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. 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. 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.

中文翻译：

---

在藏红花 (Crocus sativus L.) 中使用 CRISPR/Cas9 开发高效愈伤组织生产、体细胞胚胎发生和基因编辑系统

番红花是一种难以进行遗传转化和遗传改良的植物，主要​​是由于农杆菌介导的转化和营养繁殖的困难。有效的基因组编辑需要熟练的愈伤组织生产和将 Cas9 和 sgRNA 输送到植物中的有效方法。在这里，我们展示了农杆菌介导的藏红花转化。此外，我们在该植物中开发了一个基于 CRISPR-Cas9 的系统，用于在未来进行有效的基因敲除或编辑。有效的愈伤组织生产和再生为在植物中开发有能力的转化系统带来了重要的好处。藏红花球茎切片经过两步灭菌程序并在补充有 2,4-D (0.5 mg/L)、BAP (1 mg/L) 的完整 MS 培养基上生长，愈伤组织起始增殖率超过 70% ), IAA (1 mg/L)，光周期为 16/8 h，相对湿度为 45%，20 ± 2 °C。在 15 ± 2 °C 下，通过在补充有 TDZ (0.5 mg/L) + IAA (1 mg/L) + 活性炭 (0.1 g/L) 的 MS 培养基上使用成熟的体细胞胚胎，在 8 周内完成了体外蠓的产生。使用农杆菌介导的转化的尝试导致二元载体成功整合到藏红花的体细胞胚中，转化效率为 4%。PCR和Southern印迹分析证实Cas9整合到藏红花中。愈伤组织产生、体细胞胚胎发生和再生的方案是标准化的。本研究中整合 Cas9 的成功证明是开发藏红花基因操作策略的第一步，迄今为止，藏红花一直被认为是顽固的。