This is how we do it here

scolnipa at esvax.dnet.dupont.com scolnipa at esvax.dnet.dupont.com
Fri Sep 6 08:31:03 EST 1991


We modified the root transformation procedure from Valvekens et al.(1988) 
and have had good success with WS.  

ARABIDOPSIS TRANSFORMATION

1.  Seed Sterilization.  Sterilize WS (Wassilewskija) seed for 10 min in a 
solution of 50% Chlorox with 0.1% SDS in ependorf tubes.  Rinse 3 to 5 times 
with dH20.  Dry thoroughly on sterile filter paper.

2.  Root cultures.  Place 3-5 seed into each of 3-5 250 ml Belco flasks 
with 50 ml B5 medium.  Place on shaker (70-80 rpm) at 23!C, 24 hr light for 
about 3 weeks.  Roots will fill flask and provide plenty of tissue for 
transformation.  Use roots while still white and healthy.

3.  Preculture on CIM.  Removing entire root mass from flask  and cut off 
shoots and any green roots.  Using forceps, pull off small bundles of roots 
and lay them on MSKig medium.  Place several root bundles on each 25x100 
plate.  Seal dishes with filter tape (Carolina Biologicals) and incubate at 
23!C, 24 hr light for 2 to 4 days.

4.  Inoculation.  Pour 40-50 ml MSKig liquid medium into a tripour 100 5m 
filter sitting in a 25x100 petri dish.  (We make these filter baskets by 
cutting off the bottom of tripour beakers and melting a piece of 100 5m 
mesh to the bottom.)  Place Arabidopsis root bundles from MSKig into a petri 
dish and cut them into 0.5 cm segments.  Transfer root pieces into filter 
units with medium.  Pipet 1.5 to 2 ml of an Agrobacterium overnight culture 
into each filter with roots.  Mix gently and let sit a few minutes.  Blot the 
roots on sterile filter paper.  Place bundles of roots on MSKig medium 
containing 100 5M Acetosyringone  Our root bundles cover about 1 cm2.  Seal 
with filter tape and incubate at 23!C, 24 hr light for 2-3 days.

5.  Rinse and Transfer to SIM with Selection.  Place roots bundles into 
a tripour filter which is sitting in a 25x100 petri dish.  Pour 30-50 ml 
liquid MSKig medium over the roots and shake the filter unit vigorously in 
the solution.  Repeat rinse with a clean petri dish until liquid is clear.  Blot 
the roots on filter paper.  Transfer root bundles to MSg v500 containing 50 
mg/l kanamycin, 5-8 bundles per 25 x 100 petri dish.  Make sure the roots 
are in contact with the medium.  Seal with filter tape and place at 23!C, 24 
hr light for 10-20 days.

6.  Transfer to No Hormone medium.  Green nodules and the first 
indications of shoots should appear within a few weeks.  When shoots are 
first visible (they can look like dark spots to the naked eye) transfer entire 
explant, or break up explant into smaller pieces, to GM v500 k50.  Secure lid 
to dish with two pieces of tape.  Incubate 7 to 14 days at 23!C, 24 hr light.  
As the shoots develop, it is helpful to carefully break up the explants to 
allow the shoots to expand.  

7.  Root Induction:  When shoots are large enough, excise them from the 
callus and place them on MSRg v500 k50 medium in a 25x100 petri dish.  
Secure lid to dish with two pieces of tape and incubate for 2-4 days.

8.  Plantlet Growth:  Transfer shoots to GM v500 k50 medium in 25x100 
petris.  Secure lid to dish with two pieces of tape.  This ensures good air 
exchange and helps to harden off the seedlings.  Roots should form in 10-20 
days.

9A.  Transfer to Soil:  When shoots have produced a good root system, 
transfer them to potting mix as follows:  Place MetroMix in 2" pots and 
moisten with Hoaglands solution.  Carefully pull robust plants from the agar.  
Rinse well with water from squirt bottle and use fingers and/or forceps to 
gently remove agar and vitreous tissue from base of shoot.  Place root in a 
hole made in the MetroMix and gently press the soil around the root.  Water 
the plantlet into the soil using the squirt bottle.  Immediately place the 
potted plant into a Magenta box with lid.  Return the plants to the culture 
room (23!C, 24 hr light).  Within a day or two, the stems should straighten 
up and start to grow.  As quickly as possible, without allowing the plants to 
wilt, remove the lid from the Magenta box in stages, starting one to two 
days after transplanting and removing the lid within a week.  Water the 
plants as necessary to keep them moist.  We have had up to 80% success rate 
with this procedure, however, the plants must not be vitreous.  When well 
established, the plants can be placed in a growth chamber.

OR 9B.  Grow to Seed in Magenta boxes:  Transfer rooted plants to GM 
v500 k50 medium in Magenta boxes (about 100 ml medium per box).  To 
facilitate air exchange and to allow for room to grow, the lid is replaced 
with a modified Magenta box using the Coupler for Magenta Vessel (Sigma C-
0667).  The Magenta box is modified by drilling a 6 mm hole in the bottom of 
the box (which will become the top); this hole is covered by a Suncap 
Closure (Sigma C-6920), held on by a rubber band during autoclaving.  If 
condensation occurs, replace the Magenta box lid with a dry one.  About half 
of the plants will set seed but plants in soil will set seed much faster.

ARABIDOPSIS MEDIA

Basic Medium

1 pkg. Murashige and Skoog Minimal Organics Medium without Sucrose 
	(Gibco #510-3118 or Sigma # M6899)
10 ml DM Vitamin Supplement
0.05% MES   (0.5 g/l)
0.8% agar   (8 g/l)
pH 5.8

DM Vitamin Supplement - 100 X Stock

10 mg/l thiamine
50 mg/l pyridoxine
50 mg/l nicotinic acid


GM  = Germination Medium

Basic Medium
1% sucrose   (10 g/l)

MSKig - Callus Inducing Medium

Basic Medium
2% glucose   (20 g/l)
0.5 mg/l 2,4-D	2.3 5M
0.3 mg/l Kinetin	1.4 5M
5 mg/l IAA	28.5 5M

MSg - Shoot Inducing Medium

Basic Medium
2% glucose   (20 g/l)
0.15 mg/l IAA	0.86 5M
5.0 mg/l 2iP	24.6 5M

MSRg - Root Inducing Medium

Basic medium
2% glucose   (20 g/l)
12 mg/l IBA	58.8 5M
0.1 mg/l Kinetin	0.46 5M

Notes

v500 = 500 mg/l vancomycin
B5 medium = Gamborg's B5 medium (Gibco # 500-1153)
Use 50 mg/l kanamycin for selection.

Sandra H. Russell
DuPont, P.O. Box 80402, Wilmington, DE  19880-0402
                                 302-695-8480 (FAX)



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