Application of the phosphomannose-isomerase/mannose selection system in the Agrobacterium-mediated transformation of Lonicera hypoglauca Miq.

Abstract

The dried buds of Lonicera hypoglauca Miq. have antipyretic, antidotal and anti-inflammatory properties and as Flos lonicerae are widely used in Chinese patent medicine. Because of its medicinal value, there is a need to establish its transformation system in order to study the biosynthesis pathway of its active substances. The phosphomannose isomerase (PMI) gene is probably the most effective selection maker for the transformation system of plants. To establish PMI selection system for transformation of L. hypoglauca, explants were cultured on the induction medium (MS + 0.1 mg/L NAA + 1.0 mg/L 6-BA + 6 g/L agar) supplemented with different ratios of mannose and sucrose, and data on callus formation were collected. After the concentration of mannose and sucrose was determined, 172 leaf explants, 61 leafstalks and 53 stem segment explants were infected with Agrobactrium containing pCAMBIA1301-PMI. The putatively transformed calluses induced from these explants were detected by PCR, RT-PCR and chlorophenol red assay (CPR). We found that callus formation was effectively suppressed on the induction medium supplemented with mannose. The appropriate concentration of mannose and sucrose in the selection medium was 8 g/L and 22 g/L, under which the transformation frequency of leaf from L. hypoglauca was 15.96%. CPR assay for callus induced from leafstalk and stem have a moderate false negative rate when RT-PCR used as criterion. Taken together, it was concluded that PMI/mannose selection system could be used in the transformation of L. hypoglauca, and CPR assay might be suited for the preliminary screening of the transformation of L. hypoglauca.