Plant tissue culture is the technique of growing plant cell tissue organ in an artificial prepared nutrient medium under aseptic condition. Haberlandt [1898] successfully cultured somatic cells of higher plant in simple nutrient medium t.c technique is use for quick vegetative propagation some fruits,tember trees & ornamental plant. The cultured tissue lead to produce callus. The callus is a again coltured in suitable nutrient medium to induced it forming embryos which grow into plantlet. The plantlet formed into cultured can be transfer to the soil of pot or field. Overall technique of plant tissue culture require for the following culture—
Nutrient medium or cultured medium:- Nutrient medium provides nutrition & hormones as well as other important requirement for all part growing & multiplaying culture. Nutrient medium are two types—
(i) LIQUID:- in liquid medium plant are emerged either partially or completely.
(ii)SEMISOLID:- On the other hand the semiliquid plant material,is place on the surface of the medium. Generally the nutrient following —
(i) Inorganic nutrient:- Micronutrient & Macronutrient. Iron is require in smaller quantities then Macronutrient more than Micronutrient.
ORGANIC NUTRIENT:- Which are added to the nutrient solution include carbon sources ,vitamin ,amino acid, hormones.
(1)Carbon sources:- C- source provide energy to the plant growing in culture,sucrose or glucose are generally use as c-source at the conservation of 2to3%.
(2) Vitamin:-it is have catalytic function in enzyme system & are require only in trace amount generally vitamin B1,nicotinic acid , thymine & vitamin c are used in culture medium.
(3)Amino acid:- Amino acid is added in several invitro medium for plant.
(4)Growth hormones:- In culture medium growth regulator Gibberlin,Cytokinin,Auxin. Mostly auxia in tissue culture IAA[Indole Acetic Acid]
(5)PH: The pH of medium is kept b/w 5-5.8%[acidic condition].
ASEPTIC CONDITION:- Aseptic condition mean free from all micro-organisms. Propagation of plant tissue in culture requires Aseptic technique to insure speciment & aquipment are free of contaminate such as —Bacteria fungi or algae.
EXTRALIZATION BY DRY HEAT:- The seal student use in culture work[niddle forsap, petrydisc ,wash-glass etc] are red heated on the flame & deep in athyl alcohol. The glass were use in culture work are heated in an electric oven 160⁰for fee hours. The dry heat of flame are oven kills are the microorganisms or there megaspore.
(2) EXTRALIZATION BY WET HEAT:- Application of wet heat that is stream under pressure in an autochome for 20mint.id capable killing all the containing.
(3) EXTRALIZATION BY ERRADIATION:- Uv light is use for irradiation the laboratory space which is as a result is a made free from the contaminant micro-organisms.
(4) EXTRALIZATION BY CHEMICAL:- Hear are a irradiation can not be use for EXTRALIZATION of the living plant material are surface specialized by equs solution of Na,Ca,hydrochloride(CaHCl).
(5) EXTRALIZATION BY FILTRATION:- Some organic component of the nutrient media are disantigreated when astriligid under substance are by filtering then through bacterial specialized by filter.
TECHNIQUE Of CELL & TISSUE CULTURE:-
Cell and tissue culture techniques are laboratory methods used to grow and manipulate cells and tissues outside of their natural environment. These techniques are widely used in biological research, biotechnology, and medicine to study cell behavior, develop new drugs, and produce biological products.
Here are some common techniques of cell and tissue culture:
Sterilization:- Before starting a culture, all materials, including media, reagents, and equipment, must be sterilized to prevent contamination from bacteria, viruses, and fungi.
Cell isolation:- Cells are usually isolated from animal or plant tissues by enzymatic or mechanical methods. For example, digestive enzymes can be used to break down the extracellular matrix and release cells from tissue fragments.
Cell maintenance:- Once cells have been isolated, they need to be cultured in an appropriate environment that mimics their natural habitat. This typically involves providing a nutrient-rich growth medium, maintaining optimal temperature and pH, and regulating oxygen and carbon dioxide levels.
Cell passage:- Cells can grow and divide rapidly in culture, leading to overcrowding and nutrient depletion. To prevent this, cells need to be regularly subcultured or passaged, which involves removing a portion of the cells and transferring them to a fresh culture vessel with fresh medium.
Cell differentiation:- Some cell types can differentiate or specialize into different cell types depending on the signals they receive from their environment. This process can be manipulated by altering the composition of the culture medium or adding specific growth factors and signaling molecules.
Tissue engineering:- In addition to growing individual cells, tissue culture techniques can be used to grow three-dimensional tissues and organs. This involves seeding cells onto a scaffold or matrix that provides structural support and cues for tissue formation.
Overall, cell and tissue culture techniques provide powerful tools for studying and manipulating living cells and tissues in a controlled laboratory environment. These methods have revolutionized our understanding of cell biology and are increasingly being used in drug discovery, regenerative medicine, and biotechnology.
Callus formation and it’s culture:-
Callus formation is a process in which undifferentiated plant cells proliferate and differentiate into a mass of cells that have the potential to develop into different plant organs, such as roots, stems, and leaves. Callus formation can occur naturally as a response to injury or in vitro as a result of tissue culture techniques.
In tissue culture, callus formation is often used to initiate plant regeneration from a variety of plant tissues, including leaf, stem, and root segments. The tissue is first sterilized and then cultured on a nutrient medium containing plant hormones, such as auxins and cytokinins, that stimulate cell division and differentiation.
The callus tissue is usually subcultured onto fresh media at regular intervals to maintain its growth and vigor. Once the callus tissue has reached a suitable size, it can be induced to differentiate into shoots or roots by altering the hormone concentrations in the culture medium. The resulting plantlets can then be transplanted into soil for further growth and development.
Callus culture is widely used in plant biotechnology for various purposes, including plant breeding, genetic engineering, and the production of secondary metabolites. It allows for the regeneration of whole plants from a small piece of plant tissue, which can be genetically manipulated or screened for desirable traits. Callus culture can also be used to produce large quantities of plant biomass for the production of natural products or for studies on plant physiology and biochemistry.
Organogenesis and embryogenesis:-
Organogenesis and embryogenesis are two different processes that can occur in tissue culture.
Organogenesis refers to the process of generating new organs from plant tissue culture. In tissue culture, specific plant tissues, such as shoot tips or meristems, are cultured in a nutrient-rich medium that contains growth regulators such as auxins and cytokinins. These growth regulators stimulate the formation of new shoots, roots, and other plant structures from the cultured tissue. The resulting structures can then be transferred to soil or other growing media to form complete plants.
Embryogenesis, on the other hand, refers to the process of generating embryos from animal tissue culture. In tissue culture, animal embryos can be generated by culturing fertilized eggs or by using somatic cell nuclear transfer techniques. These techniques involve removing the nucleus from an egg cell and replacing it with the nucleus from a somatic cell. The resulting embryo can then be cultured in a nutrient-rich medium until it is ready to be implanted into a surrogate mother.
Both organogenesis and embryogenesis in tissue culture have important applications in plant and animal biotechnology, respectively. For example, tissue culture techniques can be used to produce large numbers of identical plants or animals for research or commercial purposes, or to generate transgenic plants or animals with desirable traits.
