Stable plastid transformation in Scoparia dulcis L.

Abstract

In the present investigation we report stable plastid transformation in L., a versatile medicinal herb via particle gun method. The vector KNTc, harbouring as a selectable marker and as a reporter gene which were under the control of synthetic promoter pNG1014a, targets inverted repeats,/ of the plastid genome. By use of this heterologous vector, recovery of transplastomic lines with suitable selection protocol have been successfully established with overall efficiency of two transgenic lines for 25 bombarded leaf explants. PCR and Southern blot analysis demonstrated stable integration of foreign gene into the target sequences. The results represent a significant advancement of the plastid transformation technology in medicinal plants, which relevantly implements a change over in enhancing and regulating of certain metabolic pathways.

Improved plastid transformation efficiency inScoparia dulcisL.

“…In our lab, the same trnA/trnI of IR region was targeted to recover transplastomic lines of Momordica charantia by a Cucumis sativus based heterologous vector (CuIA) (Narra et al 2018b) and obtained 2 transplastomic lines from 15 bombarded plates using petiole explants. Two different plastid vectors, KNTc, and pFaadAII targeting trnR/trnN and rpl32/trnL of IR and SSC regions of plastid genomes were employed to transform chloroplasts of S.dulcis (Muralikrishna et al 2016;Narra et al 2018a). Using these vectors, two transplastomic lines were recovered from 25 bombarded explants i.e 8%, whereas, in the current investigation three transplastomic lines were recovered from 25 transformed explants i.e 12%.…”

“…Each plate containing 5 explants, totally twenty-five explants were bombarded and cultured for two days on the same medium at 25 ± 2°C, photoperiod is 16/8 hrs. Later tissues were cut into desired size 1 cm 2 and placed on selection medium (MS medium, 4 mg/L BAP and 0.2 mg/L IAA and 75 mg/L spectinomycin) (Muralikrishna et al 2016), selection pressure was maintained throughout the screening process. Tissue was subcultured every two weeks.…”

“…First successful plastid transformation was reported in Chlamydomonas reindhartii (Boynton et al 1988). Later, it has been expanded to various higher plants i.etobacco (Svab et al 1990), potato (Sidorov et al 1999a), Brassica (Cheng et al 2010), Arabidopsis (Sikdar et al 1998), tomato (Ruf et al 2001), Soybean (Dufourmantel et al 2004), lettuce (Kanamoto et al 2006), Oryza sativa (Lee et al 2006;Wang et al 2018), cotton (Kumar et al 2004b), brinjal (Singh et al 2010), Scoparia (Muralikrishna et al 2016;Narra et al 2018a), Maize (Sidorov et al 2019) and Capsicum (Kota et al 2019),etc. Flanking regions of insertional sequences and selectable markers are the crucial elements to develop species-specific chloroplast transformation vector (Wang et al 2009).…”

“…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). The present study is focused to improve plastid transformation efficiency by targeting a new insertion site of plastid genome IR region of S. dulcis.…”

Plastid Transformation: How Does it Work? Can it Be Applied to Crops? What Can it Offer?

“…To date, the plastids of over 20 flowering plants have been transformed [15] (Table 1). In addition to the crops mentioned above, recent successes in plastid transformation have been reported in the plant species bitter melon [16], and the medicinal plant sweet wormwood (Artemisia annua) [17] and licorice weed (Scoparia dulcis) [18,19] (Table 1). Based on these successful cases, plastid transformation should be applicable to many plant families, whether they are monocots or dicots.…”