This document summarizes a project completed by Miss Othima Sharma for her M.Sc in Biotechnology from Thapar University, Patiala, Punjab, India. The project was conducted under the guidance of Professor Sunil D. Purohit at the Plant Biotechnology Laboratory of Mohanlal Sukhadia University in Udaipur, Rajasthan, India. The project evaluated the effects of using liquid culture medium as an alternative to agar-gelled semi-solid medium for plant tissue culture of Gerbera species. Plantlets cultured on liquid medium showed better growth and higher biochemical characteristics compared to those cultured on semi-solid medium.

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 Laboratory
Department of Botany, University Collage of Science
Mohanlal Sukhadia University, Udaipur

2. Lake City :
Udaipur

3. Mohanlal Sukhadiya University,
Udaipur

4. Plant Tissue Culture & Mol. Biology Laboratory (MLSU),
Udaipur
Incharge : Prof. S.D. Purohit
Plant Molecular Genetic
Micropropagation Characterization Transformation
UGC- Major Research Project: 12 lacs
DST Major Research Project: 45 lacs
DST WOS –A Project: 20 lacs
Total: 77 lacs

5. CONTENTS
• Why this project
• Introduction to project
• Materials and method
• Results
• Conclusion
• Discussion

6. Why this
Project?
To evaluate the effects of liquid
culture as an alternative to
Agar- Gelled medium for
Plant Tissue Culture.
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7. Introduction to
project work,
In present piece of
base cultures 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
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8. Materials and
Method
• Choice of Material
• Physiological Assays
• Biochemical Assays
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9. Choice of
Material
Plant : Gerbera sp.
Culture Media: Murashige and Skoog
(1962) supplemented
with 2.0 mg/l BAP
Culture 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.
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10. Work Plan and
PlantletsMethodologies
from Base Culture were sub-cultured on
semi-solid and liquid media
Total of 60 flasks, 30
Growth 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
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11. Results
As shown in following
photographs and graphs
of,
Liquid Media resulted to
be better than Semi-solid
Media

12. Base
Culture
of
Gerbera
sp.
(Source
of

13. After 15 Days
Plantlets sub-cultured on Semi- Plantlets sub-cultured on Liquid
Solid medium medium

14. After 30 Days
Plantlets sub-cultured on Semi- Plantlets sub-cultured on
Solid medium Liquid medium

15. Comparison Between
Gerbera Grown on Semi-
Solid and Liquid Media
After 15 days

16. Comparison Between
Gerbera Grown on Semi-
Solid and Liquid Media
After 30 days

17. Comparison of Shoot
Length of Gerbera
Grown on Semi-solid
and Liquid Media

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 ation
agar-gelled media(15 days) 5.39 2.65 2.50 59.85 11.04 21.80 15.95 6.30 1.69 0.94 9.07
agar-gelled media(30 days) 11.85 4.81 3.46 69.59 13.19 21.80 15.95 9.64 3.39 1.98 2.55
liquid media(15days) 8.63 3.31 4.80 74.22 14.69 25.89 18.70 8.28 4.36 2.15 6.52
liquid 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. 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 0.48
agar-gelled media(30 days) 2.29 1.18 0.81
liquid media(15days) 3.82 15.20 0.93
liquid media(30days) 7.01 27.53 2.24

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.
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21. Discussio
ns

22. REFERRENCES
Aswath 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 Plants
Chu 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, India
Dave, 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: 167-176
Dillen 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. Thank You

24. Gerbera plant: Gerbera is a genus of ornamental plants from
the sunflower family (Asteraceae). It was named in honour of
German Botanist and naturalist Traugott Gerber (1749) who
travelled extensively in Russia and was a friend of Carlous
Linnaeus.
Kingdom: Plantae
(Unranked): Angiosperms
(Unranked): Eudicots
(Unranked): Asterids
Order: Asterales
Family: Asteraceae
Subfamily: Mutisioideae
Tribe: Mutisieae
Genus: Gerbera

25. 6-Benzylaminopurine, benzyladenine or BAP is a
first-generation synthetic cytokinin that
elicits plant growth and development
responses, setting blossoms and stimulating fruit
richness by stimulating cell division. It is an
inhibitor of respiratory kinase in plants, and
increases post-harvest life of green vegetables.
6-Benzylaminopurine was first synthesized and
tested in the laboratories of plant
physiologist Folke K. Skoog.
Molecular formula C12H11N5
Molar mass 225.25 g mol−1
Appearance White to off-white
powder
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26. FRESH WEIGHT:
Plant samples (shoot clusters with leaves) were
weighed on the balance after removing any
adhering agar.
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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.
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28. PERCENT DRY WEIGHT:
Percent dry weight of the plant samples was
calculated by the following formula:
Percent Dry Weight =
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29. Kimberly Wong Stomata Leaf Peel
1.
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.
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30. The amount of chlorophyll was determined with the
help of following formula
Chlorophyll ‘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.
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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
0
A 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
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