Introduction
- Plant tissue culture has a great significance in plant biotechnology especially in the crop improvement programs. The term tissue culture may be defined as the process of in-vitro culture of explants (pieces of living differentiated tissues) in nutrient medium under aseptic conditions. However, in general, the tissue culture includes the term tissue culture as well as cell culture, organ culture and suspension culture also.
- Plant tissue culture is fundamental to most aspects of biotechnology of plants. It is evident now that plant biotechnology is one of the most beneficial of all the sciences. The products of plant biotechnology are being transferred rapidly from laboratories to the fields.
History
- G. Haberlandt, a German botanist, in 1902 cultured fully differentiated plant cells isolated from different plants. This was the very first step for the beginning of plant cell and tissue culture. Further contributions were made by the Cell Doctrine which admitted that a cell is capable of showing totipotency.
- Three other scientists Gautheret, White and Nobecourt also made valuable contributions to the development of plant tissue culture techniques.
- The first plant from a mature plant cell was regenerated by Braun in 1959. Foundation of commercial plant tissue culture was laid in 1960 with the discovery for a million fold increase in the multiplication of Cymbidium (an orchid) which was accomplished by G. M. Morel.
- In India, the work on tissue culture was initiated during 1950s at University of Delhi by Shri Panchanan Maheshwari who was working there in the Department of Botany. Discovery of haploid production was a land-mark in the development of in-vitro culturing of plants.
- Shri S.C. Maheshwari and Sipra Guha made a remarkable contribution in the development of plant tissue culture in India.
- Gottleib Haberlandt was the first person to make attempts for plant tissue culture, i.e., he developed the concept of in-vitro culture of plant cells and is aptly regarded as the father of tissue culture. Thereafter, there happened some dramatic advances in tissue culture techniques.
Basic Requirements and Techniques of Plant Tissue Culture:
The main requirements of plant tissue culture are:
- Laboratory Organisation
- Culture Media
- Aseptic Conditions
1. Laboratory Organisation:
In a standard tissue culture lab, there must be a few basic facilities like:
- A Media Room for preparation, sterilization and storage of culture media.
- Facilities for washing of lab-wares, explants, etc.
- Space for storage of lab-wares.
- Culture rooms or incubators where conditions of temperature, humidity and light etc. can be maintained.
- Observation and Data Collection area.
2. Culture Media:
- The formulation or the medium on which the explant is cultured is called culture medium. It is composed of various nutrients required for proper culturing. Different types of plants and organs need different compositions of culture media. A number of media have been devised for specific tissues and organs. Some important of them are:
- MS (Murashige and Skoog) Medium
- LS (Linsmaier and Skoog) Medium
- B5 (Gamborg’s) Medium
- White’s Medium, etc.
Important constituents of a culture medium are:
(i) Organic supplements:
- Vitamins like thiamine (B1), Pyridoxin (B6), Nicotinic Acid (B3), etc.
- Antibiotics like Streptomycin, Kanamycin;
- Amino Acids like Arginine, Asparagine
(ii) Inorganic Nutrients:
- Micronutrients as Iron (Fe), Manganese (Mn), Zinc (Zn), Molybdenum (Mo), Copper (Cu), Boron (Bo).
- Macronutrients include six major elements as Nitrogen (N), Sulphur (S), Phosphorus (P), Potassium (K), Calcium (Ca), Magnesium (Mg).
- Several trace elements are required for the growth of plant cells, tissues and organs. Iodide, though not essential, stimulates cell growth. Iron is added in a chelated form either as an EDTA salt or as agricultural chelates e.g. Sequestrene 330 Fe.
- The pH of the medium drops when ammonium source is used and rise when nitrate source is used. Though some species use ammonium or glutamine, high concentra-tions of these can be toxic due to shifts in pH. Phosphates provide phosphorus and also act as buffer.
(iii) Carbon and Energy Source:
- Most preferred carbon source is Sucrose. Others include lactose, maltose, galactose, raffinose, cellobiose, etc.
- In most of the cultures, sucrose is readily taken up in one or two days and converted to starch granules within the cells. Some media contain m-inositol which is a beneficial component for cell wall metabolism.
(iv) Growth Hormones:
- Auxins: Mainly for inducing cell division.
- Cytokinins: Mainly for modifying apical dominance and shoot differentiation.
- Abscisic Acid (ABA): Used occasionally.
- Gibberellins: Used occasionally.
- Other vitamins like biotin, pantothenic acid, folic acid, choline chloride, p-aminobenzoic acid, riboflavin, vitamin B12 and ascorbic acid are added at 1mg/Iitre or less in some instances, especially when cells or protoplasts are cultured.
(v) Gelling Agents:
- These are added to media to make them semisolid or solid. Agar, Gelatin, Alginate etc. are common solidifying or gelling agents.
(vi) Other Organic Extracts:
- Sometimes culture media are supplemented with some complex organic ingredients like coconut milk, orange juice, tomato juice, potato extract, yeast extract, fruit juices and casein hydrolysates are used. They provide amino acids, vitamins, growth regulators etc. which can enhance plant cell growth.
- Mannitol, sorbitol or combinations of these are used as an osmoticum (increase osmolarity).
3. Aseptic Conditions:
- Maintenance of aseptic conditions is the most critical and difficult aspect of in-vitro culturing experiments. Aseptic condition means the conditions free from any type of microorganisms (so as to prevent the loss of experiment by contamination). For this, sterilization (i.e., complete removal or killing of microbes) is done. The most common contaminants in culture are fungi and bacteria.
Measures to be taken for maintaining asepsis during tissue culture are:
- Sterilization of the culture vessels using detergents, autoclaves, etc.
- Sterilization of instruments like forceps, needles etc. by flame sterilization.
- Sterilization of culture medium using filter sterilization or autoclaving methods.
- Surface sterilization of explants using surface disinfectants like Silver Nitrate (1%), H2O2 (10-12%), Bromine water (1-2%), Sodium hypochlorite solution (0.3-0.6%), etc.
- The whole procedure of plant tissue culture is to be carried out essentially under aseptic conditions. So, the overall design of the laboratory must focus on the maintenance of aseptic conditions. Secondly, the worker is also required to have proper knowledge of operating various equipment’s like pH meter, balance, laminar air flow, microscope, etc.
- While performing the tissue culture experiments there must present the first aid kits and fire extinguishers in the laboratory to avoid any mishap or accident. In addition, proper attention should be given while handling the toxic chemicals and all the chemicals should be kept in correct labelled containers and bottles
General Technique of Plant Tissue Culture:
General technique of plant cell, tissue and organ culture is almost the same with a little variation for different plant materials. There are certain basic steps for the regeneration of a complete plant from an explant cultured on the nutrient medium.
- Selection and Sterilisation of Explant: Suitable explant is selected and is then excised from the donor plant. Explant is then sterilized using disinfectants.
- Preparation and Sterilisation of Culture Medium: A suitable culture medium is prepared with special attention towards the objectives of culture and type of explant to be cultured. Prepared culture medium is transferred into sterilized vessels and then sterilized in autoclave.
- Inoculation: Sterilized explant is inoculated (transferred) on the culture medium under aseptic conditions.
- Incubation: Cultures are then incubated in the culture room where appropriate conditions of light, temperature and humidity are provided for successful culturing.
- Sub culturing: Cultured cells are transferred to a fresh nutrient medium to obtain the plantlets.
- Transfer of Plantlets: After the hardening process (i.e., acclimatization of plantlet to the environment), the plantlets are transferred to green house or in pots.
Equipment in Tissue Culture Lab:
Cellular Totipotency:
- The potential of a plant cell to grow and develop into a whole new multicellular plant is described as cellular totipotency. In other words, the property of a single cell for differentiating into many other cell types is called as totipotency.
- This is the property which is found only in living plant cells and not in animal cells (exception being stems cells in animals).
- The term totipotency was coined in 1901 by Morgan. During culture practice, an explant is taken from a differentiated, mature tissue. It means, the cells in explants are generally non-dividing and quiescent in nature.
- To show totipotency, such mature, non-dividing cells undergo changes which revert them into a meristematic state (usually a callus state). This phenomenon of reverting back of mature tells to dividing state is called dedifferentiation.
- Now, these dedifferentiated cells have the ability to form a whole plant or plant organ. This phenomenon is termed as re-differentiation.
- Dedifferentiation and re-differentiation are the two inherent phenomena involved in the cellular totipotency. Regarding this, it is clear that the cell differentiation is the basic event for development of plants and it is also referred to as cytodifferentiation.
- To express its totipotency, a differentiated cell first undergoes the phenomenon of dedifferentiation and then undergoes the re-differentiation phenomenon (Fig). Usually the dedifferentiation of the explant leads to the formation of a callus. However, the embryonic explants, sometimes, result in the differentiation of roots or shoots without an intermediary callus state.
Various applications of cellular totipotency are:
- It has potential applications in the crop plant improvement.
- Micro-propagation of commercially important plants.
- Production of artificial or synthetic seeds.
- It helps in conservation of germplasm (genetic resources).
- This ability is utilized for haploid productions.
- Applied in producing somatic hybrids and cybrids.
- Helps in cultivation of those plants whose seeds are very minute and difficult to germinate.
- Also helps to study the cytological and histological differentiations.
- For high scale and efficient production of secondary metabolites.
- The genotypic modifications can also be possible.
Methods in Plant Tissue Culture:
- There are different methods of culturing plant material. These methods differ on the basis of explants used and their resultant products.
- Some of the most popular and advantageous methods in plant tissue culture are discussed below:
1. Cell Culture:
- Cell culture is actually, the process of producing clones of a single cell. The clones of cell are the cells which have been derived from the single cell through mitosis and are identical to each other as well as to parental cell.
- First attempts for cell culture were made by Haberlandt in 1902. However, he failed to culture single cell but his attempts stimulated other workers to achieve success in this direction.
- The method of cell culture is meritorious over other methods of culturing because it serves as the best way to analyze and understand the cell metabolism and effects of different chemical substances on the cellular responses. Single cell culturing is of immense help in crop improvement programs through the extension of genetic engineering techniques in higher plants.
The method of cell culture is done by following three main steps:
- Isolation of single cell from the intact plant by using some enzymatic or mechanical methods.
- In-vitro culturing of the single cell utilizing micro chamber technique, or micro drop method or Bergmann cell plating technique (Fig).
- Testing of cell viability done with the phase contrast microscopy or certain special dyes.
It is important to note here that the cell cultures require a suitably enriched nutrient medium and it should be done in dark because light may deteriorate the cell culture. Large scale culturing of plant cells under in-vitro conditions provides a suitable method for production of large varieties of commercially important phytochemicals.
2. Suspension Culture:
- A culture which consists of cells or cell aggregates initiated by placing callus tissues in an agitated liquid medium is called as a suspension culture. The continuous agitation of the liquid medium during a suspension culture is done by using a suitable device called as shaker, most common being the platform/orbital shaker.
- Agitation with shaker is important because it breaks the cell aggregates into single cell or smaller groups of cells and it helps in maintaining the uniform distribution of single cell and groups of cells in the liquid medium.
- A good suspension is the one which has high proportion of single cells than the groups of cells. Changes in the nutritional composition of medium may also serve as a useful technique for breakage of larger cell clumps (Fig).
- The general technique of suspension culture involves basically two types of cultures: batch culture and continuous cultures.
- A batch culture is a suspension culture in which cells grow in a finite volume of the culture medium and as a result, medium gradually depletes.
- On the other hand, a continuous suspension culture is the one which is continuously supplied with nutrients by the inflow of fresh medium but the culture volume is normally constant.
3. Root Culture:
- Pioneering attempts for root culture were made by Robbins and Kotte during 1920s. Later on, many workers tried for achieving successful root cultures. In 1934, it was White who successfully cultured the continuously growing tomato root tips.
- Subsequently, root culturing of a number of plant species of angiosperms as well as gymnosperms has been done successfully. Root cultures are usually not helpful for giving rise to complete plants but they have importance of their own. They provide beneficial information regarding the nutritional needs, physiological activities, nodulations, infections by different pathogenic bacteria or other microbes, etc.
4. Shoot Culture:
- Shoot cultures have great applicability in the fields of horticulture, agriculture and forestry. The practical application of this method was proposed by Morel and Martin (1952) after they successfully recovered the complete Dahalia plant from shoot-tips cultures.
- Later on, Morel realized that the technique of shoot culturing can prove to be a potent method for rapid propagation of plants (i.e. Micro propagation). In this technique, the shoot apical meristem is cultured on a suitable nutrient medium. This is also referred to as Meristem Culture (Fig).
- The apical meristem of a shoot is the portion which is lying beyond the youngest leaf primordium. Meristem tip culture is also beneficial for recovery of pathogen-free specially virus-free plants through the tissue culture techniques. Various stages in this culture process are the initiation of culture, shoot multiplication, rooting of shoots and finally the transfer of plantlets to the pots or fields.
Applications of Plant Tissue Culture:
- Germplasm conservation mainly in the form of cryopreservation of somatic embryos or shoot apices, etc. by cryopreservation, and slow growth cultures.
- Large scale production of useful compounds and secondary metabolites by using genetically engineered plant tissue cultures e.g. alkaloids, steroids, phenolics, variety of flavors and perfumes.
- Technique of micro propagation for enhancing the rate of multiplication of economically important plants.
- Eradication of systemic diseases in plants and raising disease free plants. e.g. Production of virus free plant (by thermotherapy, chryotherapy, chemotherapy, and meristem culture).
- Somaclonal variations are useful sources of introduction of valuable genetic variations in plants.
- Helps plants in imparting resistance to antibiotics, drought, salinity, diseases, etc.
- Somatic hybrids and cybrids overcome species barriers and sexual incompatibility and produce hybrid plants with desired combination of traits.
- Embryo culture helps in overcoming seed sterility and dormancy.
- Haploid production in culture helps to solve various problems of genetic studies and thus aids the plant breeders for producing new varieties.
- Production of synthetic seeds via somatic embryo differentiation for commercially important plants helps to achieve increased agricultural production. These seeds are easier to handle, transport and store.
- Large scale production of biomass energy.
- Plant tissue culture aids in producing the genetically transformed plants.
- Early flowering can be induced by in-vitro culturing of plants so as to attain commercial benefits.
- Triploids as well as polyploid plants can also be produced by tissue culture techniques for uses in plant breeding, horticulture and forestry.
- Seedless fruits and vegetables can be produced by following the endosperm culture method which adds to their commercial values.
- Increased Nitrogen fixation ability can be achieved through association of tissue culture techniques with genetic engineering.
- Callus cultures are useful in plant pathology as they act as an effective tool in the study of mechanism of disease resistance and susceptibility.
- Different tissue culture techniques help us to study various biosynthetic processes, physiological changes and cytogenetic changes