This ppt will give a brief idea about how to control viral diseases in ornamental crops. Viral diseases are difficult to control as there are no specific chemicals developed to control viral diseases to date. So, many scientists developed several strategies to decrease the disease spread; the strategies are mentioned in the ppt.
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Strategies for controlling viral diseases in ornamental crops 1
1.
2.
3. UNIVERSITY OF HORTICULTURAL SCIENCES, BAGALKOT
3rd SEMINAR
Strategies for controlling viral diseases in ornamental
crops
Takhellambam Henny Chanu
UHS18PGD253
Department of FLA
5. INTRODUCTION
➢ Nucleic acid surrounded by a protein coat
➢ Virus: set of one or more nucleic acid template molecule,
normally encased in protective protein coat (Mathews, 1981)
➢ Obligate intracellular parasites
➢ Small infectious agent that replicates only inside the
living cells of other organisms
➢ Sub microscopic
➢ Transmit – vectors
➢ 1st - TMV (Tobacco mosaic virus) – discover
4/22/2021 Department of FLA 5
6. Types
Type of genome Virus
ss RNA Tobacco mosaic virus, Carnation latent virus,
Carnation mottle virus, Carnation ring spot virus,
Cucumber mosaic virus
ds RNA Rice dwarf virus, Reovirus
ss DNA Bean yellow mosaic virus, Banana bunchy top
virus
ds DNA Cauliflower mosaic virus, Petunia vein clearing
virus
4/22/2021 Department of FLA 6
Katoch et al., 2002
13. CULTURAL METHODS
➢ Use of Virus Free Planting Materials
➢ (a) occurrence of infection as soon as possible in young developing
seedling
➢ (b) infected seedling act as source of initial virus inoculum for subsequent
further spread.
➢ Avoidance of Collateral Hosts and Volunteers
➢ Wherever healthy planting materials were used, the virus infection comes
from the weed hosts or other infected crops
➢ Roguing - main crop is the primary/sole source of virus infection - not effective
the virus has wider host range, the spread is relatively fast.
➢ Plant density - higher planting density lessens the number of infections per unit
area
4/22/2021 Department of FLA 13
Tripathi and Verma, 2017
14. ➢ Trap crop and barrier crops – fast growing and taller than the
main crop and it should not be susceptible to the viruses and
their vectors of the main crop – maize, marigold (Rose -
Pelargonium geraniums as trap crop )
➢ Use of plastic mulch – avoid landing of insect on crop plants,
minimize; UV, IR light
➢ Use of physical barriers – fine mesh screen
➢ Sowing/Planting Dates - avoid - highest number of
viruliferous insect vectors. Planting time – virus migration
➢ Remove plant debris from plastic tunnel houses and
glasshouses
➢ Treatment of implements with 3% trisodium orthophosphate
4/22/2021 Department of FLA 14
Tripathi and Verma, 2017
15. Methods for obtaining virus free stocks
Thermotherapy
Electrotherapy
Meristem
culture
Chemotherapy
Cryotherapy
Virus free
stocks
Naturally
occurring virus
free material
Department of FLA 15
4/22/2021
16. (A) MERISTEM TIP CULTURE:
➢ An efficient tool for regeneration, elimination of viruses from infected plants
and production of virus-free seed material
➢ Absence of virus in meristem - high metabolic activities, usually accompanied
by elevated endogenous auxin content in shoot apices, lack of vascular system
and virus inactivating system may inhibit virus multiplication
➢ The production of virus-free plants - determination of optimal size range of
meristem/shoot tips (0.3-1.5 mm) and a high rate of plant regeneration
➢ Morel and Martin (1952) – eliminate viruses from dahlia using meristem
culture
➢ Number of virus-free plantlets obtainable is inversely proportional to the size
of the cultured tips (Faccioli and Marani,1998)
Bhat et al., 2020
Department of FLA 16
4/22/2021
18. Scientia Horticulturae, 2009, 119: 108–112
Elimination of mixed infection of Cucumber mosaic and
Tomato aspermy virus from Chrysanthemum morifolium
Ramat. cv. Pooja by shoot meristem culture
Kumar, S., Khan, M.S., Raj, S.K. and Sharma, A.K.
➢ Eliminate CMV and TAV at a time by culturing shoot meristems of the
naturally infected chrysanthemum cv. Pooja
➢ To produce virus-free chrysanthemum plants
18
4/22/2021 Department of FLA
19. MATERIALS AND METHODS
➢ Naturally infected C. morifolium Ramat. cv. Pooja meristem size 0.3 mm -
culture on MS medium
➢ 25 ± 2°C – 16/8 hr photoperiod - forthnightly
➢ DAC-ELISA - performed by using antiserum of CMV (PVAS 242a) and TAV
(PVAS 24)
➢ RT-PCR using specific primers of CMV(AM180922 and AM180923) &
(J566128), (AJ566129) primer for TAV
➢ RT-PCR Process:-denaturation at 94 oC for 5 min there were 25 cycles of
amplification with denaturation at 94 for 1 min, annealing at 52 oC for 1min
(CMV) and 56 oC for 1 min (TAV) and extension at 72 oC for 1 min following
the final extension of 10 min at 72 °C.
Department of FLA 19
4/22/2021 Kumar et al., 2009
20. Figure 1: Gel electrophoresis of RT-PCR products showing 657 bp
amplicons in naturally infected chrysanthemum samples
obtained by CMV-specific primers (a) and TAV specific primers
(b). PCR products from naturally infected and healthy
chrysanthemum plants.
Department of FLA 20
4/22/2021 Kumar et al., 2009
21. Figure 2: Optimization of growth regulator concentrations(NAA and BAP)
supplemented to MS media with 0.3 mm shoot meristem explants of
healthy chrysanthemum Pooja cultivar
Department of FLA 21
4/22/2021 Kumar et al., 2009
22. Figure 3 (a and b): Multiple shoots
regenerated from the mild callus at
the end of excised shoot meristem in
20–25 days
Figure 3 (c): Well developed
shootlets excised after 55–60 days
and positioned onto rooting medium
which developed roots after 6 - 7
days
Figure 3 (d): Acclimatized
chrysanthemum cv. Pooja plants in
glasshouse
Department of FLA 22
4/22/2021 Kumar et al., 2009
23. Table 1: Indexing for CMV- and TAV-free plants from Chrysanthemum cv. Pooja
shoot meristems
Table 2: Performance of in vitro-raised virus-free chrysanthemum cv. Pooja plants as
compared to diseased plants with respect to growth parameters
Department of FLA 23
4/22/2021
Virus-free plants Percentage of virus-free plants (virus-free/total) obtained as tested by
ELISA RT-PCR Total
CMV-free 81.2% (26/32) 84.0% (21/25) 65.6% (21/32)
TAV-free 87.5% (28/32) 88.0% (22/25) 68.7% (22/32)
Both CMV and TAV-free 78.1% (25/32) 84.0% (21/25) 65.6% (21/32)
Sl.
No.
Explant type Growth parameters
Plant height
(cm)
No. of
branches per
plant
Length of
branch (cm)
No. of
flowers per
plant
1. CMV and TAV-free 34.40 ± 1.58* 9.05 ± 0.39* 26.60 ± 0.75* 38.05 ± 0.94*
2. Control (doubly infected
plant)
17.33 ± 0.58* 4.33 ± 0.33* 12.00 ± 1.35* 24.33 ± 0.61*
3. Increase in performance over
control, % (infected)
50.20 52.15 34.88 36.05
Kumar et al., 2009
24. INFERENCE
• Shoot meristem culture method – generate TAV-free and CMV-free
chrysanthemums
• Reasons for virus elimination: growth regulators action particularly
cytokinin- loss of enzymes necessary for viral replication, RNA
degradation due to cell injury
Department of FLA 24
4/22/2021
25. (B) THERMOTHERAPY:
➢ 35–42 °C for 4–6 weeks is applied to the target plant, depending on virus type
and plant species and virus-host combination
➢ Higher the temperature and the longer the exposure duration are, the higher the
virus-eradication frequency is.
➢ Kassanis (1949) eliminate PLRV - potato tubers – 37ºC and 25 days
➢ Mechanisms –
➢ (1) Prevent virus movement toward the meristematic cells of the treated shoots
- result in production of larger virus free areas of the infected shoot tips, thus
helping virus eradication
➢ (2) Inhibit viral replication or caused virus RNA degradation
➢ (3) Biogenesis of vsiRNAs and inhibited viral RNA accumulation, by
upregulating the expression of key genes in the RNA silencing pathway
➢ Virus-free - carnation, narcissus, chrysanthemum, geranium
4/22/2021 Department of FLA 25
Wang et al., 2018
26. Table 3: Some examples of in vitro thermotherapy followed by shoot tip culture
for virus eradication
4/22/2021 Department of FLA 26
Wang et al., 2018
Plant species and cultivars
or genotypes
Viruses Temperatur
e and
duration
Size of
shoot
tips (in
mm)
Virus-free
frequency
Chrysanthemum ‘Regol Time’ Chrysanthemum B
virus (CVB)
38 °C, 30
days
0.3–1.0 12
Lilium × elegans geno- types
409 and 599
Lilium symptomless
virus (LSV)
35 °C, 42
days
0.3 100
Lilium Asiatic hybrid
‘Vis- conti’, and LA hybrids
‘Fangio’ and ‘Lacorno’
LSV, lily mottle virus
(LMoV) and
cucumber mosaic
virus (CMV)
35 °C, 5
weeks
NS 100 (LSV)
100 (CMV)
100 (LMoV
Begonia Prunus necrotic
ringspot virus
(PNRSV)
38 °C/22 °C,
(day/night),
35 days
2-3 cm 37.5
27. (C) CHEMOTHERAPY:
➢ Incorporation of antiviral compounds into explant and meristem culture media
to obtain higher percentage of virus free progeny plants
➢ Synthetic nucleoside : tiazofurin, selenazofurin ,benzamide riboside
➢ Non-nucleosides : micophenolic acid were tested against CMV
➢ Antibiotic – streptothricin, terramycin, achromycin, agrimycin, aureomycin
hydrochloride; dyes - malachite green and methylene blue; nicotinic acid,
amino acids, IAA, 2, 4-D, hydroquinone
➢ Ribavirin/ virazole - 9 families
➢ Limitations - narrow antiviral spectrum, ineffectiveness against the latent
virus, development of drug-resistant mutants and toxic side effects
4/22/2021 Department of FLA 27
Erik, 1996
28. (D) ELECTROTHERAPY:
➢ Degrade viral
nucleoprotein and
eliminate its virulence
➢ First - eliminate Potato
virus X (PVX) from
potato -15 mA for 5
min - 60–100 %
elimination of PVX
(Lozoya-Saldana et
al., 1996)
4/22/2021 Department of FLA 28
Figure 4: Electrotherapy treatments steps and
equipment used to produce virus-free plants
Natrium
chloride solution
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
Kaur, C., Raj, R., Kumar, S., Purshottam, D.K., Agrawal, L., Chauhan, P.S. and Raj,
S.K.
Objective:- to establish an effective and facile BYMV management strategy for
quality improvement of gladiolus.
Department of FLA 29
4/22/2021
30. MATERIAL AND METHODS
➢ CSIR-NBRI, Lucknow
➢ Aldebaran, Tiger Flame and Vink’s Glory
➢ 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.
Department of FLA 30
4/22/2021 Kaur et al., 2019
31. Figure 5: a Leaf and corm samples of symptomatic showing mild to severe virus-
like mosaic symptoms on leaflets and color breaking on florets b image
of RT-PCR with CP-specific primers to check BYMV c Morphology of
healthy and infected plants
Department of FLA 31
4/22/2021 Kaur et al., 2019
32. Figure 6: 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
Department of FLA 32
4/22/2021 Kaur et al., 2019
33. Figure 7: Graphical representation of RE, rate of BYMV elimination and TEI of three
gladiolus cultivars treated with various treatments. a RE (%); b rate of BYMV
elimination (%); and c % TEI
Department of FLA 33
4/22/2021 Kaur et al., 2019
34. Table 4: Results obtained in various treatments using thermotherapy, chemotherapy,
electrotherapy and the combination of electro- with chemotherapy in three
gladiolus cultivars and their respective germination efficiencies in MSg
medium and virus-free plants
Department of FLA 34
4/22/2021 Kaur et al., 2019
35. Figure 8: Comparison of cormels and root systems of “Aldebaran” cultivar of
gladiolus developed through various treatments with control
Department of FLA 35
4/22/2021 Kaur et al., 2019
36. Table 5: Performance of BYMV-free gladiolus corms developed through
various treatments as compared to the control
Department of FLA 36
4/22/2021
Cultivars Av. no. of cormels produced
per plant
Average size of cormels
produced (in cm3)
Control BYMV-free Control BYMV-free
Aldebaran 2.0 ± 0.50 5.0 ± 0.50 0.35 ± 0.04 0.56 ± 0.07
Tiger Flame 1.0 ± 0.31 4.0 ± 0.31 0.30 ± 0.01 0.55 ± 0.02
Vink’s Glory 2.0 ± 0.57 6.0 ± 0.57 0.33 ± 0.07 0.51 ± 0.05
Kaur et al., 2019
37. INFERENCE
➢ Therapies like thermo-, chemo- and electrotherapies alone and in the
combination of chemo- and electrotherapies.
➢ Electrotherapy coupled with chemotherapy in three commercially important
gladiolus cultivars (Aldebaran, Tiger Flame and Vink’s Glory cultivars)
➢ This therapy - effective than other treatments and can be recommended for
better quality production and improvement of gladiolus, valuable crops.
Department of FLA 37
4/22/2021
38. (E) CRYOTHERAPY:
➢ Liquid nitrogen (-196ºC) or liquid nitrogen vapour (-165 to -190ºC)
➢ Infected cells are eliminated by the lethal effects of the ultra-low temperature
➢ Plum Pox Virus (PPV), CMV, PLRV
➢ Chrysanthemum morifolium - chrysanthemum stunt viroid (CSVd). Jeon et al.
(2016)
➢ Brison et al. (1997) - eliminate Plum pox virus (PPV) - Prunus rootstock
4/22/2021 Department of FLA 38
Wang et al., 2008
39. Springer-Verlag Wien, 2015, 1-10
Elimination of chrysanthemum stunt viroid and chrysanthemum
chlorotic mottle viroid from infected chrysanthemum by
cryopreservation
Jeon, S.M., Naing, A.H., Kim, H.H., Chung, M.Y., Ki Byung Lim, K.B. and Kim,
C.K.
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 viroids by cryopreservation
Department of FLA 39
4/22/2021
40. MATERIALS AND METHODS
Figure 9: Disease symptoms on Chrysanthemum morifolium cultivars
Department of FLA 40
4/22/2021
Figure 10: Viroid detection by RT-PCR. a Chrysanthemum morifolium.
252-bp for CSVd and 316-bp for CChMVd
Jeon et al., 2015
41. 4/22/2021 Department of FLA 41
Figure 11: Shoot regeneration from explants treated
with PVS3 (a) and PVS2 (b)
Figure 12: Comparison of the surface structures of
shoot tips treated with PVS2 or PVS3
▪ 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).
Jeon et al., 2015
42. Vitrification solution Viroid elimination %
Detected by RT-PCR Detected by nested PCR
Control 0b 0b
PVS2 20a 13.3a
PVS3 0b 0b
4/22/2021 Department of FLA 42
Table 6: Effects of PVS2 and PVS3 on elimination of CSVd
Duration of exposure to
LN (hr)
Viroid elimination %
Detected by RT-PCR Detected by nested PCR
1 20a 13.3a
3 20a 13.3a
5 13.3b 6.7b
7 13.3b 6.7b
10 13.3b 6.7b
Table 7: Effects of duration of exposure to LN on CSVd elimination
252 bp
252 bp
Jeon et al., 2015
43. Table 8: 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 1-3 20a 13.3c
LP 3-4 13.3b 6.7b
4/22/2021 Department of FLA 43
Low temperature Viroid elimination %
Detected by RT-PCR Detected by nested PCR
Control 20b 13.3b
-20ºC for 1hr 6.7c 0d
4ºC for 4 weeks 26.7a 20a
4ºC for 8 weeks 20b 6.7c
Table 9: Effects of low-temperature treatment on CSVd elimination from
infected chrysanthemum ‘Borami’ following cryopreservation
Jeon et al., 2015
44. INFERENCE
➢ Low-temperature pretreatment – eliminate viroid
➢ Nested PCR > RT-PCR
➢ Cryopreservation > conventional methods
➢ 85 days/ 120 days
➢ Production of pathogen-free plants and for long-term storage
➢ Cryopreserved stocks could be a means to decrease inadvertent dispersal of
plant pathogens.
4/22/2021 Department of FLA 44
46. CONTROL OF AIR BORNE VECTORS
4/22/2021 Department of FLA 46
natural
pesticides
• nicotine
• pyrethrum
• rotenone-
based
compound
• Dioscorea
floribunda
(TMV)
• Azadirachta
indica
• Boerhaavia
diffusa
Synthetic
pesticides
• chlorinated
hydrocarbons
• organophosphor
us compounds
• carbamates
• (imidachloprid,
carbofuran,
monocrotophos,
dicofol,
tetradifon)
Oil
sprays
• mineral oil
• silicon oil
• plant oil
47. Table 10: Evaluation of different plants extracts and a chemical pesticide
against aphids on sunflower Hysun-33
Treatments 24 HBS 24 HAS 48 HAS 72 HAS 168 HAS
Emamectin benzoate 11.48 b 1.75 h 1.38 h 1.02 g 0.75 h
A. indica oil 11.62 a 3.02 f 2.80 f 2.56 e 2.31 f
A. indica seed extract 11.45 b 3.63 e 3.27 e 3.05 d 2.63 e
A. sativum extract 11.58 a 4.97 c 4.78 c 4.40 c 4.55 c
Parthenium extract 11.73 a 5.82 b 5.30 b 5.01 b 5.22 b
D. alba seed extract 11.88 a 2.62 g 2.00 g 1.55 f 1.27 g
C. longa extract 11.15 b 4.32 d 3.87 d 3.85 d 3.53 d
Control 11.46 b 11.61 a 12.00 a 12.28 a 12.71 a
4/22/2021 Department of FLA 47
Said et al., 2015
48. EXCLUSION OF SOIL BORNE VECTORS
NEMATODE
➢ Fumigants & nematicides (methyl
bromide, DD aldicarb)
➢ Clean planting stocks
➢ Crop rotation with break crops
➢ Nematode resistance variety
➢ Exclusion of nematode by
quarantine
FUNGUS
➢ Fungicide application (Bavistin &
copper fungicides)
➢ Soil Sterilants and Disinfectants (to
reduce active and resting spores)
➢ E.g-Chloropicrin, (PCNB), methyl
bromide, and (D-D)
4/22/2021 Department of FLA 48
49. BIOLOGICAL CONTROL
➢ Entomo-pathogens, parasitoids and predators –
reduce
4/22/2021 Department of FLA 49
Dogan et al., 2019
Vectors Control agents
Aphids (Aphididae) Coccinellids, Aphidolates aphidimyza, Adalia
bipunctata and Aphidius colemani; Aphidiidae
and Aphelinidae; Verticillium lecanii , Beauveria
bassiana and Lecanicillium lecanii; Macrolophus
caliginosus
Leaf miner (Liriomyza trifolii) parasitoids (Diglyphus isaea, Chrysonotomyia
formosa, Hemiptarsenus varicornis)
Two-spotted spider mite
(Tetranychus urticae)
Phytoseilus persimilis
Flower thrips (Thrips spp.) Orius spp., Predator mites
50. Table 11: Usage dosages of some biological control agents and storage conditions
4/22/2021 Department of FLA 50
Dogan et al., 2019
51. CROSS-PROTECTION
➢ “pre-immunization”
➢ A plant is deliberately infected with a mild strain of a virus - to protect the
plant against damage caused by a more severe strain of the same virus
➢ Closely related strains of the same virus
➢ The protective strain preventing the challenge strain from uncoating upon
entering the plant cell. The coat protein from the protective mild strain may
recoat the challenge strain and prevent its replication
➢ Isle of Wight, 1965 –Tomato mosaic virus
➢ Property: induce milder symptoms, alter marketable properties of crops,
genetically stable, provide protection against widest possible range of strains
and easy & inexpensive to produce
Pechinger et al., 2019
Department of FLA 51
4/22/2021
52. TRANSGENIC RESISTANCE
➢ Deployment of virus resistant transgenic plants has become an important
strategy to implement effective virus management
➢ Expression of nucleotide sequences of viral and non-viral origin - virus
resistance
Department of FLA 52
4/22/2021
Transgenic
approach
Pathogen derived
resistance
Pathogen
targeted resistance
Host resistance (R)
Baulcombe, 1996
53. Pathogen derived resistance
➢ Integration of pathogen components that interfere with the normal life cycle of
the virus
➢ Hamilton (1980)
➢ PDR against TMV by expression of TMV coat protein (Powel-abel et al.,1986)
4/22/2021 53
Pathogen-
derived
resistance
Coat
protein
mediated
resistance
MP
mediated
resistance
RNAi
mediated
virus
resistance
Replicase
mediated
resistance
Baulcombe, 1996
54. Coat protein mediated resistance:-
➢ Resistance of transgenic plants that express genes encoding viral CPs to infection
by the viruses from which the genes are derived
➢ Introduce CP genes, which will be expressed in genetically engineered plants in
order to protect from virus infection
➢ Accumulation of the CP confers resistance to infection and/or disease development
by the virus from which the CP gene was derived and by related viruses
Movement protein mediated resistance:-
➢ Resistance - transgene specified a dysfunctional MP
➢ Expression of defective movement protein confer resistance against viruses
➢ Reduces virus movement throughout the infected plants
➢ Transgenic plants - contain dMP from TMV – tobamoviruses (Lapidot et al. 1993)
➢ MP is non-structural viral protein known to function in viral movement.
4/22/2021 Department of FLA 54
Baulcombe, 1996
55. Transgenic Research, 2005, 14: 41–46
Transgenic resistance to Cymbidium mosaic virus in
Dendrobium expressing the viral capsid protein gene
Chang, C., Chen, Y.C., Hsu, Y.H., Wu, J.T., Hu, C.C., Chang, W.C. and Lin, N.S.
Objective: To develop transgenic resistance against Cymbidium mosaic virus in
Dendrobium through coat-protein mediated resistance
KRC
College
of
Horticulture,
Arabhavi
55
4/22/2021
56. MATERIALS AND METHODS
➢ RNA extraction from CymMV isolate, cDNA synthesis of CP gene and
sequencing
➢ (RT-PCR) was performed for the synthesis of cDNA of CymMV CP gene
➢ Plasmid construction and transformation – pGEM-T
➢ pGEMCymMVCP contained the full-length CP gene of CymMV.
➢ pKYLX vector
➢ Plasmid pCYCP11 (CaMV 35S promoter, CymMV CP gene, and the hygR
gene)
➢ 15 days - Dendrobium protocorms were bombarded with 1.0 µm diameter gold
particles coated with pCYCP11 (5 µg pCYCP11/1.25 mg gold particle).
Department of FLA 56
4/22/2021 Chang et al., 2005
57. Figure 13: Regeneration of transgenic Dendrobium after particle bombardment (A)
Flowering Dendrobium Hickam Deb. B) Protocorm-like bodies proliferating
on medium supplemented with 20 mg/L hygromycin after 6 month selection
(C) Transgenic lines planted on selection medium, showing green leaves and
healthy roots (D) Transgenic plants transferred to growth chamber
Department of FLA 57
4/22/2021
Chang et al., 2005
58. Figure 15: (A) Northern blot
analysis of CP gene expression in
transgenic Dendrobium.
(B) Western blot analysis using
anti-CymMV serum for the
detection of CP protein from wild
type (lanes CK1 and CK2) and
transgenic lines (lanes 15-9-1, 15-
4-3, 15-2-2, 9-1-3, 4-1-1, 3-4-2
and 3-4-1).
(C and D) Resistance to CymMV
infection as analyzed using
ELISA
Department of FLA 58
4/22/2021
1 µl/ml
10 µl/ml
Chang et al., 2005
59. INFERENCE
➢ Transform the CymMV CP gene into Dendrobium protocorms through
biolistic transformation.
➢ CPMP provides a good protection from CymMV infection in Dendrobium
➢ CymMV-CS isolate - the production of transgenic orchids expressing this CP
gene has a great potential to be resistant to infection by various CymMV
isolates
Department of FLA 59
4/22/2021
60. RNAi mediated virus resistance:-
➢ Involves RNA silencing in which sequence-specific RNA degradation occurs
➢ Waterhouse et al. (1998) against PVY in transgenic tobacco plants
➢ Resistance based on RNA - durable - protein mediated resistance
Replicase-Mediated Resistance (Rep-MR):-
➢ Replicase (Rep) protein
➢ Golemboski et al. (1990) - tobacco against TMV
➢ Resistance may be protein based (Rep-MR to PVY) or post transcriptional
gene silencing (Rep-MR to PVX, Cowpea mosaic virus)
➢ Gives immune type reaction and there is a substantial inhibition of virus
replication
Kawazu et al., 2009
4/22/2021 Department of FLA 60
61. Plant Cell Rep, 2008, 27:1027–1038.
Agrobacterium tumefaciens-mediated transformation of poinsettia, Euphorbia
pulcherrima, with virus-derived hairpin RNA constructs confers resistance to
Poinsettia mosaic virus
Clarke, J.L., Spetz, C., Haugslien, S., Xing, S., Dees, M.W., Moe, R. and Blystad, D.R.
Objective:- to develop transgenic PnMV resistant poinsettia plants carrying PnMV
derived hpRNA
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62. MATERIAL AND METHODS
➢ Poinsettia cv. Millenium
➢ Heat therapy – eliminate PnMV
➢ A. tumefaciens strain LBA4404
➢ 3 HpRNA constructs – pCP (viral coat protein), pR2 and pR3 (RNA-
dependant RNA-polymerase (RdRp))
➢ 1-1.5 mm explant
➢ Screening of transgenic poinsettia plants
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4/22/2021 Charke et al., 2008
63. Figure 14: Somatic embryogenesis in poinsettia cv. Millenium
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Charke et al., 2008
64. Table 12: Summary of five Agrobacterium-mediated transformation
experiments on poinsettia cv. Millenium with pCP, pR2 and
pR3 constructs
Experiment a No. of
explants
No. of
regenerate
d plants
No. of
transformantsb
Transformation
efficiency (%)c
1 (CP) 185 54 3 1.6
2 (R3) 132 69 3 2.3
3 (R3) 172 66 6 3.5
4 (R2) 254 21 3 1.2
5 (R2) 125 18 3 2.4
a CP, R3, and R2 represent transformation experiments with constructs pCP, pR2 and
pR3, respectively
b Transformants verified by PCR and Southern blot analysis
c Number of transformants/total number of explants transformed
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868 228 18
9
6
Charke et al., 2008
2.1
65. Figure 15: PCR analysis. PCR positive
transformants detected with primer pairs for (a)
CP, (b) R2 and (c) R3 fragments respectively.
Figure 16: Southern blot analysis
transformants carrying
pCP, pR2 and pR3
constructs.
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Charke et al., 2008
66. Table 13: Immunological detection of PnMV on non-transformed and
transformed poinsettia plants inoculated with PnMV
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Construct Transgenic plant a ELISAb
6 wpi 10wpi
R2 72-1 0.1 0.1
72-2 0.1 0.2
R3 18-1 0.1 0.2
40B 0.8 2.4
41-2 0.5 2.6
62-1 0.1 0.2
41-5 0.7 2.8
CP 11-1 0.1 0.2
3-1 0.1 0.1
Control + 1 0.8 2.8
2 0.8 2.7
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67. INFERENCE
➢ Develop Agrobacterium tumefaciens-mediated transformation of poinsettia,
Euphorbia pulcherrima
➢ Transgenic PnMV resistant transgenic lines expressing PnMV-derived hairpin
RNA constructs
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68. ➢ Pathogen targeted resistance
➢ Integration of components that
specifically target viral genes and
their products
➢ Achieved by:
➢ Ribosome inactivating protein
➢ Interferons
➢ Ribozymes
➢ Host resistance
➢ Integration of existing host
resistance (R) genes into non-
resistant hosts
Figure 17: Antiviral activities of
interferon`
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Kawazu et al., 2009
69. Viruses that Enhance the Aesthetics of Some Ornamental Plants
69
Tulip (TBV) Camellia (CYMV) Nandina (NSPV) Canna (CYMV)
71. Table 14: List of selected viruses that enhance the aesthetics of some ornamental
plants
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Rodrigo et al., 2012
72. 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 ornamental crops.
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