Welcome

Cisgenesis strongly improves Introgression and Induced

Translocation breeding of plants.

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Introduction

Why cisgenic approach?

Prerequisites for cisgenic approach

Method to develop cisgenic plant

Conclusion

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Concept of cisgenesis introduced by Dutch

researchers Schouten, Krens and Jacobsen

(2006)

How can we go for genetic improvement?

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Cla

ssic

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GMO REGULATION

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PLANT VARIETIES

Modern insights

&techniques including

biotechnology

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Overview of existing Technologies

Key terms

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• Cisgenesis is the genetic modification of a recipient plant with a natural gene (includes its introns and is flanked by its native promoter and terminator in the normal sense orientation) from a crossable sexually compatible plant

• A cisgene is a natural gene, coding for an (agricultural) trait, from the crop plant itself or from a sexually compatible donor plant that can be used in conventional breeding.

Contd...

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The gene pool for cisgenesis is identical to the gene pool

available for classical breeding.

Cisgenic plants can harbor one or more cisgenes, do not

contain any transgenes.

No foreign DNA, such as selection marker genes and vector-

backbone sequences, should remain in the final cisgenic

plant.

The author of ‘Invasion of Genes - Genetic Heritage of India’ Dr B. S. Ahloowalia said:

“Cisgenics removes all fears associated with transgenic crops.”

Contd…

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• Transgenesis is the genetic modification of a recipient plant with one or more genes from any non-plant organism, or from a donor plant that is sexually incompatible with the recipient plant.

• This includes gene sequences of any origin in the anti-sense orientation, any artificial combination of a coding sequence and a regulatory sequence, such as a promoter from another gene, or a synthetic gene.

Contd…

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• Traditional or conventional breeding: uses all classical and modern insights and techniques, including those related to biotechnology, however, without genetic modification.

• It includes a number of techniques that are not regarded as genetic modification or that are exempted from the GMO-legislation.

• Particularly the European Directive 2001/18/EC on the deliberate release of GMOs into the environment

(European Parliament, 2001)

Contd…

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• Translocations or interchanges: structural changes in chromosomes, where segments of non homologous chromosomes have exchanged positions.

Why interchanges important for plant breeders, geneticists & evolutionists?

Interchanges bring about changes in linkage relationship, changes in chromosome structure & behavior by which variability is created.

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Traditional breeding 

Why cisgenic approach?

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Some plant spp. difficult to breed by classical method

e.g. woody plants- don’t flower for many years, intolerant

to inbreeding, highly heterozygous.

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

desired and commercially widespread clone whose

genotype needs to remain intact.

e.g. potato, apple, grape, and banana.

To appreciate cisgenesis……

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1st we need to understand the problems related to…

1.Transgenic approach

2.Traditional breeding and

3.Translocation breeding.

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Transferred gene usually derives from an alien species.

• Extends the gene pool of the recipient species.

• Such a novel gene might provide the target plant with a new trait that neither occurs in the recipient species in nature nor can be introduced through traditional breeding.

What is the problem with transgenesis?

B.t

Contd…

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In recipient species fitness may change in various ways:

Through gene flow between a GM crop and its wild relatives

potentially creating shifts in natural vegetation.

The generation of these new ‘unnatural’ gene combinations is

regarded as both unethical and having potential long-term risks

for health and environment.(non-targeted organisms/soil

ecosystems)

den Nijs et.al., 2004

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Target plant

GENE FLOW

FITNESS OF THE POPULATION

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How cisgenic plants can overcome problems of transgenic plants?

Transgenesis

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High-quality genotype (cultivar) X wild plant (gene of

interest).

The wild plant, passes genes of interest to the progeny, but also deleterious genes.

This ‘linkage drag’ tremendously slow down the breeding process, esp. if the gene of interest is genetically tightly linked to one or more deleterious genes.

Quality of crop is ruined.

To reduce linkage drag, need successive generations of recurrent backcrossing with the cultivated plant and simultaneous selection for the trait.

What is the problem with introgression breeding?

Contd..

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As apple cultivars are self-incompatible and highly

heterozygous, the phenotype of a cultivar is unique and

breeding produces genotypes with new and distinct

characteristics.

Limited Popularity of the new cultivars carrying disease

resistance genes since originality of cultivar lost.

(Gardiner et al., 2007)

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How cisgenesis can overcome problems of introgression breeding?

Introgression breeding

Induced Translocation breeding

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Chromosome pairing and recombination in common wheat is

largely governed by the gene Phi, located on the long arm of

chromosome 5B, which ensures that only homologous

chromosomes can pair and recombine.

Sears (1956) used ionizing radiation treatment to induce

chromosome breaks and transferred a gene conditioning resistance

to:

leaf rust caused by Puccinia recondita f. sp. tritici from

Ae. umbellulata Zhuk. to wheat.Riley & Chapman,1958; Sears & Okamoto, 1958;Sears, 1976;

Major problem in induced translocation breeding.

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Radiation treatment causes random chromosome breaks.

The majority of translocations resulting from radiation

treatments were formed between non-homoeologous

chromosome arms.

These non-compensating translocations are genetically

unbalanced, and lead to reduced agronomic performance.

The radiation-induced Sr26 transfer, derived from A. elongatum,

the translocation causes a reduction of about 10% in yield

The et al., 1988

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Particularly efficient method for cross-fertilizing

heterozygous plants that propagate vegetatively, such as

potato, apple and banana.

Cisgenesis might also supplement classical breeding for

improving traits with. limited natural allelic variation in

cultivars and wild species

E.g. expression of an endogenous phytase gene in barley

through the insertion of extra gene copies of the

endogenous phytase gene isolated from barley itself

Is Cisgenesis efficient method in crops? Yes…

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Sequence information of the plant.

The isolation and characterization of genes of interest from

crossable relatives.

The prerequisites for cisgenesis….

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Web site address for data base info..

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Solutions via cisgenesis

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Development of a cisgenic apple plant.

Thalia Vanblaere et.al.,2011

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earl

y sy

mp

tom

sPresent day Apple

(Malus x domestica) cultivarsare susceptible to Apple scab

which is caused by thefungus Venturia inaequalis

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Spraying fungicides in Apple orchard

Apple growers spray on an average 15 times in one season to control the disease.

Patocchi et al., 2004

Any natural resistance? Yes……..

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Source natural resistance to diseases is known .

Classical breeding has developed scab resistant cultivars, mostly by introgression of Vf resistance from Malus floribunda 821

(Lespinasse,1989; MacHardy, 1996)

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Identified and were mapped on different linkage groups of the Apple genome.

Several apple scab resistance genes

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Impact of Vf genes in conventionally bred Apple

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Only Vf locus has been positionally cloned

( Vinatzer et al., 2001)

Proved to consists of a gene cluster with fourparalogs :

1.HcrVf12.HcrVf23.HcrVf3 and4.HcrVf4 ( Xu and Korban

2002)

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Initially, only the HcrVf2 gene was tested by overexpression using the

CaMV35S promoter & also own promoter and nos terminator,in scab

susceptible Apple plants.

Only one resistance encoding gene, HcrVf2, has been isolated and proven

functional to date in cvs. Gala and Elstar.

Belfanti et al., 2004;

Szankowski et al., 2009;

Joshi, 2010.

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In Transgenic Apple, for successful transformation marker gene nptII & other regulators added.

Joshi, (2010)

But people don’t need Transgenic Apple……..

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A large proportion of the consumers in Europe view genetically modified foods as a risk to both health and the environment

(Gaskell et al., 2000)

CISGENIC

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Cisgenic plants are produced by the same transformation techniques as transgenic plants.

Construction of vector

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17434 bp

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In order to develop marker-free plants the chemically inducible recombinase system reported in strawberry was applied to apple.

Schaart et al., (2004)

Method to develop cisgenic plants

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Technique used: 2 independent regeneration step with 1 binary vector

Transformation with stable integration using positive selection e.g. on kanamycin (nptII)

Removal of marker by chemical induction of Recombinase R activity

( Decamethosone treatment)

Selection for marker free plants using negative selection (codA) on 5-Fluro cytosine (toxic 5-Fluro uracil)

Schaart et.al.,2004

Clean vector system

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

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10 transgenic lines were regenerated through selection on Kanamycin medium

8 out of 10 lines have backbone integration (nptII)

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HcrVf2

CodA marker gene

nptII (backbone)

3 cisgenic lines are derived

Florina,classical bred cv. Vf resistance

‘Gala’ scab susceptible cultivar

pMF vector

Gene expression analysis by PCR of cDNA

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Cisgenic lines Cisgenic lines

Transgenic Transgenic

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Copy number by southern Blot

1 copy of nptII in all transgenic lines

1 copy of HcrVf2 in C7.1.49 & C 11.1.53

2 copies of HcrVf2 in T12.1 (C12.1.49)

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The new concept : HEALTHY POTATO

A cisgenic GM strategy for durable resistance based on

R and Avr genes

Haverkort, A. J. et al.,2007

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Late blight of potato deadly disease in potato.

Phytophthora resistance in potato is easily broken.

A better strategy is needded for Sustainable resistance

Many R-genes are available in crossable wild species,

enabling the development of a resistance strategy

With useful molecular knowledge Avr-genes from the

pathogen.

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List of R genes and Avr genes

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Approach:

Cloning of R genes

Marker free transformation

Cisgene field tests

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Endogenous Fungal Resistance Genes

Plants have been genetically engineered with

either:

Endogenous gene 1: V. vinifera thaumatin-like

protein gene (vvtl-1) for fungal disease resistance

or

Endogenous gene 2: 2S albumin gene (alb) from

grapevine

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Genes are expressed via bidirectional promoter system

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Cisgenic Approach for Improving the

Bioavailability of Phosphate in the Barley

Grain.

Holme I B et al.,2012

leading to water pollution…….

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Two types: HvPAPhy_a and HvPAPhy_b.

HvPAPhy_a is preferentially synthesized during seed development and stored as preformed phytase in the mature grain,

HvPAPhy_b is preferentially synthesized during germination.

Therefore ,the HvPAPhy_a gene as a candidate for our cisgenic approach.

PAPhy (purple acid phosphatase)

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Phytase Activities in Cisgenic Barley Seeds

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Revealed a positive

correlation between

gene dosage and gene

expression of the

phytase gene.

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Modifying Plant Growth using GA-associated cisgenes.

POPLAR TREES

Han K et.al.,2010

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The transfer of entire native genes that play roles

in biosynthesis or signaling of gibberellic acids

(GAs), including their 5’ and 3’ proximal regulatory

regions, impart changes in growth rate and stature

in poplar trees.

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Five different cisgenes :

GA20ox7,GA2ox2, GAI1, RGL 1_1, and RGL 1_2 . GA20oxidase catalyzes the penultimate step in the

biosynthetic GA pathway, in turn promote cell division and elongation.

The other genes tend to repress or attenuate active GA actions.

a) GA degradation by GA2ox2.

b) DELLA domain proteins that attenuate GA signals.

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Plants transformed with GA20ox7 cisgene had a higher rate and frequency of regeneration of transgenic shoots during antibiotic selection.

The average stem volume of the GA20ox7 transformed plants increased by 40% compared with the transgenic (empty vector) controls.

GA20ox7 gene expression was also statistically associated with the growth enhancement.

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Thank you