**1. Introduction**

Plants, like any other living organisms on planet Earth, are strongly influenced by environmental cues. Unlike animals, plants are sessile and at the mercy of their surrounding environment. Consequently, they have evolved mechanisms that perceive and respond to environmental changes and adapt their development and growth accordingly. Light plays a pivotal role in a plant's life, not only for photosynthetic energy production but also for its regulative role of molecular, biochemical and morphological processes that underlie plant growth and development [1–3]. Fluence rate, regions of wavelength electromagnetic spectrum, duration and direction are the key attributes of light that drive photosynthesis and photomorphogenesis through mechanisms including the selective activation of various light receptors [4–9]. Plant light photoreceptors have evolved in articulated biochemistry structure that capture photons and detect many of the light physical properties. Subsequently, through interactive pathways the photoreceptors interpret information from incoming light and traduce them in biochemical and biological responses able to regulate plant growth and development. A discrete number of photosensor families have evolved in plants. The phytochrome (PHY) family receptors monitor the red (R, 600–700 nm) and far red (FR, 700–750 nm) light regions [10–12]. PHY can be present in two states and the active state (Pfr) is formed due to absorption of red light by the inactive state (Pr) [13]. The wavelength region of light from UV-A to blue (B, 320–500 nm) is perceived by three small families of photoreceptors [14] that mediate plant responses. All three photoreceptor families contain flavin adenine dinucleotide (FAD) as a chromophore: three cryptochromes (CRY) with CRY1 and CRY2 acting in the nucleus, whereas CRY3 is probably acting in

**Citation:** Cavallaro, V.; Pellegrino, A.; Muleo, R.; Forgione, I. Light and Plant Growth Regulators on In Vitro Proliferation. *Plants* **2022**, *11*, 844. https://doi.org/10.3390/ plants11070844

Academic Editors: Paul Devlin and Eva Darko

Received: 14 February 2022 Accepted: 17 March 2022 Published: 22 March 2022

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the mitochondrion and chloroplast [15,16], two phototropins (PHOT) [9,11,17] and the members of the *Zeitlupe* family (ztl, fkf1 and lkp2) [18]. In addition, PHY has also been found to mediate various blue responses [19]. The UV Resistance Locus 8, monitoring ultraviolet B wavelengths (UV-B, 280–315 nm), regulates both developmental and UV-protective outcomes [20–22].

PHYs act in detecting mutual plant shading through the change in the R:FR ratio and appropriately redirect growth and development through the modulation of apical dominance and of axillary meristems formation according to survival [23–25]. CRY1 is thought to be the CRY responsible for the B high-irradiance response, inhibiting stem plant growth and reducing internode elongation, whereas CRY2 is likely responsible for the inhibition because of the B low-irradiance response [19]; collectively, in plants they perform important traits such as flowering and plant stem elongation [26]. PHOT1 and PHOT2 are involved in auxin polar transport, modulation of auxin sensing and phototropism [27–29].

*Micropropagation* is considered an effective large-scale in vitro plant multiplication of selected insect/disease/virus-free plants in a short time, all year round, and is a reliable method for in vitro preservation of threatened plant species. The micropropagation technology differs strongly from all other agamic propagation methods since the plants, cultured frequently as microcuttings, can remain under constant environmental conditions for a long time. The habitat of an in vitro culture is strongly restricted, and plants switch from an ontogenetic processing that starts from similar juvenility traits to a much deeper juvenility state [30]. Photoperiod, light intensity, light quality, temperature and relative humidity are factors that in the in vitro habitat are subjected to scarce fluctuations that alter the periodic and oscillator systems upon which plants depend; therefore, plants remain under largely invariable conditions. Although, currently, we cannot establish whether the mutations that are detected in the genomes of in vitro growing plants appear during in vitro culture, however, we could hypothesize that under pressure of these unnatural conditions, plants develop adaptive mechanisms to survive in limited spaces. These adaptive mechanisms involve epigenetic modifications that are programmed to confer plasticity to in vitro plants [31].

Tissue culture is also used in genetic improvement procedures with the aim of selecting subjects under the conditions of selected stress pressure, although in most cases the conditions do not reproduce the real ones. Evolution, in fact, diversifies and adapts species to better achieve suitability to the environmental conditions prevailing at a given time and habitat; a chain of genetic adjustments is selected at the same time as the periodic physiological events that generally occur during plant's life [32].

In vitro propagation proved to be particularly valuable for vegetatively propagated plants such as *Solanum tuberosum* L., *Allium sativum* L., *Musa acuminata*, *Saccharum officinarum* L., different ornamentals, orchids and fruit trees and energy crops [33,34]. Currently, micropropagation has also attracted growing attention from researchers as an efficient alternative way for rapid and controlled production of bioactive phyto-chemicals or food ingredients from medicinal and aromatic plants.

However, the effectiveness of a micropropagation protocol depends on the proliferation rate and stability, i.e., the number of explants, such as microshoots and single nodes, obtained from a single donor plant [35]. In addition, adventitious roots induction and the subsequent extra vitro acclimation of plantlets determine the success of a commercial propagation protocol [2]. The multiplication of shoots is based on the concomitance of two iterative processes: the induction and formation of phytomer, which includes lateral meristems formation (axillary buds) from the apical meristem (apex) and the subsequent outgrowth of the axillary buds into new shoots [36]. In this contest, artificial light plays a crucial role in successful in vitro plant production, together with other factors such as medium composition, gas exchange in the culture vessel, temperature and specific physiological outcomes of plant explant, i.e., the species-specific physiologic adaptation to the in vitro conditions previously described. Illumination should provide light in the appropriate spectral regions for promoting photosynthetic metabolism and photomorphogenic responses [37,38]. Controlling light quality (wavelength ranges), irradiances (photon flux) and light regime (photoperiod) enables the production of plants with desired characteristics [35,39].

From the outset, the lighting systems used in in vitro plant growth had been fluorescent tubes (Fls), high pressure sodium (HPS), metal halide (MH) and incandescent lamps (IL) with varying wavelengths from 400 to 700 nm. Among these, Fls have been the most popular in tissue culture rooms and consume approximately 65% of total electricity in tissue culture labs [40]. The Fls have high amounts of photons in the infrared and red ranges, gradually dropping toward blue. Due to the presence of phosphor coating, white FLs also have a continuous visible spectrum with peaks near 400–450 nm (violet-blue), 540–560 nm (green-yellow) and 620–630 nm (orange-red). The main inconveniences tied to the use of these lamps are: (i) a significant portion of the spectral output emitted (from 350 to 750 nm) [41] is not utilized by the plant cultures since they are abundant in green (G) and yellow (Y) light, which are less efficient for plants and usually lack FR light [35,41], (ii) light irradiation may cause photo-inhibition of growth and differentiation [42] and photooxidative damage in plants [43] and (iii) the dissipation of a large amount of energy as heat [44].

In recent years, light-emitting diodes (LEDs) have attracted increasing attention as potential light sources for various applications of plant tissue culture [40]. The advantages of LED lights over conventional lighting systems mainly consist in the higher photosynthetic photon efficacy (PPE) as compared to the previously used HPS or Fls. The maximum PAR efficiency of LED lamps ranges between 80 and 100%, while Fls provide only 20–30% [45,46]. The precision in converting electrical energy to photons of specific wavelengths at the desired photosynthetic photon flux density (PPFD) with negligible heat loss makes LEDs more energy-efficient than all other available artificial lighting sources. Based on the manufacturers' specifications, the LED lamps require about 32% less energy than the Fls per µmol m<sup>2</sup> s <sup>−</sup><sup>1</sup> of photons delivered to the plants [34] and 10–25% total energy saving can be realized when considering climate modification by the transition from HPS to LED [47]. Moreover, LED lamps possess a longer operating lifetime (>50,000 h), negligible heat emissions and, consequently, an indirect reduction in refrigeration costs, a more robust and easy-to-handle plastic body, no emissions of greenhouse gases (CO2) for their production and they produce no mercury pollution [46,48].

The narrow waveband emission and dynamic control of light intensity in LED-based illumination systems allow the choice of spectral quality to match the absorption range of a specific photoreceptor and thus to regulate the photosynthetically and photomorphogenic responses required for the cultivation of each species in vitro [41]. For these reasons, the use of LED lamps in the in vitro culture systems is a useful tool for photobiological studies since they allow the control of irradiance and the emission of specific spectral patterns [41]. With the rapid advancement of the technology, the reduction of LED prices and the diverse studies that show more vigorous in vitro plants cultivated under these lighting conditions, the replacement of Fls with LED lamps has attracted considerable attention around the globe [9].

Numerous studies reported the applications of LEDs, as compared to white Fls, in promoting in vitro organogenesis, growth and morphogenesis from various plant species such as *Gossypium hirsutum*, *Anthurium andreanum*, *Brassica napus*, *Musa acuminata* and so on [49–52]. The impact of LED lighting on somatic embryogenesis has also been explored for a few plant species [53–58].

Although there are a discrete number of studies, many tissue culture laboratories hesitate to replace conventional lighting systems with LEDs out of apprehension of an unpredictable and aberrant in vitro, which may damage consolidated production protocols [59].

Moreover, light quality influences the biological effectiveness of the growth regulators added to the culture substrate, as well as the endogenous hormonal balance in

the tissues [60], which must be readdressed after the substitution of the old ones with LED lamps.

Keeping this in mind, in this review, the attention will focus on the literature on the effects of light on shoot proliferation, a main process of in vitro propagation. The effects of the light spectrum on the balance of endogenous growth regulators will be also presented.
