Plant Tissue Culture

Class 12 | Biology — Botany | Chapter 5

Class 12 Unit 5 Biology

Chapter 5: Plant Tissue Culture

Learning Objectives

The learner will be able to:

• Perceive the concepts of tissue culture.
• Cognize the steps of tissue culture techniques and its types.
• Understand the protoplast culture in detail.
• Elicit the list of secondary metabolites obtained through cell suspension culture.
• Learn plant regeneration pathway.
• Appreciate the uses of micro propagation, somatic hybridization, shoot meristem culture and germplasm conservation.
• Acquire the knowledge of patenting Biosafety and Bioethics.

Chapter Outline

• 5.1 Basic concepts in plant tissue culture
• 5.2 Plant tissue culture techniques and types
• 5.3 Plant regeneration pathway
• 5.4 Applications of plant tissue culture
• 5.5 Conservation of plant genetic resources
• 5.6 Intellectual Property Rights (IPR), Biosafety and Bioethics
• 5.7 Future Biotechnology

Growing plant protoplasts, cells, tissues or organs away from their natural or normal environment, under artificial condition, is known as Tissue Culture. It is also known as in vitro (In vitro is a Latin word, it means that - in glass or in test-tube) growth of plant protoplasts, cells, tissues and organs. A single explant can be multiplied into several thousand plants in a short duration and space under controlled conditions. Tissue culture techniques are often used for commercial production of plants as well as for plant research. Plant tissue culture serves as an indispensable tool for regeneration of transgenic plants. Apart from this some of the main applications of Plant tissue culture are clonal propagation of elite varieties, conservation of endangered plants, production of virus-free plants, germplasm preservation, industrial production of secondary metabolites, etc.

In this chapter let us discuss the history, techniques, types, applications of plant tissue culture and get awareness on ethical issues.

Gottlieb Haberlandt

Gottlieb Haberlandt (1902), the German Botanist proposed the concept Totipotency and he was also the first person to culture plant cells in artificial conditions using the mesophyll cells of Lamium purpureum in culture medium and obtained cell proliferation. He is regarded as the father of tissue culture.

5.1 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.

5.2 Plant Tissue Culture (PTC)

Plant tissue culture is used to describe the in vitro and aseptic growth of any plant part on a tissue culture medium. This technology is based on three fundamental principles:

• The plant part or explant must be selected and isolated from the rest of plant body.
• The explant must be maintained in controlled physically (environmental) and chemically defined (nutrient medium) conditions.

Explant

The tissue taken from a selected plant transferred to a culture medium often to establish a new plant.

5.2.1 Laboratory Facilities for PTC

For PTC, the laboratory must have the following facilities:

• Washing facility for glassware and ovens for drying glassware.
• Medium preparation room with autoclave, electronic balance and pH meter.
• Transfer area sterile room with laminar air-flow bench and a positive pressure ventilation unit called High Efficiency Particulate Air (HEPA) filter to maintain aseptic condition.
• Culture facility: Growing the explant inoculated into culture tubes at 22 to 28°C with illumination of light 2400 lux, with a photoperiod of 8 to 16 hours and a relative humidity of about 60%.

5.2.2 Technique Involved in PTC

1. Sterilization

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

1. Maintenance of Aseptic Environment: During in vitro tissue culture maintenance of aseptic environmental condition should be followed, that is, sterilization of glassware, forceps, scalpels, and all accessories in wet steam sterilization by autoclaving at 15 psi (121°C) for 15 to 30 minutes or dipping in 70% ethanol followed by flaming and cooling.
2. Sterilization of culture room: Floor and walls are washed first with detergent and then with 2% sodium hypochlorite or 95% ethanol. The cabinet of laminar airflow is sterilized by clearing the work surface with 95% ethanol and then exposure of UV radiation for 15 minutes.
3. Sterilization of Nutrient Media: 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. The plant extracts, vitamins, amino acids and hormones are sterilized by passing through Millipore filter with 0.2 mm pore diameter and then added to sterilized culture medium inside Laminar Airflow Chamber under sterile condition.
4. Sterilization of Explants: The plant materials to be used for tissue culture should be surface sterilized by first exposing the material in running tap water and then treating it in surface sterilization agents like 0.1% mercuric chloride, 70% ethanol under aseptic condition inside the Laminar Air Flow Chamber.

2. Media Preparation

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. No single medium is capable of maintaining optimum growth of all plant tissues. Suitable nutrient medium as per the principle of tissue culture is prepared and used. MS nutrient medium (Murashige and Skoog 1962) is commonly used. It has carbon sources, with suitable vitamins and hormones. The media formulations available for plant tissue culture other than MS are B5 medium (Gamborg et al. 1968), White medium (White 1943), Nitsch's medium (Nitsch & Nitsch 1969). A medium may be solid or semisolid or liquid. For solidification, a gelling agent such as agar is added.

Agar: A complex mucilaginous polysaccharide obtained from marine algae (sea weeds) used as solidifying agent in media preparation.

3. Culture Condition

pH: The pH of medium is normally adjusted between 5.6 to 6.0 for the best result.

Temperature: The cultures should be incubated normally at constant temperature of 25°C for optimal growth.

Humidity and Light Intensity: The cultures require 50 to 60% relative humidity and 16 hours of photoperiod by the illumination of cool white fluorescent tubes of approximately 1000 lux.

Aeration: Aeration to the culture can be provided by shaking the flasks or tubes of liquid culture on automatic shaker or aeration of the medium by passing with filter-sterilized air.

4. Induction of Callus

Explant of 1 to 2 cm sterile segment selected from leaf, stem, tuber or root is inoculated (transferring the explants to sterile glass tube containing nutrient medium) in the MS nutrient medium supplemented with auxins and incubated at 25°C in an alternate light and dark period of 12 hours to induce cell division and soon the upper surface of explant develops into callus.

Callus is a mass of unorganized growth of plant cells or tissues in in vitro culture medium.

5. Embryogenesis

The callus cells undergoes differentiation and produces somatic embryos, known as Embryoids. The embryoids are sub-cultured to produce plantlets.

6. Hardening

The plantlets developed in vitro require a hardening period and so are transferred to greenhouse or hardening chamber and then to normal environmental conditions. 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.

5.2.3 Types of Plant Tissue Cultures

Based on the type of explants other plant tissue culture types are:

1. Organ culture
2. Meristem culture
3. Protoplast culture
4. Cell suspension culture

1. Organ Culture

The culture of embryos, anthers, ovaries, roots, shoots or other organs of plants on culture media.

2. Meristem Culture

The culture of any plant meristematic tissue on culture media. Older leaves are removed from a cutting; after removal of larger leaves, the shoot tip with growing tip after removal of enclosing leaves is used. This involves paper wick, callus and plantlets.

3. Protoplast Culture

Protoplasts are cells without a cell wall, but bound by a cell membrane or plasma membrane. Using protoplasts, it is possible to regenerate whole plants from single cells and also develop somatic hybrids.

Steps involved in protoplast culture:

1. 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 over-night 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.
2. 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.
3. 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.
4. Selection of somatic hybrid cells: The fusion product of protoplasts without nucleus of different cells is called a cybrid. Following this nuclear fusion takes place. This process is called somatic hybridization.

4. 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.

Production of Secondary Metabolites: Cell suspension culture can be useful for the production of secondary metabolites like 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 but are produced in the plant as 'byproducts' of cell metabolism.

For Example: Biosynthesis and isolation of indole alkaloids from Catharanthus roseus plant cell culture.

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.

Few examples of industrially important plant secondary metabolites are listed below:

Secondary Metabolites	Plant Source	Uses
Digoxin	Digitalis purpurea	Cardiac tonic
Codeine	Papaver somniferum	Analgesic
Capsaicin	Capsicum annuum	Rheumatic pain treatment
Vincristine	Catharanthus roseus	Anticarcinogenic
Quinine	Cinchona officinalis	Antimalarial

Table 5.1: Secondary metabolites and its plant resources

5.3 Plant Regeneration Pathway

From the explants, plants can be regenerated by somatic embryogenesis or organogenesis.

Flow Chart of Plant Regeneration Pathway

• Explant →
  • Direct embryogenesis → Embryoids → Plantlets → Hardening → Transfer to field
  • Meristem → Multiple shoot induction (Micropropagation) → Root induction → Plantlets → Hardening → Transfer to field
  • Shoot apex / Stem / Leaf / Root → Callus →
    • Indirect embryogenesis → Shoot induction → Root induction → Plantlets → Hardening → Transfer to field
    • Direct embryogenesis → Embryoids → Plantlets → Hardening → Transfer to field
• Direct organogenesis: Explants → Direct somatic embryogenesis → Callus → Somatic embryo → Rooting; Root organogenesis / Shoot organogenesis → Rooting → Plantlets → Indirect embryogenesis

5.3.1 Somatic Embryogenesis

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 of Somatic Embryogenesis

• Somatic embryogenesis provides potential plantlets which after 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.
• Synthetic seeds are produced by encapsulation of embryoids in agarose gel or calcium alginate.

5.3.2 Organogenesis

The morphological changes occur in the callus leading to the formation of shoot and roots is called organogenesis. Organogenesis can be induced in vitro by introducing plant growth regulators in the MS medium. Auxin and cytokinins induce shoot and root formation.

5.4 Applications of Plant Tissue Culture

Plant tissue culture techniques have several applications such as:

1. Improved hybrids production through somatic hybridization.
2. Somatic embryoids can be encapsulated into synthetic seeds (synseeds). These encapsulated seeds or synthetic seeds help in conservation of plant biodiversity.
3. Production of disease resistant plants through meristem and shoot tip culture.
4. Production of stress resistant plants like herbicide tolerant, heat tolerant plants.
5. 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.
6. Production of secondary metabolites from cell culture utilized in pharmaceutical, cosmetic and food industries.

Somaclonal Variations

Somatic variations found in plants regenerated in vitro (that is, variations found in leaf, stem, root, tuber or propagule).

Gametoclonal Variations

Gametophytic variations found in plants regenerated in vitro of gametic origin (i.e. variations found in gametes and gametophytes).

5.4.1 Micropropagation of Banana

Micropropagation of plants at industrial level maintains high standards of homogeneity in plants like pineapple, banana, strawberry and potato.

5.4.2 Artificial Seed

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

Advantages of Artificial Seeds

Artificial seeds have many advantages over the true seeds:

• Millions of artificial seeds can be produced at any time at low cost.
• They provide an easy method to produce genetically engineered plants with desirable traits.
• It is easy to test the genotype of plants.
• They can potentially be stored for long time under cryopreservation method.
• Artificial seeds produce identical plants.
• The period of dormancy of artificial seeds is greatly reduced, hence growth is faster with a shortened life cycle.

5.4.3 Virus-free Plants

The field grown plants like perennial crops, usually are infected by variety of pathogens like fungi, bacteria, mycoplasma, viruses which cause considerable economic losses. Chemical methods can be used to control fungal and bacterial pathogens, but not viruses generally. Shoot meristem tip culture is the method to produce virus-free plants, because the shoot meristem tip is always free from viruses.

5.5 Conservation of Plant Genetic Resources

5.5.1 Germplasm Conservation

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. 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. 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.

5.5.2 Cryopreservation (–195.5°C)

Cryopreservation, also known as Cryoconservation, 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. Protective agents like dimethyl sulphoxide, glycerol or sucrose are added before cryopreservation process. These protective agents are called cryoprotectants, since they protect the cells, or tissues from the stress of freezing temperature.

5.6 Intellectual Property Right (IPR)

Intellectual property right (IPR) is a category of rights that includes intangible creation of the human intellect, and primarily consists of copyrights, patents, and trademarks. It also includes other types of rights, such as trade secrets, publicity rights, moral rights, and rights against unfair competition. In biotechnology, the transformed microorganisms and plants and technologies for the production of commercial products are exclusively the property of the discoverer. The discoverer has the full rights on his property. It should not be neglected by the others without legal permission. The right of discoverer must be protected and it does by certain laws framed by a country. The IPR is protected by different ways like patents, copyrights, trade secrets and trademarks, designs and geographical indications.

5.6.1 Patents

It is a special right to the discoverer/inventor that has been granted by the government through legislation for trading new articles. A patent is a personal property which can be licensed or sold by the person or organisation just like any other property. Patent terms give the inventor the rights to exclude others from making, using or selling his invention.

5.6.2 Biosafety and Bioethics

Advances in biotechnology and their applications deals with genetic manipulation.

Biosafety

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. 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.

Bioethics — Ethical, Legal and Social Implications (ELSI)

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. Bioethicists are concerned with the ethical questions that arise in the relationships among life sciences, biotechnology and medicine. It includes the study of values relating to primary care and other branches of medicine. The scope of bioethics is directly related to biotechnology, including cloning, gene therapy, life extension, human genetic engineering, astroethics life in space, and manipulation of basic biology through altered DNA, RNA and proteins. These developments in biotechnology will affect future evolution, and may require new principles, such as biotic ethics, that values life and its basic biological characters and structures.

The Ethical, Legal, and Social Implications (ELSI) program was founded in 1990 as an integral part of the Human Genome Project. The mission of the ELSI program was to identify and address issues raised by genomic research that would affect individuals, families, and society. A percentage of the Human Genome Project budget at the National Institutes of Health and the U.S. Department of Energy was devoted to ELSI research.

Genetic Engineering Appraisal Committee (GEAC)

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.

5.7 Future of Biotechnology

Biotechnology has become a comprehensive scientific venture from the point of academic and commercial angles, within a short time with the sequencing of human genome and genome of some important organisms. The future developments in biotechnology will be exciting. Thus the development in biotechnology will lead to a new scientific revolution that would change the lives and future of people. Like industrial and computer revolution, biotechnological revolution will also promise major changes in many aspects of modern life.

Summary

Summary

• Tissue culture is the in vitro aseptic culture of cells, tissues or organs into whole plants under controlled nutritional and environmental conditions.
• A German physiologist Gottlieb Haberlandt in 1902 for the first time attempted to culture plant cells in artificial medium, hence he was regarded as father of Tissue culture.
• Tissue culture mainly based on the concepts totipotency, differentiation, redifferentiation and dedifferentiation.
• Plant tissue culture technique involves selection of explants, sterilization, media preparation, maintaining culture condition, callus formation, embryogenesis or organogenesis and hardening.
• Based on the explants chosen the types of tissue culture are organ culture, meristem culture, protoplast culture and cell suspension culture.
• From the explants, plants can be regenerated by somatic embryogenesis or organogenesis is said to be plant regeneration pathway.
• Some of the main applications of tissue culture are production of somatic hybrids, artificial seeds, disease resistant and stress resistant plants, germplasm conservation, micropropagation and production of secondary metabolites.
• Intellectual Property Right (IPR) is primarily aimed at patents, copyrights, trade secret and trademark given to the discoverer/inventor for the commercial production of transformed micro organisms or plants.
• Biosafety is the prevention mechanism to protect harmful incidents due to biohazards or pathogens.
• Bioethics dealt with ethical issue emerging from biotechnological advancement.
• ELSI program addresses issues related to genomic research.
• GEAC (Genetic Engineering Appraisal Committee) is a regulatory authority for release of genetically modified products or organisms into the environment.

Evaluation

Exercise Choose the Correct Answer

1. Totipotency refers to

a. capacity to generate genetically identical plants. b. capacity to generate a whole plant from any plant cell/explant. c. capacity to generate hybrid protoplasts. d. recovery of healthy plants from diseased plants.

2. Micro propagation involves

a. vegetative multiplication of plants by using micro-organisms. b. vegetative multiplication of plants by using small explants. c. vegetative multiplication of plants by using microspores. d. Non-vegetative multiplication of plants by using microspores and megaspores.

3. Match the following:

Column A	Column B
1. Totipotency	A. Reversion of mature cells into meristem
2. Dedifferentiation	B. Biochemical and structural changes of cells
3. Explant	C. Properties of living cells develops into entire plant
4. Differentiation	D. Selected plant tissue transferred to culture medium

a. 1-C, 2-A, 3-D, 4-B b. 1-A, 2-C, 3-B, 4-D c. 1-B, 2-A, 3-D, 4-C d. 1-D, 2-B, 3-C, 4-A

4. The time duration for sterilization process by using autoclave is __ minutes and the temperature is __

a. 10 to 30 minutes and 125°C b. 15 to 30 minutes and 121°C c. 15 to 20 minutes and 125°C d. 10 to 20 minutes and 121°C

5. Which of the following statement is correct?

a. Agar is not extracted from marine algae such as seaweeds. b. Callus undergoes differentiation and produces somatic embryoids. c. Surface sterilization of explants is done by using mercuric bromide. d. pH of the culture medium is 5.0 to 6.0.

6. Select the incorrect statement from given statement

a. A tonic used for cardiac arrest is obtained from Digitalis purpurea. b. Medicine used to treat Rheumatic pain is extracted from Capsicum annuum. c. An anti malarial drug is isolated from Cinchona officinalis. d. Anti-carcinogenic property is not seen in Catharanthus roseus.

7. Virus free plants are developed from

a. Organ culture b. Meristem culture c. Protoplast culture d. Cell suspension culture

8. The prevention of large scale loss of biological integrity

a. Biopatent b. Bioethics c. Biosafety d. Biofuel

9. Cryopreservation means it is a process to preserve plant cells, tissues or organs

a. at very low temperature by using ether. b. at very high temperature by using liquid nitrogen. c. at very low temperature of –196°C by using liquid nitrogen. d. at very low temperature by using liquid nitrogen.

10. Solidifying agent used in plant tissue culture is

a. Nicotinic acid b. Cobaltous chloride c. EDTA d. Agar Exercise 11. Short Answer

11. What is the name of the process given below? Write its 4 types.

Exercise 12. Short Answer

12. How will you avoid the growing of microbes in nutrient medium during culture process? What are the techniques used to remove the microbes?

Exercise 13. Short Answer

13. Write the various steps involved in cell suspension culture.

Exercise 14. Short Answer

14. What do you mean by Embryoids? Write its application.

Exercise 15. Short Answer

15. Give the examples for micro propagation performed plants.

Exercise 16. Long Answer

16. Explain the basic concepts involved in plant tissue culture.

Exercise 17. Long Answer

17. Based on the material used, how will you classify the culture technology? Explain it.

Exercise 18. Long Answer

18. Give an account on Cryopreservation.

Exercise 19. Long Answer

19. What do you know about Germplasm conservation? Describe it.

Exercise 20. Long Answer

20. Write the protocol for artificial seed preparation.

Glossary

Aseptic condition Preparation of materials free from microbes in in vitro cultures. Cell Culture Growing of cells in vitro, including the culture of single cells or small aggregates of cells in a liquid medium. Chemically defined medium A nutritive medium used for culturing cells or tissue; each chemical of this medium is known and defined. Cybrid 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. Organogenesis The process of initiation and development of shoot or root through in vitro culture particularly from callus.

APPENDIX: Composition of MS (Murashige and Skoog) Medium

Component	Quantity
Macronutrients
Ammonium nitrate (NH₄NO₃)	1650.0 mg/l
Potassium nitrate (KNO₃)	1900.0 mg/l
Calcium chloride (CaCl₂·2H₂O)	440.0 mg/l
Magnesium sulphate (MgSO₄·6H₂O)	370.0 mg/l
Potassium dihydrogen phosphate (KH₂PO₄)	170.0 mg/l
Micronutrients
Manganese sulphate (MnSO₄·4H₂O)	22.3 mg/l
Zinc sulphate (ZnSO₄·4H₂O)	8.6 mg/l
Boric acid (H₃BO₃)	6.2 mg/l
Potassium iodide (KI)	0.83 mg/l
Minor Nutrients
Sodium molybdate (Na₂MO₄·2H₂O)	0.250 mg/l
Cupric sulphate (CuSO₄·5H₂O)	0.025 mg/l
Cobaltous chloride (CoCl₂·6H₂O)	0.025 mg/l
Iron Stock
Na EDTA	37.25 mg/l
Ferrous Sulphate (FeSO₄·7H₂O)	27.85 mg/l
Vitamins
Glycine	2.0 mg/l
Nicotinic acid	0.5 mg/l
Pyridoxin HCl	0.5 mg/l
Thiamine HCl	0.1 mg/l
Growth Hormones
IAA	1.30 mg/l
Kinetin	0.4–10.0 mg/l
Myo-inositol	100.0 mg/l
Sucrose	30.0 g/l
Solidifying Agent
Agar	8.0 g/l

5.1 Milestones in Plant Tissue Culture

Historical Milestones

• Haberlandt (1902): Cultured plant cells in artificial condition called in vitro (inside glass) in culture medium (Knop's salt solution) containing glucose and peptone and developed callus (unorganized growth of cells and tissue) and proposed the concept Totipotency — the development of whole plant from isolated cells or tissue in in vitro condition.
• P.R. White (1934): Developed root cultures, used Knop's solution along with three vitamins like pyridoxine, thiamine and nicotinic acid.
• F.C. Steward (1948): Used coconut water in plant tissue culture work and obtained cell proliferation from carrot explants (Cellular totipotency).
• Morel and Martin (1952, 1955): Developed virus-free Dahlia and potato plants using shoot meristem culture.
• Murashige and Skoog (1962): Formulated tissue culture medium, a landmark in plant tissue culture and it is the most frequently used medium for all kinds of tissue culture work.
• Kanta et al. (1962): Produced test-tube fertilization in flowering plants.
• Yamada et al. (1963): Produced calli and free cells in tissue culture of Tradescantia reflexa.
• Guha and Maheshwari (1964): Developed in vitro production of haploid embryos from anthers of Datura.
• Vasil and Hildebrandt (1965): Achieved differentiation of tobacco plants from single, isolated cells in micro propagation.
• Takebe et al. (1971): Regenerated tobacco plants from isolated mesophyll protoplasts.
• Carlson and co-workers (1971): Obtained protoplast fusion between Nicotiana glauca and Nicotiana langsdorffii and developed first interspecific somatic hybrid.
• Melchers and co-workers (1978): Developed intergenic hybrid between potato and tomato called pomato.
• Chilton (1983): Produced transformed tobacco plants from single cell transformation and gene insertion.
• Horsch et al. (1984): Developed transgenic tobacco by Agrobacterium mediated gene transfer.

Knop's Solution

Nutrient solution used in growth experiments of plants which contains:

Component	Quantity
Calcium nitrate	3.0 g
Magnesium sulfate	1.0 g
Potassium nitrate	1.0 g
Dibasic Potassium phosphate	1.0 g
Sucrose (optional)	50.0 g
Deionized water	1000.0 ml

Book-back Exercises — Questions & Answers

Answers generated strictly from the Samacheer Kalvi textbook content. Attempt each question first, then click "Show Answer" to check.

I. Choose the Correct Answer

1. Totipotency refers to

a) capacity to generate genetically identical plants. b) capacity to generate a whole plant from any plant cell/explant. c) capacity to generate hybrid protoplasts. d) recovery of healthy plants from diseased plants.

Answer: b) capacity to generate a whole plant from any plant cell/explant.

Explanation: Totipotency is 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.

2. Micro propagation involves

a) vegetative multiplication of plants by using micro-organisms. b) vegetative multiplication of plants by using small explants. c) vegetative multiplication of plants by using microspores. d) Non-vegetative multiplication of plants by using microspores and megaspores.

Answer: b) vegetative multiplication of plants by using small explants.

Explanation: Micropropagation technique is used to obtain large numbers of plantlets of both crop and tree species using small explants within a short span of time.

3. Match the following:

Column A	Column B
1. Totipotency	A. Reversion of mature cells into meristem
2. Dedifferentiation	B. Biochemical and structural changes of cells
3. Explant	C. Properties of living cells develops into entire plant
4. Differentiation	D. Selected plant tissue transferred to culture medium

a) C A D B b) A C B D c) B A D C d) D B C A

Answer: a) C A D B

Explanation: 1-C: Totipotency is the property of living cells to develop into an entire plant; 2-A: Dedifferentiation is the reversion of mature cells to meristematic state; 3-D: Explant is the selected plant tissue transferred to culture medium; 4-B: Differentiation involves biochemical and structural changes of cells.

4. The time duration for sterilization process by using autoclave is ___ minutes and the temperature is ___

a) 10 to 30 minutes and 125°C b) 15 to 30 minutes and 121°C c) 15 to 20 minutes and 125°C d) 10 to 20 minutes and 121°C

Answer: b) 15 to 30 minutes and 121°C

Explanation: Sterilization of glassware and accessories is done in wet steam sterilization by autoclaving at 15 psi (121°C) for 15 to 30 minutes.

5. Which of the following statement is correct

a) Agar is not extracted from marine algae such as seaweeds. b) Callus undergoes differentiation and produces somatic embryoids. c) Surface sterilization of explants is done by using mercuric bromide. d) pH of the culture medium is 5.0 to 6.0

Answer: b) Callus undergoes differentiation and produces somatic embryoids.

Explanation: The callus cells undergo differentiation and produce somatic embryos, known as Embryoids. Agar is obtained from marine algae (seaweeds), and surface sterilization uses mercuric chloride (not bromide), and pH is adjusted between 5.6 to 6.0.

6. Select the incorrect statement from given statement

a) A tonic used for cardiac arrest is obtained from Digitalis purpurea b) Medicine used to treat Rheumatic pain is extracted from Capsicum annuum c) An anti malarial drug is isolated from Cinchona officinalis. d) Anti-carcinogenic property is not seen in Catharanthus roseus.

Answer: d) Anti-carcinogenic property is not seen in Catharanthus roseus.

Explanation: According to the chapter, Vincristine obtained from Catharanthus roseus is anticarcinogenic, so the statement that anti-carcinogenic property is NOT seen in Catharanthus roseus is incorrect.

7. Virus free plants are developed from

a) Organ culture b) Meristem culture c) Protoplast culture d) Cell suspension culture

Answer: b) Meristem culture

Explanation: Shoot meristem tip culture is the method to produce virus-free plants, because the shoot meristem tip is always free from viruses.

8. The prevention of large scale loss of biological integrity

a) Biopatent b) Bioethics c) Biosafety d) Biofuel

Answer: c) Biosafety

Explanation: Biosafety is the prevention of large-scale loss of biological integrity, focusing both on ecology and human health.

9. Cryopreservation means it is a process to preserve plant cells, tissues or organs

a) at very low temperature by using ether. b) at very high temperature by using liquid nitrogen c) at very low temperature of -196 by using liquid nitrogen d) at very low temperature by using liquid nitrogen

Answer: c) at very low temperature of -196°C by using liquid nitrogen

Explanation: Cryopreservation is a process by which biological materials are preserved by cooling to very low temperature of –196°C using liquid nitrogen.

10. Solidifying agent used in plant tissue culture is

a) Nicotinic acid b) Cobaltous chloride c) EDTA d) Agar

Answer: d) Agar

Explanation: Agar is a complex mucilaginous polysaccharide obtained from marine algae (seaweeds) used as solidifying agent in media preparation.

II. Short Answer Questions

11. What is the name of the process given below? Write its 4 types.

Answer: The process is Plant Tissue Culture (PTC). Based on the type of explants, the four types of plant tissue cultures are: 1. Organ culture – culture of embryos, anthers, ovaries, roots or shoots; 2. Meristem culture – culture of any plant meristematic tissue; 3. Protoplast culture – culture using cells without a cell wall; 4. Cell suspension culture – growing single cells or small aggregates of cells in liquid medium.

12. How will you avoid the growing of microbes in nutrient medium during culture process? What are the techniques used to remove the microbes?

Answer: Microbes are avoided by sterilization. The techniques used are: (1) Autoclaving glassware and media at 15 psi (121°C) for 15–30 minutes; (2) Sterilizing culture room floors and walls with 2% sodium hypochlorite or 95% ethanol; (3) Sterilizing the laminar airflow cabinet with 95% ethanol and UV radiation for 15 minutes; (4) Surface sterilizing explants with 0.1% mercuric chloride or 70% ethanol; (5) Filtering heat-sensitive substances like vitamins and hormones through Millipore filter (0.2 mm pore diameter).

13. Write the various steps involved in cell suspension culture.

Answer: Cell suspension culture involves: (1) A portion of callus is transferred to liquid medium; (2) The liquid medium is agitated using a rotary shaker instrument; (3) Cells are separated from callus tissue and used for suspension culture; (4) The culture can be used for production of secondary metabolites like alkaloids, flavonoids, terpenoids, phenolic compounds and recombinant proteins; (5) The process can be scaled up using bio-reactors for commercial production using strategies like biotransformation, elicitation and immobilization.

14. What do you mean by Embryoids? Write its application.

Answer: Embryoids are somatic embryos formed from callus tissue directly through differentiation, or formed directly from in vitro cells that differentiate into pre-embryonic cells. Applications: (1) Embryoids provide potential plantlets which after hardening can establish into plants; (2) Somatic embryoids can be used for the production of synthetic seeds; (3) Somatic embryogenesis is now reported in many plants such as Allium sativum, Hordeum vulgare, Oryza sativa and Zea mays.

15. Give the examples for micro propagation performed plants.

Answer: Micropropagation at industrial level maintains high standards of homogeneity in plants like pineapple, banana, strawberry and potato. The technique obtains large numbers of plantlets of both crop and tree species useful in forestry within a short span of time and all through the year.

III. Answer in Detail

16. Explain the basic concepts involved in plant tissue culture.

Answer: The 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.

Dedifferentiation: The phenomenon of the reversion of mature cells to the meristematic state leading to the formation of callus.

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.

The two phenomena of redifferentiation and dedifferentiation are the inherent capacities of living plant cells or tissue, described collectively as totipotency.

17. Based on the material used, how will you classify the culture technology? Explain it.

Answer: Based on the type of explants, plant tissue culture is classified into four types:

1. Organ culture: The culture of embryos, anthers, ovaries, roots, shoots or other organs of plants on culture media.

2. Meristem Culture: The culture of any plant meristematic tissue on culture media. It is used to produce virus-free plants since the shoot meristem tip is always free from viruses.

3. Protoplast Culture: Protoplasts are cells without a cell wall but bound by a plasma membrane. Isolation is done using enzymes (Macrozyme and Onozuka cellulase). Fusion is done using PEG (Polyethylene Glycol). This can regenerate whole plants from single cells and develop somatic hybrids. The fusion product of protoplasts from different cells is called a cybrid; after nuclear fusion, this is called somatic hybridization.

4. Cell Suspension Culture: The growing of cells including the culture of single cells or small aggregates of cells in vitro in liquid medium. The cell suspension is prepared by transferring callus to liquid medium and agitating using a rotary shaker. It is used for production of secondary metabolites like alkaloids, flavonoids, terpenoids, phenolic compounds, and recombinant proteins.

18. Give an account on Cryopreservation.

Answer: Cryopreservation, also known as Cryoconservation, 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 extremely low temperature, any enzymatic or chemical activity of the biological material will be totally stopped, and this leads to preservation of material in a 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.

19. What do you know about Germplasm conservation? Describe it.

Answer: 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. 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. 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. Encapsulated seeds or synthetic seeds also help in conservation of plant biodiversity.

20. Write the protocol for artificial seed preparation.

Answer: Artificial seeds or synthetic seeds (synseeds) are produced by using embryoids (somatic embryos) obtained through in vitro culture. They may be derived from single cells from any part of the plant that divide to form a cell mass containing dense cytoplasm, large nucleus, starch grains, proteins, and oils. To prepare artificial seeds, different inert materials are used for coating the somatic embryoids like agarose and sodium alginate (calcium alginate). Somatic embryoids are encapsulated in agarose gel or calcium alginate.

Advantages: Millions can be produced at low cost; they allow production of genetically engineered plants with desirable traits; easy genotype testing; can be stored for long time under cryopreservation; they produce identical plants; and the period of dormancy is greatly reduced, hence growth is faster with a shortened life cycle.

📝 Summary

1. Plant Tissue Culture (PTC) is the in vitro, aseptic growth of plant protoplasts, cells, tissues, or organs on a nutrient medium under artificial and controlled conditions, a concept first proposed by German Botanist Gottlieb Haberlandt in 1902, who is regarded as the father of tissue culture.
2. The fundamental basic concepts of plant tissue culture include Totipotency (the genetic potential of a live plant cell to give rise to a complete individual plant), Differentiation, Dedifferentiation (reversion of mature cells to meristematic state forming callus), and Redifferentiation (further differentiation of an already differentiated cell into another type).
3. Plant tissue culture is based on three fundamental principles: selecting and isolating an explant from the plant body, maintaining the explant in controlled physical (environmental) conditions, and providing chemically defined nutrient medium conditions.
4. A single explant can be multiplied into several thousand plants in a short duration and space, making tissue culture techniques valuable for commercial production, plant research, and regeneration of transgenic plants.
5. Key applications of Plant Tissue Culture include clonal propagation of elite varieties, conservation of endangered plants, production of virus-free plants, germplasm preservation, industrial production of secondary metabolites, micro propagation, and somatic hybridization.