Virus free plant

VIRUS FREE PLANTS

1. DR AKANKSHA JAIN

2. SYNOPSIS
INTRODUCTION
HISTORY
TRANSMISSION OF VIRUS
VARIOUS REASON ATTRIBUTED TO THE ESCAPE OF MERISTEM TO VIRUS
PRODUCTION OF VIRUS FREE PLANT
THERMOTHERAPY
MERISTEM TIP CULTURE
CRYOTHERAPY
CHEMOTHERAPY
MICROGRAFTING
CALLUS CULTURE
VIRUS INDEXING
MAINTENANCE OF VIRUS FREE STOCK
APPLICATION & LIMITATION
CONCLUSION
REFERENCES

3. Viruses are very small, infectious particles
composed of protein coat & a nucleic acid core.
The application of shoot meristem culture mostly
lies in production of disease free plant.
Most of the horticultural & forest crops are
infected by systematic disease caused by fungi,
viruses, bacteria, mycoplasma & nematodes.
Pathogen attack does not always lead to the death
of the plant but very often the infection caused by
pathogens reduces the yield & quality of crops.
Cassava brown streak virus disease(CBSVD),
cassava mosaic virus disease(CMVD), & sweet
potato virus disease(SPVD),are nearly always
transferred in plants through vegetative
propagation from one season to another.

4. In 1880 Mayeridentified an infectious agent in
tobacco leaves that could transmit the infections into
a healthy new plant.
In 1892 a Russian scientist Dmitri ivanofsky
discovered that infectious agent of tobacco leaves
was filterable claiming that agents are smaller than
bacteria.
In 1898 Beijerinckreferred to this new disease agent
as a contagious living liquid.
In 1935 Wendell Stanley crystallized tobacco mosaic
virus to demonstrate that viruses had regular shape
& in 1939 tobacco mosaic virus was 1
st
visualized
using the electron microscope.

5. SEEDS
VEGETATIVE PROPAGATION/GRAFTING
VECTORS
BACTERIA(e.gAgrobacterium tumefaciens)
INSECTS

6. VECTOR : INSECTS

7. VARIOUS REASON ATTRIBUTED TO THE
ESCAPE OF MERISTEM BY VIRUS
INVASION
Viruses move viaa vascular system
Apical meristem is generally free & carry low
concentration of virus
High concentration of auxin in meristem
High metabolic activity does not allow replication
of virus

8. Virus distribution in shoot tip
encouraged by Holmes(1948) to
obtain virus free plant from infected
Dahlia
Morel & Martin (1952) applied tissue
culture technique for elimination of
viral infection in plant

9. FIG: DahliaPLANT

10. GENERAL PROCEDURE OF
CREATING A PLANT TISSUE
CULTURE

11. 1.) THERMOTHERAPY
Heat treatment is given through hot water or hot
air.
Hot air treatment has given better elimination of
virus and better survival of the host in actively
growing shoots.
Hot –air treatment is easy to apply; actively
growing plants are transferred to a thermotherapy
chamber and exposed to a temperature of 35-48.c
The duration of the treatment varies from few
minsto several months.
Humidity 85-95%. And light should be maintained
E.g. carnation shoot tip.
Potato shoot tip

12. MERISTEM
MERISTEM
CULTURE

14. MERISTEM TIP
CULTURE
REQUIREMENT
PROCEDURE
1.) The explants has been taken from the mother
plant of green house
2.) Wash with running tap water
3.) After washing surface sterilized with surface
disinfectant
4.)Rinsed several times in sterile water
5.)Exposed meristem tip appears as a shiny dome
when we dissect under microscope
6.) Explants is transferred to a nutrient medium in a
culture tube

15. FACTOR AFFECTING
VIRUS ERADICATION BY
SHOOT TIP CULTURE
CULTURE MEDIUM
EXPLANT SIZE
INCUBATION CONDITION
PHYSIOLOGICAL CONDITION
THERMOTHERAPY

16. E.g.-Shoot tip culture from
Chrysenthemumplants treated with
50c for 4 month yields 67% plant
free from Chrysenthemum stunt
virus (csv) & 22% plants free from
Chrysenthemumchlorotic mottle
virus (ccmv).

17. Chrysenthemum stunt
virus (csv)
Chrysenthemum chlorotic
mottle virus (ccmv)

18. Some chemical e.g. virazole(ribavirin),
cycloheximide, actinomycin D etc interferes
virus multiplication added into culture
medium for curing the shoot tip of virus
For e.g. virazole eradicate potato virus x (pvx)
Pretreatment of cymbidium tip with antiserum
increased frequency of virus free plant
Combination of thermotherapy & meristem
culture used for virus free plant production
e.g.
cassava,banana,citruses,strawberry,potato
apple, chrysenthemum

19. Sometimes the shoot developed in shoot tip
culture do not form root
In such case it may possible to graft the shoot
onto virus free rootstock
The concept by MOREL & MARTIN studies to
raise virus free plant of Dahlia

20. In case of tobacco virus free shoot tip are
taken & callus have been separated
mechanically
Only 40% of single cell mechanically
separated from TMV infected tobacco callus
contained the virus
Some cell were actually free of virus was
confirmed by regeneration of many TMV free
plant from infected calli
But this technique is limited use because high
chance of somaclonal variation & genetic
variation

21. FIG: TOMATO CALLUS

22. Every meristem tip & callus derived plant
should therefore be tested for specific viruses
before using it as a mother plant to produce
virus free stock
The most common used serological test is
ELISA
This test relies the use of antibodies prepared
against viral coat protein require only a small
amount of antiserum
Most agricultural crops can now be routinely
tested with ELISA

23. LIMITATION-It is not applicable to viroid
Involves several related lute virus such as
plant leaf role virus all virus may not react
with antiserum against virus
Such agent can be detected through
cDNA/CRNA probes
The basic principle is the detection based on
nucleic acid hybridization

24. Microplate ELISA:
coloured
wells indicate reactivity
ELISA
KIT

25. MAINTENANCE OF VIRUS
FREE
STOCKS
Plant freed from viruses are maintained in
insect free glasshouse
A large scale multiplication of such plant can
be done in areas where& during season when
chance of infection of virus are minimal
The best and cheapest means of maintaining
virus free plant is in vitro culture

26. GREEN HOUSE
CULTIVATION

27. VIRUS ELIMINATION GENERALLY IMPROVES
THE YIELD BY 20-90% OVER INFECTED
CONTROL
HOWEVER ELIMINATION OF VIRUS MAY
INCREASE THE SUSCEPTIBILITY OF PLANT
TO OTHER MORE SEVERE VIRUS
VIRUS FREE PLANT OBTAINED MAY RAPIDLY
BECOME INFECTED BY SAME VIRUS
USED IN BREEDING PROGRAMME &
GERMPLASM EXCHANGE

28. CONCLUSION

29. REFERENCES
BIOTECHNOLOGY -BY B.D SINGH EXPANDING
HORIZON, 1
ST
EDITION 2004
PLANT TISSUE CULTURE –BY K.K DEY
INTRODUCTION TO PLANT BIOTECHNOLOGY -BY
H.S CHAWLA, 2
nd
EDITION 2002
PLANT TISSUE CULTURE –BY M.K RAJDAN & S.S
BHOJWANI,2
ND
EDITION 2005
INTERNET
www.google.com
www.wikipedia.com
Photos from www.dpvweb.net