Soma t i cHy br i di z a t i on -I nt e r ge ne r i cSoma t i cHy br i di z a t i onBe t we e nBr as s i c anap u sL . a ndSi nap i sal b aL .Thepur pos eof t hi ss t udywa st opr e s e ntt hee ffic i e nc yofe l e c t r of us i onf ort hepr oduc t i on ofB. nap u s( Bn)a ndS. al b a( Sa )hy br i dswi t hahi ghe rc onc e nt r a t i onoff a t t ya c i dsa ndwi t h be t t e ri ns e c t -a nddi s e a s e r e s i s t a nc ec ha r a c t e r i s t i c s .
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Pr ot opl as tI s ol at i on Pr ot opl as t= a l lt hec ompone nt sofa pl a ntc e l l e x c l udi ngt hec e l l wa l l
s e e ds
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e t ha nol -1mi n bl e a c h-2 5mi n
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me di um unde rfluor e s c e ntl i ght
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Pr ot opl a s ti s ol a t i onc a nbedonebyus i ng ame c ha ni c a lora ne nz y ma t i cme t hodhe r et hel a s tonewa sus e d.
Cut
Fort hee nz y ma t i cme t hodami x t ur eof ma c e r oz y mea ndc e l l ul a s ei sus e df ort he de gr a da t i onoft hec e l l wa l l .
I nc uba t i on
Ma nni t oli sa dde di nt hee nz y memi xi n or de rt os t a bi l i z et hepr ot opl a s t s .
Pr e i nc uba t i on
c e l l ul a s e 2 0mi n ma c e r oz y me s or bi t ol +Ca Cl 2 ma nni t ol r e l e a s eofpr ot opl a s t pl a s mol y s e dc e l l
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Themi x t ur eobt a i ne da f t e rt hee nz y ma t i c i s ol a t i on of pr ot opl a s t s c ont a i ns s ubc e l l ul a r de br i s , undi ge s t e d c e l l s , br ok e na ndv i a bl epr ot opl a s t s . Thepur i fic a t i oni sdonebyac ombi na t i on offil t r a t i on, c e nt r i f uga t i ona ndwa s hi g.
3 AC
El ec t r of us i on Pr ot opl a s t sa r epl a c e di nt oas ma l l c ul t ur e c e l l c ont a i ni nge l e c t r ode s . Thi sf us i ont e hni quec ons i s t si nt wos t e ps :
El e c t r of us i on
1 )a na l t e r na t i ngc ur r e nt( AC)fie l dof 1 800k Hzi sa ppl i e dwhi c hde t e r mi ne st he pr ot opl a s t st oa t t a c ht oe a c hot he ra l ong t hefie l dl i ne s ;
Mi x 1Bn: 2Sa
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2)adi r e c tc ur r e nt( DC)fie l dof1 5 01 60Vi sa ppl i e df or1 5μswhi c hha st he e ffe c t of br e a k i ng down t he c e l l me mbr a ne si nt he c ont a c ta r e a s of a dj a c e nt c e l l sr e s ul t i ng i nf us i on a nd me mbr a ner e or ga ni z a t i on.
DC
Pl antRegener at i on f us e da ndunf us e d pr ot opl a s t s
Fort her e ge ne r a t i onoft hene w pl a nt s t hemi c r oc a l l iwe r et r a ns f e r e dfir s tont oa s ol i dme di um a nda f t e r4we e k st he ywe r e t r a s ffe r e d a ga i n t o a di ffe r e nt i a t i on me di um.
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Af t e r3-4we e k st hene ws hoot swe r e t r a ns f e r r e dt o a MS me di um wi t hout hor mone sf orpl a nt l e tde v e l opme nt .
Cel l Cul t ur e The f us e d a nd unf us e d pr ot opl a s t s obt a i ne da f t e re l e c t r of us i ona r ec ul t ur e di n i nas pe c i a lme di um a tr oom t e mpe r a t ur e a ndi nda r k ne s s .
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Fr e s hme di um wa sa dde de v e r y5da y s unt i lmi c r oc a l l iof3 2 -64 c e l l s we r e obt a i ne d.
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mi c r oc a l l i c ul t ur e
Hor mone f r e e Me di um
Whe nami c r oc a l l usf or ms ,s omeofi t s c e l l sma yor ga ni z ei nt ogr owi ngpoi nt s , s omeofwhi c hi nt ur ngi v er i s et or oot s whi l eot he r spr oduc es t e msa ndl e a v e s . Thusi tma ybec a pa bl eofr e ge ne r a t i nga n e nt i r epl a nt .
pl a nt
dsDet er mi nat i on 6 Hybri For de t e r mi ni ng t he r a t e of hy br i ds f or ma t i ont wot e hni que swe r eut i l i s e d: 1 ) Nuc l e a rDNACont e ntt ha ta na l y s e dt he nuc l e iofl e a v e sf r om bot hpa r e ntpl a nt s a ndf r omt hene wone s ; 2) Si mpl e Se que nc e Re pe a tAna l y s i s whe r ege nomi cDNAwa si s ol a t e df r omt he y oungl e a v e s ;t hi st e hni quei sba s e don PCR. I nt he e nd,7outof68 pl a nt swe r e c onfir me dt obehy br i ds( 1 0. 3 %oft het ot a l numbe rofr e ge ne r a t e dpl a nt s ) .
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Bi bl i ogr a phy : [ 1 ]Y . P .Wa ng,K.Sonnt a g,E.Rudl off,a ndJ . M.Che n," I nt e r ge ne r i cSoma t i cHy br i di z a t i onBe t we e nBr a s s i c ana pusL.a ndSi na pi sa l baL, "J our na lof I nt e gr a t i v ePl a ntBi ol ogy , v ol . 47 , no. 1 , pp. 8491 , 2 005 / 01 / 012 005 , doi : 1 0. 1 1 1 1 / j . 1 7 447 909. 2 005 . 00009. x [ 2] Y . P . Wa ng, K. Sonnt a g, a ndE. Rudl off, " De v e l opme ntofr a pe s e e dwi t hhi ghe r uc i ca c i dc ont e ntbya s y mme t r i cs oma t i chy br i di z a t i onbe t we e nBr a s s i c a na pusa ndCr a mbea by s s i ni c a , " The or Appl Ge ne t , v ol . 1 06, no. 7 , pp. 1 1 47 5 5 , Ma y2 00 3 , doi : 1 0. 1 007 / s 001 2 200 21 1 7 6x [ 3 ] H. Cha wl a , I nt r oduc t i ont oPl a ntBi ot e c hnol ogy . 2 00 2, p. 5 3 8