Reducing the chilling injury on water spinach (ipomoea aquatic) by using the low light treatment with shade net

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

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

DOCUMENTATION PAGE WITH ABSTRACT

Thai Nguyen University Of Agriculture And Forestry

Degree Program: Bachelor of Environmental Science and Management

Thesis Title:

REDUCING THE CHILLING INJURY ON WATER SPINACH

(IPOMOEA AQUATIC) BY USING THE LOW LIGHT

TREATMENT WITH SHADE NET

This report is a part of 100495 Cooperative Education in Agriculture

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 6 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

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

Date of submission: 09/25/2018

Supervisor’s

signature

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 do 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

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

1.1 Research Rationale 1

1.2 Research’s Objectives 3

1.3 Research’s questions and hypothesis 3

1.4 Definitions 4

PART 2: LITERATURE REVIEW 5

2.1 Water spinach: 5

2.1.1: Cultivation Characteristics 5

2.1.2: Chemical composition, nutritive and medicinal value 5

2.1.3: Antioxidant activity 7

2.1.4: Water spinach in Taiwan 8

2.2: Chilling injury (CI) 9

2.2.1: Symptoms of chilling injury 9

2.2.2: Mineral nutrition 10

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

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

LIST OF TABLES

Table 1: Nutritional value per 100 g (3.5 oz) of Water spinach, raw (USDA Nutrient

Database) 6

Table 2 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

LIST OF ABBREVIATIONS

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

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

are generally sensitive to environmental extremes Extreme variation in temperature and limited soil moisture are the major causes of low yields as they greatly affect several physiological and biochemical processes like reduced photosynthetic activity, altered metabolism and enzymatic activity, thermal injury to the tissues, reduced pollination and fruit set etc These will further be magnified by climate change which can exact a heavy toll on the vegetable production Under changing climatic situations, crop failures, shortage of yields, reduction in quality and increasing pest and disease problems are common, which render the vegetable cultivation unprofitable (Koundinya et al., 2014)

In Taiwan, vulnerable area to climate change, Water spinach (Ipomoea aquatic) has become an important leafy vegetable because of storm-tolerant rain, rapid growth and high yields beside their nutritional, medical value According to statistics from Taiwan's Agricultural Annual Report, the cultivated area is more than 2,000 hectares per year Nevertheless, the cultivation of Water spinach has been facing up to CI (Chilling injury) for the first time The production and quality of Water spinach, reduced by CI became a new challenge for Taiwanese farmers Therefore, it is important to establish treatments that can reduce CI of Water spinach There are several techniques are available to either reduce the development of CI symptoms or increase the resistance to CI such as heat-treatment, intermittent warming, controlled atmosphere storage, treatments with calcium, chemicals, waxing, film packaging, genetic modification, and applications with ethylene, abscisic acid, polyamines, or other natural compounds However, these treatments are not suitable and economical

for the condition of cultivating Water spinach in Taiwan because most of its products have been cultivated in the fields which directly are under the climate change effects

Therefore, in this research, the low light treatment by using the shade nets which is easy to apply on the fields and has economic value for the farmers was used

to lessen the effects of light, an important factor on Photosynthesis process, for avoiding the impacts of Chilling injury on Water spinach

1.2 Research’s Objectives

The main purpose of this research is to evaluate the potential of low light treatment by using the Shade nets for preventing the Chilling injury on Water spinach

as well as other vegetables

1.3 Research’s questions and hypothesis

• Research’s questions:

 Is there any impact of chilling temperature on water spinach?

 Which temperature affects significantly water spinach?

 What are the differences between normal light treatment and low light treatment with shade net?

 Is it possible to apply shade net as a solution for farmers?

1.4 Definitions

• Water spinach (Ipomoea aquatic) is a member of the Convolvulaceae

(Morning glory) family and the same genus as the sweet potato (Ipomoea batatas)

Water spinach is an herbaceous aquatic or semi-aquatic perennial plant of the tropics

and subtropics (Wikipedia, 2005)

• Chilling injury (CI) is damage to plant parts caused by

temperatures above the freezing point (32°F, 0°C) Plants of tropical or subtropical

origin are most susceptible Chilling injured leaves may become purple or reddish and

in some cases wilt Both flowers and fruit of sensitive species can be injured

PART 2: LITERATURE REVIEW 2.1 Water spinach:

2.1.1: Cultivation Characteristics

Water spinach will grow in a slightly acidic (5.5-7.5) and fertile soil rich in organic matter Water spinach is not adapted to climates with mean temperatures

below 10°C and the optimal temperature is around 20°C – 30°C It is grown

year-round in the tropics Flowering occurs under short-day conditions and commences from mid-summer onwards Water spinach is perennial in warm climates, but an annual under cooler growing conditions It tolerates very high rainfall, but not frost Water spinach can be grown outside in summer In cool areas, it can be grown in unheated greenhouses in summer, but will require heated greenhouses for a spring crop It prefers full sun but where summer temperatures are very high, it is sometimes grown as a ground cover beneath climbing plants Water spinach should be sheltered from strong winds (Rubatzky, V (1990))

2.1.2: Chemical composition, nutritive and medicinal value

Water spinach is ranked among world’s healthiest food During the 1930’s and post it, the vegetable became widely loved and demanded in the market by parents as well as kids after the famous comic and cartoon Popeye This is a rich source of nutrients, vitamins and minerals that are highly beneficial for skin eyes and brain The spinach contains abundance of water, iron, vitamin C and Vitamin A among other rich nutrients This leaf is also a source of calcium and fiber Water spinach helps us keep ourselves healthy and young complemented with beautiful and radiant skin and strong teeth bones and hair It reduces cholesterol, cures

anemia, controls diabetes, treats jaundice and liver problems, increases metabolism and provides solution for constipation and indigestion, helpful for heart disease patients, prevents cancer, beneficial for eyes, rejuvenates skin and maintains

healthy scalp and hair, also famous for its anti-ageing benefits

Table 2.1: Nutritional value per 100 g (3.5 oz) of Water spinach, raw (USDA

Nutrient Database)

Nutrient Unit 1Value per 100

g

1 cup, chopped = 56.0g

1 shoot = 13.0g

Besides, Water spinach is highly effective in treating ulcers, menstrual pains, toothache, urination, nosebleed etc It also acts as a sedative for people suffering from insomnia or sleeping difficulty (Burkill, 1966) Its juice mixed with water is usable as a cold compress to treat fever Being anti-venom, it is in use to promote vomiting in case of poisoning It treats skin diseases such as ringworm; athlete’s foot etc helps in the prevention of skin cancer and is usable in the treatment of acne, eczema and psoriasis Due to its healing and detoxifying properties, it helps

in providing relief in case of skin itching or insect bites

2.1.3: Antioxidant activity

It is by now commonly accepted that under situations of oxidative stress, reactive oxygen species such as superoxide (O2·-), hydroxyl (OH·-), and peroxyl (·OOH, ROO·) radicals are generated These reactive oxygen species play an important role in degenerative or pathological processes, such as aging (Burns et al., 2001), cancer, coronary heart disease, Alzheimer's disease (Ames, 1983), neurodegenerative disorders, atherosclerosis, cataracts, and inflammation (Aruoma, 1998) The use of traditional medicine is widespread, and plants are still a large source of natural antioxidants that might serve as leads for the development of novel drugs Several anti-inflammatory, digestive, antinecrotic, neuroprotective, and hepatoprotective drugs have recently been shown to have an antioxidant and/or radical scavenging mechanism

as part of their activity (Perry et al., 1999; Lin and Huang, 2002; Repetto and Llesuy, 2002) In searching for novel natural antioxidants, some plants have been extensively studied in the past few years for their antioxidant and radical scavenging components

These include echinacoside in Echinaceae root (Hu and Kitts, 2000), anthocyanin

(Espin et al., 2000), phenolic compounds (Rice-Evans et al., 1997), water extracts of

roasted Cassia tora (Yen and Chuang, 2000), and whey proteins (Allen and Wrieden,

1982)

The aquatic plant water spinach (Ipomoea aquatica Forsk) grows wild and is

cultivated throughout Southeast Asia and is a widely consumed vegetable in the

region Many of the waters where I aquatica grows serve as recipients for domestic

and other types of waste water Because these waters contain not only nutrients, but often also a wide variety of pollutants, such as heavy metals from various human activities, many people risk poisoning Water spinach is also supposed to possess an insulin-like activity according to indigenous medicine in Sri Lanka (Malalavidhane et al., 2000)

2.1.4: Water spinach in Taiwan

In Taiwan, they turned this wild plant into an important cultivated vegetable crop Mainland people were settling on the island of Taiwan by A.D 1167 and maybe before Taiwan is dominated by dry land and aquatic cultivation The drylands are cultivated throughout the province, mainly in Taipei, Taoyuan, Taichung, Changhua, Nantou, Yunlin, Chiayi, Tainan and Pingtung Aquatic cultivation is mainly carried out in Jiaoxi Township of Yilan County, Dali City and Wufeng Township of Taizhong County, Ming Jian Township and Zhushan Township of Nantou County Because of its high heat resistance and moisture resistance, it grows rapidly in the hot and humid seasons and can be harvested from 18 to 28 days after sowing In the past, during the growth period, it was less likely to be harmed by small gold flower worms and white

rust, and few other pests occurred Therefore, it can be harvested about 10 times a year

in Taiwan

2.2: Chilling injury (CI)

2.2.1: Symptoms of chilling injury

Vegetables are sensitive to chilling and after prolonged storage in these temperatures external symptoms of injury are developed and death of the organism occurs (Table1) Plants, which have the visual injuries at temperatures above 15°C, are called “very sensitive to chilling” (Raison, Lyons, 1986) A number of tropical or subtropical plants, such as rice, maize, tomato, cucumber, cotton, soybeans, etc., introduced in the higher latitudes have not acquired substantial resistance to chilling, despite the long history of cultivation in temperate regions (Wilson, 1985)

According to Skog (1998), potential symptoms of chilling injury are surface lesions, water soaking of tissues, water loss, desiccation or shriveling, internal discoloration, tissue breakdown, failure of fruit to ripen, or uneven or slow ripening, accelerated senescence and ethylene production, shortened storage or shelf life, compositional changes, loss of growth or sprouting capability, wilting and increased decay due to leakage of plant metabolites, which encourage growth of microorganisms, especially fungi

Table 2.2 The list of the vegetables, sensitive to chilling temperatures, the lowest safe

storage/handling temperature and the symptoms of chilling injury (DeEll, 2004)

lesions, decay

seed blackening

areas, pitting, decay

acids and proteins, and a drop in the proportion of organic phosphorus and an increase

in inorganic P content (Holobrada et al., 1981; Zia et al., 1994), which is a consequence of a breach of phosphorylation and enhanced decomposition of organic P compounds Mechanisms to reduce the absorption of nutrients by chilling temperatures include depression of respiration and/or oxidative phosphorylation, impair enzymatic transport systems acted with conformational proteins changes in membranes, changes

in membrane potential, reducing the supply of ATP to H+-transporting ATPase, as well as lowering the permeability coefficients for ions (Alexander S LUKATKIN et al., 2012)

2.2.3: Light and Photosynthesis

During and after chilling, the rate of photosynthesis in the leaves of chilling sensitive plants decreased and this is more related to decreasing temperature and lengthening of chilling period and persisted for a long time after transfer of chilled plants in the heat The physiological reasons for the suppression of photosynthesis are the inhibition of phloem transport of carbohydrates from the leaves, stomatal limitation, destruction of the photosynthetic apparatus, damage to water-splitting complex of photosystem I, inhibiting electron transport, and uncoupling of electron transfer and energy storage, changes in the activity and inhibition of synthesis of key enzymes of the Calvin cycle and C4-way (Yordanov, 1992) Cold-sensitive crop species have smaller temperature homeostasis of leaf photosynthesis than cold-tolerant species

Chilling of sensitive plants in light had much stronger effects on the photosynthetic apparatus than chilling in the dark (Alam, Jacob, 2002) It is considered

that a disturbance of photosynthesis due to the light chilling is largely a result of photo inhibition and photo oxidation occurring in the chilling-sensitive plants (but not cold-resistant), as a result of the excess energy of excitation obtained by photosynthetic apparatus Photo inhibition of photosynthesis is the lowering of photosynthetic activity under excessive illumination during chilling It increases with decreasing temperature and increasing light intensity Primary site photo inhibition is the photosystem II However, it was discovered that photo inhibition occurs at relatively low light and low temperature, and the main site of damage is photosystem I Decrease of photosynthesis

at chilling temperatures may be a consequence of photo-oxidative damage to the photosystems in the membranes of chloroplasts, which is manifested by increased lipid peroxidation, degradation of chlorophyll, carotene, and xanthophyll It was caused by activated oxygen species and was associated with reduced antioxidant activity of tissues

2.2.4: Cell membrane changes

Low temperatures alter the physical properties of cell membranes Chilling of sensitive plants leads to multiple changes in their membranes, namely reduce the membrane elasticity, decreasing their compliance and preventing lipid inclusion in their composition, lower lipid fluidity, thereby reducing the activity of several membrane-bound enzymes, including H+ ATPase, increase the lateral diffusion of phospholipids, sterols and proteins in the plasma membrane The phase transition of cell membranes occurs at chilling temperatures in chilling-sensitive plants (but not cold-resistant), and membranes from flexible liquid-crystal turn into solid-gel

enzyme activity (Raison et al., 1971; Lyons, 1973) It is believed that the phase transitions of even small fractions of membrane lipids result in the formation of solid domains that cause cell membrane and cell damage The phase separation of the membrane components is linked with phase transition This phase separation is characterized by the appearance of gel-like sites in the plane of the bilayer lipid These sites are partially or completely free of proteins When the cells were not damaged, the formation of these micro-domains was of a temporary nature The disturbances became irreversible with long-term chilling, and coincided with the appearance of visual symptoms of damage (Thomson, 1989) A number of species of tropical origin have the lateral phase separation temperature some higher (15°C) than in plants from temperate zones (6–8°C) suggesting that plants reduce the freezing point of membranes with the distance from zone of tropical origin