Highly efficient production of transgenic Scoparia dulcis L. mediated by Agrobacterium tumefaciens: plant regeneration via shoot organogenesis

Abstract

Efficient Agrobacterium-mediated genetic transformation of Scoparia dulcis L. was developed using Agrobacterium tumefaciens strain LBA4404 harboring the binary vector pCAMBIA1301 with b-glucuronidase (GUS) (uidA) and hygromycin phosphotransferase (hpt) genes. Two-day precultured leaf segments of in vitro shoot culture were found to be suitable for cocultivation with the Agrobacterium strain, and acetosyringone was able to promote the transformation process. After selection on shoot organogenesis medium with appropriate concentrations of hygromycin and carbenicillin, adventitious shoots were developed on elongation medium by twice subculturing under the same selection scheme. The elongated hygromycin-resistant shoots were subsequently rooted on the MS medium supplemented with 1 mg l -1 indole-3-butyric acid and 15 mg l -1 hygromycin. Successful transformation was confirmed by PCR analysis using uidA-and hpt-specific primers and monitored by histochemical assay for b-GUS activity during shoot organogenesis. Integration of hpt gene into the genome of transgenic plants was also verified by Southern blot analysis. High transformation efficiency at a rate of 54.6% with an average of 3.9 ± 0.39 transgenic plantlets per explant was achieved in the present transformation system. It took only 2-3 months from seed germination to positive transformants transplanted to soil. Therefore, an efficient and fast genetic transformation system was developed for S. dulcis using an Agrobacteriummediated approach and plant regeneration via shoot organogenesis, which provides a useful platform for future genetic engineering studies in this medicinally important plant.

Biolistic transformation of Scoparia dulcis L.

“…In conclusion, we for the first time established a simple and efficient protocol for recovery of transgenic S. dulcis generated via biolistic-mediated nuclear transformation. Although the stable transformation efficiency (52.0 %) reported in the present study is less than that of earlier report (54.6 %) that which was attained by Agrobacterium mediated transformation (Aileni et al 2011a). However, biolistic method is the most efficient way to adopt towards plastid transformation in S. dulcis.…”

“…The availability of biolistic mediated-nuclear transformation in S. dulcis, has significant interest in developing transplastomic plants for metabolic engineering of certain regulatory gene traits and also in extensive development of molecular farming for production of biopharmaceuticals (Bock 2007). In previous transformation studies of S. dulcis 3.9 ± 0.39 transgenic plantlets per leaf explant were recorded (Aileni et al 2011a). While there was no remarkable differences observed in the present investigation (3.7 ± 0.24 transgenic plantlets per leaf/explant).…”

“…Plant genetic manipulation is more reliable for the production of specific metabolites in controlled (in vitro) conditions (Ludwig-Müller et al 2014). Interestingly, highly efficient transformation (Aileni et al 2011a;Yamazaki et al 1996) along with profused in vitro response (Aileni et al 2011b;Aileni et al 2008) was noticed in S. dulcis, that found to be similar to model plants like Nicotiana tabacum and Arabidopsis thaliana.…”

Improved plastid transformation efficiency inScoparia dulcisL.

“…In our lab, a reliable regeneration, Agrobacterium and biolistic mediated genetic transformation using uidA, hpt and gus and nptII genes respectively in S. dulcis (Aileni et al 2011a;Srinivas et al 2016). In Scoparia dulcis, efficient plant regeneration system comparable to model plant system like tobacco using leaf explant made more convenient to achieve reliable chloroplast transformation by targeting rpl32/trnL and trnR/trnN insertional sites of SSC and IR regions of plastid genome using pFaadAII and KNTc heterologous vectors respectively (Muralikrishna et al 2016;Narra et al 2018a).…”

Stable plastid transformation in Scoparia dulcis L.

“…Stable genetic transformation in S. dulcis was achevied through Agrobacterium using uidA and hpt genes (Aileni et al 2011) and efficient biolistic transformation using uidA and npt II genes (Srinivas et al 2016). Development of chloroplast transformation in this plant system will provide new opportunities towards plastid based metabolic engineering of certain valuable natural product entities and to delve this technology towards enhanced metabolite production and extension in engineering of biosynthetic pathways by plastid transformation.…”