*2.1. Red Light Effects*

2.1.1. Red Light Effects on Shoot Proliferation

Some authors agree on the positive role of RL [123], and high-ratio RL:FRL [35] on shoot proliferation [135]. RL significantly enhanced the adventitious bud formation and development in *Gerbera jamesonii* [136], in *Lactuca sativa*. [137], in *Spathiphyllum cannifolium* [83], in *Stevia rebaudiana* [114] and in *Mirtus communis* [120]. RL was effective for bud formation and outgrowth in *Pseudotsuga menziesii* embryo cultures [122]. In contrast, as compared to the cultivation under WL or combined RL with BL, under monochromatic RL or BL, Bello-Bello et al. [106] observed a decrease in the proliferation ratio in *Vanilla planifolia* Andrews and Estrada et coll. [111] and Lotfi et al. [59] found the same decrease in *Anthurium andreanum* and in *Pyrus communis* L., respectively. Somatic embryo germination and conversion of three southern pine species [53] and *Cydonia oblonga* [126] were positively affected by application of RL.

Positive effects of RL illumination have been ascertained in many orchids. In *Cymbidium Waltz* 'cv Idol', the highest protocorm-like bodies (PLBs) formation rate (100%) was found in the culture media containing 0.01 and 0.1 mg L−<sup>1</sup> N- acetylglucosamine (NAG) under RL, although a promotive role was observed under GL, but at 1 mg L−<sup>1</sup> NAG [100]. In a study of Mengxi et al. [90], the highest PLBs induction rate, propagation coefficient and fresh weight of *Oncidium Gower Ramsey* were observed under RL treatment, which agrees with observations on the Cattleya hybrid [138]. However, in this last species, monochromatic RL resulted in an impaired leaf growth and chlorophyll content. Moreover, in *Oncidium Gower Ramsey*, even if R-LEDs promoted PLB induction, it was observed that

BL emitted by LEDs promoted a differentiation of PLBs [90]. Hamada et al. [88] found that R fluorescent lamps increased the PLB proliferation of *Cymbidium finlaysonianum*, even if used only during the early stage of the culture. The R spectrum was effective for *Cymbidium* callus proliferation [80] but not for the successive propagation. The combination of RL and FRL wavelengths determined the highest number of somatic embryos in *Doritaenopsis 'Happy Valentine'* [54].

The action mechanisms promoted by RL has been investigated by different authors. In *Vitis vinifera*, the axillary shoot development could be due to the release of apical dominance caused by BL, as suggested by Chée [68] and Chée and Pool [70]. Similarly, Burritt and Leung [79] observed that the inhibitory influence of FRL on shoot proliferation is reversible, whereas exposure to BL permanently reduces explant's competence for new shoot formation. They suggested that PHY and an independent BL photoreceptor, probably CRY, regulate shoot production from *Begonia* × *erythrophylla* petiole explants. RL has been shown to exert effects on plants proliferation through the PHY, which, in the active form, would alter the endogenous hormonal balance increasing in the quantity of cytokinin (CK) in tissue, counteracting the action of auxin and thus determining an increase in the development of lateral shoots [139,140].

Moreover, research on the effects of PHY on in vitro multiplication of shoots of the *Prunus domestica* rootstock GF655-2 [141] demonstrated that the actions of WL, BL and FRL on shoot proliferation were fluence-rate dependent, while RL was effective both at 37 µmol m−<sup>2</sup> s <sup>−</sup><sup>1</sup> and at 9 µmol m−<sup>2</sup> s −1 . The increase in light intensity had, instead, a positive effect on the production of axillary shoots in a *Prunus domestica* Mr.S.2/5 shoot exposed to RL and BL. However, if the number of shoots produced was expressed as a percentage of the total number of axillary buds, the rate of bud outgrowth for each shoot under RL was significantly higher than that detected under BL [142].

The effects of RL on proliferation are also largely dependent on the growth regulators, mainly cytokinins (CKs) applied to the culture medium, and they were found to be indispensable in the outgrowth of lateral buds in *Prunus domestica* rootstock shoots [142]. The same was true for *Spiraea nipponica* where the interaction between CKs and RL resulted in an enhancement of the shoot proliferation rate [123]. Plantlets of this species exposed to RL and FRL resulted in more marked growth than under WL [123]. Interesting interactions resulted from the growth of this species under low RL:FRL photon fluence followed by high-fluence WL and the benzyl aminopurine (BA) levels [123]. More detailed information on the interactions between light and growth regulators will be provided in paragraph 5.
