ROLE OF BIOTECHNOLOGY IN DISEASE

MANAGEMENT

Biotechnology: It is defined as manipulation of genetic

makeup of living organisms through novel techniques such
as tissue culture, genetic engineering, recombinant DNA
technology, resulting in production of improved organisms
& products that can be used to develop resistance against
pests.
Ex: High milk producing cows --- ova are removed – in
vitro fertilization with good quality sperm – Introduction
in local breeds – Produce top quality calves
Ex: Anther – Haploid – Homozygous diploid are produced
through tissue culture in large no which are virus free
 • USES OF BIOTECHNOLOGY
• Increased production of plants through rapid clonal
propagation
• Pathogen free mother plants & subsequent protection
of coming generation ex: Meristem culture of citrus in
south.
• To look for possible vehicles for gene transfer
ex: Agrobacterium tumefaciens, Cauliflower mosaic
virus
• Study of genes for resistance in the host or virulence
in case of pathogens
• Use of transgenic organisms.
Tools of Molecular Biotechnology
1 Diagnosis of plant diseases:
Nucleic acid based diagnosis methods are used
which overcome microscopic to serological
detection.
 A)Hybridisation based diagnosis:
• It involves the use of Nucleic acid probes to detect the
pathogen specific sequence. Nucleic acid probes are the
sequence of DNA or RNA (usually DNA) of target
pathogen that are labelled with radio active / non radio
active substances & used to detect the complementary
sequences by hybridisation.
• Dot blot or spot hybridisation: It is commonly used by
taking pathogen infected leaf extract containing NA is
spotted on to nitrocellulose membrane and fixed by
heating. After several blocking steps hybridisation with a
labelled probe is allowed to occur. Hybridisation of probe
with text sample give the indication of the presence of
pathogen.
 B)Polymerase chain reaction based diagnosis:
• The technique of PCR was developed by Karry
Mullis et al in 1988. In this selected DNA
fragment can be amplified by in vitro DNA
synthesis with the help of flanking primers and
a thermo stable DNA polymerase. The key of
success of PCR lies in the design of pathogen
specific primers. Primers exhibiting specificity
at the generic, specific of race level can be
designed from the sequence that is unique to
organism.
• For bacterial pathogens it is plasmid &
subsequently used in A. tumefaciens, Xanthomonas
campestris pv phaseoli, Erwinia amylovora.
• Other primer used include the gene for
pathogenecity or toxin production.
• Ex. Bean – Pseudomonsa syringae – ethylene
forming gene (efe)
• Potato – Pythium ultimum – Internal transcribed
spacer (ITS) region
• Wheat – Triticum indica – mt DNA (mitochondrial
DNA)
2 Characterisation of variability in pathogen:
All the management strategies based on host
resistance require knowledge of pathogenic
variability. Traditionally differential hosts are
used for studying variability in plant pathogens.
– Hybridisation based DNA fingerprinting (RFLP)
– PCR- based fingerprinting RAPD, AFLP etc.
• Potato – Phytophthora infestans – Random
genomic DNA
• Chickpea – Ascochyta rabiei simple repetitive
sequence DNA
3 Develop of disease resistance germplasm:
Tissue culture and other biotechnological
approaches are being extensively used to
develop germplasm with new resistance & to
transfer them in crop plants. These technique
are :
A )In vitro selection for disease resistance:
• Cell & callus are grown in media containing selection
agent i.e. pathogen toxin or culture filterate or pathogen
itself. The resistant or tolerant are isolated to regenerate
plants.
• Selection with chemicals: methionine sulfoximine of
selection agent in wild five resistance tobacco plants.
• Plant pathogen as selection agent
• Selection with fungal metabolites.
B)Transfer of resistance from alien species:
• The wild spp. of crop plants are important
reservoir of useful genes, including resistance
to diseases. The desirable genes from the
related alien spp can be transferred to crop
plants. The 1st step is to obtain viable F 1
hybrids, however, certain barriers (gene action,
genomic incompatibility, ploidy level of spp.)
are encountered in Interspecific / Intergeneric
hybrid which can be overcome by following
techniques.
1)Embryo rescue :
• Culturing of embryos in medium :
• 1st used in Linum perenme × L. astriecum by Laiback
(1925)
• Bacterial blight resistance gene form Oryza minuta have
been transferred to O. sativa (Amante – Beadeos et al ,
1992)
• A number of interspecific classes in rice have acquired
bacterial blight resistant from O. brachyantha (Brad
et.al. 1996) by embryo rescue
 2)Ovary & Ovule culture
• Ovary & ovule culture are important to culture
early stage embryo of Inter specific /
Intergeneric crosses
3)In vitro pollination / fertilisation/Somatic
cell hybridisation / Para sexual hybridisation
4) Generation of disease resistance
somaclonal variants: Soma clone are the plants
regenerated from somatic cell or tissue
cultured in vitro medium.
 C)Anther culture : 1st haploid from anther culture
in Datura monia by Guha Maheshurari (1964).
Culturing of anthers on semi solid medium
produce culture or embryoids which can be
regenerated into complete plants. Anthers culture
has been used to develop disease resistance variety.
• Ex. Winter wheat – Junghua No. 1 and Florin
--- Resistant to powdery mildew and stripe rust
4 Strain improvement of biocontrol agents: It
has the following advantages
1 Expanding the range of target species
2 Restricting the range of non-target species
3 To improve the survival ability or rhizosphere
competence
4 Expanding the bio-agents environmental
range beyond its congenial habitat
5 Development of fungicide tolerant strains
5 Transgenics for plant disease management
I Coat protein mediated resistance for papaya
ring spot virus in Hawaii islands
II Cloning of resistance genes, viz., Xa 21,
bacterial blight resistance gene isolated
from African rice, Oryza longistaminata was
introduced into cultivable rice, Oryza sativa
6 Determination of biochemical nature and
the signals involved in plants reaction to
pathogen invasion and disease development.
• Ex: Host-pathogen interaction has been
studied in rice blast disease incited by
Magnaporthe grisea.
7 Manipulation of resistance of host by
expression of PR-proteins, antifungal
peptides.
• Ex: Expression of multiple Pk-proteins
(Chitinases and B-l,3 glucanases) in rice
enhanced disease resistance to rice sheath
blight pathogen, Rhizoctonia solani.
• PLANT TiSSUE CULTURE
In vitro culture of plant, cells, tissues as well as
organs. Totipotency is the ability -of a plant
cell to perform all the functions of
development which are characteristic of
zygote, i.e., its ability to develop in to a
complete plant.
 IMPORTANT TISSUE CULTURE TECHNIQUES OF
IMPORTANCE TO PLANT PATHOLOGY:

I Meristem tip culture

2 Protoplast culture
A. Production of virus free plants through plant tissue
culture

• Meristem tip culture: Cultivation of axillary or apical

meristems, particularly of shoot apical meristem, is known
as meristem culture.
• 1. Explant: the explant must consist of the meristernatic
dome of cells together with atleast one leaf primordial.
Meristem tips varying in size from 0.1 to 2.0 mm in
diameter (usually 0.3-l.5 mm) can be used for meristem tip
culture. The infected parent plant or organ of the plant from
which explant is excised is generally subjected to
thermotherapy in a temperature controlled cabinet at 300C
to 40 C for six to twelve weeks to inactivate the virus. •
2. Culture initiation on suitable medium:
In general Murashige and Skoog medium has been
found satisfactory for most plant species. But for
some species, a much lower salt concentration
maybe adequate or even necessary since the high
salt concentration of MS medium may be
deleterious or even toxic. Culture initiation consists
of surface sterilization of explants and establishing
them in vitro on culture medium. Culture initiation
often involves anti-metabolite chemicals such as
ribavirin (virazole) In the tissue culture medium.
3. Shoot multiplication: After 2-3 weeks, the
cultures are transferred to a shoot
multiplication medium designed to promote
axillary branching. This medium generally
contains cytokinins, either alone or in
combination with an auxin. Higher
concentration of cytokinins induces
adventitious buds. During culture initiation
and shoot multiplication phases, the cultures
are generally kept at 250C.
4. Rooting of shoots: In general, the rooting
medium has low salt and sugar levels. But in
most species, 0.1-1 mg/I Naphthalene Acetic
Acid (NAA) or IndoleButyric acid (IBA) is
required for rooting. Rooting takes about 10-
15 days depending on species.
5. Transfer of plantlets to soil: Rooted shoots are
removed from the medium, agar
sticking to roots is washed with tap water, and
they are transplanted into plastic cups
containing a suitable potting mixture, Plants
are kept in high (>90%) humidity and initially
low light intensities. The humidity is generally
decreased to the ambient level after about 7 -
15 days, and the light intensity is increased.
The plants are finally exposed 'to greenhouse
conditions (hardening).
6. Indexing, clone selection and stock maintenance:
Virus indexing* is done several times during first year and the virus
free plantlet is used as a nuclear stock material for commercial
multiplication, Virus indexing is generally "made by Enzyme
Linked Immune-Sorbent Assay (ELISA) or Immune Sorbent
Electron Microscopy (ISEM).
* Virus indexing is the testing of plants for the presence or absence of
viruses. Each meristem tip or callus or callus-derived plantlets must
be tested before using it as mother plant to produce virus-free stock.
Following are the methods for virus indexing:
• Sap Transmission Test:
• It is the most sensitive method which is performed
on commercial scale. Cell sap from the test plant
(i.e. plant to be tested for presence of virus) is
obtained after grinding the leaves. Filtered leaf sap
(extract) is put on the indicator plant (i.e. a plant
susceptible to a specific virus or group of viruses).
• If the indicator plant develops symptoms, the leaf
extract contains viral particles. If no symptoms
develop, it means that the plant is free from viruses.
B. Protoplast culture: Fungal protoplasts are
important tools in physiological and genetic
research. Interspecific, intraspecific and
intrageneric hybridization could be done by
this technique for strain improvement of
biocontrol agents to enhance the biocontrol
potential for the management of pathogenic
fungi. Isolation and self-fusion of protoplasts
were achieved in Trichoderma harzianum and
T. viride. .
 Steps in protoplast fusion

1. Isolation of protoplasts is achieved by treating cells with a suitable mixture of cell

wall degrading enzymes.

2. The pH of enzyme solution is adjusted between 4.7 and 6.0 and temperature is
kept around 25-30°C. The osmotic concentration of enzyme mixture and of
subsequent media is elevated to stabilize the protoplasts and to prevent them
from bursting. Usually, 50-100 . m moll CaCh is added to the osmoticum as it
improves plasma membrane stability.

3. The protoplasts of different strains are treated with 28-50% Poly Ethylene Glycol
(fusogen) for 15-30 min followed by gradual washing of the protoplasts to
remove PEG. The washing medium may be alkaline and contain high calcium
ion concentration (50 m molll). Protoplast fusion occurs during washing step.

4. Selection of hybrid cells and culturing in suitable medium.