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Wed Feb 3 13:31:46 EST 1993

We have combined and modified procedures as described below.  Note that
some superscripts have been lost (40C should be 4C, etc).  This method
works extremely well for us.  Using very young roots (1 week) seems to 
be the key to getting lots of shoots! 

Diana Polisensky (Braam lab)
Rice University
dhp at bioc.rice.edu

Arabidopsis  Root Transformation 

Generation of sterile roots.

1.	Vernalize  RLD or NO-O seed at 4C one week.
2.	Sterilize seed in a 50 ml. conical tube by adding approximately 25-50 ml
of 190 		proof ethanol and incubating for 1 minute.
	Pour out ethanol and add approximately 25- 50 ml of 100% clorox and 0.25
or 0.5  		ml, respectively, of sterile 10% SDS (to lower surface tension). 
	Incubate for 15-20 min with periodic mixing.
	Allow seeds to sediment,
	Discard solution.
	Wash seed three times with 50 ml of sterile water.
	(Sterile seed can be stored in sterile water at 40C for several
weeks.(PMBR Vol. 10 		#4  p375)
3.	Germinate seed by placing 5-10 seed in sterile Belco Flasks containing 
25ml. of 		seed media (SM) plus silver thiosulphate 
	Shake at room temperature for 7 to 10 days.  Roots must be young, white
(no 		greening) and healthy  for adequate regeneration.

Note:	1.	The effects of silver are two fold:
			Reduce growth-inhibitory effects of ethylene
			Prevent overgrowth of tissue by Agro. (p185 PMBR Vol 10 #2)
	2.	Protocols for media from PMBR Vol 10, #2 are preferred as hormone
			content is less than that of Valvekens

Silver Thiosulphate  Seed Germination Media (1 liter).
			             	Final Conc.				per liter
M.S. salts			   1/2 X				         2.215g
Sucrose			       1%					         10 g
	pH to 5.8 with 1M KOH 
	autoclave 250 for  20 minutes
	cool then add 4ml of 
 				Final Conc.		Stock			per liter
Silver Thiosulphate	5 mg/l		1.25 mg/ml.	 	  4 ml

	1.	Alternatively, one can add 100ul silver thiosulphate per Belco flask of
25 		ml of SM media 
	2.	Preparation of Silver thiosulphate.
		Add dropwise a solution of AgNO3 (2.5 mg/ml.) to an equal volume of Na
			thiosulphate pentahydrate (14.6 mg/ml.)
		Filter sterilize.
		Store 4 ml. and  1 ml. aliquots at -20oC

Preparation of  Agrobacterium  Innoculum.

1.	Streak out Agrobacteria from a frozen stock onto a LB + 50 mg/l
Kanamycin (or 	other appropriate antibiotic) plate.  Grow for 2 days at
300C.  This plate can be used 	for 1 week if stored at 40C .
	(For a negative control use Agro lacking Kan r)
2.	Start a 2-ml culture from a single discrete colony in LB + 50 mg/l Kan.
	Grow with shaking 1-2 days
 3.	Dilute culture to an  O.D of 0.1 at 600 nm. with Agro dilution media. 
Make final 		volume of innoculum  20 ml.

Agro Culture Dilution Media.
		              	Final conc.		per liter.
Gamborgs B 5	   1X			           3.1
Glucose		        2%			           20g
MES		           	0.05%		         0.5g

Adjust pH to 5.7 with 1M KOH
Store in 50 ml and 200 ml. aliquots in 40C room.

Preculture of Roots.

1.	Cut roots from the shoots, taking care to remove all greenery.  Presence
of shoot 	tissue will inhibit regeneration.
2.	Preculture the intact roots on CIM (no selection) for four hours before
	innoculating. (Reference page 255, Koncz)
	Seal plates with breathable tape.

Callus Inducing Media
			Stock		      	Final		        	per liter
Gamborgs B5				  1X 		          	3.1g
MES					        	0.05%	         	0.5 g
Glucose					     2%		           	20 g
2,4-D		10mg/ml in 1N NaOH	0.5mg/ l    		50  l
Kinetin 	3 mg/ml in 1N NaOH	0.05mg/l     		16.6  l
	pH  to 5.8 with 1M KOH
	Add 8.0g agar  (Difco, Bacto.)    
	Cool, then add 4 ml of 1.25mg/ml silver thiosulphate (Stored in 4 ml.
aliquots in 		sterile tubes -20C )  	

Co-cultivation of  Roots and Agrobacterium
1.	Add Acetosyringone to a CIM plate and allow it to absorb into the plate
about 10 	minutes.  (final concentration of 100 M = 30  l of a 100mM stock
(filter sterilized) 	kept at -20C.
2.	Cut roots with sharp sterile surgical blade
3.	Place in sterile tripour filter (made by heat fusing a piece of 100 5M
mesh gauze to 	the base of a 250ml plastic beaker with the bottom cut
out.).  The tripour filter 	should be placed in a pertri dish so that the
roots can be suspended in culture 	media.
4.	Pour over 20 ml. of Agro culture and suspend the roots in it and mix
gently 	periodically  for 2 minutes.
5.	Blot off excess with sterile paper towel.  As much excess liquid as
possible should 	be removed.
6.	Apply root bundles to CIM +Acetosyringone plate, taking care to spread
the roots 	thinly so that as much surface as possible is in direct contact
with the media.

Wash and Transfer  to SIM.
1.	Wash root bundles  by placing them  in a tripour filter sitting in a
25x100 petri 	dish.  Pour 30-50 ml. Agro dilution media over the roots and
shake filter unit 	vigorously in the solution.  Repeat rinse with clean
petri dish until liquid is clear.
2.	Blot with sterile paper towel.  Again, remove  all  excess liquid.
3.	Suspend the roots in 10 ml SOM +100 mg/l Timentin (i.e., 10 ul of 100
mg/ml 	stock);  50 mg/l kanamycin (i.e., 14.2 ul of  35 mg/ml stock)
overlay that has been 	cooled to approx. 300C.  Use approx. 0.1 g. tissue
for 10 ml. SOM. 
	(SOM is optional but ensures complete contact with media, even
distribution of 	individual explants, and prevents the production of toxic
phenolic compounds 	which inhibit regeneration. PMBR Vol 10 #2  p. 186.)
4.	Pour suspension evenly onto SIM plates, that have been spread with fresh
	timentin (i.e., 30 ul of 100 mg/ml stock.final conc. 100 mg/l) .  Fresh
plates work 	best.  
5.	Seal plates with breathable tape.
6.  	Incubate at 240C.  Calli should develop within 6-8 days; shoots should
develop 	within a week or two after calli formation.
7.	Optional:  When green calli have grown to one or two mm in diameter,
they can 	be transferred to a fresh SIM plate to encourage faster growth. 
	Large calli(greater than 5 mm.) can also be broken up to yield more

Shoot Inducing Media.
				Stock			    	               final 		       	per liter
Gamborgs B-5 (Sigma) .				      	1X			            3.1g 
MES							                      	0.05%		         0.5g
glucose					                    		2%			          20g
2 iP 		 	20 mg/ml in 1N NaOH		5 mg/ml 		  250  l
IAA 			5 mg /ml in I N NaOH		0.15 mg/l 		  30 ul
	pH  to 5.8 with 1M KOH
	Add 8.0g agar  (Difco, Bacto.)    
	Cool, then add kanamycin to final concentration of 50mg/l (1.43ml of
35mg/ml		stock solution)
	Store plates at 4C
	Immediately before use spread 30 l of 100mg/ml timentin onto each plate.
	The timentin is unstable, the stock keeps one week at 4C.

Shoot Overlay Media.

Add 0.8 g  low melting point agarose (Sea Plaque G.T  FMC) in 100 ml.  SIM
media Dispense 10 ml  into sterile 50 ml. conical tubes.
Just before use, melt agar in double boiler.
Cool to 370C  
Add 14.2 l of kanamycin (35mg/ml stock) and 10 l timentin (100mg/ml stock)

Transfer of Shoots to SEM

Shoots will develop from green calli.  Relatively normal looking shoots
(cluster of 2 or more leaves with central meristem) should be completely
separated from callus and placed on SEM media.  Multiple shoot clusters
should be pulled apart at the base (using two pairs of forceps at the base
of the material) and transferred to SEM plates with 30mg/l Kanamycin.  All
callus material should be removed with a pair of fine point forceps. The
lower Kan concentration is to encourage differentiation; kanamycin is a
strong inhibitor of differentiation (p255 Koncz).  After one week, the
explants should be transferred to new plates (SEM or RIM, depending on size
of shoots) lacking selection.  From now on, the taller petri plates should
be used to provide more space for the growing shoots; ethylene will be
produced if the shoots touch the top of the plate.  If multiple shoots
develop, they should be promptly separated into single shoots.
If necessary add 30 ul of  100 mg/ml timentin to eliminate Agro growth.
Note: If callus material cannot be totally removed during transfer, be sure
to remove it later before putting onto RIM.

Shoot Elongation Media.
				            Final		         	per liter
M.S. Salts + MSMO 	1/2 X 		     2.215 g
Sucrose			10g/l		               	10g
	pH to 5.8 with KOH
	Add 8.0 g Bacto Agar (Difco)/liter, autoclave, and pour 25x 150 mm plates.

Transfer to RIM.

When the single shoots (free of all callus material) are  4-10 mm in size
or begin to show signs of bolting, they should be transferred to RIM plates
for 2 to 4 days (p381  
PMBR, Vol.10, No 4).  No selection is necessary, but timentin can be used
if contamination is a problem.
Place plates on the verticle so shoots will grow up without touching the
top of the petri dish and roots will grow down on the surface of the agar
(not into it) to facilitate removing of the plants for transplantation to
soil. )  Optimally, 15-17 plantlets should be on each plate (pg. 379  PMBR 
Vol 10, #4 - "it seems possible that a  root-promoting substance 

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