THAI NGUYEN UNIVERSITY OF AGRICULTURE AND FORESTRY NGUYEN SON HA REDUCING THE CHILLING INJURY ON WATER SPINACH (IPOMOEA AQUATIC) BY USING THE LOW LIGHT TREATMENT WITH SHADE NET BACHELOR THESIS Study Mode: Full-Time Major: Environmental Science and Management Faculty: The Advanced Education Program Batch: 2014 – 2018 Thai Nguyen, 09/25/2018 DOCUMENTATION PAGE WITH ABSTRACT Thai Nguyen University Of Agriculture And Forestry Degree Program: Bachelor of Environmental Science and Management Student name: Nguyen Son Ha Student ID: DTN 1454290010 REDUCING THE CHILLING INJURY ON WATER SPINACH Thesis Title: (IPOMOEA AQUATIC) BY USING THE LOW LIGHT TREATMENT WITH SHADE NET This report is a part of 100495 Cooperative Education in Agriculture Assist Professor I – Chun Pan, National Chung Hsing Supervisor (s): University, Taiwan Dr Nguyen Thanh Hai - Thai Nguyen University of Agriculture and Forestry, Vietnam Abstract: Water spinach (Ipomoea aquatic), one of the most sensitive vegetables with temperature, faced the cold growing condition as the first time in Taiwan, that brings many negative impacts to the farmers The aim was defined the hazard of the chilling injury (CI) with the critical low temperature and time as well as finding a potential treatment within shade net to reduce the CI on water spinach The growth plants were transferred to the growth chamber at 6oC, 8oC and 10oC for hours before using the electronical conductivity (EC), leaf color and 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging to estimate the chilling injury index by comparing the samples in normal light- growing condition with the low light-growing condition with shade net i The results indicate that at 6oC with shade net, water spinach had the lowest EC and DPPH while the converse was true for 10oC Hence, the low light treatment by using shade net is potential and available to apply for reducing the chilling injury Key-words: Water spinach, chilling injury, shade net, EC, DPPH Number of pages: 36 Date of submission: 09/25/2018 Supervisor’s signature ii ACKNOWLEDGEMENTS I would like firstly to emphasize the sincere appreciation to lecturers in the Advanced Education Program as well as lecturers in Thai Nguyen University of Agricultural and Forestry, who have lectured me profound knowledge not only for my subjects but also for my soft skills and gave me a chance to my thesis abroad In addition, I would like to thank all supports and help from Department of Horticulture, National Chung Hsing University for the time I conducted my research in Taiwan It is my pleasure to work with a profound supervisor – Assistant Professor I – Chun Pan, who always helped me any time She also gave me the best conditions, supported all materials for my research and discussed any problems I got whenever I did experiments in her Molecular Physiology Laboratory I would like to give special thanks to Dr Nguyen Thanh Hai, who always supported and cheered me up whole the time I worked oversea He also helps me a lot in spending much time checking my thesis report I consider it is an honor to work with Ms Nicole, who particularly helpful in guiding me toward a qualitative methodology and inspiring me for the whole period of internship time She is always helpful, friendly and very kind to me Without her guidance, I cannot accomplish this thesis Finally, I would like to express my gratitude to my family and friends, who always beside me all the time Their help supports and encouragements created the pump leading me to success Sincerely, Nguyen Son Ha iii TABLE OF CONTENTS DOCUMENTATION PAGE WITH ABSTRACT i ACKNOWLEDGEMENTS iii TABLE OF CONTENTS iv LIST OF FIGURES .vi LIST OF TABLES vii LIST OF ABBREVIATIONS viii PART 1: INTRODUCTION 1.1 Research Rationale 1.2 Research’s Objectives 1.3 Research’s questions and hypothesis 1.4 Definitions PART 2: LITERATURE REVIEW 2.1 Water spinach: 2.1.1: Cultivation Characteristics 2.1.2: Chemical composition, nutritive and medicinal value 2.1.3: Antioxidant activity 2.1.4: Water spinach in Taiwan 2.2: Chilling injury (CI) 2.2.1: Symptoms of chilling injury 2.2.2: Mineral nutrition 10 2.2.3: Light and Photosynthesis 11 2.2.4: Cell membrane changes 12 iv PART 3: MATERIALS AND METHODS 14 3.1: Materials 14 3.1.1 List of instruments 14 3.1.2 List of chemicals 15 3.1.3: Design 16 3.2: Methods 17 3.2.1: Electrical Conductivity Measurements (EC) 17 3.2.2: Analysis of the leaf color 18 3.2.3: Evaluating the damage level of water spinach under chilling stress after three recovering days 18 3.2.4: Determination of antioxidant activity by DPPH- scavenging assay 19 3.2.5: Statistical design 20 PART 4: RESULTS AND DISCUSSION 21 4.1: Electrolyte leakage 21 4.2: Leaf color 23 4.3: Damage level 24 4.4: Scavenging Activity of DPPH radicals 28 PART 4: CONCLUSION 31 REFERENCES 32 v LIST OF FIGURES Figure1: Effect of different treatments on cell membrane permeability of water spinach after 6hrs in different temperatures .21 Figure2: Effect of different treatments on cell membrane permeability of water spinach at particular temperatures after 6hrs (a) In the normal light condition; (b) in the low light condition within shade net 23 Figure 3: The symptom of chilling injury on water spinach during the period: (a) after 6hrs treatment, (b) after three- recovering days and (c) control group 26 Figure 4: The damage level of chilling on water spinach after three recovering day 27 Figure 5: DPPH radical scavenging activity of water spinach extracts 29 Figure 6: DPPH radical scavenging activity of water spinach extracts at particular temperatures after 6hrs (a) In the normal light condition; (b) in the low light condition within shade net 30 vi LIST OF TABLES Table 1: Nutritional value per 100 g (3.5 oz) of Water spinach, raw (USDA Nutrient Database) Table The list of the vegetables, sensitive to chilling temperatures, the lowest safe storage/handling temperature and the symptoms of chilling injury (DeEll, 2004) 10 Table 3.1: Name and commercial company of all instruments used in this study 14 Table 3.2: Properties of liquid nitrogen 15 Table 3.3: Properties of methanol 15 Table 3.4: Properties of 1, 1-diphenyl-2-picrylhydrazyl (DPPH) 16 Table 3.5: Severity level of Chilling injury 19 Table 4: The changes of leaf color after 6hrs in different temperatures 24 Table 5: The distribution of damage level on the leaf surface of water spinach of different treatments at particular temperatures after three recovering days .28 vii LIST OF ABBREVIATIONS CI Chilling injury a* Chromaticity coordinate (redness or greenness) b* Chromaticity coordinate (blueness or yellowness) L* Chromaticity coefficient (lightness) °C Degrees Celsius (unit for temperature measurement) g Grams (unit of Mass) hrs Hours (unit of time) CK Control condition DPPH 1, 1-diphenyl-2-picrylhydrazyl viii PART 1: INTRODUCTION 1.1 Research Rationale According to FAO, United Nation report 2002, the world agricultural growth has slowed down from an average 2.2 percent annually over the past 30 years to 1.5 per cent year until 2030 On the other hand, the world population growth will be growing at an average of 1.1 percent a year up to 2030, compared to 1.7 percent annually over the past 30 years This will put an increased pressure on producing more food to fulfill the requirement of the growing population all over the world Fruit and vegetable production is lower than grain production; however, they contribute important nutrients to the diet, including vitamins A and C, folic acid, potassium, and dietary fiber Among many types of vegetables, Leafy vegetables, such as Kale, Swiss chard, Water spinach, Cabbage, play an important role in Asian daily diet because of their nutritional and medical value Green leafy vegetables occupy an important place among food crops as these provide adequate amounts of crude fiber, carotene, a precursor of vitamin A, vitamin C, riboflavin, folic acid and mineral salts like calcium, iron, phosphorus etc They form cheap and best source of food Green leafy vegetables are highly seasonal and are available in plenty at a particular season and can be easily cooked In recent years, climate change is a “hot” issue in the world It becomes more and more seriously day by day Hence, Climate change affects directly on agriculture, especially on the cultivation of vegetables Little change in the climate will disturb the whole ecology and in-turn the traditional pattern of growing vegetables Vegetables PART 4: RESULTS AND DISCUSSION 4.1: Electrolyte leakage Membrane permeability changes during storage were analyzed by determining the relative electrolyte leakage The leakage enhanced when the vegetables suffered chilling injury The changes of electricity conductivity when using shade net to reduce the light on water spinach for comparing with the samples in the normal light growth condition in the growth chamber in different temperature 6°C, 8°C and 10°C for 6hrs (Figure 1) Figure1: Effect of different treatments on cell membrane permeability of water spinach after 6hrs in different temperatures 21 There was a general trend for all treatments that the percentages of electrolyte leakage of water spinach under the shade net condition were smaller than that under the normal light condition during the chilling stress and the EC of 6°C was significant higher than that of 8°C and 10°C in both treatments, being higher than CK Chilling temperature of 6°C had the most damage on water spinach in both normal light and low light treatment with a peak of 20.85% and 17.32% respectively while the EC of other temperatures had no significant different at 0.05 level with the lowest points belonging to 10°C (Figure 2) Membranes are generally considered the primary sites affected by CI, in which the occurrence of a phase transition leads to increased ion leakage (Murata, 1990; Wang, 1982) (a) 22 (b) Figure 2: Effect of different treatments on cell membrane permeability of water spinach at particular temperatures after 6hrs (a) In the normal light condition; (b) in the low light condition within shade net * Means the same letters are not significantly different at the 0.05 level 4.2: Leaf color Table illustrates the changes of leaf color in different temperature tests by using the shade net as the treatment After 6hrs under chilling stress, water spinach leaves were brighter (greater L* value), slightly less green (greater a* value) and yellower (greater b* value), compared with the water spinach in control condition Be specific, the color in the shade net treatment was less bright and less yellow than the normal light treatment, among the different temperatures tested The shade net treatment at 10°C was brighter with slightly less green color than at 6°C and 8°C with a* value of -12.56, while the water spinach at 6°C had the greenest color However, the yellowest color of this treatment belonged to 6°C with 32.38 By contrast, in the 23 normal light treatment, water spinach at 8°C had the brightest color with L* value of 45.16, while the leaf color was yellowest with b* value of 33.39 In addition, there was no significant different among these treating temperatures at 0.05 level Table 4: The changes of leaf color after 6hrs in different temperatures Normal light 6°C 8°C 10°C CK L* 43.88a 45.16a 44.72a 42.94a a* -11.48a -11.42a -11.27a -11.93a b* 30.55a 33.39a 30.35a 29.38a L* 44.85a 43.74a 45.22a 44.78a a* -12.56a -12.32a -12.05a -12.76a b* 32.38a 29.43a 29.87a 31.17a Shade net *Means in the same row with the same letters are not significantly different at the 0.05 level 4.3: Damage level After 6hrs in chilling temperatures, water spinach had the significant changes in both leaf and stem (Figure 3a) They became weaker and softer, so that the leaves were deformed and the stems could not stand straight, compared with the control plants As observation, the plants of 6°C group seemed to be weakest in both treatments while the plants of low light treatment looked slightly stronger than the rest treatment 24 (a) (b) 25 (c) Figure 3: The symptom of chilling injury on water spinach during the period: (a) after 6hrs treatment, (b) after three- recovering days and (c) control group By contrast, the treated plants remained rapidly after three recovering days to be stronger but brighter and yellower, compared with the control group (figure 3b&c) Besides, there were some visible symptoms of CI appearing on the leaves such as pitting white spots and leaf deformation, which continued to progress over time The chilling-induced injuries observed after chilling at low temperatures were evaluated visually after three recovering days The CI index scale ranged from level to level (Figure 4) The result of observation illustrates the difference between normal light treatment and low light treatment with shad net, compared with the control group (table 5) To be specific, level and level just appeared in the control group with the former overweighting the later (84.62 % and 15.38% respectively) Besides, the water spinach in shade net seemed to be more than 10% of normal leaves as higher as that in normal light treatment in general among different temperatures in which the plants at 10°C were the strongest while the converse was true for the plants 26 at 6°C in both treatments In addition, there was not any leaf having damage more than 31% in shade net treatment, whereas 1.11% of leaves at level were accounted in normal light treatment at 6°C, followed by 0.42% of 10°C group and 0.33% of 8°Cgroup There were no more than 50% of leaves belonging level at both 6°C and 8°C, which were much lower than the group of 10°C with over 65% in normal light condition In conclusion, the low temperature damaged dramatically the growth of water spinach while the shade net had the significant effect on protecting this plant during the low temperature time Figure 4: The damage level of chilling on water spinach after three recovering day 27 Table 5: The distribution of damage level on the leaf surface of water spinach of different treatments at particular temperatures after three recovering days Damage level 6°C CK 8°C 10°C Normal Shade Normal Shade Normal Shade light net light net light net Level 84.62% 44.01% 60.31% 47.10% 69.90% 65.29% 72.55% Level 15.38% 39.62% 37.39% 34.18% 27.51% 30.96% 27.45% level 0% 17.61% 2.30% 16.05% 2.59% 3.01% 0% Level 0% 1.11% 0% 0.33% 0% 0.42% 0% Level 0% 0% 0% 0% 0% 0% 0% 4.4: Scavenging Activity of DPPH radicals The 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radical was widely used in the model system to investigate the scavenging activities of several natural compounds such as phenolic and anthocyanins or crude mixtures such as the ethanol extract of plants DPPH radical is scavenged by antioxidants through the donation of a proton forming the reduced DPPH The color changes from purple to yellow after reduction, which can be quantified by its decrease of absorbance at wavelength 517 nm Radicalscavenging activity increased with increasing percentage of the free radical inhibition Figure shows the average proportion of scavenging activity of DPPH radical of water spinach during three recovering days in which the control group showed the excellent DPPH free radical scavenging activity (58.14%), followed by the group of 10°C in both normal and low light treatment with 40.53% and 45.68% respectively While the radical-scavenging ability of low- light group was higher than that of the remaining treatment in general Besides, the radical-scavenging activity decreased with the decreasing of temperature in both treatments with the lowest activity belonging to the groups of 6°C (19.93% for normal light treatment and 36.16% for low 28 light treatment within shade net) In addition, there is significant different between 6°C and the others at 0.05 level Figure 5: DPPH radical scavenging activity of water spinach extracts To be specific, figure illustrates the changes in the radical-scavenging activity of water spinach during three recovering days In normal light treatment, there was a slight decrease in the percentage of radical- scavenging activity of group 8°C and 6°Cafter three days with the former falling from 45.87% at first day to 32.9% at the last day and the latter decreasing slightly to 19.22%, while the converse was true for 10°C with reaching a peak of 43.51% at day By contrast, in the low light treatment, although water spinaches at 6°C had the lowest percentage of DPPH activity, there was an upward trend in this figure before reaching a peak of 43.71% on the last day While the percentages of DPPH in the remaining temperatures dropped dramatically to 33.97% for 8°C and 43.04% for 10°C 29 (a) (b) Figure 6: DPPH radical scavenging activity of water spinach extracts at particular temperatures after 6hrs (a) In the normal light condition; (b) in the low light condition within shade net 30 PART 4: CONCLUSION The present study was conducted to assess and compare the ability of low light treatments within shade net in reducing the incidence of CI on water spinach Quality of treated water spinach was assessed and compared with different chilling temperatures These results of this study answered clearly the research’s questions, demonstrating that chilling temperatures, specially ºC with the highest percentage of electrolyte leakage and the lowest DPPH radical scavenging activity, have significant effects on the growth of water spinach However, the water spinaches in low light treatment with shade net showed the better appearance and stronger and healthier growth, being significant difference with the normal light treatment among the treated temperatures Hence, this study demonstrated that with approaching low light treatments with shade net, water spinach could be protected successfully from chilling injury, which would increase the potential growth life of this vegetable Besides, the farmer could get information from weather forecast to be willing 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