**4. Effects of Photoperiod**

An organism's life has evolved adaptation mechanisms that are related to environmental variations. Some of these variations exhibit regular cyclicality such as light:dark cycles, others fluctuate, such as temperature; however, all of them induce significant changes in the physiology and metabolism of most organisms, occurring in their life trajectory as characterized by the night and day cycle [232,233]. Plants possess the circadian clock, an endogenous time-keeping device that triggers and regulates physiological events in accordance with predicted daily changes in the environment. The input of light into the circadian clock is led by a set of photoreceptors such as the ZTL-type and UVR8 receptors [234].

Photosynthesis and stomatal movements are controlled by the circadian clock [235,236]. Among several physiological processes that include chromatin-regulation, diurnal rhythmic gene expression generates networks of genes that act specifically throughout the day or the night [237–240]. The circadian clock is an endogenous oscillator with a duration of approximately 24 h, and it is coordinated by external factors such as temperature and light. These external factors are relatively constant during the micropropagation procedure since there is no change in photoperiodism and thermoperiodism. During the shoot multiplication phase of in vitro cultures, photoperiod regimes of 16 h of light and 8h of dark are usually adopted. Plantlets in vitro are mixotrophic organisms, therefore nutrients such as carbohydrates are absorbed from the medium. In plantlets exposed to a 16:8 h photoperiod, the photosynthetic activity is intense at the onset of the light cycle and decreases rapidly thereafter. The block of CO<sup>2</sup> assimilation depends on the rapid and progressive lower concentration of CO<sup>2</sup> in the culture vessels. The CO<sup>2</sup> availably in the culture vessels is largely generated during the respiration of sucrose supplied with the growth medium, since the gas exchange between the inside and the outside the vessel is almost absent (Abbot and [220,230,241,242]. The modification of the photoperiodic regime from a 16 h photoperiod cycle to a 4 h photoperiod cycle promoted the increment of fresh and dry weights of shoot clusters, and the number of neo-formed shoots from initial shoot explants in two *Prunus persica* rootstocks [243]. An analogous response was found in the *Prunus persica* cultivars Suncrest, Belle of Georgia and Evergreen when cultured in the presence of 10µM of BA in the medium [244]. However, Morini et al. [245] have found that the photosynthetic activity was only extended until 4 h after the beginning of the illumination, although the concentration of CO2, (under the 16/8 h regime) was not a limiting factor since at the end of the light period its availability was still much higher than that outside the vessel. From the same authors, the reduction of photosynthetic capacity was attributed to a reduced efficiency of the chloroplasts coupled with the lengthening of the light period. 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.

However, in studies carried out on other species, subjected to a 16 h photoperiod, low concentration of CO<sup>2</sup> into the vessels was observed: *Pfaffia glomerata* [246], Solanum tuberosum [247,248], *Carica papaya* [249], *Castanea sativa* [227], *Vitis vinifera* [250,251], *Fragaria x ananassa* [252], *Hyptis marrubioides* and *Hancornia speciosa* [253].
