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After reading this article you will learn about the meaning and system of plant tissue culture.
Meaning of Tissue Culture:
Tissue culture is a term, used to indicate the aseptic culture in vitro of a wide range of excised plant parts. It is used for both propagation and modification of genotypes (production of somaclonal variation and double haploids), biomass production of biochemical products, plant pathology, preservation and storage of germplasm, etc.
These activities come under the term biotechnology, in general and in broader sense, micropropagation and tissue culture are also used interchangeably.
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Tissue culture beings with the excision of a small piece of plant, freeing it from microorganisms and placing it into aseptic culture. The term used for small piece of plant initially excised is ‘explant’. Five others terms have been used to describe kinds of vegetative (somatic) regeneration used in tissue culture.
These are:
Meristem-Tip Culture:
The tissue culture utilizing very small part of a shoot tip including subtending rudimentary leaves is known as meristem-tip culture.
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Axillary Shoot Proliferation:
This type of tissue culture includes an expanded shoot of terminal and lateral growing points, where elongation of the terminal shoot is suppressed.
Adventitious Shoot induction:
This type involves the initiation of adventitious shoots produced on the explant after its placement into the culture.
Organogenesis:
The process by which adventitious shoots and/or roots develop from within masses of callus cells is known as organogenesis.
Somatic Embryogenesis:
The development of a complete embryo from vegetative cells produced from various sources of explants grown in culture is known as somatic embryogenesis.
System of Tissue Culture:
Various systems could be classified as follows:
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Regeneration of New Plants from Vegetative Structures (Micropropagation):
This process utilizes the smallest part of shoot tip as the explant and is aimed at producing a rooted micro-plant through isolation of a meristem which is free from pathogens. This is being commercially utilised in carnation, chrysanthemum, orchid, geranium, potato, sweet potato, cassava, banana, etc. The overall process involves 4 stages of production.
Establishment:
In this stage sterile explant is planted on a sterile basal medium (agar + mineral salts + vitamins + sucrose + plant growth regulators (PGR) like cytokinin, auxin, gibberellin, etc.), to establish it in culture, to remove contamination and to proliferate mass of micro-shoots. Repeated sub-culturing is done to have uniform well growing culture.
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Multiplication:
Multiplication of axillary shoots is promoted by controlled inhibition of shoot elongation and stimulation of lateral shoots primarily by adjusting the ratio of cytokinin to auxin.
Pre-transplant:
In this stage, the micro-shoots are transferred from the aseptic environment of the test tube to the outdoor conditions of the green house. This allows rooting of individual micro-shoots in a medium in which the auxin levels has been increased and the cytokinin concentration decreased or omitted. Several modifications in the medium are followed to induce rooting.
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Transplanting and Acclimatization:
This stage includes shifting the micro-plant from heterotrophic to an autotrophic (free- living) condition and allowing the plant to remain in the outdoor environment under high humidity and gradually exposing them to conditions similar to those available to normal transplanted plants.
Anther/Pollen Culture, Somaclonal Variation and Embryo Culture:
S. Guha and S.C. Maheshwari discovered in 1964 that pollen grains in Datura could be developed into embryos. This was later on realised in tobacco by J.P. Nitsch and his colleagues in 1969. Since then whole plants (haploids) have been produced in many species (Table 9.1).
This technique has been put to use in breeding for developing double haploids/hybrid sorting. Two desirable parental cultivars (say A and B) are crossed. Anthers from hybrid plants are cultured to produce haploids which are doubled by colchicine treatment to produce double haploids which are subjected to selection and evaluation for development of new cultivars (Fig. 9.2).
A few commercial releases utilizing this technique are Hua Yu 1 and 2 (rice in China), Jingdan 2288 (wheat), Danyu 1 (tobacco).
Somaclonal variations are the variations detected in plants derived in any form of cell culture. However, somaclonals are particularly used to refer plants regenerated from cell cultures originated from somatic tissues in contrast to gametocolones to refer to plants regenerated from cell cultures originated from gametic tissues. Plants regenerated from cell culture irrespective of tissue of origin are referred to as R or R0 plants.
The self-fertilized progeny of R plant are referred to as R1 plants. Subsequent generations produced by self-fertilization are termed R2, R3, R4 etc. Somaclonal variations could be utilised in breeding programmes (Fig. 9.3) as suggested by Evans (1984).
Changes in chromosome number or structure, single gene nuclear mutations, cytoplasmic DNA changes, small chromosome rearrangements, gene conversion, mitotic crossing over, and transposable elements have been reported as possible genetic causes of somaclonal variations.
Embryo culture is the excision of embryos from individual ovules and their eventual germination in aseptic culture. This technique is also known as ’embryo rescue’ as it is used to rescue embryos that would have aborted within the seed before maturity. This may be required in case of inter-specific hybridization.
Regeneration of Plants from Tissue and Cell Culture:
Callus (undifferentiated mass of cells) produced on explants in vitro as a result of wounding and in response to hormones is important from the viewpoints that it can be sub-cultured and maintained almost indefinitely and callus mediated regenerated plants show somaclonal variations.
The most common culture medium is the Murashige-Skoog (MS) medium. There are other media also to suit to varying conditions and new kind of plants. The basic ingredients of the culture media are inorganic salts, organic compounds, complex natural ingredients and inert supports. Table 9.2 shows 4 representative media as summarised by Hartmann (1993).
