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In vitro advances for virus elimination in Lilium (Lilium Longiflorum)
Narendra Singh BhandariICAR SRF Ph. D Scholar (IARI)
Division of Ornamental Crops, IIHR
• 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
ImportanceBorderplants
Potplants
Landscape Cut flower
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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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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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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Sharma et al., 2005
Specific primer pairs for detection of CMV, LMoV, LSV, and LVX by RT-PCR.
Masuda et al., 2011
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Why in vitro 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 banksBreeding 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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In vitro culture techniques for virus elimination
Meristem tip culture Thermotherapy Cryotherapy Chemotherapy Electrotherapy Combination of these methods
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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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Elimination of viruses from ornamental plants by shoot meristem culture or its combination with other biotechnological procedures
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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Effect of Sub-culturing
Kim et al., 2015
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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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Plants are exposed to temperature between 35 to 40°CGrowing 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
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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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.
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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AdvantagesIt 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 possibleFewer regenerated shoots need to be pathogen-
indexedTime period required to implement the whole
cryotherapy procedure was shortest compared to thermotherapy and thermotherapy followed by shoot tip culture
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Wang and Valkonen, 2008
Chemotherapy and Meristem tip culture1. 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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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
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.
BioreactorIn
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of H
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Conclusion
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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Thank you
