2.3.2. Blue and Red Light Effects on Plantlet Morphology

Many studies confirmed the effectiveness of R- and B-LEDs in enhancing growth and photosynthesis in many plant species. B- and R-LEDs were developed to grow in vitro plants because chlorophyll a and b show a maximum absorption at their respective wavelengths (460 and 660 nm). The same light ratios were effective on proliferation and in promoting the quality of plantlet characteristics.

*Plantlet elongation:* Various combinations of R- and B-LEDs proved to determine the best results for stem length and leaf growth for *Saccharum officinarum* [112], *Stevia rebaudiana* [114], *Populus x euramericana* cv 'Dorskamp' [131], *Pyrus communis* [59], *Fragaria x ananassa* [104] and *Dendrobium officinale* [92]. Sivakumar et al. [179] showed that continuous RL plus BL or intermittent BL significantly stimulated shoot elongation of sweet Solanum tuberosum plantlets in vitro. Hahn et al. [146], on *Rehmannia glutinosa*, found that shoot lengths under either B- or R-LEDs were greater than under mixed LED or Fls, but the plantlets overgrew and appeared fragile, whereas plantlets under mixed LED or Fls were healthy, with normal shoot lengths. Thus, normal plant growth was clearly related to the presence of monochromatic BL or RL. According to some authors, the synergistic interactions between CRY and PHY could either promote or inhibit the shoot elongation in different plant species.

*Plantlet growth:* The composite spectra of R- and B-LEDs positively regulated fresh and, in most cases, also dry matter accumulation. As compared to the cultures raised under Fls or monochromatic lights, in most cases LEDs supplying higher RL ratios (from 70–90%) as compared to the BL ones were effective in enhancing the in vitro growth of different species

such as banana [180], grape [145], *Fragaria x ananassa* a [149], *Vaccinium corymbosum* [147], *Tripterospermum japonicum* [62], *Eucalyptus citriodora* [181], *Phoenix dactylifera* [133] and *Lippia alba* [66]. Highest growth was observed under Fl and under a mixture of BL and RL in *Withania somnifera* plantlets [182]. Highest fresh and dry weights were obtained when plantlets were cultured under an equal BL and RL combination (50:50) in different species such as *Chrysanthemum* [42], *Lilium* [78], *Doritaenopsis* [63], *Pyrus communis* [59], *Saccharum officinarum* ([112], upland *Gossypium hirsutum* L. [50], *Vanilla planifolia* [106] and *Solanum tuberosum* [183]. As for proliferation, higher BL rates as compared to the other species are necessary to obtain the best growth in *Brassica napus* [51]. Similarly, to proliferation, higher RL ratios enhanced plant growth and the development of different orchids: *Cymbidium* [148] and *Phalaenopsis* [86]. RL plus BL and FRL or RL plus FRL light significantly enhanced the fresh and dry weights of *Oncidium* plantlets [89].

Differently from other cultures in which the same lights resulted in optimal proliferation and plantlet growth, according to Mengxi et al. [90], in *Oncidium*, the highest induction rate, propagation and fresh weight appeared in the RL treatment, whereas the largest dry weight per plantlet were obtained under B:R = 20%:80% and B:R = 30%:70%, respectively. Differently from other orchids, the in vitro growth of plantlets of the *Calanthe* hybrid was efficiently enhanced under a mixture of BL plus RL (0.7:1) and inhibited by RL plus FRL [184].

*Leaf number and area:* In *Gerbera jamesonii* [118], monochromatic RL and BL treatments resulted in a reduced leaf area, whereas leaf number was enhanced by exposure to RL:BL = 1:1.

R and B mixed LED treatments in various combinations improved leaf number and sometimes length of in vitro cultures of *Fragaria x ananassa* [149] and *Doritaenopsis* [63], leaf area of *Populus x euramericana* [131] and leaf growth of *Stevia rebaudiana* [114].

*Photosynthetic pigment levels*: Many studies showed that optimizing the RL:BL ratio may improve photosynthesis. The positive effect of the appropriate B-:R-LEDs combination on the synthesis of photosynthetic pigments was reported in several studies [51,92]. An appropriate mixture of B- and R-LEDs, compared with solely monochromatic BL or RL, is more effective to increase the chlorophyll a/b ratio and/or carotenoids content of the in vitro grown plants of *Tripterospermum. japonicum* [62], *Lippia alba* [66] and *Staphylea pinnata* [113]. On *Fragaria x ananassa* mixotrophic cultures, the chlorophyll content was the greatest under RL:BL = 70:30 and the least under 100% RL [149].

Plant growth and development caused by increasing the net photosynthetic rate was also observed in *Chrysanthemum* (*Dendranthema grandiflorum*) under mixed R-:B-LED treatments and has been attributed to the adjustment of the spectral energy distribution of RL:BL to chlorophyll absorption [42]. RL or BL plus RL treatments were found more effective in grape for net photosynthetic rates [145] as compared to BL alone. Differences in chlorophyll content in *Artemisia* and *Nicotiana tabacum* plants were ascertained. In plants grown under WL, significantly less chlorophyll content than plants growing in RL:BL (3:1) or RL:BL (1:1) was determined [34]. In *Gossypium hirsutum* L., chlorophyll content, leaf thickness and leaf and stomata area were higher in plantlets cultured under BL; however, the best growth was provided by BL:RL = 1:1 [50]. In addition, in the Colt rootstock of *Prunus avium* exposed to BL and BL plus RL dichromatic light, the leaves had a greater accumulation of chlorophyll [170].

A ratio of BL:RL = 1:1 emitted by LED light facilitated the growth and produced the highest chlorophyll, carotenoid contents and photosynthetic rates in *Oryza sativa* seedlings, but not callus proliferation, differentiation and regeneration, which were enhanced by BL [121].

Different from the other species, higher BL rates as compared to RL (3:1) are necessary in *Brassica napus* L. (cv Westar) to increase chlorophyll concentrations compared to the other LED treatments and Fl. Therefore, the response of chlorophyll content of in vitro plantlets to different light qualities may vary among plant species or cultivars [51].

In different orchid species, BL plus RL was reported as the most efficient treatment on the synthesis of photosynthetic pigments. Shin et al. [63], in *Doritaenopsis*, showed that mixtures of RL plus BL stimulated photosynthesis and chlorophyll accumulation. In *Dendrobium officinale*, chlorophyll a and b and carotenoid contents were the highest in protocorm-like bodies incubated under RL:BL LEDs = 66.6:33.3 [92]. Moreover, in *Oncidium* plantlets, it was demonstrated that the RL and BL combined with FRL or RL plus FRL radiation significantly enhanced chlorophyll content [89].
