A robust workflow for indirect somatic embryogenesis and cormlet production in saffron (Crocus sativus L.) and its wild allies; C. caspius and C. speciosus

Saffron (Crocus sativus L.) and its wild relatives, Crocus caspius and Crocus speciosus are of considerable significance in the pharmaceutical, nutraceutical, and ornamental bulbs industry. Towards the ultimate goal of the conservation of wild Crocus species and establishment of an efficient workflow for in vitro production of Crocuses, efficient protocols were developed for disinfection and in vitro production of cormlets in C. sativus and its wild allies C. caspius and C. speciosus. Moreover, the differential expression of the Somatic Embryogenesis Receptor-like Kinase (SERK) gene was evaluated as a potential molecular marker during embryogenesis between embryogenic and non-embryogenic calli. A highly efficient disinfection recipe and a low-cost TDZ-free protocol have been successfully developed for in vitro cormlet production in three Crocus species. MS medium containing 10.18 μM 2, 4-D + 4.44 μM BAP was most efficiently induced callus and somatic embryo formation. The highest conversion frequency and maximum cormlet weight were achieved in MS containing 5.37 μM NAA + 8.88 μM BAP. The SERK expression was significantly much higher in embryogenic calli than non-embryogenic in all Crocus species. The current low-cost and easy-to-use recipe suggests a promising in vitro propagation workflow for mass production of uniform pathogen-free cormlets of Crocus species, as well as a platform to better conservation of wild Crocus species and effective gene and genome editing using CRISPR-Cas9 in future studies. Callogenesis; Conservation; CRISPR-Cas9; Disinfection; Pathogen-free; TDZ-free; Transformation.