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general procedure for ptc.pptx

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Sujata Koundal

The general procedure for plant tissue culture involves sterilizing glassware and tools, preparing and sterilizing explant tissue samples, producing callus growth from the explants on nutrient media, proliferating the callus through subculture, and establishing suspension cultures. Key steps include surface sterilizing explants using chemicals like sodium hypochlorite, transferring sterilized explants to growth media, incubating to produce initial callus, subculturing callus periodically to fresh media, and creating suspension cultures by transferring callus to liquid shaking media.

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General procedure for plant tissue culture
• Sterilization of glassware tools/vessels • Preparation and sterilization of explants • Production of callus from explants • Proliferation of cultured callus • Sub culturing of callus • Suspension culture
• Sterilization of glassware tools/vessels- - All the glassware should be of Pyrex or corning. All the glassware should be kept overnight dipped in sodium dichromate-sulphuric acid solution. - Then glassware should be washed with fresh running tap water, followed by distilled water and placed in inverted position in plastic bucket or trays to remove the extra water. - For drying the glassware, it is placed in hot air oven at high temperature about 120degree celsius for ½- 1 h.
• In the case of plastic lab ware, washing should be carried out with a mild nonabrasive detergent followed by washing under tap water • the plastic ware after general washing with dilute sodium bicarbonate and water followed by drainage of extra water, rinsed with an organic solvent such as alcohol, acetone and chloroform. • Washed and dried glassware or plastic ware should be stored in dust proof cupboards.
• To prevent reinfection following sterilization, empty containers are wrapped with aluminium foil. • Stainless steel, metal tools (knives, scalpels, forceps, etc) are also wrapped with aluminium foil placed in an aluminium or stainless steel box. The period of sterilization usually ranges between 1 and 4 hour.
2. Preparation of Explant- • Explants can be defined as a portion of plant body, which has been taken from the plant to establish a culture. • Explant can be obtained from plants, which are grown in controlled environmental conditions. Such plants will be usually free from pathogens and are homozygous in nature. • Explant may be taken from any part of the plant like root, stem, leaf or meristematic tissue like cambium, floral parts like anthers, stamens etc.
• Age of the explant is also an important factor in callus production. Young tissues are more suitable than mature tissues. • A suitable portion from the plant is removed with the help of sharp knife, and the dried and mature portion are separated from young tissue. • When seeds and grains are used for explants preparation, they are directly sterilized and put in nutrient medium. After germination, the obtained seedlings are to be used for explants preparation.
3. Surface sterilization of explant- • For the surface sterilization of the explant, chromic acid, mercuric chloride(0.11%), calcium hypochlorite(1-2%) and alcohol(70%) are used. • Usually the tissue is immersed in the solution of sterilizing agent for 10 s to 15 min, and they are washed with distilled water. Repeat the treatment with sodium hypochlorite for 20 min, and the tissue is finally washed with sterile water to remove sodium hypochlorite. Such tissue is used for inoculation
• The explants are sterilized by exposing to aqueous sterilized solution of different concentration. • Name of chemical Concentration% • Bromine water 1-2 • Benzalkonium chloride 0.01-0.1 • Sodium hypochlorite 0.5-51 • Calcium hypochlorite 9-10 • Mercuric chloride 1-2
Procedure to be followed for respective explants is as follows: 1. Seeds- • Dip the seeds into absolute ethyl alcohol for 10s and rinse with purified water. • Expose seeds for 20-30 min to 10% w/v aqueous calcium hypochlorite or for 5 min in a 1% solution of bromine water. • Wash the treated seeds with sterile water followed by germination on damp sterile filter paper.
2. Fruits- • Rinse the fruit with absolute alcohol. • Submerge into 2% (w/v) solution sodium hypochlorite for 10 min. • Washing repeated with sterile water and remove seeds of interior tissue. 3. Stem- • Clean the explants with running tap water followed by rinsing with pure alcohol. • Submerge into 2% (w/v) solution sodium hypochlorite for 15-30 min. • Wash three times with sterile water.
4. Leaves- • Clean the leaf explant with purified water to make it free from dirt and rub the surface with absolute ethyl alcohol. Dip the explants in 0.1%(w/v) mercuric chloride solution, wash with sterile water to make it free from chloride and then dry the surface with sterile tissue paper.
Production of callus from explants- • The sterilized explant is transferred aseptically onto defined medium contained in flasks. The flasks are transferred to BOD incubator for maintenance of culture. • The temperature is adjusted to 25±2 degree celsius. Some amount of light is necessary for callus (undifferentiated amorphous cell mass) production. Usually sufficient amount of callus is produced within 3-8 days of incubation.
Proliferation of callus- • If callus is well developed, it should be cut into small pieces and transferred to another fresh medium containing an altered composition of hormone, which supports growth. The medium used for production of more amount of callus is called proliferation medium.
Sub culturing of callus- • After sufficient growth of callus, it should be periodically transferred to fresh medium to maintain the viability of cells. This sub culturing will be done at an interval of 4-6 weeks.
Suspension culture- • Suspension culture contains a uniform suspension of separate cells in liquid medium. For the preparation of suspension culture, callus is transferred to liquid medium, which is agitated continuously to keep the cells separate. Agitation can be achieved by rotary shaker system attached within the incubator at a rate of 50-150 rpm. After the production of sufficient number of cells sub-culturing can be done.

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general procedure for ptc.pptx

  • 1. General procedure for plant tissue culture
  • 2. • Sterilization of glassware tools/vessels • Preparation and sterilization of explants • Production of callus from explants • Proliferation of cultured callus • Sub culturing of callus • Suspension culture
  • 3. • Sterilization of glassware tools/vessels- - All the glassware should be of Pyrex or corning. All the glassware should be kept overnight dipped in sodium dichromate-sulphuric acid solution. - Then glassware should be washed with fresh running tap water, followed by distilled water and placed in inverted position in plastic bucket or trays to remove the extra water. - For drying the glassware, it is placed in hot air oven at high temperature about 120degree celsius for ½- 1 h.
  • 4. • In the case of plastic lab ware, washing should be carried out with a mild nonabrasive detergent followed by washing under tap water • the plastic ware after general washing with dilute sodium bicarbonate and water followed by drainage of extra water, rinsed with an organic solvent such as alcohol, acetone and chloroform. • Washed and dried glassware or plastic ware should be stored in dust proof cupboards.
  • 5. • To prevent reinfection following sterilization, empty containers are wrapped with aluminium foil. • Stainless steel, metal tools (knives, scalpels, forceps, etc) are also wrapped with aluminium foil placed in an aluminium or stainless steel box. The period of sterilization usually ranges between 1 and 4 hour.
  • 6. 2. Preparation of Explant- • Explants can be defined as a portion of plant body, which has been taken from the plant to establish a culture. • Explant can be obtained from plants, which are grown in controlled environmental conditions. Such plants will be usually free from pathogens and are homozygous in nature. • Explant may be taken from any part of the plant like root, stem, leaf or meristematic tissue like cambium, floral parts like anthers, stamens etc.
  • 7. • Age of the explant is also an important factor in callus production. Young tissues are more suitable than mature tissues. • A suitable portion from the plant is removed with the help of sharp knife, and the dried and mature portion are separated from young tissue. • When seeds and grains are used for explants preparation, they are directly sterilized and put in nutrient medium. After germination, the obtained seedlings are to be used for explants preparation.
  • 8. 3. Surface sterilization of explant- • For the surface sterilization of the explant, chromic acid, mercuric chloride(0.11%), calcium hypochlorite(1-2%) and alcohol(70%) are used. • Usually the tissue is immersed in the solution of sterilizing agent for 10 s to 15 min, and they are washed with distilled water. Repeat the treatment with sodium hypochlorite for 20 min, and the tissue is finally washed with sterile water to remove sodium hypochlorite. Such tissue is used for inoculation
  • 9. • The explants are sterilized by exposing to aqueous sterilized solution of different concentration. • Name of chemical Concentration% • Bromine water 1-2 • Benzalkonium chloride 0.01-0.1 • Sodium hypochlorite 0.5-51 • Calcium hypochlorite 9-10 • Mercuric chloride 1-2
  • 10. Procedure to be followed for respective explants is as follows: 1. Seeds- • Dip the seeds into absolute ethyl alcohol for 10s and rinse with purified water. • Expose seeds for 20-30 min to 10% w/v aqueous calcium hypochlorite or for 5 min in a 1% solution of bromine water. • Wash the treated seeds with sterile water followed by germination on damp sterile filter paper.
  • 11. 2. Fruits- • Rinse the fruit with absolute alcohol. • Submerge into 2% (w/v) solution sodium hypochlorite for 10 min. • Washing repeated with sterile water and remove seeds of interior tissue. 3. Stem- • Clean the explants with running tap water followed by rinsing with pure alcohol. • Submerge into 2% (w/v) solution sodium hypochlorite for 15-30 min. • Wash three times with sterile water.
  • 12. 4. Leaves- • Clean the leaf explant with purified water to make it free from dirt and rub the surface with absolute ethyl alcohol. Dip the explants in 0.1%(w/v) mercuric chloride solution, wash with sterile water to make it free from chloride and then dry the surface with sterile tissue paper.
  • 13. Production of callus from explants- • The sterilized explant is transferred aseptically onto defined medium contained in flasks. The flasks are transferred to BOD incubator for maintenance of culture. • The temperature is adjusted to 25±2 degree celsius. Some amount of light is necessary for callus (undifferentiated amorphous cell mass) production. Usually sufficient amount of callus is produced within 3-8 days of incubation.
  • 14. Proliferation of callus- • If callus is well developed, it should be cut into small pieces and transferred to another fresh medium containing an altered composition of hormone, which supports growth. The medium used for production of more amount of callus is called proliferation medium.
  • 15. Sub culturing of callus- • After sufficient growth of callus, it should be periodically transferred to fresh medium to maintain the viability of cells. This sub culturing will be done at an interval of 4-6 weeks.
  • 16. Suspension culture- • Suspension culture contains a uniform suspension of separate cells in liquid medium. For the preparation of suspension culture, callus is transferred to liquid medium, which is agitated continuously to keep the cells separate. Agitation can be achieved by rotary shaker system attached within the incubator at a rate of 50-150 rpm. After the production of sufficient number of cells sub-culturing can be done.