In this article, we will discuss the history, principles and significance of the callus culture.
Brief Past History of Callus Culture:
R. J. Gautheret (France) (1934-1937):
He first succeeded in promoting the development of callus tissue from excised cambial tissue of Salix capraea and other woody species. He was able to promote the growth of the callus tissue using simple nutrient medium supplemented with three vitamins (thiamine, pyridoxine and nicotinic acid) and indole-3-acetic acid (IAA) newly discovered by F W Went and K V Thimann (1937).
P. Nobecourt (France) (1939):
He first established the callus culture capable of potentially unlimited growth on semisolid agar medium. He started his work using the tap root explant of Daucus carota. He was also to maintain the culture by simply transferring portions of the callus to fresh medium at regular interval of four to six weeks.
J. Van Overbeck, M. E. Conklin and A. F. Blakeslee (1941):
They first reported the importance of coconut milk in callus culture.
S. M. Caplin and F. C. Steward (1948):
They first succeeded in obtaining the growth of differentiated non-cambial cells isolated from Daucus carota using coconut milk in medium. Later, they .used coconut milk in combination with synthetic auxin such as 2, 4-dichlorophenoxy acetic acid in medium and were able to induce the division of cells in species which had previously been difficult to grow.
F. Skoog (1954-1955):
He was able to produce the callus culture from the cut piece of stem of Nicotiana tabacum in nutrient medium containing auxins. The callus remained active for some time but failed to grow. Later, he discovered kinetin from old sample of herring sperm DNA. After the addition of kinetin in culture medium, it was possible to renew the growth of tobacco stem callus tissue.
F. Skoog and C. O. Miller (1957):
They first put forward the concept of hormonal control of organ formation from callus tissue. They also suggested that equal concentration of auxin and kinetin induced the continuous growth of callus tissue. The inclusion of kinetin in culture media has made it possible to produce callus culture from a large number of plant species.
What is the Meaning of Callus Tissue?
Callus tissue means an unorganised proliferative mass of cells produced from isolated plant cells, tissues or organs when grown aseptically on artificial nutrient medium in glass vials under controlled experimental conditions.
Principles of Callus Culture:
For successful initiation of callus culture, three important criteria should be accomplished:
(i) Aseptic preparation of plant material,
(ii) Selection of suitable nutrient medium supplemented with appropriate ratio of auxins and cytokinins or only appropriate auxin, and
(iii) Incubation of culture under controlled physical condition.
Different plant parts carry a number of surface borne micro-organisms-like bacteria, fungus etc. So, before attempting to initiate a callus culture, it is necessary to surface sterilize the plant parts which are to be cultured. Typical plant parts may be segments of root or stem, pieces of leaf lamina, flower petals etc. The excised plant parts called explants are at first washed with liquid detergent (generally 5% v/v ‘Teepol’).
Then the explants are surface sterilized by the most commonly used chemicals such as 0.1% w/v mercuric chloride (HgCl2) or Sodium hypochlorite (0.8% to 1.6% available chlorine) for a limited time (generally 10-15 minutes). After surface sterilization, the explants are repeatedly rinsed with autoclaved distilled water.
The surface sterilized plant material is cut aseptically into small segments (a few millimeters in size). Size of explants is a critical factor for the induction of callus tissue. The explants are finally transferred aseptically on a suitable nutrient medium solidified with agar.
Agar solidified or semi-solid nutrient medium after its preparation and sterilization by autoclave at 15 lbs. pressure for 15 minutes is used for the induction of callus tissue. In most cases successful callus culture depends upon the inclusion of plant growth hormones in the nutrient medium and for healthy callus growth usually both an auxin and a cytokinin are required.
Incubation of culture under controlled physical conditions such as temperature, light, and humidity is indispensible for the proper initiation of callus tissue. The suitable temperature for in vitro callus initiation and growth is usually 25 ±2°C. In some plant materials initiation and growth of the callus tissue take place in totally dark condition.
However, in case of other plant materials, a particular photoperiod (16 hrs. light and 8 hrs. dark) is necessary for the initiation and growth of callus tissue. Approximately 2,000 to 3,000 lux artificial light intensity is needed. Cool, white fluorescent lamps (4 ft. 2 No.) are generally used for providing light. Generally 55% to 60% relative humidity is maintained in the culture room.
Once the growth of the callus tissue is well established, portions of the callus tissue can be removed and transferred directly onto fresh nutrient medium to continue growth. In this manner, callus cultures can be continuously maintained by serial subcultures.
Protocol of Callus Culture:
Callus tissue can be induced from different plant tissues of many plant species. Carrot is a highly standardized material.
So the callus culture from excised tap root of carrot is described here by the following procedure:
(1) A fresh tap root of carrot is taken and washed thoroughly under running tap water to remove all surface detritus (Fig 3.1).
(2) The tap root is then dipped into 5% ‘Teepol’ for 10 minutes and then the root is washed.
The carrot root, sterilized forceps, scalpels, other instruments, autoclaved nutrient medium petridishes are then transferred to laminar air flow or inoculation chamber. Throughout the manipulation sequence forceps, scalpels must be kept in 95% ethanol and flamed thoroughly before use.
(3) The tap root is surface sterilized by immersing in 70% v/v ethanol for 60 seconds, followed by 20-25 minutes in sodium hypochlorite (0.8% available chlorine).
(4) The root is washed three times with sterile distilled water to remove completely the hypochlorite.
(5) The carrot is then transferred to a sterilized petridish containing a filter paper. A series of transverse slice 1 mm in thickness is cut from the tap root using a sharp scalpel.
(6) Each piece is transferred to another sterile petridish. Each piece contains a whitish circular ring of cambium around the pith. An area of 4mm2 across the cambium is cut from each piece so that each small piece contains part of the phloem, cambium and xylem. Size and thickness of the explants should be uniform.
(7) Always the lid of petridish is replaced after each manipulation.
(8) The closure (cotton plug) from a culture tube is removed and flamed the uppermost 20 mm of the open end. While holding the tube at an angle of 45°, an explants is transferred using forceps onto the surface of the agarified nutrient medium. Nutrient medium is Gamborg’s B5 or MS medium supplemented with 0.5 mg/L 2, 4-D.
(9) The closure is immediately placed on the open mouth of each tube. The forceps are always flamed before and after use. Date, medium and name of the plant are written on the culture tube by a glass marking pen or pencil.
(10) Culture tubes after inoculation are taken to the culture room where they are placed in the racks. Cultures are incubated in dark at 25°C.
(11) Usually, after 4 weeks in culture the ex- plants incubated on medium with 2, 4-D will form a substantial callus. The whole callus mass is taken out aseptically on a sterile petridish and should be divided into two or three pieces.
(12) Each piece of callus tissue is transferred to a tube containing fresh same medium.
(13) Prolonged culture of carrot tissue produces large calluses.
Significance of Callus Culture:
Callus culture as such has no major importance unless and until it is used for other experimental objectives.
Still, callus culture has got some importance:
(i) The whole plant can be regenerated in large number from callus tissue through manipulation of the nutrient and hormonal constituents in the culture medium. This phenomenon is known as plant regeneration or organogenesis or morphogenesis.
Similarly, by manipulation of nutrient and hormonal constituents, cluster of embryos can be achieved directly from the somatic cells of callus tissue. These embryos are called somatic embryos. This phenomenon is known as somatic embryogenesis. Somatic embryo directly gives rise the whole plant.
(ii) Callus tissue is good source of genetic or karyotype variability, so it may be possible to regenerate a plant from genetically variable cells of the callus tissue.
(iii) Cell suspension culture in moving liquid medium can be initiated from callus culture.
(iv) Callus culture is very useful to obtain commercially important secondary metabolites. If a bit of tissue from a medicinally important plant is grown in vitro and produced callus culture, then secondary metabolites or drug can be directly extracted from the callus tissue without sacrificing the whole plant. So, this alternative technique helps the conservation of medicinal plants in nature.
(v) Several biochemical assays can be performed from callus culture.