**3. Effects of Light Intensity**

The selection of the optimal light intensity to support in vitro proliferation and growth is also important for an optimization of the processes. Among others, light intensity regulates the dimension of leaves and stems, as well as their morphogenic pathway, and is involved in pigment formation and hyperhydricity [208].

In vitro cultures are subjected to a much lower light intensity as compared to those grown under open field conditions. The permanent low light conditions in vitro have been considered a limiting factor for photosynthesis and for supporting plant morphogenesis in vitro, so it is necessary, in most cases, to supply sucrose to the medium [209]. In vitro plants are also very susceptible to high light conditions [210] and prone to photoinhibition [211]. Too high irradiation can severely damage the photosynthetic apparatus and photosynthetic pigment synthesis [48,212], leading to the formation of harmful free oxygen radicals and damage to cells [213].

In Table 3, the research that mainly addressed the effects of different light intensities is shown, but only in a few of the studies shoot proliferation is examined.


**Table 3.** Effects of different light intensities on shoot proliferation in increasing light-intensity order.


**Table 3.** *Cont.*

The optimal value of the PFD for plantlets changes from species to species and the predominant in vivo light conditions may give an indication of the requirements for optimal culture growth in vitro [75]. In *Alocasia amazonica* [222] and *Momordica grosvenori* [219], shoot length increased with the reduction in light intensity, an adaptation mechanism indicating that these species can survive in low light-intensity environments. In *Lippia gracilis*, the weight increase of plantlets grown under high light intensities indicates that this species originates in a semiarid environment where high irradiance (HI) incoming light occurs [119]. Evidence has been previously presented [178] that plants adapted to an environment with incoming HI present better photosynthetic rates and high growth rates under intense light. In an extensive study on the photosynthetic pigments, Lazzarini et al. [119] concluded that the increase in chlorophyll b content under low irradiance (LI) is indicated as an important marker of plant adaptation to shaded environments because this pigment is more efficient for capturing the photons of the higher wavelengths of the spectrum that are mainly present. Furthermore, it is worth noting that the type of explant also influences the amount of photosynthetic pigment: leaves of plantlets generated from apical explants had higher amounts of chlorophyll a, total chlorophyll and carotenoids regardless of light conditions, whereas the amount of chlorophyll b resulted in more plantlets generated from the lateral buds of nodal segments. Moreover, an increase in the synthesis of carotenoids was observed in plants grown under high light intensities and was associated with the photoprotection exerted by these pigments within the photosystems. In *Lippia gracilis*, this increase led to better efficiency of the photosynthetic activity and, hence, the higher production of dry weight observed under these conditions [119]. In three different species, *Disanthus cercidifolius*, *Rhododendron* cultivars and *Crataegus oxyacantha*, low levels of irradiance (11 µmol m−<sup>2</sup> s −1 ) were optimal for in vitro growth, while higher irradiance determined a decrease in shoot development and leaf chlorophyll content in *Disanthus* and

*Rhododendron* cultivars, which are shade-tolerant species in their natural habitat. Plantlets of *Crataegus* generated from in vivo plants adapted to higher levels of irradiance resulted in tolerance to a wide range of irradiances in vitro. Only shoot extension was inhibited at the highest levels tested, whereas leaf chlorophyll content was unaffected. These differences were attributed to a differential adaptation to light determined by the natural habitats of these plants and of the possible direct effect of irradiance upon plant growth regulators in the culture system [75]. Different effects of rising light intensity were observed in *Plectranthus amboinicus* grown in vitro. In this species, intensities below or above the optimum (69 µmol m−<sup>2</sup> s −1 ) led to the lowest growth. In fact, photosynthesis was inefficient under low light intensity (26 µmol m−<sup>2</sup> s −1 ) but increased light intensities led to reduced concentrations of a, b and total chlorophyll, and carotenoids and thus of growth [48]. In *Withania somnifera* and *Achillea millefolium*, the treatments with the highest light intensity (60 and 69 µmol m−<sup>2</sup> s −1 , respectively) showed the highest levels of photosynthetic pigments but not the highest growth. Alvarenga et al. [172] concluded that the significant increase observed in chlorophyll and carotenoids under high light conditions would indicate that these pigments have the photoprotective function, as assumed by Biswal et al. [223], since they may be inefficient in absorbing light and increasing photosynthetic efficiency. They also attributed the damage of excess light to the photosynthetic apparatus to the production of free radicals, which may degrade these pigments [45,213]. Kurilˇcik et al. [174] on *Chrysanthemum (Chrysanthemum morifolium*), noticed that the maximal PFD (85 µmol m−<sup>2</sup> s −1 ) used in their experiment induces light abnormalities on the leaf surface. In ginger [224], the growth was restrained when the light reached 180 µmol m−<sup>2</sup> s <sup>−</sup><sup>1</sup> and the chlorophyll content decreased as the light intensity increased. of irradiance resulted in tolerance to a wide range of irradiances in vitro. Only shoot extension was inhibited at the highest levels tested, whereas leaf chlorophyll content was unaffected. These differences were attributed to a differential adaptation to light determined by the natural habitats of these plants and of the possible direct effect of irradiance upon plant growth regulators in the culture system [75]. Different effects of rising light intensity were observed in *Plectranthus amboinicus* grown in vitro. In this species, intensities below or above the optimum (69 μmol m−2 s −1) led to the lowest growth. In fact, photosynthesis was inefficient under low light intensity (26 μmol m−2 s −1) but increased light intensities led to reduced concentrations of a, b and total chlorophyll, and carotenoids and thus of growth [48]. In *Withania somnifera* and *Achillea millefolium*, the treatments with the highest light intensity (60 and 69 µmol m−2 s −1, respectively) showed the highest levels of photosynthetic pigments but not the highest growth. Alvarenga et al. [172] concluded that the significant increase observed in chlorophyll and carotenoids under high light conditions would indicate that these pigments have the photoprotective function, as assumed by Biswal et al. [223], since they may be inefficient in absorbing light and increasing photosynthetic efficiency. They also attributed the damage of excess light to the photosynthetic apparatus to the production of free radicals, which may degrade these pigments [45,213]. Kurilčik et al. [174] on *Chrysanthemum (Chrysanthemum morifolium*), noticed that the maximal PFD (85 μmol m−2 s −1) used in their experiment induces light abnormalities on the leaf surface. In ginger [224], the growth was restrained when the light reached 180 μmol m−2 s −1 and the chlorophyll content decreased as the light intensity increased. However, a different sensibility to light intensity seems to affect proliferation rate and the

higher irradiance determined a decrease in shoot development and leaf chlorophyll content in *Disanthus* and *Rhododendron* cultivars, which are shade-tolerant species in their natural habitat. Plantlets of *Crataegus* generated from in vivo plants adapted to higher levels

*Plants* **2022**, *11*, x 29 of 45

However, a different sensibility to light intensity seems to affect proliferation rate and the plantlet growth, and in most cases lower plant intensities are required for proliferation. plantlet growth, and in most cases lower plant intensities are required for proliferation.

Based on the observation of the examined papers for this review, in Figure 1, the light intensities were grouped in ranges and the frequency of their use is shown. From this study, it emerged that 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 ). Based on the observation of the examined papers for this review, in Figure 1, the light intensities were grouped in ranges and the frequency of their use is shown. From this study, it emerged that whatever the light spectrum, the most used light intensities range from 20 to 80 µmoles m−2 s −1 and the most used intensity for proliferation is 50 (µmoles m−2 s −1).

**Figure 1.** Frequency of light intensities used in literature for proliferation. **Figure 1.** Frequency of light intensities used in literature for proliferation.

In *Rubus* spp, rising WL fluence rates from 0 to 81 µmol m−2 s −1 did not improve the organogenesis from cotyledons [225]. In *Vaccinium corymbosum*, exposure at rising intensities from 55 up to 210 µmol m−2 s −1 improved proliferation and rooting ratios only with In *Rubus* spp, rising WL fluence rates from 0 to 81 µmol m−<sup>2</sup> s <sup>−</sup><sup>1</sup> did not improve the organogenesis from cotyledons [225]. In *Vaccinium corymbosum*, exposure at rising intensities from 55 up to 210 µmol m−<sup>2</sup> s −1 improved proliferation and rooting ratios only

with short time applications (7 days). Longer exposure of the leaves (14 and 28 days) determined inhibition of growth and the red color of leaves and sprouts, and less vigorous plants after in vivo transferring [215].

However, a better multiplication under increasing irradiance, from 10 to 80 µmol m−<sup>2</sup> s −1 , resulted in *Pyrus communis* [218], in *L. gracilis* at 94 µmol m−<sup>2</sup> s −1 [119] and in *Rosa hybrida* from 4 to 148 µmol m−<sup>2</sup> s −1 [221]. In this last species, higher irradiance (66 and 148 µmol m−<sup>2</sup> s −1 ) showed better effects on shoot proliferation, but leaf chlorosis was observed and better results on shoot growth were obtained at 17 µmol m−<sup>2</sup> s −1 [221]. The chlorosis occurring at the higher levels of irradiance may be due to photochemical oxidation, photoinhibition or chloroplast damage [226].

In *Castanea sativa*, Sáez et al. [227] highlighted a correlation between light intensity and the addition of sugar to the growth medium. They demonstrated that HI (150 µmol m−<sup>2</sup> s −1 ) and high sugar amounts (30 g L−<sup>1</sup> ) produced an increase in photosynthetic activity and chlorophyll content and determined a higher proliferation rate and biomass production. However, a high proliferation rate was obtained even under LI with a higher sugar content in the medium. Thus, HI but also LI may be beneficial during the in vitro culture, but this is only possible in the presence of sucrose added to the culture medium.

Kozai [228], in *Cymbidium*, doubled in vitro growth by adding CO<sup>2</sup> to the culture vessels at high PFD (230 µmol m−<sup>2</sup> s −1 ), demonstrating that CO<sup>2</sup> limitation may have a relevant role in enhancing the growth when high PFDs are adopted. The same was also true for *Actinidia deliciosa* where the proliferation rate and dry and fresh weight increased up to 120 µmol m−<sup>2</sup> s <sup>−</sup><sup>1</sup> but decreased at higher rates. The biomass produced was also affected by light intensity, since both dry and fresh weight increased at the PPFD up to 120 µmol m−<sup>2</sup> s −1 , while only dry weight increases thereafter up to the highest value of 250 µmol m−<sup>2</sup> s −1 .

The photosynthetic rate was nearly four times higher when raising CO<sup>2</sup> up to 1450 and 4500 µL L−<sup>1</sup> compared to the lowest CO<sup>2</sup> concentration tested (330 µL L−<sup>1</sup> ) [220].

In fact, it has been shown that, just a few hours after the light was turned on, CO<sup>2</sup> underwent a drastic reduction in concentration and sub-optimal CO<sup>2</sup> availability has been correlated with reduced photosynthetic ability [229]. Thus, exogenous enrichments of this gas in the culture vessels improves photosynthesis at high PFDs [230,231].

Finally, most studies on the effects of light intensities have been carried out under Fl or W-LED. However, some studies revealed a relationship between the light spectrum and the intensity that affects plant growth and development. In the presence of BA, WL, BL and FRL, action on proliferation was dependent on the fluence rate [141].

Phytochrome has been shown to induce a high-irradiance response and low-irradiance response in *Prunus domestica* rootstock Mr.S. 2/5 [142]. Similar results were also obtained with the rootstock GF 677 in which the newly formed shoots were fewer but longer under the two intensities of RL (15 and 40 µmol m−<sup>2</sup> s −1 ) than those treated with WL. In addition, the low intensity RL (15 µmol m−<sup>2</sup> s −1 ) induced higher shoot multiplication as compared to the higher irradiance (40 µmol m−<sup>2</sup> s −1 ). The formation of new shoots in the two species was affected differently by the increase in the RL irradiance, and shoot formation was found to increase in the cultures of Mr.S. 2/5 and decrease in those of GF 677. This result could be related to a species-specific response on which would depend different PHY regulation strategies [2].
