Tissue culture consists of growing plants cells as relatively on organized masses of cells on an agar medium (callus culture) or as a suspension of free cells and small cell masses in a liquid medium (suspension culture). Tissue culture is used for vegetative multiplication of many species and in some cases for recovery of virus-free plants. It has potential application in production of somatic hybrids, organelle and cytoplasm transfer, genetic transformation and germplasm storage through freeze-preservation.
The in vitro cultures of cells, tissues and organs of plants has a great potential to generate improved Crop plants and Ornamentals. The improvements is aimed at
The cultural techniques together with recombinant DNA technology are suited for both. Two most popular misconcepts are associated with these techniques:
Plant tissue culture:
Tissue culture is the process whereby small pieces of living tissue (explants) are isolated from an organism and grown aseptically for indefinite periods on a nutrient medium. For good success in plant tissue culture, it is good to start with explant that is meristems because these cells are capable of rapid proliferaflon. The used explants include buds, root tips, nodal segments or germinating seeds and these are placed on suitable culture media where they grow into an undifferentiated mass known as Callus.
Because the nutrient media used in the technique support the growth of microorganisms, the explant is first washed with disinfectant such as sodium hydrochloride, hydrogen peroxide or meseuric chloride. Once established, the callus can be propagated indefinitely by sub-division. The nutrient media contains growth nutrient/substances and hormones i.e. auxin, cytokinin and gibberddin. The absolute amounts of hormones vary for different tissue, tissue explants from different parts of the same plant and for similar explants from different plants.
There is no ideal medium, most of the in common use consist of inorganic salts, trace metals, vitamins, organic nitrogen sources, insitol, sucrose, and growth regulators. Organic nutrients cush as Casein hydrolysate or yeast extract and getting agent and optional extras. One of the composition of typical growth medium is given below:-
Composition of Murashige and Skoog (MS) Culture medium.
Let us know something more about culturing of cells and plant organs.
When placed in a suitable medium is necessary. This medium is prepared by culturing high densities of the same or different species in fresh medium for few days and then removing the cells by filter sterilization. The filterate contains many essential substances for growth . By placing singe cell on such medium, lines from single cells can be placed as in the case of micro organisms. Plant cells, however, instead of following colonies, proliferate in Callus. The occurrence of such single line of cells, different in characters, built from cell mutants is known as somatic variations.
These are cells minus their cell walls which form useful material for cell manipulations as under certain conditions, contrasting cell types can be fused to yield somatic hybrids; a process known as protoplast fusion. Protoplasts can be produced from suspension cultures, callus or intact tissues by mechanical disruption or treatment with enzymes. Pectinase breaks cell aggregates into individual cells and cellulose removes the cell wall from these protoplast suspension, by washing and plating on nutrient medium, protoplasts form new cell wall in 5-10 days and initiate cell division.
Plant Organ culture: This is the most common type known as Tissue Culture. Here shoot tips are after surface sterilization are placed in growth medium lacking hormones where these develop into single seedling like shoots. If instead cytokinin is used in the medium, axillary shoots will emerge and produce a shoot culture. These can be further subdivided into smaller clumps or separate shoots, which will in turn form similar clusters when subcultured on fresh medium. Provided basic nutrient formulation is adequate for normal growth this subdivision process known as micropropogation can be undertaken every 4-8 weeks for a quite long time say up to 10-12 sub-cultures in case of banana. Micropropogation indefinate cultures of plant roots can be achieved in the similar manner, with use of auxin in the medium and no cytokinnin many commercial companies are using micropropogation of crop plants like Banana, Sugarcane, spices crops and Ornamentals like carnation, gerbera, and antherium. Uniformity of quality, free from virus infection, high yield etc. are some of the advantages of micropropagation of commercial crops. Since the process is laborous and time taking in many countries, the same has been fully automated.
Regeneration of Plants: Numerous species of plants can be maintained in cell or callus culture. In this state new genotypes can be isolated by somaclonal variation; protoplast fusion, or mutagenesis. These cells to be useful must be converted into an intact plant. The cells capable of developing into intact plants are said to be totipotent; totipotency being the property of undetermined cells. Undetermined cells can be defined as those, with capacity to switch to different development pathways depending on environment in which they grow undetermined cells most readily develop into a callus.
It should be remembered that for callus formation auxin and cytokinin both are required; whereas only a cytokinin both are required; whereas only a cytokinin is required for shoot culture and only an auxin for root culture. Increasing levels of Cytokinin or auxin in aculture, shoot or root formation can be induced and promoted in culture. The formation of roots or shoots in culture is known as Organogenesis.
Under certain cultural conditions callus can be induced to undergo different development process known as somatic embryogenesis. This is a pattern of differentiation, similar to that seen in zygotes after fertilization, to produce embryos. These cells are embryo like but produced from somatin cells, and not from fusion of two germ cells. These embryos can develop into fully functional plants without the need to induce root and shoot formation on artificial media Embryos formation is stimulated by transferring callus grown in media containing auxin. 2,4-d in to a 2,4-D free media containing reduced sources of on nitrogen i.e.ammonium salts.
Anther and Pollen culture: Pollen grains form pollen tube and male gametes, when placed on suitable nutrient media like MS, most pollen grain follow normal development, but a few grains will form a callus instead. In a similar manner, anthers can be cultured to form embryos directly from pollen grains. These embryos can be induced to develop true haploid plants. With optimal nutrient conditions for donor plants and explants are provided, it is possible to obtain several hundred haploid plants from a single anther. For successful anther culture if is necessary to excise flower buds at first mitotic division in the uninucleated microspores tetrads. Other treatments like growing on different nutrient media, cold storage, for few days to several weeks, etc. can increase the frequency of successful embryos.
Application of cell, tissue and organ culture:
in oil palm
ii. production of minituber
All these culture techniques not only are related to plant bio-technology but often form the part of it. More we get involved in exploiting these techniques on commercial scale, more we will be close to harvest the advantages of various aspects of plant biotechnology.