LEDS APPLICATION IN EX VITRO ROOTING AND ACCLIMATIZATION OF CHRYSANTHEMUM (CHRYSANTHEMUM X GRANDIFLORUM /RAMAT./ KITAM.)

Anita Woźny, Natalia Miler
Department of Ornamental Plants and Vegetable Crops, Faculty of Agriculture and Biotechnology, University of Science and Technology, Bydgoszcz, Poland

ABSTRACT

Plants of four chrysanthemum cultivars propagated in vitro were tested in terms of survival and plantlets quality after acclimatization under fluorescent lamps (FLs) and light-emitting diodes (LEDs). The plantlets were acclimatized directly or after in vitro rooting. Two out of four cultivars showed lowered survival capability after direct acclimatization; only a few plantlets of ‘Bingo’ and ‘Cool Time’ survived, while most plantlets representing ‘Euro’ and ‘Vymini’ were properly acclimatized, rooted and growing. The survival rate in plantlets acclimatized after in vitro rooting was high, from 85–95% in all the cultivars. The light source did not influence the survival rates. Plantlets quality after acclimatization under LEDs or FLs differed in particular cultivars. In ‘Bingo’ and ‘Vymini’ acclimatized under LEDs the average height of plantlets and fresh weight was higher than under FL. The length of roots was only affected by light source in ‘Vymini’; the roots produced under LEDs were longer. Chlorophyll content was higher under LEDs in ‘Cool Time’ and ‘Euro’. Chrysanthemum acclimatization can be successfully conducted under LEDs and FLs, however it should be forgone by in vitro rooting.

Key words: direct rooting, light-emitting diodes, micropropagation.

INTRODUCTION

Plant growth and developmental processes are regulated by light quality (color, wavelength), quantity and photoperiod (duration of illumination). Red and blue light are most important for plant growth, while far-red light influences the development of specific plant characteristics. Manipulating the lights profile provides an opportunity to apply the optimum light recipe at every stage of plant growth. Artificial light is commonly used to increase the yield and quality of ornamental plants. Light sources such as fluorescent and high-pressure sodium lamps are generally used for plants growing under greenhouse conditions as well as in vitro laboratory. Nowadays, Light Emitting Diodes (LEDs) are gaining importance, mostly due to their potentially higher energy efficiency. In comparison to traditional lamps, the improved LEDs characteristics include smaller size and weight, solid-state construction, long lifetime, low temperature emission [3, 7]. Since the usage of LEDs allows to strictly control the spectral composition and the adjustment of light intensity, it becomes possible to simulate the changes of sunlight intensity along the day.

The physiological and morphological effects of LEDs usage have been widely studied for some species of ornamental plants [2, 13–15, 18, 21]. Light emission with well-defined wavelength optimal for a given plant species allow to improve micropropagation technology for the species that play a fundamental role in horticultural production [11, 17].

Acclimatization is one of the most crucial stages in plants micropropagation and light is one of the most important factors affecting plants acclimatization [20]. Plantlets in vitro grow usually under relatively low light intensity (1200–3000 lx) and stable temperature conditions. Specific culture conditions lead to many morphological, anatomical and physiological disorders of in vitro produced plants [19]. The transfer of plants from ex vitro to greenhouse conditions should be carried out gradually, since many abiotic (variable, intensive and broad spectrum sunlight, variable temperature) and biotic stress factors influence plants at that stage and the probability of plants loss is high [6]. Since LEDs do not heat plants, they seem to be suitable for use during acclimatization [4].

Since the process of rooting in vitro is labour-intensive and expensive (the cost of rooting varies from 35 to 75% of the total micropropagation cost), many commercial laboratories tend to omit rooting stage in vitro and conduct rooting and acclimatization of plantlets simultaneously [6, 22]. For chrysanthemum, direct rooting after the multiplication stage was successfully conducted in four cultivars rooted and acclimatized in various substrates [25].

The aim of our study has been to evaluate the usefulness of LEDs during acclimatization and direct rooting of micropropagated chrysanthemum plants. We have tested four chrysanthemum cultivars in terms of plants quality and survival after rooting and acclimatization under fluorescent light and light emitting diodes.

MATERIAL AND METHODS

The experiment was performed at the Laboratory of Biotechnology and the glasshouse of the Department of Ornamental Plants and Vegetable Crops of the University of Technology and Life Sciences in Bydgoszcz. Four chrysanthemum cultivars (Chrysanthemum × grandiflorum [Ramat.] Kitam.), 'Bingo', 'Cool Time', 'Euro' and 'Vymini' were used in the experiment.

Multiplication in vitro
Chrysanthemums were propagated in vitro using single-node explants on a modified medium according to Murashige and Skoog (MS, 1962) [16], with half-increased content of calcium and iron, 3% sucrose and solidified with 0.8% agar. At the stage of multiplication no plant growth regulators were used in the medium. The pH was established at 5.8, prior to autoclaving. The explants were placed in 350 ml glass jars, six explants per jar, the volume of medium in each jar was 40 ml. The plantlets were cultured under daylight-emitting fluorescent tubes (TLD 36W/54, Koninklijke Philips Electronics N.V., the Netherlands), Photosynthetically active radiation (PAR) was 32 μmol m^-2·s^-1, the photoperiod: 16 h /8 h light/dark and temperature: 24±1°C. Half of the four-week-old plantlets were purposed for acclimatization directly after the stage of in vitro multiplication, while the other half – for acclimatization following roots induction under in vitro conditions. Plantlets intended for direct acclimatization or roots induction were, on average, 2.5 cm in height and showed from 4 to 6 leaves.

Direct acclimatization
The plantlets were taken out from the medium, the basal part of each plantlet with an initial explant and some in vitro regenerated roots were cut off, and the plantlets were planted into propagation trays (28 ml individual cell volume) filled with a propagation substrate composed of peat substrate and coarse perlite (2:1, v:v). The plantlets were covered with a perforated film and regularly sprinkled with water and aired. Air temperature in the growth chamber was established at 21°C and relative humidity at 68%. Substrate temperature was measured and recorded daily. The average substrate temperature was 20°C under fluorescent lamps and 18.6°C under light-emitting diodes. Plants were cultivated in a growth chamber under 16h/8h light/dark photoperiod. Twenty plantlets representing each cultivar were grown under fluorescent lamps and twenty – under LEDs.

The fluorescent light was delivered from six fluorescent lamps emitting white light (TLD 36W/54, Koninklijke Philips Electronics N.V., the Netherlands). Photosynthetically active radiation (PAR) was 55 µmol m^-2·s^-1, measured at the level of plantlets using the photometer (Optel FR-10, Poland). Light intensity under fluorescent tubes was 5200 lx, measured at the level of plantlets applying the luxometer (Lutron LX-107, Poland). The LED light was provided by two LED modules (Philips GreenPower LED module HF, Koninklijke Philips Electronics N.V., the Netherlands). Each LED module consisted of three aluminum bars containing five LEDs, same colour (red – 660 nm, blue – 470 nm, and far-red – 740 nm; ratios 10 : 7.5 : 1.0, respectively). PAR under LEDs was 65 µmol m-2·s-1, measured at the level of plantlets with the use of photometer (model Optel FR-10, Poland). Light intensity under LEDs was 5200 lx, measured at the level of plantlets with the luxometer (Lutron LX-107, Germany). Acclimatization took 2 weeks.

Acclimatization after in vitro rooting
Following 4 weeks of multiplication, the plantlets, after the basal part of each plantlet with an initial explant and some in vitro regenerated roots were cut off, of approximately 3–4 cm in height with 3–4 leaves weresubcultured onto the rooting medium, based on MS with the addition of 2.0 mg dm^-3 indoleacetic acid (IAA). The culture room conditions were the same as during the multiplication stage. After ten days of in vitro roots induction, the plantlets were taken out from the culture medium, planted into propagation trays and transferred to the growth chamber. Roots were only slightly visible as a 1 mm insets, being induced but not elongated. The growing conditions were the same as described above. Twenty plantlets from each cultivar were placed under fluorescent lamps and twenty – under LED. Light conditions were as described above. Acclimatization took two weeks.

Data collection
After two weeks of acclimatization the plantlets were removed from propagation trays and the substrate was gently rinsed off the roots. Acclimatization efficiency was evaluated as the percentage of vital growing plants. The percentage of rooted and acclimatized plants was estimated. The length of the longest root of each plant and height were measured. The fresh weight of plantlets and roots were studied after acclimatization. The readings of chlorophyll content index (CCI) in leaves were performed with a portable chlorophyll meter (model CCM-Plus, Opti-Sciences. Inc. USA) in healthy and fully developed leaves. The CCM – Plus uses the ratio of optical absorbance at 655 nm to that at 940 nm to calculate the chlorophyll content index (CCI).

The results were statistically verified with the analysis of variance of the one-factor experiment in a completely randomized design and the differences between the means were estimated using the Tukey HSD test at α=0.05. A single experimental combination covered five replications, four plants each.

RESULTS AND DISCUSSION

The acclimatization survival rates varied considerably between in vitro rooted and unrooted plantlets of ‘Bingo’ and ‘Cool Time’ (Tab. 1). Almost all in vitro rooted plantlets of the two cultivars survived the acclimatization process, with only one or three plantlets lost while most plantlets acclimatized directly – died. Losses in ‘Euro’ and ‘Vymini’ acclimatized without rooting were not that high yet also higher than in the group of plantlets acclimatized foregone by in vitro rooting. Interestingly, in the experiment reported by Tymoszuk [25], not such high percentage of chrysanthemum plants lost after direct acclimatization was observed. In our opinion, cutting off the basal parts of plantlets, in order to adjust the plantlets height, was an additional stress factor which negatively influenced the plant survival capability; some cultivar dependence was observed in our study. In strawberry, direct acclimatization was efficient and, in comparison to in vitro rooted plantlets, directly acclimatized plantlets showed better quality in terms of roots parameters and the number of runners [1]. It was found that the concentration as well as the type of auxins in the medium was essential for the survival of Vitis vinifera plantlets during acclimatization, both direct and foregone by in vitro rooting; the survival rate ranged from 39.2 to 93.3% [5]. We observed an influence of light source on survival rates neither in directly acclimatized plantlets nor in the previously rooted ones.

The ‘Euro’ plantlets directly acclimatized and rooted under diodes or fluorescent tubes did not differ in quality (Tab. 2). As for ‘Vymini’ there was noted a positive effect of the light emitted by fluorescent lamps on the height and fresh weight of plantlets. However, there was noted no effect of the factor on the length and weight of roots and the content of chlorophyll in leaves. In the second part of the research it was observed that the plantlets of ‘Bingo‘ and ‘Vymini’ acclimatized under diodes were longer than those grown exposed to the light of fluorescent lamps. In ‘Euro’ there was observed an opposite reaction, while in ‘Cool Time’ there were noted no differences between the light sources. Numerous researches have demonstrated that the development of chrysanthemum plantlets and the pattern of acclimatisation depend on the photon flux density as well as the light spectrum applied. Kim et al. [9] did not demonstrate any differences in the length of stems among the chrysanthemums grown under conditions in vitro in the light emitted by blue and red diodes and glow lamps. On the other hand, supplementary lighting provided to plantlets with diodes red in colour connected with far-red resulted in an excessive elongation of the third internode. According to Jeong et al. [8] who performed research into the effect of light on the growth of Dendranthema grandiflora, 'Zembla' cultivar, adding blue light to red light emitted by LEDs can enhance the growth of stems and internodes elongation. Kurilčik et al. [11], on the other hand, who in supplementary lighting of culture in vitro ofDendranthema grandiflora, 'Ellen' cultivar, used the light emitted by LEDs with quantum irradiation of 43 μmol·m^-2·s^-1, observed an inhibiting effect of blue light on stem growth. Treder et al. [24] reported that after seven weeks of acclimatization, plantlets of 'Pink Rosa' strawberry plants treated with blue light in connection with red and near infrared were higher, as compared with the plants placed under sodium lamps.

In the present experiment the fresh weight of ‘Bingo‘ and ‘Vymini’ plantlets acclimatized after in vitro rooting under diodes was higher than the plants under fluorescent lamps (Tab. 3). There were observed no such differences in chrysanthemums ‘Cool Time’ and ‘Euro’. Kim et al. [9] observed that chrysanthemums grown in vitro under LEDs emitting red light in connection with blue colour and those the growth of which occurred under fluorescent lamps did not differ in terms of fresh weight. Throughout the acclimatization of 'Pink Rosa' strawberry plantlets, the fresh weight of the underground plant part was higher than when their growth took place under LEDs, as compared with sodium lamps [24]. Nhut et al. [17], on the other hand, who provided supplemental light to culture in vitro of 'Akihime' strawberry with LEDs and fluorescent lamps, demonstrated that the fresh weight of the underground plant part was highest for the cultivation performed under red diodes, lower when combining red and blue light as well as exposed to fluorescent light, and lowest – in the blue light emitted by diodes.

‘Vymini’ plantlets growing under the light of diodes showed longer roots, as compared with the plants acclimatized under fluorescent lamps. In the other cultivars the application of various sources of light did not affect the length of the longest root. A favourable effect of diodes on the weight of the root system was observed in ‘Bingo’ and ‘Vymini’ plantlets (Fig. 1). Kurlčik et al. [11] reported on 'Ellen' chrysanthemum plantlets grown in cultures in vitro, exposed to mixed light (blue, red and far red), with longer roots, as compared with chrysanthemums provided with supplemental red light. A considerable increase in the share of blue light was inhibiting the formation of roots. In their opinion, the roots are more sensitive to the effect of light, as compared with the underground part of chrysanthemums. The authors also add that a simultaneous effect of blue, red and far-red light results in the interactions between photoreceptors; phytochrome and cryptochrome which can be responsible for inducing the process of rhizogenesis. In another experiment performed in the same chrysanthemum cultivar there was reported a positive effect of far red on the root length, however, the higher the photon flux density in that range of spectrum, the lower the rate of rooting [12]. Treder et al. [24] demonstrated that the acclimatization of unrooted strawberry plantlets under  LEDs emitting red, blue light and near infrared increased the root length. The plantlets in which that stage took place applying red diodes with a decreased share of blue diodes showed a higher number of the roots and thicker root necks.

Light has a significant effect on the formation of chloroplasts and their photosynthetic activity. Its deficit can result in changes in the content of chlorophyll which is responsible for an efficient operation of photosynethetic apparatus [10]. In the present experiment the content of chlorophyll in the leaves of ‘Cool Time’ and ‘Euro’ plantlets grown in the light of diodes was higher than in the plants acclimatized under fluorescent lamps. The opposite reaction was observed in ‘Bingo’, whereas ‘Vymini’ plantlets exposed to light of a varied spectral composition showed a similar chlorophyll content. According to many authors, blue light enhances the synthesis of chlorophyll and so its presence is considered indispensable. Kurilčik et al. [11] demonstrated that a high share of blue light increases the concentration of photosynthetic pigments in chrysanthemum plantlets. The pigments content can be decreased by far red [12]. Treder et al. [23] report on ‘Shiny Rose’ iresine and ‘Beacon’ fuchsia grown exposed to light emitted by diodes and a high share in blue irradiation being better coloured, as compared with the plants provided with supplemental traditional light sources.

LEDs can be successful as a light source at the stage of chrysanthemum acclimatisation. The use of the light emitted by LEDs can be an effective non-chemical method of supporting the development of the chrysanthemum root system and, at the same time, their faster adaptation to conditions in vivo.

CONCLUSION

• The application of diodes in acclimatization of chrysanthemum positively affects the height of plantlets as well as fresh weight of shoots and roots in ‘Bingo’ and ‘Vymini’.
• Light emitted by diodes enhances chlorophyll content in leaves of plantlets of cultivars ‘Cool Time’ and ‘Euro’.
• Diodes can effectively replace traditional sources of artificial lighting during the acclimatization of in vitro rooted chrysanthemums.

REFERENCES

Accepted for print: 7.10.2016

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