Samacheer Class 12 Bio Botany - Plant Tissue Culture

These are the basic steps in plat Tissue culture technology
The process is plant tissue culture. Based on the explants, plant tissue culture is classified as:
* Organ culture
* Meristem culture
* Protoplast culture
* Cell culture

The microbial growth in the culture medium can be overcome by autoclaving the medium at Plant Tissue Culture II 121°C (15 psi) for 15 to 30 minutes.
Chemical sterilization using chemicals, sterilizing using UV radiation. Alcoholic sterilization using ethanol, autoclaving and filtration, etc., are the various techniques used to remove microbes.

Definition: The culture of single cells or small aggregate of cells invitro in liquid medium is called cells suspension culture.
Preparation steps:
Production of Secondary Metabolites:
* Alkaloids, flavonoids, terpenoids, phenolic compounds, and recombinant proteins.
* secondary metabolites are chemical compounds that are not required by the plant for normal growth and development.
* The process of production of secondary metabolites can be scaled up and automated using bio-reactors for commercial production.
* Many strategies such as biotransformation, elicitation, and immobilization have been used to make cell suspension cultures more efficient in the production of secondary metabolites.

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:
* Somatic embryogenesis provides potential plantlets which after the hardening period can establish into plants.
* Somatic embryoids can be used for the production of synthetic seeds.
* 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.

Basic concepts of plant tissue culture are totipotency, differentiation, differentiation, and redifferentiation.
1. Totipotency: The property of live plant cells that they have the genetic potential when cultured in a nutrient medium to give rise to a complete individual plant.
2. Differentiation: The process of biochemical and structural changes by which cells become specialized in form and function.
3. 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.
4. 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.

Based on the explants some other plant tissue culture types are:
1. The culture of embryos
* anthers
* ovaries
* roots
* shoots etc
2. Meristem culture
The culture of any plant meristematic tissue on culture media.
3. Protoplast culture
* Protoplasts (cells without a cell wall, but plasma membrane) are used to regenerate whole plants from single-cell protoplasts of 2 different plants fused into hybrids – later by PTC – develop into many plantlets.
* This process of formation of somatic hybrids into somatic hybridization.
4. Cell culture
* The formation of cell suspension from the callus
* The cells are separated from the callus tissue and used for cell suspension culture

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 a very low-temperature of-196°C using liquid nitrogen. At this extremely low temperature, any enzymatic or chemical activity of the biological material will be totally stopped and this leads to the preservation of material in dormant status.
Later these materials can be activated by bringing to room temperature slowly for any experimental work. Protective agents like dimethyl sulphoxide, glycerol, or sucrose are added before the cryopreservation process. These protective agents are called cryoprotectants since they protect the cells, or tissues from the stress of freezing temperature.

Living genetic resources such as pollen, seeds, or plant tissue materials are preserved in living conditions for future use for many hybridization crop improvement research works. Eg. Pollen banks, Seedbanks
Purpose
* To maintain viability and Fertility for future use
* Gene bank, DNA bank of elite plants are maintained to keep
* biological diversity
* food security

Later these seeds are grown in vitro medium and converted into plantlets. These plantlets require a hardening period (either greenhouse or hardening chamber) and then shifted to normal environmental conditions.
12th Bio Botany Guide Plant Tissue Culture Additional Important Questions and Answers
I. Choose the correct answer

• An autoclave is a device used to do wet steam sterilization.
• Autoclaving at 15 psi (121°C) for 15-30 minutes.
• Glassware, forceps, scalpels, and all accessories are subjected to autoclaving for

Hardening slowly steadily helps the plantlets from the conditions of readymade medium, light & temperature of the laboratory, to which they were used, to the conditions of light, temperature & soil in the natural environment.

The chosen leaf tissue is immersed in the following solutions.
* 0.5% macrozyme. 2% onozuka cellulose enzyme dissolved in 13% sorbitol or mannitol kept at pH 5.4 at 25°c incubated during the night.
* After a gentle teasing of the cells, the protoplasts are obtained.
* Then they are transferred to 20% sucrose solution to retain viability.
* Finally by centrifuging the protoplasts are isolated.

• The morphological changes in the callus leading to the formation of the shoot, root, and then plantlets. The plantlets formation has 2 steps
• Root formation is known as Rhizogenesis
• Shoot formation is known as Caulogenesis.

Callus:
It is the mass of unorganized growth of plant cells or tissues in in-vitro -culture medium.
Clone:
The clone develops from callus – which gets differentiated into many plantlets known as clones (i.e) genetically uniform population.

• Hardening is the gradual exposure of invitro developed plantlets in humid chambers in diffused light – or transferred to – greenhouse setup.
• This enables them to get acclimatized to grow under normal field conditions.

* Somatic embryoids – can be used in the production of syn seeds.
* They are nothing but somatic embryoids encapsulated in Agarose gel or calcium alginate/sodium
alginate.

PTA – Plant Tissue Culture
HEPA – High-Efficiency Particulate Air
RCGM – Review Committee on Genetic Manipulation
GEAC – Genetic Engineering Approval Committee
ELSI – Ethical Legal and Social Implications
GMO – Genetically Modified Organism
GEM – Genetically Engineered Micro Organism

Organ culture:
The culture of embryos anthers, ovaries, roots, shoots
Meristem culture:
The culture of plant meristematic tissue on culture media
Give the tabulation of few secondary metaboltes a their plant sources.

Somatic embryogenesis is the formation of embryos from the callus tissue directly and these embryos are called Embryoids or from the pre-embryonic cells which differentiate into embryoids.
VII. Three Marks

• The inherent genetic potential of any living plant cell, when cultured in the nutrient medium can develop into a complete individual plant.
• One of the basic concepts exploited in tissue culture.

I. It contains various salts dissolved in Sucrose solution
* Calcium Chloride: 3.0 gm
* Potassium Nitrate: 1.0 gm
* Magnesium Sulphate: 1.0 gm
* Dibasic Potassium Phosphate: 1.0 gm
II. Sucrose: 50 gm(optimal)
III. Deionized Water: 1000ml

* M.S. Nutrient Medium (Muroshige & Skoog -1992)
It has carbon sources, suitable vitamins & hormones
* B5 – Medium (Gamborg.et.al 1968)
* White Medium (White 1943)
* Nitsch’s Medium (Nitsch & Nitsch 1969)
The medium may be solid or semisolid or liquid – For solidification, a gelling agent such as agar is added.

Steps
I) Inoculation: Sterile segment of leaf, stem, tuber or root or (explant) is transferred to the sterile nutrient medium (MS – medium – + Auxins)
II) Incubation: The inoculated medium + auxins are incubated at 25 °C ± 2°C in an alternate light & dark period of 12 hours.
III) Induction of Callus:
The cell division occurs & the upper surface of the explant develop into a callus.
Callus – is a mass of unorganized growth of plant cells/tissue in-vitro – culture medium

Somaclonal Variations:
Variation found in somatic parts such as
* Leaf, stem
* root, tuber
* propagule etc
Gametoclonal variations:
Variations found in plants regenerated in vitro by gametes & gametophytes

• Chemicals can be used to control fungal and bacterial mycoplasma pathogens but not viruses generally.
• Viral pathogens also cause great economic loss to the crops.
• Shoot meristem culture – help to produce virus-free plants because shoot meristem is free of viruses.

• Number/ time / cost – Millions of seeds produced / at any time / cheaper cost.
• Method – Easy method to produce genetically engineered plants.
• Quality – Seeds with desirable traits are produced.
• Storage – can be stored for long time use by Cryopreservation method.
• Nature of plants – Plants – Produced are identical
• Period of dormancy – greatly reduced
• Growth & Lifespan – grow faster, plants have a shorter life span

• Somatic hybridization → Improve hybrids produced
• Somatic embryoids → develop into syn – seeds help to conserve biodiversity
• Meristem & Shoot tip culture → production of Disease Resistant Varieties
• Production of plants → Stress resistant → herbicide tolerant → Drought tolerant
• Micropropagation → Large number of plantlets produced in a short time & throughout the year of both
• crop plants & true species – Used in Forestry

• Plant tissue culture is the conventional method which is also known as micropropagation.
• In this method, we take the meristematic tissue of the plant, referred to as explant is cultured over the given conditions of temperature and humidity, which makes the plant disease resistant.

It has 3 parts
* The grant
* The specifications
* The claim
The grant
* It is a signed document (actually agreement) that grants patent rights to the inventor.
* It is filled at the patent office, (not published)
The Specifications
*
* It is a narrative describing the invention & how it was carried out.
* Specifications & their claims are published from the patent office.
The Claim
The scope of the invention to be protected by the patent, preventing others from practicing it.

* It is a category of properly include products created through one’s knowledge, research & creativity.
* It includes v Copyrights v Patents & v Trademarks
* It also includes v trade secrets v publicity rights v moral rights v rights against unfair competitions
* It also includes – designs & geographical indications
Other Various aspects:
The above-mentioned property of the discovery should not be exploited by others without legal permission or by getting proper authorization.
Rights – must be protected by the enforcement of laws framed by a country.

• It will bring in a great revolution like the computer revolution.
• It will lead to new scientific – revolutions that would change the lives & future of people.
• Major challenges will be met and major changes incomprehensible in many aspects of modern life.

• It regulates -manufacturing, use, import, export, and storage of hazardous microbes or genetically modified organisms (GMOs) and cells in the country.
• It approves – activities involving large-scale use of hazardous microbes and recombinants in research & Industrial production.
• It is responsible – for approval of proposals relating to the release of GEO and products into the environment including experimental field trials (Biosafety Research Level – trial – I and II are known as BRL – I and BRL – II)

• Privacy and fairness in the use of genetic information, including the potential for genetic discrimination in employment and insurance.
• The integration of new genetic technologies such as genetic testing, into the practice of clinical medicine.
• Ethical issues surrounding the design and conduct of genetic research with people, including the process of informed consent.

Washing facility for glassware and ovens for drying glassware.
Medium preparation room with autoclave, electronic balance, and PH meter.
Culture facility:
Growing the plant inoculated into culture tubes at 22-280C with the illumination of light 2400 lux, with a photoperiod of 8 -16 hours and relative humidity of about 60%
VIII. Five Marks.

• Haberlandt (1902) – In-vitro culture of plant cells – (using knop’s salt solution + glucose & peptone)
• He proposed the Totipotency concept.
• P.R.White (1934) – In Knop’s solution + 3 vitamins (Pyridine, thiamine & nicotinic acid → developed root culture)
• F.C.Steward (1948) – used coconut water → produced cell proliferates from carrot explants.
• Morel & Martin (1952, 55) – Produced virus-free plants by shoot meristem culture → Eg. Dahlia, Potato.
• Murashige & Skoog (1962) – Most frequently used culture medium for all kinds of tissue culture work.
• Guha & Maheswari (1964) – developed in-vitro production of haploid embryos from another of Datura.
• Vasil & Hildbrandt (1965) – developed a tobacco plant by micropropagation.

PH:
* PH of medium – should lie between 5.6 to 6 – Temperature
* Incubation of culture normally at temperature 25°C ± 2°C for optimal growth.
Humidity & Light Intensity
* 50-60% relative humidity
* 16-hours of photo period by the illumination of cool white fluorescent tubes of approximately 1000 lux
Aeration:
* Provided by shaking of flasks or tubes of liquid culture of Automatic shaker
* Aeration of the medium bypassing with filter-sterilized air.

Washing & drying facility (oven) for the glassware
Medium preparation room with
* autoclave
* electronic balance
* PH meter etc., Maintain aseptic condition in,
a) Laminar air flow bench a positive pressure ventilation, unit
* (High-Efficiency Particulate Air (HEPA) filter to maintain the aseptic condition.
* Culture facility
* growing the ex-plant – inoculate into culture tube at 22 – 28°C with the illumination of light 2000 lux with 8-6 hours photoperiod, the relative humidity of about 60%

Protoplasts are cells without a cell wall but with a cell membrane or plasma membrane.
1. Isolation of protoplast
2. Fusion of protoplast (Agglutination & Fusion)
Protoplast (A) + Protoplast (B) – fused in to one in the presence of Fusogenic agent PEG in 25 – 30% concentration (Poly Ethylene Glycol) with Ca++ ions.
3. Culture of protoplast:
Protoplast viability is tested with Fluorescein diacetate – before culture.
MS – Medium – used – (with some modifications) droplet, plating or Micro drop array technique.
a. Incubation: done in continuous light (1000 – 2000 lux) at 25°C.
The cell wall formation occurs within (24-48 hrs).
The first division of new cells occurs between 2-7 days of culture.
4. Selection of somatic hybrid cells:
The fusion product of protoplasts without a nucleus of different cells – (cybrid)
Cybrid is also known as Somatic hybrid the process is known as somatic hybridization

• It deals with the application of knowledge, techniques & equipment with strict guidelines in biological laboratories & related industries,
• to prevent large scale loss of
• biological integrity
• ecology
• human health aspects
• to minimize human error and technical flaws & failures which contribute to unnecessary.
• exposures & disposal of – pathogenic microbes & hazardous chemicals, to regularise, risk management assessment and to set in best safeguard measures as per need.

* ELSI – represents Ethical legal and social Implications.
* Advancements in biotechnology such as,
* In Agriculture – Transgenic plants
* In the pharmaceutical Industry – genotherapy
* Advancements of medicine etc.,
* The biotechnological applications have raised controversies, hurting social beliefs, raising legal
issues certain ecological principles & moral values.
* So it is high time to regularise legally the modern biotechnological applications & manipulation as Bioethics, for the welfare of humanity & other plant & animal communities of our world.

• Pathogenicity – of living organisms & viruses natural or genetically modified to infect i) humans, ii) animals, iii) plants causing diseases
• Toxicity of allergy – associated with microbial production.
• Antibiotic-Resistant Microbes – increasing in number day by day.
• Disposal problem – regard to spent microbial biomass & purification of effluents.
• Safety aspects – regard to – i) contamination, ii) infection, iii) mutant strains
• regard to industrial use of microorganisms containing invitro recombinants.

IBSCs – Institutional Bio-Safety Committees monitor the research activity at the institutional level.
RCGM – The Review Committee on Genetic manipulation, functioning in the Department of Biotechnology (DBT) monitors the risky research activities in the laboratories.
GEAC – Genetic Engineering Approval Committee
– (Ministry of Environment and Forest)
– has the power to use GMO at a commercial level and open field trials of transgenic
* crops
* industrial product
* health care products