INTRODUCTION
Research Rationale
According to a 2002 FAO report, global agricultural growth has declined from an average of 2.2% annually over the past 30 years to just 1.5% per year projected until 2030 In contrast, the world population is expected to grow at an average rate of 1.1% annually until 2030, down from 1.7% over the previous three decades This trend will intensify the demand for food production to meet the needs of a growing global population While fruit and vegetable production lags behind grain production, these crops are vital for providing essential nutrients such as vitamins A and C, folic acid, potassium, and dietary fiber.
Leafy vegetables, such as Kale, Swiss chard, Water spinach, and Cabbage, are essential in the Asian diet due to their significant nutritional and medicinal benefits These green vegetables are rich in crude fiber, carotene (a vitamin A precursor), vitamin C, riboflavin, folic acid, and essential minerals like calcium, iron, and phosphorus, making them a cost-effective and nutritious food source They are highly seasonal, readily available during specific times of the year, and can be easily prepared for meals.
Climate change has emerged as a critical global issue, increasingly impacting agriculture, particularly vegetable cultivation Even minor shifts in climate can disrupt ecological balance and alter traditional vegetable growing practices.
Vegetable crops are highly sensitive to environmental extremes, particularly temperature fluctuations and limited soil moisture, which significantly reduce yields by impacting essential physiological and biochemical processes These effects include decreased photosynthetic activity, altered metabolism, thermal injury to plant tissues, and reduced pollination and fruit set Climate change exacerbates these challenges, leading to crop failures, lower yields, diminished quality, and heightened pest and disease issues, ultimately making vegetable cultivation less profitable (Koundinya et al., 2014).
In Taiwan, where climate change poses significant challenges, Water spinach (Ipomoea aquatic) has emerged as a vital leafy vegetable due to its storm resilience, rapid growth, and high nutritional value, with over 2,000 hectares cultivated annually However, Taiwanese farmers are now confronting chilling injury (CI) for the first time, which adversely affects the production and quality of Water spinach To combat this issue, it is crucial to develop effective treatments that mitigate CI Various methods, including heat treatment, intermittent warming, controlled atmosphere storage, calcium treatments, and genetic modification, have been explored; however, many of these solutions are not practical or cost-effective for farmers.
3 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
This research explores the use of shade nets as a practical and cost-effective method for reducing light exposure in agricultural fields By mitigating light intensity, this approach aims to enhance the Photosynthesis process and minimize the risk of Chilling injury in Water spinach crops.
Research’s Objectives
This research aims to assess the effectiveness of low light treatment using shade nets to prevent chilling injury in water spinach and other vegetables.
Research’s questions and hypothesis
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?
Null hypothesis: there is no significant difference between normal light treatment and low light treatment with shade net
Alternative hypothesis: there are significant difference between normal light treatment and low light treatment with shade net Therefore, Shade net is possible to avoid chilling injury on water spinach
Definitions
Water spinach (Ipomoea aquatic), belonging to the Convolvulaceae family, is closely related to sweet potato (Ipomoea batatas) This herbaceous perennial plant thrives in aquatic or semi-aquatic environments, primarily found in tropical and subtropical regions.
Chilling injury (CI) refers to the damage inflicted on plant parts by temperatures above freezing (32°F, 0°C), particularly affecting tropical and subtropical plants Symptoms of chilling injury include the development of purple or reddish hues on leaves, as well as wilting Additionally, both flowers and fruits of susceptible species may also experience injury.
LITERATURE REVIEW
Water spinach
Water spinach thrives in slightly acidic, fertile soil rich in organic matter, with a pH range of 5.5 to 7.5 It prefers temperatures between 20°C and 30°C and is not suited for climates with average temperatures below 10°C This plant can be cultivated year-round in tropical regions and flowers during short-day conditions starting from mid-summer While it is perennial in warm climates, it behaves as an annual in cooler areas Water spinach can withstand high rainfall but is sensitive to frost In summer, it can be grown outdoors, and in cooler regions, unheated greenhouses can be used, although heated greenhouses are necessary for spring crops It thrives in full sun but may be grown as ground cover under climbing plants in extremely hot conditions, and should be protected from strong winds.
2.1.2: Chemical composition, nutritive and medicinal value
Water spinach, recognized as one of the healthiest foods globally, gained immense popularity in the 1930s, particularly among families, thanks to the iconic cartoon character Popeye This nutrient-rich vegetable is loaded with vitamins and minerals that support skin, eye, and brain health Packed with water, iron, vitamin C, and vitamin A, water spinach also provides essential calcium and fiber Regular consumption of this leafy green promotes overall health, contributing to youthful skin, strong teeth, bones, and hair, while also helping to reduce cholesterol levels.
This powerful remedy addresses various health issues, including anemia, diabetes, jaundice, and liver problems It boosts metabolism, alleviates constipation and indigestion, and supports heart disease patients Additionally, it plays a role in cancer prevention, promotes eye health, rejuvenates the skin, and maintains a healthy scalp and hair Known for its anti-aging properties, this solution offers comprehensive benefits for overall well-being.
Table 2.1 : Nutritional value per 100 g (3.5 oz) of Water spinach, raw (USDA
Vitamin C, total ascorbic acid mg 55 30.8 7.2
Water spinach is known for its effectiveness in treating various ailments, including ulcers, menstrual pain, toothache, and urination issues, as well as providing relief from insomnia Its juice, when mixed with water, serves as a cold compress for fever, while its anti-venom properties aid in inducing vomiting during poisoning incidents Additionally, water spinach is beneficial for skin conditions such as ringworm and athlete’s foot, and it may help prevent skin cancer It is also useful in treating acne, eczema, and psoriasis, offering relief from skin itching and insect bites due to its healing and detoxifying properties.
Oxidative stress leads to the generation of reactive oxygen species (ROS) such as superoxide, hydroxyl, and peroxyl radicals, which are implicated in various degenerative diseases including aging, cancer, coronary heart disease, Alzheimer's, neurodegenerative disorders, atherosclerosis, cataracts, and inflammation Traditional medicine continues to be a vital source of natural antioxidants, potentially guiding the development of new drugs Recent studies have highlighted that several drugs with anti-inflammatory, digestive, antinecrotic, neuroprotective, and hepatoprotective properties exhibit antioxidant and radical scavenging mechanisms as part of their therapeutic effects.
Recent studies have focused on various plants as potential natural antioxidants, highlighting their antioxidant and radical scavenging properties Notable examples include echinacoside found in Echinacea root and anthocyanins, which are recognized for their beneficial effects.
(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,
Water spinach (Ipomoea aquatica Forsk) is a widely consumed aquatic plant cultivated across Southeast Asia, thriving in waters often contaminated with domestic and industrial waste These environments provide nutrients but also expose the plant to various pollutants, including heavy metals, posing potential health risks to consumers Additionally, traditional medicine in Sri Lanka suggests that water spinach may have insulin-like properties, highlighting its significance beyond mere nutrition.
In Taiwan, a wild plant has been transformed into a vital cultivated vegetable crop, with evidence of mainland settlers on the island as early as A.D 1167 The region is characterized by both dry land and aquatic cultivation, with drylands primarily found in cities such as Taipei, Taoyuan, and Tainan, while aquatic farming is concentrated in areas like Jiaoxi Township and Dali City This vegetable thrives in Taiwan's hot and humid climate, exhibiting high heat and moisture resistance, and can be harvested within 18 to 28 days of sowing Historically, it has shown resilience against pests such as small gold flower worms and whiteflies during its growth period.
9 rust, and few other pests occurred Therefore, it can be harvested about 10 times a year in Taiwan.
Chilling injury (CI)
Vegetables are highly susceptible to chilling injuries, which can lead to visible damage and ultimately the death of the plant after extended exposure to low temperatures Plants exhibiting visual injuries at temperatures exceeding 15°C are classified as "very sensitive to chilling." Despite being cultivated for a long time in temperate regions, many tropical and subtropical plants, such as rice, maize, tomato, cucumber, cotton, and soybeans, have not developed significant resistance to chilling.
Chilling injury can manifest through various symptoms, including surface lesions, water-soaked tissues, desiccation, and internal discoloration Other signs include tissue breakdown, failure of fruit to ripen properly, uneven or slow ripening, and accelerated senescence along with increased ethylene production Additionally, chilling injury leads to a shortened storage life, compositional changes, loss of growth capability, wilting, and heightened decay due to the leakage of plant metabolites that promote microbial growth, particularly 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)
Crop Lowest safe temperature °C Chilling injury symptoms
Asparagus 0-2 Dull, gray-green, limp tips
Bean ( snap) 7 Pitting and russeting
Cucumber 7 Pitting, water- soaked lesions, decay
Eggplant 7 Surface scald, alternaria rot, seed blackening
Okra 7 Discoloration, water-soaked areas, pitting, decay
Pepper 7 Pitting, alternaria rot, seed blackening
Pumpkin 10 Decay, especially alternaria rot
Squash 10 Decay, especially alternaria rot
Sweet potato 10 Decay, pitting, internal discoloration
Tomato( ripe) 7-10 Water-soaking, softening, decay Tomato ( mature- green)
13 Poor color when ripe, alternaria rot
Low temperatures adversely affect the mineral nutrition of plants by hindering the absorption of ions by roots and their movement within the plant This disruption results in an uneven distribution of nutrients among plant organs and an overall decrease in nutrient content Additionally, chilling conditions reduce the activity of nitrate reductase, leading to diminished nitrogen incorporation into amino acids.
Chilling temperatures lead to a decrease in nutrient absorption due to several mechanisms, including reduced respiration and oxidative phosphorylation, which impair enzymatic transport systems These changes are associated with alterations in membrane proteins and potential, resulting in decreased ATP supply to H+-transporting ATPase and lower ion permeability Additionally, there is a notable shift in biochemical composition, characterized by an increase in inorganic phosphorus and a decrease in organic phosphorus, attributed to disrupted phosphorylation and enhanced decomposition of organic compounds (Holobrada et al., 1981; Zia et al., 1994; Alexander S LUKATKIN et al., 2012).
Chilling-sensitive plants experience a significant decrease in photosynthesis rates during and after chilling periods, primarily due to lower temperatures and prolonged chilling durations This reduction persists even after the plants are returned to warmer conditions Key physiological factors contributing to this suppression include inhibited phloem transport of carbohydrates, stomatal limitations, damage to the photosynthetic apparatus, and disruption of the water-splitting complex in photosystem I Additionally, there are changes in enzyme activity and synthesis within the Calvin cycle and C4 pathway Notably, cold-sensitive crop species exhibit a narrower temperature homeostasis for leaf photosynthesis compared to their cold-tolerant counterparts.
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
Chilling-sensitive plants experience disturbances in photosynthesis primarily due to photo-inhibition and photo-oxidation, resulting from excess excitation energy in their photosynthetic apparatus Photo-inhibition, which reduces photosynthetic activity under excessive light during chilling, intensifies with lower temperatures and higher light intensity, predominantly affecting photosystem II However, research indicates that photo-inhibition can also occur at lower light and temperature levels, with significant damage to photosystem I The decrease in photosynthesis at chilling temperatures is linked to photo-oxidative damage to chloroplast membranes, leading to increased lipid peroxidation and degradation of essential pigments like chlorophyll, carotene, and xanthophyll, caused by reactive oxygen species and diminished antioxidant activity in plant tissues.
Low temperatures significantly impact the physical properties of cell membranes in chilling-sensitive plants These chilling effects lead to reduced membrane elasticity and compliance, hinder lipid incorporation, and decrease lipid fluidity Consequently, the activity of membrane-bound enzymes, such as H+ ATPase, is diminished Additionally, chilling temperatures increase the lateral diffusion of phospholipids, sterols, and proteins within the plasma membrane This phase transition from a flexible liquid-crystal state to a solid-gel structure alters the overall properties of the membranes, affecting their functionality.
Phase transitions in membrane lipids can lead to the formation of solid domains that damage cell membranes (Raison et al., 1971; Lyons, 1973) This phase separation results in gel-like sites within the lipid bilayer, which may be free of proteins While these micro-domains are temporary in undamaged cells, prolonged chilling can cause irreversible disturbances and visible damage symptoms (Thomson, 1989) Additionally, tropical plant species exhibit higher lateral phase separation temperatures (around 15°C) compared to temperate plants (6–8°C), indicating that membrane freezing points decrease with increasing distance from tropical regions.
MATERIALS AND METHODS
Materials
All equipment and machines used in this study are listed in Table 3.1 below
Table 3.1: Name and commercial company of all instruments used in this study
Model GC-560 Firstek Scientific Instruments Co.,
Hunter lab Miniscan XE Plus D/8-S Color
Mettler Toledo™, EL20 Benchtop pH Meter Switzerland
Orbital shaker os701 Kansin instruments Co., Taiwan
High-Speed Refrigerated Centrifuge, CR
All chemicals used in this study are shown below
Table 3.2 : Properties of liquid nitrogen
Appearance Colorless gas or liquid
Table 3.4 : Properties of 1, 1-diphenyl-2-picrylhydrazyl (DPPH)
394.32 g/mol Black to green powder, purple in solution Melting point
Water spinach (Ipomoea aquatic cultivar “Taoyuan no.1”), the most popular variety in Taiwan, was cultivated in a greenhouse at National Chung Hsing University The research involved three repeated experiments, where seeds were soaked in water for three days to promote germination before being planted directly in a growth medium composed of a 50:50 mix of soil and perlite in 20-cm plastic boxes Each box accommodated sixteen seedlings, serving as a replication unit The average temperatures during the day and night were maintained at 25°C and 19°C, respectively, with plants receiving water twice daily and fertilized using an N-P-K nutrient solution.
After three weeks of pre-culture, all plants developed over six fully expanded true leaves, and subsequently, groups of seedlings were relocated to a growth chamber to undergo varying degrees of light conditions for six hours.
All plants were transferred to a greenhouse with optimal growth conditions (26°C/20°C, day/night) for three days to recover Subsequently, samples subjected to chilling stress were prepared for measurements, alongside control samples for comparison.
Methods
The electrical conductivity of water from frozen leaves serves as an indicator of cold injury (Shawky I et al, 1983), while electrolyte leakage is utilized to evaluate membrane integrity in plant tissues (Whitlow H.As et al, 1992) Significant variations in the total electrolyte content can occur among different samples, leading to the proposal of expressing electrolyte leakage as a percentage of the total electrolytes released following heat treatment.
For this experiment, the fourth true leaf of water spinach was randomly selected from each sample Each treatment involved soaking the leaves in a plastic tube containing 20ml of deionized water, with three replicates per treatment using two leaves of similar size (0.5cm diameter) Prior to measuring the initial electrical conductivity (EC0) with a digital conductivity meter, the treatments were shaken using an ORBITAL SAKER at 100rpm for 90 minutes Subsequently, all treatments were frozen at -50°C for one day before being shaken for an additional four hours to measure the final electrical conductivity (EC1).
The percentages of electrolytes were calculated as follows:
3.2.2: Analysis of the leaf color
On the third day following chilling stress, five true leaves from each sample per treatment were randomly selected for color analysis using the Hunter Lab Miniscan XE Plus D/8-S Color Analyzer This colorimeter, equipped with a 5 mm diameter measuring area, reported measurements in the CIE L*a*b color space The results were expressed in terms of L* (whiteness/darkness on a scale from 0 to 100, with 100 being the lightest), a* (redness as a positive value and greenness as a negative value), and b* (yellowness as a positive value and blueness as a negative value) (McGuire, 1992).
3.2.3: Evaluating the damage level of water spinach under chilling stress after three recovering days
The treated plants were transferred to normal temperature for recovering during
3 days The first to sixth true leaves were taken and visually observed to classify the
19 severity of chilling injury (SCI), which was divided into 5 levels based on the damage on total leaf surface
Table 3.5: Severity level of Chilling injury
Level Severity of cold injury
1 The damaged leaf is 0-5% of leaf surface
2 The damaged leaf is 6-15% of leaf surface
3 The damaged leaf is 16-30% of leaf surface
4 The damaged leaf is 31-50% of leaf surface
5 The damaged leaf is over 51% of leaf surface
3.2.4: Determination of antioxidant activity by DPPH- scavenging assay
The free radical scavenging activity of the water spinach was investigated using
The DPPH radical scavenging method involved mixing 0.05 mL of a 100 μg/L extract with 9.95 mL of distilled water The assay included 0.2 mL of a 100 mmol/L DPPH radical solution in methanol and 1 mL of the extract solution, with a blank consisting of 0.2 mL of methanol and 1 mL of the extract solution Absorbance was measured at 517 nm using a UV/Vis spectrophotometer, and the percentage of radical scavenging was calculated using a specific formula.
Where Acsorbance of control at 517 nm; As= Absorbance of sample (Huang,2004)
Experiments utilized a completely randomized design, with data presented as mean ± SE from triplicate determinations for each sample One-way analysis of variance (ANOVA) was applied to the analytical data, followed by Tukey’s HSD test for mean comparisons to identify significant differences at p < 0.05 All statistical analyses were conducted using SPSS version 22.0 for Windows.
RESULTS AND DISCUSSION
Electrolyte leakage
During storage, membrane permeability changes were assessed by measuring relative electrolyte leakage, which increased significantly in vegetables exhibiting chilling injury A comparison of electrical conductivity was conducted using a shade net to limit light exposure on water spinach, contrasting it with samples grown under normal light conditions This analysis was performed at various temperatures of 6°C, 8°C, and 10°C over a duration of 6 hours.
Figure1: Effect of different treatments on cell membrane permeability of water spinach after 6hrs in different temperatures
Under chilling stress, water spinach exhibited lower electrolyte leakage percentages when grown under shade net conditions compared to normal light conditions The electrolyte conductivity (EC) was significantly higher at 6°C than at 8°C and 10°C in both treatments, surpassing the control (CK) The chilling temperature of 6°C caused the most severe damage to water spinach, with peak electrolyte leakage at 20.85% in normal light and 17.32% in low light, while other temperatures showed no significant differences at the 0.05 level, with the lowest leakage at 10°C Membrane integrity is primarily affected by chilling injury, as phase transitions lead to increased ion leakage.
The study examined the impact of various treatments on the cell membrane permeability of water spinach at specific temperatures after six hours Results were recorded under normal light conditions and low light conditions within a shade net Statistical analysis indicated that treatments sharing the same letters were not significantly different at the 0.05 level.
Leaf color
Table 4 highlights the impact of temperature on leaf color changes in water spinach under shade net treatment After 6 hours of chilling stress, the leaves exhibited a brighter appearance (higher L* value), a slight reduction in greenness (higher a* value), and increased yellowness (higher b* value) compared to the control group Specifically, the shade net treatment resulted in leaves that were less bright and less yellow than those exposed to normal light At 10°C, the leaves were brighter and slightly less green (a* value of -12.56) than at 6°C and 8°C, with 6°C producing the greenest leaves Interestingly, the 6°C treatment also displayed the highest yellowness with a b* value of 32.38.
Under 24-hour normal light treatment at 8°C, water spinach exhibited the brightest color with an L* value of 45.16, and the leaf color was notably yellow with a b* value of 33.39 Furthermore, there were no significant differences observed among the treatment temperatures at the 0.05 level.
Table 4: The changes of leaf color after 6hrs in different temperatures
*Means in the same row with the same letters are not significantly different at the 0.05 level.
Damage level
After six hours in cold temperatures, water spinach exhibited notable changes in both leaves and stems, becoming weaker and softer The leaves became deformed, and the stems lost their upright posture compared to the control plants Notably, the plants exposed to 6°C appeared the weakest across both treatments, while those under low light conditions showed slightly greater resilience than the other treatments.
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
The treated plants exhibited notable strength and brightness, displaying a yellower hue compared to the control group after three days of recovery However, visible symptoms of chlorosis, including pitting white spots and leaf deformation, emerged on the leaves and worsened over time.
After three days of recovery, chilling-induced injuries were visually evaluated using a CI index scale from level 1 to level 5 Observations revealed significant differences between normal light treatment and low light treatment with shade nets, compared to the control group Specifically, levels 1 and 2 of injury were only present in the control group, with level 1 occurring at 84.62% and level 2 at 15.38% Additionally, water spinach grown under shade nets exhibited over 10% more normal leaves compared to those in normal light treatment, with plants at 10°C showing the strongest growth, while the opposite was true for plants at higher temperatures.
At 6°C, water spinach experienced significant growth damage, with no leaves in the shade net treatment showing damage exceeding 31% In contrast, normal light conditions resulted in 1.11% of leaves at level 4 being damaged at 6°C, along with 0.42% in the 10°C group and 0.33% in the 8°C group Additionally, less than 50% of leaves were classified as level 1 at both 6°C and 8°C, compared to over 65% in the 10°C group under normal light Overall, the study concluded that low temperatures severely hindered water spinach growth, while the use of shade nets provided significant protection during these colder periods.
Figure 4: The damage level of chilling on water spinach after three recovering day
Table 5 : The distribution of damage level on the leaf surface of water spinach of different treatments at particular temperatures after three recovering days
Scavenging Activity of DPPH radicals
The DPPH radical is a key model system for assessing the scavenging activities of natural compounds like phenolics and anthocyanins, as well as crude plant extracts Antioxidants neutralize the DPPH radical by donating protons, resulting in a color change from purple to yellow, measurable by absorbance at 517 nm The scavenging activity increases with higher percentages of free radical inhibition In a study on water spinach, the control group exhibited the highest DPPH scavenging activity at 58.14%, while treatments at 10°C showed activities of 40.53% and 45.68% under normal and low light conditions, respectively Notably, the low-light group demonstrated superior radical-scavenging ability compared to other treatments, with the lowest activity observed at 6°C (19.93% in normal light and 36.16% in low light).
29 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
Figure 6 demonstrates the changes in radical-scavenging activity of water spinach over three recovery days Under normal light conditions, the radical-scavenging activity of water spinach at 8°C decreased from 45.87% on the first day to 32.9% by the last day, while the 6°C group also saw a slight decline to 19.22% In contrast, the 10°C group exhibited an increase, peaking at 43.51% on day three Conversely, in low light treatment, water spinach at 6°C initially showed the lowest DPPH activity but experienced an upward trend, reaching a peak of 43.71% on the final day Meanwhile, the DPPH percentages at 8°C and 10°C dropped significantly to 33.97% and 43.04%, respectively.
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
CONCLUSION
This study evaluated the effectiveness of low light treatments under shade nets in reducing chilling injury (CI) in water spinach It compared the quality of treated water spinach with various chilling temperatures, revealing that 6 ºC resulted in the highest electrolyte leakage and lowest DPPH radical scavenging activity, significantly affecting growth In contrast, water spinach exposed to low light treatment under shade nets exhibited better appearance and healthier growth compared to those under normal light conditions, highlighting a significant difference among the treated temperatures.
This study highlights that using shade nets can effectively protect water spinach from chilling injury under low light conditions, thereby extending its growth life Additionally, farmers can utilize weather forecasts to implement practical strategies, such as installing shade nets, to prevent chilling injury in their crops.
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