Effects of R/B ratio on the growth and chlorophyll concentration ofZantedeschia plantlets in vitro using light emitting diodes
Ruey-Chi Jao1and Wei Fang2
- Assistant Professor, Dept. of Information Management, Chien Kuo Institute of Technology
- Professor, Dept. of Bio-Industrial Mechatronics Engineering, NationalTaiwanUniversity
Corresponding author: Ruey-Chi Jao
No. 1,Jieshou N. Rd., Changhua City, Taiwan500, R.O.C.
Dept. of Information Management
NationalTaiwanUniversity
TEL: 886-4-7111111
FAX: 886-4-7111170
E-mail:
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ABSTRACT
Effects of varying quantum ratio of red/blue (R/B) light during cultural period using light-emitting diodes (LEDs) on the photomixotrophic growth of Zantedeschia plantlets (Zantedeschia jucundacv. ‘Sensation’) in vitrowere investigated. Alleight treatments had the same photosynthetic photon flux (PPF, 80 μmol·m-2·s-1)and photoperiod (16 hours daytime/8 hours nighttime), leading to the same daily light integral (DLI), but with different R/B ratios. Results showed that the dry weight accumulation of Zantedeschia plantlets in vitro had no significant differences but with significant differences on chlorophyll content and plant height among treatments. Providingblue light alone resulted in tall/slim plantletswith less fully expanded leaves. In contrast, providing red light alone leads to dwarf/sturdy with all leaves fully expanded and lower chlorophyll concentration.Other treatments with varying R/B ratios during the 5 weeks cultivation period showed trend of shorter plant height and less chlorophyll contents on leaves with larger accumulative R/B ratios. Results also showed that one week difference of R/B ratio was sufficient to influence the plantlet height, but has no significant difference on chlorophyll development. At present, blue LEDs costs much more than that of red LEDs, results of this study suggested that using red LEDs alone is feasible for the commercial production of Zantedeschia plantlets in vitro.
Additional index words. LED, light quality, Zantedeschia, bluelight, redlight
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INTRODUCTION
Light-emitting diodes (LEDs) are a potential alternative to tubular fluorescent lamps (TFLs) due to their low forward current, small, wavelength specific, solid state construction, low degradation and long life (Bula et al., 1991; Brown et al., 1995; Fang and Jao, 2000).A light source for young plants production using blue (B) and red (R) LEDs was developed with the capabilities of altering blue and/or red light intensity, R/B ratio, frequency and duty ratio of light (Jao and Fang, 2003a).Using such device, authors found that continuous light from LEDs and TFLs had no significant difference to the growth of potato (Solanum tuberosum L. Kennebec) plantlets in vitro. However, intermittent/pulse light promotes the growth of potato plantlets significantly (Jao and Fang, 2003b). Concurrent red and blue light enhances growth of potato plantlets when compare with alternating red and blue light under same daily light integral (DLI, 5.53 mol·m-2) and 16/8 photoperiod (Jao and Fang, 2003c).
Plant growth and development are affected by light intensity, light quality, duration and photoperiod (Taiz and Zeiger, 1991). Growth, morphology and differentiation of in vitro plantlets are also affected by light quality(Econmou and Read, 1987). Red light was shown significantly enhance stem elongation of Pelargonium plantlets in vitro while blue light inhibited shoot length(Appelgren, 1991). Aksenova et al. (1994) found that in vitro potato plantlets produced longer stems and higher root/shoot ratio in red than in blue. By using TFLs as the main,and red and far-red (FR) LEDs as the supplemental light source, Iwanami et al. (1992) found that the growth of potato plantlets in vitrocan be promoted even when the relative proportion of supplemental light was very small. By using red LEDs as the main and using TFLs asthe supplemental light source, Miyashita et al. (1994) found that the morphology rather than the growth (dry weight and leaf area) of potato plantlets (Solanum tuberosum L. cv. Benimaru) was affected under same PPF. In all of the experiments mentioned above, the R/B and R/FR ratios were fixed throughout the cultivation period.
The focus of this study was to compare effects ofvarying R/B ratios throughout the cultivation period on the photomixotrophic growth of Zantedeschia plantletsin vitro under the same PPF and photoperiod.
Materials and Methods
Plant materials and culture conditions. Plantlets of calla lily (Zantedeschia jucundacv. ‘Sensation’) at final stage were provided by Sunrise Biotech. Co. Ltd., Taiwan (fresh weight = 101.7 ± 11.2 mg and dry weight = 22.7 ± 4.4 mg) andcultured in 10 × 10 cm, 785 ml cylindrical poly vinyl chloride (PVC) containers (Yu-Ping Corp., Taiwan). Eachvessel contained 10 plantlets. Vessels were incubated in acontrolled environmentat 25 ± 2oC and 50 ± 10 % RH.
Light treatments. All cultures were illuminated using red and blue LEDs programmed to provide 16 hr/8 hr (day/night) photoperiod and 80 μmol·m-2·s-1 (measured using LI-1800, LI-COR Inc., USA) of PPF, leading to the daily light integral (DLI) of 4.608 mol·m-2. The LED lighting system used was described previously by Jao and Fang (2003a, b).
Quantum ratios of red vs. blue light (R/B) werevaried among 8 treatments as listed in Table 1. The total cultivation period was 5 weeks. Treatments 1 (T1) and 2 (T2) provided with blue light alone (R/B= 0) and red light alone (R/B= infinity), respectively. Treatment 3 (T3) provided with constant R/B ratio (1.5) and treatments 4 to 8 provided with varying R/B ratios. In T4, T5 and T6, R/B ratios equaled 0.5 for the first 2 weeks and 3.0 for the last 2 weeks. In T7 and T8, R/B ratios equaled 1.5 and 3.0 for the first 2 and the last 2 weeks, respectively. On week 3, R/B ratios equals 0.5, 1.5, 3.0, 1.5 and 3.0 for T4, T5, T6, T7 and T8, respectively. The amount of total accumulative red (RACC) and blue (BACC) light among treatmentswere different and were listed in columns 5 and 6 of Table 1. The RACC/BACC ratio of all treatments was listed in the last column of Table 1.
Treatments were arranged in a completely randomized design of 10 individual samples/treatment (80 samples total). Each treatment was conducted three times.
Experimental design and statistical analysis. Plant height, chlorophyll content, fresh/dry weight of shoots and roots were measured for each treatment of all samples 35 days after planting. During each sampling, chlorophyll content of the youngest fully expanded leaf was measured using portable chlorophyll meter (Minolta SPAD 502, Spectrum Technologies, U.S.A.).
Percentage of dry matter and shoot/root ratio (S/R, calculated using dry weight) were calculated. Data were analyzed using Duncan’s multiple range test and orthogonal contrasts using SAS (SAS Inst., Cary, N.C.).
Results and Discussion
Table 2 showed that providing the same PPF and photoperiod with varying R/B ratios caused the differences inZantedeschia plantlet height, chlorophyll content, shoot/root dry weight ratios but had no significant effects on total dry weight during 5 weeks of cultivation period.Orthogonal contrasts also revealed that there were no significant differences on dry weight between treatments. Similar result was reported by Miyashita et al. (1994)showing that influences of R/B ratio on the morphology rather than the growth of potato plantlets in vitro.
Percentage of drymatter and S/R ratio of plantlets were different within treatments (Table 2). Treatment 8 had the highest dry matter content (~10%), followed by treatments 5, 6 and 7 (~8%) and treatments 1 to 4 (~7%) were the worse. Treatment 4 had the highest S/R ratio, plantlet height and chlorophyll content on leaf and treatment 2 had the lowest.
Contrasts HEI1 to HEI4 (Table 3), having T1 or T2 as one of the contrasting factor, showed significant differences on plantlet height with other contracting factor(s). It is quite obvious that red and blue light has different control mechanism over plant height. Results showed that Zantedeschia plantlet illuminated with red light alone (T2) had the lowest plant height (Table 2) and this was contradict with the results of Appelgren (1991) and Aksenova et al. (1994) on the growth of Pelargonium and potato plantlets, respectively.
Providing red and blue light together, regardless of fixed or varying R/B ratios during the cultivation period, had no significant difference on plantlet height as shown in contrast HEI5 (Table3), which might be misleading. The only difference of treatments 4, 5 and 6was on the R/B ratio of week 3 out of 5 weeks of cultivation period. Comparison among treatments 4, 5 and 6 (contrasts HEI6 to HEI8, Table 3) showed significant difference, indicating that one week difference of R/B ratio was sufficient to influence the plant height of Zantedeschia plantlets in vitro. However, no significant difference in chlorophyll content was found for this one week difference of R/B ratio among treatments 4, 5 and 6 (contrasts CHL4 to CHL6, Table 4).
Figure 1-1, top view of the cultural vessel, showed little fully expanded leaves when illuminated with blue light alone (T1). Chlorophyll content was not measured for this treatment. Other treatments had fully expanded leaves after 35 days of cultivation period (Figures 1-2 to 1-8). Plantlets grown under treatments 4 and 2 had the highest and lowest SPAD value, respectively (Table 2). Contrast CHL1 and CHL2 (Table 4) showed that treatment of red light alone (T2) had significant difference on the chlorophyll development of Zantedeschia plantlets in vitrowhen compare with other treatments. Contrast CHL3 showed that providing red and blue light together, regardless of fixed/varying R/B ratios, no significant difference was found on the chlorophyll development.
Figure 2 showed the trend of decreasing plantlet height and chlorophyll content following the increase of accumulative R/B ratio can be found. Dramatic change in plantlet height can be found when R/B ratio changed in the region near unity, which is close to the value 1.15 for the regular daylight.
In conclusion, morphology of plantlets can be regulated by light quality using LEDs. Blue light alone leads to more compact leaves, suggesting that red light is needed for the fully expansion of leaves. Red light alone leads to dwarf plant and less greenish leaves, suggesting that blue light also regulating the plant height and the chlorophyll development.
At present, the cost of blue LEDs can be 10 times more than that of red LEDs. Results of this study showed that growth of Zantedeschia plantlets in vitro using only red LEDs can develop fully expanded leaves but looksless greenish and dwarf (Figure 1-2),however it has no significant difference in dry weight when compared with other treatments.Further investigation will be conducted to see whether there exist differences or not using plantlets from T2 and T4 to grow in greenhouse after transplanting. If no difference can be found, it will be economically viable to use only red light for the production of the Zantedeschia plantlets in vitro.
Literature Cited
Appelgren, M. 1991. Effects of light quality on stem elongation of Pelargonium in vitro. Scientia Horticulture. 45:345-351.
Aksenova, N.P., T. N. Konstantinova, L.I. Sergeeva, I. Machackova and S.A. Golyanovskaya. 1994. Morphogenesis of potato plants in vitro. I. Effect of light quality and hormones. J. Plant Growth Regul. 13:143-146.
Brown, C. S., A. C. Schuerger and J. C. Sager. 1995. Growth and photomorphogenesis of pepper plants under red light-emitting diodes with supplemental blue or far-red lighting. J. Amer. Soc. Hort. Sci. 120:808-813.
Bula, R. J., R. C. Morrow, T. W. Tibbitts, D. J. Barta, R. W. Ignatus and T. S. Martin. 1991. Light-emitting diodes as a radiation source for plants. HortScience. 26:203-205.
Economou, A. S. and P. E. Read. 1987. Light treatments to improve efficiency of in vitro propagation system. HortScience. 22(5):751-754.
Fang, W. and R.C. Jao. 2000. A review on artificial lighting of tissue cultures and transplants. p. 108-113 In: C. Kubota and C. Chun (eds.), Transplant Production in the 21st Century. Kluwer Academic Publishers.Netherlands.
Iwanami, Y., T. Kozai, Y. Kitaya and S. Kino. 1992. Effects of supplemental red and far-red lighting using light emitting diode on stem elongation and growth of potato plantlets in vitro. p. 183. In Abstr. Intl. Symp. Transplant Production Sytems. 21-26 July 1992. YokohamaJapan.
Jao, R. C. and W. Fang. 2003a. Anadjustablelightsourceforphoto-phytorelated researchandyoungplantproduction.Appl.Eng. Agric.(accepted).
Jao, R. C. and W. Fang. 2003b. Effects of frequencyand duty ratio on the growthof potato plantletsin vitrousinglight emitting diodes. HortScience (in press).
Jao, R. C. and W. Fang. 2003c. Growth of Potato Plantlets in vitro is Different when Provided Concurrent versus Alternating Red and Blue Light Photoperiods. HortScience (accepted).
Miyashita, Y., T. Kimura, Y. Kitaya and T. Kozai. 1994. Effects of red light on the growth and morphology of potato plantlets in vitro: using light emitting diodes (LEDs) as light source for micropropagation. Acta Horticulturae.418:169-173.
Taiz, L. and E. Zeiger. 1991. Plant Physiology. 1st ed., 179-264. New York: Benjamin/Cummings Publishing Company, Inc.
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Table 1. Summary of eight treatments. (Treatments T4 to T8 have changing R/B ratios).
Treatments / Weeks 1,2 / Week 3 / Weeks 4,5 / RAcc ** / BAcc *** / RAcc/BAccR/B ratio / mol·m-2 / mol·m-2
T1 / Blue light alone (0/80) / 0.0 / 161.3 / 0.00
T2 / Red light alone (80/0) / 161.3 / 0.0 / Infinity
T3 / 1.5 (48/32) * / 1.5 / 1.5 / 96.8 / 64.5 / 1.50
T4 / 0.5 (26/54) / 0.5 / 3.0 (60/20) / 79.8 / 81.5 / 0.98
T5 / 0.5 / 1.5 / 3.0 / 88.7 / 72.6 / 1.22
T6 / 0.5 / 3.0 / 3.0 / 93.5 / 67.8 / 1.38
T7 / 1.5 / 1.5 / 3.0 / 106.4 / 54.9 / 1.94
T8 / 1.5 / 3.0 / 3.0 / 111.3 / 50.0 / 2.23
* Values before bracket represent the R/B ratio and values inside the bracket were the absolute PPF of red vs. blue (R/B) provided on each treatment.
** Total accumulative red light provided to plantlets over 35 days on each treatment.
*** Total accumulative blue light provided to plantlets over 35 days on each treatment.
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Table 2. Plant height, chlorophyll content, shoot/root dry/fresh weight per plantlet, percentage of dry matter, shoot/root dry weight ratio (S/R ratio) on a dry weight basis of the Zantedeschia plantlets in vitro on days 35 after treatments.
Treatments / Plant Height(cm) / Chlorophyll
Content
(SPAD) / Fresh Weight
(g/plantlet) / Dry Weight
(g/plantlet) / Dry matter
% / S/R
ratio
Shoot / Root / Total / Shoot / Root / Total
T1 / 7.6 a* / N/A / 0.295 bc / 0.603 ab / 0.898 bc / 0.017 bc / 0.055 a / 0.071 ab / 7.94 / 0.30
T2 / 5.3 d / 19.84 c / 0.234 c / 0.636 ab / 0.869 c / 0.013 c / 0.056 a / 0.068 ab / 7.86 / 0.23
T3 / 6.3 bc / 25.70 b / 0.326 b / 0.733 c / 1.059 ab / 0.020 b / 0.059 a / 0.079 a / 7.48 / 0.34
T4 / 8.0 a / 30.88 a / 0.402 a / 0.692 ab / 1.094 a / 0.025 a / 0.059 a / 0.084 a / 7.69 / 0.42
T5 / 5.7 cd / 28.03 ab / 0.229 c / 0.572 b / 0.800 cd / 0.014 c / 0.054 a / 0.067 ab / 8.42 / 0.25
T6 / 6.7 b / 26.63 b / 0.254 c / 0.563 b / 0.817 cd / 0.015 c / 0.056 a / 0.071 ab / 8.70 / 0.27
T7 / 6.1 bc / 24.29 b / 0.256 c / 0.418 c / 0.674 d / 0.015 c / 0.040 b / 0.055 b / 8.17 / 0.38
T8 / 6.4 b / 26.99 ab / 0.279 bc / 0.453 c / 0.732 cd / 0.018 bc / 0.059 a / 0.077 a / 10.49 / 0.30
* Means within a column followed by the same letters are not significantly different at the 5% level of Duncan’s multiple range test
Table 3. Analysis of variance summary for the plant height of Zantedeschia plantlets. Data were analyzed using procedures for a completely randomized design with treatment comparisons made using orthogonal contrasts.
Source / DF / Sums of squares / Mean square / F-valueTreatment / 7 / 56.118 / 8.016 / 13.98 **
Error / 72 / 41.283 / 0.573
Corrected total / 79 / 97.402
contrasts
HEI1 a / 1 / 11.589 / 11.589 / 20.21 **
HEI2 b / 1 / 16.115 / 16.115 / 28.11 **
HEI3 c / 1 / 6.742 / 6.742 / 11.76 **
HEI4 d / 1 / 5.479 / 5.479 / 9.56 **
HEI5 e / 1 / 0.546 / 0.546 / 0.95 ns
HEI6 f / 1 / 27.144 / 27.144 / 47.34 **
HEI7 g / 1 / 9.248 / 9.248 / 16.13 **
HEI8 h / 1 / 4.704 / 4.704 / 8.20 **
** Significant contrast at the 0.01 level; ns = non-significant.
a Treatment 1 vs. others : Treatment of blue light alone vs. all other treatments.
b Treatment 2 vs. others : Treatment of red light alone vs. all other treatments.
c Treatment 1 vs. 3 : Treatment of blue light alonevs. fixed R/B ratio treatment.
d Treatment 2 vs. 3 : Treatment of red light alonevs. fixed R/B ratio treatment.
e Treatment 3 vs. 4, 5, 6, 7, 8 : Treatment of fixed R/B ratio vs. treatments of varying R/B ratios.
f Treatment 4 vs. 5
g Treatment 4 vs. 6
h Treatment 5 vs. 6
Table 4. Analysis of variance summary for the chlorophyll content of Zantedeschia plantlets. Data were analyzed using procedures for a completely randomized design with treatment comparisons made using orthogonal contrasts.
Source / DF / Sums of squares / Mean square / F-valueTreatment / 6 / 702.534 / 117.089 / 7.31 **
Error / 63 / 1008.980 / 16.015
Corrected total / 69 / 1711.514
contrasts
CHL1 a / 1 / 450.121 / 450.121 / 28.11 **
CHL2 b / 1 / 171.698 / 171.698 / 10.72 **
CHL3 c / 1 / 23.074 / 23.074 / 1.44 ns
CHL4 d / 1 / 40.612 / 40.612 / 2.54 ns
CHL5 e / 1 / 90.332 / 90.332 / 5.64 ns
CHL6 f / 1 / 9.800 / 9.800 / 0.61 ns
** Significant contrast at the 0.01 level; ns = non-significant.
a Treatment 2 vs. others : Treatment of red light alonevs. treatments 3 to 8.
b Treatment 2 vs. 3 : Treatment of red light alonevs. fixed R/B ratio treatment.
c Treatment 3 vs. 4, 5, 6, 7, 8 : Treatment of fixed R/B ratio vs. treatments of varying R/B ratios.
d Treatment 4 vs. 5
e Treatment 4 vs. 6
f Treatment 5 vs. 6
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Fig. 1. Top view of uncapped culture vessel for eight treatments after 5 weeks of growth. T1:illuminated with blue light alone, T2:illuminated with red light alone, T3:illuminated with fixed R/B ratio (=1.5) for 5 weeks, T4: illuminated with varying R/B ratios (=0.5, 0.5, 0.5, 3.0, 3.0 for week 1 to 5, respectively), T5:illuminated with varyingR/B ratios (=0.5, 0.5, 1.5, 3.0, 3.0 for week 1 to 5, respectively),T6:illuminated with varying R/B ratios (=0.5, 0.5, 3.0, 3.0, 3.0 for week 1 to 5, respectively),T7:illuminated with varyingR/B ratios (=1.5, 1.5, 1.5, 3.0, 3.0 for week 1 to 5, respectively), T8:illuminated with varyingR/B ratios (=1.5, 1.5, 3.0, 3.0, 3.0 for week 1 to 5, respectively).
Fig. 2. Plant height and SPAD value of leaves of Zantedeschia plantlets vs. accumulative R/B ratio of all treatments.
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