*3.4. Development of Bulblets in Somatic Embryos of F. cirrhosa*

Bulblet formation was observed in all three types of somatic embryos taken from cultures under LED lights CW5000K or WW2700K, as described in Section 2.3 (Table 4; Figure 6a–c; Figure 7a–b). However, the response percentage and number of bulblets varied depending upon the developmental stage of the somatic embryos. The highest response of bulblet formation (90%) was recorded with single embryos with an average of 4.7 bulblets/SE. In the case of a cluster of five embryos, the percentage of response and the average number of bulblets were 86.7 and 3.3 bulblets/SE, respectively. The lowest response (43.6%) and the least number of bulblets (1.1/SE) were recorded in SEs with cotyledonary leaf. However, in this case, some secondary somatic embryos at the base of cotyledonary leaf were observed, though cotyledonary leaf did not grow further, and withered and dried. Single embryos grew further in size and developed multiple bulblets without much callus growth. The cluster of five embryos grew in size and developed further embryogenic callus and secondary somatic embryos.

Since bulbs constitute the most critical parts of *Fritillaria* plants and are the primary source of isosteroidal alkaloids in *Fritillaria* species used in traditional Chinese medicine (TCM), the production of bulbs in *F. cirrhosa* by tissue culture technology is highly desirable. The objective of the present study was to investigate the effects of LED lights on the growth and development of embryogenic callus and the analysis of alkaloid contents in cultures; however, the development of bulblets in somatic embryos is an important observation in the study because in natural conditions, one bulb typically develops into a single seedling and it takes about 5–6 years to grow into an appropriate size [46]. It has also been reported that isosteroidal alkaloids in *F. cirrhosa* bulbs are greatly influenced by environmental conditions, plant age, and harvest times [47]. Recently, Chang and co-workers in our laboratory reported an efficient micropropagation method of bulblet production in *F. cirrhosa* and also the presence of some isosteroidal alkaloids in tissue culture-derived bulblets and callus [10].

*Plants* **2020**, *9*, x FOR PEER REVIEW 12 of 15

*Plants* **2020**, *9*, x FOR PEER REVIEW 12 of 15

**Figure 6.** Development of bulblets in somatic embryos of *F. cirrhosa* D. Don (photographs taken after three months of incubation): (**a**) Bulblets from a single embryo, (**b**) bulblets in a cluster of five embryos, (**c**) bulblets in embryo with cotyledon leaf. bu: bulblet; se: somatic embryo; sse: secondary somatic embryo; ec: embryogenic callus. Bar = 0.8 cm. **Figure 6.** Development of bulblets in somatic embryos of *F. cirrhosa* D. Don (photographs taken after three months of incubation): (**a**) Bulblets from a single embryo, (**b**) bulblets in a cluster of five embryos, (**c**) bulblets in embryo with cotyledon leaf. bu: bulblet; se: somatic embryo; sse: secondary somatic embryo; ec: embryogenic callus. Bar = 0.8 cm. **Figure 6.** Development of bulblets in somatic embryos of *F. cirrhosa* D. Don (photographs taken after three months of incubation): (**a**) Bulblets from a single embryo, (**b**) bulblets in a cluster of five embryos, (**c**) bulblets in embryo with cotyledon leaf. bu: bulblet; se: somatic embryo; sse: secondary somatic embryo; ec: embryogenic callus. Bar = 0.8 cm.

**Figure 7.** Development of bulblets from somatic embryos of *F. cirrhosa* D. Don*:* (**a**) Scanning electron microscopy (SEM) of bulblets (bar = 0.77 mm), (**b**) different stages of growth of culture-derived bulblets of *F. cirrhosa* (bar = 1.0 cm). **Figure 7.** Development of bulblets from somatic embryos of *F. cirrhosa* D. Don*:* (**a**) Scanning electron microscopy (SEM) of bulblets (bar = 0.77 mm), (**b**) different stages of growth of culture-derived bulblets of *F. cirrhosa* (bar = 1.0 cm). **Figure 7.** Development of bulblets from somatic embryos of *F. cirrhosa* D. Don: (**a**) Scanning electron microscopy (SEM) of bulblets (bar = 0.77 mm), (**b**) different stages of growth of culture-derived bulblets of *F. cirrhosa* (bar = 1.0 cm).

Since bulbs constitute the most critical parts of *Fritillaria* plants and are the primary source of isosteroidal alkaloids in *Fritillaria* species used in traditional Chinese medicine (TCM), the production of bulbs in *F. cirrhosa* by tissue culture technology is highly desirable. The objective of the present study was to investigate the effects of LED lights on the growth and development of embryogenic callus and the analysis of alkaloid contents in cultures; however, the development of bulblets in

Since bulbs constitute the most critical parts of *Fritillaria* plants and are the primary source of isosteroidal alkaloids in *Fritillaria* species used in traditional Chinese medicine (TCM), the production of bulbs in *F. cirrhosa* by tissue culture technology is highly desirable. The objective of the present study was to investigate the effects of LED lights on the growth and development of embryogenic callus and the analysis of alkaloid contents in cultures; however, the development of bulblets in
