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PLANT TISSUE CULTURE

Presenter: Dr. M. IRSHAD


Contents
• Definition
• History
• Types of growth
• Steps involved in plant tissue culture
• Common problems
• Applications
• Advantages
Definition:
Plant tissue culture is the science of growing
plant cells, tissues or organs isolated from the
Mother plant, on artificial media in vitro (in
glass) under controlled conditions

Edwin F. George, M. A. H., Geert-Jan De Klerk (2008). "Plant Propagation by Tissue


Culture 3rd Edition." Volume 1. The Background.
Pioneers of PTC
History
Contin…
H. Haberlandt (1902) attempted to culture isolated
mesophyll cells but not succeeded.
R.J. Guatheret (1939) callus culture of carrot.
F. Skoog and C.O. Miller (1957) put forth the Hormone
hypothesis

S.G. Guha and S.C. Maheshwari (1966) cultured


pollens to obtain haploid plant.
A.F. Mascarens (1991) induced flowering in bamboo plant
by tissue culture technique.
Types of Growth
Organized Growth
It occurs when plant cell or tissue (explant) are
transferred to culture media and continue to grow
with their structure preserved. e.g shoot, root
Unorganized Growth
It occurs when pieces of whole plants are cultured in
vitro and the cells aggregate and typically lack any
recognizable structure.
e.g. Callus and Cell Suspension cultures.
Edwin F. George, M. A. H., Geert-Jan De Klerk (2008). "Plant Propagation by Tissue Culture 3rd Edition." Volume 1. The Background.
Contin…

Organised growth Unorganized growth Unorganized growth


“Callus” “Suspension”
Steps involved in plant tissue culture
Cleaning of glassware

Preparation of nutrient medium

Selection and sterilization of explant.

Inoculation of aseptic explant in to nutrient medium.

Proliferation of shoots on a multiplication medium.

Transfer of shoots for sub-culturing.

Rooting and hardening of plantlets

Field trials.
Cleaning of glassware

Borosilicate glassware (Corning/Pyrex) is used. Graduated measuring cylinders,


conical flasks beakers

petridishes, pipettes (2 ml, 5 ml and 10 ml) glass rods

centrifuge tubes culture vials, culture tubes

bottles
Medium preparation
• Solid
• Semisolid
• Liquid
(with use of Agar Powdered)
Nutrient medium

MS medium is the most commonly used for plant tissue culture


Selection and sterilization of explants

Explant
Cell, tissue or organ of a plant that is used to start in vitro cultures
Sterilization
Aseptic environment, use of some chemicals, Hgcl2, NaOCl, Methanol

cultures
Inoculation of aseptic explant in to
nutrient medium
• Depends on our aims and objectives
Two main types
• Indirect organogenesis ( callus stage is there)
• Direct organogenesis (No callus involved)
Indirect organogenesis

Kumar et al 2016
Direct organogenesis
How this is possible?

• Two major hormones affect Plant Differentiation


Auxins: Stimulates Root Development
Cytokinin: Stimulates Shoot Development
• Generally, the ratio of these two hormones can
determine plant development
• – ↑ Auxin ↓Cytokinin = Root Development
• – ↑ Cytokinin ↓Auxin = Shoot Development
Plant hormones VS PGRs
Plant Hormones are naturally occurring chemicals that influence plant
growth.
Plant Growth Regulators are synthetic versions of hormones

Auxin : IAA,IBA,NAA
Cytokinin: BAP,BA,TDZ
The action of the different hormones / regulators is not consistent:

• Different plants will respond to the same chemical differently. Different


plant parts from the same plant can respond differently
PGRs balance in Tissue
culture
• Auxin Cytokinin

High Low
Root formation on cuttings
Adventitious root formation in callus
Callus initiation
Adventitious shoot formation
Axillary shoot growth

• Low High
Shoot proliferation and sub culturing

• Multiplication of shoots with PGRs manipulation


Rooting and acclimatization
Field Trails
Schematic diagram
Contin……
Common challenges

Contamination Hyperheidcity Browning


Tissue Culture Applications
• Micropropagation
• Germplasm preservation
• Somaclonal variation
• Haploid & dihaploid production
• In vitro hybridization – protoplast fusion
Advantages
• From one to many propagules rapidly
• Multiplication in controlled laboratorium
conditions
• Continuous propagation year round
• Potential for disease-free propagules
• Inexpensive per plant once established
• Precise crop production scheduling
• Reduce stock plant space
Limitations

• Specialized equipment/facilities required


• More technical expertise required
• Protocols not optimized for all species
• Plants produced may not fit industry standards
• Relatively expensive to set up