Plant tissue culture and micropropagation
Plant tissue culture explained: totipotency, MS medium, hormone ratios, micropropagation stages, meristem culture, anther culture and somaclonal variation.
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A banana field in Maharashtra planted with tissue-culture plants looks strikingly uniform. The plants are of equal height, flower within days of each other and are free of the viruses that plague sucker-propagated plantations. Every plant in that field may have come from a single selected mother plant, multiplied in glass jars in a laboratory.
Plant tissue culture is growing plant cells, tissues or organs on a nutrient medium under aseptic (sterile) conditions. It rests on totipotency: the ability of a single plant cell to regenerate a whole plant. Gottlieb Haberlandt proposed the idea in 1902. Tissue culture is now used commercially for mass propagation, disease-free planting material, rapid breeding and conservation.
The culture medium
The most widely used medium is Murashige and Skoog (MS) medium (1962). Media contain:
- macronutrients (N, P, K, Ca, Mg, S) and micronutrients (Fe, Mn, Zn, B, Cu, Mo, Co, I);
- a carbon source, usually sucrose at 2–3%, because cultures photosynthesise little;
- vitamins, such as thiamine, nicotinic acid, pyridoxine and myo-inositol;
- plant growth regulators: auxins (IAA, IBA, NAA, 2,4-D) and cytokinins (BAP, kinetin, zeatin, TDZ);
- a gelling agent, usually agar at 0.6–0.8%, or the medium is used as a liquid;
- a pH adjusted to about 5.6–5.8 before autoclaving.
Skoog and Miller (1957) showed that the ratio of auxin to cytokinin controls development. A high cytokinin to auxin ratio gives shoots, a high auxin to cytokinin ratio gives roots, and intermediate ratios give callus, an unorganised mass of dividing cells. 2,4-D is the auxin most used to induce callus and somatic embryos.
Aseptic technique
Microbes grow far faster than plant tissues on a sugar-rich medium, so everything must be sterile.
- Media and tools are sterilised in an autoclave at 121 °C and 15 psi (about 1.05 kg per cm2) for 15–20 minutes. Heat-sensitive compounds are filter-sterilised.
- The explant is surface-sterilised. Typical treatments are 70% ethanol for 30–60 seconds, then sodium hypochlorite (1–2%) or mercuric chloride (0.1%), followed by several rinses in sterile water.
- Work is done in a laminar air-flow cabinet that blows HEPA-filtered air over the bench.
- Cultures are kept in growth rooms at about 25 ± 2 °C with a 16-hour photoperiod.
Types of culture
| Type | Material | Main use |
|---|---|---|
| Callus culture | Any tissue induced to form callus | Regeneration, somaclonal variation, secondary metabolites |
| Cell suspension | Callus broken up in liquid medium | Secondary metabolites, cell selection |
| Meristem culture | Shoot apex of 0.1–0.5 mm | Virus-free plants |
| Embryo culture | Immature embryos | Embryo rescue in wide crosses; overcoming seed dormancy |
| Anther and pollen culture | Anthers or microspores | Haploids and, after doubling, doubled haploids |
| Protoplast culture and fusion | Cells with walls removed by enzymes | Somatic hybrids, such as the pomato (potato + tomato) |
| Organ culture | Roots, shoots, ovaries | Physiology studies; in vitro pollination |
Regeneration happens by one of two routes. In organogenesis, shoots and roots form separately, either directly from the explant or via callus. In somatic embryogenesis, cells form embryo-like structures, each with a shoot and root pole, that grow directly into plantlets. Encapsulating somatic embryos in alginate beads gives synthetic seeds.
Micropropagation
Micropropagation is the clonal mass multiplication of plants in vitro. It follows the stages in the figure:
- Stage 0: selecting and preparing a healthy, true-to-type mother plant that has been tested (indexed) for viruses.
- Stage I: establishing a clean culture from the explant.
- Stage II: multiplying shoots by repeated subculture on a medium with cytokinin.
- Stage III: rooting the shoots on a medium with auxin.
- Stage IV: hardening (acclimatisation), when plantlets adapt from the humid jar to the greenhouse and then the field.
How fast can micropropagation multiply a plant?
One banana shoot tip is established in culture. Each four-week subculture multiplies shoots fourfold. After eight subcultures, about eight months, there could be 48 = 65,536 shoots. If 85% root and 90% of these survive hardening, about 65,536 × 0.85 × 0.90 ≈ 50,000 plants come from one mother plant. Laboratories usually limit the number of subcultures because the risk of somaclonal variation rises with each one.
Micropropagation is commercial for banana, potato, sugarcane, strawberry, date palm, orchids and other ornamentals, teak and bamboo. In India, the National Certification System for Tissue Culture Raised Plants (NCS-TCP), run under the Department of Biotechnology, certifies laboratories and tests plants for viruses and genetic fidelity.
Virus elimination
Viruses are often absent from the tiny dome of the apical meristem. Cells there divide faster than the virus can move in, and there are no vascular connections. Culturing a meristem of 0.1–0.5 mm, sometimes combined with thermotherapy (35–38 °C) or chemotherapy, produces virus-free plants. Morel and Martin first showed this in dahlia in 1952. It is now standard for potato, sweet potato, banana, sugarcane, citrus and garlic.
Somaclonal variation
Plants regenerated from culture, especially through callus, sometimes differ from the parent. This somaclonal variation (named by Larkin and Scowcroft in 1981) comes from chromosome changes, mutations and epigenetic changes. It is a problem in clonal propagation, where uniformity is required, but it can be a source of new variants for breeding. Disease-tolerant somaclones of sugarcane and banana have been selected this way.
Key points
- Tissue culture grows plant parts aseptically on nutrient media and relies on totipotency (Haberlandt, 1902).
- MS medium contains salts, sucrose, vitamins, growth regulators and agar, at pH 5.6–5.8.
- Skoog and Miller: high cytokinin to auxin gives shoots, high auxin to cytokinin gives roots, and intermediate ratios give callus.
- Micropropagation has five stages: mother plant, establishment, multiplication, rooting and hardening.
- Meristem culture produces virus-free plants; anther culture gives haploids; embryo rescue enables wide crosses; protoplast fusion creates somatic hybrids.
- Somaclonal variation is a risk for clonal uniformity but also a source of variation.
What the exams ask
- Define totipotency, callus, explant and somaclonal variation.
- List the components of MS medium and the roles of auxin and cytokinin.
- Describe the stages of micropropagation.
- Explain how meristem culture produces virus-free plants.
- Give uses of anther culture, embryo rescue and protoplast fusion.
Described in general terms: the kind of examination, not a past paper. Laisha never reproduces exam questions.
Questions people ask
Are tissue-culture plants genetically modified?
No. Tissue culture multiplies existing plants without changing their DNA, apart from occasional somaclonal variation. It is, however, an essential step in making transgenic and gene-edited plants, because transformed cells must be regenerated into whole plants.
Why are tissue-culture banana plants more expensive than suckers?
They require laboratory infrastructure, skilled staff, sterile media, electricity for climate-controlled growth rooms, and weeks of hardening. The extra cost usually pays back through uniform growth, earlier and synchronised harvest, freedom from bunchy top and other diseases, and higher yields.
Can any plant be micropropagated?
Most can, but protocols differ widely. Some woody trees, many conifers and certain cereals are hard to regenerate, and some species brown and die from phenolic compounds released by cut tissues. Media, hormone levels and explant choice must be optimised for each species, and sometimes for each variety.
Sources
- Bhojwani, S. S. and Dantu, P. K. Plant Tissue Culture: An Introductory Text. Springer, 2013.
- Murashige, T. and Skoog, F. "A revised medium for rapid growth and bio assays with tobacco tissue cultures." Physiologia Plantarum 15, 473–497 (1962).
- Skoog, F. and Miller, C. O. "Chemical regulation of growth and organ formation in plant tissues cultured in vitro." Symposia of the Society for Experimental Biology 11, 118–131 (1957).
- Larkin, P. J. and Scowcroft, W. R. "Somaclonal variation: a novel source of variability from cell cultures for plant improvement." Theoretical and Applied Genetics 60, 197–214 (1981).
- George, E. F., Hall, M. A. and De Klerk, G.-J. (eds) Plant Propagation by Tissue Culture, 3rd edition. Springer, 2008.
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