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Regeneration of plants and application of plant tissue culture
- 1. Regeneration Of Plants& Pharmaceutical Applications Of Plant Tissue Culture Subject :- Pharmaceutical Biotechnology DEPARTMENT OF PHARMACEUTICAL SCIENCES Dr. HARISINGH GOUR VISHWAVIDYALAYA Sagar ( M.P)- 470003, India (A Central University ) SUBMITTED BY :- Suruchi Dahiya ( M Pharm Sem I ) Roll No;- Y22254028 SUBMITTED TO :- Prof. UMESH K PATIL (Professor, DOPS) DR. UDITAAGARWAL (Guest Faculty, DOPS)
- 2. CONTENTS •Introduction •Regeneration Of Plants •Typesof regeneration in plant •Plant tissue culture •Types of tissue culture •Application of tissue culture •Advantages & Disadvantages of tissue culture •References
- 3. Introduction ▪ Regeneration:- Regenerationis the natural process of replacing or restoring damaged or missing cells, tissues, organs, and even entire body parts to full function in plants and animals. ▪ The process of growing an entire plant from a single cell or group of cells. ▪ Regeneration is possible because plant cells can be made totipotent using hormones. ▪ Differentiated tissue: stems, leaves, roots, etc. ▪ Undifferentiated (embryonic) cells are totipotent: can become a whole new plant by differentiating into a whole new plant.
- 4. THE PLANTS CANBE REGENERATED BY :- • ORGANOGENESIS :- It refers to the formation of organs from the cultured explants. The shoot buds or monopolar structures are formed by manipulating the ratio of cytokinin's to auxins in the cultures. • SOMATIC EMBRYOGENESIS :- In Somatic embryogenesis, the totipotent cells may undergo embryogenic pathway to form somatic embryos which are grown to regenerate into complete plants.
- 5. REGENERATION OF PLANTLETS 1.Preparation of Suitable Nutrient Medium: Suitable nutrient medium as per objective of culture is prepared and transferred into suitable containers. 2. Selection of Explants: Section of explants such as shoot tip should be done. 3. Sterilization of Explants: Surface sterilization of the explants by disinfectants and then washing the explants with sterile distilled water is essential. 4. Inoculation: Inoculation (transfer) of the explants into the suitable nutrient medium (which is sterilized by filter-sterilized to avoid microbial contamination) in culture vessels under sterile conditions is done.
- 6. 5. Incubation: Growingthe culture in the growth chamber or plant tissue culture room, having the appropriate physical condition (i.e., artificial light; 16 hours of photoperiod), temperature (-26°C) and relative humidity (50-60%) is required. 6. Regeneration: Regeneration of plants from cultured plant tissues is carried out. 7. Hardening: Hardening is gradual exposure of plantlets to an environmental conditions. 8. Plantlet Transfer :After hardening plantlets transferred to the green house or field conditions following acclimatization (hardening) of regenerated plants
- 8. Plant tissue culture •Plant tissue culture is a collection of techniques used to maintain or grow plant cells, tissues or organs under sterile conditions on a nutrient culture medium of known composition. • It is widely used to produce clones of a plant in a method known as micropropagation. • Plant tissue culture relies on the fact that many plant cells have the ability to regenerate a whole plant (Cellular totipotency) • single cells, plant cells without cell walls (protoplasts), pieces of leaves, stems or roots can often be used to generate a new plant on culture media given the required nutrients and plant hormones. • Preparation of plant tissue for tissue culture is performed under aseptic conditions under HEPA filtered air provided by a laminar flow cabinet.
- 9. • It hasproved beneficial for the production of disease-free plants and increase plant yield in developing countries • It only requires a sterile workplace, greenhouse, trained manpower, and a nursery. Following are the main categories of Cultures:- Primary Culture:- These model the natural function of the Tissue and are generally mortal. They consist of natural Tissues excised from the living organisms by biopsy. Culture of Established Cell Lines:- These are derived from tumor biopsies, or from the primary cells that had undergone mutation and continued to replicate.
- 10. Types of TissueCulture • Some types of Tissue Culture techniques:- Seed Culture:- In seed Culture, explants are obtained from an in- vitro derived plant and hence are introduced into a laboratory where they proliferate. To prevent the plants from Tissue damage it should be sterilized. Embryo Culture:- Embryo Culture involves the in-vitro development of an embryo. For this process, an embryo is isolated from and living organism, both a mature and an immature embryo can be used. Mature embryos can be obtained from ripe seeds whereas immature embryos are obtained from the seeds that failed to germinate. The ovule, seed, or fruit has already been sterilized, hence there is no need to sterilize them.
- 11. • Callus Culture:-A callus can be defined as an unorganized, dividing mass of cells. A callus is the explants are Cultured in a proper medium good. The growth of callus is followed by organ differentiation. This Culture is grown on a gel-like medium composed of agar and specific nutrients which are required for the growth of the cells. • Organ Culture:- In organ Culture, any organ of the plant such as a shoot, the leaf can be used as an explant. Many methods can be used for the organ Culture such as the plasma clot method, raft method, the grid method, and Agar gel method. This method can be used to preserve the structure and functions of an organism.
- 12. • Protoplast Culture:-It can be defined as a cell without a cell wall. The hanging-drop method or micro-Culture chambers can be used to Culture a protoplast. A number of phases can be observed in protoplast Culture, development of cell walls, cell division, regeneration of a whole plant. • Suspension Culture:- suspension Culture can be defined as a form of Culture in which single cells or small aggregates of cells multiply while suspended in an agitated liquid medium. It can also be called cell Culture or cell suspension Culture.
- 13. • Meristem Culture:-meristems have the main function of the production of new cells and the synthesis of protoplasm. Shoot meristem consists of a group of certain actively dividing cells that are being protected by the developing leaves. Steps of Tissue Culture :- • Following are the steps of Tissue Culture:- Initiation Phase:- • This is a stage when the Tissue is initiated into the Culture. To prevent the process from any contamination the Tissue of interest is obtained, introduced, and sterilized.
- 14. Multiplication Phase:- • Inthe multiplication stage, The sterilized ex-plant is introduced into the medium which consists of growth regulators and appropriate nutrients, they are responsible for the multiplication of cells. Hence this undifferentiated mass of cells is known as a callus. Root Formation:- • This is the stage when the root starts forming. To initiate the formation of root plant growth hormones are added. Consequently, complete plantlets are obtained.
- 15. Shoot Formation:- • Forthe formation of the shoot, plant growth hormones are added and growth is observed for a week. Acclimatization:- • When the plant starts to develop, the plant is transferred to a greenhouse for it to develop under controlled environmental conditions. Thereafter it is finally transferred to the nurseries for its growth under natural environmental conditions.
- 16. • HORMONES USEDIN PLANT TISSUE CULTURE :- 1. Auxins 2. Cytokinin's 3. Gibberellins 4. Abscisic Acid 5. Polyamines
- 17. • Application ofplant tissue culture • Plant tissue culture technology has been used in almost all the field of biosciences. Its applications include • Production of phytopharmaceuticals and secondary metabolites. a) Biotransformation (Biochemical Conversion) b) Plant cell immobilization) c) Genetic transformation (Transgenic plant) d) Elicitors • Micropropagation (Clonal Propagation) • Synthetic seed Protoplast culture and somatic hybridization
- 18. Extracts from blackcarrot tissue culture as potent anticancer agents. Enhanced production of tropane alkaloids in transgenic Scopolia parviflora hairy root cultures over-expressing putrescine N-methyl transferase (PMT) and hyoscyamine-6B-hydroxylase (H6H). Taxus globosa S. cell lines: initiation, selection and characterization in terms of growth, and of baccatin III and paclitaxel production. Production of camptothecin in cultures of Chonemorpha grandiflora. Regeneration, in vitro glycoalkaloids production and evaluation of bioactivity of callus methanolic extract of Solanum tuberosum L. The influence of medium composition on alkaloid biosynthesis by Penicillium citrinum.
- 19. A Differential productionof tropane alkaloids in hairy roots and in vitro cultured two accessions of Atropa belladonna L. under nitrate treatments. Increased vincristine production from Agrobacterium tumefaciens C58 induced shooty teratomas of Catharanthus roseus G. Don. Enhancement of taxane production in hairy root culture of Taxus x media var. Hicksii An endophytic taxol-producing fungus from Taxus media, Cladosporium cladosporioides MD2. Optimized nutrient medium for galanthamine production in Leucojum aestivum L. in vitro shoot system. Salinity stress enhances production of solasodine in Solanum nigrum L.
- 20. REFERENCES • D.Dumet, A.Adeyemi,O.B.Ojuederie (2008). Yam invitro genebanking. Genebank manual. http://www.iita.org/genebank/manual • D.Dumet, A.Adeyemi, O.B.Ojuederie (2008). Cassava in vitro processing and the genebanking. Genebank manual. http://www.iita.org/genebank/manual • HORT689/AGRO689 Biotechniques in Plant Breeding • H.S Chawla .2002 Introduction to Plant Biotechnology 2nd edition. Oxford & IBH Publishing C./ Pvt. Ltd New Delhi India • Murashige T. and Skoog F. (1962). A revised medium for rapid growth and bioassay with Tobacco tissue culture. Physiologia plantarum 15: 473-497.