evaluation of sucrose in reducing browning effect on harumanis tissue culture
Post on 10-May-2015
659 Views
Preview:
DESCRIPTION
TRANSCRIPT
EVALUATION OF CARBON SOURCES IN REDUCING BROWNING EFFECT ON HARUMANIS TISSUE CULTURE
CIK SOLEHAH BINTI SAEDON2011152061
SUPERVISED BY:PUAN NOOR ZUHAIRAH BINTI SAMSUDDIN
INTRODUCTIONAccording 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).
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.
Objective
To identify the effectiveness of sucrose in reducing browning effect
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.
LITERATURE REVIEW
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.
Tissue culture method
Browning in tissue cultureBrowning 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.
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
METHODOLOGYThere are two processes involved in media preparation which are sterilization and preparation of media.
STERILIZATIONChemical that are needed will be ethanol, Clorox and hypochlorite (NaOCl2).
PREPARATION OF MEDIAThe 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).
The different concentration of sucrose in each solutions.
Treatment 10g/L sucrose
+ 1mg/L IAA
+ 3mg/L Kn
+ 1% PVP
+ 7g/L Gelrite
Treatment 210g/L
sucrose +
1mg/L IAA +
3mg/L Kn +
1% PVP +
7g/L Gelrite
Treatment 320g/L
sucrose +
1mg/L IAA +
3mgL Kn +
1% PVP +
7g/L Gelrite
Treatment 430g/L
sucrose +
1mg/L IAA +
3mg/L Kn +
1% PVP +
7g/L Gelrite
Treatment 540g/L
sucrose+
1mg/L IAA +
3mg/L Kn +
1% PVP +
7g/L Gelrite
Composition of MS mediumMacroelements Concentration in medium
(mg/L)
Ammonium nitrate (NH4 NO3) 1650.00
Potassium nitrate (KNO3) 1900.00
Calcium chloride anhydrous (CaCl2 2H2O)
440.00
Magnesium sulphate (MgSO4
7H2O)370.00
Potassium phosphate monobasic (KH2 PO4)
170.00
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
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
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.
Apparatus and equipment
Growth chamber
Media preparation5000ml of distilled
water will be kept into 10
Schott bottles
Prepare the different
concentration of sucrose
(0g/L, 10g/L, 20g/L, 30g/L and 40g/L)
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
pH media will be adjusted
to 5.8 by using 1M of
Sodium hydroxide
(NaOH) or 1M of
(Hydrochloric acid) HCl.
7g/L of agar Gelrite will be
added into the solution
Sterilize it under
autoclave (121oC at
1.05kg cm-2) for 15
minutes
The medium solution will be poured
into petri dish sterile
container after it have been cooled
about 45oC in Laminar Air
Flow
Surface sterilization
Explants part for tissue culture
21
43
5 6
•Young bud•Matured leaf
Experimental design CRD Factorial with 7 replications and 5 treatments
BT4R4
BT1R2
LT1R2
BT5R1
LT5R3
LT2R6
LT3R6
BT2R1
LT4R2
LT3R4
LT1R4
LT2R5
BT4R6
BT5R7
BT3R1
BT4R2
BT1R6
BT3R4
LT4R6
BT3R6
LT2R4
BT4R1
BT3R2
LT1R5
BT1R1
BT4R5
BT2R6
BT4R7
BT1R4
LT1R7
BT5R3
LT5R1
BT2R7
BT5R6
LT2R2
LT1R1
BT1R3
LT1R3
BT2R4
BT1R5
LT4R5
BT2R5
LT2R1
BT5R5
LT2R3
BT3R7
LT1R6
BT3R3
LT2R7
LT3R1
LT4R3
LT5R5
BT2R3
LT3R5
BT1R7
LT3R7
LT4R1
BT2R2
LT3R2
LT3R3
BT5R2
BT3R5
LT4R7
BT5R4
LT5R2
BT4R3
LT4R4
LT5R4
LT5R6
LT5R7
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.
Data collection
TreatmentTypes of carbon
source/explant
Total healthy
callus
Total browning
callus
T1
Sucrose/young
buds
T2
T3
T4
T5
T1
Sucrose/matured
leaves
T2
T3
T4
T5
The data will be collected every week
Treatment Average percentage (%) of callus growth on MS media
containing sucrose
Young buds Matured leaves
T1
T2
T3
T4
T5
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
Expected outcome
Healthy calli Browning calli
Carbon source Concentration
(g/L)
Types of
explants
Fresh calli (g) Dried calli (g) Wet calli
(g)
Dried calli
(g)
sucrose
0 Young buds
10
20
30
40
0 Matured
leaves10
20
30
40
The effect of sucrose on sub-cultured calli of Harumanis
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 forhealthy 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
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 in soil. (Plazek, 2010)
ReferencesBanerjee, 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.539-560Khosroushashi, 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., 37-45.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); 383-388.Plazek, A. and Dubert, F. 2010. Improvement of Medium for Miscanthus giganteus Callus Induction and Plant Regeneration. Acta Biologica Cracoviensia Series Botanica. 52(1); 105- 110Singh, H. P., Parthasarathy, V. A., Babu, K. N. 2011. Advances in Horticulture Biotechnolgy- Regenerations 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: Enzymatic Browning and Phenolics Production. Biotechnology Progress.18 (4); 862-866.
Gantt Chart Year 2013 2014
Activities/Month Sept Oct Nov Dec Jan Feb Mac April May Jun JulyDecide 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 Submission Proposal Proposal Presentation
Start Lab Experiment Statistical Analysis Writing Final Report Submission Final Report
Thank You
top related