AGROBACTERIUM-MEDIATED TRANSFORMATION OF TOBACCO, NIGHTSHADE, TOMATO, POTATO AND ALFALFA By Martha L. Orozco-Cr !"a# U"$%!r#$ty o& Ca'$&or"$a - R$%!

r#$ ! A. GENERAL PROTOCOLS (. Gro)$"* Agrobacterium Small aliquots of frozen transformed Agrobacterium tumefaciens are used to initiate fresh overnight cultures at 28oC in medium No. 1; overnight cultures are diluted with liquid MS medium No. 2 to 108 Agrobacterium cells ml for the infection of !lant tissues "co# cultivation$. +. Pr!,arat$o" o& to-acco &!! !r ,'at!# %o&acco sus!ension#cell cultures "'ine N%#1$ are su&cultured to fresh medium No. ( and incu&ated four da)s at 2*oC under dar+. %hen, ( ml of the fresh sus!ension cells !er !etri dish are !lated over the same medium No. (, &ut with 0.-. agarose. %he cells are incu&ated as &efore, for another two da)s. /hen read) to &e used, a sterile filter !a!er "/hatman No. 0$ !re#soa+ed in liquid medium No. 1 is !laced in each !etri dish, over the fine la)er of to&acco cells well s!read over the surface of the medium. .. E/,'a"t# ,r!,arat$o" ..(. S!! *!r0$"at$o". Seeds can &e sterilized with 1*. commercial &leach containing a few dro!s of tween 20 for 20 minutes. %hen rinse four times with sterile#distilled water. Seeds are germinated in MS medium No. 1 and incu&ated at 28oC with 11 hours dail) of light. ..+. Mat!r$a' &ro0 th! *r!!"ho1#!. 2lant material from the greenhouse can &e sterilized using a 10. solution of commercial &leach with few dro!s of tween 20 during 10 minutes. %hen the) are washed si3 times with sterile#distilled water. .... Coty'! o"#. 4fter -#10 da)s, when the first true leaves a!!ear, ta+e the cot)ledons out of seedlings, and cut their edges with a scal!el, leaving cot)ledon squares of a&out 0.2#0* cm2. ..2. L!a& $#c#. 'eaf sam!les from in vitro !lantlets, no more than one#month#old, or from active#growing vigorous greenhouse !lants, can &e used. Square sections of leaf tissue "a!!ro3imatel) 0.* cm2$ are o&tained using a scal!el. ..3. T1-!r $#c#. %u&ers are !eeled, washed &riefl) with distilled water and surface sterilized for 1* minutes in 10. commercial &leach with tween 20. %hen, the) are washed si3 times with sterile#distilled water, and finall) immersed in liquid MS medium

No. 1. Columnar sections of !otato tu&ers are o&tained using a sterile 1#cm diameter cor+#&orer, and then the) are sliced into thin discs "a!!ro3imatel) 2#( mm thic+$ using a scal!el. 2. Pr!-co" $t$o"$"* o& !/,'a"t#. %omato, !otato and alfalfa e3!lants are usuall) !re#conditioned &) incu&ating them in to&acco feeder !lates for two to three da)s at 2*oC under dar+ conditions, &efore their treatment with Agrobacterium. 3. Tr!at0!"t )$th Agrobacterium 4co-c1't$%at$o"5. 2re#conditioned e3!lants are infected with Agrobacterium &) soa+ing them in 20 ml of sterile liquid MS medium No. 1, containing 108 Agrobacterium cells ml, during 20 minutes. 5efore or during the co#cultivation, the e3!lants are carefull) !unched with a s)ringe needle, or scal!el &lade. %hen, the e3!lants are &lot dr) with sterile filter !a!er, and incu&ated again in the to&acco feeder !lates for another two da)s. %o&acco and nightshade e3!lants are incu&ated in ( ml of liquid medium No. 1 with Agrobacterium, at 28oC under dar+ conditions for three da)s. 4fter the co#cultivation, the e3!lants are washed three times with sterile liquid medium No. 1 and finall) rinsed with the same medium &ut containing *00 mg l of cefota3ime. %hen the e3!lants are &lot dr) with sterile filter !a!er and !laced on the res!ective selective medium. 6. S1-c1't1r! to &r!#h 0! $10. 6t is im!ortant to transfer the e3!lants to fresh medium after the first three da)s of culture and wee+l) "no more than 10 da)s$, to get high efficienc) in !lant regeneration and transformation. /hen calli or shoots a!!ear, the) must &e se!arated from the rest of the e3!lants, and !laced in direct contact with the medium. Shoots "(#0 cm high$ are usuall) rooted in a different medium "rooting medium$.

M! $10 No. (. Gro)$"* Agrobacterium 7east 83tract 5acto 2e!tone NaCl 9anam)cin %etrac)cline 4cetos)ringone "*0 mM$ 10.0 g l 10.0 g l *.0 g l 10.0 mg l (0.0 mg l :.81 g l M! $10 No. +. S!! *!r0$"at$o" Murashige and S+oog "MS$ salt mi3 Sucrose %hiamine 6nositol !; 2h)toagar 0.( g l 20.0 g l 1.0 mg l 100 mg l *.8 -.0 g l

M! $10 No. .. To-acco &!! !r ,'at!# MS salts Sucrose 6nositol %hiamine 2,0#< 9;22=0 !; 0.( g l (0.0 g l 100 mg l 1.0 mg l 0.2 mg l 0.18 g l *.8

M! $10 No 2. To-acco Ca''1#7#hoot#+ 0*7' 0,(00.0 10.0 1.0 1.0 100.0 (0,000.0 1.0 0.1 00.0 *00.0 *.8 -,000.0 200.0 2*0.0 *00.0 # Root$"*. 0*7' 0,(00.0 10.0 1.0 1.0 100.0 20,000.0 # # # # *.8 -,000.0 *0 # # *00.0

MS salts %hiamine Nicotinic acid 2)rido3ine 6nositol Sucrose 542 644( 4denine M8S !; 2h)toagar 9anam)cin0 Cefota3ime0 Car&enicillin0 >ancom)cin0

Not!8 Neom)cin !hos!hotransferase "n!t$ gene fusion confers resistance to +anam)cin "selecta&le mar+er$. 4ll hormones are added !rior to autoclaving and all anti&iotics are added !ost autoclaving.
(7 +7

?irst 10 da)s 0#1 wee+s .7 (#0 wee+s 27 4nti&iotics for &acteria control. %he anti&iotics should &e sterilized and stored at #20oC.

M! $10 No.3. N$*ht#ha ! Shoot#( 0*7' MS salts %hiamine Nicotinic acid 2)rido3ine 6nositol Sucrose 542 644 N44 4denine M8S !; !h)toagar Cefota3ime Car&enicillin 9anam)cin >ancom)cin
(7 +7

Root$"*+ 0*7' 0,(00.0 10.0 1.0 1.0 100.0 20,000.0 # # 1.0 # # *.8 -,000.0 # # *0.0 *00.0

0,(00.0 10.0 1.0 1.0 100.0 (0,000.0 1.0 0.1 # 00.0 *00.0 *.8 -,000.0 2*0.0 *00.0 100.0 #

0#1 wee+s (#0 wee+s

M! $10 No. 6. To0ato Shoot#( 0*7' MS salts %hiamine Nicotinic acid 2)rido3ine 6nositol Sucrose 542 644 !; !h)toagar Cefota3ime Car&enicillin 9anam)cin >ancom)cin
(7 +7

Root$"*+ 0*7' 0,(00.0 10.0 1.0 1.0 100.0 20,000.0 # # *.8 -,000.0 # # 20.0 *00.0

0,(00.0 10.0 1.0 1.0 100.0 (0,000.0 2.* 1.0 *.8 -,000.0 2*0.0 *00.0 100.0 #

1#8 wee+s (#0 wee+s

M! $10 No.9. Potato Shoot#( 0*7' MS salts %hiamine Nicotinic acid 2)rido3ine 6nositol Sucrose @eatin 644 !; !h)toagar Cefota3ime Car&enicillin 9anam)cin >ancom)cin
(7 +7

Root$"*+ 0*7' 0,(00.0 10.0 1.0 1.0 100.0 20,000.0 # 0.0* *.8 -,000.0 # # *0.0 *00.0

0,(00.0 10.0 1.0 1.0 100.0 (0,000.0 1.0 0.* *.8 -,000.0 2*0.0 *00.0 100.0 #

0#1 wee+s (#0 wee+s

M! $10 No. :. A'&a'&a Ca''1#( 0*7' 9N=( MgS=0 N;0;22=0 Na;22=0 "N;0$2S=0 CaCl2.2;2= MnS=0.;2= ;(5=( @nS=0.-;2= CuS=0.*;2= Na2Mo=0.2;2= CoCl2.1;2= ?eS=0.-;2= Na28<%4 96 %hiamine Nicotinic acid 2)rido3ine 6nositol Sucrose Casein ;)drol)sate N44 96N 2,0#< A4( !; 4garose Cefota3ime Car&enicillin 9anam)cin
( + .

E0-ryo I" 1ct$o"+ 0*7' 2,*00.0 000.0 (00.0 0 0 200.0 10.0 (.0 1.0 0.2 0.1 0.1 1*.0 20.0 1.0 *.0 *.0 0.* 1,000.0 (0,000.0 0 0 1.011.0 0 *.8 -,000.0 2*0.0 *00.0 *0.0

P'a"t'!t#. 0*7' 2,*00.0 000.0 0 (*8.8 1,(21.0 200.0 10.0 (.0 1.0 0.2 0.1 0.1 1*.0 20.0 1.0 *.0 *.0 0.* 1,000.0 20,000.0 2,000.0 0 0 0 0.1 *.8 -,000.0 2*0.0 *00.0 *0.0

2,*00.0 000.0 (00.0 0 0 200.0 10.0 (.0 1.0 0.2 0.1 0.1 1*.0 20.0 1.0 *.0 *.0 0.* 1,000.0 (0,000.0 0 0.1 2.1* 0 0 *,8 -,000.0 2*0.0 *00.0 *0.0

0#1 wee+s ( da)s 0#1 wee+s

B. SUMMAR; Protoco' To-acco Nicotiana tabacum var.Banthi No 'eaves ( da)s 542, 644 MS N$*ht#ha ! Solanum nigrum No 'eaves ( da)s 542, 644, N44 MS To0ato Lycorpersico n esculentum cv. 5etter &o) 7es Cot)ledons 20 minutes 542, 644 MS Potato Solanum tuberosum cv. <esiree 7es 'eaves %u&er discs 20 minutes @eatin, 644 MS A'&a'&a Medicago sativa cv. C4( 7es Cot)ledons 2etioles 20 minutes N44, 96N, 2,0#<, A4( S;

?eeder !lates 83!lants 4grobacteriu m Cocultivation 1 ;ormones 5asal salts

(

Hor0o"!#

542D 1#5enz)lamino!urine 644D 6ndole#(#acetic 4cid N44D E#Na!hthaleneacetic 4cid 96ND 9inetin 2,0#<D 2,0#<ichloro!heno3)acetic 4cid A4( D Ai&&erellic 4cid R!&!r!"c!#. 1. NarvFez#>Fsquez, G., Orozco-Cr !"a#, M.L., and C)an, C.4. "1::2$. <ifferential e3!ression of a chimeric CaM>#tomato !roteinase inhi&itor 6 gene in leaves of transformed nightshade, to&acco and alfalfa !lants. 2lant Mol. 5iol. +<, 110:#11*-. 2. McAurl, 5., 2earce A., Orozco-Cr !"a#, M.L. and C)an, C.4, "1::2$. Structure, e3!ression and antisense inhi&ition of the s)stemin !recursor gene. Science. ++3, 1*-0# 1*-(. (. Orozco-Cr !"a#, M.L. McAurl, 5. C)an, C.4. "1::($. 83!ression of an antisense !ros)stemin gene in tomato !lants reduces resistance toward Manduca sexta larvae. 2roc. Natl. 4cad. Sci. HS4. =<, 82-(#82-1. 0. McAurl, 5., Orozco-Cr !"a#, M.L., 2earce, A., C)an, C.4. "1::0$. =vere3!ression of the !ros)stemin gene in transgenic tomato !lants generate a s)stemic signal that constitutivel) induces !roteinase inhi&itor s)nthesis. 2roc. Natl. 4cad. Sci. HS4. =(, :-::#:802.

1. Orozco-Cr !"a#, M.L. and C)an, C.4. 2ol)galacturase I#su&unit antisense gene e3!ression in tomato !lants leads to a !rogressive enhanced wound res!onse and necrosis in leaves and a&scission of develo!ing flowers. "200($. 2lant !h)siol. (.., 1:(#-01.