Arabidopsis and maize transformation

Arabidopsis transformation was carried out using the floral dip method according to Clough and Bent [28]. Mature seeds were harvested, dried down, and stored until selection. Seeds were treated with 70 % ethanol for 1 min, 1 % sodium hypochlorite for 15 min, followed by five rinses with sterile water. Sterilized seeds were planted on kanamycin selection plates (75 μg mL⁻¹) and grown in a chamber at 21 °C under 16 h of lighting (50–100 μ Einsteins m⁻²s⁻¹) for 10 days. Kanamycin-resistant seedlings with green leaves and well-established roots were selected from the plates and planted into moistened potting soil.

The maize calli derived from immature Hi-II zygotic embryos were used as target tissues for bombardment. The bombardment and subsequent selection of bar-resistant calli were performed according to Songstad [29]. Briefly, type II calli derived from Hi-II were transferred to osmotic medium and then bombarded two times. Bombarded calli were incubated at 28 °C in the dark. After selection and regeneration, plantlets grew out from bar-resistant calli. The genomic DNA of putative transgenic maize plantlets was isolated, and PCR was performed to confirm the presence of the transgene (Table 1). The T₀ transgenic VHb plants were used as the donors, and two maize inbred lines (Zheng58 and CML50) were used as recurrent parents. After six cycles of marker-aided backcrossing and one instance of self-pollination, we obtained three homozygous Zheng58 (VHb) and five homozygous CML50 (VHb) lines. These transgenic lines were named Zheng58 (VHb) 1 to 3, and CML50 (VHb) 1 to 5. The expression levels of VHb in these homozygous lines were determined by qRT-PCR. In the next generation, the lines including Zheng58 (VHb)1-3, Zheng58 (VHb)2-5, CML50 (VHb)3-2, and CML50 (VHb)5-5 that expressed high levels of VHb and produced enough seeds were chosen for further research.

Primers used in this study