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Cryotherapy of Potato Shoot Tips for Efficient

Elimination of Potato Leafroll Virus (PLRV) and
Potato Virus Y (PVY)

Article in Potato Research · July 2006

DOI: 10.1007/s11540-006-9011-4

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Potato Research (2006) 49:119–129
DOI 10.1007/s11540-006-9011-4

Cryotherapy of Potato Shoot Tips for Efficient

Elimination of Potato Leafroll Virus (PLRV)
and Potato Virus Y (PVY)

Qiaochun Wang & Yong Liu & Yonghong Xie & Misa You

Received: 5 February 2006 / Accepted: 29 September 2006 /

Published online: 5 January 2007
# EAPR 2007

Abstract Viral diseases constitute a major constraint to high yield and high quality
production of potato. Potato leafroll virus (PLRV) and Potato virus Y (PVY) are among the
most damaging potato viruses and are prevalent in most potato growing areas. In the
present study, attempts were made to eliminate PLRV and PVY by three cryogenic
protocols, i.e., encapsulation-dehydration, encapsulation-vitrification and droplet. Results
showed that both PLRV and PVY could be efficiently eliminated by cryogenic treatments
with 83–86% and 91–95% of frequencies of virus-free plantlets obtained for the former and
latter, respectively. Frequencies of virus-free plantlets produced by cryogenic treatments
were higher than those by meristem culture (56% for PLRV and 62% for PVY) and
thermotherapy (50% for PLRV and 65% for PVY), and similar to those by thermotherapy
followed by meristem culture (90% for PLRV and 93% for PVY). Survival (75–85%) and
regrowth (83–89%) from cryo-treated shoot tips were higher than those from meristem
culture (50–55%) and thermotherapy followed by meristem culture (40–50%), but similar
to those from thermotherapy (80–87%). The morphology of the plantlets regenerated from
cryo-treated shoot tips was similar to that of non-treated plantlets. Thus, cryotherapy would
provide an alternative method for efficient elimination of potato viruses, and can be
simultaneously used for long-term storage of potato germplasm and for production of virus-
free plants.

Q. Wang
Y. Xie
M. You

Plant Biotechnology Laboratory, Chengdu Center of Food Test and Analysis, Jingju Shi Lu 19,
Chengdu 610066 Sichuan, People_s Republic of China

Present Address:
Q. Wang (*)
Department of Applied Biology, University of Helsinki, P.O. Box 27, 00014 Helsinki, Finland
e-mail: qiaochunwang@hotmail.com

Y. Liu
Instittute of Crop Protection, Sichuan Academy of Agricultural Science,
Chengdu 610066 Sichuan, People_s Republic of China
120 Potato Research (2006) 49:119–129

Keywords cryotherapy . meristem culture . potato leafroll virus . potato virus Y .

Solanum tuberosum L. . thermotherapy

Abbreviations

BSV Banana streak virus

BAP Benzylaminopurine
CMV Cucumber mosaic virus
DMSO Dimethylsulfoxide
GA3 Gibberellic acid 3`
GVA Grapevine virus A
LSD Least significant difference
LN Liquid nitrogen
MS Murashige and Skoog medium
PVS2 Plant vitrification solution 2
PPV Plum Pox Potyvirus
PLRV Potato leafroll virus
PVY Potato virus Y
RAPD Random amplified polymorphic DNA

Introduction

Potato (Solanum tuberosum L.) is globally the fourth largest staple crop after rice, wheat
and maize in the world. Viral diseases constitute a major constraint to high yield and high
quality production of potato (Waterworth and Hadidi 1998). Potato leafroll virus (PLRV)
and Potato virus Y (PVY) are among the most damaging potato viruses and are prevalent in
most potato growing areas (Loebenstein 2001; Brunt 2001). Experimental data showed that
plants from PLRV-infected seed tubers produced at least 60% less total yield and 88% less
marketable yield (tubers >85 g) than plants from healthy seed tubers (Hamm and Hane
1999). PVY infection of seed tubers caused 49 and 65% reductions in total yield and
marketable yield, respectively, compared with healthy plants (Hane and Hamm 1999). Both
PLRV and PVY are transmitted by aphids.
Control of potato viruses is based on the production of healthy plants and use of virus-
resistant cultivars. Meristem culture, thermotherapy and thermotherapy followed by
meristem culture were the most often employed methods for obtaining virus-free potato
plants (Slack and Singh 1998; Faccioli 2001).
Recently, it has been demonstrated that plant viruses could be effectively eradicated by
cryotherapy of shoot tips (Brison et al. 1997; Helliot et al. 2002; Wang et al. 2003).
Cryopreservation of potato shoot tips has been extensively studied and a routine procedure
for cryopreservation of a large number of genotypes (at least 125) has been developed
(Schäfer-Menuhr et al. 1996). Therefore, cryotherapy of potato shoot tips may be used
simultaneously for virus elimination and long-term storage of potato germplasm.
In the present study, we report for the first time efficient elimination of PLRV and PVY
by cryotherapy of in vitro-grown shoot tips. Comparisons were also made in survival,
regrowth and virus elimination frequency between cryogenic treatments and the traditional
methods such as meristem culture, thermotherapy and thermotherapy followed by meri-
stem culture.
Potato Research (2006) 49:119–129 121

Materials and Methods

Plant Material

Naturally PLRV- and PVY-infected plants of Solanum tuberosum genotype 117 from seed
tubers were used in the present study. Stems were taken from potato plants showing viral
symptoms and grown on the Experimental Station of Sichuan Academy of Agricultural
Science, Chengdu, PR China. Single nodal segments (about 0.5 cm in length) were excised
and used as explants for establishment of stock cultures in vitro. After surface disinfection
using 1% hypochlorite solution for 10 min and washing for three times in sterile distilled
water, the explants were grown on solid Murashige and Skoog (1962) medium (MS)
supplemented with 30 g l−1 sucrose and solidified with 2.6 g l−1 Gelrite. The pH was
adjusted to 5.8 prior to autoclaving at 121 °C for 20 min. Stock cultures were maintained at
a temperature of 22±1 °C under a 16-h photoperiod with a light intensity of 50 μE s−1 m−1
provided by cool-white fluorescent tubes. Subculturing was performed every four weeks.

Encapsulation-dehydration Cryotherapy

The encapsulation-dehydration procedure was based on the protocol developed by Bouafia

et al. (1996) with some modifications. Shoot tips (1–1.5 mm in size, Fig. 1a) were excised
from the stock cultures and suspended in MS inorganic medium supplemented with 2.5%
(w/v) Na-alginate, 2 M glycerol and 0.4 M sucrose. The mixture, including shoot tips, was
dropped with a sterile pipette into 0.1 M CaCl2 solution containing 2 M glycerol and
0.4 M sucrose and left for 20 min to form beads about 4 mm in diameter, each bead

Fig. 1 PLRV- and PVY-free plant regeneration from cryotherapy-treated shoot tips of Solanum tuberosum
genotype 117. a A typically excised shoot tip used for cryotherapy. b Shoot tips in 3.5 μl droplets of DMSO
solution. c Surviving shoot tip by droplet procedure 10 days after post-culture. d Surviving shoot tip by
encapsulation-vitrification procedure 10 days after post-culture. e Regrowth of shoot tips from droplet
procedure 4 weeks after survival assessment. f PLRV- and PVY-free plantlets in vitro 4 weeks after rooting
122 Potato Research (2006) 49:119–129

containing one shoot tip. The beads were stepwise precultured on increasing sucrose
concentrations of 0.25, 0.5 and 0.75 M, with one day for each concentration. Following
preculture, the beads were rapidly surface-dried by blotting on cellulose tissue and
dehydrated for different durations of time on sterile filter paper in a laminar flow at room
temperature and moisture. The water content of the beads was measured by weighing them
prior and after drying in an oven at 80°C for 48 h. Ten dehydrated beads were then
transferred into a 1.8-ml cryotube and immersed directly in liquid nitrogen (LN) for 1 h.
The cryotubes that had been stored in LN were rapidly thawed at 40°C for 3 min. Thawed
beads were post-cultured on MS medium supplemented with 1.0 mg l−1 gibberellic acid 3
(GA3) and 0.4 mg l−1 benzylaminopurine (BAP). The cultures were maintained in the dark
at 22±1°C for three days and then transferred to light conditions as described for stock
cultures, for survival and regrowth.

Encapsulation-vitrification Cryotherapy

The encapsulation-vitrification protocol described by Hirai and Sakai (1999) was used in
the present study. The encapsulation and preculture were the same as for the encapsulation-
dehydration procedure. The precultured beads were loaded with a loading solution
composed of MS supplanted with 0.4 M sucrose and 2 M glycerol for 90 min at room
temperature, followed by dehydration with plant vitrification solution 2 (PVS2) (Sakai et al.
1990) in a 50 ml plastic tube at 0 °C for different lengths of time. PVS2 contained
30% (w/v) glycerol, 15% (w/v) ethylene glycol, 15% (w/v) dimethylsulfoxide (DMSO) and
0.4 M sucrose in MS medium. After dehydration, 10 beads were transferred into a 1.8-ml
cryotube and immersed directly in LN for 1 h. Thawed shoot tips were washed with 1.0 M
sucrose for 30 min at room temperature and then processed as described for encapsulation-
dehydration, for survival and regrowth.

Droplet Cryotherapy

The droplet protocol of Schäfer-Menuhr et al. (1996) was employed in this study. Excised
shoot tips were precultured as in the encapsulation-dehydration procedure, but without
encapsulation. The precultured shoot tips were transferred into a cryoprotective solution
composed of 10% DMSO in MS medium and incubated for various periods of time.
Thereafter, 3.5 μl droplets of cryoprotectant were pipetted onto sterile 0.03 mm-thick
aluminium foil (0.5×3 cm) at five drops per foil. One shoot tip was transferred onto each
drop (Fig. 1b) and the foils were immersed directly into LN for 1 h. Thawed shoot tips were
washed with 1.0 M sucrose for 30 min at room temperature and then processed as described
for encapsulation-dehydration, for survival and regrowth.

Meristem Culture

Meristem culture was performed according to Faccioli and Colombarini (1996) with some
modifications. Meristems (about 0.2 mm) with one or two leaf primordia were excised from
stock cultures and cultured on MS medium supplemented with 0.4 mg l−1 BAP in the dark
for three days at 22±1°C and then transferred to light conditions as described for stock
cultures, for survival. Surviving meristems were then placed on MS medium containing
1 mg l−1 GA3 and 0.4 mg l−1 BAP, for shoot regrowth.
Potato Research (2006) 49:119–129 123

Thermotherapy

Thermotherapy was carried out according to Faccioli and Colombarini (1996) with some
modifications described as followings. In vitro plantlets were grown for four weeks in a
growth chamber at a temperature of 36±1°C under a 16-h photoperiod with a light intensity
of 50 μE s−1 m−1 provided by cool white fluorescent tubes. Following heat treatment, shoot
tips (about 1 mm) with three or four leaf primordia were excised and cultured on MS
medium supplemented with 0.4 mg/l BAP. The cultures were placed in the dark for three
days at 22±1°C and then transferred to light conditions as described for stock cultures, for
survival. Surviving meristems were then placed on MS medium containing 1.0 mg l−1 GA3
and 0.4 mg l−1 BAP, for regrowth.

Thermotherapy Followed by Meristem Culture

The procedure was based on that of Faccioli and Colombarini (1996) with the following
modifications. In vitro plantlets were incubated for 4 weeks in a growth chamber at a
temperature of 36±1°C under a 16-h photoperiod with a light intensity of 50 μE s−1m−2
provided by cool white fluorescent tubes. Following heat treatment, meristems (about
0.2 mm) with one or two leaf primordia were excised as explants and cultured on MS
medium supplemented with 0.4 mg l−1 BAP. The cultures were placed in the dark for three
days at 22±1°C and then transferred to light conditions as described for stock cultures, for
survival. Surviving meristems were then placed on MS medium containing 1.0 mg l−1 GA3
and 0.4 mg l−1 BAP, for regrowth.

Plant Regeneration

Survival was identified by green colour formation in post-cultured materials following 10

days of post-culture. Regrowth was defined as the shoot tips or meristems that developed
shoots. For rooting, shoots longer than 1 cm were transferred to hormone-free MS medium.
Subculture was done every four weeks.

Detection of Viruses in Plant Materials

Viruses were tested at different stages of the productions using DAS-ELISA (Clark and
Adams 1977). Antisera against PLRV and PVY (Loewe Biochemica GmbH, Germany),
respectively, each at the concentration of 1:1,000, was used. The cultures initially
established in vitro were tested and only samples showing both PLRV- and PVY-positive
reactions were further used for multiplication as the stock cultures. At the beginning of the
experiments, stock cultures that would be used for cryopreservation, meristem culture and
thermotherapy, respectively, were tested again to ensure that only double PLRV- and PVY-
infected shoots were used for the designed experiments. The health status of in vitro
plantlets regenerated from different procedures was checked after two months of plant
regeneration. After three months of establishment in greenhouse conditions, plants were
again tested by DAS-ELISA, to further confirm sanitary status of plants regenerated from
different procedures. Our preliminary studies showed that data on the production of virus-
free plants were consistent between in vitro plantlets and plants established in greenhouse
conditions. Therefore, virus-free percentage was calculated from results of testing in vitro
plantlets.
124 Potato Research (2006) 49:119–129

Statistical Analysis

Unless stated otherwise, the experiments were carried out as completely randomized
designs and repeated at least twice with 30 samples per treatment in every experiment. The
data were compared by Student’s t-test. Least significant difference (LSD) was calculated at
P = 0.05.

Results

Survival of Cryo-treated Shoot Tips by Encapsulation-dehydration

Survival of cryo-treated shoot tips as a function of changes in the water content of the
precultured beads during dehydration by air drying is presented in Fig. 2. The initial water
content of the precultured beads was 67.1% on fresh weight basis. Water content decreased
to 20.4% at 5 h and was 15.1% following 8 h of dehydration. Survival of cryo-treated
shoot tips was observed after at least 2 h of dehydration and reached a maximum (78%) at
5 h of dehydration.

Survival of Cryo-treated Shoot Tips by Encapsulation-vitrification

Survival of cryo-treated shoot tips required a minimum time of 30 min of vitrification in

PVS2, was the highest (75%) following 180 min vitrification time, and then decreased with
longer vitrification periods (Fig. 3).

Survival of Cryo-treated Shoot Tips by Droplet

Survival of cryo-treated shoot tips required a minimum of 20 min of DMSO treatment,

reached maximum (85%) at 120 min, and then decreased (Fig. 4).

Fig. 2 Survival of cryotherapy- 80 90

treated shoot tips of Solanum
tuberosum genotype 117 as a 70 water content 80
function of changes in the water survival
content of beads by encapsula- 60
70
tion-dehydration. Data of water
content were collected from two 60
Water content (%)

50
independent experiments and
Survival (%)

presented as mean. The bar indi- 50

40
cates LSD at P=0.05 for survival
LSD0.05 40
30
30
20
20
10 10

0 0
0 1 2 3 4 5 6 7 8
Dehydration time (h)
Potato Research (2006) 49:119–129 125

Fig. 3 Effect of dehydration 100

with PVS2 solution on survival
of cryotherapy-treated shoot tips 90
of Solanum tuberosum genotype
117 by encapsulation-vitrifica- 80
tion. The bar indicates LSD LSD0.05
70
at P=0.05

Survival (%)
60

50

40

30

20

10

0
0 30 60 90 120 150 180 210 240
Time of exposure to PVS2 (min)

Effect of Cryogenic Treatments, Meristem Culture, Thermotherapy and Thermotherapy

Followed by Meristem Culture on Survival, Regrowth and Virus Elimination

All three cryogenic procedures and thermotherapy produced similar rates of survival and
regrowth, which were much higher than those from meristem culture and thermotherapy
followed by meristem culture (Table 1). The lowest survival (40 and 50%) and regrowth
(55 and 50%) were observed in meristem culture and meristem cultures following
thermotherapy, respectively. All three cryogenic procedures and thermotherapy followed by

Fig. 4 Effect of DMSO treat- 90

ment on survival of cryotherapy-
treated shoot tips of Solanum 80
tuberosum genotype 117 by LSD0.05
droplet. The bar indicates LSD 70
at P=0.05
60
Survival (%)

50

40

30

20

10

0
0 20 40 60 80 100 120 140 160

Time of exposure to DMSO (min)

126 Potato Research (2006) 49:119–129

Table 1 A comparison of effects of cryogenic treatments, meristem culture, thermotherapy, and

thermotherapy followed by meristem culture on survival, regrowth and virus elimination

Methods No. of total Survival Regrowth Virus-free

samples (%) (%) (%)

PLRV PVY

Encapsulation-dehydration 60 78a 85a 85a 91a

Encapsulation-vitrification 60 75a 83a 83a 93a
Droplet 60 85a 89a 86a 95a
Meristem culture 90 50b 55b 56b 62b
Thermotherapy 90 80a 87a 50b 65b
Thermotherapy followed by meristem 90 40b 50b 90a 93a
culture

Data in the same column with different letters are significantly different at P < 0.05 (Student’s test).

meristem culture resulted in the highest frequency for elimination of PLRV (83–90%) and
PVY (91–95%). Both meristem culture and thermotherapy were less effective in eradication
of PLRV (50–56%) and PVY (62–65%).

Analysis of Time Period Involved in Different Stages of Cryotherapy, Meristem Culture,

Thermotherapy and Thermotherapy followed by Meristem Culture

Cryo-treated shoot tips (Fig. 1c, d) and all other cultures from meristem culture,
thermotherapy and thermotherapy followed by meristem culture turned green in about 10
days of post-culture (Table 2). Regrowth of cryo-treated shoot tips (Fig. 1e) occurred in
about 10 days of post-culture, which was faster than any other procedures. Regrowth of
cryo-treated shoot tips was direct without intermediary callus formation (Fig. 1e). Root
formation of regrown shoot tips or meristems from all procedures occurred in about two
weeks after transfer to rooting medium, thus leading to regeneration of virus-free plantlets
(Fig. 1f). Morphologies of plantlets regenerated from cryo-treated shoot tips were identical
to those of non-treated plantlets (data not shown). Time period of the whole procedure

Table 2 Analysis of time period for virus-free plant production required by cryotherapy, meristem culture,
thermotherapy and thermotherapy followed by meristem culture

Methods Time of Time of Time needed Time needed Time needed Total time
preculture thermotherapy for survival for regrowth for root needed
(days) (days) (days) (days) formation (days)
(days)

Cryotherapy 3 – 10 28 14 55
Meristem culture – – 10 35 14 59
Thermotherapy – 28 10 35 14 87
Thermotherapy – 28 10 35 14 87
followed by
meristem culture
Potato Research (2006) 49:119–129 127

required by cryogenic procedures was shortest (about 55 days), while thermotherapy and
thermotherapy followed by meristem culture needed about 87 days to complete the whole
procedure (Table 2).

Discussion

Conventional methods such as meristem culture, thermotherapy and thermotherapy followed

by meristem culture have been successfully applied to obtain virus-free plants of potato
(Lozoya-Saldaña and Dawson 1982; Faccioli and Rubies-Autonell 1982; Lozoya-Saldaña
and Merlin-Lara 1984; Faccioli et al. 1988; Griffiths and Slack 1990; Sanchez et al. 1991;
Faccioli and Colombarini 1996; Slack and Singh 1998; Faccioli 2001). Results of the
present study confirm previous reports that PLRV and PVY could be eradicated by
meristem culture, thermotherapy and thermotherapy followed by meristem culture.
The application of meristem culture to eliminate virus was initially based on the concept
of meristem “immunity” towards viruses (Morel 1948). When meristem culture is applied
for virus eradication, care must be taken to excise meristems that are small enough (usually ≤
0.3 mm in size for potato), because the frequency of success in virus eradication is
inversely proportional to the size of excised meristems (Faccioli and Marani 1998).
However, culturing of such small meristems makes plant regeneration difficult, because the
regeneration ability is positively proportional to the size of the meristems (Faccioli and
Marani 1998). The size of excised meristem must, therefore, accomplish both needs:
regeneration into a complete plant and virus elimination.
When thermotherapy and thermotherapy followed by meristem culture are employed,
infected stock plants have to be heat-treated by growing at high temperatures for several
weeks (Faccioli and Rubies-Autonell 1982; Lozoya-Saldaña and Dawson 1982; Lozoya-
Saldaña and Merlin-Lara 1984; Griffiths and Slack 1990; Faccioli and Colombarini 1996).
For some heat-stable potato virus strains (Mellor and Stace-Smith 1970, 1971),
thermotherapy alone might not be sufficient to eradicate them. Some potato cultivars were
sensitive to heat treatment (Faccioli and Rubies-Autonell 1982; Lozoya-Saldaña and
Dawson 1982; Lozoya-Saldaña and Merlin-Lara 1984; Sanchez et al. 1991) and,
subsequently, could not withstand high temperature required for virus elimination. In
addition, thermotherapy is time-consuming.
With the idea that cryopreservation, which requires the use of shoot tips, might be
considered as a possible combined method for virus elimination and conservation of
germplasm, Brison et al. (1997) were the first to successfully eliminate Plum Pox Potyvirus
(PPV) from an interspecific Prunus rootstock, using cryopreservation of shoot tips. Later,
Helliot et al. (2002) reported the successful elimination of Cucumber mosaic virus (CMV)
and Banana streak virus (BSV) from Musa spp. by cryopreservation of shoot tips. More
recently, Wang et al. (2003) applied cryopreservation of shoot tips to efficiently eliminate
Grapevine virus A (GVA). Studies of Brison et al. (1997) and Wang et al. (2003) showed
that frequency of virus elimination was not affected by the size of shoot tips (0.3–2 mm)
used for cryopreservation. The present study also showed that both PLRV and PVY could
be efficiently eradicated by cryopreservation of shoot tips
It is well-known that the distribution of viruses in plants is uneven. In infected plants the
meristematic dome is generally either free or contains a very low concentration of the
viruses, while the virus titre increases with increasing distance from the meristem dome.
Cells of the meristematic dome are characterized by their small size, a high nucleo-
128 Potato Research (2006) 49:119–129

cytoplasm ratio and dense cytoplasm containing small vacuoles (Helliot et al. 2002). The
nucleo-cytoplasm ratios decreased progressively with increasing distance from the
meristematic dome. When shoot tips were subject to freezing in liquid nitrogen, large
cells with bigger vacuoles and containing more water, which are likely to be infected by the
virus, were killed. Only small cells with dense cytoplasm, which are located in the top
layers of meristem and are likely to be free from virus infection, survived freezing in liquid
nitrogen (Helliot et al. 2002, Wang et al. 2005). Thus, plants regenerated from cryotherapy
could be virus-free.
When plant materials are stored in liquid nitrogen, all cellular divisions and metabolic
processes cease, and in theory, any variation of materials treated in this way can be
prevented (Engelmann 1997). However, since cryogenic procedures involve not only
freezing in liquid nitrogen but also in vitro tissue culture and regeneration processes, plants
derived from cryogenic treatments may be subject to somaclonal variation induced during
these stages, leading subsequently to distinct differences in their genotype/phenotype
profiles (Harding 2004). To date, a great number of studies have shown that cryostorage-
derived plants were genetically stable (Harding 2004; Wang and Perl 2006). With potato,
genetic stabilities of plants recovered from cryopreserved shoot tips were evaluated at the
morphological (Benson et al. 1996), histological (Benson et al. 1996) and molecular
(Harding 1991, Schäfer-Menuhr et al. 1996, Hirai and Sakai 1999) levels. All of the above
studies showed no differences in plants recovered between cryopreservation and control.
All procedures used in the present study including tissue culture, cryotherapy and plant
regeneration were quite similar to those of Schäfer-Menuhr et al. (1996) and Hirai and
Sakai (1999). Our results also showed that morphologies of plantlets regenerated from cryo-
treated shoot tips were identical to those of non-treated plantlets.
In conclusion, PLRV and PYV could be efficiently eradicated by different cryogenic
procedures. Compared with meristem culture, thermotherapy and thermotherapy followed
by meristem culture, several distinct advantages are found in cryotherapy: easier handling;
simpler procedures for plant regeneration; higher rates of survival, regrowth and virus
elimination; shorter time for production of virus-free plants. Cryotherapy would provide an
alternative method for efficient elimination of potato viruses. Thus, cryopreservation can be
simultaneously used for the long-term storage of biodiversity of potato germplasm and for
production of virus-free plants.

Acknowledgments This study is financially supported by Department of Science & Technology of Sichuan
Government, Sichuan, PR China. Plant materials were kindly provided by Dr. Wei He from Institute of Field
Crops of Sichuan Academy of Agricultural Science, China. Thanks are given to Prof. Gad Loebenstein and
Prof. Jari P. T. Valkonen for their critical reading of the manuscript.

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