INTRODUCTION AND HISTORY OF

PLANT TISSUE CULTURE

SIR SOHAIL AHMED

Course: Animal , Plant Tissue Culture Lab
(APTC)
SZABIST
 WHAT IS PLANT TISSUE CULTURE ?

It is a technique of growing cells, tissues, organs or whole organism in

vitro (in glass) on artificial culture medium under aseptic and
controlled conditions.
 WHO IS FATHER OF PLANT TISSUE CULTURE

Gottlieb Haberlandt (Austrian botanist) (1854-1945) attempted to

cultivate plant tissue culture cells in vitro.

He is regarded as the father of Plant Tissue Culture.

 TIMELINE OF PLANT TISSUE CULTURE (PTC)
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: otte was successful in obtaining growth from isolated root tips on inorganic media. Robbins
reported similar success from root tip and stem tip.
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.
1939: Tobacco crown gall culture, callus obtained: called as Plant Cancer.
1940: Coconut milk used in plant cultures to obtain heart-shaped embryos.
1950s: Skoog used adenine sulfate to obtain buds on tobacco segments:
1958: 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

1970: Discovery of restriction endonuclease (Daniell Nathan).

1972-73: First recombinant molecule created by Stanley Cohen.

1974: Discovery of Ti plasmid in Agrobacterium tumefaciens (by Zaenen)

1983: First transgenic plant. (Monsanto).

1985: Leaf disk transformation method (Monsanto)

LANDMARK ACHIEVERS OF PTC
FUNDAMENTAL PRINCIPLES OF PLANT TISSUE CULTURE

Plant Tissue Culture depends upon following;

Totipotency :- It is the ability of plant cells to regenerate into a whole plant

Plasticity :- It is the ability of plants to alter their metabolism, growth and

development to best suit their environment

Dedifferentiation :- Capacity of mature cells to return to meristematic condition and

development of a new growing point, follow by redifferentiation which is the ability to
reorganize into new organ
 BASIC PHYTOHORMONES & THEIR FUNCTION

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

PLANT GROWTH REGULATORS USED IN PLANT TISSUE CULTURE
MEDIA WITH MOLECULAR WEIGHTS (MW)
STEPS IN PLANT TISSUE CULTURE TECHNIQUE

Selection of plant
↓
Isolation of explant
↓
Sterilization of explant
↓
Inoculation of explant
↓
Incubation
 ↓
Initiation of callus
↓
Sub culturing
↓
Regenaration
↓
Hardening
↓
Transfer of plantlets to Green house or
open field
 WHAT IS EXPLANT ?

Plant tissue cultures are generally initiated from multicellular tissue fragments,
called explants, obtained from living plants. Explants may originate from a
wide range of plant tissues, such as…

leaf, stem, root, petiole, hypocotyl, cotyledon, embryo, or meristem

 SELECTION OF EXPLANT

The explant is selected it is either haploid or diploid explant

The plant growth can be achieved in two ways:

1. Shoots directly by appropriate media

2. By somatic embryogenesis
 ENVIRONMENTAL CONDITIONS

There are Three important aspects in vitro culture

Nutrient medium

Aseptic condition

Aeration of the tissue

 NUTRIENT MEDIUM

The nutrient medium commonly contains

• Macronutrient, micronutrient, vitamins, Ferron & carbon source
• The optimum culture medium may vary with the species, the genotype
within the species, and the origin and age of the cultured tissue.
• The preferred physical state of the culture medium, whether a liquid medium
or a solid agar gel, may vary with the species and the culture environment.
• pH- 5.8
 BASIC ELEMENT & THEIR FUNCTION

M
A
C
R
O

M
I
C
R
O
 ASEPTIC CONDITION

• Nutrient medium contains sugar which increases growth of microbes

• These microbes compete with growing tissue and finally kill it.

• It is important to maintain aseptic condition.

• Sterilization is very important to stop the growth of microbes.

 STERILIZATION TECHNIQUES

sterilization is achieved by one of the following approaches:

(i) Dry heat treatment (i.e. microwave)
(ii) Flame sterilization
(iii) Autoclaving
(iv) Filter sterilization
(v) Wiping with 70% ethanol
(vi) Surface sterilization.
 AERATION OF THE TISSUE

• Proper aeration of the cultured tissue is also an important aspect of culture

technique.

• It is achieved by occasionally stirring the medium by stirring or by automatic

shaker
 CULTURE MEDIA

Explants are then usually placed on the surface of a solid culture medium, but
are sometimes placed directly into a liquid medium, when cell suspension
cultures are desired.

Culture media are generally composed of inorganic salts plus a few organic
nutrients, vitamins and plant hormones.
 MAJOR TYPES OF MEDIA

MS medium - formulated by Murashige and Skoog (MS) is most widely used for
many types of culture systems

White’s medium - is one of the earliest plant tissue culture media

B5 medium - developed by Gamborg for cell suspension and callus cultures and
at present it’s modified form used for protoplast culture

N6 medium - formulated by Chu and used for cereal anther culture

Nitsch’s medium - developed by Nitsch and Nitsch and used for anther culture
TYPE OF PLANT TISSUE CULTURE
 PLANT TISSUE CULTURE IN DEVELOPMENT OF MEDICINE

• The elucidation of the biosynthetic pathways of secondary metabolites

with isolation of corresponding enzymes.

• Discovery of new secondary metabolites in vitro.

• The commercial production of expensive secondary metabolites .

• They have also been used for metabolic and genetic studies.

• The selection of superior strains of medicinal plants.

 APPLICATION OF PLANT TISSUE CULTURE

• The commercial production of plants used as potting, landscape, and florist

subjects

• To conserve rare or endangered plant species.

• To screen cells rather than plants for advantageous characters, e.g.

herbicide resistance/tolerance.

• Large-scale growth of plant cells in liquid culture

• Bioreactors for production of valuable compounds, like plant-derived
secondary metabolites and recombinant proteins used as
biopharmaceuticals

• To cross distantly related species by protoplast fusion and regeneration of

the novel hybrid.

• To produce clean plant material from stock infected by viruses or other

pathogens.

• Production of identical sterile hybrid species can be obtained

 LIMITATIONS OF PLANT TISSUE CULTURE
• Specialized equipment required as; Laminar flow cabinets, Autoclave, Water
purification systems, Glassware etc.
• High labor cost is the most limiting factor as; Skilled labor required
• Contamination risks
• Maintenance of aseptic (sterile) environment difficult.
• Rapid spread of contaminants = widespread loss.
• Risk of mutation arising

⚫ Artificial environment induces mutations.

⚫ Responses to tissue culture conditions varies

⚫ Trial and error to determine optimum media or conditions

 FACTORS AFFECTING TISSUE CULTURE EFFICIENCY

Plant regeneration from tissue culture varies with the following parameters:

Plant species

Genotype within the species

Source of the cultured tissue

Age and health of the donor plant

Nutrient medium
QUESTIONS , COMMENTS & FEEDBACK