Development of a system for efficient callus production, somatic embryogenesis and gene editing using CRISPR/Cas9 in Saffron (Crocus sativus L.)

Abstract

Background: Crocus sativus is a recalcitrant plant for genetic transformation and genetic improvement, largely due to difficulties in Agrobacterium mediated transformation and vegetative reproduction. Effective genome editing requires proficient callus production and an efficient method to deliver Cas9 and sgRNAs into the plant. Here, we demonstrate Agrobacterium-mediated transformation of saffron. Further, we developed a CRISPR-Cas9 based system in this plant, for efficient gene knockout or edits in future. Results: Efficient callus production and regeneration confers important benefits in developing competent transformation system in plants. More than 70% multiplication rate of callus initiation was achieved from corm slices of saffron subjected to a two-step sterilization procedure and grown on complete MS medium supplemented with 2,4-D (0.5 mg/L), BAP (1 mg/L), IAA (1 mg/L), photoperiod of 16/8 h and 45% relative humidity at 20 ± 2 °C. In vitro cormlet generation was accomplished in 8 weeks by using mature somatic embryos on MS medium supplemented with TDZ (0.5 mg/L) + IAA (1 mg/L) + Activated charcoal (0.1 g/L) at 15 ± 2 °C. The attempt of using Agrobacterium-mediated transformation resulted in successful integration of the binary vector into the somatic embryos of saffron with a transformation efficiency of 4%. PCR and Southern blot analysis confirmed the integration of Cas9 into saffron. Conclusion: The protocol for callus production, somatic embryogenesis and regeneration was standardised. Successful demonstration of integrated Cas9 in this study constitutes first step in developing strategies for genetic manipulation of saffron, which has so far been considered recalcitrant. Furthering the development of this technology holds significant potential for advancing genetic research in saffron by integrating multigene targeting and/or use of recyclable cassettes.

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

“…Approximately 81% of survival rate was reported in a similar study utilizing 0.1% (w/v) carbendazim, 0.2% (w/v) mancozeb followed by 50% (v/v) sodium hypochlorite and 1.6% (w/v) mercuric chloride in C. sativus (Yasmin and Nehvi 2014). The use of 0.1% mercuric chloride followed by 4% sodium hypochlorite resulted in 86% survival rate in C. sativus (Chib et al, 2020). Results showed that the development of disinfection protocols for wild crocus species is more challenging than C. sativus, maybe because of their recalcitrant nature.…”

“…It was reported that 0.25 mg/L 2,4-D þ 1 mg/L BAP showed the best results for callus initiation and callus growth in Crocus (Zeybek et al, 2012); while 4 mg/L NAA þ 4 mg/L TDZ was proved to successfully induce callus in five Crocus species (Verma et al, 2016). In another report, over 70% callus induction was achieved using MS medium containing 0.5 mg/L 2,4-D, 1 mg/L BAP and 1 mg/L IAA (Chib et al, 2020). It is also reported that higher levels of auxin relative to cytokinin had increased somatic embryogenesis in the Ajowan calli (Carum copticum L.) (Niazian et al, 2017).…”

“…Moreover, saffron sterility culminated in poor genetic variation and as a result, limited the use of conventional breeding approaches; so, researchers are now trying to use alternative techniques such as biotechnological approaches. Recently, a successful CRISPR/Cas9 system was reported for gene editing in saffron (Chib et al, 2020). Altogether, the use of tissue culture for mass production of cormlets is inevitable; it also provides the requisite platform for the improvement of saffron traits through molecular breeding.…”

CRISPR/Cas9 System: A Potential Tool for Genetic Improvement in Floricultural Crops

“…Only the f3ha-f3hb mutant plants had visible changes, pale purplish-pink flower color, while the rest, including plants with a single-copy gene knockout, had purple-violet flowers that resembled wild-type petunia. Chib et al [ 128 ] established a CRISPR/Cas9-based system for future research advancements in saffron ( Crocus sativus L.). Zhang et al [ 129 ] presented evidence for redefining DPL’s role (DPL: THE R2R3-MYB transcription factor, which is connected to anthocyanin, has been linked to the regulation of corolla tube venation) in Petunia plants.…”

Computational screening of miRNAs and their targets in saffron (Crocus sativus L.) by transcriptome mining

“…Crocus sativus L., also known as saffron, is the most precious spice in the world (Chib et al 2020). This species spreads from central Europe to western China (Harpke et al 2013).…”