What is it?

• Tissue culture is the term used for “the process of growing cells artificially in the laboratory”

• Tissue culture involves both plant and animal cells

• Tissue culture produces clones, in which all product cells have the same genotype (unless affected by mutation during culture)

What’s the Background?

• Tissue culture had its origins at the beginning of the 20th century with the work of Gottleib Haberlandt (plants) and Alexis Carrel (animals)

Haberlandt

Carrel

The Background, II• The first commercial

use of plant clonal propagation on artificial media was in the germination and growth of orchid plants, in the 1920’s

• In the 1950’s and 60’s there was a great deal of research, but it was only after the development of a reliable artificial medium (Murashige & Skoog, 1962) that plant tissue culture really ‘took off’ commercially

Young cymbidium orchids

The Background, III

• A more recent advance is the use of plant and animal tissue culture along with genetic modification using viral and bacterial vectors and gene guns to create genetically engineered organisms

Plant Tissue Culture: historical highlights

1902: Haberlandt attempted to the culture mesophyll tissue and root hair cells. This was the first attempt of in vitro culture.1904: Haning attempted to culture excised embryos from mature seeds.1922: Kotte was successful in obtaining growth from isolated root tips on inorganic media. Robbins reported similar success from root tip and stem tip.1920-40: First PGR, IAA, discovered by experiments on oat seedlings (Fritz Went).1934: Used yeast extract (vit B) with inorganic salts to repeatedly culture root tips of tomato.1935: Importance of B vitamins and PGRs in culture of mesophyll cells.1936: Haning experiment was repeated with IAA: works !!!1939: Tobacco crown gall culture, callus obtained: called as Plant Cancer.1940: WWII. Coconut milk used in plant cultures to obtain heart-shaped embyos.1950s:Skoog used adenine sulfate to obtain buds on tobacco segments: PGR #2 identified: kinetin1958: Stewart and Reinert obtained somatic embryos from carrot cells using PGRs.1950-60s: Botanists turned to plant tissue culture to study plant development.

1960: Cocking isolated protoplasts from cultured cells.1962: Murashige and Skoog developed MS media for tobacco.1966: Guha and Maheshwari obtained first haploid plants (Delhi Univ., India)1970: Discovery of restriction endonuclease (Daniell Nathan). Plasmids were already known.1972-73: First recombinant molecule created by Stanley Cohen, Stanford Univ.1974: Discovery of Ti plasmid in Agrobacterium tumefaciens (by Zaenen in Ghent

Univ., Belgium)1970-80s:Ti plasmid analysis (Nester, Seattle; Van Montagu, Ghent)1983: First transgenic plant. (Monsanto, Ghent, Washington Univ).1985: Leaf disk transformation method (Monsanto)

Tissue Culture: De-differentiation to Regeneration

seeds

Wheat inflorescence

+ auxinCallus- auxin/ +cytokinin

embryos

Totipotency: ability of a cell or tissue or organ to grow and develop into a fully differentiated organism.

Plant cells are totipotent

What is needed?Tissue culture, both plant and animal has

several critical requirements:

• Appropriate tissue (some tissues culture better than others)

• A suitable growth medium containing energy sources and inorganic salts to supply cell growth needs. This can be liquid or semisolid

• Aseptic (sterile) conditions, as microorganisms grow much more quickly than plant and animal tissue and can over run a culture

What is Needed, II

• Growth regulators - in plants, both auxins & cytokinins. In animals, this is not as well defined and the growth substances are provided in serum from the cell types of interest

• Frequent subculturing to ensure adequate nutrition and to avoid the build up of waste metabolites

A. The physical factors:

• Temperature

• pH

• The gaseous environment

• Light (quality and duration)

• Osmotic pressure

All these factors have to be maintained within acceptable limits

for the success of plant tissue culture

The culture environment

• Nitrogen (N)• Potassium (K)• Phosphorous (P)• Calcium (Ca)• Magnesium (Mg)• Sulfur (S)

• Iron(Fe)• Manganese (Mn)• Zinc (Zn)• Boron (B)• Cupper(Cu)• Molybdenum(Mo)• Cobalt (Co)• Iodine (I)

2. Organic supplement

a) Vitamins:

• Only thiamine (vitamin B1) is essential for most plant cultures, it is required

for carbohydrate metabolism and the biosynthesis of some amino acids,

b) Myo-inositol

• Although it is not essential for growth of many plant species, its effect on

growth is significant.

c) Complex organics

• Such as coconut milk, coconut water, yeast extract, fruit juices and fruit

pulps.

3. Source of carbon

Sugars

• Most plant tissue cultures are not highly autotrophic due to

limitation of CO2. Therefore, sugar is added to the medium as an

energy source.

• Sucrose is the most common sugar added, although glucose,

fructose, and sorbitol are also used in certain instances.

• The concentration of sugars in nutrient media generally ranges

from 20 to 40 g/l.

• Sugars also contribute to the osmotic potential in the culture

4) Gelling agents

• When semi-solid or solid culture media are required, gelling

agents are used.

a. Agar

• Agar is the most commonly used gelling agent.

•Composition: Agar consists of 2 components

1.Agarose is an alternating D-galactose and 3,6-anhydro-L-galactose with side

chains of 6-methyl-D-galactose residues.

2.Agaropectin is like agarose but additionally contains sulfate ester side chains

and D-glucuronic acid.

•Agar tertiary structure is a double helix the central cavity of which can

accommodate water molecules

• Advantages:

Agar is an inert component, form a gel in water that melt at 100 ° C

and solidify at nearly 45 ° C

Concentrations commonly used in plant culture media range between

0.5% and 1%

If necessary, agar can be washed to remove inhibitory organic and

inorganic impurities.

• Disadvantages:

Agar does not gel well under acidic conditions (pH <4.5).

The inclusion of activated charcoal in media may also inhibit gelling

of agar.

b) Agarose

• It is extracted from agar leaving

behind agaropectin and its sulfate

groups.

• It is used when the impurities of

agar are a major disadvantage.

c) Gelrite™

• Gelrite consists of a polysaccharide

produced by the bacterium Pseudomonas elodea.

• It gives clear-solidified medium that

leads to detection of contamination at an

early stage.

• Gelrite requires more stirring than agar.

• Concentration of divalent cations such as calcium and

magnesium must be within the range of 4-8 mM/L or the

medium will not solidify

d) Phytagel™

• It is an agar substitute produced from a bacterial substrate

composed of glucuronic acid, rhamnose and glucose.

• It produces a clear, colorless, high-strength gel, which aids in

detection of microbial contamination.

• It is used at a concentration of 1.5-2.5 g/L.

• It should be prepared with rapid stirring to prevent clumping.

Commertial Media Formulations:

• Murashige and Skoog (MS)

• Linsmaier and Skoog (LS)

• White Medium

• Gamborg medium

• Schenk and Hildebrandt medium

• Nitsch and Nitsch Medium

• Lloyd and McCown Woody plant medium

• Knudson’s medium

C. Plant growth regulators:

Definition:

Plant hormone or

Phytohormone or

Plant growth regulator (PGR):

• They are small organic molecule that elicits a physiological

response at very low concentrations.

Characteristics:

1. Synthesized by plants.

2. Show specific activity at very low concentrations

3. Display multiple functions in plants.

4. Play a role in regulating physiological phenomena in vivo in a dose-

dependent manner

5. They may interact, either synergistically or antagonistically, to

produce a particular effect.

Growth Regulators

Plant Hormones

• Natural (made by plants) – also called hormones

• Synthetic (man made)• Also called PGRs (plant growth

regulators)• Purposes: start growth, stop

growth, modify growth & development

5 Known Plant Hormones:

• Auxins (ox ins)• Gibberellins (jib ber ill ins)• Cytokinins (site oh kine ins)• Ethylene (eth el een)• Abscisic acid (ab sis ick)

Hormones may act individually or together

Auxins

• Stem elongation• Produced in tips

of stems (“B” in photo)

• Migrate from cell to cell in stems

Phototropism – ability to bend towards light

• Auxins - responsible for plants bending towards light.

• Auxins - move down shaded side of the stem and cause cells to elongate

Gravitropism (geotropism) – plant response to gravity

• Auxins – responsible for plant response to gravity

• Auxins – move to lowest side and cause stem tissue to elongate – stem curves upwards

Apical dominance

• Auxins – move down the stem from the terminal bud and inhibit growth of side shoots

Pinching

• Pinching = removing the terminal bud

• Pinching - stops flow of auxins down the stem and allows side shoots to develop

• Produces bushy, well-branched crops

Root development

• Auxins encourage root development in cuttings

• Some plants produce plenty of auxins to make rooting cuttings easy

• Other plants need synthetic auxins such as IBA

Gibberellins• Cell elongation and cell

division• Stimulate development

of flowers (as in “gibbing” camelias)

• Cause internodes to stretch

• Produced in stem and root apical meristems, seed embryos, young leaves

Internode Elongation• Gibberellins cause

internodes to stretch in relation to light intensity.

• High light intensity = no stretch

• Low light intensity = long internodes. Leaves are raised to capture light

Problems with Internode Elongation

• Greenhouse problem – plants spaced too closely to one another

• Plants shade one another – results in stretching, less compact plants, weaker stems, loss in value $$$

• B-Nine is a growth regulator that inhibits gibberellin and controls plant height in bedding plants

Cytokinins

• Cell division (used in tissue culture)• Cell differentiation (used in tissue culture for plant

organ formation)• Formation of callus tissue• Delay aging process in plants• Produced in roots• Transported through xylem• Still researched

Cytokinins vs. Auxins

• In stems – auxins inhibit lateral shoots, cytokinins promote lateral shoots

• In roots – Auxins promote root branching, cytokinins inhibit root branching

• Work together to control cell differentiation and cell division

Ethylene Gas

• Colorless gas• Produced in nodes of stems, ripening

fruits, dying leaves

Ethylene exposure

• Thickens stems• Breaks down

chlorophyll• Weakens cell

membranes• Softens cell

walls

Abscisic Acid – The Plant Stress Hormone

• Growth inhibiting hormone• Responsible for seed dormancy• Responsible for closing stomata

during drought

Synthetic Growth Regulators• Rooting Compounds –

• increase rooting %• speed rooting• increase number

and quantity of roots

• increase uniformity of roots

Rooting compounds• Liquid or mixed with talc

Growth Retardants• Widely used in the greenhouse industry• Inhibit action of gibberellins on stem

elongation

• Explants : Sterile pieces of a whole plant from which cultures

are generally initiated

• Types of explant:

Generally all plant cells can be used as an explant, however

young and rapidly growing tissue (or tissue at an early stage of

development) are preferred.

Culture Types

a) Root tip:

• Root cultures can be established from explants of the root tip of

either primary or lateral roots.

b) Shoot tip:

• The shoot apical meristem from either axillary or adventitious buds

can be cultured in vitro.

c) Embryo:

• Both immature and mature embryos can be used as explants to

generate callus cultures or somatic embryos.

• Immature, embryo-derived callus is the most popular method of

monocot plant regeneration.

d) Haploid tissue

• Male gametophyte (Pollen in anthers) or female gametophyte

(the ovule) can be used as an explant.

• Haploid tissue cultures can produce haploid or di-haploid

plants due to doubling of chromosomes during the culture

periods.

A. Callus:

• Definition: It is an unspecialized and

unorganized, growing and dividing mass

of cells, produced when explants are

cultured on the appropriate solid medium,

with both an auxin and a cytokinin and

correct conditions.

• During callus formation there is some

degree of dedifferentiation both in

morphology and metabolism, resulting in

the lose the ability to photosynthesis.

• This necessitates the addition of other components e.g.: vitamins and, a

carbon source to the culture medium, in addition to the usual mineral

nutrients.

• Habituation: it is the lose of the requirement for auxin and/or cytokinin

by the culture during long-term culture.

• Callus cultures may be compact or friable.

Compact callus shows densely aggregated cells

Friable callus shows loosely associated cells and the callus becomes

soft and breaks apart easily.

B. Cell-suspension cultures

• When friable callus is placed into the appropriate liquid medium and

agitated, single cells and/or small clumps of cells are released into the

medium and continue to grow and divide, producing a cell-suspension

culture.

• The inoculum used to initiate cell suspension culture should neither be too

small to affect cells numbers nor too large too allow the build up of toxic

products or stressed cells to lethal levels.

• Cell suspension culture techniques are very important for plant

biotransformation and plant genetic engineering.

Culturing (micropropagating) Plant Tissue - the steps

• Selection of the plant tissue (explant) from a healthy vigorous ‘mother plant’ - this is often the apical bud, but can be other tissue

• This tissue must be sterilized to remove microbial contaminants

The Steps, II• Establishment of the

explant in a culture medium. The medium sustains the plant cells and encourages cell division. It can be solid or liquid

• Each plant species (and sometimes the variety within a species) has particular medium requirements that must be established by trial and error

The Steps, III

• Multiplication- The explant gives rise to a callus (a mass of loosely arranged cells) which is manipulated by varying sugar concentrations and the auxin (low): cytokinin (high) ratios to form multiple shoots

• The callus may be subdivided a number of times

Dividing shoots

Warmth and good light are essential

Multiplication

• Two Hormones Affect Plant Differentiation:– Auxin: 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– Auxin = Cytokinin = Callus Development

What is Callus development

• A callus is a blob of tissue – (mostly undifferentiated cells)

• A callus is naturally developed on a plant as a result of a wound

• This callus can be left to develop or can be further divided

The Steps, IV

• Root formation - The shoots are transferred to a growth medium with relatively higher auxin: cytokinin ratios

The bottles on these racks are young banana plants and aregrowing roots

Tissue culture plants sold toa nursery & then potted up

The Steps, V

• The rooted shoots are potted up (deflasked) and ‘hardened off’ by gradually decreasing the humidity

• This is necessary as many young tissue culture plants have no waxy cuticle to prevent water loss

Why do Plant Tissue Culture?

• A single explant can be multiplied into several thousand plants in less than a year - this allows fast commercial propagation of new cultivars

• Taking an explant does not usually destroy the mother plant, so rare and endangered plants can be cloned safely

• Once established, a plant tissue culture line can give a continuous supply of young plants throughout the year

Why do Plant Tissue Culture, II

• In plants prone to virus diseases, virus free explants (new meristem tissue is usually virus free) can be cultivated to provide virus free plants

• Plant ‘tissue banks’ can be frozen, then regenerated through tissue culture

• Plant cultures in approved media are easier to export than are soil-grown plants, as they are pathogen free and take up little space (most current plant export is now done in this manner)

Why do Plant Tissue Culture, III

• Tissue culture allows fast selection for crop improvement - explants are chosen from superior plants, then cloned

• Tissue culture clones are ‘true to type’ as compared with seedlings, which show greater variability