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  1. 1. PROJECT REPORT ON Micro propagation Protocol Development of Bacopa monnieri( Linn ) <ul><li>Submitted in partial fulfillment of requirement s for the </li></ul><ul><li>DEGREE OF </li></ul><ul><li>MASTER OF SCIENCE IN BIOTECHNOLOGY </li></ul><ul><li>BY: </li></ul><ul><li>SHUBHAM RASTOGI ; </li></ul><ul><li>M.Sc.BIOTECHNOLOGY(4 TH Semester ) </li></ul><ul><li>Under the Supervision of : </li></ul><ul><li>Dr. A. K. Johri Dr. Seema Saxena </li></ul><ul><li>Head of Agrobiotechnology HOD Biotechnology </li></ul><ul><li>Dabur Research Fo undation G.I.C.T.s. College </li></ul><ul><li>Sahibabad U.P. Jiwaji university </li></ul><ul><li>Gwalior M.P . </li></ul>
  2. 2. Contents: <ul><li>1. INTRODUCTION </li></ul><ul><li>2. TECHNIQUE </li></ul><ul><li>3. REVIEW OF LITERATURE </li></ul><ul><li>4. MATERIALS AND METHODS </li></ul><ul><li>5. SCOPE AND OBJECTIVES </li></ul><ul><li>6. APPLICATIONS OF BRAHMI </li></ul><ul><li>7. RESULTS AND DISCUSSION </li></ul><ul><li>8. CONCLUSION </li></ul><ul><li>9. BIBLIOGRAPHY </li></ul>
  3. 3. INTRODUCTION <ul><li>AYURVEDA= Ayu+ Veda </li></ul><ul><li>Brahmi having “ Madhya Rasayan” properties that is effective herbs to enhance memory and learning process </li></ul><ul><li>Main contents are: Herpestine,Brahmine,Bacoside-A,Bacoside-B From leaves other contents are Betulic acid ,mannitol,stigmasterol and b-Sitostirol. </li></ul>
  4. 4. History of Plant Tissue Culture <ul><li>But in a strict science, tissue culture denotes the invitro cultivation of plant cells in an unorganized mass. </li></ul><ul><li>Tissue culture technique can be divided into two broad categories. </li></ul><ul><li>1. Culture of unorganized tissue. </li></ul><ul><li>A) Callus culture </li></ul><ul><li>B) Cell culture </li></ul><ul><li>C) Protoplast culture </li></ul><ul><li>D) Anther culture </li></ul>
  6. 6. <ul><ul><ul><li>2. Culture of organized structure </li></ul></ul></ul><ul><ul><ul><li>a) Meristem and shoot tip culture </li></ul></ul></ul><ul><ul><ul><li>b) Embryo culture </li></ul></ul></ul><ul><ul><ul><li>c) Isolated root culture </li></ul></ul></ul>
  7. 7. Meristem Culture
  8. 8. TECHNIQUE <ul><li>Plant tissue culture is a technique in which microbe-free plant tissue is maintained in an aseptic (sterile) environment, such as sterilized nutrient medium in a test tube. </li></ul><ul><li>Totipotency : the concept underlying tissue culture </li></ul><ul><li>It is possible to regenerate new plants from small pieces of plant tissue because each cell of a given plant has the same genetic makeup. This is totipotent, that i.e. capable to develop along a &quot;programmed&quot; pathway leading to the formation of an entire plant that is genetically identical to the plant from which it was derived (a clone). </li></ul>
  9. 9. BENEFITS OF TISSUE CULTURE <ul><li>Faster growth rates </li></ul><ul><li>multiplied easily and economically </li></ul><ul><li>Conservation of germplasm of rare species can be achieved by this technique. </li></ul><ul><li>large number of genetically identical clones </li></ul><ul><li>Tissue culture techniques allow virus eradication, genetic manipulation, somatic hybridization and other procedures that contribute to plant improvement and benefit propagation . </li></ul>
  10. 10. Micro propagation <ul><ul><li>Micro propagation is a specialized technique of propagation in which very small pieces of plant tissue are regenerated in an artificial medium under sterile conditions. Usually it is embraces the development of shoot and axillary buds etc. The shoot tip/axillary buds is the most commonly cultured plant part and its culture and regeneration is therefore the main subject. </li></ul></ul><ul><li>Pioneer workers in micropropagation were F.Skoog and T.Murashige, with the latter continuing the work at the University of California at Riverside. </li></ul>
  11. 11. <ul><li>Different stages of micropropagation </li></ul><ul><li>Stage 0(Pre-Initiation of Culture) </li></ul><ul><li>Stage-I (Initiation of Culture) </li></ul><ul><li>Stage-II (Multiplication) </li></ul><ul><li>Stage-III (Rooting of. Shoots) </li></ul><ul><li>Stage-IV (Transplantation /Acclimatization)a </li></ul>
  12. 13. <ul><ul><ul><ul><ul><li>Advantages of micropropagation : - </li></ul></ul></ul></ul></ul><ul><li>1. virus free plants </li></ul><ul><li>2.As a very effective and rapid method to multiply clonal material. It is particularly useful to increase material in following situation. </li></ul><ul><li>For rapid bulking up of plants. </li></ul><ul><li>For plants that are normally difficult to grow </li></ul><ul><li>3. As a specialized method of raising plants from seed </li></ul><ul><li>As a means of radically both the number of stock plants and the size of stock plant beds because the mother stock invitro at the laboratory. </li></ul>
  13. 14. Limitation of Micropropagation: - <ul><li>The initial capital investment in laboratory and equipment can be high. </li></ul><ul><li>Additional salary costs. </li></ul><ul><li>It is not yet commercially successful with a variety of species, including most confers fruit like mango etc. </li></ul><ul><li>The major problems have been with obtaining a sufficient root system, controlling disease infection, and obtaining rooting and growing on soil media and overall field survivability of it vitro plants. </li></ul>
  14. 15. General Plant profile: <ul><li>BOTENICAL NAME : Bacopa monnieri </li></ul><ul><li>COMMON NAME : Brahmi </li></ul><ul><li>PART USED : Whole plant </li></ul><ul><li>DISTRIBUTION : Found in damp or marshy areas near streams or on the border of ponds, throughout India </li></ul><ul><li>PROPAGATION : 4-5cm long Stem Cuttings </li></ul><ul><li>CHEMICAL </li></ul><ul><li>CONSTITUENTS : Bacoside -A & B </li></ul><ul><li>* Hersaponin (CNS depressant activity) </li></ul>
  15. 16. <ul><li>CHEMICAL CONSTITUENTS: Bacoside-A& B,Hersaponin (CNS depressant activity) </li></ul><ul><li>USES: Plant is astringent, bitter, sweet, cooling purgative, intellect promoting </li></ul><ul><li>Used against epilepsy, insanity, neuralgia, tumors, unclear voice, sleeplessness, indigestion, splenomegaly skin diseases, fever, convulsions & asthma. </li></ul>
  16. 17. MATERIALS AND METHODS <ul><ul><ul><ul><ul><li>MATERIALS </li></ul></ul></ul></ul></ul><ul><li>. Explant Collection: - Explants of brahmi (“GREEN HOUSE” of Dabur Research Foundation,) </li></ul><ul><li>. Glassware’s:- Culture bottles with caps, Small beaker, Spatula, </li></ul><ul><li>. Chemicals: - Basal salts for nutrient medium nitrates, Phosphates, sulphate, etc. Sucrose, DM waters (Deminrelized water), Myo-inositol, Vitamins and plant growth regulators or natural complexes. </li></ul>
  17. 18. <ul><li>Sterilants used:- </li></ul><ul><ul><li>Bavistin(Carbendazim-50% , Fungicide) </li></ul></ul><ul><ul><li>Antiseptic:-Savlon </li></ul></ul><ul><ul><li>HgCl2 used as sterilizing agents for every type of contamination </li></ul></ul><ul><ul><li>Gelling agent:- Phytagel </li></ul></ul><ul><ul><li>Instruments:- Weighing balance, pH meter,Oven(BPL-SANYO), Horizontal Cylindrical High Pressure semiautomatic Autoclave,Shaker, Laminar air flow, temperature controlled cultured room( Microprocessor). </li></ul></ul>
  18. 19. PROTOCOL OF SURFACE STERLIZATION <ul><li>Material from green house </li></ul><ul><li>2-3 wash with tap water(15min) </li></ul><ul><li>Explant leave in tap water having labolene detergent </li></ul><ul><li>Wash again with tap water (3times) </li></ul><ul><li>Explant now dip in a 200ml bottle having a mixture of sterilizing agent {Incidur(1ml),Bavistin(1%)} </li></ul>
  19. 20. <ul><li>Bottle placed in orbital shaker(for10min) </li></ul><ul><li>Decant the solution from the bottle </li></ul><ul><li>Add HPLC water 200ml having 1%HgCl2 </li></ul><ul><li>3-4 washing of explant with HPLC water </li></ul><ul><li>Use as Explant </li></ul>
  20. 21. PROTOCOL of media preparation: - For 2lit <ul><li>Approx. 500 ml HPLC water </li></ul><ul><li>Add 60 gm Sucrose </li></ul><ul><li>Myoinositol, 200mg </li></ul><ul><li>MS- macro, 50 ml </li></ul><ul><li>MS- CaCl2, 20 ml </li></ul><ul><li>MS- Iron, 10ml </li></ul>
  21. 22. <ul><li>MS- Micro –I, 2 ml </li></ul><ul><li>MS-Micro-II, 2ml </li></ul><ul><li>Vitamin, 2ml </li></ul><ul><li>Growth Regulator (viz.: BAP, TDZ, 2ml for 1-% concentration) </li></ul><ul><li>Set the pH between 5.8-5.9 with the help of NaOH or HCl </li></ul><ul><li>Add 5.8 gm phytagel </li></ul>
  22. 23. Murashige and Skoog medium (1962) <ul><li>CHEMICAL CONSTITUENT(mg/liter) </li></ul><ul><li>Macro-nutrients </li></ul><ul><li>NH 4 NO 3 (1650), KNO 3 (1900) </li></ul><ul><li>CaCl 2 .2H 2 O(400), MgSO 4 .7H 2 O(370) </li></ul><ul><li>KH 2 PO 4 ( 170) </li></ul><ul><li>Micro-nutrients </li></ul><ul><li>KI( 0.83),H 3 BO 3 ( 6.2),MnSO 4 .4H 2 O( 22.3),ZnSO 4 .7H 2 O(8.6) and </li></ul>
  23. 24. <ul><li>Na 2 MoO 4 .2H 2 O(0.25),CuSO 4 .5H 2 O(0.025),CoCl 2 .6H 2 O (0.025) </li></ul><ul><li>Iron Source </li></ul><ul><li>FeSO 4 .7H 2 O(27.8), NaEDTA.2H 2 O (37.3) </li></ul><ul><li>Vitamins </li></ul><ul><li>Glycine( 2), Myo-inositol( 100) Nicotinic acid (0.5), Pyridoxine.HCl(0.5), Thiamine. HCl (0.1) </li></ul>
  24. 25. METHOD <ul><li>1- Initiation of cultures </li></ul><ul><li>i ) MS Medium (BASAL), </li></ul><ul><li>ii) MS Medium + BAP ( 0.5%), </li></ul><ul><li>iii) MS Medium + BAP (1%), </li></ul><ul><li>iv) MS Medium + BAP (2%) </li></ul><ul><li>v) MS Medium + TDZ ( 0.5%), </li></ul><ul><li>vi) MS Medium + TDZ ( 1.0%), </li></ul><ul><li>MS Medium + TDZ (2%) </li></ul>
  25. 26. 2. Sub-culturing <ul><li>must be regularly replenished to avoid drying due to depleted nutrients and pH change also and to get rid of products of cell metabolism excreted into old media. </li></ul><ul><ul><li>we saw that some treatments are not good for proliferation of Bacopa like all treatment of TDZ. These treatments were good for callogenesis. </li></ul></ul>
  26. 27. <ul><li>Precautions for Sub culturing : - </li></ul><ul><li>1. Condition should be aseptic. </li></ul><ul><li>2. Presence of part of callus gives good result </li></ul><ul><li>3. Here cutting of explant should be slightly laterally also. </li></ul>
  27. 28. Procedure for Culturing
  28. 29. 3. Rooting <ul><li>This is the main step. Following treatment are used for the detection of good media for rooting. </li></ul><ul><li>1.MS NAA (0.1) </li></ul><ul><li>2. MS NAA (0.1) + BAP 1% </li></ul><ul><li>3.MS IAA (0.1) </li></ul><ul><li>4.MS IAA (0.1) + BAP 1% </li></ul><ul><li>The best media for rooting give healthy adventitious roots after few days. </li></ul>
  29. 30. 4. Hardening <ul><li>First we transfer the plant into the trays or small pots containing sterilized mud and different type of fertilizer e.g. Neopete . Newly arisen roots will start taking the nutrients from the mud. These trays initially kept in green house (for 8-12days). This stage is adaptive stage also.After 8-15 days we transfer the plants into field from the green house where it grow naturally. </li></ul>
  30. 31. APPLICATIONS OF BRAHMI <ul><li>Bacopa monnieri used in various product of DABUR. Some of these products are </li></ul><ul><li>1. Brahmi Bati (Gold): </li></ul><ul><li>In this product of Dabur Brahmi have 8 part . </li></ul><ul><li>Indication: General derangement of vat and quick recovery in convalescence . </li></ul>
  31. 32. <ul><li>2. Brahmi Bati (ORD): </li></ul><ul><li>In this product of Dabur Brahmi have 5 part and other main constituents are pippali, Red sulphide, Vidhanga, Kala mirch etc. </li></ul><ul><li>Indication: This medicine is useful in removing weakness after fever and promote quick recovery. It is also useful in mental weakness and deficient memory. </li></ul>
  32. 33. <ul><li>3.Brahmi Ghirt: </li></ul><ul><li>In this product Brahmi have 512 part Indication: Useful in epilepsy, insanity and promoting memory. </li></ul><ul><li>4.Saraswatarishta: </li></ul><ul><li>In this product of Dabur Brahmi have 80 part . </li></ul><ul><li>Indication: Recommended for improvising vitality, memory and sound sleep </li></ul>
  33. 34. RESULTS AND DISCUSSION <ul><li>PERCENTAGE OF GERMINATION = </li></ul><ul><li>No. of shoots sprouted X 100 </li></ul><ul><li>Total No. Of initiated cultures </li></ul>
  34. 35. SHOOT LENGTH <ul><li>The results revealed that the shoot length on the final day were in the order MS-BASAL > MS BAP (2%) > MS BAP (1%) > MS BAP (0.5%). </li></ul><ul><li>In different concentration of TDZ length was negligible in comparison to Concentration of BAP or we can say no shoots. Hence we don’t consider the MS-TDZ because here callogenesis occurs. </li></ul>
  35. 38. Comparison
  36. 39. <ul><ul><li>the most suitable media for shoot length was concerned; we had four best media to compare. They were MS BAP (2%) > MS BAP (1%) > MS BAP 0.5%. This was satisfactory, but there was a need to analyze how significantly different they were from each other and from the rest of the media involved. </li></ul></ul>
  37. 40. SHOOT NUMBER: <ul><li>The results revealed that the shoot length on the final day were in the order of MS-BASAL > MS BAP (2%) > MS BAP (1%) > MS BAP (0.5%). In different concentration of TDZ length was negligible in comparison to Concentration of BAP or we can say no shoots. Hence we don’t consider the TDZ because here callogenesis occurs. </li></ul>
  38. 42. <ul><ul><li>These statistical tests helped us infer that as far as the most suitable media for shoot length was concerned; we had four best media to compare. They were MS BAP (2%) > MS BAP (1%) > MS BAP 0.5%. This was satisfactory, but there was a need to analyze how significantly different they were from each other and from the rest of the media involved. </li></ul></ul>
  39. 44. Percentage of Root germination <ul><li>NAA0.1 >75% </li></ul><ul><li>NAA0.1%+BAP1% 75% </li></ul><ul><li>IAA0.1% 75% </li></ul><ul><li>IAA0.1%+BAP 1% 75% </li></ul>
  40. 46. ROOT NUMBER
  41. 47. Rooting
  42. 48. Conclusion <ul><li>MS BAP 2% stands our best choice from the final results at the end of 32 days. </li></ul><ul><ul><ul><li>Percentage of germination was best in All concentration of MS-BAP (6,Benzyle aminopurine) while in different concentration of MS- TDZ Percentage of germination was less. It was seen that MS-TDZ growth regulator responsible for callogenesis that is shoot number and leaves does not appear or very frizzle. </li></ul></ul></ul>
  43. 49. <ul><ul><ul><li>For the shoot length MS-BASAL media was best but in the final result it was seen that leaves of the plant in this media were very frizzle and number of shoot also very less in comparison to MS-BAP2%. Hence we should use MS-BAP2% to get good result in all aspects. </li></ul></ul></ul><ul><ul><ul><li>Number of shoot and leaves on them were best in concentration of growth regulator BAP2%. Though it was seen that number of shoot and leaves are also well in MS-BAP1%. But the statically observation shows that MS-BAP 2% is best in all aspects. </li></ul></ul></ul><ul><li>Root was best in NAA-0.1% </li></ul>