2.2.1. Blue Light Effects on Shoot Proliferation

The effects of BL are often reported to be antagonistic of RL ones, although the studies reported in literature concerning the role played by BL on new meristem formation are not always consistent. The positive effects of BL on the stimulation of shoot production and growth of *Nicotiana tabacum* during in vitro culture were reported, but at a higher light intensity [67], and the authors hypothesized photoinactivation of IAA. Five weeks of exposure to BL induced the highest shoot production from *Nicotiana tabacum* callus [159].

Monochromatic BL increased shoot number in *Ficus benjamina* [94], the number of shoots and nodes in *Vitis vinifera* L. *hybrid* [68,70], the number of adventitious buds in *Hyacinthus orientalis* L. [160] and the percentage of organogenesis and the mean number of buds per explant in *Curculigo orchioides* [103]. Higher percentages of BL in the light spectrum were also effective on in vitro shoot induction and proliferation of *Anthurium andreanum* [49], *Gerbera jamesonii* 'Rosalin' [107], *Remnania glutinosa* [65] and *Saintpaulia ionantha* [69]. In various species, positive results on proliferation from adding different ratios of B to the R spectrum have been described and will be widely discussed in sub-paragraph 2.3.1. The proliferation rate was greater in *Brassica napus* plantlets when cultured under monocromatic BL and BL plus RL [51]. In lavandin, on a BA-free medium, shoot number was enhanced under BL, WL and RL at low photon fluence rates [72]. In *Oryza sativa* [121] under B-LED illumination, the time required for callus proliferation, differentiation and regeneration was the shortest and the frequency of plantlet initiation, differentiation and regeneration was the highest. Concerning orchids, in *Dendrobium officinale*, the monochromatic BL and RL:BL (1:2) emitted by LEDs determined a higher percentage of protocorm-like bodies (PLBs) producing a higher number (1.5 fold) of shoots [92], in *Cattleya intermedia* × *C. aurantiaca* the number of shoots regenerated from PLBs was enhanced by BL [161]. In *Oncidium*, RL promoted PLB induction from shoot apex and the higher content of carbohydrate but the lowest differentiation rate, while the highest differentiation rate and protein content were observed under B-LED [87]. BL increased node and total shoot number as compared to RL, FRL and dark in *Prunus avium* cv 'Hedelfinger' and one of its somaclones [127]. In contrast, on *Begonia erythrophylla* petiole explants, RL played a role in meristem initiation and BL and FRL were antagonistic to meristem formation, but BL was important for primordia development [79]. In *Gerbera jamesonii* [118], inhibition of shoot multiplication and a reduced plant height was observed under BL compared to what resulted from all other light treatments, and a decrease of lateral shoots number was observed on *Malus domestica* [135] as compared to RL. The same study demonstrated that BL inhibited the rate of proliferation, increasing the apical dominance. Inhibition of meristematic tissue proliferation by BL has also been observed for the embryogenic tissue of Norway spruce [162]. The conflicting reports found in the literature might not only be attributed to species effects, but also to the different types of explants and to the stage of the organogenic process. Hunter and Burritt [81], working on different *Lactuca sativa* L. genotypes, observed a significant decrease under monochromatic BL in shoot proliferation as compared to RL or WL. They argued that RL is required for the formation of shoot primordia, whereas BL is inhibitory to primordia initiation. The effects of RL and BL on this species depended on the stage of the organogenic process in which *Lactuca sativa* plantlets were exposed to the different lights. Exposure to BL during the critical first few days of culture, when meristems are being initiated, results in a significant reduction in the number of shoots produced as compared to exposure to RL and WL. Furthermore, this suppression of meristem initiation is permanent and not reversible afterward by culturing plants under RL. Observations with a scanning electron microscope (SEM) clarified that the lowest shoot development under BL was attributable to the production of much more callus as compared to those cultured under WL or RL, demonstrating that rapid cell division occurred, although the organized center of cell division required for primordia formation was reduced. Moreover, the same authors observed that explants exposed to continuous RL developed numerous small shoot primordia, which occurred more slowly than those detected on tissue exposed to WL. Based on the literature, they stated that the stimulatory effects of RL as compared to WL is genotype dependent, but the inhibitory effect of BL is more widely diffused. Callus formation as affected by continuous BL illumination was observed also in *Pyrus communis*, where callus weight doubled as compared to BL plus RL and BL plus FRL [59]. In *Ficus benjamina*, BL induced a huge formation of callus at the basal section of shoots [94]. Other studies have shown that the timing of exposure to different light regimes is also critical for shoot development *in vitro*. For example, at least 2 wks under RL were required to improve shoot numbers from *Pseudotsuga menziesii* callus, and the length of time in which

RL promoted shoot production lasted only 2–3 wks [122]. It was suggested that PHY plays an inductive role in organogenesis of *Lactuca sativa* L., as suggested by Kadkade and Seibert [137], in contrast to antagonistic role of BL, probably via CRYs.

In a series of research projects carried out with different rootstocks of *Malus domestica*, *Prunus domestica* and *Prunus persica*, M9, MM106, Mr.S.2/5, and GF677, respectively [125,128,142,163], it was demonstrated that BL induced, in the starting explant and in the developed shoots, a greater number of nodes with shorter internodes than those observed in RL and in dark. It should be noted that the percentage of nodes that formed lateral shoots was higher in the presence of RL as compared to the BL one. In the *Malus domestica* M9 rootstock, the percentage of sprouted buds under RL was double that under BL [135].

Based on these results, shoot multiplication can be defined as the result of two events: the induction and formation of new buds from the apical meristem and their sprouting through the reduction or the suppression of apical dominance [2,36]. BL would increase the number of axillary buds but, in contrast, it exerts an inhibitory action on buds sprouting (increase in apical dominance). RL, on the other hand, would reduce the apical dominance even though it reduces the formation of new axillary buds. The lower outgrowth of buds in the presence of BL compared to RL would indicate a role in a specific photoreceptor(s) of BL, which would act as an antagonist of the PHY. Photomorphogenetic events detected in the presence of RL and BL would agree with an antagonistic model of stem branching, modulated by light through the PHYs and the photoreceptors of BL, which would interact with each other according to a dynamic model. Moreover, Muleo et al. [142] also showed that the internode extension inhibition under BL exposure and the concomitant positive effect of BL in enhancing axillary bud formation (neoformed nodes) was dependent on the photon fluence rate, but not on PHY photoequilibrium or on concomitant exposure to RL. A quantitative BL threshold was found near 30 µmol m−<sup>2</sup> s −1 (400–500 nm); up to this value, internode extension decreased [142].

Plants, thus, possess a complex and dynamic light response and memory system that involves reactive oxygen species and hormonal signaling, which are used to optimize light acclimation and immune defenses [164]. Thus, regulating the spectral quality, particularly by the B-LED, improves the antioxidant defense line and is directly correlated with the enhancement of phytochemicals in *Rehmannia glutinosa* [65]. Mengxi et al. [90] found higher values of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) activities in leaves under B-spectrum irradiance and concluded that B-LED may be more satisfactory for activating different defensive systems to reduce excessive amounts of reactive oxygen species. However, in two important *Dianthus caryophyllus* cultivars, 'Green Beauty' and 'Purple Beauty', RL treatment also increased the activities of antioxidant enzymes and nutrient contents [165]. The B-LED illumination also significantly increased the antioxidant enzyme activities in leaves and roots in *Amaranthus tricolor* and *Brassica rapa* L. subsp. *oleifera* [166]. In the in vitro cultured *Pyrus communis* plantlets, it was detected that the gene encoding the pathogenesis-related protein PR10 is regulated daily by the body clock of a plant, while *PR1* was expressed without clear evidence of circadian regulation [167]. In the same studies, a specific function was played by PHYB and CRY1 photoreceptors, considering that in transgenic plants the first photoreceptor enhanced the gene expression of *PR1* 5- to 15-fold, and CRY1 enhanced plant resistance to the *Erwinia amylovora* bacterial infection [167]. *Prunus avium* rootstock plantlets, overexpressing the PHYA gene and grown in vitro, displayed a strong resistance to bacterial canker (*Pseudomonas syringae* pv. morsprunorum), highlighting a role of light quality and quantity in the regulation of plant resistance to bacterial disease [168]. Therefore, light quality through the regulative network of photoreceptors plays a relevant role in the endogenous rhythms of gene expression and pathogen attacks.
