In this article, we will discuss about the totipotency and its relation to tissue culture studies in plants.
It is now generally considered that every living cell in a plant possesses the potentially of producing an entire new plant or in other words it is totipotent. This phenomenon of capability of such a totipotent cell in generating an entire new plant is called as totipotency.
Tissue culture studies in higher plants have provided most convincing evidence of the existence of totipotency. Cultures of tissues from pith, cortex, cambium, leaf parenchyama and storage parenchyama in a suitable aseptic (sterile) medium grow readily by cell division and cell enlargement to produce a mass of undifferentiated cells called as callus.
The individual cells isolated from the callus tissue cultures under proper environmental conditions can be shown to behave like zygotes and to produce entire new plants. Therefore, such totipotent cells have capability in recapitulating the embryonic stages of development with the eventual formation of mature plants. The culture medium usually consists of inorganic salts, sucrose, vitamins and an organic supplement such as yeast extract, malt extract or coconut milk.
Experiments with callus tissue cultures in showing totipotency of cells have been more successfully carried out in some plants of the families Umbelliferae and Solanaceae such as carrot, tobacco etc., than in plants of other species.
Most significant results in this direction have been obtained by Steward et al in their extensive experiments and studies with the growth of carrot root tissue cultures. Small segments of tissues (‘explants’) from sec. phloem of carrot were taken and grown on a suitable sterile medium.
The individual cells were isolated from the carrot root callus tissue by ‘washing technique’ and cultured. Many of these cells began to behave like zygotes and produced cell masses (’embryoids’) that resembled various stages of development of embryos. These continued to grow producing roots and shoots and ultimately developed into normally appearing carrot plants.
Steward considered the sec. phloem cells of carrot root as totipotent for which two things were essential:
(i) Each individual cell has to be completely freed (separated) from the neighbours and
(ii) Proper environment such as that found in embryo-sac was needed.
Coconut milk provides such an environment (coconut milk is a complex mixture of carbohydrates, cyclitols, amino acids, gibberellins, cytokinins, phenolics and other substances). The tissue culture techniques such as that mentioned above have been successful in growing ‘test tube plants’ and also in cultivation of many plants such as orchids, fruit trees etc., through embryoids formation, so much so that ‘germplasm banks’ have now been established in laboratories in many countries all over the world.
The main advantage with such an idea is that callus tissue cultures can be maintained for long periods of time and that too on a minimal medium and space. When embryoids are needed for plantlets production, these cultures are transferred to a suitable medium containing appropriate growth factors. The plantlets then can be planted in soil and raised to maturity. Germ plasm banks may also be successful in saving many endangered plant species from extinction.
Under suitable culture conditions, the pollen grains within the anther can be induced to form embryoids and eventually entire plants which are haploid. This production of haploid plants by the germination of young pollen grains inside the anther is called as androgenizes and is also a very important aspect of totipotency. Because the pollen grains contain x number of chromosomes, the plants generated from them are haploid.
The anthers are removed from the flowers usually at a stage when nucleus of the pollen grain has undergone its first division. They are then cultured under sterile conditions on a medium that contains minerals, vitamins, sugar and other growth factors.
Considering the enormous number of pollen grains inside the anther, the significance of the anther culture technique in obtaining plantlets is quite obvious. These plantlets can be planted in soil after suitable period of development and raised to full maturity.
Although, the plants obtained through this technique are haploid and therefore, sterile, but such haploids are ideal for obtaining mutants by a variety of mutagenic agents. By the use of colchicine or other means, however, their chromosome number can be doubled and thus homozygous diploid pure lines can be obtained for various crop plants.
Such diploid plants are fertile. By inducing mutations and doubling of chromosome number in haploid plants obtained by another culture technique, new vistas have become open to increase disease resistance and consequently better yield of important food crops.
Originally, the anther culture technique was limited successfully only to a few closely related species of dicot plants such as Datura and tobacco (both of the family solanaceae), but now this technique has successfully been applied in more than 200 plants species including the monocots. This is significant because monocots contain important cereal crops. Much work in this direction has been done in China, Japan and Philippines. In Philippines, another culture technique is being employed for improving the rice crops at IRRI (International Rice Research Institute) at Manila.
A very significant achievement in the cell culture technique in recent past has been to remove the cell walls from cultured cells in some plants and to obtain the protoplasts. The protoplasts have successfully been cultured and fusion among different protoplasts observed. Such protoplasts have been made to undergo embryogenesis and to form plantlets and eventually entire plants.
Many procedures have been devised to obtain protoplasts from various tissues and cells such as leaves, pollen, callus, suspension cells etc. The sterilized tissues or cells are at first placed in a sterile medium containing mannitol, where they get slightly plasmolysed (so that protoplasts get detached from the cell walls).
Then they are transferred to an enzyme mixture containing purified preparations of cell wall degrading enzymes celluloses, hemicelluloses and or pectinases which dissolve their cell walls. After fusion among them, the protoplasts are washed and made free of these enzymes and placed in a suitable sterile medium so that regeneration of cell walls around them takes place. By using appropriate growth factors in the medium, the regenerated cells are made to divide to form cell masses and plantlets and then entire plants.
If haploid cells are used then diploid plants might be produced by such technique. By fusing protoplasts of two different species, novel hybrid plants can be generated. One such exciting example is the successful production of pomato which has been developed by fusing the protoplasts of potato and tomato plants.
Attempts are being made to introduce nucleic acids from different sources into the cultured protoplasts and alter their genetic makeup. It is also possible to introduce sub-cellular organelles such as chloroplasts and mitochondria into the protoplasts. Chances of getting novel plants by applying genetic complementation techniques and fusion of protoplasts are enormous.