Lecture - 11 Plant tissue culture: Introduction, history, tools, techniques and applications

Plant tissue culture: Introduction, history, tools, techniques and applications:-

History of Plant Tissue Culture:-

· Haberlandt (1902):- ‘’It is possible to grow embryos from somatic cells.’’ This idea was visualized by him in 1902. He tried to grow the mature leaf cells of Lamium purpureum and hair cells of Tradescantia and Pulmonaria in knop’s solution containing 1 – 5% sucrose without success.

· Robbins and Kotte (1922):- They independently were able to grow the root tips of maize and pea for a limited period.

· White (1922):- He successfully grew the root tips of tomato (Lycopersicum esculentum) continuously for a prolonged period in liquid medium containing inorganic salts, yeast extract and sucrose.

· Kogl (1934):- He reported that the growth substance invented by Went (1926) was IAA (Indole Acetic Acid).

· Snow (1935):- He demonstrated the ability of IAA to stimulate cambial activity.

· White, Nobecourt and Goutheret (1939):- They simultaneously and independently reported establishment of tomato, Salix capraea and Populus nigra callus cultures for prolonged period.

· Nobecourt (1939):- He reported the differentiation of roots in callus cultures of carrot.

· White (1939):- He successfully induced leafy shoot buds in tobacco stem cultures.

· Since 1939 it was possible to culture:-

i. Embryos (Van Overbeck - 1941)

ii. Single isolated cell (Muir - 1954)

iii. To establish suspension cultures (Steward and Shantz - 1955)

· Dawson (1942):- He demonstrated the innate capacity of plant tissue cultures to biosynthesize important metabolites in vitro which led to the production of many secondary metabolites of vital importance.

· Miller and Skoog (1955):-

Ø Miller discovered the kinetin.

Ø Miller and Skoog demonstrated the concept of hormonal control of organ formation using tobacco tissue cultures.

· Steward (1958):- He revealed the inherent capacity of a somatic cell to regenerate into complete plant which proved the totipotency of cells.

· Steward and Rienert (1958):- They reported somatic embryogenesis in carrot cultures.

· Morel (1960):- He produced pathogen free orchid plants through meristem culture and this method was later exploited in many plant species.

· Guha and Maheshwari (1964):- He induced haploid embryos and subsequently plantlets from pollen or anther cultures of Datura inoxa.

· Cocking (1960):- He reported enzymatic isolation of protoplasts from root tips of tomato (Lycopersicum esculentum).

· Power (1970):- He gave the idea of protoplast fusion.

· Takebe (1971):- He reported successful regeneration of plantlets from protoplasts isolated from tobacco mesophyll cells.

· Since 1971 many reports are available on the production of interspecific and intergeneric hyrids.(Bhojwani and Rajdan - 1983)

· Heinz and Mee (1969, 1971):- Their observation of somaclonal variants encouraged many researchers to work in this direction and allowed them to release many varieties with desired characters.

· Redenbaugh (1984):- He reported the encapsulation of somatic embryos with a view to produce artificial seeds.

· Kitto and Janick (1985) and Ow (1986):- They successfully demonstrated the genetic transformation and obtained transgenic tobacco plants by transformation of fire fly luciferase gene.

Plant tissue-culture:- Plant tissue culture is ideally done in 7 steps: -

1. Selection of Explant:-

• Explant:- The part of the plant which is selected for culturing on the medium is called explant.

2. Sterilization of Explant:-

• To destroy the spores of bacteria or fungi present on the surface of explant, the following sterilizing agents can be used:-

i. NaOCl = 1-1.4%

ii. Ca (OCl)2 = 9-10%

iii. H2O2 = 10-12%

iv. AgNO3 = 1%

v. Hg2Cl2 = 0.01 - 1%

3. Sterilization of Glasswares:-

• Glass wares are sterilised by dry hot air in the oven.

4. Sterilization of Culture medium:-

• The culture medium is sterilised at 121 ° C temperature and 15 p.s.i. pressure in autoclave for 30 minutes. Sterilization occurs by hot steam in the autoclave.

5. Pouring and Inoculation:-

• The LAF cabinet is used for this step. [LAF = Laminar Air Flow]

• In LAF first of all the internal environment is sterilised by UV light for 15 minutes.

• The air in the LAF flows through the HEPA filter to the user.

[HEPA = High Efficiency Particulate Air]

• The pore size of HEPA filter is 0.3µm through which 99.97% of airborne particles cannot pass out.

• Pouring:- The medium is removed from the autoclave and brought to the LAF cabinet. Now, we pour the medium in the culture vessel, in which the explant is to be cultured. This is called Pouring.

• Inoculation:- When the medium cools and freezes, the explants are transferred on to the medium in the culture vessel. This is called Inoculation.

6. Incubation:-

• Culture vessels are sealed with cotton plugs and placed in the Incubator.

• A suitable amount of temperature, light and humidity is provided to the explant in the incubator.

• In a few weeks, the seedlings are developed from explants.

7. Hardening and Acclimatization:-

• The plant is removed from the adapted environment of the incubator and transferred to the green house. Where the plant has to face some adverse conditions.