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CISGENESIS ANDINTRAGENESIS :NEW TOOLS

IN CROP IMPROVEMENT

Presented By:

SAURABH PANDEY

PALB-3!

Sr" M"S#"$A%"&

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Seminar Flow

Introduction

Why Cisgenesis/Intragenesis

Pre-requisites of Cisgenesis/Intragenesis approach

Method to develop Cisgenic/Intragenic plant

Crops and traits currently modified y theCisgenesis/Intragenesis

Case study Conclusion

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Intrd2#t0n

! concept named cisgenesis was introduced y"ochemsen and Schouten in #$$$ in the oo%&'oetsen en egren(en) *enethische enpolitie%e eoordeling van de moderneiotechnologie+)

,Cisgenesis is the introduction of isolatedgenes with their native promoters fromcrossale species or from the crop plant itself

.Schouten 0)") et al)/ #$$12)

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Intragenesis

3y 4ommens in #$$5)

allows for the design of cassettes comining

specific genetic elements from plants elonging to

the same se6ually compatile gene pool)

,Intragenesis refers to 7M8s where the

introduced intragene also originates from the

same species or a crossale species ut in

contrast to cisgenes intragenes are hyrid

genes which can have genetic elements from

di erent genes and loci . Rommens et al.,20072)

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Holme et al., (2013)

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9ifferent gene pools for plant improvement

(Michelmore,2003)

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Cisgenesis/Intragenesis as !lternative approach

Some plant spp) difficult to reed y classical method

e)g) woody plants- don+t flower for many years intolerant

to inreeding highly hetero(ygous)

Some plant spp) are naturally sterile / are part of a highly

desired and commercially widespread clone whose genotypeneeds to remain intact)

e)g) potato apple grape and anana)


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:stwe need to understand the

prolems related to;

:) 'ransgenic approach

#) 'raditional reeding and

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'ransferred gene usuallyderives from an alien species)

*6tends the gene pool of therecipient species)

Such a novel gene might provide

the target plant with a newtrait that neither occurs in therecipient species in nature norcan e introduced throughtraditional reeding)

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What is the problem with transgenesis?

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,

B+#ter0+

An0,+/s V0r2se

s

Et#"

3)t

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In recipient species fitness may change in

various ways=

'hrough gene flow etween a 7M crop andits wild relatives potentially creating shifts

in natural vegetation)

'he generation of these new &unnatural+

gene cominations is regarded as oth

unethical and having potential long-term

ris%s for health and environment).non-

targeted organisms/soil ecosystems2

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Den Nis et.al., 200!

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"ene #low #itness o#pop$lation.

Plant%ingdom

3acteria

!nimals8ther

sources

>ocalpopulation

"%N% &'(W

ITNESS O THE POPULATION

Targetplant

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Concerns

Inse#t res0st+n#e

S2*er ;eeds

P//en dr0.t

H+r, t ;0/d/0.e

H+r, t s0/

H+r, t 12,+n 1e+/t1

H0dden +//er%ensRe/0%02s +nd ,r+/#ns0der+t0nsAnt0

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'(')! De*+rt,ent . P/+nt B0te#1n/%y )!

Nochangein#itness

No ris)*on

non targetorg.,ecos+stem

No alterin genepool

Noaitional

traits inrecipientspp.

Transgenesis-isgenesis

ow cisgenic plants can oercome problemso# transgenic plants?

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0igh-quality genotype .cultivar2 ? wild plant.gene of interest2)

'he wild plant passes genes of interest to theprogeny ut also deleterious genes)

'his &lin%age drag+ tremendously slow down the

reeding process esp) if the gene of interestis genetically tightly lin%ed to one or moredeleterious genes)

$alit+ o# crop is r$ine)

To re$ce lin)age rag need successivegenerations of recurrent ac%crossing with thecultivated plant and simultaneous selection forthe trait)

What is the problem with introgression breeing?

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!s apple cultivars are self-incompatile and highly hetero(ygous

the phenotype of a cultivar is unique and reeding produces

genotypes with new and distinct characteristics)

>imited Popularity of the new cultivars carrying disease resistance

genes since originality of cultivar lost)

.7ardiner et al) #$$@2

-ont..

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-omparable with

traitionalintrogressionresistancebreeing $singsame gene pool.

%nhance thebreeingspee toobtain$rable

m$ltigenicresistance

1ingle stepinsertion

omestication o#nat$ral R geneslin)age rag

#ree.

Introgressionbreeing

-isgenesis

ow cisgenesis can oercome problems o# introgressionbreeing?

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Chromosome pairing and recomination in common wheat is largely

governed y the gene Philocated on the long arm of chromosome A3

which ensures that only homologous chromosomes can pair and

recomine)

Sears .:BA12 used ioni(ing radiation treatment to induce chromosome

rea%s and transferred a gene conditioning resistance to=

leaf rust caused y Puccinia recondita f) sp) tritici from

!e) umellulata hu%) to wheat)

Induced 'ranslocation reeding

Rile+ -hapman,34561ears ()amoto, 34561ears, 3478

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4adiation treatment causes random chromosome rea%s)

'he maDority of translocations resulting from radiationtreatments were formed etween non-homologous chromosome

arms)

'hese non-compensating translocations are geneticallyunalanced and lead to reduced agronomic performance)

'he radiation-induced Sr#1 transfer derived from !)

elongatum the translocation causes a reduction of aout :$E in

yield


MaDor prolem in induced translocation reeding)

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Particularly efficient method for cross-fertili(ing

hetero(ygous plants that propagate vegetatively such as

potato apple and anana)

Cisgenesis might also supplement classical reeding for

improving traits with) limited natural allelic variation in

cultivars and wild species

*)g) e6pression of an endogenous phytase gene in arley

through the insertion of e6tra gene copies of the

endogenous phytase gene isolated from arley itself

Importance o# -isgenesis

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Prerequisite of Cisgenic/Intragenic approach

Sequence information of the plant)

'he isolation and characteri(ation of genes of

interest from crossale relatives)

Intragenic vectors

P-9!Chimeric right '-9! order


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Web site aress #or ata base in#o..

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Minimum requirements are=

plant-derived '-9!-li%e region containing order

sequence and a multiple cloning site 8rigin of replication

Selectale mar%er

.maintenance of vector in *) coli and !groacterium2

.Conner et al)/ #$$@2

Intragenic ectors

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Plant-derived '-9!-li%e region

referred to as P-9!.

Identification of a

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8nly the first

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Plant derived acterial ori=

'he smallest %nown pro%aryotic origins of replication are the

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&ig

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ot contain regulatory elements such as promoters

ot e derived from the heterochromatic regions

recommended that a significant length of :-#% ofintragenic 9! occurs outside the left order

should comprise of a minimal numer of genetic elements=mimic naturally occurring 9! rearrangements in plant

genomes)

selectale mar%er genes= Plant endogenous genes conferringresistance to hericides or antiiotics

-onsierations #or proper esign o# intragenic ectors

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'echniquesused ingene rationofCisgenic/I ntragenic plant

'echni ques usedi n generati onofCi sgeni c/Intrageni cpl ant

Mar%er free transformation

sing super virulent strains of !groacterium=

.0igh amylopectin potato G de Hetten et al)/ #$$

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%bin$ma et al.,92003: =lant cell reports

Co-transformation .Seed e6pression of arley phytase gene G 0olme et al)/ #$:#2

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4ecominase induced e6cision

(Apple scab resistance Joshi et al., 2011)

*6cision y homologous recomination

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'ransposan ased e6icision

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*6amples of mar%er free method usedin cisgenesis


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'echnique used= # independent regeneration step with :inary vector

'ransformation with stale integration using positive selection e)g) on%anamycin .nptII2

4emoval of mar%er y chemical induction of 4ecominase 4 activity

. 9ecamethosone treatment2

Selection for mar%er free plants using negative selection .cod!2 on A-Flurocytosine .to6ic A-Fluro uracil2

Method to develop Cisgenic/ Intragenic plants

1chaart et.al.,200!

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Cisgenic plants are produced y the same transformationtechniques as transgenic plants)

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Construction of vector

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-lean ector s+stem

T-DNA insert in transgenic line

RB

RB

RB

LB

LB

LB

HcrVf2

HcrVf2

HcrVf2

RS

RS

RS

RS

RS

R-LBD Recombinase

R-LBD Recombinase

prom

prom

prom

term

term

term

CodA-NptII fusion

CodA-NptII fusion

Recombination

T-DNA insert after cisgenic line

(8

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Crops and traits currently modified y the

Cisgenesis/Intragenesis

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Type Gene Trait Author

Potato Epression R-genes !ate bli"htresistance

Ha#er$ort et al%&(200')

Apple Epression HcrVf2 cab resistance anblaere et al%&(2011)

Grape#ine Epression VVTL-1, NtpII *un"al +isease

resistance

,he$ney et al%&

(2011)

Poplar -#erepression Genes in#ol#e+in "ro.th&PAT

,i//erent "ro.thtypes

Han et al., (2011)

arley -#erepression HvPAPhya mpro#e+ "rainphytase acti#ity

Holme et al%&(2012)

,urum .heat Epression 1y10 mpro#e+ ba$in"uality

Ga+aleta et al.,(200)

Holme et al., 2013

-isgenesis

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Ty*e Gene Tr+0t A2t1r

Pt+t S0/en#0n% GBSS H0%1 +,y/*e#t0n de Vetten et al"> $883&

Pt+t S0/en#0n% Ppo Pre?ent0n% $8)&

Str+;$88(&

A/.+/.+ S0/en#0n% Comt Red2#ed /e?e/s ./0%n0n

Wee9s et al"> $886&

Perenn0+/

rye%r+ss

O?ere@*ress0n Lpvp1 Dr2%1t t/er+n#e B++ et al., $886&

A**/e E@*ress0n HcrVf2 S#+< res0st+n#e s10 et al.> $8))&

Intragenesis

Holme et al., 2013

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Field trials with Intragenic/Cisgenic crops

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C+se St2dy

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Materials and Methods

Potato Harieties= !tlantic 3intDe PotaeB Phytophthora infestans and late light resistance

tests=

Five P) infestans isolates were used-the *uropean

isolates IP8-C and B$:#JK 'he !merican isolates*C: and picBB:JBK and 'he Lorean isolate 909::)

9etached >eaf !ssays were used

Introduction method= 'ransformation y mar%er-freeinary vectors.p3I!W#2 and susequentregeneration in medium without selective antiioticsfollowed y PC4-ased selection of transformationevents)


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!groacterium tumefaciens strain used= !7>: vir7

'-9! si(e= :: % of 4pi-vnt :=4pi-sto :

Potato transformation= Mar%er assistedtransformation.%anamycin mediated2

Mar%er-free transformation

. 4emoval of a selectale mar%er gene= y site specific

recomination2

Functional tests of resistance.42 genes= !groinfiltration

9! e6traction and PC4= 7enomic 9! isolation y followingFulton et al)/method).:BBA2

PC4 reaction= B5$C for 1$s followed y

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AGROINILTRATION

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4esults and 9iscussion=

Selection of two cisgenic events in !tlantic five in3intDe and one in PotaeB containing and functionallye6pressing a stac% of two late light 4genes)

!verage mar%er free transformation frequency= :)

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'(')!

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'ale=Phenotypic characteri(ation of vector ac%one free.cisgenic2 events in different potatovarieties carrying the 4pi-vnt: and 4pi-sto : genes

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-;''%N"%1 ;ND 'I>IT;TI(N1 (& -I1"%N%1I1 ;NDINTR;"%N%1I1

8nly those of the traits in se6ually compatile genepool can e transferred to a crop)

'he gene of interest or fragments of genes may note readily availale ut need to e isolated from these6ually compatile gene pool

9evelopment of mar%er and vector ac%one free plants)

Position effect


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Summary

Cisgenesis and intragenesis have created an opportunity todiscuss aout a new group of genetically modified crops whichare &consumer friendly+)

'he author of &Invasion of 7enes - 7enetic 0eritage of India+

9r 3) S) !hloowalia said=,Cisgenics removes all fears associated with transgenic

crops)


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cisgenesis

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