**6. Discussion and Conclusions**

Several papers on different species concern the effects of light on in vitro proliferation to assess the light properties capable of enhancing the efficiency of the micropropagation process, also ensuring consistent energy savings, as compared to traditionally used Fls lamps, or the broad range of WL. However, the results are often conflicting. Many authors ascribe these results to the different responses to light of plant species, cultivars or even explant types [119], plant stage development [122], medium composition [143] and microenvironmental characteristics such as PPFD [174] and vessel ventilation [146]. However, a large cause of variability may be tied also to the difficulty in applying uniform intensities along the shelves, and/or the use of the right spectral composition for each light quality.

Moreover, the lack of sufficient in vitro experimental protocols like those available for in vivo study, which would make the effects of light clearer, limits the comparability of the experiments [34]. The issues of major concern, among others, in this regard are (i) the short timescale in which these experiments are carried out (mostly a propagation cycle), (ii) the quality and quantity of exogenous applied growth regulators, (iii) the narrow range of light intensity values within which the efficiency of axillary multiplication of explants occurs and (iv) the mixotrophic state of plantlets. Concerning the first issue, the short-time experiments strongly limit the comprehension of the effects of light spectra on the stability of proliferation and plantlet growth during subsequent multiplication cycles (see particularly the RL effects). Concerning the second one, due to the interaction of light with endogenous growth regulators (particularly CKs), attention must be paid to the doses of the exogenous growth regulators applied. It seems from the examined literature, in fact, that RL effects are visible under low CK concentrations in the medium, whereas WL effects are even visible under high CKs doses [83]. Too high CKs quantities mask the effects of RL or may determine growth alteration. Concerning light intensities, excessive LIs or HIs may

determine low growth rates, photoinhibition and may mask light spectra effects. Moreover, information on how the mixotrophic metabolism of a plantlet grown in vitro affects the development and morphology of the microcutting is scarce.

In this review, several research are presented regarding the different response of species and cultivars to different light spectra, intensities and photoperiod and it seems that some general indications arise from the different studies. Concerning the optimal irradiance intensity, it has been hypothesized that the prevailing light conditions under the natural habitats of some species can be used to indicate their requirements for optimal in vitro growth [75]. Evidence have been presented that plants adapted to an environment characterized by high light intensities present better photosynthetic rates and high growth rates under in vitro intense light, whereas shade-tolerant plants are damaged by high intensities. A survey of the tested literature revealed that in most species, whatever the light spectrum, the most used light intensities range from 20 to 80 µMoles m−<sup>2</sup> s <sup>−</sup><sup>1</sup> and the most used intensity for proliferation is 50 µMoles m−<sup>2</sup> s −1 . Better growth, however, have been registered especially in plants adapted to high intensities (see *Saccharum officinarum*, *Actinidia deliciosa*, *Lippia gracilis*, etc.) at intensities up to or exceeding 80 µMoles m−<sup>2</sup> s −1 . Significant improvements on in vitro fresh and dry weights of shoot clusters, and the number of neo-formed shoots from initial shoot explants were obtained, also modifying the photoperiodic regime from a 16 h photoperiod to a 4 h photoperiod cycle, thus permitting the plantlets to replace the CO<sup>2</sup> [243,244]. In fact, in plantlets exposed to the 16:8 h photoperiod, the photosynthetic activity is intense at the onset of the light cycle and decrease rapidly thereafter because of the rapid and progressive lower concentration of CO<sup>2</sup> in the culture vessels. Moreover, the promotive role of the 4 h photoperiod cycle on the shoot proliferation rate was hypothesized to be dependent on the diverse regime of photo-equilibrium of photoreceptors that promoted the reduction in apical dominance and development of axillary buds [243]. In this view, also adding CO<sup>2</sup> [220] or aerating the vessels [146] proved to be effective in enhancing in vitro growth.

Concerning light spectra, RL alone or high RL:FRL ratios seem to enhance shoot proliferation, as well as PLB and callus formation, in many species. The main effects of RL are tied to the promotive role of phytochrome in the synthesis of CK in tissue, which counteracts the actions of auxins, increasing the development of lateral shoots. RL also regulates the synthesis of carotenoids and, in particular, strigolactones that seem to regulate apical dominance by modification of auxin fluxes [271]. The stimulatory effects of RL seem to be exerted during the beginning of the multiplication phases. However, different reports indicated that RL alone is not able to activate the pathway of chlorophyll synthesis and may determine excessive stem elongation and leaf disorders, the so-called Red Light Syndrome [36]. In fact, when plants are grown under 100% monochromatic RL a strong decrease in photosynthetic capacity, rates of electron transport, dark-adapted Fv/Fm and leaf thickness, as well as unresponsive stomata and reduced leaf pigmentation occurs [272]. BL is effective in increasing callus formation and the number of axillary buds but exerts an inhibitory action on buds sprouting (increase in apical dominance). It has been demonstrated that this light mostly controls some morphological characteristics such as shoot length and enhances chlorophyll synthesis and chloroplast development. RL, on the other hand, would remove the apical dominance but seem to reduce the formation of new axillary buds. Hence, a minimum threshold of BL is necessary for normal plant growth [146]. Moreover, regulating the spectral quality particularly by the BL improves the antioxidant defense line and is directly correlated with the enhancement of phytochemicals [65,90,166] or with the regulation of gene expression [167]. All these reasons would explain why the RL:BL illumination resulted effectively in a wide range of species. Moreover, more recently, an abundance of evidence has indicated the role of GL in carrying information about the environment to the plants, because it is involved in the shade avoidance response, but also in regulating different biological, morphological and biological processes in vitro and in vivo [189]. The addition of GL to the combined RL and BL contributed to the proliferation, the growth and development of some in vitro cultures. In a few cases, even the addition of

YL seems to improve plant proliferation and growth. In addition, the absence of ultraviolet light may determine foliar intumescence and could become a serious limitation for some crops lighted solely by narrow-band LEDs [273]. Thus, the use of monochromatic or combined R- or B-LEDS may determine a mismatch with the photosynthetic spectrum. The application of the broad band WL may overcome this problem [44]. In some species, better results have been obtained under W-LEDs [109,112,130]. Even if WL is not as effective as RL in overcoming apical dominance, high proliferation rates are obtained when CKs are added to the medium. In most cases, the best propagation was obtained at higher CK ratio [141]. It seems that the CK ratio may be enhanced in woody species under WL to obtain high and stable proliferation. However, in some species, after long-time cultivation under WL the rate of newly formed sprouts was reduced regardless of the CK concentration but increased when RL was applied to the crops [2]. Thus, in some cases, an early phase of RL irradiation of at least 2 weeks [122], followed by growth under a WL, may be advisable. The use of an initial stimulatory effect of RL or RL enriched followed by the WL may also improve proliferation and somatogenesis [126] in species that are particularly difficult to regenerate in vitro and/or with an high sensibility to higher concentration of CKs in the medium, such as *Euphorbia milii* and *Ceratonia siliqua* L. (Cavallaro et al., unpublished data). Moreover, the exposition to a period of RL:FRL followed by the WL may enable a reduction in exogenous growth regulator concentrations, mainly CKs added to the medium [124], which may be unnaturally high in vitro. This reduction may be favorable for enhancing the following phases of the in vitro process (rooting and acclimation). Finally, currently, lamps with a more optimal spectral composition of WL enriched in the most useful wavelengths (BL, RL and GL) are already available on the market [185,274] for vertical farming systems and could be interesting for in vitro production after appropriate investigation.

**Author Contributions:** Conceptualization, V.C. and R.M. contributed to the conception and the design of the review; V.C. and A.P., contributed to the drafting of tables; writing—original draft Preparation, V.C. and R.M. contributed to define the original draft; writing—review & editing, V.C., I.F., A.P. and R.M. wrote and edited the final version of the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Conflicts of Interest:** The authors declare no conflict of interest.
