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TECHNIQUES IN PLANT VIROLOGY

CIP Training Manual


4.0 CONTROL

Section 4.2
Virus Eradication: Tissue
Culture of Meristems,
Thermotherapy, and
Chemotherapy

Introduction

Phytopathogens such as nematodes, fungis, bacteria, phytoplasmas,


viruses and viroids, can be transmitted from infected to healthy potato
plants. Nevertheless, not all the cells become infected; sometimes
meristematic tissues are free of pathogens, which allows recovery of
healthy plants through techniques of in vitro meristem culture.

Thermotherapy is also applied for virus eradication. This technique has


been successfully used for many years in virus eradication for carnation,
geranium, strawberry, and citrus; plants are treated at high temperatures,
in screenhouses or growth chambers. The standard method for virus
eradication in many vegetatively propagated crops is thermotherapy
combined with meristem culture.

Due to the fact that it takes many months for meristems to become
plantlets, some researchers have tested the application of chemical
products (chemotherapy) that reduce or inhibit viral multiplication. In the
case of potato, three of the most important viruses, PVX, PVS, and PVY,
have been eradicated by adding Ribavirin to the culture media and
isolating the axillary buds (Griffiths et al., 1990).

This chapter describes the methods that can be used to eliminate


phytopathogens from infected material to produce pathogen-free plants
for international distribution and propagation in potato seed production
programs.

Nature of the phytopathogens

Pathogens that affect potato plants can be transmitted from diseased to


healthy plants through vectors or seed. The relative size of these
pathogens is variable. Among phytopathogens, nematodes are the
biggest and can be easily observed with a stereoscopic microscopy.
Virus and viroids are the smallest, so an electronic microscope is needed
for their observation.

The disease does not depend only on the presence of the host, but on
environmental conditions as well, especially humidity and temperature,
which play an important role. The disease can be defined as the product
of the interaction among the host, pathogen, and environment.

The distribution of different pathogens in a diseased plant also varies. For


example, Pseudomonas solanacearum, potato leaf roll virus (PLRV), and
phytoplasmas are restricted to vascular tissue of the plant. Erwinia
carotovora and potato virus X (PVX) invade the vascular tissue as well as
the rest of the other tissues of the plant.

Not all the cells in a diseased plant become infected with pathogens. The
meristematic tissue of the root and the terminal sprouts of an infected
plant are sometimes pathogen-free. Sometimes, such as in potato with
PVX and TRV (tobacco rattle virus), only the apical dome and the first
young primordial leaves are free of virus. The reason for this is unknown.
Nevertheless, it is believed that one or more of the following factors are
responsible:

• High metabolic activity. Viruses multiply according to the


metabolism of the host plant. Due to the high metabolic activity in
meristematic cells, the viruses are unable to take over control of
the host biosynthetic machinery.

• Lack of vascular tissue. Viruses are rapidly disseminated


through the vascular system. Those located in the phloem (e.g.,
PLRV) cannot invade the meristematic tissues because there is no
cell differentiation in this zone. Viruses that infect non-vascular
tissues are disseminated from cell to cell through intercell conduits

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(plasmodesmata). This is a slow process, which makes it relatively
difficult for viruses to infect the rapidly dividing cells.

• High auxin concentration. Plant meristematic tissues have a


higher auxin concentration than other plant tissues. Some authors
indicate that these auxin inhibit virus multiplication.

Thermotherapy

Experiments carried out with viruses and their host plants have shown
that when plants are treated at high temperatures (thermotherapy) the
virus concentration is reduced (Kassanis, 1957; Quak, 1977). There are
different explanations for this phenomenon. One explanation is that
competition among the rapidly dividing host cells and the virus particles
for the places where nucleic acids and proteins synthesize results in a
change in the balance between the synthesis and degradation of virus
particles. Another explanation is that under high temperatures, the union
of the protein subunits that protect the nucleic acid of the virus becomes
weaker and temporal fissures appear, allowing the attack of nucleases,
which inactivate the virus and decreases its concentration.

Thermotheraphy has been applied to potato tubers in dormancy. A


reduction in virus concentration has been observed, especially in potato
leaf roll virus, which has been successfully eliminated only with
thermotherapy.

Thermotherapy applied to the whole plant, as well as to sprouted tubers,


followed by meristems culture, has been successfully used as a standard
procedure for the eradication of many potato viruses (Stace-Smith and
Mellor, 1970; Pennazio and Redolfi, 1973).

In the standard procedure used at the International Potato Center (CIP,


Lima, Peru), the best results have been obtained when the plant is cut
before being treated with thermotherapy, and the axillary buds continue
growing while receiving high temperature treatment. A regimen of a daily
temperature of 36°C for 16 hours, and 30°C for 8 hours, under a high
intensity continued light (5000 lux), improved the score of virus
eradication. The plants are kept under these conditions for four weeks.
The meristems, either of the axillary buds or the topical buds, are isolated
or cultivated as shown later.

Thermotherapy can be applied to in vitro plantlets. One-bud nodes (20


nodes/box) are placed in plastic boxes (Magenta GA-7) containing a
semisolid propagation media (Espinoza et al., 1991). The boxes are
incubated under adequate conditions (Espinoza et al., 1991) and sealed
with adhesive tape when the plants reach 3-cm height and have
developed a root system. Then they are treated with thermotherapy as
explained before. A month later, apical meristems are isolated and
cultivated in an appropriate culture media.

Culture of meristems

Culture of meristems includes a process of surface sterilization, the


excision or isolation of the meristem and its culture in a media under
adequate conditions. When the vegetative material comes from in vitro
plants, sterilization of the surface is not necessary.

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1. Surface sterilization.

It is necessary to sterilize the surface of the vegetative material to prevent


contamination with pathogens or saprophites. Some contaminants grow
very rapidly and can kill the plant tissue introduced in vitro.

Most of the tissue surface contaminants can be eliminated from the


vegetative material with an adequate sterilizing agent. Under aseptic
conditions, the sterilizing solution is normally applied for 10–15 minutes.
This is eliminated, and the material is washed with sterile distilled water 3
or 4 times for 5 minutes each time. Washing is very important to remove
excess of the sterilizing agent, which could inhibit the growth of the plant.

Alcohol (ethanol). This is a common surface-sterilizing agent to


eliminate bacteria and fungus; it is frequently used for brief washes (30
minutes) before applying other sterilizing treatments. It has a low
superficial tension, which enables it to easily penetrate into the hairy foliar
and moisten the surface of the plant. Ethanol 70% is more effective as a
sterilizing agent than 95–100%.

• Sodium or calcium hypochlorite. The surface of the vegetative


material can also be sterilized with aqueous solutions of sodium
hypochlorite (NaOCl) or calcium hypochlorite (CaOCl). Calcium
salt is preferred because it is less phytotoxic. Many laboratories
use sodium hypochlorite of domestic use (Clorox). These
commercial products normally contain 5.25% of NaOCl as the
active ingredient. When diluted in water (10% of Clorox and 90%
of water), the new sterilizing solution should have not less than
0.5% of NaOCl. Due to a complete disassociation, the hypochlorite
has a relatively low activity at a pH superior to 8.0, and is more
effective when the solution has a pH of 6.0.
• The surface of the tissue, recently dissected and completely
submerged in the sodium or calcium hypochlorite, is sterilized after
an exposure of 10 to 15 minutes. The treated material should be
carefully washed many times with distilled water to remove the
disinfectant completely.

• Mercury bichloride. Mercury bichloride (HgCl2) is used as


disinfectant, although it is extremely toxic. This solution is volatile
at environmental temperature and can produce mercury poisoning.
Therefore, it is not recommended as sterilizing agent.

• Bactericides and fungicides. Highly contaminated material should


be washed in a commercial mix of bactericide/fungicide before
sterilizing the surface. This treatment has no effect in systemic
infections.

2. Isolation and culture of the meristem

The meristem is the active growing point of the plant shoot. It is a small
zone composed of cells (meristematic), which divide very fast.

The dome of the apical bud contains the real meristematic cells and is
surrounded by leaf primordia and primary leaves. Due to the fact that the
most differentiated vascular tissues are far away from the meristems

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(toward the older tissues of the stem), the vascular elements of the
primordium leaves are incipient and have not yet made contact with the
principal part of the stem’s vascular system. For this reason, the virus
particles present in the vascular system can only reach the top
meristematic zone by moving slowly from cell to cell. This is one of the
main reasons why virus concentration in an infected plant decreases
from the base towards the meristems, either in the top or in the axillary
buds.

Isolation of the meristematic point in aseptic conditions and its culture in


an adequate aseptic nutrient medium leads to the development of
plantlets. The development of the meristem in in vitro conditions similar
to that of a normal plant. The meristematic cells divide and the
differentiation of new tissues continues. The artificial media gives the
necessary nutrition to the tissues of the dissected section. The aseptic
dissection of the meristem is a delicate process and requires a lot of
practice.

Eradication of potato viruses

The process used at CIP for the eradication of potato viruses includes the
following steps (Fig. 1):

a. Thermotherapy: Plants (two months old) or in vitro plantlets (one


month old) are kept at temperatures of 36°C for 16 hours and 30°C
for 8 hours daily for 30 days, under a high intensity continued light
(5000 lux).

b. Disinfecting vegetal material: Plant stems are cut in uninodal


segments with their axillary bud. Leaves are carefully removed and
it is recommended that, before the disinfection, stem cuttings be
treated with a wide spectrum acaricide (Morestan-Bayer 0.5% for
10 minutes). The explants are treated with alcohol (70%) for 30
seconds and with calcium hypochlorite (2.5%) for 15 minutes. After
this, the stems should be washed 4 times with sterilized distilled
water, 5 minutes each time to eliminate the excess of hypochlorite.

c. Excision of meristems: Under a binocular dissection microscope,


leaflets surrounding the growth point are removed, until only the
apical bud’s dome and a few primordium leaves remain (usually
two).

d. Meristem culture: The dome and two primordium leaves are


dissected and transferred to the culture media. The dissected
meristem is transferred weekly to a new media. After 6–8 weeks
plantlets are obtained; these should be propagated and then
indexed.

The culture media used for this purpose is based in Murashige and
Skoog salts (1962), supplemented with 2 mg/l glycine, 0.5 mg/l
nicotinic acid, 0.5 mg/l piridoxine, 0.4 mg/l thiamine, 0.1 mg/l
gibberellic acid, 0.04 mg/l kinetine, and 2.5% sucrose. The media
is turned to a gel with agar (0.6%) and then sterilized in an
autoclave at 15 pounds pressure and 121°C for 15 minutes.

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e) Indexing: Plants grown from meristems are tested to detect any
remaining virus infection. Tests carried out at CIP include: NASH
for PSTVd detection, serological ELISA test, and indicator host
plants tests (International Potato Center, 1993). Search of viral
particles through electronic microscope is optional. All these tests
can be carried out in a two-month period.

Figure 1. Schematic representation of virus eradication process for potato.

Chemotherapy

Chemotherapy is being used in potato as an alternative to thermotherapy.


An analog to the nucleoside, virazole, known for its wide spectrum
against ADN and ARN of viruses affecting animals, has shown variable
results when applied to potato plants by aspersion or in hydroponic
culture, followed by a meristem culture. Addition of 100 mg/l of virazole to
the meristem culture media has been successful for the eradication of
PVS, PVX, and PVY, but not of PLRV (Griffiths, 1990).

Although problems in genetic variation in potato treated with antiviral


chemotherapy have not been reported, the tendency to use this kind of
products has decreased because some reports show that antiviral
chemicals can cause mutations in plants. Currently, thermotherapy is the
preferred method of pre-treatment, followed by meristem culture.

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Antibiotics are only used in vegetative tissue cultures when the
microorganisms can not be eliminated using other methods. These are
expensive, and none of them are effective enough to control all the
contaminant organisms. Before using antibiotics, elimination of all
possible sources of contamination is recommended (Reed and
Tanprasert 1995).

The following combinations of antibiotics have been applied successfully


in plant tissue culture: Cefotaxime, Gentamycin, Rifampicin, Nystatin +
Carbenicillin, Gentamycin + Amphotericin B, Vancomycin HCI +
Mycostatin, and Streptomycin + Carbenicillin. There have been several
reports of toxicity caused in some tissues by: Penicillin, Streptomycin,
Bactericin, and Sparsomycin (Lizárraga et al., 1991).

At CIP, infections with bacteria and yeast can be eliminated satisfactorily.


For this purpose, antibiotics are added to the culture media in the
concentrations indicated below. Rifampicina is used for bacteria
(Rimactan 300, CIBA) at 60 mg/l or Sodium Cefatoxim (Claforan,
Rousell) at 200 mg/l. Cefatoxim (Mefoxin, Merck) can also be used at
200 mg/l. With yeast infection, 0.25–0.5 mg/l of Amphotericin B is added
to the culture media.

Recommended Literature
CIAT (Centro Internacional de Agricultura Tropical). 1991. Cultivo de
tejidos en la agricultura: Fundamentos y aplicaciones. W.M. Roca
and L.A. Mroginski (eds.). Cali, Colombia. P. xii, 970.
Espinoza, N., R. Lizárraga, C. Sigueñas, F. Buitrón and J.H. Dodds.
1991. Cultivo de tejidos: Micropropagación, conservación y
exportación de germoplasma de papa. Guía de Investigación CIP 1.
Centro Internacional de la Papa. Lima, Perú. 19 p.
Griffiths, H.M., S.A. Slack and J.H. Dodds. 1990. Effect of chemical and
heat therapy on virus concentration in in vitro plantlets. Can. J. Bot.
68:1515–1521.
Kassanis, B. 1957. The use of tissue culture to produce virus-free clones
from infected potato varieties. Annals of Applied Biology 45:422–
427.
International Potato Center (CIP). 1993. Basic techniques in plant
virology. U. Jayasinghe y L.F. Salazar (eds.). CIP, Lima (Technical
Training Unit 1).
Lizárraga, R., A. Panta, U. Jayasinghe and J.H. Dodds. 1991. Tissue
culture for elimination of pathogens. CIP Research Guide 3.
International Potato Center (CIP). Lima, Peru.
Lizárraga, R.E., L.F. Salazar, W.H. Roca and L. Schilde-Rentscheler.
1980. Elimination of potato spindle tuber viroid by low temperature
and meristem culture. Phytopathology 70:754–755.
Lizárraga, R.E. and L.F. Salazar. 1982. Effect of meristem size on
eradication of potato spindle tuber viroid. In: Hooker, W.J. (ed.).
Research for the potato in the year 2000. International Potato
Center. Lima, Peru. P. 118–119.
Mellor, F.C. and R. Stace-Smith. 1977. Virus-free potatoes by tissue
culture. In: Plant cell, tissue, and organ culture. J. Reinert and Y.P.S.
Bajaj (eds.). Springer-Verlag, Berlin. P. 616–635.

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Morel, G.M. and C. Martin. 1952. Guerison de dahlias atteints d’une
maladie virus. Comptes rendus hebdomadaire des séances del
l’Academie des Sciences, Paris. 235:1324–1325.
Murashige, T. and F.C. Skoog. 1962. A revised medium for rapid growth
and bioessays with tobacco tissue cultures. Physiologia Plantarum
15:473–497.
Pennazio, S. and P. Redolfi. 1973. Factors affecting the in vitro culture of
potato meristem tips. Potato Research 16:20–29.
Quak, F. 1977. Meristem culture and virus-free plants. In: J. Reinert and
Y.P.S. Bajaj (eds.). Applied and fundamental aspects of plant cells,
tissue, and organ culture. Springer, Berlin. pp. 598–615.
Reed, B.M. and P. Tanprasert. 1995. Detection and control of bacterial
contaminants of plant tissue cultures. A review of recent literature.
Plant Tissue Culture and Biotechnology 1:137–142.
Stace-Smith, R. and F.C. Mellor. 1970. Eradication of potato spindle
tuber virus by thermotheraphy and axillary bud culture.
Phytophathology 60:1957–1958.
Zamora, A.B., C.N. Paet, and E.C. Altoveros. 1994. Micropropagation
and virus elimination procedures in potato for conservation,
dissemination, and production in the humid tropics. Southeast Asian
Program for Potato Research and Development (SAPPRAD) CIP
c/o PCARRD; and College of Agriculture, University of The
Philippines, Los Baños College, Los Baños, Laguna, Philippines.
105 p.

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