Artificial Light Source Using Light-emitting Diodes (LEDs) in the Efficient Micropropagation of Spathiphyllum lets Duong Tan Nhut 1,2, T. Takamura 1, H. Watanabe 3 and M. Tanaka 1 1 Faculty of Agriculture, Kagawa University, Miki-cho, Kagawa 761-0795, Japan 2 Dalat Institute of Biology, 116 Xo Viet Nghe Tinh, Dalat, Lam Dong, Vietnam 3 Mitsubishi Chemical Corp., Yokohama Research Center, Yokohama 227-8520, Japan Keywords: Acclimatization, in vitro growth, irradiation level Abstract Spathiphyllum plantlets were cultured in Culture Pack, a rockwool system, with CO 2 enrichment (3000 μmol mol -l ) under different blue to red LED ratios and different LED irradiation levels. Growth was compared to that under plant growth fluorescent lamps (PGF) at 45 μmol m -2 s -1 PPF for 60 days. The highest total shoot and root fresh weight of plantlets was obtained under 80% red + 20% blue LED as compared to others. Total shoot and root fresh weight of in vitro plantlets grown under 60 μmol m -2 s -1 was higher than that under 45, 75 μmol m -2 s -1 and PGF. Attempts were also made to examine whether the light source during in vitro culture affected the subsequent growth of plantlets 90 days after transferring to soil. The fresh shoot and root weight of in vitro plantlets grown under 80 % red + 20% blue LED was highest compared to others. Fresh shoot and root weights of in vitro plantlets grown under 60 μmol m -2 s -1 and 75 μmol m -2 s -1 were higher than those under 45 μmol m -2 s -1 and PGF. These results suggested that a culture system using LED and film is advantageous for the micropropagation of Spathiphyllum plantlets. INTRODUCTION Spathiphyllum is an ornamental plant. Due to its long-lasting, showy, white flowers and ease of growing, it has gained popularity in the tropical foliage industry. Because of greater grower demands for uniformity and higher quality plants, tissue culture is being used more. In commercial tissue culture laboratories, much concern has been placed on the culture media, optimal culture conditions, and culture techniques for such elite plants. Light source is one of the important factors controlling plant growth and development. The use of light-emitting diodes (LEDs) as a radiation source for plants has attracted considerable interest in recent years because of its vast potential for commercial application. The most attractive features of LEDs are their small mass, volume, and long life (Bula et al., 1991; Brown et al., 1995). Due to these unique characteristics, there is the suggestion that LEDs may be suitable for the culture of plants in a controlled environment such as a space based plant culture system (Bula et al., 1991, Barta et al., 1992). Enhanced growth of Cymbidium plantlets cultured in vitro under superbright red and blue LEDs has been reported (Tanaka et al., 1998) and both in vitro and subsequent growth of banana plantlets were improved under blue and red LED (Nhut et al., 2002). The objectives were to find out the optimal red and blue LED ratio for in vitro growth of Spathiphyllum plantlets that might then reduce the cost for its micropropagation. Attempts were also made to examine whether the light source during in vitro culture affected the subsequent growth of plantlets. MATERIALS AND METHODS Unrooted Spathiphyllum shoots that had three leaves and were 30-35 mm in, were used as explants in all treatments. These shoots were excised from shoot masses derived from shoot-tip cultures on Murashige and Skoog (MS) (1962), agar medium supplemented with 3% sucrose and 1 mg/l 6-benzylaminopurine. Sixteen shoots were cultured in the Culture Pack -rockwool [CP.RW] (4 x 4 blocks, Grodan Rockwool Multiblock TM AO 18/30, Grodania A/S, Denmark), containing 120 ml MS Proc. IInd IS on Biotech. of Trop & Subtrop. Species Eds: W.-C. Chang and R. Drew Acta Hort 692, ISHS 2005 137
medium. The CP (7.5 x 7.5 x 10.5 cm, the outer size of stainless frame) was made of fluorocarbon polymer film (Neoflon PFA films, 25 μm in thickness, Daikin Industries, Japan) as described by Tanaka et al. (1988) and Tanaka (1991). The RW was presterilized in a dry sterilizer (150ºC, 1h), and placed in the CP. These culture vessels were placed in the LED PACK 3 (Ryusho Industrial Co., Japan) in which the irradiation level can be adjusted blue to red LED ratios or LED irradiation levels (Okamoto et al., 1996). The LAD PACK 3 was placed in a temperature-controlled culture room (1.8 m x 1.8 m x 2.2 m) at 25ºC and a 16 h photoperiod (45 μmol m -2 s -1, Homo-Lux, National, Japan) in which the CO 2 concentration was maintained at 3000 ppm. To clarify the optimum blue to red LED ratios, shoots were cultured under different blue to red LED ratios: (1) 100% red LED, (2) 90% red + l0% blue LED, (3) 80% red + 20% blue LED and 70% red + 30% blue LED at 45 μmol m -2 s -1. In order to examine the effect of LED irradiation levels, shoots were cultured under 45, 60, 75 μmol m -2 s -1 in which the red to blue LED ratio was 90% red + 10% blue as compared to that under PGF at 45 μmol m -2 s -1. For acclimatization, 16 plantlets cultured in two CP RW systems for 60 days for each treatment were transferred to a soilless mixture (Metro-Mix 350, Scottsco, USA), in plastic pots (5 x 5 cm) and placed in the greenhouse under natural light. The plant, number of leaves and roots, root, and fresh shoot and root weight of plantlets were recorded 60 days after planting. Chlorophyll content in the third leaf of the plantlets, counting from the top downwards, was measured as SPAD value by a Chlorophyll meter (SPAD-502, Minolta Co., Ltd., Japan). The acclimatized plants were analyzed after 90 days of cultivation. Explants in experiments were arranged in a randomized complete block design with 16 shoots; data was analyzed for significance by analysis of variance with the mean separation by Duncan's Multiple Range Test (Duncan, 1995). RESULTS AND DISCUSSION Effects of Various Blue to Red LED Ratios on the Growth of Spathiphyllum lets on Sugar-free Medium under CO 2 Enrichment Effects of various blue to red LED ratios on the growth of Spathiphyllum plantlets on sugar-free medium under CO 2 enrichment are shown in Table 1 and Figure 1. The number of plantlet leaves was higher under PGF while the others were equal. The plantlet was lowest under PGF while the others were equal. The number of roots was lowest under 80% red + 20% blue LED. The root of plantlets was equal among treatments. The SPAD value of plantlet leaves was highest under 70% red + 30% blue LED. The highest fresh top weight was observed in plantlets cultured under 80% red + 20% blue LED while the fresh root weight was equal among treatments. lets cultured under 80% red + 20% blue LED were vigorous and uniform. Effects of Different Irradiation Levels on the Growth of Spathiphyllum lets on Sugar-free Medium under CO 2 Enrichment Effects of different irradiation levels on the growth of Spathiphyllum plantlets on sugar-free medium under CO 2 enrichment is shown in Table 2. let growth was normal in all treatments. The number of leaves was equal among treatments. The plantlet was greater under 45 and 60 μmol m -2 s -1 as compared to 75 μmol m -2 s -1. The number of roots was slightly higher under 75 μmol m -2 s -1 while the root was almost equal among treatments. The SPAD value of plantlet leaves under 45 μmol m -2 s -1 was higher than at 60 and 75 μmol m -2 s -1. The fresh top weight of plantlets under 60 μmol m -2 s -1 was higher than that under 45 and 75 μmol m -2 s -1 while the fresh root weight was equal among treatments. The effects of light generated by superbright blue and red LEDs on the growth of Cymbidium plantlets cultured in vitro have been studied (Tanaka et al., 1998). Leaf growth, chlorophyll content and fresh shoot and root weights were affected by LED 138
irradiations. Red light promoted leaf growth but decreased chlorophyll content (Tanaka et al., 1998). This effect was reversed by blue light. The growth of plantlets in terms of increase in total shoot and root weight was comparable under red and blue LEDs and fluorescent lamps. The optimum blue to red ratios for banana plantlets has been studied (Nhut et al., 2002). The banana plantlets were enhanced under 80% red + 20% blue LED (Nhut et al., 2002). On the other hand, the results suggested that plantlets grown under superbright blue to red LED were affected by blue LED. In the absence of blue LEDs, plantlets were abnormal. Normal plantlet growth is clearly related to the presence of blue LEDs and plant quality is a function of the amount of blue LEDs. Subsequent Growth of Spathiphyllum lets after Transferring to Soil for 90 Days Culture (lets Cultured under Different Blue to Red LED Ratios) Subsequent growth of Spathiphyllum plantlets after transferring to soil for 90 days is shown in Table 3. The plantlets showed normal growth in all treatments. lets do not elongate under 100% red LED. The number of leaves of plantlets cultured under 100% red LED, 90% red + 10% blue LED and 80% red + 20% blue LED was higher than that under 70% red + 30% blue LED and PGF. lets were highest under 90% red + 10% blue LED and 80% red + 20% blue LED as compared to others. The number of roots was highest under 80% red + 20% blue LED and the lowest was obtained under 70% red + 30% blue LED and PGF. Highest root was observed in plantlets cultured under 90% red + 10% blue LED while lowest root was obtained under 70% red + 30% blue LED. The SPAD value of plantlet leaves was higher under PGF and lower under 100% red LED. Fresh shoot and root weight were highest under 80% red + 20% blue LED. Subsequent Growth of Spathiphyllum lets after Transferring to Soil for 90 Days (lets Cultured under Different LED Irradiation Levels) Subsequent growth of Spathiphyllum plantlets after transferring to soil for 90 days is shown in Table 4 and Figure 2. The plantlets showed normal growth and were vigorous. The number of plantlet leaves was higher under 45 and 60 μmol m -2 s -1 than at of 75 μmol m -2 s -1. The plant under 45 μmol m -2 s -1 was higher than that of 60 and 75 μmol m -2 s -1. The number of roots was equal among treatments while the root was higher under 75 μmol m -2 s -1. The SPAD value of leaves of plantlets was highest under 60 μmol m -2 s -1. Highest fresh top weight was obtained under 60 and 75 μmol m -2 s -1 while highest fresh root weight was obtained under 45 and 75 μmol m -2 s -1. Subsequent growth of in vitro Spathiphyllum plantlets was enhanced after transferring to soil. This study is in agreement with the data reported for strawberry by Nhut et al. (2000). Tanaka et al. (1996) reported that growth and development in the rooting stage of plantlets of some horticultural plants were remarkably improved by using the novel film culture systems. In this study, the film culture system (CP RW) and LED irradiation for Spathiphyllum were used. They were shown to improve the growth and development of plantlets during culture. These results demonstrate the effectiveness of a total radiation system using LEDs for micropropagation of Spathiphyllum and subsequent acclimatization in ex vitro conditions. LEDs have a long life and changing the light source is therefore less frequent, resulting in a reduction in labour cost. Also LEDs generate very little heat, thereby minimizing the need for an extensive cooling system in plant growth facilities. The low energy consumed by LEDs makes commercial clonal propagation of plant tissue more cost effective. The compactness of LEDs is another attractive feature. With further improvements to be made, it is reasonable to expect that the total LED irradiation system will be a major light source for a wide range of plant culture systems in the future. 139
Literature Cited Barta, D.J., Tibbitts, T.W., Bula, R.J. and Merrow, T.W. 1992. Evaluation of lightemitting diodes characteristics for a space-based plant irradiation source. Advances in Space Research. 12:141-149. Brown, C.S., Schuerger, A.C. and Sagar, J.C. 1995. Growth and photomorphogenesis of pepper plants under red light-emitting diodes with supplemental blue or far-red light. Journal of American Society for Horticultural Science 120:808-813. Bula, R.J., Merrow, 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 26:203-205. Duncan, D.B. 1995. Multiple range and multiple F test. Biometrics 11:1-42. Murashige, T. and Skoog, E. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. 15:473-497. Nhut, D.T., Anh Hong, L.T., Watanabe, H., Goi, M. and Tanaka, M. 2002. Growth of banana plantlets cultured in vitro under red and blue light-emitting diode (LED) irradiation source. Acta Hort. 575(1):117-124. Nhut, D.T., Takamura, T., Watanabe, H. and Tanaka, M. 2000. Light emitting diodes (LEDs) as a radiation source for micropropagation of strawberry. p.114-118. In: C. Kubota and C. Chun (eds.), Transplant Production in the 21 st Century. Kluwer Academic Publishers. Okamoto, K., Yanagi, T., Takita, S., Tanaka, M., Higuchi, T., Ushida, Y. and Watanabe, H. 1996. Development of growth apparatus using blue and red LED as artificial light source. Acta Hort. 440:111-116. Tanaka, M. 1991. Disposable film culture vessels. p.212-228. In: Y.P.S. Bajai (ed.), Biotechnology in Agriculture and Forestry, Vol. 17, High-Tech and Micropropagation I. Springer-Verlag, Berlin. Tanaka, M., Goi, M. and Higashiura, T. 1988. A novel disposable culture vessel made of fluorocarbon polymer films for micropropagation. Acta Hort. 226:663-670. Tanaka, M., Nagae, S., Takamura, T., Kusanagi, N., Ujike, M. and Goi, M. 1996. Efficiency and application of film culture systems in the in vitro production of plantlets in some horticultural plants. J. Sci. High Tech. Agr. 8:280-285. Tanaka, M., Takamura, T., Watanabe, H., Endo, M., Yanagi, T. and Okamoto, K. 1998. In vitro growth of Cymbidium plantlets cultured under super red and blue lightemitting diodes (LEDs). Journal of Horticulture Science and Technology 73:39-44. Tables Table 1. Effects of various blue to red LED ratio on the growth of Spathiphyllum shoots on the sugar-free medium under CO 2 enrichment. Lighting source leaves roots SPAD W value Top Total 100%R X +0%B Y 6.6ab 6.1a 4.2a 7.0a 30.2c 288.9b 135.8a 429.6b 90%R+10%B 6.3b 6.4a 3.6bc 6.3a 36.3b 334.1b 123.7a 456.2ab 80%R+20%B 6.4ab 6.4a 3.3c 6.4a 36.4b 416.1a 111.9a 530.0a 70%R+30%B 6.4ab 6.2a 3.8abc 6.3a 40.1a 321.2b 132.7a 456.9ab Control(PGF Z ) 6.9a 6.9b 4.1ab 6.5a 35.9b 341.9b 100.2a 445.5ab a Different letter within a column indicate significant differences at P = 0.05 by Duncan s multiple range test W Chlorophyll content in the third leaf, counted from the top downward, of the plantlets X Blue LED, Y Red LED, Z Growth Fluorescent Lamps 140
Table 2. Effects of LED irradiation level on the growth of Spathiphvllum shoots on the sugar-free medium under CO 2 enrichment. Irradiation level z (μmol m -2 s -1 ) leaves roots SPAD Y value Top Total 45 6.4ab 6.2a 3.8abc 6.3a 40.la 321.2b 132.7a 456.9ab 60 6.4ab 6.4a 3.3c 6.4a 36.4b 416. la 111.9a 530.0a 75 6.9a 4.9b 4.lab 6.5a 35.9b 341.9b 100.2a 445.5ab a Different letter within a column indicate significant differences at P = 0.05 by Duncan's multiple range test Y Chlorophyll content in the third leaf, counted from the top downward, of the plantlets X 90% Red + 10% Blue LED Table 3. Subsequent growth of Spathiphyllum plantlets three months after transferring to soil. Light source leaves roots SPAD W of leaves Shoot Total 100%R X +0%B Y 6.8a 12.4b 8.4b 8.7b 43.0c 2225.1c 798.5c 3023.6c 90%R+ 10%B 7.0a 13.2a 8.5b 9.6a 47.0b 2568.8b 895.9b 3464.7b 80%R+ 20%B 7.2a 13.6a 9.0a 8.9b 45.3b 2768.2a 1201.6a 3969.8a 70%R+ 30%B 6.0b 10.7c 7.4c 8.lc 44.3b 2028.9d 578.9d 2607.8d Control (PGF Z ) 6.1b 12.3b 7.5c 8.8b 48.9a 2250.2c 843.4b 3093.6c a Different letter within a column indicate significant differences at P = 0.05 by Duncan's multiple range test W, X, Y, Z See Table 1. Table 4. Subsequent growth of Spathiphyllum plantlets three months after transferring to soil. Irradiation level z (μmol m -2 s -1 ) No. of leaves No. of roots SPAD Y of leaves Shoot Total 45 60 7.0a 7.3a 13.2a 12.0c 8.5a 8.9a 9.6b 10.0b 47.0b 49.5a 2568.8b 2661.0a 895.9a 3464.7b 850.3b 3541.3a 75 6.5b 12.7b 8.5a 10.6a 47.7b 2649.9a 870.9a 3520.8a a Different letter within a column indicate significant differences at P = 0.05 by Duncan's multiple range test Y, Z See Table 2. 141
Figures Fig. 1. In vitro growth of Spathiphyllum plantlets cultured under 80% red + 20% blue LED and PGF. Fig. 2. Subsequent growth of Spathiphyllum plantlets cultured under 60 μmol m -2 s -1 90 days after transferring to soil. 142