Overall comments:
The protocol described for somatic embryogenesis of soybean can be used for particle bombardment-mediated transformation. Note that it is essential to use recently established cell lines to avoid problems with sterility in any resulting transgenics.
Pre-Bombardment
- The best target is repetitive embryos growing on MSD20 medium, selected for the presence of small, compact embryos, and which are preferably 1-3 months old, but never more than 9 months old.
- About 100 mg of embryogenic tissue is transferred onto a plate with MSD20 on which they will be shot, arranging them in a 3-cm disc in the center of the dish, 4 days prior to the the actual bombardment:
- The photos above show soybean embryogenic tissue prepared for bombardment
- Prior to bombardment, the lids are removed from the Petri dishes,and the embryos allowed to air-dry for 20 minutes in a laminar flow hood.
Bombardment
We shoot using this protocol for DNA preparation, and shooting parameters that consist of:Use of 0.6µm gold particlesEach shot delivers ~50 ng DNA precipitated on to 667µg goldShoot only once at 1100 psi6 mm gap distance6 cm flying distanceThe photo above shows soybean embryogenic tissue 2 days after bombardment. Lines in the background are 1 mm apart.
Selection
- Leave tissues on the plate upon which they were shot for 1 week.
- Then place approximately 0.25 g of tissue per 125-ml Erlenmeyer flask containing about 35 ml of Finer and Nagasawa Lite medium supplemented with 20 mg/L hygromycin.
- One shot plate yields enough embryos for 4 flasks
- Flasks are placed on a shaker at 125 rpm, in dim light (5-10µE m-2 s-1).
- The medium is replaced at weekly intervals by pipeting the spent medium out of the flask and replacing with fresh medium.
- Pick green colonies after 6 to 8 weeks in selection.
