evaluation of sucrose in reducing browning effect on harumanis tissue culture


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the problem of browning in callus culture

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evaluation of sucrose in reducing browning effect on harumanis tissue culture

  2. 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. 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. 4. Objective To identify the effectiveness of sucrose in reducing browning effect
  5. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 15. Apparatus and equipment
  16. 16. Growth chamber
  17. 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. 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. 19. Explants part for tissue culture •Young bud •Matured leaf
  20. 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. 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. 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. 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. 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. 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. 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. 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. 28. Thank You