Tissue Culture

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

1. to induce variations and selection and

2. Maintenance and propagation of desired varieties

The cultural techniques together with recombinant DNA technology are suited for both. Two most popular misconcepts are associated with these techniques:

1. These new techniques will shorten the time taken to introduce a new plant variety to the market. It is not so, varieties developed in the laboratory take considerably long time in field testing and

2. Plant cell, tissue and organ culture are not synonymous, these are quite distinct with many features in common.

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.

1. Plant Cell Culture: By transfer of callus culture to a liquid medium and agitated, the cell mass breaks to give a suspension of isolated cells with few small cell clusters and aggregates, the latter being extremely heterogenous. The cells can be subculture and maintained in vitro indefinately. Genetic instability is added to the heterogenitity. Some plants like nicofiana tabacum (tobacco) and Glycine max (Soyabean) yield very fiable celluses and the cell lines obtained from these species are more homogenous.

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.

Protoplasts:

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:

1. Production of fine chemicals like Codeine, Diosgenin, Sitostero, Quinine, Vincristine, Atropine, Pyrethrin, Saffoon and Methanol which are presently produced from plant sources can be produced from cell culture. However, the commercial success has been obtained in production of `Shikonin' from cells of plant lithospermusm erythorhizon.

2. Production of pathogen -free plants : Crops and ornamental plants can be infected by a wide variety microbial pests; Viroids, viruses, mycoplasms, bacteria, fungi and nematodes. The infections greatly affect yield and quality of plants. In some cases, infection can be eliminated by micropropagation of unaffected parts of the plants. The basic method of obtaining virus-free plants is culture of apical meristems. If a small enough piece is cultured preferably first half mm or so, of the growing tip, there is possibility to obtain virus free plants which can be further multiplied. There are other methods like high temp treatment, incubation of explants on suitable culture media containing malachite green or thiouracil which can reduce virus infection and replication.

3. Large scale plant propagation: In certain situation large scale vegetative multiplication of crop plants/fruit trees/ ornamentals is useful to save time get uniform quality and disease free plants. Such plants can be obtained by

1. i. In vitro methods of propogation through callus and cell culture as

in oil palm

ii. production of minituber

2. iii. Micropropagation as in case of Banana.

3. iv. Germplasm conservation through seed storage and multiplication has many problems. The current method of germplasm maintenance is being replaced by methods like cell, tissue and organ culture. In this methods, the advantages are

1. extremely slow growth involving depression of metabolism and]

2. Slow growth which is obtained by cryopreservation of cell and organ culture. However, the basic requirement of gene bank or germplasm preservation is that original characters of the genotype should not be changed. This can not be guaranteed in invitro preservation, plant cell, organ and tissue culture, are all developing technologies. All the three has a role to play in academic as well as in applied often commercial fields. Plant tissue culture has already proved to be a regular technique for propagation and quick multiplication of many crop plants as well as ornamentals. The success and performance of these techniques are dependent on many factors like development of fool-proof techniques to suit the needs of a plant variety, cost of production -skills of operators, and continuos research support for improving the techniques and finally rigorous testing of the micropropagated plants for their being free from diseases and also for their yield and quality. All these need a time-bound research and development programme having the involvement of private and public sector.

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.