Effect of low temperature on the germination of cucumber seeds

11and CU-74 were used to perform in four different temperatures 10℃, 15℃, 20℃ and 25℃, to compare the germination and cold tolerance of seeds under low temperature conditions.. Key words

THAI NGUYEN UNIVERSITY

UNIVERSITY OF AGRICULTURE AND FORESTRY

Study mode: Full-time

Major: Environmental Science and Management

Faculty: Advanced Education Program Office

Batch: 2014 - 2018

Thai Nguyen, 16/09/2018

DOCUMENT PAGE WITH ABSTRACT

Thai Nguyen University of Agriculture and Forestry Degree program Bachelor of Environmental Science and Management Student name Do Thi Quynh Trang

signature (s)

Abstract: Poor seed germination is a common phenomenon at sub-optimal temperatures which causes a great concern for farmers Several priming treatments have been reported to test for the seed resistance at different temperatures, particularly at low temperatures In this study, six cucumber cultivars (CU-87, CU-

127, Cuigu, Kappa summer no 7, Kappa summer no 11and CU-74) were used to perform in four different temperatures (10℃, 15℃, 20℃ and 25℃), to compare the germination and cold tolerance of seeds under low temperature conditions

There are five experiments were performed and each experiment was run in three replications The results showed that cucumber was not suitable at low temperature and tolerance of cucumber in poor chilling conditions The germination was highest

at 25℃ and 20℃ but seeds also germinated readily at 15℃ and no germination was observed at 10℃ Pre-treatment of seeds in hot water for 10 minutes had only minor

effect on germination rate

Key words: Low temperature, chilling, germination, low treatment, rate

root, cucumber

Number Pages: 45

Date of Submission: 16/09/2018

ACKNOWDLEDGEMENT

I would like firstly to emphasize the sincere appreciation to teachers in Advance Education Program Office as well as teachers in Thai Nguyen University of Agricultural and Forestry, who have taught me knowledge not only for my subjects but also for my living 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 did my research in Taiwan

It is my pleasure to work with a great teacher - Assistant Professor San-Gwang Hwang, who always helped me any time He also gave me the best conditions, supported all materials for my research and discussed about any problems I got whenever I did experiments in his Vegetable Laboratory Indeed, without his help this study would not have been possible I am thankful that he provided me an opportunity

I consider it is an honor to work with Mr Wayne, a master student, who particularly helpful in guiding me toward a qualitative methodology and inspiring me

in whole period of internship time He was always helpful, friendly and very kind with

me Without his guidance, I cannot complete this thesis

Finally, I would like to express my gratitude to my family and friends, who always beside me all the time Their helps, supports and encouragement created the pump leading me to success

Sincerely,

Do Thi Quynh Trang

TABLE OF CONTENT

ACKNOWDLEDGEMENT iv

TABLE OF CONTENT vi

LIST OF FIGURES viii

LIST OF TABLES viii

LIST OF ABBREVIATIONS ix

PART I INTRODUCTION 1

1.1 Research rationale 1

1.2 Research objectives 3

1.3 Research questions and hypothesis 3

1.3.1 Research questions 3

1.3.2 Hypothesis 3

1.4 Limitations 3

1.5 Definition 3

PART II LITERATURE REVIEW 6

2.1 An overview about the experiment 6

2.2 Factors influence the germination of seeds 8

2.2.1 Temperature 8

2.2.2 Ethylene (C2H4) and respiration production (CO2) 10

2.2.3 Cell membrane on the germination of seeds 12

PART III METHODS 15

3.1 Materials 15

3.2 Methods 16

3.2.1 Germination test 16

3.2.2 Root growth (Radicle length) 17

3.2.3 Relative electrical conductivity (EC) 17

3.2.4 Respiration measurement (CO2) and ethylene measurement (C2H4) 17

3.2.5 Statistics 18

PART IV RESULT 19

4.1 Final germination percentage (FGP), days to 50% germination and 80% germination 19

4.2 Root growth (Radicle length) 21

4.3 Relative electrical conductivity (EC) 27

4.4 Respiratory production rate (CO2) 29

4.5 Ethylene production rate (C2H4) 32

PART V DISCUSSION AND CONCLUSION 35

5.1 Discussion……… ….35

5.2 Conclusion……… ……36

REFERENCES 38

LIST OF FIGURES

Figure 4.1 Effect of temperature on root growth for 7 days 24

Figure 4.2 The root of 6 cultivars at 10℃ after 7 days 25

Figure 4.3 The root of 6 cultivars at 15℃ after 7 days 25

Figure 4.4 The root of 6 cultivars at 20℃ after 7 days 26

Figure 4.5 The root of 6 cultivars at 25℃ after 7 days 26

Figure 4.6 Effects of tolerance on relativity electrolyte conductivity 29

Figure 4.7 Seeds respiration rates at different temperature 31

Figure 4.8 Ethylene production rate change in seed within low temperature 34

LIST OF TABLES

Table 3.1 Cucumber seeds information 16 Table 4.1 Germination ability at four temperatures in six cucumber cultivars 19 Table 4.2 Effect of germination temperature on the time to 50% germination 21 Table 4.3 Effect of germination temperature on the time to 80% germination 21

LIST OF ABBREVIATIONS

CO 2 The respiration

C 2 H 4 The ethylene

EC Electrical conductivity

EC 0 Electrical conductivity of the seeds after put in 4 different temperature

levels for 24 hours

EC 1 Electrical conductivity of the seeds after bath in 95℃ hot water for 1 hour

PART I INTRODUCTION 1.1 Research rationale

The phrases climate change and global warming and more recently global cooling are now part of our life Climate change has come upon us in a relatively short space of time and is accelerating with alarming speed It is perhaps the most serious problem that the civilized world has had to face Earth’s climate is in a nonstop state of change; it is inherent in the dynamic nature of our planet Changes in the basic components that influence the state of the Earth’s climatic system can occur externally (from extraterrestrial systems) or internally (from oceans, atmosphere, and land systems) through any one of the described components For example, an external change may include a variation in the sun’s production which would externally vary the amount of solar radiation received Internal variations in the Earth's climate system may be due to changes in atmospheric concentrations, mountain formation, volcanic activity, and surface or atmospheric changes These forces will continue to have a major effect on our future climate

The influence of global warming is now can be seen in many parts of the world Abnormality in climate patterns, caused by rapid warming, have begun to affect a catchment specific hydrologic cycle Scientists believe that rapid warming in the last several decades are mostly due to human induced changes in the atmosphere, on top of some natural variations Impacts of climate change are complex as they can be both direct and indirect, the biggest casualty being natural resources such as agriculture and horticulture High temperatures lead to a high rate of evaporation and dry conditions in some areas in the world Severe weather events are now more common Environmental

stresses shows the most important limiting conditions for horticultural productivity and plant exploitation worldwide Climate change has led to erratic weather, where long droughts and severe freezing occur greatly affecting the growth of crops, one of those

is cucumbers

Cucumber (Cucumis sativus L.), a warm-season crop, germinates at optimal temperatures of 24 to 28°C (Staub and Wehner 1996) Fluctuating temperatures encountered in many production areas during the early part of the growing season may negatively influence germination and seedling growth in cucumber (Kłosińska et al., 2013) Cultivars with low temperature tolerance are currently unavailable, although differences in response to chilling temperatures were found among the cultivars present on the market Cucumber cultigens differ in their requirements for the optimal and minimal temperature or germination (Kłosińska et al., 2013) This might be associated with the geographic region they originated from Lower (1975) demonstrated that 11 cucumber cultivars exhibited differences in germination speeds

at temperatures between 14 and 17°C Cultivars developed in the northern part of the United States were better cold germinators than those developed in the southern part of the U.S The exception was ‘Pixie’, with good cold germination ability even it originating from the South Similarly, Wehner (1981, 1982) found differences in days

to germination at 15℃ among 203 cucumber breeding lines and cultivars (from 3.5 to 17.3), but not at 20°C Significant differences were found also among 15 cucumber cultivars when germinated at 15°C, but not at 25°C

In this study, we wanted to conduct experiments on 6 cultivars originating from Japan, which is: CU-87, CU-127, Cuigu, Kappa Summer no 7, Kappa Summer no 11,

and CU-74 To test how the low temperature affects the germination of seeds of cucumber and which temperature levels are appropriate for the development of this plant

1.2 Research objectives

The main purpose of this research is to identify how low temperature affected the germination and growth of cucumber seeds and where is suitable temperature for the development of these type of seeds

1.3 Research questions and hypothesis

1.3.1 Research questions

The study aims to address the following questions:

- Does the low temperature might not affect the germination and growth of cucumber seeds?

- What are the ideal temperatures for the grow of cucumber seeds?

1.3.2 Hypothesis

- H0 (Null hypothesis): Cucumber seeds will not affect by low temperature

- Ha (Alternative hypothesis): Cucumber seed will affect by low temperature

1.4 Limitations

Japanese cucumber seeds were grown under cold conditions Because individual seed adapted differently to the environmental temperature, therefore some of experimental results were not completely accurate

1.5 Definition

- Cucumber (Cucumis sativus L.)

Cucumber is a type of flowering plant that regarding to the pumpkin family This plant originates from South Asia Cultivation of cucumber started 3000 years ago in India and it quickly spread out to the rest of the world Cucumber was popular and often used type of vegetable in the ancient Egypt and Rome It is still one of the most widely used plants in human meal Cucumber grows best in warm condition, subtropical climate, in sunny areas on a fertile and well drained soil People plant cucumbers mostly as a source of food Some kinds of cucumber are cultivated in ornamental purposes

Most seeds of the cucumber (Cucumis sativus L.) produce long, slender cucumbers that have skins that are thin enough to be eaten without being peeled While almost seeds produce either slicing or pickling cucumbers, there are hybrid and foreign varieties that may notice the home gardener Do not gather seeds for planting from cucumbers that are at the eating stage These seeds are not mature and will not germinate Let the cucumbers developed before you collect the seeds Collect cucumbers to select seeds at the end of the growing season

- Electrical conductivity (EC):

EC stands for electrical conductivity, which determine the potential for a material

to conduct electricity Even though most farmers are familiar with measuring the amount of feeding that they have to give in ounces per gallon, grams per liter, or any other measuring units used, EC goes a little further than this When growing, it is important to have a good understanding of what EC is all about and its significance to the farmer

In this study EC is based on the fact that seeds, when soaked in water, exude ions, sugars and other metabolites, from the starting of the soaking period, due to change in the integrity of the cell membranes, as a function of water amount and of the level of seed deterioration (Fessel et al., 2006) In deteriorated seeds, the repair mechanism is absent or inefficient, or the membranes are completely damaged, thus permitting leaching of larger electrolyte amounts

PART II LITERATURE REVIEW

The quality of seeds germination is determined by all its performance determining properties, such as temperature, ethylene and respiration production, cell membrane Many factors at each process unit which can influence the germination quality These factors can be measured by various techniques

2.1 An overview about the experiment

Chilling injury is a physiological disorder that occurs in sensitive plants subjected to non-freezing temperatures below 12°C (Saltveit and Morris 1990) Sensitive plants include those of tropical and subtropical origin (e.g., avocados, bananas, cotton, maize and rice) and some temperate plants (e.g., asparagus and some apple cultivars) Symptoms of chilling injury include stunted growth, reduced photosynthetic capacity, necrosis and discoloration, abnormal ripening and increased disease susceptibility (Mary E Mangrich and Mikal E Saltveit 2000) Chilling sensitivity restricts the length of the growing season, limits the geographical area available for cultivation and requires the storage temperature of harvested commodities to be at least 10°C higher than the optimum 0°C storage temperature recommended for most chilling-tolerant crop (Mary E Mangrich and Mikal E Saltveit 2000) While many hypotheses have been offered to explain the cellular basis for chilling injury, a phase transition of cellular membranes is still thought to be the initial step in a chain of events that result in chilling injury (Lyons and Raison 1970)

Radicle root growth after chilling is a sensitive indicator of chilling stress, more sensitive than the frequently used measure of ion leakage (Saltveit and Morris 1990) This difference in sensitivity is shown by comparing the incremental decrease in

subsequent elongation of the cucumber (Cucumis sativus L.) radicle after a few days of

chilling with the significant increase in the rate of ion leakage from the radicle that only occurs after 3 days of chilling (Jennings and Saltveit 1994) Above zero temperatures need to occur over at least 6 weeks for angiosperm survival (Körner, 2011) Results of earlier works have indicated that tissue formation, irrespective of whether above or below ground, becomes very slow at or below 5°C (ALVAREZ‐URIA P and KÖRNER C 2007); (Körner, 2008); (Sebastian et al., 2016) and was never observed at or below 0°C, a temperature that still permits CO2 uptake

at ca 30% of photosynthetic capacity In addition to the factors mentioned above the role of ethylene in seed germination has been extensively studied in several species ("Index," 1992) and it was observed that ethylene may be involved in the removal of inhibitors and thus derepress germination (Bewley J et al., 2013), especially under stress conditions A possible mechanism of seed priming in circumventing thermo inhibition of carrot seeds would be by increasing ethylene production during germination at high temperatures because in low temperature there is no ethylene production

Seed vigor has been defined as ‘a physiological property determined by the genotype and modified by the environment, which governs the ability of a seed to produce a seedling rapidly in soil and the extent to which that seed tolerates a range of environmental factors’ (Mary E Mangrich and Mikal E Saltveit 2000) concluded that seeds with high vigor are more tolerant of environmental stress

The present work was undertaken to investigate the effect of heat shock on the subsequent growth of cucumber radicles chilled for different lengths of time We also

examine the relationship between the vigor of germinating seeds and their response to chilling temperatures

2.2 Factors influence the germination of seeds

To obtain a good quality in the term of seeds germination, there several factors which control the quality of its temperature, the ethylene and respiration production, and cell membrane

2.2.1 Temperature

Appropriate temperature is probably the most important factor in adjusting germination (Nerson, 2007) Temperature affects the germination capacity, the germination rate and the germination frequency alongside the incubation time (Kurtar, 2010) Germination speed frequently increases until the temperature reaches 30 - 35°C (Roberts and Ellis 1989) Temperature has significant influence on the onset, potential and rate of germination (Flores and Briones 2001)

The thermal limits for germination are defined by the minimum, optimum and maximum temperatures which can determine some of the ecological limitations for the geographic distribution of the species Optimum temperature is the temperature value which results in the highest germination speed (Håkansson et al., 2002) Ideal temperatures produce both the most rapid seed germination and plant growth The ideal temperature for germination rate is typically higher than that required to reach maximum percentage germination in partially dormant or partially deteriorated seed populations

The effects of temperature on seed germination and emergence for some species have been examined through modelling, as well as under field or laboratory

conditions Kevseroglu et al., (2000) carried out studies using some industrial plants,

in sugar beet (Dürr et al., 2001), in some vegetable crops, in peach (Malcolm et al., 2003), in Legume crops (Kurtar et al., 2004), in wheat (Jame and Cutforth 2004), in tobacco (Cırak et al., 2007), in grain legumes and cereals (Odabas and Mut 2007), in some brassica species (Balkaya et al., 2008), in corn Models have been consumed by many researchers to determine plant growth potential, development and yield (Prusinkiewicz, 2004); (Yang et al., 2004), as well as seed germination, seedling emergence times, and seedling growth potential, in recent years (S P Hardegree et al., 2003); (Stuart P Hardegree, 2006) These models can be developed for different environmental conditions and different agricultural crops and can be used for accurately forecasting long term crop responses

Low temperature is detrimental to plant growth and development and thus affects the productivity of important warm-season food crops worldwide Cucumbers (Cucumis sativus), like all members of the pumpkin family, need a long, warm growing season to produce juicy, high quality fruit Their small oval seeds germinate quickly in warm weather but may germinate slowly or are susceptible to low temperatures throughout its growth cycle In general, cucumbers are best cultivated in late spring or early summer Numerous mechanisms have been suggested to account for chilling or tolerance in plants Some of the changes related to cold temperature stress involve alterations in gene expression, proteins, lipids, carbohydrate composition, membrane properties, solute leakage, mitochondrial respiration, and photosynthesis

2.2.2 Ethylene (C 2 H 4 ) and respiration production (CO 2 )

The growth and development of higher plants, from the earliest to the most advanced stages of the life cycle, are strictly regulated by phytohormones, such as ethylene (Fluhr and Mattoo 1996) Germination, flowering, maturation, senescence and response to pathogens are some of the processes that involve this two-carbon alkene Ethylene is a naturally produced, simple two carbons gaseous plant growth regulator that has numerous effects on the growth, development and storage life of many fruits, vegetables and ornamental crops This powerful plant hormone is effective at part-per-million (ppm, ml l−1) to part-per-billion (ppb, ml l−1) concentrations

Both the synthesis and action of C2H4 involve complicated metabolic processes, which require oxygen and are sensitive to elevated concentrations of carbon dioxide Endogenous sensitivity to C2H4 changes during plant development, as does its rate of synthesis and loss by diffusion from the plant (Matilla, 2007)

Although the great majority of seeds produce ethylene during the germination process, it is not yet clear whether this gas acts as a phytohormone in the chain of germination events, or whether C2H4 production is a result of, rather than a requirement for, germination and consequently does not alter the pattern of events prior to or during the breaking of the seed coat (Nascimento et al., 2013) A number of papers will be reviewed to shed more light on this enigma Various studies have demonstrated that C2H4 production in certain seeds increased before radicle protrusion, and this protrusion was reduced on trapping the ethylene produced

Seed germination involves a series of hormonally regulated metabolic processes Consequently, as germination involves the revival of the growth of the organ that breaks the seed coat, this part of the seed may contain the true target cells for certain phytohormones (e.g ethylene; Kieber, 1997) In lettuce, it has been suggested that ethylene is necessary for germination at supra-optimal temperatures (Nascimento et al., 2000); (Kozareva et al., 2004)

As well as C2H4, the CO2 of seeds during germination also plays an important role in the ability of the seed to survive in extreme climatic conditions Germination of

a seed starts with water absorb by the seed This is an essential stage for the seed to germinate The total water takes up is about 2-3 times the weight of the seed Whether

or not a viable seed will germinate depends on a various factor The chemical environment of the seed must be suitable Water must be available, oxygen has to be present since the seed must respire and dangerous chemicals should not be present The physical environment must also be favorable The temperature must be proper as well as the light quality and quantity

Many techniques have been studied to improve germination and emergence at low temperature One successful seed treatment that increases seed vigor is osmoconditioning or priming Promising results have been observed in vegetable crops Germination is an energy-requiring process, dependent on the respiration of the seed (Mayer and Poljakoff-Mayber 1965) Respiratory activity is initiated upon imbibition, but may be delayed or reduced by low temperatures or reduced water potentials (Dahal et al., 1996) Respiration rates of seeds in the early stages of germination have been positively correlated with seedling development

(Woodstock W and Pollock M., 1965) However, little information is available on seed respiratory characteristics of different cultivars in relation to their capacity to germinate under low temperatures Furthermore, these relation-ships are not clear, e.g

in soybean and maize (Van de Venter A and Grobbelaar 1985; Ismail M et al., 1989) Low temperature positively affected seed respiration in spinach, kohlrabi and eggplant (Mazor et al., 1984), but contrasting results were obtained among different pepper cultivars

2.2.3 Cell membrane on the germination of seeds

As the first essential step of the plant life cycle, seed germination is an important part of crop growth Depending on the rate of water uptake, the time course of germination and subsequent growth is divided into three phases (Xiaomei et al., 2015)

In phase I, which is also called imbibition, water uptake is rapid; in phase II, water uptake is much slower and reaches a plateau; in phase III (post-germination), there is

an increase in water uptake (Bewley J D., 1997; Weitbrecht et al., 2011) Imbibition is negatively affected by high rates of water uptake and low temperatures Low temperatures reduce the speed and/or rate of germination through imbibition chilling injury Imbibition chilling injury is a common problem in agriculture (Cheng et al., 2009) and has been reported for a wide range of crops, including bean and lima bean (Pollock, 1969), cotton (Christiansen, 1967), pea, cucumber (Makeen et al., 2006), and corn and soybean (Hobbs and Obendorf 1972) Imbibition has attracted wide and long-lasting interest from researchers

The most important event during imbibition is probably membrane reorganization, given that it occurs before other events and is a precondition for most

cellular process (W., 1974) Adoption of the hexagonal II phase of membrane (Ishibashi et al., 2013), contributes to substantial cytoplasmic leakage upon hydration (Lyons, 1973) Membranes must quickly reorganize from a hexagonal II phase to a lamellar phase in order to restore normal function and to terminate leakage of cellular components (Villa-Hernández et al., 2013) Against this background, the widely accepted model for imbibition chilling injury is that a low temperature disrupts membrane reorganization

Plant membranes mainly consist of glycerolipids, including six classes of phospholipids: phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserin, phosphatidic acid, and phosphatidylglycerol, and two classes of galactolipids: monogalactosyldiacylglycerol and digalactosyldiacylglycerol, which are plastid lipids In comparison with the lipid composition in leaves, seed membranes have low levels of both monogalactosyldiacylglycerol and digalactosyldiacylglycerol, and high levels of phosphatidic acid (Crowe and Crowe 1992) The low levels of monogalactosyldiacylglycerol and digalactosyldiacylglycerol result from the lack of plastids in seeds The high level of phosphatidic acid probably results from the drying during seed maturation because desiccation usually induces its formation (Devaiah et al., 2006; Francisco et al., 2013), moreover, a high level of phosphatidic acid in membranes favors formation of the hexagonal II phase (Verkleij et al., 1982) Besides its specific role in membrane structure, phosphatidic acid is also a central intermediary (phosphatidic acid pool) in lipid metabolism pathways for both storage and membrane lipids (Francisco et al., 2013) Given the importance of storage lipid catalysis, the phosphatidic acid pool could have critical functions during germination Under

conditions of dehydration and low temperature, phosphatidic acid is derived mainly from phospholipase D-mediated hydrolysis of phospholipids (Zhang et al., 2013)

PART III METHODS 3.1 Materials

Seeds material: The cucumber is almost annual production in Taiwan, so the farmer prefers to choose which have the following characteristics, like parthenocarpy, high gynoecium (female flower) at main vine or side vine, high production and which can be planted throughout the year, like the CU series and the Kappa summer series

- CU series: Japanese cucumber, the fruit color was dark green with luster, high yield, and good storability (CU-87 have high temperature resistance; the main vine production can be harvest earlier CU-127 has downy mildew and low temperature resistance, which is suitable for autumn and winter cultivation)

- Kappa summer: Japanese cucumber, the fruit color was light green with white thorns, have heat resistance (Kappa summer no.11 have powdery mildew resistance, one fruit position can form 2 female flowers)

- Cuigu: Taiwanese cucumber, the fruit color was dark green with luster, high yield, suitable for summer cultivation

Table 3.1 Cucumber seeds information

Cultivars

Rate of female flower

Country of origin

Fruit length (cm)

3.2.2 Root growth (Radicle length)

Seeds were used 60℃ hot water disinfection for 10 min and then cooling immediately Soaked the seeds for 6 hours and then germinated in the dark condition

at 25℃ for 24 hours Radicles were approximately 0.1~0.2 cm in long after 24 hours’ treatment Each experiment was run in 3 replicates with 10 seeds per replicate The germinated seeds were then placed in vertical plastic boards with filter papers and cultured at 10, 15, 20 and 25℃ for 7 days, keep the medium moist during the experiment (Jennings, P Saltveit, M.E, 1994)

3.2.3 Relative electrical conductivity (EC)

Seeds were soaked in glass tube contained with 10 ml of deionized water for 24 hours at 10, 15, 20 and 25℃ Each experiment was run in 3 replicates with 3 seeds per replicate The steeped water from soaked seeds was collected and the electrical conductivity (EC0) of seed the leachate was measured in digital conductivity meter After that, using 95℃ hot water bath for 1 hour and cool down to room temperature All tubes contain seeds after being cooled will in turn be onto the vibrator; vibrate until the vortex appears in the tube (Hu, Wenhai et al., 2006) Then the electrical conductivity (EC1) was determined form each test tube by electrical conductivity meter The EC of seed sample per tube was then computed using the following formula:

EC (%) = (EC0/EC1) x100%

3.2.4 Respiration measurement (CO2) and ethylene measurement (C2H4)

Seeds were used 60℃ hot water disinfection for 10 min and then cooling immediately Cultured in the dark condition in 9-cm petric dishes on Whatman No 1

filter paper imbibed with 3.0 ml deionized water for 48 hours at 10, 15, 20 and 25℃ Each treatment was run in 3 replicates with 3 seeds per replicate After 48 hours cultured, transferred the seeds to 14 tubes for 1 hour, and then used the rubber stopper

to seal tight the tube The rates of C2H4 and CO2 evolution from each of tube were calculated from an analysis of 1 ml samples taken from the headspace gas that accumulated during 1 hour, Patanè, C., et al (2006)

3.2.5 Statistics

There are 3 independent series of each experiment were performed and resulted

in similar tendencies Data were analyzed as means for each treatment combination (petri plate) using the means, when ratios were significant, means were separated by the Least Significant Difference (LSD) test, F-test, procedures of SAS (Statistical Analysis Software) SAS can understand any type of data and it can access data from any software and any format Logical operation can also be performed in SAS by using if –then statement