Micropropagation & Cryopreservation: A Deep Dive

Posted on Nov 12, 2024 in Biology

Micropropagation: Definition, Merits & Demerits

Micropropagation is the practice of rapidly multiplying stock plant material to produce a large number of progeny plants using modern plant tissue culture.

Merits of Micropropagation

  • Clonal Mass Propagation: Extremely large numbers of plants can be produced.
  • Culture Initiation: Culture is initialized from small parts of plants.
  • Disease and Virus-Free Plantlets: This leads to simplification of international exchange of plants.
  • Increased Production of Slow-Growing Plants: Enables increased production of plants that normally propagate very slowly, such as narcissus.
  • Vegetative Propagation of Sterile Hybrids: Can be used as parent plants for seed production (e.g., cabbage).
  • Rapid Cloning of Disease-Free Trees: One of the rapid methods for cloning.
  • Long-Term Storage: In vitro cultures can be stored for a long time through cryopreservation.
  • Shortened Breeding Cycle: Enables a shorter breeding cycle.

Demerits of Micropropagation

  • Cost: Expensive laboratory equipment and service.
  • Lack of Mechanization: No possibility of using mechanization.
  • Non-Autotrophic Plants: Plants are not autotrophic.
  • Poor Acclimatization: Cannot be done with all crops to date; in cereals, much less success is achieved.
  • Mass Propagation Limitations: Cannot be done with all crops to date; in cereals, much less success is achieved.
  • Rooting Problems: More problems in inducing rooting.
  • Growth Uniformity: May not get uniform growth or the original plant from tissue culture.

Cryopreservation

Cryo is a Greek word (kryos – frost). It literally means preservation in a “frozen” state. The principle is to bring plant cells or tissues to a zero metabolism and non-dividing state by reducing the temperature in the presence of a cryoprotectant.

Cryopreservation is non-lethal storage of biological material at ultra-low temperatures. At the temperature of liquid nitrogen (-196°C), almost all metabolic activities of cells cease, and the sample can be preserved for extended periods. However, only some biological materials can be frozen to -196°C without affecting cell viability. Liquid nitrogen is chemically inert, relatively low cost, non-toxic, non-flammable, and readily available; therefore, it is the most widely used material for cryopreservation. Dry ice can also be used.

Mechanism of Cryopreservation

Freeze preservation is based on transferring water present in the cells from a liquid to a solid state. Due to the presence of salts and organic molecules in the cells, cell water requires a much lower temperature to freeze (even up to -68°C) compared to the freezing point of pure water (around 0°C).

When stored at low temperatures, metabolism and biological deterioration in the cells/tissues almost come to a standstill. A cryoprotectant protects biological tissue from freezing damage due to ice formation. Several cryoprotectants include DMSO, glycerol, ethylene, propylene, sucrose, mannose, glucose, proline, and acetamide. Among these, DMSO, sucrose, and glycerol are most widely used.

Mechanism of Cryopreservation: Selection of Plant Material -> Addition of Cryoprotectant -> Verification -> Cryoprotection Dehydration -> Encapsulation and Dehydration -> Freezing -> Storage -> Thawing -> Determination of Survival or Viability.

Applications of Cryopreservation

  • Long-Term Conservation: Ideal method for long-term conservation of material.
  • Disease-Free Plant Conservation: Conserves and propagates disease-free plants.
  • Recalcitrant Seed Maintenance: Maintains recalcitrant seeds for a long time.
  • Endangered Species Maintenance: Maintains endangered species.
  • Pollen Maintenance: Maintains pollens to increase longevity.
  • Germplasm Storage: Stores rare germplasm and other genetically manipulated material.

Ex-Situ Conservation of Germplasm

Germplasm

Germplasm is a collection of genetic resources for an organism. For plants, germplasm may be stored as seeds, stems, callus, or whole plants in nutrients. In the case of animals, genes and body parts are stored in gene banks/cryobanks.

Germplasm Conservation

Plant germplasm is genetic source material in the form of seeds, cultured cells, callus, or pollen. The in situ/ex situ preservation of these materials is known as germplasm conservation. Germplasm provides the raw material (genes) that breeders use to develop commercial crop varieties.

Methods of Germplasm Conservation

  1. In situ Conservation
  2. Ex situ Conservation

Ex situ Conservation

Maintains biological material outside their natural habitats. This includes storage in seed banks, gene collections, in vitro collections, and botanical gardens. Ex situ conservation is a viable way of saving plants from extinction, and in some cases, it is the only possible strategy to conserve certain species. In vitro conservation is especially important for vegetatively propagated and non-orthodox seed plant species.

In vitro techniques used to achieve medium-term conservation allow storage of biological material from several months to 2-3 years without subculture, depending on the technique used. Plant material growth reduction is generally attained by modifying the culture medium and/or the environmental conditions. Modification of the culture medium can include dilution of mineral elements, reduction of sugar concentration, and other changes.

Types of Culture

Plant tissue culture is a technique of growing a complete plant in vitro from a part of a plant, such as a leaf, root, shoot, or stem. Plant tissue culture is mainly based on totipotency. Totipotency is the capacity of plants to regenerate a complete plant from any part of the plant.

Types of Cultures Involved in Plant Tissue Culture

  1. Seed Culture
  2. Embryo Culture
  3. Root Culture
  4. Callus Culture
  5. Cell Suspension Culture
  6. Protoplast Culture
  7. Anther Culture

Seed Culture

Seed culture is a technique in which a complete plant or seedling can be generated in vitro by taking the seed as an explant. This technique is primarily used for orchid plants, as the seeds of these plants do not germinate well in vivo.

Embryo Culture

Embryo culture is a technique involving the isolation and development of mature or immature embryos in vitro to produce a complete plant. There are two types of culture:

  1. Mature Culture
  2. Immature Culture

Embryo culture is used in crop improvement programs to obtain hybrids.

Root Culture

Root culture is a culture of apical or lateral root tips to produce an in vitro root system. They are mainly based on inoculation with Agrobacterium rhizogenes to produce secondary metabolites.

Callus Culture

Callus culture is a technique through which a complete plant is formed from an undifferentiated mass of cells under aseptic conditions on nutrient media. Callus culture is a slow-growth plant culture system that enables several studies on plants, like growth, differentiation, and metabolism.

Cell Suspension Culture

Cell suspension culture consists of cells…