Table Of Content:
Introduction
- Plant tissue culture is an in vitro technique in biotechnology that is generally used for the aseptic isolation and culture of single cells, protoplasts, tissues, organs and their components on artificial media under aseptic conditions, so that these parts can grow and develop into complete plants
- The theoretical framework of plant tissue culture is based on the unique property of cellular totipotency and regeneration
- Cellular totipotency is the capacity of the plant cell to regenerate into entire plant.
- The experimental evidence of totipotency was first time obtained by Gottlieb Haberlandt in 1902.
- Haberlandt grew palisade cells from leaves of various plants (Lamium purpureum, Pulmonaria, Urtica etc.) in Knop’s salt solution in hope of regenerating whole plant from it.
- The cells increased in size, survived for several months, but failed to divide. Later the cultures were lost due to microbial contamination.
- On the basis of his pioneering experimentation, Haberlandt is credited as the founder of plant tissue culture.
- After that, during the period 1902 to 1930, attempts were made to culture isolated plant tissues and organs such as the root, shoot, apex, and embryos.
- During this period, different types of nutrient media were evaluated for the culture and differentiation of organs and tissues.
- Lots of experiments were carried out to formulate suitable nutrient media and standardize culture conditions including various plant-hormone combinations on in vitro morphogenesis.
Plant Tissue Culture Medium
- Plant tissue culture medium contains all the nutrients required for the normal growth and development of plants.
- It is mainly composed of macronutrients, micronutrients, vitamins, other organic components, plant growth regulators, carbon source and some gelling agents in case of solid medium.
- Murashige and Skoog medium (MS Medium) is most extensively used for the vegetative propagation of many plant species in vitro .
- The pH of the media is also important that affects both the growth of plants and activity of plant growth regulators.
- It is adjusted between 5.4 - 5.8.
Role of Plant growth regulators (PGR’s) in Tissue Culture
- Plant growth regulators (PGR’s) play an essential role in determining the development pathway of plant cells and tissues in culture medium.
- The type and the concentration of hormones used depend mainly on the species of the plant and the tissue or organ cultured.
- Auxins and cytokinins are most widely used plant growth regulators in plant tissue culture and their amount determines the type of culture established or regenerated.
- The high concentration of auxins generally favors root formation.
- Whereas the high concentration of cytokinins promotes shoot regeneration.
- A combination of both auxin and cytokinin leads to the development of mass of undifferentiated cells known as callus.
Types of Cultures
Tissue culture or micropropagation may be:
1. Organ culture
2. Explant culture
3. Callus culture
4. Cell suspension culture
5. Protoplast culture
1. Organ Cultures
Culturing isolated organs in an artificial media under controlled conditions are known as organ culture.
The following are the various types of organ culture and its specific purpose:
- Seed culture: It Increases the efficiency of germination of seeds that are difficult to germinate in vivo, it involves the precocious germination by application of plant-growth regulators, and production of clean seedlings for explants or meristem culture.
- Embryo culture: It helps to overcome embryo abortion due to incompatibility barriers, overcome seed dormancy and self-sterility of seeds, and embryo rescue in distant hybridization where endosperm development is poor. It also helps in shortening of breeding cycle.
- Ovary or ovule culture: Ovary is a common explant for the production of haploid plants. Ovary culture helps in overcoming abortion of embryos of wide hybrids at very early stages of development due to incompatibility.
- Anther and Microspore Culture: Anther and Microspore Culture is used for production of haploid plants. Production of homozygous diploid lines through chromosome doubling, thus reducing the time required to produce inbred lines, and it also helps in uncovering mutations or recessive phenotypes.
2. Explant Culture
- Culturing of any excised tissue or plant parts such as leaf tissue, stem parts, cotyledon, hypocotyls, root parts, etc., is called explant culture.
- The primary purpose of explant culturing is to induce callus cultures or to regenerate whole plantlets directly from it without the formation of callus.
- Shoot apical meristem culture is an example, and its important uses are:
- Production of virus-free germplasm or plantlets.
- mass production of desirable genotypes.
- cryopreservation (cold storage) or in vitro conservation of germplasm.
3. Callus Culture
- Callus represents an unorganized or undifferentiated mass of cells
- When an explant is cultured in a medium supplemented with sufficient amount of plant hormones, it starts producing mass of cells from the surface of the explant.
- Callus cultures can be maintained for a very long time by intermittent sub-culturing to a fresh medium.
- Callus cultures can be used for:
- regeneration of plantlets.
- preparation of single cells or suspension cultures, or for protoplasts preparation.
- genetic transformation studies.
- the generation of useful somaclonal variants.
4. Cell-Suspension Cultures
- Single-cell cultures and suspension cultures can be established from callus cultures by transferring a piece of callus tissue into liquid medium and subjecting it to continuous shaking.
- The growth rate of the suspension-cultured cells is generally higher than that of the solid culture.
- By subculturing for several generations, a fine cell suspension culture containing small-cell aggregates and single cells is established.
- The cells in suspension are also used for a large-scale culture with jar-fermentors and tanks.
- The suspension cultures can be grown either as batch cultures or as continuous cultures for producing phytochemicals in airlift bioreactors.
- The cell-suspension cultures can be used for:
- inducing somatic embryogenesis and the preparation of artificial seeds.
- induction of somatic mutation, and selection of mutants by screening the cells just like microbial cultures.
- The cells can also be used for genetic transformation experiments to produce transgenic plants.
5. Protoplast Cultures
- Protoplasts are plant cells without cell walls.
- The cells may be taken from the leaf tissue or from any other part of the plant or from the suspension cultures.
- These cells are incubated in an enzyme mixture consisting of cellulase, hemicellulase, and pectinase for a specific period of time.
- The enzyme mixture acts on the cell wall and is completely digested, so that the underlying cell membrane is exposed.
- The plant protoplasts can be used for various biochemical and metabolic studies.
- It can be used for the somatic cell fusion to create somatic hybrids.
- Formation of cybrids, in which there is no fusion of nucleus, but fusion in between the cytoplasm.
- Protoplasts can also be used for genetic transformation studies with biolistic methods, electroporation techniques, by PEG-mediated DNA transfer or by direct injection of DNA into the nucleus of the protoplast using microsyringes.
Plant Tissue Culture Procedure
The following are the major steps involved in plant-tissue culture:
1. Selection of explant
2. Surface sterilization of explant
3. Initiation of culture
4. Shoot formation
5. Root formation
6. Hardening
Step 1. Selection of Explants
- Leaves, stems, or any other suitable plant part may be cut from healthy plants.
- The explants should be cut to suitable sizes.
- Old tissue parts should be avoided.
- Wash the dust off the tissue in a beaker of distilled water, which should be surface sterilized.
Step 2. Surface Sterilization of Explants
- This part of the procedure should be carried out in a laminar flow hood cabinet.
- The leaf or any other tissue should be immersed in 70% ethanol for 30 seconds, and then transferred to a sterile petri dish.
- Sterile scissors and forceps are then used to cut the tissues into small suitable sizes of explants.
- The explants are transferred into a 10% hypochlorite bleach solution or mercuric chloride for five minutes, gently agitating once or twice during this time.
- They are then washed thoroughly by immersing in sterile distilled water for two to three minutes.
Step 3. Initiation of Culture
- The surface sterilized explant is implanted into a suitable nutrient medium with the correct type of growth hormones in appropriate concentrations.
- Inoculated cultures are transferred to growth chambers or tissue-culture rooms having appropriate physical conditions such as the right temperature (26-28°C), relative humidity (50-60%), and fluorescent light (16 h photoperiod).
- Depending on the nutrient conditions and hormone combination, the explant either starts developing callus or directly produces shoot buds and roots to form a large number of plantlets.
- Shoot formation. The healthy calli are transferred to shoot induction medium having cytokinins for shoot initiation.
- Root formation. After development of shoots from callus, they are excised and transferred to a rooting medium having auxins. Shoots after development of roots are ready for being transferred to the greenhouse for hardening.
Step 4. Transplantation and Hardening
- The plantlets that develop roots are transferred to special pots with sterilized sand for hardening under greenhouse conditions.
- Hardening is a process by which the in vitro generated plants are acclimatized to the greenhouse conditions.
- Then they can be transferred to the field.
Plant Regeneration Pathways
There are two methods of whole plant regeneration:
1. Organogenesis: Plant regeneration through the formation of shoots and roots is known as plant regeneration through organogenesis.
2. Somatic Embryogenesis: The cells under a particular hormonal combination, change into zygotes (somatic zygotes) and follow an embryonic path of development to form somatic embryos. These somatic embryos are similar to normal embryos and can develop into a complete plant.
