CryoLetters 36 (1), 51-59 (2015)

CryoLetters, businessoffice@cryoletters.org

CRYOPRESERVATION OF SUGARCANE USING

THE V CRYO-PLATE TECHNIQUE
Tariq Rafique1, 2*, Shin-ichi Yamamoto1, Kuniaki Fukui1, Zahid Mahmood2 and Takao Niino1, 3
1

Genebank, National Institute of Agrobiological Sciences (NIAS), 2-1-2 Kannondai, Tsukuba 3058602, Japan.
2
Plant Genetic Resource Institute, National Agricultural Research Center, Islamabad 45500, Pakistan.
3
University of Tsukuba, 1-1-1 Tennodai, Tsukuba 305-0006, Japan
*Corresponding author enail: tariqabp@gmail.com
Abstract
BACKGROUND: Sugarcane is a tropical crop of major importance primarily for its high
sucrose content. It is difficult to conserve it in the field or in vitro because of biotic and abiotic
stresses. Cryopreservation of sugarcane germplasm is an appropriate approach for conserving its
genetic diversity. OBJECTIVE: This study was carried out to develop an efficient and practical
cryopreservation protocol for sugarcane with high post-cryopreservation recovery. MATERIALS
AND METHODS: Factors affecting regrowth after cryopreservation using the V cryo-plate method
including preculture medium, size of shoot tips, sucrose concentration in loading solution, exposure
time to PVS2, light conditions after liquid nitrogen exposure, presence and absence of alginate gel and
recovery medium composition were studied. RESULTS: Shoot tips with a length of 1.5 to 2.0 mm,
precultured on semi-solid 1/2 MS medium for 1 day and semi-solid MS medium with 0.5 M sucrose
for 1 day, treated with LS containing 2.0 M glycerol + 1.6 M sucrose for 30 min and exposed to PVS2
for 30 min showed maximum (100%) recovery after cryopreservation. It was also observed that
removing the alginate gel and keeping the cultures in the dark for 7 days after cryopreservation
significantly improved recovery. After optimizing the cryopreservation conditions using sugarcane
variety Ni-1, 10 additional varieties were cryopreserved using the optimized protocol, with regrowth
ranging from 56.7% to 100%. CONCLUSION: This study showed that V cryo-plate is an efficient
and practical method for cryopreservation of sugarcane shoot tips in genebanks.
Keywords: cryopreservation, PVS2, sugarcane, V cryo-plate, vitrification.

its genetic structure intact and after 15 years

culture the plants need to be replaced. This is
why in many parts of the world sugarcane
germplasm is conserved as field collections of
plants. In Japan 1975 accessions including 592
wild species of sugarcane are conserved in field
genebanks at different locations. The drawback
of sugarcane field conservation is that it makes
the germplasm exposed to various biotic and
abiotic stresses, and germplasm maintenance is
costly (3). Another way to conserve sugarcane
germplasm is the use of in vitro methodology.
CIRAD-CA Montpellier has more than 650
sugarcane varieties conserved in in vitro culture

INTRODUCTION
Sugarcane has become an increasingly
important crop in recent years, not only because
it is the raw material for sugar industries, but
also because of its importance in industries
producing alcohol, acetic acid, butanol, paper,
plywood, industrial enzymes and animal feed
(1). Sugarcane includes various genotypes, some
of which are sterile and others that produce
orthodox seeds. However, orthodox seeds are
generally heterozygous, which makes them
unstable for conservation (23). Sugarcane is
propagated asexually (vegetatively) to maintain

51

under slow growth conditions (21). However,

maintaining a large in vitro collection is
laborious because of the need to periodically reculture to maintain the germplasm (31) and there
is also the risk of contamination. The main
concern in genetic resources conservation is the
maintenance of genetic stability. There is also
the risk of somaclonal variation when sugarcane
germplasm has been conserved in tissue culture
for extended periods (20).
To overcome all these difficulties,
cryopreservation of sugarcane is an alternative
solution for long-term conservation as it is safe
and
cost-effective.
In
cryopreservation,
germplasm is stored in liquid nitrogen (LN, 196oC), which virtually stops all metabolic
activities in stored explants (4). Research has
been conducted recently to develop effective
cryopreservation methods for sugarcane (13).
The V cryo-plate method using aluminium cryoplates has been reported for strawberry (32),
Dalmatian chrysanthemum (33), mint (35),
mulberry (34), carnation (27) and mat rush (18)
shoot tips/buds. This method produced very high
regrowth after cryopreservation with the tested
plant species. The advantages of this method are
its simplicity and user-friendliness. It also
ensures very high cooling and warming rates of
treated explants, which protects them from
freezing damage.
The present study was carried out to
develop cryopreservation of sugarcane shoot tips
using the V cryo-plate method for practical use
in genebanks.

placed on 1/2 MS semi-solid medium (15)

containing 3% sucrose, 0.01 mg/l naphthalene
acetic acid (NAA) and 0.1 mg/l benzyladenine
(BA) in test tubes (9). Initially all the cultures
were kept in the dark at room temperature (RT)
for 7 days and after that incubated at 252oC
with 16 h light/8 h dark photoperiod under
fluorescent light (52 mol/m2/s, standard
condition). In vitro shootlets were then
transferred to 1/2 MS semi-solid medium
containing 3% sucrose and 1 ml/l Plant
Preservative Mixture (PPM, Plant Cell
Technology, Inc, Canada) in 80 x 102 mm jars.
Preconditioning and preculture
After removal of dead leaves, in vitro grown
shoots of sugarcane containing shoot tips were
cut to a size of 57 mm (Fig. 1A) and plated on
semi-solid 1/2 MS medium containing 3%
sucrose in Petri dishes for one week to obtain
uniform material for shoot tip excision. The
shoot tips used were 1.52.0 mm long with a
basal part (Fig. 1B). The excised shoot tips were
cultured on semi-solid 1/2 MS medium
containing 3% sucrose for 1 day and precultured
on semi-solid MS medium containing 0.5 M
sucrose for 1 day at RT. For the experiment
studying the effect of shoot tip size, three
different sizes were used: 0.51.0, 1.01.5 and
1.52.0 mm. For the preculture experiment,
shoot tips were precultured on semi-solid MS
medium containing 0.3 M or 0.5 M sucrose for 1
or 2 days at RT.
V cryo-plate procedure
The successive steps of the V cryo-plate
procedure developed by Yamamoto et al. (35)
were as follows 1) aluminium cryo-plates No. 2 (33) were
placed in a Petri dish and 2.02.5 l of a 2%
(w/v) sodium alginate solution in a calcium-free
MS basal medium were poured in each well of
the cryo-plate.
2) precultured shoot tips were placed in the
well, one by one, with the tip of a scalpel blade
and the shoot tips lightly pressed to make them
fit into the plates well (Fig. 1C).
3) subsequently, calcium solution (0.1 M
calcium chloride in basal MS medium) was
poured on the section of the aluminium plates
where the shoot tips were located until they were
covered and left for 15 min to achieve complete
polymerization of alginate gel at RT (Fig. 1D).
4) calcium solution was removed from the cryoplate by sucking it up gently with a micropipette.

MATERIALS AND METHODS

Plant materials and tissue culture protocols
Sugarcane germplasm was acquired from
sub-genebanks of Japan. Sugarcane variety Ni-1
was used to optimize the conditions of the
successive steps of the V cryo-plate method.
After optimization of the protocol, 10 additional
varieties were also tested using the
cryopreservation protocol developed with
variety Ni-1. Lateral buds from fully grown
canes were excised, washed with Muse
medicated liquid soap under tap water and
surface-sterilized with 70% ethanol for 1 min
and sodium hypochlorite solution with 2% active
chlorine for 15 min. The buds were then washed
three times with sterile water and from these
sterilized buds, 24 mm shoot tips were excised
under the laminar flow hood using a
stereomicroscope. Excised shoot tips were

52

The shoot tips adhered to the cryo-plate through

the alginate gels distributed in the wells.
5) cryo-plates with shoot tips were then placed
in a 25 ml pipetting reservoir filled with about

20 ml LS (19), which contained 2.0 M glycerol

and 0.8, 1.2 or 1.6 M sucrose in liquid MS basal
medium (Fig. 1E). The shoot tips were
osmoprotected at 25oC for 30 min.

Figure 1. V cryo-plate procedure for sugarcane and regrowth after cryopreservation.

A) Preconditioning of shoots on 1/2 MS medium to get uniform shoot tips;
B) Preculture on semi-solid MS medium with 0.5 M sucrose;
C) Shoot tips mounted on cryo-plate with sodium alginate solution;
D) Hardening of gel with calcium solution;
E) Shoot tip treatment with LS and PVS2;
F) Immersion of cryotubes containing cryo-plates in LN;
G) Warming in 1.0 M sucrose solution after LN exposure;
H) Regenerated shoot tips after LN exposure (3 weeks after rewarming).

53

6) subsequently, cryo-plates were placed in a

25 ml pipetting reservoir filled with about 20 ml
PVS2 (25). The shoot tips were treated with
PVS2 at 25oC for 20, 30 or 40 min. One
experiment was performed to compare the effect
of treatment with PVS2 and PVS3 (2)
vitrification solutions.
7) after dehydration, cryo-plates were transferred
to uncapped 2 ml plastic cryotubes held on a
cryo-cane, and directly plunged in LN where
they were kept for at least 30 min (Fig. 1F).
8) the cryotubes were retrieved from LN for
regeneration and the cryo-plates were immersed
in 2 ml of 1.0 M sucrose solution in 2 ml
cryotubes (Fig. 1G). The shoot tips were
incubated in this solution for 15 min at RT.
9) alginate gel was removed from shoot tips
prior to transfer to semi-solid 1/2 MS medium
containing 3% sucrose, 0.01 mg/l NAA and 0.1
mg/l BA in Petri dishes for regrowth (Fig. 1H).
10) initially all the cultures were kept in the dark
for 7 days and then transferred into light under
standard conditions. In one experiment aiming at
observing the effect of light exposure, the shoot
tips were placed under light directly after
transfer to regrowth medium.
11) in another experiment, the effect of the
growth medium on regrowth of cryopreserved
shoot tips was tested. Shoot tips were transferred
on medium without growth regulators, with 0.01
mg/l NAA + 0.1 mg/l BA or with 0.01 mg/l
NAA + 0.1 mg/l BA + 0.01 mg/l
polyvinylpyrolidone (PVP).

Preconditioning and preculture

The regrowth of cryopreserved shoot tips
was affected by the size of shoot tips. Regrowth
significantly increased in line with increasing
shoot tip size (Table 1). Shoot tips of a size of
1.52.0 mm displayed 100% regrowth after
cryopreservation.
Preculture of shoot tips on medium with
high sucrose is an important step to prepare them
for cryostorage and to help them acquire the
capacity
for
high
regrowth
after
cryopreservation. When shoot tips were
precultured on medium with 0.5 M sucrose for 1
day at RT, regrowth was maximum (100%) after
cryopreservation whereas regrowth was lower
after preculture with 0.3 M sucrose (Table 2).
With both sucrose concentrations, regrowth
decreased when preculture duration increased to
2 days.
V cryo-plate procedure (osmoprotection and
dehydration)
After preculture of shoot tips on semi-solid
MS medium with 0.5 or 0.3 M sucrose, the
effect of osmoprotection with LS containing
different sucrose concentrations and of PVS2
treatment duration was evaluated (Tables 3 and
4). When shoot tips were precultured on medium
with 0.5 M sucrose, high regrowth was obtained
with LS containing 2.0 M glycerol + 1.6 M
sucrose for 30 min regardless of the PVS2
exposure duration (Table 3). By contrast, when
Table 1. Effect of shoot tip size on regrowth
after cryopreservation using the V cryo-plate.

Data analysis
Regrowth (shoot elongation with or without root
formation) of cryopreserved shoot tips was
evaluated 4 weeks after rewarming. Each
experimental treatment was replicated three
times with each replication containing 10 shoot
tips. Statistical analysis was performed using
ANOVA and then Tukeys honest significant
difference (HSD) was used to compare the
means. Significant differences were set at
P0.05. Means of replicates with standard error
are presented in Tables and Figures.

Shoot tip size (mm)

0.5 1.0
1.0 1.5
1.5 2.0

Regrowth (% SD)
13.3 5.8 c
60.0 10.0 b
100.0 0.0 a

Mean values followed by different letters differ

significantly at the 0.05 probability level.
Table 2. Effect of sucrose concentration in
preculture medium and of preculture duration on
regrowth (%) of shoot tips cryopreserved using
the V cryo-plate.

RESULTS

Sucrose concentration
Regrowth
and preculture duration
(% SD)
0.3 M sucrose, 1 day
76.7 5.8 ab
0.3 M sucrose, 2 days
36.7 11.5 c
0.5 M sucrose, 1 day
100.0 0.0 a
0.5 M sucrose, 2 days
70.0 17.3 b
Mean values followed by different letters differ
significantly at the 0.05 probability level.

In
the
V
cryo-plate
procedure,
preconditioning, preculture, osmoprotection with
LS, dehydration with PVS2 and post-rewarming
handling of shoot tips are key parameters for
successful cryopreservation.

54

shoot tips were precultured on medium

containing 0.3 M sucrose for 1 day, regrowth
was variable, even though maximum regrowth
(100%) was obtained with shoot tips
osmoprotected with LS containing 1.2 M and 0.8
M sucrose and exposed to PVS2 for 20 and 30
min, respectively (Table 4). The combination
including preculture with 0.5 M sucrose, LS
treatment with 1.6 M sucrose and PVS2
treatment for 30 min was selected as optimal
based on the higher vigour of regrowing plants

compared to other experimental conditions.

When comparing the effect of PVS2 and
PVS3 vitrification solutions on shoot tip
regrowth, it appeared that regrowth following
PVS2 treatment was twice that measured after
PVS3 treatment (Table 5).
Post-warming regrowth
Post-warming manipulations are very
important to obtain high regrowth of
cryopreserved shoot tips. Removing alginate gel
from shoot tips significantly increased post-LN
regrowth compared to shoot tips covered with
alginate gel (Table 6).
The initial light conditions after rewarming
affected regrowth of cryopreserved shoot tips.
When cryopreserved shoot tips were placed in
standard light conditions, their regrowth was
significantly lower compared to shoot tips,
which were kept in the dark for 7 days (Table 7).
The composition of the culture medium
also affected regrowth after cryopreservation.
The addition of 0.01 mg/l NAA and 0.1 mg/l BA
in the culture medium produced 100% regrowth,
whereas it was only 80% on medium containing
0.01 mg/l NAA + 0.1 mg/l BA + 0.01 mg/l PVP
and 67% on medium devoid of growth
regulators (Table 8).

Table 3. Effect of sucrose concentration in LS

o
and of duration of exposure to PVS2 at 25 C on
regrowth (%) of sugarcane Ni-1 shoot tips
cryopreserved using the V cryo-plate procedure.
Preculture medium contained 0.5 M sucrose.
Sucrose
conc. in LS
0.8 M

1.2 M

1.6 M

Exposure time
in PVS2
20 min
30 min
40 min

Regrowth
(% SD)
70.0 20.0 b
90.0 0.0 ab
78.0 11.5 ab

20 min

90.0 10.0 ab

30 min

83.3 11.5 b

40 min

93.3 5.8 ab

20 min

96.7 5.8 a

30 min

100.0 0.0 a

40 min

96.7 5.8 a

Optimal V cryo-plate procedure

The optimal conditions established for Ni-1
were as follows: 1.5 to 2.0 mm long shoot tips
were placed for 1 day on semi-solid 1/2 MS
medium with 3% sucrose, precultured on semisolid MS medium containing 0.5 M sucrose for
1 day, osmoprotected with LS containing 2.0 M
glycerol + 1.6 M sucrose for 30 min at 25 oC and
dehydrated with PVS2 for 30 min at 25oC before
direct immersion in LN. For regeneration,
cryopreserved shoot tips were rewarmed in 1.0
M sucrose solution for 15 min at room
temperature. The shoot tips were plated on semisolid 1/2 MS medium containing 3% sucrose,
0.01 mg/l NAA and 0.1 mg/l BA after removal
of the alginate gel. Cultures were kept in the
dark for 7 days and then transferred under
standard light conditions.
These optimal conditions were applied to
10 additional sugarcane varieties. Regrowth
ranged from 56.7 to 100%, with an average of
70.3% for the eleven varieties tested (Fig. 2).

Mean values followed by different letters differ

significantly at the 0.05 probability level.
Table 4. Effect of sucrose concentration in LS
o
and duration of exposure to PVS2 at 25 C on
regrowth (%) of sugarcane Ni-1 shoot tips
cryopreserved using the V cryo-plate procedure.
Preculture medium contained 0.3 M sucrose.
Sucrose
conc. in LS
0.8 M

1.2 M

1.6 M

Exposure
time in PVS2
20 min
30 min
40 min

Regrowth
(% SD)
86.7 5.8 ab
100.0 0.0 a
86.7 5.8 ab

20 min

100.0 0.0 a

30 min

73.3 11.5 b

40 min

80.0 10.0 ab

20 min

73.3 11.5 b

30 min

73.3 5.8 b

40 min

76.7 5.8 b

Mean values followed by different letters differ

significantly at the 0.05 probability level.

55

Table 5. Effect of PVS2 and PVS3 treatment

(duration: 30 min) on regrowth (%) of sugarcane
shoot tips cryopreserved using the V cryo-plate
procedure.
Vitrification solution
Regrowth (% SD)
PVS2
100.0 0.0 a
PVS3
50.0 17.3 b
Mean values followed by different letters differ
significantly at the 0.05 probability level.
Table 6. Effect presence or absence of alginate
gel on regrowth (%) of shoot tips cryopreserved
using the V cryo-plate procedure.
Alginate gel
Regrowth (% SD)
Presence
56.7 15.3 b
Absence
100.0 0.0 a
Mean values followed by different letters differ
significantly at the 0.05 probability level.

Figure 2. Regrowth (%) of shoot tips of 11

sugarcane varieties cryopreserved using the
optimized V cryo-plate procedure.

DISCUSSION
Table 7. Effect of light conditions during the first
7 days after rewarming on regrowth (%) of shoot
tips cryopreserved using the V cryo-plate
procedure.
Light conditions

Regrowth (% SD)

Dark

100.0 0.0 a

Light

56.7 15.3 b

Cryopreservation has been developed for

various sugarcane accessions using in vitro shoot
tips, cell suspensions, embryogenic callus and
somatic embryos (13). However, there are only a
few papers reporting the use of in vitro shoot
tips. Successful cryopreservation was first
achieved using encapsulation-dehydration (7,
22). Recently, in vitro shoot tips of two clones of
sugarcane were successfully cryopreserved using
encapsulation-dehydration
and
dropletvitrification, with recovery ranging between 53
60% and 2037%, respectively (2).
In this study, using 11 sugarcane varieties
regrowth between 56.7 and 100%, with an
average of 70.3%, was achieved using the V
cryo-plate procedure. This high regrowth was
due to the cryopreservation procedure and also
to the use of vigorous specimens and of specific
post-rewarming handling.
Preconditioning is necessary to obtain
uniform and actively growing shoot tips for
cryopreservation experiments (18). In sugarcane,
in vitro grown shoots containing shoot tips were
cut to a size of 57 mm and plated on semi-solid
1/2MS medium containing 3% sucrose in Petri
dishes for 1 week to obtain uniform material.
The size of shoot tips is also crucial for high
regrowth. Martinez-Montero et al. (13)
suggested that vitrification-based procedures
allow using samples of relatively large size
(shoot tips of 0.5 to 2.0-3.0 mm), which can
regrow directly without any difficulty. In our
experiments we tested various sizes of shoot tips

Mean values followed by different letters differ

significantly at the 0.05 probability level.
Table 8. Effect of composition of the culture
medium on regrowth (%) of shoot tips
cryopreserved using the V cryo-plate procedure.
Medium
Regrowth
(Semi-solid 1/2 MS + 30
(% SD)
g/l sucrose)
No growth regulators
67.0 15.2 b
0.01 mg/l NAA + 0.1 mg/l
100.0 0.0 a
BA
0.01 mg/l NAA + 0.1 mg/l
80.0 10.0 ab
BA + 0.01 mg/l PVP
Mean values followed by different letters differ
significantly at the 0.05 probability level.

56

from 0.5-1.0 to 1.5-2.0 mm. Larger shoot tips

produced higher regrowth, showing that a small
change in the size of shoot tips used for
cryopreservation can have a great effect on
regrowth. This was one of parameters which
proved to be of critical importance for the
optimization of the protocol.
Preculture on medium with high sucrose
concentration and osmoprotection were effective
to induce tolerance to PVS2 (16, 17). In this
study, it was necessary to place the dissected
shoot tips on standard medium for 1 day before
transferring them on medium with high sucrose
concentration to obtain high regrowth after
cryopreservation.
The
optimal
sucrose
concentration in preculture medium and duration
of preculture vary with plant species and variety
(6, 30). Gonzalez-Arnao and Engelmann (8)
found that preculture of sugarcane shoot tips on
medium containing 0.75 M sucrose for 24 h
produced the best results. In this study, 1 day
preculture on medium with 0.5 M sucrose
produced the highest regrowth with the varieties
tested. Preculture on medium with 0.3 M sucrose
followed by LS treatment with 2.0 M glycerol +
1.6 M sucrose tended to decrease regrowth of
cryopreserved shoot tips. On the other hand,
shoot tips precultured on medium with 0.5 M
sucrose followed by LS treatment with 2.0 M
glycerol + 1.6 M sucrose achieved very high
regrowth. These differences in the regrowth with
preculture medium after LN treatment might be
caused by rapid osmotic stress changes.
However, with both sucrose concentrations in
the preculture medium, regrowth was over 70 %
regardless of the LS used and PVS2 treatment
duration tested.
An osmoprotective treatment is essential to
achieve post-warming regrowth. The LS
treatment is very effective to induce dehydration
tolerance and to produce vigorous growth
recovery (14, 26). Our study showed that a LS
containing 1.6 M sucrose led to maximum
regrowth of sugarcane shoot tips. Hirai and
Sakai (11) indicated that the success of
cryopreservation by vitrification depends on
preconditioning
meristems
to
induce
osmotolerance to PVS2. For cryopreservation of
sweet potato shoot tips using encapsulationvitrification, a high sucrose concentration (1.6
M) in LS and a longer period of osmoprotection
with PVS2 (3 h at 25C) were necessary to
increase osmotolerance (11). However, direct
exposure of shoot tips to PVS2 caused harmful
effects due to osmotic stress (10). When

developing alternative LSs employed in a

droplet-vitrification procedure, Kim et al. (12)
indicated that the loading treatment might act as
an osmotic stress neutralizer and/or induce a
physiological adaptation of tissues and cells
before both dehydration and vitrification. They
also pointed out that an appropriate LS should be
selected for plant species, which are highly
sensitive to the cytotoxicity of PVSs, in order to
reduce their detrimental effect. In the case of
Dalmatian chrysanthemum, a LS solution
containing 1.4 M sucrose was effective in
increasing osmotolerance (33). Meristematic
cells, which have been treated with LS are
osmotically dehydrated and might be
plasmolyzed to a considerable extent (28). These
cells have then to be dehydrated with PVS until
they reach the optimal cytosolic concentration
required
for
vitrification.
Successful
cryopreservation by vitrification might lay with
the establishment of precisely controlled
dehydration procedures and combination of
preculture, LS and PVS treatment, while
preventing injury by chemical toxicity or
excessive osmotic stress during treatment with
PVS.
PVS2 protects plant cells from fatal
intercellular freezing that can occur during rapid
cooling in LN (24). So successful
cryopreservation using a vitrification-based
method depends on the definition of the optimal
time of exposure to PVS2 (30). Barraco et al. (2)
reported that 20 and 40 min were optimal PVS2
exposure times for two sugarcane varieties, H70144 and CP68-1026, respectively, cryopreserved
using a droplet-vitrification procedure. In our
study, exposure of shoot tips to PVS2 for 30 min
was optimal.
To achieve high recovery after LN
exposure, an efficient tissue culture procedure
and post-rewarming handling system are
necessary. According to Tarique et al. (29), the
addition of 1.0 mg/l BAP + 0.5 mg/l NAA to the
culture medium was optimal to achieve
multiplication of sugarcane shoots. The same
growth regulators at lower concentration were
used in this study.
When cryopreserving sugarcane shoot tips
using encapsulation-dehydration, GonzalezArnao and Engelmann (8) indicated that it was
beneficial to perform the post-warming recovery
in the dark for a short period (around 1 week) to
avoid detrimental photo-oxidation. In sugarcane,
our experiments showed that performing the
initial culture immediately after rewarming in

57

the dark for 7 days was very effective for

regrowth. Also, removal of alginate gel from
shoot tips enhanced post LN regrowth in
sugarcane. Using encapsulation-dehydration, it
was necessary for some materials including
microspore embryos of oilseed rape, shoot tips
of grape and mulberry to extract the explants
from the beads and to place them directly on the
recovery medium to ensure their regrowth (5).
Recently, the V cryo-plate method has been
successfully applied for long-term conservation
of various plant species using cryopreservation.
This study shows the possibility of using the V
cryo-plate method for sugarcane shoot tip
cryopreservation. The V cryo-plate appeasr as an
efficient
and
practical
method
for
cryopreservation of sugarcane shoot tips in
genebanks.

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Acknowledgements: We are grateful to Shigeki

Nakayama for providing germplasm for the
experiments and to Ms. Naoko Nohara, Ms.
Akiko Nishiuchi, Ms.Noriko Ishikura and Ms.
Yumi Sato for their technical assistance. This
work was mainly supported by a grant from
Ministry of Agriculture, Forestry, and Fisheries
of Japan (Genomics-based Technology for
Agricultural Improvement, CRS-1001).

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