BANGLADESH RESEARCH PUBLICATIONS JOURNAL ISSN: 1998-2003, Volume: 10, Issue: 2, Page: 223-227, July - October, 2014
EFFECTS OF DIFFERENT LIGHT QUALITY ON GROWTH AND DEVELOPMENT OF PROTOCORM-LIKE BODIES (PLBs) IN Dendrobium kingianum CULTURED In vitro Sultana Umma Habiba1*, Shimasaki Kazuhiko2, Md. Monjurul Ahasan3 and Md. Meskatul Alam4 Sultana Umma Habiba, Shimasaki Kazuhiko, Md. Monjurul Ahasan and Md. Meskatul Alam (2014). Effects of Different Light Quality on Growth and Development of Protocorm-Like Bodies (PLBs) in Dendrobium Kingianum Cultured In Vitro. Bangladesh Res. Pub. J. 10(2): 223-227. Retrieve from http://www.bdresearchpublications.com/admin/journal/upload/1410028/1410028.pdf
Abstract Light is one of the most important abiotic factors, acting on plants not only as the sole source of energy, but also as the source of external information, affecting their growth and development of a plant culture. Advances in plant tissue culture methods with regard to lighting requirements are currently focused on the improved features of light emitting diodes(LEDs).The effects of light generated by red, blue, red plus blue, green and white light emitting diodes on the in vitro organogenesis from PLBs of Dendrobium kingianum were studied and responses were compared with explants grown under fluorescent light ( 16h per day).The average number of PLBs (12.33) and chlorophyll percentage (4.96) were highest under blue LEDs. In contrast, the percentage of shoot formation (2.6) and fresh weight were highest under red LEDs amongst explants cultured under other LEDs. These results suggest that this newly developed light source could be used as an energy efficient light source for the organogenesis of Dendrobium kingianum PLBs in vitro and also bule and red LED plays an important role of PLBs and shoot formation.
Key words: Light emitting diodes (LED), Dendrobium kingianum, Protocorm–like bodies (PLBs). Introduction Orchids are an important group of ornamental plants comprising several thousand species and hybrids. Orchids attract almost every kind of individual including professional breeders, amateurs and normal collectors because of their naturally beautiful and uniquely shaped flowers that come in a wide spectrum of vibrant colors. In the year 2005 alone, orchids held 8% share of the worldwide floriculture trade (Martin and Madassery, 2006). Dendrobium kingianum is a neat, compact, attractive plant, even when not in bloom, and it is easy to grow and rewards one with an array of beautiful, long lasting, fragrant flowers. In orchid, tissue culture has been used for conservation and rapid multiplication. Several external and internal factors regulate in vitro plant growth and development and among them light is most important. Light is the major energy source for photosynthesis and regulates plant morphogenic (Mark et al.2000) and gene expression (Ma et al. 2001). Light is also one of the most important abiotic factors that influences the successful establishment and subsequent development of a plant culture. Tissue culture and growth rooms have long been using artificial light sources , including fluorescent lamps, high pressure sodium lamps, metal halide lamps, incandescent lamps etc. Among these, fluorescent lamps have been the most popular in tissue culture rooms. Fluorescent lamps having different spectral emissions composed of many wavelengths from 350 to 750nm are main light sources for maintaining tissue cultures (Economou and Read, 1987). *Corresponding Author Email:
[email protected] 1The United Graduate School of Agricultural Sciences, Ehime University, 3-5-7 Tarami, Matsumaya, Ehime 7908556, Japan. 2Faculty of Agriculture, Kochi University, B200 Monobe, Nankoku, Kochi 783-8520, Japan 3Department of Molecular Genetics, Graduate School of Medical Science, Kanazawa University, Takaramachi Kanazawa, Japan 4Department of Computer Science and Engineering, Dhaka International University, Dhaka, Bangladesh
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Recently, LEDs as a new light source have drawn considerable interest as an alternative light source for in vitro propagation. Light emitting diodes (LEDs) have been considered as a novel radiation source for growth and development of plant species because of several characteristics in practical use (Tanaka et al., 1998; Barreiro et al., 1992). The most attractive features of LEDs are small mass, volume, solid state construction and long life (Bula et al., 1991). Because of these unique characteristics, LEDs may be suitable for the culture of plants in a tightly controlled environment as space-based plant culture systems (Bula et al., 1991; Barta et al., 1998). The reaction to different lighting conditions differs between species and their various growth stages. The effects of LEDs on plantlets cultured in vitro were reported in potato (Miyashita et al., 1995), Cymbidium (Tanaka et al., 1998), Rehmannia glutinose (Hahn et al., 2000) and strawberry (Nhut et al., 2003). However, there is very little information on the effects of LEDs on cultured plant cells and tissues. The purpose of this study was to investigate the effect of light quality on organogenesis by protocorm-like bodies and to find a light regime effective in improving the growth and development of PLBs in Dendrobium kingianum in vitro. Materials and Methods Plant material and culture medium PLBs of Dendrobium kingianum were proliferated in the modified Murashige and Skoog (Shimasaki and Uemoto, 1990) medium by transferring to a new medium. After excision of PLB into singles, each PLB used as an explant. MS medium supplemented with 412.5 mg/L ammonium nitrate, 950 mg/L potassium nitrate, 20gm/L sucrose and 2.2 gm/L phytagel (Sigma) were used as a culture medium. Five explants cultured in one vessel and three vessels were used for each treatment. Jars of 250ml (UM culture bottle, as one, Japan) with plastic caps containing 30ml of medium were used for culture vessels. The pH of the medium was adjusted to 5.5-5.8 using 0.1mM 2- (N- morpholino) ethanesulfonic acid sodium salt (MES-Na) before autoclaving at 1210 C for 15 min. Lighting conditions To elucidate the effect of different light conditions on the in vitro PLBs growth of Dendrobium kingianum, the cultures were established and grown under different light conditions of photon flux density (PFD) of 50 µmol m-2 s -1. There were six radiation treatments : (1) fluorescent lamp (National FL20SS as control), (2) red LEDs (Jefcom, P18WE1701-R, peak wavelength: 640nm), (3)blue LEDs (Jefcom, P18W-E1701-B, peak wavelength: 450nm), (4) green LEDs (Jefcom, P18W-E1701-G, peak wavelength: 510nm), (5) white LEDs (Jefcom, P18W-E1701-W, peak wavelength: broad spectrum),(6) blue LEDs plus red LEDs(1:1).All treatments were maintained at 25 + 10 C under different LEDs during 16 h photoperiods for four weeks. Data analysis Experimental data were collected by counting the number of PLBs, number of shoots, fresh weight (gm) and their chlorophyll (%) were measured. The data were statistically analyzed by calculating standard errors of the means (means ±SE) and significant differences assessed by Tukey HSD test (P< 0.05).` Results and discussion Effect of light quality on PLBs and shoot formation The growth and development of protocorm-like bodies of D. kingianum were significantly affected different light treatments in vitro. After four weeks of culture, the average number of PLBs per explant was maximum (12.33) under blue LEDs and the minimum (8.4) was under white fluorescent lamp. Among the six different light treatments, blue LEDs, red LEDs and blue +red (1:1) LEDs showed the promotive effect on PLB formation.
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Table 1: Effect of different light quality on organogenesis in PLBs cultures of Dendrobium kingianum Light conditions
White Fluorescent White LED Blue LED Red LED Green LED Blue+ red (1:1) LED
Avg. No. of PLBs
Shoot
8.4±1.8a 8.5±1.4a 12.3±1.5a 11.9±1.7a 9.6±1.2a
Avg. Number 2.2±0.9a 2.1±0.7a 1.6±0.6a 2.6±0.8a 1.5±0.6a
10.3±1.5a
1.1±0.5b
Rate (%) 53.3 53.3 46.7 60 33.3 40
Fresh Weight (gm)
Chlorophyll (%)
0.1±0.03a 0.12±0.02a 0.14±0.02a 0.16±0.02a 0.11±0.02a
4.28±0.7a 2.67±0.4ab 4.96±0.8a 1.52±0.3b 3.6±0.7a
0.11±0.02a
4.72±0.7a
Values represent means ± SE followed by the different superscript letters show significant differences by Tukey HSD test (p< 0.05) The highest number of shoots (2.6) was recorded on the medium treated under red LEDs and was significantly different with blue + red (1:1) LEDs. PLBs showed normal chlorophyll formation when grown under blue LED, green LED and fluorescent lamp. Table 1 showed that Chlorophyll (%) was highest under blue LED compared with other treatments. In addition, the fresh weight of PLBs was recorded. The maximum fresh weight of PLBs under all six different light treatments was 0.16g under red LED, and minimum fresh weight was 0.1 gm under white fluorescent lamp.
Fig.1: Effect of different light quality on organogenesis in PLB cultures of D. kingianum. A: White fluorescent lamp; B: White LED; C: Blue LED; D: Red LED; E: Green LED; F: Blue LED + Red LED; Bars: 1cm Figure 1 showed that white LED and Red LED promoted shoot formation compared with other LEDs.On the other hand, blue LED had promotive effect on PLBs formation. But in Green LED, the quality of PLBs on the basis of color and size were good compared with other LED. Light is one of the most important factors regulating plant development through photoreceptors active under specific wavelength of light (Lee et al., 2007). In recent years, with the technological advancement of LEDs, numerous studies have been carried out on a variety of plant species to investigate the effects of LEDs as an alternative light source for in vitro culture. The aim of this study was mainly to investigate the effects of monochromatic LEDs applied singly or in combination (blue + red LEDs) on organogenesis of PLBs in Dendrobium kingianum. In previous studies, PLBs cultured under red LEDs showed the lowest differentiation rate, compared to blue LEDs, which resulted in the highest differentiation rate in cultures of Oncidium in vitro (Xu et al. 2009) .White light enhanced http://www.bdresearchpublications.com/journal/
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growth and betacyanin accumulation of S. salsa calli (Zhao et al. 2010). This study showed the results that blue LEDs had good effects on new PLBs formation. Blue LEDs might play an important role in stimulating PLB formation from callus as PLBs formed very well in blue LED treatments of Cymbidium orchid (Van et al. 2004). In Phalaenopsis, blue LEDs proved to be effective for PLB formation from cultured PLB segments (Tanaka et al., 2001). In the present study red LEDs has induced better performance on shoot formation and increasing fresh weight. Red light induced the buds to elongate and to develop a morphology characteristic of etiolated shoots (Bonnett 1972) and increased shoot height, internode length and rooting frequency. Shoot morphogenesis in Anthurium, was promoted by exposure to higer percentages of red than blue illumination. However, the number of shoots was more when exposed to higher percentages of blue than red LEDs (Budiarto 2010). Fresh and dry weights of upland cotton plantlet were greatest when cultured under blue and red LEDs in equal proportion (Li et al. 2002). Red plus blue LEDs also promoted bulblet growth, producing bigger bulblets with higher fresh weights, dry weights, and dry matter accumulation (Lian et al. 2002). In present study, highest fresh weight was found on the medium cultured under red LEDs. Blue LED might be essential for chlorophyll synthesis by accumulating chlorophyll in PLBs in vitro (Lin et al. 2011). Blue light encouraged chlorophyll synthesis and stomatal development (Poudel et al. 2008). Same result found in present study. In vitro, Betula pendula grew larger leaves and showed increased photosynthetic activity under blue light compared with red light (Sab et al. 1995).Previous studies reported that the primary photosynthetic pigments in higher plants, chlorophylls a and b, have absorption in the blue and red regions and have very little absorption in the green region. Thus, photosynthesis and overall productivity of plant material could be enhanced by increasing the amount of blue and red light present in the growth environment. Using LEDbased light sources, it is possible to regulate and control the physiological aspects of PLBs growth and development, such as photosynthesis and/or photomorphogenesis. Conclusion Based on our present study, we conclude that blue Led promote the proliferation of protocorm-like bodies of Dendrobium kingianum in vitro by increasing the number of PLBs and are effective in promoting chlorophyll production with blue plus red LED. But red LED increase the number of shoot and fresh weight of protocorm-like bodies of D. kingianum. It should therefore be possible to increase PLBs number and improve shoot production and the quality of shoot of D. kingianum by using blue and red LED respectively. Acknoledgements The authors are grateful to the Kochi University, Faculty of Agriculture, for providing the research work. This research was supported by the Monbukagakusho: MEXT, Japanese Government scholarship. References Barreiro, R., Guiamet, J. J., Beltrano, J. and Montaldi, E. R. (1992). Regulation of the photosynthetic capacity of primary bean leaves by the red:far-red ratio and photosynthetic photon flux density of incident light. Physiol.Plant. 85, 97-101. Barta, D.J., Tibbitts, T. W., bula, R.J. & Merrow, T. W. (1992). Evaluation of light-emitting diodes characteristics for a space based plant irradiation source. Advances in Space Research 12, 141-149. Bonnett, H.T. (1972) Phytochrome regulation of endogenous bud development on root cultures of Convolvulus arvensis. Planta 106, 325–330. Budiarto, K. (2010). Spectral quality affects morphogenesis on Anthurium plantlet during in vitro culture. Agrivita, 32, 234-240. Bula, R. J., Morrow, T.W., Tibbitts, T.W., Barta, D.J., Ignatius, R. W. and Martin, T.S. (1991). Light-emitting diodes as a radiation source for plants. HortScience, 120, 808-813. Economou, A. S., & Read, P. E. (1987). Light treatments to improve efficiency of in vitro propagation systems. HortScience, 22, 751-754.
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