End-of-day Far-red Light Quality and Dose Effects on Elongation of Tomato Rootstock Seedling Hypocotyls

End-of-day Far-red Light Quality and Dose Effects on Elongation of Tomato Rootstock Seedling Hypocotyls Item Type text; Electronic Thesis Authors Chia, Po-Lung Publisher The University of Arizona. Rights Copyright is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author. Download date 12/07/2018 20:36:52 Link to Item http://hdl.handle.net/10150/193454

END-OF-DAY FAR-RED LIGHT QUALITY AND DOSE EFFECTS ON ELONGATION OF TOMATO ROOTSTOCK SEEDLING HYPOCOTYLS by Po-Lung Chia Copyright Po-Lung Chia 2009 A thesis submitted to the Faculty of the SCHOOL OF PLANT SCIENCES In Partial Fulfillment of the Requirements For the degree of MASTER OF SCIENCE In the Graduate College THE UNIVERSITY OF ARIZONA 2009

2 STATEMENT BY AUTHOR This thesis has been submitted in partial fulfillment of requirements for an advanced degree at the University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library. Brief quotations from this thesis are allowable without special permission, provided that accurate acknowledgment of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the copyright holder. Po-Lung Chia APPROVAL BY THESIS DIRECTOR This thesis has been approved on the date shown below: Dr. Chieri Kubota Date

3 ACKNOWLEDGEMENTS Special thanks to my advisor Dr. Chieri Kubota for giving, without fail or delay, expertise, guidance, mentorship and critical encouragement, and especially for her belief in my abilities at the most crucial moments. Without these this work would have been impossible. Thanks to the member of my committee Drs. Dennis Ray and Guangyao (Sam) Wang for their guidance and direction. Thanks to Dr Mohammad Torabi for assistance in statistical analysis of the raw research data. Thanks to Mark Kroggel for assistance in various areas. Thanks to Ian Justus for his valuable help, critical in the crucial task of data collection. Thanks to members of the CEAC Dr. Murat Kacira for his suggestions and advice on possible applications and opportunities to demonstrate before my colleagues.

4 TABLE OF CONTENTS LIST OF TABLES...5 ABSTRACT....6 INTRODUCTION..7 LITERATURE REVIEW. 11 PRESENT STUDY...18 REFERENCES 22 APPENDIX A: END-OF-DAY FAR RED LIGHT QUALITY AND DOSE EFFECTS ON ELONGATION OF TOMATO ROOTSTOCK SEEDLING HYPOCOTYLS... 26 Abstract. 28 Materials and Methods.. 32 Results and discussion...37 Conclusions...42 References. 44

5 LIST OF TABLES TABLE 1: List of EOD-FR treatments with the source, intensity and duration used by various experiments...21

6 ABSTRACT Methyl bromide fumigation, a common method used to combat soil borne pathogens in commercial tomato cultivation, was to be limited by the Montreal Protocol due to concerns of ozone depletion. Alternative methods to protect tomatoes against diseases include grafting. However, short grafted transplants may expose the scion to the soil. To avoid scion exposure, hypocotyl elongation of two tomato rootstocks Maxifort and Aloha via end-of-day far-red (EOD-FR) was examined in terms of light quality (red to far-red ratio, or R/FR) and dose (product of far-red intensity and duration). In EOD-FR light quality experiments, Aloha seedlings were exposed to unfiltered and filtered incandescent light with an R/FR of 0.5 and 0.05 respectively. The resulting hypocotyl elongation was higher in filtered light than either the unfiltered light or the untreated control. Hypocotyl elongation response to EOD-FR dose in Aloha and Maxifort was affected by both far-red intensity and treatment duration. A saturating response was also found within a far-red dose between 0 8 mmol m -2 d -1 and modeled using non-linear regression with a three parameter Michaelis-Menten equation to estimate the far-red dose required to obtain near-maximum hypocotyl elongation for Aloha and Maxifort. The far-red dose required was affected by cultivar and experimental period. None of the EOD-FR treatments affected plant mass or stem diameter. To conclude, for maximum hypocotyl elongation using EOD-FR, the lower R/FR would increase the effectiveness of the treatment. The far-red dose should preferably be at 4 8 mmol m -2 d -1.

7 INTRODUCTION As a result of the Montreal Protocol, traditional means of combating soil borne pathogens using methyl bromide fumigation was to be restricted and phased out in developed countries (UNEP, 1995). Alternative fumigants were not as effective (Locascio et al., 1997), and as a result, horticultural practices such as grafting became increasingly attractive with U.S. growers. Grafting provides resistance to soil pathogens such as Fusarium wilt to otherwise vulnerable but productive scions (Lee, 1994). Short hypocotyls, however, are more difficult to graft and there is a risk that the vulnerable scion would root when exposed to the soil during transplanting (personal communication, De Ruiter seeds). This is particularly the case when producers are to handle large quality of grafted seedlings by untrained workers or the seedlings are mechanically transplanted. To overcome the issue of scion exposure, rootstock hypocotyl length could be extended. Several means such as use of plant growth regulators and water management had limitations. In contrast, end-of-day (EOD) light quality treatments, described below, seemed to be more flexible and affordable for most commercial growers. EOD treatments involves artificial provision of light with either high (>5) or low (<0.5) red to far-red ratios (R/FR) to induce an EOD phytochrome-mediated response such as stem elongation. EOD treatments are detected by photo-sensitive proteins called phytochromes within the plant which also act as the signal transducer. Encased within the each phytochrome protein is a chromophore molecule which has two inter-convertible isoforms with different light absorption spectra in the red (wavelengths of 600-700 nm)

8 and far-red (wavelengths of 700-800 nm) regions (Hendricks et al., 1962; Siegelman et al., 1966). A change in light environment such as being under the shade of a neighboring plant usually comes with a change in R/FR. Hence, when plants experience a change in the light environment, phytochrome isoform ratio will also change, triggering various physiological responses such as germination (Mancinelli et al., 1966), stem elongation and pigment synthesis (Hendricks and Borthwick., 1967). EOD treatment was used experimentally to prove that taller growth within rows of field cultivated plants compared to the edge of the field was a result of lower R/FR due to shading from neighboring plants (Kasperbauer, 1971). By providing lab-grown plants with a short duration of light rich in far-red (and low in R/FR) at the end of the photoperiod, the experimenters were able to induce taller growth than plants that were exposed to light rich in red (Kasperbauer, 1971). EOD treatments became widely used as assays to test for phytochrome involvement in various morphological responses (Mancineli et al., 1966). Understanding the mechanism of plant responses under EOD treatment has been done at the molecular level. Mutants deficient or over-expressing genes encoding the phytochrome protein or components of the phytochrome signal transduction pathway were exposed to EOD and the response, usually stem elongation, was compared to that of the wild-type plants. Plant models assayed using EOD treatments include Arabidopsis thaliana, cucumber (Cucumis sativus) and aspen (Populus tremula x tremloides). By utilizing EOD treatments and observing the response, investigators were able to identify

9 various phytochrome genes and crucial intermediate molecules such as gibberellic acid in the phytochrome signal transduction pathway (Nagatani et al., 1991; Olsen and Junttila, 2002; Moe et al., 2003). Given the potential of EOD treatments in extending stem lengths in a wide variety of plant species under experimental conditions, the treatment could perhaps be used to obtain a desired stem length. EOD treatments reversed the shortened stems of chrysanthemum (Dendranthema x grandiflorum) grown under sunlight with far-red light removed (Rajapakse et al., 1993), demonstrating potential in practical applications under ambient conditions. Our experiment focused on application of EOD treatments in a greenhouse setting for tomato rootstocks. In order to design a practical system for commercialization, both light quality (R/FR) and the dose (defined as far-red intensity multiplied by duration) of the EOD treatment need to be investigated using commercially available materials. As of now, there is no study investigating both light quality and dose for the purpose of tomato rootstock hypocotyl elongation. Previous experiments with tomato seedlings ( Mountain Pride ) used high doses of far-red (up to 40 mmol m -2 d -1 ) and confirmed that end-of-day far-red (EOD-FR) produced longer stem lengths compared to end-of-day red (EOD-R) but not the untreated control, though hypocotyl lengths were not explicitly measured (Decoteau et al., 1988; Decoteau and Friend, 1991). Other far-red dose experiments used specialist far-red fluorescent light bulbs and spectral filters investigated a dose ranging from 1-100 mmol m -2 for anthocyanin degradation and mesocotyl elongation response in corn

10 seedlings. Saturating far-red levels were close to 8-10 mmol m -2 d -1. The dose was achieved by varying treatment duration from 2 s to 35 min at a constant far-red intensity of 32 mol m -2 s -1 (Gorton and Briggs, 1980). Our experimental design therefore utilized commercially available incandescent light, red and far-red LED and acrylic filters to investigate both EOD-FR light quality (R/FR) and dose for hypocotyl elongation response.

11 LITERATURE REVIEW Phytochromes Light affects the plant in many ways. Besides being the main driving force behind photosynthesis, light also affects plant morphology. Germination, flowering and etiolation are classical light dependent responses that plant physiologists have investigated. In order to perceive light, a sensor or a receptor has to be present. A photo-sensitive protein was identified and given the name phytochrome (Hendricks et al., 1962) and later successfully isolated (Daniels and Quail, 1984) for characterization. The phytochrome molecule contains a chromophore which interchanges between two isoforms with differential light absorption specifically of the red (wavelength 600-700 nm) and far-red (wavelength 700-800 nm) spectra (Siegelman et al., 1966). Irradiation with far-red or red light would change phytochrome isoform ratio and alter biochemical and physiological responses such as germination (Mancinelli et al., 1966), stem elongation and pigment synthesis (Hendricks and Borthwick, 1967) in various plant species. In addition, red light, far-red light and the red to far-red ratio (R/FR) varies throughout the time of the year as well as the latitude (Gorski, 1980). The conclusion was that plants may sense the period of the year via the R/FR using their phytochromes. Thus, with phytochromes, plants are able to sense the external light environment and respond in various physiological and morphological means. Plant Responses to End-of-Day Far-Red Light

12 In end-of-day (EOD) treatments, plants respond to relatively short durations of either red or far-red light at the end of the photoperiod, after which the dark period would continue as per normal. The phenomenon was first reported in morning glory (Ipomoea nil). The investigators exposed the plants to four minutes of far red at the end of different photoperiods and observed that flowering of the short-day plant was hampered by EOD-FR (Fredericq, 1964). In tobacco plants (Nicotiana tobacum), it was observed that field grown plants within the rows grew taller than those at end of the rows. Spectral analysis showed that plants within rows were shaded by neighboring plants. R/FR levels were 0.14, 0.15 and 0.24 below the canopy, within the canopy and under a single leaf respectively as far-red light penetrated the canopy and leaves more easily than red light. The observation gave the speculation that the taller growth was a result of low R/FR within the rows. EOD was carried out in a controlled setting and the seedlings exposed to EOD-FR were discovered to be significantly taller than those exposed to EOD-R (Kasperbauer, 1971). After the initial discovery and description of EOD treatments, other investigators had utilized EOD in other species. In tomatoes EOD-FR increased hypocotyl lengths (Decoteau et al., 1988; Decoteau and Friend, 1991) compared to EOD-R. In chrysanthemums (Dendranthema x grandiflorum) EOD-FR reversed the stem-shortening effect of the far-red-removing CuSO 4 filter (Rajapakse et al., 1993). EOD-FR also increased stem length in watermelon (Citrullus lanatus), cucumbers (Cucumis sativa), cowpeas (Vigna sinensis), aspen (Populus tremula x tremloides) and Arabidopsis thaliana (Hatt Graham and Decoteau, 1997; Xiong et al., 2002;

13 Martinez-Garcia et al., 2000; Olsen and Junttila, 2002; Nagatani et al., 1991 and Moe et al., 2003) compared to EOD-R treatments. Due to the relative ease of setup, EOD irradiation became useful as a phytochrome-activity assay to many molecular biologists and plant physiologists investigating the various components of the phytochrome signaling pathway (Smith and Whitelam, 1990). Plant mutants that exhibit an atypical EOD-FR response tend to be either deficient or over-expressing phytochromes (Nagatani et al., 1991; Olsen and Junttila, 2002) or deficient in part of the phytochrome signal transduction pathway (Chen, 2005). Gibberellic acid (GA) was also found a component of the EOD-FR stem elongation response pathway after reviewing GA and GA catabolite levels in cowpeas (Martinez-Garcia et al., 2000). Plants may also modulate its phytochrome levels in response to EOD treatments. EOD-FR treated Avena plants had more phytochromes while EOD-R had less (Steward et al., 1992), suggesting a feedback response loop. EOD irradiation had proven useful as an assay, but its practical applications in both floriculture and horticulture have not been fully studied. However, with the increased availability of affordable far-red containing such as LED, it may be possible to discover new avenues to use EOD irradiation effectively. Tomato cultivation Tomato (Solanum lycopersicum) is a plant that originated in Central and South America (Jenkins, 1948). The cultivation of the plant radiated from its native region

14 during the 16 th century as Europeans colonized the Americas and brought the plant to other parts of the world (Jenkins, 1948). The fruit of the tomato plant became popular whether fresh or processed as sauces or puree. World production in 2007 reached 126 million ton with China, US and Turkey leading the production at 33.6 million, 11.5 million and 9.9 million ton produced in the same year respectively (FAOSTAT, 2006). Tomato cultivation in the US had an estimated value of USD 2.35 billion in 2007. Of which, the fresh-market tomato production was worth USD 1.4 billion while processed tomatoes was worth USD 0.95 billion. The area devoted to fresh tomato cultivation in 2008 was at 109,200 acres, with the states of Florida and California leading in terms of both production and acreage. Processed tomato production in 2008 occupied an even larger area of 296,500 acres (USDA NASS, 2009). Fresh-market tomato cultivation, having a much higher crop value compared to those destined for processing, is the focus of many new horticultural research. Methyl Bromide and Grafting Tomatoes are affected by many diseases, and fresh-market tomatoes are more vulnerable as consumers tend are more selective to appearance, shape and size of the tomatoes, which are easily affected by pests and diseases (Lucier et al., 2000). Common tomato diseases include Fusarium wilt. The disease is caused by a soil-borne pathogen Fusarium oxysporum which may either attack the vascular system or cause crown or root rot. It is widespread in tomato growing areas, such as Florida, and causes significant

15 damage (Jones et al., 1991). In order to combat these pathogens, methyl bromide (MB) fumigation was used. However, with article 2H of the Montreal Protocol (UNEP, 1995), MB would be phased out due to its ozone depletion properties. Alternatives to MB proved to be not as effective (Locascio et al., 1997), and complete phasing out of MB would cause an estimated 46% loss of tomato crop in Florida alone (Gilreath et al., 1994). Due to the inability to replace MB as a solution against the various pathogens, critical use exemptions were still provided in 2009, with tomato cultivation consuming 25% (1245 ton) of the total allocations (5000 ton) set at 20% of 1991 levels (BUNNI, 2006). However, long term solutions must still be sought. One of these solutions is grafting, which is commonly used in Asian (Oda et al., 1993) and Mediterranean (Besri, 2007) countries to combat soil borne diseases. One of the most common grafting procedures is tube grafting, which involves the removal of the portion above the cotyledons of rootstock and scion seedlings by performing a 45 o or sharper cut with a razor blade. The cut surfaces of the scions and rootstocks are then spliced together and held usually with a silicon clip or tube until the cut surfaces have healed. The grafted plant would have the resistant root system of the rootstock and the shoot system of the productive scion. Additionally, grafting may offer other benefits such as increased capacity for salinity (Rivero et al., 2003) and heat tolerance (Abdelmageed et al., 2007). The cost of each grafted seedling is high as the procedure is laborious. Also, when transplanting the grafted seedlings, there is a risk of exposing the scion to the soil, causing it to root and defeat the purpose of grafting (personal communication with De

16 Ruiter Seeds). Use of EOD, EOD light sources, treatment duration and dose In order to overcome the problem of the vulnerable scion being exposed to the soil, it may be advantageous to enhance the length of the rootstock hypocotyls without compromising stem diameter and strength. Methods include chemical means such as gibberellic acid applications (Lang, 1956). Difference between day temperature and night temperature (DIF) may also be used (Erwin et al., 1989). However, chemical means may delay flowering and hence fruit production (Marth et al., 1956), and most tomato transplant propagators do not have access to precise temperature control systems, making these options less attractive. On the other hand, EOD-FR applications have been known to cause stem elongation in various other plants (Decoteau and Friend, 1991; Hatt Graham and Decoteau, 1997) and seem to be easier to setup and implement without any change to either plant mass, flowering time or yield (Decoteau and Friend, 1991). Since far-red light sources could be obtained at a reasonable price commercially, and that light intensities required for EOD treatments are very small compared to what is needed for supplemental photosynthetic lighting, we believe that the method had greater potential and hence became the focus of our investigation. Previously reported EOD-FR experiments have used a variety of light sources for their EOD-FR experiments, of which incandescent light with a spectral cut filter seemed to be the most common. The investigators used total far-red dose (a product of far-red

17 intensity and duration) with a range of 0.3 mmol m -2 d -1 to 133 mmol m -2 d -1 (Table 1). However, as the experiments largely utilized EOD-FR as an assay to either elucidate components of the phytochrome signal transduction pathway or the effects of EOD-FR on plant growth, most of the doses utilized were probably at saturating levels. There were a few experiments that investigated the relationship between physiological response and EOD-FR dose. In corn (Zea mays) seedlings, anthocyanin degradation and lengths of the coleoptile and mesocotyl were plotted against far-red dose (Gorton and Briggs, 1980), the dose utilized was 1-100 mmol m -2 d -1 with the response saturation at 10-16 mmol m -2 d -1. In oat seedlings (Avena sativa), morphological measurements relative to a darkness control was also plotted against far-red dose ranging from <1 to 1000 mmol m -2 d -1, with saturation at 0.02 and 10.00 mmol m -2 d -1 for coleoptile and mesocotyl elongation respectively (Mandoli and Briggs, 1980). Dose could be altered by either extending the duration of the EOD-FR treatment (Gorton and Briggs, 1980), the intensity of the far-red light source or both (Mandoli and Briggs, 1980). In both cases, the investigators did not attempt to use a known model to make any extrapolation or prediction. Further studies would compare the effects of both treatment duration and far-red intensity, and determine which of these two is more important on the EOD-FR induced response.

18 PRESENT STUDY END-OF-DAY FAR-RED LIGHT QUALITY AND DOSE ON ELONGATING TWO TOMATO ROOTSTOCK CULTIVAR HYPOCOTYLS As grafting becomes more attractive as an alternative to methyl bromide fumigation, challenges in using grafted transplants should be met. Short hypocotyls which benefited stand establishment were known to have higher incidences of scion exposure to the soil, allowing pathogens to infect the vulnerable scion directly (personal communication, De Ruiter Seeds). The objective of this study was to evaluate EOD-FR as a means to elongate hypocotyl in tomato rootstocks. Both the R/FR and the far-red light dose (a product of treatment duration and far-red intensity) of the EOD-FR treatments were investigated. The results would provide information on the suitability of incandescent light as an EOD-FR light source as well as the minimum dosage for maximum hypocotyl extension. Two tomato rootstock cultivars, Aloha and Maxifort, were used in the study. Seedlings were germinated in growth chambers and then grown under greenhouse conditions. When the cotyledons had fully expanded, the seedlings were exposed to 14 consecutive days of EOD-FR treatment inside the greenhouse. In the R/FR experiment, Aloha seedlings were exposed to both unfiltered and filtered incandescent light. The unfiltered incandescent light had an R/FR of 0.5 showed significant hypocotyl elongation compared to the untreated control seedlings (22%).

19 However, hypocotyl elongation was further enhanced (44%) by lowering R/FR to 0.05 with a spectral cut filter. In the far-red dose experiment, both Aloha and Maxifort seedlings were exposed to EOD-FR with different far-red doses by altering both treatment duration and far-red intensity. The experiment was repeated twice in March, April and May of 2009. When comparing between treatments with an EOD-FR dose of 1 mmol m -2 d -1 and 2 mmol m -2 d -1 (achieved either by doubling treatment duration or far-red intensity), the higher dose treatment exhibited longer hypocotyl lengths. When comparing between the two treatments with EOD-FR dose of 2 mmol m -2 d -1 that differed in both treatment time and far-red intensity, there was no significant difference. We conclude that far-red dose response could be altered by both treatment intensity and duration. Seedlings exposed to the same dose despite having different treatment duration or light intensity had similar hypocotyl lengths. Also, for doses between 2-8 mmol m -2 d -1, hypocotyl lengths were also similar to each other, suggesting a saturating response. Non-linear regression using a Michaelis-Menten model was also carried out to illustrate a relationship between far-red dose and hypocotyl lengths. The resulting equation was used to calculate the theoretical dose required to achieve 90% of maximum elongation. Aloha had a saturating dose of 5-14 mmol m -2 d -1 while Maxifort had a saturating dose of 8-15 mmol m -2 d -1, with the dose varying by month. The results suggest that EOD-FR hypocotyl elongation response is affected by far-red intensity, treatment duration as well as the environment. The results of the study provided reference information for EOD-FR applications in

20 commercial scale. Far-red light sources would require a sufficiently small R/FR as well as have the intensity to provide the required dose to achieve significant elongation.

21 Table 1. List of EOD-FR treatments with the source, intensity and duration used by various experiments. Intensity and Paper Plant Model Far-red light source duration Fredericq, 1964 Martinez-Garcia et al., 2002 Decoteau et al., 1991 Hatt Graham and Decoteau, 1997 Rajapakse et al., 2003 Moe et al., 2003 Nagatani et al., 1991 Morning glory (Pharbitis nil) Cowpeas (Vigna sinensis) Tomato (Solanum esculentum) Watermelon (Citrullus lanatus) Chrysanthemum (Dendrathema x grandiflorum) Cucumber (Cucumis sativus) Arabidopsis thaliana Arabidopsis thaliana 150-w incandescent flood lamps filtered with 5 cm of water and 2 layers each of dark blue and red cellophane 500 W reflector flood lamp filtered through 5 cm layer of water Two 150-W internal reflector incandescent filament lamps filtered through a polyacrylic sheet of cast acrylic #2711, dark red Two reflector incandescent filament bulbs filtered through a polyacrylic sheet of cast acrylic #2711, dark red Far-red fluorescent light tubes wrapped with Primary Red filters (No.106, LEE filters) and green filters (dark green, No. 124) 2x150 W incandescent bulbs filtered through 5 cm of water and one layer of far-red-perspex (Plexiglas type FRF 700) -1 20 W cm -2 nm at 740 nm for 4 minutes 222 mol m -2 s -1, 10 minutes -1 43.7 mol m -2 s in 700-780 nm, 15 minutes 12.0 W m -2 in 700-780 nm, 15 minutes 0.8 mol m -2 s -1 at 700-800 nm, 30 minutes 26 mol m -2 s -1, 20 min Dosage z (mmol m -2 d -1 ) 0.3 133.0 z Dosage is the product of far-red intensity and duration of the treatment. Conversion from irradiance unit of W m -2 to mmol m -2 d -1 was carried out using the Planck-Einstein equation (E = hc/ where E = energy, h = Planck s constant, c = speed of light and = wavelength), assuming the average far-red wavelength was 750 nm if not provided already by the investigators. 39.3 67.7 1.44 31.2

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25 http://www.nass.usda.gov/quickstats/index2.jsp Xiong, J.Q., G.G. Patil and R. Moe. 2002. Effect of DIF and end-of-day light quality on stem elongation in Cucumis sativus. Scientia Hort. 94:219-229.

26 APPENDIX A: END-OF-DAY FAR RED LIGHT QUALITY AND DOSE EFFECTS ON ELONGATION OF TOMATO ROOTSTOCK SEEDLING HYPOCOTYLS

27 End-of-Day Far-Red Light Quality and Dose Effects on Elongation of Tomato Rootstock Seedling Hypocotyls Po-Lung Chia 1 and Chieri Kubota School of Plant Sciences, Division of Horticultural and Crop Sciences, The University of Arizona, Tucson, AZ 85721-0036 1 To whom reprint requests should be addressed. Email address: sephodwyrm@gmail.com

28 Subject Category: Environmental Stress Physiology End-of-Day Far Red Light Quality and Dose Effects on Elongation of Tomato Rootstock Seedling Hypocotyls Additional Index Words. Phytochrome, LED, incandescent light, transplant production, grafting, controlled environment Abstract As a result of the Montreal Protocol, methyl bromide fumigation would be limited and alternatives such as grafting became viable. For the purpose of preventing scion exposure to the soil in grafted tomato transplants, effects of end-of-day far-red (EOD-FR) on tomato rootstock hypocotyl elongation were investigated. Two commercially available rootstock cultivars Maxifort (Solanum lycopersicum S. habrochaites) and Aloha (S. lycopersicum) were used for the experiments examining responses to EOD-FR light quality (red to far-red ratio, or R/FR) and EOD-FR dose in a greenhouse environment. In the EOD-FR light quality experiment using Aloha seedlings, incandescent light (R/FR = 0.5) induced significant hypocotyl elongation (22%) compared to the untreated control. Incandescent light with a spectral cut filter (reducing R/FR to 0.05 with no change to the far-red intensity) induced a much greater hypocotyl elongation (44%), confirming the importance of lower R/FR in EOD-FR treatments. In the experiment on EOD-FR dose response, hypocotyl elongation of both Aloha and Maxifort was affected by far-red

29 intensity as well as far-red treatment duration. The increase in hypocotyl length was well described using a Michaelis-Menten type model with far-red dose (in mmol m -2 d -1 ) as an independent variable. The model based estimation of saturating far-red dose for Aloha and Maxifort was 5-14 mmol m -2 d -1 and 8-15 mmol m -2 d -1 respectively, although practically a near saturation dose seems to be 2-4 mmol m -2 d -1 for both cultivars. None of the EOD-FR treatments affected stem mass and diameter. Hence, elongation was achieved without compromising growth and development. EOD-FR was shown to have a potential use in transplant propagation industry which hopes to provide long and strong hypocotyls for grafting use. Introduction Phytochromes are photoreceptor proteins made by plants to perceive the external light environment. The light reception region within phytochrome is the chromophore which has two inter-convertible isoforms with different light absorption spectra in the red (wavelengths of 600-700 nm) and far-red (wavelengths of 700-800 nm) regions (Hendricks et al., 1962; Siegelman et al., 1966). Irradiation with far-red or red light would change phytochrome isoform ratio and alter biochemical and physiological responses such as germination (Mancinelli et al., 1966), stem elongation and pigment synthesis (Hendricks and Borthwick, 1967). These experiments were given the name end-of-day (EOD) as the provision of far-red or red light was usually carried out at the end of the photoperiod for a duration of between 5-30 min.

30 EOD treatments were widely used as assays to test far-red induced morphological and growth responses. Tobacco (Nicotiana tabacum) seedlings were treated to EOD-FR and enhanced stem length was observed, confirming that taller growth within field rows was due to higher far-red environment (Kasperbauer, 1971). EOD-FR was also used in investigating phytochrome signal transduction pathway in Arabidopsis thaliana, cucumber (Cucumis sativus) and other species such as aspen (Populus tremula x tremloides) where stem elongation under EOD-FR was used as a positive-test in identifying phytochrome genes and intermediate molecules such as gibberellic acid (Nagatani et al., 1991; Olsen and Junttila, 2002; Moe et al., 2003). EOD-FR treatment also reversed the shortened stems of chrysanthemum (Dendranthema x grandiflorum) grown under sunlight with the far-red radiation removed (Rajapakse et al., 1993), demonstrating potential in practical applications in horticultural plant production systems. However, specific information necessary for application in industry, such as far-red light quality, intensity or duration of EOD treatment has not been investigated fully. Our study focuses on two tomato rootstocks, Aloha and Maxifort, used in grafting. The rootstocks confer resistance to soil pathogens and could help reduce the reliance on methyl bromide fumigation which came under restriction as a result of the Montreal Protocol. However, the short stems of the grafted transplants may lead to exposure of the vulnerable scion to the soil. Longer hypocotyl lengths would both allow easier grafting and reduce the risk of scion exposure. EOD-FR was chosen as the means to extend stem length as it involves neither chemicals nor overwatering. Earlier experiments which also

31 utilized tomato (S. lycopersicum) seedlings confirmed that EOD-FR produced longer stem lengths compared to EOD-R but not the untreated control, though hypocotyl lengths were not explicitly measured (Decoteau et al., 1988; Decoteau and Friend, 1991). Since earlier experiments could not provide satisfactory answers to questions in practical applications, we carried out an EOD-FR light quality (R/FR) experiment. Treatments include both filtered and unfiltered incandescent lights as well as an untreated control which helped to determine the effects of R/FR in the EOD-FR treatments. EOD-FR dose, a product of far-red intensity and treatment duration, must be investigated in order to design a commercial system. To decide on the choice of far-red light for EOD-FR treatments, the minimum far-red dose required for maximum hypocotyl extension must be found. An earlier far-red dose experiment using far-red fluorescent light bulbs and far-red spectral filter in corn (Zea mays) seedlings investigated a dose ranging from 1-100 mmol m -2 d -1 for anthocyanin degradation and coleoptile elongation response. The saturation doses were close to 8-10 mmol m -2 d -1 in both cases. In the experiment, different doses were achieved by varying treatment duration from 2 s to 35 min at a constant far-red intensity of 32 mol m -2 s -1 (Gorton and Briggs, 1980). With this in mind, we carried out an EOD-FR dose experiment to find out the relationship between hypocotyl length and EOD-FR dose (1-8 mmol m -2 d -1 ) using filtered-incandescent and LED lights as far-red light source. Dose was varied using both intensity and duration to find out whether the response was intensity or duration dependent.

32 Materials and Methods Plant Materials and growth conditions. The tomato rootstock cultivars Aloha (Solanum lycopersicum) (Ameriacan Takii, Salinas, California, U.S.) and Maxifort (S. lycopersicum S. habrochaites) (De Ruiter Seeds, Bergshenhoek, The Netherlands) were used in the experiment. Aloha was used in EOD-FR light quality experiment while both Aloha and Maxifort were used for the EOD-FR dose experiment. Approximately 300 seeds of each cultivar were sown over either moist vermiculite or moist filter paper in a plastic tray covered with a thin plastic film. The seeded trays were placed for two days under darkness in growth chambers (VWR International, Model 2015, Cornelius, Oregon, U.S.) controlled at 29 o C and 23 o C for Aloha and Maxifort respectively. Uniform seedlings with 2-3 cm long radicles were selected at three to five days after seeding and transplanted into a 98-cell seedling tray (tray size: 28 cm x 55 cm; one seedling per cell) filled with commercial substrate (SunGro Sunshine Professional Mix 3, Bellevue, Washington, U.S.). The plants were placed in the greenhouse and irrigated daily throughout the treatment with nutrient solution (EC 1.2-1.5 ds m -1, ph 6.3-6.5) containing 104 NO 3 -N, 23 P, 177 K, 127 Ca, 30 Mg, 1 Fe as well as micro nutrients in mg/l. When the cotyledons had fully expanded, ten uniform seedlings were selected (planting area: 22.9 x 15.2 cm) for each treatment and cultivar. The greenhouse (Tucson, Arizona, U.S.) used in the present experiments was covered by a double-layered polyethylene roof and transparent polycarbonate walls, and equipped with pad-and-fan cooling and over-head gas heating systems. Daytime and

33 nighttime set points were 24 o C and 20 o C respectively and was controlled using a greenhouse environment control system (Argus, White Rock, British Columbia, Can.). Shade screen was deployed when solar radiation in the greenhouse exceeded 800 W m -2. EOD-FR light treatments. After 5 7 days of seeding, the rootstock seedlings were subject to daily EOD-FR light treatments. EOD treatments were initiated at 6:30 PM at varied far-red light intensities and durations (Table 1) every day for 14 days inside an opaque cardboard box (Officemax, Cleveland, Ohio, U.S.) that prevented light contamination. The plants were kept inside the boxes during the night and taken out of the boxes at dawn (6:30 AM ) of the following day. Air temperature within each treatment box was monitored using a thermocouple (Type T, 0.75 mm in diameter) and recorded using a CR-23X datalogger (Campbell Scientific, Logan, Utah, U.S.). Table 1 shows the light source, R/FR ratio, phytochrome photostationary state (PPS), far-red light intensity, duration and dosage examined in the experiment. Our dose experiment study used a dose range of 1-8 mmol m -2 d -1, varying both treatment duration as well as far-red intensity (Table 1) to see if either duration or intensity was more effective in inducing the EOD-FR hypocotyl elongation response. There were five treatments and an untreated control in EOD-FR dose experiment. The treatments were labeleds S01T12D1, S01T24D2, S03T12D2, S03T24D4 and S40T03D8, with S and the representing the far-red photon flux (to the closest mol m -2 s -1 ), T representing duration of the treatment (in min) and D representing the dose (in mmol m -2 d -1 ). Hence, S01T12D1 would be the treatment where

34 far-red photon flux was 1 mol m -2 s -1, treatment duration was 12 min and dose was 1 mmol m -2 d -1. Control treatments did not provide any EOD-FR treatment although the plants were still kept in the same box from 6:30 PM to 6:30 AM to prevent light contamination from external light sources. The EOD treatment light source was small incandescent light bulbs (input voltage: 2.5 V, ACE #9826009, Oakbrook, Illinois, U.S.) distributed over a horizontal surface 22-25 cm above the plant canopy inside the EOD box. Tinted transparent acrylic sheets (Ridout Plastics Plexiglas blue, 0.32 mm thick, #2424, San Diego, California, U.S.) were used as a spectral cut filter to remove a significant portion of R light (600 nm to 700 nm) and lower the R/FR ratio and phytochrome photostationary state (PPS) value (Sager et al., 1988) from 0.5 to 0.05 and from 0.62 to 0.22 respectively. A layer of plastic shade and dimmers (Lutron Electronics Co. TT-300-NLH, Coopersburg, Pennsylvania, U.S.) were used to reduce the light intensity as needed for incandescent lamps. For the far-red dose response experiment, an LED panel (ISL series, CCS Inc, Kyoto, Japan) mounted with red and far-red LEDs (peak wavelengths at 660 nm and 735 nm, respectively) attached to a digital controller (CCS Inc, Kyoto, Japan) was also used as a light source to provide the highest intensity of 40 mol m -2 s -1 far-red photon flux. R/FR ratio and far-red intensity of the light sources in both experiments (Table 1) were measured using a spectroradiometer (Model PS-100, Apogee Instruments Inc., Logan, Utah, U.S.). Measurements were averages of five readings at plant canopy height across the growing area (23 x 15 cm) under the center of the light source. Three

35 measurements were carried out on days 1, 7 and 14 of the treatment. Fig. 1 shows the spectral profile of the light sources used in the present experiments. Plant Measurements. After 14 days of EOD-FR treatment, lengths of hypocotyl, epicotyl and leaves were measured using a stainless steel ruler. Stem diameter was measured using an electronic digital caliper while stem (hypocotyl and epicotyl) and leaf fresh weights of the seedlings were measured using an electronic balance. After drying at 80 o C for three days, stem dry weight and leaf dry weight were also measured. plastochron index (Erickson and Michelini, 1957) was computed using the following formula from leaf number and leaf lengths: Plastochro n log Ln log R Index n.... Eq.[1] log L n log L n 1 where n is the number of leaves with lengths (from the nodal end of petiole to the tip of the youngest leaflet) greater than R, the reference length (10 mm). L n is the length of leaf n while L n+1 is the length of the first leaf younger than leaf n. Saturation Far-Red Dose Analysis. As EOD-FR was perceived by the plants via phytochrome receptors present in limited amounts in the plant, a Michaelis-Menten model used often to illustrate enzyme substrate reaction was utilized to represent the relationship. In this case, the enzyme would be the phytochrome molecule, the

36 substrate was the far-red dose provided and the response would be hypocotyl elongation. To find the saturation far-red dose, the EOD-FR dose response was fit with an equation based on three parameter Michaelis-Menten model for each of the three repeated experiments. The equation used was: H L V K max M D H D o.eq. [2] Where H L was hypocotyl length; V max was theoretical maximum hypocotyl elongation; D was the far-red dose applied; K M, the Michaelis-Menten constant, was the far-red dose required to reach 50% of the maximum theoretical elongation (when D = K M, the multiplier [D / K M + D] of V max becomes 1/2 and eq. [2] becomes H L = 1/2 V max + H o ); and H o was the hypocotyl length with no EOD-FR treatment (D = 0 mmol m -2 d -1 ). The model parameters were estimated using a non-linear regression program (JMP, SAS Institute, Cary, North Carolina, U.S.). The resulting curve was used to calculate K M and FD 90, the D (dose) required to obtain a near-saturating far-red dose that would provide 90% of V max. FD 90 would essentially be nine times of K M as the V max multiplier in eq. [2] becomes 0.9. Experimental Design. The EOD light quality experiment was carried out in 12-26 Sept. 2008 and 15-28 Oct. 2008 (seeding dates were 9 Sept. 2008 and 11 Oct. 2008 respectively). Each repetition consisted of a non-treated control and two treatments of

37 different R/FR (0.5 and 0.05). One EOD-FR treatment box was used for each treatment. The far-red dose response experiment was conducted for 14-27 Mar. 2009 and repeated twice for 8-21 Apr. 2009 and 1-14 May 2009 (seeding dates: 11 Mar. 2009, 6 Apr. 2009 and 30 Apr. 2009 respectively). Within each experimental period, the treatments were repeated three times and grouped into three blocks. For a randomized block design, the position of the blocks as well as the treatments were randomized. Mean values of each plant physical characteristic were pooled together and analyzed for means separation since two-way ANOVA did not show interaction in either month nor block (for EOD-FR dose experiments).the results were analyzed across the month using JMP (SAS institute, Cary, North Carolina, U.S.) Results and Discussion EOD-FR light quality effects on plants. An EOD-FR of R/FR 0.05 (filtered incandescent lamps) significantly increased hypocotyl length compared to both the R/FR of 0.5 (unfiltered) and the untreated control. Aloha seedling hypocotyl length was increased by 8 mm (22%) and 18 mm (44%) for R/FR of 0.5 and 0.05 respectively (Table 2). Aloha seedling epicotyl lengths were significantly increased by 15 mm (42%) for R/FR of 0.05, but not for R/FR of 0.5. As a result of the stem elongation, stem fresh weight also increased significantly by 0.109 g (36%) and 0.239 g (79%) for R/FR of 0.5 and 0.05 respectively. Stem dry weight, however, did not show any significant difference between the treatments. The fresh and dry weights of the leaf, stem diameter and plastochron

38 index were also not significantly affected by the treatment (Table 2). Maxifort seedlings were grown in October 2008 for a preliminary analysis, in which the same general trend was observed (data not shown). With regards to the stem growth under EOD-FR treatments, similar observations of enhanced stem elongation were observed in other experiments using light with reduced R component (and hence lower R/FR). EOD-FR produced plants with longer stems than the untreated control (Hatt Graham and Decoteau, 1997; Martinez-Garcia et al., 2000; Rajapakse et al., 1993; Xiong et al., 2003). Previously reported experiments done on Mountain Pride tomatoes either did not compare EOD-FR with an untreated control (Decoteau et al., 1988) or found no significant difference in plant height (sum of hypocotyl and epicotyl lengths) between the untreated control and the EOD-FR treated plants (Decoteau and Friend, 1991). The difference could be that previous experiments began EOD-FR treatment 18 days after seeding compared to 5 days after seeding in our experiment, and since hypocotyl length was not accounted for the earlier results did not fully contradict our findings. The R/FR only describes the light quality of the EOD-FR source. Phytochrome photostationary state (PPS) would additionally describe the effect of the EOD-FR treatment on the plant (Sager et al., 1988). PPS is the relative amount of active phytochromes compared to total phytochromes in the plant (P fr /P total ). Filtered incandescent light treatment, with a lower R/FR, produced a lower PPS compared to the unfiltered treatment (0.22 compared to 0.62). The degree of hypotocyl elongation could