Bangladesh J.

SugarcaDe, 29

1-6

.lt1ly,:'l)(),'

Transformation of Foreign Gene in Sugarcane Variety Isd 28

{.Jsin

g Agrob acte

riu

m-mediated Meth od

M. A. Hossaln, M. M. Shaikr, N. lslam, H. M. Faruqueer and M. A. S. [,]iah

Bangladesh Sugarcane Research lnstitute, lshu rdi, Pabna, Bangladesh.

ABSTRACT
The sugarcane variely lsd 28 and Agrabacleium slraln LBA4404 conlainn! brnary vector pCAMBlA1301 was used ln this experimenl. The ptasnid contarns hyqromycf

phosphotransferase (hpl) gene and a B-grucuronrdase (GUs) gene rn rhe T-DNA reg on, and carried neomycin phosphorransferase (nprll) gene oulsde T-DNA region. unexpanclecr reaf

sheath segments were used as explants for transformation and direcl regeneraton was obtained aiter inlection, co-culltvatlon and selection to avoid sonlaclo|.l variatro,. Acetosyrlngone (2.0 mgl-r) was used ro enhance lransrorrnalon in sugarcane. Infccl on and
rnediurn was used as seleclion medium conlalned hygromycln (30.0 mgt.i). Begeneraled shools from lransforrned explanls were rooled in neclum coaaposed of MS satls supplerne.ted with sucrose 3Ogl L, agar 69 1 a.cJ 5.0 mgi I ol NAA. To obscrve ltre lransforrnation, histochen'icaj GUS assay was made alle. co-cullvatlon and serecr on. GUS positive results was showed pulative blue colour at surlace level as well as cellu ar level tn lransforn'red tissues. confirmation of transfoin-ralron was done by pcF melhocl. Fcsirlls revealed the genomic level lransformation of ioreign gene in sugarcane var ety lsd 2g. Key words: Sugarcane, Transforrnalon, GUS,assay, Foreign gene, Gcnorn c lovcl.
co-cultivaton was done using direct shoot regenerailon medium, l\,4s salls supprementecl vr'ith 7.5 mgl-1 NAA, 0.5 mgl'1 Kn, 30 gml r sucrose ancl 6 gml l agar. Shoot and Toot regenerat on

INTRODUCTION

Traditional plant breeding techniques, together with chemicaJ and biorechnologica approaches, have been extensively used to increase crop yields by seecting mproved varlies which are more productive and resistant to diseases anci palhogens. Howcver, convef ror.rai sugarcane breeding requlres 10-15 years to identify and release a new clone as varjety unfodunately some irnporlanl traits such as resistant to insect pest and to some herbicicle. appears to be absent from the genelic pool of sugarcane cultivars (Arencibia el a/., 1997) lncorporation of desired gene(s) from foreign source is the prime neecis 10 overcome the
n

bortle

eck.

Transgenlc sugarcane lines resistant

lo stem-borer attack transferring Agrabacteriurn.

by a team of scientists from the center for Genetic Engineering and Botechnoloqy, l-.lavana, Cuba and the lnstitute of Bio-Agriculture, Science Academia Sinica, Nan Kang, Taipel, Taiwan. The new nlethod increases the possibilities for lntroducing usefui traits inlo many sugarcane cullivars of economic importance (Arencibia el a/., 1998).
1. Deparlment of Biotechnology and Genetic Engineering.

mediated gene to plants recently has been repoded by Arencrbia et al. (tgg7). The first effjcieft protocol for sugarcane transformation mediated by Agrobacterium tumefaciens has been reporecl

lslamic

niversity, Kushtia, Bangladesh.

Eang lades lt J.

S u ga rca n

uiy 2tri)l

Curenlly lhe most wde y used methocl for transferring ge|es rllo p ants rs Agrobacle um--r,ediated gene lransfer. Agrobacle riun tLrnifacieDs mediatecl translont-ral on rnusl be an optimal system in order to meet up the transformaton lechnology of varous gcnes. Therefore, the present study has been under'taken to transfer forergn gene in sugarcane var ety lsd 28 using Agrabacleum-mecliated genet c translormation.
T,IATEBIALS AND IV,]ETHODS

Unexpanded eaf shealh segmenls were excised lrom sp ndles ol 4-5 ntonils old rcid grown plant of the sugarcane variely lsd 2B and were used as explants for transformatton. Ouler whoris of spindles were rernoved and ster ized in 1o% savlon for I 0 rnnutes lollo!,/ed by 3,4 times washed with sterilized dstlled vrater. Then the spndles were immersecl in 0.1 % Hgciz ior i 5 riin!tes and then washed 3-4 tlmes with slerlized distl led water lo rernove ster lant. Tl-en lhc unexpanded leaf sheaths $ere cui ,o'aru]1 ,plecs' (i cmx0.5cm) and userl as explants for
transformation.

Agrobacle unt strain LBA4404 contaring binary veclor pCAllBlA1301 \r,/as usecl r llls experimen. The plasmid contains hygromycin phosphotransierase (hpt) gene ancl a B-glucuronidase (GUS) gene ln the T-DNA region, and carriecl neomycln phospholranslerase (npil| gene outside T-DNA region. LBA44O4pCAlvl B lA 1301 was grown at 2BoC lar 4 days Ln l. liquid medium (LB) supplemented wth kanamycin 50 mgl lt was shaken at 150 rpm usir'g a horizontal shaker. Alter 48 hours, oplical denslly (OD) of bacterial s!spension cu ture at 600 rrnr was taken with the help of spectropholomele r. When oplical densjty (OD) reading was 1.0 thcrl
the culture was ready for inection.

lnfeclion medlum was prepared by aCdng liquid direct shoot induction rnediunr ([4S + NAA 7.5 mgi-r + Kn 0.5 mgl'r wilhout agar) 10 bacter al suspensron cuLture (LB . kanamycrn 50mgl'1) of strain LBA4404pCANl BIA 1 301 wrth 1:1 ralio ancl 2 mgl'r ol acetosyringone was adciecj and mixed well. Steriiized explants of the variety Lsd 2B were nfectecl with Agrabactettunt sa n LBA4404pCAN4B 1A 1301 for 60 min. Co-cullivation medium was, lv4s salts supplcmcnlcd w ttr l), 30 gm .r sucrose and 6 gm 1 1 agar. The pH was r) NAA (7.5 mgl and Kn (0,5 mgl adjusted ro 5.8. ln co-cultivation medjum no anl biotlc and no acetosyringone were used. Co'cu tlvation was done in 16h light per day al25'2BoC for 14 days.

Co-cultivated leaf segments were washeci v.rith 500 mclr cefolaxLme, and iheI transferred to seleclion medium, semisolid medium conlained N4S saLls sUpp cnrcnlcd with 7 5 r mgl-1 NAA, 0,5 mgl-r Kn, 30 gml'' sucrose, 6 gml agar and 30 mgl'1 hygrornycln. Se ectron was done n the 16 hours pholoperjod at 25-280C for 4 weeks. The survlved explants were then lransferred to regeneration mediurn. Shooting medlum composed ol N,4S salls supplemenled '../ith l) r) sucrose 30 gl'1, agar 6 gl'r and NAA (7.5 mgl + hn (0.5 mgl '1 and hygromyc n (30 mgl and incubated al 25-28"C for 4 weeks. Regenerated shots were transferred 10 rooirng rnedium l r composed of l\4S salts suppiementecj with sucrose 30 gl'1, agar 6 gl and 5.0 mgl of NAA anc
incubate at 25-2BoC for.+ weeks.

For the conflrmalion ol transformation, histochcmical GUS assay !r,as rrade aller -fhe randomly selecleci lissues were plaled co-cultivation and selectjon (Jefferson et al. 1987). over sterile discs of paper for dryjng, and then incubated in X-gluc solution in eppendon iub'es lor 1Lrl/r(./,rL/L/rr 24 f\\na arl 370C Ar Coflr()l llrrj ::rlll() rJir:i (lr)f(i willr cxl) iUrl:, wtlru!t
co-cullivation.

Trnsformalion of Foreign Gene n Sugarcane ........... AgrcbaclerlulTr, mecliated lVetlrod Plants regenerated from GUS positive explants were used for DNA iso atlon fo ow nq the melhod reponcd by Hossain ct r/. (2006) to confirrn of lranslorrnlion by PCn mr.thod Prilror pairs, Foruard 5'-TTT GCA AGT GGT GAA TCC CGA CCT-3-and Reverse 5'-AGT TTA CGC GTT GCT TCC GCC AGT-3' (Gambley et a/., 1993) were used. PCR amplificat on was clone n an oil-free thermal cycler (Master Cycler Gradient, Eppendorf) fo lowing the PCR profile oi gsqc or 3 minules (intial denaturation) followed by 35 cycles of 1 minute denaluralton at 95eC, l minute annealing at 54eC and elongalion or exlenson at 7zec la( 1 mlnute. After the last cycle a final step of 5 minutes at 72eC was added to allow complele exlension ol al ampl f ed fragircrts. After completion of cycling programme, reactions were held al 4aC. PCR reaclions wcre performed on each DNA sample in a 10 pl reaclion rn xture conlainng 1.0 // of 1OXA Taq bLrf f er (Tris with l5 mM \4gClr), 1.O ul ol 2.5 mM DNTPs, 2.25 p af 1.0 !M each of orward and reversc Prmer, 0.3 !l of 3U/A/l Ampli Taq DNA polyrerase (Bangajore Genei Pvt. Ltd. lndia), ,3 1tl oi 25 ng/pl genomic DNA and a suitable amount (0.2 fl) of sterile deionized distil ed r/ater. After

pl loading dye was added to the arnDliflcation prodLrct ior separalon !sing 1.4'.! r) e eclrophoresis. Eleclrophores s vias agarose gel (containing Ethidum Bronride 0.8 pgm performed at 100V for 2.5 hours. DNA iadder 100 bp (Bangalore Genei Pvt. Ltd., ndia) was rur aongside the reactions. Expected amplilied DNA fragment was observed on Uv'lrans lunr nator
amplificaton 2

in

Gel Documentation System (uvitech, DBT-2000LS), prnled and saved as sofl copy or l.1cr.l

us e.

RESULTS AND DISCUSSION

investigatlon, Agrobacte um strain LBA4404pCAII4 B lA 1 301 ancr sugarcane varjety lsd 28 were used. unexpanded sprndle eaf sheath segmenls were usec as transformation explants. Fesults of transformation, regeneraton of transformed explants, bioassay, histochemlcal GUS-assay and final y molecular assay by PCF were dscussed as
oliows.

ln the present

After inlection for 60 minutes exp arLs were co-cullivaLcd lor 14 days. CUS ;rs:iry r'vi-; done among randomly selecled leaf sheath segrnents after co-cultivation and select cf. GUS' assay revealed prominence positive results shownO putalive lllLre colour in BB.99| sc cclc.l lo.i sheath segmenls (Fig. 1,2 and 3). Unexpanded spindle leaf segments of sugarcane variely lsd 28 were used for translormation using jnducing chernical acetosyringone. Acetosyrlngone (2.0 mgl'1) was used cluring infection time in rneciium. Perhaps the use of aceiosyringone enhance(l the transformation and explants showed good GUS posilive resu ls. The mporlance of acetosyringone for lransfolmalion of monocot 9s sugarcane were descrlbed by At-mor-be ei ,/. :Hie'el;t (1994);:Komari et at (1989)i Bidny er'a). (1 992);1.oekema er ai-'ltsos); 119961t Nauerby el a/. (1997)t Klee (2000). They studied the transfer f T-DNA and rts integralon nlo the pLant genome is influenced by several A. lumefaciens and pant tlssue specrfc ficiors. lleso include plant genotypes, explants, vectors-plasrnd, bacteria strains, addiiion of vir-gene rnducing synlhetic phenolics cmpounds, c!lture.media cornposition, tissue damage, suppresson an.l elimination al A. tumeaciens nfection after co-cultlvation. Southgale el a/. (1996) reported llat

Bangladesh J. Sugarcane

July,2a07

supplemented at Agrabacterium culture with approprlate antbiotics and 100 pN,l acetosyringone increase transformation efficiency in monocot. stacheL et a/. (1985) reported thal A. luntcl.cic|ts infects only wounded, aclively dividing plant ceLls, which excrete phenolic compounds, such as acetosyringone and hydroxy-acetosyringone. These phenolics act bolh as chemo-attractan ts ior Agrobacterium and inducers of the virulence genes (Stachel e/ a/., t9B5).

Fegeneration responses were showed good results after transforrnalion. After lransformation 66.7% af explants produced shoots. All shoots produced roois n the rooling
medium (Figu re 4). For the confirmation of transformation at genornic level, genomic DNA was isolated frorn

the GUS postive putative transgenc plantets regenerated from unexpanded leaf sheath segments of variety lsd 28 after iransformation with L8A4404 pcA[/8141301, using a method

already established with simple modificaton from the method of Aljanabl et a/., j 999. cenomic DNA from non-transformed plants was also isolated. The purity of total DNA was checked for PCR operation by running in '1.0 % electrophoresis gel. By viewing on Gel documentalion system DNA was selected for PcR amplification. The GUS gene in genomic level was detecred ily amplifying using primer pair reported by cambley and co-workers (1993). prmer par amp if ies 600 bp DNA segment from GUS gene. The transformed pian ets arnplified DNA segment of fhe expected size 600 bp by the primer pair for the GUS fragment at the same position. No band was produced in DNA isolated from the non-transformed sugarcane control plants. The pcF resulis were shown in fig.5. Matsuoka et al. (2000) conducted Agrabacterium-nediated transformalion in

sugarcane. Hygfomycin-resistant, GUS-positive callr were selected from the inlected ce suspension culture. Hygromycin-resistant, GUS-positive shoots were regenerated from the selected calli. The hpt gene was detected from these ca and shoots by pCR anatysis.
PCR amplification of selected segments from GUS reporter gene was done using DNA solated from transformed regenerated plantslets in lsd 28. selected ampllfied DNA band was found against GUS primer pair. Thus the foreign gene was stably transferred to the genome oi sugarcane variety lsd 28.

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4
1

Transformation of Foreign Gene in Sugarcane ........... Agtobaclaurmcdiatcd Mcthod

Figure 1. GIJS expresson showing bl!e colo!r of lsd 28 in slce of lransformed leai segment at surface leve

Figure 2. GUS expression shorlng blue mlour of lsd 2B jn lhe cross section of kanslormed leafsegment at cellular level.

FrcJre 3. CUS e)(oress'on sowinq olLe co oLI ol I o 28 in loigituo.nel secrior of lns'ormeo leaf seg-renI al cell-lar le\e

Figure 4 Rogeneralion of shools lrom lransformed eaf segr.enl of sd 28 infected wlih LBA4404 (pcAMBlA 1301) 60 mLnutes.

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geromic DNA isoLaled from lransgenic sugarcane variery isd 28 Lane 1 10O n" :, q nomic DNA isotared f rorn vanslormed varlety rs d 28; La rs 4, genomic DNA nottt,tnslornd sugarcanevaiety lsd 28; Lane5, l00bpLadder i.-oruni;
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