*3.1. In Vitro Establishment of Saffron Aseptic Culture and Flower Emergence*

Preliminary trials were conducted in October 2020 to test the surface disinfection of saffron corms using sodium hypochlorite, mercuric chloride, and hydrogen peroxide for different durations and at various concentrations following the protocols described in previous studies [9,17–19]. Despite careful cleaning during corm preparation (Figure 1a–c), pathogen contamination was a serious challenge during the establishment of aseptic saffron culture (Figure 1d,e). Only 10% of the corms survived fungal and bacterial contamination, and we were unable to establish sufficient aseptic corms in this season. In the following season, we surface-disinfected saffron corms in July 2021 using the protocol described in the Materials and Methods Section, and a high percentage of aseptic corms (70%) was obtained. Teixeira da Silva et al. [20] reviewed various saffron corm disinfection methods, highlighting the numerous factors that can influence the efficiency of disinfection: cultivation conditions; the physiological state of the stock plant; the size, age, and type of the explant; the type and concentration of disinfectant; and the time and temperature of exposure. In the present study, establishing the aseptic saffron culture after the corm harvest in July, rather than in later months, increased the efficiency of corm disinfection. Bud sprouts during corm storage can increase microorganism abundance within the saffron corm, making it difficult to eradicate contaminants.

The growth of the main apical bud and the emergence of roots occurred within 1 week (Figure 1f), and all lateral buds grew within 3 weeks of culture (Figure 1g). Saffron flower buds appeared 30 days after culture (Figure 1h,i). Interestingly, the sizes of the flowers and stigmas produced in vitro and via hydroponics were similar (Figure 1j,k; Table 1). Additionally, only large saffron corms (≥2.8 cm in diameter) produced flowers in vitro; small corms did not produce flowers.


**Table 1.** Comparison of stigma and flower size of saffron produced under in vitro culture and via a hydroponic system (Dewir and Alsadon [16]).

NS = non-significant according to *t*-test.

**Figure 1.** Surface disinfection and the establishment of in vitro saffron aseptic culture: (**a**–**c**) Cleaning, surface disinfection, and culture of corms on half-strength MS medium (Murashige and Skoog, 1962) supplemented with 3% sucrose; (**d**,**e**) Fungal contamination of in-vitro-cultured corms 1 week after incubation; (**f**) Bud break and rooting of saffron corms 7 days after incubation at 16 ◦C; (**g**) Shoot emergence and growth of saffron buds 3 weeks after incubation; (**h**,**i**) In vitro flowering of saffron corms 30 days after incubation; (**j**) Normal size and structure of in-vitro-produced saffron flowers; and (**k**) Harvested stigma of in-vitro-produced saffron.

In vitro flowering systems are considered convenient tools for studying the transition to flowering. They can be used to study specific aspects of flowering, as well as the mechanisms underlying the reproductive process [21]. Both developmental cues and environmental signals control the timing of flowering, and genetic studies have revealed the complexity of the mechanisms underlying flowering [22]. A combination of environmental, developmental, hormonal, and genetic factors determines the eventual transition to flowering [23]. However, saffron corms have no cold requirement to break dormancy or to complete flower formation, which is often the case in geophytes [24,25].

Under field conditions, floral initiation usually occurs during summer, coinciding with the revival of meristematic activity in the apical bud. Thus, corms can be cultivated directly in the field to force flowering, or they can be stored for up to 60 days at 2 ◦C to extend and facilitate harvesting [26,27]. Temperature, among several environmental parameters, plays a pivotal role in saffron flower induction [4,28,29]. Therefore, saffron corms are incubated at a moderately high temperature (23 ◦C–27 ◦C) to induce flowering before they are exposed to a moderately low temperature (17 ◦C) for floral emergence. In the present study, floral emergence occurred in vitro 30 days after the incubation of the saffron corms at 15 ◦C ± 1 ◦C. These in-vitro-produced saffron stigma can be utilized as a source of biochemicals and pharmaceuticals. They also can be explored as initial explants for callus formation and cell cultures. In-vitro-formed flowers are generally undersized compared with ex-vitro-formed flowers, as reported previously for *Phyllanthus niruri* [30] and *Spathiphyllum cannifolium* [31]. However, some in-vitro-grown plant species form normal-size flowers, e.g., *Chamomilla recutita* [32] and *Euphorbia milii* [33]. Comparing the in vitro and hydroponic flowering of saffron, the flowers formed in vitro had a similar size compared with those formed ex vitro. This may have been due to the early initiation of the saffron floral buds and the available nutrient supply from the mother corm supporting floral bud emergence and flower development.

#### *3.2. Effects of Explant Type and Solid/Liquid Culture on Saffron Daughter Corm Formation*

The use of intact mother corms or apical buds as initial explants (Figure 2a,e) influenced saffron daughter corm formation. In vitro, daughter corms were formed successfully after 10 weeks of culture (Figure 2b–g). After 14 weeks of culture, the life cycle of saffron was completed in vitro; the leaves were dried, and the saffron daughter corms were harvested (Figure 2d,g). All corms and apical bud explants formed daughter corms. However, the use of intact corms was more effective for corm production than the use of apical buds. More corms were produced, and the average fresh weight and diameter of corms were higher in mother corm explants than in apical bud explants. The number of cormlets, cormlet fresh weight, and cormlet diameter were also influenced by the culture type (i.e., continuous immersion bioreactors, ebb and flow bioreactors, and solid culture) (Figure 2h,i and Figure 3). Intact corms produced a higher number of corms than were produced by apical bud explants. The average number of cormlets per intact corm was 2.0–4.5 cormlets, owing to the presence of several lateral buds in the mother corms, whereas the apical buds produced single cormlets. For both intact corms and apical buds, liquid cultures using bioreactors resulted in corms with a higher fresh weight and diameter than those grown via solid culture. The average fresh weight and diameter of cormlets was the highest under the ebb and flow system although a low number of cormlets was obtained. Conversely, a higher number of cormlets with a smaller size was obtained using the immersion system.

Liquid medium is known to promote nutrient uptake and encourage growth and proliferation in vitro. The bioreactors designed for plant cultures provide optimal aeration and mixing with oxygen, without subjecting the propagules to shear stress [11]. Several plant species exhibit better growth in liquid/bioreactor cultures than in solid cultures, e.g., *E. milli* [34,35], *S. cannifolium* [36], *Gentiana triflora* [37], *Lessertia frutescens* [38], *Kalopanax septemlobus* [39], and *Philodendron bipinnatifidum* [40]. Piqueras et al. [41] highlighted the improved development of the cormogenic nodules and microcorms of saffron grown in liquid cultures relative to solid cultures. In the present study, the high number of cormlets and fresh mass of saffron corms grown in a bioreactor system might be attributable to enhanced water and nutrient uptake, as well as aeration, i.e., conditions that favor better growth compared with those under a nonaerated, solid culture system. Compared with continuous immersion, the ebb and flow system produced fewer cormlets, but they were larger. The higher efficacy of the ebb and flow system, in terms of fostering optimum cormlet growth, is probably due to culture ventilation and the intermittent contact between the entire tissue surface and the liquid medium, two features that are not usually combined in other liquid culture procedures [42,43].

**Figure 2.** Effects of explant type (intact corms and apical buds) and culture system (solid culture, continuous immersion bioreactors, and ebb and flow bioreactors) on in-vitro-produced daughter saffron corm formation: (**a**) Established saffron aseptic culture; (**b**–**d**) Daughter corm formation following 10, 14, and 16 weeks, respectively, of solid culture using intact corm explants; (**e**) Bud break and shoot development of apical buds after 2 weeks of culture; (**f**,**g**) Daughter corm formation following 14 and 16 weeks, respectively, of solid culture using intact corm explants; (**h**) Layout of continuous immersion and ebb and flow bioreactors; (**i**–**k**) Daughter corm formation using intact corms in continuous immersion bioreactors, ebb and flow bioreactors, and solid culture, respectively, after 16 weeks of culture; (**l**) Daughter corm formation using apical buds in continuous immersion bioreactors, ebb and flow bioreactors, and solid culture, respectively, after 16 weeks of culture.

**Figure 3.** Effects of explant type (intact corms and apical buds) and culture type (solid culture, continuous immersion bioreactors, and ebb and flow bioreactors) on number of cormlets (**a**), cormlet fresh weight (**b**), and cormlet diameter (**c**) of saffron. Different letters show significant differences at *p* ≤ 0.05.
