Shoot - tip meristem

(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.

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

The specification part is narrative in which the subject matter of invention is described as how the invention was carried out.
The claim specifically defines the scope of the invention to be protected by the patent which the others may not practice.

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

The success of tissue culture lies in the composition of the growth medium, plant growth regulators and culture conditions such as temperature, pH, light and humidity.

Culture media are dispensed in glass containers, plugged with non-absorbent cotton or sealed with plastic closures and then sterilized using autoclave at 15 psi (121°C) for 15 to 30 minutes.

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.

Sterilization of Nutrient Media:
1. Culture media are dispensed in glass containers, plugged with non-absorbent cotton or sealed with plastic closures and then sterilized using autoclave at 15 psi (121^o(C)  for 15 to 30 minutes.
2. The plant extracts, vitamins, amino acids and hormones are sterilized by passing through Millipore filter with 0.2 mm pore diameter.
3. Then added to sterilized culture medium inside Laminar Airflow Chamber under sterile condition.
Other techniques used to remove the microbes
(a) Maintenance of aseptic environment
(b) Sterilization of culture room
(c) Sterilization of explants

Redifferentiation and Dedifferentiation.

Bioethics refers to the study of ethical issues emerging from advances in biology and medicine. It is also a moral discernment as it relates to medical policy and practice.

Grant is a signed document, actually the agreement that grants patent right to the inventor. It is filled at patent office and not published.

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

Based on the explants some other plant tissue culture types are:
1. Organ culture
2. Meristem culture
3. Protoplast culture
4. Cell culture

(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.

Basic concepts of plant tissue culture are totipotency, differentiation, dedifferentiation and redifferentiation.

Pineapple, banana, strawberry and potato.

Advantages of Artificial seeds
Artificial seeds have many advantages over the true seeds as follows:
1. Millions of artificial seeds can be produced at any time at low cost.
2. They provide an easy method to produce genetically engineered plants with desirable traits.
3. It is easy to test the genotype of plants.
4. They can potentially stored for long time under cryopreservation method.
5. Artificial seeds produce identical plants .
6. The period of dormancy of artificial seeds is greatly reduced, hence growth is faster with a shortened life cycle.

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.

Types of Plant tissue cultures
Based on the explants some other plant tissue culture types are
(a) Organ culture
(b) Meristem culture
(c) Protoplast culture
(d) Cell culture

(a) Organ culture
The culture of embryos, anthers, ovaries roots, shoots or other organs of plants on culture media.
(b) Meristem Culture:
The culture of any plant meristematic tissue on culture media.

(c) Protoplast Culture:
1. Protoplasts are cells without a cell wall, but bounded by a cell membrane or plasma membrane.
2. Using protoplasts, it is possible to regenerate whole plants from single cells and also develop somatic hybrids.
The steps involved in protoplast culture
(i) Isolation of protoplast:
1. Small bits of plant tissue like leaf tissue are used for isolation of protoplast.
2. The leaf tissue is immersed in 0.5% Macrozyme and 2% Onozuka cellulase enzymes dissolved in 13% sorbitol or mannitol at pH 5.4.
3. It is then incubated over-night at 25^oC.
4. 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
1. It is done through the use of a suitable fusogen. This is normally PEG (Polyethylene Glycol).
2. 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^oC. 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.
(d) Cell Suspension Culture
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. 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.