INTRODUCTION

1.0 Plants evolved from bacterial clusters over 3,600 Mya and have adapted to use energy from light as their key source of nourishment. Over this period, plants have specialised and developed a need for certain portions of the electromagnetic spectrum. As many plant scientists are aware, plants use blue and red light to anabolism and catabolism molecules for growth, timing of germination and flowering (Delmar, 2008; Kratz, 2011). By manipulating these light wavelengths growers are able to control plant growth and timing of fruiting and flowering. Growers can use a wide range of techniques to focus the spectrum and to concentrate the frequencies required. The latest techniques such as photoselective films, L.E.Ds and specialised bulbs are available to manipulate plant development (Apple, 2012; Raff, 2009). All these techniques come with their advantages and disadvantages but all have in common high start-up and short-term costs. A possible solution to this problem could be chemiluminescence in the form of ‘light sticks’.

1.1 The key reaction starts with a mixture of dye and diphenyl oxalate which is isolated from hydrogen peroxide. By mixing the peroxide with the diphenyl a reaction occurs yielding 2 molecules of phenol and peroxyacid ester (Roda, 2010). The peroxyacid decomposes to carbon dioxide releasing energy that excites the dye resulting in the release of a photon or a particle of light.

1.2 The aim of this study is to investigate if chemiluminescence could be used to sustain plant growth. Conclusions have been drawn from the data collected from the trials and analysed by using various statistical tests. 3 trials of 18 Helianthus annuus plants have been grown under 2 light sticks at a time for 3 months, to see if they develop normally when compared to the control which will also have 18 specimens.

1.3 The experiment was held in a dark room with all natural light blocked out. In order to form a control Helianthus was grown under white L.E.D. light in the same darkened room as the test group. In the discussion 4 previous papers were analysed with their results compared to this experiment. Possible explanations for the results were summarised with recommendation for future research.

HYPOTHESES

2.0 The purpose of this paper is to prove the hypotheses and to investigate what processes are involved in chemiluminescence, how they may directly or indirectly affect growth. The fundamental aim of this investigation is to see if plant growth could be sustained. There are 2 principle hypotheses that will be investigated through this study:

1. H₁ There will be no significant difference in height when Helianthus is grown under Chemiluminescence

2. H₀ There will be a significant difference in height when Helianthus is grown under Chemiluminescence

3. H₁ Under chemiluminescence there will be no significant difference in root elongation

4. H₀ Under chemiluminescence there will be a significant difference in root elongation

5. H₁ Under chemiluminescence there will be no significant difference in dry weight to the control

6. H₀ There will be a significant difference in dry weight between the control and chemiluminescence

LITERATURE REVIEW

3.0 The aim of this study was to review the processes involved in chemiluminescence and how photoreceptors respond to the stimuli. This paper aimed to determine if chemiluminescence could be used to sustain plant growth, by utilising secondary research to form a valid experiment.

Process of Chemiluminescence

3.1 Since their invention 25 years ago, light sticks have become a useful source of inexpensive and reliable light used within many industries throughout the world such as: the military, emergency services and marine science (Kaplan, 1999; Garcia-Campana, 2001). However these sticks have not been considered within the horticultural industry, even though they have been proven to emit sections of P.A.R. (500-70nm) (Briggs and Onley, 2001). Refer to appendix 1 for spectral comparisons. Comparable to natural light, artificial light is a form of subatomic particle or a photon which can be produced through 3 key processes: incandescence (emission of light due to heat), phosphorescence (emission of light in response to chemicals) and laser generation (emission of photons from excited atoms) (Roda, 2010; Galstyna and Guseman, 1989).

3.2 Chemiluminescence reactions begin by mixing multiple chemical compounds. An outside chemical reaction excites atoms, resulting in them releasing photons. When atoms become excited they begin to ‘speed up’, as they speed up more collisions occur resulting with a greater force (Eisenbraun, 2009). If atoms within the chemicals become excited enough, the collisions will transfer energy to some of the atom's electrons. When this occurs an electron will be temporarily boosted to a higher energy level, moving away from the atom's nucleus (Harris, 2011). When it eventually falls back to its original level it releases energy in the form of photons (Brown, 2012. Pers. Comm. Ms Ros Brown chief technician of microbiology at Newcastle University).

3.3 The chemical reaction in chemiluminescence involves 5 key processes. A typical light stick contains a hydrogen peroxide solution, a solution containing a phenyl oxalate ester and a fluorescent dye. The hydrogen peroxide oxidizes the phenyl oxalate ester, resulting in a chemical called phenol and an unstable peroxyacid ester. Subsequently an unstable peroxyacid ester compound decomposes, resulting in additional phenol and a cyclic peroxy compound. The cyclic peroxy compound decomposes to carbon dioxide (Fardy and Yang, 2008; Harris, 2011). This decomposition releases energy to an added dye. The particular dye used in the chemical solution gives the light a distinctive colour for instance ‘9, 10-diphenylanthracene’ emitting blue light (450nm) and ‘16, 17-(1, 2-ethylenedioxy) violanthrone’ emitting red light (680nm) (Galland, 2011). The electrons in the dye atoms jump to a higher state and then fall back down releasing energy in the form of light. These reactions can be summarised as:

Diphenyl oxalate + H₂H0₂ → 2OH + 1, 2-dioxetanedione

1, 2-dioxetanedione + 9, 10-diphenylanthracene → 2C0₂ + dye + photon

3.4 Within a light stick 2 solutions are contained in separate chambers. The phenyl oxalate ester and dye solution fills most of the plastic tubing. While the hydrogen peroxide solution called the ‘activator’; is contained in a fragile glass vial in the centre of the tubing (Allaby, 2006). When the tubing is bent the vial breaks and the 2 solutions fuse. The chemicals immediately react to one another and the atoms begin producing light. Depending on which compounds are used the chemical reaction may last for several minutes or several hours. If you heat the solutions, the extra energy will accelerate the reaction and the stick will glow brighter for a shorter amount of time (Roda, 2010). If you cool the stick the reaction will slow down and the light will reduce.

3.5 Light sticks are just one application of a natural occurrence of luminescence. Luminescence is an emission of light that is not caused by heating solutions. Practical applications of luminescence include: televisions, neon lights and glow-in-the-dark substances (Hoe et al, 2002). Correspondingly this is the key principle that makes fireflies glow and some fungi phosphoresce which is known as bioluminescence.

Photoreceptors and Growth Responses

3.6 Plants detect photons by using a system of 3 photoreceptors: phytochromes, cryptochromes and phototropins. Phytochromes are sensitive to the red and far-red regions of the spectrum. Angiosperms use this to regulate the time of flowering based on photoperiodism and to set circadian rhythms (Franklin et al, 2005). It also regulates other responses including: seed germination or photoblasty, elongation of seedlings, size, shape and number of leaves, the synthesis of chlorophyll and the straightening of the hypocotyl of dicotyledon seedlings (Hazina et al, 2005; Cooper, 2009).

3.7 A unique feature of phytochrome is that it exhibits ‘photoreversibility’. Phytochrome exists in 2 forms that are interchangeable. (Pr) red light absorbing form and (Pfr) far-red light absorbing form. When Pr absorbs red light (660nm) it is converted into Pfr. When Pfr absorbs far red light (730nm) it is converted into Pr. In summary phytochrome acts like a ‘light switch’ (Abelson et al, 1998). This can be demonstrated as:

Pr ↔ Pfr

However not all the phytochrome is interconverted equally. This is because the absorption spectra for the 2 pigments overlap and begin to compete. A measure of the efficiency conversion is the ratio of the Pfr to the total which is expressed as follows:

Efficiency = Pfr/(Pr + Pfr = phytochrome total)

In Pr = 0.85; Pfr 720nm = 0.03 this varies with the environment

(Ke, 2001)

There are a series of intermediate phases in the conversion from Pr to Pfr. There are 3 intermediate stages in conversion of Pr to Pfr and 2 stages in conversion of Pfr to Pr, which can be expressed as:

Pr → red light → Pr* → 1 → 2 → 3 → Pfr → far red → Pfr* 1 → 2 → Pr

Where * represents photon absorption

The synthesis of phytochrome can be expressed as:

phy gene → mRNA → Pr ↔ Pfr

Where mRNA presents the building proteins

Where phy presents gene expression of phytochrome

The breakdown of phytochrome can be expressed as:

phy gene → mRNA → Pr ↔ Pfr → destruction

(Papageorgiou and Govindjee, 2009)

3.8 Cryptochromes are blue light receptors that mediate various light induced responses in plants. They share sequence similarity to photolyase, flavoproteins that catalyse the repair of UV light-damaged DNA, but do not have photolyase activity (Koller, 1990). Cryptochromes work in partnership with the red and far-red light receptor phytochromes to regulate various light responses including the regulation of cell: elongation, photoperiodic flowering and to regulate stomatal opening and closing (Beerling, 2008; Mauseth; 2008).

3.9 Phototropins are photoreceptor proteins that mediate phototropism responses in higher plants. Along with cryptochromes and phytochromes they allow plants to respond and alter their growth in response to the light environment. Phototropins are auto-phosphorylating protein kinases (key enzyme in phototropism) that are active in response to blue light (Willis and McElwain, 2002). When blue light reaches the phototropin protein in the cell membrane, the phototropin protein will unfold and undergo phosphorylation. This can cause a cascade of events inside the cell (Arsovski and Anahit, 2012; Wolken, 1995). Phototropins are part of the phototropic sensory system in flora which causes various environmental responses in plants.

3.10 When phytochromes absorb light they emit a chemical signal that produces a hormone known as ‘auxin’. Upon release from the cells in the apical meristem, auxin collects primarily on the darker side of the stem and stimulates cell elongation (Lawlor, 2000; Kendrick and Kronenborg, 1998). Thus the cells on the side not directly exposed to light will grow much faster than the opposing side. The resulting stem will begin to curve towards the light source which is the process of ‘phototropism’. Different organs of the plant exhibit different phototropic reactions to different wavelengths of light (Devlin, 2007). Stem tips exhibit positive phototropic reactions to blue light. While root tips exhibit negative phototropic reactions to blue light (Blankenship, 2001). Both root tips and stem tips exhibit positive phototropism to red light.

3.11 Auxins are hormones that have a variety of functions. In regards to phototropism, auxins are responsible for expelling protons through activating proton pumps which decrease the pH in the cells on the dark side of the plant. This acidification of the cell wall activates enzymes known as ‘expansins’ which break bonds in the cell wall, resulting in the cell wall becoming less rigid (Taiz and Zeiger, 2010; Oxlade and Graham, 2007). In addition, the acidic environment causes disruption of hydrogen bonds in the cellulose that make up the cell wall. The decrease in strength causes the cells to swell, exerting the mechanical pressure that drives phototropism.

3.12 A similar tropic response towards light is called heliotropism which tracks the light. This ‘solar tracking’ mechanism is controlled by specialised organs called ‘pulvini’. These organs are swollen parts of the petiole that occur where it joins the stem or the leaf blade (Salisbury and Ross, 1991; Hall and Rao, 1999). They contain motor cells that utilise Ca⁺ and K⁺ to generate mechanical forces, which control the orientation of the petiole leading to movement of the leaf blade. The forces are produced by changes in the turgor in the pulvini stimulated by the cryptochromes and phytochromes. The cells of the pulvinus have highly elastic cell walls that allow them readily to modify their size and shape (Christie et al, 1998; Journal of Horticulture Science and Biotechnology, 2010). The cells of the upper pulvinus have the capability of increasing their turgidity with water uptake, while the lower pulvinus can lose water very easily. This results in a force that moves the petiole (Ramsay, 2012. Pers. Comm. Mr Paul Ramsay professor of plant biology at Plymouth University).

Research Review

3.13 Fluorescence is a type of chemiluminescence which has been demonstrated to sustain plant growth. Fluorescent lights utilise trace amounts of mercury, argon and phosphorous powder and have a similar function to light sticks with the major difference being the use of electrodes. Helson (2000) conducted a study comparing the efficiency of fluorescent and incandescent lamps utilised within grow cabinets. The study used 20 Lycopersicum esculentum in 3 different Percival growth cabinets with side lights; 1 having fluorescent bulbs, 1 with incandescent and the other having a 50/50 mix of both. Lycopersicum were germinated within their growth chambers and grown for a further 2 months, this formed the first trial. 12 hours of daylight at 16˚C were programmed with a 6˚C decrease in temperature during the nights. This change was reduced in stages during the ‘twilight’ period. As plants were watered humidity increased, as water evaporated humidity decreased. This contributed to the difficulty controlling humidity.

3.131 When the wavelengths were measured the florescent bulbs were shown to produce P.A.R. with the peak occurring within the red light region. Lycopersicum grown under fluorescence were shown to sustain plant growth however, when compared to incandescence internodes became extended by up to 20mm. Plants also showed a 44% increase in root dry weight, 73% increase in leaf area and 80% increase in plant height when grown under fluorescence (Helson, 2000).

3.132 Many technicians and research scientists are aware, growth chambers cannot be programmed and expected to function to those specifications immediately (Langhans and Tibbitt, 2010). There is usually a 5% margin of error when programming these machines. Manufacturers recommend that cabinets are programmed and monitored for up to a month to ensure specifications are correct. Without this check which can lead to more expense, specifications may not be accurate. A 5% margin of error may impede greatly upon the outcome of the test groups.

3.14 Tamulaitis et al (2007) directed a study to investigate the effects of short-wavelength light on Lactuca sativa and Raphanus sativus growth and nutritional quality. The purpose of the experiment was to compare 4 sources of short-wavelengths of light by using: blue, green, cyan (blue-green combination) and near U.V. L.E.Ds. Combinations of red and short-wavelength components and high pressure sodium lamps enabled the researchers to elucidate the influence of light in short-wavelength form, visible and near-UV regions on plant development. 10 Lactuca and Raphanus were used in all groups including a control. 3 trials were grown to provide a more accurate outcome, totalling 150 plants. Plants were grown within an automatic glasshouse which had L.E.D. panels attached to the roof and shading screens attached to the sides. This was to prevent contamination and loss of light.

3.141 The paper revealed an ideal combination for maximum photosynthetic output of 90% red light and 10% blue light. The most interesting aspect of this study demonstrated that cyan light which is close to the green spectrum has a positive biological effect on growth. According to the results, supplemental lighting with cyan light emitting diodes at 505nm can significantly affected carbohydrate and nitrate metabolism in Lactuca and slighting improved Raphanus development (Tamulaitis et al, 2007). As a comparison chemiluminescence peaks between 505-550nm, supporting the theory that light sticks could sustain Helianthus annuus development.

3.142 The study concluded that the introduction of short-wavelength components to the spectrum of illumination for plant cultivation, is insufficient to compensate the stress caused by excessive illumination in the red region (Tamulaitis et al, 2007). Spectral position of the short-wavelength component in the region 385-495nm has no crucial influence on plant development, though illumination in cyan region (505nm) is more favourable for biomass accumulation than illumination at shorter wavelengths.

3.143 When plants are grown within a glasshouse they receive varying amounts of light and varied day and night temperatures. When cooling or heating is applied to stabilise the temperature there is usually a gradient of temperature from the source. If experiments do not account for this gradient it can introduce serious variability into the experiment. Side fans used for forced ventilation can also develop a gradient of atmospheric conditions inside the greenhouse (Lovelidge, 2012). Although the glasshouse in this experiment was automated, it cannot prevent all of these variations. On average it takes 20 minutes for a control system to react to variations in environmental specifications (Bongers, 2012). This could unavoidably impact plant growth, thus affecting the outcome of this experiment. A more controlled environment such as a growth chamber would significantly reduce these variations however; as more controls are introduced cost will inevitably increase.

3.15 The effects of artificial lighting on photomorphogensis of callus-induced embryo and plantlet generation have been a topic of debt within the propagation industry for some years. The problem of identifying the most efficient light source was discovered by Nhut et al (2006). Nhut’s team used 8 different sources: 3-U compact, white 1-U compact, red 1-U compact, green 1-U compact, blue 1-U compact all types of fluorescent lamps and red and blue L.E.D.s. 5 Phalaenopsis amabilis calli were placed into 250ml flask containing: 40ml of Hyponex (6N 6P 19K) media supplemented with a vitamin solution, 30g l-1 sucrose, 15% coconut water and 9g of common agar. This was repeated for the other 7 groups and then further repeated 3 times to form 3 trials, each trial lasted 10 times.

3.151 The control was a commonly used fluorescent bulb. After 10 weeks of culture the somatic embryogenesis and the plantlet regeneration were shown to be influenced by the lighting source quality. Almost all plantlets appeared dark-green with epidermal hairs under compact white, 3-U compact and compact red which were hardly observed below other light environments. There was an increasing tendency in the number and weight of embryos under all light environments however; the highest values were under white compact fluorescence.

3.152 Embryo development was lowest when grown under blue L.E.Ds and compact blue, contrary to the highest rate under red L.E.Ds, compact white and 3-U compact (Nhut et al, 2006). Embryos under 3U compact, white compact and red L.E.Ds exhibited optimal number of plantlets and leaves, fresh weight, dry weight and regeneration rate. Leaf length under compact red and red L.E.Ds increased significantly; leaf span under compact blue and blue L.E.Ds enlarged rapidly in comparison with that of other light treatments. As a concluding mark, only compact blue florescence demonstrated chimeric leaves (differentiated leaf cells).

3.153 All calli were placed into a growth room and were divided by using metal screens to prevent light contamination emitted from the other test groups. A common problem associated with growing specimens within a growth room is the air flow. In a typical growth room 2 fans are allocated at the upper back and lower back wall. Having 2 fans in those positions creates an ‘Eddie Effect’ increasing air circulation (Global Specifications; 2012; Biochambers, 2012). Additionally the metal screens preventing light pollution may have restrict air flow. With air flow restricted humidity will inevitably increase the likelihood of moulds and bacterial diseases. An increase in plant illness would greatly affect the validity of the above experiment. As growth room and chambers are expensive to rent experimenters must keep to their schedule, if not the cost of the experiment would increase (Leegood and Lea, 1998). In the event of damaged plants, the researcher would have to increase the amount of time to compensate for the effect on validity caused by the damaged plants.

3.16 A study originally published in 1975 was revisited by Cathey and Campbell (1999). The aim of this research was to identify the impacts of using chemical security and street lighting on 22 common plant species. 2 types of chemical lighting were used, high pressure sodium and mercury vapour. The groups were set-up within an automated glasshouse with the supplementary lighting attached to the roof. 22 common landscaping epitypes were used in each of the 2 groups. Each of the 22 species were repeated 3 times to gain an accurate outcome. Before the experiment occurred species were matured to approximately 2 years and grown in multi-purpose peat based compost. Each trial lasted for 8 weeks and in regards to the overall experiment 132 plants were utilised.

3.161 The study revealed that all lighting that extend day length on plants exert the same growth control of promoting the formation of new leaves and the elongation of the distances between the leaves. Mercury vapour emitted low amounts of red light which delayed flowering only on glasshouse crops such as Poinsettia pulcherrima and promoted continuous vegetative growth only on highly responsive trees including Betula pendula, Acer pseudoplatanus and Ulmus glabra.

3.162 Only 7 out of the 22 tested exhibited growth responses to mercury vapour however, high pressure sodium lamps altered the growth responses of 16. The study established that supplementary lighting at night caused by mercury and high pressure sodium could alter the way the plant receives their signals, from the environment and adjusted their growth characteristics according to the seasons (Cathey and Campbell, 1999). In effect plants could continuously grow even when frosts develop, thus damaging or killing some species.

3.163 The experiment used 2 groups to identify which kinds of commonly used artificial light sources significantly influence plant development. The experiment concluded that fluorescence influenced the signals received by the photoreceptors from their environment. An area of concern would be that there was no control. The control was derived from previous research. Having a control based on other studies may not necessarily be appropriate as experiments occur at different locations and environments. Most researchers are aware that plants do not particularly proliferate during periods of dark however there are natural sources of light which are present such as reflected sunlight off the Luna surface (Botha et al, 2008). Other studies may not have included these factors and this may have impeded with this investigation.

3.17 It is apparent from the research which has been reviewed, that adapt controls need to be in place to ensure this investigation can draw precise conclusions. To ensure environmental specifications are met an environmental box with overhead lighting has been used. Growing specimens in a glasshouse would increase the amount of confounding variable. Furthermore this would safeguard against variation in the wavebands being received by the plants. To increase validity of this experiment, 3 trials have been completed to gain a more accurate outcome. Plants have been sexually reproduced which would increase disease resistance within the majority of the specimens. However this has come at some cost as there will be a lot more diversity within the plants. Additional controls have been implemented from the secondary research which has been conducted. 2 seeds have been planted per pot so it could protect against seed development failure as suggested by Nhut et al (2006).

METHODOLOGY

4.0 The aim of this study is to determine if plants could sustain growth under chemiluminescence by measuring height, root length and biomass. Observations were also recorded to monitor plant health. Specimens were grown in cardboard boxes within a darkened room. The average room day and night temperatures were recorded to determine suitability for this experiment. Tests occurred 2 weeks prior to this investigation. Illumination was provided from above to spread the light equally by means of 4 light sticks alternatively placed which were all activated at the same time, 2 red and 2 blue and for the control 6 white L.E.D lights.

4.1 36 x 7.6cm sized pots were filled with peat based multi-purpose compost (John Innes) and 2 seeds of Helianthus annuus were sown in and then watered. In each group, 6 Helianthus were grown for a month, 12 for each trial and 36 overall. As reinforcement, 2 seeds were planted in each pot reducing further confounding variables. When 2 seeds developed 1 was withdrawn. 12 plant pots were placed in 4 medium sized drip proof trays. 2 trays were placed into each group. 2 with blue and red chemiluminescence and 2 with white L.E.Ds. Subjects were then placed into environmental boxes 15cms away from the source of light to ensure maximum output. Refer to appendix 2 for trial design.

4.2 Helianthus had been germinated under glass for 2 weeks until the first hypocotyls developed and then they were placed in the environmental boxes. During the time the specimens were grown, temperature and light readings were collected to monitor environmental specifications. See appendix 3 for environmental specifications.

4.3 There were 3 trials to gain an accurate outcome. Each trial consisted of 2 groups to test if Helianthus could sustain growth under chemiluminescence. Plants under white L.E.Ds were used as the control. Each group was given 11 hours of light. Refer below for the trial layout.

Table 1 Trial Layout

4.4 Specimens were irrigated when required and supplemented with Phostrogen multi-purpose fertiliser to enrich the substrate, after 2 weeks within the boxes. The application has been uniform throughout the trials at 4.5 grams per 4 litres, refer below for details.

Table 2 Nutrient Make up

(Source: Phostrogen, 2011)

4.5 Specimens had their height measured daily followed by an observation log. Helianthus were measured inside the boxes to ensure environmental constancy within the experiment. After each trial specimens were measured from stem base to apical meristem and from stem base to root-meristem, photographed, observations documented and dry weight recorded.

4.6 To gain dry weight, specimens were removed from the substrate and briefly washed. Plants were then blotted to remove any free surface moisture. Specimens were placed into a drying oven at 100°C for 48 hours. Plants were left to cool for 3 hours and weighed on a scale which measured in milligrams (Carpentier, 2004). This trial process was repeated twice during subsequent weeks.

RESULTS

5.0 From the descriptive statistics that were analysed it was found that when grown under Chemiluminescence Helianthus annuus grew on average 1.61cm taller, as demonstrated in the table below.

Table 3 Growth rates above substrate (cm) n²=18

Below is a chart illustrating the differences in height. Note 1 presents the chemiluminescence and 2 the control.

Figure 1 Growth rates above substrate (cm) n²=18

Three inferential analyses were then completed. The Anderson-Darling test provided a P-value of <0.0001 signifying normal distribution. The Levene’s test provided a P-value of 0.453 which demonstrates equal variance within data. A Man Whitney U Test was then complete to further investigate if there was a significant difference between the groups. The test result was:

Man Whitney U Test: F=0.5, P <0.0001 (N²=18)

This demonstrates a significant difference within the height of specimens.

5.1 Specimens which developed under chemiluminescence had an increase in root elongation of 0.20cm, as shown in the table below.

Table 4 Root elongation rates (cm) N²=18

Below is a chart illustrating the differences in height.

Figure 2 Root elongation rates (cm) N²=18

Three inferential analyses were then finished. The Anderson-Darling test provided a P-value of 0.336 indicating that the data is not normally distributed. The Levene’s test provided a P-value of 0.119 which shows equal variance within data. A Man Whitney U Test was then complete to further examine if there was a significant difference among the groups. The test outcome was as follows:

Man Whitney U Test: F=0.5, P 0.458 (N²=18)

This establishes that there is no significant difference within the root length of specimens.

5.2 Specimens which developed under chemiluminescence had a major increase in root elongation of 0.02cm, as shown in the table below.

Table 5 Dry weight (g)

Below is a chart illustrating the differences in height.

Figure 3 Dry weight (g) n²=18

Three inferential analyses were then completed. The Anderson-Darling test provided a P-value of 0.171indicating that the data is not normally distributed. The Levene’s test presented a P-value of 0.664 which is an indication of equal variance within data. A Man Whitney U Test was then complete to further investigate if there was a significant difference among the groups. The test outcome was as follows:

Man Whitney U Test: F=0.5, P 0.716 (N²=18)

This demonstrates that there is no significant difference within the root length of specimens.

5.3 The following charts are comparing the overall results for each of the readings to clearly demonstrate differences in growth patterns for each of the groups.

Figure 4 Comparison of results (cm and g) N²=18. Green representing chemiluminescence and red presenting no light.

The chart below represents the percentage differential in growth for all the readings that were taken. Note the similarly patterns in growth in both groups.

Figure 5 Percentage differentials in growth (%) N²=18

Refer to appendix 4 for raw data.

See appendix 5 for observation log, labelling system and photographic account.

DISCUSSION

6.0 There was a significant difference found in the height of plants when under the environmental boxes. When further investigated, there was an increase of 1.61cm when grown under chemiluminescence. Refer to page 22, table 3. The P-value (P <0.0001) establishes that there was a significant variance and from this, the H₀ can be accepted. This may be due to the fact that chemiluminescence emits sections of P.A.R. which plants used to grow. This was further reinforced by the sustained positive phototropic responses to the blue light within 2 days of starting the tests.

6.1 Tamulaitis et al (2007) study conclusively supported the findings of this research. His team discovered that cyan light (a combination of blue-green light) is emitted from sources of chemiluminescence and peaks at 505-550nm (Bekefi and Barrett, 2005). His experiment proved that cyan light had a beneficial effect on plant growth and even increase carbohydrate and nitrate contents of Lactuca sativa and Raphanus sativus. Although cyan light has been proven to sustain growth a combination of blue and red light is still required for healthy growth. A ratio of 90:10 was expressed from the tests that were completed by Tamulaitis et al (2007). Chemiluminescence peaks at cyan light but also emits a combination of blue and red. This combination may not meet the ideal ratio for long term sustained growth however, it could be utilised as a supplement to increase this new beneficial part of the spectrum. This experiment demonstrated that plant growth could be sustained without any abnormalities or discolouration caused by chemiluminescence. Conversely, this study found that plant growth could be improved as shown by the P-value.

6.2 It is still unclear why the specimens had grown much faster within the first week when compared to outdoor Helianthus, which have steady growth. Plant growth increase drastically within the first several days. This was an indication that the light being emitted was not being detected by the plants effectively. However after this period of fast development, plants began to exhibit a positive phototropic response to the blue light. This was not shown in Tamulaitis et al (2007) study and suggests that plants need to be much closer to the source to be able to detect it.

6.3 This could highlight that chemiluminescence gives off far less light when compared to other sources which use a form of chemical energy, such as incandescence or florescence. The above study utilised a form of fluorescence which contains similar compounds and protocols to achieve a luminescence. This further supports the theory behind chemiluminescent sustainability of plant growth. As a concluding remark, plant growth was sustained for a month and was found to have been improved when compared to the control. However growth was slower when compared to outdoor grown Helianthus which was highlighted from the research.

6.4 It was revealed that the root length was unaffected when grown under the chemiluminescence, on average there was a differential of 0.20cm. See page 23, table 4. Blue light has been identified to manipulate the root shoot ratio as the energy transfer channels are sensitive to this radiation. As blue light decreases so does the efficiency of photosynthesis (Devlin, 2007; Ke, 2001). This reduces root length from the lower amounts of energy available. The Anderson-Darling test calculated a P-value of 0.336 which indicates that the data was not normally distributed. This indicates that there was a lot of variation in the results, most likely due to the reduced amount of blue light. However from the P-value of 0.458 there was no significant difference found in root length. This results in the H₁ being accepted as both groups elongated at a similar rate.

6.5 Helson (2000) also demonstrated that there was no significant difference when plants were grown under 3 types of fluoresces. The root elongation rate varied by 0.17cm. This supports the conclusions of this experiment as fluorescence is a form of chemiluminescence. In contrast to Helson’s (2000), test groups peaked at red the part of the spectrum (650nm) whereas this experiment peaked at the cyan part of the spectrum (550nm). Although red light is not known to directly affect root growth, it influences the genes which in turn influence the signal transduction pathways for growth (Briggs and Olney, 2001). A good comparison with both experiments would be the conclusion that there were no significant differences in growth between the test groups and the control.

6.6 Nhut et al (2006) exposed calli to different wavelengths of compact florescent lights for 10 weeks. The plantlets exhibited similar root elongation rates as this experiment. There was low variation in root elongation (0.05cm) comparable to Helson (2000) and this experiment supports the theory that roots are not affected by changes in the chemical light.

6.7 From the observations that were made Nhut et al (2006) and Helson (2000) studies indicated that root growth exhibited regular negative-phototropic responses. Conversely, this study revealed that roots had shown small positive phototropic responses. Blue light is known to negatively impact upon root growth, as roots need to grow down to anchor the plant in and to take in nutrients and water. This illustrates that the blue light levels were low supporting the argument in 6.2. As previously stated, this method could be used as a supplement due to this shortcoming.

6.8 There was no significant difference found in the dry weight when under the environmental groups. When further examined, there was a slight increase of 0.02g when grown under the control. Refer to page 24, table 5. The P-value of 0.716 clearly demonstrates that there is a close relationship between the 2 groups. This establishes that there was no significant difference and therefore the H₁ can be accepted. However there was a large amount of variation within the results, showing that the plants were individually affected.

6.9 Helson’s (2000) study also demonstrated that there was no significant difference when Lycopersicum esculentum were grown under 3 types of fluorescence. Cathey and Campbell’s (1999) paper revealed a similar result when 22 common plant species were grown under 2 types of chemical lighting. These epitypes were fully mature and were left to grow for 8 weeks. The study verified that there was a slight increase in dry weight within the control. A study conducted by Nhut et al (2006) demonstrated that were was again no significant difference in dry weight when callus-induced Phalaenopsis amabilis plantlets were grown under 8 different types of fluorescence and L.E.Ds. Another similarity to this experiment was the slight increase in the control of 0.07g.

6.10 The following papers suggested that when plants were grown under different types of light sources other than natural or white light, had a small reduction in dry weight. With the increases in height when grown under chemiluminescence, this shows that plants may be slightly weakened when compared to plants grown under white light. Healthy plants would have higher dry weight as there would be higher amounts of plant matter. Higher amounts of plant matter would be required for taller plants. During the first few days of this experiment a fast increase in plant growth was shown. This indicates the specimens were attempting to detect the light. Plants detected the light after day 3 due to exhibiting positive phototrophic responses. This then indicated that the plants were receiving the light.

6.11 This conclusion was supported by Nhut et al (2006) and Helson’s (2000) results which on average had a 2-4 day period where plants had a fast increase in growth. Phalaenopsis amabilis saw the most growth in this period. As a concluding remark, the papers that were reviewed revealed similar findings to this experiment. Their results supported this experiment demonstrating that plant height had a significant increase, root length had similar growth to the control and finally that their specimens had a slight reduction in dry weight when grown under their test groups.

6.12 The most difficult aspect to this research was ensuring that the environmental specifications were kept within parameters. The day and night temperatures and humidity were the most difficult. Without the use of a growth cabinet this experiment had to increase its environmental margin of error, refer to appendix 3. The use of a growth cabinet would have reduced this margin of error by to up 50%. These growth chambers can accurately control the environment (Global Specifications, 2012). If a growth cabinet was available only 1 box would have been required. This box would house the chemiluminescence group which would be covered up to prevent light pollution, see appendix 2. However the use of a growth cabinet would have vastly increased expenditure. There was little funding available for this experiment and the environment controls had to be manually adjusted and monitored.

6.13 One simple adjustment could have been implemented to the enviro-boxes to increase the light intensity of the light sticks and L.E.Ds. A reflected surface such as stainless steel or mirrors placed in the interior of the boxes could have increase light intensity without adding extra light by up to 50% (Biochambers, 2012). Tamulaitis et al (2007) used this idea to resolve the problem of absorption. Materials which do not emit or reflect light absorb it which can cause luminosity to decrease. This is why growth cabinet manufacturers use reflective material, so that light can be reflected rather than be absorbed.

6.14 One major problem which was highlighted during the experiment was a pest which resides in the compost. When the compost is placed in a warm damp area, the eggs begin to hatch. The eggs are not detectable as they are small and black (Cranshaw, 1998). When this pest hatched, it caused some leaf damage (11.6% coverage) which led to further complications refer to appendix 5, page 12. A possible solution to this problem could have been to sterilise the compost. 2 strategic methods of sterilisation include mechanical, placing soil under steam pressure at 120˚C or by adding chemical (Plaster, 2008). On the other hand, sterilising the compost would have removed the majority of the nutrients. This sterilised compost would need constant fertilising before and during this experiment to be able to sustain healthy growth. The constant need for fertilisation could have caused diseases to increase through the watering. As watering is increased, so does humidity and in an environment other than a growth cabinet the likelihood of diseases such as grey would have increased.

6.15 More research is required to overcome the lack of blue light emitted from the light sticks and how to specialise them for horticultural purposes. This would require the use of different fluorescent pigments and dyes. Lacking blue light causes plants to increase growth before receiving the light they need for photosynthesis, resulting in weakened growth. Another associated problem includes slight positive phototropic responses in the roots; resulting in roots to be stunted or even to grow up. Finding a better dye which gives off more blue light and retains the red light would resolve this issue. Further refinements of the chemicals contained in these sticks are necessary. Scientists have a good understanding of the process involved in photosynthesis which would unlock a better use of chemiluminescence within the horticultural industry.

6.16 Another area of research required would be the use of chemiluminescence as a form of supplementary lighting. Cyan has been proven to have beneficial effects on plant development. This would need more research within a glasshouse to further understand the effects and possible commercial applications. One question which still remains unanswered is how cyan light can be detected by plants. This fundamental process needs a better understanding, so it can be improved for the use in nurseries.

6.17 A last area of future research for the use of chemiluminescence would be the commercial application. It would be costly and wasteful using light sticks within a nursery. A system needs to be developed to deliver this supplementary lighting. Once refinement of the chemicals has been complete the commercial application must be established. Once both of these issues have been rectified, then chemiluminescence would most likely be available for growers to use as a form of supplementary light or as a back-up system in case of emergencies.

6.18 To summarise, there was a significant difference in plant height and no significant difference in root elongation and dry weight. This proves that chemiluminescence can sustain plant growth. However the use of chemiluminescence does have its limits such as the lack of blue light. With this lack, plants were found to have grown more efficiently so they could reach their light source faster. The roots were also found to have been influenced by this lack of blue light which translates as roots not being able to grow down as much as they should.

CONCLUSION

7.0 Chemiluminescence has been used to investigate the effects on Helianthus annuus development. This was achieved by using blue and red alternatively placed light sticks, placed above the specimens. 3 measurements were then recorded to monitor and track developments within the specimens. From the research it is clear that chemiluminescence can sustain growth as in the case of height which increased by 8.8%. In regards to root length and dry weight there was no significant difference which also supports the hypotheses. This research suggests that chemiluminescence could be available to use within the horticultural industry. Chemiluminescence could be used as a source of supplementary lighting in the form of cyan light or could be applied to nursery backup systems in case of emergencies.

7.1 One of the shortcomings of this experiment was the pest issue, which caused 11.6% damage to the specimen health. Sciarid fly naturally reside within the soil and compost and hatch from small black eggs which are undetectable by Human vision. By using steam sterilisation, the pests would be unable to survive due to the pressure and temperature. This would have prevented them from causing a confounding variable within this experiment. Furthermore, the surface of the interior of the enviro-boxes could have been reflected by utilising stainless steel or mirrors. By making the surface more reflective the light being emitted would have increased intensity. This would have reduced further confounding variables.

7.2 More research is required to overcome the lack of blue light emitted from the light sticks and how to specialise them for the horticultural market. This lack of blue light can result in plants to produce fast elongated growth and for roots to grow up or to weaken. This area of research would need to improve and specialised the use of fluorescent pigments or dyes. Additionally chemiluminescence needs more exploration as a form of supplementary lighting. Cyan light has been proven to be advantageous to improve plant health and yield. Further understanding of the effects and possible commercial applications are also required.

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ACRONYMS USED

2OH – 2 Hydroxide ions

C02 – Carbon dioxide

Ca+ - Calcium ion

DNA – Deoxyribonucleic acid

H₂H0₂ – Hydrogen peroxide

H2H02 – Phenyl oxalate ester

K – Potassium

K+ - Potassium ion

L.E.D. – Light emitting diode

mm – millimetres

mRNA – Messenger ribonucleic acid

Mya – Million years ago

N – Nitrogen

Nm – Nanometres

P- Phosphorous

PAR – Photosynthetically active radiation

Pfr – Far Red light

phy gene – Phytochrome gene

Pr – Red light

UV – Ultra violet

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