Introduction

Micropropagation is the practice of rapidly multiplying stock plant material to produce a large number of progeny plants, using modern plant tissue culture methods. Success in producing large numbers of plantlets requires the most sterile facility, an ideal cultural media, and an experienced expert to oversee stringent protocol.

Plant tissue culture: 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. Different techniques in plant tissue culture may offer certain advantages over traditional methods of propagation, including:

• 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, pest, 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.

Plant tissue culture relies on the fact that many plant cells have the ability to regenerate a whole plant. Using just a single cell, a pieces of a leaf, or (less commonly) roots can be used to generate a new plant on specific culture medium given the required nutrients and plant hormones. Having experienced, expert staff, is key to determining the required culture medium.

Plant Tissue Culture Techniques

Establishing Growth and Shoots

Micropropagation begins with the selection of plant material to be propagated. Clean stock materials that are free of viruses and fungi are important in the production of the healthiest plants. Living plant materials from an outside environment are naturally contaminated on their surfaces (and sometimes interiors) with microorganisms, so surface sterilization of starting materials (explants) in chemical solutions is required. Modern plant tissue culture must be performed under aseptic conditions in an environment with filtered air. The explant material is then surface sterilized, usually in multiple courses of bleach and alcohol washes and finally rinsed in sterilized water. The small portion of plant tissue, sometimes only a single cell, is placed on a growth medium, typically containing sucrose as an energy source and one or more plant growth regulators. Usually the medium is thickened with agar to create a gel which supports the explants during growth. Some plants are easily grown on simple media but others require more complicated media for successful growth. The plant tissue grows and differentiates into new tissues depending on the medium.

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 unorganized growth of cells, but the morphology of the outgrowth will depend on the plant species as well as the medium composition. As shoots emerge from a culture, they may be sliced off and rooted with auxin to produce plantlets. The skill and experience of the tissue culturist are important in judging which pieces to culture and which to discard. Following this stage, shoots must then experience a ‘hardening’ stage, this involves a transition towards a more natural growing environment.

Pretransplant

This stage involves treating the plantlets/shoots produced to encourage root growth and “hardening.” It is performed in vitro, or in a sterile “test tube” environment.

“Hardening” refers to the preparation of the plants for a natural growth environment. Until this stage, the plantlets have been grown in “ideal” conditions, designed to encourage rapid growth. Due to lack of necessity, the plants are likely to be highly susceptible to disease and often do not have fully functional dermal coverings and will be inefficient in their use of water and energy. In vitro conditions are high in humidity and plants grown under these conditions do not form a working cuticle and stomata that keep the plant from drying out. When taken out of culture the plantlets need time to adjust to more natural environmental conditions. Hardening typically involves slowly weaning the plantlets from a high-humidity; low light, warm environment to what would be considered a normal growth environment for the species in question. This is done by moving the plants to a location high in humidity,

In the final stage of plant micropropagation, the plantlets are removed from the plant media and transferred to soil or (more commonly) potting compost for continued growth by conventional methods.

Advantages

Micropropagation has a number of advantages over traditional plant propagation techniques:

• Micropropagation often produces more robust plants, leading to accelerated growth compared to similar plants produced by conventional methods – like seeds or cuttings.
• Micropropagation produces rooted plantlets ready for growth, saving time for the commercial grower when seeds or cuttings are slow to establish or grow.
• Micropropagation can produce disease-free plants.
• Micropropagation is the re-production or cloning of one plant with particular characteristics and traits beneficial to optimal growth and success.
• It can have an extraordinarily high fecundity rate, producing thousands of propagules while conventional techniques might only produce a fraction of this number.
• 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.
• A greater number of plants can be produced per square meter and the propagules can be stored longer and in a smaller area.

Disadvantages

Micropropagation is not always the perfect means of multiplying plants. Conditions that limits its use include:

• The entire process is very labor intensive.
• 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. However, this can make disease prevention more logistical.
• An infected plant sample can produce infected progeny. This is uncommon if 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.

Summary

Micropropagation is the practice of rapidly multiplying stock plant material to produce a large number of progeny plants, using modern plant tissue culture methods. The factors that have identified in contributing to the highest rate of success include an extremely sterile facility, determining the cultural media, and  an experienced expert.