In this article we will discuss about the Callus Culture:- 1. Meaning of Callus Culture 2. Nutrient Medium of Callus Culture 3. Methods 4. Development 5. Nature 6. Significance.
Meaning of Callus Culture:
Callus is formed by the proliferation of the parent tissue. The cells of a callus are parenchymatous, amorphous and unorganised. Generally callus is formed as a result of injury at the cut ends of a stem or a root. Localised centres of activity is recorded in a callus.
When tissues on culture produce unorganised mass of callus with no regular form then it is called callus culture. Callus formation from isolated stem segments of Populous was first observed by Rechinger in 1893. Working with cambial tissues of carrot and tobacco first prolonged callus culture were simultaneously reported by Gautheret in Paris, Nobecourt in England and White in Princeton, USA in 1930.
Gautheret cultured explants of carrot root on a medium containing inorganic salts, sugar (glucose), thiamine, cystine and IAA. On culture the explants grew forming undifferentiated tissue or callus. By repeated sub-culturing this callus was maintained for a prolonged period. White (’39) cultured the stem pro-cambium of hybrid Nicotiana glauca x N- langsdorfii on agar medium where callus was formed.
Callus may initiate from explants of any multicellular plant. Explants from stem, root, leaf, flower, fruit or seed etc. may be taken for culture. Callus formation has been recorded from storage parenchyma, pericyclic cells of roots, cambial cells of vascular bundles, provascular cells, secondary phloem, pith cells, mesophyll cells and cotyledons.
Usually large pieces of tissue are selected for culture. Minimum size of the ex- plant is generally determined by the average cell size of the tissue to be cultured. 3.8 mg carrot explant having about 25,000 cells is viable.
But as the cells of Jerusalem artichoke is much larger, the minimum size of the explant is found to be 8 mg having about 20,000 cells. But Caplin (’63) successfully cultured much smaller explant of artichoke.
For sub-culturing the inoculum should not be very small, as very small inoculum fails to grow or shows little growth. According to Street (’69) the inoculum should be 0.5—1 cm and weighing about 20—100 mg.
The callus is sub-cultured because:
(a) The nutrient may be exhausted,
(b) Agar may be desiccated, or
(c) Cell metabolites may accumulate and cause toxicity. Active growth can be maintained even after several subcultures. Repeated sub-culturing can be avoided by freeze preservation of the culture (Withers ’79).
Nutrient Medium of Callus Culture:
Some standard media, such as, Murashige and Skoog’s medium can be successfully used for callus culture. For initiation and maintaining callus kinetin is widely used in the medium.
For callus initiation usually an exogenous supply of hormone is required. But explants having cambial cells do not require a supply of hormone. According to hormone requirements callus culture may be of five types.
(a) Auxin requiring cultures,
(b) Cytokinin requiring cultures,
(c) Cultures requiring both auxin and cytokinin,
(d) Gibberellin requiring cultures. In some plants, such as tobacco, presence of gibberellin and N6 2 isopentenyl adenine in the medium favours callus growth. But gibberellin inhibits growth of callus tissue in monocots, (e) cultures requiring other natural extracts, such as, yeast extract, coconut milk, casein hydrolysate or tomato juice etc.
Methods of Callus Culture:
Usually explants from suitable materials (such as, carrot root, potato or sweet potato tuber, stem of tobacco, hypocotyl and cotyledon of soya bean etc..) are taken. The explants is first surface sterilised with 1.6% sodium hypochlorite solution or 0.1% mercuric chloride solution or 1% aqueous solution of bromine.
Then the inner uncontaminated tissue is excised. If the excised tissue (such as, root, hypocotyl, cotyledon etc.) is taken from a seedling then the seed before germination is surface sterilised and allowed to germinate under aseptic conditions.
Carrot Root Culture:
(1) Fresh and healthy carrot root is selected. It is thoroughly washed in running tap water.
(2) External 1-2 mm is scraped. Upper 1 cm of carrot root is discarded and then it is cut into 0.5 cm thick slices (Fig. 19).
(3) These slices are placed immediately in a beaker containing water.
(4) These are then transferred to a beaker containing sodium hypochlorite solution and kept there for 10 minutes.
(5) Slices are taken out with a sterile force from the hypochlorite solution and washed successively in 3 beakers containing double distilled water keeping the slices for 20—30 seconds in each. The slices are kept in the third beaker.
(6) A carrot slice is taken and is placed on a petridish. Tissue cylinders are cut out from the cambial region by a sterilised cork borer, after cutting maximum number of tissue cylinders from the cambial region remaining portion of the slice is discarded
(7) Tissue cylinders are placed in a petridish containing double distilled water.
(8) A tissue cylinder is transferred to a petridish and its two sides are trimmed with a sterile scalpel and discarded.
(9) Remaining cylinder is cut into explants measuring 5 mm diameter and 2 mm thickness.
(10) These explants are placed in a petridish containing double distilled water.
(11) Explants are then transferred with a sterile forceps on the surface of a sterile filter paper on a petridish. The upper and lower surfaces of each explants are blotted.
(12) One such explants is transferred to each culture tube containing the nutrient medium.
(13) Culture tubes are kept in a glass storage jar, wrapped in aluminium foil and placed in an incubator at 25°C.
(14) The surface of the explants after few days becomes somewhat rough, indicating initiation of the callus. Callus can be maintained from few weeks to three months depending on the rate of growth.
(15) Generally after 6-8 weeks the callus is sub-cultured. The callus is divided into small parts of 100 mg approximately.
(16) Each piece is transferred to a new flask containing 30 c.c. of culture medium and sub-cultured at a temperature of 25°C or above.
Development of a Callus Culture:
Callus formation from an explants occurs in three stages:
(a) Induction stage:
Metabolism is stimulated and the cells prepare to divide. Cell size remains unchanged.
(b) Cell division stage:
Cells divide actively and the cell size decreases. Cell division is mainly periclinal and occurs towards the periphery giving rise to wound cambial cells.
Cells differentiate by expansion and maturation. Rapidly growing calluses are more or less alike but as the growth rate decreases the calluses show their characteristic structures and forms. But all calluses have some similarities. They all contain nodular or sheet meristems in groups or scattered throughout the tissue.
Vascular nodules or meristemoids are formed in a callus from small groups of meristematic cells. The nodules may not differentiate or may form root or shoot primordia or embryos.
These meristemoids resemble vascular bundles and consist of xylem, phloem and cambium. Measurement of growth in a callus culture is based on fresh weight or dry weight or cell number counts. The callus may be weighed directly under aseptic conditions.
Nature of Callus Tissue:
Morphological nature of the callus tissue varies considerably. Some cultures are hard and anatomically consists of compactly arranged small cells without intercellular spaces. Such callus may be composed of lignified cells.
Other callus may be friable and anatomically consists of large loosely arranged cells with intercellular spaces. Friable callus is fragile and easily breaks up. These are suitable for suspension culture, where the tissue can be dispersed by mechanical agitation.
Reinert and White (’56) working with Picea glauca noted that hard compact callus can easily give rise to friable callus but the friable type cannot form compact type. But Blakesly and Steward (’61) working with Haplopappus gracilis and Grant and Fuller (’68) working with Vicia faba showed that friable callus and hard callus are inter-convertible.
The colour of the callus also varies and usually depends on the colour of the tissue from which it is taken. The callus may be colourless or green containing chlorophyll or yellow having carotenoids or flavonoids or it may be purple due to the presence of anthocyanin. Callus may be uniformly or partially coloured.
Calluses derived from green explants are not always autotropic. Chlorophyll containing callus requires an exogenous supply of sugar (Hildebrandt and et al) and light. Carrot root explants cultured on a medium supplemented with IAA, inositol, kinetin in presence of light become autotropic.
After repeated subculture the colour of the callus may change. Concentration of sugar, dextrose and soluble starch, deficiency of nitrogen, light, temperature and exogenous auxin etc. influence the colour of the callus.
Anatomically, a callus may be homogenous, consisting of uniform parenchyma cells or it may be heterogenous, having differentiation into tracheids, seive tubes, trichomas, secretory cells and suberized cells etc.
Callus which grows actively may be composed of large vacuolated parenchyma cells and small closely arranged dividing cells. Such variations among the cells of the callus tissue is due to the origin and age of the callus and the composition of the culture medium.
Grant and Fuller (’68) noted that friable callus contains larger amount of cell wall polysaccharides than compact callus, but a lower amount of cellulose compared with hemicellulose and pectic substances. In a callus culture phenotypic variations may occur due to epigenetic changes. Such changes are reversible and occur due to selective gene expression as in cytokinin habituation.
Callus on prolonged culture shows changes in the nuclear cytology of the cells. Changes both in chromosome structure and chromosome number have been noted. Chromosomal aberrations, nuclear fragmentation or endopolyploidy etc. are observed.
With the age of the callus culture these nuclear abnormalities increase and certain aneuploid or polyploid cells divide more rapidly than normal cells and form the dominant cell line. Within few months of the callus culture nuclear changes may occur. But certain callus is comparatively stable. In Crepis capillaris and Helianthus annus callus remains stable for about two years.
Significance of Callus Culture:
(1) Yeoman and Mitchell (’70j described a method to determine the total amount of nucleic acids (i.e., DNA and RNA) rapidly from callus culture.
(2) By using tracer elements in callus culture the path of various metabolic processes has been determined. For determination of duration and extent of DNA tritiated thymidine was used (Harland ’71). In rose callus culture Dougall (’65) used C14 amino acids to understand the pathway of protein synthesis. In callus culture of the Jerusalem artichoke Fraser (’68) used P32 phosphate to investigate the metabolism of RNA.
(3) Friable callus is suitable for cell suspension culture.