Introduction to Plant Tissue Culture

Introduction

Tissue culture involves the use of small pieces of plant tissue (explants) which are cultured in a nutrient medium under sterile conditions. Using the appropriate growing conditions for each explant type, plants can be induced to rapidly produce new shoots, and, with the addition of suitable hormones new roots. These plantlets can also be divided, usually at the shoot stage, to produce large numbers of new plantlets. The new plants can then be placed in soil and grown in the normal manner.
Plants usually reproduce by forming seeds through sexual reproduction. That is, egg cells in the flowers are fertilized by pollen from the stamens of the plants. Each of these sexual cells contains genetic material in the form of DNA. During sexual reproduction, DNA from both parents is combined in new and unpredictable ways, creating unique plants.
This unpredictability is a problem for plant breeders as it can take several years of careful greenhouse work to breed a plant with desirable characteristics. Many of us think that all plants grow from seeds. However, researchers have now developed several methods of growing exact copies of plants without seeds. And they are now doing this through a method called “tissue culture”.
Tissue culture (TC) is the cultivation of plant cells, tissues, or organs on specially formulated nutrient media. Under the right conditions, an entire plant can be regenerated from a single cell. Plant tissue culture is a technique that has been around for more than 30 years. Tissue culture is seen as an important technology for developing countries for the production of disease-free, high quality planting material and the rapid production of many uniform plants.
Micropropagation, which is a form of tissue culture, increases the amount of planting material to facilitate distribution and large scale planting. In this way, thousands of copies of a plant can be produced in a short time. Micropropagated plants are observed to establish more quickly, grow more vigorously and are taller, have a shorter and more uniform production cycle, and produce higher yields than conventional propagules.
Plant tissue culture is a straightforward technique and many developing countries have already mastered it. Its application only requires a sterile workplace, nursery, and green house, and trained manpower. Unfortunately, tissue culture is labor intensive, time consuming, and can be costly. Plants important to developing countries that have been grown in tissue culture are oil palm, plantain, pine, banana, date, eggplant, jojoba, pineapple, rubber tree, cassava, yam, sweet potato, and tomato. This application is the most commonly applied form of traditional biotechnology in Africa.

Types of Tissue Culture

1. Seed Culture

Seed culture is the type of tissue culture that is primarily used for plants such as orchids. For this method, explants (tissue from the plant) are obtained from an in-vitro derived plant and introduced in to an artificial environment, where they get to proliferate. In the event that a plant material is used directly for this process, then it has to be sterilized to prevent tissue damage and ensure optimum regeneration.

2. Embryo Culture

Embryo culture is the type of tissue culture that involves the isolation of an embryo from a given organism for in vitro growth.
Note, the term embryo culture is used to refer to sexually produced zygotic embryo culture.
Embryo culture may involve the use of a mature of immature embryo. Whereas mature embryos for culture are essentially obtained from ripe seeds, immature embryo (embryo rescue) involves the use of immature embryos from unripe/hybrid seeds that failed to germinate. In doing so, the embryo is ultimately able to produce a viable plant.
For embryo culture, the ovule, seed or fruit from which the embryo is to be obtained is sterilized, and therefore the embryo does not have to be sterilized again. Salt sucrose may be used to provide the embryo with nutrients. The culture is enriched with organic or inorganic compounds, inorganic salts as well as growth regulators.

3. Callus Culture

Callus – This is the term used to refer to unspecialized, unorganized and a dividing mass of cells. A callus is produced when explants (cells) are cultured in an appropriate medium – A good example of this is the tumor tissue that grows out of the wounds of differentiated tissues/organs.
In practice, callus culture involves the growth of a callus (composed of differentiated and non- differentiated cells), which is the followed by a procedure that induces organ differentiation.
For this type of tissue culture, the culture is often sustained on a gel medium, which is composed of agar and a mixture of given macro and micronutrients depending on the type of cells. Different types of basal salt mixtures such as murashige and skoog medium are also used in addition to vitamins to enhance growth

4. Organ Culture

Organ culture is a type of tissue culture that involves isolating an organ for in vitro growth. Here, any organ plant can be used as an explant for the culture process (Shoot, root, leaf, and flower).
With organ culture, or as is with their various tissue components, the method is used for preserve their structure or functions, which allows the organ to still resemble and retain the characteristics they would have in vivo. Here, new growth (differentiated structures) continues given that the organ retains its physiological features. As such, an organ helps provide information on patterns of growth, differentiation as well as development.
There are number of methods that can be used for organ culture. These include:

Plasma clot method – Here, the method involves the use of a clot that is composed of plasma and chick embryo extract (or any other extract) in a watch glass. This method is particularly used for the purposes of studying morphogenesis in embryonic organ rudiments and more recently for studying the actions of various hormones, vitamins and carcinogens of adult mammalian tissues.
Raft method – For this method, the explant is placed on a raft of lens paper/rayon acetate and floated on a serum in a watch glass.
Agar gel method – The medium used for this method is composed of a salt solution, serum as well as the embryo extract or a mixture of various amino acids and vitamin with 1 percent agar. The explant has to be subcultured every 5 to 7 days. The method is largely used for the study of developmental aspects of normal organs and tumors.
Grid method – Grid method, as the name suggests involves the use of perforated stainless steel sheet, on which the tissue of interest is placed before being placed in a culture chamber containing fluid medium.

5. Protoplast Culture

Protoplasts are cells without cell walls. A protoplast is the term used to refer to cell (fungi, bacteria, plant cells etc) in which the cell wall has been removed, which is why they are also referred to as naked cells.
Protoplasts may be cultured in the following ways:

Hanging-drop cultures
Micro culture chambers
Soft agars matrix

Once a protoplast has regenerated a cell wall, then it goes through the process of cell division to form a callus, which may then be subcultured for continued growth.
Protoplast culture is an important method that provides numerous cells (single cells) that can be used for various studies. These include:

Protoplast culture regenerated into a whole plant
Development of hybrids
Cell cloning
Genetic transformations
Membrane studies

In protoplast culture, a number of phases can be observed. These include:

Development of a cell wall
Cell division
Continuous growth or regeneration to a whole plant

For plants, some of the special requirements include:

Less amounts of iron and zinc and no ammonium
Higher concentration of calcium
High auxin/kinetin ratio for cell division and high kinetin/auxin ration for regeneration
Glucose and vitamins

6. Others

Some of the other types of tissue culture include:

Single cell culture
Suspension culture
Anther culture
Pollen culture
Somatic Embryogenesis

Major Steps of Tissue Culture (Plants)

1. Initiation Phase (Stage 1)

The initiation phase is the first phase of tissue culture. Here, the tissue of interest is obtained and introduced and sterilized in order to prevent any microorganism from negatively affecting the process. It is during this stage that the tissue is initiated in to culture.

2. Multiplication Phase (Stage 2)

The multiplication phase is the second step of tissue culture where the in vitro plant material is re- divided and then introduced in to the medium. Here, the medium is composed of appropriate components for growth including regulators and nutrients. These are responsible for the proliferation of the tissue and the production of multiple shoots.
This step is often repeated several times in order to obtain the desired number of plants

3. Root formation (Stage 3)

It is at this phase that roots are formed. Here, hormones are required in order to induce rooting, and consequently complete plantlets.

Plant Tissue Culture

Tissue culture is applied in plant research for such purposes as the growing of new plants, which in some cases undergo genetic alterations. Here, the plant of interest is taken through the tissue culture process and grown in a controlled environment.

The Process of Plant Tissue Culture

This process involves the use of small pieces of a given plant tissue (plant of interest). Once the tissue is obtained, it is then cultured in the appropriate medium under sterile conditions so as to prevent various types of microorganisms from affecting the process.
The following is a general procedure for plant tissue culture:

A. Medium preparation

The appropriate mixture (such as the MS mixture) is mixed with distilled water and stirred while adding the appropriate amount of sugar and sugar mixture. Here, sodium hydroxide or hydrochloric acid is used to adjust the pH – Contents used here will depend on the plant to be cultured and the number of tissues to be cultured.
Agar is added to the mixture, heat and stirred to dissolve
After cooling, the warm medium is poured into polycarbonate tubes (to a depth of about 4 cm)
With lids sitting on the tubes, the tubes are placed in a pressure cooker and sterilized for 20 minutes

B. Plant preparation

Cut the plant part in to small pieces (e.g. cauliflower can be cut to florets of about 1cm across). On the other hand, such parts as the African violet leaves can be used as a whole.
Using detergent and water, wash the plant part for about 20 minutes
Transfer the plant part in to sterilizing Clorox solution, shake for a minute and leave to sock for 20 minutes
Using a lid, gently discard the Clorox and retain the plant part in the container and then cap the container

C. Transferring the plant material to a tissue culture medium

70 percent alcohol should be used for the sterilization of the equipment used and containers

Open the container and pour sterile water to cover half the container
Cover with a sterile lid again and shake the container for 2 to 3 minutes in order to wash the tissue and remove the bleach
Pour the water and repeat this three times
Using sterilized gloves, remove the plant part from the container and on to a sterile Petri dish
Using a sterile blade cut the plant material to smaller pieces of about 2 to 3 mm across avoiding the parts that have been damaged by bleach
Using sterile forceps, place a section of the plant in to the medium

Cauliflower – partly submerged in medium with flower bud facing up
Rose with shoots at level with medium surface
African violet leaf laid directly in surface of medium
Depending on the plant used, it is important to check and find out how it should be placed in the medium.
Replace the lid/cap and close tightly

This procedure will result in the development of a callus, which then produces shoots after a few weeks. Once the shoots develop, then the plant section may be placed in the right environment (well lit, warmth etc) for further growth.
Plant materials should be sterilized so as to remove any bacteria or spores that may be present.
For plants, the medium culture acts as a greenhouse that provides the explant with the idea environment for optimum growth. This includes being free of microorganisms, nutrients as well as the right balance of chemicals and hormones. Such media as BAP, TDZ are used while such hormones as IBA and IAA are used to induce growth.

Some of the major reasons tissue culture is used for plants include:

To produce large quantities of a given plant
To accelerate the production of new varieties of a plant
To maintain a virus free stock of the plant of interest

Technique for Plant In Vitro Culture

1. Micropropagation – This technique is used for the purposes of developing high- quality clonal plants (a clone is a group of identical cells). This has the potential to provide rapid and large scale propagation of new genotypes.
2. Somatic cell genetics – Used for haploid production and somatic hybridization
3. Transgenic plants – Used for expression of mammalian genes or plant genes for various species. It has proved beneficial for the engineering of species that are resistant against viruses and insects.