1. Effect of Controlled and Modified Atmosphere Storage on Qualities and Senescence of Dendrobium Orchid Mr.Warinthon Yimyong ID: 109618

2. Effect of Controlled and Modified Atmosphere Storage on Qualities and Senescence of Dendrobium Orchid Mr.Warinthon Yimyong ID: 109618 Examination Committee: Asst. Prof. Dr. Peeyush Soni (Chairperson) Prof. Athapol Noomhorm (Member) Prof. Nguyen Thi Kim Oanh (Member) Assoc. Prof. Dr. Danai Boonyakiat (External Expert)

4. Objective

5. 1. To study trends and prospects of current controlled atmosphere storage and modified atmosphere packaging (MAP) for Dendrobium orchid. 2. To investigate the optimum conditions of controlled atmosphere storage for Dendrobium orchid in Thailand. 3. To investigate the suitable material use for modified atmosphere packaging (MAP) of Dendrobium orchid. 4. To investigate the effective of controlled and modified atmosphere treatments on the changes of physiological and biochemical qualities and senescence of Dendrobium orchid during storage.

6. MATERIALS AND METHODS

7. Experiment 1 To investigate the optimum conditions of controlled atmosphere storage for Dendrobium orchid Experiment 2 To investigate the suitable material use for modified atmosphere packaging (MAP) of Dendrobium orchid Experiment 3 Effects of controlled and modified atmosphere on biochemical changes involved in petal senescence of Dendrobium orchid during storage

8. Experiment 1 To investigate the optimum conditions of controlled atmosphere storage for Dendrobium orchid To investigate the optimum conditions of controlled atmosphere storage for Dendrobium orchid flowers which had the combination of 5% CO 2 and 2, 4, 6, and 8% O 2 and the combination of 10% CO 2 and 2, 4, 6, and 8% O 2 compare with Dendrobium orchid flowers storage under normal atmosphere (21% O 2 and 0.03% CO 2 ) on the storage qualities of Dendrobium orchid flowers at record temperatures 13°C with 95% RH. There were tree replications for each treatment.

9. Set up controlled atmosphere systems with storage room Testing System / Pretest Testing with Dendrobium Orchids which packed in cardboard box Investigate effect of controlled atmosphere on qualities of orchids -Storage life -Freshness of inflorescence -Weight loss -Respiration rate -Ethylene production -Petal color -Anthocyanin content Examine every 5 days Storage in control temperature at 13 o C with 95% RH Atmosphere Conditions: - Normal atmospheres (21% O 2 and 0.03% CO 2 ) - 5% CO 2 + 2% O 2 - 5% CO 2 + 4% O 2 - 5% CO 2 + 6% O 2 - 5% CO 2 + 8% O 2 - 10% CO 2 + 2% O 2 - 10% CO 2 + 4% O 2 - 10% CO 2 + 6% O 2 - 10% CO 2 + 8% O 2 1 box filled with 40 of orchid inflorescence Schematic diagram of experiment to investigate the optimum conditions of CA storage for Dendrobium orchid flower

13. Table 1 Effect of controlled atmosphere on storage life of dendrobium orchid Treatment Storage life (Days)* 5% CO 2 + 2% O 2 28.33 a ± 2.07 5% CO 2 + 4% O 2 25.33 b ± 0.97 5% CO 2 + 6% O 2 25.33 b ± 1.05 5% CO 2 + 8% O 2 23.33 c ± 1.15 10% CO 2 + 2% O 2 18.33 d ± 1.15 10% CO 2 + 4% O 2 18.33 d ± 0.57 10% CO 2 + 6% O 2 16.67 e ± 1.15 10% CO 2 + 8% O 2 15.00 f ± 0.57 Control 11.67 g ± 0.57 CV (%) 3.57 LSD 0.85 * Means (Mean ± S.D.) of different superscripts differ significantly at p <0.05

16. Fig.1 Effects of controlled atmosphere storage on weight loss of Dendrobium orchid stored at 13 o C with 95% relative humidity (RH) (Error bars represent S.D.) Weight loss

17. Fig.4.1.3 Effects of controlled atmosphere storage on total anthocytanin content of Dendrobium orchid stored at 13 o C with 95% relative humidity (RH) (Error bars represent S.D.) Anthocyanin Content

18. Fig.4.1.4 Effects of controlled atmosphere storage on respiration rates of Dendrobium orchid stored at 13 o C with 95% relative humidity (RH) (Error bars represent S.D.) Respiration rate

19. Fig.4.1.5 Effects of controlled atmosphere storage on ethylene production of Dendrobium orchid stored at 13 o C with 95% relative humidity (RH) (Error bars represent S.D.) Ethylene Production

20. Fig.4.1.6 Effects of controlled atmosphere storage on petal color in lightness (L*) of Dendrobium orchid stored at 13 o C with 95% relative humidity (RH) (Error bars represent S.D.) Petal Color

21. Fig.4.1.7 Effects of controlled atmosphere storage on petal color in chroma (C*) of Dendrobium orchid stored at 13 o C with 95% relative humidity (RH) (Error bars represent S.D.)

22. Fig.4.1.8 Effects of controlled atmosphere storage on petal color in hue angle (H  ) of Dendrobium orchid stored at 13 o C with 95% relative humidity (RH) (Error bars represent S.D.)

24. Fig.4.1.9 Effects of controlled atmosphere storage on freshness of Dendrobium orchid stored at 13 o C with 95% relative humidity (RH) (Error bars represent S.D.) Freshness (Visual appearance)

25. The aim of undertaking and inclusion of this concise financial feasibility analysis herein is to evaluate the commercial viability of the proposed CA storage. The discount rate of 12%, as stipulated by the Office of the National Economics and Social Development Board, was used in this financial analysis. The CA-generating machine was anticipated to last 5 years with no salvage value at the end of its life. As the straight-line depreciation method was applied to the machine, annual non-cash depreciation expense was THB 40,000 at the end of years 1 to 5. (In this case studied on small orchid farm in Pathumthani province, Thailand) Annual sales revenue was that from sales of orchid flowers for a period of six months each year, the duration of which is the period of high demand for orchid flowers. One obvious advantage of the CA-generating machine is its ability to prolong the flower shelf life and thereby increase availability of orchid flowers. During the period of high demand, orchid flowers typically command 5 Thai baht (THB 32/USD) extra per bunch. In this research work, a CA-generating machine with daily capability of 150 bunches was used. The investment outlays, monthly sales revenue and major expenditure items are listed below: Financial feasibility analysis

26. Sales revenue Monthly revenue during high orchid demand = 150 bunches x 5 baht x 30 days = 22,500 baht Sales revenue per year (i.e., 6 months of high orchid demand) = 22,500 x 6 =135,000 baht Costs of investment & major expenditure items Cost of machine = 200,000 baht (Depreciate straight-line over 5 years, giving rise to annual depreciation expense of THB 40,000 at end of years 1 – 5) Gas/month = 3,080 baht Electricity/month = 960 baht Cost of electricity and Gas for 6 months = 4,040 x 6 = 24,240 baht/6 months

27. Year Investment Cash Outflow Operating Cash Outflow Total Cash Outflow Cash Inflow Operating Cash Flow Deprecia- tion Net Cash Flow Discount Factor @12% Present Value (THB) 0 200,000.00 - 200,000.00 1 1 24,240.00 135,000.00110,760.0040,000.00150,760.000.8929134,613.60 2 24,240.00 135,000.00110,760.0040,000.00150,760.000.7972120,185.87 3 24,240.00 135,000.00110,760.0040,000.00150,760.000.7118107,310.97 4 24,240.00 135,000.00110,760.0040,000.00150,760.000.635595,807.98 5 24,240.00 135,000.00110,760.0040,000.00150,760.000.567485,541.22 Net Present Value (NPV) 343,459.65 The details of calculation of net present value (NPV) of investment in the machine are tabulated below.

28. The NPV from investment in the machine is THB 343,459.65 and the rule of thumb is that an investment project that yields a positive NPV is worthy of investment as the positive NPV will increase the overall value of the firm. In other words, the positive NPV indicates that the net return on the investment is greater than the machine cost, operating costs and opportunity cost of 12 percent combined. In addition, if the CA-generating machine were improved to better facilitate commercial scale operation, the users would gain from lower unit cost of production with economies of scale. Since the emphasis of this dissertation is on determination of the optimum controlled atmosphere of the orchid flowers, the cost figures are possibly be conservatively estimated, i.e. the cost figures are likely to be on the high side.

29. Experiment 2 To investigate the suitable material use for modified atmosphere packaging (MAP) of Dendrobium orchid To investigate the suitable material use for modified atmosphere packaging (MAP) of Dendrobium orchid flower which had different wrapping materials for cardboard box under normal atmosphere and optimum conditions on extensibility of the shelf life of Dendrobium orchid flower from experiment 1.

30. Testing with Dendrobium Orchids which packed in cardboard box Storage in control temperature at 13 o C with 95% RH Atmosphere Conditions: Normal atmospheres (21% O 2 and 0.03% CO 2 ) - Cardboard box - (PE) film - (HDPE) film - (LDPE) film -(PP) film - (PVC) film 1 box filled with 40 of orchid inflorescence Packing in cardboard box with different thickness of wrapping materials Atmosphere Conditions: Optimum atmosphere from exp. 1 - (PE) film - (HDPE) film - (LDPE) film -(PP) film - (PVC) film To investigate the modified atmosphere packaging (MAP) of Dendrobium orchid under simulated shipment Schematic diagram of experiment to investigate the suitable material use for modified atmosphere packaging (MAP) of Dendrobium orchid

31. Schematic diagram of experiment to investigate the suitable material use for modified atmosphere packaging (MAP) of Dendrobium orchid (continued) Investigate effect of modified atmosphere on qualities of orchids Examine every 3 days Internal atmosphere of packaging -Storage life -Freshness of inflorescence -Weight loss -Respiration rate -Ethylene production -Petal color -Anthocyanin content The qualities parameters were examined same as the Experiment 1.

36. Treatment Storage life (Days)* PE + 5% CO 2 + 2% O 2 15 b ± 2.07 HDPE + 5% CO 2 + 2% O 2 15 b ± 0.97 LDPE + 5% CO 2 + 2% O 2 15 b ± 1.05 PP + 5% CO 2 + 2% O 2 15 b ± 1.15 PVC + 5% CO 2 + 2% O 2 15 b ± 1.15 PE 12 c ± 0.57 LDPE 15 b ± 1.15 HDPE 15 b ± 0.57 PP 18 a ± 1.15 PVC 12 c ± 0.57 Control 9 d ± 0.57 CV (%) 5.95 LSD 1.16 Table 2 Effects of modified atmosphere packaging on storage life of dendrobium orchid * Means (Mean ± S.D.) of different superscripts differ significantly at p <0.05

37. Fig.4.2.10 Effects of modified atmosphere packaging on concentration of oxygen inside the packaging of Dendrobium orchid stored at 13 o C with 95% relative humidity (RH) (Error bars represent S.D.) Internal O 2

38. Fig.4.2.11 Effects of modified atmosphere packaging on concentration of carbon dioxide inside the packaging of Dendrobium orchid stored at 13 o C with 95% relative humidity (RH) (Error bars represent S.D.) Internal CO 2

39. Fig.4.2.2 Effects of modified atmosphere packaging on weight loss of Dendrobium orchid stored at 13 o C with 95% relative humidity (RH) (Error bars represent S.D.) Weight loss

40. Fig.4.2.3 Effects of modified atmosphere packaging on total anthocyanin content of Dendrobium orchid stored at 13 o C with 95% relative humidity (RH) (Error bars represent S.D.) Anthocyanin Content

41. Fig.4.2.4 Effects of modified atmosphere packaging on respiration rate of Dendrobium orchid stored at 13 o C with 95% relative humidity (RH) (Error bars represent S.D.) Respiration Rate

42. Fig.4.2.5 Effects of modified atmosphere packaging on ethylene production of Dendrobium orchid stored at 13 o C with 95% relative humidity (RH) (Error bars represent S.D.) Ethylene production

43. Fig.4.2.6 Effects of modified atmosphere packaging on petal color in lightness (L*) of Dendrobium orchid stored at 13 o C with 95% relative humidity (RH) (Error bars represent S.D.) Petal Color

44. Fig.4.2.7 Effects of modified atmosphere packaging on petal color in chroma (C*) of Dendrobium orchid stored at 13 o C with 95% relative humidity (RH) (Error bars represent S.D.)

45. Fig.4.2.8 Effects of modified atmosphere packaging on petal color in hue angle (H  ) of Dendrobium orchid stored at 13 o C with 95% relative humidity (RH) (Error bars represent S.D.)

46. Fig.4.2.9 Effects of modified atmosphere packaging on freshness of Dendrobium orchid stored at 13 o C with 95% relative humidity (RH) (Error bars represent S.D.) Freshness (Visual appearance)

47. Financial feasibility analysis Prior to the application of PP plastic film MAP on a commercial scale to prolong the storage life of the orchid flowers, it makes an economic sense to estimate the unit cost of the MAP. One pack of PP plastic film is priced at 1,000 baht and can wrap 120 boxes of orchids, each box containing 30 bunches of orchid flowers. As such, the ratio of orchid flower bunches to PP plastic film is 3,600:1, resulting in the unit cost (per bunch) of 0.28 Thai baht. When taking into consideration the unit sellable price (per bunch) of orchid flowers during the high demand season of 5.00 Thai baht, the cost of the PP film MAP accounts for merely 5.6 percent (i.e., 0.28 out of 5.00 baht), still leaving exporters with sufficient profits. However, when the ineffectiveness to prolong freshness of orchid flowers of PP film MAP is factored into the cost-benefit equation, its attractiveness is lost and even pales in comparison with the CA-generating machine.

48. Experiment 3 Effects of controlled and modified atmosphere on biochemical changes involved in petal senescence of Dendrobium orchid during storage. To investigate effect of controlled and modified atmosphere on biochemical changes involved in petal senescence of Dendrobium orchid with the optimum condition from Exp. 1 and Exp.2 compare with Dendrobium orchid kept in cardboard boxes under normal atmospheres.

49. The following parameters were recorded : ACC synthase activity ACC oxidase activity Protein degradation of Dendrobium orchid during ethylene-induced Senescence Protease activity of Dendrobium orchid during ethylene-induced senescence Permeability of membrane: Determination of electrolyte leakage

50. To investigate effect of controlled and modified atmosphere on biochemical changes involved in petal senescence of Dendrobium orchid Testing with Dendrobium Orchids which packed in cardboard box Investigate biochemical change involved in petal senescence of orchids Examine every 3 days - Ethylene production - ACC synthase activity - ACC oxidase activity - Protein quantification - Protease essay - Permeability of membrane Storage in control temperature at 13 o C with 95% RH Atmosphere Conditions: - Normal atmospheres (21% O 2 and 0.03% CO 2 ) - Optimum condition from Experiment 1 - Optimum condition from Experiment 2 1 box filled with 40 of orchid inflorescence Schematic diagram of experiment to investigate effects of controlled and modified atmosphere on biochemical changes involved in petal senescence of Dendrobium orchid during storage.

54. Fig.4.3.1 Effects of controlled atmosphere (5% CO 2 + 2% O 2 ) and modified atmosphere packaging (PP film) on activities of ACC synthase of Dendrobium orchid stored at 13 o C with 95% relative humidity (RH) (Error bars represent S.D.) ACC synthase activities

55. Fig.4.3.2 Effects of controlled atmosphere (5% CO 2 + 2% O 2 ) and modified atmosphere packaging (PP film) on activities of ACC oxidase of Dendrobium orchid stored at 13 o C with 95% relative humidity (RH) (Error bars represent S.D.) ACC oxidase activities

56. Fig.4.3.3 Effects of controlled atmosphere (5% CO 2 + 2% O 2 ) and modified atmosphere packaging (PP film) on total protein of Dendrobium orchid stored at 13 o C with 95% relative humidity (RH) (Error bars represent S.D.) Total Protein

57. Fig.4.3.4 Effects of controlled atmosphere (5% CO 2 + 2% O 2 ) and modified atmosphere packaging (PP film) on activity of protease enzyme of Dendrobium orchid stored at 13 o C with 95% relative humidity (RH) (Error bars represent S.D.) Protease activity

58. Fig.4.3.5 Effects of controlled atmosphere ( 5% CO 2 + 2% O 2 ) and modified atmosphere packaging (PP film) on permeability of membrane of Dendrobium orchid stored at 13 o C with 95% relative humidity (RH) (Error bars represent S.D.) Permeability of membrane

59. CONCLUSIONS The experiments associated with the effect of controlled and modified atmosphere storage on quality and senescence of Dendrobium ‘Queen Pink’ orchid flowers in comparison with the control treatment of the same flower type lead to the following conclusions: 1. Application of controlled atmosphere storage (CA) could prolong storage life of Dendrobium orchid flowers and delay senescence of Dendrobium ‘Queen Pink’ when compared with the control treatment. The optimum condition of controlled atmosphere storage for Dendrobium orchid ‘Queen Pink’ was 5% CO 2 + 2% O 2. Under such a condition, storage life was longest at 28.33 days on average and the flower quality was better retained than in other treatments.

60. 2. Use of modified atmosphere packaging (MAP) could prolong storage life of Dendrobium orchid flowers, retain most flower quality, and delay senescence of Dendrobium ‘Queen Pink’ when compared to the control treatment. The most suitable material for modified atmosphere packaging (MAP) of Dendrobium orchid ‘Queen Pink’ was polypropylene film (PP) as this film type offered the longest storage life of 18.00 days and could retain most quality of flowers better than other film types.

61. 3.Controlled atmosphere storage (CA) and modified atmosphere packaging (MAP) could prolong storage life and maintain quality of Dendrobium orchid ‘Queen Pink’ by reducing weight loss, inhibiting ethylene production, reducing respiration rate and delaying the degradation of color and anthocyanin pigment. 4.Controlled atmosphere storage (CA) under 5% CO 2 + 2% O 2 condition and modified atmosphere packaging (MAP) of polypropylene film (PP) act positively in delaying aging and senescence in biochemical changes involved in petal senescence of Dendrobium orchid ‘Queen Pink’. The delayed aging and senescence are attributable to the decreasing rates of ACC synthase activity and ACC oxidation activity, delayed degradation of total protein, decreasing rate of protease activity, and reduced electrolyte leakage from the cells as it influences loss of turgor and visible wilting.

62. RECOMMENDATIONS FOR FURTHER STUDY The following suggestions are recommended for further research: 1.The optimum condition of CO 2 concentration less than 5% on quality and senescence of Dendrobium orchid during storage need to be investigate. 2. Need to study the effects of controlled (CA) and/or modified atmosphere packing (MAP) storage on quality and senescence of Dendrobium orchids in difference color such as white, blue, purple red and dark pink.

63. Thank You