CHAPTER 7 : INNOVATION AND CHALLENGES IN MALAYSIAN AGRICULTURE (TISSUE CULTURE)
Monday 30 November 2015 @ 00:32

TISSUE CULTURE

1. DEFINITION OF TISSUE CULTURE
Tissue culture is the growth of tissues or cells separate from the organism. This is typically facilitated via use of a liquid, semi-solid, or solid growth medium, such as broth or agar. 
Tissue culture commonly refers to the culture of animal cells and tissues, with the more specific term plant tissue culture being used for plants. The term "tissue culture" was coined by American pathologist Montrose Thomas Burrows, M.D.

2. TYPES OF TISSUE CULTURE
  • Cell Culture
  • Organ Culture
  • Plant Tissue Culture (We're focusing on this topic.)
PLANT TISSUE CULTURE
1. DEFINITION OF PLANT TISSUE CULTURE
Plant tissue culture is a collection of techniques used to maintain or grow plant cells, tissues or organs under sterile conditions on a nutrient culture medium of known composition. 
Plant tissue culture is widely used to produce clones of a plant in a method known as micropropagation. 
Micropropagation is the practice of rapidly multiplying stock plant material to produce a large number of progeny plants, using modern plant tissue culture methods. 
Micropropagation is used to multiply noble plants such as those that have been genetically modified or bred through conventional plant breeding methods. It is also used to provide a sufficient number of plantlets for planting from a stock plant which does not produce seeds, or does not respond well to vegetative reproduction.
  • STAGE OF MICROPROPAGATION 
Establishment => Multiplication => Pretransplant => Transfer from culture
  • ADVANTAGES OF MICROPROPAGATION
- Micropropagation can be used to produce disease-free plants.
- It can have an extraordinarily high fecundity rate, producing thousands of propagules while conventional techniques might only produce a fraction of this a number.
- It is the only viable method of regenerating genetically modified cells or cells after protoplast fusion.
- It is useful in multiplying plants which produce seeds in uneconomical amounts, or when plants are sterile and do not produce viable seeds or when seed cannot be stored (see recalcitrant seeds).
- Micropropagation often produces more robust plants, leading to accelerated growth compared to similar plants produced by conventional methods - like seeds or cuttings.
- Some plants with very small seeds, including most orchids, are most reliably grown from seed in sterile culture.
- A greater number of plants can be produced per square meter and the propagules can be stored longer and in a smaller area.
  • DISADVANTAGES OF MICROPROPAGATION
- It is very expensive, and can have a labour cost of more than 70%
- A monoculture is produced after micropropagation, leading to a lack of overall disease resilience, as all progeny plants may be vulnerable to the same infections.
- An infected plant sample can produce infected progeny. This is uncommon as the stock plants are carefully screened and vetted to prevent culturing plants infected with virus or fungus.
- Not all plants can be successfully tissue cultured, often because the proper medium for growth is not known or the plants produce secondary metabolic chemicals that stunt or kill the explant.
- Sometimes plants or cultivars do not come true to type after being tissue cultured. This is often dependent on the type of explant material utilized during the initiation phase or the result of the age of the cell or propagule line.
- Some plants are very difficult to disinfect of fungal organisms.

2. TECHNIQUES OF PLANT TISSUE CULTURE
Modern plant tissue culture is performed under aseptic conditions under HEPA filtered air provided by a laminar flow cabinet. Living plant materials from the environment are naturally contaminated on their surfaces (and sometimes interiors) with microorganisms, so surface sterilization of starting material (explants) in chemical solutions (usually alcohol and sodium or calcium hypochlorite) is required. Explants are then usually placed on the surface of a solid culture medium, but are sometimes placed directly into a liquid medium, particularly when cell suspension cultures are desired. Solid and liquid media are generally composed of inorganic salts plus a few organic nutrients, vitamins and plant hormones. Solid media are prepared from liquid media with the addition of a gelling agent, usually purified agar.

The composition of the medium, particularly the plant hormones and the nitrogen source (nitrate versus ammonium salts or amino acids) have profound effects on the morphology of the tissues that grow from the initial explant. For example, an excess of auxin will often result in a proliferation of roots, while an excess of cytokinin may yield shoots. A balance of both auxin and cytokinin will often produce an unorganised growth of cells, or callus, but the morphology of the outgrowth will depend on the plant species as well as the medium composition. As cultures grow, pieces are typically sliced off and transferred to new media (subcultured) to allow for growth or to alter the morphology of the culture. The skill and experience of the tissue culturist are important in judging which pieces to culture and which to discard.
As shoots emerge from a culture, they may be sliced off and rooted with auxin to produce plantlets which, when mature, can be transferred to potting soil for further growth in the greenhouse as normal plants.

3. REGENERATION PATHWAYS
The specific differences in the regeneration potential of different organs and explants have various explanations. The significant factors include differences in the stage of the cells in the cell cycle, the availability of or ability to transport endogenous growth regulators, and the metabolic capabilities of the cells. The most commonly used tissue explants are the meristematic ends of the plants like the stem tip, auxiliary bud tip and root tip. These tissues have high rates of cell division and either concentrate or produce required growth regulating substances including auxins and cytokinins. Shoot regeneration efficiency in tissue culture is usually a quantitative trait that often varies between plant species and within a plant species among subspecies, varieties, cultivars, or ecotypes. Therefore, tissue culture regeneration can become complicated especially when many regeneration procedures have to be developed for different genotypes within the same species.

The three common pathways of plant tissue culture regeneration are;
(i) propagation from preexisting meristems (shoot culture or nodal culture)
(ii) organogenesis
(iii) non-zygotic (somatic) embryogenesis

organogenesis

The propagation of shoots or nodal segments is usually performed in four stages for mass production of plantlets through in vitro vegetative multiplication but organogenesis is a common method of micropropagation that involves tissue regeneration of adventitious organs or axillary buds directly or indirectly from the explants. Non-zygotic embryogenesis is a noteworthy developmental pathway that is highly comparable to that of zygotic embryos and it is an important pathway for producing somaclonal variants, developing artificial seeds, and synthesizing metabolites. Due to the single cell origin of non-zygotic embryos, they are preferred in several regeneration systems for micropropagation, ploidy manipulation, gene transfer, and synthetic seed production. Nonetheless, tissue regeneration via organogenesis has also proved to be advantageous for studying regulatory mechanisms of plant development.

4. APPLICATIONS OF PLANT TISSUE CULTURE
plants as bioreactors

Plant tissue culture is used widely in the plant sciences, forestry, and in horticulture. Applications include:
- The commercial production of plants used as potting, landscape, and florist subjects, which uses meristem and shoot culture to produce large numbers of identical individuals.
- To conserve rare or endangered plant species.
- A plant breeder may use tissue culture to screen cells rather than plants for advantageous characters, e.g. herbicide resistance/tolerance.
- Large-scale growth of plant cells in liquid culture in bioreactors for production of valuable compounds, like plant-derived secondary metabolites and recombinant proteins used as biopharmaceuticals.
- To cross distantly related species by protoplast fusion and regeneration of the novel hybrid.
- To rapidly study the molecular basis for physiological, biochemical, and reproductive mechanisms in plants, for example in vitro selection for stress tolerant plants, and in vitro flowering studies.
- To cross-pollinate distantly related species and then tissue culture the resulting embryo which would otherwise normally die (Embryo Rescue).
 - For chromosome doubling and induction of polyploidy, for example doubled haploids, tetraploids, and other forms of polyploids. This is usually achieved by application of antimitotic agents such as colchicine or oryzalin.
- As a tissue for transformation, followed by either short-term testing of genetic constructs or regeneration of transgenic plants.
- Certain techniques such as meristem tip culture can be used to produce clean plant material from virused stock, such as potatoes and many species of soft fruit.
- Production of identical sterile hybrid species can be obtained.

5. ADVANTAGES OF PLANT TISSUE CULTURE
  • The production of exact copies of plants that produce particularly good flowers, fruits, or have other desirable traits.
  • To quickly produce mature plants.
  • The production of multiples of plants in the absence of seeds or necessary pollinators to produce seeds.
  • The regeneration of whole plants from plant cells that have been genetically modified.
  • The production of plants in sterile containers that allows them to be moved with greatly reduced chances of transmitting diseases, pests, and pathogens.
  • The production of plants from seeds that otherwise have very low chances of germinating and growing, i.e.: orchids and Nepenthes.
  • To clean particular plants of viral and other infections and to quickly multiply these plants as 'cleaned stock' for horticulture and agriculture.