Amer J of Potato Res (2004) 81:171-176 171

Salicylic Acid Enhances Heat Tolerance and Potato Virus X (PVX)

Elimination during Thermotherapy of Potato Microplants

H. L 6 p e z - D e l g a d o ' * , M. E. M o r a - H e r r e r a ' , H. A. Z a v a l e t a - M a n c e r a 2, M. C a d e n a - H i n o j o s a , a n d I. M. S c o t t 3

1Programa Nacionalde Papa, Instituto Nacional de [nvestigaciones Forestales Agricolasy Pecuarias (INIFAP),Metepec, Mdx. 52142, Mdxico
2Programa de Bot&rdca, Instituto de Recursos Naturales IRENAT,Colegiode Postgraduados, Montecillo,Texcoco, Edo. M~x. 25630, M6xico
3Institute of BiologicalSciences, University of Wales, Aberystwyth,Ceredigion,SY233DA UK
*Correspondingauthor: Tel: 722 232 98 33 Fax: +72-22320089;E-mail:humlopde@prodigy.net.mx

ABSTRACT e n m i c r o p l a n t a s t r a t a d a s con SA: de siete genotipos al

A modification o f t h e s t a n d a r d t h e r m o t h e r a p y u s e d
t o e l i m i n a t e virus from p o t a t o v i r u s X (PVX)-infected
Solanum tuberosum microplants of t h e Mexican
N a t i o n a l P o t a t o P r o g r a m is d e s c r i b e d here. M i c r o p l a n t s
were c u l t u r e d with or w i t h o u t 104 M salicylic acid (SA)
for 4 wk, t h e n s u b c u l t u r e d w i t h o u t SA a n d e x p o s e d t o 42
C for 30 days. Survival was more c o n s i s t e n t i n SA-
t r e a t e d microplants: a m o n g s e v e n genotypes, 40% to
100% of SA-treated m i c r o p l a n t s a n d 0% to 96% o f con-
t r o i s survived at the e n d of thermotherapy. SA also
improved survival o f the p o s t - t h e r m o t h e r a p y subcul-
t u r e . A m o n g surviving m i c r o p l a n t s , SA i n c r e a s e d t h e
virus-free yield to 100% from 40% to 65% i n the con-
trois. I n a n a d d i t i o n a l 30 PVX(+) genotypes, 98% of sur-
viving S A - t r e a t e d m i c r o p l a n t s were virus-free a f t e r
thermotherapy, compared t o 75% of controls. S A - t r e a t e d
m i c r o p l a n t s had lower c a t a l a s e activity a n d higher
h y d r o g e n peroxide (HzO2) levels.
final de l a t e r m o t e r a p i a , 40% a 100% s o b r e v i v i e r o n e n
m i c r o p l a n t a s t r a t a d a s con S A y 0% a 96% e n los testigos.
SA t a m b i ~ n m e j o r 6 la s o b r e v i v e n c i a e n el subcultivo
p o s t e r i o r a la t e r m o t e r a p i a . E n t r e las m i c r o p l a n t a s
s o b r e v i v i e n t e s , SA i n c r e m e n t 6 el n d m e r o de p l a n t a s
fibres de v i r u s a 100% comparado c o n 40% a 65% e n los
testigos. P o s t e r i o r m e n t e e n 30 genotipos (+) a PVX, 98%
de las m i c r o p l a n t a s t r a t a d a s con SA f u e r o n libres de
virus despu~s de t e r m o t e r a p i a , comparado con 75% de
los testigos. M i c r o p l a n t a s t r a t a d a s con SA, t u v i e r o n
m e n o r actividad c a t a l a s a y niveles mas altos de peroxido
de hidr6geno (H~O2).
INTRODUCTION
This paper reports a new application of SA in potato tis-
sue culture technology. The modern production systems of
basic and certified seed potato require the micropropagation
of virus-free material and the adoption of fast virus-eradication
methods (Faccioli and Colombarini 1996). In systemically

RESUMEN infected plants, viruses tend to be absent from the meristem-

Se empleo t e r m o t e r a p i a p a r a e l i m i n a r el virus X de
la p a p a (PVX), ell p l a n t a s i n f e c t a d a s de S o l a n u m t u b e r o -
s u m del P r o g r a m a Nacional de P a p a de M@xico. Se culti-
v a r o n m i c r o p l a n t a s e n p r e s e n c i a o a u s e n c i a de 10 ~ M de
ficido salicilico (SA) por c u a t r o semanas, p o s t e r i o r m e n t e
se s u b c u l t i v a r o n e n a u s e n c i a de SA y se e x p u s i e r o n a
42 C por 30 dias. La s o b r e v i v e n c i a fue mas c o n s i s t e n t e
atic tissues and young primordial leaves, so that meristem cul-
ture forms the basis of standard virus-eradication methods in
potato (Salazar and Jayasinghe 1996). The long-standing
empirical observation that virus concentration can be reduced
when plants are heat-treated has resulted in thermotherapy
becoming a common additional step in virus eradication in
potato (Sgmchez et al. 1991; Salazar and Jayasinghe 1996;
Fletcher et al. 1998; Rosenberg 2000), and other vegetatively

ABBREVIATIONS:ELISA, Enzyme-linkedimmunosorbent assay; H202,

Accepted for publication 23 December 2003. hydrogen peroxide; MS, Murashige and Skoog; SA, salicylic acid; PVX,
ADDITIONALKEYWORDS:salicylate,virus-freeplants potato virus X; PVX(+), PVX detected
172 AMERICAN JOURNAL OF POTATO RESEARCH
propagated species (Gella and Errea 1998; Zaitlin and
Palukaitis 2000; Shiboleth et al. 2001).
Enhanced procedures for thermotherapy of microplants
would be useful in potato biotechnology. The thermotherapy
treatment depends on the virus present in the plants and the
sensitivity of the cultivar to heat. Often plants do not tolerate
the high temperatures for periods long enough to inactivate
the virus, and this limits the number of regenerated individuals
available for the virus detection tests (Sfinchez et al. 1991;
Rosenberg 2000). We previously demonstrated that acetyl-SA
treatment enhanced thermotolerance in potato microplants
(L6pez-Delgado et al. 1998). Also, SA is known to play an
important role in plant defence responses to viral infection
(Mur et al. 1997; Murphy and Carr 2002; Takahashi et al. 2002).
As a natural hormone, SA is unlikely to raise concerns as a
chemotherapeutic agent. We therefore reasoned that SA held
promise as a medium supplement to improve the efficiency of
virus eradication in potato microplants. This paper demon-
strates the beneficial effects of culture in the presence of SA
on both the survival of potato microplants subjected to ther-
motherapy and on the number of virus-free plants obtained.
MATERIALS AND METHODS
Plant Materials
Sterile Solanum tuberosum L. microplant advanced
clones from the Breeding Project of the National Potato Pro-
gram of the National Institute for Agriculture and Livestock
(INIFAP), in Toluca, Mexico, were used. The origins of most of
these materials are Mexican wild species and Mexican cuiti-
vars. The heat tolerance of the genotypes was unknown prior
to the study. The following 34 advanced clones with resistance
to late blight and three cultivars were used as part of the seed
production scheme of the National Potato Program: T-95-20-
116, T-93-2-84,T-92-18-225, 92-367-48, T-95-47-129,T-95-5-120, T-
92-8-11, T-91-35-9, T-92-42-2, T-95-35-141, T-95-29-138, 79-15-39,
T-91-3-12, T-93-7-35, T-96-11-21, T-90-5-136, T-90-2-21, T-94-4-20,
970802, T-92-16-12, T-95-32-117, T-94-3-15, T-96-13-11, T-93-1-2,
T-95-42-128, T-94-17-17, T-98-31-4, T-92-3-20,T-95-57-139, T-94-4-
18, T-90-2-27, T-92-8-11, T-92-4-73, T-92-4-113, Alpha, Atlantic,
Tollocan.
The presence of PVX in these genotypes was cortfi_rmed
by ELISA (Clark and Adams 1977). Tissue samples (c. 0.3 g)
consisting of stems and leaves were ground in phosphate
buffer-Tween + 2% of polyvinylpyrrolidone+ 0.2% egg albumin.
Vol. 81
Antibodies to PVX (Agdia) were used. The presence of PVX
was recorded visually by the yellow color of the reaction.
Microplants indexed as positive for PVX were micropropa-
gated by the method of Espinoza et al. (1986). Microplants
were cut into single nodes with a leaf and subcultured in vitro
on solid MS propagation medium without plant growth reguia-
tors (Murashige and Skoog 1962), containing sucrose 3% (w/v),
and agar 0.6% (w/v), pH 5.7, sterilized for 20 min at 120 C.
Microplants were cultured in jars (57 x 68 nun, Sigma) at 20 _+
1C under a 16-h photoperiod (fluorescent lights, 35 pmol m 2
sec -~, 400 to 700 nm). Surviving plantlets were subcultured,
transplanted to peat moss, and placed in the greenhouse (23 C
day, 10 C night). ELISA was performed before starting the
experiments, 30 days after the post-thermotherapy subculture
and 30 days after transplanting to greenhouse. Tubers were
harvested after 90 days of culture.
E f f e c t o f S A o n Thermotherapy
Before thermotherapy began, nodal cuttings were culti-
vated for 4 wk on propagation medium either in the presence
or absence of SA, which was added to the media for a final
concentration of 10-0 M under the conditions described above
(L6pez-Delgado et al. 1998). This concentration was based on
previous work with acetyl-SA, which was effective in enhanc-
ing thermotolerance of potato microplants at concentration of
10~ to 10~ M (L6pez-Delgado et al. 1998). Twenty-four hours
prior to thermotherapy for 30 days, both treatments of nodal
cuttings were subcultured on SA-free propagation medium in
tubes (12 x 100 mm, Sigma), and incubated as described. For
each genotype, thermotherapy was then applied to 30 nodal
cutting at 42 C, in a Biotronette Mark ]]] (Lab-Line) environ-
mental chamber under fluorescent lights (35 pmol m 2 sec -~,
400-700 nm). After thermotherapy, surviving microplants of
each genotype were subcultured to propagation medium. After
a further 30 days, survival assessment and ELISA for PVX were
performed (n = 8 to 30). Subcultures of surviving plants were
transplanted to the greenhouse for subsequent virus indexing
as described above. Plants were also indexed 30 days after
transplanting to greenhouse.
Catalase Activity
Tissue samples (c. 0.1 g), consisting of microplants with-
out roots, were frozen in liquid nitrogen, ground to fine pow-
der, and allowed to thaw in 0.4 mL buffer (100 mM
HEPES-KOH buffer; pH 7.4). Following centrifugation at
2004 L6PEZ-DELGADO et al.: MICROPIANT THERMOTHERAPY 173

11,000 g for 10 rain at 4 C, supernatants were assayed for cata- Statistics

lase activity at 20 C in a liquid phase oxygen electrode (Hansat- Significance of data was tested by analysis of variance
ech, UK). O 2 evolution was followed in a total reaction volume and Duncan's Multiple Range Test (Duncan 1955).
of I mL consisting of I-I202 (530 mM), HEPES-KOH buffer (pH
7.4), and extract (10 to 40 pL). Catalase activity was expressed
RESULTS
per g fresh weight tissue.
InitiaUy, seven genotypes of PVX(+) potato microplant
H202 Measurement clones, from the INIFAP National Potato Program, were cul-
H202 was measured in microplants without roots. Tissue tured for 4 wk on medium with or without 10~ M SA, then sub-
samples (c. 0.2 g) frozen in liquid nitrogen were ground to a cultured onto medium without SA for 24 h and exposed to a
fine powder, extracted in 1.2 mL ice-cold 5% (w/v) trichloro- standard antiviral thermotherapy treatment at 42 C. At the end
acetic acid (TCA), and centrifuged (10 rain, 11,000 g). Samples of 30 days of thermotherapy, microplants incubated previously
(0.5 mL) of the supernatant fractions were passed through in SA showed better survival than the control microplants,
Dowex-1 resin (0.5 g, Fluka) followed with 3.5 mL of 5% TCA. with the protective effects of SA being most apparent in the
H202 was measured in the eluates using the luminol-dependent most thermosensitive genotypes (Table 1). Genotypes incu-
chemiluminescence method of Warm and Laties (1982): 0.5 mL bated in SA showed sum~ival rates of at least 40% and up to
of eluate was added to 0.5 mL of 0.5 mM luminol (Sigma), the 100%, while among the control microplants, three genotypes
volume was made up to 4.5 mL with 0.2 M NH4OH (pH 9), and died completely (n = 30). Immediately after thermotherapy,
0.452 mL of this mixture in a polysterene tube (12 x 75 mm, surviving nu'croplants were subcultured and their survival re-
Fisher) was analyzed using an Optocomp P luminometer assessed after a further 30 days (Table 1). In the three ther-
(MGM Instruments, USA). moseusitive genotypes in which all control nficroplants had
Chemiluminescence was initiated by injecting 50 llL of 0.5 already died in thermotherapy (T-95-29-138, T-96-11-21,
mM potassium ferricyanide in 0.2 M of NH4OH and emitted 970802), 63% to 100% of the SA-treated microplants that sur-
photons were counted over 5 s. A parallel sample of each ini- vived thermotherapy (n > 12) were still growing 30 days after
tial extract was processed after addition of a known concen- the subculture. In all genotypes in which some control
tration of H202 to provide a recovery correction factor (Warm microplants had survived thermotherapy, more than 93% of the
and Laties 1982). SA-treated population (n > 12), but as few as 53% of the con-
trois (n > 8), still survived
30 days after subculture
TABLE 1--Resistance to thermotherapy (42 C, 30 days) and percentage of PVX-free plants
(Table 1). Therefore, SA
of 7 genotypes of potato mic~vplants, subcultured from. in vitro plants grown
enhanced not only toler-
4 wk on propagation medium :kSA (10 ~ M). A. Survival of nodal cuttings,
ance to the 42 C thermo-
subcultured on to SA-free medium for 24 h and then subjected to thermotherapy
therapy, but also to
(n = 30). B. Survival of nodal cuttings, subcultured from surviving microptants
recovery at 20 C after the
after thermotherapy, assessed afl~vr30 days of culture (n = 8 to 30).
subsequent subculture.
% Survival. Moreove5 as Table 1
B. 30 days after subculture of shows, 30 days after the
Genotypes A. After thermotherapy microplants surviving from A % of PVX-FREE PLANTS
post-thermotherapy sub-
With SA Without SA With SA Without SA With SA Without SA
culture, 100% of the surviv-
T-95-20-116 100 33 93 63 100 50
T-95-5-120 83 93 100 90 100 62 ing SA-treated microplants,
T-95-29-138 56 0 83 n.d. 1 100 n.d. but only 40 to 65% of the
T-96-11-21 40 0 63 n.d. 100 n.d. controls, were found by
970802 73 0 100 n.d. 100 n.d.
T-96-13-11 100 26 I00 53 100 65 EUSA to be free of PVX
T-98-31-4 40 40 97 73 100 40 (n = 8 to 30).
~n.d. = no data
174 A M E R I C A N J O U R N A L O F POTATO R E S E A R C H Vol. 81

Additional e x p e r i m e n t s w e r e c o n d u c t e d to c o n f i r m t h e
e n h a n c e m e n t of t h e efficiency of virus e r a d i c a t i o n b y SA. In
t h e s e experiments, 15 m i c r o p l a n t s of e a c h of a f u r t h e r 30
PVX(+) genotypes w e r e s u b j e c t e d to t h e r m o t h e r a p y w i t h or
w i t h o u t p r i o r culture o n SA as d e s c r i b e d above. T h e s e geno-
types w e r e o b s e r v e d to b e m o r e t h e r m o t o l e r a n t t h a n t h e pre-
v i o u s group, with 58.4 _+ 1.0% of controls surviving after 30
days at 42 C. However, SA t r e a t m e n t still i m p r o v e d survival,
w i t h a n o b s e r v e d survival r a t e of 64.0 _+0.9% of t h e genotypes.
Survival r a t e s of e a c h g e n o t y p e are s h o w n in Table 2. As
o b s e r v e d w i t h the p r e v i o u s sets of cultivars, t h e SA t r e a t m e n t
p r i o r to t h e r m o t h e r a p y r e s u l t e d in a near-total e l i m i n a t i o n of
PVX, with 98 -+ 1.0% o f t h e m i c r o p l a n t s t h a t survived ther-
m o t h e r a p y a n d s u b s e q u e n t culture for 30 days i n d e x e d as free
of PVX infection, while only 75 + 0.7% of c o n t r o l p l a n t s w e r e
i n d e x e d as PVX-free (Table 2).
M e a s u r e m e n t s w e r e m a d e of t h e effects o f culture o n 10-~
M SA for 4 w k o n t h e H~O 2 c o n t e n t a n d c a t a l a s e activity of
microplants, in t h r e e g e n o t y p e s u s e d in the p r e v i o u s experi-
m e n t s of t h e r m o t h e r a p y (Alpha, Atlantic a n d Tollocan). H202
c o n t e n t s w e r e f o u n d to b e 19% to 46% greater in t h e SA-treated
p l a n t s (Figure la), w h i l e c a t a l a s e activities w e r e 21% to 38%
l o w e r (Figure lb).
DISCUSSION
SA a p p e a r s to offer a d o u b l e b e n e f i t as a m e d i u m supple-
m e n t in p o t a t o tissue culture t h e r m o t h e r a p y , e n h a n c i n g b o t h
nficroplant survival a n d virus e r a d i c a t i o n rates. The e n h a n c e d
survival rate w a s especially p r o n o u n c e d in cultivars i n t o l e r a n t
to t h e r m o t h e r a p y .
Ldpez-Delgado et al. (1998) d e m o n s t r a t e d t h a t acetyl-SA
i n d u c e d t h e r m o t o l e r a n c e in p o t a t o microplants. The results
p r e s e n t e d in this p a p e r s u p p o r t this o b s e r v a t i o n using t h e
related c o m p o u n d SA, w h i c h is a naturally p r o d u c e d h o r m o n e
in p l a n t s (Mur et al. 1997). We t e s t e d SA bearing in m i n d liter-
a t u r e suggesting similar effects for b o t h salicylates o n induc-
tion o f t h e r m o t o l e r a n c e (L6pez-Delgado et al. 1998; Dat et al.
1998, 2000).
We also d e m o n s t r a t e h o w SA c a n b e integrated into a n
effective antiviral t r e a t m e n t for r e m o v i n g PVX from infected
microplants, b y e n h a n c i n g t h e i r survival during t h e r m o t h e r a p y
a n d t h e r e c o v e r y p e r i o d following t h e p o s t - t h e r m o t h e r a p y
subculture. E n h a n c e m e n t of h e a t t o l e r a n c e b y SA or acetyl-SA
in p o t a t o h a s also b e e n d e m o n s t r a t e d to o c c u r in a r a n g e o f
o t h e r p l a n t species (Dat et al. 1998, 2000; S e n a r a t n a et al. 2000;
Larkindale a n d Knight 2002). In t h e p r e s e n t r e s e a r c h the p l a n t s
were t r e a t e d w i t h SA prior to t h e t h e r m o t h e r a p y , b e c a u s e
L6pez-Delgado et al. (1998) f o u n d t h a t t h e p e r c e n t a g e of sur-
vival w a s i m p r o v e d if t h e acetyl-SA w a s n o t p r e s e n t during t h e

TABLE 2--Resista~we to thermotherapy (42 C, 30 days) and percentage of PVX-free plants of 30 genotypes of potato
mieroplants, subcultured f r o m in v i t r o plants grown 4 wk on propagation m e d i u m +_SA (10 -5 M). Nodal
cuttings were subcultured on to SA-free m e d i u m for 24 h and then subjected to thermotherapy (n = 15).
*** +_SA values significantly different (P < 0.001).
% Survival % of PVX-Free Plants % Survival % of PVX-Free Plants
Genotype With SA Without SA With SA Without SA Genotype With SA Without SA With SA Without SA
T-95-35-141 75 50 100 75 %95-47-129 70 67 100 75
%95-32-117 75 55 100 75 T-95-42-128 60 57 100 75
%9242-2 67 50 100 75 T-94-4-20 60 57 100 71
T-94-4-18 67 53 99 70 T-95-57-139 65 63 100 75
T-94-3-15 64 53 100 80 T-93-1-2 52 50 80 70
T-91-3-12 68 57 100 70 Alpha 66 64 100 80
Atlantic 68 57 99 74 T-92-18-225 54 53 100 80
%92-16-12 67 57 100 72 T-92-8-11 63 63 100 75
%90-5-136 60 50 100 79 79-15-39 60 60 100 80
T-93-7-35 68 60 99 80 T-924-73 65 67 100 75
92-367-48 61 53 100 72 T-924-113 64 67 100 85
T-92-8-11 60 53 98 70 T-90-2-21 63 67 97 75
Tollocan 66 60 100 75 T-93-2-84 62 67 100 75
T-91-35-9 63 57 100 81 T-92-3-20 53 60 75 70
%90-2-27 65 60 97 70 Mean 64.03 58.43*** 98 75***
%94-17-17 70 66 99 75 _+SE _+0.99 _+1.05 _+1.04 _+0.7
 2004 LOPEZ-DELGADO et al.: MICROPLANT THERMOTHERAPY 175

erature on SA as a plant defence signal during virus infection

1.6
~SA nC (Mur et al. 1997; Murphy and Cart 2002; Takahashi et al. 2002).
Despite its well-documented roles in nature, however, there
o
1.2 are still relatively few direct agrochemical applications of SA.
o
o However, other potential uses have been envisaged, e.g., pre-
o
"o treatment of tomato fruit with methyl-SA induces the synthe-
=o 0.8 sis of stress proteins, leading to increased chilling tolerance
o
o. and resistance to pathogens, thereby decreasing the incidence
r
o of decay (Ding et al. 2002).
,9o o.4 It has been suggested that H202 has a central role as a dif-
-r fusible signal for the induction of defence genes against
stresses (Neill et al. 2002; Pastori and Foyer 2002), including
i
virus infection (Hernfindez et al. 2001). In the present study,
Alpha Atlantic Tollocan
SA-treated nficroplants were found to have increased levels of
H~O2, which correlates to the results reported for acetyl-SA
treatment by LSpez-Delgado et al. (1998). One factor con-
[] SA [] Controls b tributing to the observed increased in H202 levels recorded by
16 T our assay may have been the observation that catalase activity
3- is reduced in SA-treated plants. Several previous reports indi-
cated that SA treatments reduce catalase activity (LSpez-Del-
12 =g
gado et al. 1998; Dat et al. 1998, 2000; Srivastava and Dwivedi
1998) as well as catalase mRNA accumulation (Ding et al.
o 2002) in several plant species. Moreover, reductions in cata-
8
lase activity have also been observed in virus-challenged

tO
4 84
0 I I I
Alpha Atlantic Tollocan
Genotypes
FIGURE 1.
E f f e c t s o f S A o n ( a ) h y d r o g e n p e r o x i d e c o n t e n t (pmol g-' f r e s h
weight), and (b) c a t a l a s e a c t i v i t y (pmol 02 min-' g-' f r e s h
weight), of microplants of t h r e e p o t a t o g e n o t y p e s . P l a n t s w e r e
c u l t u r e d f o r 4 w k o n MS m e d i u m • S A 104 M. D a t a a r e m e a n s
+ s t a n d a r d e r r o r b a r s (n -- 3) of t h r e e e x p e r i m e n t s , e a c h with
t h r e e s a m p l e s o f e a c h g e n o t y p e m e a s u r e d in t r i p l i c a t e . * S A
v a l u e s s i g n i f i c a n t l y d i f f e r e n t f r o m c o n t r o l s ( P < 0.05).
heat shock treatment. This report demonstrates for the first
time that SA increases the number of virus-free microplants
among thermotherapy survivors, which is a second major ben-
efit of SA treatment.
Our hypothesis that SA might be effective as an antiviral
agent in potato biotechnology was based on the extensive lit-
plants, e.g., apricot inoculated with plum pox virus (Hern~n-
dez et al. 2001). The results presented in this report should be
interesting to virus researchers who may derive new concepts
of the interaction between salicylates, antioxidants, H202, and
virus infection. LSpez-Delgado et al. (1998) demonstrated the
induction of thermotolerance in potato by SA and H202 so the
potential application of H202 and antioxidants in virus cleaning
is worthy of further research.
The combination of SA and thermotherapy was found to
be effective for recovering PVX-free plants in this report, but
these treatments should be tested for other potato viruses,
alone or in combination, as well as for other plant host-virus
systems.
Sixty years of therapy of virus-infected plants by ther-
motherapy, chemotherapy, and meristem tip culture have
made possible the micropropagation of marketable virus-free
germplasm, but have only slightly improved our understanding
of virus biology (Pennazio 1997). Nonetheless, the practical
utility of heat and SA treatment as demonstrated in the present
report is strong justification for continued investigation of the
interactions between these factors and plant viruses.
176 A M E R I C A N J O U R N A L O F POTATO R E S E A R C H
AKNOWLEDGMENTS
We gratefully a c k n o w l e d g e Martha Alvarado Ordbnez for
h e r technical a s s i s t a n c e in tissue culture. The a u t h o r s t h a n k
the INIFAP and the Alianza p a r a el Campo P r o g r a m for their
support.
LITERATURE CITED
Clark MF, and AN Adams. 1977: Characterististics of the microplate
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