Cisgenics for crop improvement

CREDIT SEMINAR

ON

Vivek Kumar Singh

Admn. No. : 2013A42D

Email: vks.slay@gmail.com

Presented by:

Department of Genetics and Plant Breeding

COA, CCS HAU, HISAR

Introduction

Cisgenesis

Why cisgenic approach

Prerequisites for cisgenic approach

Methods to develop cisgenic plant

Case study

Limitations

Future trends

Conclusion

Concept of cisgenesis introduced by Dutch researchers Schouten,

Krens and Jacobsen (2006).

According to this concept, genetic material transferred to the plant should originate from the plant itself or from closely related species capable of sexual hybridization.

The gene pool available for cisgenesis is accordingly identical to the gene pool available for classical breeding.

This is in contrast to transgenesis in which genetic material can be transferred and mixed between any species.

Henk J. Schouten

Frans A. Krens

Evert Jacobsen

Cisgenesis

Schouten et al. (2006) definition of ‘cisgenic plant’:

“A crop plant that has been genetically modified with one or more genes (containing introns and flanking regions such as native promoter and terminator regions in a sense orientation) isolated from a crossable donor plant”.

i.e.: It has all the necessary regulatory elements of a natural gene (cisgene). (Espinoza et al., 2013)

• Examples: - Cisgenic apple which confer scab resistance (Vanblaere et al., 2011)

- Cisgenic barley with improved phytase activity (Holme et al., 2012)

Cisgenesis report, 2012

Transgene: gene from outside the sexual compatible group Could be from any organism May contain marker genes of any origin for selection

Intragenics: Gene, regulatory elements and components from the plant itself or from

crossable species Silencing approaches possible Use of plant-derived sequence for gene transfer (P-DNA) via Agrobaterium Selection markers are removed

Cisgene: Contiguous gene from the plant itself or from crossable species Gene with all native components including promoter, introns and terminator

regions Use of Agrobacterium sequence for gene transfer (T-DNA) Selection markers are removed

Major characteristics of different GM concepts

Why cisgenesis ?

Escape of foreign genes via pollen flow to natural vegetation can be a problem for transgenesis. However, in case of cisgenesis the genes are taken from wild relatives.

“Cisgenesis is as safer as conventional breeding” (EFSA journal 2012, (10) 2561.)

To overcome the problem of linkage drag

Genetic make-up of the original cultivar is preserved. Only one or few genes added.

Specially important for outbreeding, vegetatively propogated plants ( apple, potato etc.)

To appreciate cisgenesis……

1st we need to understand the problems related to…

Transgenic approach and

Traditional breeding

o Transferred gene usually derives from an alien species.

o 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.

Plant kingdom

Bacteria

Animals Viruses

Etc.

B.t.

What is the problem with transgenesis ?

Contd… .. .

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

How cisgenic plants can overcome problems of transgenic plants ?

No change in fitness

No risk-on non target org., ecosystem

No alter in gene pool

No additional traits in recipient spp.

Transgenesis

What is the problem with traditional breeding ?

o Time taking

o Linkage drag

o Plants contain undesired genetic elements, which is modified by classical breeding.

eg.- Breeding of apple for scab resistance took 40 years.

o Change vigour, generating a change in the natural vegetation.

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.

eg. - 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

Genome sequence information of the plant.

The isolation and characterization of genes of interest from

crossable relatives.

Clean vector technology

The prerequisites for cisgenesis….

Clean vector technology aims to produce GM plants with only

the gene-of-interest as newly introduced gene function without

any regulatory gene sequences.

Primarily, the goal is to avoid the use or the continued presence

of antibiotic resistance genes as selectable markers.

Four approaches to achieve this can be followed:

Clean vector technology

A. Co-transformation

Based on Agrobacterium- or biolistic mediated transformation in which a SMG and gene of interest are on separate construct.

Approaches for co-transformation:

1) Two T-DNA in separate Agrobacterium 2) Two T-DNAs carried by different replicons within the same

Agrobacterium 3) Two T-DNAs located on the same replicon within an Agrobacterium

SMGs can subsequently be removed from the plant genome during segregation and recombination

Selection schemes are required to select the stable integration on antibiotic or herbicide-containing media

B. Site-specific recombination

It takes place only between defined excision sites in the phage and in the bacterial chromosome.

Positions of the site-specific recombination in the bacterial and phage DNA are called the bacterial and phage attachment sites, respectively.

Different site-specific recombination systems:

a) Cre/loxP system (bacteriophage P1)

b) FLP/FRT system (Saccharomyces cerevisiae)

c) R/RS system (Zygosaccharomyces rouxii)

Cre/loxP system …

C. Transposon-based marker method

D. chloroplast marker gene

Explants + Agrobacterium (carrying vectors)

Inoculation

The steps for the production of marker-free GM plants:

Positive selection (Selection of transgenic material)

Recombinase Dexamethasone

Negative selection On 5- Fluorocytosine

Molecular Characterization

CASE STUDY

Development of a cisgenic apple plant

Apple Cultivar Gala is susceptible to Apple scab which is caused by the fungus Venturia inaequalis

Vanblaere et al.,2011, Journal of Biotechnology

Source of natural resistance...

Source of natural resistance to scab diseases is known .

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

(Lespinasse,1989; MacHardy, 1996)

HcrVf (Homologues of Cladosporium fulvum

resistance gene of Vf region)

Patocchi et al., 1999

Method • The idea of this technique is to develop a cisgenic plant through 2

independent regeneration steps with 1 binary vector (Schaart et al. 2004).

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

↓ Removal of marker by chemical induction of Recombinase R

activity (Dexamethasone treatment) ↓

Selection for marker-free plants using negative selection (cod A) on 5-FluoroCytosine

Vector: pMF1 (HcrVf2)

HcrVf2

HcrVf2

Results 10 transgenic lines were regenerated through selection on

Kanamycin medium.

Analysis of genomic DNA (presence/ absence of gene):

8 out of 10 lines have backbone integration (nptIII)

210 bp

Fig: Backbone integration, PCR analysis using primers specific for nptIII to detect backbone integration

Genomic DNA analysis

HcrVf2

Cod A

marker gene

Npt III (backbone)

226 bp

856 bp

210 bp

3 transgenic lines 2 lines without backbone

integration and 1 line with

backbone integration as control

3 derived cislines Florina, classical bred Vf cutivar

Gala, scab susceptible cultivar

Gene Expression analysis by PCR

HcrVf2

856 bp

226 bp

Cod A

marker gene

Development of late blight resistant potatoes

by cisgene stacking. Jo et al., 2014, BMC Biotechnology

Introduction:

• Phytophthora infestans, causing late blight in potato.

• The introduction of multiple resistance (R) genes with different spectra from crossable species into potato varieties is required.

Plant Material: Atlantic (America),

Bintje (Dutch) &

Potae9 (Korea)

Vector construct

R genes: Rpi-sto1 – Solanum stoloniferum & Rpi-vnt1.1 - Solanum venturii

Results

Table-2: Marker-free transformation of two R genes (Rpi-vnt1.1:Rpi-sto1) to different potato varieties; Marker-free transformation frequencies

Variety explants shoots PCR frequency % bbf bbf %

Atlantic 200 497 0/0/12 2.4 9 75

Bintje 200 590 2/0/6 1.0 5 83

Potae9 200 428 0/0/7 1.6 5 71

total 600 1515 2/0/25 1.7 19 76

PCR - Rpi-sto1, Rpi-vnt1.1, or both genes

bbf - number of vector backbone free events; % bbf - percentage of vector backbone c carrying both Rpi-sto1 and Rpi-vnt1.1

Conclusion: The susceptibility or the narrow late blight resistance spectra of the selected varieties were upgraded to broad spectrum resistance after the successful introduction of two cisgenic late blight R genes.

The cisgenic potatoes, will offer a safe, eco-friendly.

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Limitations

• Characters outside the sexually compatible gene pool cannot be introduced.

• Sequence information of the plant.

• Low transformation efficiencies to create large number of transformants.

• The production of marker free plants usually requires the development of innovative protocols, since such protocols may not be readily available for the crop.

Future trend

Future developments regarding the generation and commercialization of intragenic and cisgenic crops will depend on application of less stringent regulation to these crops worldwide.

Cisgenic crops are acceptable to more number of people than transgenic crops

Conclusion

Despite success, the Classical methods of alien gene transfer have disadvantages and difficulties, particularly linkage drag, that require time-consuming backcrosses and simultaneous selection steps.

It is a single-step gene transfer without linkage drag

Specific: only wanted alleles inserted

Stacking of (resistance) genes is more feasible

Existing varieties can be improved directly using genes from the gene pool of breeders

Preferred by consumers compared to transgenics

Cisgenesis is a powerful alternative: