Effects of encapsulated ethylene on germination rate and size of mung bean sprouts

By soaking mung bean seeds in 4 hours with ICs complex powder, this study was established to determine the effects of released ethylene gas on the germination rate and quality of mung be

Graduated thesis

EFFECTS OF ENCAPSULATED ETHYLENE

ON GERMINATION RATE AND SIZE OF

MUNG BEAN SPROUTS

School of Agriculture and Food Science

DECLARATION

I, Nghia Khang Tran, hereby state that this graduate thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis

I have clearly stated the contribution of others to my graduate thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, and any other original research work used or reported in my graduate thesis The content of my graduate thesis is the result of work I have carried out since the commencement of my research higher degree candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution I have clearly stated which parts of graduate thesis, if any, have been submitted to qualify for another award

I acknowledge that copyright of all material contained in my graduate thesis resides with the copyright holder(s) of that material Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this graduate thesis

-

Nghia Khang Tran

Date: / /2012

ABSTRACT

Encapsulated ethylene is inclusion complex form of ethylene gas and α cyclodextrin (ICs complex powder) The ICs complex powder can be dissolved into water to release completely ethylene gas which plays a role as third hormone in plant Ethylene gas has been concerned as an advantaged factor which could improve the germination rate and hypocotyl diameter of many plants By soaking mung bean seeds in 4 hours with ICs complex powder, this study was established to determine the effects of released ethylene gas on the germination rate and quality of mung bean sprouts The results indicated that released ethylene gas retains in soaking water and the highest ethylene concentration was 18.85 mg ethylene/kg water at ratio 52.5 mg ICs complex/100 mL water However, the concentration declines over time and this decline reflect differences

of quantity ICs complex Soaking mung beans seeds with ICs complex powder at the level of 0.525 mg/100 mL water enhanced the germination rate by 15% in limited water condition (30mL water/50 seeds) Diameters of bean hypocotyl were significantly improved if the seeds were soaked at level 5.25 mg and 52.5 mg ICs/100 mL water and the radial respectively was 2.57 and 2.94 The 52.5 mg ICs sample had more uniform sprout in length and the smallest standard deviation in comparison with the rest samples However, the average length of sprouts in this sample was decreased under effect of ethylene gas

I would like to send my thanks to Dr Honest Madziva and Dr Lesleigh Force and

Ms Tuyen Thuc Truong for their helps in technical supports, laboratory introduction and laboratory equipment during my research periods

I would like to send my honest thanks to Dr John Schiller for his great helps and his patients in editing my graduate thesis

I would like to thank my parents and my wife for their helps, support and continuous encouragements in all the time of my studying

I would like to send my sincere thanks to Australian Development Scholarships, their helps and support greatly help in accomplishing my Master degree

Finally, I would like to thank all PhD and Master Students who work in same building with me for being friendly and giving helpful advices during my research period

Table of Contents

DECLARATION 1

ACKNOWLEDGEMENT 3

List of tables 6

List of Figures 7

1 Introduction 8

2 Literature Review 9

2.1 Mung beans sprouts introduction 9

2.1.1 Mung beans background 9

2.1.2 Production of mung bean sprouts 10

2.1.3 Mung bean sprouts production process 12

2.1.4 Mung bean sprout quality 14

2.2 Seed dormancy and germination background 15

2.2.1 Seed dormancy 15

2.2.2 Seed germination 15

2.3 Ethylene 17

2.3.1 Ethylene gas properties 17

2.3.2 Role of ethylene gas and other plant hormones during seed germination 17

2.3.3 The effects of ethylene on bean sprouts production 20

2.4 Ethylene-α-cyclodextrin inclusion complexes (ethylene-α-CD ICs) 20

3 Materials and methods 21

3.1 Materials 21

3.2 Methods 21

3.2.1 Determination of the concentration of ethylene gas in soaking water 21

3.2.2 Quantification ethylene gas concentration 22

3.2.3 Determination of the effects of released ethylene at different concentrations on mung bean germination rates in different water conditions 22

3.2.4 Determination of the effects of released ethylene at different concentrations on the size of mung bean spouts 23

3.2.5 Replication and statistical analysis 24

4 Results and Discussion 24

4.2 Effects of released ethylene at different concentrations on mung bean germination rates in

different water conditions 28

4.3 The effects of released ethylene at different concentrations on the size of mung bean spouts 32

5 Conclusions and Recommendations 37

5.1 Conclusions 37

5.2 Recommendations 38

REFERENCES 39

APPENDIX 44

1 The concentration of retained ethylene in soaking water 44

2 Effects of released ethylene at different concentrations on mung bean germination rates in different water conditions 46

3 The effects of released ethylene at different concentrations on the size of mung bean spouts 49

List of tables

Table 1 Production methods and consumption forms of three types of sprouts 11

Table 2 Statistical analyst STDEV in length of mung bean sprout treated at different weights of ICs complex powder 34

Table 3 Released ethylene concentration in soaking water at different weight of ICs complex (mg ethylene/kg water) 44

Table 4 Average concentration of released ethylene gas at different weight of ICs complex powder (mg ethylene/kg water) 45

Table 5 Standard deviation of ethylene concentration at different weight of ICs 45

Table 6 Differences average concentration of released ethylene in soaking water, with and without beans 46

Table 7 Average percent (%) of geminated seeds over soaking time (full water condition) 46

Table 8 Stdev of average percent geminated seeds (full water condition) 46

Table 9 Average percent (%) of geminated seeds over soaking time (limited water condition) 47

Table 10 Stdev of average percent geminated seeds (limited water condition) 47

Table 11 One-way ANOVA: Ethylene concentrations 52.5 mg versus soaking conditions 48

Table 12 One-way ANOVA: Ethylene concentrations 5.25 mg versus soaking conditions 48

Table 13 Germination rate (21 hrs.) versus water condition 48

Table 14 Germination rate (27 hrs.) versus treatment (limited water) condition) 49

Table 15 Average length and diameter of bean sprout hypocotyls 49

Table 16 Final length and diameter of bean sprout hypocotyls 50

Table 17 Distribution (%) in length of mung bean sprouts 50

Table 18 Average distribution (%) in lenght of mung bean sprouts 51

Table 19 Stdev of average distribution 51

Table 20 Thickness (mm) versus Treatment 51

Table 21 Length (mm) versus Treatment 52

Table 22 STDEV (Length) versus Treatment 52

List of Figures

Figure 1 Typical production steps during the production of seed sprouts (Food

Standards Australia New Zealand 2010) 10 Figure 2 Growth rate of sprouts (Bari et al 2011) 13 Figure 3 The schematic of determining the morphological indexes of mung bean sprout (Rui et al 2011) 14 Figure 4 Germinating seed structure (Graeber et al 2010) 16 Figure 5 Triple responses on Arabidopsis 3-day-old seedling (Guzmán & Ecker 1990) 19 Figure 6 Experiment design to determine the effects of released ethylene at different concentrations on mung bean germination rates in different water conditions 22 Figure 7 Measurement methods for length and radical diameter 23 Figure 8 Concentration of retained ethylene in soaking water for 0.525 mg ICs

complex/100 ml water 24 Figure 9 Concentration of retained ethylene in soaking water for 5.25 mg ICs

complex/100 ml water 25 Figure 10 Concentration of retained ethylene in soaking water for 52.5 mg ICs

complex/100 ml water 26 Figure 11 Concentrations of retained ethylene in soaking water 525 mg ICs

complex/100 ml water 26 Figure 12 Differences in ethylene concentration after 4 hours of soaking in water, with and without mung beans 29 Figure 13 Germination rates of mung bean seeds in full water conditions 30 Figure 14 Germination rates of mung bean seeds in limited water conditions 31 Figure 15 The average diameter of bean sprout hypocotyls in different weights of ICs complex powder 33 Figure 16 The average length of bean sprouts for different weights of ICs complex powder 34 Figure 17 The distribution of length of bean sprouts at different weights of ICs complex 35 Figure 18 Mung bean sprouts at different treatments 36

1 Introduction

Ethylene (H2C=CH2) is the simplest alkene which has known as a plant hormone (Hart 2007) Much early research has implied that exogenous ethylene can promote the germination of many non-dormant seeds, and also overcome dormancy in dormant seeds at concentrations from 0.1 to 200 µl.l-1 (Kepczynski & Kepczynska 1997; Matilla

2000; Nascimento 2003) Furthermore, early studies on Arabidopsis plant showed that,

under effects of ethylene in dark growing condition , the stems displayed short roots, an exaggerated apical hook and a short and thick hypocotyl (Guzmán & Ecker 1990; Larsen & Chang 2001) This phenomenon has been known as triple responses of plants under ethylene gas effect Since its biological functions, ethylene has been widely studied in plant such as ripening fruit, growing plant or even sprouting beans to produce bean sprouts

According to a report from Food Standard Australian and New Zealand (FSANZ 2010a) mung bean sprouts are the most common bean sprouts in Asian countries and Australia This vegetable usually is eaten in raw as components of salads or slightly cooked in various dishes (Bari et al 2011) Bean sprout producing is very simple, mung beans are soaked then sprouting for bean sprout (FSANZ 2010a) Quantity of beans sprout production can be assessed by main criteria as germination rate, length and diameter of hypocotyl, root length and yield of sprouts By flushing ethylene gas or using ethephon (an ethylene-releasing compound in liquid form) during sprouting, Tajiri (1982) and Ahmad (1993) achieved successes in improving either mung bean germination and sprout diameter However because of high volatility, difficult storage and flammable risk (Zimmermann & Walzl 2000), convenience in use of ethylene gas is a big issue

For improving quality and usability of ethylene gas, Ho Joyce and Bhandari (2011a) successfully encapsulated ethylene gas into the form of inclusion complexes with α cyclodextrin (ICs complex powder) This powder can be dissolved in water for completely releasing ethylene gas (Ho, Joyce & Bhandari 2011b) The released ethylene gas from ICs complex powder promisingly can have similar effects on mung bean sprout as reported in the early researches However, while the early studies paid more attention on determining effects of ethylene on bean sprout in sprouting period,

soaking is also an essential period which can effect on the germination rate (Bayindirli 2010; Beyer & Morgan 1970; Chen, Breene & Schowalter 1987; Ross 1995) Therefore, this research aim to determine effects of released ethylene gas on germination rate and quality of mung bean sprouts by soaking bean seeds with ICs complex powder

To archive the objective, this research was established to determine the concentration of released ethylene in soaking water and its change over soaking Effects of released ethylene on germination rate and hypocotyls size of mung bean sprout also will be determined in this study The results not only could widen applications of ICs complex powder in mung bean sprout production but also could be extended for further research on other plants

2 Literature Review

2.1 Mung beans sprouts introduction

2.1.1 Mung beans background

Mung bean (Vigna radiata or Phaseolus aureus) is a common name of legume species (Fabaceae family) which has its origins in Northeastern India or Myanmar (Li et

al 2010) Mung beans is a short-season, indeterminate, small-seeded tropical pulse crop, and its seeds are classified as being non-endospermic seeds (Brink & Belay 2006) There are more than 2,000 types of mung beans which have very broad plant types, forms and adaption (Australian Mungbean Association 2010) Normally, mung beans can be classified into two basic types; (i) green gram is grown mainly for human food (for cooking or as fresh bean sprouts), and (ii) golden or yellow gram which is mainly grown for use as animal food, or as a green manure or cover crop (Winch 2006) Since it contains high levels of protein (27%), minerals and vitamins, mung beans are widely grown in India, Thailand, Indonesia, Bangladesh, Philippines, Africa, Australia and the Americas (Li et al 2010; Winch 2006)

According to a report of FSANZ (2010b), mung beans are produced for sale as whole beans, sprouted or processed into flour; they are also exported Within Australia, mung beans are mainly grown in parts of central Australia, southern Queensland and northern New South Wales, with annual production ranging from 30,000 to 50,000 tons

The mung beans produced in Australia are classified into three grades - sprouting grade beans, cooking grade beans or processing grade beans Sprouting grade beans have

the potential to return the highest profit, with 1,375/38,974 tons of mung beans being used to produce bean sprouts during 2006-2007 (FSANZ 2010b) According to the Australian mung beans classification (2010), six varieties of mung beans can be used for the production of bean sprouts in Australia, these varieties being Berken, Crystal, Emerald, Regur, Satin II and White Gold

2.1.2 Production of mung bean sprouts 2.1.2.1 Background information

Sprouts is the name given to sprouted seeds which are grown from seed and harvested before formation of the first leaves (Food Safety Authority of Ireland 2011) Sprouts, especially bean sprouts, are widely consumed in some parts of Asia as traditional foods In the period 2000 to 2010 the consumption of seed sprouts (especially mung bean sprouts) as a vegetable, has increased in Western Europe and America, reflecting the fact that they are the good and inexpensive sources of

vitamins(Mbithi et al 2001; Peñas et al 2010) In addition, the consumption of bean sprouts in Asian dishes

is increasing, because they are considered as a natural healthy food and due to culinary factors or dietary supplements (FSANZ 2010a; Peñas et al 2008) Seed nutrition also can also be improved by germination, since the anti-nutritional factor in legumes can be reduced or eliminated during germination (Mbithi et al 2001)

Figure 1 Typical

production steps during

the production of seed

sprouts (Food Standards

Australia New Zealand

2010)

2.1.2.2 Sprout market

Sprouts vary in texture and taste because many types of seed can be sprouted, including Adzuki, alfalfa, buckwheat, cabbage, clover, cress, garbanzo, green peas, lentils, mung beans, radish, rye, sesame, wheat, and triticale (FSANZ 2010a; Schrader 2002) Some are spicy (e.g radish and onions), some are used in Asian foods (e.g mung beans), and others are delicate (e.g alfalfa) or are used in salads and sandwiches to add texture (Schrader 2002) Bari (2011) listed three main types of sprouts which can be found in markets, these being the bean sprouts type, young sprouts/mini sprouts and green sprout/micro-green sprouts These sprout types are classified based on production quantity, production method, and consumption style (Table 1)

Approximately 40 million tons of mung bean, black matpe bean, and soy bean sprouts are produced annually world-wide A large proportion of the bean sprouts are consumed in Asian countries, while there is also increasing use of them in western countries, especially in Europe (Bari et al 2011) Bari (2011) also estimated that between 30 and 100 tons of bean sprouts are provided daily by ten large-scale factory producers in Europe According to the food standards of Australian and New Zealand (2010), the annual revenue of the sprouting industry in these two countries is

Table 1 Production methods and consumption forms of three types of sprouts* (Bari et

al., 2011)

approximately $AUD 30 million, with the industry creating about 300 jobs

Mung bean sprouts are the most common form of bean sprouts in most Asian countries They are usually eaten raw as components of salads, or slightly cooked in various food dishes For example, uncooked mung bean sprouts are used in Vietnamese spring rolls and in a variety of Malaysian and Thai dishes (Bari et al 2011) About 400,000 tons/year of bean sprouts are consumed in Japan (Bari et al 2011) Mung bean sprouts are the predominant bean sprouts product in Australia (Food Standards Australia New Zealand 2010)

2.1.3 Mung bean sprouts production process

Mung bean sprout production is a simple germination process that requires neither sunlight nor soil The process can be completed in a period of from 4 to 8 days, without seasonal limitations (Lal & Shanmugasundaram 2001) Typical steps in the production of commercial bean sprouts are shown in Figure 1 In the sprout production process, there are three main steps which can potentially have a direct effect on bean

or mung bean germination, these steps being: selection of seed, initial soaking, and sprouting of the seed (Food Standards Australia New Zealand 2010)

2.1.3.1 Seed selection

For processing mung bean sprouts, the Berken variety is the most suitable mung bean variety for producing bean sprouts, because it can produce the large sprouts preferred by buyers (Food Standards Australia New Zealand 2010) The mung bean seeds should be medium in size and have a smooth surface, because small hard-seeded mung bean seeds are often associated with poor germination and weak sprout growth (Bari et al 2011) Mung bean seed needs to be washed to reduce the risk of contamination before moving to the initial soaking step (Beyer & Morgan 1970) Lal and Shanmugasundaram (2001) have also suggested that mung bean seeds (15% moisture) be stored at 100C or below in dry conditions (relative humility <85%)

2.1.3.2 Initial soaking (Pre-germination)

Water is an essential resource for bean seed germination The water absorbance process of mung bean can activate the mung bean germination process (Bayindirli 2010; Beyer & Morgan 1970) The volume of water used to soak mung bean sprouts is five time higher than the volume of the mung bean seed layer, while the time required for soaking is from 8 to 12 hours at room temperature (Chen, Breene & Schowalter 1987) Other research (Ross 1995) has showed that when mung bean seeds are soaked in water at 30 0C for 5 to 7 hours and then sprinkled with water at a temperature at 30 0C for 15 minutes every 4 hours, a germination rate of about 90% can be achieved for mung bean

2.1.3.3 Sprouting (germination)

2.1.3.3.1 Germination container

Since mung beans increase in size about 6 fold by the time sprouting is completed, the size of the chamber used for the sprouting process should be at least 6 times greater than the mung bean layer Drainage is an essential requirement of the germination container to prevent the development of anaerobic conditions, which can lead to death of seedlings and accelerate microbial spoilage Thick layers of cotton or paper can be used as the drainage layers Light is not necessary in the germination

process, because it can create a green color in the primary mung bean leaf To prevent this defect, the germination container should

be made of, or covered by, light

placed in dark environment The relative humidity in the container should be remained around 90% (El-Adawy et al 2003; Schrader 2002)

Figure 2 Growth rate of sprouts (Bari et al 2011)

2.1.3.3.2 Germination conditions

Mung bean seeds fully germinate within 2-3 days in dark conditions at room temperature, and sprouts can achieve a length of at least 5 cm after 4-5 days (Figure 3) (Lal & Shanmugasundaram 2001) In many countries, the marketable length of mung bean sprouts is at least 5 cm, while the maximum time for mung bean sprouting ranges from 6 to 8 days During sprouting, water should be applied by sprinkler irrigation every

4 to 6 hours to provide adequate moisture and to prevent mold growth (Chen, Breene & Schowalter 1987; Lal & Shanmugasundaram 2001) The temperature of the sprinkled water also plays an important role in the mung bean germination process Research of Tajiri (1980) has shown that mung bean seeds have a high germination rate if they are sprinkled with water at 25 - 300C A low temperature of sprinkled water can lead to a decrease in the length and thickness of the hypocotyls of beans, while the hypocotyls can become dark in color when the temperature of the sprinkled water is excessive (Tajiri 1980)

2.1.4 Mung bean sprout quality

Mung bean sprout quality is assessed in terms of the length and diameter of the

hypocotyl, root length, yield of sprouts and color of the sprouts Chen (1987) and Tajiri (1982) have reported that the most desirable sprouts have short roots (radicals) (15-25 mm), and relatively long (5-8 cm) and wide diameter hypocotyls (2- 3 mm) The thickness of the hypocotyl is important

unattractiveness (i.e color) of the roots can decrease the appeal of mung bean (Chen, Breene & Schowalter 1987)

The yield of sprouts is expressed as the weight (g) of sprouts per 100g of dry (unsoaked) mung beans (Chen, Breene & Schowalter 1987) Tajiri (1982) also provided

Figure 3 The schematic of determining

the morphological indexes of mung bean

sprout (Rui et al 2011)

reasonable parameters of color and hypocotyl hardness for commercial mung bean sprouts; the preferred color is called ‘milk white’, with the ranges of L, a and b are 12-

18, (-2.3)–(-1.7) and 1.9–2.8, respectively The marketability standard for the hypocotyl hardness is 600-650 dyne/mm2

2.2 Seed dormancy and germination background

2.2.1 Seed dormancy

According to Baskin (2004), Copeland and McDonald (2001), seed dormancy is defined as a state in which seed does not have the capacity to germinate, even under environmental conditions normally favorable for germination Seed dormancy is a genetically inherited trait which ensures plants are able to survive and adapt to their environment (Copeland & McDonald 2001) There are two mechanisms of dormancy in dormant seed, primary and secondary dormancy However, not all plant species produce dormant seeds There are a number of non-dormant seed species (and some species without full physiological dormancy), which have the capacity to germinate over

a wide range of normal physical environmental conditions (temperature, light/dark, etc.) (Baskin & Baskin 2004) Non-dormant seed also will not germinate if the growing environment is lacking in one or more physical factors including water, appropriate temperature and light (Copeland & McDonald 2001) This state is the resting state of the seed, and is referred to as the quiescence state of non-dormant seeds Seed in the quiescence state will germinate when they experience appropriate environmental conditions within their range of germination requirements (Baskin & Baskin 2004) According to M.Brink and G.Belay (2006), mung beans have no deep seed dormancy, but the germination of the seed can be affected by a hard seed coat

2.2.2 Seed germination

Although germination is a period in the growing process of every plant, a universally useful biochemical marker of the progress of germination has not yet been found (Ross 1995) According to Deno (1993), the time when the radicle is first detected breaking through the seed coat can be taken as the point of completion of germination of seed Expressed in other terms, germination commences with the

water imbibition of the seed and ends with the start of elongation by the embryonic axis, usually the radicle (Ross 1995)

There are a number of

internal and external factors which

can affect the germination process

Water, temperature and light are the

important environmental factors that

can have a considerable impact on

seed germination (Ross 1995)

Copeland (2001), water is a basic

requirement for the germination of

imbibition can activate enzymes that are essential for the commencement of the germination process In addition, these authors indicate that a high-humidity environment, as a result of sprinkling with water, is also an optimum condition for the sprouting of seed In relation to temperature, each plant species has a specific temperature range for germination Generally, seed of plants from temperature climates require a lower temperature than seed of plants in tropical regions The optimum temperature for most seed is between 15 and 300C, with the maximum temperature for most seed being between 30 and 400C (Copeland & McDonald 2001; Ross 1995)

The germination of seed can be described and assessed by germination rates and germination curves A germination curve is plotted in terms of the germination rate versus germination time (days) (Deno 1993) The germination rate is usually expressed

in percentage terms, which is normally determined at specific time intervals over the course of germination (Ross 1995) It is calculated as the number of germinated seeds divided by the total number of seeds (Bari et al 2011)

Figure 4 Germinating seed structure (Graeber et

al 2010)

2.3 Ethylene

2.3.1 Ethylene gas properties

Ethylene which is also known as ethane, has the structural formula of H2C=CH2,

and molecular weight of 28.52 Ethylene is a colorless flammable gas with a sweet odor The lower limit explosive level of ethylene gas in air at 0.1 MPa and 200C is 2.75 vol%

or 34.6 g/cm, and the upper explosive level is 28.6 vol% or 360.1 g/cm The ethylene ignition temperature is between 425 and 5270C It is the largest-volume petrochemical produced worldwide, and is produced mainly from petroleum-based feed stocks by thermal cracking (Zimmermann & Walzl 2000)

The solubility of ethylene in water is five times greater than that of oxygen (Abeles, Morgan & Mikal Saltve 1992) The solubility of ethylene at 00C and 101 kPa is 0.026 mL/mL H2O (Sundaram, Shreehan & Olszewski 2000) and at 200C is 131 mg/l (Screening Information Data Set 1998) The solubility of ethylene in water decreases with increasing temperature (Bradbury et al 1952) The solubility of ethylene also decreases significantly in response to adding ammonium sulfate ((NH4)2S04) at saturated levels (Beyer & Morgan 1970) Ethylene gas concentration can be measured

by gas chromatography with a flame ionization detector, or by using gas chromatography combined with mass spectrometry (Lawton 1991)

2.3.2 Role of ethylene gas and other plant hormones during seed germination

Together with the environmental factors, both the germination and dormancy of seed are affected by the plant hormones, abscisic acid (ABA), gibberellins (GA) and ethylene Although the concentrations of these hormones are very low, they and their interactions play a crucial role in the regulation of seed dormancy and germination (Kucera, Cohn & Leubner-Metzger 2005)

2.3.2.1 Role of ABA and GA

There appears to be general agreement among botanical scientists that abscisic acid (ABA) and gibberellins (GA) are the main hormones among plant growth regulators that are involved in the mechanisms of seed dormancy and germination In the

hormone-balance model, ABA (inhibitor) and GA (promoter) simultaneously and antagonistically regulate the onset, maintenance and termination of dormancy (Baskin & Baskin 2004)

The increase in ABA biosynthesis can enhance seed dormancy or delay germination ABA is responsible for maintaining the dormant stage in plant seeds, and also inhibits seed germination (Frey et al 2004; Koornneef, Bentsink & Hilhorst 2002) Primary dormancy in seed cannot occur without the presence of this hormone (Kucera, Cohn & Leubner-Metzger 2005) The role of ABA in inhibiting germination has been described by Schopfer and Plachy (1984) and Frey (2004) ABA inhibits the extension of the embryo, which can lead to the rupturing of the endosperm during the water uptake stage of seeds This effect of ABA is by regulating the ion-channel activities or changing the water uptake tissue (Kucera, Cohn & Leubner-Metzger 2005) In addition, ABA is believed to be one of the factors that inhibit root elongation in some of plants (Subbiah & Reddy 2010)

Contrary to the effects of ABA, GA can release the seed from a state of dormancy and counteract the ABA effects indirectly or directly (Kucera, Cohn & Leubner-Metzger 2005) In addition, the most important function of the GA hormone is

to promote and maintain seed germination (Koornneef, Bentsink & Hilhorst 2002) This hormone promotes the elongation of the embryo and weakens the endosperm cap, leading to an improvement in the germination rate

2.3.2.2 Ethylene gas as a plant hormone

According to Baskin (2004), ethylene plays a role as the third plant hormone involved in the regulation of seed dormancy and germination of many plant species Much early research has implied that exogenous ethylene can promote the germination

of many non-dormant seeds, and also overcome dormancy in dormant seeds (Kepczynski & Kepczynska 1997; Matilla 2000; Nascimento 2003) For example, ethylene can remove primary dormancy in the seed of peanut, apple, redroot, pigweed and cocklebur (Kepczynski & Kepczynska 1997) It is also an essential factor for the

germination of lettuce seed under favorable conditions, and for the germination of A

caudatus and C arietinum seeds and tobacco (Kucera, Cohn & Leubner-Metzger 2005;

Matilla 2000) Ethylene at concentrations from 0.1 to 200 µl.l-1 is usually sufficient to promote seed germination of most plant species However, the concentration level of exogenous ethylene has to be limited, for excessive concentrations of exogenous ethylene can result in the inhibition of germination of dormant seed (Kepczynski & Kepczynska 1997)

In ABA hormone interactions, the exogenous ethylene counteracts the effects of ABA hormone, to overcome the inhibition of germination in dormant and non-dormant seeds (Bradford & Nonogaki 2007) Studies have suggested that ethylene may enhance the vigor and stimulate the metabolism of some plant species (Nascimento 2003), or increase the respiration in seeds Surprisingly, although ethylene interferes with the ABA hormone in germination, ethylene has the same effects as ABA in reducing the length of the radical of seeds (Kucera, Cohn & Leubner-Metzger 2005)

In GA hormone interactions, ethylene acts in concert with GA to promote dormancy termination and germination in seeds (Bradford & Nonogaki 2007) A number of studies have showed that GA and ethylene or ethephon, an ethylene-releasing compound, can speed up dormancy release These hormones also support embryo extension and weaken the tissues surrounding the radicle, which facilitates seed germination in beechnut and tobacco (Calvo, A P et al 2003; Calvo, Angel Pablo, Nicolás, Lorenzo, et al 2004; Calvo, Angel Pablo, Nicolás, Nicolás, et al 2004; Kucera, Cohn & Leubner-Metzger 2005)

A number of researchers have shown the effects of ethylene on hypocotyls, roots and stems during germination, which are called triple responses (Figure 5) According to

Guzmán & Ecker (1990), in seedlings grown in dark conditions, Arabidopsis displayed

short roots, an exaggerated apical hook and a short and thick hypocotyl, due to the

effects of ethylene In dark conditions, ethylene is involved in the re-orientation of cell expansion of hypocotyls This phenomenon leads to a decrease in cell elongation and increases the radical thickness (Larsen & Chang 2001; Le et al 2005)

2.3.3 The effects of ethylene on bean sprouts production

There has been considerable recent research relating to the effects of several effects of ethylene gas on bean sprout production By flushing ethylene gas through mung bean seeds during sprouting, Tajiri (1982) showed that ethylene gas can enhance the germination of mung bean and the sprout qualities Research undertaken

by Tajiri has suggested that 50ppm of ethylene, when used to flush mung bean seeds

at 1.0l/min for 60 minutes once a day starting 12 hours after planting, can improve the elongation and thickening of the hypocotyl of mung bean sprouts, and increases the weight and vitamin C content of sprouts as well In addition, the cultivation sprouting period was shortened by 2 to 3 days

Ethephon (2-chloroethyl phosphonic acid), an ethylene-releasing compound (Robbins et al 1985), has also been used to enhance the qualities of mung bean sprouts Ahmad & Abdullah (1993) noted that treating mung bean sprouts with ethephon can increase the hypocotyl diameter and decrease the length of root It can be said that, the majority of past researches on the effects of ethylene gas have focused on the sprouting period There has been limited research on the effects of ethylene gas on the germination and qualities of mung bean in soaking period

2.4 Ethylene-α-cyclodextrin inclusion complexes (ethylene-α-CD ICs)

Since ethylene gas is flammable, of high volatility and exploitable (Zimmermann

& Walzl 2000), one of important issues in relation to its potential use relate to the issues of safety in its storage and distribution The quality and usability of ethylene gas can be improved by using an encapsulation technique which can capsulate volatile compounds into encapsulating materials (Bhandari, D'Arc & Padukka 1999) Early research has indicated that cyclodextrins (CDs) (most popular α -, β - and γ –CD) can

be have used as encapsulation materials (Hedges, Shieh & Sikorski 1993)

Recently, Ho (2011a) achieved success in encapsulating ethylene gas into inclusion complexes with α cyclodextrin under pressure from 0.2–1.5 MPa for 12–120 h This powder, which is known as ethylene-α-cyclodextrin inclusion complex (α-CD ICs or ICs complex powder), can be dissolved in water for complete release of the ethylene gas (Ho, Joyce & Bhandari 2011a) The author also indicated that the ethylene gas release speed is affected by relative humidity and ambient temperature In addition, a number of researches have shown that dissolved ethylene can remain in water at different concentrations in different conditions at ambient temperature (Beyer & Morgan 1970; Bradbury et al 1952; Ho, Joyce & Bhandari 2011a, 2011b) In comparison with ethylene gas, the ICs complex powder is more stable and convenient, less volatile and easier for storage or packaging (Ho, Joyce & Bhandari 2011a) Reflecting the potential benefits of encapsulated ethylene powder, the applications of ethylene gas have shown promise in the form of this new product

3 Materials and methods

3.1 Materials

Ethylene-α-cyclodextrins inclusion complex (ICs complex) was prepared and provided by Ho (Ho, Joyce & Bhandari 2011a) Ammonium sulfate was obtained from Sigma – Aldrich (USA) The Berken mung bean variety which was used for the sprouting study was provided by the Hertzberg mung bean company Mung bean seeds were germinated on the basement of a desiccator lined with sponge cloths

3.2 Methods

3.2.1 Determination of the concentration of ethylene gas in soaking water

Four different weights of ICs complex: 1.05 mg, 10.5 mg, 105 mg and 1050 mg were put in 250 ml beakers containing 200 ml of distilled water; 5ml water samples were taken in 5 different positions after 1, 2, 3, 4, 6, 8, 12, 24, 48, 72 and 96 hours of soaking, using a 5 ml syringe A 16.5 mL vial containing 4 gram of ammonium sulfate was prepared and sealed with a septum; a 5 ml sample of the soaking solution was injected into the vial According to Beyer and Morgan (1970), a saturated solution of ammonium sulfate (NH4)2S04 can significantly reduce the solubility of ethylene in water

Therefore, ammonium sulfate was used to extract the dissolved ethylene from the soaking water for concentration analysis The vial was then shaken for 20 minutes at a speed of 400 Five hundred microliter in vial headspace was collected and analyzed for the concentration of ethylene gas by using GC The concentrations of retained ethylene

in the soaking water were calculated by the results of ethylene gas concentration in the vial head space

3.2.2 Quantification ethylene gas concentration

The quantification of ethylene gas in the head space of the vials was analyzed using gas chromatography (GC) (Shimadzu 17A, Tokyo, Japan) The GC was fitted with

a stainless steel column (3 m x 1.2 mm) packed with Porapak N (100–120 Mesh) (Waters, Milford, MA, USA), and run with operating conditions of helium carrier gas at

40 ml/min The temperatures of the oven, detector (FID) and injector temperature were

900C, 1050C and 1500C, respectively Five hundred microliters of headspace gas was manually taken using an airtight syringe (SGE Pty Ltd, Australia) and injected into the

GC The ethylene gas quantity was determined based on ethylene standard calibration using CLASS-GC10 Version1.6 software (Ho, Joyce & Bhandari 2011a)

3.2.3 Determination of the effects of released ethylene at different concentrations

on mung bean germination rates in different water conditions

Four 250 ml breakers with 200 ml distilled water were repaired/prepared?? Thirty five grams of mung bean seed were soaked in each breaker Four different

Control

sample

Soaked with 52.5 mg ICs sample

Soaked with

0.525 mg ICs

ICs

Soaked with 5.25 mg ICs

Control sample

Soaked with 52.5 mg ICs sample

Soaked with 0.525 mg ICs ICs

Soaked with 5.25 mg ICs

Germinating in full water condition

Germinating in limited water

condition

Count germinated seeds

Figure 6 Experiment design to determine the effects of released ethylene at different

concentrations on mung bean germination rates in different water conditions

weights of ICs complex powder, which are 0 (control sample), 0.525 mg/100 mil, 5.25 mg/100 mil and 52.5 mg/100 mil respectively, were added into four soaking water breakers The seed in these treatments was soaked for 4 hours at 250C in uncovered condition

After soaking, 50 x 2 ungerminated seeds from each treatment were placed on a

12 x 8 cm cloth to germinate under different conditions (full water conditions and limited (30 ml) conditions) The cloths were put into unsealed desiccators and placed in an incubator (Sanyo Incubator MIR-253) at 250C in dark conditions The number of germinated seeds in each sample was counted in every 3 hours for 30 hours The experiment was designed as summarized in the Figure 6 A seed was defined as having germinated when the first radicle had broken through the seed coat (Deno 1993) Mung bean germination rates in the treatments were calculated by the number

of germinated seeds divided by the total number of seeds (Bari et al 2011)

3.2.4 Determination of the effects of released ethylene at different concentrations

on the size of mung bean spouts

Four 250 ml breakers with 200 ml distilled water were prepared Thirty five grams of mung bean seed were soaked in each breaker Four different weights of ICs complex powder, 0 (control sample), 0.525

mg/100 mil, 5.25 mg/100 mil and 52.5 mg/100

mil respectively, were added to the four

soaking water breakers The seeds in these

treatments were soaked for 4 hours at 25 0C in

uncovered conditions

After soaking, 50 x 2 ungeminated

seeds of each treatment were transferred to a

12 x 8 cm cloth The cloths were put into

unsealed desiccators and placed in an

incubator (Sanyo Incubator MIR-253) at 25 0C

under dark conditions Mung bean seeds were

sprouted for 5 days in the incubator Length

Length of hypocotyl Diameter of hypocotyl

(Radial of hypocotyl)

Figure 7 Measurement methods for

length and radical diameter

and radial of beans sprout hypocotyls in all the treatments were measured at the end of sprouting process (Figure 7)

3.2.5 Replication and statistical analysis

All measurements were duplicated with individual treatments replicated three times The data on seed germination and size of the bean sprout hypocotyls were analyzed using Minitab 16 software using one-way ANOVA

4 Results and Discussion

4.1 The concentration of retained ethylene in soaking water

According to Ho (2011a) the encapsulated ethylene is soluble in water and can completely release trapped ethylene during dissolution In addition, ethylene gas is a soluble gas, 200mg/l at 150C (SIDS assessment report 2010) Figures 8, 9, 10 and 11 show that the released ethylene gas was retained in soaking water for all samples, but there were differences in the ethylene concentrations The highest concentrations of retained ethylene were significantly different (p< 0.001) and reflected differences in the

complex/100 ml water

weights of the ICs complex powders used The highest weight ICs sample (525 mg/100ml, Fig 11) had the highest ethylene concentration of 18.85 ppm after 3 hours of soaking However, the highest concentration of ethylene in the minimum weight ICs sample (0.525 mg/100 mL, Fig 8) reached only 0.07 ppm after 1 hour of soaking The highest ethylene concentrations of other samples, 52.5 mg and 5.25 mg ICs complexes, were 3.95 ppm and 0.56 ppm, respectively (Figures 9 and 10)

complex/100 ml water