Plant tissue culture and its application

Plant tissue culture

Growing the plant cells, tissues and organs on a artificial, synthetic medium under controlled conditions is called plant tissue culture. Plant tissue culture has become a major thrust area in plant biotechnology.

Concept

The basic concept of plant tissue culture is totipotency, differentiation, dedifferentiation and redifferentiation.
Totipotency
The inherent potential of any living plant cell to develop into entire organism is called totipotency. This is unique to plant cells.

Differentiation

The meristematic tissue is differentiated into simple or complex tissues. Dedifferentiation
Reversion of mature tissue into meristematic state leading to the formation of callus is called dedifferentiation.

Redifferentiation

The ability of the callus to develop into shoot or root or embryoid.

The origin and development of plant tissue culture

The beginning of plant tissue culture was made as early as 1898, when a German Botanist G. Haberlandt successfully cultured individual plant cells, isolated from different tissues. But only during 1934 to 1939, a foundation of plant tissue culture was laid down by three scientists (Gauthret, White and Nobecourt) due to discovery of plant growth regulators such as auxins and vitamins.

During next twenty years (1940 to 1960) a variety of growth regulator such as cytokinins were identified for their effect on cell division, growth and differentiation.
After 1960, in vitro culture of plant cells, tissues and organs was reasonably well developed. Research in this area was initiated in early
1960s by Prof. P. Maheshwari and Prof. S. Narayanaswamy at the Department of Botany, University of Delhi in India. Consequently, media and culture techniques for a variety of plant materials became known, which are now extensively utilised in all areas of plant improvement programmes.

Basic techniques of plant tissue culture

1. Culture vessels

The culture vessels used for plant tissue studies includes Erylenmayer flask (conical flask), petri plates and culture tubes (25 x 150mm).

2. Culture medium

The important media used for all purpose experiment are Murashige and Skoog medium (MS medium), Gamborg medium (B5 medium), White medium (W medium) and Nitsch medium. The culture medium is closed with cotton plug/ or aluminium foil sheet. The pH of the medium is adjusted to 5.8 (acidic range).

3. Sterilization

Sterilization is the technique employed to get rid of the microbes such as bacteria and fungi in the culture medium and plant tissues. So, it is important to sterilize the culture medium and plant tissue.

The culture medium can be sterilised by keeping it in an autoclave and maintaining the temperature of 121’C for 15 minutes. The plant tissue (inoculum) is to be surface sterilised.
Chemical sterilization
By treating the inoculum in any one of the chemical sterilizant such as Sodium hypochlorite, Calcium hypochlorite, Mercury chloride for 15 to 20 minutes followed by repeated washing in sterile water upto 3 to 5 times.

4. Inoculation

Transfer of explant (root, stem, leaf, etc.) on to a culture medium is called inoculation. The inoculation is carried out under aseptic condition for which an apparatus called laminar air flow chamber is used. Flamed and cooled forceps are used for transfer of plant materials to different culture media kept in glasswares.

5. Incubation

The culture medium with the inoculum is incubated at 26 + 20C with the light intensity at 2000 to 4000 lux (unit of intensity of light) and allowing photoperiod of 16 hour of light and 8 hours of darkness.

6. Induction of callus

Due to activity of auxins and cytokinins, the explant is induced to form callus. The callus is an unorganized mass of undifferentiated tissue. The mechanism of callus formation is that auxin induce cell elongation and cytokinin induces cell division as a result of which masses of cells are formed.

7. Morphogenesis

Formation of new organs from the callus under the influence of auxin and cytokinin is called morphogenesis. Roots and shoots are differentiated from the callus. Such embryos are called somatic embryos result in the formation of young plantlet.
There are two types of morphogenesis

a. Organogenesis

Formation of new organs such as shoot and root is known as organogenesis. The development of shoot from the callus is called caulogenesis and formation of root is called rhizogenesis respectively.

b. Embryogenesis

Formation of embryos (ie. bipolar structure having shoot and root) from the callus is called embryogenesis. These embryos arise from somatic callus tissue and are called somatic embryos or embryoids or somaclonal embryos.

8. Hardening

Exposing the plantlets to the natural environment in a stepwise manner is known as hardening. Finally the plantlets are gradually transferred to the soil.