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Plant Tissue Culture

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Plant tissue culture involves growing plant cells, tissues or organs in sterile conditions on nutrient media. It allows for the rapid propagation of plants through micropropagation. Some key applications of plant tissue culture include large scale multiplication of plants, genetic transformation, production of hybrids and haploids, conservation of plant germplasm, and synthesis of secondary metabolites.

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PLANT TISSUE CULTURE Presented By: Sushant Mangesh Gawali (A701118421007) 1st Sem M.Tech Food Biotech.

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CONTENTS • Plant Tissue Culture • General technique of Plant Tissue Culture • Culture medium • Types of Plant Tissue Culture • Micropropagation • Advantages and disadvantages • Applications of Plant Tissue Culture 2

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PLANT TISSUE CULTURE • Tissue culture is in-vitro cultivation of plant cell or tissue under aseptic and controlled environment conditions, in liquid or on semisolid well-defined nutrient medium for the production of primary and secondary metabolite or to regenerate plant. • Plant tissue culture relies on the fact that many plant cells have the ability to regenerate a whole plant (totipotency). • Gottlieb Haberlandt, a German botanist, in 1902 cultured fully differentiated plant cells isolated from different plants. This was the very first step for the beginning of plant cell and tissue culture. 3

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Cont… • The mostly used MS media was invented early by T. Murashige and F. Skoog in 1962 during the invention of plant growth regulator. • In India, Shri S. C. Maheshwari and Sipra Guha made a remarkable contribution in the development of anther culture in 1970, that opened the new era of androgenesis. • The main requirements of plant tissue culture are: (1) Laboratory Organization (2) Culture Media (3) Aseptic Conditions 4

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Fig. 1: General Technique of Plant Tissue Culture 5

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Selection and Sterilization of Explant Suitable explant is selected and is then excised from the donor plant. Explant is then sterilized using disinfectants. Preparation and Sterilization of Culture Medium A suitable culture medium is prepared with special attention towards the objectives of culture and type of explant to be cultured. Prepared culture medium is transferred into sterilized vessels and then sterilized in autoclave. 6 Detail steps involved in PTC

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Inoculation Sterilized explant is inoculated (transferred) on the culture medium under aseptic conditions. Incubation Cultures are then incubated in the culture room where appropriate conditions of light, temperature and humidity are provided for successful culturing. 7

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Sub culturing Cultured cells are transferred to a fresh nutrient medium to obtain the plantlets. Transfer of Plantlets After the hardening process (i.e. acclimatization of plantlet to the environment), the plantlets are transferred to green house or in pots. 8

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9 Fig. 2: Flow chart of plant tissue culture technique

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A Laminar air flow i.e. LAF hood/cabinet is an enclosed workstation that is used to create a contamination - free work environment through filters to capture all the particles entering the cabinet. Laminar air flow or LAF Fig. 3: LAF cabinet 10

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11 The formulation or the medium on which the explant is cultured is called culture medium. It is composed of various nutrients required for proper culturing. A number of media have been devised for specific tissues and organs. Some important of them are: 1. MS (Murashige and Skoog) Medium 2. LS (Linsmaier and Skoog) Medium 3. B5 (Gamborg’s) Medium 4. White’s Medium, etc. CULTURE MEDIUM

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Important constituents of a culture medium are: (i) Organic supplements: • Vitamins like thiamine (B1), Pyridoxin (B6), Nicotinic Acid (B3), etc. • Antibiotics like Streptomycin, Kanamycin, etc. • Amino Acids like Arginine, Asparagine, etc. (ii) Inorganic Nutrients: • Micronutrients as Iron (Fe), Manganese (Mn), Zinc (Zn), Molybdenum (Mo), Copper (Cu), Boron (B). • Macronutrients include six major elements as Nitrogen (N), Phosphorus (P), Potassium (K), Sulphur (S), Calcium (Ca), Magnesium (Mg). (iii) Carbon and Energy Source: • Most preferred carbon source is Sucrose. • Others include lactose, maltose, galactose, raffinose, cellobiose, etc. 12

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(iv) Growth Hormones: • Auxins - mainly for inducing cell division. • Cytokinins - mainly for modifying apical dominance and shoot differentiation. • Abscisic Acid (ABA) • Gibberellic acid (GA) (v) Gelling Agents: • Added to media to make them semisolid or solid. • Agar, Gelatin, Alginate etc. (vi) Other Organic Extracts: • Sometimes culture media are supplemented with some organic extracts also like coconut milk, orange juice, tomato juice, potato extract, etc. 13

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The culture room means the room for keeping or incubating the culture under controlled temperature, light and humidity. The relative humidity of the culture room is maintained above 50%. The optimum temperature in growth room is about 20°C to 30°C and the light intensity required is about 1000-2000 lux. Fig. 4: Culture growth room 14

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15 Cell or suspension culture Shoot culture Root culture Protoplast culture Embryo culture Endosperm Culture (Triploid Production) Anther and pollen culture (Haploid Production) TYPES OF PLANT TISSUE CULTURE

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Cell Culture: • Cell culture is actually, the process of producing clones of a single cell. First attempts for cell culture were made by Haberlandt in 1902. • It is important that the cell cultures require a suitably enriched nutrient medium and it should be done in dark because light may deteriorate the cell culture. Suspension Culture: • A culture which consists of cells or cell aggregates initiated by placing callus tissues in an agitated liquid medium is called as a suspension culture. • The general technique of suspension culture involves basically two types of cultures: batch culture and continuous cultures. 16 Fig. 5: Cell culture Fig. 6: Suspension culture

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17 Shoot Culture: • The practical application of this method was proposed by Morel and Martin (1952) after they successfully recovered the complete Dahalia plant from shoot-tips cultures. • In this technique, the shoot apical meristem is cultured on a suitable nutrient medium. This is also referred to as Meristem Culture. Fig. 7: Shoot culture Root Culture: • Pioneering attempts for root culture were made by Robbins and Kotte during 1920s. Root culturing of a number of plant species of angiosperms as well as gymnosperms has been done successfully. • Root cultures are usually not helpful for giving rise to complete plants but they have importance’s of their own. Fig. 8: Root culture

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18 Protoplast Culture: • A protoplast is described as a plasma membrane bound vesicle which consists of a naked cell formed as a result of removal of cell wall. • At present, there are available a number of enzymes which have enabled the isolation of protoplasts from almost every plant tissue. • Production of hybrid plants through the fusion of protoplast of two different plant species or varieties is called somatic hybridization and the hybrid plant obtained is known as somatic hybrid • It not only serves for genetic manipulations in plants but also for biochemical and metabolic studies in plants. • In-vitro culturing of protoplasts has immense applications in the field of plant biotechnology. Fig. 9: Protoplast culture

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Endosperm Culture (Triploid Production): • Endosperm tissue is triploid therefore the plantlets originating by the culture of endosperm are also triploid. Endosperm is formed after the double fertilization of one male nucleus with two polar nuclei. • The triploid plants are usually seedless therefore this technique is most beneficial for increasing the commercial value of fruits like apple, mango, grapes, watermelon, etc. • Endosperm culture is helpful for studying biosynthesis and metabolism of certain natural products also. Embryo Culture: • The technique of embryo culture involves the isolation and growth of an embryo under in-vitro conditions to obtain a complete viable plant. • First success for embryo culture was made by Hannig in 1904. • Embryo culture is advantageous for in-vitro micro propagation of plants, overcoming seed dormancy and for production of beneficial haploid plants. Fig. 10: Embryo culture

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20 Anther and Pollen Culture (Haploid Production): • Haploid plants are those which contain half the number of chromosomes (denoted by n). • There are two approaches for in-vitro haploid production and these are: (a) Androgenesis: The technique of production of haploids through anther or microspore culture is termed as androgenesis. It is achieved either by another culture or by microspore (pollen) culture. Androgenesis is preferred over gynogenesis. (b) Gynogenesis: It is an alternative source of in-vitro haploid production. It refers to the production of haploid plant from ovary culture or ovule culture. Fig. 11: Anther culture

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21 • Tissue culture helps in the rapid propagation of plants by the technique of micro- propagation or clonal propagation in-vitro. The asexually produced progeny of a cell or individual is called as clone and the clones have an identical genotype. • Micropropagation is the technique of in-vitro production of the clones of plants i.e., it produces the progeny plants which have an identical genotype as their parents, by cell, tissue or organ culture. • It is nothing but the use of plant tissue culture at large scale. It serves as an alternate method to conventional vegetative propagation methods. • Micro propagation may be achieved by shoot tips, axillary buds, adventitious buds, bulbs or somatic embryos. MICRO-PROPAGATION

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The stages in general Micro-propagation technique Stage I - Initiation This stage also involves the preparatory process for achieving better establishment of aseptic cultures of explant. Suitable explant is selected from the mother plant. Then, the explant is sterilized and transferred to the nutrient medium for culture. State II - Multiplication This is the most important stage of micro propagation. In this stage, there occurs the proliferation or multiplication of shoots (or embryoids) from the explant on medium. It occurs either by the formation of an intermediary callus or by induction of adventitious buds directly from the explant. Stage III - Sub-culturing The shoots are transferred to rooting medium (sub-cultured) to form roots. As a result, complete plantlets are obtained. 22

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Stage IV - Transplantation In this stage, the regenerated plantlets are transferred out of culture. These are grown in pots followed by field trials. 23 Fig 12: Micropropagation in Banana

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• The plantlets are obtained in a very short time with a small amount of plant tissue. • The new plants produced are disease-free. • The plants can be grown throughout the year, irrespective of the season. • A large space is not required to grow plants by tissue culture technique. • The production of new varieties in the market place speeds up. • This technique is being used for the production of ornamental plants such as dahlia, chrysanthemum, orchids, etc. ADVANTAGES 24

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• Complex and time consuming • Dependent to plant genotype • Require efficient in-vitro plant regeneration protocol • Requires complete sterile conditions throughout the culturing • Labor intensive • High risk of contamination, etc. DISADVANTAGES 25

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26 1. Large scale and fast multiplication 2. Clonal propagation 3. Germplasm conservation in form of cryopreservation 4. Genetic transformation 5. Synthetic seeds 6. Somatic hybrids and cybrids 7. Early flowering 8. Seedless fruits and vegetables 9. Breaking dormancy and overcoming male sterility 10. Secondary metabolite production, etc. APPLICATIONS OF PLANT TISSUE CULTURE

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Plant Tissue Culture

More Related Content

Plant Tissue Culture

  • 1. PLANT TISSUE CULTURE Presented By: Sushant Mangesh Gawali (A701118421007) 1st Sem M.Tech Food Biotech.
  • 2. CONTENTS • Plant Tissue Culture • General technique of Plant Tissue Culture • Culture medium • Types of Plant Tissue Culture • Micropropagation • Advantages and disadvantages • Applications of Plant Tissue Culture 2
  • 3. PLANT TISSUE CULTURE • Tissue culture is in-vitro cultivation of plant cell or tissue under aseptic and controlled environment conditions, in liquid or on semisolid well-defined nutrient medium for the production of primary and secondary metabolite or to regenerate plant. • Plant tissue culture relies on the fact that many plant cells have the ability to regenerate a whole plant (totipotency). • Gottlieb Haberlandt, a German botanist, in 1902 cultured fully differentiated plant cells isolated from different plants. This was the very first step for the beginning of plant cell and tissue culture. 3
  • 4. Cont… • The mostly used MS media was invented early by T. Murashige and F. Skoog in 1962 during the invention of plant growth regulator. • In India, Shri S. C. Maheshwari and Sipra Guha made a remarkable contribution in the development of anther culture in 1970, that opened the new era of androgenesis. • The main requirements of plant tissue culture are: (1) Laboratory Organization (2) Culture Media (3) Aseptic Conditions 4
  • 5. Fig. 1: General Technique of Plant Tissue Culture 5
  • 6. Selection and Sterilization of Explant Suitable explant is selected and is then excised from the donor plant. Explant is then sterilized using disinfectants. Preparation and Sterilization of Culture Medium A suitable culture medium is prepared with special attention towards the objectives of culture and type of explant to be cultured. Prepared culture medium is transferred into sterilized vessels and then sterilized in autoclave. 6 Detail steps involved in PTC
  • 7. Inoculation Sterilized explant is inoculated (transferred) on the culture medium under aseptic conditions. Incubation Cultures are then incubated in the culture room where appropriate conditions of light, temperature and humidity are provided for successful culturing. 7
  • 8. Sub culturing Cultured cells are transferred to a fresh nutrient medium to obtain the plantlets. Transfer of Plantlets After the hardening process (i.e. acclimatization of plantlet to the environment), the plantlets are transferred to green house or in pots. 8
  • 9. 9 Fig. 2: Flow chart of plant tissue culture technique
  • 10. A Laminar air flow i.e. LAF hood/cabinet is an enclosed workstation that is used to create a contamination - free work environment through filters to capture all the particles entering the cabinet. Laminar air flow or LAF Fig. 3: LAF cabinet 10
  • 11. 11 The formulation or the medium on which the explant is cultured is called culture medium. It is composed of various nutrients required for proper culturing. A number of media have been devised for specific tissues and organs. Some important of them are: 1. MS (Murashige and Skoog) Medium 2. LS (Linsmaier and Skoog) Medium 3. B5 (Gamborg’s) Medium 4. White’s Medium, etc. CULTURE MEDIUM
  • 12. Important constituents of a culture medium are: (i) Organic supplements: • Vitamins like thiamine (B1), Pyridoxin (B6), Nicotinic Acid (B3), etc. • Antibiotics like Streptomycin, Kanamycin, etc. • Amino Acids like Arginine, Asparagine, etc. (ii) Inorganic Nutrients: • Micronutrients as Iron (Fe), Manganese (Mn), Zinc (Zn), Molybdenum (Mo), Copper (Cu), Boron (B). • Macronutrients include six major elements as Nitrogen (N), Phosphorus (P), Potassium (K), Sulphur (S), Calcium (Ca), Magnesium (Mg). (iii) Carbon and Energy Source: • Most preferred carbon source is Sucrose. • Others include lactose, maltose, galactose, raffinose, cellobiose, etc. 12
  • 13. (iv) Growth Hormones: • Auxins - mainly for inducing cell division. • Cytokinins - mainly for modifying apical dominance and shoot differentiation. • Abscisic Acid (ABA) • Gibberellic acid (GA) (v) Gelling Agents: • Added to media to make them semisolid or solid. • Agar, Gelatin, Alginate etc. (vi) Other Organic Extracts: • Sometimes culture media are supplemented with some organic extracts also like coconut milk, orange juice, tomato juice, potato extract, etc. 13
  • 14. The culture room means the room for keeping or incubating the culture under controlled temperature, light and humidity. The relative humidity of the culture room is maintained above 50%. The optimum temperature in growth room is about 20°C to 30°C and the light intensity required is about 1000-2000 lux. Fig. 4: Culture growth room 14
  • 15. 15 Cell or suspension culture Shoot culture Root culture Protoplast culture Embryo culture Endosperm Culture (Triploid Production) Anther and pollen culture (Haploid Production) TYPES OF PLANT TISSUE CULTURE
  • 16. Cell Culture: • Cell culture is actually, the process of producing clones of a single cell. First attempts for cell culture were made by Haberlandt in 1902. • It is important that the cell cultures require a suitably enriched nutrient medium and it should be done in dark because light may deteriorate the cell culture. Suspension Culture: • A culture which consists of cells or cell aggregates initiated by placing callus tissues in an agitated liquid medium is called as a suspension culture. • The general technique of suspension culture involves basically two types of cultures: batch culture and continuous cultures. 16 Fig. 5: Cell culture Fig. 6: Suspension culture
  • 17. 17 Shoot Culture: • The practical application of this method was proposed by Morel and Martin (1952) after they successfully recovered the complete Dahalia plant from shoot-tips cultures. • In this technique, the shoot apical meristem is cultured on a suitable nutrient medium. This is also referred to as Meristem Culture. Fig. 7: Shoot culture Root Culture: • Pioneering attempts for root culture were made by Robbins and Kotte during 1920s. Root culturing of a number of plant species of angiosperms as well as gymnosperms has been done successfully. • Root cultures are usually not helpful for giving rise to complete plants but they have importance’s of their own. Fig. 8: Root culture
  • 18. 18 Protoplast Culture: • A protoplast is described as a plasma membrane bound vesicle which consists of a naked cell formed as a result of removal of cell wall. • At present, there are available a number of enzymes which have enabled the isolation of protoplasts from almost every plant tissue. • Production of hybrid plants through the fusion of protoplast of two different plant species or varieties is called somatic hybridization and the hybrid plant obtained is known as somatic hybrid • It not only serves for genetic manipulations in plants but also for biochemical and metabolic studies in plants. • In-vitro culturing of protoplasts has immense applications in the field of plant biotechnology. Fig. 9: Protoplast culture
  • 19. Endosperm Culture (Triploid Production): • Endosperm tissue is triploid therefore the plantlets originating by the culture of endosperm are also triploid. Endosperm is formed after the double fertilization of one male nucleus with two polar nuclei. • The triploid plants are usually seedless therefore this technique is most beneficial for increasing the commercial value of fruits like apple, mango, grapes, watermelon, etc. • Endosperm culture is helpful for studying biosynthesis and metabolism of certain natural products also. Embryo Culture: • The technique of embryo culture involves the isolation and growth of an embryo under in-vitro conditions to obtain a complete viable plant. • First success for embryo culture was made by Hannig in 1904. • Embryo culture is advantageous for in-vitro micro propagation of plants, overcoming seed dormancy and for production of beneficial haploid plants. Fig. 10: Embryo culture
  • 20. 20 Anther and Pollen Culture (Haploid Production): • Haploid plants are those which contain half the number of chromosomes (denoted by n). • There are two approaches for in-vitro haploid production and these are: (a) Androgenesis: The technique of production of haploids through anther or microspore culture is termed as androgenesis. It is achieved either by another culture or by microspore (pollen) culture. Androgenesis is preferred over gynogenesis. (b) Gynogenesis: It is an alternative source of in-vitro haploid production. It refers to the production of haploid plant from ovary culture or ovule culture. Fig. 11: Anther culture
  • 21. 21 • Tissue culture helps in the rapid propagation of plants by the technique of micro- propagation or clonal propagation in-vitro. The asexually produced progeny of a cell or individual is called as clone and the clones have an identical genotype. • Micropropagation is the technique of in-vitro production of the clones of plants i.e., it produces the progeny plants which have an identical genotype as their parents, by cell, tissue or organ culture. • It is nothing but the use of plant tissue culture at large scale. It serves as an alternate method to conventional vegetative propagation methods. • Micro propagation may be achieved by shoot tips, axillary buds, adventitious buds, bulbs or somatic embryos. MICRO-PROPAGATION
  • 22. The stages in general Micro-propagation technique Stage I - Initiation This stage also involves the preparatory process for achieving better establishment of aseptic cultures of explant. Suitable explant is selected from the mother plant. Then, the explant is sterilized and transferred to the nutrient medium for culture. State II - Multiplication This is the most important stage of micro propagation. In this stage, there occurs the proliferation or multiplication of shoots (or embryoids) from the explant on medium. It occurs either by the formation of an intermediary callus or by induction of adventitious buds directly from the explant. Stage III - Sub-culturing The shoots are transferred to rooting medium (sub-cultured) to form roots. As a result, complete plantlets are obtained. 22
  • 23. Stage IV - Transplantation In this stage, the regenerated plantlets are transferred out of culture. These are grown in pots followed by field trials. 23 Fig 12: Micropropagation in Banana
  • 24. • The plantlets are obtained in a very short time with a small amount of plant tissue. • The new plants produced are disease-free. • The plants can be grown throughout the year, irrespective of the season. • A large space is not required to grow plants by tissue culture technique. • The production of new varieties in the market place speeds up. • This technique is being used for the production of ornamental plants such as dahlia, chrysanthemum, orchids, etc. ADVANTAGES 24
  • 25. • Complex and time consuming • Dependent to plant genotype • Require efficient in-vitro plant regeneration protocol • Requires complete sterile conditions throughout the culturing • Labor intensive • High risk of contamination, etc. DISADVANTAGES 25
  • 26. 26 1. Large scale and fast multiplication 2. Clonal propagation 3. Germplasm conservation in form of cryopreservation 4. Genetic transformation 5. Synthetic seeds 6. Somatic hybrids and cybrids 7. Early flowering 8. Seedless fruits and vegetables 9. Breaking dormancy and overcoming male sterility 10. Secondary metabolite production, etc. APPLICATIONS OF PLANT TISSUE CULTURE