ELSI which represents ethical, legal and social implications of biotechnology broadly covers the relationship between biotechnology and society with particular reference to ethical and legal aspects.

(a) Dimethyl sulphoxide
(b) Glycerol

The morphological changes occur in the callus leading to the formation of shoot and roots is called organogenesis.

(a) Digoxin is obtained from Digitalis purpuria.
(b) Vincristine is obtained from Catharanthus roseus.

Hardening is the gradual exposure of in vitro developed plantlets in humid chambers in diffused light for acclimatization so as to enable them to grow under normal field conditions.

(a) Murashige and Skoog medium
(b) Gamborg medium
(c) White medium
(d) Nitsch's medium

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.

Name of the process-Plant tissue culture Types:
(a) Organ culture
(b) Meristem culture
(c) Protoplast culture
(d) Cell culture

(a) Glass wares are sterilized by auto claving.
(b) Nutrient medium is sterilized by auto claving.
(c) Laminar air flow chamber is sterilized by UV radiation.
(d) Explant is surface sterilized using chemicals.

(a) A - Differentiation
(b) B - Redifferentiation

Protocol for virus free meristem tip culture
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Apical meristem tip with 1 or 2 leaf primordia are excised in sterile condition from the explant
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In vitro culture in 10 ml of solid MS medium supplemented with growth hormones
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Cultures are maintained at 24±1°C in dark for 3 days followed by normal illumination of 2400Lux
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Plantlets developed from meristem tip culture after organogenesis process, transferred to hardening process
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Transferred to field condition

GEAC is an apex body under Ministry of Environment, Forests and Climate change for regulating manufacturing, use, import, export and storage of hazardous microbes or genetically modified organisms (GMOs) and cells in the country. It was established as an apex body to accord approval of activities involving large scale use of hazardous microorganisms and recombinants in research and industrial production. The GEAC is also responsible for approval of proposals relating to release of genetically engineered organisms and products into the environment including experimental field trials (Biosafety Research Level trial-I and II known as BRL-I and BRL-II).

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.

Cryopreservation, also known as Cryo-conservation, is 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°C using liquid nitrogen. 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. Later these materials can be activated by bringing to room temperature slowly for any experimental work.

Somatic embryogenesis is the formation of embryos from the callus tissue directly and these embryos are called Embryoids.
Applications:
1. Somatic embryogenesis provides potential plantlets which after hardening period can establish into plants.
2. Somatic ,embryoids can be used for the production of synthetic seeds.

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.

Macronutrients:
(a) Ammonium nitrate
(b) Potassium nitrate
(c) Calcium chloride
Micronutrients:
(a) Manganese sulphate
(b) Zinc sulphate
(c) Potassium iodide

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

Germplasm Conservation
1. Germplasm conservation refers to the conservation of living genetic resources like pollen, seeds or tissue of plant material maintained for the purpose of selective plant breeding, preservation in live condition and used for many research works.
2. Germplasm conservation resources is a part of collection of seeds and pollen that are stored in seed or pollen banks, so as to maintain their viability and fertility for any later use such as hybridization and crop improvement.
3. Germplasm conservation may also involve a gene bank, DNA bank of elite breeding lines of plant resources for the maintenance of biological diversity and also for food security.

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.

Micropropagation protocol for banana
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In vitro micropropagation of banana (Musa sp.)
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Suckers surface sterilized with 1% NaOCl for 30 minutes
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Apical meristems were isolated - cultured on MS basal medium supplemented with Benzylaminopurine (BAP) 10.0 mg/l and Indole Acetic Acid (IAA) 1.0 mg/l
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Induction of shoots within 168 days
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Kinetin 2.0 mg/l and Naphthalene Acetic Acid (NAA) 0.5 mg/l for root induction
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Acclimatization in polyhouse
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Followed by shade-house under 50% light for hardening process
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Tested for genetic Uniformity
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Transferred to field

Plant tissue culture techniques have several applications such as:
i. Improved hybrids production through somatic hybridization.
ii. Somatic embryoids can be encapsulated into synthetic seeds (synseeds). These encapsulated seeds or synthetic seeds help in conservation of plant biodiversity.
iii. Production of disease resistant plants through meristem and shoot tip culture.
iv. Production of stress resistant plants like herbicide tolerant, heat tolerant plants.
v. Micropropagation technique to obtain large numbers of plantlets of both crop and tree species useful in forestry within a short span of time and all through the year.
vi. Production of secondary metabolites from cell culture utilized in pharmaceutical, cosmetic and food industries.

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.

Basic concepts of Tissue Culture
Basic concepts of plant tissue culture are totipotency, differentiation, dedifferentiation and redifferentiation.
Totipotency
The property of live plant cells that they have the genetic potential when cultured in nutrient medium to give rise to a complete individual plant.
Differentiation
The process of biochemical and structural changes by which cells become specialized in form and function.

Redifferentiation
The further differentiation of already differentiated cell into another type of cell. For example, when the component cells of callus have the ability to form a whole plant in a nutrient medium, the phenomenon is called redifferentiation.
Dedifferentiation
The phenomenon of the reversion of mature cells to the meristematic state leading to the formation of callus is called dedifferentiation. These two phenomena of redifferentiation and dedifferentiation are the inherent capacities of living plant cells or tissue. This is described as totipotency.