plant tissue culture are used to cultivate plants or plant cells on a nutritional medium in a sterile, controlled setting. This technique has numerous benefits for plant study, conservation, and agriculture by enabling the growth of plants from a small amount of tissue or cells.

Steps in Plant Tissue Culturing

Plant tissue culture requires a sterile and optimal growth conditions. The procedure involves a series of well defined steps as follows:

1. Selection and Preparation of Explant

The tissue or cells taken from a plant (e.g., leaf, stem, root, meristem) for culturing are called explants. The explant is chosen based on the purpose of the culture.To guarantee effective growth, only healthy, disease-free plants are chosen.

2. Sterilization
a. Explant Sterilization : To get rid of dirt, the explant is cleansed under running water. Chemicals like 70% ethanol (short rinse)  and Hydrogen peroxide or sodium hypochlorite (for a few minutes)are used to sterilize surfaces.
To get rid of these sterilizing chemicals, the explant is repeatedly rinsed with sterile distilled water.
b. Sterilizing Equipment : Culture vessels and instruments (such as scalpels and forceps) are sterilized, usually for 15 to 20 minutes at 121°C in an autoclave. The workspace is then sanitized using alcohol or UV light under a laminar airflow hood.

3. Nutrient Medium Preparation
Macronutrients (such as potassium, phosphorus, and nitrogen) are included in a nutrient medium (such as Murashige and Skoog [MS] medium). Micronutrients, such as zinc and iron; Vitamins (such as nicotinic acid and thiamine) ; plant growth regulators  such as auxins for root growth , cytokinins for shoot growth; source of carbon (such as sucrose) are also included in the medium. If a solid medium is needed, use a gelling agent (such as agar) is used. The medium is then sterilized by autoclaving.

4. Inoculation

Inside a laminar airflow cabinet, the sterile explant is placed on the nutritive medium in an aseptic environment.
During handling, precautions are taken to prevent contamination.

5. Incubation
A controlled atmosphere is used to incubate the culture. Depending on the species, the temperature ranges from 22 to 28°C. Controlled Photoperiods using fluorescent or LED lights (e.g., 16 hours of light and 8 hours of dark) and
Optimal humidity levels are maintained. The type of culture and growth regulators utilized determine whether callus, branches, or roots form.

6. Subculturing

The growing tissues are moved to new media as the explant grows or the medium is depleted for the nutrient factors. This process is repeated at regular intervals until adequate growth is attained.

7. Regeneration

The callus or cultured tissues are used to regenerate shoots, roots, or plantlets. The medium’s growth regulators are modified to promote regeneration.

8. Hardening

In this step the plantlets are acclimatized to natural conditions by moving them  to pots filled with compost or sterile soil and placed in a greenhouse with indirect light with high humidity. The plants are then gradually exposure to external environments to ensure their survival

9. Field Transfer

Fully acclimatized plants are then transplanted to the field or garden for their further growth.

Techniques in Plant Tissue Culture

1. Micropropagation: This is a specific method in plant tissue culturing. This method is used to quickly multiply plants in sterile environments. In order to create many genetically identical plants, or clones,  plant tissues, organs, or cells are cultivated  in a controlled setting. This method is frequently employed in conservation, horticulture, and agriculture. By micropropagation large numbers of genetically identical plants are produced rapidly.This is referred to as clonal propagation. This method helps to conserve plant species that are at risk of extinction. Further it is used for mass production of ornamental plants, fruit trees, and vegetables. micropropagation facilitates the propagation of plants with desirable traits, such as higher yield or pest resistance.“Micropropagation” by mw-parser-This image has been created during “DensityDesign Integrated Course Final Synthesis Studio” at Politecnico di Milano, organized by DensityDesign Research Lab., creativecommons.org, via Wikimedia Commons is licensed under CC BY-SA 4.0
2. Callus Culture: A cluster of unorganized, undifferentiated plant cells that forms in response to wounding or hormonal signals is called as Callus . Callus can be induced from various plant tissues such as leaves, stems, roots, or meristems. Callus culture is a plant tissue culture technique where the callus is grown on a nutrient medium under sterile conditions for the purpose of  plant research, genetic engineering, and plant propagation. Callus can differentiate into complete plants via organogenesis or somatic embryogenesis.                 Watch the video from PRINCIPIA on callus culture
3. Somatic embryogenesis : Using the plant tissue culture technique known as “somatic embryogenesis,” somatic (non-reproductive) cells can grow into embryos that have the potential to become whole plants. This process, which usually takes place in controlled in vitro settings, starts with somatic cells and resembles zygotic embryogenesis, which happens in seeds.Somatic cells from explants  such as from leaves, roots, or immature embryos  are placed on a nutrient medium containing plant growth regulators, such as auxins .The auxins induce dedifferentiation.  This makes the somatic cells to become totipotent (capable of developing into any plant structure).These cells either grow directly into embryogenic cells or divide to produce a callus.  The two types of Somatic Embryogenesis are :
   • Direct Somatic Embryogenesis- Here the somatic embryos do not go through a callus phase before forming from the explant.This method is quicker and more reliable.
   • Indirect Somatic Embryogenesis– In this type  the callus phase, which starts in the explant, gives rise to somatic embryos. Higher rates of multiplication are possible, but somaclonal variation may be introduced.
4. Somaclonal Variation : Genetic or phenotypic changes that arise in plants regenerated from somatic cells during plant tissue culture are referred to as somaclonal variation. This phenomenon is brought on by genetic or epigenetic alterations that take place during the tissue culture procedure, especially when plants are regenerated using suspension or callus cultures.Somaclonal variation is used for producing  improved yield, disease resistance, drought tolerant plants . this method creates genetic diversity without the need for crossbreeding and also useful for introducing desirable traits into plants.
5. Embryo Culture: A plant embryo is cultivated in vitro (apart from the seed or ovule) on a nutritional medium in a sterile environment using the embryo cultivation technique. The main purpose of this technique is to save embryos that might not normally grow into adult plants because of problems including seed dormancy, hybrid incompatibility, or adverse environmental circumstances. the two types of Embryo Culture are
   • Mature Embryo Culture: involves growing mature seeds into fully formed embryos. this is used to help seedlings that have viability or dormancy problems during germination.
   • Immature Embryo Culture ( also known as Embryo Rescue): This  involves growing immature embryos from seeds that wouldn’t make it under typical circumstances. this method is used to save embryos from incompatible crossings in hybridization projects.
6. Protoplast Culture: Plant cells without their cell walls are called as protoplast .Protoplast culture is a plant tissue culture technique where isolated protoplasts are cultured in vitro under sterile condition to carry out additional cellular functions like somatic hybridization or to grow back into whole plants. The cell walls can be physically or enzymatically removed to produce protoplasts. The process of protoplast culturing involves isolation of protoplasts from leaf mesophyll, suspension cultures, or callus tissues. This includes enzymatic digestion using: Cellulase: To degrade cellulose and Pectinase: To break down pectin in the middle lamella. The cells are treated with these enzymes in an osmotic solution (e.g., mannitol or sorbitol) to prevent protoplast lysis.Protoplasts are then separated from debris by filtration and centrifugation in a density  gradient. Protoplasts divide to form a small cell mass. This cell mass grows into a callus.
7. Anther/Pollen Culture: Anther or pollen culture is a method of plant tissue culture in which isolated pollen grains (microspores) or anthers (male reproductive organs) are cultivated in vitro under sterile conditions to create haploid plants. Because they only have one set of chromosomes, these haploid plants are useful for genetic study and plant breeding. Immature flower buds are chosen when pollen grains are at the right developmental stage (e.g., uninucleate or early binucleate).Flower buds are sterilized with disinfectants (e.g., ethanol, sodium hypochlorite) to ensure aseptic conditions.For anther cultures ,anthers are excised and placed on the nutrient medium and for pollen cultures the pollen grains (microspores) are isolated from the anthers and cultured separately.

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