Tissue culture plays a major role in the propagation of plants as varied as ornamental foliage crops, medicinal plants, and woody trees with desirable characteristics . Several external and internal factors regulate in vitro plant growth and development. Light is the most important of these factors because the illumination system used can affect photosynthesis and the photomorphogenic response [2, 3]. The light environment includes the photon flux density, light quality and photoperiod. Currently, light-emitting diode (LED) technology is widely used as a flexible artificial lighting source with several unique advantages compared to conventional lighting systems like fluorescent lamps, high-pressure sodium lamps, metal halide lamps, and incandescent lamps. LED lighting systems exhibit wavelength specificity, durability, small sizes, long operating lifetimes, and relatively cool emitting surfaces. Because the photon output is linearly related to the electrical input, the spectral composition can be controlled [3, 4]. Several plant species grow well under LEDs, including seedlings of lettuce, pepper, cucumber, wheat, and spinach; plantlets of potato; and in vitro cultures of Rehmannia glutinosa [5, 6, 7, 8]. Therefore, LED lights are ideal instruments for use in lighting designs for in vitro plant production.
Acacia melanoxylon is an evergreen tree species belonging to the family Mimosaceae and the genus Acacia Mill. It is fast-growing, is tolerant to both drought and barren soil, and has notable nitrogen fixation abilities. It has excellent wood material and is used for superior furniture. Due to its high economic and ecological value, it has been planted in many areas in China ever since it was introduced from Australia in the early 1990s . Because of some serious difficulties with seed breeding, there has been increased attention paid to clonal afforestation. Clonal afforestation can not only keep the parent’s excellent characteristics, but also form a stable forest. Plantlets in tissue culture have high somaclonal variations, are not restricted by external conditions, can produce plantlets several times in one year, and can cover small areas with high propagation coefficients. Therefore, tissue culture propagation is currently a main method of asexual propagation of this species. There have been many reports relating to the establishment of an efficient tissue culture system for rapid propagation of Acacia melanoxylon [10, 11]. For example, Hu et al.  used aseptic leaves, petioles, and stems of geminated axillaries of Acacia melanoxylon to induce calluses and established high-frequency regeneration systems. He found that the optimal material for callus formation was the stem and the optimal medium for induction was Murashige and Skoog basal medium + 1.5 mg L-1 6-benzyladenine + 0.2 mg L-1 naphthalene acetic acid + 3% sucrose. Dou et al.  reported the genetic diversity of 174 isolates of symbiotic bacteria associated with Acacia melanoxylon from different sites in China. However, there has been little research on the effects of the light environment on tissue propagation of Acacia melanoxylon [14, 15]. Light intensity, quality, and photoperiod may control the growth and development of tissue organ cultures by triggering physiological reactions.
The purpose of this research was to study the effects of light environment on bud proliferation of in vitro tissue cultures of Acacia melanoxylon. We hypothesized that (1) LED light intensity, quality, and photoperiod would have effects on bud proliferation and plantlet growth, and (2) LED light intensity and photoperiod would have effects on root growth of Acacia melanoxylon. We attempted to determine the optimal light environment and provide some theoretical and technical support for providing this type of lighting to tissue cultures of Acacia melanoxylon.
2 Materials and Methods
The research materials were five clones of Acacia melanoxylon designated FM1, FM2, FM4, FM5, and FM10. They were provided by the Chinese Fir Engineering Research Center of the State Forestry Administration, Fujian Agriculture and Forestry University, Fuzhou, Fujian Province.
2.2 Experimental design
The experiment was divided into two parts. First, we designed three in vitro experiments to test the effects of LED light intensity, quality and photoperiod on bud proliferation and plantlet growth. Second, we designed two ex vitro experiments to test the effects of LED light intensity and photoperiod on root growth.
2.2.1 Effects of LED light environment on bud growth and plantlet growth in vitro
We selected strong and healthy buds of similar size from the each of the five clones of Acacia melanoxylon. Four tissue clumps were transplanted onto Murashige and Skoog basal medium with 0.1 mg L-1 6-benzyladenine and 0.5 mg L-1 naphthalene acetic acid and cultured under different light treatments in vitro for 40 days at ~25°C. Six buds formed one tissue clump, and there was 4 tissue clumps in each replicate. There were 10 replicates for each treatment.
The three experiments in vitro were as follows: (1) The effects of LED light intensity on bud proliferation and plantlet growth. Plants were cultured under white LED light with a wavelength of 400–700 nm and intensity of 45 μmol m-2 s-1, 90 μmol m-2 s-1, or 135 μmol m-2 s-1 respectively. (2) The effects of LED light quality on bud proliferation and plantlet growth. Because plants need to grow under photosynthetic active radiation between 400 nm and 700 nm, and because photosynthetic efficiency is highest at wavelengths of 450 nm (blue) and 650 nm (red), we designed experimental LED light combinations using red and blue wavelengths. The ratio of red and blue LEDs were 1: 1, 1: 4, and 4: 1, all with an intensity of 135 μmol m-2 s-1. There was also a control with light provided by a fluorescent lamp. (3) The effects of LED photoperiod on bud proliferation and plantlet growth. We used two photoperiods, 16 h light and 8 h dark or 12 h light and 12 h dark, under 135 μmol m-2 s-1 white LED light. For each treatment of LED light intensity, light quality, and light photoperiod, the tissues were cultivated under controlled light condition in sterile tissue culture room.
After 40 days, we calculated the bud proliferation coefficient (K) and observe plantlet growth for each clone under the different LED intensities, qualities, and photoperiods. The formula was K = the number of buds after 40 days/the number of buds at the initiation of the experiment.
2.2.2 Effects of LED light environment on root growth ex vitro
The ex vitro experiments were based on a substrate of peat, vermiculite, and perlite at a mass ratio of 5: 4: 1. After the substrates were thoroughly mixed, they were poured into 0.1% potassium permanganate solution, mixed, and transferred to pots. The substrates were then washed with water after 3 d, separated into cultivation pots and compressed, then watered as needed. After samples were air-dried and finely ground, pH measurements were made, and each sample was adjusted to pH 5.8. To study light intensity and light photoperiod influences on root growth ex vitro, for each light treatment, 30 healthy plantlets without roots (average height ca. 3 cm) from FM1, FM2, FM4, FM5, and FM10 clones in vitro tissue cultures were transferred to the substrate described above. The base roots of samples were not treated using rooting accelerator treatments. All they were then placed in controlled light environment in sterile tissue culture room.
The two experiments ex vitro were as follows: (1) The effects of LED light intensity on root growth. We used three light intensities, 45 μmol m-2 s-1, 90 μmol m-2·s-1, and 135 μmol m-2 s-1, with white LED light of wavelength 400–700 nm. The photoperiod was 16 h light and 8 h dark. (2) The effects of LED light photoperiod on root growth. We used two photoperiods, 12 h light and 12 h dark or 16 h light and 8 h dark, with white LED light intensity of 135 μmol·m-2·s-1 and wavelength of 400–700 nm.
After 30 days, we calculated average rooting rate, average root length, and average number of roots (root number) per plant. The average root length was calculated as the total root length divided by the total number of plants with root formation. Rooting rate was calculated as the number of plants with root formation divided by the total number of plants.
2.3 Statistical analysis
A one-way ANOVA was used to compare the differences between treatments and to evaluate the effects of LED light intensity, quality, and photoperiod on bud proliferation coefficient, average root length, and rooting rate. Mean comparisons were performed with the least significant difference (LSD) test at p = 0.05. All statistical analyses were performed with the SPSS statistical package (SPSS 12.0, SPSS Inc., IL., USA).
3.1 The effects of LED light intensity on bud proliferation and plantlet growth in vitro
The effects of LED light intensity on bud proliferation of Acacia melanoxylon are shown in Table 1. The LED light intensity had significant effects on FM2, FM4, and FM5 (p< 0.01), as well as on FM1 (p< 0.05). LED light intensity had no significant effects on FM10 (p = 0.8001). For FM1 and FM2, the bud proliferation coefficient decreased with increasing light intensity, and it was significantly highest with light intensity of 45 μmol m-2 s-1. For FM4, the coefficient varied significantly among the three light intensities, and the maximum bud proliferation coefficient appear at the light intensity of 45 μmol m-2 s-1. For FM5, the coefficient was significantly higher with the light intensity of 90 μmol m-2 s-1 than with the other two intensities. For FM10, there was no significant difference among the three light intensities. Therefore, bud proliferation coefficient was highest under the light intensity of 45 μmol m-2 s-1 for all the clones except for FM5.
We found that plantlet growth was well-supported under all tested LED light intensities (Figure 1). All plantlets were strong and grew to an average length of 2–3 cm and all plantlets grew roots, though the plantlets under 135 μmol m-2 s-1 had more root number and rooting rate. Therefore, the Acacia melanoxylon plantlets grew stronger and higher with the increased light intensities, but with lower bud proliferation coefficient.
3.2 The effects of LED light quality on bud proliferation and plantlet growth in vitro
The effects of red and blue LED light combinations on bud proliferation of Acacia melanoxylon are shown in Table 2. The bud proliferation coefficient decreased from the red/ blue light ratio combination of 1:1 to 4:1 and 1:4, and the average proliferation coefficient was all lower than the control treatment. However, there were only significantly lower with the red/blue ratio of 1:4 compared to other two red/blue combinations on the bud proliferation coefficient. Under the treatment of a red/blue ratio of 4: 1, the plantlets grew higher and with a small amount of root growth; however under the treatment of a red/blue ratio of 1: 4, the plantlets grew with a produced green leaves but without root growth. Therefore, compared to the fluorescent lamp control treatment, the plantlets under the red/blue LEDs grew well, but bud proliferation was poor (Table 2).
3.3 The effects of LED light photoperiod on bud proliferation and plantlet growth in vitro
For FM2 and FM4, the effects of LED light photoperiod on bud proliferation were highly significant (p < 0.01); for FM5, the effects were significant (p < 0.05); and for FM1 and FM10, the effects were not significant (p = 0.05) (Table 3). The bud propagation coefficient was significantly higher in the treatment with the photoperiod of 12 h light and 12 h dark (p < 0.05) for FM2, FM4, and FM5. We found that the photoperiod of 12 h light was beneficial for the bud proliferation; however, the photoperiod of 16 h light may promote a small number of roots growth and promote greater plantlet height (Figure 3). Therefore, prolonged the photoperiod of light illumination may decreased bud proliferation, but improve the height growth and root growth of plantlets.
3.4 The effects of light intensity on root growth ex vitro
We found that the average root number and rooting rate significantly increased with increasing light intensity (Table 4) (p< 0.05), while the average root length decreased, though there were no significant differences for FM1 and FM10 (p= 0.05). The average root number was 5–13 among the five clones. The average root length was highest in FM4 at 2.57 cm, while the average rooting rate was highest in FM2 at 90%.
3.5 The effects of photoperiod on root growth ex vitro
We found that a prolonged photoperiod did not have significant effects on average root length or average rooting rate (Table 5) (p= 0.05) but did have a significant effect on average root numbers (p< 0.05). Prolonging the light photoperiod significantly increased average root numbers, but to some extent decreased average root length. The effects of light photoperiod on root numbers were more strongly observed in FM5 and FM10 compared to the other clones. In particular, the highest average root number was 13 in FM10 with 16 h light and 8 dark.
Our results partly verified our hypothesis that LED light intensity, quality, and photoperiod would have great effects on bud proliferation and plantlet growth of Acacia melanoxylon. We found that LED light intensity had significant effects on bud proliferation of clones FM1, FM2, FM4, and FM5 (p< 0.05) and the maximum proliferation coefficient was under the light intensity of 45 μmol m-2 s-1. The proliferation coefficient decreased with increasing light intensity, but the plantlets grew well with green leaves and strong stems under increased light intensity. Our results were consistent with other reports. Lin  reported that high light intensity had negative effects on bud differentiation, perhaps due to light-induced stress. However, compared to our result that the best light intensity for Acacia melanoxylon growth was 135 μmol m-2 s-1, Li et al.  found that stem diameter, fresh weight, average diameter, root volume, and root tips of Dendrobium officinale Kimura et Migo in vitro were significantly higher under light intensity of 75 μmol m-2 s-1 than under 50 μmol m-2 s-1 or 100 μmol m-2 s-1. The difference between the responses of these two species may be that Acacia melanoxylon is a light-demanding species and Dendrobium officinale is a shade-demanding species.
In our study, we found that bud proliferation was poor under red/blue LED light compared to fluorescent lamp treatments, but plantlets under the red/blue ratio 4:1 grew taller and produced some roots (Table 2, Figure 2). Moon et al.  reported rooting is promoted by red LEDs and inhibited by blue LEDs during the study of LEDs effects on Tsuru-rindo cultured in vitro. Red LEDs had a positive effect on plantlet height and root growth according to Kong et al.  who studied the effects of light quality on the growth of in vitro cultured Doritaenopsis plants. Our results were in accord with Qiu et al. , who reported that LED light combinations were beneficial for axillary bud height and had significant effects on maximum leaf width and fresh leaf mass, while fluorescent light was suitable for Tectonagrandis cultures and rapid differentiation of axillary buds. Zhou et al.  similarly found that red LED light was advantageous to the growth of aerial parts of plants, and that blue light had effects on both dwarfed and strong tissue culture seedlings of Anoectochilus roxburghii. It has been reported that a mixture of blue and red LEDs enhanced plant growth as well as fresh and dry weights compared to monochromatic LEDs [22, 23]. Chen et al.  indicated that leaf length, blade width, and root number reached their maximums with a red/blue light ratio of 8:2, which is consistent to our findings (the plantlets under a red/blue ratio of 4: 1 grew higher and with a small amount of root growth). Similar results with Red:Blue (1:1) were also obtained from in vitro plants of Lillium , banana  and strawberry . We found the red/blue combination of LED lights (1: 1) was beneficial for the bud proliferation coefficient but increasing the red ratio (R: B = 4:1) could enhance plantlets height and root growth. Nhut and Nam  reported that the blue LEDs under 10% (30% in some cases) may promote plant height growth. Further studies need to focus on identifying an optimal red/blue combination of LEDs for both bud proliferation and plantlet growth for Acacia melanoxylon.
We found that extending the photoperiod could decrease the proliferation coefficient of buds of Acacia melanoxylon (Table 3). However, we also found that longer illumination was beneficial for plantlet height and root growth. Our results were in agreement with Zhang et al. , who reported that the average height, average root collar diameter, and average numbers of lateral buds and lateral shoots of Abies concolor were all greatest with 22 h light at 50 μmol m-2 s-1. Some studies found that plants grown at more Northern latitudes, high elevation, and in a continental climate were very sensitive to photoperiod. Tinus  reported that extending the photoperiod in the greenhouse was beneficial for the height growth of southern Rocky Mountain Engelmann Spruce and Douglas-fir.
In our study, the average root number and average rooting rate increased with increasing light intensity, and the optimal light intensity was 135 μmol·m-2·s-1 (Table 4). In root growth experiments, plantlets changed from photoheterotrophic growth to photoautotrophic growth. The light intensity was neither excessively high nor low; strong light intensity may destroy the chlorophyll . We found that increased photoperiod could increase average root numbers but decrease root length (Table 5). These results were similar to Li et al. , who indicated that 10 h light per day was enough for sweet pepper plantlets, but prolonging the light was beneficial for root growth. However, Fan et al.  found that if the photoperiod was longer or shorter than 16 h, the shoot-rooting of Paulownia plants was inhibited. Kurilcik et al.  studied the effects of illumination spectrum on the chrysanthemum plantlets grown using LED lights, and found that blue LEDs enhanced plantlet extension and roots formation. Therefore, we will continue to study the effects of light quality (blue, red and far-red) on Acacia melanoxylon propagation in the future.
In our study, we found that the optimal light intensity for bud proliferation of the clones FM1, FM2, FM4, FM5, and FM10 of Chinese fir Acacia melanoxylon was 45 μmol m-2 s-1. Higher light intensity was not beneficial for bud proliferation, but with increasing light intensity, plantlet growth was improved. The effects of red/blue combinations of LEDs on bud proliferation were not significant, but plantlet and root growth was better under red/blue LEDs ratio 4:1 than under a fluorescent lamp. The extended photoperiod was not beneficial for bud proliferation, but good for the plantlet growth of Acacia melanoxylon in vitro. Increased light intensity greatly increased root number and the rooting rate ex vitro, while a prolonged photoperiod greatly increased root number. In short, increases in light intensity and photoperiod were beneficial for plantlet growth and root numbers, but much less so for bud proliferation; a red/blue LED ratio of 4:1 was advantageous to plantlet and root growth.
This work was funded by the Science and Technology Major Project of Fujian Province, China (No. 2018NZ0001-1), the Science and Technology Innovation Special Project of Fujian Agriculture and Forestry University (No. CXZX2017106), the National Natural Science Foundation of China (No. 31800532), the National Natural Science Foundation of Fujian Province (No. 2018J05059), the Educational and Scientific Research Project of Fujian Provincial Educational Department (No. JAT170464) and the Forestry Science and Technology Project of Fujian Province (No. 16, 2019). We would express our appreciation on Zhihui Ma for the valuable suggestions on experimental design. We also thank for Editage Company for editing and the constructive suggestions on the manuscript writing. We are also grateful to Chinese Fir Engineering Research Center of the State Forestry Administration, Fujian Agriculture and Forestry University, Fuzhou, Fujian Province for support and help.
Khan U.W., Ahmed R., Shahzadi I., Shah M.M., Some important factors influencing tissue culture response in wheat., Sarhad J. Agric., 2015, 31, 199–209 Google Scholar
Guo F., Liu H.P., Li B.G., Zhang X.M., Qi G.H., Li Y.C., Effect of light quality on growth and partial physiological and biochemical characteristics of red raspberry tissue culture plantlets, North. Hortic., 2016, 22, 15–19 (in Chinese) Google Scholar
Gupta S.D., Jatothu B., Fundamentals and applications of light-emitting diodes (LEDs) in in vitro plant growth and morphogenesis, Plant Biotechnol. Rep., 2013, 7, 211–220 CrossrefWeb of ScienceGoogle Scholar
Li H.M., Xu Z.G., Tang C.M., Effect of light-emitting diodes on growth and morphogenesis of upland cotton Gossypium hirsutum L.) plantlets in vitro Plant Cell Tiss. Organ Cult., 2010, 103, 155–163 Web of ScienceCrossrefGoogle Scholar
Nhut D.T., Takamura T., Watanabe H., Tanaka M., Efficiency of a novel culture system by using light-emitting diode (LED) on in vitro and subsequent growth of micropropagated banana plantlets, Acta Hortic., 2003a, 616, 121–127 Google Scholar
Nhut D.T., Takamura T., Watanabe H., Okamoto K., Tanaka M., Responses of strawberry plantlets cultured in vitro under superbright red and blue light-emitting diodes (LEDs), Plant Cell Tiss. Organ Cult., 2003b, 73, 43–52 CrossrefGoogle Scholar
Wang X.Y., Xu X.M., Cui J., The importance of blue light for leaf area expansion, development of photosynthetic apparatus, and chloroplast ultrastructure of Cucumis sativus grown under weak light, Photosynthetica, 2015, 53, 213–222 CrossrefWeb of ScienceGoogle Scholar
He B., Wei S.H., Zhang W., Tang T., Biological cycling of nutrients in Acacia melanoxylon plantation, J. NE. Forestry Univ, 2012, 40, 9–12 (in Chinese) Google Scholar
Jones C., Smith D., Effect of 6-benzylaminopurine and 1-naphthlacetic acid on in vitro axillary bud development of mature Acacia melanoxylon Proc. Int. Plant Prop. Soc., 1988, 38, 389–393 Google Scholar
Dou Y.J., Lu J.K., Kang L.H., Wang S.K., Jiang Y.G., Liao S.B., Biodiversity of Rhizobia associated with Acacia melanoxylon grown in South China, Acta Microbiol. Sin., 2012, 52, 1439–1448 Google Scholar
Ding G.C., Gao S.H., Lin S.Z., Chen Y., Xu J.Y., Li C.Q., Research on open-ended photoautotrophic micropropagation technology of Acacia melanoxylon J. Fujian Coll. For., 2010, 30, 11–14 (in Chinese) Google Scholar
Lin L.S., Study on the Acacia melanoxylon rapid propagation technology in vitro. Doctoral dissertation, Fujian Agriculture and Forestry University, 2008 (in Chinese). Google Scholar
Li W., Zheng J.R., Mo Z.W., Li Z.J, Jiang L., Qiu D.S., Cai S.K., Liu X.J., Effect of light intensity on seedling growth and physiological characteristics of Dendrobium officinale Kimura et Migo in vitro Chin. J. Trop. Crop. 2014, 35: 121–125. (in Chinese) Google Scholar
Moon H.K., Park S.K., Kim Y.W., Kim C.S., Growth of Tsuru-rindo Tripterospermum japonicum cultured in vitro under various sources of light-emitting diode (LED) irradiation, J Plant Biol., 2006, 49, 174–179 CrossrefGoogle Scholar
Kong, S.S., Murhy H.N., Heo J.W., Hahn E.J., Paek K.Y., The effect of light quality on the growth and development of in vitro cultured Doritaenopsis plants, Acta Physiol. Plant., 2008, 30, 339–343 Google Scholar
Qiu Z.F., Zeng B.D., Guo G.S., Liu Y., Effects of various LED lights on growth and development of Tectona grandis plantlets in vitro Guihaia, 2017, 37, 592–598 (in Chinese) Google Scholar
Zhou J.Y., Ding G.C., He J.Z., Cao G.Q., Li X.L., Bu Z.Y., Effects of light quality on the growth of tissue culture chlorophyll and chlorophyll fluorescence in Anoectochilus roxburghii J. Agric., 2015, 5, 67–72 (in Chinese) Google Scholar
Li H., Xu Z., Tang C., Effect of light-emitting diodes on growth and morphogenesis of upland cotton Gossypium hirsutum L.) plantlets in vitro Plant Cell Tiss. Organ Cult., 2010, 103, 155–163 Web of ScienceCrossrefGoogle Scholar
Poudel R.P., Kataoka I., Mochioka R., Effect of red-and blue-light emitting diodes on growth and morphogenesis of grapes, Plant Cell Tiss. Organ Cult., 2008, 92, 147–153 Web of ScienceCrossrefGoogle Scholar
Chen X.X., Shao X.L., He S.L., Effect of different light quality ratios of LED on growth of Spathiphyllum in vitro North. Hortic., 2015, 6, 86–89 (in Chinese) Google Scholar
Lian M.L., Murthy H.N., Paek K.Y., Effects of light emitting diodes (LED) on the in vitro induction and growth of bulblets of Lilium oriental hybrid ‘Pesaro’, Sci Hortic., 2002, 94, 365–370 CrossrefGoogle Scholar
Duong T.N., Hong L.T.A., Watanabe H., Goi M., Tanaka M., Efficiency of a novel culture system by using light-emitting diode (LED) on in vitro and subsequent growth of micropropagated banana plantlets, Acta Hortic., 2003, 616, 21–127 Google Scholar
Nhut D.T., Nam N.B., Light emitting diodes (LEDs): an artificial lighting source for biological studies. In: Proceedings of the 3rd international conference of the development of BME in Vietnam, 11–14 January 2010
Zhang J.F., Zhang H.L., Wang J.F., Chen Y.G., Hu W.Z., Effects of extended phtotoperiod on the growth of Abies concolor seedlings, J. Beijing Forest. Univ., 2006, 28, 107–110 (in Chinese) Google Scholar
Tinus R.W.. Effects of extended photoperiod on Southern Rocky Mountain Engelmann spruce and Douglas-fir, Tree Plant. Notes U.S. Forest Service., 1981, 32, 9–12 Google Scholar
Hazrati S., Tahmasebi-Sarvestani Z., Modarres-Sanavy S.A., Mokhtassi-Bidgoli A., Nicola, S., Effects of water stress and light intensity on chlorophyll fluorescence parameters and pigments of Aloe vera L., Plant Physiol Bioch., 2016, 106, 141–148 Web of ScienceCrossrefGoogle Scholar
Li J., Gu H., Xu F.M., Effects of environmental factors on root cultivation of bell peppers Capsicum annuum, J. China Capsicum, 2004, 4, 36–37 (in Chinese) Google Scholar
Fan G.Q., Dong Z.Q., Li F.W., Jia F., Effects of photoperiod on regrowth of Paulownia plant following artificial propagation, Acta Bot. Boreal-Occident. Sin., 2007, 27, 104–109 (in Chinese) Google Scholar
Kurilcik A., Canova M.R., Dapkuniene S., Zilinskaite S., Kurilcik G., Tamulaitis G., Duchovskis P., Zukauskas A., In vitro culture of Chrysanthemum plantlets using light emitting diodes, Cent Eur J Biol, 2008, 3, 161–167 Web of ScienceGoogle Scholar
About the article
Published Online: 2019-07-22
Conflict of InterestConflicts of Interest: The authors declare no conflict of interest.