*2.2. Saffron Flowering in In Vitro Culture and Hydroponic Culture*

In vitro aseptic corms were grown in MS medium supplemented with 3% sucrose and solidified with 0.8% agar–agar. The pH of the medium was adjusted to 5.8 prior to autoclaving at 121 ◦C and 1.2 kg cm−<sup>2</sup> pressure for 20 min. The environmental conditions of the growth chamber were adjusted to 15 ◦<sup>C</sup> ± <sup>1</sup> ◦C and 70 <sup>μ</sup>mol m−<sup>2</sup> <sup>s</sup>−<sup>1</sup> photosynthetic photon flux density (PPFD) for a 16 h photoperiod using cool white fluorescent lamps. The flowering characteristics, i.e., the number of flowers per plant, stigma length, and stigma fresh and dry weights, under in vitro culture were compared with those under an aerated, volcanic-rock-based hydroponic system as described by Dewir and Alsadon [16]. The corms were obtained from the same source (Bloembollenbedrijf J.C.Koot) and were of the same size grade (3.2–3.5 cm diameters).

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

Two types of liquid culture systems, temporary immersion (ebb and flow) and continuous immersion (with a net), were tested to select a suitable method for saffron cormlet production in liquid media and then compared with solid culture. Intact corms or apical buds (15 explants per bioreactor) were transferred toa3L balloon-type bubble bioreactor with 1.2 L of MS liquid medium supplemented with 30 g L−<sup>1</sup> of sucrose and 0.5 mg L−<sup>1</sup> of NAA. The pH of the medium was adjusted to 5.8 before autoclaving at 121 ◦C and 1.2 kg cm−<sup>2</sup> pressure for 30 min. In the immersion-type bioreactor, a supporting net was used to hold the plant material to avoid the complete submersion of explants in the liquid medium. The volume of air input was adjusted to 0.2 vvm (air volume/culture volume min−1). The ebb and flow system (PLT Scientific SDN BHD, Puchong, Selangor D.E., Malaysia) was programmed to immerse the plantlets in medium four times per day for 60 min per immersion. All the bioreactors and culture vessels were maintained at 15 ◦C ± 1 ◦C and 70 μmol m−<sup>2</sup> s−<sup>1</sup> PPFD for a 16 h photoperiod using cool white fluorescent lamps. There were 15 explants in each treatment, and the diameter, fresh weight, and number of daughter corms were recorded after 14 weeks of culture from 9 randomly selected explants.
