Plant Tissue Culture Techniques and Applications

The term plant tissue culture is commonly used to describe in vitro (in glassware) and aseptic cultivation of any plant part on a nutrient medium. Tissue culture technique has provided an efficient tool for modern plant breeding.

Plant tissue Culture

Plant Tissue Culture Techniques and Applications

Plant tissue culture technology is based on three fundamental principles.
1. The plant part (ex-plant) must be isolated from the rest of the plant body.
2. The ex-plant must be maintained in a controlled, preferable defined, nutrient medium.
3. Aseptic conditions must be maintained.

Types of Plant Tissue Culture

Plant cultures may be callus culture, cell suspension cultures, anther cultures, ovule culture, embryo culture, protoplast cultures or micro-propagation.

Callus Culture Method
Callus Culture Method

Callus Culture Method

Haberlandt in 1902 was first to attempt cultivation of isolated plant cells in vitro on a nutrient medium. However, his attempt failed. White in 1934 first successfully cultured excised tomato root tips in nutritive medium. Later, Gautheret in 1939 and Nobecourt and White in 1939 independently obtained plant tissue cultures in the form of callus cultures from cambial tissues isolated from tobacco and carrot. Then Van Overbeek and his coworkers in 1941 demonstrated highly nutritive property of coconut milk in culture medium in culture of isolated embryos of Datura.
White and Braun in 1942 initiated studies on crown gall and tumor formation in plants using tissue culture method. Skoog in 1944 started work organogenesis in tobacco callus. Steward and coworkers obtained a completely regenerated carrot plant from cultured secondary phloem cells from tap root.

Suspension Culture Method

Muir in 1953 started work on suspension culture. Morel and Wetmore 1951 and Ball in 1950 obtained cultured tissues of gymnosperms Sepia.

Anther Culture Method
Anther Culture Method

Anther Culture Method

Guha and Maheshwari (1966, 1967) demonstrated potentiality of anthers in producing vast number of haploid embryos in culture (anther culture). Nitsch (1974, 1977) developed technique of isolated microspores of Tobacco and Datum.

Protoplast Culture Method

Cocking (1960) pioneered protoplast technique. Takebe (1971) demonstrated plantlet formation from cultured protoplast.

Plant Tissue Culture Technique

When an excised and isolated piece of tissue (ex-plant) is cultured on a nutrient medium, an unrecognized mass of cells appear. It is called callus. With age, such as callus may show meristemetic strands and individual or groups of trachieds and pigmented cells. This culture is transferred onto different media to regenerate plants. This technique is called callus culture.
Regeneration of plants though callus culture provides genetic variability which is important for successful plant breeding. This variation induced in plants regenerated through tissue culture are called somaclonal variation. Many agriculturally useful variants have been obtained through somaclonal variation.

Cell Suspension Culture Technique

It is the culture of isolated cells or very small cell aggregates dispersed in liquid medium. The cell suspension is obtained by agitating pieces of callus in a liquid medium on a rotatory shaker.
Cells suspension culture is mostly used to study the physiological, biochemical and metabolic characteristics of plant cells. It is also used to isolate cells showing tolerance to different stresses such as salts, herbicides, metals and drought conditions. Cells suspension culture is particularly useful in inducing genetic variability in cells through different physical and chemical mutagens.

Protoplast Culture Technique
Protoplast Culture Technique

Protoplast Culture Technique

The technique involves isolation, culture and fusion of protoplasts. It is one of the most significant and recent developments in the field of plant tissue culture. The protoplasts are usually isolated from cultured cells or leaf mesophyll cells by treating them with enzyme solutions. The isolated protoplasts may be used to regenerate the plants directly or for the production of somatic hybrids through fusion.
Protoplasts culture technique is important in studies of plant improvement , cell modifications and somatic hybridization. It is used for the induction of mutations and genetic variability and also for the production of somatic hybrids. The process is successfully, particularly in those species where sexual hybridization is impossible or very difficult.

Embryo Culture Technique

The embryos are isolated from young seeds and placed on a solid medium containing nutrients and vitamins. The embryo are cultured at 25 °C, first in dark until seedlings are about 2 cm long and root formation has started, than in the light until the seedlings can be planted in soil.

Anther Culture Technique

Anther culture is a means to produce plants with a gametic number of chromosomes by aseptic culture of anthers. The technique give rise to haploid plants either directly or through formation of a haploid callus. The technique quickens the breeding cycle by achieving homozygosity (double haploid) in one step through diplodization of the genetic chromosome number.
New cultivars obtained by anther culture are constantly being reported from China in crops of economic importance such as rice, wheat and tobacco.

Ovule Culture Technique
Ovule Culture Technique

Ovule Culture Technique

Ovule culture technique is an important technique in modern plant breeding. It is much easier to culture whole ovule than to isolate a single embryo, especially in small seeded plants. Ovule culture allows direct hybridization of various species such as Glycine, Gossypium (cotton), Nicotiarta (tobacco), Brassica, etc.

Micropropagation
Micropropagation

Micropropagation

Micropropagation may be defined as on vitro asexual propagation of crop plants. This technique is advantageous over the conventional practice of asexual propagation as only a small amount of plant is needed, species highly resistant to conventional bulk propagation can be propagated by this method, and it is non-season dependent.
Micro-propagation is used for rapid multiplication of stocks, elimination of disease, germ plasm preservation, physiological studies and induction of mutation.

In Vitro Fertilization and Pollination

This method makes it possible to study in detail the process of fertilization and embryogenesis in controlled conditions, to obtain viable seeds, to raise hybrids when normal hybridization fails, an to induce parthenogenesis and the development off haploid embryos and plants.
In vitro self-pollination of placentas with ovule has been performed successfully with about 35 species, usually those whose ovaries contained a large number of ovules.

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