Seeds sterilization Overall the sterilization procedure takes about
Seeds sterilization (Overall the sterilization procedure takes about 30 minutes) 1 Two spoons of seeds (50 -60 ul volume in 1. 5 ml tube) 2 3 Shake for 10 min at 1200 rpm Add 1 ml of 70% (v/v) ethanol 6 4 Centrifuge for 3 sec at 6, 000 rpm (for seeds down) Add 1 ml of sterile distilled water 7 5 Shake for 10 min at 1200 rpm Remove 70% ethanol using vacuum Suction REPEAT 4 times 10 8 Centrifuge for 3 sec at 6, 000 rpm (for seeds down) 9 Remove the water using vacuum suction 5 -6 cm distance between tte the pipe the tip and be top of tu Add 1 ml of sterile distilled water using pipette 11 Wait 30 sec until the seeds down at the bottom of tube - No shaking - No centrifuge 12 Remove the water using vacuum suction
Seeds Plating Water surrounding seeds 2 1 Sterilized sees Add 200 ul of sterile distilled water Plate seeds using pipette (blue tip) 3 Remove the surround seeds vacuum suction 30 ml of MS solid medium containing 0. 8 % agar 4 Dry seeds in the inside of sterilized bench for 2 hrs without lid water using
Seed dissection *** Prepare a new MS solid plate containing 0. 8% agar After 4 h imbibition of seed on MS medium, transfer seeds onto the Watman 3 MM paper using forceps 1. 5 cm 1. 0 cm Two pieces of autoclaved Watman 3 MM paper Seed dissection [working under binocular (10 X 3. 2)] 2 Cut the seed coat (testa + endosperm) of seed using syringe needle 1 A forceps with ultrasharp tips (#5) Fix the seed with the truncated forceps BD Micro-Fine™+ 1 ml syringe 0. 33 mm (29 G) x 12. 7 mm 3 4 Cut the sharp tips of forceps Truncated forceps Separated embryo and seed coats Push the seed smoothly using truncated forceps
Seed Coat Bedding Assay (SCBA) *** Prepare a new MS solid plate containing 0. 8% agar Dissected seeds 3. 5 cm 5. 0 cm EMBRYOS Put an autoclaved piece of Nylon Mesh (SEFAR NITEX 03 -50/31) SEED COATS 4 Truncated forceps Transfer embryos onto the middle of the bed of seed coat 3 Make a bed of seed coat as single layer 1 To transfer embryos and seed coats without damage, embryos and seed coats were moved to the side of forceps using syringe needle carefully. 2 Truncated forceps Transfer seed coats onto the Nylon Mesh
GROWTH CONDITIONS • For dormancy and low GA conditions (ga 1 background and PAC condition): 22 ℃ and continuous light (40 μmol·m-2·sec-1) • For phytochrome studies: 22 ℃ and darkness (by wrapping plate in several layers of aluminum foil) SEEDS EMBRYOS + SEED COATS A piece (5. 0 X 3. 5 m) of Nylon Mesh (SEFAR NITEX 03 -50/31) MS solid plate containing 0. 8% agar
- Slides: 5