In vitro advances for virus elimination in Lilium (Lilium Longiflorum)

1. In vitro advances for virus
elimination in Lilium (Lilium
Longiflorum)
Narendra Singh Bhandari
ICAR SRF Ph. D Scholar (IARI)
Division of Ornamental Crops, IIHR

2. • Liliaceae.
• Monocot bulbous flower
• Genus contains 110 species
• 7 groups but famous ones
are: Asiatic, L A Hybrids,
Oriental lilies and O T
hybrids.
• Due to its size, beauty,
aroma and longevity, holds
4th position among top 10
cut flowers.
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Introduction

3. Importance
Border
plants
Pot
plants
Landscape Cut flower

4. Lilium viruses
20 species of virus have been reported to infect
lily plants worldwide.
Lily mottle virus (LMoV), Lily symptomless
virus (LSV), Cucumber mosaic virus (CMV) and
Lily X viruses (LVX) (Derks, 1995).
Tobacco ring spot (Travis and Brierley, 1957)
Lily mild mosaic, Narcissus mosaic (Lee et al.,
1996)
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5. Symptoms…
 Lily mottle virus (LMoV) and
Cucumber mosaic virus (CMV)
were identified first time in the
field of IHBT, Palampur,
Himachal Pradesh, India
 These plants were found to
exhibit various types of
symptoms, viz. deformed leaves,
deformed flowers, stunting of
the plants and deformed bulbs
Sharma et al., 2005
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6. Detection and characterization
 Bioassay
 Double antibody sandwich-Enzyme linked
immunosorbent assay (DAS-ELISA)
 Electron microscopy
 Immunosorbent electron microscopy
 Reverse transcription polymerase chain
reaction (RT-PCR) and sequencing.
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7. IndianInstituteofHorticulturalResearch
Sharma et al., 2005

8. Specific primerpairs fordetection of CMV, LMoV, LSV, and
LVX by RT-PCR.
Masuda et al., 2011
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9.  Pathogen-free stocks of planting materials: Pivotal for
productivity and value of ornamental plants
 Pathogen-free planting materials is required for
 Commercial cultivation
 Germplasm collections in gene banks
Breeding new cultivars for future needs
 Tissue culture methods for producing virus-free (VF) lilies
have been known and applied for over 20 years.
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10. In vitro culture techniques for virus elimination
 Meristem tip culture
 Thermotherapy
 Cryotherapy
 Chemotherapy
 Electrotherapy
 Combination of these methods
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11.  The youngest meristematic cells are usually free of viruses/ phytoplasma/
bacteria. Excision and regeneration might result in pathogen-free plants
 Regeneration ability: Proportional to size of the shoot tip, but pathogen
eradication is more efficient using small shoot tips (0.2–0.4 mm)
Meristem culture and associated problems
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12. Elimination of viruses from ornamental plants by shoot meristem
culture or its combination with other biotechnological procedures

13. Hosokawa et al., (2004) presented a new method for
production of virus-free plants, for which a root tip of
chrysanthemum, petunia and carnation used apart from the
apical shoot meristem.
Root apex is considered to be a suitable organ for meristem
culture, since root meristem has a high potential of cell
division.
The main reason for use of the meristem culture is the fact
that most viruses do not attack the meristem of shoots, since
the multiplication of meristem cells is faster than replication of
viruses
 Foster et al., 2002 studies have shown that the mechanism of
gene silencing is in fact the main reason.
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14. Effect of Sub-culturing
Kim et al., 2015
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15. Virus eradication using meristem culture is challenged by
nnumerous obstacles, such as:
 Meristem size
 Meristem position on a plant
 Protocol applied during detection (different
sensitiveness of the applied methods)
Chaum et al., 2006
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16. Plants are exposed to temperature between 35 to 40°C
Growing host plants at higher temperatures significantly
reduces replication of many plant viruses by disrupting viral
ssRNA and dsRNA synthesis.
Effective against iso-metric and thread –like viruses.
Hot air treatment appears to be more successful in
eliminating virus than the hot water method and causes less
damage to the plant tissue.
Thermo-therapy and Meristem Culture
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Ram et al., 2005

17. The principal alterations in viral particles as a result of
thermal treatment above 35°C are related to the rupture of
hydrogen and disulfide bonds of capsid protein, followed
by nucleic acid phosphodiester covalent bonds
Wang et al., (2008) combined thermotherapy (38°C) and
cryotherapy treatments and reported that close
relationship between temperature and RNA silencing
which seems to act as a means to increase the degradation
of virus RNA.
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18. Plant survival and LSV elimination in lilium oriental hybrid Casa
Blanca when treated with thermotherapy during tissue culture
Nesi et al., 2015 suggested that thermotherapy given to in vitro
growing bulblets effectively eliminated the virus and induced a fast
and efficient micropropagation technique for virus-free mother plant
stock.

19. Cryotherapy
 It eliminates plant pathogens like viruses/ phytoplasmas/ bacteria by briefly
treating shoot tips in liquid nitrogen using cryopreservation protocols
 In cryotherapy, infected cells are eliminated by the lethal effects of the ultra-
low temperature and/or subsequent warming
 Healthy plants are regenerated from the surviving pathogen-free
meristematic tissue
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20. Advantages
It facilitates treatment of large numbers of samples
because pathogen eradication is independent of the
size of shoot tips
Easier excision of larger shoot tips is possible
Fewer regenerated shoots need to be pathogen-
indexed
Time period required to implement the whole
cryotherapy procedure was shortest compared to
thermotherapy and thermotherapy followed by shoot
tip culture
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21. IndianInstituteofHorticulturalResearch
Wang andValkonen, 2008

22. Chemotherapy and Meristem tip culture
1. Chemotherapy can be used with meristem-tip culture to
increase the incidence of VF plants (Hollings, 1965;
Walkey 1985).
2. Chemotherapy uses compounds such as ribavirin
(Virazole) or vidarabine (Vira A), Actinomycin-D,
Cyclohexamide, that affect the virus multiplication added
into the culture medium for curing the shoot tips virus.
3. The principle of chemotherapy is similar to that of heat
treatment in that the compound allows the virus to degrade
and not allow it to multiply.
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23. Plant survival and LSV elimination in lilium oriental hybrid
Casa Blanca when treated with antiviral agents during tissue
culture
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Nesi et al., 2015

24. 1. Bioreactor combined with these techniques may offer a
promising tool for virus free mass propagation system
in future.
2. Bioreactor can easily be applied on liquid suspension
culture systems for large scale propagation of lilium
bulbs.
3. Bioreactors enable further process automation, quality
control system.
Bioreactor
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25.  Very less work has been done on ornamental crops, so
focus should be given
 Specific protocols, need to developed for different
species/genotypes
 Combination of techniques for virus elimination is more
effective
 Bioreactor combined with these techniques may offer a
promising tool for virus free mass propagation system in
future.
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