*3.1. Callus Proliferation*

Callus of *F. cirrhosa* cultured in Murashige and Skoog's liquid medium with 2,4-D, under agitated conditions in dark incubation for six weeks, proliferated readily. Callus not only grew in volume but also became embryogenic since early stages of embryos were observed (Figure 4). There are several reports from our laboratory where various secondary metabolites have been obtained from different culture systems [25], including callus cultures of several medicinally important plant species, e.g., *Salvia miltiorrhiza* Bunge [26] and *Saussurea involucrata* Kar. et Kir. [27]. These and numerous other reports demonstrate that callus cultures have tremendous potential for the sustainable and large-scale production of secondary metabolites used in pharmaceuticals. Biotechnological applications of plant callus cultures have been recently reviewed [28], and, according to the author, the full potential of callus plant culture technology has not yet been exploited. Callus cultures and suspension cell cultures offer a wide range of applications in agriculture and horticulture, including for Chinese medicinal plants. Genetically transformed callus cultures cannot only be used for the synthesis of bioactive compounds but also for the development of plants with traits [28].

of plants with traits [28].

according to the author, the full potential of callus plant culture technology has not yet been exploited. Callus cultures and suspension cell cultures offer a wide range of applications in agriculture and horticulture, including for Chinese medicinal plants. Genetically transformed callus

**Figure 4.** Embryogenic calli (EC) of *F. cirrhosa* D. Don growing in Murashige and Skoog's liquid basal medium supplemented with 2,4-D (0.5 mg/mL) and 2% sucrose. The image is a photograph taken by a scanner of the bottom of the culture flask after six weeks of incubation (bar = 1.2 cm). **Figure 4.** Embryogenic calli (EC) of *F. cirrhosa* D. Don growing in Murashige and Skoog's liquid basal medium supplemented with 2,4-D (0.5 mg/mL) and 2% sucrose. The image is a photograph taken by a scanner of the bottom of the culture flask after six weeks of incubation (bar = 1.2 cm).

#### *3.2. Influence of LED Lights on Morphogenesis of Embryogenic Calli (EC) of F. cirrhosa 3.2. Influence of LED Lights on Morphogenesis of Embryogenic Calli (EC) of F. cirrhosa*

The eight LED lights had significant effects on the growth and development of embryogenic calli (EC) of *F. cirrhosa* (Table 2). Although the development of somatic embryos (SEs) in embryogenic calli were recorded under all the light treatments, the maximum number of SEs was recorded under red (9R, 223.7), infrared (9IR, 231.3), and a combination of red+blue+infrared light (3R3B3IR, 230.7), respectively. Among the red, infrared, and a combination of red+blue+infrared LED lights, there was a low significant difference concerning the number of SEs and the number of SEs with cotyledonary leaves (Table 2). LED lights also influenced the number of embryos with the development of cotyledonary leaves. Calli exposed to white light (WW-2700K) developed a maximum number of SEs with cotyledonary leaves (22.3), followed by 8R1B (16.7) and CW-5000K (12.0), respectively. SEs under LED lights 9B, 9R, 9IR, and 3R3B3IR developed a reduced number of cotyledonary leaves (in the range of 3.7 to 5.3). There was a low significant difference in the total fresh weight of EC among different LED light treatments. The maximum total fresh weights (FW) of EC biomass, i.e., 17.92 g and 16.67 g, were recorded with treatments of red (9R) and a combination of red+blue+infrared light (3R3B3IR), respectively. Eight LED lights also influenced the growth and development, and morphological features of somatic embryos. Embryogenic calli exposed to different LED lights developed somatic embryos in different developmental stages from early globular to mature SEs with cotyledonary leaves (Figure 5a–i). However, the average number of SEs varied depending upon the LED light. LED light WW-2700K developed the maximum number of SEs with cotyledonary leaves (22.3), followed by 8R1B (16.7) (Table 2). Different LED lights also affected the overall color of the embryogenic calli mass (Figure 5a–h). Under red (9R) and infrared (9IR) treatments, cultures turned white (Figure 5f,g), while under red+blue+infrared (3R3B3IR), these were dark green (Figure 5h). The eight LED lights had significant effects on the growth and development of embryogenic calli (EC) of *F. cirrhosa* (Table 2). Although the development of somatic embryos (SEs) in embryogenic calli were recorded under all the light treatments, the maximum number of SEs was recorded under red (9R, 223.7), infrared (9IR, 231.3), and a combination of red+blue+infrared light (3R3B3IR, 230.7), respectively. Among the red, infrared, and a combination of red+blue+infrared LED lights, there was a low significant difference concerning the number of SEs and the number of SEs with cotyledonary leaves (Table 2). LED lights also influenced the number of embryos with the development of cotyledonary leaves. Calli exposed to white light (WW-2700K) developed a maximum number of SEs with cotyledonary leaves (22.3), followed by 8R1B (16.7) and CW-5000K (12.0), respectively. SEs under LED lights 9B, 9R, 9IR, and 3R3B3IR developed a reduced number of cotyledonary leaves (in the range of 3.7 to 5.3). There was a low significant difference in the total fresh weight of EC among different LED light treatments. The maximum total fresh weights (FW) of EC biomass, i.e., 17.92 g and 16.67 g, were recorded with treatments of red (9R) and a combination of red+blue+infrared light (3R3B3IR), respectively. Eight LED lights also influenced the growth and development, and morphological features of somatic embryos. Embryogenic calli exposed to different LED lights developed somatic embryos in different developmental stages from early globular to mature SEs with cotyledonary leaves (Figure 5a–i). However, the average number of SEs varied depending upon the LED light. LED light WW-2700K developed the maximum number of SEs with cotyledonary leaves (22.3), followed by 8R1B (16.7) (Table 2). Different LED lights also affected the overall color of the embryogenic calli mass (Figure 5a–h). Under red (9R) and infrared (9IR) treatments, cultures turned white (Figure 5f,g), while under red+blue+infrared (3R3B3IR), these were dark green (Figure 5h).

Light is one of the essential components required by plants for photosynthesis. However, its quantity and duration (photoperiod) drastically affect plant growth and development [29]. Fluorescent tubes are the most common lighting source in a culture room in a typical tissue culture set up. However, in the recent past, due to several advantages, more advanced light-emitting diodes (LEDs) have been used as a source of light. LEDs are relatively cool, emit light of specific wavelengths (spectra), are much smaller in size, and are more durable compared to conventional ones [30]. Due to their efficiency in growth and development, LED lights have been used for micropropagation of many horticultural and agricultural crops [31–33]. Depending on requirements, different types of growth chambers equipped with LED lights can be designed. Since the supply of specific light spectra can be controlled in plant tissue culture systems via LED lights, the effects of individual or combinations of light spectra on plant growth and development can be investigated appropriately as in the present study. Several studies on the influence of LED lighting on plant growth, physiology, and

secondary metabolism have been reviewed [11,34]. Recently, Pedmale and co-workers reported that the quality of light affects plant growth and development by the regulation of different mechanisms, including the selective activation of light receptors, such as phytochromes by red and far-red light, cryptochromes and phototropins by blue light, and UV-B receptors by ultraviolet light [35]. In a previous study in our laboratory, LED lights affected the development of somatic embryos and callus proliferation (fresh weight) in *Peucedanum japonicum* Thunb [22]. Several studies have demonstrated that the quality, duration, and intensity of red, infrared, blue, and ultraviolet light can have a profound influence on plants by activating or deactivating physiological reactions and controlling their growth and development [36–38]. These studies confirm with many other reports that LED lights are more efficient in plant growth compared to fluorescent lamps. Similar to our results, the beneficial effects of some LED light sources on the induction of embryogenesis in *Oncidium* have been reported [39]. Due to these beneficial effects, LED light systems are being increasingly used to boost the horticulture industry in Taiwan and several other countries [40–42]. *Plants* **2020**, *9*, x FOR PEER REVIEW 10 of 15

**Figure 5.** Influence of different LED lights on the growth and development of embryogenic calli of *F. cirrhosa* D. Don: (**a**) CW-5000K; (**b**) WW-2700K; (**c**) 7R1G1B; (**d**) 8R1B; (**e**) 9B; (**f**) 9R; (**g**) 9IR; (**h**) 3R3B3IR; (**i**) Different stages of somatic embryos. For microphotographs, cultures are taken out of the bottles and transferred to sterilized Petri dishes. g: globular embryos; m: mature embryos; c: cotyledonary leaf; ec: embryogenic callus. (a–f, h, bar = 0.57 cm; g, bar = 1.4 cm). Culture medium is Murashige and Skoog's basal medium supplemented with 2% sucrose and 0.4% GPP. Culture vessels are 650 mL glass bottles, each containing 100 mL medium. Observations recorded after three months of incubation in a specially designed LED light growth chamber. **Figure 5.** Influence of different LED lights on the growth and development of embryogenic calli of *F. cirrhosa* D. Don: (**a**) CW-5000K; (**b**) WW-2700K; (**c**) 7R1G1B; (**d**) 8R1B; (**e**) 9B; (**f**) 9R; (**g**) 9IR; (**h**) 3R3B3IR; (**i**) Different stages of somatic embryos. For microphotographs, cultures are taken out of the bottles and transferred to sterilized Petri dishes. g: globular embryos; m: mature embryos; c: cotyledonary leaf; ec: embryogenic callus. (a–f, h, bar = 0.57 cm; g, bar = 1.4 cm). Culture medium is Murashige and Skoog's basal medium supplemented with 2% sucrose and 0.4% GPP. Culture vessels are 650 mL glass bottles, each containing 100 mL medium. Observations recorded after three months of incubation in a specially designed LED light growth chamber.

their efficiency in growth and development, LED lights have been used for micropropagation of many horticultural and agricultural crops [31–33]. Depending on requirements, different types of growth chambers equipped with LED lights can be designed. Since the supply of specific light spectra can be controlled in plant tissue culture systems via LED lights, the effects of individual or combinations of light spectra on plant growth and development can be investigated appropriately as in the present study. Several studies on the influence of LED lighting on plant growth, physiology, and secondary metabolism have been reviewed [11,34]. Recently, Pedmale and co-workers reported that the quality of light affects plant growth and development by the regulation of different mechanisms, including the selective activation of light receptors, such as phytochromes by red and

Light is one of the essential components required by plants for photosynthesis. However, its quantity and duration (photoperiod) drastically affect plant growth and development [29]. Fluorescent tubes are the most common lighting source in a culture room in a typical tissue culture
