*3.3. Effects of Salicylic Acid, Glutamine, and Jasmonic Acid on Saffron Daughter Corm Formation In Vitro*

The apical buds of saffron cultured on MS medium supplemented with different additives, i.e., salicylic acid, glutamine, and jasmonic acid, showed variation in cormlet diameter and fresh weight (Table 2; Figure 4). With the exception of 2 mg L−<sup>1</sup> of jasmonic acid, all other treatments increased cormlet diameter and fresh weight relative to the control treatment, with salicylic acid at 75 mg L−<sup>1</sup> and glutamine at 600 mg L−<sup>1</sup> providing the highest and second highest increases, respectively, increasing the corm diameter by around 0.4 cm and nearly doubling the fresh weight compared with the control treatment. Salicylic acid is a phytohormone that plays important roles in many aspects of plant life, including seed germination, physiological and biochemical processes, flowering, and fruit yield [44,45]. Under field conditions, salicylic acid is effective for improving plant and bulb growth. In a plastic house experiment, soaking *Iris hollandica* bulbs in salicylic acid (200 mg L−<sup>1</sup> for 4 h) improved plant vegetative growth [46]. In another study, the exogenous application of salicylic acid (250 mg L−<sup>1</sup> at 30, 45, and 60 days after transplanting) increased not only the vegetative growth, but also the bulb weight, diameter, and yield of onion (*Allium cepa* 'ALR') [47]. In a study on saffron, corm dipping in a solution of salicylic acid (2 mM for 6 h) improved vegetative and reproductive characteristics, as well as yield quality [48]. Similarly, the application of salicylic acid (1–2 mM) to saffron, either during corm priming or foliar application, improved the vegetative growth and physiological and biochemical characteristics [49]. Under in vitro conditions, the exogenous application of salicylic acid is

useful for the growth and development of plants. Salicylic acid has been reported to enhance microtuber formation [50] and increase starch percentage [51] in potato plants (*Solanum tuberosum*) propagated in vitro. Glutamine is an amino acid and nitrogen source that supports several metabolic processes. The addition of amino acids provides a readily available primary source of nitrogen in tissue culture systems, and uptake occurs more rapidly than the uptake of inorganic nitrogen in the same medium [52]. Glutamine has been reported to have positive effects in plant tissue cultures [53,54]. Jasmonic acid is an endogenous plant-growth regulating substance. In the present study, jasmonic acid at 1 mg L−<sup>1</sup> increased corm size compared with corm size in the control, but 2 mg L−<sup>1</sup> of jasmonic acid did not produce positive effects. Exogenous jasmonic acid is considered to play an important role in bulb formation, as shown in studies on garlic [55] and onion [56]. In addition, exogenous jasmonic acid supported the microtuberization of three food yam (*Dioscorea*) species [57] and promoted the enlargement of in-vitro-grown bulbs in shoot cultures of *Narcissus* plants [58] and the daughter bulb development of *Tulipa gesneriana* [59]. However, the positive or negative effects of jasmonic acid are species- and concentration-dependent.

**Table 2.** Effects of salicylic acid, glutamine, and jasmonic acid treatments on saffron daughter corm diameter and fresh weight after 14 weeks of culture.


Different letters within a set of values denote significant differences at *p* ≤ 0.05 according to Tukey's test.

**Figure 4.** Influence of salicylic acid, glutamine, and jasmonic acid on saffron daughter corm formation using apical bud explants: (**a**) Surface disinfection and bud break of saffron apical buds cultured on MS medium supplemented with 3% sucrose and1gL−<sup>1</sup> of activated charcoal 2 weeks after incubation; (**b**) Shoot development of saffron apical buds and growth 6 weeks after incubation; (**c**,**d**) Daughter corm formation 12 and 16 weeks, respectively, after treatment with salicylic acid (75 and 150 mg L<sup>−</sup>1), glutamine (600 and 1200 mg L<sup>−</sup>1), and jasmonic acid (1 and 2 mg L−1).

## **4. Conclusions**

The present results indicate that saffron flowering can be induced in vitro, and the harvested stigma of these flowers could be used as a source of spice or pharmaceuticals. Compared with solid culture, liquid cultures/bioreactors improved daughter corm diameter and fresh weight. Moreover, salicylic acid at 75 mg L−<sup>1</sup> and glutamine at 600 mg L−<sup>1</sup> increased corm diameter and fresh weight. These findings will help improve in vitro cormogenesis toward the production of saffron in a controlled environment. However, further investigations on optimal growth conditions, e.g., medium composition, light intensity and quality, and incubation temperature, are required to optimize in vitro cormogenesis.

**Author Contributions:** Conceptualization, Y.H.D. and A.A.; methodology, Y.H.D., A.A. and A.A.A.- A.; formal analysis, Y.H.D., A.A. and A.A.A.-A.; investigation and data curation, Y.H.D., A.A., A.A.A.-A. and M.A.-M.; validation, Y.H.D., A.A., A.A.A.-A. and M.A.-M.; visualization, Y.H.D., A.A., A.A.A.-A. and M.A.-M.; writing—original draft preparation, Y.H.D. and A.A.; writing—review and editing, Y.H.D., A.A., A.A.A.-A. and M.A.-M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This project was funded by the National Plan for Science, Technology and Innovation (MAARIFAH), King Abdulaziz City for Science and Technology, Kingdom of Saudi Arabia, Award Number (15-AGR3704-02).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** All data are presented within the article.

**Acknowledgments:** This project was funded by the National Plan for Science, Technology and Innovation (MAARIFAH), King Abdulaziz City for Science and Technology, Kingdom of Saudi Arabia, Award Number (15-AGR3704-02).

**Conflicts of Interest:** The authors declare no conflict of interest.
