Soybean"Cultivar"

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!‘The!Transformation!of!Elena’!

A"GM"Soybean"Cultivar"for"the"UK"Market!

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Chris!D’Agorne!

Presented!as!final!requirement!for!the!degree!of!Master!of!Biotechnology! Oxford!Brookes!University!! School!of!Life!Sciences! September!2014!

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The!Transformation!of!‘Elena’:!A"GM"Soybean"Cultivar"for"the"UK"Market! Chris!D’Agorne!!!|!!!Student!No:!13089167! line!

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TABLE"OF"CONTENTS"

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! Acknowledgements! ! Declaration!of!Individual!Authorship! ! Abstract! ! Table!of!Abbreviations! ! ! INTRODUCTION! ! Preface! ! Soybean!Agriculture! ! GMO!Perception!in!the!UK! ! Two!Transformation!Techniques! ! Biolistics!or!Agrobacterium?! ! Quantifying!Transformation!with!Gus! ! Selection!of!a!Soybean!Cultivar! ! The!Problems!with!Dairy!Milk! ! Bioengineering!a!Vegan!Cheese! ! PostWTranslational!Modifications! ! Genetic!Modification!of!Soy!Milk!Flavour! ! SeedWSpecific!Promoters! ! Localising!Expression!with!GFP!&!tdTomato! ! Outline!of!Research!Goals! ! ! METHODS! ! Bleach!Protocol! !

MSc!Biotechnology"Project"Report!

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Determining!the!Appropriate!Tissue!for!Infiltration! ! Infiltration!of!Soybean!Cotyledons! ! Protoplasting! ! Gus!Gateway!Cloning!and!Transformation! ! Gus!Infiltration!and!Staining!Protocol! ! Biolistic!Infiltration! ! ! RESULTS! ! Bleach!Protocol!Germination! ! Bleach!Protocol!Contamination! ! Cotyledon!Infiltration! ! Protoplasting! ! Gus!Staining! ! Biolistic!Transformation! ! ! DISCUSSION! ! Bleach!Protocol! ! Cotyledon!Infiltration! ! Protoplasting! ! Gus!Staining! ! Biolistic!Transformation! ! Conclusions!and!Future!Research! ! ! REFERENCES!&!APPENDICES! ! References! ! Appendices! !


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Title!/!Section! ! ! INTRODUCTION! ! Biolistic!Particle!Delivery!System!(no!annotations)! ! Gus!staining!of!Arabidopsis!ecotypes! ! ‘Elena’!soybean!seeds! ! International!supply!and!demand!for!milk!products! ! Workflow!for!yeast!transformation! ! GmMYB39!overexpression!in!transformed!soybeans! ! βWconglycinin!suppression!and!GFPWkdel!expression! ! Explant!regeneration!in!Acacia!mearnsii!after!bleaching! ! METHODS! ! Soybean!seeds!in!MS!media!under!growth!lights! ! Cropped!PNG!images!after!processing!in!Lightroom! ! Screenshot!of!threshold!colour!analysis!in!Fiji! ! Soybean!seedling!annotated!schematic! ! Soybean!cotyledon!infiltration!illustration! ! Vector!maps!of!the!pEntrWgus!entry!vector! ! Mounted!GusWstained!leaf!discs!scanned!image! ! Autofluorescence!of!soybean!leaf!hairs!under!543nm!laser! ! Biolistic!Particle!Delivery!System!annotated!schematic! ! RESULTS! ! Graph:!Bleaching!duration/Percentage!germination!


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! Chart:!Bleaching!condition/Average!seedling!height! ! Chart:!Bleaching!condition/Percentage!contamination! ! Confocal!micrograph!z!series!projection!of!infiltrated!cotyledon! ! Confocal!micrograph!of!normal!autofluorescence!in!cotyledon! ! Confocal!micrograph!of!GFP!HDEL!transgenic!tobacco!leaf! ! Light!micrograph!of!single!soybean!protoplast! ! Light!micrograph!of!soybean!protoplast!agglomeration! ! Chart:!Gus!infiltration!condition/Surface!area!of!Gus!staining! ! Micrographs:!Soybean!leaves!after!bombardment!with!LTI6b!tdTomato! ! Micrographs:!Individual!LTI6b!tdTomato!transformed!cells!! ! DISCUSSION! ! Chart:!Bleach!Conditions/Contamination/Percentage!Germination! ! Spread!of!bacteria!and!fungus!in!unbleached!vs!bleached!MS!condition! ! Cotyledon!leaf!discs!showing!blue!staining! ! Chart:!Average!staining!intensity/Bleach!Conditions! ! GFP!LTI6b!expression!in!Arabidopsis!visualised!by!confocal!microscopy! ! LTI6b!tdTomato!fluorescence!distribution!in!tobacco!and!soybean! ! Strong!autofluorescence!in!GFP!emission!spectrum!of!cotyledon! !


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Title!/!Section! ! ! METHODS! ! Lightroom!parameters!for!uncompressed!TIFF!enhancement! ! Threshold!values!for!contamination!quantification!in!Fiji! ! Threshold!values!for!Gus!staining!quantification!in!Fiji! ! RESULTS! ! Blue!staining!in!initial!Gus!protocol! ! Blue!staining!in!final!Gus!protocol! ! !


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1.0:!ACKNOWLEDGEMENTS! ! I!would!like!to!take!this!opportunity!to!thank!those!who!have!helped!me!on!my!way!through!this!Masters! course!and!those!who!inspired!me!to!reNenter!academia.!! ! With"thanks"to…" Frances!Tolmie!and!Joe!McKenna!PhD!for!teaching!me!many!different!lab!techniques!and!for!being!patient,! respectful!and!understanding!when!I!needed!assistance!with!the!project.! John!Runions!PhD!for!assisting!with!microscopy,!advising!me,!and!inspiring!the!design!of!the!project.! Katja!Graumann!PhD!for!demonstrating!the!biolistic!particle!delivery!system.! All!those!in!the!Health!and!Life!Sciences!Department!who!aided!me!with!my!project!in!other!ways.! XXXXXXXXXXX!of!XXXXXX!(industry!expert)!who!afforded!me!an!invaluable!insight!into!soybean!agriculture.! ! Alice!Baker!for!inspiring!me!to!get!back!into!academia!and!for!being!a!great!friend.! Charles!Cowdale!for!giving!me!some!friendly!competition!which!inspired!me!to!work!harder.! Lynn!Rogers!PhD!&!Sue!Mansfield!MS!at!the!WRI!in!Minnesota,!for!inspiring!me!with!their!bear!research.! ! Special"thanks"to…" My!parents!and!family,!for!supporting!me!both!emotionally!and!financially!through!an!eventful!year!! ! And"lastly," My!wife,!Kelly!D’Agorne!and!my!unborn!son,!Casey!Bear!D’Agorne!for!inspiring!me!to!work!towards!a!better! future!for!my!new!family!and!providing!some!muchWneeded!comic!relief!in!my!free!time.!

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My"parents,"grandparents,"Kelly,"my"son,"and"I"on"my"wedding"day"at"Bournemouth"Beach" !July!28th!2014! !


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1.1:!DECLARATION!OF!INDIVIDUAL!AUTHORSHIP! ! Unless!otherwise!acknowledged,!the!work!presented!in!this!thesis!is!my!own!and!has!not!been!submitted!for! another!degree!at!Oxford!Brookes!University!or!any!other!institute!of!learning.! ! ! 1.2:!ABSTRACT! ! Soy!milk!is!a!widelyWconsumed,!vegan!alternative!to!cow’s!milk!with!ethical,!environmental!and!health!benefits! over!its!mainstream!competition.!The!taste!of!soy!milk!is!widely!regarded!as!unpleasant,!but!removing!this! bad!flavour!has!recently!been!proven!possible!through!advances!in!genetic!modification.!A!genetically! modified!(GM)!soybean!cultivar!for!the!UK!market!is!not!available,!as!the!political!and!public!perception!of!GM! has!historically!been!negative,!but!this!is!now!changing.!Glycine"max"‘Elena’!is!an!existing!UK!soybean!cultivar! which!has!not!previously!been!the!subject!of!GM!research;!the!susceptibility!of!this!plant!to!transformation! was!assessed!using!three!different!reporter!genes.! ! Agrobacterium"tumefaciens!was!transformed!with!a!vector!comprising!GFP!HDEL!Wa!fluorescent!marker!of!the! Endoplasmic!ReticulumW!under!the!control!of!the!35S!promoter!from!cauliflower!mosaic!virus.!This!cell!culture! was!suspended!in!infiltration!buffer!and!injected!directly!into!mature!soybean!cotyledons.!No!transformation! events!were!observed!when!cotyledons!were!subsequently!examined!under!a!confocal!microscope.!In! subsequent!trials,!variations!on!this!initial!experiment!included!modifying!the!concentration!of!A."tumefaciens! and!Acetosyringone,!and!adding!vectors!containing!the!gene!P19;!an!RNAi!suppressor.!! ! Unfortunately,!even!with!experiments!using!2!different!GFP!HDEL!constructs,!transformation!of!cotyledons! was!not!successful,!thus,!protoplasts!were!generated!from!Glycine"max"‘Elena’!and!used!as!an!alternative! tissue!for!infiltration.!While!protoplasts!were!successfully!produced!for!the!first!time!in!this!cultivar,! transformation!was!not!achieved,!potentially!due!to!a!flawed!methodology.!Another!reporter!gene,!Gus,! expresses!betaWglucorinidase!enzymes,!which!catalyse!the!cleavage!of!beta!glucorinides,!causing!a!clear!‘XW Gluc’!solution!to!turn!a!vivid!blue,!and!stain!transformed!tissue.!2!Gus!constructs!were!cloned!and!transformed! into!A."tumefaciens,!then!used!to!infiltrate!further!soybean!cotyledons.!While!these!cotyledons!were! successfully!stained!blue,!this!strand!of!research!was!inconclusive,!as!both!experimental!and!negative!control! leaf!discs!were!stained.!Previous!studies!have!shown!that!soybean!cotyledons!are!high!in!levels!of!intrinsic! GusWlike!compounds,!which!can!cause!natural!cleavage!of!beta!glucorinides.! ! The!final!reporter!gene!exploited!in!this!soybean!research!was!LTI6b!tdTomato;!this!construct!encodes!a! vibrant!red!fluorescent!protein!which!localises!to!the!cell!membrane.!A!biolistic!particle!delivery!system!was! used!to!blast!vectors!comprising!LTI6b!tdTomato!under!the!control!of!the!35S!promoter!into!unifoliate!and! trifoliate!Glycine"max"‘Elena’!leaves.!Successful!transformation!of!isolated!cells!seemed!to!have!occurred!in! more!than!half!of!target!leaves,!with!the!resulting!intracellular!fluorescence!distribution!matching!that! observed!in!a!Tobacco!(Nicotiana"tabacum)!leaf!which!underwent!the!same!biolistic!protocol.!


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! Transient!transformation!of!the!‘Elena’!cultivar!is!a!step!on!the!road!to!engineering!a!GM!soybean!for!the!UK!in! which!isoflavonoid!and!saponin!production!is!suppressed.!These!compounds!have!been!identified!as!a!source! of!the!bitterness!and!beany!flavours!of!soy!milk,!and!a!viable!target!for!suppression!using!genetic!modification! of!metabolic!pathways.!The!resulting!soy!milk!would!have!an!enhanced!palatability,!and!its!myriad! environmental!and!ethical!advantages!over!the!current!status!quo!could!be!a!key!selling!point.!The! replacement!of!cow’s!milk!with!a!plantWderived!alternative!would!have!beneficial!implications!for!climate! change,!the!welfare!of!livestock,!food!security,!the!suppression!of!antibioticWresistance!in!bacteria,!and!public! perception!of!science.! ! !

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1.3:!TABLE!OF!ABBREVIATIONS! ! Elena!

Glycine"max"‘Elena’"soybean"cultivar"

GFP!

Green"Fluorescent"Protein"

GM!

Genetically"modified"

GMO!

Genetically"modified"organism"

Gus!PB!

Agrobacterium"tumefaciens"cell"culture"with"Gus"gene"contained"in"the"PB7FWG2M"Orange"2"vector"

Gus!PG!

Agrobacterium"tumefaciens"cell"culture"with"Gus"gene"contained"in"the"PGWB2"vector!

IB!

Infiltration"Buffer"

PPS!

Protoplasting"Solution"

Siverka!

Glycine"max"‘Siverka’"soybean"cultivar"

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Introduction! CONTENTS"of"INTRODUCTION" " " "

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Preface! ! Soybean!Agriculture! ! GMO!Perception!in!the!UK! ! Two!Transformation!Techniques! ! Biolistics!or!Agrobacterium?! ! Quantifying!Transformation!with!Gus! ! Selection!of!a!Soybean!Cultivar! ! The!Problems!with!Dairy!Milk! ! Bioengineering!a!Vegan!Cheese! ! PostWTranslational!Modifications! ! Genetic!Modification!of!Soy!Milk!Flavour! ! SeedWSpecific!Promoters! ! Localising!Expression!with!GFP!&!tdTomato! ! Outline!of!Research!Goals! !

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2.0:!PREFACE! ! ! This!research!project!started!with!a!simple!thought:!“Can!we!use!techniques!from!biotechnology!to!enhance! the!flavour!of!soy!milk?”.!My!wife’s!lactose!intolerance!had!introduced!me!to!this!cow’s!milk!substitute,!and!I,! like!many!consumers!(Alpaslan!&!Hayta!2007)!was!disgusted!by!the!taste.!In!the!pursuit!of!a!betterWtasting!soy! milk,!I!read!papers!which!reported!advances!in!our!understanding!of!metabolic!pathways;!these!advances! have!created!opportunities!for!manipulation!of!fruit!flavour!(e.g.!DavidovichWRikanati!et!al!2007).!However,!I! soon!realised!that!a!successfullyWimplemented!strategy!for!replacing!cow’s!milk!might!be!beneficial!for!food! security!and!our!environment,!but!it!could!wreak!havoc!on!the!UK!dairy!industry.!This!notion,!and!a! preliminary!literature!review!inspired!the!evolution!of!a!more!specific!question:! !

“Can!we!produce!a!GM!soybean,!with!enhanced!milk!flavour,!which!grows!in!the!UK!climate?”! ! This!clarification!addressed!the!potential!economic!implications!to!UK!farmers!of!a!popular!GM!soy!milk! product;!farmers!could!replace!their!cows!in!their!fields!with!GM!soybeans.!In!this!thesis,!I!examine!past! studies!in!soybean!genetic!modification,!and!identify!opportunities!for!improving!the!taste!of!soy!milk.! Through!my!own!research,!and!a!review!of!relevant!papers,!I!will!attempt!to!determine!the!answer!to!this! soybean!conundrum.! 2.1:!SOYBEAN!AGRICULTURE! ! The!soybean!(Glycine"max)!is!a!legume!with!nitrogenWfixing!roots,!which!can!be!grown!in!temperate!and! tropical!climates,!to!produce!pods!of!seeds!(Giller!&!Dashiell!2007).!The!high!protein!content!of!these!seeds! has!made!them!popular!for!use!as!a!basic!ingredient!in!many!different!foods!and!drinks;!including!tofu!and! milk!substitutes,!while!the!oil!content!of!the!seeds!is!harnessed!in!the!production!of!vegetable!oil!and! biodiesel!(TandangWSilvas!et!al!2011,!Goettel!et!al!2014).!Soybeans!are!grown!in!equatorial!regions,!and!as!far! North!and!South!as!the!55th!parallel!(Southern!Scotland/Southern!Argentina),!which!means!that!cultivars!are! available!which!are!suited!to!a!UK!climate.!! ! In!the!UK,!soybean!is!usually!grown!as!a!‘Spring!Break’!crop;!the!nitrogen!fixing!roots!enrich!the!soil!and,!the! cover!of!plants!during!a!period!when!fields!are!often!left!uncultivated!has!been!found!to!suppress!the!growth! of!weeds.!In!the!UK,!Spring!Break!crops!have!been!found!to!reduce!the!growth!of!blackgrass!and!wild!oat! (two!prominent!weed!species)!by!as!much!as!90%!(Moss!2010).!Farming!experts!describe!how!this! suppressant!effect!reduces!the!levels!of!herbicides!required!to!prevent!weeds!in!cereal!crops!grown!later!in! the!season!(Moss!2010).!However,!XXXXXXXXX!of!XXXXXX,!(interviewed!by!phone!on!19th!December!2013)! described!how!lupins!can!also!perform!this!function,!and!are!more!popular!in!the!UK!market.!This!popularity!is! due!to!the!palatability!of!lupins!as!livestock!fodder;!in!contrast,!soybean!are!rendered!inedible!by!toxic!trypsin! inhibitors,!which!must!be!destroyed!by!heat!treatment!before!the!seeds!are!fed!to!cattle.!! !


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Worldwide,!however,!soybeans!continue!to!increase!in!popularity;!in!2012/13!the!global!production!was!268! million!metric!tons,!but!is!estimated!to!reach!284m!in!2013/14,!and!the!USDA!projects!a!further!increase!in!the! following!year.!The!USA!is!the!largest!producer,!and!is!expected!to!account!for!22%!of!global!soybean!output! in!2014/15!(USDA!2014b).!Of!the!soybeans!farmed!in!the!USA,!94%!were!‘Biotechnology!Varieties’,!with!intrinsic! herbicide!resistance!(USDA!2014a).!Globally,!geneticallyWmodified!(GM)!varieties!of!soybean!are!estimated!to! make!up!at!least!75%!of!total!production,!and!the!GM!company!Monsanto!claims!that!its!‘Roundup!Ready®’! herbicideWresistant!strain!is!the!most!widely!adopted!biotechnology!trait!(Monsanto!2014).!! ! 2.2:!GMO!PERCEPTION!IN!THE!UK! ! Monsanto’s!Roundup!Ready®!soybeans!will!lose!their!patent!protection!in!2015,!but!the!liberties!taken!by! Monsanto!during!this!20!year!period!are!widely!regarded!to!have!reflected!poorly!on!all!genetically!modified! crops!(Scott!2000).!Indeed,!the!media!portrayal!of!genetically!modified!organisms!(GMOs)!has!been! overwhelmingly!negative!in!the!UK,!although!it!is!unclear!whether!this!is!the!cause!of,!or!a!response!to,!the! public’s!aversion!to!GM.!The!organic!certification!body,!the!Soil!Association,!actively!campaigns!against! commercially!grown!GM!crops,!and!a!recent!YouGov!poll!indicated!that!only!21%!of!the!public!support!GM! food,!while!35%!oppose!it!(YouGov!2013).!However,!as!with!much!of!the!coverage!of!GMOs!in!the!media,!this! survey!gave!credence!to!unsubstantiated!concerns,!with!a!leading!question.!The!text!of!this!survey!implied! GMOs!‘have!a!negative!effect!on!wildlife,!encourage!the!growth!of!new,!more!resistant!superWweeds!or!pests! and!are!potentially!harmful!to!humans’.!! ! Given!the!lack!of!public!support!for!GMOs,!and!the!corresponding!danger!to!politicians!of!backing!GM! research!in!the!UK,!it!is!necessary!to!determine!whether!there!is!a!future!for!GMOs!here,!before!proceeding! with!research!into!a!UK!soybean!cultivar.!Surprisingly,!it!seems!that!the!signs!are!good;!the!minister! responsible!for!farming!in!the!UK,!Liz!Truss,!recently!voiced!her!support;!“I!want!us!to!have!a!competitive,! productive!food!and!farming!sector!and!we!absolutely!need!to!have!GM!as!a!part!of!that”!(Midgley!2014).!In!a! recent!report!on!food!security!for!the!House!of!Commons,!a!government!representative!also!suggested!that! GMOs!posed!little!risk!to!the!public;!“the!European!Food!Safety!Authority,!does!an!enormous!amount!of!work! to!license!GM!crops…!the!consensus!is!there!is!not!really!a!threat!to!food!safety.”!(House!of!Commons!2014W 15).!In!addition,!media!coverage!of!GM!trials!in!the!UK!(Omega!3Wenriched!Camelina"sativa)!also!indicates!a! softening!of!attitudes!toward!GMOs,!even!in!the!conservative!press!(O’Callaghan!2014,!Knapton!2014).!! ! With!political!will!clearly!shifting!in!favour!of!future!developments!in!GM!crops,!now!is!an!excellent!time!to! investigate!whether!a!UK!soybean!cultivar!is!susceptible!to!genetic!modification,!and!to!determine!the!most! appropriate!technique!for!this!process.! !

2.3:!TWO!TRANSFORMATION!TECHNIQUES! ! Since!the!1980s,!plant!scientists!have!investigated!different!methods!for!both!transient!and!stable! transformation!of!soybean!varieties,!with!varying!degrees!of!success.!Soybeans!have!generally!been!very!


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resistant!to!transformation,!with!methods!relying!on!the!susceptibility!to!transformation!of!a!few!cultivars,! and!many!early!papers!relying!on!a!single!cultivar;!‘Jack’!(Trick!et!al!1997,!Yamada!et!al!2012).!Two!methods! have!tended!to!dominate!soybean!transformations;!AgrobacteriumWmediated!infiltration!and!biolistic!particle! delivery!systems.!These!two!techniques!are!so!highly!dissimilar!that!it!is!perhaps!surprising!that!both!were! successfully!implemented!for!the!first!time!in!the!same!year;!1988!(Trick!et!al!1997).!! ! Biolistic!Particle!Delivery!System! ! A!biolistic!particle!delivery!system!is!a!device!designed!for!blasting!transgenic!DNA!into!cells!at!extremely!high! velocities!(shown!in!figure!1).!In!this!method,!gold!(or!similar)!particles!are!coated!with!a!delivery!vector,! containing!a!transgene(s)!of!interest;!this!genetic!material!is!shot!through!cell!walls!and!may!accumulate!in! the!nucleus,!where!it!can!be!expressed!(Homrich!et!al!2012).!The!speed!of!the!particles!is!determined!both!by! the!level!of!hydraulic!pressure!which!initiates!the!blast,!and!the!strength!of!the!rupture!disc!–!an!internal! component!which!splits!under!pressure,!to!direct!the!release!of!gas!through!the! system!(see!section!3.6).!This!gas!propels!a!particleWcoated!microcarrier!disc! rapidly!into!a!mesh!stopping!screen;!on!impact!with!this!mesh,!the!DNAWcoated! particles!are!separated!from!the!disc!and!driven!across!a!narrow!gap! and!into!the!target!plant!cells!(BioWrad!2014).!! ! The!air!within!the!system!is!evacuated!using!a!vacuum! pump!prior!to!‘bombardment’!to!ensure!that!particles!are! not!disrupted!by!air!resistance.!Shelves!allow!the!placement! of!plant!tissue!at!varying!distances!from!the!stopping! screen;!the!height!also!determines!the!diameter!of! bombardment,!as!the!particles!spread!out!as!they! propagate!through!the!vacuum!chamber.!At! slower!speeds,!particles!may!bounce!off!the!cell! wall,!while!at!higher!speeds,!tissue!can!be!shredded,! particularly!if!the!diameter!of!the!target!region!is! narrow.! !

Figure!1!–!Biolistic!Particle!Delivery!System!with!a!leaf! ready!to!be!bombarded;!an!annotated!version!of!this! figure!is!in!section!3.6!

The!challenge!of!biolistics!is!the!fragile!balancing! act!between!the!speed!of!particles,!diameter!of!the!blast!and!type!of!plant!tissue!bombarded,!in!order!to! achieve!a!high!transformation!rate!with!minimal!tissue!damage.!According!to!a!technician!in!the!Runions!lab,! who!is!familiar!with!biolistics,!the!strength!of!epidermis!varies!between!tissues,!and!over!time,!so!that!an! immature!leaf!may!be!easier!to!transform!than!an!adult!leaf.!Early!experiments!showed!stable!transformation! was!rare,!but!possible,!if!the!gene!was!blasted!into,!or!copied!into,!the!host!cell!DNA!(McCabe!et!al!1988).!


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However,!over!the!past!three!decades,!techniques!have!been!improved!and!regeneration!of!fertile!transgenic! soybeans!has!been!achieved!on!multiple!occasions!(Yamada!et!al!2012).!The!vacuum!chamber!within!which! bombardment!takes!place!is!restricted!and!narrow,!which!makes!it!unsuitable!for!whole,!living!plants;!thus,! biolistic!infiltration!of!soybeans!is!usually!performed!with!plant!tissue,!rather!than!entire!living!plants.!! ! AgrobacteriumNmediated!Transformation! ! Agrobacterium"tumefaciens!is!a!bacterium!which!is!naturally!found!in!soil;!after!landing!on!a!host!plant,!this! species!stimulates!the!formation!of!a!tumour!if!it!is!able!to!penetrate!the!cell!wall.!The!bacteria!relies!on! compounds!produced!in!response!to!cellular!damage,!in!order!to!trigger!the!expression!of!certain!virulence! proteins!(Pitzschke!&!Hirt!2010).!These!proteins!are!then!taken!into!the!plant!cell!through!the!cell!wall!by!a! bacterial!secretion!system,!which!forms!in!response!to!plant!signals.!The!virulence!proteins!effectively!hijack! the!host!cell!and!suppress!its!immune!system,!facilitating!the!insertion!of!tDNA!into!the!plant!cell! chromosome!(Paz!et!al!2004,!Pitzschke!&!Hirt!2010).!This!inserted!DNA!then!initiates!tumour!formation,!thus! providing!an!ideal!environment!for!bacterial!growth!and!replication.!! ! Scientists!have!genetically!modified!this!bacterium,!rendering!it!benign!to!the!host!plant!and!creating!an! expression!system!which!has!been!widely!used!to!both!examine!plant!cell!function!and!engineer!transgenic! plant!lines.!The!bacterial!genome!has!been!edited!to!remove!some!of!the!genes!associated!with!tumour! formation,!while!retaining!genes!for!expression!of!a!foreign!protein!within!the!host!cell!(Pitzschke!&!Hirt! 2010).!Thus,!rather!than!initiating!tumour!development,!A."tumefaciens!which!successfully!infiltrates!a!plant! cell!will!promote!expression!of!a!transgene!carried!in!a!modified!vector.!Crucially,!this!gene!becomes! integrated!into!the!plant!cell!DNA,!rather!than!floating!freely!in!the!nucleus.!Thus,!if!the!tissue!infiltrated!is! embryonic!in!nature!or!is!induced!to!form!callus,!a!new!transgenic!plant!line!can!be!regenerated!(Yamada!et!al! 2012).!! ! 2.4:!BIOLISTICS!OR!AGROBACTERIUM?! ! The!first!use!of!the!biolistic!technique!in!soybeans!was!by!McCabe!et!al!(1988),!in!which!shoot!meristems!were! transformed;!the!research!was!published!in!Nature"Biotechnology.!In!the!same!edition,!Hinchee!et!al!(1988)! focused!their!attention!on!the!cotyledonary!node;!their!gene!of!interest,!‘betaWglucorinidase’!(see!section!2.5),! was!found!to!be!expressed!3!weeks!after!this!embryonic!tissue!had!been!infiltrated!with!Agrobacterium.!Given! that!the!two!techniques!are!both!known!to!yield!transgenic!soybeans,!it!is!important!to!determine!which!is! most!appropriate!for!this!study.!The!end!goal!of!this!research!is!to!produce!a!stably!transformed!plant!line! which!expresses!a!gene!or!combination!of!genes!that!enhances!soy!milk!flavour.!Thus,!stable!transformation! should!be!a!priority,!although!to!a!lesser!extent,!use!of!a!technique!which!facilitates!rapid!transformation!is! important,!due!to!time!constraints!on!the!project.! ! A!review!by!Yamada!et!al!(2012)!showed!that!AgrobacteriumWmediated!infiltration!had!been!successful!in! regenerating!26!fertile!transgenic!plant!lines,!while!biolistic!techniques!had!generated!just!8!fertile!transgenic!


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lines!over!the!preceding!24!years.!Additionally,!ElWShemy!et!al!(2004)!describes!how!Agrobacterium!infiltration! is!often!preferable!in!soybeans,!as!it!can!avoid!multiWlocus!gene!insertions!which!are!characteristic!of!biolistic! particle!delivery;!however,!the!opposite!effect!was!found!by!Dai!et!al!(2001)!in!a!study!of!rice.!When!multiple! copies!of!the!same!gene!are!present!within!one!cell,!this!can!lead!to!gene!silencing.!Dai!et!al!(2001)!ran!a!direct! comparison!of!Agrobacterium!and!biolistic!transformation!techniques!and!found!that!stable!transformation! was!more!likely!with!Agrobacterium.!Side!by!side!comparisons!of!biolistic!and!Agrobacterium!systems!are! difficult!to!find,!and!it!is!important!not!to!draw!conclusions!from!an!isolated!study,!particularly!given!that!Dai! et!al!(2001)!uses!rice!plants!rather!than!soybean.!However,!the!above!indications!generally!point!towards! AgrobacteriumWmediated!transformation!as!a!more!suitable!system!for!regeneration!of!stable!transgenic!plant! lines!from!transformed!soybean!tissue.! !

2.5:!QUANTIFYING!TRANSFORMATION!WITH!GUS!

! Much!research!has!already!been!conducted!into!the!‘transformation!efficiency’!of!soybean!cultivars!(their! susceptibility!to!transformation!techniques),!including!developing!varieties!with!enhanced!transformation! efficiency!(Kita!et!al!2007).!As!previously!mentioned,!the!cultivar!‘Jack’!is!highly!susceptible!to!transformation,! leading!to!its!widespread!use!in!soybean!studies.!However,!reliance!on!a!single!cultivar!can!create!problems;! qualities!like!drought!tolerance,!frost!hardiness!and!seed!size!vary!across!cultivars!(Yamada!et!al!2012,!Takada! et!al!2013).!It!is!therefore!agronomically!beneficial!to!plant!a!variety!of!soybean!which!has!proven!to!be!highW yielding!under!local!climatic!conditions.!Scientists!have!generated!new!cultivars!of!soybean!by!backcrossing! with!‘Jack’!in!order!to!facilitate!regeneration!of!stable!plant!lines,!but!these!may!not!be!suitable!for!all!climates! (Kita!et!al!2007).! ! Studies!in!China!and!South!Africa!have!been!conducted!in!order!to!establish!the!local!cultivar!with!the!highest! transformation!efficiency!(Song!et!al!2013,!McKenzie!&!Cress!1992).!The!Chinese!study!was!conducted!with!the! use!of!the!GusWstaining!technique,!harnessing!this!genetic!marker!which!can!quantitatively!assess! transformation!efficiency.!In!this!technique,!the!host!plant!is!infiltrated!with!Agrobacterium,!which!carries!a! vector!containing!the!betaWglucorinidase!(‘Gus’)!reporter!gene!(Wroblewski!et!al!2005).!The!resulting!Gus! enzyme!is!a!hydrolase!which!is!derived!from!Escherichia"coli,!and!catalyses!the!cleavage!of!beta!glucorinides;! colourless!compounds!which!turn!blue!when!cleaved.!Early!research!indicated!that!betaWglucorinideWcleaving! enzymes!were!not!naturally!present!in!higher!plants!(Jefferson!et!al!1987),!which!meant!blue!staining!of!tissue! was!thought!to!be!a!reliable!indicator!of!transformation.!However,!as!discussed!in!section!5.3,!Hu!et!al!(1990)! found!that!GusWlike!compounds!were!naturally!present!in!some!plant!tissues,!which!led!later!scientists!to! design!more!rigorous!Gus!protocols.!! ! Gus!staining!occurs!on!a!cellular!level,!with!individual!cells!stained!blue!when!immersed!in!a!betaWglucorinide! solution!(‘XWGluc’);!this!specificity!allows!Gus!expression!to!be!effectively!localized!and!quantified.!One! example!is!shown!in!figure!2!–staining!of!Arabidopsis"thaliana!leaves!can!provide!a!quantifiable!measure!of!the!


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transformation!efficiency!of!different!Agrobacterium!varieties.!The!table!also!reveals!a!difference!in!staining! between!different!ecotypes!of!Arabidopsis.! !

! Figure"2!–!Gus!staining!allows!quantification!of!transformation!efficiency,!as!in!these!leaves!of! Arabidopsis!thaliana!ecotypes!(Ws,!ColN0,!VanN0),!which!were!infiltrated!with!8!different!GusN carrying!Agrobacterium!species!(from!Wroblewski!et!al!2005)"

!

The!transformation!efficiency!of!Chinese!soybeans!in!Song!et!al!(2013)!was!determined!using!a!similar!Gus! protocol;!it!varied!more!than!tenfold!between!cultivars.!Both!transient!and!stable!transformation!rates!were! assessed!in!Song!et!al!(2013),!with!PCR!detection!of!a!marker!gene!and!a!herbicide!assay!of!a!resistance!gene! used!alongside!Gus!staining.!Song!et!al!(2013)!also!determined!regeneration!rates;!this!measure!comprises!the! number!of!transformed!explants!which!successfully!grew!into!seedlings!on!plant!growth!media.!If!a!study!is! designed!to!assess!the!potential!for!the!production!of!fertile!transgenic!plant!lines,!the!survival!of! transformed!plants!is!clearly!an!important!criterion.!4!of!the!20!Chinese!soybean!cultivars!were!found!to!be! not!only!susceptible!to!transformation,!but!also!produce!viable!seedlings!from!infiltrated!explants.!In!the! South!African!study!(a!much!earlier!paper),!susceptibility!to!Agrobacterium!was!determined!by!tumour! growth;!all!ten!cultivars!were!found!to!produce!tumours!after!infiltration!(McKenzie!&!Cress!1992).!Song!et!al! (2013)!reported!that!both!traditional!American!soybean!cultivars!(‘Jack’!and!‘Williams!82’)!were!successfully! transformed,!with!effective!regeneration!of!seedlings.! ! 2.6:!SELECTION!OF!A!SOYBEAN!CULTIVAR! ! Alongside!determining!the!most!appropriate!transformation!technique!for!this!project!and!investigating! methods!for!quantifying!transformation!efficiency,!an!experimental!soybean!cultivar!must!be!identified.!If!it!is! to!be!appropriate!for!production!of!GM!soy!milk!in!the!UK,!this!variety!must!match!several!different!criteria:! ! 1.

Suitable!for!growing!in!UK!climate!

2.

Available!to!this!project!in!large!quantities!


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3.

Proven!success!in!UK!market!

4.

Appropriate!for!soy!milk!production!

5.

Average!or!aboveWaverage!yield!

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! In!the!UK!market,!the!‘flagship’!soybean!cultivar!of!the!leading!soybean!importer,!‘Soya!UK’,!is!‘Elena’!(Soya! UK!2014).!This!variety!conforms!to!4!of!the!5!criteria,!with!the!added!benefit!of!a!particularly!high!yield;! however!there!is!one!crucial!exception.!The!large!black!hilum!(see!figure!3)!renders!this!product!unsuitable!for! the!production!of!soy!milk,!according!to!XXXXXXXXXXX!from!XXXXXX!(via!phone!on!December!19th!2013),!as!it! will!colour!the!milk!an!unappetising!grey.!XXXXX!revealed!that!‘Elena’!is!primarily!used!for!agricultural!feed,! where!the!high!yield!is!of!great!importance!and!its!unsuitability!for!soy!milk!is!inconsequential.!! ! Unfortunately,!the!clear!hilum!cultivar!ordered!from!Soya!UK!for!this!study,!‘Siverka’,!was!not!available!in!2014! due!to!political!instability!in!Ukraine,!where!it!was!produced.!However,!Siverka!is!reportedly!a!close!relative!of! Elena,!which!opens!up!the!possibility!that!these!cultivars!share!a!similar!susceptibility!to!transformation.!Once! a!technique!for!transformation!is!established!using!Elena,!it!could!easily!be!repeated!with!Siverka!plants.! ! Given!that!the!Siverka!cultivar! was!unavailable,!it!might! seem!strange!that!the!focus! of!this!research!project!is!on! the!enhancement!of!soy!milk.! However,!the!size!of!the! potential!global!market!for!a! better!tasting!soy!milk,!the! huge!environmental!and! ethical!benefits!such!a! product!would!confer,!and!

Figure"3"–"Two!soybean!seeds!produced!in!the!greenhouse!during!the! course!of!this!research!from!mature!Glycine!max!‘Elena’!plants;!note!the! dark!hilum!(or!‘eye’)!on!the!near!side!of!the!seeds."

the!advantages!of!soy!milk!over!other!milk!substitutes!combine!to!justify!the!pursuit!of!this!goal!(details!in! section!2.7).!Additionally,!although!the!goal!is!to!transform!soybeans!with!a!gene!which!would!enhance!the! flavour!of!the!milk,!a!more!immediate!aim!is!simply!to!identify!such!a!gene!through!a!literature!review.!! ! 2.7:!THE!PROBLEMS!WITH!DAIRY!MILK! ! The!world!demand!for!milk!is!continuing!to!rise,!with!a!recent!report!by!leading!dairy!processor,!‘Fonterra’! suggesting!that!demand!significantly!outstrips!supply!across!many!global!markets!(Fonterra!2014a!and!shown! in!Figure!4).!!The!dairy!market!as!a!whole!was!worth!over!$300bn!in!2011,!with!this!figure!also!projected!to!rise,! and!increased!demand!even!from!countries!where!the!population!is!genetically!predisposed!to!lactose! intolerance!(citation"needed).!However,!lactose!intolerance!is!just!one!of!the!problems!faced!by!the!global! population!when!considering!the!impact!of!cow’s!milk.!!


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! The!digestive!system!of!cows!is!complex,! consisting!of!multiple!stomachs,!and!an! awkward!system!of!regurgitation!and! repetitive!chewing!which!breaks!down! the!tough!cell!walls!of!grass!and!similar! food!plants.!Cows!and!other!ruminants! possess!a!large!chamber,!known!as!the! ‘rumen’,!in!which!cellulose!is!digested!in! enteric!fermentation;!a!process!which! emits!methane!(Raven!et!al!2005).!In!the! USA,!the!gas!produced!by!cows! comprises!25%!of!methane!emissions,! although!this!figure!also!includes!other! livestock,!including!beef!cattle!and!goats! (EPA!2014).!Methane!is!a!potent! greenhouse!gas!which!may!have! contributed!as!much!as!a!third!towards! the!climate!change!experienced!in!the! past!quarter!millennium!(Ramanujan!2005).!!

Figure"4!–!International!supply!and!demand!for!milk!products! (Fonterra!2014a)"

! The!potential!impact!of!dairy!farming!on!climate!change!is!mitigated!slightly!by!the!reliance!of!many! consumers!on!locallyWproduced!milk,!reducing!transportation!emissions.!This!practice!is!so!widespread!that! industry!experts!coined!the!term!‘milkshed’!for!the!area!in!which!milk!for!one!city!is!produced,!in!reference!to! the!geographical!term!‘watershed’!(Dupuis!2002).!However,!in!our!globalised!economy,!shipping!of!powdered! milk!has!increased,!with!Fonterra!reporting!250,000!metric!tonnes!shipped!each!year!from!just!one!New! Zealand!factory!(Fonterra!2014b).! ! In!addition!to!the!potential!impact!of!milk!production!on!climate,!there!are!other!risks!inherent!in!our!current! system!of!dairy!farming.!Esiobu!et!al!(2002)!reported!a!high!prevalence!of!antibioticWresistance!in!bacteria!on! dairy!farms,!which!was!attributed!to!the!overuse!of!antibiotics!in!cattle.!Mastitis!(painful!inflammation!of!the! udders)!is!frequent!among!dairy!cows!due!to!regular!irritation!during!milking!and!the!abundance!of!thick! faecal!deposits!Wbreeding!grounds!for!bacteriaW!in!areas!such!as!milking!sheds!(Vernelli!2005).!In!2002,!it!was! estimated!that!over!70%!of!global!antibiotic!production!went!into!agriculture,!potentially!creating!a!huge! reservoir!of!antibioticWresistant!bacteria!(Esiobu!et!al!2002).!This!problem,!however,!could!soon!be!reduced,! as,!in!late!2013,!the!U.S.!Food!and!Drug!Administration!legislated!against!the!use!of!certain!antibiotics!in!cattle! (Kastrenakes!2013).! !


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Research!has!recently!been!carried!out!into!the!use!of!genetic!modification!to!reduce!the!effects!of!mastitis,! which!could!potentially!lessen!farmers’!reliance!on!antibiotics.!Yang!et!al!(2011)!describe!a!study!in!which! embryonic!fibroblasts!(and!other!cells)! from!Holstein!cows!were!transformed! using!an!electrotransformation!process.! This!process!introduced!an!expression! vector!into!the!cells,!which!contained!the! ! VEGAN!MILK!ALTERNATIVES! ! Many!plant!products!can!be!processed!to!produce!milk,!so! why!are!soybeans!most!suitable!for!this!study?!It!seems!that! there!are!major!disadvantages!to!other!milk!substitutes…!

!

! Almond! Grown!in!Mediterranean!regions,!where!water!for!agriculture! is!already!scarce!(Rodrigues!et!al!2012)! ! Coconut! Destruction!of!mangrove!swamps!to!reclaim!land!for!coconut! growing!has!removed!natural!barriers!to!storm!surges!and! tsunamis!along!tropical!coastlines!(Hogan!2013)! ! Hazelnut! Requires!large!trees!which!take!6!years!to!mature,!giving! farmers!little!flexibility!to!respond!to!short!term!market! trends!(HNP!2013)! ! Rice! High!methane!output!due!to!growing!in!stagnant!water! (Allen!et!al!2003)! ! …!in!contrast,!soybeans!are!a!nitrogen!fixing!crop,!suitable! for!a!huge!variety!of!climates!and!grown!in!a!season!when!the! ground!is!frequently!left!uncultivated!(see"section"2.1)!

!

human!lysozyme!(HLZ)!gene.!HLZ!acts!as!a! nonWspecific!immune!factor,!and!is! naturally!present!in!human!breastmilk!at!a! typical!concentration!of!200!to!400µg/ml.! The!typical!concentration!of!HLZ!in!cows’! milk!is!just!0.05!to!0.22µg/ml,!but!this! enzyme!may!assist!in!the!suppression!of! mastitisWcausing!bacteria!(Yang!et!al!2011).! While!Yang!et!al!(2011)!were!ultimately! successful!in!upregulating!the!production! of!HLZ!in!cows,!their!methodology!is! controversial;!the!use!of!genetic! modification!in!animals!is!ethically! challenging.!Of!312!embryos!transplanted! by!Yang!et!al!(2011)!into!cows,!only!37! calves!were!carried!to!term!and!just!24! survived!the!first!6!months!of!life;!a!35%! mortality!rate.!In!comparison,!a!Canadian! study!found!a!normal!mortality!rate!of!just! 6%!among!1,968!calves!over!a!6!month! season!(Waltnertoews!et!al!1986).!

! 2.8:!BIOENGINEERING!A!VEGAN!CHEESE! ! Clearly,!there!are!ethical!and!environmental!issues!with!relying!on!cows!for!the!production!of!milk!on!a!large! scale,!but!what!are!the!alternatives?!Recently,!while!investigating!the!possibilities!for!soybean!milk! modification,!I!stumbled!across!a!crowdfunding!campaign,!which!sought!to!manufacture!cheese!by! transforming!baker’s!yeast!(Saccharomyces"cerevisiae).!After!corresponding!with!the!founders!of!this!project,!I! was!given!access!to!their!research,!although,!given!the!‘biohacker’!nature!of!their!experiment,!this!material! was!not!peerWreviewed.!There!were,!however,!experienced!geneticists!on!the!team,!and!the!methodology!


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with!which!they!were!proposing!to!transform!S."cerevisiae!with!a!bovine!milk!gene!was!of!great!relevance!to! this!project.!!

! Figure!5!–!The!workflow!for!yeast!transformation!from!the!‘Real!Vegan!Cheese’!campaign!

!

The!biohackers’!primary!goal!is!to!insert!the!gene!for!production!of!Kappa!casein!(KWcasein)!into!S."cerevisiae;! this!protein!is!the!substrate!for!the!protease!chymosin,!which!splices!KWcasein!into!two!halves.!The!split!KW casein!acts!as!a!clarifying!agent,!causing!a!milk!solution!within!which!it!is!suspended!to!‘clot’.!Clotting!is! scientifically!known!as!micellar!flocculation,!and!it!is!this!process,!whereby!suspended!lipids!are!induced!to! precipitate,!which!initiates!solid!cheese!formation!from!a!liquid!milk!product!(Fox!1999).!Clearly,!KWcasein! alone!is!not!enough!to!create!a!‘vegan!cheese’!–!it!is!but!one!ingredient!in!a!complex!solution,!which!also! comprises!other!casein!proteins;!key!constituents!of!cow’s!milk.!However,!the!public!interest!in!this!project! was!noteworthy,!with!$37,369!in!funding!from!696!backers,!and!exposure!in!many!internationally!renowned! publications!(Indiegogo!2014).! ! The!marketing!surrounding!this!campaign!is!somewhat!disingenuous,!suggesting!that!the!product!is!just! around!the!corner,!despite!the!many!and!varied!technical!obstacles!which!are!yet!to!be!overcome.!However,! there!are!interesting!parallels!between!this!campaign!and!my!pursuit!of!an!improved!soy!milk,!including!the! focus!on!an!short!term!research!goal!which!will!have!no!immediate!impact!on!the!dairy!industry.!It!is!worth! noting!that!the!claimed!environmental!benefits!of!yeast!may!also!be!somewhat!misleading.!Yeast!bioreactors! require!an!input!of!energy,!an!enclosed!space!with!climate!controls!and!the!input!of!plantWderived!sugars,! increasing!the!environmental!footprint!of!the!product!(Raven!et!al!2005).! ! 2.9:!POSTWTRANSLATIONAL!MODIFICATIONS! ! The!vegan!cheese!campaign!and!its!associated!research!has!shown!us!that!using!another!organism!to!produce! a!form!of!cow’s!milk!by!genetic!modification!is!far!from!a!simple!process.!Indeed,!even!if!we!were!to!focus! purely!on!the!casein!proteins,!there!may!be!significant!obstacles!to!progress!in!the!form!of!postWtranslational! modifications!which!are!unique!to!mammals!(Maughan!et!al!1999).!This!is!a!frequent!issue!when!producing! animal!proteins!in!a!species!from!a!different!kingdom,!as!Saccharomyces"cerevisiae!is!a!species!of!fungi!(Pollard! et!al!2007).!Plants!are,!taxonomicallyWspeaking,!even!further!removed!from!cows!than!yeast,!and!expressing!a! transgene!in!plants!has!already!been!found!to!present!issues!with!postWtranslational!modification!(Schmidt!&! Herman!2008).!! !


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Bovine!casein!has!in!fact!previously!been!expressed!in!the!soybean,!in!an!unrelated!experiment!by!Maughan! et!al!(1999).!This!study!was!not!intended!to!alter!the!flavour!of!soy!milk,!but!rather,!to!provide!a!proof!of! concept!for!integration!of!mammalian!transgenes!into!this!plant!species.!Issues!with!postWtranslational! modifications!formed!one!of!the!key!findings!of!this!paper;!the!recombinant!protein!was!significantly!shorter! in!soybean!than!in!cow’s!milk.!Maughan!et!al!(1999)!speculated!that!this!difference!in!size!was!due!to!disparity! in!phosphorylation!and!glycosylation!of!the!protein.!It!seems,!therefore,!that!the!best!route!to!production!of! an!enhanced!cow’s!milk!substitute!in!soybeans!might!be!to!modify!the!existing!biochemical!pathways,!rather! than!to!throw!further!transgenes!into!the!mix.!The!resulting!soy!milk!might!also!be!more!appealing!to! consumers,!than!a!chimeric!mammal/plant!alternative.! ! 2.10:!GENETIC!MODIFICATION!OF!SOY!MILK!FLAVOUR! ! There!has!been!much!research!in!recent!years!into!the!manipulation!of!existing!biochemical!pathways!in!order! to!enhance!the!flavour!or!odour!of!fruit!and!vegetables!(DavidovichWRikanati!et!al!2007,!Klee!&!Tieman!2013,! Morris!et!al!2011).!However,!soybeans!are!viewed!as!difficult!to!transform,!compared!with!other!crops! (Yamada!et!al!2012),!which!may!reduced!their!appeal!to!scientists!involved!in!flavour!modification.! ! The!bitter!and!distasteful!components!of!soybean!flavour!have!long!been!attributed!to!two!classes!of! compound;!isoflavonoids!and!saponins!(Fukushima!2001).!The!effect!of!isoflavonoids!(or!‘isoflavones’)!on! taste!is!compounded!by!their!hydrolysis!into!aglycones!by!the!enzymes!known!as!βWglucosidases!(Fukushima! 2001).!A!single!transgene!which!completely!arrests!isoflavonoid!production!in!soybeans!has!not!been! identified,!despite!the!best!efforts!of!plant!scientists!(Liu!et!al!2013).!Furthermore,!research!into!suitable! techniques!for!destruction!of!these!compounds!during!the!processing!of!soybeans!into!milk!has!also!been! unsuccessful!(Fukushima!2001).!However,!recent!research!has!identified!both!a!mutant!line!of!soybeans! lacking!some!saponins!and!a!gene!which!inhibits!production!of!isoflavonoids.! ! Takada!et!al!(2013)!reports!that,!although!there!are!potential!health!benefits!of!many!types!of!saponin,!a! certain!group;!‘A!acetyl!saponins’!are!a!source!of!bitter!taste!in!soybeans!and!are!not!necessary!to!the! development!of!a!healthy!plant.!While!backcrossing!was!able!to!produce!a!plant!line!naturally!low!in!A!acetyl! saponins;!‘Kinusayaka’,!not!all!of!this!compound!was!completely!eliminated,!although!the!variety!was!adopted! for!use!in!Japan!(Kato!et!al!2007).!! ! Takada!et!al!(2012)!examined!germplasm!of!hundreds!of!different!soybean!genotypes!and!isolated!one! cultivar,!‘B01082’,!with!a!natural!mutation,!which!knocked!out!A!acetyl!saponin!synthesis.!A!recessive!allele! was!identified!which!caused!this!phenotype;!this!allele!was!also!present!in!the!Kinusayaka!cultivar.!However,! the!B01082!cultivar!also!differed!significantly!from!other!soybean!phenotypes!in!its!small!seed!size!and!other! undesirable!traits.!The!negative!phenotypic!traits!of!B01082!were!traced!back!to!an!absence!of!soyasapogenol! A;!thus,!Takada!et!al!(2012)!crossed!B01082!with!another!cultivar!to!rectify!this!genetic!abnormality!and! produce!a!novel!plant!line;!‘Tohoku!152’.!This!line!produced!soybeans!with!economically!desirable!traits!and!


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milk!with!a!pleasant!flavour,!lacking!the!characteristic!soy!milk!bitterness.!This!research!shows!that!traditional! breeding!techniques!can!be!combined!with!modern!genetic!tools!to!enhance!soy!milk!flavour.! ! In!contrast!to!Takada!et!al’s!(2012)!blend!of!the!traditional!and!the!modern,!Liu!et!al!(2013)!relied!entirely!on! transformation!to!modulate!soybean!biochemical!pathways.!Liu!et!al!(2013)!describes!how!isoflavonoids!have! been!found!to!play!an!important!role!in!the!development!of!root!nodules;!they!establish!the!relationship! between!root!cells!and!rhizobial!bacteria.!Thus,!any!modification!of!pathways!related!to!these!compounds! must!be!carried!out!with!great!care,!so!as!to!avoid!damaging!the!intrinsic!benefit!of!soybeans!over!many!other! crops;!nitrogen!fixation.!Any!modification!of!isoflavonoid!production!should!be!localised!to!the!seed,!and! must!not!affect!synthesis!in!the!roots.!Liu!et!al!(2013)!identified!a!gene,!GmMYB39,!which!was!found!to! interact!with!the!CHS!isoflavonoid! promoter!in!a!coWtransfected!plant;! when!under!the!control!of!the!CHS! promoter,!Gus!expression!was!visibly! reduced!by!the!presence!of!GmMYB39.! When!GmMYB39!was!transformed!into! Agrobacterium"rhizogenes!K599!and! used!to!transfect!soybean!cotyledons,! isoflavonoid!production!in!root!tissue! from!regenerated!transgenic!plant! Figure"6!–!Transformed!soybean!lines!in!which!GmMYB39!is! overexpressed!have!reduced!isoflavonoid!content!(Liu!et!al!2013)"

lines!was!suppressed!(see!figure!6).! However,!GmMYB39!was!only!

expressed!at!low!levels!in!the!soybean!seed,!suggesting!that!could!be!some!potential!for!redirecting!the! expression!to!regulate!isoflavonoid!production!in!soybean!seeds.!! ! 2.11:!SEEDWSPECIFIC!PROMOTERS! ! Within!a!plant,!it!is!important!for!some!gene!expression!to!be!localised!exclusively!to!the!developing!seed.! This!tissueWspecificity!is!achieved!by!promoters,!which!drive!gene!expression,!and!which!may!only!be!active!at! certain!points!in!a!plant!lifecycle!or!in!specific!parts!of!the!plant.!In!contrast,!the!35S!promoter,!which!was! used!throughout!the!research!in!this!thesis,!causes!high!expression!levels!in!most!tissues,!throughout! development!(Jopcik!et!al!2014).!35S!is!a!promoter!from!the!cauliflower!mosaic!virus;!other!promoters!have! been!found!which!are!specific!to!the!developing!seed;!these!would!be!of!potential!value!for!driving!expression! of!the!GmMYB39!gene!away!from!root!tissue.!! ! Kita!et!al!(2010)!describe!a!glycinin!promoter!which!is!seedWspecific;!similar!promoters!have!been!used!in!other! research.!In!Schmidt!&!Herman!(2008),!synthesis!of!a!βWconglycinin!α!subunit!was!suppressed!in!order!to! upregulate!production!of!a!GFP!construct!with!a!glycinin!promoter.!Suppressing!βWconglycinin!α!synthesis!


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increased!the!production!of!glycinin;!an!effect!which!was!harnessed!to!drive!a!fourfold!increase!of!GFP! production!in!the!seed!(Kinney!et!al!2001,!Schmidt!&!Herman!2008),!as!shown!in!figure!7.!Therefore,! suppression!of!βWconglycinin!α!synthesis!in!a!transgenic!plant!which!expresses!a!GmMYB39!transgene!under! the!control!of!a!glycinin!promoter,!could!significantly!reduce!the!production!of!isoflavonoids!exclusively!in!the! seed.!Soy!milk!produced!from!this!transgenic!line!would!have!an!enhanced!flavour,!without!deleterious! consequences!for!nitrogen!fixing!root!nodules.!

! Figure"7!–!βCS!=!seed!with!suppression!of!βNconglycinin!(from!research!by!Kinney!et!al!2001)!GFP!=!seed!with! GFPNkdel!linked!to!a!seedNspecific!promoter!(glycinin)!βCS!x!GFP!=!seed!with!both!phenotypes!expressed! together.!Left!image!is!under!visible!light,!right!image!shows!fluorescence!under!blue!light.! !

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2.12:!LOCALISING!EXPRESSION!WITH!GFP!&!tdTOMATO! ! A!primary!goal!of!this!research!project!is!to!transform!Glycine"max!‘Elena’!with!a!transgene,!to!demonstrate! that!this!cultivar!is!susceptible!to!transformation.!As!previously!mentioned,!one!method!for!identifying!the! transformation!efficiency!of!a!cultivar!is!the!use!of!a!reporter!gene,!like!Gus.!However,!there!are!many! problems!with!Gus!staining!(see!section!5.3),!not!the!least!of!which!is!that!live!plant!tissue!cannot!be! examined!(Haseloff!et!al!1997).!A!better!alternative,!which!will!be!used!alongside!GusWstaining!in!this!research,! is!a!fluorescent!protein!marker,!which!can!be!monitored!in!living!tissue!without!the!long!staining!process! necessary!for!Gus.!! ! A!plant!transformed!with!a!fluorescent!protein!can!be!taken!straight!from!the!greenhouse!and!a!tissue!sample! examined!under!the!microscope!within!minutes.!This!marker!can!also!highlight!the!exact!region!of!a!cell!in! which!it!is!expressed.!For!example!the!GFP!HDEL!construct!used!in!this!study!(functionally!identical!to!the!GFPW kdel!construct!from!Schmidt!&!Herman!2008)!is!localised!to!the!endoplasmic!reticulum!(ER);!the!HDEL! sequence!is!a!‘retention!signal’!which!instructs!the!cell!to!retain!an!associated!protein!within!the!ER! (Matsushima!et!al!2002).!The!other!localisation!signal!used!in!this!project!forms!part!of!the!protein!LTI6b,! which!is!delivered!to!the!cell!membrane!(in!this!case,!it!is!connected!to!the!fluorescent!tdTomato!marker).!In! the!case!of!LTI6b,!the!protein!may!accumulate!enroute!to!the!membrane;!this!is!described!as!a!‘backup’!of! fluorescence!(Unpublished,!J.!McKenna!2014).!!


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Haseloff!et!al!(1997)!describe!how!Arabidopsis!with!GFP!localised!to!the!ER!were!more!likely!to!form! regenerated!transgenic!plants;!additionally,!GFP!HDEL!is!widely!used!in!the!Runions!lab;!these!properties! make!the!construct!ideal!for!this!research!project.!LTI6b!has!been!frequently!used!by!Joe!McKenna;!a!postW doctoral!student!who!assisted!with!this!project;!it!was!first!identified!by!Cutler!et!al!(2000).!A!fluorescent! protein!Tagging!a!fluorescent!marker!onto!LTI6b!causes!transformed!cells!to!glow!with!a!highly!visible!outline;! which!makes!it!ideal!for!identification!of!widely!spaced!transformation!events,!such!as!with!biolistic! transformation.! ! 2.13:!OUTLINE!OF!RESEARCH!GOALS! ! On!commencing!this!investigation!into!the!transformation!of!Glycine"max!‘Elena’,!there!were!several!strands! of!research!to!be!pursued,!which!are!all!related!to!the!eventual!goal;!a!transgenic!plant!line!with! enhanced!soy!milk!flavour,!adapted!to!the!UK!climate.!It!was!therefore!vital!to!select!a!technique!for! transformation!which!was!likely!to!produce!stably!transformed!fertile!plants.!This!requirement!led!us!to! initially!focus!on!AgrobacteriumWmediated!transformation,!which,!as!discussed!in!section!2.4,!has!a!proven! track!record!of!producing!stably!transformed!cultivars.! ! The!second!priority!of!this!research!was!to!identify!the!most!suitable!tissue!for!infiltration!in!Glycine"max! ‘Elena’,!due!to!a!lack!of!previous!studies!using!this!cultivar.!This!strand!of!research!would!rely!on!both!existing! papers!on!soybean!transformation,!and!novel!techniques,!adapted!from!the!Tobacco!and!Arabidopsis!plants! more!frequently!studied!in!the! Runions!lab.!Multiple!methods! of!infiltration!would!be!trialled,! with!the!goal!of!identifying!a! method!for!transformation! which!was!quick!and!easy,!and!a! tissue!with!high!transformation! efficiency.! ! Alongside!the!more!complex! genetic!modification!of! soybeans,!a!third!strand!of! research!would!determine!the! most!appropriate!means!of! sterilising!seeds.!This!is!a!vital!step!in! any!transformation!experiment,!as!

Figure"8!–!The!rate!of!explant!regeneration!in!Acacia!mearnsii!after! sterilisation!with!bleach!at!different!concentrations!and!two!other! sterilants!(Jik!=!Household!Bleach).!From!Thompson!et!al!(2009).!

!

long!periods!of!sterilisation!can!prevent!germination,!while!short!periods!might!result!in!contamination!of! explants.!This!protocol!would!use!bleach;!Thompson!et!al!(2009)!showed!that!household!bleach!is!just!as! effective!for!sterilising!explants!of!Acacia"mearnsii!as!HgCl2!(Mercuric!chloride)!and!better!than!CaCl2O2,!with!a!


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far!higher!rate!of!regeneration,!postWsterilisation!(see!figure!8).!Seeds!were!the!focus!of!this!experiment! rather!than!another!plant!tissue,!as!most!successful!AgrobacteriumWmediated!transformation!so!far!has!been! conducted!using!seed!tissue!(Yamada!et!al!2012).!! ! ! ! ! ! !

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Methods! CONTENTS"of"METHODS" " " " "

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3.0!

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3.1!

33!

3.2!

35!

3.3!

38!

3.4!

40!

3.5!

44!

3.6!

Bleach!Protocol! ! Determining!the!Appropriate!Tissue!for!Infiltration! ! Infiltration!of!Soybean!Cotyledons! ! Protoplasting! ! Gus!Gateway!Cloning!and!Transformation! ! Gus!Infiltration!and!Staining!Protocol! ! Biolistic!Infiltration! ! "

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3.0:!BLEACH!PROTOCOL! ! Background!to!Bleach!Protocol! ! Over!the!initial!3!months!of!research,!various!bleaching!durations!were!tested!for!their!effects!on!the! sterilisation!and!germination!of!seeds.!Thompson!et!al!(2009)!designated!the!growth!of!plants!and!the! resulting!sterility!of!seeds!postWbleaching!as!the!two!primary!metrics!of!success!for!bleaching!explants.!The! results!of!early!trials!were!used!to!inform!the!design!of!a!final,!large!scale!sterilisation!trial,!with!450!seeds! planted!in!3!different!substrates!across!6!different!conditions.!This!final!protocol,!detailed!below,!was!used!to! determine!an!optimal!duration!for!bleaching!of!Glycine"max"‘Elena’!seeds.! ! Bleaching!Process! ! Over!600!soybean!seeds!were!poured!into!a!50ml!beaker,!which!was!placed!in!a!sterile!laminar!flow!hood.!! Approximately!100!seeds!were!poured!into!each!of!6!separate!50ml!Falcon!tubes,!which!were!labelled!per! condition:! BLEACH!PROTOCOL!CONDITIONS! ! Label:!0B/0W! 0"mins"bleaching,"0"minutes"soaking"in"water" ! Label:!0B/45W! 0"mins"bleaching,"45"mins"soaking"in"water" ! Label:!2B/43W! 2"mins"bleaching,"43"mins"soaking"in"water" ! Label:!5B/40W! 5"mins"bleaching,"40"mins"soaking"in"water" ! Label:!15B/30W! 15"mins"bleaching,"30"mins"soaking"in"water" ! Label:!30B/15W! 30"mins"bleaching,"15"mins"soaking"in"water" ! ! ! To!the!0B/45W!tube,!sterile!dH2O!was!added!until!it!reached!the!40ml!mark!on!the!tube!exterior.!To!the!2,5,15! and!30B/15W!tubes,!‘50:50!bleach’!(50%!dH2O!and!50%!sodium!hypochlorite!solution)!was!added!until!it! reached!the!same!40ml!mark.!A!timer!was!started,!and!all!tubes,!including!0B/0W!were!sealed!and!placed!on!a! lab!bench!rocker!at!approximately!40RPM.! ! After!2!minutes!had!elapsed,!the!dH2O!from!the!2/45!tube!was!poured!out!(while!the!lid!was!partly!closed,!to! retain!the!seeds!within)!and!replaced!to!the!40ml!mark!with!sterile!dH2O.!This!process!was!repeated!at!the!5! mins!point!for!the!5B/40W!tube,!at!15!mins!for!the!15B/30W!tube!and!30mins!for!the!30B/15W!tube.!After!45! mins!had!elapsed!on!the!timer,!all!6!tubes!were!drained!of!dH2O,!the!lids!firmly!closed!and!left!at!room! temperature.!


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Planting!on!Paper!Towels! ! A!standard!laboratory!paper!towel!sheet!(approximately!40cm!x!30cm)!was!flattened,!inside!the!sterile! laminar!flow!hood.!It!was!sprayed!with!70%!industrial!methylated!spirits!(IMS)!until!the!whole!of!the!towel!was! visibly!darkened!from!IMS!absorption;!at!this!point,!the!towel!was!folded!to!fit!into!the!base!of!a!square!Petri! dish!(125mm!x!125mm).!This!was!repeated!for!four!dishes,!which!were!labelled!0B/0W,!5B/40W,!30B/15W!and! ‘Ctrl’.!! ! Within!the!hood,!sterile!forceps!were!used!to!evenly!place!18!seeds!in!3!rows!across!the!surface!of!the!towel!in! each!dish;!seeds!were!taken!from!the!corresponding!50ml!Falcon!tube.!No!seeds!were!placed!in!the!‘Ctrl’! plate,!but!the!forceps!were!lightly!pressed!into!the!towel!18!times,!in!positions!corresponding!to!the!seeds! within!the!other!plates.!This!control!plate!was!prepared!in!order!to!provide!a!baseline!mould/bacterial!growth! level!against!which!the!other!conditions!could!later!be!compared.! ! Planting!on!Murashige!and!Skoog!Media! ! Two!500ml!bottles,!each!containing!400ml!of!MS!rooting!medium!(see!below),!were!autoclaved!and!allowed! to!cool!to!approximately!40°C.!13!square!Petri!dishes!were!labelled;!2!per!condition!(arbitrarily!labelled!‘A’!and! ‘B’)!and!1!‘Ctrl’;!40ml!of!MS!rooting!medium!was!poured!into!each!dish.!Before!the!medium!set!firm,!18!seeds! were!placed!onto!each!dish!(from!the!appropriate!50ml!Falcon!tube),!using!sterile!forceps,!in!3!rows!of!6! seeds!(see!Figure!9).!The!‘Ctrl’!dish!contained!no!seeds,!but!the!media!was!lightly!prodded!18!times,!as! described!in!Planting!on!Paper!Towels.! ! Murashige!&!Skoog!(MS)!Rooting!Medium! ! 4g"sucrose"and"1.18g"Murashige"&"Skoog"powder"were"added"to"a"500ml"bottle,"then"topped"up"with" dH2O"to"the"350ml"mark."Using"a"pH"meter"and"acid/alkaline"solutions,"pH"of"the"MS"solution"was"adjusted" to"pH5.8"and"then"2.4g"of"agar"powder"was"added,"before"the"bottle"was"filled"to"400ml"mark"with"dH2O."

! ! Germination!Conditions!for!Paper!Towel!and!MS!Petri!Dishes! ! Plates!were!closed!and!sealed!with!Parafilm,!before!being!placed,!flat,!in!three!rows!under!a!grow!light!in!a! plant!room!with!24h!light!at!25°C.!The!position!of!plates!within!the!rows!was!randomised!to!avoid!variation!in! light!levels!from!compromising!the!validity!of!this!germination!assay.!The!fluorescent!tube!growth!light!ran! longitudinally!along!the!centre!of!the!second!row,!at!a!height!of!approximately!60cm.! !


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! Figure"9!–!Square!Petri!dishes!containing!MS!media!and!soybean!seeds!under!growth!lights.!Labels!in!the! experiment!differed!slightly!from!this!protocol!(0/45"="oB/45W;""5/45"="5B/40W;"30/45"="30B/15W"etc.)! ! ! Quantification!of!Fungal/Bacterial!Growth!with!Scanned!Images! ! 6!days!after!seeds!were!planted!in!MS!media,!the!petri!dishes!were!placed,!2!at!a!time,!on!a!flatbed!scanner,!

!

MS!side!downwards!as!per!in!the!growth!room.!Each!pair!of!dishes!were!scanned!as!one!200ppi!RGB!image! with!dimensions!of!approximately!1300x2000!pixels!in!uncompressed!TIFF!format.! ! The!uncompressed!TIFF!was!opened!in!Adobe!Lightroom!(version!5.5!on!Mac!OSX!10.9.4),!where!the!image! parameters!were!adjusted!as!a!batch!to!enhance!contrast!in!both!luminance!and!saturation:! ! Table"1"

Original!Value!

Modified!Value!

Exposure!

0!

W0.85!

Contrast!

0!

+100!

Highlights!

0!

+100!

Shadows!

0!

W100!

Clarity!

0!

+100!

Saturation!

0!

+100!

! ! A!JPEG!image!(of!equivalent!scale,!but!under!5MB!in!size)!was!exported!from!Adobe!Lightroom!after!these! parameters!were!altered,!and!then!the!file!was!duplicated,!so!that!each!dish!had!a!separate,!dedicated!file.! These!files!were!then!opened!in!the!Mac!application!‘Preview’;!at!this!point,!the!approximate!outline!of!the! Petri!Dish!was!traced!using!the!Lasso!Selection!tool,!and!the!image!was!cropped!to!this!shape!(in!the!process,! it!was!converted!to!a!PNG!file!to!preserve!the!irregular!image!margins).!These!shapes!were!somewhat!inexact,!


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although!the!range!of!sizes!varied!by!less!than!6%!across!all!samples;!this!cropping!defined!a!region!within! which!to!determine!surface!area!covered!by!fungal/bacterial!growth!(i.e.!contamination).!!

! Figure!10!–!Some!examples!of!cropped!PNG!images!after!adjustment!in!Adobe!Lightroom!!

! The!PNG!files!were!opened!in!Fiji!–!an!enhanced!version!of!the!image!analysis!software,!‘Image!J’!(version!2).!

!

Three!slides!were!chosen!which!varied!visibly!in!the!surface!area!covered!by!fungal/bacterial!colonies;!these! were!used!to!determine!threshold!values!for!the!hue,!saturation!and!brightness!of!the!fungus/bacteria! colonies!by!experimentation.!This!process!also!ensured!that!the!area!of!the!image!covered!by!the!soybean! seeds!and!roots!was!not!inaccurately!classified!as!fungal!growth.!Unfortunately,!the!edges!of!the!Petri!dish! and!Parafilm!were!such!a!similar!colouration!to!the!fungal/bacterial!growth!that!these!were!unavoidably! included!within!the!threshold!colour!values.!This!was!mitigated!by!the!ubiquity!of!these!features!between! conditions!(as!shown!in!Figure!10).!! ! The!threshold!values!used!for!determining!the!surface!area!of!fungal/bacterial!growth!were!as!follows:! ! Table"2"

Min!value!

Max!value!(of!256)!

Hue!

23!

108!

Saturation!

28!

177!

Brightness!

89!

204!

! !


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! Before!thresholded!surface!area!was!recorded,!a!ruler!which!was!scanned!alongside!the!Petri!Dishes!was!used! to!calibrate!the!analysis!software!in!millimetres.!The!threshold!values!were!then!set!for!each!image,!the! thresholdWspecified!region!was!selected,!and!its!surface!area!determined!using!the!Measure!tool.!Both!the! total!surface!area!of!each!cropped!image!and"the!surface!area!covered!by!fungal/bacterial!colonies!(according! to!the!threshold!values)!were!recorded,!to!enable!a!final!value!of!‘percentage!contamination’!to!be!calculated.! ! Percentage!Contamination!=!Surface!Area!Specified!by!Threshold!Parameters!in!Cropped!Image! ! ! ! ! ! Total!Surface!Area!of!Cropped!Image! !

Figure!11!–!Screenshot!of!Fiji!software!before!and!after!running!the!Threshold!Colour!analysis;!pixels!which! matched!the!threshold!parameters!are!highlighted!in!red!and!recorded!using!the!Measure!tool.!The!edges!of! the!dish!were!included!in!the!threshold!area!due!to!similarity!in!pixel!parameters,!while!some!colonies!of! bacteria!were!unintentionally!excluded.!

!

! 3.1:!DETERMINING!THE!APPROPRIATE!TISSUE!FOR!INFILTRATION!

! Over!the!course!of!the!first!three!soybean!infiltration!procedures!(not!detailed!in!this!report),!a!successful! method!for!infiltration!of!the!plants!was!established.!The!Runions!lab!usually!works!with!tobacco!plants,! which!can!be!infiltrated!by!making!a!small!hole!in!a!mature!leaf,!into!which!infiltration!buffer!is!injected!(as!per! Sparkes!et!al!2006).!The!reverse!of!this!hole!is!sealed!with!a!gloved!finger!during!the!injection,!to!avoid!the! solution!passing!straight!through!the!leaf.!A!similar!system!was!attempted!in!initial!trials!on!unifoliate!and! trifoliate!soybean!leaves!without!success;!the!leaf!was!impenetrable!and!the!solution!leaked!out!sideways! from!the!point!of!injection.! ! Injection!into!the!hypocotyl!resulted!in!significant!damage!to!the!seedling,!and!injecting!immature!cotyledons! (less!than!10!days!old)!was!also!unsuccessful.!However,!cotyledons!at!approximately!15!days!after!planting! were!easy!to!infiltrate,!with!the!solution!rapidly!absorbed!by!this!spongy!tissue.!!


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! 3.2:!INFILTRATION!OF!SOYBEAN!COTYLEDONS! ! Background!to!Infiltration!Protocol! ! This!following!protocol!was!followed,!with!minor! modifications,!throughout!all!infiltration!experiments;!this!was! adapted!from!Sparkes!et!al!(2006).!The!protocol!was!standard! practice!within!the!Runions!lab!for!successful!transformation!of! tobacco!plants;!Nicotiana"tabacum.!The!early!experiments!in! this!study!were!conducted!using!Agrobacterium"tumefaciens! strain!GV3101,!with!GFP!HDEL!under!the!control!of!the!35S! promoter!in!a!PVKH18EN6!binary!vector.!Throughout!this! document,!this!specific!construct/cell!culture!is!referred!to! simply!as!GFP!HDEL!1.! ! Between!27th!May!and!27th!June,!an!alternative!strain!of! Agrobacterium!was!trialled;!Agrobacterium"tumefaciens!strain! EHA105,!with!the!GFP!HDEL!also!under!the!control!of!the!35S! promoter.!This!GFP!HDEL!construct!and!associated! Agrobacterium!cell!culture!are!referred!to!as!GFP!HDEL!2! throughout!this!thesis.!In!July,!a!lack!of!successful! transformations!with!GFP!HDEL!2,!and!reWexamination!of! earlier!micrographs,!compelled!a!switch!back!to!GFP!HDEL!1.!

Figure!12!–!Annotated!anatomy!of!a!15!day! old!Glycine!max!‘Elena’!seedling;!note!the! emerging!unifoliate!leaves,!which!are! followed!in!development!by!a!set!of! trifoliate!leaves.!NB.!The"epicotyl,"hypocotyl" and"radicle"are"truncated"and"the"hilum" enlarged"for"visibility"in"this"schematic" diagram.!

! Preparation!of!Cell!Culture! ! Glycerol!stocks!of!GFP!HDEL!1!and!GFP!HDEL!2!were!already!present!in!the!lab,!so!it!was!not!necessary!to! transform!Agrobacterium"with!these!constructs.!The!rest!of!this!protocol!details!the!steps!necessary!for!the! infiltration!of!cotyledons!with!Agrobacterium!carrying!either!of!the!two!GFP!HDEL!genes,!the!P19!gene!(which! supresses!RNAi)!or!the!Gus!constructs.! ! In!a!sterile!laminar!flow!hood,!a!P200!pipette!tip!was!firmly!pushed!into!the!glycerol!stock!and!twisted,!to! collect!a!sample!of!cells,!before!being!dropped!into!a!20ml!Sterilin!tube!containing!5ml!of!media.!This!media! was!mixed!in!advance!with!the!antibiotics!appropriate!for!the!construct!and!cell!culture!in!use!(see!box!on! following!page).!The!20ml!tube!was!labelled!and!placed!in!a!rack!within!a!28°C!rotating!incubator!at! approximately!150RPM!overnight.! !


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Antibiotics"and"Media"for"Cell"Cultures" " Construct:"GFP!HDEL!1" Media:"50ml"YEB"Agar" Antibiotics:"50µl"Rifampicin,"5µl"Gentamicin,"25µl"Kanamycin" " Construct:"GFP!HDEL!2" Media:"50ml"YEB"Agar" Antibiotics:"50µl"Rifampicin,"25µl"Kanamycin" " Construct:"Gus!PB"(see"section!3.3)" Media:"50ml"LB"Agar" Antibiotics:"50µl"Rifampicin,"25µl"Spectinomycin,"5µl"Gentamicin" " Construct:"Gus!PG"(see"section!3.3)" Media:"50ml"LB"Agar" Antibiotics:"50µl"Rifampicin,"25µl"Kanamycin,"5µl"Gentamicin,"50µl"Hygromycin" " Construct:"P19! Media:"50ml"LB"Agar" Antibiotics:"5µl"Gentamicin,"25µl"Kanamycin!

! Preparing!the!Infiltration!Buffer! ! 1000µl!of!an!overnight!cell!culture!was!pipetted!under!nonWsterile!conditions!into!a!2ml!Eppendorf!tube.!A! further!1000µl!was!pipetted!into!a!second!2ml!tube!and!both!tubes!were!sealed,!labelled!and!placed!into!a! centrifuge.!The!samples!were!spun!at!5000RPM!for!5!minutes,!before!the!supernatant!was!poured!off!and!the! cells!resuspended!in!1000µl!Infiltration!Buffer!(see!below!for!details).!The!centrifugation!step!was!repeated! and!the!supernatant!again!poured!off,!to!ensure!that!any!antibiotics!were!washed!from!the!cell!culture.!The! cells!were!again!resuspended!in!1000µl!of!Infiltration!Buffer!before!the!optical!density!(OD)!of!these!cells!at! 600nm!was!recorded!using!a!Nanodrop!spectrophotometer.! " Infiltration"Buffer" " 250mg"dhGlucose"was"added"into"a"50ml"Falcon"tube,"followed"by"5µl"Acetosyringone,"and"5ml"of"20mM" Sodium"Phosphate"(Na3PO4),"then"5ml"0.5M"MES"(C6H12NNaO4S)."The"tube"was"topped"up"to"50ml"with" dH2O,"and"labelled"as"Infiltration!Buffer"(IB)"then"kept"refrigerated"until"it"was"required."! !

! !


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Calculating!the!Required!Optical!Density! ! The!following!formula!was!used!to!determine!the!volume!of!resuspended!cell!culture!to!add!per!1ml!of! Infiltration!Buffer:! ! OPTICAL!DENSITY!EQUATION!! for"OD"of"0.01"at"600nm" ! α!=!y!÷!(OD!x!10)! ! α:!µl!of!resuspended!cell!culture!to!add!(after!removing!α!µl!of!IB)! y:!value!which!differs!between!constructs;!for!P19,!this!value!=!25! and!for!all!other!constructs!in!this!project,!this!value!=!5! OD:!optical!density!measured!at!600nm!by!spectrophotometer!

! ! ! ! For!many!of!these!experiments,!more!than!1ml!of!infiltration!buffer!was!required,!so!the!α!value!would!be! multiplied!by!the!number!of!ml!required.!Later!experiments!also!involved!manipulating!the!optical!density!of! the!infiltration!buffer;!this!would!require!modifying!the!formula!above!by!multiplication.! ! Infiltrating!Soybean!Cotyledons! ! A!hypodermic!needle!was!used!to!pierce!a!primary!hole!in!the! upper!epidermis!of!the!cotyledon!which!extended!¾!of!the!tissue! depth,!but!did!not!break!the!lower!epidermis.!The!location!of!this! primary!hole!and!two!secondary!holes,!which!only!extended!to!up! to!½!the!tissue!depth!is!shown!in!figure!13.!A!1ml!syringe!was!then! filled!with!the!infiltration!solution!(IB!with!suspended!cell!culture)! and!the!syringe!tip!was!pressed!firmly!against!the!primary!hole!in! the!upper!epidermis.!A!gloved!finger!was!placed!beneath!this! primary!hole!and!the!syringe!was!gently!depressed!to!gradually! infiltrate!the!cotyledon.!Once!this!solution!had!reached!the!two! secondary!holes,!it!emerged!in!the!form!of!two!small!droplets;!

Figure"13""–"Soybean!Cotyledon,!with!primary! hole!for!injection!and!secondary!holes!for! monitoring!throughflow!to!determine!extent! of!infiltration;!blue!arrows!show!infiltration! solution!route."

these!were!wiped!away!using!a!paper!towel.!Any!excess!solution!was!also!wiped!away!to!avoid!contamination! of!adjacent!cotyledons!or!droplets!reaching!the!plant!roots.!Infiltrated!plants!were!watered!and!kept!in!a!light! incubation!chamber!to!encourage!optimal!growth!until!required.! ! 3.3:!PROTOPLASTING! ! Background!to!Infiltration!Protocol! ! A!common!procedure!used!to!transform!plants!which!have!low!susceptibility!to!transformation!is!to!dissolve! the!plant!cell!walls,!creating!‘protoplasts’,!which!are!less!resistant!to!penetration!by!Agrobacterium.!After! research!showed!that!the!Elena!cultivar!was!extremely!resistant!to!transformation!by!leaf!infiltration,!a!


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protoplasting!procedure!was!pursued,!following!2!established!methods!which!were!previously!used!on! Arabidopsis!and!Glycine"max"plants.!! ! Preparation!of!Plant!Tissue!–!First!Trial! ! For!the!initial!experiment,!several!different!tissue!types!were!used,!in!order!to!establish!the!most!efficient! method!for!generating!protoplasts.!Two!different!protoplasting!solutions!were!used!in!order!to!separate!the! cells!and!dissolve!their!cell!walls;!the!first!type!is!referred!to!here!as!‘Runions!Protoplasting!Solution’!(Runions! PPS);!the!second!type!is!referred!to!as!‘Soybean!Protoplasting!Solution’!(Soybean!PPS);!details!of!these! solutions!are!provided!below.! ! The!roots!of!soybean!plants!grown!for!12!days!in!MS!rooting!medium!(see!section!3.0)!were!cut!from!the!main! body!of!the!plant!using!a!scalpel.!These!roots!were!placed!in!2!Petri!dishes!in!a!nonWsterile!environment!and! sliced!transversely!in!order!to!create!a!bundle!of!10!approximately!50mmWlong!root!fragments.!Approximately! 10!cotyledons!were!sliced!into!thirds!transversely!and!added!to!2!further!dishes!and!a!final!2!dishes!contained! 15mm!long!hypocotyl!fragments.! ! 7ml!of!Soybean!PPS!was!added!to!half!of!the!dishes!(1!per!tissue!type),!while!the!other!half!had!7ml!of!Runions! PPS;!the!solution!was!poured!over!the!samples!before!the!dishes!were!sealed!with!Parafilm.!Petri!dishes!were! left!at!room!temperature!on!a!lab!bench!rocker!at!approximately!40RPM!overnight.! ! Soybean"PPS"–"adapted"from"Baldes"et"al"(1987)" " 50ml"of"a"0.45M"DhMannitol"solution"was"autoclaved"and"allowed"to"cool"to"room"temperature."Taking" care"not"to"inhale"the"powders,"750mg"Cellulase,"100mg"Macerozyme"and"25µl"Pectinate"were"added"to"a" 50ml"Falcon"tube;"this"was"toppedhup"to"50ml"with"the"0.45M"DhMannitol"in"a"sterile"laminar"flow"hood."A" vortex"was"then"used"to"suspend"the"hormones"in"the"solution." ! ! Runions"PPS"–"standard"lab"protocol" " A"solution"comprising"0.4M"M"Mannitol,"1mM"CaCl2,"20mM"KCl"and"10mM"MES"(C6H12NNaO4S)was"mixed" and"then,"using"a"pH"meter"and"acid/alkaline"solutions,"the"pH"adjusted"to"5.6."This"solution"(‘Runions" PPS’)"was"then"autoclaved"in"aliquots"of"50ml"and"allowed"to"cool"to"room"temperature."500mg"Cellulase" and"50mg"Macerozyme"were"added"to"a"50ml"Falcon"tube,"which"was"then"topped"up"to"50ml"with" Runions"PPS"in"a"sterile"laminar"flow"hood."A"vortex"was"then"used"to"resuspend"the"hormones." ! ! ! Preparation!of!Plant!Tissue!–!Second!Trial! ! For!the!second!trial,!unifoliate!leaves!were!used!in!every!condition;!2!dishes!contained!leaves!harvested!from! plants!which,!immediately!prior!to!harvesting,!were!kept!for!a!week!in!the!plant!incubation!chamber.!A!further!


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2!dishes!contained!leaves!from!plants!grown!exclusively!in!the!greenhouse.!Petri!dishes!were!filled!to!a!depth! of!2!leaves,!before!these!were!sliced!with!a!scalpel!into!a!series!of!strips!approximately!1mm!across,!extending! from!the!central!leaf!vein!to!the!edge!of!the!leaf.!! ! 10ml!of!Soybean!PPS!was!added!to!half!of!the!dishes!(1!per!condition),!while!the!other!half!had!10ml!of! Runions!PPS;!this!was!poured!over!the!samples!before!the!dishes!were!sealed!with!Parafilm.!Petri!dishes!were! left!at!room!temperature!on!a!lab!bench!rocker!at!approximately!40RPM!for!22!hours.! ! Sterile!Preparation!of!Plant!Tissue!–!Third!Trial! ! Using!scissors,!unifoliate!leaves!were!cut!from!14!day!old!soybean!seedlings!and!stacked,!before!25!were! placed!each!of!two!50ml!Falcon!tubes.!In!a!sterile!laminar!flow!hood,!the!tubes!were!each!filled!up!to!the!40ml! mark!with!50:50!bleach.!Tubes!were!shaken!vigorously!for!30!seconds!and!moderately!for!a!further!60! seconds,!to!sterilise!the!leaves!and!cause!some!lacerations!to!the!epidermis.!The!50:50!bleach!was!then! poured!off!and!replaced!to!the!40ml!mark!with!sterile!dH2O;!tubes!were!again!shaken!vigorously!for!30! seconds,!before!the!dH2O!was!poured!off.!All!leaves!within!the!2!tubes!were!emptied!into!a!sterile!Petri!dish! and!distributed!evenly!between!4!further!dishes;!the!leaves!were!then!sliced!as!per!the!second!trial.!! ! PPS!solutions!were!filtered!through!a!sterile!20ml!syringe!filter!before!10ml!was!poured!over!each!dish.!These! dishes!were!sealed!and!placed!on!a!lab!bench!rocker!at!approximately!40RPM!for!20!hours.! ! Sterile!Preparation!of!Plant!Tissue!&!Subsequent!Infiltration!–!Fourth!Trial! ! Both!unifoliate!and!trifoliate!leaves!were!used!in!this!trial!(from!22!day!old!seedlings),!with!the!slicing!of!leaves! modified!to!the!extent!that!only!shallow!cuts!were!made!in!the!lower!epidermis.!Four!Petri!dishes!were! divided!between!four!different!conditions;!only!Soybean!PPS!was!used,!but!half!of!dishes!contained!no! macerozyme;!additionally,!half!of!dishes!(1!per!macerozyme!condition)!were!run!through!a!vacuum!infiltration! process!for!2!minutes.!20ml!of!Soybean!PPS!was!used!per!dish,!with!the!leaves!placed!topsideWup!to!allow! penetration!of!PPS!through!stomata.!The!vacuum!infiltration!protocol!is!described!in!section!3.4;!sterile! technique!was!used!in!this!trial!as!per!the!third!trial.! ! After!being!left!on!a!lab!bench!rocker!overnight,!the!dishes!were!lightly!shaken!and!the!Soybean!PPS!was! poured!into!4x!50ml!Falcon!tubes.!These!were!labelled!per!condition!and!centrifuged!at!300RPM!for!10! minutes!to!pellet!protoplasts;!a!visible!green!film!at!the!bottom!of!the!tubes!indicated!successful!protoplast! production!in!all!4!conditions.!The!Soybean!PPS!supernatant!was!poured!off!to!leave!approximately!1ml,! before!the!protoplasts!were!resuspended!in!a!further!1ml!of!Incubation!Solution!(see!overleaf)!as!per!Mazarei! et!al!(2008).!Tubes!were!centrifuged!at!250RPM!for!5!mins!then!the!remaining!supernatant!was!removed!with! a!pipette.!5ml!of!incubation!solution!with!GFP!HDEL!1!at!an!OD!of!1.0!was!prepared!as!per!section!3.2!and!the! tube!containing!the!most!protoplasts!was!selected;!the!GFP!HDEL!1!solution!was!then!used!to!resuspend!the!


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cells!in!this!tube.!The!same!procedure!was!followed!for!a!second!tube,!without!any!Agrobacterium!added,!to! act!as!a!control;!these!two!tubes!were!left!in!a!29°C!incubator!overnight!as!per!Mazarei!et!al!(2008).! ! Incubation"solution"–Mazarei"et"al"2008" " 10ml"of"0.5M"MES"at"pH5.7"was"mixed"and"0.4ml"of"this"was"added"to"a"50ml"Falcon"tube."67µl"of"0.45M" Mannitol"and"14.8mg"of"1M"KCl"was"added"to"the"same"50ml"tube,"which"was"then"topped"up"to"50ml"with" dH2O"and"vortexed"to"mix."

!

!

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3.4:!GUS!GATEWAY!CLONING!&!TRANSFORMATION! ! PB7FWG2M,!PGWB2!and!pEntr™ogus! ! As!previously!mentioned,!the!Runions!lab!already!maintains!two!separate!GFP!HDEL!constructs!in!transformed! Agrobacterium!glycerol!stocks.!However,!there!were!no!Agrobacterium!cultures!transformed!with!a!Gus! construct.!A!GusWcontaining!entry!vector!was!present,!but!was!maintained!in!cold!storage;!it!was!therefore! necessary!to!carry!out!a!Gateway!reaction!in!order!to!transfer!this!entry!vector!into!a!destination!vector!and! then!transform!Agrobacterium.! ! Two!destination!vectors!were!used!as!part!of!this!protocol;!PB7FWG2M!Orange!2!(henceforth!truncated!to! ‘PB7FWG2M’)!and!PGWB2;!these!two!alternatives!were!used!to!maximise!the!possibility!of!success!in!the!LR! Gateway!reaction,!rather!than!for!any!specific!property!of!the!vectors!themselves.!For!the!purposes!of!this! experiment,!these!two!vectors!were!functionally!identical,!containing!a!35S!promoter,!and!were!both!to!be! transformed!with!the!same!Gus!entry!vector,!‘pEntr™ogus’!(shown!in!figure!14).!Despite!the!functional! similarity!of!PB7FWG2M!and!PGWB2,!both!were!used!throughout!the!experiments!in!this!research.!To! facilitate!differentiation!between!Agrobacterium!transformed!with!the!pEntrWgusWcontaining!PGWB2!vector! and!the!PB7FWG2M!alternative,!these!two!cell!cultures!are!referred!to!in!this!document!as!‘Gus!PG’!and!‘Gus! PB’.!!

! Figure"14!–!Vector!maps!of!the!pENTRNgus!entry!vector!from!the!software!Serial!Cloner!(left)!and!the! supplier,!Invitrogen!(right)."


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PCR!of!Vectors! ! 3µl!of!pEntrWgus,!1µl!Clonase!and!1µl!of!either!PB7FWG2M!or!PGWB2!were!added!into!two!separate!0.2ml! Eppendorf!tubes!and!centrifuged!at!3000RPM!for!10!seconds.!The!reagents!were!kept!on!ice!at!all!times;!after! centrifugation,!they!were!mixed!by!briefly!pipetting,!before!both!tubes!were!placed!in!a!PCR!machine! overnight.!This!process!allows!the!vectors!to!multiply!and!the!Gus!gene!from!the!entry!vector!to!be!linearised! and!ligated!into!the!destination!vectors.! ! Transformation!of!E.!coli!Culture! ! SOC!Outgrowth!Media!was!heated!in!a!microwave!until!melted!and!aliquotted!into!2x!50ml!Falcon!tubes.! When!these!tubes!reached!approximately!40°C,!within!a!sterile!laminar!flow!hood,!25µl!of!Spectinomycin!was! added!to!the!first!tube!and!50µl!Hygromycin!with!25µl!Kanamycin!to!the!second;!after!adding!these! antibiotics,!the!tubes!were!inverted!8!times!to!mix.!Within!the!sterile!hood,!25ml!of!the!first!tube!was!poured! into!a!plate!labelled!‘PB7FWG2M’,!and!25ml!into!another!labelled!‘Spec!Negative!Control’.!Then!this!process! was!repeated!with!the!second!tube!and!the!third!and!fourth!plates!labelled!as!‘PGWB2’!and!‘Hyg!&!Kan! Negative!Control’.!These!plates!were!then!closed!and!placed!on!a!shelf!in!the!37°C!incubator.! ! Tubes!were!removed!from!the!PCR!machine!and!0.5µl!proteinase!K!was!added!to!each!tube,!to!stop!the! reaction!within.!A!P20!pipette!set!to!3µl!was!used!to!mix!the!solution!inside!the!tubes,!before!they!were! incubated!for!a!further!10!minutes!in!a!PCR!machine.!Two!2ml!Eppendorf!tubes!containing!Escherichia"coli! culture!were!removed!from!the!W80°C!freezer!and!put!on!ice,!before!labelling!as!PB7FWG2M!and!PGWB2.! These!tubes!were!then!manually!spun!to!collect!the!culture!at!the!bottom!of!the!tube.!After!the!E."coli!tubes! had!been!on!ice!for!30!minutes,!they!were!placed!in!a!42°C!water!bath!for!30!seconds,!before!being!put!back! on!ice!and!transferred!into!the!sterile!hood.!After!these!tubes!were!on!ice!for!5!minutes,!they!were!topped!up! to!1ml!with!SOC!media!at!37°C,!before!being!placed!inside!a!box!in!the!rotating!incubator!at!37°C.!After!60! minutes!had!elapsed,!the!tubes!were!removed!and!culture!pipetted!into!the!centre!of!the!correspondingly! labelled!plate;!this!culture!was!then!distributed!evenly!using!a!spreader.!Negative!control!plates!were!not! inoculated!in!this!way!and!remained!closed.!All!plates!were!placed!in!the!37°C!incubator!overnight.! ! The!temperature!changes!in!this!protocol!were!designed!to!induce!susceptibility!to!transformation!in!the!E.! coli.!This!bacterium!will!replicate!the!destination!vector!to!a!high!copy!number!before!these!multiplied!vectors! are!isolated!and!transformed!into!Agrobacterium"tumefaciens.! ! ColonyoPicking!and!MinioPrep! ! Twelve!15ml!Falcon!tubes!were!labelled!PB1WPB6!and!PG1WPG6!and!placed!in!a!sterile!hood,!within!which!the! rest!of!this!protocol!was!carried!out.!50µl!Hygromycin,!25µl!Kanamycin!!and!50ml!LB!Media!were!added!to!a! 50ml!Falcon!tube!(for!PB)!and!25µl!Spectinomycin!with!50ml!LB!Media!was!added!to!a!second!(for!PG).!5ml!of! the!50ml!PB!solution!was!poured!into!each!15ml!PB!tube;!5ml!of!the!50ml!PG!solution!was!poured!into!the!


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remaining!6!PG!15ml!tubes.!Into!each!15ml!tube!was!placed!a!pipette!tip!which!had!been!lightly!dipped!into!an! individual!(visually!distinct)!colony!from!the!corresponding!overnight!plate!(e.g.!PG!from!PGWB2!plate;!see! above).!The!15ml!Falcon!tubes!PB1W6!and!PG1W6!were!sealed!and!placed!in!a!shaking!37°C!incubator!for!6!hours.! ! From!each!15ml!Falcon!tube!cell!culture,!1ml!was!pipetted!into!a!separate!1.5ml!labelled!Eppendorf!tube.!These! tubes!were!centrifuged!at!11,000RPM!for!30!seconds,!then!the!supernatant!was!poured!off.!The!cell!cultures! were!then!washed!and!lysed,!before!the!vectors!were!eluted,!using!the!MiniPrep!kit!from!MacheryWNagel! (MacheryWNagel!2012).!This!eluted!DNA!was!stored!in!a!W20°C!freezer!to!preserve!it!for!a!few!days!until!the!next! stage!in!this!protocol.! ! Transformation!of!Agrobacterium! ! The!optical!density!of!DNA!samples!stored!in!the!W20°C!freezer!was!determined!using!the!Nanodrop! spectrophotometer.!The!required!optical!density!of!samples!was!500ng!per!20µl!Agrobacterium;!an!A." tumefaciens!culture!was!retrieved!from!storage!at!W80°C!and!mixed!with!the!DNA!at!this!calculated! concentration.!These!mixed!samples!were!left!on!ice!for!5!minutes,!then!at!W80°C!for!a!further!five!minutes,! before!heatWshocking!at!37°C!for!a!final!five!minutes.!1ml!of!LB!Agar!at!37°C!was!added!to!each!tube!and!this! was!left!in!the!28°C!shaking!incubator!for!2!hours.! ! 5µl!Gentamycin,!50µl!Rifampicin,!25µl!Spectinomycin!and!50ml!LB!Agar!(melted!then!left!to!stand!until!it! reached!37°C)!were!added!to!each!of!four!50ml!Falcon!tubes!for!the!PB7FWG2M!culture.!A!second,!identical! set!of!four!tubes!was!prepared!with!the!Spectinomycin!substituted!for!50µl!Hygromycin!and!25µl!Kanamycin.! 25ml!of!this!media!was!then!poured!into!each!of!14!plates!(7!per!condition)!in!a!sterile!hood,!then!the!plates! were!labelled!PB1W6!(and!one!PB!control)!and!PG1W6!(and!a!control).!After!this!media!had!set!firm,!the! corresponding!tubes!of!cell!culture!were!removed!from!the!28°C!shaking!incubator!and!poured!in,!with!even! distribution!achieved!using!a!spreader.!Plates!were!sealed!and!incubated!at!27°C!for!2!days.! ! 50ml!of!media!with!antibiotics!in!two!50ml!Falcon!tubes!was!prepared!as!per!the!plates!above,!substituting!LB! Media!for!LB!Agar.!10ml!of!these!solutions!was!poured!into!each!of!six!50ml!Falcon!tubes!(3!per!condition);! then!the!colony!picking!technique!from!earlier!was!repeated!(for!3!of!the!6!plates),!with!a!pipette!tip!used!to! inoculate!each!tube!with!cells!from!the!corresponding!plate!colony!in!the!27°C!incubator.! ! Thus,!6!tubes!contained!3!samples!each!of!Gus!PB!or!Gus!PG;!these!Agrobacterium!cell!cultures!were!then! grown!overnight!and!glycerol!stocks!were!produced!for!longer!term!storage.! ! !

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3.5:!GUS!INFILTRATION!&!STAINING!PROTOCOL! ! Gus!and!GFP!HDEL!1!Infiltration! ! Separate!cell!cultures!for!Gus!PB!and!Gus!PG!were!grown!up,!infiltration!buffer!prepared!and!the!optical! density!of!cultures!calculated!as!per!section!3.2.!The!cell!cultures!were!then!added!to!5ml!of!infiltration!buffer,! to!make!up!final!optical!densities!of!both!0.2!and!0.02.!5ml!of!infiltration!buffer!only!was!also!added!to!a!tube;! thus!there!were!five!different!tubes:! ! GUS!PROTOCOL!CONDITIONS*! ! Label:!PB!0.2! PB!at!OD!of!0.2!in!Infiltration!Buffer! ! Label:!PB!0.02! PB!at!OD!of!0.02!in!Infiltration!Buffer! ! Label:!PG!0.2! PG!at!OD!of!0.2!in!Infiltration!Buffer! ! Label:!PG!0.02! PG!at!OD!of!0.02!in!Infiltration!Buffer! ! Label:!IB! Infiltration!Buffer!only! ! *Cotyledons!labelled!‘None’!were!not!injected!with!any!solution! ! ! Cotyledons!were!infiltrated!as!described!in!section!3.2,!with!one!syringe!used!per!condition!and!the! cotyledons!of!15!day!old!seedlings!the!target!tissue.!Two!cotyledons!were!infiltrated!per!condition,!except!for! IB,!in!which!3!cotyledons!were!infiltrated;!more!than!5!cotyledons!were!not!infiltrated!at!all;!these!were!later! labelled!‘None’.!The!plants!were!watered!and!placed!in!a!plant!incubation!chamber.! ! XoGluc!Staining! ! After!8!days,!the!plants!were!retrieved!from!the!incubation!chamber;!infiltrated!cotyledons!were!detached! from!the!stem!and!two!sections!of!the!tissue!were!extracted!from!each,!between!the!infiltration!holes,!with!a! cork!borer.!These!tissue!samples!(leaf!discs)!were!placed!individually!in!2ml!Eppendorf!tubes;!480µl!Gus! staining!solution,!(see!box!overleaf),!was!then!added!to!each!tube.!The!tubes!were!left!unsealed!in!order!to! prepare!for!vacuum!infiltration,!which!forces!the!solution!into!air!spaces!within!the!tissue,!increasing!the! surface!area!of!tissue!in!contact!with!solutes.!! !


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Gus"Staining"Solution"–"for"50ml" " 3.1ml"of"1M"Dipotassium"Phosphate"(K2HPO4)"and"1.9ml"of"1M"Monopotassium"Phosphate"(KH2PO4)"were" added"to"a"50ml"Falcon"Tube;"then"it"was"topped"up"to"the"40ml"mark"with"dH2O"and"this"PO4"buffer" solution"was"verified"to"be"ph7.0"using"a"pH"meter"(otherwise,"adjust"pH"to"7.0"using"acid"and"alkali" solutions)."When"this"solution"reached"pH7.0,"2.5ml"of"it"was"pipetted"into"a"second"50ml"Falcon"tube;"the" original"PO4"buffer"stock"solution"was"labelled"and"stored"at"room"temperature"for"future"use." " 250µl"of"0.1M"Ferricyanide"and"250µl"of"0.1M"Ferrocyanide"were"added"to"the"second"50ml"Falcon"tube," followed"by"1ml"of"0.5M"EDTA."To"this"tube,"50µl"of"Triton"X100"was"added,"before"the"solution"was" topped"up"to"the"49ml"mark"with"dH2O." " In"a"fume"hood,"25mg"of"XhGluc"(5hbromoh4hchloroh3hindolylhβhDhglucuronic"acid,"cyclohexylammonium" salt)"was"added"to"1ml"of"DMF"(dimethylformamide)"in"a"2ml"Eppendorf"tube."This"solution"was"then" shaken"to"mix"and"added"to"the"50ml"Falcon"tube"containing"the"other"solutes.""

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! Vacuum!Infiltration! ! The!2ml!Eppendorf!tubes!were!placed!in!a!rack!inside!a!vacuum!infiltration!chamber!consisting!of!a!simple!

!

clear!plastic!dome!over!a!circular!plastic!bowl.!A!vacuum!was!generated!using!water!pressure!through!a! connected!series!of!valves!and!pipes;!once!a!vacuum!was!established,!the!tubes!were!kept!in!the!infiltration! chamber!for!2!mins.!During!this!time,!they!were!visually!inspected!through!the!dome,!to!ensure!that!bubbles! were!forming!on!the!top!of!the!solution!within!the!tubes.!The!vacuum!was!then!slowly!released;!after!1! minute,!the!vacuum!was!reapplied!for!a!further!2!mins,!before!being!released!in!the!same!gradual!manner.! ! The!tube!rack!was!then!left!in!a!37°C!incubator!for!20!hours!and!at!room!temperature!for!a!further!2!hours,!in! order!to!allow!the!staining!to!develop.!Leaf!discs!were!then!temporarily!removed!with!forceps,!one!at!a!time,! from!the!tubes,!which!were!refilled!to!1ml!with!clear!70%!IMS.!! ! Note:!‘Old!IB’!Condition! " One"previous"Gus"trial"had"been"run,"which"resulted"in"no"visible"staining"across"PB"and"PG"conditions"in"either" tobacco"or"soybean"leaf"discs"at"this"point"in"the"protocol,"with"the"exception"of"1"soybean"leaf"disc."This"isolated" result"was"from"a"cotyledon"in"the"IB"condition;"thus,"this"disc"was"added"to"the"other"leaf"discs"(in"a"separate"2ml" tube)."From"this"point"in"the"protocol,"all"of"the"following"steps"incorporated"this"single"additional"leaf"disc,"which" was"labelled"as"‘Old!IB’." ! Fixing!the!Leaf!Disc!Samples! ! After!a!further!22!hours!in!the!37°C!incubator,!leaf!discs!were!removed!with!forceps!from!the!70%!IMS!and!reW immersed!in!250µl!Chloryl!Hydrate!within!2ml!tubes!for!4!hours!in!a!37°C!incubator.!The!discs!were!then!


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removed!from!this!solution!using!forceps,!briefly!dried!on!paper!towel,!before!being!placed!on!a!glass! microscope!slide.!A!fixative!containing!PVA!was!pipetted!onto!the!glass!slide!around!the!disc,!then!this!was! used!to!hold!in!place!a!5x2mm!cover!slip;!slides!were!then!labelled.!! ! Imaging!Leaf!Discs!with!Microscopes! ! Immediately!after!mounting!on!slides,!each!leaf!disc!was!individually!imaged!using!a!colour!camera!mounted! on!a!Leica!dissecting!microscope.!The!micrographs!were!taken!with!the!objective!at!minimum!magnification! (1.0x)!in!order!to!include!as!much!of!the!leaf!disc!surface!as!possible;!one!image!was!recorded!per!specimen,! with!software!parameters!remaining!constant!across!all!images.!After!5!days!at!room!temperature,!a! representative!selection!of!slides!from!each!condition!was!then!imaged!using!a!colour!camera!attached!to!a! compound!microscope!with!10x!and!5x!objectives.!The!resulting!images!were!named!according!to!the! condition!and!examined!to!determine!the!distribution!of!any!staining!at!a!cellular!level.!! ! Quantification!of!GusoStaining!with!Scanned!Images! ! 5!days!after!the!slides!were!mounted,!they!were!all!simultaneously!inverted!on!a!flatbed!scanner,!and!covered! with!a!sheet!of!white!A4!paper!to!provide!a!contrasting!background.!The!slides!were!then!scanned!at!1200ppi! to!an!RGB!image!with!dimensions!of!8381x12640!pixels!in!uncompressed!TIFF!format!(see!Figure!15).! !

! Figure"15!–!GusNstained!leaf!disc!slides!with!ruler!for!scale!on!flatbed!scanner"

! ! The!TIFF!was!opened!in!Fiji!(see!also!section!3.0)!and!an!individual!uncompressed!TIFF!was!saved!for!each! slide,!cropped!to!the!slide’s!dimensions,!to!derive!images!with!a!small!file!size,!which!were!easier!to!process!


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individually!(the!original!image!was!318.1MB!in!size).!Three!slides!were!chosen!which!varied!significantly!in! contrast,!area!of!blue!staining!and!artefacts!(such!as!a!visible!meniscus);!these!were!used!to!determine!a! threshold!for!the!hue,!saturation!and!brightness!of!the!average!blueWstained!tissue.!Threshold!levels!were! derived!by!experimentation!with!sliders,!to!avoid!collateral!selection!of!meniscus!or!unstained!tissue:! ! Table"3"

Min!value!

Max!value!(of!256)!

Hue!

111!

137!

Saturation!

54!

255!

Brightness!

0!

237!

! Each!individual!slide!image!was!then!processed!sequentially,!with!slider!values!set!to!the!thresholds! determined!above,!and!the!selected!areas!quantified!using!the!Measure!tool.!A!ruler!(seen!in!Figure!15),!was! used!to!set!the!scale!of!these!images,!with!the!crossWsectional!area!of!blueWstained!tissue!recorded!in!mm2.!! ! 3.6:!BIOLISTIC!INFILTRATION! ! Background!to!Infiltration!Protocol! ! AgrobacteriumWmediated!infiltration!was!chosen!as!the!method!of!transformation!for!this!research!due!to!its! proven!record!at!producing!fertile!transgenic!soybean!plant!lines.!However,!after!a!complete!lack!of! transformation!in!the!initial!three!months!of!this!project,!biolistic!transformation!was!attempted!using!the! LTI6b!tdTomato!construct.!The!vector!containing!this!construct!was!in!W20°C!storage!in!the!lab!and!had! previously!been!used!to!successfully!transform!Nicotiana"tabacum!plants!(Unpublished!by!J.!McKenna).! ! tdTomato!is!a!DsRedWderived!fluorescent!protein!with! excitation!at!554nm!and!emission!at!581nm!(Shaner!et!al! 2004);!this!puts!its!emission!maxima!beyond!the! wavelength!of!most!soybean!autofluorescence.!The! emission!wavelength!of!tdTomato!was!found!in! preliminary!microscopy!not!to!conflict!with!existing! strong!soybean!autofluorescence,!although!leaf!hairs! did!autofluoresce!at!this!wavelength!(see!figure!16).!! ! Preparation!of!Carrier!Particles! ! A!1.5ml!Eppendorf!tube!suspension!of!34mg!gold! particles!in!680µl!dH2O!(50mg/ml)!was!vortexed!for!2! Figure!16!–!Red!autofluorescence!of!soybean!leaf! hairs!after!excitation!with!543nm!laser!

minutes,!then!centrifuged!at!14,000!RPM!for!a!further! minute.!The!supernatant!was!discarded!and!the!particles!

were!then!washed!with!100%!ethanol,!with!the!same!vortexing,!centrifuging!and!pouring!off!supernatant! process!repeated!3!times!with!100%!ethanol,!before!a!final!cycle!where!the!ethanol!was!substituted!for!680µl!


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dH2O.!The!solution!was!then!vortexed,!while!aliquots!of!25µl!were!removed!using!a!pipette!and!added!to!a! separate!1.5ml!tube,!which!was!stored!at!4°C.! ! Aliquots!of!gold!were!resuspended!by!vortexing,!before!1.28µg!of!LTI6b!tdTomato!vector!was!added!to!each! tube!and!the!tube!was!vortexed!briefly.!25µl!of!2.5M!CaCl2!was!added!to!each!tube!and!the!solution!was! resuspended!by!pipetting!5!to!10!times;!this!step!precipitated!DNA!onto!the!gold!particles.!10µl!of!0.1M! spermidine!was!then!added!to!protect!the!DNA!from!cellular!DNAses;!the!solution!was!again!mixed!by! pipetting!and!then!vortexed!for!2!minutes!at!a!low!speed.!The!tubes!were!then!incubated!on!ice!for!30! minutes!before!centrifuging!at!5000!RPM!for!5!seconds;!following!this!step,!the!supernatant!was!poured!off! and!the!particles!resuspended!in!180µl!of!100%!ethanol.!The!tubes!were!placed!in!a!sonicator!bath!and! resuspended!to!wash,!before!pelleting!again!in!a!centrifuge!as!before!and!discarding!the!supernatant.!The! particles!were!finally!resuspended!in!100µl!of!100%!ethanol!and!sonicated!again,!before!being!stored!at!W20°C.!

! Figure!17!–!Biolistic!Particle!Delivery!System!with!unifoliate!soybean!leaf!on!specimen!platform! !


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Preparation!of!Leaves! ! Unifoliate!and!trifoliate!leaves!from!soybeans!(Glycine"max"‘Elena’)!grown!in!the!greenhouse!for!over!20!days! were!removed!at!the!base!(stem!end)!of!the!petiole!using!scissors.!12!Petri!dishes!containing!30ml!dH2O!each! were!used!to!hold!11!separate!trifoliate!and!unifoliate!soybean!leaves,!and!one!mature!leaf!from!a!Tobacco! plant!(Nicotiana"tabacum)."In!the!case!of!trifoliate!leaves,!all!three!leaves!were!left!connected,!to!minimise! damage!to!the!target,!central,!leaf.!! ! Biolistic!System!Operation! ! Prior!to!running!through!the!following!procedure!with!a!leaf!specimen,!it!is!best!practice!to!clear!the!system! by!firing!it!without!any!microcarrier!launch!disc!or!specimen!present.!The!protocol!listed!below!describes!the! steps!required!for!infiltrating!a!specimen!once!this!‘dry!run’!has!been!perfomed.! ! The!tap!on!the!helium!bottle!was!opened!and!the!pressure!set!to!1250PSI,!before!the!PDSW1000!Biolistic! Particle!Delivery!System!(henceforth!‘Biolistic!System’)!was!switched!on.!The!door!of!the!biolistic!system!was! opened!and!the!microcarrier!launch!assembly!was!pulled!out!and!a!new!mesh!and!microcarrier!disc!were! inserted!inside.!The!gold!particles!in!the!vector!solution!(see!Preparation!of!Carrier!Particles)!were! resuspended!by!vibration,!immediately!before!a!P10!pipette!was!used!to!transfer!10µl!of!the!solution!onto!the! exposed!lower!surface!of!the!microcarrier!disc.!The!solution!was!rotated!slowly!to!evenly!coat!the!gold!across! the!exposed!surface;!after!the!ethanol!in!the!solution!had!dried!for!20!seconds,!the!saucer!was!inverted!and! screwed!back!into!the!assembly.!! ! The!rupture!disc!holder!within!the!biolistic!system!was!unscrewed!and!a!new!1100PSI!rupture!disc!was!placed! into!the!holder.!The!holder!was!then!screwed!back!into!the!biolistic!system!and!firmly!tightened!using!the! biolistic!torque!wrench.!The!microcarrier!launch!assembly!was!then!pushed!back!into!the!chamber!in!the! uppermost!slot,!before!the!specimen!platform!was!pulled!out.!! ! A!Petri!dish!lid!was!placed!onto!the!circular!holder!on!the!specimen!platform,!and!a!leaf!was!inverted!onto!this! lid,!with!the!lower!epidermis!exposed.!The!position!of!the!leaf!was!then!adjusted!so!that!the!bulk!of!the!leaf! lay!in!the!centre!of!the!dish;!the!specimen!platform!was!then!pushed!back!into!the!chamber.!The!height!of!the! specimen!platform!was!switched!between!the!top!slot!(level!1),!the!second!slot!(level!2),!and!the!bottom!slot! (level!4)!over!a!number!of!firings,!in!order!to!determine!the!optimum!position!for!soybean!leaves.! ! A!separate,!connected!vacuum!pump!was!switched!on!and!the!air!evacuated!from!the!chamber!in!the!biolistic! system;!once!the!pressure!inside!reached!W13.78PSI!(W0.95!bar),!the!chamber!was!sealed.!The!helium!was!then! allowed!to!flow!into!the!rupture!disc!holder!(by!depression!of!the!‘fire’!button),!building!pressure!rapidly!to! 1100PSI!before!the!disc!ruptured.!This!released!a!blast!of!helium!into!the!microcarrier!launch!disc;!which!was!


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ejected!from!its!holder!and!impacted!the!stopping!screen,!whereupon!the!vectorWcarrying!gold!particles!were! thrust!through!the!vacuum!into!the!lower!epidermis!of!the!soybean!leaf,!as!described!in!section!2.3.! ! The!vacuum!in!the!chamber!was!slowly!released!before!the!door!was!opened!and!the!specimen!removed;!if! the!infiltration!site!was!not!visible,!the!edges!were!marked!out!using!a!permanent!pen.!The!specimen!was! then!returned!to!the!Petri!dish!containing!dH2O!and!the!lid!was!closed!and!labelled!with!the!level!at!which!the! infiltration!took!place!and!the!volume!of!vector!solution!used.!These!dishes!were!stored!overnight!in!a!plant! incubation!chamber,!and!imaged!using!a!543nm!laser!under!a!Zeiss!confocal!microscope!within!24!hours!of! particle!bombardment.! ! ! ! !

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Bleach!Protocol!Germination! ! Bleach!Protocol!Contamination! ! Cotyledon!Infiltration! ! Protoplasting! ! Gus!Staining! ! Biolistic!Transformation! ! "

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4.0:!BLEACH!PROTOCOL!GERMINATION! ! Percentage!Germination!and!Bleach!Duration! ! Of!the!450!seeds!which!were!planted!across!15!conditions,!174!successfully!germinated;!percentage! germination!was!more!than!twice!as!high!in!seeds!planted!on!MS!than!in!the!other!two!conditions.!However,!a! similar!trend!seemed!to!emerge!across!the!two!primary!mediums!–!compost!(‘soil’)!and!MS!rooting!media! (‘MS’).!This!trend!is!shown!in!figure!18,!which!also!shows!that!seeds!soaked!in!water!(0B/45W)!were!less!than! half!as!likely!to!germinate!as!dry!seeds!(0B/0W)!in!the!soil!condition.!Germination!rates!for!MS!were!measured! on!day!6;!after!this!point,!fungal/bacterial!growth!obscured!the!seeds.!Germination!rates!in!soil!were! measured!on!day!11,!after!which,!fungal!growth!began!to!damage!germinating!seeds.!The!fluctuations!in! temperature!and!sunlight!hours!in!the!greenhouse!may!have!contributed!to!the!low!germination!rate!in!soil,! plants!were!also!exposed!to!overW!and!underwatering.!However,!this!treatment!was!consistent!across!all!the! seeds!in!the!soil!treatment,!as!they!were!contained!within!one!seed!tray.!It!is!therefore!expected!that!this! would!not!affect!the!validity!of!comparisons!within!soil!or!MS!conditions!although!comparisons!between!MS! and!soil!might!be!invalid.!

! Figure"18!–!The!relationship!between!bleaching!duration!and!percentage!germination!seems!similar!across! both!the!MS!and!Compost!conditions.!Very!few!seeds!germinated!in!the!paper!towel!condition." " The!sample!size!in!the!soil!condition!was!so!low!as!to!negate!the!use!of!any!statistical!analysis;!however,!a!chiW

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square!test!was!run!to!determine!whether!or!not!bleaching!duration!had!an!effect!on!percentage!germination! in!the!MS!condition.!There!was!no!significant!difference!between!the!0B/45W!and!5B/40W!conditions!(chi2!=! 0.54!,!df=1,!ns)!or!between!the!5B/40W!and!30B/15W!conditions!(chi2!=!0.72!,!df=1,!ns).!These!were!the!most! MSc!Biotechnology"Project"Report!

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extreme!variations!within!this!sample;!thus,!no!effect!of!bleaching!duration!was!found!on!percentage! germination.! " Seedling!Height!&!Bleach!Duration! ! The!height!of!seedlings!was!measured!at!13!and!20!days!after!planting!in!the!soil!condition;!the!distribution!of! height!across!categories!on!both!dates!is!almost!identical!(see!figure!19).!30!seeds!were!planted!in!each! condition,!but!the!germination!rates!for!these!plants!were!low,!so!the!final!data!set!(at!20!days)!represents! only!33!seedlings;!just!18%!of!the!total!planted!(some!germinated!but!then!succumbed!to!fungal!growth).!In! the!30B/15W!condition,!no!plants!germinated,!so!concluding!anything!from!the!height!data!here!may!be! contentious.!The!least!vigorous!seedlings!were!in!the!2B/43W!condition,!while!the!most!vigorous!were!in!the! 0B/0W!condition;!i.e.!those!which!had!not!been!bleached!or!soaked!at!all.!The!difference!between!these!two! conditions!falls!outside!of!the!standard!error!(as!shown!by!the!error!bars!in!figure!19),!although!the!tiny! sample!size!(for!0B/0W,!n=2)!makes!it!unfeasible!to!test!the!statistical!significance!of!this!result.!!

! Figure"19!–!Variation!in!average!height!of!seedlings!which!successfully!germinated!in!soil;!the!sample!size!of! 2B/43W!and!15B/30W!is!just!2,!making!it!difficult!to!draw!scientifically!valid!conclusions!from!these!data.! ! !

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4.1:!BLEACH!PROTOCOL!CONTAMINATION! ! " Image!Analysis!of!Contamination! ! There!was!a!very!close!similarity!in!luminance,!hue!and!saturation!between!pixels!showing!fungal!or!bacterial! colonies!and!those!showing!the!edge!of!the!dish,!reflections!and!smudges!on!the!surface!of!the!Petri!dish.!This! similarity!resulted!in!a!high!false!positive!rate!in!the!identification!of!‘contamination’!by!the!image!analysis! software!‘Fiji’.!! MSc!Biotechnology"Project"Report!

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Visual!assessment!of!contamination!showed!no!fungal/bacterial!growth!in!any!conditions!except!nonW bleached!seeds!(both!0B/0W!and!0B/45W!conditions).!The!output!from!Fiji!in!terms!of!‘contamination!surface! area’!must!be!interpreted!in!the!light!of!this!observation;!maximum,!minimum!and!average!values!for!dishes! without!visible!contamination!are!presented!alongside!percentage!contamination!in!figure!20.!Despite!the! issues!with!the!thresholding!process,!the!visible!contamination!of!the!0B/0W!seed!and!0B/45W!conditions!is! still!apparent!in!the!data.!Furthermore,!there!is!a!consistently!higher!rate!of!contamination!in!0B/0W!seeds,! compared!to!those!in!the!0B/45W!condition!(the!latter!could!be!described!as!a!45!minute!‘wash’!in!sterile! dH2O).!

Bleach'Condition' " Figure"20!–!Variation!in!contaminated!surface!area!assessed!by!image!analysis,!in!Petri!dishes!containing!18! soybean!seeds!after!a!6!day!incubation!period.!The!2!dishes!per!condition!are!differentiated!by!the!suffix!A!and! B;!dark!portion!of!bar!shows!percentage!of!Petri!dish!clear!of!contamination.!Pale!portion!shows!surface!area!of! dish!falling!within!threshold!values!of!fungal/bacterial!colony!colouration,!including!artefacts!(e.g.! condensation!was!similar!to!fungal!colony!colouration!in!30B/15W!B).! ! ! 4.2:!COTYLEDON!INFILTRATION! ! The!GFP!HDEL!1!and!2!constructs!were!infiltrated!into!cotyledons!numerous!times!across!multiple!trials,!with!

manipulation!of!many!conditions!from!acetosyringone!concentration!to!P19!presence!or!absence!(P19! supresses!RNAi)!and!optical!density!of!cell!cultures.!Each!different!trial!was!imaged!under!the!confocal! microscope,!with!over!1000!micrographs!taken!of!over!100!cotyledon!sections.!No!conclusive!evidence!of! transformed!tissue!was!uncovered!in!this!way,!although!many!micrographs!showed!interesting! autofluorescence!patterns,!which!prompted!further!investigation.!! !


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The!following!four! micrographs!show!the!typical! pattern!of!soybean!cotyledon! autofluorescence;!an!unusual! distribution!of! autofluorescence!in!the!same! tissue!and!the!appearance!of!a! transformed!tobacco!leaf!for! comparison.! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !

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Figure"21"–"Projection!of!Z!series!image!stack!of!a!soybean!cotyledon! infiltrated!with!GFP!HDEL!1.!This!micrograph!is!an!isolated!example!of! what!was!initially!interpreted!as!transformation!of!cells.!It!prompted!a! repetition!of!the!protocol,!with!later!imaging!suggesting!that!this!

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Figure"22"–"Micrograph!showing!normal!pattern!of!autofluorescence!in! control!soybean!cotyledon!cells!under!488nm!laser,!with!reflection!from! cell!walls!likely!to!be!the!source!of!the!most!intense!green!colouration." MSc!Biotechnology"Project"Report!

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! Figure"23"–"Confocal!micrograph!of!GFP!HDEL!1!Tobacco!transgenic!leaf!with!laser!(left)!and!transmitted!light! (right)!channels.!This!image!was!split!into!separate!channels!and!then!shown!split!down!the!middle,!to!make! features!of!the!leaf!more!visible;!scale!bar!applies!to!both!sides!of!this!image." ! ! ! !

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4.3:!PROTOPLASTING! ! Trial!1!(Roots,!Hypocotyls!and!Cotyledons)! ! Protoplasting!solution!removed!from!the!experimental!dishes!and!examined!under!the!Leica!stereoscopic! microscope!revealed!no!protoplasts!in!any!of!the!conditions!tested.!! ! Trial!2!(Unifoliate!&!Trifoliate!Leaves)! Protoplasting!solution!was!examined!under!both!the!stereoscopic!and!compound!microscopes;!despite! significant!bacterial!growth,!there!were!visible!protoplasts!in!all!4!conditions.!The!highest!density!of! protoplasts!were!found!in!Soybean!PPS!with!unifoliate!leaves!from!plants!grown!in!the!light!incubation! chamber.!However,!most!of!these!protoplasts!seemed!to!have!become!lysed!in!the!solution!(see!lower!right! cell!in!figure!25);!the!protoplasts!with!the!healthiest!appearance!were!in!the!Soybean!PPS/Greenhouse! condition.!

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Figure"24"h"A!single!protoplast!in!Soybean!PPS;!cell!originated!from!leaves!on!plants!grown!partially!in!the! light!incubation!chamber.!Image!taken!at!40x!magnification!with!a!Zeiss!Compound!Light!Microscope.!Note! the!abundant!bacteria!visible!in!the!PPS!solution;!these!appear!as!small!white!objects." !

! Figure"25"–"A!raft!of!protoplasts!in!Soybean!PPS;!cells!originated!from!leaves!on!plants!grown!in!the! greenhouse.!Image!taken!at!40x!magnification!with!a!Zeiss!Compound!Light!Microscope." !


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Trial!3!(Sterile!Protocol)! ! No!protoplasts!were!found!using!either!the!compound!or!stereoscopic!microscopes!in!this!trial.! ! Trial!4!(Sterile!Protocol!with!Infiltration)! ! The!apparent!rim!of!green!protoplasts!visible!during!the!preparation!of!infiltration!tubes!in!this!protocol!(see! Methods)!had!entirely!dissipated!by!the!following!day.!No!protoplasts!were!visible!after!the!Agrobacterium! infiltration.! ! 4.4:!GUSWSTAINING! ! As!mentioned!in!the!Methods!section,!an!early!trial!resulted!in!the!staining!of!only!one!leaf!disc;!the!following! table!outlines!which!species!were!infiltrated!with!which!construct!and!the!presence!or!absence!of!visible!blue! staining:! ! Species! Tobacco"(Nicotiana"tabacum)" Tobacco! Tobacco" Soybean"(Glycine"max!‘Elena’)! Soybean"! Soybean"!

Table"4" Construct! Gus!PB! Gus!PG! None!(IB!only)! Gus!PB! Gus!PG! None!(IB!only)!

Sample!Size! 8! 8! 4! 4! 4! 2!

%!Samples!Stained! 0! 0! 0! 0! 0! 50!

! ! The!second!trial!was!more!rigorous!and!followed!the!protocol!outlined!in!the!Methods!section;!the!staining!in! this!trial!was!also!visually!assessed;!the!table!below!reports!the!visible!blue!staining!after!mounting!on!slides.! All!soybeans!were!Glycine"max"‘Elena’!as!before;!in!this!trial,!the!optical!density!of!cell!cultures!in!infiltration! buffer!was!altered!between!0.2!and!0.02.! ! Species! Soybean! Soybean! Soybean! Soybean! Soybean! Soybean!

Construct! Gus!PB! Gus!PB! Gus!PG! Gus!PG! None!(IB!only)! None!(no!IB)!

Table"5! Optical!Density!(at!600nm)! 0.2! 0.02! 0.2! 0.02! N/A! N/A!

Sample!Size! 3! 3! 2! 2! 5! 5!

%!Samples!Stained! 100! 100! 100! 100! 80! 100!

! In!the!second!trial,!all!but!one!sample!appeared!to!be!stained!under!visual!inspection;!thus!the!variation!in! intensity!of!blue!staining!was!then!quantified!using!scanned!images!and!image!analysis!software.!The!chart! overleaf!illustrates!this!range!of!staining!intensity!(approximate!surface!area!of!blue!staining).! ! ! ! ! ! ! MSc!Biotechnology"Project"Report!

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! Figure"26"–"Staining!intensity!was!highest!in!leaf!discs!injected!with!Infiltration!Buffer!only;!the!level!of! staining!in!Gus!PB!and!Gus!PG!conditions!was!similarly!low,!while!leaf!discs!which!were!not!infiltrated!had! low!levels!of!staining.!The!single!stained!leaf!disc!from!the!first!trial!(‘Old!IB’)!showed!the!highest!staining! intensity!of!all!categories!–!note!the!truncated!y!axis!scale" ! ! ! !

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4.5:!BIOLISTIC!TRANSFORMATION! ! The!following!micrographs!show!tissue!from!soybean!and!tobacco!leaves!infiltrated!with!LTI6b!tdTomato! using!the!biolistic!particle!delivery!system;!controls!which!were!not!infiltrated!are!also!shown.! !

Nonoinfiltrated!Control!Soybean!at!10x!magnification! with!transmitted!light!channel! !

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LTI6b!tdTomato!Infiltrated!Soybean!also!at!10x!with! transmitted!light!channel;!note!the!green!cells.! !

! Nonoinfiltrated!Control!Soybean!at!40x!magnification!

LTI6b!tdTomato!Infiltrated!Soybean!also!at!40x! magnification;!note!the!single!green!cell! !

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Figure"27!–!Comparison!of!nonNinfiltrated!soybean!leaves!with!leaves!infiltrated!via!the!biolistic!particle!delivery! system.!Green!channel!is!560N615nm!and!red!channel!is!above!650nm;!tdTomato!emission!maxima!is!at!580nm.! Fluorescent!green!cells!are!therefore!thought!to!be!transformed!with!LTI6b!tdTomato;!note!the!concentration!of! fluorescence!in!the!membranes;!red!channel!is!chloroplast!autofluorescence.!

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40x!Soybean!Level!1!10ml!&!Level!2!5ml!


63x!Soybean!Level!2!10ml!

63x!Tobacco!Level!2!10ml!x!2!

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" Figure"28!–Confocal!micrographs!of! individual!LTI6b!tdTomato! transformed!cells!within!soybean!and! tobacco!leaves.!All!scale!bars!are!30µl! in!length;!images!have!been! brightened!and!contrast!decreased! by!the!same!values;!all!micrographs! were!cropped.!Transmitted!light! channel!has!been!excluded!for! enhanced!visibility!of!cellular! structure.!Channels!otherwise! ! allocated!as!per!previous!figure.!

63x!Tobacco!Level!2!10ml!x!2!


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Discussion! CONTENTS"of"DISCUSSION" " " " "

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Bleach!Protocol! ! Cotyledon!Infiltration! ! Protoplasting! ! Gus!Staining! ! Biolistic!Transformation! ! Conclusions!and!Future!Research! ! "

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5.0:!BLEACH!PROTOCOL! ! Germination!! !! ! Despite!the!inconsistency!in!percentage!germination!between!MS!and!soil,!the!relationship!between! bleaching/soaking!duration!and!percentage!germination!initially!appeared!remarkably!similar!across!the!2! conditions.!In!both!conditions,!percentage!germination!were!fairly!constant!between!the!0B/45!and!2B/43W! treatments,!appearing!to!drop!off!as!the!bleaching!duration!was!increased!beyond!5!minutes!(see!figure!29).! However,!Chi!Square!tests!have!shown!that!these!effects!were!either!statistically!undetectable!at!such!a!small! sample!size,!or!simply!a!misinterpretation!of!random!data.!Unfortunately!it!seems!that,!despite!the!450!seeds! planted!for!this!experiment,!a!statisticallyWsignificant!result!was!not!forthcoming.!Additionally,!it!transpired! that!AgrobacteriumWmediated!transformation!of!Glycine!max!‘Elena’!was!not!successful,!so!seed!bleaching! might!in!fact!be!of!little!value!to!regeneration!of!transgenic!plant!lines.!Sterilisation!of!leaf!tissue!may!be!of! greater!importance,!as!this!was!the!only!section!of!the!soybean!plant!to!be!transformed!during!this!research! (see!section!5.4).! ! It!is,!however,!notable!that,!even!after!seeds!were!bleached!for!30!minutes,!germination!was!not!only! possible,!but!50%!likely!in!the!MS!condition.!This!result!confirms!the!resilience!of!these!seeds!to!sterilisation,! when!they!are!grown!under!ideal!conditions.!

! Figure"29"h!No!statistically!significant!relationship!between!bleach!duration!and!percentage!germination!was! uncovered.!Note!that!no!data!was!recorded!for!Towel!germination!in!0B/45W,!2B/43W!or!15B/30W! conditions;!no!germination!occurred!in!0B/0W!condition.!Red!box!shows!conditions!with!visible! contamination!in!MS!media;!no!fungal!or!bacterial!growth!was!visible!in!conditions!outside!of!this!box." ! !

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Sterilisation! ! The!analysis!of!contamination!performed!using!the!Fiji!software!was!somewhat!ambiguous,!due!to!collateral! selection!of!nonWcontaminated!areas!during!the!threshold!process.!However,!a!visual!assessment!of!the!dishes! was!enough!to!determine!that!bleaching!seemed!to!be!successful!in!destroying!any!fungal!spores!or!residual! bacteria!present!on!the!exterior!of!soybean!seeds!(see!figure!30).!Further!trials!would!of!course!be!necessary! in!order!to!confirm!this!assessment,!but!not!a!single!bleached!soybean!showed!any!signs!of!bacterial!or!fungal! growth!under!close!examination.!It!would,!however,!be!difficult!to!quantify!exactly!how!many!unbleached! seeds!were!the!source!of!fungal!or!bacterial!colonies!in!the!MS!media!due!to!the!rapidity!of!this!growth;!the! contamination!had!blanketed!these!dishes!within!5!days!of!planting.!This!would!make!a!statistical!analysis!of! the!contamination!problematic;!however,!multiple!points!of!origin!were!visible!for!colony!growth!in!all! unbleached!dishes,!as!seen!in!figure!30.! !

0B/0W!Condition!

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2/45!Condition!

" Figure"30"–"The!spread!of!fungus!and!bacterial!colonies!across!MS!media!after!6!days;!both!control!plates! containing!no!seeds!were!clear!of!contamination" ! ! Seedling!Growth!Rate!&!Height! ! It!would!be!optimistic!to!conclude!anything!from!the!data!gathered!for!seedling!height,!due!to!the!small! sample!size!and!distinctly!unequal!distribution!of!seedlings!between!conditions.!However,!it!is!perhaps! interesting!to!note!that!average!seedling!height!varies!as!much!as!threefold!between!conditions.!As!the! vigour!of!germinated!seedlings!is!key!to!the!success!of!transformed!plants,!it!would!be!important!to!gather! further!data!on!this!before!proceeding!with!any!bleaching!duration!in!a!larger!scale!trial.! ! Optimising!the!Bleaching!Duration! ! Despite!the!scale!of!this!final!bleaching!protocol,!with!nearly!500!seeds!planted,!the!results!were!insufficient! to!recommend!a!specific!bleaching!duration.!We!can,!however,!conclude!from!the!data!that!bleaching!seeds,!


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even!for!30!minutes,!is!not!enough!to!destroy!them.!Additionally,!144!seeds!in!the!bleached!MS!condition! showed!no!signs!of!contamination!by!fungus!or!bacteria!after!6!days!growing!in!a!humid,!nutrientWrich! environment.!83!of!these!seeds!successfully!germinated;!these!results!would!appear!to!support!the!use!of!MS! rooting!medium!as!a!substrate!for!future!research!into!Glycine!max!‘Elena’.!!As!noted!in!the!Results!section,! however,!the!disparity!between!percentage!germination!in!MS!and!Soil!might!have!been!caused!by!an! inadequate!watering!schedule!in!the!Soil!condition;!direct!comparison!of!these!conditions!is!therefore!illW advised.! ! 5.1:!COTYLEDON!INFILTRATION! ! The!methodology!used!for!much!of!the!previous!genetic!modification!of!soybeans!relied!on!embryonic! infiltration!and!subsequent!growth!of!explants!on!specific!coWcultivation!media!(Liu!et!al!2008,!Zeng!et!al!2004,! Zia!et!al!2010).!Over!the!years,!the!process!behind!preparing!explants!and!the!constituents!of!the!coW cultivation!media!have!been!refined,!but!the!resulting!recommended!protocols!are!elaborate!and!timeW consuming!(Yamada!et!al!2012).!! ! A!recent!paper!by!Zia!et!al!(2011)!in!which!soybean!pods!were!directly!infiltrated!with!Agrobacterium!was!the! inspiration!behind!the!methodology!used!in!this!research!project.!Large!scale!experiments!require!the! transformation!of!many!different!plants,!in!order!to!produce!scientifically!valid,!repeatable!results;!however,! the!time!and!financial!cost!associated!with!transformation!of!soybeans!has!been!prohibitive.!The!typical! infiltration!of!cotyledonary!nodes!requires!many!complex!steps!in!sterile!conditions,!and!is!somewhat!reliant! on!cultivars!with!an!intrinsically!high!transformation!efficiency!like!‘Jack’!and!‘Williams!82’!(Yamada!et!al!2012).! Zia!et!al!(2011)!were!able!to!transform!soybeans!relatively!easily!via!pods,!but!their!method!required!growing! the!plants!to!maturity!before!infiltration.!In!this!research!project,!I!attempted!to!transform!a!tissue!(the! cotyledon)!which!is!receptive!to!infiltration!from!just!2!weeks!after!planting,!with!an!easy!methodology!for! preparation!of!Agrobacterium!and!rudimentary!growth!conditions.!Were!this!method!to!have!proven! successful,!with!high!transformation!efficiency,!it!would!have!greatly!reduced!the!cost!and!time!intrinsic!in! soybean!transformation.! ! Unfortunately,!as!described!in!section!4.2,!while!there!were!many!different!types!of!fluorescence!distribution! recorded!during!the!cotyledon!infiltration!microscopy,!no!conclusive!evidence!of!transformation!was!found.! Indeed,!most!of!this!fluorescence!could!be!quickly!dismissed!as!either!the!reflection!of!light!from!cell!walls!or! autofluorescence!during!cell!death!and!damage,!as!noted!in!Holliday!et!al!(1981).!Despite!repeated! modifications!of!the!experimental!protocol,!there!were!no!successful!transformation!events!using!this!novel! infiltration!method!with!either!GFP!HDEL!construct.!This!failure!was!sufficiently!dramatic!that,!by!two!months! into!the!investigation,!a!Gus!staining!protocol!was!instigated!alongside!the!HDEL!experiments.!Additionally,!in! order!to!determine!whether!this!soybean!cultivar!was!at!all!susceptible!to!AgrobacteriumWmediated! transformation,!a!protoplast!infiltration!technique!was!attempted.! !


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5.2:!PROTOPLASTING! ! While!protoplasting!was!established!as!a!successful!method!for!transforming!soybean!cells!in!Baldes!et!al! (1987),!this!study!required!the!use!of!an!existing!soybean!suspension!culture.!In!their!protoplast! transformation!experiment,!the!tissue!from!which!the!suspension!culture!was!produced!was!that!of!soybean! roots.!Thus,!the!first!plant!tissue!used!in!our!research!was!from!the!root;!unfortunately,!this!tissue!did!not! shed!protoplasts!when!immersed!in!the!PPS,!while!unifoliate!and!trifoliate!leaves!in!a!subsequent!experiment! produced!a!substantial!quantity!of!protoplasts!under!the!same!conditions.!After!closer!examination!under!the! confocal!microscope,!it!transpired,!however,!that!leaf!protoplasts!suspended!in!this!solution!had!mostly! become!lysed.!It!was!predicted!that!a!larger!trial!in!which!more!leaf!tissue!was!used!might!result!in!a!higher! quantity!of!healthy!protoplasts.!Indeed,!trial!4!resulted!in!a!much!larger!volume!of!protoplasts,!although!the! subsequent!attempted!transformation!of!these!cells!was!unsuccessful.! ! Agrobacterium!suspended!in!incubation!solution!was!used!to!transform!these!cells,!but!after!a!day’s! incubation,!no!protoplasts!were!found!remaining!in!suspension.!The!constituents!of!this!incubation!solution! were!based!on!a!paper!by!Mazarei!et!al!(2008),!in!which!protoplasts!were!successfully!transformed.!However,! the!subject!of!transformation!in!this!experiment!was!a!monocot!plant;!switchgrass!(Panicum"virgatum"L.).!It! would!have!been!preferable!to!continue!following!the!protocol!detailed!by!Baldes!et!al!(1987),!but!time! constraints!prevented!this.!! ! Due!to!the!dissimilarity!of!soybean!and!switchgrass,!it!would!be!difficult!to!conclude!anything!from!the!lack!of! transformation!in!this!experiment.!However,!the!susceptibility!of!Glycine!max!‘Elena’!to!cell!wall!degradation! and!subsequent!detachment!of!cells!in!the!protoplasting!solution!from!Baldes!et!al!(1987)!is!promising,!and! presents!a!potential!avenue!for!future!research.!This!is!also!noteworthy!as!there!appears!to!be!no!prior! instance!of!protoplast!generation!using!the!Elena!soybean!cultivar.! ! 5.3:!GUS!STAINING! ! The!intensity!of!the!staining!in!the!‘Old!IB’!leaf!disc!from!the!first!trial!was!made!more!visible!after!the!tissue! was!cleared!with!IMS,!and!even!more!so!after!Chloryl!Hydrate!was!applied!(see!figure!31!and!section!4.3).!This!

‘Old!IB’!

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‘No!Infiltration’!

!

‘PB!OD!0.02’!

!

!

‘PG!OD!0.2’!

" Figure"31"–!4!scanned!leaf!discs!after!treatment!with!Chloryl!Hydrate;!note!staining!intensity!of!‘Old!IB’! !!


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leaf!disc!was!the!most!intensely!stained!by!such!a!significant!margin!that!it!was!initially!assumed!to!have!been! mixed!up!during!the!first!trial!with!a!leaf!disc!from!an!experimental!cotyledon.!However,!the!second!trial,! which!was!more!rigorous,!resulted!in!a!conclusive!and!surprising!discovery.! ! ! During!this!second!GusWstaining!protocol,!it!was!found!that!95%!of!soybean!leaf!discs!were!stained!blue!when! exposed!to!XWGluc!and!subsequently!soaked!in!IMS,!then!Chloryl!Hydrate.!The!ubiquity!of!this!blue!staining,! across!both!experimental!and!control!samples!(see!Figure!32),!led!to!the!obvious!conclusion!that!this!staining! was!not!a!reliable! indicator!of! transgene! expression.! Interestingly,!the! staining!appeared! to!be!most!visible!in! leaf!discs!which!had! been!pierced!and! subsequently! infiltrated;!far!more! so!than!in! cotyledons!which!had! not!been!infiltrated! (although!the!small!

Figure"32!–!There!is!a!significantly!reduced!average!staining!intensity!in!the!four! experimental!conditions!(grey)!than!in!control!leaf!discs!infiltrated!only!with! infiltration!buffer!(black)"

sample!size!prevented!us!from!determining!if!this!difference!was!significant).!Thus,!it!might!be!interesting!to! explore!the!relationship!between!intensity!of!the!blue!staining!and!degree!of!damage!to!the!cotyledon.!! ! An!early!investigation!into!the!use!of!GusWstaining!as!a!transgene!expression!assay!found!that!there!are!natural! compounds!in!certain!plants!which!mimic!the!activity!of!the!betaWglucorinidase!enzyme!(Hu!et!al!1990).!This! activity!results!in!the!breakdown!of!betaWglucorinides,!thus!causing!a!blueWstaining!effect!which!parallels!the! effect!expected!with!expression!of!a!Gus!transgene.!Hu!et!al!(1990)!found!that!this!effect!was!particularly! intense!in!soybean!cotyledons;!more!so!than!many!of!the!other!plant!tissues!studied!in!this!investigation.!This! simple!confounding!effect!would!explain!the!high!levels!of!blue!staining!found!in!this!experiment;!however,! given!that!all!the!leaf!discs!were!exposed!to!the!same!concentration!of!beta!glucorinides,!it!is!interesting!to! note!that!experimental!leaf!discs!have!a!consistently!lower!staining!intensity!than!those!infiltrated!with!IB! only.! ! A!paper!by!Li!et!al!(2002)!might!hold!an!intriguing!explanation!for!the!reduced!Gus!staining!in!the! experimental!conditions.!The!authors!found!that!increasing!the!copy!number!of!the!Gus!gene!in!transgenic!


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plants!resulted!in!a!reduction!in!the!intensity!of!blue!staining!to!such!a!degree!that!high!copy!number! transgenic!plants!showed!no!staining!at!all.!This!study!was!supported!by!similar!evidence!from!earlier!papers! by!Hobbs!et!al!(1990)!and!Assaad!et!al!(1993),!and!Li!et!al!(2002)!believed!that!this!reduction!may!be!caused!by! geneWsilencing.!If!this!is!true,!then!we!might!expect!a!reduction!in!staining!intensity!with!successfully! transformed!cotyledons,!due!to!the!high!level!of!intrinsic!GusWlike!activity.!Indeed,!we!found!that!when! infiltration!was!attempted!with!a!tenfold!higher!concentration!of!Gus!PB,!the!cotyledon!had!an!approximately! tenfold!reduction!in!staining!intensity.!However,!it!would!be!premature!to!conclude!anything!from!such!a! small!sample!size!(n