TECHNIQUES IN PLANT VIROLOGYCIP Training Manual4.0 CONTROL

Section 4.2Virus Eradication: TissueCulture of Meristems,Thermotherapy, andChemotherapy

Introduction

Phytopathogens such as nematodes, fungis, bacteria, phytoplasmas,viruses and viroids, can be transmitted from infected to healthy potatoplants. Nevertheless, not all the cells become infected; sometimesmeristematic tissues are free of pathogens, which allows recovery ofhealthy plants through techniques of in vitro meristem culture.

Thermotherapy is also applied for virus eradication. This technique hasbeen successfully used for many years in virus eradication for carnation,

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geranium, strawberry, and citrus; plants are treated at high temperatures,in screenhouses or growth chambers. The standard method for viruseradication in many vegetatively propagated crops is thermotherapycombined with meristem culture.

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

This chapter describes the methods that can be used to eliminatephytopathogens from infected material to produce pathogen-free plantsfor international distribution and propagation in potato seed productionprograms.

Nature of the phytopathogens

Pathogens that affect potato plants can be transmitted from diseased tohealthy plants through vectors or seed. The relative size of thesepathogens is variable. Among phytopathogens, nematodes are thebiggest and can be easily observed with a stereoscopic microscopy.Virus and viroids are the smallest, so an electronic microscope is neededfor their observation.

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

The distribution of different pathogens in a diseased plant also varies. Forexample, Pseudomonas solanacearum, potato leaf roll virus (PLRV), andphytoplasmas are restricted to vascular tissue of the plant. Erwiniacarotovora and potato virus X (PVX) invade the vascular tissue as well asthe rest of the other tissues of the plant.

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

• High metabolic activity. Viruses multiply according to themetabolism of the host plant. Due to the high metabolic activity inmeristematic cells, the viruses are unable to take over control ofthe host biosynthetic machinery.

• Lack of vascular tissue. Viruses are rapidly disseminatedthrough the vascular system. Those located in the phloem (e.g.,PLRV) cannot invade the meristematic tissues because there is nocell differentiation in this zone. Viruses that infect non-vasculartissues are disseminated from cell to cell through intercell conduits

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

• High auxin concentration. Plant meristematic tissues have ahigher auxin concentration than other plant tissues. Some authorsindicate that these auxin inhibit virus multiplication.

Thermotherapy

Experiments carried out with viruses and their host plants have shownthat when plants are treated at high temperatures (thermotherapy) thevirus concentration is reduced (Kassanis, 1957; Quak, 1977). There aredifferent explanations for this phenomenon. One explanation is thatcompetition among the rapidly dividing host cells and the virus particlesfor the places where nucleic acids and proteins synthesize results in achange in the balance between the synthesis and degradation of virusparticles. Another explanation is that under high temperatures, the unionof the protein subunits that protect the nucleic acid of the virus becomesweaker 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. Areduction in virus concentration has been observed, especially in potatoleaf roll virus, which has been successfully eliminated only withthermotherapy.

Thermotherapy applied to the whole plant, as well as to sprouted tubers,followed by meristems culture, has been successfully used as a standardprocedure for the eradication of many potato viruses (Stace-Smith andMellor, 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 cutbefore being treated with thermotherapy, and the axillary buds continuegrowing while receiving high temperature treatment. A regimen of a dailytemperature of 36°C for 16 hours, and 30°C for 8 hours, under a highintensity continued light (5000 lux), improved the score of viruseradication. The plants are kept under these conditions for four weeks.The meristems, either of the axillary buds or the topical buds, are isolatedor cultivated as shown later.

Thermotherapy can be applied to in vitro plantlets. One-bud nodes (20nodes/box) are placed in plastic boxes (Magenta GA-7) containing asemisolid propagation media (Espinoza et al., 1991). The boxes areincubated under adequate conditions (Espinoza et al., 1991) and sealedwith adhesive tape when the plants reach 3-cm height and havedeveloped a root system. Then they are treated with thermotherapy asexplained before. A month later, apical meristems are isolated andcultivated in an appropriate culture media.

Culture of meristems

Culture of meristems includes a process of surface sterilization, theexcision or isolation of the meristem and its culture in a media underadequate conditions. When the vegetative material comes from in vitroplants, 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 preventcontamination with pathogens or saprophites. Some contaminants growvery rapidly and can kill the plant tissue introduced in vitro.

Most of the tissue surface contaminants can be eliminated from thevegetative material with an adequate sterilizing agent. Under asepticconditions, the sterilizing solution is normally applied for 10–15 minutes.This is eliminated, and the material is washed with sterile distilled water 3or 4 times for 5 minutes each time. Washing is very important to removeexcess of the sterilizing agent, which could inhibit the growth of the plant.

Alcohol (ethanol). This is a common surface-sterilizing agent toeliminate bacteria and fungus; it is frequently used for brief washes (30minutes) before applying other sterilizing treatments. It has a lowsuperficial tension, which enables it to easily penetrate into the hairy foliarand moisten the surface of the plant. Ethanol 70% is more effective as asterilizing agent than 95–100%.

• Sodium or calcium hypochlorite. The surface of the vegetativematerial can also be sterilized with aqueous solutions of sodiumhypochlorite (NaOCl) or calcium hypochlorite (CaOCl). Calciumsalt is preferred because it is less phytotoxic. Many laboratoriesuse sodium hypochlorite of domestic use (Clorox). Thesecommercial products normally contain 5.25% of NaOCl as theactive ingredient. When diluted in water (10% of Clorox and 90%of water), the new sterilizing solution should have not less than0.5% of NaOCl. Due to a complete disassociation, the hypochloritehas a relatively low activity at a pH superior to 8.0, and is moreeffective when the solution has a pH of 6.0.

• The surface of the tissue, recently dissected and completelysubmerged in the sodium or calcium hypochlorite, is sterilized afteran exposure of 10 to 15 minutes. The treated material should becarefully washed many times with distilled water to remove thedisinfectant completely.

• Mercury bichloride. Mercury bichloride (HgCl2) is used asdisinfectant, although it is extremely toxic. This solution is volatileat environmental temperature and can produce mercury poisoning.Therefore, it is not recommended as sterilizing agent.

• Bactericides and fungicides. Highly contaminated material shouldbe washed in a commercial mix of bactericide/fungicide beforesterilizing the surface. This treatment has no effect in systemicinfections.

2. Isolation and culture of the meristem

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

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

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(toward the older tissues of the stem), the vascular elements of theprimordium leaves are incipient and have not yet made contact with theprincipal part of the stem’s vascular system. For this reason, the virusparticles present in the vascular system can only reach the topmeristematic zone by moving slowly from cell to cell. This is one of themain reasons why virus concentration in an infected plant decreasesfrom the base towards the meristems, either in the top or in the axillarybuds.

Isolation of the meristematic point in aseptic conditions and its culture inan adequate aseptic nutrient medium leads to the development ofplantlets. The development of the meristem in in vitro conditions similarto that of a normal plant. The meristematic cells divide and thedifferentiation of new tissues continues. The artificial media gives thenecessary nutrition to the tissues of the dissected section. The asepticdissection of the meristem is a delicate process and requires a lot ofpractice.

Eradication of potato viruses

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

a. Thermotherapy: Plants (two months old) or in vitro plantlets (onemonth old) are kept at temperatures of 36°C for 16 hours and 30°Cfor 8 hours daily for 30 days, under a high intensity continued light(5000 lux).

b. Disinfecting vegetal material: Plant stems are cut in uninodalsegments with their axillary bud. Leaves are carefully removed andit is recommended that, before the disinfection, stem cuttings betreated with a wide spectrum acaricide (Morestan-Bayer 0.5% for10 minutes). The explants are treated with alcohol (70%) for 30seconds and with calcium hypochlorite (2.5%) for 15 minutes. Afterthis, the stems should be washed 4 times with sterilized distilledwater, 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 theapical bud’s dome and a few primordium leaves remain (usuallytwo).

d. Meristem culture: The dome and two primordium leaves aredissected and transferred to the culture media. The dissectedmeristem is transferred weekly to a new media. After 6–8 weeksplantlets are obtained; these should be propagated and thenindexed.

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

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e) Indexing: Plants grown from meristems are tested to detect anyremaining virus infection. Tests carried out at CIP include: NASHfor PSTVd detection, serological ELISA test, and indicator hostplants tests (International Potato Center, 1993). Search of viralparticles through electronic microscope is optional. All these testscan 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 spectrumagainst ADN and ARN of viruses affecting animals, has shown variableresults when applied to potato plants by aspersion or in hydroponicculture, followed by a meristem culture. Addition of 100 mg/l of virazole tothe meristem culture media has been successful for the eradication ofPVS, PVX, and PVY, but not of PLRV (Griffiths, 1990).

Although problems in genetic variation in potato treated with antiviralchemotherapy have not been reported, the tendency to use this kind ofproducts has decreased because some reports show that antiviralchemicals can cause mutations in plants. Currently, thermotherapy is thepreferred method of pre-treatment, followed by meristem culture.

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Antibiotics are only used in vegetative tissue cultures when themicroorganisms can not be eliminated using other methods. These areexpensive, and none of them are effective enough to control all thecontaminant organisms. Before using antibiotics, elimination of allpossible sources of contamination is recommended (Reed andTanprasert 1995).

The following combinations of antibiotics have been applied successfullyin plant tissue culture: Cefotaxime, Gentamycin, Rifampicin, Nystatin +Carbenicillin, Gentamycin + Amphotericin B, Vancomycin HCI +Mycostatin, and Streptomycin + Carbenicillin. There have been severalreports 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 theconcentrations 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 at200 mg/l. With yeast infection, 0.25–0.5 mg/l of Amphotericin B is addedto the culture media.

Recommended LiteratureCIAT (Centro Internacional de Agricultura Tropical). 1991. Cultivo de

tejidos en la agricultura: Fundamentos y aplicaciones. W.M. Rocaand 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 yexportación 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 andheat 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 clonesfrom infected potato varieties. Annals of Applied Biology 45:422–427.

International Potato Center (CIP). 1993. Basic techniques in plantvirology. U. Jayasinghe y L.F. Salazar (eds.). CIP, Lima (TechnicalTraining Unit 1).

Lizárraga, R., A. Panta, U. Jayasinghe and J.H. Dodds. 1991. Tissueculture 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 temperatureand meristem culture. Phytopathology 70:754–755.

Lizárraga, R.E. and L.F. Salazar. 1982. Effect of meristem size oneradication of potato spindle tuber viroid. In: Hooker, W.J. (ed.).Research for the potato in the year 2000. International PotatoCenter. Lima, Peru. P. 118–119.

Mellor, F.C. and R. Stace-Smith. 1977. Virus-free potatoes by tissueculture. 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’unemaladie virus. Comptes rendus hebdomadaire des séances dell’Academie des Sciences, Paris. 235:1324–1325.

Murashige, T. and F.C. Skoog. 1962. A revised medium for rapid growthand bioessays with tobacco tissue cultures. Physiologia Plantarum15:473–497.

Pennazio, S. and P. Redolfi. 1973. Factors affecting the in vitro culture ofpotato meristem tips. Potato Research 16:20–29.

Quak, F. 1977. Meristem culture and virus-free plants. In: J. Reinert andY.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 bacterialcontaminants 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 spindletuber virus by thermotheraphy and axillary bud culture.Phytophathology 60:1957–1958.

Zamora, A.B., C.N. Paet, and E.C. Altoveros. 1994. Micropropagationand virus elimination procedures in potato for conservation,dissemination, and production in the humid tropics. Southeast AsianProgram for Potato Research and Development (SAPPRAD) CIPc/o PCARRD; and College of Agriculture, University of ThePhilippines, Los Baños College, Los Baños, Laguna, Philippines.105 p.