Plant breeding,

1. Lecture 13 & 14
Non –Conventional Breeding: Clonal
Propagation
Plant Breeding
BBT 2208
Ms. Sarah Stephenie John

2. Introduction
• Tissue culture is the cultivation of plant cells, tissues, or
organs on specially formulated nutrient media. Under the
right conditions, an entire plant can be regenerated from a
single cell.
• Micropopagation is a tissue culture method developed for
the production of disease-free, high quality planting material
and for rapid production of many uniform plants.
• Actively-dividing young cells (meristem) are placed in a
special medium and treated with plant hormones to
produce many similar sister plantlets. Since the meristem
divides faster than disease-causing virus, clean materials are
propagated and hundreds of uniform plantlets are produced
in a short time.

3. Applications of In Vitro Culture Technology in
Plant Breeding
• In vitro culture is the culturing of cells, tissues and organs under
aseptic laboratory conditions in culture media.
• Plant parts known as explants are cultured in the nutrient medium.
• The explants are surface sterilized, disinfectant - sodium
hypochlorite (10 – 50% w/v; 10-30 minutes) or mercury chloride
(0.1% w/v; 5-10 minutes), washed with sterile (autoclaved) water
and cultured in the nutrient media at 25 ± 1℃.
• Commonly used plant tissue culture media: Murashige and Skoog,
1962 (MS), White 1963, Gamborg et al. 1968 (B5) and Chu 1978
(N6).
• TC media contains – major elements, micro elements, vitamins,
amino acids, carbohydrates (sucrose) and growth regulators (auxins,
cytokinins)

4. Applications of In Vitro Culture Technology in
Plant Breeding
• Placement of explant on suitable growth medium –
dedifferentiation.
• The renewned growth on agar gelled medium gives an unorganized
mass of cells (callus).
• The cells might be cultured in liquid medium – suspension culture.
• The callus is subcultured after every 3-4 weeks.
• Ultimately such cell tissues are to be used to obtain organized
structures – roots, shoots, flower buds etc. through the process
known as organogenesis.

5. Applications of In Vitro Culture Technology in
Plant Breeding
• Somatic embryogenesis can also be attempted in callus culture.
• It is the development of embryo-like structures from cell culture.
Such somatic embryos can be encapsulated in a suitable matrix like
sodium alginate and synthetic seeds can be produced.
• Synthetic seeds can be stored for several years and used as natural
seeds. The major applications of in vitro culture technology include
plant regeneration, micropropagation, somatic embryogenesis,
exploitation of somaclonal variation, meristem culture, anther
culture, pollen culture, embryo culture, protoplast culture,
cryopreservation of germplasm, secondary metabolite production
and in vitro mutagenesis from plant cell culture.

7. Tissue Culture Manipulations

8. Micropropagation
“… the art and
science of multiplying
plants in vitro.”

9. MICROPROPAGATION
• Involves in production of plants from very small (0.2 – 1.0mm) plant
parts through tissue culture techniques.
• Micropropagation of selected plant species is one of the best and most
successful examples of commercial application of tissue culture
technology for mass multiplication of plants that has following 4
distinct advantages:
a) It is independent of seasonal and regional constraints hence ensures
year round rapid propagation.
b) The plants produced in this way are true to type.
c) Production of disease free plants
d) Exhibit vigorous growth, better quality and higher yield.
• Uniform behavior of the clonal crop is highly advantageous in terms
of agronomic and harvest practices. But the chances of susceptibility
to new strains of pathogens and adverse environmental conditions are
always associated with such genetically uniform crop populations

10. Clone
Genetically identical assemblage of individuals
propagated entirely by vegetative means from a
single plant.

11. • The 4 distinct stages of micropropagation are;
a) Establishment of an aseptic culture
b) Multiplication of propagules
c) Pre-transplant
d) Transplant

12. Stage 1
Establishment of an aseptic culture

13. • Surface-sterilization of explant tissues.
• Isolation of explant under sterile conditions.
• Medium must contain all components necessary to make the explant
perform as desired (medium composition and PGRs).
• Adjust the environmental conditions (Light, Temperature, Relative
humidity, etc)

15. Stage 2
Multiplication of propagules

16. • Maintenance & multiplication of the propagules for later rooting to a
plantlet stage.
• Repeated enhanced shoot production by high cytokinin in the medium,
alone or with a smaller amount of auxin.
• Shoots must be transferred to fresh medium at regular intervals.
• A choice for type of medium.
• Modification in medium

18. Stage 3
Pre-transplant

19. • Main function of this stage – prepare propagule for transplanting from
in vitro to in vivo conditions.
• It is rooting of shoots or shoot clusters in vitro.
• Auxins are important for root initiation in vitro
• Advantages of rooting after removal from culture
Reduced costs
Structurally and physiologically better
Avoid damage to roots that occur during transplanting

20. Stage 4
Transplant

21. • Acclimatization: a process of physiologically and anatomically
adjustment from in vitro to ex vitro conditions.
• Relatively slow process, may take weeks.
• Must adjust from high to lower relative humidity (e.g. from 98-99%
to 20 - 60%): development of sufficient defenses to control water
loss.
• Must adjust from low light to high light: from low photosynthetic
competence (heterotrophic nutrition) to photosynthetic competence.

22. An enhanced transplantation success has been reported through
• Maintenance of high relative humidity (90-100%) around the newly
transferred plants for initial 10 – 15 days.
• Partial defoliation and application of anti transpirants such as Acropol (1%
v/v)
• Use of loose, aerated and well drained rooting medium.
• Use of fungicides such as bavistin (0.5-1.0%) during watering for first 1 – 3
days.
• Application of thin film of 50% aqueous glycerol, paraffin (melting point 52
– 54°C) or grease dissolved in equal amounts of diethylether on the surface
of leaves before transplantation.
• In plant species which undergo dormancy, chilling of plantlets at 0 - 4°C for
1-2 minutes, inclusion of gibberellic acid (1-2mg/l) in the rooting medium or
spraying the plantlets with gibberellic acid (200mg/l) after transfer.
• Induction of corms, bulbs, tubers in vitro and their transfer to the soil rather
then transferring delicate plants.

24. Some potential uses of clonal propagation in agronomic crops
are:
• large-scale increase of a heterozygous genotype,
• increase of a self-incompatible genotype,
• increase of a male-sterile parent in a hybrid-breeding program,
• propagation of disease-free genetic stocks, and
• preservation and international exchange of germplasm.

25. Micropropagation Advantages
• From one to many propagules rapidly
• Shoot tips cloned from axillary buds or meristem tissue
produce fewer genetic variants
• Multiplication in controlled lab conditions
• Continuous propagation year round
• Potential for disease-free propagules

26. Micropropagation Limitations
• Equipment/facility intensive operation
• Technical expertise in management positions
• Protocols not optimized for all species
• Liners may not fit industry standard
• Propagules may be too expensive

27. Problems encountered in Micropropagation
• Microbial contamination
Bacterial & fungal contamination in the cultures normally do not allow
the propagules to grow and the contaminated cultures have to be usually
discarded. Such problem can be overcome by growing the donor plants
in the growth chambers, by effective sterilization of the explants,
performing inoculation in the Laminar Air Flow Cabinets fitted with
HEPA filters (0.2um) and by using sterilized surgical instruments.
Fumigation of inoculation room using dilute formaldehyde solution also
helps to minimize this problem.
• Browning of culture medium
Certain cultured explant e.g sugarcane secrete phenolic substances into
the medium which cause browning of the medium and reduction in the
growth of cultured explants. Use of activated charcoal (0.1-0.2%), citric
acid or ascorbic acid (500-1000 mg/l) and polyvinylpyrrolidone (pvp)
into the culture medium helps to check the browning.

28. • Callusing
Production of undifferentiated mass of cells from the propagules is
highly undesirable as it affects the normal development of shoots and
roots and may lead to variability among the regenerated plants. Addition
of triiodobenzoic acid, phloroglucinol and phloridzin into culture
medium or reduction of the inorganic salt concentrations help in
overcoming this problem.
• Vitrification
The shoots/plantlets being multiplied in vitro appear abnormal because
of abnormal leaf morphology, poor photosynthetic efficiency,
malfunctioning of stoma and reduced epicuticular waxes. It can be
minimized by making in vitro growth conditions comparable to in vivo
growth environment which could be achieved by creating sapstream in
the plants by reducing the relative humidity in the culture vessels.

29. • Vulnerability of Micropropagated Plants to Transplantation Shock
High mortality rates upon transferring tissue culture derived plants to soil
continues to be a major bottleneck in micropropagation of many plant
species. Conservation of moisture by creating high humidity around the
plants, partial defoliation, application of antitranspirants has met with
good success.
• Tissue culture induced variation
It may be good from breeding point of view but is highly undesirable
while propagating selected plants in their true-to-type form. Therefore
the variation can be controlled by careful selection of initial explant i.e
selecting meristems and controlling the cultural environment favouring
relatively slow multiplication rates.

30. Micropropagation Applications
•Rapid build up of stock of new,
superior variety
•Elimination of endophytic disease
•Better suited to morphology/growth
characteristics (few offshoots/sprouts;
date palms, ferns, nandinas)
•Propagules have enhanced growth
features (multibranched character;
Ficus, Syngonium)