PRODUCTION OF VIRUS FREE PLANTS

By
KAUSHAL KUMAR SAHU
Assistant Professor (Ad Hoc)
Department of Biotechnology

Govt. Digvijay Autonomous P. G. College

Raj-Nandgaon ( C. G. )
CONTENTS-

1.What is plant tissue culture?

2.Production of virus free plants.
3.History.
4.Virus elimination by heat treatment.
5.Virus elimination by Meristem Tip culture.
6.Factor affecting virus eradication by Meristem Tip
culture.
7.Chemotherapy.
8.Virus elimination through in vitro shoot-tip Grafting.
9.Virus Indexing.
10.Conclusion .
11.References .
What is plant tissue culture?

Plant tissue culture is a method or technique to

isolate parts of plants (protoplasm, cells, tissues,
and organs) and grow them on artificial media in
aseptic conditions in a controlled space so that parts
of these plants can grow and develop into complete
plants.
PRODUCTION OF VIRUS FREE
PLANTS
Plant infected with bacteria and fungi may respond to
treatments with bactericidal and fungicidal compounds ,
there is no commercially available treatment to cure
virus infected plants.

Virus free plants can be produce by plant tissue

culture.

Methods to produce virus free plants-

1.Heat treatment .
2.Meristem tip culture.
3.Chemical treatment.
4Other in vitro methods .
History:

The knowledge of the gradient of virus

distribution in the shoot tip enabled Holmes in 1948
to obtain virus free plant from infected individuals
of Dahelia through shoot tip cutting.

Morel & Mortin in 1952 developed the

technique of shoot tip culture .

In 1962 Morel developing micropropagation

technique for production of large number of plant by
shoot tip culture.
VIRUS ELIMINATION BY HEAT
TREATMENT
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

hotwater 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
 PROSEDURE

For hot-air treatment, which is comparatively easy to apply,

actively growing plants are transferred to a thermotherapy chamber
and exposed to a temperature of 35-40°C for a suitable period.

The duration of the treatment varies from a few minutes to

several weeks.

Baker and Kinnaman (1973) eradicated all viruses from Carnation

shoot tips by continuous treatment of plants at 38°C for 2 months.
On the other hand, Potato Virus X (PVX) required several months
of treatment at 35°C to obtain some virus-free tips (Stace-Smith and
Mellor, 1968).

Small cuttings are taken from the shoot tips immediately after the
heat treatment and grafted onto healthy root stock
 THERMOTHERAPY

HOST VIRUS TEMPRATURE

ELIMINATED
Chrysanthemum Chrysanthemum B 35 to 38°C
virus
Carnation Carnation ringspot 35 to 40°C
virus, Carnation vein
mottle virus
Banana Cucumber mosaic 35 to 43°C
virus
Goose berry Gooseberry vein 35°C
banding virus
Potato Potato virus Y, S, X 33 to 38°C
 LIMITATION

A major limitation of thermotherapy for virus

eradication has been that not all viruses are sensitive to the
treatment.

For example, in potato only Leaf Roll Virus could be

eradicated by this technique.
 MERISTEM CULTURE

Apical meristem culture is second

method for producing virus free plants.

Apical meristem free from diseases or

its carry very low concentration of viruses
because virus moves via vascular system
which is absent in meristems.

High auxin level in meristem inhibit viruses.

Meristems also free from Mycoplasma, Bacteria, and

Fungi.
 METHOD
As with other types of tissue cultures, the first
essential step in meristem-tip cultures
is to obtain explants.

Surfase sterilization.

At the time of dissecting out meristem-tips

the explant is held under the microscope in
one hand with the help of a fine pair of forceps, and
leaves and leaf primordia are removed using fine needles.

Transfer the tip to culture medium.

 Continue……

The shoots derived from meristem-tips often root in the

original medium but if this does not happen a different
treatment has to be tried.

Occasionally, as experienced with Dahlia by Morel

and Martin (1952), the shoots developed in cultures may
fail to root under any treatment. In such cases whole
virus-free plants have been obtained by grafting the
virus-free shoots onto healthy rootstocks.
 FACTOR AFFECTING VIRUS ERADICATION
BY MERISTEM TIP CULTURE

1.CULTURE MEDIUM-

MS medium is most known and suitable medium for meristem tip

culture.

Both liquid and semi-solid media have been tried for meristem-tip
culture but, because of the convenience in handling, agar medium is
generally preferred. However, in those cultures where agar medium
induces callusing of the explants the use of a liquid medium is
recommended.
For the liquid cultures a
filter- paper bridge is
prepared and inserted
into the culture tube.
 2.EXPLANT SIZE
The larger the explant, the greater are the chances of
plant regeneration.

Explants should be small enough to eradicate viruses and large

enough to be able to develop into a complete plant.
3.INCUBATION CONDITION-
16 hrs light and 8 hrs dark.

The cultures are generally stored under standard culture-room

temperature 25 ± 2ºc.
4.PHYSIOLOGICAL CONDITION-
Meristem tips should, preferably, be taken from actively growing
buds.
Terminal and Apical buds are free from viruses.
The time of excision of buds is also an important factor.
meristem tips should be cultured during the spring.

5.THERMOTHERAPY

In Carnation 33% of the terminal portion of shoot tips carried

Carnation Mottle Virus.

Other viruses known to invade the meristematic region of shoot

tips are TMV,CMV,PMV etc.

In such cases also it has been possible to obtain virus-free plants

by combining meristem-tip culture with thermotherapy.
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
 INVITRO SHHOT TIP GRAFTING

Sometimes the shoots developed in shoot-tip

cultures do not form roots.

In some such cases it may be possible to graft

the shoots onto a virus-free rootstock derived
from seedlings.

This technique first used for Dahlia.

Murashige et al. (1972) were able to obtain a

few citrus plants by grafting shoot tips from
diseased plants on young rootstock seedlings
growing in vitro.
 CALLUS CULTUR

In calli derived from infected tissues not all cells uniformly carry the
pathogen.

Only 40% of the single cells mechanically separated from TMV-

infected tobacco callus contained the virus.

Virus free plants may be produced via callus culture because

(a)Virus replication is unable to keep pace with cell

proliferation, and
(b) Some cells acquire resistance to virus infection
through mutagenesis.
-
 VIRUS INDEXING-
1.By the symptoms-
 2.BY ELISA-

The most widely used plant virus indexing technique since 1977..

1. Sensitive
2. Specific
3. Efficient
4. Reproducibe
5. Objective
6. Cost effective
BY PCR(POLYMERASE CHAIN REACTION)

Virus can be detected via probes or primers.

1.Blotting of tissue on membrane

2. Dissolving of membrane or mixing

with PCR reaction mixture.

3. PCR amplification

4. Visualization of bands
 Conclusion
From the various example given in this topic it is apparent
that tissue culture techniques have a useful role in the
eradication of systemic disease in plants caused by viruses.
Meristem tip culture seems the most reliable method for
pathogen elimination.
 REFERENSES-

s.n Book name Edition Author Page no.

o. name
1 Introduction to 3rd H.S.Chawla 51-63
plant edition
biotechnology

2 Plant cell & 2nd I.K.Vasil 37-63

tissue culture edition

3 Plant tissue - S.S.Bhojwan 451-435

culture i
M.K.Razdan