Establishment of Agrobacterium rhizogenes-mediated hairy root transformation of Crocus sativus L

Abstract

Efficient transformation system for genetic improvement is essential in Crocus sativus, as it lacks sexual reproduction. This is the first report wherein an efficient protocol is developed for the transformation of Crocus sativus L. by Agrobacterium rhizogenes strain ARqua1 with a transformation efficiency of 78.51%. The ARqua1 strain harboring both Ri plasmid and binary vector plasmid pSITE-4NB, and marker genes for red fluorescent protein (RFP) and a β-glucuronidase (GUS) reporter gene were used for selection. Transformation was confirmed by RFP signal, GUS reporter assay and polymerase chain reaction (PCR) analysis of the test samples after 21 days post inoculation. These results confirm the establishment of protocol for hairy root transformation in C. sativus that can be further used for gene transfer or gene editing in Crocus for its genetic improvement.

Keywords: Agrobacterium rhizogenes; Crocus sativus; Hairy root; Monocot; Saffron; Transformation.

Fig. 1

Hairy root induction in C. sativus basal plate explants by A. rhizogenes at 21 days post inoculation (dpi) on MS media. a Small roots observed in the control sample without A. rhizogenes inoculation, b, c hairy root induction observed in samples inoculated with A. rhizogenes

Fig. 2

Fluorescence microscopy images of C. sativus roots showing RFP fluorescence signal at Ex.532 nm/Em.580 nm using Evos inverted fluorescent microscope. a Control or non-transformed root, b transformed saffron root harboring RFP gene, and c cross-section of the transformed root

Fig. 3

GUS histochemical staining of the control and transformed roots of C. sativus. a Non-transformed control plant roots and b transformed roots showing indigogenic product of the GUS assay.

Fig. 4

Agarose gel showing PCR amplification of GUS gene in the transgenic hairy roots. A. rhizogenes harboring GUS gene used as positive control and non-transformed roots were used as negative control. T1, T2 and T3 represent three different transformed plant roots