Comprehensive in vitro regeneration study with SCoT marker assisted clonal stability assessment and flow cytometric genome size analysis of Carthamus tinctorius L.: an important medicinal plant

Key message An efficient, reproducible in vitro regeneration protocol was achieved for safflower via direct, callus mediated organogenesis and somatic embryogenesis. Histology and SEM study revealed typical dicot embryogenic stages. SCoT molecular marker and flow cytometric 2C genome size analysis were further applied for evaluating genetic fidelity of in vitro regenerated safflower. An efficacious and reproducible in vitro regeneration technique for safflower was established using varying concentrations and composition of plant growth regulators (PGRs) supplemented Murashige and Skoog (MS) medium. Successful in vitro seed germination in half strength MS (H-MS) with 1.4 mu M GA(3) resulted in procurement of sterile explants (cotyledons, apical meristems) for in vitro study. Callogenesis (2.2 mu M BAP + 2.7 mu M NAA), indirect organogenesis of shoot buds (0.54 mu M NAA + 9.08 mu M TDZ), somatic embryogenesis (2.2 mu M BAP + 5.4 mu M NAA) and somatic embryo germinated plantlets (H-MS + 1.4 mu M GA(3) + 2.2 mu M BAP + 5.4 mu M NAA) were successfully obtained. Histological study and scanning electron micrographs of embryogenic callus revealed pre-globular, heart-shaped and torpedo stages of dicot embryogeny. H-MS + 8 mu M NAA showed maximum rhizogenic response with a mean root and shoot length of 17.5 mm and 48.50 mm respectively in 2.2 mu M BAP + 0.54 mu M NAA bearing an average of 9 capitula per plantlet with 70% post transplantation survival rate. True to type nature of the regenerates was confirmed using Start Codon Targeted (SCoT) marker, exhibiting 100% and 97.3% monomorphic bands for direct and somatic embryo regenerated plants respectively. Flow cytometry method (FCM) was employed for 2C DNA content analysis. The histogram peaks of 2C nuclear DNA content of in vitro regenerated safflower (direct and embryo derived) were similar to the peak of field grown donor plant. 2C nuclear DNA content of field grown, direct and somatic embryo regenerated C. tinctorius was 2.65 +/- 0.04 pg, 2.62 +/- 0.06 pg and 2.68 +/- 0.04 pg respectively, further verifying genetic homogeneity. All things considered, the above protocol is insusceptible to genetic alteration and can be used for large scale production and sustainable utilization of desired genotype.

Automated summary

An efficient and reproducible in vitro regeneration protocol was established for safflower using direct and callus-mediated organogenesis and somatic embryogenesis. Various techniques such as histology, molecular markers, and flow cytometry were utilized to confirm the genetic fidelity of the regenerated plants, demonstrating their potential for large-scale production and sustainable utilization.