2.2.2. Blue Light Effects on Plantlet Morphology

BL is mostly considered to be able to increase leaf growth, photosynthetic pigment synthesis, chloroplast development and stomatal opening, soluble proteins and carbohydrates and dry matter content and to inhibit stem and root elongation, while RL enhances stem growth and carbohydrate accumulation [41,50,87,158]. In *Scrophularia kakudensis*, BL imposed a stressful environment that resulted in the activation of several proteins related to stress tolerance, photosynthesis, gene regulation, post-translational modification and secondary metabolism [169]. The improvement in the leaf characteristics induced by the addition of BL to RL seem to indicate a better quality of micropropagated plantlets, which in turn may also improve acclimation [2,170].

*Plant height:* A few papers report positive effects of BL on shoot length, while most studies agree on its inhibition of plantlet elongation. The blue spectrum was recognized to inhibit stem growth in *Oncidium* [90], in *Pelargonium* × *hortorum* [144], in *Dendranthema grandiflorum* [42] and in *Zantedeschia jucunda* [171], especially as compared to RL or RL:FRL. In different tree species, *Prunus domestica* Mr.S.2/5 and *Malus domestica* MM106 and M9, inhibition of internode elongation was also detected [128,135,142]. In contrast, BL (470 nm) and RL (660 nm) illumination were found effective for increasing shoot length in *Achillea millefolium* [172] and *Dendrobium Sonia*, where, however, BL significantly reduced multiplication as compared to YL [116].

In some cases, BL is necessary to contrast the excessive effects of RL on shoot length assuring good plantlet development. Nhut et al. [149] observed that *Fragaria x ananassa* plantlet growth was inhibited under BL, whereas an irregular plantlet growth and development was observed in the absence of BL. In the experiment of Jao et al. [171], a shorter stem of plant and a higher chlorophyll content was found in the RL plus BL treatment, highlighting that BL may be involved in the regulation of both plant height and chlorophyll development.

BL induces the production of short shoots with good leaf development and many micro-tubers in *Solanum tuberosum*. Under BL, kinetin not only strongly stimulated tuber formation, but also increased the total fresh weight and root(+stolons)/shoot ratio [71].

*Fresh and dry weight:* In *Dendrobium officinale*, compared to other light treatments (dark, Fl and R-LEDs), B-LEDs, alone or with R-LEDs (1:2), induced higher dry matter accumulations of PLBs and shoots [92]. Increased biomass production in cultures of *A. millefolium* [172] was noted under monochromatic B-LED or R + B-LEDs. Monochromatic BL determined higher fresh and dry weight and leaf number per plantlets in *Euphorbia milii*, *Spathiphyllum cannifolium* [83] and *Rehmannia glutinosa* [146].

It is noteworthy that monochromatic BL had a negative effect on the dry matter production of *Lippia gracilis* [119], *Plectranthus amboinicus* [48], *Gossypium hirsutum* [50] and *Vanilla planifolia* [106], as well as in the sensitive cv Dopey of *Rhododendron* where it also reduced leaf chlorophyll content [75]. In most cases, however, RL was the most effective in all these species.

Many authors, however, agree on the most positive effects obtained on fresh and/or dry weight of plantlets by adding different ratios of BL to RL as compared to only monochromatic BL (see the next chapter) [62,65,90,173,174]. Moreover, Kurilˇcik et al. [174] demonstrated that the influence on shoot length and weight of the BL component of a mixed light is tied to the photon flux density (PFD) of the FRL component. Once more, these results indicate the species-specific effects of BL on in vitro plantlet growth [51]. Cio´c et al. [120] evidenced the relationship of BL and growth regulators. B-LED illumination and a high BA content in the substrates stimulated the growth of a greater number of *Mirtus communis* L. leaves (BL and RL plus BL) and increased the fresh weight as compared to Fls, but did not affect the dry weight, whereas RL with low amount of BA enhanced both proliferation and shoot growth. Moreover, in *Oncidium*, the amounts of soluble protein in the PLBs and leaves were the highest in the BL treatment, which suggests that the B spectrum was advantageous for protein synthesis [87,90].

*Leaf morphology and functionality:* BL is considered an important regulator of leaf expansion; however, differences have been ascertained among the different species. BL induced the largest number of leaves per plant, and the largest leaf thickness and area in *Altenanthera brasiliana* [175] and *Platycodon grandiflorum* [158] and a similar response on leaf area was demonstrated in *Gossypium hirsutum* [50] and *Brassica napus* [51]. BL enhanced leaf chloroplast area and the translocation of carbohydrates from chloroplasts in *Betula pendula* [154]. In contrast, less leaf area was observed in *Pyrus communis* under monochromatic BL, as compared to RL, RL plus FRL and RL plus BL [59] and in *Azorina vidalii* [74], as compared to RL plus FRL. Furthermore, CRYs are known to regulate chloroplast development in response to BL [176].

*Photosynthetic pigments accumulation:* Several studies have reported that B irradiation resulted in higher chlorophyll contents and carotenoids in the in vitro plantlets as compared to RL and FL. Cultures of *Euphorbia milii* [61], *Doritaenopsis* [63], *Oncidium* [16,87], *Stevia rebaudiana* [114], *Dendrobium officinale* [92], *Prunus avium* cv 'Hedelfinger' and in its somatoclone [127], *Zantedeschia jucunda* [171], *Tripterospermum japonicum* [62], *Chrysanthemum* [174], *Anthurium andreanum* [111], *Phalaenopsisis* [177], *Brassica napus* [51] and *Vaccinium ashei reade* [147] exhibited higher total chlorophyll content under monochromatic B-LEDs or combinations of R- plus B-LEDs as compared to cultures exposed to R-LED or Fls treatments. The chlorophyll content, leaf and stomata number per explant were also highest on plants cultured under BL in *Vitis vinifera* [85] and in *Gossypium hirsutum* [50].

BL and UV irradiation enhanced chlorophyll content in *Hyacinthus orientalis* L. [160] and chlorophyll a+b content, but not the carotenoid content, in leaves of *Pyrus communis* [59]. Photosynthetic capacity was highest in *Betula pendula* Roth [154] and in chrysanthemum (*Dendranthema grandiflorum*) [42] when the plantlets were exposed to BL as compared to RL. In *Dendrobium kingianum*, the average number of PLBs and the chlorophyll content were highest under B-LEDs, in contrast to the explants cultured under R-LEDs where the highest shoot formation and fresh weight were observed [99]. Likewise, a study of *Oncidium* PLBs by Mengxi et al. [90] showed that chlorophyll a and b and carotenoid levels and the greatest growth were detected under B-LEDs. On the contrary, a reduction in chlorophyll levels in plants grown under BL was observed in *Vanilla planifolia* [106]. Thus, according to Li et al. [51], the chlorophyll content of in vitro plantlets grown under different light qualities varies within plant species or cultivars. Moreover, even if BL, as compared to RL or different RL:BL ratios, reduced leaf expansion and hence leaf area in *Azorina vitalii*, the chlorophyll and carotenoid content per unit leaf area was higher than RL:FRL [74].

Changes in chlorophyll biosynthesis induced by changes in spectral quality may provide advantages regarding plant growth [178]. The species-specific responses to the B spectrum, in terms of photosynthetic pigments, are probably tied to the different environments in which the different species developed and to the type of explant used for in vitro initiation. In *Lippia gracilis*, plantlets that originated from apical explants had higher pigment production under the BL spectrum, whereas those from nodal explants showed higher production under WL, followed by the BL conditions [119]. These studies indicate that BL provides important environmental information and mostly promotes higher photosynthetic efficiency.
