Sugar Tech (May-June 2017) 19(3):331–336 DOI 10.1007/s12355-016-0459-5

RESEARCH ARTICLE

The Effect of Light Quality on Growth and Development of In Vitro Plantlet of Stevia rebaudiana Bertoni Marco A. Ramı´rez-Mosqueda1 • Lourdes G. Iglesias-Andreu1 • Jose´ R. Bautista-Aguilar1

Received: 11 March 2016 / Accepted: 31 May 2016 / Published online: 13 June 2016 Ó Society for Sugar Research & Promotion 2016

Abstract Fluorescent lamps are the most commonly used light source for the in vitro culture of various plant species. However, there are other sources of illumination, such as light-emitting diodes (LEDs), which have proven to be more efficient for in vitro culture. In the present study, we evaluated the effect of LEDs on the in vitro morphogenesis, proliferation of shoots, growth and rooting of Stevia rebaudiana Bertoni. For that, five different sources of light were tested under a 16-h photoperiod: fluorescent lamps (Fl), white LEDs (W), red LEDs (R), blue LEDs (B) and a combination of blue and red LEDs (B/R, 1:1). The proliferation rate was higher with R LEDs compared with Fl light, although shoots have a lower length under R LEDs. Under B/R LEDs, maximum shoot elongation was obtained. During rooting, LEDs did not improve the rooting of shoots but increased the content of photosynthetic pigments, which contributed to the acclimation process of in vitro plantlets. Our results revealed that the spectrum of different light sources produced different effects during the in vitro culture of S. rebaudiana. Keywords LEDs  Chlorophyll  Light quality  Micropropagation

& Marco A. Ramı´rez-Mosqueda [email protected] & Lourdes G. Iglesias-Andreu [email protected] 1

Instituto de Biotecnologı´a y Ecologı´a Aplicada (INBIOTECA), Universidad Veracruzana, Av. de las Culturas Veracruzanas No. 101, Campus para la Cultura, las Artes y el Deporte, Col. Emiliano Zapata, C.P. 91090 Xalapa, Veracruz, Mexico

Abbreviations M&S Murashige and Skoog medium BA Benzyladenine LED Light-emitting diodes PGR Plant growth regulators

Introduction The leaves of Stevia rebaudiana Bertoni contain glycosides such as steviosides and rebaudiosides, which are 200–300 times sweeter than sucrose or cane sugar (Geuns 2003). In addition, this plant species has medicinal properties and prevents both high blood pressure and dental cavities; it is also widely used for the regulation of blood sugar levels (Kinghorn and Soejarto 2002; Singh and Rao 2005). The seeds of S. rebaudiana are characterized by a low viability coupled with a poor germination rate (Mitra and Pal 2007). Besides, vegetative propagation is limited to a low number of specimens that acclimatize to soil (Mitra and Pal 2007). In vitro micropropagation techniques are an attractive option to propagate large numbers of plants in a short time (Ramı´rez-Mosqueda and Iglesias-Andreu 2015). Factor that affects growth and development of different plant species cultivated in vitro is light (spectral quality, photon flux and photoperiod) (Huges 1981; Gupta and Jatothu 2013). The light source typically used for in vitro culture is tubular fluorescent lamps (FLs). FLs emit a broad spectrum that ranges from 350 to 750 nm, including unnecessary wavelengths that are of low quality for promoting growth (Kim et al. 2004). Light-emitting diodes (LEDs) are a potential alternative light source for in vitro culturing, due to their wavelength specificity, narrow

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bandwidth, low amount of thermal emissions, low degradation and long life (Bull et al. 1991; Gupta and Jatothu 2013). Particularly, the evaluation of the effect of different LED wavelengths on the in vitro growth and development of plantlets of various species has raised interest recently (Gupta and Jatothu 2013). However, despite the economic importance of S. rebaudiana, the effect of different LED wavelengths on the morphogenesis process and growth of this species in vitro has not been evaluated. Therefore, this study investigates the effect on morphogenesis and growth of S. rebaudiana cultivated in vitro under LEDs of different wavelengths.

Materials and Methods Plant Material This investigation used in vitro plantlets of S. rebaudiana variety Morita II established in the Plant Tissue Culture Laboratory of Instituto de Biotecnologı´a y Ecologı´a Aplicada (INBIOTECA) at the Universidad Veracruzana. Light Irradiation and Growth Conditions Light-quality experiments were performed in a culture room with relative humidity of 80 ± 5 %, photoperiod of 16 h light/8 h dark and temperature of 25 ± 2 °C. The radiation intensity of artificial light was set to 40–50 lmol m-2 s-1. Light sources used in this experiment were: 1. 2. 3. 4. 5.

FL (control): Fluorescent lamps emitting light at broad wavelengths of 400–700 nm. W: White LEDs with a wavelength of 420 nm. R: Red LEDs with a wavelength of 660 nm. B: Blue LEDs with a wavelength of 460 nm. B/R = 1:1: 50 % blue light at a wavelength of 460 nm and 50 % red light at a wavelength of 660 nm.

Explants in the shoot proliferation and rooting phases were incubated under the different LED light treatments (IP65 model, SMD 5050 RBG supplying 12 V and 1 W per module, Techno LiteÒ, Zapopan, Jalisco).

autoclaving at 121 °C and 124 kPa for 15 min. After 8 weeks of culture, the number of shoots, shoot size and number of leaves were evaluated. Effect of Light Quality on Rooting Individual shoots measuring 1–2 cm long were incubated in semisolid MS medium at half its concentration added to half the amount of vitamins used in the proliferation phase and 100 mg l-1 myoinositol with no PGR supplementation. The pH of the medium was adjusted to 5.8 ± 0.2; then, the medium was solidified with 2.5 g l-1 GelriteTM (Sigma-Aldrich, St. Louis, MO) before autoclaving at 121 °C and 124 kPa for 15 min. After 8 weeks of culture, the number of roots, root size, leaf number and content of photosynthetic pigments were determined (Arnon 1949). Chlorophyll Assessment The chlorophyll content of the third leaf from the apex was determined by extracting the pigment with 80 % acetone (Arnon 1949). Leaves of each treatment were washed with sterile distilled water, and moisture was removed with blotting paper. Known weight (0.2 g) of freshly chopped leaves was ground in 20 ml of a 1:1 (v/v) mixture of 80 % acetone and absolute ethyl alcohol in a 25-ml stoppered test tube and centrifuged at 6000 rpm for 12 min (using 15-ml polypropylene centrifuge tubes). Absorbance (OD) was measured at 663 nm (Chl. a), 645 nm (Chl. b) and 440.5 nm (carotenes) with a Spectrophotometer (VelabÒ UV/Vis)) using 80 % acetone as blank. The contents were determined using the following formulas: Chl a ¼ ½ð12:25  A663 2:25  A645 Þ  V=100  W Chl b ¼ ½ð20:30  A645 4:91  A663 Þ  V=100  W Chl a þ b ¼ ½ð7:34  A663 þ 17:76  A645 Þ  V=100  W Car ¼ ½ð4:46  A441 Chl a þ Chl bÞ  V=100  W where V is the total volume of acetone extract (ml) and W is the fresh weight (g) of the sample.

Effect of Light Quality on Shoot Proliferation

Hardening

Nodal segments measuring 0.5–1 cm in length were incubated in semisolid MS medium (Murashiges and Skoog 1962) supplemented with 0.1 mg l-1 thiamine HCl, 0.5 mg l-1 nicotinic acid, 0.5 mg l-1 pyridoxine HCl, 2 mg l-1 glycine, 100 mg l-1 myoinositol, 1 mg l-1 benzyladenine (BA) and 30 g l-1 sucrose. The pH of the medium was adjusted to 5.8 ± 0.2 and solidified with 2.5 g l-1 GelriteTM (Sigma-Aldrich, St. Louis, MO) before

Individual shoots (10–15 cm), previously rooted in vitro, were thoroughly washed with tap water and planted under greenhouse conditions in separate pots (5 9 5 9 8 cm) containing a sterile substrate consisting of a 1:1 mixture of peat mossTM and agroliteTM. Plantlets were maintained under 50 % shaded conditions. Irrigation was performed manually by spraying tap water so as to maintain a relative humidity between 80 and 95 %.

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Statistical Analysis For all assays, the experimental design was completely random with ten replicates, each consisting of a flask with 20 ml of culture medium and five explants (nodal segments or individual shoots). The data obtained were analyzed using analysis of variance (ANOVA) followed by a Tukey’s test (p B 0.05) using the IBM software SPSS statistics (version 21 for Windows Inc., Chicago, IL).

Results and Discussion Effect of Light Quality on Shoot Proliferation The different LED wavelengths produced a positive effect on the in vitro formation of shoots of S. rebaudiana. After 4 weeks of culture, significant differences were observed between the different LED wavelengths (Table 1). The largest number of shoots (9.11 shoots per explant) was

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obtained under treatment R, followed by treatments B and W (7.09 and 6.25 shoots per explant, respectively) (Fig. 1); however, treatment R produced shorter shoots with less leaves relative to the other LED treatments (Fig. 1). The control treatment Fl produced the fewest number of shoots (5.40) and leaves (6.19) per explant, evidencing that LEDs exert a positive effect on the variables evaluated during the in vitro formation of shoots in S. rebaudiana. Longer shoots were observed under the B/R light treatment (Fig. 1). The B/R LED combination has been reported to lead to a larger number of shoots per explant in Brassica napus and Dendrobium officinale (Lin et al. 2011; Li et al. 2013). In contrast, the results obtained in this study show that R LEDs produce a larger number of shoots per explant in S. rebaudiana. However, under the test conditions, R LEDs led to short shoots. Regarding the number of shoots obtained in S. rebaudiana using fluorescent-lamp light, Modi et al. (2012) reported an average of 5.5 shoots using nodes as explants in modified MS medium and 1 % (w/v) sucrose with no PGR supplementation.

Table 1 Effect of different LED wavelengths on the in vitro formation of shoots in Stevia rebaudiana after 4 weeks of culture Light quality

Number of shoots per explant

Shoot length (cm)

Number of leaves per shoot

Fl

5.40 ± 0.21bc

1.90 ± 0.11c

6.19 ± 0.28c

W

6.25 ± 0.75

b a

ab

6.55 ± 0.54bc

c

2.65 ± 0.58

R B

9.11 ± 0.85 7.09 ± 0.51b

2.05 ± 0.15 2.75 ± 0.26ab

5.24 ± 0.44c 8.13 ± 0.59ab

B/R

5.60 ± 0.32bc

2.99 ± 0.21a

8.92 ± 0.52a

Figures represent the mean ± SE (standard error). Means with different letters are significantly different (Tukey, p B 0.05) Fl fluorescent lamps, LEDs: W white, R red, B blue, blue ? red = B/R (1:1)

Fig. 1 Effect of different LED wavelengths on the in vitro propagation of Stevia rebaudiana. Fl fluorescent lamps, LEDs: W white, R red, B blue, blue ? red = B/R (1:1)

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In our study, higher shoot length was obtained under the combination of R and B LEDs (1:1) in S. rebaudiana. However, R LEDs proved to be more effective in shoot elongation in D. officinale (Li et al. 2013). In some plant species such as Alternanthera brasiliana, B LEDs produce a larger number of leaves per shoot (Macedo et al. 2011), whereas in S. rebaudiana, the number of leaves increases when shoots cultivated under the B/R LED combination. Effect of Light Quality on Rooting At 4 weeks of culture, significant differences were observed in rooting responses under different LED wavelengths (Table 2).

Fl light produced more roots per explant (12.30) and higher length (2.13 cm), contrasting with the results observed with the different types of LEDs (Fig. 2). This indicated that the different LEDs did not enhance, the in vitro rooting of S. rebaudiana; however, the number of leaves increased under different LED treatments (Table 2). Under B/R LEDs, a higher number of leaves per shoot (8.9) were obtained (Fig. 2). The use of LEDs has contributed to increase the number and length of roots per explant in different plant species grown in vitro (Shin et al. 2008; Li et al. 2013, Lim and Eom 2013). However, the different LED treatments did not positively influence these variables in S. rebaudiana. Some reports indicate that B LEDs stimulate in vitro formation and elongation of roots in various plant species (Lim and

Table 2 Effect of different LED wavelengths on the in vitro rooting in Stevia rebaudiana after 4 weeks of culture Light quality

Number of roots per explant

Root length (cm)

Number of leaves

Fl

12.30 ± 0.24a

2.13 ± 0.16a

6.1 ± 0.28c

W R B B/R

bc

5.20 ± 0.25

c

6.60 ± 0.07

c

2.75 ± 0.06

bc

5.46 ± 0.26

1.16 ± 0.07

bc

6.5 ± 0.54bc

1.33 ± 0.06

b

5.2 ± 0.44c

0.71 ± 0.04

c

8.1 ± 0.59ab

1.14 ± 0.12

bc

8.9 ± 0.52a

Figures represent the mean ± SE (standard error). Means with different letters are significantly different (Tukey, p B 0.05) Fl fluorescent lamps, LEDs: W white, R red, B blue, blue ? red = B/R (1:1)

Fig. 2 Effect of different LED wavelengths on the in vitro rooting of Stevia rebaudiana. Fl fluorescent lamps, LEDs: W white, R red, B blue, blue ? red = B/R (1:1)

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Table 3 Effect of different LED wavelengths on the content of photosynthetic pigments in Stevia rebaudiana Light quality Fl W

Chlorophyll a (lg g-1 FW) 1.19 ± 0.014f c

8.09 ± 2.35

d

Chlorophyll b (lg g-1 FW)

Chlorophyll a ? b (lg g-1 FW)

Carotenes (lg g-1 FW)

0.34 ± 0.14f

1.87 ± 0.19f

0.06 ± 0.06f

c

14.60 ± 0.11

c

1.02 ± 0.01c

d

0.78 ± 0.01d

6.51 ± 0.25

d

R

5.14 ± 2.31

6.50 ± 0.07

11.64 ± 0.10

B

2131 ± 0.09b

2.23 ± 0.06b

2354 ± 0.01b

2.55 ± 0.02b

a

a

a

6.25 ± 0.01a

B/R

26.42 ± 0.93

11.96 ± 0.26 to

38.39 ± 0.01

Figures represent the mean ± SE (standard error). Means with different letters are significantly different (Tukey, p B 0.05) Fl fluorescent lamps, LEDs: W white, R red, B blue, blue ? red = B/R (1:1)

Fig. 3 S. rebaudiana plantlets at different stages of the acclimation process. a Plants 4 weeks after transfer to the greenhouse, b Stevia plantlets after 6 weeks of acclimation

Eom 2013). The best results earlier have been obtained by combining blue and red LEDs (Shin et al. 2008; Li et al. 2013), which contrasts with the findings of the present study in S. rebaudiana. It was previously reported that B produced a larger number of leaves per shoot in Alternanthera brasiliana (Macedo et al. 2011). These responses were improved in S. rebaudiana by combining blue and red (B/R) LEDs. Chlorophyll and Carotene Content Regarding the content of photosynthetic pigments, higher chlorophyll a, b, a ? b and carotene contents were obtained under B/R light (Table 3), which contrasts with the low content obtained under Fl light. This suggests that LEDs increase the content of photosynthetic pigments in the in vitro rooting phase in S. rebaudiana and possibly had positive effects during the acclimation phase of plantlets. Our results indicate that the combination of B/R LEDs results in higher content of photosynthetic pigments. These

results agree with those reported in various plant species, such as the tree peony (Paeonia lactiflora) (Ding et al. 2010), chrysanthemum (Dendranthema grandiflorum) (Kim et al. 2004) and some orchid species (Shin et al. 2008; Chung et al. 2010; Lin et al. 2011). These results are in agreement with the present observation in S. rebaudiana, since the B/R combination was associated with the highest photosynthetic pigment levels. Additionally, a seemingly inhibitory effect on chlorophyll synthesis was observed in S. rebaudiana in the R LED treatment, similar to findings reported by Chen and Hsu (2009) in cultivars of the genus Phalaenopsis. Acclimation Differences in percent survival of in vitro plantlets were observed associated with the different LED treatments. The highest percent acclimation (95 %) was observed with B/R LEDs (Fig. 3a), followed by 75 % obtained with F, R, B and W LEDs.

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The observed survival percentages (75–95 %) were similar to those previously reported for this species (80–95 %) by various authors (Hwang 2006; Modi et al. 2012). After 6 weeks in acclimation (Fig. 3b), plantlets were transplanted to the field. The protocol used ensured an adequate percent survival in the field of this species of commercial interest.

Conclusions This study demonstrates that light (spectral quality) is an important factor that influences in growth and development of in vitro plants of S. rebaudiana. The use of R LEDs stimulated shoot formation; however, the combination of B/R LEDs produced longer shoots with a higher number of leaves. For in vitro rooting, the B/R LED combination stimulated chlorophyll content only, which favored the acclimation of in vitro plantlets. Therefore, the use of this procedure can be useful for the micropropagation of S. rebaudiana. Acknowledgments We thank to the project ‘‘Plan breeding of Vanilla planifolia Jacks.’’ from the ‘‘Program for Teacher Professional Development’’ (PRODEP), SEP Conv. 2011 (CA-UVER-234), for the financial support to this research. Authors would like to thank M. E. Sa´nchez-Salazar for translation of the manuscript. Author Contribution Statement LGIA and MARM conceived and designed research. MARM conducted experiments. MARM and JRBA analyzed and reviewed the statistical analysis. MARM and LGIA wrote the manuscript. All authors read and approved the manuscript.

Compliance with Ethical Standards Conflict of interest All the authors declare that they have no conflict of interest.

References Arnon, D.I. 1949. Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant Physiology 24: 1–15. Bull, R.J., R.C. Morrow, T.W. Tibbitts, D.J. Barta, R.W. Ingnatius, and T.S. Martin. 1991. Light-emitting diodes as a radiation source for plants. HortScience 26: 203–205. Chen, C., and H.C. Hsu. 2009. Statistical technique for the evaluation of the effect of light quality on growing characteristics of in vitro cultures. Biosystems Engineering 103(2): 57–264. Chung, J.P., C.Y. Huang, and T.E.A. Dai. 2010. Spectral effects on go through a somatic embryogenesis process and plantlet growth of

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Sugar Tech (May-June 2017) 19(3):331–336 Oncidium Gower ‘Ramsey’. Scientia Horticulturae 124: 511–516. Ding, Y., S. He, J.A. Teixeira da Silva, G. Li, and M. Tanaka. 2010. Effects of a new light source (cold cathode fluorescent lamps) on the growth of tree peony in vitro plantlets. Scientia Horticulturae 125: 167–169. Geuns, J.M.C. 2003. Molecules of interest: stevioside. Phytochemistry 64: 913–921. Gupta, S.D., and B. Jatothu. 2013. Fundamentals and applications of light-emitting diodes LEDs in in vitro plant growth and morphogenesis. Plant Biotechnology Reports 7: 211–220. Huges, K.W. 1981. In-vitro ecology: Exogenous factors effecting growth and morphogenesis in plant culture system. Environmental and Experimental Botany 21: 281–288. Hwang, S.J. 2006. Rapid in vitro propagation and enhanced stevioside accumulation in Stevia rebaudiana Bert. Journal of Plant Biology 49: 267–270. Kim, S.J., E.J. Hahn, J.W. Heo, and K.Y. Paek. 2004. Effects of LEDs on net photosynthetic rate, growth and leaf stomata of chrysanthemum plantlets in vitro. Scientia Horticulturae 101: 143–151. Kinghorn, A.D., and D.D. Soejarto. 2002. Discovery of terpenoid and phenolic nonnutritive sweeteners from plants. Pure and Applied Chemistry 74: 1169–1179. Li, H., C. Tang, and Z. Xu. 2013. The effects of different light qualities on) cannot be used (Brassica napus L.) plantlet growth and morphogenesis in vitro. Scientia Horticulturae 150: 117–124. Lim, Y.J., and S.H. Eom. 2013. Effects of different light types on root formation of Ocimum basilicum L. cuttings. Scientia Horticulturae 164: 552–555. Lin, Y., J. Li, B. Li, T. He, and Z. Chun. 2011. Effects of light quality on growth and development of protocorm-like bodies of Dendrobium officinale in vitro. Plant Cell, Tissue and Organ Culture 105: 329–335. Macedo, A.F., M.V. Leal-Costa, E.S. Tavares, C.L. Salgueiro-Lage, and M.A. Esquibel. 2011. The effect of light quality on leaf production and development of in vitro plants of breaks and rearrangements Alternanthera brasiliana Kuntze. Environmental and Experimental Botany 70: 43–50. Mitra, A., and A. Pal. 2007. In-vitro regeneration of Stevia rebaudiana (Bert.) from nodal explants. Journal of Plant Biochemistry and Biotechnology 16: 59–62. Modi, A.R., G. Patil, N. Kumar, A.S. Singh, and N. Subhash. 2012. A simple and efficient in vitro mass multiplication procedure for Stevia rebaudiana Bertoni and analysis of genetic fidelity of in vitro raised plants through RAPD. Sugar Tech 14: 391–397. Murashige, T., and F. Skoog. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum 15: 473–497. Ramı´rez-Mosqueda, M.A., and L.G. Iglesias-Andreu. 2015. Direct organogenesis of Stevia rebaudiana Bertoni using thin cell layer (TCL) method. Sugar Tech. doi:10.1007/s12355-015-0391-0. Shin, K.S., H.N. Murthy, J.W. Heo, E.J. Hahn, and K.Y. Paek. 2008. The effect of light quality on the growth and development of in vitro plants Act breaks and rearrangements Doritaenopsis. Plant Physiology 30: 339–343. Singh, S.D., and G.P. Rao. 2005. Stevia: The herbal sugar of 21st century. Sugar Tech 7: 17–24.