Plant regeneration and stable transformation in the floricultural plant Cleome spinosa, a C3 plant closely related to the C4 plant C. gynandra

Abstract

Cleome spinosa is widely used as a garden ornamental in many countries. Here we determined the optimal conditions for plant regeneration from different tissue explants grown in vitro. Induction medium containing MS salts, MS vitamins, 3% sucrose, 1 mg l⁻¹ BA, 200 mg l⁻¹ timentin, and 0.8% agar was sufficient for shoot regeneration of all the tissue explants examined, including leaf, hypocotyl, and cotyledon. Subsequently, an Agrobacterium tumefaciens-mediated method was developed to transform the vector pCHS, which carries the transgenes Petunia chalcone synthase (chs) and selection marker neomycin phosphotransferase II (nptII), into C. spinosa. From a total of 368 cotyledon explants, 13 putative transgenic lines were regenerated from selection medium supplemented with 50 mg l⁻¹ kanamycin and 200 mg l⁻¹ timentin, and transferred to the greenhouse. Genomic PCR and Southern blot analyses revealed that the nptII transgene was present in all 13 transgenic plants. Similarly, when the Petunia chs transgene was used as a probe in Southern blot analysis, single or multiple hybridization bands were detected in 12 out of the 13 transgenic plants. In addition, T₁ progeny assay from selected transformants showed that the nptII transgene can be transmitted in a Mendelian manner from transgenic parents into their progeny. This is the first report of stable transformation of the C₃ dicotyledon C. spinosa, which will facilitate functional comparison of cell-type specific genes with counterpart C₄ dicotyledon C. gynandra using transgenic approaches.

Transformation and characterization of transgenic rice and Cleome spinosa plants carrying the maize phosphoenolpyruvate carboxylase genomic DNA

“…Freshly collected seeds from white flowering plants were sterilized, and germinated on germination medium as previously described (Tsai et al 2012). In vitro cotyledons were used in transformation experiments.…”

“…Protocols and media for C. spinosa regarding co-culture, Agrobacterium infection, kanamycin selection and plant regeneration were as previously described (Tsai et al 2012). …”

“…After electrophoresis on 0.8% agarose gel in TAE buffer, the gel was washed, denatured, and transferred onto membrane as previously described (Tsai et al 2012). To prepare the non-radioactive PCR DIG probe (Roche Applied Science, Germany), equal amounts (20 pmol) of primers ZmPEPC-f2 and ZmPEPC-r2 were used to amplify the foreign gene maize pepc DNA fragment (753 bp) from the expression vector PEPCgenome/pBIH2 (1 µg) and the reaction mixture as previously described (Tsai et al 2012). For PCR amplification of the probe, hybridization, and detection were as previously described (Tsai et al 2012).…”

“…After electrophoresis and transfer onto membrane, DIG-labelled PCR products from two gene-specific primer sets, Kan-F and Kan-R for amplification of the 795-bp-long DNA fragment corresponding to the full-length selection marker nptII gene, and ZmPEPCf2 and ZmPEPC-r2 for the 753-bp-long DNA fragment corresponding to the maize pepc gene were used as probes in hybridization experiments. Detailed protocols were described previously (Tsai et al 2012).…”

“…In this study, an Agrobacterium-mediated method established in our laboratory was adopted to transform the PEPCgenome/pBIH2 vector into the japonica rice cultivar Tainung 67 (TNG67), an important rice cultivar in Taiwan, and a C 3 dicot plant Cleome spinosa (Tsai et al 2012). The genus Cleome is closely related to Arabidopsis; but more interestingly, contains C 3 and C 4 species.…”