Effect of supplemental lighting on primary gas exchanges of Chrysanthemum morifolium Ramat cultivar White Reagan By Xiao Ma A Thesis Presented to University of Guelph In partial fulfillment of requirements for the degree of Master of Science in Plant Agriculture Guelph, Ontario, Canada Xiao Ma, October 217
ABSTRACT Effect of supplemental lighting on primary gas exchanges of Chrysanthemums morifolium Ramat cultivar White Reagan Xiao Ma University of Guelph, 217 Advisor: Professor Bernard Grodzinski Flower greenhouse production is reduced in winter, which is due to the short photoperiod and low light levels. Studies indicate that LEDs could increase the quality and yield of ornamental crops. However, there is very little research about LED and HPS effects on chrysanthemums during long day (LD) and short day (SD). Plants were grown in the research greenhouse and growth chambers at the University of Guelph. Plants were subjected to a 16h photoperiod (LD) and a subsequent 12h photoperiod (SD). Four different light treatments were compared: 1) Natural light (Amb), 2) Amb supplemented with Red/white LEDs, 3) Amb supplemented with Red/blue LEDs and 4) Amb supplemented with HPS. Plant height, leaf area, dry matter of different parts, SPAD reading, and leaf and whole-plant level photosynthesis, respiration, transpiration, water use efficiency and daily carbon gain during both long day and short day periods were measured. We concluded that chrysanthemums showed different response to light spectrum quality between LD and SD at the leaf level but not the whole plant level.
Acknowledgements I would first like to sincerely thank my thesis advisor Professor Bernard Grodzinski, who is not only teach me in my master program but also help me a lot during my foreign life. The door to Prof. Bernard office was always open whenever I ran into a trouble spot or had a question about my research or writing. He consistently allowed this paper to be my own work, but steered me in the right the direction whenever he thought I needed it. I would also to thank my committee member: Professor Michael Dixon and Professor Barry J. Micallef for their passionate participation and help which helped me to improve my thesis and experiment design. I would like to thank Dr. Evangelos Demosthenes Leonardos for his friendship and help with the technical aspects during experimental set up and data analysis. Naheed Rana for her technical support in the as well as Ron Dutton and David Kerec for their assistance with LED lighting and growth chambers. Finally, I am grateful to my fellow graduate students, Jason Lanoue and Jonathan Stemeroff, for their friendship and advice throughout my masters. I also want to thank Theo Slaman for his supporting. I must express my very profound gratitude to my parents and my friends in both Canada and China for providing me with unfailing support and continuous encouragement throughout my years of study and through the process of researching and writing this thesis. This accomplishment would not have been possible without them. iii
Table of contents ABSTRACT... ii Acknowledgements... iii Table of contents... iv List of Figures... vi List of Tables... viii List of Abbreviations and Definitions... ix 1. Introduction... 1 2. Literature Review... 3 3. Materials and Methods... 6 3.1 Plant materials and growth conditions... 6 3.1.1 Research greenhouse chrysanthemums... 6 3.1.2 Growth chamber chrysanthemums... 6 3.1.3 Burford commercial greenhouse chrysanthemums... 7 3.2 Measurements... 7 3.2.1 Leaf gas exchange measurement... 9 3.2.1.1 Research greenhouse chrysanthemums leaf gas exchange... 12 3.2.1.2 Growth Chamber chrysanthemums leaf gas exchange... 12 3.2.2 Whole plants gas exchange measurement... 12 3.2.2.1 Research greenhouse chrysanthemums whole plants gas exchange... 17 3.2.2.2 Growth Chamber chrysanthemums whole plants gas exchange... 17 4. Results... 18 4.1 Research greenhouse chrysanthemums... 18 4.1.1 Chrysanthemums growth and development in research greenhouse... 18 4.1.2 Chrysanthemum whole plant gas exchange of research greenhouse grown plants... 23 4.1.3 Chrysanthemum leaf gas exchange of research greenhouse grown plants... 42 4.2 Growth chamber chrysanthemums... 51 4.2.1 Chrysanthemums whole plant gas exchange in growth chamber... 51 4.2.2 Chrysanthemums leaf gas exchange in growth chamber... 61 iv
5. Discussion... 66 5.1 Whole plant diurnal patterns of gas exchange and growth during LD and SD... 66 5.1.1 LD and SD under conventional HPS... 66 5.1.2 LD and SD under newer LED systems... 67 5.2 Leaf gas exchange during LD and SD... 69 5.3 Summary and implications... 69 Reference... 72 Appendix... 77 v
List of Figures Figure 3.1: Light spectrum of W, R, B, RB, RW and HPS used in leaf gas exchange measurement... 1 Figure 3.2: Examples of leaf gas exchange measurements.11 Figure 3.3: Light spectrum of HPS, RB and RW LED used in the whole plant gas exchange system...15 Figure 3.4: Overview of whole plant gas exchange system....16 Figure 4.1: Height of chrysanthemums grown in the research greenhouse. 19 Figure 4.2: SPAD reading of chrysanthemums grown in the research greenhouse....2 Figure 4.3: Leaf area and open flower number of chrysanthemums grown in the research greenhouse at final harvest..21 Figure 4.4: Dry weight of different parts of chrysanthemums grown in the research greenhouse at final harvest....22 Figure 4.5: Hourly whole plant net carbon exchange rate, transpiration rate and water use efficiency of non-acclimated and acclimated plants grown in the research greenhouse during LD... 26 Figure 4.6: Daytime and nighttime average whole plant NCER of non-acclimated and acclimated plants grown in the research greenhouse during LD....27 Figure 4.7: Daily whole plant carbon gain of non-acclimated and acclimated plants grown in the research greenhouse during LD.. 29 Figure 4.8: Average whole plant transpiration and WUE of non-acclimated and acclimated plants grown in the research greenhouse during LD...... 31 Figure 4.9: Hourly whole plant NCER, transpiration and WUE of acclimated plants grown in the research greenhouse during LD and SD......34 Figure 4.1: Daytime and nighttime average whole plant NCER of acclimated plants grown in the research greenhouse during LD and SD......35 vi
Figure 4.11: Daily whole plant carbon gain of acclimated plants grown in the research greenhouse during LD and SD...37 Figure 4.12: Average whole plant transpiration and WUE of acclimated plants grown in the research greenhouse during LD and SD......39 Figure 4.13: Light curves for leaf NCER, transpiration and WUE of research greenhouse grown chrysanthemums during LD under the different supplemental lights.. 43 Figure 4.14: Leaf NCER light response curves of research greenhouse grown chrysanthemums during LD under different supplemental light sources 45 Figure 4.15: Light curves for leaf NCER, transpiration and WUE of research greenhouse grown chrysanthemums during LD and SD.......48 Figure 4.16: Leaf NCER light response curves of research greenhouse grown chrysanthemums during LD and SD...49 Figure 4.17: Hourly whole plant NCER, transpiration and WUE of plants grown in the growth chamber during LD and SD 53 Figure 4.18: Daytime and nighttime average NCER of plants grown in the growth chamber during LD and SD..54 Figure 4.19: Carbon gain of plants grown in the growth chamber during LD and SD 56 Figure 4.2: Average transpiration and WUE of plants grown in the growth chamber during LD and SD.....58 Figure 4.21: Light curves for leaf NCER, transpiration and WUE of growth chamber grown chrysanthemums during LD and SD...62 Figure 4.22: Leaf NCER light response curves of growth chamber grown chrysanthemums during LD and SD under different light sources.63 Appendix Figure 1: Commercial greenhouse chrysanthemums vs Research greenhouse chrysanthemums final height..76 Appendix Figure 2: Commercial greenhouse chrysanthemums vs Research greenhouse chrysanthemums final SPAD reading.....78 vii
List of Tables Table 3.1: Time line for leaf and whole plant gas exchange measurements for growth chamber and research greenhouse chrysanthemums......8 Table 4.1: Whole plant leaf area, dry weight and specific leaf weight of research greenhouse plants used for whole plant gas exchange measurements during LD and SD..... 41 Table 4.2: A summary of the major physiological traits determined by analysis of leaf gas exchanges of research greenhouse chrysanthemums during LD. 46 Table 4.3: A summary of the major physiological traits determined by analysis of leaf gas exchanges of research greenhouse grown chrysanthemums during LD and SD.. 5 Table 4.4: Leaf area, dry weight and specific leaf weight of growth chamber plants used for whole plant gas exchange measurements during LD and SD.....6 Table 4.5: A summary of the major physiological traits determined by analysis of leaf gas exchanges of growth chamber chrysanthemums during LD and SD...64 viii
List of Abbreviations and Definitions Amb: Ambient condition (no supplemental light) B: Blue LED DW: Dry weight (g) HID: High-intensity discharge HPS: High pressure sodium LA: Leaf area (m 2 ) LCP: Light compensation point. The light intensity level when the plant photosynthetic rate is equal to the respiration rate so that the NCER is equal to. LD: Long day period LED: Light-emitting diode NCER: Net carbon exchange rate (μmol m -2 s -1 ) PAR: Photosynthetically active radiation Pn: Photosynthetic rate (μmol m -2 s -1 ) PPFD: The photosynthetic photon flux density (μmol photon m -2 s -1 ) R: Red LED RB: Red-blue LED Rd: Respiration rate (μmol m -2 s -1 ) RW: Red-white LED SD: Short day period SLW: Specific leaf weight, dry weight per leaf area (g m -2 ) W: White LED WUE: Water use efficiency, the rate of Pn to E (μmol CO 2 mmol -1 H 2O) ix
Y Q: Maximum quantum yield. An estimate of the maximum slope given by the nonlinear equation. CO 2 fixed per photon absorbed under PPFD limited condition. x
1. Introduction Greenhouse production is one of the biggest industries in Canada. Not only various vegetables are grown year-round, but also many varieties of flower crops are grown commercially in greenhouses. From the data of Statistic Canada of 215(Statistical Overview of the Canadian Ornamental Industry-215), the area of greenhouse flower production was approximately 7.8 million square meters and the sales of the ornamental greenhouse products was approximately $2.2 billion, which increased from $1.93 billion in 214. Ontario is still the largest province of greenhouse flowers and plants, taking a 44% share of the total production area, followed by British Columbia (25%) and Quebec (16%). Indoor production could provide the optimum environment, where the temperature, humidity and CO 2 level are suitable for plants to grow. In the greenhouse production, there are many factors that are related to yield, such as plant density, temperature, CO 2 concentration, humidity and light conditions. Supplemental lighting systems are usually used in the greenhouse to provide extra light intensity not only during no-sunshine weather but also low light seasons (fall and winter). Because of the huge effect on final yield, farmers need to consider about the light system very carefully and spend a lot of money on it, when they are preparing to build a greenhouse. In accordance to that, it is valuable for us to consider many aspects of a lighting system and especially light quality. There are many types of lamps which have been used in lighting systems for growing plants. The most commonly used light sources include fluorescent, high intensity discharge and incandescent lamps. For example, fluorescent lights are often used over germination shelves because they can be placed close to plants without overheating them. But they are usually not used for supplemental lighting in the greenhouse as their fixture causes excessive shading. High intensity discharge (HID) lamps are most often used for greenhouse supplemental light because of their high light output and relatively little shading there are two main types: high pressure sodium (HPS) which look yellow/orange and metal halide which look bluish. All these lamps were originally designed for human lighting application. Since humans have different photoreceptors from plants, these light sources have various limitations and are not very optimum for growing plants. For instance, HPS was introduced around 197 and have been used as the main technology. The advantages of HPS include long lifetime and relatively moderate installation cost (Van Ieperen and Trouwborst, 27). However, HPS lamps still have shortcomings. It is a wide range spectrum lamp that cannot provide the specific narrow wavelength lighting, which is more significant for plant 1
photosynthesis. To solve this problem, a new type of lighting system is introducing, light emitting diodes (LEDs). LEDs are semiconductor devices that produce narrow-spectrum light. The wavelength of LEDs can be from the UVC band (25nm) to infrared (1nm). The type of semiconductor material determines the color of the light emitted. The commonly cited advantages of LEDs are efficiency and lifetime. LEDs are more efficient (radiation output divided by electric input) than fluorescent and incandescent lamps and are roughly equivalent to the newest HPS lamps (Fig.1 Bourget, 28). The lifetime of LEDs is two to three times better than HPS or fluorescent lamps (Fig.2 Bourget, 28). Although the cost for setting up the LEDs is larger than that of conventional lighting system such as HPS, LEDs are becoming more affordable and of higher output with the development of light technology in recent years. In addition, the most attractive aspect of LEDs in the plant growth field is that LEDs could provide specific narrow spectrum and are often red and blue to target spectra where photosynthesis is slightly more efficient. Considering this advantage, LEDs are thought as an alternative lighting system replacing the conventional ones, such as HPS. In my experiments, I suppose to compare HPS, which is commonly used in the greenhouse industry, and LED, which is believed to be the future of greenhouse lighting system, in ornamental greenhouse production-chrysanthemum. Chrysanthemum is a major cutting flower produced in Canada. In 215, there were 3.9 million cutting chrysanthemums being sold, taking the fourth position of flower production (Statistical Overview of the Canadian Ornamental Industry-215). The production of chrysanthemums is variable depending on the cultivar, but common procedures are as bellow: Cuttings are taken from the stock plants when it is optimal. The cuttings should be planted on a misting bed (in the well-watered soil and then irrigated with a liquid fertilizer) to root. Long day photoperiod (LD; 16h day/8h night) should be applied to them from the first day. After one or two weeks, the rooted cuttings are transplanted into the ground in the greenhouse and stay in the long day period. Three weeks later, when chrysanthemums have reached the desired stem length (around 35-5cm), the photoperiod is changed to the short day (SD; 12h day/12h night), which will induce flowering. Black curtains are usually used to cover the plants and ensure the SD conditions. It normally requests five or more weeks to develop flowers and harvest the crop. 2
2. Literature Review With the development of lighting technologies, LEDs have been proved as a new source for supplemental lighting in both research and commercial purposes (Nakamura et al., 1994; Tepperman et al., 24). Comparing with the conventional HPS, LEDs have the several characteristics: (i) be able to provide specific wavelength lighting, (ii) a cooler light emitting surface, (iii) be able to use internal of the canopy (Nakamura et al., 1994; Nelson and Bugbee, 214). For commercial usage of LEDs, it is important to understand how plant production can be optimized by receiving both natural and LED lighting at the different seasons of the year (Runkle and Heins, 21; Kim et al., 24b; Gomez and Mitchell, 215; Rabara et al., 217). Most of the studies of LED and photosynthesis are based on leaf gas exchange (Goins et al., 1997; Kim et al., 24a; Hogewoning et al., 21; Liu et al., 212). Leaf physiological traits have been studied as a representative of whole plant functions under different environment conditions (Liu et al., 29). However, it is well known that the physiology of a single organ such as a leaf differs from the traits exhibited at the whole plant level because of mutual shading, different ages of leaves and canopy architecture (Davis and McCree, 1978; De Vries, 1982; Dutton et al., 1988). Totally saying, whole plant gas exchange cannot be simply predicted by leaf gas exchange data. Whole plant gas exchange data also can be used to show the daily growth patterns of the plant exposed to different physiological conditions (Dutton et al., 1988; Leonardos et al., 214). Specific wavelength LEDs have been used to study chrysanthemum growth and development. For example, one study illustrated that light quality affected the flowering inhibition of Chrysanthemum morifolium when light was used during the night break, because of the phytochrome responses in the flowering response (Higuchi et al., 212). Also, light quality could affect micro propagation of chrysanthemum (Dendranthema grandiflora cv. Tzvelev). Green light increased length of stem, internodes and fresh weight. Blue (B) light produced the shortest stems. Red (R), green and white light affected the root system of chrysanthemum (Miler and Zalewska, 24). Shimizu et al. (25) compared the effect of blue light and fluorescent lamp on chrysanthemums (Dendranthema grandiflorum cv. Reagan) height. The results showed that blue light could be used to inhibit the stem elongation. One experiment showed the red LED (663nm) was related to chrysanthemums floral bud differentiation (Fukui et al., 29). Some Chinese researcher found the usage of LED for growth of chrysanthemum (Dendranthema morifolium cv. Tzvel.) plantlets in vitro. They claimed that red LEDs effected height and the synthesis of soluble 3
sugar, while the chlorophyll content was significant increased by blue light (Huan et al., 21). One previous study discovered that the quality of LED light affected not only the flowering but also the leaf polyphenol production of Chrysanthemum morifolium (Jeong et al., 212). In a separate experiment, they indicated that blue LED could increase the stem elongation, without the inhibition of flower bud formation (Jeong et al., 214). Far-red light reduced the growth processes of Chrysanthemum morifolium Ramat. Ellen in vitro but promoted the growth of root length. Farred light could decrease the content of chlorophyll as well. Thus, the chrysanthemum growth processes were able to be controlled by far-red light (Kurilčik et al., 211). On the other hand, some researchers focused on the light combination comparison. One study showed the comparison of fluorescent light vs different LED lights in chrysanthemum (Dendranthema grandiflorum Kitam Cheonsu ), in terms of net photosynthetic rate, fresh/dry weight, leaf area and the size and number of leaf stomata (Kim et al., 24). Moreover, combination of different lights was also involved with chrysanthemum (Chrysanthemum morifolium Ramat. Coral Charm ) growth. Low red to far-red ratio light could promote the height of plants, while it did not affect the stem diameter, dry matter and internodes number (Lund et al., 27). Interestingly, there is surprisingly little research about light quality and effects on photosynthesis and virtually no data on transpiration or water use efficiency (WUE). One Chinese team (Zhou et al., 213) found that maximum net photosynthetic rate, light compensation point and light saturation point were different among chrysanthemum cultivars, but this study was not about spectral quality but an analysis of leaf photosynthesis of different cultivars. At the whole plant level, there appears to be no data regarding photosynthesis or water gas exchange for chrysanthemums. Recently, however our group reported the effects of LEDs on two other crop plants, lisianthus, and ornamental cut flower, and tomato (Lanoue et al., 217). Our results indicated that at the whole plants level, plants grown under red-blue and red-white LEDs had lower WUE than those irradiated with HPS. The net biomass gain and the daily carbon budget under HPS and LED systems were similar. These studies have lead me to my own experiments on chrysanthemums, a SD commercial greenhouse crop. My two, working hypotheses are a) use of selected LEDs (i.e., Red-blue and Red-white) as the supplemental lights will produce chrysanthemum cut flowers of similar commercial quality and at the same rate as those grown traditionally under HPS, and b) the two selected LEDs (i.e., Redblue and Red-white) supplemental lights can replace conventional HPS lighting that provides photosynthetically active radiation during both LD and SD production cycle. To our knowledge, little is known regarding photosynthesis, growth and morphology, specifically comparing LD and 4
SD as influenced by HPS. To date no information is available regarding the newer, electrically more efficient LED lights that would be deployed to supplement natural solar radiation during fall and winter in Canada. In summary, the primary objective of my studies was to provide fundamental physiological data regarding whole plant and leaf responses of chrysanthemums during LD (vegetative) and SD (flowering) stages to supplemental HPS and LED systems. 5
3. Materials and Methods 3.1 Plant materials and growth conditions 3.1.1 Research greenhouse chrysanthemums The chrysanthemum cultivar (White Reagan) was grown in the Bovey greenhouse in the winter of 217. Rooted cuttings were transplanted from plug trays to 14cm diameter pots after getting them from the supporting grower s greenhouse (Slaman s Quality Flowers, Burford, Ontario, Canada). Sungro professional growing mix #1 (Soba Beach, AB, Canada) was used as medium, which contains Canadian sphagnum peat moss, coarse perlite and dolomitic limestone. All the pots were put on 4 benches based on a complete randomized block design in the Bovey research greenhouse. Four lighting treatments were set during the experiment, HPS, Red-blue (RB) LED, Red-white (RW) LED and ambient (Amb, no supplemental lighting). Considering about the irradiance obtain with HPS lamp in supporting grower s greenhouse, the light intensity of three supplemental lights above was set at 1±25 μmol m -2 s -1, Photosynthetic active radiation(par). The LEDs we used were 39W fixtures, provided by The Light Science Group Corporation (LSGC; Warwick, RI, USA) and the HPS lighting were a 4W HPS lights from Philips (Markham, ON, Canada). This research greenhouse experiment was a light-acclimation experiment: all the chrysanthemums were grown under the supplemental light during their whole life cycle, not just exposed to the different light for a short period. The growing conditions were controlled by greenhouse control program. The temperature was 2±2 o C. Watering was manual with a fertilizer solution (2-8-2) and was done 3 times per week. The photoperiod was set as 16 hours/8 hours (day/night) for long day (LD) period and 12 hours/12 hours for short day (SD) period. Plants were kept growing in LD period for 3 weeks and then changed into SD period. 3.1.2 Growth chamber chrysanthemums Rooted cuttings of chrysanthemum (White Reagan) was transplanted from plug tray to 1cm diameters pots in both summer and fall of 216 and the winter of 217. The plant material was provided by Slaman s Quality Flowers (Burford, Ontario, Canada). Sungro professional growing mix #1 (Soba Beach, AB, Canada) was used as medium, which contains Canadian sphagnum peat moss, coarse perlite and dolomitic limestone. All the pots were placed into the growth chamber (GC-2 Bigfoot series, Biochambers, Winnipeg, MB, Canada) in the Bovey building at the University of Guelph. The growing conditions were controlled by the chamber 6
control program. The daytime temperature was 22 o C and the nighttime temperature was 19 o C. The humidity was constantly 7%. Watering was manual with a fertilizer solution (2-8-2) and was done 3 times per week. The fluorescent tubes and incandescent bulbs were set as the light source in the growth chamber and the light intensity was 3±5 μmol m -2 s -1 PAR at the top of the plants. The photoperiod was set as 16 hours/8 hours for LD period and 12 hours/12 hours for SD period. The chrysanthemums were grown under LD period for 3 weeks then changed into SD period. 3.1.3 Burford commercial greenhouse chrysanthemums The same cultivar of chrysanthemums (White Reagan) was planted into ground on Feb 23 rd of 217 in a commercial greenhouse in Burford (Slaman s Quality Flowers). The temperature was 2±2. And the humidity was set at 75±5%. The same four light sources as my description in research greenhouse, HPS, RB, RW and Amb, were set above the different rows in the greenhouse. The light intensity of all the supplemental lights was 1±25 μmol m -2 s -1 PAR at the top of the plants. The photoperiod was set as 16 hours/8 hours for LD period and 12 hours/12 hours for SD period. The photoperiod was changed to SD on March 9 th. 3.2 Measurements Plants height and SPAD reading measurement were taken every week in the research greenhouse during the whole experiment. After 2 weeks of growing during LD, both growth chamber and research greenhouse chrysanthemums began to be tested by a leaf (Licor64) and a whole plant gas exchange system. After 1 week of growing under SD, both growth chamber and research greenhouse chrysanthemums began to be tested by the leaf (Licor64) and the whole plant gas exchange system. The leaf area was measured by a leaf area meter (LI-31, LI-COR, Lincoln, NE, USA) after plants were taken out from the whole plants gas exchange system. The leaves, stems and clean-washed roots were collected separately and put into an oven at 7 o C for 48h to get the dry weight of different parts. The research greenhouse chrysanthemums provided the final harvest data, which included final height, SPAD reading and open flower number. 7
Table 3.1: Time line for leaf and whole plant gas exchange measurements for growth chamber and research greenhouse chrysanthemums. Week means the week after transplanting. Research greenhouse chrysanthemums photoperiod LD SD week 1 2 3 4 5 6 7 8 9 1 WPS WPS measurement leaf study leaf study Growth chamber chrysanthemums photoperiod LD SD week 1 2 3 4 5 6 7 WPS WPS measurement leaf study leaf study 8
3.2.1 Leaf gas exchange measurement For the leaf gas exchange measurements, we used the Licor 64 portable unit (Lincoln, NE, USA) with the different light sources. Well-watered plants were randomly selected and the 5 th highest, most expanded leaf was used for analysis. The leaf was put into the leaf chamber of a Licor 64 portable unit (Lincoln, NE, USA). The relative humidity was set steady at 7±5% by the desiccant. The concentration of CO 2 was held at 42±1 μmol mol -1 by Soda Lime. The light I used were the same to that in the lisianthus paper (Lanoue et al., 217). The spectrum of the different light was showed in Figure 3.1. 9
Figure 3.1: Light spectrum of W, R, B, RB, RW and HPS used in leaf gas exchange measurement. The data comes from the previous work on tomato and lisianthus (Lanoue et al., 217). 1 Spectral Composition (%) 8 6 4 2 4 5 6 7 Red-Blue Red-White White Red Blue HPS Wavelength (nm) 1
Figure 3.2: Examples of leaf gas exchange measurements. Leaf gas exchange measurements under the Licor 64 RB LED light source (A) and leaf gas exchange measurements under the different LEDs (e.g., RB) (B). 11
3.2.1.1 Research greenhouse chrysanthemums leaf gas exchange Research greenhouse chrysanthemums leaf CO 2 and H 2O gas exchange measurements began from the third week of LD period and the second week of SD period. The leaf was put into the leaf chamber of a Li-COR 64 portable unit (Lincoln, NE, USA) with a RB light source from Li-COR or different external LED light sources. Light curves using the Licor RB light source were produced by using the Licor 64 auto curve feature starting from a high light intensity and decreasing step by step down to no light. Light curves using the different external light source (LED lamb and HPS) were produced by using the Licor 64 but changing the light intensity manually, from a high intensity to no light. 3.2.1.2 Growth Chamber chrysanthemums leaf gas exchange Growth chamber chrysanthemums leaf CO 2 and H 2O gas exchange measurement began from the third week of LD period and the second week of SD period. The leaf was put into the head chamber of a Li-COR 64 portable unit (Lincoln, NE, USA) with different external light sources. Light curves using the different external light source (LED lamb and HPS) were produced by using the Licor 64 but changing the light intensity manually, from a high intensity to no light. 3.2.2 Whole plants gas exchange measurement A whole plant gas exchange system can monitor the CO 2 exchange and water use of the whole plant rather than just an individual leaf. The whole plant system used resembles an earlier design used by Dutton et al. (1988). The system was controlled by LabView 29 software (National Instruments Canada, Vaudreuil-Dorion, QC, Canada) running on a Dell, Precision 49 (Dell Computers, Round Rock, TX, USA) computer. This LabView 29 software allowed for the CO 2 concentration control, relative humidity control, temperature control, and light intensity control in the chambers. There were six clear polycarbonate plant chambers which measure.81m x.46m x.46m with a glass top giving a total chamber volume of 2L. During experiments using smaller plants, boxes of known volume were used to decrease the volumes of chamber, which was necessary to insure CO 2 depletion was within the systems detection limits. Two chambers used illumination with 39W RW LED fixtures from LSGC, two chambers used illumination with 39W RB LED fixtures from LSGC, and two chambers used illumination with 1W HPS lights from Philips. The light I used were the same to that in the lisianthus paper (Lanoue et al., 217). The spectrum of the light was shown in Figure 3.3. At the top of the chambers, which were illuminated by LED lights, there was a dimmable setting which allowed the light intensity to be variable. For 12
the chambers under HPS, light intensity was set by lifting the lights higher away from the chambers or by adding shade clothes to the top of the chambers. Also, there was a water bath placed between the HPS light and the chamber in order to avoid overheating of the chamber. All chambers were covered with aluminum foil on the outside to prevent light from other lights from entering the chambers and to prevent light loss lower in the chambers. Li-COR quantum sensors (LI-19SA, Li-COR Inc. Lincoln, NE, USA) were placed at the top of the plant canopy to determine the light intensity. The chambers were sealed by a polycarbonate door with 16 wing nut screws after the light intensity was set. There were 2 modes of the system: an open and closed mode. At the beginning, compressed air was passed through a purge gas generator (CO 2 Adsorber, Puregas, Broomfield, CO, USA), which scrubbed part of the CO 2. Then, the desired concentration CO 2 was established by adding pure CO 2 back into the air stream in that experiment and the mixed air was directed into the chambers. Every 2 seconds, CO 2 and relative humidity levels were checked in the chambers (1 to 6) by an infrared gas analyzer (IRGA; Li-COR CO 2/H 2O Gas analyzer 84, Lincoln, NE, USA). Each chamber was then sampled in sequence every 9 seconds with the first 3 seconds being used to flush the sample lines to prevent carry over effects from the previous chamber. The next 6 seconds of the sampling period was used for the net carbon exchange rate calculation (Equation 2.1): where Vol is the chamber volume (L); Cinitial is the initial CO 2 concentration during NCER measurement (μl L -1 ); Cfinal is the final CO 2 concentration (μl L -1 );.821 s the gas constant (L K -1 mol -1 ); T is the temperature of the chamber air ( K); and Δt is the elapse time during sampling (s) (Dutton et al., 1988). Equation 2.1: NCER= (Cinitial Cfinal)/.821 T Δt In the whole plant gas exchange experiment, the photoperiod was set to 16/8h for LD and 12/12h for SD, with the light level of 5±1 μmol m -2 s -1 PAR. Relative humidity was kept constantly at 5±5% during the whole experiment. The temperature was set to 22/19 C in day and night respectively. Plants were placed into the test chambers around 3pm and able to acclimate for the rest of the day and night period. The next morning, lights would turn on at 6am and shut off at 1pm for LD 16/8h period or turn on at 6am and shut off at 6pm for SD 12/12h period. The next day, plants were taken out of the test chamber and the leaf area would be measured by the leaf area meter. The roots were cleaned by washing and then the leaves, stems and roots were dried in the oven at 7 C for 48h. The dry weight of different parts was measured using a balance. 13
An electronic balance was placed inside the chamber to measure continuously the mass of the plants and growing media. Changes in mass over time were due to the water losses through evaporation from the media and plant transpiration. Repeat experiments without plants using the pots and media alone at the same conditions were conducted to estimate the evaporation loses. These estimates were subtracted from the total water loses to calculate the transpiration rate and water use efficiency. 14
Figure 3.3: Light spectrum of HPS, RB and RW LED used in the whole plant gas exchange system. The data comes from the previous work on tomato and lisianthus (Lanoue et al., 217). 1 Spectral Composition (%) 8 6 4 2 4 5 6 7 HPS Red-Blue LED Red-White LED Wavelength (nm) 15
Figure 3.4: Overview of whole plant gas exchange system. 16
3.2.2.1 Research greenhouse chrysanthemums whole plants gas exchange Research greenhouse chrysanthemums whole plants gas exchange experiments began on Jan 27 th, 217 and continued every 2 days until Feb 8 th, 217 for LD plants. The SD plants were tested from Feb 13 th to Feb 19 th. Acclimated plants, which were plants grown under 3 types of supplemental light (i.e., HPS, RB and RW), were randomly selected from research greenhouse and put into whole plant gas exchange chamber with the same light source. Non-acclimated plants, which were plants grown under ambient, were tested in the whole plant gas exchange system under all 3 light conditions (i.e., HPS, RB and RW). 3.2.2.2 Growth Chamber chrysanthemums whole plants gas exchange Growth Chamber chrysanthemums whole plants gas exchange experiments were from July 7 th to 27 th of 216 and from Jan 5 th to Feb 21 st. For LD, chrysanthemums were tested in the third week. For SD, chrysanthemums were tested after the 2 weeks changing into the SD photoperiod. Plants were randomly selected from growth chamber and put into whole plant gas exchange chamber under 3 light treatments (i.e., HPS, RB and RW). 17
4. Results 4.1 Research greenhouse chrysanthemums 4.1.1 Chrysanthemums growth and development in research greenhouse The heights taken every week show that plants under all the light treatments grew similarly during the first 2 weeks (Fig.4.1). After the 3 rd week, plants under the supplemental lights began to show the enhanced growth in terms of the plant height which continued almost until the end of the experiment. Among the supplemental light treatments (i.e., HPS, RB and RW), all the light treatments provided the similar enhanced effect on the plant height throughout the whole experiment. Figure 4.2 shows the SPAD readings of every week during the research greenhouse experiment. Generally, the overall trends of the different supplemental light treatments were similar. Under HPS, RB and RW, the SPAD reading was slowly increasing in LD, from the start to the third week. After changing into SD, it stayed stable for one week and increased more quickly from then on. In contrast, for Amb chrysanthemums, there was a dramatic difference during week 1 to week 3, which was illustrated by a significant decrease in SPAD readings during this period. Both height and SPAD reading show the greatest effects between second to fifth week, and that might because during that time, the weather was almost cloudy so that there was low light period. At final harvest, destructive measurements were done to get the leaf area, weight of different parts and the number of flowers (Fig.4.3). Plants under RB had the largest total leaf area, followed by the leaf area of RW grown plants. In the contrast, the leaf area of plants under HPS was not significant different to that of ambient grown plants, which showed that HPS did not enhance the expansion of leaves compared to the ambient plants. When comparing the open flower number, all 3 types of artificial lights dramatically increased the number of open flowers versus that of the ambient plants. There was no significant difference among flower number under HPS and the 2 LEDs. Figure 4.4 shows the dry weight (DW) both for total and the different parts of the plants. In total dry weight (E), plants under RB were the heaviest one, followed by plants under HPS and RW. Plants that grew under ambient light had the least biomass. The same pattern also appeared for all the different parts of the plants. RB provided the most enhanced effect in weight of plant stem (B) and total weight (E). RW and HPS resulted in similar weight in these four parts. 18
Figure 4.1: Height of chrysanthemums grown in the research greenhouse. The height of chrysanthemums grown under Amb, HPS, RB and RW lighting was measured every week after transplanting. Every point is the mean of 6 plants ± standard error (SE). The top bars show the different photoperiods (LD and SD). 1. LD SD.8 SD gas exchange Height (m).6.4 LD gas exchange Final harvest.2 Amb HPS RB RW. 1 2 3 4 5 6 7 8 9 1 Time after Transplant (Weeks) 19
Figure 4.2: SPAD reading of chrysanthemums grown in research greenhouse. The SPAD reading of chrysanthemums grow under Amb, HPS, RB and RW lighting was measured every week after transplanting. Every point is the mean of 6 plants ± SE. The top bars show the different photoperiods (LD and SD). 8 LD SD 7 SD gas exchange SPAD reading 6 5 LD gas exchange Final harvest 4 Amb HPS RB RW 3 1 2 3 4 5 6 7 8 9 1 Time after Transplant (weeks) 2
Figure 4.3: Leaf area (A) and open flower number (B) of chrysanthemums grown in the research greenhouse at final harvest. Every bar shows the mean of 1 plants ± SE under different light treatments. Letters (a, b) represent the statistical difference among light treatments based on a oneway ANOVA with a Tukey Kramer adjustment at p<.5..16 A a.14.12 b b ab Leaf Area (m 2 ).1.8.6.4.2. 16 B Amb HPS a a RB RW a 14 b Open flower number 12 1 8 6 4 2 Amb 21
Figure 4.4: Dry weight of different parts of chrysanthemums grown in the research greenhouse at final harvest. Every bar shows the mean of 1 plants ± SE under different light treatments. Letters (a, b, c) represent the statistical difference among light treatments based on a one-way ANOVA with a Tukey Kramer adjustment at p<.5. 25 25 25 25 25 A B C D E a 2 2 2 2 2 b b Weight (g) 15 1 5 c b a ab 15 1 5 b b a b 15 1 5 c ab a bc 15 1 5 c ab a Y Data bc 15 1 5 c Amb Amb Amb Amb Amb Root Stem Leaf Flower Total 22
4.1.2 Chrysanthemum whole plant gas exchange of research greenhouse grown plants Figure 4.5 illustrates a comparison of long day non-acclimated plants versus acclimated plants in terms of net carbon exchange rate (NCER), transpiration and WUE. Both non-acclimated and acclimated plants had similar patterns of NCER under all 3 light treatments during the 24 hours period: NCER increased a little from the first hour to the second, then remained at similar level during the rest of the daytime (Fig.4.5 A and B). During daytime, plants under HPS had the highest NCER in both non-acclimated and acclimated plants, while the 2 LED light treatments had similar NCERs. After the lights were shut down at the end of the sixteenth hour, the NCER dramatically dropped from positive to the negative values, and remained steady during the nighttime. During nighttime, the NCER of all 3 supplemental light treatments were similar and stable. The nonacclimated plants also had similar transpiration diel pattern with the acclimated plants (Fig.4.5 C and D). During daytime, transpiration kept increasing from the first hour to the fifth hour, then stayed at a peak for 4 hours. A decrease in transpiration was followed from the ninth hour to the end of daytime. During nighttime, transpiration was stable. However, a difference in transpiration appeared between non-acclimated and acclimated plants. For non-acclimated plants, RW treatment had the highest transpiration during the daytime, followed with RB and HPS. In the contrast, HPS treatment had the highest transpiration during the daytime in the acclimated plants, followed by the RB and RW treatments. WUE showed similar diel patterns in both non-acclimated and acclimated plants (Fig.4.5 E and F). There was a dramatic decrease in WUE from the first hour to the second hour, then WUE stayed more less stable with a little decrease until the afternoon and a slight increase towards the end of light period. The difference between non-acclimated and acclimated plants was that WUE of non-acclimated plants under HPS had the highest rate, compared to those under the RW and RB treatments, while the 3 supplemental light treatments resulted in similar WUE for the acclimated plants. Figure 4.6 shows NCER of non-acclimated and acclimated plants on 3 different bases: per plant, per dry weight (g) and per leaf area (m 2 ). The positive bars illustrate the average daytime NCER values, which are the photosynthetic rates (Pn), while the negative bars represent the average nighttime NCER values, which are the dark respiration rates (Rd). In general, there were no significant differences among different light treatments in both non-acclimated and acclimated plants on a per plant basis (Fig.4.6 A and B). Also, the values were similar under the same light treatment between non-acclimated and acclimated plants (Fig.4.6 A and B) on a per plant basis. On a per dry matter basis (Fig.4.6 C and D), NCER still did not show the significant differences among the three supplemental light treatments within the same growth condition (non-acclimated or 23
acclimated). But there was a significant difference between non-acclimated and acclimated chrysanthemums under the same light treatment. The acclimated plants had the higher photosynthesis than those of non-acclimated plants. Furthermore, NCER based on leaf area (Fig.4.6 E and F) showed a different pattern between non-acclimated and acclimated plants: plants grown under HPS had higher daytime average values than the other 2 LED light treatments grown plants, although the nighttime average values were similar. Moreover, although acclimated plants had similar values on a per plant basis NCER with those of the non-acclimated plants (Fig.4.6 A and B), plants in acclimated condition generated higher values under the 3 light treatments respectively, on a per dry matter (Fig.4.6 C and D) and a per leaf area bases (Fig.4.6 E and F), than nonacclimated ones. Figure 4.7 shows the daily carbon gain on a per plant, dry matter and leaf area bases. For nonacclimated or acclimated plants only, there were no significant differences in carbon gain among the three supplemental light treatments on a per plant basis (Fig.4.7 A and B). Also, there were no significant differences in carbon gain between non-acclimated and acclimated plants under the same light treatment. Carbon gain on a dry matter basis also showed no significant differences among the three supplemental light treatments both in either non-acclimated or acclimated plants (Fig.4.7 C and D). However, there was a significant difference between non-acclimated and acclimated plants under the same light treatment (Fig.4.7 C and D). Acclimated plants had the higher carbon gain than that of non-acclimated plants on a dry weight basis. Carbon gain on a per leaf area basis is illustrated in panel E and F of Figure 4.7. Generally, HPS treatment enhanced carbon gain on a leaf area basis, followed by the 2 LED light treatments in acclimated plants (Fig.4.7 F). But there was no significant difference among the three supplemental light treatments in non-acclimated plants (Fig.4.7 E). When comparing panel E and F in Figure 4.7, non-acclimated and acclimated plants had similar C gain under the HPS and RW treatments, but acclimated plants had higher values under the RB light treatment. Figure 4.8 illustrates the average transpiration and WUE during LD. For both non-acclimated and acclimated plants there were no significant differences in transpiration among the three supplemental light treatments (Fig.4.8 A and B). However, acclimated plants under HPS had the highest transpiration rates (Fig.4.8 B). In contrast, compared to the responses of transpiration above, WUE showed different patterns between non-acclimated and acclimated plants (Fig.4.8 C and D). Non-acclimated plants showed the highest WUE under HPS treatment, followed by RB and RW treatments (Fig.4.8 C). However, acclimated plants had similar WUE values among the three 24
supplemental light treatments (Fig.4.8 D). There were no differences between non-acclimated and acclimated plants under each light (Fig.4.8 C and D). 25
Figure 4.5: Hourly whole plant NCER, transpiration and WUE of non-acclimated (A, C and E) and acclimated (B, D and F) plants grown in the research greenhouse during LD. Non-acclimated plants, chrysanthemums grown under ambient light, were tested in the whole plant system under the 3 supplemental light treatments. Acclimated plants, chrysanthemums grown under the 3 supplemental light treatments, were tested in the whole plant systems under the same growth lights. The light intensity was set at 5±1 μmol m -2 s -1 PAR for 16h (top white bar) followed by an 8h (top black bar) dark period. Every point represents the mean of 6 replicates ± SE for panel A, C and E and 8 replicates ± SE for panel B, D and F. 2 A 2 B NCER ( mol CO 2 m -2 s -1 ) 15 1 5 NCER ( mol CO 2 m -2 s -1 ) 15 1 5 Transpiration (mmol H 2 m -2 s -1 ) WUE ( mol CO 2 / mmol H 2 O) 2.5-5 2.5-5 241519.278 241519.3125 241519.3542 241519.3958 241519.4375 241519.4792 241519.528 241519.5625 241519.642 241519.6458 241519.6875 241519.7292 241519.778 241519.8125 241519.8542 241519.8958 241519.9375 241519.9792 24152.28 24152.625 24152.142 24152.1458 24152.1875 24152.2292 241519.278 241519.3125 241519.3542 241519.3958 241519.4375 241519.4792 241519.528 241519.5625 241519.642 241519.6458 241519.6875 241519.7292 241519.778 241519.8125 241519.8542 241519.8958 241519.9375 241519.9792 24152.28 24152.625 24152.142 24152.1458 24152.1875 24152.2292 2. 1.5 1..5 Transpiration (mmol H 2 m -2 s -1 ) 2. 1.5 1..5. 16. 16 241519.278 241519.3125 241519.3542 241519.3958 241519.4375 241519.4792 241519.528 241519.5625 241519.642 241519.6458 241519.6875 241519.7292 241519.778 241519.8125 241519.8542 241519.8958 241519.9375 241519.9792 24152.28 24152.625 24152.142 24152.1458 24152.1875 24152.2292 241519.278 241519.3125 241519.3542 241519.3958 241519.4375 241519.4792 241519.528 241519.5625 241519.642 241519.6458 241519.6875 241519.7292 241519.778 241519.8125 241519.8542 241519.8958 241519.9375 241519.9792 24152.28 24152.625 24152.142 24152.1458 24152.1875 24152.2292 N.a.N. 14 12 1 8 6 4 2 C E TIME WUE ( mol CO 2 / mmol H 2 O) HPS RB RW 14 12 1 8 6 4 2 D F TIME HPS RB RW 6:: 1:: 14:: 18:: 22:: 2:: 6:: 1:: 14:: 18:: 22:: 2:: 6:: TIME (hh:mm:ss) TIME (hh:mm:ss) 26
Figure 4.6: Daytime and nighttime average whole plant NCER of non-acclimated (A, C and E) and acclimated (B, D and F) plants grown in the research greenhouse during LD. NCERs are shown on a per plant (A, B), dry weight (C, D) and leaf area (E, F) basis. Plants were placed in the whole plant NCER system under either HPS, RB, or RW lights with a light intensity of 5±1 μmol m -2 s -1 for LD (16h light period followed by an 8h dark period). Values represent the daytime or nighttime means of 6 replicates ± SE for panel A, C and E and 8 replicates ± SE for panel B, D and F. Upper case letters (A, B, or X, Y) represent statistical significances in daytime or nighttime NCER among light treatments within each panel. Lower case letters (a, b, or x, y) represent statistical significances between non-acclimated and acclimated under the same light treatment. Statistical differences were determined by a one-way ANOVA with a Tukey s-kramer adjustment (p<.5). 27
1.5 A 1.5 B NCER( mol CO 2 plant -1 s -1 ) 1..5. 1..5. Xy Xx Xx Xx Xx Xx.2 C Ab Ab Ab.2 D HPS RB RW NCER( mol CO 2 g -1 s -1 ).15.1.5..15.1.5. -.5 Xx Xx Xx -.5 Xx Xx Xy 15 E Ab 15 F Ba Ba NCER( mol CO 2 m -2 s -1 ) 1 5 1 5 Xx Xx Xx Xx Xx Xx 28
Figure 4.7: Daily whole plant carbon gain of non-acclimated (A, C and E) and acclimated (B, D and F) plants grown in the research greenhouse during LD. Carbon gains are shown in plant (A, B), dry weight (C, D) and leaf area (E, F) basis. Plants were placed in the whole plant NCER system under either HPS, RB, or RW lights with a light intensity of 5±1 μmol m -2 s -1 for LD (16h followed by an 8h dark period). Values represent the means of 6 replicates ± SE for panel A, C and E and 8 replicates ± SE for panel B, D and F. Upper case letters (A, B) represent statistical significances among light treatments within each panel. Lower case letters (a, b) represent statistical significances between non-acclimated and acclimated plants under the same light treatment. Statistic differences were determined by a one-way ANOVA with a Tukey s-kramer adjustment (p<.5). 29