The document discusses several methods for producing virus-free plants, including meristem culture, heat treatment, chemotherapy, and electrotherapy. It provides details on experiments conducted on potato, black raspberry, sugarcane, and gladiolus plants infected with various viruses. For potato, meristem culture was used to produce virus-free plantlets of three varieties infected with potato virus Y. Heat treatment eliminated the black raspberry necrosis virus from black raspberry explants. In sugarcane, combining meristem culture and chemotherapy with ribavirin helped eliminate mosaic viruses. Experiments on gladiolus used thermo, electro, and chemotherapy methods to eliminate bean yellow mosaic virus from three cultivars. RT-PCR testing confirmed

1. Virus Elimination Methods
– Case Studies
Course Teachers
Dr. E. Kokiladevi
Dr. S. Varanavasiappan
VIGNESH. P
2020615014

2. Production of virus free plants
• Plant infected with bacteria and fungi may respond to treatments with
bactericidal and fungicidal compounds, there is not much commercially
available treatment to cure virus infected plants.
• Several types of therapies are used to eliminate viruses and viroids from a
plant. Virus free plants can be produce by plant tissue culture. Methods to
produce virus free plants
Heat treatment.
Meristem tip culture.
Chemical treatment.
Other in vitro methods.
have been used either alone or in combination to eliminate viruses.

3. History of virus free plant culture
• Morel and Martin (1952) developed meristem culture technique and
recovered Dahlia shoots, free from viruses, by meristem tip culture. In
1955, they recovered virus free potato.
• This attained wide application of plant tissue culture to raise virus
free plants in Agriculture.

4. Why are viruses important?
• Cause many important plant diseases.
• Responsible for huge losses in crop production and quality in all parts of the world.
• Virus is restricted to certain parts of the plant (e.g. the vascular system; discrete
spots on the leaf) or spreads throughout the plant causing a systemic infection.
• Infection does not always result in visible symptoms
Infected plants may show a range of symptoms :
- leaf yellowing (either of the whole leaf or in a pattern of stripes or blotches),
- leaf distortion (e.g. curling)
- and/or other growth distortions (e.g. stunting of the whole plant, abnormalities in
flower or fruit formation).

5. Which part of the plant is best suited for making virus-free
plants
• The apical and axillary meristems of plants are the best parts of the plant to make
virus-free plants.
• This is because the rate of division of meristematic cell is higher than the rate of
multiplication of virus and viruses are unable to invade newly formed
meristematic cells.
• Meristematic cells are free of virus although the whole plant is infected with the
virus. With the use of meristem, a healthy plant can be recovered from the
diseased plant through the micropropagation method.

6. Meristem culture
• The use of meristem culture consists of culturing on a nutrient medium a small
(0.1–0.5 mm) piece of tissue removed from the meristematic area.
• Shoot tips, root tissue are suitable organs for meristem culture, for their high
potential of cell division. This technique is considered as a routine method and as
the base of standard virus eradication (Sastry and Zitter 2014)
• Meristems also free from Mycoplasma, Bacteria, and Fungi.
• The size of excised meristems, crop cultivar, plant species and virus species are
main factors influences success in plant virus elimination.

7. Meristem culture - Why meristems are virus free?
Failure to invade meristem is due to:
1. Lack of a vascular system: Spreading cell to cell via
plasmodesmata which are too small to allow the
passage of virus particles.
2. High metabolic activity: active mitosis – the
synthesis of RNA for viral multiplication may
suppressed. Active metabolic process which is not
suitable for virus multiplication
3. High auxin concentration in meristematic cells
inhibit virus multiplication : interfere nucleic acid
metabolism
4. Competition for nutrients enzymes for virus
replication

8. Virus elimination by heat treatment
• Thermotherapy : 35-400C.
• Heat treatment was originally applied by Kassanis in 1949 (Parmessur and
Saumtally, 2001) to eliminate viruses from plant tissue.
• Thermotherapy has been effectively used for a long time to obtain virus- free
plants from infected individuals of diverse plant species (Hollings, 1965).
• The basic principle behind heat eradication of viruses is that at temperatures
higher than normal many viruses in plant tissues are partially or completely
inactivated with little or no injury to the host tissues (Baker, 1962)
• Heat treatment is given through hot water or hot air; whereas hot water
treatment has proved better for dormant buds, hot-air treatment has generally
given better elimination of viruses and better survival of the host in actively
growing shoots.

9. • Inactivation of intact virus particles by breakage of their RNA.
• Disruption of virus particle with enzymatic degradation of its components.
• The duration of the treatment varies from few mins to several months.
• E.g. carnation shoot tip. Potato shoot tip

10. Chemotherapy
• The use of chemicals to suppress virus symptoms and multiplication in infected
plants.
• Use of antiviral compounds- Ribavirin/Virazole, DTH
• Growth promoting chemicals- Cytokinins
• Antimetabolite chemicals- Azaguanine, Thiouracil
• Plants viruses elimination using antiviral chemicals turns out to be an important
technique for the production of virus-free plantlets (Khurana 2004).
• Literatures have shown ribavirin to be the most promising antiviral chemicals
against potato plant viruses.
• The effectiveness of viral elimination by antiviral agents is proportional to their
concentrations (Cordeiro 2003).

11. In Vitro Virus Elimination by Electrotherapy
• Use of electrotherapy as a simple method that uses electric current to mitigate
virulence through degrading nucleoprotein of the virus (Sastry and Zitter 2014)
• Compared to the conventional technique of thermotherapy and meristem
culture, electrotherapy was found to be the most efficient in term of virus
elimination rate (Lozoya-Saldaña et al. 1996).
• But studies showed that virus structures and plants genotypes respond
differently to the technique (Emami et al. 2011).
• The intensity of the electric current, the duration of the technique are the factors
that influence significantly the capacity of this technique.
• Example PVX, PVY, PSTVd and PLRV to be eliminated by the technique.

12. Steps Involved in Meristem Tissue Culture

13. Plant Materials:
• Potato varieties (Alaska, Spunta and Safrane) were used in this study.
• Seventy tubers from each cultivar were subjected to double antibody sandwich DAS-ELISA.
• Virus infected tubers were chosen and used for sprouts production.
Establishment of Meristem Culture:
• Thirty PVY infected sprouts of each variety were used as explants for meristem culture.
• Sterilized in 0.1% hypochlorite and 3 drops of Tween-20 for 10 mins, followed by 3 times washing.
• The tip and sub-tending leaf primordial were removed.
• Murashige and Skoog medium free of hormone was used as basal culture medium.
• The size of the meristem was about 0.3 mm.
• Naphthalene acetic acid and Indole butyric acid

14. Shoots and Roots Development:
After 4 weeks of meristem culture,
• 10 of the developed meristems from each cultivar were sub cultured on MS
hormone free medium and
• 10 on MS medium supplemented with (0.5 mg/l) of Naphthalene acetic acid
(NAA) and
• 10 on MS medium supplemented with (0.5 mg/l) of Indole butyric acid (IBA).
• Cultures were incubated in the culture room at 25±2°C and 16/8 light/dark
photoperiod;
• After 5 weeks, developed plantlets were examined for various parameters:
1. Shoot length, 3. Number of leaves,
2. Root length, 4. Number of roots and shoots3..

16. • Spunta cultivar highest mean of 6.88cm. Shoots length was the highest on IBA medium with mean 7.71cm.
The shortest shoots length on hormone free medium (control) with mean equals to 2.54 cm.
For interaction b/w cultivars and media, the highest shoots length was 9.30 cm for Spunta cultivar on IBA, while the shortest shoots length was obtained at
hormone free medium mean equals 2.11 cm for Alaska.
Results
• The highest response of roots length was in Spunta cultivar with mean equal to 7.88 cm.
Medium containing IBA was found to be the most effective and produced the longest roots length with Spunta cultivar (9.41 cm).
For interaction, the roots length ranged from 1.22 cm at hormone free medium with Alaska cultivar to 12.87 cm at medium containing IBA with Spunta cultivar.
• The means of shoots number for cultivars Spunta, Alaska and Safrane were 2.66, 1.93 and 1.53 respectively.
Medium supplemented with IBA was found to be the most effective one with mean 2.60, while hormone free media was the least effect and produced 1.36
shoots. For interactions, highest number of shoots (3.60) was observed at the 0.5 mg/l IBA followed by 0.5 mg/l NAA with mean 2.80 with Spunta cultivar. The
lowest mean number of shoots (1.10) was observed in Alaska cultivar at hormone free medium.

17. Acclimatization
• Plantlets from the three cultivars were gradually acclimatized. Potato plantlets
pots were covered with plastic bags, the bags were removed within two weeks.
• After four weeks, plantlets developed and their leaves increased in size. 90%
survival plantlets were produced from Spunta cultivar and 80% from Alaska and
Safrane cultivars.
• The plantlets were transferred to the green house in which they continue
growing.
Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR):
• Ten plantlets from each cultivar were subjected to RT-PCR.
• The results of RT-PCR produced from in vitro plantlets for the three cultivars
revealed the absence of PVY virus in the plantlets that developed by meristem
culture.

18. Detection of PVY in Spunta cultivar by RT-PCR.
• Lane M 1Kb DNA Marker,
• lane 2 positive control,
• lane 3 PVY infected sample,
• lane 4 negative control,
• lanes 5-14 Spunta potato plantlets developed from the meristem
culture.

19. Materials and methods
• Stem cuttings from BRNV-infected black raspberry plants.
• Presence of the virus in the mother plants was confirmed by RT-PCR.
• The cuttings were surface-sterilized with 1% NaOCl for 10 min, 70% ethanol for 5
min and rinsed three times with double sterilized water.
• Explants, each containing a single axillary bud, were placed on corresponding
shoot initiation medium (8 ml) in glass tubes of 150 × 25 mm.

20. Thermotherapy
• Heat treatment was performed on in vitro axillary bud cultures in a growth
chamber where the temperature was alternated every 4 h between 29°C and
38°C to avoid continuous extreme condition that results plant death.
• Explants were cultivated for 5 weeks under this high temperature regime in the
initial experiment.
• In a second experiment, various periods (1 to 5 weeks) under the high
temperature regime were investigated.
• Explants were removed from the heat treatment every week and transferred to
regular temperature (22°C) for recovery. After 4 weeks of the recovery culture,
samples of each treatment were collected for virus detection.

21. Root induction and acclimatization in greenhouse.
• RT-PCR-negative plantlets were transferred onto root-induction medium.
• The plantlets were maintained under low light for one week and then transferred
to regular light conditions. Within 4–8 weeks, rooted plantlets of 3 cm or higher
were transferred to soil covered with clear plastic cups and maintained in a
growth chamber at 22°C with 16-h photoperiod.
• Four to six weeks later, acclimatized plants were transferred to a greenhouse and
maintained there for 2 months.

22. • Virus detection. Leaf tissues were collected from: regenerated shoots 5–
9 weeks after in vitro culture; plantlets after the recovery growth; and
plants after the 3-month dormancy period.
Comparison of in vitro shoots of R. occidentalis between control plants at 22°C and heat-treated
(29/38°C) after 5 weeks in culture

23. Results
• In the first experiment, all (100%) of the regenerated shoots under 5 week-heat
treatment were negative for BRNV, which draw the second experiment
investigating the effect of the thermotherapy duration (1–5 weeks). Heat-treated
explants from infected black raspberry produced shorter shoots and smaller
leaves when compared with those of control explants grown at 22°C.
• Regeneration rates (ratio of the number of elongated shoots to the total number
of cultured explants) of heat treated explants were slightly lower than control
explants.
• The result showed that heat treatment greatly affected the replication of BRNV.
Growing host plants at elevated temperature can inhibit virus replication and
movement. It was recently shown that viral RNA of RBDV, a virus that enters
meristem tissues, was disorganized in leaves and shoot tips in plants growing at
high temperatures (38°C) (Wang et al. 2008).

24. Schematic diagram of the complete thermotherapy process and culture of apical meristems:
1) Infected plant in the field; 2) Establishment in vitro cultures; 3) Isolation of apical shoots and culture; 4) Shoots
multiplication; 5) Application of gradual heat therapy; 6) Isolation and culture of apical meristems; 7) New shoots
development and DAS-ELISA and RT-PCR analysis; 8) Plants free of viruses.

25. Material and Methods
• Co85004, Co 91010, Co 86032 are the varieties chosen for the experiment.
• Young shoots of sugarcane were collected from different varieties of the
sugarcane plants from the field of Sugarcane Breeding Institute.
• After washing the shoots were sterilized thoroughly using ethyl alcohol.
• The chemotherapeutant used to eradicate viruses from sugarcane meristems
tissues was ribavirin.
• The presence of the virus in the plants regenerated from the meristems was
assayed and proved using RT PCR.

26. The MS (Murashige and Skoog) were prepared with different
concentrations of Ribavirin
Treatments Concentrations (mg/l)
T 0(Control) -
T 1 2.5
T 2 5.0
T 3 7.5
T 4 10.0
T 5 12.5
T 6 15.0

27. Results
Shoot initiation, elongation and multiplication:
• The shoot tips started growing from the meristem within 3 to 4 days and it
attained two to three leaf within 3 to 4 weeks and it is transferred to
multiplication medium and after 2 to 3 weeks, young plantlets are observed for
illustration.

28. • In the present study it is investigated that the chemotherapy at lower
concentrations (2.5,5.0,7.5mg/l) has no influence on the initiation and growth of
the meristem, shoot multiplication as well as the elongation of shoots. However,
there was no elimination of virus.
• Higher concentrations resulted in phytotoxicity and found to be negatively
affecting the shoot multiplication and growth.
• Combined method of antiviral chemotherapy and meristem tip culture was found
to be more effective in sugarcane mosaic virus elimination. Amending the MS
medium with 10mg/l of ribavirin increased the SCSMV and SCMV elimination
from the meristem tip and it did not affect the shoot emergence.

29. Biotech, 2019, 3(9): 153-162
Elimination of Bean yellow mosaic virus from infected cormels of
three cultivars of gladiolus using thermo-, electro and chemotherapy
Material and methods
• 3 gladiolus cultivars: Aldebaran, Tiger flame and Vink’s glory, CSIR-NBRI, lucknow.
• The cormel explants were first indexed for the presence of bymv by rt-pcr.
• Indexed cormels (ten cormels/replication of each cultivar) of 0.3–0.5 cm3 size
• Thermotherapy - 37 ± 2 °C for 30 days
• Chemotherapy - 30, 40, 50 and 60 mg/L of ribavirin
• Electrotherapy - immerse in 1X TAE buffer in an electrophoresis tank - electric
currents of 10, 20 and 30 ma for 20 min using a power supply
• Combination of chemotherapy and electrotherapy
• 23–25 °C with 16 h light.
• Regenerated plantlets – test for the presence or absence of BYMV - RT-PCR.

30. Fig. 1.
a) Leaf and corm samples of symptomatic showing mild to severe virus-like mosaic symptoms on leaflets and
color breaking on florets.
b) A representative image of RT-PCR to check BYMV infection in one plant of Aldebaran.
c) Morphology of healthy and infected plants

31. Different stages of production of BYMV-free gladiolus plants by combination of chemotherapy (30 mg/L) with
electrotherapy (30 mA for 20 min).
a) Infected gladiolus mother b) cormels getting electrotherapy in electrophoretic tank;
c) cormels explants in MSc media amended with ribavirin; d) germination after 30 days;
e) proliferation in MSp medium; f) harvesting of cormels after drying; and
g) acclimatization of gladiolus plantlets

33. • The combination of electro- and chemotherapies has given the best response as
compared to other treatments.
• Among the individual therapies, electrotherapy (30 mA/20 min) was found to be
the best for and production of BYMV-free gladiolus plants (44–46%) with
moderate regeneration efficiency (54–58%) followed by chemotherapy and
thermotherapy.
• However, the cormels obtained from a combination of electro- and
chemotherapy treatment (30 mA/20 min + 30 mg/L) has given highest virus free
(46–52%) and highest therapy efficiency indices (56%) as compared to other
treatments.

34. Elimination of Chrysanthemum stunt viroid and Chrysanthemum chlorotic mottle
viroid from infected chrysanthemum by cryopreservation
Objective:-
• To estimate the effect of various factors - Vitrification solution, duration of
exposure to LN, shoot-tip size, and low-temperature treatment, on the
elimination of the viroid by cryopreservation

35. Materials and Methods
• Cultivars 'Borami' and 'Secret Pink’ - Stunt disease and Yellow Cap - Chlorotic
mottle (Chilgok, Gumi, and Gyeongsan in Korea).
• Young apical shoots, Liquid Nitrogen.
• Real-time PCR, Nested PCR
• Murashige and Skoog (MS) basal medium

36. Disease symptoms on Chrysanthemum morifolium cultivars
Viroid detection by RT-PCR.
A. Chrysanthemum morifolium 'Borami' infected with CSVd, B. Secret Pink' infected with CSVd, C. Yellow
Cap' infected with CChMVd. N negative control (viroid-free plant), P positive control. 252-bp for CSVd and
316-bp for CChMVd.

37. Shoot regeneration from explants treated with PVS3 (a) and PVS2 (b) using cryotherapy.
Comparison of the surface structures of shoot tips treated with PVS2 or PVS3 by SEM:
A. Untreated shoot tip (control), B. Shoot tip treated with PVS2, C. Shoot tip treated
with PVS3, D, E, F, are the Magnification
PVS2 - 30% (w/v) glycerol,
15% (w/v) ethylene glycol,
15% (w/v) DMSO, and 0.4 M
sucrose (Sakai et al.1990).
PVS3 - 30% (w/v) glycerol
and 0.4 M sucrose Jeon et
al.(2015).

38. Results
CSVd and CChMVd concentrations in chrysanthemums differ according to the region of
origin.
• Visual observation of the severity of disease symptoms suggested that the viroid titers
differed from region to region.
• The real-time PCR results supported the occurrence of different viroid titers in the
different regions of origin: Higher titers were observed for both viroids in all three
cultivars from Chilgok, followed by Gyeongsan and Gumi.
• Based on these results. 'Borami' from the Gumi region that showed the lowest
concentration of CSVd was chosen for assessment of viroid elimination by
cryopreservation.
Optimization of the cryopreservation protocol for the elimination of viroids.
• The cryopreservation protocol developed by Jeon et aI. (2015) was applied to eliminate
CSVd from the cultivar 'Borami' . First, we examined the effects of different plant
vitrification solutions (PVS2 and PVS3), durations of exposure to LN. shoot-tip sizes, and
low-temperature treatments.

39. Effects of PVS2 and PVS3 on elimination of CSVd from infected shoot tips of
chrysanthemum 'Borami' following cryopreservation
Vitrification solution Viroid elimination %
Detected by RT-PCR Detected by nested PCR
Control 0b 0b
PVS2 20a 13.3a
PVS3 0b 0b
Effects of duration of exposure to LN on CSVd elimination from infected shoot tips
of chrysanthemum ‘Borami' following cryopreservation
Duration of exposure to LN (h) Viroid elimination %
Detected by RT-PCR Detected by nested PCR
1 20a 13.3a
3 20a 13.3a
5 13.3a 6.7b
7 13.3a 6.7b
10 13.3a 6.7b

40. Effects of shoot-tip size on CSVd elimination from infected chrysanthemum 'Borami'
following cryopreservation
Shoot-tip size {mm} Viroid elimination %
Detected by RT-PCR Detected by nested PCR
LP 0 6.7c 6.7b
LP 1-2 20a 13.3c
LP 3-4 13.3a 6.7b
Effects of low-temperature treatment on CSVd elimination from infected chrysanthemum
'Borami' following cryopreservation
Low temperature Viroid elimination %
Detected by RT-PCR Detected by nested PCR
Control 20b 13.3c
-20°C for I h 6.7c 0d
4°C 4 weeks 26.7a 20a
4°C 8 weeks 20b 6.7b

41. • In conclusion, we showed that low-temperature pretreatment is required for
effective viroid elimination by cryopreservation.
• In addition, the success of viroid elimination by cryopreservation depends on the
initial concentration of viroid in the shoot tips as well as the plant genotype.
• Furthermore, nested PCR is more sensitive and reliable than RT-PCR for viroid
detection. The advantage of cryopreservation over conventional methods is that
this procedure is less time-consuming:
• It generally takes 85 days compared to 120 days for shoot tip culture (Savitri et aI.
(2013).
• In addition, it can be used for the production of pathogen-free plants and for
long-term storage ofplant germplasm simultaneously.

42. 1. Viruses that are included in a particular group behave similarly with respect to
ease of eradication.
2. Viruses with isometric particles are more readily inactivated than those with rod
shaped particles.
3. For those viruses with isometric particles, the ilarviruses are most readily
inactivated and the comoviruses are the most difficult.
4. For those viruses with rod-shaped particles, the long flexuous rods (potyviruses
and carlaviruses) are more readily eradicated than viruses of intermediate length
(potexviruses and carlaviruses).
5. The most difficult viruses to eradicate belong to the tobamovirus group. These
viruses appear to be even more refractory than the viroid.

43. Factors influencing Virus elimination
Virus elimination depends on:
• Meristem explant size
• Bud location : terminal bud >have stronger growth potential than lateral.
• Season: early spring & early autumn > winter & summer; after dormancy of
storage organs
• Heat treatment : in water or in air
• Culture media : high concentrations of growth hormones inhibit virus growth

44. Conclusion
• Preventive measures are the most general approach to control viral diseases.
• Indirect methods - use of modified cultural practices, use of virus-free planting
materials, application of insecticides and oils for control, transgenic approach,
cross protection etc.
• A single approach to control is unwise; integrated disease management is the
best strategy to combat viral diseases in Agricultural crops.

45. References
• Al-Taleb, Miassar M., Dhia S. Hassawi, and Saeid M. Abu-Romman. "Production of virus free potato plants using meristem
culture from cultivars grown under Jordanian environment." American-Eurasian Journal of Agricultural & Environmental
Sciences 11, no. 4 (2011): 467-472.
• Cheong, E. J., A. R. Jeon, J. W. Kang, R. Mock, G. Kinard, and R. Li. "In vitro elimination of Black raspberry necrosis virus
from black raspberry (Rubus occidentalis)-Short Communication." Horticultural Science 41, no. 2 (2014): 95-99.
• Lizárraga, Analí, Javier Ascasíbar, and María Luz González. "Fast and Effective Thermotherapy Treatment for In Vitro Virus
Eradication in Apple and Pear Trees." American Journal of Plant Sciences 8, no. 10 (2017): 2474-2482.
• Neelamathi, D., Manuel, J. and George, P., 2014. Influence of apical meristem and chemotherapy on production of virus
free sugarcane plants. Research Journal of Recent Sciences. ISSN, 2277, p.2502.
• Kaur, C., Raj, R., Kumar, S., Purshottam, D.K., Agrawal, L., Chauhan, P.S. and Raj, S.K., 2019. Elimination of Bean yellow
mosaic virus from infected cormels of three cultivars of gladiolus using thermo-, electro-and chemotherapy. 3
Biotech, 9(4), pp.1-10.
• Jeon, S.M., Naing, A.H., Kim, H.H., Chung, M.Y., Lim, K.B. and Kim, C.K., 2016. Elimination of chrysanthemum stunt viroid
and chrysanthemum chlorotic mottle viroid from infected chrysanthemum by cryopreservation. Protoplasma, 253(4),
pp.1135-1144.