A seed coat bedding assay to genetically explore in vitro how the endosperm controls seed germination in Arabidopsis thaliana

Two spoons of seeds (50-60ul volume in 1.5ml tube)

Shake for 10 min at 1200rpm

Centrifuge for 3 sec at 6,000rpm (for seeds down)

Remove 70% ethanol us- ing vacuum Suction

Add 1ml of sterile distilled water

Shake for 10 min at 1200rpm

1 2

3

4

5 6

7

Centrifuge for 3 sec at 6,000rpm (for seeds down)

8

Remove the water using vacuum suction

9

Seeds sterilization ( Overall the sterilization procedure takes about 30 minutes)

Add 1ml of 70% (v/v) ethanol

Wait 30 sec un- til the seeds down at the bot- tom of tube - No shaking - No centrifuge Add 1ml of sterile

distilled water us- ing pipette

10 11

Remove the water using vacuum suction

12 REPEAT 4 times

5-6cm distance

between the pipette

tip and the top of tube

Seeds Plating

Sterilized sees

Add 200ul of sterile distilled water

Plate seeds using pipette (blue tip)

30 ml of MS solid medium containing 0.8 % agar

Water surrounding seeds

Remove the water sur- round seeds using vacuum suction

Dry seeds in the inside of sterilized bench for 2 hrs without lid

1

2

3

4

Seed dissection

Two pieces of autoclaved Watman 3MM paper

*** Prepare a new MS solid plate containing 0.8% agar

After 4 h imbibition of seed on MS medium, transfer seeds onto the Watman 3MM paper using forceps

1 2

Cut the sharp tips of forceps

A forceps with ultra- sharp tips (#5)

Truncated for- ceps

Fix the seed with the truncated forceps Cut the seed coat (testa + endosperm)

of seed using syringe needle

BD Micro-Fine™+ 1ml syringe 0.33mm (29G) x 12.7mm

Seed dissection [working under binocular (10 X 3.2)]

3

Push the seed smoothly using truncated forceps

4

Separated embryo and seed coats

1.5cm

1.0cm

*** Prepare a new MS solid plate containing 0.8% agar

Put an autoclaved piece of Nylon Mesh (SEFAR NITEX 03-50/31)

5.0 cm

3.5 cm

Seed Coat Bedding Assay (SCBA)

EMBRYOS SEED COATS

Dissected seeds

Truncated for- ceps

Truncated for- ceps

2

To transfer embryos and seed coats without damage, embryos and seed coats were moved to the side of forceps using syringe needle carefully.

Transfer seed coats onto the Nylon Mesh

1

3

Make a bed of seed coat as single layer

4

SEEDS EMBRYOS EMBRYOS + SEED COATS

GROWTH CONDITIONS

• For dormancy and low GA conditions (ga1 background and PAC condition): 22 ℃ and continuous light (40 μmol·m

·sec

)

• For phytochrome studies: 22 and darkness (by wrapping plate in ℃ several layers of aluminum foil)

MS solid plate containing 0.8% agar

A piece (5.0 X 3.5m) of Nylon Mesh

(SEFAR NITEX 03-50/31)