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.

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