Overexpression of TcCHS Increases Pyrethrin Content When Using a Genotype-Independent Transformation System in Pyrethrum (Tanacetum cinerariifolium)

Abstract

Pyrethrum (Tanacetum cinerariifolium) is one of the most important industrial crops for the extraction of pyrethrins, which are natural insecticidal compounds. Progress in pyrethrum molecular breeding with the objective of increasing pyrethrin content has been slow for lack of a suitable gene transfer system. Regeneration recalcitrance is a crucial barrier to establishing a genetic transformation system in pyrethrum. Therefore, in this study, an Agrobacterium-mediated transformation system in pyrethrum was developed using shoot apical meristems from germinated seedlings. Factors affecting transformation efficiency were optimized. Optimal conditions included explants at the “no true leaf” stage with a half apical meristem, an Agrobacterium tumefaciens cell density of OD600 = 0.5, two days of cocultivation, and the incorporation of 1.5 mg L−1 6-BA and 30 mg L−1 kanamycin into the selection medium. Under the optimized conditions, two expression cassettes (proTcCHS-GUS and proRbcS-TcCHS) were successfully transformed into pyrethrum. Polymerase chain reaction (PCR), Southern blotting, reverse-transcription quantitative PCR (RT-qPCR), and histochemical staining confirmed the identity of proTcCHS-GUS transgenic plants. PCR and RT-qPCR analyses confirmed the identity of proRbcS-TcCHS transgenic plants. The transformation efficiency was 0.83% (5 transgenic lines/600 infected explants). The relative concentration of pyrethrins in proRbcS-TcCHS transformants (OX T0-1: 1.50% or OX T0-2: 1.24%) was higher than that in nontransformed plants (WT: 0.76%). Thus, the genetic transformation system overcame the low regeneration efficiency and integrated a foreign gene into the pyrethrum genome. The new system is a suitable and effective tool for creating high-yielding cultivars of pyrethrum.