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Plant tissue culture

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Callus cultures are initiated from a small part of an organ or tissue segment called the explants on a growth supporting solidified nutrient medium under sterile conditions. Any part of the plant organ or tissues may be used as the explants.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. Plant tissue culture is widely used to produce clones of a plant in a method known as micropropagation.Tissue culture commonly refers to the culture of animal cells and tissues, with the more specific term plant tissue culture being used for plants.Plant Tissue Culture products include Murashige and Skoog media, plant growth regulators, plant growth hormones, plant transformation systems,

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Plant tissue culture

  1. 1. PLANT TISSUE CULTURE K.Sudheer Kumar, Assistant professor. Dept.of Pharmacognosy Chilkur Balaji college of Pharmacy Hyderabad. E-mail: sudheer.y2k8@gmail.com K.Sudheer Kumar, Assistant professor. Dept.of Pharmacognosy Chilkur Balaji college of Pharmacy Hyderabad. E-mail: sudheer.y2k8@gmail.com
  2. 2. Plant Tissue Culture Plant tissue culture broadly refers to the in-vitro cultivation of plants, seeds and various parts of the plants (organs, embryos, tissues, single cells, protoplasts). The cultivation process is invariably carried out in a nutrient culture medium under aseptic conditions. Plant cells have certain advantages over animal cells in culture systems. Unlike animal cells, highly mature and differentiated plant cells retain the ability of totipotency i.e. the ability of change to meristematic state and differentiate into a whole plant.
  3. 3. Plant research often involves growing new plants in a controlled environment. These may be plants that we have genetically altered in some way or may be plants of which we need many copies all exactly alike. Plant tissue culture, or the aseptic culture of cells, tissues, organs, and their components under defined physical and chemical conditions in vitro, is an important tool in both basic and applied studies as well as in commercial application. Tissue culture is the term used for “the process of growing cells artificially in the laboratory". Tissue culture involves both plant and animal cells. Tissue culture produces clones, in which all product cells have the same genotype.
  4. 4. It was the availability of these techniques that led to the application of tissue plant culture as follows .. Cell behavior Cytology Nutrition Metabolism Morphogenesis Embryogenesis Pathology plant modification and improvement pathogen-free plants and germplasm storage, clonal propagation, and product (mainly secondary metabolite) formation.
  5. 5. Cell cultures have remained an important tool in the study of basic areas of plant biology and biochemistry and have assumed major significance in studies in molecular biology and agricultural biotechnology. (Tissue culture had its origins at the beginning of the 20th century with the work of Gottleib Haberlandt (plants) and Alexis Carrel (animals)) Haberland clearly established the concept of totipotency. Haberland is justifiably recognized as the father of plant tissue culture. Haberland Carrel
  6. 6. STERILIZATION TECHNIQUES Sterilization Methods Used in Tissue Culture Laboratory The materials, e.g., vessels, instruments, medium, plant material, etc., used in culture work must be freed from microbes. This is achieved by one of the following approaches: (i) Dry heat treatment, (ii) Flame sterilization, (iii) Autoclaving, (iv) Filter sterilization, (v) Wiping with 70% ethanol (vi) Surface sterilization
  7. 7. Defined medium for the growth of cell cultures consists of following components. 1.Inorganic cells: concentration of potassium and nitrate at least 50-25 mM and phosphate ,sulphate 1-3mM and magnesium appear to be adaquate.Ammonium is essential 8Mm,and other micronutrients like iodide, boric acid,zinc,manganese,copper,cobalt,iron. 2.Vitamins:Thaimine ,pyridoxine,myo-inositol,nicotinic acid improves cell growth. 3.Carbon source:sucrose,2-4% 4.Growth regulators: Growth regulators induce cell division most commonly used regulator is NAA naphthalene acetic acid, and 2,4-dichlrophenoxy acetic acid(2,4-D), 5.Organic supplements: protein hydrolyzates,yeast,malt extract and coconut milk used for enhancement in growth rate. CULTURE MEDIUM
  8. 8. Chemicals are dissolved in distilled water. the stock solution of vitamins, micro-nutrients and growth regulators and hormones are added and pH adjusted to 5.5-6.5. The solution is made to volume 50 to 100 ml quantities distributed in to 250 ml Erlenmeyer flask. Flasks are supported with cotton plug and autoclaved at 120°C 15 min ,all media are stored at 10°C. Media preparation
  9. 9. Explant An excised piece of differentiated tissue or organ is regarded as an explant. The explant may be taken from any part of the plant body e.g., leaf, stem, root. Callus The unorganized and undifferentiated mass of plant cells is referred to as callus. Generally, when plant cells are cultured in a suitable medium, they divide to form callus i.e., a mass of parenchymatous cells. De-differentiation The phenomenon of mature cells reverting to meristematic state to produce callus is dedifferentiation. Re-differentiation The ability of the callus cells to differentiate into a plant organ or a whole plant is regarded as re-differentiation. Totipotency The ability of an individual cell to develop into a whole plant is referred to as totipotency. Terms Used in Tissue Culture
  10. 10. Explant Preparation and Surface Sterilization Surface sterilization treatments applied on the explants were:  Dipping in Ethyl alcohol 70% for 5 minutes.  Dipping in Chlorox 3% for 20 mins..  Dipping in undiluted Chlorox for 20 mins. .  Dipping in Ethyl alcohol 70% for 5 mins. then in Chlorox 3% (plus Tween 20) for 20 mins.  dipping in Ethyl alcohol 70% for 5 mins. then in Mercuric chloride 0.3% for 5 mins. After each treatment explants were rinsed three times in autoclaved distilled water.
  11. 11. Tissue culture involves the use of small pieces of plant tissue (explants) which are cultured in a nutrient medium under sterile conditions. Using the appropriate growing conditions for each explant type, plants can be induced to rapidly produce new shoots, and, with the addition of suitable hormones new roots. These plantlets can also be divided, usually at the shoot stage, to produce large numbers of new plantlets. The new plants can then be placed in soil and grown in the normal manner. Plant preparation plant material must first be surface sterilized to remove any bacteria or fungal spores that are present.aim is to kill all microorganisms, but at the same time not cause any adverse damage to the plant material.
  12. 12. Wash the prepared plant material in a detergent-water mixture for about 20 minutes. If trying hairy plant material scrub with a soft brush this will help remove fungi etc., and the detergent will help wet the material and remove air bubbles that may be trapped between tiny hairs on a plant. Transfer the washed plant material to the sterilizing chlorox solution. Shake the mixture for 1 minute and then leave to soak for 10-20 minutes. Carefully pour off the bleach solution using the lid to keep the plant tissue from coming out and then carefully cap the container. Using the appropriate growing conditions for each explant type, plants can be induced to rapidly produce new shoots, and, with the addition of suitable hormones.
  13. 13. 1.Place the plant material still in the chlorox bleach sterilizing container, the containers of sterile water, the sterilized forceps and blades, some sterile paper towel to use as a cutting surface and enough tubes containing sterile medium into the sterile aquarium. 2.The outside surfaces of the containers, the capped tubes and the aluminum wrapped supplies should be briefly sprayed with 70% alcohol before moving them into the chamber. 3.The gloves can be sprayed with a 70% alcohol solution and hands rubbed together to spread the alcohol just prior to placing hands into the chamber. TRANSFER OF PLANT MATERIAL TO TISSUE CULTURE MEDIUM
  14. 14. 4. Carefully open the container with the plant material and pour in enough sterile water to half fill the container. Replace the lid and gently shake the container to wash tissue pieces (explants) thoroughly for 2-3 minutes to remove the bleach. Pour off the water and repeat the washing process 3 more times. 5. Remove the sterilized plant material from the sterile water, place on the paper towel or sterile Petri dish. Cut the material into smaller pieces about 2 to 3 mm across. If using rose cut a piece of stem about 10 mm in length with an attached bud. Be sure to avoid any tissue that has been damaged by the bleach, which is apparent by its' pale color. 6.Take a prepared section of plant material in sterile forceps and place into the medium in the polycarbonate tube. 6.Replace the cap tightly on the tube.
  15. 15. Figure : The small explant develops callus which then produces shoots a few weeks after being placed into tissue culture media
  16. 16. Figure : Roots are fully developed prior to moving plants to pots of soil Once roots are well formed the plants are ready to be transferred into soil.
  17. 17.  Each plant should be carefully removed from its tube of media and planted into a small pot containing a clean light potting mix. Gently wash off all the agar medium prior to planting. The plants will still need to be protected at this stage. Place all of the pots onto a tray and cover lightly with a plastic dome or tent. Place the plants in an area with 12-16 hours of light (either natural or artificial) but not direct sunlight. After a week the cover can be gradually removed and the plants acclimated to stronger light and drier atmospheric conditions. Some of these tests could include looking at plant responses to low light levels, to drought or to saline soil conditions.
  18. 18. (A) Callus and suspension cultures. (B) Type of explant:  Single cell culture  Shoot and root cultures  Somatic embryo culture  Meristem culture  Anther culture and haploid production  Embryo culture  Ovule culture  Ovary culture. Methods of Plant Tissue CultureMethods of Plant Tissue CultureMethods of Plant Tissue CultureMethods of Plant Tissue Culture
  19. 19. Cell division in explant forms a callus. Callus is irregular unorganized and undifferentiated mass of actively dividing cells. The medium ordinarily contains the growth regulators Auxin, 2,4-D, (2, 4- Dichlorophenoxy acetic acid) and often a Cytokinin like BAP (Benzyl aminopurine). This stimulates cell divison in explant Callus is obtained within 2-3 weeks. A suspension culture consists of single cells and small groups of cells suspended in a liquid medium. Usually, the medium contains the auxin 2,4-D. Suspension cultures must be constantly agitated at 100-250 rpm (revolutions per minute). Suspension cultures grow much faster than callus culture. Callus and Suspension CulturesCallus and Suspension Cultures
  20. 20. 1. Bergmann’s Plating Technique: The cells are sus-pended in a liquid medium. Sterilized agar medium is kept malted in a water bath at 35°C. Equal volumes of the liquid and agar media are mixed and spread in 1 mm thick layer in a petridish. The cells remain embedded in the soft agar medium which is observable under a microscope. When large colonies develop they are isolated and cultured separately. There are two popular techniques for Single cell cultureSingle cell culture
  21. 21. 2. Filter Paper Raft Nurse Tissue Technique: Single cells are placed on small pieces (8×8 mm) of filter paper, which are placed on top of callus cultures several days in advance. This allows the filter papers to be wetted by the callus tissues. The single cells placed on the filter paper derive their nutrition from the callus. The cells divide and form macroscopic colonies on the filters. The colonies are isolated and cultured.
  22. 22. Shoot culture is promoted by a cytokinins and auxins like NAA (naphthalene acetic acid). The shoot and root cultures are generally controlled by auxin-cytokinin balance. Usually, an excess of auxin promotes root culture, whereas that of cytokinin promotes shoot culture. Roots culture from the lower end of these shoots to give complete plantlets. ShootShoot and Root Cultures
  23. 23. A somatic embryo develops from a somatic cell. The pattern of development of a somatic embryo is comparable to that of a zygotic embryo. Somatic embryo culture is induced by a high concentration of an auxin, such as 2,4-D. These embryos develop into mature embryos. Mature somatic embryos or embryoids germinate to give complete plantlets. Somatic Embryo CultureSomatic Embryo Culture
  24. 24. Meristem is a localized group of cells, which are actively dividing and undifferentiated but ultimately giving rise to permanent tissue. Cultivation of axillary or apical shoot meristems is called meristem culture. The apical or axillary meristems are generally free from virus. The explants commonly used in meristem culture are shoot tips and nodal segments. These explants are cultured on a medium containing a cytokinin. Meristem culture is carried out in Potato, Banana, Cardamom, Sugar-cane, Strawberry, Sweet Potato, etc. Applications: (i)Production of virus-free plants (ii) Germplasm conservation, (iii) Production of transgenic plants, (iv) Rapid clonal multiplication. Meristem CultureMeristem Culture
  25. 25. An individual/cell having the chromosome number found in the gametes of the species is called haploid. Thus haploid individuals arise from the gametes. A haploid has only one copy of each chromosome. The chromosome number of these haploid plants is doubled by using colchicine to obtain homozygous plants. In nature, haploid plants originate from unfertilized egg cells, but in laboratory, they can be produced from both male and female gametes. When anthers of some plants are cultured on a suitable medium to produce haploid plants, it is called anther culture. It is also useful for immediate expression of mutations and quick formation of purelines. Anther Culture and Haploid ProductionAnther Culture and Haploid Production
  26. 26. Culturing young embryos on a nutrient medium is called embryo culture. Young embryos are obtained from the developing seeds. The embryos complete their development on the medium and grow into seedlings. In general, older embryos are more easily cultured in vitro than young embryos. Embryo culture is useful as follows: (i) Embryo culture in orchids can be applied for rapid clonal propagation. (ii) In certain species, inhibitors present in the endosperm or seed coat make the seed dormant. Such embryos can escape dormancy by culturing on a suitable medium. (iv)Haploid wheat plants have also been successfully obtained through culture of hybrid embryos from wheat x maize crosses Embryo CultureEmbryo Culture
  27. 27. Ovule culture technique is utilized for raising hybrids which normally fail to develop due to the abortion of the embryos at an early stage. Ovules can easily be excised from the ovary and cultured on the basal medium. The loss of a hybrid embryo due to premature abscission of fruits may be prevented by ovule culture. In some cases, addition of fruit/vegetable juice increase the initial growth. Ovule CultureOvule Culture
  28. 28. Ovary culture technique has also been successfully employed to raise interspecific hybrids between sexually incompatible species. Ovaries are excised from the flowers and cultured at the zygote or two-celled proembryo stage for obtaining normal development on culture medium. Sometimes coconut milk when used as a supplement to the medium promote formation of fruits that are larger than those formed in vivo (within the living organism). In Anethum, addition of kinetin in the medium caused polyembryony which gave rise to multiple shoots. Ovary cultureOvary culture
  29. 29. 1.It helps in rapid multiplication of plants. 2. A large number of plantlets are obtained within a short period. 3. Plants are obtained throughout the year under controlled conditions, independent of seasons. 4.. It is an easy, safe and economical method for plant propagation. 5. In case of ornamentals, tissue culture plants give better growth, more flowers and less fall-out. 6. Genetically similar plants are formed by this method. 7. The rare plant and species are multiplied by this method and such plants are saved. APPLICATIONS OF PLANT TISSUE CULTURE
  30. 30. 8.Development of Transgenic Plants 9. Induction and Selection of Mutations 10. Resistance to Weedicides , development of resistance against pathogens Synthetic seeds  Prepared by encapsulating the somatic embryos obtained from tissue culture in a protective jelly capsule, which is usually prepared with sodium alginate.  From the synthetic seeds whole plant can be recovered under in vitro, green house and field conditions.
  31. 31. Taxol (plaxitaxol), a complex diterpene alkaloid found in the bark of the Taxus tree, is one of the most promising anticancer agents known due to its unique mode of action on the micro tubular cell system. Ketchum and Gibson (1996) reported that addition of carbohydrate during the growth cycle increased the production rate of paclitaxel, In order to increase the taxoid production in these cultures, the addition of different amino acids to the culture medium were studied, and phenylalanine was found to assist in maximum taxol production The influence of biotic and abiotic elicitors was also studied to improve the production and accumulation of taxol through tissue cultures The production of taxol from nodule cultures containing cohesive multicultural units displaying a high degree of differentiation has been achieved from cultured needles of seven Taxus cultivars PRODUCTION OF SECONDARY METABOLITES BY PLANT TISSUE CULTURE (PTC)
  32. 32. Morphine and Codeine Latex from the opium poppy, Papaver somniferum, is a commercial source of the analgesics, morphine and codeine. Callus and suspension cultures of P.somniferum are being investigated as an alternative means for production of these compounds. The root of Panax ginseng so-called ginseng, has been widely used as a tonic and highly prized medicine since ancient times. Ginseng has been recognized as a miraculous promoter of health and longevity. In recent years ginseng cell culture has been explored as a potentially more efficient method of producing ginsenosides. The effect of medium components like carbon, nitrogen and phosphate concentrations and plant growth hormones were thoroughly studied to increase the production of ginsenosides Concentration of plant growth regulators in the medium influences the cell growth and ginsenoside production in the suspension cultures
  33. 33. The productivity of berberine was increased in cell cultures by optimizing the nutrients in the growth medium and the levels of phytohormones Researchers found that carbon and nitrogen levels greatly influenced diosgenin accumulation in one cell line. Capsaicin, an alkaloid, is used mainly as a pungent food additive in formulated foods. It is obtained from fruits of green pepper (Capsicum spp.). Capsaicin is also used in pharmaceutical preparations as a digestive stimulant and for rheumatic disorders. Suspension cultures of Capsicum frutescens produce low levels of capsaicin, but immobilizing the cells in reticulated polyurethane foam can increase production approximately 100 fold further improvements in productivity can be brought about by supplying precursors such as isocapric acid.
  34. 34. The dimeric indole alkaloids vincristine and vinblastine have become valuable drugs in cancer chemotherapy due to their potent antitumor activity against various leukemias and solid tumors. These compounds are extracted commercially from large quantities of Catharanthus roseus. Since the intact plant contains low concentrations (0.0005%), plant cell cultures have been employed as an alternative to produce large amounts of these alkaloids.  Podophyllotoxin is an antitumor found in Podophyllum hexandrum. To increase the yield of podophyllotoxin used a complex of a precursor, coniferyl alcohol, and b-cyclodextrin to P. hexandrum cell suspension cultures. The addition coniferyl alcohol complex yielded 0.013% podophyllotoxin on a dry weight basis.
  35. 35. THANK YOU

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