190 1. Introduction Light is important for plants, not only as an energy source but also as an environmental signal. Plants respond appropriately to light in their environment, as in seed ger- mination and seedling growth (Godo et al., 2011), floral transition (Cerny et al., 2003), phototropism (Palmer et al., 1993), and so on. Light wavelength is also involved in cell elongation (Braidwood et al., 2014). Pollen tube elongation is of one of the fastest grow- ing plant cell types. The mechanism of pollen tube growth is a multi-stepped one, and it is co-regulated by a variety of essential cellular processes, including exocytosis, actin cytoskeleton organization and activity, calcium and proton physiology, and cellular energetics (Hepler et al., 2013). The main factor that attracts the growth tip of the pollen tube is considered to be chemo-attractants from the female gametophyte (Higashiyama and Hamamura, 2008). In vitro, the effects of light wavelength on pollen ger- mination or pollen tube elongation have been studied in Pinus roxburghii (Dhawan and Malik, 1981): pollen tube growth decreased in white light compared to the dark, and increased in red light, although this was counteracted by far red light. In a study of two cultivars of wheat × maize crosses, pollen tube growth was significantly affected by light intensity in one cultivar but not in the other (Camp- bell et al., 2001). Furthermore, the directional growth of pollen tubes of Nicotiana alata occurred in cultures incu- bated in the dark as well as in the light (Lush et al., 1998). These results highlight the effects of different wavelengths of light on pollen germination and pollen tube elongation, which vary between species, while few studies have de- scribed these effects in detail. The aim of this study was to verify the effect of dif- ferent wavelengths of light-emitting diode (LED) light on pollen germination and direction of pollen tube elonga- tion in Cyrtanthus mackenii using an in vitro experimental system for pollen germination developed by Hirano and Hoshino (2010). 2. Materials and Methods Plant materials and pollen culture Mature pollen with anthers that showed dehiscence was collected from C. mackenii Hook.f. (Amaryllidaceae) and stored at -20°C. Pollen culture was performed using 1% (w/v) agar, medium that contained 0.01% (w/v) CaCl 2 , 0.01% (w/v) H 3 BO 3 , 0.0007% (w/v) KH 2 PO 4 , 10% (w/v) sucrose, and 0.01% (w/v) yeast extract at pH 5.8 (Hirano and Hoshino, 2009, 2010). The culture medium was steril- ized by autoclaving at 121°C for 15 min. Effects of different wavelengths of LED light on pollen germination and direction of pollen tube elongation in Cyrtanthus mackenii Y. Hoyo*, K. Fujiwara**, Y. Hoshino*(1) * Field Science Center for Northern Biosphere, Hokkaido University, Kita 11, Nishi 10, Kita-Ku, Sap- poro 060-0811, Japan. ** Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo, Tokyo 113-8657, Japan. Key words: Cyrtanthus mackenii, direction of pollen tube elongation, germination rate, light emitting diode, light wavelength. Abstract: The effects of different light wavelengths on pollen germination and determination of the direction of pollen tube elongation of Cyrtanthus mackenii was examined in vitro using light-emitting diodes (LED) with five peak wavelengths: 405 nm (violet), 465 nm (blue), 630 nm (orange), 660 nm (red), and 735 nm (far-red). Pollen grains were cultured on a medium solidified with agar, and maintained at 21°C in an incubator under 3-4 h of continuous lighting or darkness. Neither the pollen germinated under LED lighting and dark conditions nor the rate of pollen germination differed among light conditions, includ- ing darkness. However, pollen tubes elongated less in the direction toward light under LED, and elongated with no directional trend in darkness. Moreover, pollen tubes did not elongate toward far-red LEDs. These results suggest that light exerts effects on the factors determining the direction of pollen tube elongation, but not on those controlling pollen germination. Adv. Hort. Sci., 2014 28(4): 190-194 (1) Corresponding author: hoshino@fsc.hokudai.ac.jp Received for publication 17 September 2014 Accepted for publication 3 October 2014 191 The effect of light condition on pollen germination rate Five spots of culture medium with a diameter of 1 cm were put on a glass slide at ca. 5 mm intervals. A tiny amount of frozen pollen grains were sown on each coagulated spot of medium using a paintbrush. Two or three prepared slides were horizontally positioned in a petri dish, which was kept at high humidity (Fig. 1A), with toothpicks between the slides to avoid overlapping. Samples were then cultured at 21°C in an incubator (BIO- TRON LPH200, NK system, Japan) for 3-4 h under dif- ferent light conditions. The incubator was divided into five rooms fitted with overhead LED panels (40×10 cm) fabricated using 2,800 indicator-type LEDs of 3 mm di- ameter (Fujiwara et al., 2011; Yano and Fujiwara, 2012). The LEDs were of five types: violet (L405R-36; Epitex Inc., Kyoto, Japan), blue (L460-36; Epitex Inc., Kyoto, Ja- pan), orange-red (L630-36; Epitex Inc., Kyoto, Japan), red (SRK3-3A80-LE; Toricon Co., Shimane, Japan), and far- red (L735-36AU; Epitex Inc., Kyoto, Japan). Each LED emitted a specific peak wavelength (λ p ) of light: 405 nm, 460 nm, 630 nm, 660 nm, and 735 nm, respectively. The petri dishes were set 2.5 cm below the LED panels. In ad- dition to these treatments, lightproof petri dishes wrapped in aluminum foil were set in a room to confirm whether pollen germination was stimulated or suppressed under dark conditions. Five replicated culture slides were pre- pared per treatment (Fig. 1B). After culturing, all slides were observed using a micro- scope (Axiovert 40 CFL, Carl Zeiss, Germany), and ger- minated pollen grains were counted. Germinated pollen grains with pollen tubes that were shorter than the pollen grain diameter were not counted. We repeated the experi- ment three times under the same conditions, and are re- ferred to as G1, G2, and G3. The effects of light condition on the direction of pollen tube elongation Cover glasses placed over spots of medium with a diam- eter of 1 cm were prepared. Frozen pollen grains were sown on the spots of medium in the same manner as for the pollen germination experiment. Five cover glasses were vertically set in a humid petri dish using a paper stand to prevent the glasses from sticking. The petri dishes were wrapped in alu- minum foil with a slit (8 cm × 0.5 cm) on the upper side for LED light irradiation, and were then cultured at 21°C in the previously described incubator for 3-4 h. To verify the effects of light intensity on pollen tube elongation, two petri dishes were prepared for each light condition: one was placed 3 cm below the LED panel (strong light) and the other was placed 22 cm below (weak light). After culturing, the direction of pollen elongation was observed using the previously described microscope. The direction of pollen tube elongation was determined as follows: 1. Elongation of the pollen tube tip within a 60° arc centered on the line connecting the LED panel, pol- len grain, and floor, and widening in a direction to- ward the LED light, was defined as “Light” (under LED lighting conditions) or “Upward” (under dark conditions) (Fig. 1C); 2. “Dark” or “Downward” defined the occurrence of pollen tube elongation in the opposite direction of the Light or Upward response; 3. Pollen tube elongation toward a direction that was neither Light/Upward nor Dark/Downward was de- fined as “Vertical.” 4. This experiment was also repeated three times un- der the same experimental settings and is referred to as D1, D2, and D3. Statistical analysis All statistical analyses were conducted with the statisti- cal package R (v.3.0.2) (R Foundation for Statistical Com- puting, 2014). A generalized linear mixed-effects model (GLMM) with an offset term was used to investigate de- terminants on the number of germinated pollen grains, assuming Poisson distribution. The light condition, triple experiment design, and subsequent interactions were ex- planatory variables in the model, and an individual spot of medium was a random factor that considered effects seen on an individual spot compared to other spots on the same slide. The total number of pollen grains per medium was Fig. 1 - Diagrammatic representation of the experimental systems and germinated pollen. A, Petri dish set in prepared slides for pollen germination experiment. B, Petri dishes set in cover glasses and then wrapped in aluminum foil with a slit on the upper side for pollen tube elongation experiment. C, Criterion for determining of the direction of pollen tube elongation. 192 an offset term. The Akaike information criterion (AIC) was used to select the best models of the GLMMs. To estimate the effects of light intensity and light wave- lengths on the direction of pollen tube elongation, mul- tinomial logistic regression (Venables and Ripley, 2002) was performed using the R package’s vector generalized linear and additive model (VGAM). The direction of pol- len tube elongation was considered a categorical response variable (Light/Upward, Vertical, Dark/Downward). The explanatory variables were distance from LED panel (long or short); wavelength (405, 460, 630, 660, or 735 nm); and triple experiment design in the case of LED lighting conditions. In the case of analysis for dark conditions, the experiment was the explanatory variable. 3. Results The effect of light condition on pollen germination rate The total number of C. mackenii pollen grains sown on spots of medium were 9 017 (G1), 6 769 (G2), and 4 257 (G3). The pollen germinated under both LED lightning and dark conditions (Fig. 2). The mean and standard deviation of germination rate under each light condition were 0.30±0.015 at 405 nm, 0.38±0.019 at 465 nm, 0.28±0.018 at 630 nm, 0.34±0.020 at 660 nm, and 0.31±0.023 at 735 nm peak wave- length LEDs, and 0.37±0.018 in darkness. The germination rate varied among the three experiments, but did not differ among the five types of LED and darkness (Table 1). The effect of light condition on the direction of pollen tube elongation The total number of pollen tubes elongated in the three defined directions as detailed in Fig. 1C for the three experiments was 1 410 for Light, 2 994 for Verti- cal, and 1 223 for Dark under LED lightning, and 187 for Upward, 421 for Vertical, and 161 for Downward in darkness (Fig. 3). Under LED lightning, the probabil- ity of pollen tube elongation classified as Light or Ver- tical was higher than that classified as Dark (Table 2). However, under darkness, the probability of pollen tube elongation classified as Upward was equivalent to that of Downward, although the Vertical classification was higher. Variability among the three experiments was not found in Light and Upward pollen tube elongation, but was found in Vertical elongation. There was no effect of the distance from LED panel on pollen tube elongation. Under lighting from 735 nm peak wavelength LEDs, the number of Light pollen tube elongations was lower than for Dark pollen tube elongations. 4. Discussion and Conclusions In this study, light neither inhibited nor promoted Cyr- tanthus pollen germination, and the different wavelengths Table 1 - Parameters estimated by GLMM to predict number of gemi- nated pollen in different light conditions for three times of germination experiment. The light condition was discarded explanatory variable by AIC Coefficient Z score Intercept -1.19 -15.98 *** Experiment G2 -0.37 -3.40 *** Experiment G3 0.33 3.06 ** Significance is determined by Z scores. ***: significantly different from G1 at P < 0.001 and ** P < 0.01. Fig. 2 - Relationships between light condition and each germination experiment and germination rate of pollen. Germinated pollen per total sown pollen on each medium spot under each irradi- ated LEDs and darkness: 405 nm (squares), 465 nm (circles), 630 nm (triangles), 660 nm (diamonds), 735 nm (upside-down triangles) peak wavelength of the LED, and darkness (small circles). Three lines show the estimated mean number of ger- minated pollen for three experiments by GLMM. Results of GLMM are shown in Table 1. Fig. 3 - Rates of pollen tube elongation in three directions for each irradi- ated LEDs for the two distances (3 and 22 cm) from LED panels and for shading. The three directions are expressed as follows: toward light or upward (Light or Upward, grey culms), away from light or downward (Dark or Downward, black culms), and in a direction that is neither Light/Upward nor Dark/Downward (Vertical, white culms). The rates are calculated using sum of the number of pollen tubes in five replications of all experiments. The width of culm shows the number of pollen tubes. 193 Table 2 - Coefficients estimated by multinomial logistic regression models that predict probability of pollen tube elongation toward to the Light/ Upward and Vertical directions based on Dark/Downward direction in short distance from 405 nm peak wavelength LED panel in D1 LEDs irradiation Darkness Direction Light Vertical Upward Vertical Intercept 0.23* 0.72*** -0.02 n.s 0.59 ** Experiment D2 0.05 n.s 0.30*** 0.19 n.s 0.42 n.s D3 0.03 n.s 0.11 n.s 0.27 n.s 0.54 * Distance long -0.007 n.s -0.02 n.s Wavelength 465 nm -0.09 n.s 0.14 n.s 630 nm -0.05 n.s 0.08 n.s 660 nm -0.10 n.s 0.03 n.s 735 nm -0.37** 0.10 n.s Significant effects of distance and type of LED lights on the direction of pollen tube elongation are determined by Z scores. ***: significantly different at P < 0.001, **P < 0.01, *P < 0.05 and NS: non-significant. of LED light also did not affect the pollen germination rate. However, pollen tubes were less likely to elongate toward the opposite direction of light, although they elon- gated with no directional trend in darkness. These results suggest that light has some effect on the factors determin- ing the direction of pollen tube elongation, but not on the factors controlling pollen germination. The growth direction of a pollen tube is continuous- ly reoriented by external signals and physical obstacles (Cheung and Wu, 2008). Without any chemotropic attrac- tants or physical directional control caused by configura- tion of female tissue or gametophytes, pollen tubes elon- gated randomly in vitro (Horade et al., 2013). However, the pollen tubes did not show negative phototropism under LED lighting from only one direction in the present study. Therefore, it can be assumed that there is an integrative mechanism that causes unequal elongation of the pollen tube tip, e.g. perception of the light environment (Casal, 2013), transcription factors such as phytochrome interact- ing factors (PIFs) (Chen and Chory, 2011), or hormones and growth-related genes such as for hypocotyl cells (Braidwood et al., 2014). In a unique manner, the pollen tubes did not elongate toward light from 735 nm peak wavelength LEDs. This result was consistent with previous studies that examined the effect of different light wavelengths in combination with hormones on pollen tube growth in Arachis hypogaea (Chhabra and Malik, 1978) and Pinus roxburghii (Dhawan and Malik, 1981). Considering the red:far-red ratio, phy- tochrome undergoes a conformational change from the ac- tive far-red form into the red-light-absorbing, active red form, and derepresses PIF activity, resulting in an increase in cell elongation. If the phytochrome exists in pollen tube tips and causes local cell elongation at the point irradiated with far-red light, the phenomenon of pollen tube elonga- tion away from light may be explained. In conclusion, our results demonstrate that the different wavelengths of LED light affected the direction of pollen tube elongation, but not pollen germination, in an in vi- tro experimental system. Further studies are necessary to clarify the influence of light wavelength on determination of the direction of pollen tube elongation. Acknowledgements This study was supported by grants from a Grant-in- Aid for Scientific Research from the Ministry of Educa- tion, Culture, Sports Science and Technology (MEXT), Japan. References BRAIDWOOD L., BREUER C., SUGIMOTO K., 2014 - My body is a cage: mechanisms and modulation of plant cell growth. - New Phytologist, 201(2): 388-402. CAMPBELL A.W., GRIFFIN W.B., BURRITT D.J., CONNER A.J., 2001 - The importance of light intensity for pollen tube growth and embryo survival in wheat × maize crosses. - An- nals of Botany, 87(4): 517-522. CASAL J.J., 2013 - Photoreceptor signaling networks in plant responses to shade. - Annual Review of Plant Biology, 64: 403-427. CERNY T.A., FAUST J.E., LAYNE D.R., RAJAPAKSE N.C., 2003 - Influence of photoselective films and growing season on stem growth and flowering of six plant species. - J. Amer. Soc. for Hortic. Sci., 128(4): 486-491. CHEN M., CHORY J., 2011 - Phytochrome signaling mecha- nisms and the control of plant development. - Trends in Cell Biology, 21(11): 664-671. CHEUNG A.Y., WU H.M., 2008 - Structural and signaling net- works for the polar cell growth machinery in pollen tubes. - Annual Review of Plant Biology, 59: 547-572. CHHABRA N., MALIK C.P., 1978 - Influence of spectral qual- ity of light on pollen tube elongation in Arachis hypogaea. - Annals of Botany, 42(5): 1109-1117. DHAWAN A.K., MALIK C.P., 1981 - Effect of growth regula- tors and light on pollen germination and pollen tube growth in Pinus roxburghii Sarg. - Annals of Botany, 47(2): 239-248. FUJIWARA K., YANO A., EIJIMA K., 2011 - Design and de- velopment of a plant-response experimental light-source 194 system with LEDs of five peak wavelengths. - J. of Light & Visual Environ., 35(2): 117-122. GODO T., FUJIWARA K., GUAN K., MIYOSHI K., 2011 - Effects of wavelength of LED-light on in vitro asymbiotic germination and seedling growth of Bletilla ochracea Schltr. (Orchidaceae). - Plant Biotechnology, 28(4): 397-400. HEPLER P.K., ROUNDS C.M., WINSHIP L.J., 2013 - Control of cell wall extensibility during pollen tube growth. - Mo- lecular Plant, 6(4): 998-1017. HIGASHIYAMA T., HAMAMURA Y., 2008 - Gametophytic pollen tube guidance. - Sexual Plant Reproduction, 21(1): 17-26. HIRANO T., HOSHINO Y., 2009 - Detection of changes in the nuclear phase and evaluation of male germ units by flow cy- tometry during in vitro pollen tube growth in Alstroemeria aurea. - J. Plant Res., 122(2): 225-234. HIRANO T., HOSHINO Y., 2010 - Sperm dimorphism in terms of nuclear shape and microtubule accumulation in Cyrtanthus mackenii. - Sexual Plant Reproduction, 23(2): 133-162. HORADE M., KANAOKA M.M., KUZUYA M., HIGASHIYA- MA T., KAJI N., 2013 - A microfluidic device for quantita- tive analysis of chemoattraction in plants. - RSC Advances, 3: 22301-22307. LUSH W.M., GRIESER F., WOLTERS-ARTS M., 1998 - Di- rectional guidance of Nicotiana alata pollen tubes in vitro and on the stigma. - Plant Physiology, 118(3): 733-741. PALMER J.M., SHORT T.W., BRIGGS W.R., 1993 - Correla- tion of blue light-induced phosphorylation to phototropism in Zea mays L. - Plant Physiology, 102(4): 1219-1225. R FOUNDATION FOR STATISTICAL COMPUTING, 2014 - R: a language and environment for statistical computing. - R Development Core Team. R Foundation for statistical com- puting Vienna, Austria. VENABLES W.N., RIPLEY B.D., 2002 - Modern applied sta- tistics with S. - Fourth edition. Springer, Tokyo, Japan, pp. 183-210. YANO A., FUJIWARA K., 2012 - Plant lighting system with five wavelength-band light-emitting diodes providing photon flux density and mixing ratio control. - Plant Methods, 8: 46.