A brief account of Applications of Plant tissue culture - Micropropagation, Meristem culture, Synthetic seeds, Embryo culture, In vitro mutagenesis, In vitro production of secondary metabolites

2. Applications of Plant Tissue Culture
i) Micropropagation
• A large number of plantlets can be
produced from a small part of plant by
in vitro tissue culture in a suitable
nutrient medium.
• This method can be used for the
production of large number of
plantlets in rare, endangered and
threatened species ; commercially
important ornamental plants like
orchids, Anthurium, Heliconia etc. ;
Banana; plantation crops like
Cardamom etc.

3. ii) Production of Virus free (disease
free) plants by Meristem culture (Morel
and Martin 1952)
• In vitro culture of shoot apical meristem
 Apical or lateral shoot tips are used – contains
shoot apex and several leaf primordia – at the axils
of which axillary shoots could potentially develop.
 Excised shoot tips are cultured on solid medium
with high concentration of a cytokinin hormone –
development of axillary shoots – separate each
shoot and repeat the process to rapidly multiply the
shoots. These shoots are planted into a solid
medium containing auxin (root inducing hormone)
– formation of plant let.

4. iii) Production of Artificial seeds
(Synthetic seeds/ Synseeds (Toshio
Murashiege 1978 )
–)
Uploaded by: Dr. Somdeep Ghosh, Oct
18, 2018

5.  Somatic embryos of a well known strain can be
produced by tissue culture and they can be
encapsulated in a protective gel containing
nutrients, growth regulators etc. to produce
artificial or synthetic seeds.
 Bead of gel contains
• Somatic embryo
• Nutrients (serves as artificial
endosperm)
• Growth regulators
• Pesticides
• Antibiotics

6.  Synthetic Seeds contain identical somatic
embryos of a well known strain
 Small in size since they contain only
minimum essential amount of nutrients.
Encapsulating agents
 Sodium alginate
 Polyacrylamide
 Nitrocellulose
 Agarose
 Polyoxyethylene

7. Synthetic Seed Production
1.) Desiccated systems – Somatic embryos are first
hardened to withstand desiccation and then are
encapsulated in a suitable coating material.
Steps:
 Production of somatic embryos by tissue culture
 Hardening of somatic embryos to withstand
desiccation
 Treating or coating mature somatic embryos with a
suitable polymer and drying
 Treating somatic embryos with Abscisic acid
(ABA) during maturation phase.

8.  Encapsulation in a suitable encapsulating agent
(Protective gel/ chemical membrane)
 eg. Synthetic seed production in Carrot (Kim and
Janick 1989)
 Mixed equal volumes of carrot somatic embryo
suspension and 5% solution of poly ethylene
oxide ( a water soluble resin) and dried to form
polyembryonic desiccated seeds.

9. 2.) Hydrated System
 Somatic embryos are enclosed in gels which remain
hydrated.
 Somatic embryos are prepared by tissue culture
 2% solution of sodium alginate filled in a burette is
allowed to form a drop at the tip.
 Somatic embryo is inserted into the drop with a
spatula.

10.  The drop with somatic embryo is allowed to fall
into 100m M Calcium chloride solution.
 The bead becomes hardened as Calcium alginate
is formed
 After 20 – 30 minutes , the artificial seeds are
removed, washed with water.
 A waxy coating can be provided over the seeds to
prevent the sticky nature (difficult to handle on a
large scale and dry rapidly in air)

11. Advantages of Hydrated Artificial seeds
Reduced cost of vegetatively
propagated elite lines
Direct transplantation to field
Genetic uniformity of plants

12. Significance / Advantages of
Synthetic Seeds
 A potential technology for the use of
somatic embryogenesis for large scale
propagation of plants through automation
 Somatic embryos of suitable age can be
produced in a bioreactor.

13. Disadvantages / Limitations of
Synthetic seeds
Quality of somatic embryos
Low gas exchange in coated somatic
embryos

14. In vitro culture of embryo in a suitable
medium to obtain seedlings.
Hanning tested a variety of nutrient media
containing sugars, mineral salts, plant
decoctions, certain amino acids and
gelatin.

15. Chemical composition of culture
media for Embryo culture
Carbon source – Sucrose/Glucose
Mineral salts
Amino acids
Vitamins
Growth regulators
Gelatin/Agar
Organic supplements
 Plant decoctions
 Coconut milk
 Casein hydrolysate
 Yeast extract
 pH: 5 – 7.5

16. Young embryos – need elaborate culture
medium containing many nutrients
Mature differentiated embryos – need only
a medium with minimum nutrients – a few
mineral salts and sucrose.

17. A medium containing
Rich source of sucrose/glucose
Ammonium nitrate
Vitamins
Casein hydrolysate
Plant extracts with growth regulators
pH 5 – 7.5 – show good response to
embryo culture

18. Procedure
 Surface sterilization of seeds (5-10% Chlorox or
0.45% Sodium hypochlorite for 5 – 10 minutes)
 Dissect out the embryo
 Direct inoculation on to the medium or after
surface sterilization with 70% ethanol (30-60
seconds)
 Incubation at 25  2OC  Callus  Regeneration
by organogenesis or embryoids (somatic
embryos)  Plants

19. Other methods
 In cereals, transfer of embryos to the endosperm
of other genera (Stingl, 1907)
 Culture in Knop’s solution with 2.5 – 5% cane
sugar and 1.5% Agar (Dietrich, 1924)
 Embryo placed on moist filter paper containing
sucrose/glucose (Laibach 1925, 1929)
 Embryo rescue in interspecific cross, Linum
perenne x L. austriacum)

20. Types of Embryo culture
Embryo culture involves the culture of
1. Proembryos (immature embryos)
 Culture of heart shaped, globular
proembryo in a suitable medium.
Helps to understand
– the differentiation process
– Nutritional requirements of developing
embryo
2. Intact seed containing undifferentiated
embryo. (lacking radicle and plumule,
eg: Orchids)

21. 3. Mature and intact seed embryo – helps to
determine diverse parameters of embryonic
growth
4. Dissected embryo-helps to analyze the
interrelationship of different parts of the
embryo.
5. Inviable or abortive embryos. (Embryo
rescue) – In interspecific or intergeneric
crosses.

22. Applications of Embryo culture
1. Shortening the breeding cycle – develop into
seedlings – avoid a long time of seed maturity.
2. Overcoming dormancy (in certain fruit trees)
3. Recovery of distant hybrids (Embryo rescue – in
interspecific or intergeneric crosses)
4. Propagation of orchids – young or mature
embryos are cultured.
5. Propagation of rare plants – Makapuno coconut
(has soft, solid, fatty tissue in place of liquid
endosperm).

23. v) In vitro Mutagenesis
 Used to improve cultivars of vegetatively propagated
plants.
Materials used:
 Highly regenerative cell lines.
 Protoplasts
 Shoot apical meristem

24. Use of Physical Mutagen – Gama Rays
(from 60Co)
 Irradiation of explant – shoot tips, in
vitro plant parts.
 Culture of irradiated explant in a
medium containing auxins and
cytokinins (72 -96hrs- recover from
radiation shock)
 Selection of desirable mutants –
resistant to a pathogen, herbicide, heavy
metal toxicity etc.

25. Selection of Fusarium wilt tolerant
mutants
 Add the toxin produced by the pathogen
Fusarium – Fusaric acid to the medium.
 Remove dead tissues at regular intervals to
avoid harmful effects on rate of shoot
regeneration.
 Subculture at regular intervals (3 -4 weeks) on a
fresh culture medium containing the toxin
Fusaric acid to dissociate chimeras ( two or
more genetically distinct tissues) and maintain
the stability of mutant traits.

26. Use of Chemical Mutagen – Ethyl
Methane Sulphonate (EMS)
Most common method.
 Immerse the explant in a solution of
optimum dose of EMS for 30 minutes.
 Wash the explant repeatedly in sterile
distilled water.
 Transfer the explant to liquid growth
medium.

27.  Culture on fresh media at least twice to
remove residual mutagen.
 Transfer the explant to sterile Whatman
filter paper to remove excess liquid
growth medium.
 Transfer to a semi – solid M S basal
growth medium containing growth
regulators for shoot regeneration and
finally plant regeneration.

28. A Scheme for producing induced
mutants by in vitro mutagenesis
In vitro Culture
Mutagen treatment
Regenerate induced mutants
Screening of regenerated plants
(Proto plast/ cell cultures/ adventitious buds/ somatic embryos)
(Physical or Chemical)
(By Somatic embryogenesis/ adventitious shoot formation/
axillary bud break)
Uniformly mutated
Plants (Homohistonts)
Chimeric
Plants
Reinitiate in vitro
culture
(Homohistonts)
Field Testing (confirm stability of
mutants
Release new variety

29. vi) In Vitro Secondary metabolite
production
 In vitro plant tissue cultures can be used for the
commercial production of secondary metabolites –
recognized since early 1950’s.
 Use of Batch cultures – Continuous cultures –
Techniques are available for the induction and
selection of stable genetic variants.
 Secondary Metabolites – Chemical substances
derived from primary metabolites – Alkaloids,
Terpenoids, Flavonoids, Phenolics, oils, steroids
etc.

30.  Not directly involved in the primary metabolic
processes.
 Potential sources of drugs, pigments, flavours
 High economical and pharmaceutical
importance.

31. Alkaloids
 - Natural, nitrogenous compounds derived from aminoacids
and heterocycles of pyrrole, pyrimidine etc.
 High medicinal value.
e.g. Quinine from Cinchona officianalis _ Cure for
malaria
Vincristine and vinblastin – from Catharanthus roseus –
Anticancerous potential
Taxol from Taxus brevifolia ,,
Digoxin from Digitalis purpurea - for cardiovascular
disorders
Reserpine from Rauvolfia serpentina - for hypertension

32. Terpenoids
 Isoprenoids – (Five Carbon Isoprene units) Monoterpenes
,sesquiterpines, diterpines.
 Helps in plant defense – antimicrobial (bactericidal,
fungicidal, antiviral)
 eg. Azadirachtin, Nimbin, Nimbidin etc. from Azadirachta
indica (Neem); Menthol
 Camphor
 Carotenoid pigments
 Polyterpenes- Rubber etc.
Other Effects
Cytotoxic
Spermicidal
Anticancerous

33. Flavonoids
–
Red, blue and purple pigments of plant
tissues
Antimicrobial
Insecticidal

34. Advantages of in vitro secondary
metabolite production
 Plant cells produce secondary metabolites only in
small amounts.
 Secondary metabolites have complex structure –
chemical synthesis is economically
unattractive
 Plant cells can be easily cultured under aseptic,
controlled nutritional and environmental
conditions - can avoid variations in climate and
soil.

35.  Easy to incorporate precursors in
suspension cultures (Difficult to administer
to plant growing in native).
 Commercial production by Batch cultures,
continuous cultures etc.
 Refined culture systems to improve
biochemical yields.

36. Examples
Commercial Production of Shikonin
(Naphtoquinone – an antiseptic, a dye
for silk & cosmetics), from cell cultures
of Lithospermum erythrorhizon.
Taxol – (Alkaloid – for breast and
ovarian cancer treatment ) from Taxus
brevifolia. .

37. Extraction of Secondary Metabolites
Explant
 Sterilization

Induction of Callus

Cell Suspension Culture

Cell Plating

Cell colonies

Testing for high production potential (for desirable product)

High yielding clones

Large scale culture of cells in bioreactor

Extraction of
Desirable secondary metabolites

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