There is a little variation in the methods of plant cell, tissue and organ culture, but the basic steps are almost the same.
1. Basic Steps :
(i) Preparation of Suitable Nutrient Medium:
Suitable nutrient medium as per objective of culture is prepared and transferred into suitable containers (e.g. flasks, Petri plates, culture tubes) and autoclaved at 15 psi (pound per inch square) for 30 minutes. The hormones and vitamins are sterilised millipore filter and added to the medium.
(ii) Selection of Explants:
Always healthy and material young explants must be selected.
(iii) Sterilisation of Explants:
The explants are sterilised by disinfect (e.g., sodium, hypochlorite – NaOCl, mercuric chloride- HgCl2) and washed aseptically for 6-10 times with sterilised distilled water.
(iv) Inoculation (Transfer):
The sterile explant is inoculated on the surface of solidified nutrient medium under aseptic conditions. The cabinet of laminar airflow provides the sterile conditions.
The cultures are incubated in the growth chamber/tissue culture room at 25d2°C, 50-60% relative humidity and 16 hours of photoperiod (i.e. light and dark regime is created artificially in growth chamber). After defined period callus develops on the medium or shoots/roots develop from explant.
Plantlets regenerate after transferring a portion of callus onto another medium and induction of roots and shoots or directly from explants.
Hardening is the gradual exposure of plantlets for acclimatisation to environmental conditions.
(viii) Plantlet Transfer:
After hardening process plantlets are transferred to green house or field conditions.
2. Composition of Nutrient Media :
Composition of nutrient media governs the growth and morphogenesis of plant tissue in vitro. Generally, cultured tissue requires the same nutrients as the whole plant.
But laboratory grown cultures require some special components that promote optimum growth of a tissue under laboratory conditions.
Depending on the type of plant cells or tissue used for culture the composition of nutrient media vary. During the past two decades, considerable progress has been made on the development of media for growing plant cells, tissues and organs aseptically.
The principal constituents of tissue culture media are inorganic nutrients, carbon sources, organic supplements, growth regulators and gelling agent.
(a) Inorganic Nutrients:
Several minerals (i.e. macro- and micro- nutrients) are required by the plants. Macro-nutrients are needed in the concentrations more than 0.5 mMol l-1. Micro-nutrients are required in a concentration less than 0.05 mMol 1-1.
Minerals dissolved in water are dissociated and ionised. For example, in MS medium NH4NO3 contributes NO3 – and KNO3 contributes K+ ions.
There are six major macro-nutrients such as nitrogen, phosphorus, potassium, calcium, magnesium and sulphur. But the essential or micronutrients are required in low amount. These are boron, molybdenum, cobalt, zinc, manganese, iron and copper.
(b) Carbon and Energy Sources:
Mostly sucrose is required as carbon source is converted into glucose and fructose. These carbon sources enhance cell proliferation and tissue regeneration.
(c) Organic Supplements:
There is a large number of organise supplements used for best growth of tissues. Vitamins (vitamins B1, B2, B6, B5, E, riboflavin, folic acid) are added in the range of 0.1 to 10 mg l”1.
Amino acids (casein hydrolysate, L-glutamine, L-asperagine, L-glycine, L-arginine, L-cysteine) are commonly used as nitrogen source and enhancer of cell growth. Besides, culture media are also supplemented with casein, coconut milk, yeast and malt extracts, ground banana, orange juice and tomato juice.
Activated charcoal added to culture media is known to stimulate plant growth. If required antibiotics (streptomycine or kanamycin) may be added to culture medium to avoid systemic infection of micro-organisms.
(d) Growth Regulators:
For proliferation of cultured tissues four classes of growth regulators (e.g. auxins, cytokinins, gibberellins and abscisic acid) are used. For induction of root or shoot the ratio of hormones varies considerably.
For example, auxins (e.g. indole acetic acid, 1-naphthaleneacetic acid) induce cell division and cause elongation of stem, and internodes. Cytokinins (e.g. 6-benzylaminopurine, 6-benzyladenine, zeatin) induce cell division and shoot differentiation of cultured tissues.
Different ratios of auxins and cytokinins are important in morphogenesis of callus. High ratio of auxin to cytokinin promotes embryogenesis, callus and root initiation. But high ratio of cytokinin to auxin leads to axillary and shoot promotion.
(e) Solidifying Agents:
Most commonly agar (a polysaccharide obtained from seaweed i.e. a red alga, Gelidium amansii) is used as solidifying or gelling agent. Agar gels do not react with constituents of media and not digested by plant enzymes.
Generally 0.5 – 1% agar is used to form gel. Before use of agar, gelatin (10%) had been used as gelling agent. Demerit of gelatin is that it melts at low temperature (25°C).
The pW affects the uptake of ions. Optimum pH between 5.0 to 6.0 is required for growth and development of cultured tissues. Therefore, optimum pW of the medium should be maintained before sterilisation of the medium.
All steps of media preparation should be followed carefully. Otherwise, mistakes in preparation of media can harm much as any fault in the technique. Examples of some media used in cell and tissue culture.
3. Maintenance of Aseptic Environment
During in vitro culture maintenance of aseptic environment is the most difficult task. Because the cultures are easily contaminated by fungi and bacteria present in the air.
The contaminants produce; toxic metabolites which inhibit growth of cultures plant tissues. Therefore, each stem must be handled aseptically and with great care. Following are some of the sterilisation methods for aseptic manipulation of plant tissues.
(a) Sterilisation of Glassware:
Glassware (Petri plates, vials, culture tubes, flasks, pipettes, etc.), metallic instruments are sterilised in a hot air oven at 160-180°C for 2-4 hours.
(b) Sterilisation of Instruments:
The metallic instruments (e.g. forceps, scalpels, needles, spatulas, etc.) are flame sterilised i.e. dipping them in 25% ethanol followed by flaming and cooling. It is called incineration.
(c) Sterilisation of Culture Room and Transfer Area:
Floor and walls of culture room are washed first with detergent then 2% sodium hypochlorite or 95% ethanol. Larger surface area is sterilised by exposure to UV light.
The cabinet of laminar airflow is also sterilised by exposing UV light for 30 minutes and ethanol 15 minutes before beginning of work inside the cabinet of laminar airflow.
(d) Sterilisation of Nutrient Media:
Culture media are properly dispensed in glass container, plugged with cotton or sealed with plastic closures and sterilised by autoclaving (steam sterilisation) at 15 psi (that gives 121°C) for 30 minutes. Minimum time required for autoclaving of nutrient media is given.
During autoclaving vitamins, plant extracts, amino acids and hormones are denatured. Therefore, the solution of these compounds are sterilised by using millipore filter paper which has 0.2 µm pore diameter.
(e) Sterilisation of Plant Materials:
Surface of all plant materials has microbial contaminants. Therefore, disinfectants (e.g. sodium hypochlorite, hydrogen peroxide, mercuric chloride, or ethanol) should be used to make plant materials sterile.
Then the chemicals must be washed 6-8 times using sterile distilled water. Then explants are transferred aseptically on nutrient medium inside the cabinet of laminar airflow.
4. Types of Cultures :
There are different types of cultures which are produced through cultured plant materials e.g. explant culture, callus culture, cell or suspension culture, protoplast culture, organ culture.
(a) Explant Culture:
As discussed earlier, explant cultures are the cultures of plant materials (Fig. 7.6). Any part of plant may be explant such as young and healthy pieces of leaf, stem hypocotyl, cotyledons, etc. Explant cultures are generally used for induction of callus or regeneration of plant. Fig. 7.6: Explant culture.
(b) Callus Culture:
Callus is defined as an unorganised mass of cells. Generally parenchymatous cells constitute callus. For induction of callus the growth hormone auxin is added in the medium. The types and quantity of auxin depends on the source and genotype explants.
The cut ends of explant when put on callus culture medium exhibit callusing. The callus can be maintained for a long time by sub- culturing i.e. transfers of callus to a fresh medium of same composition.
The callus culture is most applied for preparation of single cell suspension and protoplasts, plant regeneration, and stress related to genetic transformation.
(c) Cell Suspension Culture:
Gottlieb Haberlandt (1902) was the first to originate the concept of cell culture. Leaf tissue and callus are the most suitable materials to isolate a single cell. Leaf tissue contains a homogeneous population of cells.
Hence, these act as a candidate for raising controlled cell cultures on a large scale. There are two methods described for isolation of a single cell.
(i) Mechanical Method:
About 10 grams of leaves are macerated in 40 ml of buffered medium using pestle and mortar. The homogenate is filtered through muslin cloth. Cells are washed by centrifugation at low speed. Cells are collected and debris are removed.
(ii) Enzymatic Method:
Using this method, maximum amount of cells can be isolated with minimum damage and injury in the cells. It is accomplished by providing osmotic protection to the cells.
The enzyme (pectinase/macerozyme) degrades the middle lamella and cell walls of the parenchymatous tissue. Consequently, individual cells are set free.
Besides, a single cell system is most commonly obtained from the callus. The callus growing around the explant is sub-cultured on the same culture medium to get mass of cells. Repeated sub- culturing results in friability of the callus. Friability is a pre-requisite for raising a fine cell suspension in liquid medium.
The free cells isolated from mesophyll tissue, callus or cell suspension are grown as single cells in suitable suspension culture medium. The suspension culture can be grown as batch culture or continuous culture.
The cell suspension cultures are used in: (a) induction of somatic embryos and shoots, (b) in vitro mutagenesis and selection of mutants, (c) genetic transformation studies, (d) production of secondary metabolites.
(d) Mass Cultivation of Plant Cells:
After selecting cell lines for high yield, efforts are made for mass culture to achieve large scale production of metabolites. The mass culture of cells includes some biotechnological barriers such as slow grow rate and genetic instability of cells.
These barriers are: shear sensitivity, oxygen transfer, cell aggregation and cell wall growth by the adhesion of cells. Therefore, the basic properties of cells are taken into account when cell cultures are selected for mass cultivation and metabolite production.
Cell suspension grows to low densities due to availability of low amount of nutrients. Therefore, different types of fermentors (21 to 20,000 litre capacity) are designed to grow high density of plant cells. An outline for mass production of cells for production of desired metabolites is given.
(i) Airlift Bioreactors:
These are designed to supply oxygen at the surface of plant cells. Such bioreactors support biomass level of 30 g per litre dry weight. Use of a bioreactor with modified paddle type impeller resulted in shikonin cells upto 75 g per litre dry weight.
(ii) Stirred-tank Bioreactor:
Stirred-tank bioreactors with modified impellers (that import mixing under low shear) have been advocated for large scale cultivation of fragile cell suspension cultures. Oxygen plays a major role in bioenergetics of plant cells in cultures.
Some cultures require O, besides C02 to improve cell growth and synthesis of metabolites. Hence, these gases are provided in a controlled way for their optimal utilisation in bioreactor.
(e) Protoplast Culture:
Protoplasts are the naked cells i.e. cells without cell wall. There are three ways of isolating protoplasts i.e. mechanical method, sequential enzymatic (two-step) method and mixed enzymatic (simultaneous) method.
Cooking (1968) mixed two enzymes together (simultaneous method) and isolated protoplasts in one attempt.
In the mixed enzymatic method cellulases, hemicellulases or pectinases are used to isolate protoplasts. Plant leaves are the most convenient and popular source of plant protoplasts due to their uniformity.
Besides, protoplasts are also isolated from seedling, callus, pollen grains, embryo sacs, etc. Plant protoplasts are obtained following the five basic steps:
1. Sterilisation of plant materials e.g. leaves
2. Removal of epidermis
3. Pre-enzyme treatment
4. Incubation in enzyme preparation, and
5. Isolation of protoplasts by filtration and centrifugation.
There are different factors that affect protoplast culture such as osmotic pressure (equal to growth medium), nutritional requirement (vitamin B2 and carbon and nitrogen), growth regulators (as required for callus culture), protoplast density (number of protoplasts in unit volume of medium) and environmental factors (e.g. optimum dim light or darkness, 24-26°C temperature and pH range from 5.5 to 5.8).
Examples of plant species that have regenerated from protoplasts are: Cucumis sativus, Capsicum annuum, Ipomoea batata, Beta vulgaris, Helianthus annuus, Glycine max, Rosa sp., Chrysanthemum sp., etc.
The isolated protoplasts are used for various purposes as below:
1. Biochemical and metabolic studies.
2. Fusion of two different somatic cells to get somatic hybrids.
3. Fusion of nucleated (containing nucleus) and enucleated (without nucleus) cells to produce cybrid (cytoplasmic hybrid).
4. Genetic manipulation.
5. Drug sensitivity.
(f) Organ Culture:
Different types of organs (e.g. roots, ovary, ovule, endosperm, anther) are excised from the plants. Then these are separately put over the surface of solidified gelled medium. The inoculated cultures are incubated in controlled growth chamber.
The cultures are named on the basis of organs used such as root culture, ovary culture, ovule culture, endosperm culture and another culture.
5. Plant Regeneration
The steps of organogenesis and somatic embryogenesis result in regeneration of plants in the cultured tissues.
In 1944, first time F. Skoog indicated that in vitro organogenesis could be chemically regulated. Further F. Skoog and C.O. Miller (1957) found that relatively high auxin: cytokinin ratio induced root formation in callus, while a low ratio of the same hormone favoured shoot formation (i.e. caulogenesis).
Formation of floral buds, vegetative buds and roots has been demonstrated in thin layer explants by regulating auxin : cytokinin ratio, carbohydrate supply and environmental conditions.
(b) Somatic Embryogenesis:
Generally embryos are formed in flowering plants. After fertilasation of gametes, zygote differentiates into embryo.
In contrast, the totipotent somatic cells at certain conditions in culture undergo embryogenic pathways and form somatic embryos or embryoids it shows complete embryogenesis in mango. The embryoids can regenerate to form complete plant.
In 1958, for the first time F.C. Stewart and co-workers (U.S.A.) reported somatic embryogenesis in suspension culture of carrot. One year later J. Reinert (1959) in Germany independently reported somatic embryogenesis in callus of carrot grown on nutrient agar medium.
According to Sharp and co-workers (1980) somatic embryogenesis is initiated either by inducedpre-embryonic determined cells which are programmed to form embryo, or by induced embryonic cells within the callus. 2,4-D is most commonly used auxin in somatic embryogenesis. The other chemicals compounds are 2,4,5-T, picloram, dicamba, etc.
The MS medium is used for somatic embryogenesis in 70% cases. 2, 4-D is used in 57% cases for induction of somatic embryogenesis, while NAA is used in 25% cases.
Supplementing the embryos with activated charcoal facilitates embryogenesis in several cultures. Auxin and reduced nitrogen content in medium induce somatic embryogenesis. Somatic embryos have been produced in a large number of plants of different families, for example, Atropa belladona, carrot, Panicum maxima, Pennisetum purpurium, etc.
H.W. Kohlenbach (1978) proposed the following classification of embryos:
(a) Zygotic embryos: It is formed by the zygote.
(b) Non-zygotic Embryos: It is formed from the cells other than zygote. It is of the following types:
(i) Somatic Embryos. Formed from somatic cells in vitro
(ii) Parthenogenetic Embryos. Formed by unfertilised egg
(iii) Androgenic Embryos. Formed by pollen grains
Generally, somatic embryos called embryoids are similar to zygotic embryos (or seed embryos) except they originate from somatic cells and are larger in size.
(c) Factors Affecting Regeneration:
Regeneration is a complex multi-stage phenomenon. There are many factors that influence the regeneration process. The major factors affecting regeneration are given below:
(i) Sources of Explant:
The responses of explant are governed by the size of explant, physiological and ontogenetic age, season in which explant is obtained and overall quality of plant.
(ii) Nutrient Media:
The constituents of the medium influence regeneration. These are inorganic salts, organic substances and culture environment (like physical form of medium, pH of medium).
(iii) Physical Factors:
The physical factors that influence regeneration are: light quality and quantity, temperature and relative humidity.