A                Project Completed                         By               Miss Othima Sharma           [M.Sc (Final) Bio...
Lake City : Udaipur
Mohanlal Sukhadiya University,           Udaipur
Plant Tissue Culture & Mol. Biology Laboratory (MLSU),                        Udaipur                     Incharge : Prof....
CONTENTS•   Why this project•   Introduction to project•   Materials and method•   Results•   Conclusion•   Discussion
Why this      Project?To evaluate the effects of liquid   culture as an alternative to    Agar- Gelled medium for      Pla...
Introduction to      project work,In present piece ofbase cultures grown on AgarGelled semi-solid mediawere sub-cultured o...
Materials and  Method• Choice of Material• Physiological Assays• Biochemical Assays                         Bac
Choice of     Material Plant    :       Gerbera sp.Culture Media:           Murashige and Skoog (1962)                    ...
Work Plan and     PlantletsMethodologies              from Base Culture were sub-cultured on                semi-solid and...
Results As shown in followingphotographs and graphs            of,Liquid Media resulted tobe better than Semi-solid       ...
BaseCulture   ofGerbera  sp.(Source   of
After 15 DaysPlantlets sub-cultured on Semi-   Plantlets sub-cultured on Liquid         Solid medium                      ...
After 30 DaysPlantlets sub-cultured on Semi-   Plantlets sub-cultured on           Solid medium                 Liquid med...
Comparison BetweenGerbera Grown on Semi- Solid and Liquid Media      After 15 days
Comparison BetweenGerbera Grown on Semi- Solid and Liquid Media     After 30 days
Comparison of Shoot Length of GerberaGrown on Semi-solid  and Liquid Media
COMPARATIVE STUDIES ON PHYSIOLOGICAL CHARACTERISTICS OF                                  GERBERA GROWN ON LIQUID AND SEMI-...
COMPARATIVE STUDIES ON BIOCHEMICAL CHARACTERISTICS OF                           GERBERA GROWN ON LIQUID AND SEMI-SOLID MED...
Conclusion   Shoots of Gerbera grew much better in liquid phase    provided with Glass Beads as compared to those    grow...
Discussio   ns
REFERRENCESAswath CR, Choudhary ML (2002). Rapid plant regeneration from Gerbera jamesonii    Bolus callus cultures. Acta ...
Thank You
Gerbera plant: Gerbera is a genus of ornamental plants fromthe sunflower family (Asteraceae). It was named in honour ofGer...
6-Benzylaminopurine, benzyladenine or BAP is afirst-generation synthetic cytokinin thatelicits plant growth and developmen...
FRESH WEIGHT:Plant samples (shoot clusters with leaves) were  weighed on the balance after removing any  adhering agar.   ...
DRY WEIGHT:Plant samples (shoot clusters with leaves) were  wrapped in filter paper and kept in oven at  60°C for 12 hours...
PERCENT DRY WEIGHT:Percent dry weight of the plant samples was  calculated by the following formula:Percent Dry Weight =  ...
Kimberly Wong Stomata Leaf Peel1.                         Count   A fresh leaf and a bottle of nail varnish was taken.2.  ...
The amount of chlorophyll was determined with the help of following formulaChlorophyll ‘a’ mg gˉ¹ fresh leaf =Chlorophyll ...
0.35                                                                y = 0.002x + 0.025        Absorbance at 595nm         ...
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Comparative Studies on Growth & Biochemical Characteristics of Gerbera Grown on Semi-Solid and Liquid Media

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I,in order to excel in Plant Biotechnology, joined this prestigious university as a trainee under the guidance of Mr. Sunil D. Purohit for micro-propagation of plants and their phylogenetic analysis using molecular markers.

In present piece of work, base cultures of Gerbera grown on Agar Gelled semi-solid media were sub-cultured on Agar gelled semi-solid media and Glass bead/liquid media and their growth and biochemical characteristics were studied in two sets after every 15 days.

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Comparative Studies on Growth & Biochemical Characteristics of Gerbera Grown on Semi-Solid and Liquid Media

  1. 1. A Project Completed By Miss Othima Sharma [M.Sc (Final) Biotechnology] Thapar University Patiala, Punjab Under the Guidance of Sunil. D. Purohit Ph. D. Professor and Head Plant Biotechnology LaboratoryDepartment of Botany, University Collage of Science Mohanlal Sukhadia University, Udaipur
  2. 2. Lake City : Udaipur
  3. 3. Mohanlal Sukhadiya University, Udaipur
  4. 4. Plant Tissue Culture & Mol. Biology Laboratory (MLSU), Udaipur Incharge : Prof. S.D. Purohit Plant Molecular GeneticMicropropagation Characterization Transformation UGC- Major Research Project: 12 lacs DST Major Research Project: 45 lacs DST WOS –A Project: 20 lacs Total: 77 lacs
  5. 5. CONTENTS• Why this project• Introduction to project• Materials and method• Results• Conclusion• Discussion
  6. 6. Why this Project?To evaluate the effects of liquid culture as an alternative to Agar- Gelled medium for Plant Tissue Culture. Bac
  7. 7. Introduction to project work,In present piece ofbase cultures grown on AgarGelled semi-solid mediawere sub-cultured on Agargelled semi-solid media andGlass bead/liquid media andtheir growth andbiochemical characteristics Bac
  8. 8. Materials and Method• Choice of Material• Physiological Assays• Biochemical Assays Bac
  9. 9. Choice of Material Plant : Gerbera sp.Culture Media: Murashige and Skoog (1962) supplemented with 2.0 mg/l BAPCulture Conditions: 25 2 C with 16h/8h light and dark periods provided by fluorescent tubes providing 2000-3000 lux (ca. 30-40 µmol m-2s-1) light controlled by Saveer Sequential Timers. Bac
  10. 10. Work Plan and PlantletsMethodologies from Base Culture were sub-cultured on semi-solid and liquid media Total of 60 flasks, 30Growth Parameters Biochemical Parameters each for semi solid and liquid media. Each time, 6 Number of Shoot Clusters replicates were used Shoot Length Total Soluble Protein Estimation Number of Leaves Carbohydrate Estimation Fresh Weight Chlorophyll Content Dry Weight Percent Dry Weight Stomatal Studies Bac
  11. 11. Results As shown in followingphotographs and graphs of,Liquid Media resulted tobe better than Semi-solid Media
  12. 12. BaseCulture ofGerbera sp.(Source of
  13. 13. After 15 DaysPlantlets sub-cultured on Semi- Plantlets sub-cultured on Liquid Solid medium medium
  14. 14. After 30 DaysPlantlets sub-cultured on Semi- Plantlets sub-cultured on Solid medium Liquid medium
  15. 15. Comparison BetweenGerbera Grown on Semi- Solid and Liquid Media After 15 days
  16. 16. Comparison BetweenGerbera Grown on Semi- Solid and Liquid Media After 30 days
  17. 17. Comparison of Shoot Length of GerberaGrown on Semi-solid and Liquid Media
  18. 18. COMPARATIVE STUDIES ON PHYSIOLOGICAL CHARACTERISTICS OF GERBERA GROWN ON LIQUID AND SEMI-SOLID MEDIA. 120.00 100.00 80.00 60.00 40.00 20.00 0.00 no. rate of shoot Stomatal Size of Size of wet dry ofshoots shoot no. of Stomatal %water length frequenc Stomata Stomata weight weight per multiplic leaves index content (cm) y (L) (B) (g) (g) cluster ationagar-gelled media(15 days) 5.39 2.65 2.50 59.85 11.04 21.80 15.95 6.30 1.69 0.94 9.07agar-gelled media(30 days) 11.85 4.81 3.46 69.59 13.19 21.80 15.95 9.64 3.39 1.98 2.55liquid media(15days) 8.63 3.31 4.80 74.22 14.69 25.89 18.70 8.28 4.36 2.15 6.52liquid media(30days) 29.95 6.32 6.21 98.51 21.02 25.89 14.21 18.70 20.89 4.12 5.67
  19. 19. COMPARATIVE STUDIES ON BIOCHEMICAL CHARACTERISTICS OF GERBERA GROWN ON LIQUID AND SEMI-SOLID MEDIA. 30.00 25.00 20.00 15.00 10.00 5.00 0.00 Carbohydrate Content(mg/g/FW) Protein Content(mg/g/FW) Chlorophyll Content(mg/g/FW)agar-gelled media(15 days) 1.50 0.85 0.48agar-gelled media(30 days) 2.29 1.18 0.81liquid media(15days) 3.82 15.20 0.93liquid media(30days) 7.01 27.53 2.24
  20. 20. Conclusion Shoots of Gerbera grew much better in liquid phase provided with Glass Beads as compared to those grown on semi-solid media. In addition to better shoots, higher shoot length and more number of leaves with greater surface area were shown in Gerbera grown in liquid media. Higher concentration of Protein and Carbohydrate content in the shoots grown on liquid phase medium with glass beads which showed best growth facilitated by better absorption of nutrients and higher metabolic activity under hydrated condition. Liquid medium is better than semi-solid medium during in-vitro cultivation. The plantlets produced on liquid medium possessed better quality in all respects than the semi-solid ones. Bac
  21. 21. Discussio ns
  22. 22. REFERRENCESAswath CR, Choudhary ML (2002). Rapid plant regeneration from Gerbera jamesonii Bolus callus cultures. Acta Bot. Croatica, 61: 125- 134.C.H. Pearson, K. Cornish, C.M. McMahan. 2007. Using Peat Pellets in Liquid Media to Root Sunflower Tissue Culture PlantsChu CY, Knight SL, Smith MAL (1993). Effect of liquid culture on the growth and development of minature rose (Rosa chinensis Jacq. Minima. Plant Cell Tissue Organ Cult. 32: 329-334.Dave, A. 1994. In vitro multiplication of some economically important plant species of Aravallis in Rajasthan. Ph.D. Thesis, Mohanlal Sukhadia University, Udaipur, IndiaDave, A., Bilochi, G. and Purohit, S.D. 2003. Scaling-up production and field performance of micropropagated medicinal herb Safed Musli (Chlorophytum borivilianum). In Vitro Plant Cell. Dev. Biol. – Plant 39: 419-425.Dave, A., Joshi, N. and Purohit, S.D. 2004. In vitro propagation of Chlorophytum borivilianum using encapsulated shoot buds. Europe. J. Horti. Sci. 69:37-42.Debergh, P.C. and Read, P.E. 1990. Micropropagation. In: “Micropropagation, Technology and Application”. P .C. Debergh and R.H. Zimmerman (Eds.), pp. 1–13. Kluwer Academic, Dordrecht.Debergh PC, Mane LJ (1981). A scheme for commercial propagation of ornamental plants by tissue culture. Sci. Hortic. 14: 335-345.Debergh PC, Aitken-Christic J, Cohen B, Von Arnold S, Zimmerman R, Ziv M (1992). Reconsideration of the term "vitrification" as used in micropropagation. Plant Cell Tissue Organ Cult. 30: 135-140.Densco I (1987). Factors influencing vitrification of carnation and conifers. Acta Hortic. 212: 167-176Dillen W, Buysens SA (1989). Simple technique to overcome vitrification in Gypsophila paniculata L. Plant Cell Tissue Organ Cult. 19: 181- 188.Earle ED, Langhans LW (1975). Carnation propagation from shoot tip cultured in liquid medium. Horticult. Sci. 10: 608-610.El-Gengaihi, S., Taha, H.S. and Kamel, A.M. 2006. In vivo and in vitro comparative studies of Origanum species. J. Food Agric. Environ. 4:127-134.
  23. 23. Thank You
  24. 24. Gerbera plant: Gerbera is a genus of ornamental plants fromthe sunflower family (Asteraceae). It was named in honour ofGerman Botanist and naturalist Traugott Gerber (1749) whotravelled extensively in Russia and was a friend of CarlousLinnaeus.Kingdom: Plantae(Unranked): Angiosperms(Unranked): Eudicots(Unranked): AsteridsOrder: AsteralesFamily: AsteraceaeSubfamily: MutisioideaeTribe: MutisieaeGenus: Gerbera
  25. 25. 6-Benzylaminopurine, benzyladenine or BAP is afirst-generation synthetic cytokinin thatelicits plant growth and developmentresponses, setting blossoms and stimulating fruitrichness by stimulating cell division. It is aninhibitor of respiratory kinase in plants, andincreases post-harvest life of green vegetables.6-Benzylaminopurine was first synthesized andtested in the laboratories of plantphysiologist Folke K. Skoog.Molecular formula C12H11N5Molar mass 225.25 g mol−1Appearance White to off-white powder Bac
  26. 26. FRESH WEIGHT:Plant samples (shoot clusters with leaves) were weighed on the balance after removing any adhering agar. Bac
  27. 27. DRY WEIGHT:Plant samples (shoot clusters with leaves) were wrapped in filter paper and kept in oven at 60°C for 12 hours. Dry samples were allowed to equilibrate to room temperature and humidity conditions and weighed to obtain dry weight. Bac
  28. 28. PERCENT DRY WEIGHT:Percent dry weight of the plant samples was calculated by the following formula:Percent Dry Weight = Bac
  29. 29. Kimberly Wong Stomata Leaf Peel1. Count A fresh leaf and a bottle of nail varnish was taken.2. 2 squares (about 1cm2) on each side (2 on the bottom and 2 on the top) were painted.3. Those patches were allowed to dry.4. A second layer was applied when the first one was completely dry.5. While waiting for the nail varnish to dry, a microscope was taken and adjusted (it was made sure that the light was dim)6. Using forceps, a strip of nail varnish was peeled off from the upper epidermis and observed under 10x.7. The number of stomata were counted when the patch was in focus.8. Steps 6-8 were repeated with a strip of nail varnish from the lower epidermis. Bac
  30. 30. The amount of chlorophyll was determined with the help of following formulaChlorophyll ‘a’ mg gˉ¹ fresh leaf =Chlorophyll ‘b’ mg gˉ¹ fresh leaf =Chlorophyll ‘c’ mg gˉ¹ fresh leaf = a = length of light path in the cell (1 cm) V = volume of extract in ml (final volume) w= fresh weight of the sample (leaf) in g. Bac
  31. 31. 0.35 y = 0.002x + 0.025 Absorbance at 595nm 0.3 R² = 0.991 0.25 0.2 0.15 0.1 0.05 0A 0 20 40 60 80 100 120 concentration of BSA (µg) 0.7 y = 0.005x + 0.039 0.6 R² = 0.991 Absorbance at 620nm 0.5 0.4 0.3 0.2 0.1 0 0 20 40 60 80 100 120 B concentration of Glucose (µg) a) Standard Plot for Protein Estimation by Bradford Reagent b) Standard Plot for Carbohydrate Estimation by Anthrone Reagent Bac
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