Shoot - tip meristem

Biosafety is the prevention of large-scale loss of biological integrity, focusing both on ecology and human health. These prevention mechanisms include conduction of regular reviews of the biosafety in laboratory settings, as well as strict guidelines to follow. Biosafety is used to protect from harmful incidents. Many laboratories handling biohazards employ an ongoing risk management assessment and enforcement process for biosafety. Failures to follow such protocols can lead to increased risk of exposure to biohazards or pathogens. Human error and poor techniques contribute to unnecessary exposure to hazards arid compromise the best safeguards set into place for protection.

Secondary Metabolites	Medicinal use
Capsaicin	Rheumatic pain treatment
Vincristine	Anti-carcinogenic
Quinine	Anti-malarial

The cell suspension is prepared by transferring a portion of callus to the liquid medium and agitated using rotary shaker instrument. The cells are separated from the callus tissue and used for cell suspension culture.

Redifferentiation	Dedifferentiation
A process by which an already differentiated cell undergo further differentiation to form another cell type	A process of reversion of cells (differentiated cells) to meristem state leading to callus formation is called dedifferentiation it.
Differentiated cell ➝ meristem state ➝ New cells

Cryopreservation (- 195.C)
1. Cryopreservation, also known as Cryo conservation.
2. A process by which protoplasts, cells, tissues, organelles, organs, extracellular matrix, enzymes or any other biological materials are subjected to preservation by cooling to very low temperature of -196^oC using liquid nitrogen.
3. At this extreme low temperature any enzymatic or chemical activity of the biological material will be totally stopped and this leads to preservation of material in dormant status.
4. Later these materials can be activated by bringing to room temperature slowly for any experimental work.
5. Protective agents like dimethyl sulphoxide, glycerol or sucrose are added before cryopreservation Process.
6. These protective agents are called cryoprotectants, since they protect the cells, or tissues from the stress of freezing temperature.

Somatic embryogenesis is the formation of embryos from the callus tissue directly and these embryos are called Embryoids or from the in vitro cells directly form pre-embryonic cells which differentiate into embryoids
Applications:
i. Somatic embryogenesis provides potential plantlets which after hardening period can establish into plants.
ii. Somatic embryoids can be used for the production of synthetic seeds.
iii. Somatic embryogenesis is now reported in many plants such as Allium sativum, Hordeum vulgare, Oryza sativa, Zea mays and this is possible in any plant.

1. The growing of cells including the culture of single cells or small aggregates of cells in vitro in liquid medium is known as cell suspension culture.
2. The cell suspension is prepared by transferring a portion of callus to the liquid medium and agitated using rotary shaker instrument.
3. The cells are separated from the callus tissue and used for cell suspension culture.

Protoplasts are cells without a cell wall, but bounded by a cell membrane or plasma membrane. Using protoplasts, it is possible to regenerate whole plants from single cells and also develop somatic hybrids. The steps involved in protoplast culture are:
i) Isolation of protoplast: Small bits of plant tissue like leaf tissue are used for isolation of protoplast. The leaf tissue is immersed in 0.5% Macrozyme and 2% Onozuka cellulase enzymes dissolved in 13% sorbitol or mannitol at pH 5.4. It is then incubated overnight at 25°C. After a gentle teasing of cells, protoplasts are obtained, and these are then transferred to 20% sucrose solution to retain their viability. They are then centrifuged to get pure protoplasts as different from debris of cell walls.
ii) Fusion of protoplast: It is done through the use of a suitable fusogen. This is normally PEG (Polyethylene Glycol). The isolated protoplast are incubated in 25 to 30% concentration of PEG with Ca++ ions and the protoplast shows agglutination (the formation of clumps of cells) and fusion.
iii) Culture of protoplast: MS liquid medium is used with some modification in droplet, plating or micro-drop array techniques, Protoplast viability is tested with fluorescein diacetate before the culture. The cultures are incubated in continuous light 1000-2000 lux at 25°C. The cell wall formation occurs within 24-48 hours and the first division of new cells occurs between 2-7 days of culture.
iv) Selection of somatic hybrid cells: The fusion product of protoplasts without nucleus of different cells is called a cybrid. Following this nuclear fusion happen. This process is called somatic hybridization.

Artificial Seed
1. Artificial seeds or synthetic seeds (synseeds) are produced by using embryoids (somatic embryos) obtained through in vitro culture.
2. They may even be derived from single cells from any part of the plant that later divide to form cell mass containing dense cyptoplasm, large nucleus, starch grains, proteins, and oils etc.
3. To prepare the artificial seeds different inert materials are used for coating the somatic embryoids like agrose and sodium alginate.

Cybrid is a cytoplasmic hybrid obtained by the fusion of cytoplasm of cells of different parental sources; a term applied to the fusion of cytoplasms of two different protoplasts.

Sterilization is the technique employed to get rid of microbes such as bacteria and fungi in the culture medium, vessels and explants.

Growing plant protoplasts, cells, tissues or organs away from their natural or normal environment, under artificial condition, is known as Tissue Culture.