Photosynthesis

Plant tissue culture

What is Plant tissue culture?

Plant tissue culture is a collection of techniques used to maintain or grow plant cells, tissues or organs under sterile conditions on a nutrient culture medium of known composition.

Basics of Plant tissue culture

Ø  Totipotency

Ø  Morphogenic potential

Totipotency:

·         It is the ability of single cell to divide and re-divide and produce all of the differentiated cells in an organism.

·         Totipotency is the genetic potential of a plant cell to produce the entire plant.

·         Totipotency is the cell characteristic in which the potential for forming all the cell types in the adult organism is retained.

·         The cells are obtained from stem, root or other plant parts and are allowed to grow in culture medium containing mineral nutrients, vi­tamins and hormones to encourage cell division and growth.

·         The cells in culture will produce an unorganized proliferative mass of cells which is known as callus tissue.

Morphogenic potential:

It includes cell divisions and cellular differentiations:

Cell divisions:

·         Cell division is the process of splitting a parent cell into two daughter cells.

Cellular differentiations:

·         Cellular differentiation is the process of forming a variety of cell types that have specific functions.

 Cellular differentiations may be

·         Differentiation

The cells derived from root apical meristem (RAM) and shoot apical meristem (SAM) and cambium differentiate, mature to perform specific functions. This act leading to maturation is termed differentiation. They undergo a few or major structural changes both in their cell walls and protoplasm.

·         Dedifferentiation

In plants, the living differentiated cells can regain the capacity to divide mitotically under certain conditions. The sum of events, that bestow this capacity to divide once again, are termed dedifferentiation. A dedifferentiated tissue can act as meristem (e.g., interfascicular vascular cambium, wound meristem, cork cambium).

·         Redifferentiation

The products of dedifferentiated cells/tissue which lose the ability to divide are called redifferentiate cells/tissues and the event is redifferentiation.

Applications of Plant Tissue Culture

·        Micropropagation

It is a plant tissue culture technique used for production of plantlets, in which the culture of aseptic small sections of tissues and organs in vessels with defined culture medium and under controlled environmental conditions.

 Micropropagation is the technique of multiple production of plants in vitro.

·        Genetic Engineering

It is the direct genetic modification or genetic manipulation of an organism’s gene by biotechnology.

·        Polyploidy

Polyploids act as a source of genetic variation in plant tissue culture.

 Multiple genomes alter gene frequency; induce a permanent hybridity, genetic buffering and evolutionary flexibility (esp. Allopolyploids)

Autopolyploid typically have larger cell sizes, resulting in larger, lusher plants than the diploid version .

Chromosome doubling occurs naturally in all plants at low frequency as a result of mitotic failure.

 Can be induced by chemicals (colchicines from Colchicum autumnale (saffron), vincristine and vinblastine from vinea rosea) applied to meristematic tissue.

 Young zygotes respond best; vegetative tissue usually results in mixoploid chimeras.

·        Secondary metabolite production

These are not directly involved in normal growth, development and reproduction but having ecological function.

Five secondary metabolites are:

                                I.            Terpenoids and steroids

                             II.            Fatty acids derived substances and polyketides

                           III.            Alkaloids

                          IV.            No ribosomal polypeptides and

                            V.            Enzyme cofactor

TECHNIQUES OF PLANT TISSUE CULTURE 

Micro propagation

Micro propagation starts with the selection of plant tissues (ex-plant) from a healthy, vigorous mother plant.

 Any part of the plant (leaf, apical meristem, bud and root) can be used as ex-plant.

Stages of Micropropagation:

 The whole process can be summarized into the following stages:

       I.            Stage 0: Preparation of donor plant

§  Any plant tissue can be introduced in vitro.

§  To enhance the probability of success, the mother plant should be ex vitro cultivated under optimal conditions to minimize contamination in the in vitro culture.

    II.            Stage 1: Initiation stage

§  Ex-plant is surface sterilized and transferred into nutrient medium.

§  Combined application of bactericides and fungicides products is suggested.

§  Selection of products depends on the type of ex-plant to be removing contaminants with minimal damage to plant cells.

§  Disinfectants like sodium hypochlorite, calcium hypochlorite, ethanol and mercuric chloride (HgCl₂) are used.

§  Cultures are incubated in growth chamber either under light or dark conditions according to the method of propagation.

 III.            Stage 2: Multiplication stage

§  The aim of this phase is to increase the number of propagules.

§  The number of propagules is multiplied by repeated subcultures until the desired (or planned) number of plant is attained.

 IV.            Stage 3: Rooting stage

§  Rooting stage may occur simultaneously in the same culture media used for multiplication of the ex-plant.

§  In some cases it is necessary to change media, including nutritional modification and growth regulator composition to induce rooting and the development of strong root growth.

    V.            Stage 4: Acclimatization stage

§  The in vitro plants are weaned and hardened.

§  Hardening is done gradually from high to low humidity and from low light intensity to high light intensity.

§  The plants are then transferred to an appropriate substrate (sand, peat, compost etc) and gradually hardened under greenhouse.

BASIC REQUIREMENTS OF PLANT TISSUE CULTURE

In plant tissue culture techniques, there are some basic requirements,

a)      Aseptic condition

b)     Control of temperature

c)      Proper culture media

d)     Sub-culturing

Aseptic conditions

§  The tissue culture laboratory should have aseptic conditions.

§  It means it should be well sterilizes against pathogen.

§  A pathogen free environment will help in maintaining good health of the callus, cell or protoplast cultures resulting in recovery of healthy plants from such cultures.

§  The explants and glassware should be properly sterilized before their entry into the tissue culture laboratory.

Control of Temperature

§  Air conditioning of the tissue culture laboratory is essential.

§  Generally, temperature between 18-25^OC must be maintained.

§  However, this temperature varies from species to species.

§  High temperature adversely affects the growth of the callus.

§  Thermohygrometer is used to measure the temperature of the laboratory.

Proper Culture Medium

§  Culture media have been developed by various workers for different crop species.

§  The medium has to be modified as per as the requirement of a species.

§  The culture media developed by Murashige and Skoog (1962) is used with some modification in various crop species.

 Sub-Culturing

Transfer of tissues or callus from old culture media to fresh culture media is called sub-culturing.

It is essential to maintain good health of the callus or tissues, because after some period, some nutrients are depleted in the culture media and change of media become essential.

Apparatus

·         Graduated cylinder

·         Beaker

·         Spatula

·         Forceps

·         Electronic balance

·         PH meter

·         Aluminum foil

·         Jug

·         Magnetic foil

·         Micropipette

·         Magnetic heat stirrer

·         Autoclave

·         Microwave

·         Laminar air flow

·         Magnetic stirrer

·         Tip box

·         Jars

·         Indicator tape

·         Polypropylene bags

·         Rubber bands

Material

·         Sugar/sucrose

·         MS (Murashige and Skoog) media

·         Agar

·         Distilled water

Hormones

·         Cytokinin

·          Auxin

For PH maintenance

·         HCL

·         NaOH

Important steps for plant tissue culture

Plant tissue culture techniques generally consist of five important steps:

                    i.            Media preparation and sterilization

                 ii.            Ex-plants selection and collection

               iii.            Explants inoculation

               iv.            Culture incubation

                  v.            Rooting and Acclimatization