Page 1 ICSAFS Conference Proceedings 2nd International

6 downloads 0 Views 3MB Size Report
Nov 23, 2017 - [2] Direktorat Budidaya dan Pascapanen Buah., 2012. Pedoman Penanganan Pascapa- nen Pisang. Direktorat Budidaya dan Pascapanen ...
,&6$)6 &RQIHUHQFH 3URFHHGLQJV QG ,QWHUQDWLRQDO &RQIHUHQFH RQ 6XVWDLQDEOH $JULFXOWXUH DQG )RRG 6HFXULW\ $ &RPSUHKHQVLYH $SSURDFK 9ROXPH 

&RQIHUHQFH 3DSHU

Agrobacterium tumefaciensPHGLDWHG 7UDQVIRUPDWLRQ RI (PEU\RJHQLF &DOOXV DQG 6RPDWLF (PEU\RV RI WKH %DQDQD FY ȍ$PERQ /XPXWȎ Musa acuminata 7LID 5 .XVXPDVWXWL  5L]NLWD 5 (V\DQWL  DQG )HQQ\ 0 'ZLYDQ\ 

'HSDUWPHQW RI 3ODQW 6FLHQFH DQG %LRWHFKQRORJ\ 6FKRRO RI OLIH VFLHQFH DQG WHFKQRORJ\ %DQGXQJ ,QVWLWXWH RI 7HFKQRORJ\ %DQGXQJ  :HVW -DYD ,QGRQHVLD  'HSDUWPHQW RI *HQHWLFV DQG 0ROHFXODU %LRWHFKQRORJ\ 6FKRRO RI OLIH VFLHQFH DQG WHFKQRORJ\ %DQGXQJ ,QVWLWXWH RI 7HFKQRORJ\ %DQGXQJ  :HVW -DYD ,QGRQHVLD

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

SURWRFRO ZDV GHYHORSHG IRU HVWDEOLVKLQJ JHQHWLF WUDQVIRUPDWLRQ IURP HPEU\RJHQLF FDOOXV DQG VRPDWLF HPEU\RV RI WKH EDQDQD FY $PERQ /XPXW (PEU\RJHQLF FDOOXV ZDV REWDLQHG LQ ,' PHGLXP 06EDVHG PHGLXP VXSSOHPHQWHG ZLWK  PJ /−1 ,$$  PJ /−1 ' DQG  J /−1 DFWLYH FKDUFRDO (PEU\RJHQLF FDOOXV ZDV WUDQVIHUUHG LQWR OLTXLG PHGLX P WR HVWDEOLVK VRPDWLF HPEU\RV (PEU\RJHQLF FDOOXV DQG VRPDWLF HPEU\RV ZHUH XVHG IRU Agrobacterium tumefaciensPHGLDWHG WUDQVIRUPDWLRQ A. tumefaciens VWUDLQ $ */ FRQWDLQLQJ S$577(67 S ODVPLG ZLWK JIS JHQH DV D UHSRUWHU DQG &D0 96 DV D SURPRWHU ZDV XVHG IRU WUDQVIRUPDWLRQV 7KH HPEU\RJHQLF FDOOXV DQG VRPDWLF HPEU\RV ZHUH WUDQVIRUPHG XVLQJ KHDWVKRFN PHWKRG IROORZHG E\ FHQWULIXJDWLRQ  USP DQG FRFXOW LYDWLRQ LQ OLTXLG PHGLXP FRQWDLQLQJ DFHWRV\ULQJRQH  𝜇0

&UHDWLYH &RPPRQV $WWULEXWLRQ

IRU  GD\V 5HVXOWV RI WKH *)3 DQDO\VLV VKRZHG WUDQVLHQW H[SUHVVLRQ IURP gfp JHQH

/LFHQVH ZKLFK SHUPLWV

UHSRUWHU LQ WUDQVIRUPHG HPEU\RJHQLF FDOOXV DQG VRPDWLF HPEU\RV 7UDQVIRUPDWLRQ

XQUHVWULFWHG XVH DQG

HIʃFLHQF\ LQ VRPDWLF HPEU\RV  ZDV KLJKHU WKDQ WKDW LQ HPEU\RJHQLF FDOOXV

UHGLVWULEXWLRQ SURYLGHG WKDW WKH RULJLQDO DXWKRU DQG VRXUFH DUH FUHGLWHG 6HOHFWLRQ DQG 3HHUUHYLHZ XQGHU WKH UHVSRQVLELOLW\ RI WKH

  3&5 DQDO\VLV XVLQJ &D09 SULPHU VKRZHG EDQGV WKDW FRPSDWLEOH ZLWK &D096 SURPRWHU DW  ES 7KLV LV D UHSRUW VKRZLQJ HVWDEOLVPHQW RI HPEU\RJHQLF FDOOXV DQG VRPDWLF HPEU\R FXOWXUH WUDQVIRUPDWLRQ E\ XVLQJ A. tumefaciensPHGLDWHG WUDQVIRUPDWLRQ SURWRFRO RI WKH ORFDO EDQDQD FY $PERQ /XPXW 7KLV VWXG\ SURYHG

,&6$)6 &RQIHUHQFH

WKH KXJH SRWHQWLDO IRU JHQHWLF WUDQVIRUPDWLRQ RI EDQDQD FY $PERQ /XPXW IRU FURS

&RPPLWWHH

LPSURYHPHQW VXFK DV SHVW RU GLVHDVH UHVLVWDQFH DQG DELRWLF IDFWRU VWUHVV WROHUDQFH

.H\ZRUGV EDQDQD HPEU\RJHQLF FDOOXV VRPDWLF HPEU\RV

+RZ WR FLWH WKLV DUWLFOH 7LID 5 .XVXPDVWXWL 5L]NLWD 5 (V\DQWL DQG )HQQ\ 0 'ZLYDQ\   ȍAgrobacterium tumefaciensPHGLDWHG 7UDQVIRUPDWLRQ RI (PEU\RJHQLF &DOOXV DQG 6RPDWLF (PEU\RV RI WKH %DQDQD FY ȍ$PERQ /XPXWȎ Musa acuminataȎ LQ 2nd International Conference on Sustainable Agriculture and Food Security: A Comprehensive Approach .Q( /LIH 6FLHQFHV SDJHV ȇ '2, NOVYL

3DJH 

,&6$)6 &RQIHUHQFH 3URFHHGLQJV

 ,QWURGXFWLRQ %DQDQD LV RQH RI WKH PRVW LPSRUWDQW VWDSOH IRRGV LQ WKH ZRUOG 6LQFH  ,QGRQHVLD VXSSOLHG DERXW  EDQDQD LQ LQWHUQDWLRQDO WUDGHV >@ 'DWD VKRZHG WKDW EDQDQD SUR GXFWLRQ LQ ,QGRQHVLD ZDV LQFUHDVLQJ DQG UHDFKHG  PLOOLRQ WRQQHV LQ  ,W ZDV DERXW  IURP QDWLRQDO IUXLW SURGXFWLRQ >@ 8QIRUWXQDWHO\ ,QGRQHVLD VWLOO FDQQRW LQFUHDVH LWV H[SRUW GXH WR ORZ TXDOLW\ SURGXFW IRU H[SRUW VWDQGDUG &RQYHQWLRQDO EUHHG LQJ WR LPSURYH EDQDQDV TXDOLW\ KDV OLPLWDWLRQV VXFK DV VWHULOLW\ RI FXOWLYDWHG EDQDQD KLJK SRO\SORLG\ OHYHOV DQG ORQJ JHQHUDWLRQ WLPH WR JURZ QHZ FXOWLYDU ,Q WKLV VWXG\ D QHZ PHWKRG WR LPSURYH ORFDO EDQDQD TXDOLW\ XVLQJ ELRWHFKQRORJLFDO DSSURDFK ZDV GHYHORSHG *HQHWLF WUDQVIRUPDWLRQ XVLQJ Agrobacterium tumefaciens RIIHUV WKH EHVW VROXWLRQ IRU FURS LPSURYHPHQW LQ EDQDQD 5HODWLYH VXFFHVV LQ A. tumefaciens JHQHWLF WUDQVIRUPDWLRQ KDG EHHQ DFKLHYHG LQ RWKHU VWXGLHV XVLQJ GLIIHUHQW FXOWLYDUV VXFK DV $JEDJED > @ 5DVWKDOL 5REXVWD >@ *UDQG 1DLQH >@ &DYHQGLVK DQG /DG\ ʃQJHU >@ E\ LQVHUWLQJ JHQHV VXFK DV Carica papaya F\VWDWLQ &S&@ 𝛽 JOXFDQDVH DQG +XPDQ O\VR]\P +/ >@ 7KH SXUSRVH RI WKLV VWXG\ ZDV WR REWDLQ D SURWRFRO IRU A. tumefaciensPHGLDWHG WUDQVIRUPDWLRQ LQ HPEU\RJHQLF FDOOXV DQG VRPDWLF HPEU\RV RI EDQDQD FY $PERQ /XPXW WKDW KDG QHYHU EHHQ UHSRUWHG HOVHZKHUH (PEU\RJHQLF FDOOXV DQG VRPDWLF HPEU\RV ZHUH FKRVHQ DV WKH VXEMHFWV LQ WKLV VWXG\ (PEU\RJHQLF FDOOXV KDG EHHQ UHSRUWHG IRU LWV KLJKO\ PHULVPDWLF FHOOV WKDW FRXOG LQFUHDVH WUDQVIRUPDWLRQ HIʃ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gfp UHSRUWHU JHQH nptII VHOHFWDEOH PDUNHU JHQH DQG &D096 SURPRWHU &D096 ZDV SURPRWHU WKDW GHULYHG IURP FDXOLʄRZHU PRVDLF YLUXV &D09  D VSHFLʃHG SURPRWHU IRU SODQWV 7KH &D096 SUR PRWHU LV ZLGHO\ XVHG WR GULYH WUDQVJHQH H[SUHVVLRQ VLQFH LW KDV EHHQ VKRZQ WR EH DFWLYH LQ D ZLGH UDQJH RI WLVVXH DQG LQ PDQ\ FURS VSHFLHV 7KH &D09 SURPRWHU LV SUHIHUUHG EHFDXVH LW LV D PRUH SRZHUIXO SURPRWHU WKDQ RWKHUV DQG LV QRW JUHDWO\

'2, NOVYL

3DJH 

,&6$)6 &RQIHUHQFH 3URFHHGLQJV

LQʄXHQFHG E\ HQYLURQPHQWDO FRQGLWLRQV RU WLVVXH W\SHV &D09 KDV WZR SURPRWHUV 6 DQG 6 RI WKHVH WZR WKH 6 SURPRWHU LV PRUH IUHTXHQWO\ XVHG LQ ELRWHFKQRORJ\ EHFDXVH LW LV PRVW SRZHUIXO > @ 7KH XVH RI WKH JUHHQ ʄXRUHVFHQW SURWHLQ *)3 IURP Aequorea victoria DV D YLWDO PDUNHU KDV DWWUDFWHG LQWHUHVW DQG KDV EHHQ GHVFULEHG LQ GLIIHUHQW RUJDQLVPV *)3 LV D  N'D SURWHLQ WKDW ʄXRUHVFHQW JUHHQ ZKHQ H[FLWHG E\ XOWUDYLROHW RU EOXH OLJKW 7KH *)3 KDV VHYHUDO DGYDQWDJHV RYHU RWKHU YLVXDO UHSRUWHU JHQHV VXFK DV 𝛽JOXFXURQLGDVH *86  𝛽JDODFWRVLGDVH /DF= DQG OXFLIHUDVH /8&  2QH RI LPSRUWDQW DGYDQWDJHV LV WKDW *)3 GRHV QRW UHTXLUH WKH DGGLWLRQ RI D VXEVWUDWH RU FRIDFWRUV IRU ʄXRUHVFHQFH WR EH GHWHFWHG 2Q WKH FRQWUDU\ RWKHU UHSRUWHU JHQHV UHTXLUH HLWKHU WKH DGGLWLRQ RI H[RJHQRXV VXEVWUDWHV RU FRIDFWRUV DQG WKHLU DSSOLFDWLRQV DUH OLPLWHG EHFDXVH WKH DVVD\V DUH JHQHUDOO\ GHVWUXFWLYH 7KLV PDNHV *)3 WKH EHVW FKRLFH IRU UHSRUWHU JHQH LI WKH H[SODQWV RU VDPSOHV DUH OLPLWHG > @ ,Q WKLV VWXG\ SURWRFRO ZDV GHYHORSHG IRU WUDQVIRUPDWLRQ LQ OLTXLG FRFXOWLYDWLRQ PHGLXP RI EDQDQD ORFDO FXOWLYDU $PERQ /XPXW GHWHFWHG E\ XVLQJ WUDQVLHQW H[SUHVVLRQ RI gfp JHQH UHSRUWHU LQ ERWK HPEU\RJHQLF FDOOXV DQG VRPDWLF HPEU\RV RI EDQDQD ORFDO FXOWLYDU $PERQ /XPXW

 0DWHULDOV DQG 0HWKRG  0DWHULDOV  S$577(67 SODVPLG S$577(67 SODVPLG XVHG LQ WKLV VWXG\ ZDV DFTXLUHG IURP 4XHHQVODQG 8QLYHUVLW\ RI 7HFKQRORJ\ 487  4XHHQVODQG $XVWUDOLD 7KH SODVPLG KDV UHSRUWHU JHQH gfp, VHOHFWDEOH PDUNHU JHQH nptII neomycin phospotransferase DQG SURPRWHU &D096 )LJ  

 %DQDQD LQʄRUHVFHQV PDOH ʄRZHUV %DQDQD LQʄRUHVFHQFH PDOH ʄRZHUV ZHUH REWDLQHG IURP ORFDO IDUPHUV LQ %DQGXQJ :HVW -DYD ,QGRQHVLD

'2, NOVYL

3DJH 

,&6$)6 &RQIHUHQFH 3URFHHGLQJV

)LJXUH  6FKHPDWLF GLDJUDP RI S$57 7 (67  SODVPLG 7 '1$ UHJLRQ

 A. tumefaciens VWUDLQ $*/ A. tumefaciens EDFWHULXP VWUDLQ $*/ ZHUH REWDLQHG IURP *HQHWLFV DQG 0ROHFXODU /DE RUDWRU\ 6FKRRO RI OLIH VFLHQFH DQG WHFKQRORJ\ %DQGXQJ ,QVWLWXWH RI 7HFKQRORJ\

 0HWKRGV  3ODQW PDWHULDOV DQG FXOWXUH LQLWLDWLRQ 0DOH LQʄRUHVFHQFH EXG ZDV SUHSDUHG E\ UHPRYLQJ WKH EUDFWV DQG WKH KDQGV RI WKH PDOH ʄRZHU 7KH EXGV GLVLQIHFWHG ZLWK 1D2&O ULQVHG WKUHH WLPHV ZLWK VWHULOH GLVWLOOHG ZDWHU DQG IXUWKHU UHGXFHG LQ VL]H WR WLSV WKDW ZHUH DERXW  FP ORQJ ×  FP ZLGH LQ D ODPLQDU ʄRZ KRRG 7KH H[SODQWV WKH KDQGV RI LPPDWXUH PDOH ʄRZHUV ,0)V  ZHUH LVRODWHG DVHSWLFDOO\ IURP WKH EXGV E\ UHPRYLQJ WKH EUDFWV XQGHU D ELQRFXODU VWHUHRPLFURVFRSH 7KH LPPDWXUH ʄRZHU KDQGV ZHUH LVRODWHG IURP SRVLWLRQV  WR  𝑠𝑡 EHLQJ WKH KDQG FORVHVW WR WKH PHULVWHPDWLF GRPH RI WKH PDOH EXG HDFK EXG ZHUH FXOWXUHG LQ FDOOXV LQGXFWLRQ PHGLXP 1,' 06 0DFURQXWULHQW [ 06 0LFURQXWULHQW [ 06 )H('7$ [ 6XFURVH  J/−1  $FWLYDWHG FKDUFRDO  J / 1$$  PJ /−1  ,$$ PJ /−1  '  PJ /−1  $JDU  J /−1  S+   7KH FXOWXUHV ZHUH NHSW LQ D JURZWK URRP DW  ± ∘ & XQGHU WRWDO GDUNQHVV DQG VXEFXOWXUHG HYHU\ IRXU ZHHNV 1RQ HPEU\RJHQLF FDOOXV HPHUJHG IURP H[SODQWV DIWHU WKUHH WLPHV VXEFXOWXUHG XVLQJ 1,' PHGLXP 1RQ HPEU\RJHQLF FDOOXV ZHUH WKHQ WUDQVIHUUHG LQWR ,' PHGLXP 0 6 0DFURQXWULHQW [ 06 0LFURQXWULHQW [ 06 )H ('7$ [ 6XFURVH  J /−1  $FWLYDWHG FKDUFRDO  J / ,$$  PJ /−1  '  PJ /−1  $JDU  J/−1  S+   NHSW LQ D JURZWK URRP DW  ± ∘ & XQGHU WRWDO GDUNQHVV DQG VXEFXOWXUHG HYHU\ IRXU ZHHNV WR SURGXFH HPEU\RJHQLF FDOOXV '2, NOVYL

3DJH 

,&6$)6 &RQIHUHQFH 3URFHHGLQJV

6RPDWLF HPEU\RV IRU WUDQVIRUPDWLRQ ZHUH REWDLQHG E\ WUDQVIHUULQJ HPEU\RJHQLF FDOOXV LQWR  P/ HUOHQPD\HU ʄDVN FRQWDLQLQJ  PO OLTXLG PHGLXP 0( 06 0DFURQX WULHQW [ 06 0LFURQXWULHQW [ 06 )H('7$ [ 6XFURVH  J/−1  $FWLYDWHG FKDUFRDO  J /−1  1$$  PJ /−1  %$3  PJ /−1  S+   7KH FXOWXUHV ZHUH DJLWDWHG RQ DQ RUE LWDO VKDNHU DW  USP LQ D JURZWK URRP PDLQWDLQHG DW  ± ∘ & LQ WRWDO GDUNQHVV DQG VXE FXOWXUHG HYHU\ WZR ZHHNV XQWLO VRPDWLF HPEU\RV GRPLQDQW LQ JOREXODU VKDSH ZHUH REWDLQHG

 7UDQVIRUPDWLRQ DQG FRQʃUPDWLRQ RI S$57 7(67 SODVPLGV LQ A. tumefaciens $*/ S$577(67 SODVPLG WUDQVIRUPDWLRQ ZDV SHUIRUPHG RQ FRPSHWHQW $ WXPHIDFLHQV VWUDLQ $*/ &RPSHWHQW $ WXPHIDFLHQV $*/ FHOOV ZHUH PDGH XVLQJ &D&O PHWKRG DQG $ WXPHIDFLHQV SODVPLG ZDV FDUULHG RXW XVLQJ KHDWVKRFN PHWKRG >@ 7KH H[LVWHQFH RI S$577(67 SODVPLG LQ $ WXPHIDFLHQV $*/ ZDV FRQʃUPHG E\ FUXGH 3&5 PHWKRG XVLQJ WKH SDLU RI &D09 SULPHU SULPHU IRUZDUG ȋ&&7$$&$*$$&7&*&&*7$$$*$ȋDQG UHYHUVH ȋ&&&*7*77&7&7&&$$$7*$$$7*ȋ  $PSOLʃFDWLRQ RI &D096 SURPRWHU ZDV VWDUWHG ZLWK WKH  PLQXWHV LQLWLDO GHQDWXUDWLRQ DW  ∘ & WHPSHUDWXUH WKHQ SHUIRUPHG  F\FOHV FRQVLVWLQJ RI  VHFRQGV DW D WHPSHUDWXUH RI  ∘ & IRU GHQDWXUDWLRQ  VHFRQGV DW  ∘ & IRU DQQHDOLQJ DQG WZR PLQXWHV DW  ∘ & IRU HORQJDWLRQ )LQDO HORQJDWLRQ SURFHVV ZDV FDUULHG RXW IRU  PLQXWHV DW D WHPSHUDWXUH RI  ∘ & '1$ IUDJPHQWV IURP 3&5 SURGXFWV ZHUH VHSDUDWHG XVLQJ JHO HOHFWURSKRUHVLV RQ  DJDURVH FRQFHQWUDWLRQ LQ 7$( EXIIHU VROXWLRQ

 A. tumefaciens EDFWHULD FXOWXUH SUHSDUDWLRQ $ WXPHIDFLHQV $ */ FXOWXUH FRQWDLQLQJ SODVPLG S$577(67 ZDV JURZQ LQ @

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∘ & > @

 7UDQVIRUPDWLRQ DQG FRFXOWLYDWLRQ (PEU\RJHQLF FDOOXV DQG VRPDWLF HPEU\RV ZHUH LQRFXODWHG ZLWK $ WXPHIDFLHQV FXOWXUH 2'QP

  $ WRWDO RI  ) SOXRURQLF DFLG ZDV DGGHG DV D VXUIDFWDQW

(PEU\RJHQLF FDOOXV DQG VRPDWLF HPEU\RV ZHUH LQRFXODWHG ZLWK $ WXPHIDFLHQV IRO ORZHG E\ FHQWULIXJDWLRQ DW  US P IRU  PLQXWHV KRPRJHQL]HG XVLQJ D YRUWH[ IRU  VHFRQGV DQG WKHQ UHVWHG IRU  PLQXWHV >@ (PEU\RJHQLF FDOOXV DQG VRPDWLF HPEU\RV ZHUH WKHQ WUDQVIHUUHG LQWR WKH OLTXLG PHGLXP IRU FRFXOWLYDWLRQ 06 0DFURQXWULHQ [ 06 0LFURQXWULHQ [ 06 )H('7$ [ 6XFURVH  J /  DQG LQFXEDWHG IRU  GD\V DW D WHPSHUDWXUH RI  ∘ & LQ GDUN FRQGLWLRQV

 'HFRQWDPLQDWLRQ DQG UHFRYHU\ &XOWXUH ZDV UHPRYHG DVHSWLFDOO\ LQWR WKH IDOFRQ WXEHV FRQWDLQLQJ VWHULOH GLVWLOOHG ZDWHU VR WKDW DOO SDUWV RI WKH FXOWXUH ZHUH VXEPHUJHG )DOFRQ WXEH ZDV DJLWDWHG ZKLOH RFFD VLRQDOO\ KRPRJHQL]HG E\ YRUWH[ IRU RQH PLQXWH GLVWLOOHG ZDWHU ZDV WKHQ GLVFDUGHG '2, NOVYL

3DJH 

,&6$)6 &RQIHUHQFH 3URFHHGLQJV

7KH FXOWXUH ZDV DGGHG ZLWK ZDVKLQJ OLTXLG 06 PHGLXP ZKLFK KDG EHHQ DGGHG ZLWK  PJ /−1 WLPHQWLQ )DOFRQ WXEH ZDV DJLWDWHG DW D VSHHG RI  USP IRU  PLQXWHV ZKLOH RFFDVLRQDOO\ KRPRJHQL]HG E\ YRUWH[ 7KH HQWLUH ZDVK VWHS ZDV UHSHDWHG  WLPHV

 2EVHUYDWLRQV RI JIS UHSRUWHU JHQH H[SUHVVLRQ 2EVHUYDWLRQV RI JIS UHSRUWHU JHQH H[SUHVVLRQ ZDV SHUIRUPHG XVLQJ D PLFURVFRSH ʄXRUHVFHQWV 1LNRQ (FOLSVH ( &DOOXV DQG HPEU\RV WUDQVIRUPDQW DV ZHOO DV QRQ WUDQVIRUPDQW ZHUH SODFHG RQ WKH JODVV REMHFW DQG REVHUYHG XVLQJ ʄXRUHVFHQFH PLFURVFRS\ ZLWK XOWUDYLROHW OLJKW 89 DW D ZDYHOHQJWK RI  QP 3DUWV WKDW HPLWWHG JUHHQ OXPLQHVFHQFH GXULQJ REVHUYDWLRQ LQGLFDWHG WKH SUHVHQFH RI JIS UHSRUWHU JHQH WKDW ZDV H[SUHVVHG LQ WUDQVIRUPHG FXOWXUH

 (VWLPDWLRQ SHUFHQWDJH RI *)3 H[SUHVVLRQ DQG WUDQVIRUPDWLRQ HIʃFLHQF\ LQ WUDQVIRUPHG HPEU\RJHQLF FDOOXV DQG VRPDWLF HPEU\RV (VWLPDWLRQ SHUFHQWDJH RI *)3 H[SUHVVLRQ DQG WUDQVIRUPDWLRQ HIʃFLHQF\ LQ WUDQVIRUPHG HPEU\RJHQLF FDOOXV DQG VRPDWLF HPEU\RV ZDV FDUULHG XVLQJ $GREH 3KRWRVKRS &6 VRIWZDUH 7UDQVIRUPHG FDOOXV DQG VRPDWLF HPEU\RV SLFWXUHV ZHUH YLHZHG XVLQJ $GREH 3KRWRVKRS &6 SDUW WKDW HPLWWHG JUHHQ OXPLQHVFHQFH DQG QRQ HPLWWHG JUHHQ OXPLQHV FHQFH ZHUH FRXQWHG XVLQJ JULG OD\HU 5HVXOWV ZHUH FDOFXODWHG XVLQJ IRUPXOD EHORZ (VWLPDWLRQ RI SHUFHQWDJH RI 7RWDO JULG LQ WUDQVIRUPHG FXOWXUH HPLWWHG JUHHQ OXPLQHVFHQFH *)3 H[SUHVVLRQ DQG WUDQV = × 100% 7RWDO DOO JULG LQ WUDQVIRUPHG FXOWXUH IRUPDWLRQ HIʃFLHQF\

 &D09 6 SURPRWHU PROHFXODU 3&5 WHVWLQJ ,VRODWLRQ RI WUDQVIRUPHG '1$ ZDV SHUIRUPHG XVLQJ 7LDQJHQ 3ODQW *HQRPLF '1$ .LW '1$ RI HPEU\RJHQLF FDOOXV DQG VRPDWLF HPEU\RV ZHUH FRQʃUPHG E\ 3&5 XVLQJ WKH SDLU RI SULPHU IRUZDUG ȋ&&7$$&$*$$&7&*&&*7$$$*$ȋ DQG UHYHUVH ȋ &&&*7*77&7&7&&$$$7*$$$7*ȋ  $PSOLʃFDWLRQ RI &D096 SURPRWHU ZDV VWDUWHG ZLWK WKH  PLQXWHV LQLWLDO GHQDWXUDWLRQ DW  ∘ & WHPSHUDWXUH WKHQ SHUIRUPHG  F\FOHV FRQVLVWLQJ RI  VHFRQGV DW D WHPSHUDWXUH RI  ∘ & IRU GHQDWXUDWLRQ  VHFRQGV DW  ∘

& IRU DQQHDOLQJ DQG WZR PLQXWHV DW  ∘ & IRU HORQJDWLRQ )LQDO HORQJDWLRQ SURFHVV ZDV

FDUULHG RXW IRU  PLQXWHV DW D WHPSHUDWXUH RI  ∘ & '1$ IUDJPHQW V RI 3&5 SURGXFWV ZHUH VHSDUDWHG XVLQJ JHO HOHFWURSKRUHVLV RQ  DJDURVH FRQFHQWUDWLRQ LQ 7$( EXIIHU VROXWLRQ '2, NOVYL

3DJH 

,&6$)6 &RQIHUHQFH 3URFHHGLQJV

 5HVXOW DQG 'LVFXVVLRQ  (PEU\RJHQLF FDOOXV DQG VRPDWLF HPEU\RV EDQDQD DPERQ OXPXW LQLWLDWLRQ 7KH FDOOXVHV ZHUH LQLWLDWHG IURP H[SODQWV RI LPPDWXUH PDOH ʄRZHUV RI EDQDQD FY $PERQ /XPXW 0DOH ʄRZHUV KDG EHHQ ZLGHO\ XVHG DV H[SODQWV LQ VRPDWLF HPEU\RJH QHVLV LQ EDQDQDV WR REWDLQ D FXOWXUH RI HPEU\RJHQLF FDOOXV VRPDWLF HPEU\RV >@ DQG HPEU\RJHQLF FHOO VXVSHQVLRQ FXOWXUH >  @ 7KH LPPDWXUH PDOH ʄRZHUV RI EDQDQD ZDV VHOHFWHG EHFDXVH LQ WKHRU\ WKH FHOOV ZHUH VWLOO PHULVWHPDWLF HDVLO\ SUROLIHUDWHG DQG KDG D KLJK DELOLW\ WR GLYLGH 7KH FORVHU WKH SRVLWLRQ RI WKH PDOH ʄRZHUV WR WKH PHULVWHP WKH KLJKHU WKH DELOLW\ WR GLYLGH DQG VR HPEU\RJHQLF FDOOXV ZRXOG EH HDVLO\ REWDLQHG >@ &DOOXV REWDLQHG LQ ODERUDWRU\ KDG D EODFNLVK FRORU KDUG DQG QRQ HPEU\RJHQLF )LJ XUH $ &DOOXV ZHUH WKHQ VXEFXOWXUHG LQWR ,' PHGLXP HYHU\  ZHHNV WR REWDLQ HPEU\RJHQLF FDOOXV ZKLFK ZDV ZKLWH URXQG ZLWK D VPRRWK VXUIDFH )LJ % DV FRPPRQ FKDUDFWHULVWLF RI HPEU\RJHQLF FDOOXV >@ 7KH XVH RI DX[LQ LQ WKH PHGLXP ZDV WR LQFUHDVH FHOO SUROLIHUDWLRQ DQG LQGXFWLRQ RI HPEU\RJHQLF FDOOXV $X[LQ SDUWLFXODUO\ ' DQG ,$$ SOD\HG D UROH LQ WKH LQLWLDWLRQ RI VRPDWLF HPEU\RJHQHVLV LQ EDQDQDV >  @ FDUURWV >@ DOIDOID >@ DQG FKU\VDQWKHPXP >@ (PEULRJHQLF FDOOXV ZDV WUDQVIHUUHG LQWR OLTXLG PHGLXP 0( 8VH RI OLTXLG PHGLXP IRU VRPDWLF HPEU\R LQLWLDWLRQ KDV EHHQ XVHG LQ VHYHUDO VWXG\ VXFK DV WKH EDQDQD FXOWLYDU ȋ5DVWKDOLȋ >@ ȋ'ZDUI %UD]LOLDQȋ >@ DQG WKH FXOWLYDU ȋ0DVȋ >@ ,Q WKH OLT XLG PHGLXP SURHPEU\RV FHOOV WKDW IRUP QRGXOHV RQ WKH FDOOXV ZHUH VHSDUDWHG LQWR VLQJOH SURHPEU\RV ZKLFK KDG D \HOORZLVK ZKLWH FRORU )LJ &  6LQJOH SURHPEU\RV ZDV WUDQVIHUUHG LQWR D OLTXLG PHGLXP 06 ,Q WKH OLTXLG PHGLXP QRW DOO SURHPEU\RV IRUPHG JOREXODU HPEU\RV DW WKH VDPH WLPH ZKLFK PLJKW EH GXH WR VHYHUDO FRQGLWLRQV LQFOXG LQJ PHGLD FRPSRVLWLRQ VRXUFH RI H[SODQWV DQG JHQHWLF IDFWRUV >@ 6RPDWLF HPEU\R FXOWXUH ZHUH KRPRJHQL]HG E\ VWDJHV WKLV SURWRFRO KDG EHHQ GRQH E\ )XMLPXUD DQG .RPDPLQH >@RQ FDUURW HPEU\RJHQLF FHOO FXOWXUHV WR SURGXFH D XQLIRUP GHYHORSPHQW RI WKH HPEU\R LQ D FXOWXUH (PEURJHQLF FDOOXV DQG VRPDWLF HPEU\RV ZHUH WUHDWHG ZLWK KHDWVKRFN IRU  PLQ XWHV DW ∘ & +HDWVKRFN WHFKQLTXHV SHUIRUPHG RQ FDOOXV FXOWXUH DQG HPEU\R ZHUH H[SHFWHG WR GLVWXUE WKH EDODQFH RI WKH FHOO PHPEUDQH PHWDEROLVP DQG RWKHU SK\V LRORJLFDO SURFHVVHV LQ WKH FHOO > @ 7KLV WUHDWPHQW FRXOG LQGXFH FHOOV UHVSRQVH WR VWUHVV LQ WKH IRUP RI +VS/ QDPHO\ VPDOO FU\VWDOOLQH DOSKDW\SH KHDWVKRFN SURWHLQ +VS 𝛼  ,Q WKH SURFHVV RI WUDQVIRUPDWLRQ +VS/ ZDV UHTXLUHG IRU 9LU% SURWHLQ DFFXPXODWLRQ

'2, NOVYL

3DJH 

,&6$)6 &RQIHUHQFH 3URFHHGLQJV

)LJXUH  (PEU\RJHQLF FDOOXV DQG VRPDWLF HPEU\RV RI $PERQ /XPXW $ QRQ HPEU\RJHQLF FDOOXV % HPEU\RJHQLF FDOOXV RQ SHWUL GLVK & VRPDWLF HPEU\RV FXOWXUH LQ OLTXLG PHGLXP ' VRPDWLF HPEU\RV REVHUYHG XQGHU VWHUHR PLFURVFRSH

ZKLFK DOOHJHGO\ SOD\HG DQ LPSRUWDQW UROH LQ WKH WUDQVIHU RI 7'1$ E\ 9LU%  ' >@ 7UHDWPHQW RI KHDWVKRFN KDG SUHYLRXVO\ EHHQ VXFFHVVIXO LQ LQFUHDVLQJ WKH HIʃFLHQF\ RI WUDQVIRUPDWLRQ LQ HPEU\RJHQLF FHOO FXOWXUHV RI EDQDQD FXOWLYDU ȋ&DYHQGLVKȋ DQG ȋ/DG\ )LQJHUȋ >@ VRPDWLF HPEU\RV RI EDQDQDV ȋ&DYHQGLVKȋ >@ DQG WKH FXOWXUH RI U\HJUDVV DQG ULFH HPEU\RJHQLF FDOOXV >@

 &RQʃUPDWLRQ RI SODVPLG SDUWWHVW LQ A. tumefaciens VWUDLQ $*/ Agrobacterium tumefaciens VWUDLQ $*/ ZHUH JURZQ LQ @ DQG VRPDWLF HPEU\RV RI JUDSHV Vitis vinifera / FY7KRPSVRQ 6HHGOHVV >@ 6XFFHVVIXO WUDQVIRUPDQW SDUW LQ H[SUHVVLQJ *)3 SURWHLQ S URYHG WKH SRWHQWLDO RI &D096 SURPRWHU WR EH XVHG DV D SURPRWHU LQ WUDQVIRUPDWLRQ EDQDQD FXOWLYDUV $PERQ /XPXW 7UDQVLHQW H[SUHVVLRQ IURP UHSRUWHU JHQH FRXOG EH XVHG WR HVWLPDWH WKH SUR PRWHU VWUHQJWK 6WURQJ DFWLYLW\ IURP SURPRWHU FDQ OHDG WR VWURQJ H[SUHVVLRQ RI UHSRUWHU '2, NOVYL

3DJH 

,&6$)6 &RQIHUHQFH 3URFHHGLQJV

)LJXUH  2EVHUYDWLRQ RI *)3 RQ VRPDWLF HPEU\RV RI EDQDQD FXOWLYDU $PERQ /XPXW $% QRQ WUDQVIRUPHG VRPDWLF HPEU\R &' VRPDWLF HPEU\R WUDQVIRUPHG E\ A. tumefaciens VWUDLQ $*/S$577(67 

JHQH 3URPRWHU DFWLYLW\ ZDV RQH RI WKH NH\ IDFWRUV WKDW LQFOXGHG LQ JHQH H[SUHVVLRQ UHJXODWLRQ 2WKHU UHVHDUFK RI WUDQVIRUPDWLRQ LQ JUDSH VRPDWLF HPEU\RV XVLQJ WKH SUR PRWHU GRXEOH FDXOLʄRZHU PRVDLF YLUXV 6 &D096 DQG GRXEOH FDVVDYD YHLQ PRVDLF YLUXV &V909 SURGXFHG WUDQVJHQLF SODQW H[SUHVVLQJ D WUDQVJHQH ZKLFK ODVW XS WR  \HDUV >@ *)3 H[SUHVVLRQ ZDV VWDEOH XQWLO WKH SODQW VXUYLYHG DQG UHJHQHUDWHG DQG LW H[SUHVVHG LQ YDULRXV WLVVXHV DQG RUJDQV RI WKH SODQW EXGV OHDYHV URRWV SHWDO VWDPHQ  HYHQ LQ ʄRZHUV IUXLWV DQG VHHGV >@

 (VWLPDWLRQ RI SHUFHQWDJH DUHDV RI *)3 H[SUHVVLRQ DQG WUDQVIRUPDWLRQ HIʃFLHQF\ LQ HPEU\RJHQLF FDOOXV DQG VRPDWLF HPEU\RV RI $PERQ /XPXW EDQDQD (VWLPDWLRQ RI SHUFHQWDJH DUHD WKDW H[SUHVVHG *)3 LQ WUDQVIRUPHG VRPDWLF HPEU\RV ZDV KLJKHU WKDQ HPEU\RJHQLF FDOOXV (VWLPDWH QXPEHU DUHD WKDW HPLWWHG JUHHQ OXPL QHVFHQFH LQ VRPDWLF HPEU\RV WLVVXH ZDV DERXW  SHU HPEU\RV ZKLOH WUDQVIRUPHG HPEU\RJHQLF FDOOXV ZDV DERXW  SHU FDOOXV 7DEOH   7KHVH UHVXOW VKRZHG WKDW '2, NOVYL

3DJH 

,&6$)6 &RQIHUHQFH 3URFHHGLQJV

7˔˕˟˘  3HUFHQWDJH RI DUHD H[SUHVVHG *)3 LQ WUDQVIRUPHG VRPDWLF HPEU\RV DQG HPEU\RJHQLF FDOOXV

$YHUDJH WRWDO DUHD SHU FXOWXUH

$YHUDJH DUHD HPLWWHG JUHHQ OXPLQHVFHQV SHU FXOWXUH

3HUFHQWDJH RI *)3 H[SUHVVLRQ DQG WUDQVIRUPDWLRQ HIʃFLHQF\

















7RWDO WUDQVIRUPHG FXOWXUH FXOWXUH WHVWHG

6RPDWLF (PEU\RV (PEU\RJHQLF &DOOXV

1DPH

)LJXUH  5HVXOW RI '1$ LVRODWLRQ IURP HPEU\RJHQLF FDOOXV DQG VRPDWLF HPEU\RV RI EDQDQD FXOWLYDU $PERQ /XPXW / '1$ /DGGHU  NE )(50(17 $6 ( WUDQVIRUPHG VRPDWLF HPEU\RV . WUDQVIRUPHG HPEU\RJHQLF FDOOXV ( QRQ WUDQVIRUPHG VRPDWLF HPEU\RV . QRQ WUDQVIRUPHG HPEU\RJHQLF FDOOXV

WUDQVIRUPDWLRQ HIʃFLHQF\ DQG *)3 H[SUHVVLRQ LQ VRPDWLF HPEU\RV ZDV KLJKHU WKDQ HPEU\RJHQLF FDOOXV

 0ROHFXODU 3&5 WHVWLQJ RQ HPEU\RJHQLF FDOOXV DQG VRPDWLF HPEU\RV EDQDQD $PERQ /XPXW 6DPSOHV RI HPEU\RJHQLF FDOOXV DQG VRPDWLF HPEU\RV WUDQVIRUPHG DQG QRQ WUDQV IRUPHG ZHUH LVRODWHG XVLQJ 7LDQJHQ 3ODQW *HQRPLF '1$ .LW VSHFLʃFDOO\ IRU WKH LVROD WLRQ RI '1$ JHQR PLF RI SODQWV 7KLV NLW XVHV VLOLFD PHPEUDQH WHFKQRORJ\ WR HOLPLQDWH FRPSOLFDWH VWDJHV LQ WKH SURFHVV RI '1$ LVRODWLRQ (VSHFLDOO\ LI WKH VDPSOH FRQWDLQV SRO\SKHQROV UHVLQV RU RWKHU PHWDEROLWHV > @ ,VRODWHG '1$ )LJ  VKRZHG WKH '1$ EDQGV WKDW ZHUH IDLQW EXW FOHDQ RI LPSXULWLHV 7KLV UHVXOW ZDV XVHG IRU IXUWKHU WHVWLQJ WR FRQʃUP H[LVWHQFH RI S$577(67 SODVPLG XVLQJ 3&5 PHWKRG '2, NOVYL

3DJH 

,&6$)6 &RQIHUHQFH 3URFHHGLQJV

1000 bp 750 bp 500 bp 250 bp

507 bp

)LJXUH  3&5 SODVPLG S$577(67 FRQʃUPDWLRQ UHVXOW LQ HPEU\RJHQLF FDOOXV DQG VRPDWLF HPEU\RV RI $PERQ /XPXW EDQDQDV / '1$ /DGGHU  NE )(50(17$6 FR SRVLWLYH FRQWURO SULPHU &D09 S$57  7(67 . WUDQVIRUPHG HPEU\RJHQLF FDOOXV ( WUDQVIRUPHG VRPDWLF HPEU\RV . QRQ WUDQVIRUPHG HPEU\RJHQLF FDOOXV ( QRQ WUDQVIRUPHG VRPDWLF HPEU\RV FR QHJDWLYH FRQWURO +2 2

&RQʃUPDWLRQ SUHVHQFH RI WKH SODVPLG S$577(67 LQ HPEU\RJHQLF FDOOXV DQG VRPDWLF HPEU\RV ZDV GRQH XVLQJ WKH 3&5 PHWKRG ZLWK &D09 SULPHU$PSOLʃFDWLRQ UHVXOW XVLQJ &D09 SULPHU DJDLQVW &D096 SURPRWHUFRQWDLQHG LQ SODVPLG S$577(67 VKRZHG EDQGV DW  ES FRUUHVSRQGHG WR WKH SRVLWLYH FRQWURO SODVPLG S$577(67  7KHVH EDQGV ZHUH QRW IRXQG LQ QRQ WUDQVIRUPHG HPEU\RJHQLF FDOOXV DQG VRPDWLF HPEU\RV .DQG ( DV ZHOO DV QHJDWLYH FRQWURO XVLQJ ZDWHU +2 )LJ   7KH XVH RI VXUIDFWDQWV DQG WKH DGGLWLRQ RI DFHWRF\ULQJRQH FRPSRXQGV FDQ LPSURYH WKH HIʃFLHQF\ RI WUDQVIRUPDWLRQ LQ SODQWV 6XUIDFWDQWV VXFK DV 3OXURQLF DFLG ) XVHG LQ WKLV VWXG\ ZHUH DGGHG DW WUDQVIRUPDWLRQ 7KH XVH RI VXUIDFWDQW VHUYHV WR IDFLOLWDWH WKH DWWDFKPHQW RI EDFWHULD A. tumefaciens FXOWXUHV E\ UHGXFLQJ WKH VXUIDFH WHQVLRQ RI WKH VROXWLRQ 7KH DGGLWLRQ RI VXUIDFWDQW FDQ DOVR HOLPLQDWH RWKHU FRPSRXQGV WKDW LQKLELW WKH DWWDFKPHQW RI EDFWHULD WR WKH SODQW FHOO > @ $GGLWLRQ RI H[RJHQRXV FRPSRXQGV VXFK DFHWRV\ULQJRQH HVSHFLDOO\ IRU PRQR FRW\OHGRQRXV SODQWV ZHUH SURYHQ WR LPSURYH WKH HIʃFLHQF\ RI WUDQVIRUPDWLRQ $FH WRV\ULQJRQH FDQ DFWLYDWH 9LU$ SURWHLQ ORFDWHG RQ WKH FHOO VXUIDFH RI PHPEUDQH DQG WULJJHU DFWLYDWLRQ RI RWKHU vir gHQHV >@ $FHWRV\ULQJRQH FRPSRXQGV ZHUH FRPPRQO\ S URGXFHG LQ GLFRW\OHGRQRXV SODQWV EXW RQO\ D VPDOO SRUWLRQ RI PRQRFRW\OHGRQRXV SODQWV SURGXFH WKHVH FRPSRXQGV 0RQRFRW\OHGRQRXV SODQWV VXFK DV EDQDQDV UHTXLUH DGPLQLVWUDWLRQ RI H[RJHQRXV DFHWRV\ULQJRQH >@ $FHWRV\ULQJRQH VKRXOG EH DGGHG LQ RSWLPXP FRQFHQWUDWLRQ VLQFH DFHWRV\ULQJRQH FRPSRXQGV FRXOG HIIHFWLYHO\ DFWLYDWH WKH vir JHQHV DW WKH PLQLPXP FRQFHQWUDWLRQ EXW UHVXOWHG LQ EDFWHULRVWDWLF ZKHQ XVHG DW KLJK FRQFHQWUDWLRQV >@ '2, NOVYL

3DJH 

,&6$)6 &RQIHUHQFH 3URFHHGLQJV

7KH WUDQVIRUPDWLRQ SURWRFRO KDG EHHQ XVHG E\ .KDQQD HW DO >@ DQG $SUL\DQL >@ EXW LW ZDV VOLJKWO\ PRGLʃHG LQ WKLV VWXG\ $ OLTXLG PHGLXP WR LPSURYH WKH HIʃFLHQF\ RI WUDQVIRUPDWLRQ ZDV XVHG LQ FRFXOWLYDWLRQ VWHS 7KLV SURWRFRO ZDV DOVR XVHG LQ RWKHU WUDQVIRUPDWLRQ XVLQJ GLIIHUHQW W\SHV RI PHGLD RQ WKH OHDYHV RI WREDFFR SODQWV 7KH HIʃFLHQF\ RI WUDQVIRUPDWLRQ LQ WREDFFR OHDI XVLQJ D OLTXLG PHGLXP GXULQJ FRȇ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tumefaciens WKDW FRFXOWLYDWHG LQ VROLG RU VHPLVROLG PHGLXP >@

 &RQFOXVLRQ ,Q WKLV SUHVHQW VWXG\ SURWRFRO IRU WUDQVIRUPDWLRQ ZDV REWDLQHG IRU HPEU\RJHQLF FDOOXV DQG VRPDWLF HPEU\RV RI EDQDQD ORFDO FXOWLYDU $PERQ /XPXW WKDW QHYHU EHHQ UHSRUWHG HOVHZKHUH 3URWRFRO IRU EDQDQD FXOWLYDU $PERQ /XPXW SODQW UHJHQHUDWLRQ YLD LQGL UHFW VRPDWLF HPEU\RJHQHVLV GDWD QRW VKRZQ ZDV DOVR REWDLQHG 7KLV UHSRUW VKRZHG SRWHQWLDO RI &D096 SURPRWHU DQG gfp JHQH UHSRUWHU IRU WUDQVIRUPDWLRQ LQ ORFDO FXO WLYDU EDQDQD $PERQ /XPXW 7KLV UHSRUW DOVR SURYHG SRWHQWLDO RI ORFDO FXOWLYDU EDQDQD $PERQ /XPXW IRU FURS LPSURYHPHQW XVLQJ A. tumefaciens-PHGLDWHG JHQHWLF WUDQVIRU PDWLRQ DQG D VWHS WRZDUGV VWDEOH WUDQVIRUPDWLRQ LQ WUDQVJHQLF EDQDQD ORFDO FXOWLYDUV

$FNQRZOHGJHPHQWV 7KH DXWKRUV ZRXOG OLNH WR WKDQN 4XHHQVODQG 8QLYHUVLW\ 7HFKQRORJ\ 487 IRU SURYLGLQJ S$577(67 SODVPLG FRQVWUXFW

5HIHUHQFHV >@ 6PLWK 6  )DLUWUDGH %DQDQDV $ *OREDO $VVHVPHQW RI ,PSDFW ,QVWLWXWH RI 'HYHORSPHQW 6WXGLHV 8QLYHUVLW\ RI 6XVVH[ 8QLWHG .LQJGRP >@ 'LUHNWRUDW %XGLGD\D GDQ 3DVFDSDQHQ %XDK  3HGRPDQ 3HQDQJDQDQ 3DVFDSD QHQ 3LVDQJ 'LUHNWRUDW %XGLGD\D GDQ 3DVFDSDQHQ %XDK .HPHQWHULDQ 3HUWDQLDQ -DNDUWD '2, NOVYL

3DJH 

,&6$)6 &RQIHUHQFH 3URFHHGLQJV

>@ 7ULSDWKL -1 0XZRQJH $ DQG 7ULSDWKL /  (IʃFLHQW UHJHQHUDWLRQ DQG WUDQVIRUPDWLRQ RI SODQWDLQ FYȍ*RQMD PDQMD\DȎ 0XVD VSS $$% XVLQJ HPEU\RJHQLF FHOO VXVSHQVLRQV ,Q 9LWUR &HOO'HY%LRO     >@ 7ULSDWKL / 7ULSDWKL -1 DQG +XJKHV -Gȋ$  $JUREDFWHULXP WUDQVIRUPDWLRQ RI SODQWDLQ 0XVD VSS FXOWLYDU $JEDJED $IULFDQ -RXUQDO RI %LRWHFKQRORJ\     >@ *KRVK $ *DQDSDWKL 7 5 1DWK 3 DQG %DSDW 9 $  (VWDEOLVKPHQW RI HPEU\RJHQLF FHOO VXVSHQVLRQ FXOWXUHV DQG $JUREDFWHULXPPHGLDWHG WUDQVIRUPDWLRQ LQ DQ LPSRUWDQW &DYHQGLVK EDQDQD FY 5REXVWD $$$  3ODQW &HOO 7 LVV 2UJDQ &XOWXUH  ȇ >@ 6XWKHUODQG 5  *HQHWLF PRGLʃFDWLRQ RI &DYHQGLVK EDQDQDV 0XVD VSS LQ 6RXWK $IULFD 7KHVLV )DFXOW\ RI 1DWXUH DQG $JULFXOWXUDO 6FLHQFH 8QLYHUVLW\ RI 3UHWRULD 3UHWRULD 6RXWK $IULFD >@ .KDQQD + %HFNHU ' .OHLGRQ - DQG 'DOH -  &HQWULIXJDWLRQ $VVLVWHG $JUREDFWHULXP WXPHIDFLHQV PHGLDWHG 7UDQVIRUPDWLRQ &$$7 RI HPEU\RJHQLF FHOO VXVSHQVLRQV RI EDQDQD 0XVD VSS &DYHQGLVK $$$ DQG /DG\ )LQJHU $$%  0ROHFXODU %UHHGLQJ  ȇ >@ 6UHHUDPDQDQ 6 0D]LDK 0 DQG ;DYLHU 5  $ SURWRFRO IRU $JUREDFWHULXP PHGLDWHG WUDQVIRUPDWLRQ RI EDQDQD ZLWK D ULFH FKLWLQDVH JHQH (PLUDWHV -RXUQDO )RRG $JULFXOWXUH     >@ 3HL ; : &KHQ 6 . :HQ 5 0 @ 6LGKD 0 6XSUDVDQQD 3 %DSDW 9$ .XONDUQL 8* DQG 6KLQGH % 1  'HYHORSLQJ VRPDWLF HPEU\RJHQLF FXOWXUH V\VWHP DQG SODQW UHJHUDWLRQ LQ EDQDQD %$5& 1HZVOHWWHU )RXQGHUȋV GD\ LVVXH VSHFLDO ,VVXH   >@ (VFDODQW -9 7HLVVRQ & DQG &¶WH )  $PSOLʃHG VRPDWLF HPEU\RJHQHVLV IURP PDOH ʄRZHU RI WULSORLG EDQDQD DQG SODQWDLQ FXOWLYDUV 0XVD VS  ,Q 9LWUR &HOO 'HY %LRO ȇ >@ .KDOLO 6 0 &KHDK . 7 3HUH] ( $ DQG *DVNLOO ' $  5HJHUDWLRQ RI EDQDQD 0XVD VSS $$% FY'ZDUI %UD]LOLDQ YLD VHFRQGDU\ VRPDWLF HPEU\RJHQHVLV 3ODQW &HOO 5HSRUW   >@ 6WHLQEUHFKHU 5$  7KH &D09 6 3URPRWHU *RYHUQPHQW DQG &RU SRUDWH 6FLHQWLʃF ,QFRPSHWHQFH )DLOXUH WR DVVHVV WKH VDIHW\ RI *0 FURSV KWWSZZZHFRQH[XVLQIR $FFVHVVHG  -XO\  >@ =DQQDWL $  ȍ6DQJ SHQ\HOHQJJDUDȎ LWX GLVHEXW SURPRWRU %LR7UHQGV      '2, NOVYL

3DJH 

,&6$)6 &RQIHUHQFH 3URFHHGLQJV

>@ 3«UH]&OHPHQWH 5 0 3«UH]6DQMX£Q $ *DUF¯D)«UUL] / %HOWU£Q - 3 DQG &D³DV / $  7UDQVJHQLF SHDFK SODQWV 3UXQXV SHUVLFD / SURGXFHG E\ JHQHWLF WUDQVIRUPDWLRQ RI HPEU\R VHFWLRQV XVLQJ WKH JUHHQ ʄXRUHVFHQW SURWHLQ *)3 DV DQ LQ YLYR PDUNHU 0ROHFXODU %UHHGLQJ  ȇ >@ (O6KHP\ +$ .KDODIDOOD 00 DQG ,VKLPRWR 0  7KH UROH RI *UHHQ )OXRUHVFHQW 3URWHLQ *)3 LQ WUDQVJHQLF SODQWV WR UHGXFH JHQH VLOHQFLQJ SKHQRPHQD &XUU,VVXHV 0RO %LRO  6XSSO   ȇ >@ +RIJHQ 5 DQG 0LOOPLW]HU /  6WRUDJH RI FRPSHWHQW FHOOV IRU $JUREDFWHULXP WUDQVIRUPDWLRQ 1XFOHLF $FLGV 5HVHDUFK     >@ $SUL\DQL 5 .  (ʃVLHQVL WUDQVIRUPDVL SDGD NXOWXU HPEULR SLVDQJ &DYHQGLVK 0XVD DFXPLQDWD FROOD $$$ *URXS PHODOXL $JUREDFWHULXP WXPHIDFLHQV VWUDLQ $*/ GDQ *9 \DQJ PHPEDZD SODVPLG S%, 7KHVLV 'HSDUWPHQW RI %LRWHFK QRORJ\ >@ &KDQJ 6 DQG 6KX +  $ PHWKRG WR VXSUHVV WKH EURZQLQJ LQ EDQDQD 0XVD $$$ HPEU\RJHQLF FDOOXV LQGXFHG 5HVHDUFK -RXUQDO RI %LRWHFKQRORJ\     >@ :RQJ :& -DOLO 0 2QJ$EGXOODK 0 2WKPDQ 5@ ,NHXFKL 0 6XJLPRWR . DQG ,ZDVH $  3ODQW FDOOXV 0HFKDQLVPV RI LQGXFWLRQ DQG UHSUHVVLRQ 7KH 3ODQW &HOO  ȇ >@ .KDOLO 6 0 DQG (OEDQQD $ $ 0  +LJKO\ HIʃFLHQW VRPDWLF HPEU\RJHQHVLV DQG SODQW UHJHQHUDWLRQ YLD VXVSHQVLRQ FXOWXUHV RI EDQDQD 0XVD VSS  $UDE -RXUQDO %LRWHFKQRORJ\     >@ =LPPHUPDQ -/  6RPDWLF HPEU\RJHQHVLV D PRGHO IRU HDUO\ GHYHORSPHQW LQ KLJKHU SODQWV 7KH 3ODQW &HOO   >@ 3DVWHUQDN 7 3 3ULQVHQ ( $\D\GLQ ) 0LVNROF]L 3 3RWWHUV * $VDUG + 2QFNHOHQ + $ 9 'XGLWV ' DQG )HK«U $  7KH UROH RI DX[LQ S+ DQG VWUHVV LQ WKH DFWLYDWLRQRI HPEU\RJHQLF FHOO GLYLVLRQ LQ OHDI SURWRSODVW GHULYHG FHOOV RI $OIDOID 3ODQW 3K\VLRORJ\   >@ 6KLQR\DPD + 1RPXUD 'HQGUDQWKHPD [ JUDQGLʄRUXP 5DPDW .LWDPXUD@ 3ODQW %LRWHFKQRORJ\   >@ *DQDSDWKL 75 +LJJV 16 %DOLQW.XUWL 3- $UQW]HQ &- 0D\ *' DQG 9DQ (FN -0  $JUREDFWHULXPPHGLDWHG WUDQVIRUPDWLRQ RI HPEU\RJHQLF FHOO VXVSHQVLRQV RI 7 KH EDQDQD FXOWLYDU 5DVWKDOL $$%  3ODQW &HOO 5HSRUWV  ȇ >@ )XMLPXUD 7 DQG .RPDPLQH $  6\QFKURQL]DWLRQ RI VRPDWLF HPEU\RJHQHVLV LQ D FDUURW FHOO VXVSHQVLRQ FXOWXUH 3ODQW 3K\VLRORJ\   >@ :DKLG $ *HODQL 6 $VKUDI 0 DQG )RRODG 0  +HDW WROHUDQFH LQ SODQWV $Q RYHUYLHZ (QYLURQ ([S %RW   '2, NOVYL

3DJH 

,&6$)6 &RQIHUHQFH 3URFHHGLQJV

>@ 8VPDQ 0 * 5DʃL 0 @ /L = 7 'KHNQH\ 6 'XWW 0 9DQ $PDQ 0 7 DWWHUVDOO - .HOOH\ . 7 DQG *UD\ ' -  2SWLPL]LQJ $JUREDFWHULXPPHGLDWHG WUDQVIRUPDWLRQ RI JUDSHYLQH ,Q 9LWUR &HOO 'HY %LROȈ3ODQW  ȇ >@ %HUHQVPHLHU6  0DJQHWLF SDUWLFOHV IRU WKH VHSDUDWLRQDQG SXULʃFDWLRQ RI QXFOHLF DFLGV $SSO 0LFURELRO%LRWHFKQRO   >@ (VVHU .+ 0DU[ :+ DQG /LVRZVN\ 7  0D[;ERQG ʃUVW UHJHQHUDWLRQ V\VWHP IRU '1$ ELQGLQJ VLOLFD PDWULFHV >@ 2SDERGH - 7  $JUREDFWHULXP PHGLDWHG WUDQVIRUPDWLRQ RI SODQWV HPHUJLQJ IDFWRUV WKDW LQʄXHQFH HIʃFLHQF\ %LRWHFKQRORJ\ DQG 0ROHFXODU %LRORJ\ 5HYLHZ   >@ 6X]XNL 6 6XSDLEXOZDWDQD . 0LL 0 DQG 1DNDQR 0  3URGXFWLRQ RI WUDQVJHQLF SODQWV RI WKH /LOLDFHRXV RUQDPHQWDO SODQW $JDSDQWKXV SUDHFR[ VVS 2ULHQWDOLV /HLJKWRQ /HLJKWRQ YLD $JUREDFWHULXPPHGLDWHG WUDQVIRUPDWLRQ RI HPEU\RJHQLF FDOOL 3ODQW 6FLHQFH   >@ $PRDK % . :X + 6SDUNV & $ DQG -RQHV + '  )DFWRUV LQʄXHQFLQJ $JUREDFWHULXPPHGLDWHG WUDQVLHQW H[SUHVVLRQ RI XLG$ LQ ZKHDW LQʄRUHVFHQFH WLVVXH -RXUQDO RI ([SHULPHQWDO %RWDQ\   >@ 8UDQEH\ 6 6HYLPD\ &6 .D\D 0' ,SHN $ 6DQFDN & %DVDOPD ' (U & DQG 2]FDQ 6  ,QʄXHQFH RI GLIIHUHQW FRFXOWLYDWLRQ WHPSHUDWXUH SHULRGV DQG PHGLD RQ $JUREDFWHULXP WXPHIDFLHQV PHGLDWHG JHQH WUDQVIHU %LRORJLD 3ODQWDUXP     >@ :HQFN $ 5 4XLQQ 0 :KHWWHQ 5: 3XOOPDQ * DQG 6HGHURII 5  +LJK HIʃFLHQF\ $JUREDFWHULXPPHGLDWHG WUDQVIRUPDWLRQ RI 1RUZD\ VSUXFH 3LFHD DELHV DQG OREOROO\ SLQH 3LQXV WDHGD  3ODQW 0RO %LRO  ȇ

'2, NOVYL

3DJH