Plant tissue culture

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Biology project on plant tissue culture of brahmi leaves, and a gist of the general calyx induction.

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Plant tissue culture

  1. 1. PLANT TISSUE CULTURE AND ITS APPLICATIONS BIOLOGY PROJECT SUBMITTED BY NAME: VAISHNOVI SEKAR CLASS: XII C ROLL NO:
  2. 2. INDEX # AIM # CERTIFICATE # ACKNOWLEDGEMENT # DECLARATION # OBJECTIVE # INTRODUCTION TO PLANT TISSUE CULTURE # APPLICATION OF PLANT TISSUE CULTURE # PLANT TISSSUE CULTURE FOR MASS PRODUCTION OF HIGH VALUE SECONDARY METABOLITIES # THEORY # EXERCISE 1: PREPARATION OF STOCK SOLUTION     MACRONUTRIENTS STOCK SOLUTIONS MICRONUTRIENTS STOCK SOLUTION VITAMIN STOCK SOLUTION IRON STOCK SOLUTION # EXERCISE 2: PREPARATION OF MURASHIGE AND SKOOG(MS) MEDIUM # EXERCISE 3: INOCULATION OF EXPLANTS NODAL SEGMENT # EXERCISE 4: PREPARATION OF MS MEDIUM SUPPLEMENTED WITH PLANT GROWTH HORMONES FOR REGENERATION OF EXPLANTS # RESULT AND OBSERVATION # BIBLIOGRAPHY
  3. 3. AIM: Micro propagation of Brahmi (BACOPA MONNIERI L.PENNELL) BY INDIRECT ORGANOGENESIS
  4. 4. CERTIFICATE This is to certify that this project is submitted by VAISHNOVI SEKAR to the biology department, CHINMAYA VIDYALAYA, NEW DELHI, was carried out by her under the guidance and supervision of Mrs Anubha Srivastav during academic session 20102011. Date: 03 Nov. 10 Mrs Anubha Srivastav (Biology teacher)
  5. 5. ACKNOWLEDGEMENT I wish to express my deep gratitude and sincere thanks to Mrs Anubha Srivastav for her encouragement and for all the facilities that she provided for this project work. I sincerely appreciate this magnanimity by taking me into her fold for which I shall remain indebted to her. I also thank our lab assistant, Mrs. Nikhila Nair. I take this opportunity to express my deep sense of gratitude for her invaluable guidance, constant encouragement, constructive comments, sympathetic attitude and immense motivation in all stages of this project work. VAISHNOVI SEKAR
  6. 6. DECLARATION I do hereby declare that this project work has been originally carried under the guidance and supervision of Mrs Anubha Srivastav Biology teacher Chinmaya Vidyalaya VAISHNOVI SEKAR
  7. 7. OBJECTIVE The purpose of the experiment is – to grow new plant segment with the help of invitro culturing of the plant nodal segment. I got interested to this project after learning about various technical advancements in the field of growth and metabolic engineering of plants. Micro propagation of plants of medical importance and multiplication of high yielding plants is done by use of plant tissue culture and they are adapted to local atmospheric conditions by biotechnological approaches.
  8. 8. Introduction: plant tissue culture Plant tissue culture or the aseptic culture of cells, tissues, organs and their components under defined physical and chemical conditions in vitro, has become am important tool in both basic and applied research and in commercial applications. It is an outcome of the genesis of an idea of the German scientist, Haberlandt, at the beginning of the 20th century who believed in the concept of Toti potency. It relies on maintaining plant cells in aseptic conditions on a suitable nutrient medium. The culture can be sustained as a mass of undifferentiated cells for an extended period of time, or regeneration into whole plants. Designing a strategy to culture cells from a plant for the first time can still seem like a matter of trial and error, and luck. However, the commercial production of valuable horticulture crops by micro propagation shows that it exists in the routine, as well as experimental world.
  9. 9. Applications: Plant tissue culture Plant tissue culture now has direct commercial applications as well as value in basic research into cell biology, genetics and biochemistry. The techniques include culture of cells, anthers, ovules and embryos on experimental to industrial scales, protoplast isolation and fusion, cell selection, meristem and bud culture. Application include  Micro propagation using meristem and shoot culture to produce large numbers of identical individuals  Large scale growth of plant cells in liquid culture as a source of secondary products  Crossing distantly related species by protoplast fusion and regeneration of the novel hybrid  Production of dihaploid plants through pollen or anther culture to achieve homozygous lines more rapidly for breeding programmes  As a tissue for transformation for regeneration of transgenic plants  Removal of viruses by propagation from meristematic tissues  Secondary metabolites production  Triploid through endosperm culture Tissue culture offers an additional or alternative way of propagation of selective genotypes. This technique is being commercially exploited for mass propagation of herbaceous, ornamental and fruit trees.
  10. 10. High value secondary metabolites Plant tissue culture forms the backbone for plant biotechnology. It encompasses micro propagation, induction of soma clones, somatic hybridization, cry preservation and regeneration of transgenic plants. Interest in medicinal plants as a re-emerging health aid has been fuelled by the rising costs of prescription drugs in the maintenance of personal health and well being, besides the reported side effects and inability to cure chronic diseases, coupled with bio-prospecting of new plant derived drugs. In order to have standardised formulations, the chemical constituents from plants and their parts are required to be uniform both qualitatively and quantitatively. Furthermore, an ever increasing demand of uniform medicinal plant-based medicine warrants their mass cloning through plant tissue culture strategy. A good number of medical plants have been reported to regenerate in vitro from their various parts.
  11. 11. Most secondary metabolites are important medicinal compounds. Plants cells culture provides an excellent system for large scale production of these compounds. There are a wide variety of secondary metabolites such as taxol, artemisinin, morphine, quinine, vinblastine, atropine, scopolamine and digoxin that can be produced from plants. This technique represents an optimal solution that has negated problems such as the slow growth cycle of the plant source. Plant cell culture allows for the synthesis of the bioactive substances present in plants, and is often the only available source, unhampered by quantitative limitations, of active ingredients which are poorly available or difficult to manufacture by chemical synthesis. Tissue culture is an alternative way for the production of photochemical of therapeutic importance. The technique like hairy root culture, biotransformation, immobilization and elicitations are used for the increased production of secondary metabolites. By this approach, therefore, an increased production of secondary metabolites can be obtained.
  12. 12. Theory: Plant tissue cultures can be initiated from almost any part, the explants, of a plant. The physiological state of the plant does have an influence on its response. The mother plant must be healthy and free from obvious sign of disease or decay. Younger tissue contains a higher proportion of actively dividing cells and is more responsive to culture conditions. The exact conditions required to initiate and sustain plant cells in culture, or to regenerate plantlets from cultured cells, are different for each plant species. Each variety of a species will often have a particular set of culture requirements. Despite all the knowledge that has been obtained about plant tissue culture during the 20 th century, there conditions have to be identified for each variety through experimentation. In tissue culture, propagation involves five important steps: Step I: involves the initiation of culture which should be infection free. The culture may be of shoot, root, nodal and inter-nodal segments etc. care should be taken to ensure the survival of explants after sterilization process. Step II: is the rapid multiplication of culture to produce plantlets regularly. In a number of species, this stage is easily carried out by the axillary shoot
  13. 13. initiation or adventitious organ formation which is preferred because somatic embryogenesis has been utilized to produce faster seedling production. In case of axillary multiplication, plants have the same genetic pool as the maternal source. The rate of multiplication is not high. Step III: prepare the plant propagules for transfer from in vitro or in vivo condition. It requires the induction of roots and hardening of plantlets. Both the processes start when they are maintained in cultural conditions and receive in vitro treatments. Step IV: transfer of tissue culture raised plantlets to the field to adapt to natural environmental condition. A number of methods have been applied which includes the use of mist or fog, carbon-dioxide enrichment, optimization of light and temperature, etc. the transferred plantlets grow slowly at initial stages and restore normal growth later. Step V: the selection of mother plants is very important for commercial success of tissue culture propagation. It may involve the process where the mother plants are grown under strictly controlled conditions-25°C temperature with humidity of 75.
  14. 14. EXERCISE 1: STOCK SOLUTION AIM: PREPARATION OF STOCK SOLUTION PROCEDURE: PREPARE SEPARATELY THE STOCK SOLUTION OF MACRONUTRIENTS, MICRONUTRIENTS, VITAMIN AND IRON BY DISSOLVING ALL THE CONSTITUENTS INDIVIDUALLY IN CONICAL FLASK OVER A MAGNETIC STIRRER. THE COMPOSITION OF EACH STOCK IS GIVEN BELOW:- Nutrients Major nutrients concentration NH4NO3 CaCL2 MgSO4 KNO3 KH2PO4 Minor nutrients 16.5g in 100ml 4.4g in 100ml 3.7g in 100ml 19.0g in 100ml 1.7g in 100ml H3BO4 MnSO4 ZnSO4 CuSO4 CoCl2 KI Na2MoO4 0.124g in 100ml 0.312g in 100ml 0.172g in 100ml 0.025g in 100ml 0.25g 0.83g in 100ml 0.25g Iron solution FeSo4 0.895g Na2Edta Vitamin 0.932g Glycine Nicotinic acid Pyridoxine HCl Thiamine HCl 0.1g 0.025g 0.025g 0.01g in 250ml in 100ml Precautions:  Separately dissolve all salts to prevent any precipitation  Use lukewarm water for easy dissolution
  15. 15.
  16. 16. Exercise 2: MS medium Aim: Preparation of Murashige and Skoog medium Procedure: 1. Required amount of major, minor, iron and vitamin stocks for preparation of 1L of MS medium Major stock Minor stock Iron stock Vitamin 2. 100ml 50ml 10ml 2ml Take 1L beaker Dissolve 3% sucrose in 300ml double distil water Add required amount of major, minor, iron and vitamin stocks Make up the volume to 1L Adjust pH to 5.70 (With the help of 1N HCL/1N NaOH) Supplement medium with 0.63% agar Boil the medium till agar gets dissolved
  17. 17. Pour the medium in culture vessels (Test tubes/ flasks) Plug the vessels, cover them with aluminium foil Sterilize the medium by autoclaving at 121°C for 15 minutes After autoclaving, the medium is kept inside the culture room. Precautions:  the vessels should be sterilized in order to prevent the growth of micro organism like fungus, etc.
  18. 18. Exercise 3: Inoculation Aim: inoculation of explant, nodal segment Procedure: 1. sterilization of explant The nodal segments have to be treated before inoculation a. under tap water for 15 mins b. Tween 20 (2ml/100ml) for 20mins c. Fungicide (Bavistin) (0.01g/L) for 45min d. HgCl2(0.1%) for 10min 2. Preparation of laminar air flow chamber(LAFC) Wipe the laminar air flow chamber with 70% alcohol. Keep: a. Spirit lamp (filled with spirit) b. 1 flask containing absolute alcohol filled up to neck c. Another flask containing 50ml (70% alcohol) d. Match box e. Sterile blades f. Sterile water g. Vessels containing medium h. Sterile instrument pair i. Sterile pertiplate In the laminar air flow chamber Switch on UV light in the LAFC for 15 minutes
  19. 19. Switch on Air flow after 15 minutes Can start inoculation 3. Inoculation of explants Wipe the hands with 70% alcohol Fix the blade in the scalpel, flame the forceps and scalpel, After flaming, keep them back in absolute alcohol With the help of forceps, drop the explants into sterile water containing flask Flame the sterile Petri plate with 70% alcohol Wait for 1-2 minutes so that the surface of pertiplate gets cooled Take out the explants (nodal segment) from the sterile water Cut from the top and bottom so that the sterilant at the two ends doesn’t affect the result Care should be taken to maintain polarity Hence cut the lower end can be cut in slanting way so that it can be demarcated
  20. 20. Open the vessel containing medium in front of the flame Pick the nodal segments and insert the lower cut end into the medium Close the vessel by putting back the plug in front of the flame Keep the culture in the culture room Take observation periodically Culture room conditions Temperature: 25±2°C Humidity: 55±5% Photoperiod (light/dark):16hr/8hr
  21. 21. Exercise 4: MS medium supplemented with plant growth hormones 1. Preparation of stock solution Weight 50mg BAP and put it in sterile flask Dissolve BAP in 1N HCL (2ml) Make up the volume up to 50ml with the help of double distilled water Store the stock in transparent glass bottle and label the content (1mg/ml) Keep it in refrigerator at 20°C 2. PREPARATION OF MEDIA (500ml)  2.5mg/l Take 2L beaker Dissolve 15g sucrose in 500ml double distilled water (3% sucrose is used) Add required amount of major, minor, iron and vitamin stocks
  22. 22. Make up the volume to 500ml Supplement medium with 0.63% agar Pour the medium in culture vessels, label them A-F Plug the vessels, cover them with Aluminium foil Sterilize the medium by autoclaving at 121°C for 15 minutes After autoclaving, the medium is kept inside the culture room Inoculate the explants on above medium after 48 hours of media autoclaving Study regeneration process periodically
  23. 23. RESULT AND OBSERVATIONS THE ORGANOGENESIS WAS OBSERVED AS SHOWN IN PICTURES
  24. 24. Conclusion: Micro-propagation is the development of new plants in an artificial medium under aseptic conditions. This method is employed for rapid multiplication of elite plant species. Discussion: The most frequently used micro propagation method for commercial production utilizes enhanced axillary shoot proliferation from cultured meristem. Micro propagation process supports in achieving mass production of healthy plants in low or minimum space requirement. On the other hand, micro propagation process comprises of high labour costs, danger of variation and loss by contamination. Multiplication rates are tend to be slow at first, but later, if the cultural conditions are satisfactory (temp: 25 ± 2 °; humidity 55± 5%; photoperiod – light/dark: 16 hrs/8 hrs), a rapid multiplication can be achieved. Generally, the plant taken for micro propagation are the ones which are high in demand in terms of number, are in demand round the year and have to be multiplied irrespective of season, are amongst the endangered ones, have to be cloned, or have to be preserved for their special qualities.
  25. 25. BIBLOGRAPHY  Wikipedia – the free encyclopedia  Website: www.icbse.com  Biology manual

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