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

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.