Plant Tissue Culture

A whole plant can be regenerated from a small tissue or plant cells in a suitable culture medium under controlled environment.

The term plant tissue culture is generally used for the aseptic culture of cells, tissues, organs, and their components under defined physical and chemical conditions in vitro.

The development of a plant in tissue culture depends on two fundamental principles of cells which include totipotency and plasticity. Totipotency is the inherent property of the cells to give rise to several types of cells and as a result the complete plant or organ can be formed.

Plasticity is the ability of plants to alter their growth, metabolism, and development depending on their best-suited environment. These two principles decide the growth of a cell in in-vitro conditions.

Objective of Plant Tissue Cultures

• Propagation of large quantity of good quality planting material from mother plants within short time, space with minimum cost per plant.
• Obtaining disease free plants
• Rapid propagation of plants those are difficult to grow.
• Genetics improvement of commercial plants
• Somatic hybridization
• Production of multiples of plants in the absence of seeds or pollinators to produce seeds (Micropropagation)

General Steps involved in Plant Tissue Culture

• Preparation of medium and sterilization in Autoclave: The medium comprises of the nutrients such as Micro Nutrients, Macro Nutrients, Vitamins, Irons and growth regulators required for the growth of the plant. The medium is sterilized in the electrically operated Autoclave. This sterilization is important to avoid the bacterial contamination.
• Selection of a mother plant and sterilization: The mother plants are selected from the virus free areas. Healthy, disease and virus free plants are selected as mother plants. The actual plant part, which is called as ex-plant is selected for inoculation. This plant part is washed with water, liquid soap and antiseptic solution. This ex-plant is washed again with the chemical solution to avoid any fungal contamination coming from the field environment. To remove the remnants of the chemicals it is thoroughly washed by distilled water.
• Inoculation of explants on sterilized medium of known composition: The explants are inoculated on the sterilized medium. The inoculation takes place in the Inoculation room, on the Laminar Air Flow station, which maintains a continuous air current, which keeps this bench without the risk of contamination.
• Inoculation of explants on sterilized medium of known composition: The explants are inoculated on the sterilized medium. The inoculation takes place in the Inoculation room, on the Laminar Air Flow station, which maintains a continuous air current, which keeps this bench without the risk of contamination.
• Shifting of cultures to the growth room:Inoculated cultures are then shifted to the growth room where a typical temperature of Fifteen to Twenty degree centigrade is maintained. The artificial lighting arrangement is also made for the growth of the plant.
• Sub-culturing of cultured cells:Cultured cell consumes the provided nutrients over the time, so to maintain the growth of cells, fresh nutrients are required. The cells are transferred to fresh Culture media, this is called subculturing.

Types of Plant Tissue Culture

Callus Culture:

Formation of undifferentiated mass of cells (callus) and which may also employed to form differentiated plant organs by using nutrient culture media, is called callus culture.
Meristem Tip Culture:
Meristem is the mass of undifferentiated parenchyma cells found at extreme uppermost tip of the shoot and root.
Shoot Tip Culture
Shoot tip is the uppermost apical part of the shoot, which contains, apical meristem plus 1-3 leaf primordial. This shoot tip is also used to develop virus free planters through micropropagation.
For shoot tip culture this part is isolated from plant and surface sterilized and placed over solidified nutrient culture media. This leads to formation of young planters after incubation under appropriate conditions and by adding suitable plant growth regulators.
Protoplast Culture:
Cell without cell wall is called protoplasm. Protoplasm mixing is an effective method for transfer of genetic material, for genetic engineering. Mechanical or enzymatic methods the most common approaches to remove the cell wall without damaging the protoplast. Mechanical isolation, although possible, often results in low yields, poor quality, and poor performance.
Enzymatic isolation is a safe method that is usually carried out in a simple solution with a high osmotic pressure to prevent the released protoplasts from bursting (up to 15% mannitol or sorbitol).
The mixture of cellulase and pectinase enzymes must be used for this purpose. Once the protoplasts have been isolated, they are fused, hybrids selected, plantlets can be regenerate. Protoplasts prefer to be embedded in a semi-solid medium rather than cultured in a liquid medium. Agar or agarose provide the structural support facilitating cell wall development. Protoplasts will not start to divide until new walls have formed. Protoplast cultures are usually static and require a temperature of about 25–30°C under low intensity continuous illumination.
Anther culture:
Anther culture is a technique by which the developing anthers at a precise and critical stage are excised aseptically from unopened flower bud and are cultured on a nutrient medium where the microspores within the cultured anther develop into callus tissue or embryoids that give rise to haploid plantlets either though organogenesis or embryogenesis.
Pollen or microspore culture :
Pollen or microspore culture is an in vitro technique by which the pollen grains preferably at the uninucleated stage, are squeezed out aseptically from the intact anther and then cultured on nutrient medium where the microspores, without producing male gametes, develop into haploid embryoids or callus tissue that give rise to haploid plantlets.
Embryo Culture:
In this type of culture, the embryo is isolated and cultured under in vitro conditions. Embryo culture can be done either by using mature or immature embryos. The mature embryos are obtained from ripe seeds and the immature embryos are obtained from the unripened or hybrid seeds that failed to grow and couldn’t produce viable plants.
Suspension Culture:
Suspension plant tissue culture is a technique in which plant cells are grown in a liquid nutrient medium, rather than on a solid surface, allowing the cells to remain suspended and free-floating. It is usually initiated by transferring friable callus (a soft, loose type of callus) into the liquid medium. This is mainly used for
• Mass cell proliferation: For large-scale production of cells.
• Secondary metabolite production: Efficient for extracting plant-based compounds (e.g., alkaloids, flavonoids).
• Genetic studies: Easier to manipulate and transform cells in suspension.
• Protoplast isolation: Easier to extract protoplasts (cells without cell walls) from suspended cells.
Solidified Agar medium plant tissue culture:
Solidified agar medium plant tissue culture is a type of plant tissue culture technique where plant tissues are grown on a solid nutrient medium that has been gelled using agar, a jelly-like substance derived from seaweed.
This is mainly used for
• Micropropagation: Allows growth and multiplication of plantlets.
• Organogenesis: Promotes formation of roots, shoots, or entire plants from tissues.
• Callus Induction: Supports callus formation from explants.

Applications of Plant Tissue Culture

• Production of large number of plantlets can be generated through micropropagation, from a small plant tissue, this is especially significant for the prevention of endangered plant species.
• Somatic hybridization is a technique of Plant Tissue Culture, through which genetically improved plant can be develop. Protoplast culture provides this kind of opportunity, through which genetic material can be transferred.
• Meristem and Shoot tip culture cells are generally viral free cells, the explant from these tissue produces disease free and viral cells free cells, which further develop healthy plants and cells.
• Through plant tissue culture, hybridization can be achieved, which produces hybrid plants with higher qualities. New plants can be generated without pollination and inside laboratory without getting affected by environmental factors.
• Plant tissue culture is an excellent alternative way for the commercial production of Plant derived secondary metabolites. These secondary metabolites are therapeutically very useful. Genetic and metabolic engineering improves the biosynthetic capacity of cultured cells.

Plant Name	Secondary Metabolite	Therapeutic Value
Catharanthus roseus	Vinblastine, Vincristine	Anti-cancer (used in leukemia and Hodgkin’s lymphoma therapy)
Taxus spp. (Yew)	Paclitaxel (Taxol)	Anti-cancer (ovarian, breast, and lung cancers)
Artemisia annua	Artemisinin	Anti-malarial
Rauwolfia serpentina	Reserpine	Antihypertensive, sedative, antipsychotic
Digitalis purpurea	Digitoxin, Digoxin	Cardiotonic (used in heart failure and arrhythmias)
Panax ginseng	Ginsenosides	Adaptogenic, anti-fatigue, neuroprotective
Glycyrrhiza glabra	Glycyrrhizin	Anti-inflammatory, antiviral, hepatoprotective
Camptotheca acuminata	Camptothecin	Anti-cancer (topoisomerase inhibitor)
Nicotiana tabacum	Nicotine	Used in neurobiology research and as an insecticide
Withania somnifera	Withanolides	Anti-stress, anti-inflammatory, anti-cancer