TECHNIQUES OF IN VITRO CULTURE

TECHNIQUES OF IN VITRO CULTURE

In in vitro culture, plants are multiplied by cloning the plants themselves. In such a manner, we can obtain a large number of specimens identical to each other and to the specimen introduced in vitro at the beginning of the process.

Types of Cultures

Callus Culture

Callus culture may be derived from a wide variety of plant organs, roots, shoots, leaves, or specific cell types, e.g., endosperm, pollen. Thus, when any tissue or cell is cultured on an agar gel medium, it forms an unorganized growing and dividing mass of cells called callus culture.
In culture, this proliferation can be maintained more or less indefinitely by subculturing every 4-6 weeks, in view of cell growth, nutrient depletion, and medium drying. Callus cultures are easy to maintain and most widely used in biotechnology. Manipulation of the auxin-to-cytokinin ratio in the medium can lead to the development of shoots or somatic embryos from which whole plants can be produced subsequently. Callus culture can be used to initiate cell suspensions, which are used in a variety of ways in plant transformation studies.
Callus cultures broadly fall into one of the two categories:

  1. Compact
  2. Friable callus

In compact callus, the cells are densely aggregated, whereas in friable callus, the cells are only loosely associated with each other, making the callus soft and easily breakable. It provides inoculum to form cell suspension culture.

Organ Culture

Organ culture refers to the in vitro culture and maintenance of excised organ primordia or whole or part of an organ in a way that allows differentiation and preservation of the architecture and/or function.

Importance of Organ Culture

  • Organ culture provides an excellent experimental system to define the nutrients and growth factors normally received by the organ from other parts of the plant body and from its external environment.
  • It is valuable in studies of the interdependence of organs for growth hormones and other growth factors.
  • Cultured organs are ideally suited for studying specific problems in morphogenesis and for investigating the biosynthesis of specific metabolites and growth compounds.
  • Organ culture also opens up new avenues for developments in agriculture and horticulture.

Cell Suspension Culture

When friable callus is placed into a liquid medium (usually the same composition as the solid medium used for callus culture) and then agitated, single cells and/or small clumps of few to many cells are produced in the medium, called suspension culture.
Liquid cultures may be constantly agitated, generally by a gyratory shaker of 100-250 rpm, to facilitate aeration and dissociation of cell clumps into small pieces. Suspension cultures grow much faster than callus cultures, need to be subcultured weekly, and allow a more accurate determination of the nutritional requirements of cells and somatic embryos.
The suspension culture is broadly grouped into:

A) Batch Culture

A batch culture is a cell suspension culture grown in a fixed volume of nutrient culture medium. Cell suspension increases in biomass by cell division and growth until a factor in the culture environment (nutrient or oxygen availability) becomes limiting, and growth ceases. The cells in culture exhibit the following five phases of a growth cycle:

  1. Lag phase – Cells prepare to divide.
  2. Exponential phase – The rate of cell division is highest.
  3. Linear phase – Cell division slows, but the rate of cell expansion increases.
  4. Deceleration phase – The rates of cell division and elongation decrease.
  5. Stationary phase – The number and size of cells remain constant.

B) Continuous Culture

These cultures are maintained in a steady state for a long period by draining out the used (or spent) medium and adding fresh medium. Such subculture systems are either closed or open type.

Closed System –

The cells separated from used medium are taken out for replacement and added back to the suspension culture so that the cell biomass keeps increasing.

Open System –

Both cells and the used medium are taken out continuously and replaced by an equal volume of fresh medium. The replacement volume is adjusted so that cultures remain at sub-maximal growth indefinitely.

Embryo Culture

Embryo culture is a technique in which isolated embryos from immature ovules or seeds are cultured in vitro with the objective of obtaining a viable plant. Young embryos are removed from developing seeds and placed on a suitable nutrient medium to obtain seedlings. The cultured embryos generally do not complete development but germinate prematurely to give rise to seedlings.

Types of Embryo Culture

A) Mature Embryo Culture

Mature embryos are isolated from ripe seeds and cultured in vitro under the following conditions:

  1. When the embryos remain dormant for long periods.
  2. Low survival of embryos in vivo.
  3. To avoid inhibition in the seed for germination.
  4. For converting sterile seeds to viable seedlings.
B) Embryo Rescue

Embryo rescue involves the culture of immature embryos to rescue them from unripe or hybrid seeds that fail to germinate. This approach helps avoid embryo abortion and produces viable plants. The most important application of embryo rescue is the production of interspecific and intergeneric hybrids from wild plant species.

Anther/Pollen Culture

The culture of anthers or isolated microspores (pollen) to produce haploid plants is known as anther culture or pollen culture. The underlying principle is to stop the development of the pollen cell (which normally becomes a gamete) and force its development directly into a plant.
Haploid plants were first discovered in Datura stramonium by A.D. Bergner in 1921. Guha and Maheshwari (1964) pioneered embryo formation from anthers of Datura innoxia grown in vitro. Haploid plants have since been produced via anther culture in more than 170 species.

Ovule Culture

Ovule culture is an experimental system in which ovules are aseptically isolated from the ovary and grown on a chemically defined nutrient medium under controlled conditions.
An ovule contains a megaspore or an egg cell. After fertilization, a single-cell zygote forms, which leads to a mature embryo with shoot and root primordia. In vitro ovule culture helps understand factors regulating zygote development into a mature embryo. It may also allow in vitro fertilization and subsequent embryo production.

Application of In Vitro Culture

In vitro tissue culture can be applied in the following areas:

  1. Micropropagation
  2. Embryo rescue/culture
  3. Anther/pollen/ovary/ovule culture
  4. Production of secondary metabolites
  5. Genetic transformations
  6. Somatic hybridization, etc.