Plant Tissue Culture
Content
• General Introduction
• Types of Tissue Culture
• Plant Tissue Culture
Objective
At the end of the lecture
the student will able to:
• Explain the historical development of tissue culture
• Explain the role of nutrients of tissue culture media
• Describe the various nutrients required for tissue culture
technique
Plant
Tissue Culture
• Technique by which single plant cells or organised cell masses
(tissues) or unorganised group
of cells (callus) can be induced to multiply or regenerate in to a whole plant
– manipulating – chemical and physical environment of the culture medium
• In vitro cultivation of plant cell – under aseptic
condition in a liquid or solid media – for the production of secondary metabolites
or to regenerate plant
• Medium – Semisolid or liquid
• Initiated using plant or animal tissues and the tissue
used is called as an explant
• Tissue culture relies on three fundamental abilities of plant
– Totipotency, dedifferentiation and competency
• Totipotency – capacity/ability of any cell to regenerate –
differentiate into whole plant
• For successful plant tissue culture, explant rich in undermined
cells should be used
Eg: Cortex or meristem – Rapid proliferation
Historical
Development of Tissue Culture
• Research in botany – discovery of cell – cell theory
• 1839 – Schleiden and Schwann – cell basic unit of
organisms – cell capable autonomy – artificial environment regenerate in to
whole plant
• 1902 – German physiologist – Gottlieb Haberlandt –
developed concept of in vitro culture
• He isolated single fully differentiated individual plant
cell – plant species – palisade cells – leaves of Laminium purpureum – pith
cells from petioles of Eicchornia crassiples
• In the culture cells – increased in size, accumulated
starch – but failed to divide
• Totipotency
• Despite – failure – several predictions – requirements in media – induce cell division,
proliferation and embryo induction – G. Haberlandt – father of tissue culture
• From Haberlandt failure – Hanning – 1904 –embryogenesis –
excised mature embryos – seeds of several spp cruciferae – matured plant – mineral
salts and sugar solution
• 1908 – Simon – regenerated callus, buds, and roots –
popular stem segments – established the basis of callus culture
• Kolte &Robbins – 1922 – tissue culture –Meristamatic
cells
• Next 30 years – little progress – cell culture research
• 1926 – Fritz Went – discovered first plant growth hormone
–PGR – indole acetic acid
• White – introduced vitamin B as growth supplement – tissue culture media – tomato root tip
• Gautheret (1934) – culture of cambium cells – several tree
species – to produce callus
• Johannes Van Overbeek – 1941 – reported – growth of
seedlings – embryo – enriched culture media – coconut milk besides usual salts,
vitamins and other nutrients
• Ernest Ball – 1946
– method – which part of shoot meristem – raised whole plant – shoot tip
culture
• Muir – 1953 –callus tissue – liquid medium – shaking –
broke into single cell
• Skoog and Miller – 1955 – isolated – Kinetin – cell
division hormone
• Cocking – 1960 – first to isolate – protoplast by
enzymatic degradation of cell wall
• Bergmann – 1960 – plating technique – cloning – isolated
single protoplasts
• Murashige and Skoog – 1962 – developed – MS medium with
high salt concentration – 25 times than Knops salt solution – tobacco tissue
growth was enhanced 5 times
• Kanta and Maheswari – 1962 – role in plant genetic engineering
– first – test tube fertilization technique
• Steward – 1966 – demonstrated totipotency – regenerating
carrot
• Guha and Maheswari – 1966 – produced first haploid plant –
pollen grains of Datura
• Smith and Nathans – 1970 – discovered – first restriction
enzyme – Haemophilus influenza
• Baltimore – 1970 – isolated Reverse transcriptase from RNA
tumor virus– useful enzyme – genetic engineering – converts RNA to DNA hence
useful in constriction of Complementary DNA from RNA – Nobel Prize winner
• Carlson – 1972 –isolated protoplast from N. glauca × N.
langsdorfii –
• Zaenen et al – 1974 – discovered Ti plasmid – which
is tumour inducing principle of
agrobacterium
• O’Farrel – 1975 – high resolution two dimensional gel
electrophoresis
• Chilton et al 1977 successfully integrated Ti plasmid DNA
– Agrobacterium tumefaciens in plants
• Kary Mullis – 1983 – invented – polymerase chain reaction
– PCR –amplification of DNA
• Horsh et al – 1984 – developed transgenic tobacco –
transformation with Agrobacterium
• Blattner – 1997 – sequenced – E. coli genome
• Humon genome project consortium and Venter et al – 2001 – sequenced
– human genome successfully
• Under International Rice Genome Sequencing Project – 2005
– Rice genome sequenced
Plant
Tissue Culture Advantages:
• Independent of environmental factors such as climate,
geographical and seasonal variations, diseases, microbial contamination etc
• Ensures quality and yield of the product
• Possible to produce novel compounds of commercial value
• Cultured cells can be used to study the biosynthetic pathways
• Used to study biotransformation, bioconversion, and
specific modification of chemical structure of certain compounds
• Free from any microbial contamination and insect attack
• Possible to store germplasm of higher yielding plants at
refrigerated temperature more safely – than conventional method
• Protoplast fusion – two unrelated species – opened – new
method – field of plant improvement
Requirements
for Tissue Culture
• Area for media preparation
• Sterile room for inoculation – distribution of sterilized media –
plates/flasks, for transfer of explants to medium, sub culturing – Laminar air
flow
• Glassware, apparatus and instrument
• Large flask, test tubes, pipettes, petridishes, measuring cylinders
– preparation and transferring media
• Scissors, scalpels, forceps – preparation of explants from
excised plant parts – and its transfer
• Spirit lamp – aseptic transfer
• Autoclave – sterilize –media, glass wares, Scissors,
scalpels, forceps
• Incubator – constant temp – facilitate growth of tissues
• Fluorescent light – normal growth and development
• Shaker – maintain suspension culture
Basic
Requirements for Tissue Culture
1. Culture Media
• Plant cells/ tissues – proper medium – growth and development
• Inorganic elements
Macronutrients – P, S, N, K, Ca, Mg
Micronutrients – B, Fe, Cu, Mn, Zn, Mb, Al, Ni
• Organic compounds
Carbon source – growth – Sucrose or glucose – 2 to 5 %
Nitrogen source – growth and synthesis – protein – nitrates
or amino acids
Vitamins – Thiamine, pyridoxine, nicotinic acid
Phytohormones/ –
Auxins, gibberellins, ethylene, cytokinins
Auxin: Responsible
for cell elongation, Inter node elongation, leaf growth, cambial activity,
fruit growth, apical dominance, Influencing physical, chemical properties in
leaf abscission
Eg: IAA (Natural auxin), synthetic auxin – NAA, IBA, 2, 4 –
D
Cytokinins: Cell
division (cytokinesis)
• Regulate the pattern and frequency of organ production
• Kinetin – autoclaved DNA of herring sperm
• Zeatin – liquid endosperm of coconut and in maize embryos
• Promote the formation of adventitious buds and shoots from
undifferentiated cells
• Complex extracts
Natural plant extracts – Casein hydrolysate, yeast extract,
malt extract, coconut milk, potato extract, and tomato juice
• Water –
Demineralised and double distilled water
• Agar – Gelling
agent – solid media
2. Area for medium
preparation:
Separate area and should be away from the working laboratory
• Used only for media preparation
Should be equipped with
• Glass ware
• Laminar air flow bench
• Weighing balance
• PH meter
• Deep freezer
• Preparation of
culture media: Chemicals – highest grade of purity
• Prepare different stock solutions as follows
Stock solution I – Macro nutrients – distilled water 4
months – 4˚C
Stock solution II – Micro nutrients – distilled water 4
months – 4˚C
Stock solution III – Vitamins – 100 ml – d. water – stored –
refrigeration – used within short period
Stock solution IV – phytohormones – 100 ml – d. water – cool
place
• To prepare 1 litre – pipette required – stock solutions
and make up the volume to 950 ml with dist water – conical flask
Preparation of
Culture Media
• Add sucrose (3 %) and amino acids – adjust the pH to 5.6 –
0.1 M sodium hydroxide solution or 0.1 M HCl
• Finally volume – 1 litre – distilled water
• Transfered to conical flask – plugged – non adsorbent
cotton
• Solid media – 2 % agar
• Sterilize the media by autoclaving at 120 0C, 15 lb for 20
min
• Thermo labile components
– carbohydrates, vitamins,
growth regulators, plant extracts – sterilised – microfiltration –
aseptically added – sterile media – but before the solidification of agar – set
Eg: MS media (Murashige & Skoog), LS media (Leinsmair – Skoog)
White’s media, B5 media
Summary
• Tissue culture is growing of tissues or cells on a
suitable nutrient medium under aseptic conditions in vitro, may be plant or
animal tissue culture
• Types include callus, suspension, hairy root, single cell,
haploid, organogenesis, embryo culture etc
• Technique include selection and sterilization of
apparatus, media, and explant, establishment of culture and growth measurement