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ISSN : 1225-5009(Print)
ISSN : 2287-772X(Online)
Flower Research Journal Vol.25 No.4 pp.270-277
DOI : https://doi.org/10.11623/frj.2017.25.4.12

Effects of Shading on the Growth, Development, and Anthocyanin Content of Echeveria agavoides and E. marcus

Raisa Aone M. Cabahug1,2, Son Yil Soh1,2, Sang Yong Nam1,2*
1Department of Environmental Horticulture, Sahmyook University, Seoul 01795, Korea
2Natural Science Research Institute, Sahmyook University, Seoul 01795, Korea
Corresponding author: Sang Yong Nam +82-2-3399-1732namsy@syu.ac.kr
20170905 20171025 20171101

Abstract

Shading is a key element in the control of light intensity and is usually used during the summer when sunlight is at its most intense. Succulents are ornamental crops that have beextremely popular because of their leaf structure and other characteristics. A study was conducted to determine the effects of shading on the growth and development as well as the anthocyanin content of two Echeveria species: Echeveria agavoides and E. marcus. Three levels of shading were used, namely no shading/full light treatment, partially shaded conditions, and well-shaded conditions, which were achieved by placement inside controlled growing chambers for 4 weeks. The results revealed the bolting of growth in both height and diameter for both species when grown under shaded and partially shaded conditions, which were abnormal compared withthe typical development. CIELAB color results also showed thata* was significantly affected by shading levels, exhibiting a higher positive value for succulents under full light or no shading. These results were consistent with the anthocyanin analysis, where the highest contents were identifiedinthe species grown under full light. The image analysis also confirmed a higher percentage area based on the smart segmentation for red pigments compared withthat for green pigments.


초록


    Ministry of Agriculture, Food and Rural Affairs
    514006-03-1-HD040

    Introduction

    The use of succulents as indoor and landscape plants is mainly due to their ability to survive in harsh environments with minimal watering and care compared to other ornamental plants (Fischer and Schaufler 1981; Nyffeler et al. 2008; Oldfield 1997). Crassulacae family is considered as the third largest among succulent groups. This consists of plants that have a wide range of habitat adaptation and temperature tolerance. These succulents are popularly known as the ‘stonecrop’ and ‘houseleek’ family with a broadcast appeal for growers, hobbyists and collectors (Rowley 1978; Sevilla et al. 2012). Within this huge family, several subfamilies are prevalently known including Echeveria, Sedeveria, Sedum, Crassula, and Graptopetalum. They come in variety of morphological structures and their leaves evidently create unique growth patterns.

    Most of the researches for succulents that enable consumers and propagators to improve production and quality of produce are more inclined to propagation through in-vitro and leaf cuttings and the discovery of new species (Raju and Mann 1971; Ruiz et al. 2016; Ruiz and Costea 2014).

    Echeveria is considered as one of the popular genus among the succulent plants. This genus is comprised of 140 species of which 95% of the total is endemic in Mexico (Meyran and Lopez-Chavez 2003; Vazquez et al. 2013). Succulents under this genus is known for the development of gradient colors on the margins of the lower or mature leaves of the plant. The colors range from light pink to red and even deep red hues that are already close to brown or black. This change in color may be due to the presence of the anthocyanin pigments.

    One of the most noticeable classes of flavonoids is anthocyanin. They are known to be responsible for important plant pigments like red, pink, purple and blue colors in plants (Grotewold 2006). One of the widely-accepted function of anthocyanin is that they protect leaves in plants which are facing biotic or abiotic stressors and has been discussed as putative roles as anti-oxidants and sunscreens (Hughes and Yadun 2015; Landi et al. 2015; Pringsheim 1879). Environmental conditions of other ornamental flowers and foliage have been manipulated to produce attractive colors that may enhance plant attractiveness to potential consumers (Zhao and Tao 2015; Harpaz and Padowicz 2007). With these researches, determination of pigment content is deemed necessary. They keep track of actual visible colors using high-performance liquid chromatography (HPLC), however modern color instrumentation has made use of CIELAB which is more practical and easy to use (Wrolstad et al. 2005).

    Most of the succulents are being placed inside offices, schools, homes and other indoor places where the lighting conditions are shaded. Recent studies have been conducted for few succulent species on the effect of light intensity and the type of light that would intensify color change (Nam e t al. 2016; Park et al. 2016; Soh et al. 2015). However, the color quality of these ornamental plants varies, especially, if they are used as potted plants inside establishments creating shaded environments with low source of light. This paper aimed to identify how shade in Echeveria species impacts their growth, development and quality.

    Materials and Methods

    Planting materials

    Echeveria agavoides and E.cheveria marcus species were procured from Kim Succulent Nursery, Anseong, South Korea. These species have a common trait which under unknown conditions, their leaf margins will turn pinkish to red in color. Young, healthy and disease-free succulents were chosen as experimental plants which were around 60 days old and were placed inside the greenhouse of Sahmyook University, Seoul, South Korea.

    Experimental design, treatments and growth conditions

    The experiment was laid out in a completely randomized design with four replications with six plants per replication. There was a total of seventy-two plants per species. Three shading levels served as the factor which were no shading/full light, partially shaded and well-shaded conditions. To achieve shading levels, the use of two metal frames were placed inside each chamber and was lined with polyethylene nets. Each metal frame held a different light intensity because of the density of the polyethylene nets (Fig. 1). Shading levels were also secured to be consistent throughout the metal frame by determining the lux value using the Digital Lux Meter (DX-200, Centenary Materials Co., Taiwan). The control or no shading/ full light treatment had 10,000 lux followed by the partially-shaded with 5,000 lux and the well-shaded with 2,500 lux. There was a 14 hours light period and 10 hours dark period.

    All experimental plants were placed inside three plant growth chambers (KGC-175 VH, Koenic Ltd., South Korea). Each chamber was equipped with four LED tube lights (Philips F48T12/CW/VHO 110 Watt, USA) and the relative humidity was set at 65% and the temperature at 20°C.

    Hunter’s CIELAB

    Color change was determined using the Hunter’s CIELAB (Konica Minolta Spectrophotometer CM2600d) which makes use of the L*a*b* color space to indicate lightness, hue and saturation of colors. One leaf for each plant was tagged to trace the color changes. The color value was measured through two areas within the tagged leaf; margin of the top and underside of the leaf.

    Anthocyanin analysis

    Modified quantitative method for anthocyanin (Fuleki and Francis 1968) was used in this study by gathering an inch from the tip of the tagged leaves of succulent plants. One gram fresh-cut leaf samples were macerated using a mortar and pestle. The macerated sample was added with 1mL of 95% ethanol and 1.5 N HCl (85 : 15) which served as the extracting solvent. The mixed solution was transferred to a separate container. Samples were then centrifuged at 13,000 rpm at 4°C using the Micro Refrigerated Centrifuge Smart R17 (Hanil Science Co. Ltd., Seoul, South Korea). This was then stored and refrigerated overnight. Samples were placed in a microplate which was then analyzed for a full-spectrum UV/Vis absorbance at 535 nm using the Fluostar Optima Microplate Reader (BMG Labtech, Ortenberg, Germany). Results from this analysis are expressed using μg/ g of fresh weight (FW).

    Image analysis

    Photos were taken using a Canon 750D (Canon, Japan) with the same aperture, brightness and contrast at the same distance. Individual images were cropped to show the succulents alone without the pots and were processed using the Image Pro Premier ver. 9.3 (Media Cybernetics, USA). Smart segmentation was applied to individual representative images to determine the ratio of the colors green and red pigments. Colored overlays of identified colors was presented as bases for color identification.

    Statistical analysis

    Data gathering was done every two weeks for a month. Aside from the Hunter’s CIELAB and anthocyanin analysis, growth and development parameters were also collected: plant height and diameter. Statistical analyses were conducted using Statistical Product and Service Solutions for Windows, version 16.0 (SPSS Inc., Japan). The data were analyzed using analysis of variance (ANOVA), and the differences between the means were tested using Duncan’s multiple range test (P < 0.05).

    Results and Discussion

    Plant height and diameter

    Results revealed that plant height has significantly affected plant height for both Echeveria species (Table 1). Succulents that were grown under shaded conditions was significantly taller compared to those that were exposed to higher light exposure which had 44.77 mm (E. agavoides ) and 46.95 mm (E. marcus ). These were followed by those in partial shading with 42.67 mm (E. agavoides ) and 42.06 mm (E. marcus ). Those grown in partial-shaded condition did not differ from those under full-light or no shade with 41.97 mm and 41.63 mm, respectively.

    Consequent growth of the succulent’s height was also evident in the diameter with similar results. Significant differences were observed in diameter for E. agavoides having those grown in shaded conditions had the largest plants with 90.25 mm followed by partial shading and no shade with 88.42 mm and 83.61 which were comparable with each other.

    E marcus species had similar results wherein the treatment of succulents under shaded conditions (79.82 mm) gave the largest plants followed by those in partial-shading (77.39 mm) and full-light (75.88 mm). However, these were not significantly different from each other.

    These results are commonly seen in experiment as those done in germinating bean seeds in dark and light conditions. Aside from the bean family, studies of Rylski and Spigelman (1986) have found out that when sweet peppers were grown in shaded conditions had increased plant height, number of nodes and leaf size as the light intensity decreased.

    Various explanations are said about profuse growth in plants in shaded conditions such as its relation to circadian cycles as well the shade avoidance syndrome. The changes in plant body and function are the responses of shade tolerance. Studies of Casal (2012) showed that the different responses of Arabidopsis thaliana during its different stages of life cycle. In this study, it was observed that shading increases the adaptive benefits of elasticity which enables the plant to elongate its stem, spread its leaf surface area. It was also considered that the lack of light increases the production of auxin which allows more movement in the tips of the growing plants.

    Despite of these pants recording these growths are found to produce less healthy looking plants with lightness of the green pigments as well as portraying a lanky structure. Studies of Zhao et al. (2012) on herbaceous peony concluded that plants grown under shaded condition reduced its photosynthetic capacity, light saturation and compensation point because of the declined stomatal conduction. Because of those decline, it resulted to the decrease of soluble sugar, protein and malondialdehyde which in turn produced plants with delayed flowering, reduced ornamental quality and faded plant color.

    Hunter’s CIELAB

    Results of the hunter’s CIELAB revealed that E. agavoides were significantly affected by shading conditions (Table 2).

    The top portion or exposed leaf area showed that L*, a* and b* values were significantly different from each condition. L* or the brightness of the color shows that there was a lighter quality of color with 43.00 value on the scale which was significantly comparable from those of partial shading (40.67). The darkest color quality was observed from those grown under no shade or full light condition with 40.93.

    A positive a* was observed in pants that were grown under full light with the highest value of 4.98 signifying that it had a more reddish hue compared to those nearing to the 0 value or negative which was seen in succulents under partially-shaded (0.39) and shaded conditions (-2.39) signifying towards the greener hue. Saturation of colors or b* value was also seen as more intense in full-light conditions with 14.42 compared to those of partial-shading (13.51) and shaded (11.50) conditions.

    For the bottom area of the leaf not exposed to the light, there was only a significant difference for the a* value which showed a positive value for no shading treatment (7.84) these were significantly different for those with negative values for succulents grown under partial shading (-0.23) and shaded (-2.09) conditions. However, the *L and b* values were not significantly affected by the treatments.

    For E. marcus, some of the CIELAB values were significantly affected by the treatments (Table 3). These had similar results for those of E. agavoides except for the b* values. E. marus has a differently natural light blue green. In the CIELAB sphere of colors, lower values for b* signify a bluer saturation tone. Thus, in both b* values for the top and bottom portions of the leaves have lower values for the no shade treatment compared to shaded conditions.

    Anthocyanin analysis

    The anthocyanin content was significantly affected by shading treatments for both species (Table 4). Results revealed that there is more presence of anthocyanins for Echeveria succulents that were grown under full light or non-shaded conditions.

    Based on the results, the highest anthocyanin content was found in plants in full light or no shading with 1.32 μg/g FW for E. agavoides and 0.82 μg/g FW for E. marcus. These were followed by partial-shading with 0.66 μg/g FW for E. agavoides and 0.44 μg/g FW for E. marcus. The lowest anthocyanin contents were taken for shaded levels for both species.

    Succulents are considered to mature within the four weeks treatment. According to Kliewer (1970), anthocyanin pigment which are predominantly red are supposed to accumulate and be enhanced by low light intensities. However, studies of Fukuoka et al. (2014) with Gynura bicolor suggested that there was a limited increase of anthocyanin content for shaded treatments despite expansion of leaves during maturity.

    These contrasting results have been controversial in determining anthocyanin tendencies, on the other hand, studies of Jeong et al. (2004) revealed that anthocyanins can also be affected by expression of anthocyanin biosynthetic genes and the application of shading treatments suppressed the accumulation.

    Image analysis

    Smart segmentation of images coupled with the original images for three levels of shading and their corresponding histogram results is presented on Fig. 2a for E. agavoides and Fig. 2b for E. marcus.

    Based on the histogram of each level, it was evident that red pigments are more evident in succulents under full light and has slowly decline with shading. Results of the smart segmentation suggested that there is a high percentage of red color compared to green color for both species for full light or no shading. Thus, there is a negative correlation between red pigments and shading while a positive correlation between green pigments.Fig. 3Table 5

    Acknowledgement

    Succulents Export Innovation Model Development towards Chinese Market (514006-03-1-HD040)’, Ministry of Agriculture, Food and Rural Affair and Sahmyook University Research Fund.

    Figure

    FRJ-25-270_F1.gif

    Experimental set-up inside the growth chamber to achieve light conditions: A, metal frames with polyethylene (PE) nets with black cloth barriers on the side; B, PE net for shaded condition; and C, PE net for partially-shaded.

    FRJ-25-270_F2.gif

    Original image, processed segmentation image and histogram determining area ratio of green and red pigments using number of pixels of E. agavoides in response to shading levels.

    FRJ-25-270_F3.gif

    Original image, processed segmentation image and histogram determining area ratio of green and red pigments using number of pixels of E. marcus in response to shading levels.

    Table

    Average plant height and diameter (mm) of Echeveria species in response to shading levels.

    zMean separation within columns by Duncan’s multiple range test at P = 0.05.
    yNS, *, **, Nonsignificant, significant, highly significant at P = 0.05 and P = 0.01, respectively.

    Average Hunter’s CILEAB values of E. agavoides in response to shading levels.

    zMean separation within columns by Duncan’s multiple range test at P = 0.05.
    yNS, *, **, Nonsignificant, significant, highly significant at P = 0.05 and P = 0.01, respectively.

    Average Hunter’s CILEAB values of E. marcus in response to shading levels.

    zMean separation within columns by Duncan’s multiple range test at P = 0.05.
    yNS, *, **, Nonsignificant, significant, highly significant at P = 0.05 and P = 0.01, respectively.

    Average anthocyanin (μg/g FW) content of Echeveria species in response to shading levels.

    zMean separation within columns by Duncan’s multiple range test at P = 0.05.
    yNS, *, **, Nonsignificant, significant, highly significant at P = 0.05 and P = 0.01, respectively.

    Red and green pigment ratio of using smart segmentation in response to shading levels of Echeveria species.

    zMean ± standard deviation.
    yMean separation within columns by Duncan’s multiple range test at P = 0.05.

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