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Nipponbare Rice Transformation- of Rtel

Callus induction

1. Remove the husk.

2. Soak in 70% Et! for "0 sec #remove $a&.". 'ash $ith sterile !2 " times.

(. Soak in 2)% bleach #a** 2 *rops T$een-20 *eter+ent& for 20 minutes mi $ell. ,se ""% bleach "0

min.). 'ash thorou+hl $ith sterile !2 ).

7. /late onto N me*ium an* seal $ith icropore-tape.

3. 4eave them in the *ark at 23 56 an* check for an contamination. f so chan+e to

ne$ plates.8. 'ait for -3 $eeks for callus9 calli pro*uction. Rea* to use in a month. 6heck for infection

re+ularl. /ick a+ain after another t$o $eeks. iscar* after this.

Sub culture

1. Transfer the callus9calli from callus in*uction on to N: me*ia an* seal $ith

parafilm paper.2. 4eave them in *ark at 23 56 for t$o $eeks. ,se as belo$.

Transformation

1. #on*a& /ick health lookin+ callus from subculture plates an* transfer themonto ne$ N: plates #2)9"0 calli 9plate&.

2. #'e*nes*a& streak +lcerol stock #100 ul& of the a+robacterium $ith construct onto 4: me*ium

$ith Rifampicm ; fter co-cultivation +entle $ashes of 1)0m+9l Timentin. 1st$ash ver short 2n*an* "r*$ash lon+er&. 'ash until clear. :lot *r calli #filter paperAnot too lon+&.

7. /lace #$ell space*& onto N:6T ie N: ;

*ark for 2 $eeks 2-23 56.

3. Net fe$ *as $atch for a+ro +ro$thB if so transfer calli onto ne$ N:T! ; selection plates.

10. Transfer all calli onto N:!T ; selection platesC leave them in *ark for 1(-21 *as

#2927 56&.

11. >fter 1(-21 *as transfer health callus 9calli onto fresh N:T!)0 plates /R6T; selection plates

an* leave them in *ark for 2 $eeks.

12. Transfer survivors onto /R!T ;selection an* seal $ith micropore tape. 4eave them in li+ht 10-1(

*as.

1". Transfer survivors to R!T plates. 4eave in li+ht 1 month.

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1". ?rom step 1" pick up the ones +ot shoot an* root an* transfer to D S pots leave them in li+ht for10-1( *as.

1(. The ones that have establishe* both shoot an* root +o to phtotron mister usin+ iff pots -F normalpots.

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Media composition for Nipponbare/Taipei 309 tissue culture

Stock solutionsN6 macro-elements (20!

"/l

#N!(&2S( 8."

GN" ).G!2/( 3

+S(.7!2 ".76a6l2.2!2 "."

#$ micro-elements (%00!

m"/%000ml

nS(.(!2 1000

Na2o(.2!2 2)

!":" "00

HnS(.7!2 2006uS(.)!2 ".37

6o6l2.!2 2.)

G 7)

#$ &itamins (%00!

Iambor+Js vitamin solution #1000Si+ma cell culture&

'e)T*(200!

"/200ml

?erric-So*ium salt 1.(7

#Si+ma cell culture&

2+,-) (%m"/ml!

issolve 100m+ of 2(- in 1ml absolute ethanol

>** "ml of 1N G!>*ust to p! $ith 1N !6l #ver sensitive a*ust carefull after p!8&

K2(- *ichloro-phenoacetic aci*

#* (%m"/ml!

-benLl amino purine

#Si+ma cell culture solution&

N** (%m"/ml!

Naphthalene acetic aci*

#Si+ma cell culture solution&

*#* (2.$ m"/ml!

issolve 2)0m+ of >:> in 2ml of 1 Na!ake to 100ml $ith #sterile& *istille* $ater. ?inal back+roun* concentration to be 20m Na!

K>bscisic aci*

"romcin ($0m"/ml!

Roche !+romcin solution

Timetin (%$0m"/ml!

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issolve "100m+ of Timetin in 20.ml of sterile M'.

?inal concentration to be 1)0m+9ml.

>li@uot )2)l in each eppen*orf tubes.

MS salts

urashi+e minimal or+anic me*ium

N6 Macro (%0!

"/l#N!(&2S( (.

GN" 23."

G!2/( (+S(.7!2 1.8

6a6l2.2!2 1.7

N6 micro (%000!

m"/%00ml

nS(.(!2 ((0

HnS(.7!2 1)0

!":" 10G 30

N6 1itamins (%00!

m"/%00ml

Ilcine 20

Thiamine-!6l 10/ri*oine-!6l )

Nicotinic aci* )

*!. Callus induction

N6) media

amount/litre

N macro #10& 100ml

N micro #1000& 1mlN vitamins #1000& 1ml

S iron9ET> )ml

oinositol 100m+6asamino aci* "00m+

/roline 2.8+

2(- #1m+9ml& 2mlSucrose "0+

K>*ust p! to ).3 $ith 1 G!K>** "+ phto+el9litre

K>utoclave

#!. Sub culture media

N# media

amount/litre

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N macro-elements #20& )0ml

:) micro-elements #100& 10ml:) vitamins #100& 10ml

?eET> #200& )ml

2(- #1m+9ml& 2mlSucrose "0+

/roline )00m+

Ilutamine )00m+6asein enLmatic h*rolsate "00m+

#6E!&

K>*ust p! to ).3-).3) $ith 1 G!K>** "+ phto+el9litre

K>utoclave

C!. Selection media (*fter transformation!

N#

N: me*ia plus "0+9l mannitol an* "0+9l sorbitol a**e* before p! a*ustment

N#T30N: me*ia plus "0m+9l h+romcin an* Timentin 1)0m+9l a**e* after autoclave ust before pourin+#!+romcin *o$nstairs in canister )0m+9ml in fri*+e& #Timentin if fri*+e&

N#T$0

N: me*ia plus )0m+9l h+romcin an* Timentin 1)0m+9l a**e* after autoclave ust before pourin+

T$0

N: me*ia #$ith no 2+,-)& plus follo$in+ a**e* after autoclave

amount/l

:>/ 2m+9l

N>> 1m+9l>:> )m+9l

!+romcin )0m+9l

Timetin 1)0m+9ml

T$0

N: me*ia #$ith no 2+,-)& plus follo$in+ a**e* after autoclave

amount/l

:>/ "m+9l no$ use ( m+9ml

N>> 0.)m+9l!+romcin )0m+9l

Timetin 1)0m+9ml

4 MS media

amount/l

S salts an* vitamin miture 2.21+Sucrose 10+

K>** 2.) + phto+el9lK>utoclave

K>fter autoclave a** 0.0)m+9l N>> an* Timentin 1)0m+9ml