Tissue Culture Technique

What is Tissue Culture Technique?

Tissue culture is a process that involves exposing plant tissue to a specific regimen of

nutrients, hormones, and light under sterile, in vitro conditions to produce many new plants,

each a clone of the original mother plant, over a very short period of time.

HistoryHaberland (1902) attempted to culture

isolated mesophyll cells but not succeeded.Guatheret (1939) cultured callus of carrot.Miller (1957) put forth the Hormone

hypothesisS.G. Guha (1966) cultured pollens to obtain

haploid plant.A.F. Mascarens (1991) induced flowering in

bamboo plant by tissue culture technique

Basic concepts of plant tissue culture(PTC)

Two concepts1) Plasticity.

-ability to initiate cell division from almost any tissue of the plant.

-ability to regenerate lost organs.

2) Totipotency. -each cell has the capacity to regenerate the

entire plant.

Basic concepts of plant tissue culture(PTC)

Cell lines differentiate to form specialized tissues and organs.

Unlike animal cells, living plant cells re-differentiate.

Therefore, tissue can be regenerated from explants such as leaf, ovary, protoplast, petiole, root, anthers, etc.

Factors affecting Tissue Culture

Growth Media– Minerals, Growth factors, Carbon source, Hormones

Environmental Factors– Light, Temperature, Photoperiod.

Explant Source– Usually, the younger, less differentiated plant is better for tissue culture

Genetics– Different species show different ability in tissue culture. In many cases, different genotypes within a species will have variable responses to tissue culture.

three main steps to the tissue culture

process: STAGE I is the initiation phase. It concerns the establishment of plant tissue in vitro by sterilizing the

material and initiating it into culture. STAGE II is the multiplication phase. At this stage, the in

vitro plant material is re-divided and placed in a medium with plant growth regulators that induce the proliferation of multiple shoots. This process is repeated many times

until the number of plants desired is reached. STAGE III is the root formation phase. It involves the

introduction of hormones to induce rooting and the formation of complete plantlets.

Types of tissue culturePlant tissue culture(Micropropagation). Animal tissue culture (Cell Tissue Culture).

Micropropagation The art and science of multiplying plants in vitro. Its implies

- Regeneration- Multiplication

Stages of MicropropagationStage I - Selection & preparation of the mother

plant – sterilization of the plant tissue takes place. Initiation of culture – explant placed into growth media

Stage II – Multiplication – explant transferred to shoot media; shoots can be constantly divided.

Stage III - Rooting– explant transferred to root media

Stage IV - Transfer to soil– explant returned to soil; hardened off

STAGE IV: Transfer to Natural EnvironmentSTAGE III: Pretransplant (rooting)

STAGE II: Shoot ProductionSTAGE I - Sterilization

Steps involved in the in vitro micropropagation

Cleaning of glassware

Preparation of nutrient medium

Selection and sterilization of explant.

Inoculation of aseptic explant into nutrient medium.

Proliferation of shoots on a multiplication medium.

Transfer of shoots for sub-culturing.

Rooting and hardening of plantlets

Field trials.

Cleaning of glassware Graduated measuring cylinders

conical flasks

beakers

Petridishes

pipettes (2 ml, 5 ml and 10 ml)

glass rods

centrifuge tubes

culture tubes

bottles

Procedure for cleaning of glassware

Soak glassware in soap water for 1 hour.

Transfer glassware to conc. HCl and keep for 2 hours.

Rinse glassware in tap water.

Wash the glassware at least twice with distilled water.

Keep glassware for drying in oven at 100 oC for 1 hour.

And then keep glassware in oven at 140-160 oC for 2 hours.

Preparation of nutrient medium

The growth medium used depends upon the plant species to be grown.

The medium contain the following contents:- All of the minerals and vitamins required for the plant

growth and differentiation.- A carbon/energy source such as the sugar as the explant

cannot usually photosynthesize.- Various growth regulators to encourage the cell

elongation, division and differentiation.- Agar which is used to solidify the medium.

Types of medium Chemically defined nutrient medium

Chemically undefined nutrient medium:

Complex additives viz. coconut milk, Casein hydrolysate, yeast

extract, water melon juice, etc. are added in the medium.

1. Solid medium: 6-8% agar-agar

2. Semi solid medium: Less amount of agar

3. Liquid medium: Agar is not added. It is used for cell suspension

culture.

Preparation of stock solutions

It is convenient to prepare stock solutions.

When mixed together in appropriate quantities constitutes

basal medium.

It is not feasible to weigh and mix all the constituents of the

nutrient medium for the preparation of the small quantity of

the nutrient medium.

It also provides flexibility to try different combinations of the

nutrient medium.

Plant Growth regulators

Two major hormones required for the cell differentiation:

i. Auxins - which stimulates root development.ii. Cytokinin - which stimulates shoot

developmentRatio b/w these hormones

i. Auxins Cytokinin = Root developmentii. Auxins Cytokinin = Shoot development

iii. Auxins = Cytokinin = Callus

Selection and sterilization of explant.

Culture medium supports the growth of microbes e.g bacteria, fungi, etc. these grow

fast and kills the plant cells.

Microbes may come from glass vials, instruments, nutrient medium and also from the

plant material.

Therefore, the surface of plant tissue and all non-living articles including nutrient

medium must be sterilized.

Sterilization of non-living articles: The non-living articles viz. Nutrient medium,

glassware, distilled water, instruments (wrapped with brown paper) are sterilized by

autoclaving under steam at a 15 lb/inc2 and temperature 121oC for 15 min. The

glassware can also be sterilized by heating in oven at 150oC for 3-4 hrs. The

thermoilabile compounds are sterilized by passing through the bacterial filters.

Selection and sterilization of explant.

Sterilization of the plant material

(Surface) sterilization The plant material should be surface sterilized to remove the surface

borne micro-organisms.

Water

10% v/v solution of liquid detergent (Teepol) for 10-15 min.

70% ethyl alcohol for 1 min. in front of laminar air flow.

Treatment with 0.1% HgCl2 (W/V) or 5-10% sodium hypochlorite.

Incubation of culture Cultures are incubated in a culture room where light, temperature

and humidity are controlled.

For some tissues dark is essential while for some both dark and

light conditions are required.

Humidity has also some effect.

The cultures are incubated on culture rack at 25-28 oC constant

temperature. Culture tubes are placed at 35-40o inclined position.

Culture to give a light intensity of 4-10 X 103 lux for 16 hrs.

Incubation of culture

Callus FormationCallus is defined as an unorganized tissue

mass growing on solid substrate. Callus forms naturally on plants in

response to wounding, infestations, or at graft unions (Bottino, 1981).

Callus formation is central to many investigative and applied tissue culture procedures.

Effects of culture media, growth regulators and explants on Callus induction  and growth: (a-b): Callus induction from nodal explants in Murashige and

Skoog (MS) medium, (c): Callus from leaf explants in MS+coconut water, (d-f): Callus growth in MS medium+2,4 Dichlorophenoxyacetic Acid and Kinetin, (g):

Callus induction in woody plant medium; h and i: Callus growth in MS+α-Naphthalene Acetic Acid and 6-Benzyladenine purine

Subculturing Transfer of cell or tissue from old culture

medium to fresh culture medium within

definite time period.

It provides sufficient space and nutrients to

the growing plantlet.

Multiplication of the callus.

Rooting It is the induction and development of adventitious roots on

the proliferated shoots.

Root formation is induced in a medium with high auxin and

low cytokinins concentrations.

Shoot tip or single node explant is used.

Culture medium is maintained in a green house/mist chamber.

Activated charcoal is frequently added to absorb root-

inhibiting agents.

Rooting

Hardening Healthy/elite plantlets are exposed to the natural conditions in a step wise

manner.

It is a gradual acclimatization of in vitro grown plants to in vivo condition.

The plantlets are transferred to the pots/polyghene bag and immediately

irrigated with inorganic/nutrient solution.

Plants are kept in the hardening room where controlled conditions of light,

humidity and temperature are maintained.

Plants are maintained under high humidity for 10-20 days and subsequently

transferred in the field so as to grow under natural conditions. The success

rate of micropropagation depends on the survival of the plantlets when

transferred from culture to the soil (field).

Hardening

Laboratory setupSpace for washing and storage.

Sterilization room

Inoculation room

Culture room ( incubation room)

Observation and inspection room.

Data collection and management room.

Methods of micropropagationClonal propagation involves the

multiplication of genetically identical lines by asexual reproduction.

Micropropagation involves the use of bud culture, meristem and shoot-tip culture techniques to introduce plants in vitro by induction to form adventitious buds, shoots and entire plants.

Methods of micropropagation

(cont’d..)Bud culture is the culture of plant buds

which contains active meristems.

Meristem culture is the culture of apical meristems which are capable of active cell division and differentiation into specialized and permanent tissue such as shoots and roots

Methods of micropropagation

(cont’d..)Single node culture is done on a hormone

free medium.

Axillary bud culture is done using excised shoot tips cultured on medium amended with high cytokinin concentration 6 Benzyl amino purine(BAP)/Benzyl adenine(BA)

1 2 3

4 5

Surface sterilization in NaOCL

Rinsing with SDW Transfer into sterile container for 2nd sterilization

Steps in single node culture

Methods of micropropagation (cont’d..)

Micro cuttings of yam during and after subculture

3 4

Problems in Micropropagation

There is problem of genetic variability due to Somaclonal variation in some cultures.

Contamination is a major problem which could cause high losses within a short period.

It requires expensive equipments and well trained manpower.

Verification may occur which reduces the rate of growth multiplication of the plant and eventually causes death.

Benefits of micropropagation

Rapid multiplication of superior clones can be carried out through out the year, irrespective of seasonal variations.

Multiplication of disease free plants e.g. virus free plants of sweet potato (Ipomea batatas), cassava (Manihot esculenta) e.t.c.

It is a cost effective process as it requires minimum growing space.