TX430, a non-tannin sorghum variety, was used in this study. All sorghum embryo donor plants and transgenic plants were grown in a greenhouse located in Johnston, Iowa. Greenhouse temperatures averaged 29°C during the day and 20°C at night with a 12 h d/night photoperiod and supplemental lighting was provided by a 3:1 ratio of metal halide (1,000 W) and high-pressure sodium (1,000 W) lamps. The components of the media used in this study are listed in Table 1 . The Escherichia coli phosphomannose isomerase (PMI) gene (Miles and Guest 1984 (link)), which catalyzes the reversible isomerization of mannose-6-phosphate to fructose-6-phosphate (serving as a precursor for the glycolytic pathway and enabling growth on mannose-containing media) and a codon-modified phosphinothricin acetyltransferase (moPAT) gene, which confers resistance to the herbicide glufosinate-ammonium (US patent no. 6,096,947), were used as the selectable markers. The baseline transformation protocol is described in detail as “treatment C” in Zhao et al. (2000 (link)). Briefly, freshly harvested sorghum immature grains were sterilized with 50% bleach and 0.1% Tween-20 for 30 min under vacuum and then rinsed with sterile water three times. The embryos were subjected to the following five sequential steps: (1) Agrobacterium infection: embryos were incubated in an Agrobacterium suspension (OD = 0.7 at 550 nm) with PHI-I medium for 5 min; (2) co-cultivation: embryos were cultured on PHI-T medium following infection for 3 d at 25°C in the dark; (3) resting: embryos were cultured on PHI-T medium plus 100 mg/l carbenicillin for 4 d at 28°C in the dark; (4) selection: embryos were cultured on PHI-U medium for 2 wk, followed by culture on PHI-V medium for the remainder of the selection process at 28°C in the dark, using subculture intervals of 2–3 wk; (5) regeneration: callus was cultured on PHI-X medium for 2–3 wk in the dark to stimulate shoot development, followed by culture for 1 wk under conditions of 16 h light (40–120 μE m−2 s−1) and 8 h dark at 25°C, and a final subculture on PHI-Z medium for 2–3 wk under lights (16 h, 40–120 μE m−2 s−1) to stimulate root growth. Regenerated plantlets were transplanted into soil and grown in the greenhouse (Zhao et al. 2000 (link)). T0 plants were self-pollinated to produce T1 progeny for further analysis. A new optimized protocol is described in the Results section of this manuscript.
Composition of mediaa
| Medium |
|---|
| PHI-I: 4.3 g/l MS salts (Phytotechnology Laboratories, Shawnee Mission, KS, catalog number M524), 0.5 mg/l nicotinic acid, 0.5 mg/l pyridoxine HCl, 1 mg/l thiamine HCl, 0.1 g/l myo-inositol, 1 g/l casamino acids (Becton Dickinson and Company, BD Diagnostic Systems, Sparks, MD, catalog number 223050), 1.5 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), 68.5 g/l sucrose, 36 g/l glucose, pH 5.2; with 100 μM acetosyringone added before using. |
| PHI-T: PHI-I with 20 g/l sucrose, 10 g/l glucose, 2 mg/l 2,4-D, no casamino acids, 0.5 g/l MES buffer, 0.7 g/l L-proline, 10 mg/l ascorbic acid, 100 μM acetosyringone, 8 g/l agar, pH 5.8. |
| PHI-U: PHI-T with 1.5 mg/l 2,4-D 100 mg/l carbenicillin, 30 g/l sucrose, no glucose and acetosyringone; 5 mg/l PPT, pH 5.8. |
| PHI-UM: PHI-U with12.5 g/l mannose and 5 g/l maltose, no sucrose, no PPT, pH 5.8 |
| PHI-V: PHI-U with 10 mg/l PPT |
| DBC3: 4.3 g/l MS salts, 0.25 g/l myo-inositol, 1.0 g/l casein hydrolysate, 1.0 mg/l thiamine HCL, 1.0 mg/l 2,4-D, 30 g/l maltose, 0.69 g/l L-proline, 1.22 mg/l cupric sulfate, 0.5 mg/l BAP, 3.5 g/l phytagel, pH 5.8 |
| PHI-X: 4.3 g/l MS salts, 0.1 g/l myo-inositol, 5.0 ml MS vitamins stockb, 0.5 mg/l zeatin, 700 mg/l L-proline, 60 g/l sucrose, 1 mg/l indole-3-acetic acid, 0.1 μM abscisic acid, 0.1 mg/l thidiazuron, 100 mg/l carbenicillin, 5 mg/l PPT, 8 g/l agar, pH 5.6. |
| PHI-XM: PHI-X with no PPT; added 1.25 mg/l cupric sulfate, pH 5.6. |
| PHI-Z: 2.15 g/l MS salts, 0.05 g/l myo-inositol, 2.5 ml MS vitamins stockb, 20 g/l sucrose, 3 g/l phytagel, pH 5.6 |
aPHI-I, PHI-T, PHI-U, PHI-V, PHI-X, and PHI-Z media from Zhao et al. 2000 (link)
bMS vitamins stock: 0.1 g/l nicotinic acid, 0.1 g/l pyridoxine HCl, 0.02 g/l thiamine HCl, 0.4 g/l glycine.
