Clonal Micropropagation - Plant Tissue Culture

1. Micropropagation
Dr. Divya Sharma
Assistant Professor
A Biodiction (A Unit of Dr. Divya Sharma)

2. Micropropagation
 Tissue culture technique:
Increasingly popular alternative means -- Plant vegetative propagation
 Plant tissue culture involves –
o Asexual method of propagation
o Primary goal ----- crop improvement
 In-vitro selection, genetic manipulation techniques ------- Higher plant

3. Clonal Propagation
Clonal propagation ------- In-vitro selection Micropropagation
Greek word --- Clone --- Twig ---- Identical copies
Multiplication of genetically identical copies by asexual reproduction is called Clonal
Propagation
Process -- Asexual reproduction --- Multiplication occur ----- Individual
plants genetically identical copies
 Multiplication of genetically, identically individual by asexual reproduction
 Rapid process -------- Commeralization of improvement plants like apple, pear
etc.
 Micropropagation
 Apomixes (seed development without meiosis and fertilization) and vegetative
propagation (regeneration of new plants from vegetative parts)
 Short time

4. Micropropagation
 Aseptic method of clonal propagation is called as
Micropropagation
 Synonyms -- tissue culture
 Rapid multiplying stock plant material to produce
a large number of progeny plants, using modern
plant tissue culture methods
 Widely used for Orchids, Ferns, many interior
foliage plants etc.

5. Features of Micropropagation
 Relatively short time
 Easy to manipulate production cycles
 Disease free plants can be produced
 In-vitro production can be planned
 Recycled many times to produce an unlimited number of clones
 Micropropagated plants may acquire new desirable traits, like bushy habit
of ornamental plants and increased number of runners in strawberry
 Commercially viable method for horticultural crops
Eg.: Orchids, Ferns, many interior foliage plants etc.

6. Rapid Clonal in-vitro Propagation of Plants
 From cells, tissue or organs
 Cultured aseptically on defined media
 Contained in culture vessels
 Maintained under controlled conditions of light and temperature

7. Commercialization of Micropropagation
Broad commercial applications ------ 1970s and 1980s
Murashige (1974)

8. Selection of plant material
 Part of plant
 Genotype
 Physiological condition
 Season
 Position on plant
 Size of explants

9. Explants used in Micropropagation
Different kinds of explants used in micropropagation:
Eg.: In case of orchids, shoot tips (Anacamptis pyramidalis, Aranthera,
Calanthe, Dendrobium); Axillary bud (Aranda, Brassocattleya, Cattleys
Laelia); Inflorescence segment (Aranda, Ascofinetia, Neostylis, Vascostylis);
Lateral bud (Cattleya, Rhynocostylis gigantean); Leaf base (Cattleya);
Nodal segment (Dendrobium; Flower stalk segment (Dendrobium,
Phalaenopsis); and Root tips (Neottis, Vanilla)

10. Stages of Micropropagation
Stage 0: Preparation of the mother plant
Stage 1: Initiation and establishment of cultures
Stage 2: Multiplication [shoot or rapid embryo; and rooting of shoot]
Stage 3: Germination of somatic embryo for rooting of in-vitro formed
shoots
Stage 4: Transfer of plantlets -- sterilized soil ---- Hardening
to greenhouse/ field conditions (transplantation)

11. Stages of
Micropropagation  Maintained light, temperature and
moisture regimes under which the
mother plants
 Loosely covering growing branches with
polythene
 Application of growth regulators
 Watering of plant from the base
Stage 0: Preparatory
stage
Involves the preparation of
mother plants to provide
quality explants for better
establishment of aseptic
cultures

12. Stages of
Micropropagation
 Explant selection
 Sterilization treatment
 Choice of growth medium
Stage 1: Initiation of
Cultures

13. Stage 2: Multiplication
Through Callusing
Initiation of culture on high Auxin media
Development of unorganized mass of cells
called callus
Transfer small piece of callus on
regeneration media
Shoot bud regeneration
Somatic embryo

14. Stage 2: Multiplication
Adventitious Bud Formation
 Transfer to proliferation media
 Shoots can be constantly divided

15.  Small bud present
 Apical dominance is removed
 Cytokinins
 Auxins
 Cluster formation
 Off types
Stage 2: Multiplication
Enhanced Axillary Branching

16.  Somatic embryos carry a preformed radical and may develop directly into
plantlet.
 Embryos sow very poor conversion into plantlets, especially under in-vitro
conditions
 Require an additional step of maturation to acquire the capability for normal
germination
 Adventitious and axillary shoots developed in cultures in the presence of a
cytokinins generally lack roots
 Obtain full plants the shoots must be transferred to a rooting medium which
is different from the shoot multiplication medium, particularly in its
hormonal and salt compositions
 For rooting, individual shoots measuring 2 cm in length are excised and
transferred to the rooting medium
Stage 3: Rooting of shoots
Rooting & Hardening

17. Tissue culture raised plants are characterized by:
 Low photosynthetic rates
 Non-functional stomata
 Low cuticular wax
 Abnormal leaf morphology
 Accilimitization
 Green house
 2 weeks
 Temperature (±10º)
Stage 4: Transplantation & Accilimitization

18. Stages of Micropropagation

19. In-vitro regenerative protocol of V. reitzii
Nodule cluster culture formation and shoot regeneration in V. reitzii; a). Donor plant
(inflorescence of adult plant); b). Yellow nodular cultures induced in MS medium free of PGR;
c). Nodule cluster subcultureed in MS culture medium supplemented with GA3 resulted in high
proliferation rate and the subsequent development of adventitious microshoots; d).
Elongation and growth of shoot in MS culture medium free of PGR; e). Acclimatization of
plantlets; f). Growth in green house

20. Barley Propagation

21. Potato Propagation

22. Methods of Micropropagation
 Organogenesis
 Organogenesis via callus formation
 Direct adventitious organ formation
 Embryogenesis
 Direct embryogenesis
 Indirect embryogenesis
 Microcutting
 Meristem culture (Mericloning)
 Bud culture

23. Advantages of Clonal Propagation
 From one to many propagules rapidly
 Multiplication in controlled lab conditions
 Continuous propagation year round
 Potential for disease free propagules
 Inexpensive per plant once establishment

24. Disadvantages of Clonal Propagation
 Specialized equipment/ facilities required
 More technical expertise required
 Protocols not optimized for all species
 Plants produced may not fir industry standards
 Relatively expensive to set up

25. Applications of Clonal Propagation
 Produce disease and virus free plantlets
 Leads to simplification of international exchange of plants
 Increases the production of plants which are normally propagated very slowly
such as Narcissus and other bulbous crops
 Introduction of disease free new cultivars is possible through
micropropagation
 Large number of plants can be obtained in small spaces
 Rapid method for cloning of disease free trees
 Secondary metabolites
 In-vitro cultures can be stored for long time through cryopreservation
 Breeding cycle shortened
 Rare species ---- Conservation
 Production of identical sterile hybrid species. Eg.: Cabbage

26. Horticultural uses for plant tissue culture
 Clonal mass propagation
 Difficult or slow to propagate plants
 Introduction of new cultivars
 Vegetative propagation of sterile hybrids
 Pathology – eliminate viruses, bacteria, fungi etc
 Storage of germplasm

27. Thank you
A Biodiction (A Unit of Dr. Divya Sharma)