia mangostana L.after cryopreservation by vitrification

Sarah Ibrahim • M. N. Normah

Received: 15 July 2012 / Accepted: 14 February 2013 / Published online: 20 February 2013

� Springer Science+Business Media Dordrecht 2013

Abstract This report highlights the first successful cryo-

preservation protocol for shoot tips of Garcinia mangos-

tana L. achieved by using vitrification technique. We

investigated the effects of different temperatures and expo-

sure periods to a plant vitrification solution 2 (PVS2), sucrose

concentrations and preculture periods, and unloading treat-

ments in steps of the vitrification protocol on the survival of

G. mangostana shoot tips after cryopreservation. Exposure to

PVS2 for 25 min gave beneficial effects with 10.4 ± 1.8 %

survival at 0 �C with average water content of 1.1 ±

0.3 g g-1 dry mass. Survival was 13.7 ± 5.5 % when using

preculture medium with full-strength Murashige and Skoog

(MS) medium supplemented with 0.6 M sucrose for 2 days.

A significant difference was observed in survival of shoot

tips when treated with various sucrose concentrations in pre-

culture which strengthens their importance towards enhanc-

ing survival of shoot tips after cryopreservation. MS with

0.4 M sucrose and 2 M glycerol applied as an unloading

solution increased the survival of shoot tips to 44.1 ± 6.5 %.

Experiments on the effect of ascorbic acid were also con-

ducted for each step of vitrification. Our results showed

higher survival of 45.8 ± 3.8 % but there were no signifi-

cant effects compared with the control (without ascorbic

acid). Further study on the recovery dark/light period was

conducted. Survival of shoot tips significantly increased to

50.0 ± 16.7 % when subjected to 7 days in the dark before

transferring to 16 h/8 h light/dark photoperiod. These stud-

ies strengthen suggestions that cryopreservation through

vitrification is possible for ex situ conservation of germ-

plasm of this tropical recalcitrant species.

Keywords Cryopreservation � Garcinia mangostana �Liquid nitrogen � Shoot tips � Ascorbic acid � Vitrification

Introduction

Garcinia mangostana L., commonly known as mango-

steen, is a major tropical fruit species belonging to the

family Guttiferae (also known as Clusiaceae). There are

many uses of mangosteen, and it is probably the most

highly regarded tropical fruit tree. Its fruit is mostly eaten

fresh, and both the rind and bark have several applications,

as they possess anti-inflammatory, astringent, antibacterial,

anti-tumour and anti-oxidative activities (Chairrungsri

et al. 1996). Owing to these properties, various parts of the

tree are used in Southeast Asia as traditional medicines for

the treatment of abdominal pain, dysentery, wound infec-

tions, suppuration and chronic ulcers.

Mangosteen produces recalcitrant seeds that remain sen-

sitive to desiccation both during development and after they

are shed (Normah et al. 1995). Because conventional seed

storage is not possible for recalcitrant seeds, the potentially

safest method to conserve recalcitrant seeds for long periods

without change is to store them in or above liquid nitrogen, a

process known as cryopreservation (Berjak et al. 2000). At

this temperature, biological deterioration is considered to be

completely halted, and the germplasm can be stored for

unlimited periods of time without modification including

contamination (Engelmann 2011). Mangosteen seeds do not

have differentiated embryos (Normah et al. 2011); thus, the

S. Ibrahim

School of Environmental and Natural Resource Sciences,

Faculty of Science and Technology, University Kebangsaan

Malaysia, 43600 Bangi, Selangor, Malaysia

M. N. Normah (&)

Institute of Systems Biology (INBIOSIS), Universiti

Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia

e-mail: [email protected]

123

Plant Growth Regul (2013) 70:237–246

DOI 10.1007/s10725-013-9795-6

shoot tips are the ideal tissue to be used as explants for this

important species. Cryopreservation of shoot tips offers

long-term storage capability, maximum stability of the

phenotypic and genotypic characteristics of the stored

germplasm, and minimal storage space and maintenance

requirements (Engelmann 1997).

The vitrification technique is the most widely used cryo-

preservation procedure, as it is easy to perform, is highly

reproducible, and can be successfully applied to a wide range

of tissues and plant species, especially those that are sensitive

to dehydration and freezing (Reed and Uchendu 2008). Many

studies have shown a high percentage of survival when the

shoot tips were treated with Plant vitrification solution 2

(PVS2) before plunging into liquid nitrogen (LN), including

studies on apple and pear (Niino et al. 1997) and Populus

alba (Lambardi et al. 2000) shoot tips.

Optimising the time of exposure and the temperature during

exposure to PVS2 are crucial aspects in the vitrification tech-

nique. The duration of the diffusion of PVS2 through the cell

membrane has to be long enough to ensure sufficient cell

dehydration while avoiding cytotoxic effects. If cooled very

rapidly, plant organs can be successfully cryopreserved at

water contents as high as 1.1–1.6 g H2O g-1 dry mass (Wesley-

Smith et al. 1992). Apart from that, using a stepwise method has

proven to be more beneficial, especially with species that are

relatively sensitive to dehydration (Takagi et al. 1997).

Previous studies have also shown the importance of the

preculture stage in many woody plant species. Xu et al.

(2006) reported on shoot tips of Actinidia chinensis that the

vacuoles became small and that the free water content in

the cells decreased after preculture, indicating that the

freezing tolerance and dehydration capacity increased with

minimum damage while improving the resistance to cold

and enabling the cell membrane to maintain a stable

structure. Additionally, sucrose in preculture media acts as

an osmoticum to accumulate sugar content inside cells and

strengthen membrane integrity to withstand dehydration

(Uragami et al. 1993). Many studies reported that

increasing the sucrose concentration improved the survival

percentage after cryopreservation including studies on

shoot tips of Trichilia emetica (Varghese et al. 2009),

Pyrus cordata (Chang and Reed 2001) and Mokara Golden

Nugget Orchid (Safrinah et al. 2009).

Storing plants at low temperatures could result in dele-

terious effects caused by the chilling and freezing of the

cells, which can then lead to an increased production of

reactive oxygen species (ROS), often resulting in cell death

(Day et al. 2000). ROS are believed to be produced during

axis excision and in the steps of dehydration, exposure to

and retrieval from cryogenic storage (Berjak et al. 2011).

Antioxidants are believed to be able to arrest ROS before

oxidative damage can occur and, thus, improved the sur-

vival of blackberry shoot tips after cryopreservation

(Uchendu et al. 2010a). This hypothesis is supported by the

study of Chua and Normah (2011) in which ascorbic acid

was proven beneficial for the survival of shoot tips of

Nephelium ramboutan-ake after cryopreservation. In this

study, the effects of the duration and temperature of PVS2

exposure were evaluated, and the most suitable preculture

medium and the best unloading solution for cryopreserva-

tion were determined. The effect of ascorbic acid in each

step of the vitrification procedure was also examined.

In addition, dark/light period controls explant survival

and regrowth. Touchell and Walters (2000) reported that

recovery percentages increased when cultures were ini-

tially maintained in the dark as compared to light. This is

suggested to be because damaging consequences of photo-

oxidation would be avoided or minimized in cultures

maintained in dark or minimal light conditions (Withers

1988). Thus, further research was conducted to investigate

the potential effects of dark/light exposure during recovery

of the cryopreserved shoot tips.

Materials and methods

Plant materials and culture conditions

The fruits of G. mangostana were obtained from Puchong,

Selangor, Malaysia, in the months of July and August of

2010. The fleshy edible parts were peeled off the seeds, and

the rubbery testa enclosing the seeds was removed using

forceps and a blade. The seeds were then washed under

running water for 20 min before decontamination with

80 % alcohol for 1–2 min. The seeds were then disinfected

using 20 % Clorox (5.25 % sodium hypochlorite; The

Clorox Company, USA) with two drops of Tween 20 for

20 min. The seeds were then rinsed three times with sterile

distilled water, blotted dry with sterile filter paper and

placed in a Petri dish before culturing on Murashige and

Skoog (MS) medium (1962) supplemented with 4 mg l-1

benzylaminopurine (BAP) and 2.5 g l-1 gelrite. Seeds

were cut into three segments to maximise the shoot

induction area (Fig. 1). The explants were then subcultured

onto the same medium every 3 weeks. The explants were

subcultured for a week before the shoot tips of approxi-

mately 1–2 mm long were used for cryopreservation. All

cultures were incubated under fluorescent light at

23 ± 2 �C, with a light intensity of 22.26 lE cm-2 s-1

(3,000 lux) and 16 h/8 h light/dark photoperiod.

Cryopreservation procedures

Cryopreservation of the shoot tips was carried out according

to the method of Sakai et al. (1990), with slight modifica-

tions. Excised shoot tips were precultured on MS medium

238 Plant Growth Regul (2013) 70:237–246

123

supplemented with 0.3 M sucrose and 5 % DMSO for 48 h.

The shoot tips were then transferred to 1.8 ml cryovials and

treated with 1 ml of loading solution (2 M glycerol ?

0.4 M sucrose) for 20 min at 25 �C. The loading solution

was then removed, and 1 ml of PVS2 solution (30 %

glycerol, 15 % ethylene glycol, 15 % DMSO and 0.4 M

sucrose) (Sakai et al. 1990) was added followed by incu-

bation for different times at 25 �C. The PVS2 solution was

then removed, and the shoot tips were immediately trans-

ferred to a new cryovials to which 0.5 ml of fresh PVS2

solution was added before the vials were rapidly plunged

into liquid nitrogen (LN) (-196 �C) for at least 1 h. The

cryovials containing the frozen shoot tips were then rapidly

thawed in a sterile-water bath at 38 ± 2 �C for 40–45 s

with constant agitation, and the shoot tips were rinsed with

unloading solution (MS containing 0.4 M sucrose and 2 M

glycerol) at 25 �C three times (*5 min) before culturing on

recovery medium. The control, which was not immersed in

LN, was directly rinsed with unloading solution at 25 �C

three times (*5 min) and then cultured on recovery med-

ium. The recovery medium consisted of MS supplemented

with 4 mg l-1 BAP and 2.5 g l-1 gelrite. The cultures were

incubated under fluorescent light at 23 ± 2 �C, with a light

intensity of 22.26 lE cm-2 s-1 (3,000 lux) and subcultured

every week.

Effects of the plant vitrification solution (PVS2)

exposure procedure on the survival of the shoot tips

after cryopreservation

A preliminary study was conducted to study the sensitivity

of shoot tips to PVS2 exposure. Excised shoot tips were

precultured on MS medium supplemented with 0.3 M

sucrose and 5 % DMSO for 48 h. Shoot tips were then

exposed to 100 % PVS2 for 0, 5, 10, 15, 20, 25, 30, 35 and

40 min. Treated shoot tips were then directly cultured on

the recovery medium.

The best exposure period for survival of shoot tips was

used for further study on the effects of different concen-

trations of PVS2 at different periods of exposure. The shoot

tips were exposed to seven treatments at 25 �C; direct

exposure for 0, 5, 10, 15, 20 and 25 min in 100 % PVS2;

and step-wise approach for 15 min in 50 % PVS2 followed

by 10 min in 100 % PVS2. With the results of this

experiment, the effects of two different temperatures (0 and

25 �C) of PVS2 exposure on shoot tips were conducted.

Effects of preculture on the survival of the shoot tips


Studies on the preculture stage were conducted to improve

survival after cryopreservation. The effects of different

concentrations of sucrose (0, 0.3, 0.6 and 0.9 M) and the

exposure period (1 and 2 days) in the culture medium were

studied. DMSO at 5 % was added to all preculture media.

After obtaining the highest survival from this experiment,

the effects of two strengths of MS media (full and half

strengths) were then studied. The PVS2 exposure period

and temperature and sucrose concentration for preculture

were based on the above experiments.

Effects of unloading solution on the survival

of the shoot tips after cryopreservation

Two types of unloading solutions, liquid MS supplemented

with 1.2 M sucrose (Sakai et al. 1990) and MS supple-

mented with 0.4 M sucrose and 2 M glycerol (suggested by

Matsumoto et al. 1998 for tropical species), were evaluated

for their effect on the survival of the explants. The PVS2

exposure period and temperature, sucrose concentration

and MS medium strength for preculture were based on the

above experiments.

Effects of ascorbic acid on the survival of the shoot tips


Effects of ascorbic acid on each step of vitrification (pre-

culture, loading, PVS2, unloading and recovery) protocol

were studied. Ascorbic acid at 0.28 mM was added in the

vitrification solutions to study its effects on the survival of

the shoot tips after cryopreservation. The sucrose concen-

tration, MS medium strength for preculture, PVS2 expo-

sure period and temperature and the unloading solution

were ultimately selected based on the above experiments.

Effects of the dark/light period during recovery

on the survival of the shoot tips after cryopreservation

The effects of dark/light period during recovery on the sur-

vival of shoot tips were tested. After exposed to unloading

0.5 cm

(a) (b) (c)

Fig. 1 Seeds of G. mangostana: a Seed with testa b Seed without

testa c Seed that has been cut into three segments

Plant Growth Regul (2013) 70:237–246 239

123

solution, explants were cultured on the recovery medium (as

described above) and placed under dark conditions for 0, 3,

5, and 7 days before transferring to 16 h/8 h light/dark

photoperiod. All steps and procedures for cryopreservation

were based on the experiments described above.

Statistical analysis

All of the data obtained from the experiments were statisti-

cally analysed using SAS software. Data in percentiles were

arcsin transformed before subjecting them to a parametric

statistical test. The treatment means were compared using

Duncan’s Multiple Range Test (DMRT) and standard

deviation (SD). Fifteen shoot tips were used for each treat-

ment, and each treatment was replicated three times. The

survival of the shoot tips were observed and recorded every

3 days. The shoot tips were considered to have survived if

they remained green and showed indication of growth,

whereas those that turned brown were considered dead.

Results

Effects of the plant vitrification solution (PVS2)

exposure procedure on the survival of the shoot tips


A preliminary study on the sensitivity of shoot tips to PVS2

exposure was conducted. The results in Fig. 2 show that

shoot tips exposed to PVS2 at 25 �C for up to 25 min were

able to resume 100 % shoot growth. Prolonged exposure

caused death to the shoot tips. Thus, 25 min of PVS2

exposure was used for the subsequent experiments.

The results of the mean analysis of the shoot tip survival

percentage at different combinations of the direct and

stepwise PVS2 exposure period are shown in Fig. 3. The

shoot tips that were not immersed in LN showed survival

ranging from 63.9–100 %. Longer exposure to PVS2

(25 min) decreased the shoot tips survival percentage to

less than 65 %. Most shoot tips that were cryopreserved

(?LN) did not survive except for the treatment involving

25 min direct and stepwise PVS2 exposure. Stepwise PVS2

exposure for 25 min showed no significant difference

compared with shoot tips that were directly exposed to

higher concentrations of PVS2 (100 %) for the same period

(25 min), with 39.2 ± 10.1 % and 35.6 ± 11.8 % survival

respectively. The water content of explants decreased with

increasing PVS2 exposure period (Fig. 4). Prolonged

(30 min) of PVS2 exposure resulted in excessive cell

dehydration to -1.1 ± 0.1 g g-1 dry mass. The most

suitable water content, 1.1 ± 0.3 g g-1 dry mass was

obtained when explants were exposed to PVS2 for 25 min.

This is also supported by our data where survival per-

centage of 35.6 ± 11.8 % was observed when explants

were exposed to PVS2 for 25 min. Hence, exposure period

of 25 min was chosen for the subsequent experiments.

The mean analysis of the survival percentage of the

shoot tips at different PVS2 exposure temperatures fol-

lowed by LN exposure (?LN) in recovery medium is

shown in Table 1. The survival of the G. mangostana shoot

tips not treated with LN showed the highest survival,

63.3 ± 17.5 % at 0 �C and only 26.3 ± 2.2 % for the

shoot tips treated with PVS2 at 25 �C. The same response

was also shown for the shoot tips treated with LN: only

8.3 ± 1.7 % survival was recorded for the shoot tips

treated with PVS2 at 25 �C, and 10.4 ± 1.8 % survival

-50

0

50

100

0 5 10 15 20 25 30 35 40

Surv

ival

(%)

Exposure to PVS2 (min)

Fig. 2 Survival of G. mangostana shoot tips after various periods of

exposure to PVS2 at 25 �C. Bars represent standard errors

Control - Without PVS2 exposure D - 20 min in 100% PVS2A - 5 min in 100% PVS2 E - 25 min in 100% PVS2B - 10 min in 100% PVS2 F - 15 min in 50% PVS2 followed by C - 15 min in 100% PVS2 10 min in 100% PVS2 exposure

0

20

40

60

80

100

120

Control A B C D E F

Su

rviv

al (

%)

PVS2

-LN

+LN

ba a a a a

c

d d

Fig. 3 Survival of G.mangostana shoot tips after

gradual exposure to PVS2 at

25 �C. –LN indicates explants

not exposed to liquid nitrogen

(LN) and ?LN indicates

explants exposed to LN.

Different letters indicate

significant difference at

p B 0.05 using Duncan’s

multiple-range test. Mean ± SD

(n = 15) are shown


123

was recorded for the PVS2 exposure at 0 �C. Because the

treatment of the shoot tips at 0 �C showed better survival,

this treatment was chosen for the subsequent experiments.

Effects of preculture on the survival of the shoot tips


The cryopreserved shoot tips survival percentage for both

of the sucrose exposure durations ranged from 0 to 13.7 %

(Fig. 5). The highest survival percentage (13.7 ± 5.5 %)

for this experiment was observed for the treatment

involving 0.6 M sucrose with exposure duration of 2 days.

The survival percentage decreased when the sucrose con-

centration exceeded 0.6 M in the preculture treatment.

From the statistical analysis, no significant differences

were observed between the control and the treatment with

highest sucrose concentration (0.9 M): both resulted in no

survival of the shoot tips after cryopreservation. However,

there was a significant difference between the cryopre-

served shoot tips precultured on 0.3 M sucrose and 0.6 M

sucrose conditions for 2 days. As the highest survival after

LN exposure was found for the treatment with 0.6 M

sucrose for 2 days, this treatment was chosen for the sub-

sequent experiments.

Shoot tips not exposed to LN showed survival percent-

ages of 45.8 ± 5.3 % and 36.5 ± 3.3 % for the full-

strength and half-strength MS, respectively (Fig. 6). The

shoot tips without preculture (the control) showed the

lowest survival of only 10.4 ± 0.6 %. The survival of

the cryopreserved shoot tips, however, showed only a slight

difference when the full-strength MS was compared with

the half-strength MS in the preculture medium (19.4 ±

11.4 % and 21.1 ± 8.4 % survival, respectively). The

shoot tips without preculture and exposed to LN showed

the lowest survival percentage, at 2.8 ± 4.8 %. Because

the survival of the shoot tips pretreated with full-strength

and half-strength MS did not show any significant differ-

ence or benefit on the survival of the shoot tips after

cryopreservation, the full-strength MS was chosen for the

subsequent experiments.

Effects of the unloading solution on the survival

of the shoot tips after cryopreservation

The results showed that there were no significant differ-

ences between the unloading solution with 1.2 M sucrose

and that with 0.4 M sucrose and 2 M glycerol for the shoot

tips that were not exposed to LN (Table 2). The survival

percentages of the shoot tips without LN exposure for

the 1.2 M sucrose and 0.4 M sucrose supplemented with

2 M glycerol unloading solutions were 57.1 ± 10.0 % and

-2.00

0.00

2.00

4.00

6.00

8.00

10.00W

ater

Co

nte

nt

(g g

- 1d

ry m

ass)

Fig. 4 Water content of G. mangostana shoot tips after various

periods of exposure to PVS2 at 25 �C. Bars represent standard

deviation (SD) (n = 15)

Table 1 Effects of PVS2 exposure temperature on the survival per-

centage of G.mangostana shoot tips following cryopreservation

Survival percentage of shoot tips (%)

Temperature (oC)

0 25

-LN 63.3 ± 17.4a 26.3 ± 2.2b

?LN 10.4 ± 1.8b 8.3 ± 1.7b

Assessment was done after 10 days on recovery medium

Different letters indicate significant difference at p B 0.05 using

Duncan’s multiple-range test. Mean ± SD (n = 15) are shown

-5

0

5

10

15

20

25

0 0.3 0.6 0.9

Su

rviv

al (

%)

Sucrose concentration (M)

Day 1

Day 2

Fig. 5 Effects of sucrose concentration and periods of preculture

after 10 days on recovery medium on the survival of G.mangostanashoot tips following cryopreservation. Bars represent mean ± SD

(n = 15)

0

20

40

60

Without preculture Full strength Half strength

Su

rviv

al (

%)

-LN

+LN

bb

a

ab

ab

ab

Fig. 6 Effects of two MS medium strengths of the preculture

medium on survival after 10 days on recovery medium of G.mangostana shoot tips following cryopreservation.–LN indicates

explants not exposed to liquid nitrogen (LN) and ?LN indicates

explants exposed to LN. Different letters indicate significant differ-

ence at p B 0.05 using Duncan’s multiple-range test. Mean ± SD

(n = 15) are shown


123

66.7 ± 16.7 %, respectively. The cryopreserved shoot tips

also showed no significant difference between both con-

ditions, with 33.1 ± 6.9 % and 44.1 ± 6.5 % survival,

respectively. The use of the unloading solution with the

higher sucrose concentration which is the standard

unloading solution (control) did not contribute to any

improvement in the survival of the shoot tips after cryo-

preservation. Thus, the unloading solution with 0.4 M

sucrose supplemented with 2 M glycerol concentration

suggested by Matsumoto et al. (1998) for tropical species

was chosen for the subsequent experiments.

Effects of ascorbic acid on the survival of the shoot tips


Studies on the effects of ascorbic acid were also conducted

to ascertain whether the survival of shoot tips after cryo-

preservation could be improved. Each step in vitrification

protocol (preculture, loading, PVS2, unloading and recov-

ery) was studied with respect to the effect of addition of

ascorbic acid. The results of cryopreservation showed that

the addition of ascorbic acid on each step in the vitrification

protocol (preculture, loading, PVS2, unloading solutions

and recovery medium) provided no significant differences

from the control. However, cryopreserved shoot tips treated

with ascorbic acid showed signs of regrowth on the 10th day

compared to 21 days for explants not treated with ascorbic

acid. Apart from that, the highest survival (45.8 ± 3.8 %) of

shoot tips after cryopreservation in this study was observed

in the treatment that was exposed to the unloading solution

with added ascorbic acid (Fig. 7). Figure 8 shows the

cryopreserved G. mangostana shoot tips treated with the

unloading solution with added ascorbic acid after 20 days of

culture.

Effects of the dark/light period during recovery

on the survival of the shoot tips after cryopreservation

Based on the mean separation analysis that is shown in

Fig. 9, increasing the period in the dark was associated

with improvement in the survival percentage of shoot tips

after cryopreservation. Subjecting the shoot tips to the

normal culture photoperiod of 16 h/8 h light/dark photo-

period result in low survival percentage (6.7 ± 5.0 %). In

contrast, the shoot tips that were subjected to 7 days of

dark period resulted in significantly higher survival

(50.0 ± 16.7 %).

Discussion

To ensure a high percentage of plant tissue survival and

recovery after cryopreservation using the vitrification

technique, the determination of the most suitable solution

at each step of the vitrification protocol and the duration of

exposure for each should be optimized.

In the present study, preliminary results after dehydration

with PVS2 solution for 25 min were considered to be the

best PVS2 treatment for cryopreservation of G. mangostana

shoot tips (Fig. 2). Tolerance to dehydration with PVS2

varies among species and this might be because of differ-

ence in genotype and tissue water exchange properties that

are the primary determinant of the rate of osmotic dehy-

dration in any particular species (Wang et al. 2004; Vicente

et al. 2011). However, prolonged exposure of shoot tips to

the PVS2 may lead to excessive osmotic stress and chemical

toxicity (Hoong et al. 2009). This is proven in our study

where water content decreased with increasing PVS2

exposure period (Fig. 4). Exposure to high concentrations of

vitrification solution can impose extreme biophysical and

chemical stress on germplasm, thus optimizing the water

content prior to LN exposure is crucial to ensure high sur-

vival after cryopreservation (Benson 2008). Wesley-Smith

et al. (1992) suggested that plant organs can be successfully

cryopreserved at water contents range of 1.1 g–1.6 g g-1

dry mass. Based on our study, 25 min of PVS2 exposure

showed survival of 35.6 ± 11.8 % with water content of

1.1 ± 0.3 g g-1 dry mass. Prolonged PVS2 exposure

(30 min) led to excessive dehydration leading to cell death.

Therefore, balancing the water content is important to

minimize the desiccation damage and freezing injury in

order to develop a successful cryopreservation protocols

(Volk and Walters 2006).

Apart from that, the stepwise method for PVS2 exposure

was chosen to be studied because reports have shown that

the toxicity of the PVS2 exposure could be reduced by

pretreatment with a lower concentration of PVS2 followed

by the standard concentration (Hoong et al. 2009; Chua and

Normah 2011). Similar observations were made in this

study in that the recalcitrant-seeded cryopreserved shoot

tips pretreated with 50 % PVS2 for 15 min, followed by

exposure to the full concentration of PVS2 for 10 min,

showed higher survival percentages after cryopreservation

Table 2 Percentage survival of shoot tips of G. mangostana with in

relation to the different unloading solution after cryopreservation

Survival percentage of shoot tips (%)

Unloading solution

0.4 M sucrose ? 2 M glycerol 1.2 M sucrose

-LN 66.7 ± 16.7a 57.1 ± 10.0a

?LN 44.1 ± 6.5b 33.1 ± 6.9b

Assessment was done after 10 days on recovery medium

Different letters indicate significant difference at p B 0.05 using

Duncan’s multiple-range test. Mean ± SD (n = 15) are shown


123

(39.2 ± 10.1 %) compared with the other treatments

(Fig. 3).

In addition to the exposure periods, one of the keys to

successful cryopreservation by vitrification is the careful

control of dehydration and the prevention of injury from

chemical toxicity while using a suitable temperature

(Vendrame et al. 2007). In this study, PVS2 at a lower

temperature (0 �C) was shown to be more favourable com-

pared with a higher PVS2 temperature (25 �C) (Table 1).

The reason for this result may be that at a low temperature the

mobility of water molecules contributing to prevention of

formation of ice crystals might be restricted (Benson 2008).

In contrast, at the higher temperature of 25 �C, the pene-

tration of PVS2 through the cell membrane is faster and may

0

20

40

60

80

(-)Ascorbic acid (+) Ascorbic acid

Su

rviv

al (

%)

Preculture

-LN

+LN

0

20

40

60

80

(-)Ascorbic acid (+)Ascorbic acid

Su

rviv

al (

%)

Loading

0

20

40

60

80


Su

rviv

al (

%)

PVS2

0

20

40

60

80


Su

rviv

al (

%)

Unloading

-LN

0

20

40

60

80


Su

rviv

al (

%)

Recovery

b b

a

b

a a

aa

ab

b

a

ab abb

a

ab

aa

a

a

-LN

+LN

-LN

+LN

-LN

+LN

Fig. 7 The effect of ascorbic acid supplied at each of five stages

(during vitrification, unloading and recovery) on survival of G.man-gostana shoot tips following LN exposure. Explants were assessed

after 10 days on the recovery medium.–LN indicates explants not

exposed to liquid nitrogen (LN) and ?LN indicates explants exposed

to LN. Addition of ascorbic acid (?) and control treatment (-) (without

ascorbic acid). Different letters indicate significant difference at

p B 0.05 using Duncan’s multiple-range test. Mean ± SD (n = 15)

are shown

1.5 mm

Fig. 8 Cryopreserved G. mangostana shoot tip treated in unloading

solution with ascorbic acid after 20 days of observation on the

recovery medium

01020304050607080

0 3 5 7

Su

rviv

al (

%)

Dark Period (Day)

b

b b

a

Fig. 9 Effects of dark/light period on the survival of G. mangostanashoot tips following cryopreservation. Survival was assessed after

21 days on recovery medium. Different letters indicate significant

difference at p B 0.05 using Duncan’s multiple-range test. Mean ±

SD (n = 15) are shown


123

result in toxic effects or excessive dehydration of the shoot

tips (Safrinah et al. 2009). A study on Nephelium ramboutan-

ake by Chua and Normah (2011) supports our result; in their

study, the shoot tips exposed to PVS2 for 15 min at 0 �C

showed higher survival percentages compared with expo-

sure to PVS2 at 25 �C. Similarly, the protocorm-like bodies

of Dendrobium showed higher percentages of viability after

treatment with PVS2 at 0 �C followed by LN (Tiau et al.

2009).

Previous studies have shown that the preculture stage is

crucial for most woody plant species. Based on the study by

Xu et al. (2006), the reason for the importance of this stage is

that the vacuoles become small and the free water content in

the cells decreases after preculture, indicating that the

freezing tolerance and dehydration capacity increases with

minimum damage while improving the resistance to cold

and enabling the cell membrane to maintain a stable struc-

ture. The inclusion of sucrose in the preculture medium has

been known to strengthen the cell membrane before expo-

sure to the loading solution and other vitrification solutions

of high concentrations. In the present study, cryopreserved

shoot tips precultured in MS medium supplemented with

0.6 M sucrose for 2 days showed highest viability percent-

age (13.7 ± 5.5 %) (Fig. 5). There was a significant dif-

ference observed between treatments for 2 days exposure.

Preculture with a high sucrose concentration (0.6 M) may

have increased total sugar content in the treated tissues,

which could be associated with increased survival after

cryopreservation as shown by Tan et al. (2010). Prolonged

periods of exposure on preculture medium may have also

slightly increased the sucrose accumulation, which could

prevent cell injury and protect the cell membrane from ice

formation by forming an amorphous glass phase between

adjacent cells (Fujikawa and Jitsuyama 2000). However,

higher sucrose concentration (0.9 M) resulted in no survival

after cryopreservation. This may be due to the excessive

accumulation of sucrose in the cytoplasm, which can lead to

toxic effects, as suggested by Ishikawa et al. (2000).

Apart from the sucrose concentration, MS medium

strength is also important in ensuring a high percentage of G.

mangostana shoot tip survival after cryopreservation. Sakai

et al. (2000) showed that the best results for tropical plants are

obtained with preculture in half-strength MS medium. Half

strength MS medium offers a promising result for optimum

in vitro growth which increase the spearmint shoot tips sur-

vival after cryopreservation (Fadel et al. 2010). However, in

the present study, the survival of the G. mangostana shoot tips

was not favoured by preculture in half-strength MS. Many of

the studies on the cryopreservation of tropical species also

used full-strength MS medium, including the studies on Asian

Dioscorea bulbifera calli (Hua and Rong 2010).

The unloading solution is an important factor in cryo-

preservation by vitrification techniques. The unloading

solution ensures that the PVS2 is effectively removed from

the cells to avoid potentially lethal cytotoxic effects. In our

study, a survival percentage of 44.1 ± 6.5 % was obtained

with cryopreserved shoot tips that were treated with 0.4 M

sucrose supplemented with 2 M glycerol (Table 2). The

shoot tips treated with a high sucrose concentration (1.2 M)

might have undergone hypertonic effects and osmotic

stress, which resulted in their lower survival as reported by

Hirata et al. (2002). The cells in the high sucrose treatment

may also have suffered plasmolysis, which causes higher

death percentages of shoot tips after cryopreservation as

reported by Volk and Caspersen (2004).

Oxidative stress is a potential cause of damage to plant

tissues. Thus, the addition of antioxidants and anti-stress

compounds is believed to improve shoot tip regrowth after

cryopreservation by preventing or repairing any damage

(Uchendu et al. 2010a). Uchendu et al. (2010b) suggested

that ascorbic acid in vitrification solutions might reduce

malondialdehyde (MDA) formation and reduce the injury

caused by osmotic stress, thus ensuring a higher percentage

of shoot tip survival after cryopreservation. Based on the

present study, even though there were no significant dif-

ference, the addition of ascorbic acid to the unloading

solution proved to be beneficial to the survival of the

cryopreserved shoot tips of G. mangostana where higher

survival (45.8 ± 3.8 %) and faster recovery was observed

when cryopreserved shoot tips were treated with ascorbic

acid. The shoot tips also showed signs of earlier regrowth

on the 10th day compared to 21 days for explants not

treated with ascorbic acid. The reason behind the non-

significant results may be because the ascorbic acid con-

centration (0.28 mM) used in this study is not optimum for

the survival of the shoot tips after cryopreservation and

probably the use of ascorbic acid as the source of antiox-

idant is not suitable to reduce osmotic stress injury after

cryopreservation for mangosteen.

In the study of recovery in dark/light conditions, there

was a significantly higher survival (50.0 ± 16.7 %) for the

shoot tips that were subjected to 7 days of dark period

(Fig. 9). Withers (1988) suggested that cryopreserved

in vitro plantlets should be maintained under dark condi-

tions for recovery process as cultures maintained under dark

condition or minimal amount of light are able to reduce

potentially harmful photo-oxidative effects. This is then

supported by Senula et al. (2007) and Touchell et al. (2002),

who reported that gradually exposing the cryo-sample to

light after cryopreservation increased regeneration due to

removal of stress associated with photo-oxidation.

Recalcitrant seeds are difficult to manipulate; in particu-

lar, G. mangostana is known to have highly recalcitrant

characteristics. Even though cryopreservation using vitrifi-

cation is the most promising method, no previous study on

the successful cryopreservation of G. mangostana could be


123

found in the literature. For the first time, the present study

details a successful cryopreservation protocol for G. man-

gostana shoot tips using the vitrification technique that

resulted in the best survival by using preculture with MS

medium supplemented with 0.6 M sucrose for 2 days,

25 min exposure of PVS2 at 0 �C, exposure to MS supple-

mented with 0.4 M sucrose unloading solution and sub-

jecting to 7 days of dark period before transferring to normal

culture of 16 h/8 h light/dark photoperiod for recovery.

Further research is required to determine the properties of the

recovery medium and regrowth conditions to improve the

survival percentage of G. mangostana shoot tips after

cryopreservation. Studies on the different antioxidants at

different concentrations could also be studied. Apart from

that, studies on the structural changes during the recovery of

shoot tips after cryopreservation would highlight changes or

damages occurred during and after cryopreservation. These

efforts will help to ensure that the in vitro-generated shoot

tips of G. mangostana can be preserved using cryopreser-

vation by the vitrification approach.

References

Benson EE (2008) Cryopreservation of phytodiversity: a critical

appraisal of theory and practice. Crit Rev Plant Sci 27:141–219

Berjak P, Sershen, Varghese B, Pammenter NW (2011) Cathodic

amelioration of the adverse effects of oxidative stress accompa-

nying procedures necessary for cryopreservation of embryogenic

axes of recalcitrant-seeded species. Seed Sci Res 21:187–203

Berjak P, Pammenter NW, Wesley-Smith J, Kioko JI, Norris M,

Mycock DJ (2000) In: Engelmann F, Takagi H (eds) Cryopres-

ervation of tropical plant germplasm. Current research progress

and application: Rome, pp 315–319

Chairrungsri N, Takeuchi K, Ohizumi Y, Nazoe S, Abd Ohta T (1996)

A Prenyl Xanthome from Garcinia mangostana. Phytochemistry

43(5):1099–1102

Chang Y, Reed BM (2001) Preculture conditions influence cold

hardiness and regrowth of pyrus cordata shoot tips after

cryopreservation. HortScience 36:1329–1333

Chua SP, Normah MN (2011) Effects of preculture, PVS2 and

vitamin C on the survival of recalcitrant Nephelium ramboutan-

ake shoot tips after cryopreservation by vitrification. CryoLetters

32(6):506–515

Day JG, Fleck RA, Benson EE (2000) Cryopreservation-recalcitrance

in microalgae: novel approaches to identify and avoid cryo-

Injury. J Appl Phycol 12:369–377

Engelmann F (1997) In vitro Conservation methods. In: Ford-Llyod

BV, Newbury HJ, Callow JA (eds) Biotechnology and plant

genetic resources: conservation and use: UK, pp 119–162

Engelmann F (2011) Use of biotechnologies for the conservation of

plant biodiversity. In Vitro Cell Dev Biol Plant 47(1):5–16

Fadel D, Kintzios S, Economou AS, Moschopoulou G, Constantin-

idou AHI (2010) Effect of different strength of medium on

organogenesis, phenolic accumulation and Antioxidant activity

of spearmint (Melia spicata I.). The Open Hortic J 3:31–35

Fujikawa S, Jitsuyama Y (2000) Ultrastructural aspects of freezing

adaptation of cells by vitrification. In: Engelmann F, Takagi H

(eds) Cryopreservation of tropical plant germplasm: current

research progress and application: Rome, pp 36–42

Hirata K, Monthana B, Sakai A, Miyamoto K (2002) Biotechnology

in agriculture and forestry. In: Bajaj YPS (ed) Towill LE.

Cryopreservation of plant germplasm II, Japan, pp 57–65

Hoong S, Yin M, Shao X, Wang A, Xu W (2009) Cryopreservation of

embryogenic callus of Dioscorea bulbifera by vitrification.

CryoLetters 30(1):64–75

Hua YM, Rong HS (2010) A simple cryopreservation protocol of

Dioscorea bulbifera L. embryogenic calli by encapsulation-

vitrification. Plant Cells, Tissue Organ Cult 101:349–358

Ishikawa M, Hiroyuki I, Price WS, Arata Y, Kitashima T (2000)

Freezing behaviours in plant tissues as visualised by nmr

microscopy and their regulatory mechanisms. In: Engelmann F,

Takagi H (eds) Cryopreservation of tropical plant germplasm:

current research progress and application: Rome, pp 22–35

Lambardi M, Fabbri A, Caccavale A (2000) Cryopreservation of

white poplar (Populus alba L.) by vitrification of In Vitro grown

shoot tips. Plant Cell Rep 19:213–218

Matsumoto T,Sakai A, NakoY (1998)A novel preculturing forenhancing

the survival of In-vitro grown meristems of wasabi (Wasabiajaponica) cooled to -196�C by vitrification. CryoLetters 19:27–36

Murashige T, Skoong F (1962) A revised medium for rapid growth and

bioassays with tobacco tissue culture. Physiol Plantarum 15:473–497

Niino T, Tashiro K, Suzuki M, Ohuchi S, Magoshi J, Akihama T

(1997) Cryopreservation of In Vitro shoot tips of cherry and

sweet cherry by one step vitrification. Sci Hortic 70:155–163

Normah MN, Nor-Azza AB, Aliudin R (1995) Factors affecting

in vitro shoot proliferation and ex vitro establishment of

mangosteen. Plant Cell, Tissue Organ Cult 43:291–294

Normah MN, Choo WK, Yap LV, Zeti Azura MH (2011) In vitroconservation of Malaysian biodiversity-achievements, challenges

and future directions. In Vitro Cell Dev Biol Plant 47:26–36

Reed BM, Uchendu E (2008) In: Reed BM (ed) Plant cryopreserva-

tion: a practical guide. Springer Science and Bussiness Media,

New York, pp 77–92

Safrinah R, Xavier R, Rani U, Sreeramanan S (2009) Optimisation of

cryopreservation technique in Mokara golden nugget orchid

using PVS2 vitrication. Inter J Agric Res 4(7):218–227

Sakai A, Kobayashi S, Oiyama I (1990) Cryopreservation of nucellus

cells of navel orange (Citrus sinensis var. Brasilliensis Tanaka)

by vitrification. Plant Cell Rep 9:30–33

Sakai A, Matsumoto T, Hirai D, Niino T (2000) Newly developed

encapsulation-dehydation protocol for plant cryopreservation.

CryoLetters 21:53–62

Senula A, Keller ERJ, Sanduijav T, Yohannes T (2007) Cryopres-

ervation of cold acclimated mint (Mentha spp.) shoot tips using a

simple vitrification protocol. CryoLetters 28:1–12

Takagi H, TienThinh N, Islam OM, Senboku T (1997) Cryopreser-

vation of in vitro grown shoot tips of taro (colocasia esculenta l.)

by vitrification. Plant Cell Rep 16:594–599

Tan HH, Jessica J, Advina J, Ranjeeta P, Gnasekaran P, Sreeramanan

S (2010) A novel approach for preliminary pvs2 vitrification

optimization parameters of dendrobium sonia-28 orchid with

evan blue staining. Adv Environ Biol 4(2):284–290

Tiau KH, Xavier R, Chan LK, Sreeramanan S (2009) An assessment

of early factors influencing the PVS2 vitrification method using

protocorm-like bodies of Dendrobium Sonia 28. Am-Eurasian J

Sustain Agric 3(3):280–289

Touchell DH, Walters C (2000) Recovery of embryos of Zizaniapalustris following exposure to liquid nitrogen. CryoLetters

21:261–270

Touchell DH, Turner SR, Bunn E, Dixon KW (2002) Cryostorage of

somatic tissues of endangered australian species. In: Towill LE,

Bajaj YPS (eds) Cryopreservation of plant germplasm. Springer,

Heidelberg, pp 357–372

Uchendu EE, Leonard SW, Traber MG, Reed BM (2010a) Vitamins

C and E improve regrowth and reduce lipid preoxidation of


123

blackberry shoot tips following cryopreservation. Plant Cell Rep

29(1):25–35

Uchendu EE, Muminova M, Gupta S, Reed BM (2010b) Antiox-

idant and anti-stress compounds improve regrowth of cryopre-

served rubus shoot tips. In Vitro Cell Develop Biol-Plant

46:386–393

Uragami A, Lucas MO, Ralambosoa J, Renard M, Dereuaddre J

(1993) Cryopreservation of microscope embryos of oilseed rape

(Brassica napus L.) by dehydration in air with or without algine

encapsulation. CryoLetters 14:83–90

Varghese D, Berjak P, Pammeter NW (2009) Cryopreservation of

shoot tips of Trichilia emetica. A tropical recalcitrant-seeded

species. cryoletters 30(3):280–290

Vendrame WA, Carvalho VS, Dias JMM (2007) In vitro germination

and seedling development of cryopreserved dendrobium hybrid

mature seeds. Scientia Hort 114:188–193

Vicente S, Nieto AB, Hodara K (2011) Changes in structure,

rheology, and water mobility of apple tissue induced by osmotic

dehydration with glucose or trehalose. Food Bioprocess Technol

5(8):3075–3089

Volk GM, Caspersen AM (2004) The cryopreservation process

induces plasmolysis in mint shoot tips. Plasmodesmata (August

17–21, 2004) Monterey, CA, pp 118

Volk GM, Walters C (2006) Plant vitrification solution 2 lowers water

content and alters freezing behaviour in shoot tips during

cryoprotection. Cryobiology 52:48–61

Wang QC, Munir M, Nachman S, Li P, Colova-Tsolovo V, Ron G,

Ilan S, Edna T, Avihai P (2004) Cryopreservation of grapevine

(Vitis spp.) embroygenic cell suspensions by encapsulation-

vitrification. Plant Cell Tissue Org Cult 77:267–275

Wesley-Smith J, Vertucci CW, Berjak P, Pammenter NW, Crane J

(1992) Cryopreservation of dessication-sensitive axes of Camel-lia sinensis in relation to dehydration, freezing rate and the

thermal properties of tissue water. J Plant Physiol 140:596–604

Withers LA (1988) Germplasm conservation. In: Block G, March J

(eds) Application of plant cell and tissue culture. Chichester,

Wiley, NY, pp 163–177

Xu XB, Cai ZG, Gu QQ, Zhang QM (2006) Cell ultrastructure of

Kiwifruit (Actinida chinensis) shoot tips during cryopreserva-

tion. Agric Sci China 5(8):587–590


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