Table 2.
WPBS culture media and explants used in different grass species for normal growth, propagation, callus induction, transformation and regeneration. Control media were made using modified MS medium (Duchefa M0245) instead of WPBS medium. All media were autoclaved for 15 min at 121 °C, solidified with 0.03% Gelrite and adjusted to pH 5.6 unless stated otherwise
| Purpose of culture medium and species | Explant | WPBS culture medium per litre |
|---|---|---|
| 1. Shoot tip culture, rooting and maintenance medium (MSO) | Shoot tips, shoots | WPBS-A, 3% sucrose |
|
2. Avena meristem medium (OMM) (Avena sativa, Lolium perenne, L. temulentum, Festuca arundinacea) |
Shoot tips from seedlings or plantlets on MSO | WPBS-A, 3% maltose, 0.5 mg 2,4-D, 2 mg BAP |
| 3. Pre-transformation callus induction media | ||
| Lolium perenne | Shoot tips from plantlets on MSO or meristem clusters from OMM | WPBS-B, 3% maltose, 5 mg 2,4-D, 0.5 mg BAP |
| Lolium temulentum | Immature embryos 0.5-1 mm or meristem clusters from OMM | |
| Avena sativa | Meristem clusters from OMM | |
| Festuca arundinacea, | Shoot tips from plantlets on MSO or meristem clusters from OMM | WPBS-B, 3% maltose, 4 mg 2,4-D |
| Agrostis stolonifera, Deschampsia cespitosa, Festuca. rubra, Lolium multiflorum, Poa pratensis, | Shoot tips from plantlets on MSO | WPBS-B, 3% maltose, 3 mg 2,4-D |
| Phalaris arundinacea | Shoot tips from plantlets on MSO or meristem clusters from OMM | |
| Oryza sativa | Mature embryos (shoot tips and OMM not successful) | |
| Brachypodium distachyon | Immature embryos 0.2–0.5 mm (shoot tips and OMM not successful) | WPBS-B, 3% maltose, 2.5 mg 2,4-D |
| Zea mays | Immature embryos 1.5-2 mm (shoot tips not successful) | WPBS-B, 3% maltose, 2 mg 2,4-D |
| 4. Transformation media | ||
| Agrobacterium infection medium (liquid)a | Immature embryos, calli, cell suspensions | WPBS-C, 6.84% sucrose, 3.6% glucose, 2.5 mg 2,4-D, pH 5.2 |
| Agrobacterium co-cultivation medium (liquid)a | Immature embryos, calli, cell suspensions | WPBS-C, 6% maltose, 300 mg cysteine, 2.5–5 mg 2,4-D, pH 5.2 |
| Osmotic bombardment medium | Calli, cell suspensions | WPBS-A, 3% maltose, 9% sorbitol, 3 mg 2,4-D |
| 5. Post transformation media | ||
| Callus selection medium | Agrobacterium treated or bombarded calli | WPBS-A, 3% sucrose, pH 5.6, growth regulators as for calli above |
| Regeneration medium (RM) | Calli | WPBS-A, 3% maltose, 1 mg BAP, 1 mg NAA |
| Rooting medium (MSO) | Shoot tips, shoots | WPBS-A, 3% sucrose |
| 6. Miscanthus media where different for Miscanthus sinensis, M.floridulus, M.sacchariflorus | ||
| Shoot tip culture, rooting and maintenance medium (liquid) (MS1.5P) | Shoot tips, shoots | WPBS-A, 3% sucrose, 0.8 g proline (1.5 g total) |
| Propagation medium (liquid) | Shoots | WPBS-A, 3% sucrose, 0.3 mg BAP, 0.8 g proline (1.5 g total) |
| Pre-transformation callus induction medium | Shoot tips from plantlets on propagation medium | WPBS-B, 3% maltose, 3 mg 2,4-D, 0.1 mg BAP, 0.7 g proline (1.4 g total) |
| Osmotic bombardment medium | Calli | WPBS-A, 3% maltose, 5.5% mannitol, 5.5% sorbitol, 3 mg 2,4-D |
| Post-transformation callus selection medium | Agrobacterium treated or bombarded calli | WPBS-A, 3% sucrose, 3 mg 2,4-D, 0.1 mg BAP, 0.7 g proline (1.4 g total) |
aFilter sterilised through 0.2 µM filter