Magenta vessel

Nodal explants (1.5 to 2 cm length, with a single bud) were inoculated for 5 weeks to Magenta vessels (77 mm × 77 mm × 97 mm; Sigma Chemical Co., St. Louis, MO, USA) containing 60 mL MS medium, 3% sucrose, and 0.8% agar and supplemented with various concentrations of BAP (0.1, 1.0, 2.0, 3.0 mg/L), TDZ, or zeatin (Sigma Chemical Co., St. Louis, MO, USA) (0.1, 0.5, 1.0, 1.5 mg/L). The cultures were then inoculated onto MS medium without plant growth regulator (PGRs) for 4 weeks and kept under the same conditions. There were four replicates in each treatment and each replicate was represented by a Magenta vessel containing 4 nodal explants. After seven weeks of incubation, observations on the number of axillary shoots per explant, length of the longest shoot per explant, shoot fresh weight, and shoot dry weight were recorded. Dry weight was measured after drying the shoots for 48 h at 60 °C.

Arabidopsis thaliana (Arabidopsis) plants were grown on 0.5% Phytagel/0.5×MS media within Magenta Vessels (Sigma-Aldrich Chemical Co., St. Louis, MO, USA). Plants were grown under a broad-spectrum light-emitting diode (LED) light bank (100 µmol/m²) at 22°C +/− 2°C. After large leaf rosettes developed (approximately 30 days), single Arabidopsis leaves were either infiltrated with Escherichia coli (E. coli) or harvested. Those that were not infiltrated were mixed with approximately 50 µl of E. coli pellet. Combinations of these samples were then used for the optimization of the CBIO DNA extraction protocols with various lysis buffers. For each step of the extraction process, eluants were collected and used for PCR screening. Specific primers for universal bacterial 16S rRNA and Arabidopsis-specific heat shock protein 70 (HSP70) were used to determine whether the DNA came from plants or bacteria.

Alfalfa transgenic seeds were obtained by crossing transgenic plants S1, S2 and S3 with 432-19-17 genotype plants. Transgenic and wild type seeds were treated with sulfuric acid for 10 minutes, washed three times with sterile water and placed in petri dishes with 1% agar water at 16 h of light (100 μmoles/m²s) and 25°C. Germinated plantlets were transferred to MS 0.5X flasks and incubated at 25°C with 16 h photoperiod for 1 month, after which plants were transferred to 1:1 vermiculite:perlite and maintained in magenta vessels (SIGMA) to conserve the humidity. Two-month-old plants were inoculated with P. medicaginis CT1 by spraying spore suspension to all aerial tissues. Percentage of diseased leaflet was analyzed 30-day post-inoculation and the number of plants with regrowth and the percentage of highly defoliated plants was counted 60-days post-inoculation. As an additional severity parameter of plant disease, the percentage of vigor affected plants (i.e. with visual signs of turgidity loss compared with non-inoculated plants) was estimated. All experiments were carried out with at least 10 independent plants per treatment and with at least 5 non inoculated wild type plants used as control.

Gold microprojectiles coated with the transformation vectors pL3-Trxm-CT1 and pL3-Trxm/CT1 were bombarded into in vitro-grown N. tabacum var. Petite Havana SR1 leaves (National Germplasm Resources Laboratory, Beltsville, MD, USA) using a PDS1000/He (Bio-Rad, Hercules, CA, USA) biolistic device, as described previously [37 (link)]. After bombardment, the leaves were incubated in the dark for 2 days at 28 °C, then cut into small pieces (around 5 × 5 mm) and placed adaxial side up on regeneration medium (RMOP) in Magenta vessels (Sigma, St Louis, MO, USA) containing 500 mg/L spectinomycin dihydrochloride (Duchefa Biochemie, Haarlem, Netherlands) as the selecting agent. The growth conditions of the culture chamber were 28 °C, 120 μmol m⁻² s⁻¹ and a 16-h photoperiod. Spectinomycin-resistant shoots were cut into small pieces (around 2 × 2 mm) and subjected to successive rounds of regeneration in the same selection medium. Finally, resistant shoots were rooted on growth medium containing 500 mg/L of spectinomycin and transferred into soil for homoplasmy confirmation and seed production.
Transformed and WT seeds were germinated on growth medium supplemented with or without 500 mg/L of spectinomycin, respectively. After 4–6 weeks, the seedlings were transferred to pots containing soil and grown in a greenhouse under natural sunlight.

After cryostorage, the plumules were cultured in sterile plastic Petri dishes (60 mm) on Murashige and Skoog Medium (MS) [27 (link)] (Fig. 1D) supplemented with 1.0 mg/l 6-Benzylaminopurine (BAP) [29 (link)] and sucrose (30 g/l). After one week of culture, the plumules were transferred to Woody Plant Medium (WPM) [30 ] supplemented with 0.8 mg/l BAP [31 ]. The plumules were cultured under standard conditions under light, with a 16 h/8 h light/dark photoperiod at 25 °C and a light intensity of 78 µM/m^− 2s^− 1. Shoots with small leaves formed bunches (Fig. 1E), so they were separated and transferred every 3–4 weeks into magenta vessels (Sigma) onto the same medium and were subcultured (Fig. 1F). The plumules were considered viable if growth was observed after 2 weeks of in vitro culture (referred to in this study as survival). Additional observations were made after 3 months of in vitro culture (referred to in this study as recovery).