cellular totipotency and callus culture

This ppt comprises a detailed information about cellular totipotency and callus culture in plant tissue culture . It has its applications, significance and procedure described in it . This explains about the property of totipotency. It describes stages of callus culture. It also descibes history of plant tissue culture

1. Government Vidarbha Institute of Science & Humanities (Autonomous), Amravati DEPARTMENT OF BOTANY TOPIC NAME : CELLULAR TOTIPOTENCY AND CALLUS CULTURE Presented by : NAME : NEHA MOTIRAM KAKADE CLASS : BSC III RD year 2023-2024 BATCH : B1 SEM : VI TH SUB : BOTANY (PLANT BIOTECHNOLOGY AND TISSUE CULTURE)

2. Table of content Plant tissue Culture HISTORY Cellular totipotency Factors that affect cellular totipotency Significance of cellular totipotency Callus culture Nature of Callus Tissue Steps of Callus Culture Stages of callus culture development Applications of callus cultures Significance Of callus culture Conclusion References

3. Plant tissue Culture Plant tissue culture is defined as culturing plant seeds, organs, explants, tissues, cells, or protoplasts on a chemically defined synthetic nutrient media under sterile and controlled conditions of light, temperature, and humidity. G. Haberlandt  is known as the father of plant tissue culture. He was an german botanist, who was the first to separate and culture the plant cells on knop's salt solution. The basic key used in plant tissue culture is the totipotency of plant cells. With this characteristics, plant tissue culture is used to produce genetically identical plants (clones) in the absence of fertilization, pollination or seeds.

4. 1902 The idea of the totipotency of plant cell was given by Haberlandt 1937 White first time established successful root culture of tomato 1941 Vanoverbeek used coconut milk for growth and development of young Datura embryos 1957 Skoog and Miller demonstrated the role of auxin and cytokinin on root and shoot formation in tobacco - tissue 1962 Murashige and Skoog introduced the medium for tobacco culture 1987 Isolation of Bt. gene form bacterium Bacillus thuringiensis HISTORY

5. Cellular totipotency “The potential ( totipotential ) or inherent capacity of plant cell or tissue to develop into an entire plant, if suitable stimulus is provided under favourable  conditions”. In latin , “ totus ” means “entirely” and “ potens ” means “having power”. Prime examples of totipotent cells are spores and zygotes.

6. DIFFERENTIATION The process of specializing cells functions is called cell differentiation . It is accompanied by morphogenesis, the change of the cells morphology. DEDIFFERENTIATION The phenomenon of a mature cell reverting to the meristematic state and forming undifferentiated callus tissue is termed 'dedifferentiation '. REDIFFERENTIATION A multicellular explant generally comprises cells of diverse types. As a result the callus derived from it would be heterogeneous with respect to the ability of its component cells to form a whole plant or plant organs i.e. ' redifferentiation ’.

7. Stewards Experiment of cellular totipotency

8. Factors affecting cellular totipotency Source of explants : Organ that served as tissue source, physiological and ontogenic age of organ, season in which explant is obtained, size, overall quality of explant. Nutrient media and constituents : Inorganic macro- and micro- nutrients, carbon/energy source, reduced nitrogen source, plant growth regulator. Culture Environment (Physical form of medium) : Presence/absence of agar, pH of medium, light quality and quantity, temperature, relative humidity and gaseous atmosphere in the vessels.

9. Significance of cellular totipotency Regeneration Genetic Transformation Conservation of Plant Germplasm Micropropagation Plant Breeding Studying Plant Development and Differentiation Phytoremediation Medicinal plant protection

10. Callus culture Callus : Undifferentiated mass of tissue “The initiation and culture of undifferentiated cell mass (callus) on agar media is called Callus cultures”. Callus cultures may be derived from a wide variety of plant organs (e.g. roots, shoots, leaves) or specific cell types (e.g. endosperm, pollen). Chosen explants should be in a suitable 'biological state' for callus initiation. Young, tissues are more suitable than mature ones, specially of leaves.

11. Nature of Callus Tissue Some cultures are hard, consists of compactly arranged small cells without intercellular spaces, composed of lignified cells. Other callus friable consists of large loosely arranged cells with intercellular spaces Friable callus is fragile and easily breaks up These are suitable for suspension culture, where the tissue can be dispersed by mechanical agitation The color of the callus also varies and usually depends on the color of the tissue from which it is taken The callus maybe colorless or green containing chlorophyll or yellow carotenoids or flavonoids or it maybe purple due to the presence of anthocyanin Callus may be uniformly or partially colored

12. Steps of Callus Culture 1. Selection and collection of explant material 2. Selection and preparation of culture media 3. Surface sterilization of explant material 4. Preparation of explant 5. Inoculation (transfer) of explant in culture medium 6. Incubation of culture 7. Sub-culturing of culture 8. Regeneration of plant from callus (Organogenesis and Embryogenesis method)

13. 1. Selection and collection of explant material Callus may initiate from explants of any multicellular plant Explant from stem, root, leaf, flower, fruit or seed etc. may be taken for culture 2. Selection and preparation of culture media Suitable basal medium, such as Murashige and Skoog (MS) medium or Gamborg's B5 medium . These media provides essential nutrients and vitamins necessary for plant tissue culture.

14. Media Composition: The culture medium is composed of following i ) Major inorganic nutrients (Macronutrients) ii) Minor inorganic nutrients (Micronutrients) iii) Iron source - EDTA iv) Organic supplement – Vitamin and Amino acid v) Carbon source - Sucrose, glucose vi) Plant growth regulators – Auxins, Cytokinins , Gibberellins vii) pH regulators viii) Antibiotics / antifungals ix) Gelling agent- agar, gelrite Or agarose

15. 3. Surface sterilization of explant material: Explant is sterilized by using calcium hypochloride , hydrogen peroxide, ethyl and isopropyl alcohol  etc 4. Preparation for Explant Culture: Pour the sterilized medium into sterile culture vessels, such as Petri dishes or glass jars, to create a solidified layer. Place the vessels in a laminar flow hood or clean environment to minimize contamination. 5. Inoculation (transfer) of explant in culture medium: 6. Incubation of culture: Culture the explants under controlled environmental conditions including temperature, light, and humidity. This encourages callus formation and growth.

16. 7. Sub-culturing of culture: Subculturing is done because: Nutrient may be exhausted Agar may be desiccated Cell metabolites may accumulate and cause toxicity 8. Regeneration of plant from callus (Organogenesis and Embryogenesis method): Callus can regenerate entire plants by 2 routes, Somatic embryogenesis Organogenesis Organogenesis is the direct development of shoots and roots. Somatic embryogenesis is the development of vegetative tissues to embryo.

17. Stages of callus culture development 1 Initiation / Induction stage: This stage involves the induction of callus formation from explants, typically plant tissues such as leaves, stems, or roots, under controlled conditions like hormone treatments and nutrient medium. 2.Multiplication / cell division stage: In this stage, the callus is subcultured onto fresh nutrient medium to promote its growth and proliferation. This helps in increasing the biomass of the callus. 3. Differentiation stage: The callus is induced to differentiate into specific cell types or organs, such as roots, shoots, or somatic embryos. This stage often involves altering the nutrient composition and hormonal balance of the medium to promote the desired differentiation pathways.

18. Applications of callus culture Callus cultures are slow-growth plant culture in static medium. This enables to conduct several studies related to many aspects of plants (growth, differentiation and metabolism) as listed below. • Nutritional requirements of plants. • Cell and organ differentiation. • Development of suspension and protoplast cultures. • Somaclonal variations. • Genetic transformations. • Production of secondary metabolites and their regulation.

19. Significance Of callus culture 1. Plant Propagation : Callus culture enables rapid multiplication of plants from small tissue samples. 2. Genetic Transformation: It serves as a platform for introducing foreign genes into plants for genetic engineering purposes. 3. Phytochemical Production: Callus cultures can produce secondary metabolites, including pharmaceuticals and flavors. 4. Stress Response Studies: Callus cultures provide a controlled environment for studying plant responses to various stresses. 5. Crop Improvement: It facilitates the selection and breeding of plants with desirable traits, such as disease resistance or increased yield. 6. Conservation of Endangered Species : Callus culture can help conserve endangered plant species by preserving their genetic material. 7. Research Tool: It serves as a valuable tool for studying plant physiology, biochemistry, and molecular biology

20. Conclusion Callus culture works on the property of  totipotency . It is important and basic procedure to all experiments in plant tissue culture Sterilization, Hormone concentration, And Proper handling is an important step in callus culture for successful results.

21. References Biotechnology, By U Satyanarayan . • Introduction to Plant Biotechnology, By H.S.Chawla . http://www.google.com

22. Thank you