the problem of browning in callus culture

1. EVALUATION OF CARBON SOURCES IN
REDUCING BROWNING EFFECT ON
HARUMANIS TISSUE CULTURE
CIK SOLEHAH BINTI SAEDON
2011152061
SUPERVISED BY:
PUAN NOOR ZUHAIRAH BINTI SAMSUDDIN

2. INTRODUCTION
According to Anim (2013), Malaysia policies on fruit industry is to
increase the production of fruits to meet local demand, for industrial
production and also for export. It is also to increase farmer’s income
and for supply the food supplement for Malaysia citizen. The next
policies is to improve the fruit industries that are competitive based
on the popular fruit demand for international marketing either for fresh
product or processed product and to ensure the fruits supply is
sufficient at the reasonable price.
Plant propagation is the technique to create and develop new
plants either through sexual (seeds) or asexual propagation
(cutting, grafting and tissue culture). However, propagation through
sexual does not ensure the true-to-the type plant reproduction (Iyer
and Degani, 1997). As an alternative, the researchers have
developed new way to propagate plant through tissue culture method
since 1900 centuries (Herren, 2005).

3. Problem Statement
-Carbon sources has relation in browning
occurrence.
-Tissue browning occurs when the explants are
injured and release the chemical called phenols and
directly associated with hydrocarbon group to form
phenolic compound.
-Hence, it is necessary to evaluate the germination
of callus growth of two different part of explants at
different concentration of sucrose as the carbon
source.

4. Objective
To identify the effectiveness of sucrose
in reducing browning effect

5. LITERATURE REVIEW
Harumanis variety (MR128)
Harumanis variety is categorized as seasonal crop because
this clone produces inflorescences due to drought season
only and also with water irrigation requirement. This clone is
very popular in North area of peninsular Malaysia especially
in Kedah and Perlis because the quality of fruit and it
production are high (Zainal and Malik, 1996). This variety is
very commercial for economy production in Malaysia and has
been exported into Japan (FAMA, 2013).
To ensure the supply of Harumanis mango is
continuously, the application of biotechnology should be
carried out. Thus, micro propagation (tissue culture) is a
technique that can be practiced on mango crop which can
propagate more plantlets and also maintain the true variety
from mother plant.

6. Tissue culture method
Tissue culture method
(Harry, 1994)
A technique of micropropagation
by using explants such as plant
protoplast, cells tissue and plant
parts to produce more seedlings
under aseptic condition
Tissue culture method
(Singh, 2011),
Each plant cell has an entire genetic
coding in all plant part whereas it
capable to grow new plant. There are
also required several conditions that
influence cell growth such as plant
hormones, types of explants, light
intensity, temperature and nutrient
supplement.

7. Browning in tissue culture
Browning in tissue culture
(Banerjee et al. , 1996)
(Murata et al. , 2001)
(Wu and Lin, 2002)
The symptom of browning can be seen
after the oxidation process occurs with
phenolic compound. The brown colors
are appearing on those materials
Browning in tissue culture
(Khosroushahi, 2011)
the controlled supplement of carbon
sources shows the different result of
browning on the culture media
Browning in tissue culture
(Kishiko et al., 2004)
the several amyloplast containing 3-5
starch grains were observed at around of
the cell nucleus, whereas the amyloplast
could be taken into tonoplast showing in
browning in the aged cells. These results
prove that the browning phenomenon may
be related to the metabolism of
carbohydrates within the cultural cells.

8. The role in reducing carbon
sources
The role in reducing
carbon sources
(Khosroushahi, et al.
2011)
•There are three different of carbon
sources with different concentration
(sucrose, glucose and fructose) result
on the different of browning intensity
•The browning phenomenon can be
controlled through application of the
growth media with 5g/L of sucrose,
5g/L of glucose and 10g/L for
fructose. But, for the best growth
media to increase the paclitaxel
production was in the medium
contained of 10g/L glucose, 5g/L
sucrose and 5g/L fructose

9. METHODOLOGY
There are two processes involved in media preparation which are
sterilization and preparation of media.
STERILIZATION
Chemical that are needed will be ethanol, Clorox and hypochlorite
(NaOCl2).
PREPARATION OF MEDIA
The different concentration of sucrose (0g/L, 10g/L, 20g/L, 30g/L
and 40g/L) as the carbon sources that will be added in the stock
solutions.
Other chemical substances that will be used are agar, sodium
hydroxide (NaOH) or hydrochloric acid (HCl) that will be applied
during pH adjustment process, inorganic salt and plant growth
regulator such as Kinetin (Kn), Indole-3-acetic acid (IAA) and
polyvinylpyrrolidone (PVP).

10. The different concentration of
sucrose in each solutions.
Treatment 1
0g/L sucrose
+
1mg/L IAA
+
3mg/L Kn
+
1% PVP
+
7g/L Gelrite
Treatment 2
10g/L
sucrose
+
1mg/L IAA
+
3mg/L Kn
+
1% PVP
+
7g/L Gelrite
Treatment 3
20g/L
sucrose
+
1mg/L IAA
+
3mgL Kn
+
1% PVP
+
7g/L Gelrite
Treatment 4
30g/L
sucrose
+
1mg/L IAA
+
3mg/L Kn
+
1% PVP
+
7g/L Gelrite
Treatment 5
40g/L
sucrose
+
1mg/L IAA
+
3mg/L Kn
+
1% PVP
+
7g/L Gelrite

11. Composition of MS medium
Macroelements
Concentration in medium (mg/L)
Ammonium nitrate (NH4 NO3)
1650.00
Potassium nitrate (KNO3)
1900.00
Calcium chloride anhydrous (CaCl2 440.00
2H2O)
Magnesium sulphate (MgSO4
7H2O)
370.00
Potassium phosphate monobasic
(KH2 PO4)
170.00

12. Microelements
Concentration in medium (mg/L)
Potassium iodide (KI)
0.83
Boric Acid (H3 BO3)
6.20
Manganese sulphate.(MnSO4
4H2O)
22.30
Zinc sulphate.(ZnSO4 7H2O)
8.60
Molybdic acid (Na2MoO4 2H2O)
0.25
Copper sulphate.(CuSO4 5H2O)
0.025
Cobalt chloride.(CoCl2 6H2O)
0.025
Iron sources
Concentration in medium (mg/L)
Ferrous sulphate (FeSO4 7H2O)
27.80
Na2.EDTA
37.30

13. Organic supplements /
vitamins
Concentration in medium (mg/L)
Myo-Inositol
100.00
Nicotinic acid
0 .50
Pyridoxine HCL
0 .50
Thiamine HCL
0 .50
Glycine
3.00
Carbon source /
carbohydrate
Concentration in medium (mg/L)
Sucrose
0 @ 10000 @ 20000 @30000 @
40000

14. Sterilization of equipments
•Apparatus that will be used are petri dish, glass beakers,
scissors, scalpels, forceps, and filter paper
•All the apparatus will be wrapped with aluminum foil before it
being autoclaved to disinfect bacteria.
•Distilled water will kept in Schott bottle and also autoclaved
•Autoclaved at 120ºC, 118 kPa steam pressure for 20 minutes
•Laminar Air Flow surface is wiped before to do tissue culture by
using 70 % of ethanol for 10 minutes and the ultraviolet (UV) light
will turn on for 15 minutes.

15. Apparatus and equipment

16. Growth chamber

17. Media preparation
5000ml
of
distilled water will
be kept into 10
Schott bottles
7g/L
of
agar
Gelrite will be
added into the
solution
Sterilize
it
under
oC at
autoclave (121
1.05kg cm-2) for 15
minutes
Prepare
the
different
concentration
of
sucrose
(0g/L, 10g/L, 20g/L,
30g/L and 40g/L)
pH media will be
adjusted to 5.8 by
using 1M of Sodium
hydroxide (NaOH) or
1M of (Hydrochloric
acid) HCl.
The addition of plant
growth
regulator
(1.0mg/L of IAA +
3.0mg/L of Kn + 1.0% of
PVP) in MS medium
(look at composition of
MS medium)
The volume of
medium will be
added with distilled
water to reach 1
liter solution
The medium solution will be
poured into petri dish sterile
container after it have been
cooled about 45oC in Laminar Air
Flow

18. Surface sterilization
The plant material will be washed in running tap water for 2 hours
Soaked into Decon90 for 5 minutes and then rinse 5 times with sterile distilled water
Immersed into 0.2% of polyvinylpyrrolidone (PVP) for 1 hour and soaked again about 30 minutes
The explants washed again for 5 times with sterile distilled water and treated with 80% ethanol for 10
minutes
Wash the explants for 5 times with sterile distilled water and then the explants will be treated with 80%
Clorox and tween20 for 20 minutes
Rinsed 5 times with distilled water
Plant materials will be dried on the autoclaved filter paper
Explants will be cut bisected symmetrical (1cm x 1cm) to obtain equal size
All process will be carried out in Laminar Air Flow Hood.
It ready to be cultured on Murashige and skoog (MS) medium

19. Explants part for tissue
culture
•Young bud
•Matured leaf

20. Experimental design CRD
Factorial with 7 replications and
5 treatments
B
T4R4
B
T2R1
B
T3R1
B
T4R1
B
T1R4
L
T1R1
L
T2R1
L
T3R1
L
T4R1
B
T5R4
B
T1R2
L
T4R2
B
T4R2
B
T3R2
L
T1R7
B
T1R3
B
T5R5
L
T4R3
B
T2R2
L
T5R2
L
T1R2
L
T3R4
B
T1R6
L
T1R5
B
T5R3
L
T1R3
L
T2R3
L
T5R5
L
T3R2
B
T4R3
B
T5R1
L
T1R4
B
T3R4
B
T1R1
L
T5R1
B
T2R4
B
T3R7
B
T2R3
L
T3R3
L
T4R4
L
T5R3
L
T2R5
L
T4R6
B
T4R5
B
T2R7
B
T1R5
L
T1R6
L
T3R5
B
T5R2
L
T5R4
L
T2R6
B
T4R6
B
T3R6
B
T2R6
B
T5R6
L
T4R5
B
T3R3
B
T1R7
B
T3R5
L
T5R6
L
T3R6
B
T5R7
L
T2R4
B
T4R7
L
T2R2
B
T2R5
L
T2R7
L
T3R7
L
T4R7
L
T5R7

21. The experiment laid out will be arranged in CRD
Factorial with five treatments corresponding two
explants of mango (young buds and matured
leaves)
7 replicates
2 sample of explants for each treatment in 7
replication.
2 x 5 x 7 = 70 Petri dish
Total experimental unit is 70.

22. Data collection
Types of carbon
Total healthy
Total browning
source/explant
callus
callus
Treatment
T1
T2
Sucrose/young
T3
buds
T4
T5
T1
T2
Sucrose/matured
T3
leaves
T4
T5
The data will be collected every week

23. Average percentage of calli growth = Total of healthy callus x 100
Total of callus appear
*total of callus appear including total of healthy callus and total of
browning callus
Treatment
Average percentage (%) of callus growth on MS
media containing sucrose
Young buds
T1
T2
T3
T4
T5
Matured leaves

24. Expected outcome
The effect of sucrose on sub-cultured calli of Harumanis
Healthy calli
Carbon source
Concentration
Types of
(g/L)
explants
0
Fresh calli (g) Dried calli (g)
Browning calli
Wet calli
Dried calli
(g)
(g)
Young buds
10
20
30
sucrose
40
0
Matured
10
leaves
20
30
40
Notes:
T1- Control media; T2- 10g/L; T3- 20g/L; T4- 30g/L; T5- 40g/L
Data analysis of growth measurement of callus, dry weight and fresh weight for
healthy calli and wet weight and dried weight for browning calli. This analysis will be
carry out to identify the different characteristic between healthy callus and browning callus

25. Fig. 1. Callus induction and plant regeneration of Miscanthus × giganteus. (a) Callus
initiation on MS medium with 30 g dm-3 honey instead of sucrose, (b) Severe browning of
explants (immature inflorescences cut into 0.5 cm pieces) on MS with 200 mg · dm-3
chitosan and (c) with 100 mg · dm-3 cysteine, (d) Callus developing on dark browning
explants, (e) Spongy callus with many white embryo-like structures (arrowed), (f) Start of
regeneration on MS with 65 g · dm-3 BP (many roots greening in light), (g) Leaf
regeneration on MS with 0.05 mg · dm-3 KIN, (h) Leaf buds on MS with 0.2 mg · dm-3 BAP,
(i) Regenerated plantlets on MS with 0.05 mg· dm-3 KIN, (j) Regenerated Miscanthus plant

26. References
Banerjee, S., Upadhyay, N., Kukreja, A. K., Ahuja P. S., Kumar, S., Saha, G. C. 1996.
Taxanes From In
vitro cultures of the Himalayan yew Taxus wallichiana.Planta
Medica. 62 (4); 329-331.
Harry, I. S. and Thorpe, T. A. 1994. In vitro culture of Forest Trees. Vasil, I. K. and
Thorpe, T. A.
editor. In: Plant Cell and Tissue Culture. Kluwer Academic
Publisher.539-560
Khosroushashi, A. Y., Manesh, H. N., Simonsen H. T. 2011. Effect of Antioxidant and
Carbohydrates in Callus cultures of Taxus brevifolia:Evaluation of Browning,
Callus
Growth, Total Phenolics and Paclitaxel Production. Bioimpact., 3745.
Kishiko O., Sanro T., Masaya S. 2004. Electron Microscopic Observation of Plastid
Containing Taxol-like Substances in Callus Cells of Taxus cuspidata variety
Nana.
Pakistan Journal of Biological Sciences.7 (12); 2139-2148.
Murashige, T. and Skoog, F. 1962. Revised Medium for Growth and Bioessay With Tobacco
Tissue Culture. Plant Physiology. 180; 7-12.
Murata, M., Nishimura, M., Murai, N., Haruta, M., Homma, S., Itoh, Y. 2001. A Transgenic
Apple
Callus Showing Reduced Polyphenol Oxidase Activity and Lower
Browning Potential. Bioscience Biotechnology and Biochemistry. 65 (2); 383388.
Plazek, A. and Dubert, F. 2010. Improvement of Medium for Miscanthus giganteus Callus
Induction and Plant Regeneration. Acta Biologica Cracoviensia Series
Botanica. 52(1); 105110
Singh, H. P., Parthasarathy, V. A., Babu, K. N. 2011. Advances in Horticulture
BiotechnolgyRegenerations system ; Fruit Crops, Plantation Crops and Spice
(Volume1). Westville
Publishing House.
Wu, J. and Lin, L. 2002. Ultrasound-induced Stress Responses of Panax ginseng Cells:

27. Gantt Chart
Year
July
Jun
May
April
Mac
Submission Final Report
Feb
Writing Final Report
Jan
Statistical Analysis
Dec
Start Lab Experiment
Nov
Proposal Presentation
2014
Oct
Submission Proposal
Sept
Activities/Month
Decide title of thesis
Overview of Thesis
Project
Collection Related
Journals/Materials
Decide Laboratory to be
used
Proposal Writing of
Literature Review
Proposal Writing of
Methodology
2013

28. Thank You