8. CISGENIC PLANT:
“A crop plant that has been
genetically modified with one or
more genes isolated from a crossable
donor plant” (Schouten et al., 2006).
INTRAGENIC PLANT:
The intragenic concept as the
isolation of specific genetic elements
from a plant, recombination of these
elements in vitro and insertion of the
resulting expression cassettes into a
plant belonging to the same sexual
compatibility group (Rommens et al.,
2004).
8
10. Similarity and Differences
• Similarity:
• Differences:
Genes transferred are either within the species or
from related species.
Intragenesis
• P-DNA borders are
used.
• In-vitro rearrangements
are made.
Cisgenesis
• No use of P-DNA
borders
• No in-vitro
rearrangement
10
11. To appreciate CISGENESIS and
INTRAGENESIS…..
we need to understand the problems related to…..
Traditional breeding
Transgenic approach
11
12. P = Promoter
C = Coding sequence
T = Terminator
Source: Vivek., 2016
12
13. Pre-requisites of cis/intragenic plant
Complete genome sequence information of the plant.
The isolation and characterization of genes of interest
from crossable relatives.
Transformation technique
Marker free transformation/ Clean vector technology
Intragenic vectors development
13
14. 1. Website address for data base
information
Crop plant Web addresses
Grass species http://www.gramene.org/qtl/index.html
Potato http://www.scri.ac.uk/research/genetics/GeneticsAndBreeding/p
otatoes/mappingqtls
Tomato http://164.107.85.47:8004/cgi-bin/_information.pl
http://zamir.sgn.cornell.edu/Qtl/Html/home.htm
Cucurbitaceous www.icugi.org
14
15. 2. Selection, confirmation and introduction of alleles from
the breeder’s gene pool
• Examples of
traits that can be
incorporated into
a plant by either
transferring or
modifying the
expression of
native genes.
(Rommens, 2004)
15
16. 3.Methods of plant transformation
16
• INDIRECT METHOD : Agrobacterium mediated gene transfer
• DIRECT METHOD :
Particle gun method
Polyethylene glycol method
Microinjection
Macroinjection
Electroporation
Lipofection
17. 4.Clean vector technology
Aims to produce GM plants with only the gene-of-
interest .
The goal is to avoid the use or the continued presence of
antibiotic resistance genes as selectable markers.
This can be done by the following approaches:
17
18. Methods to produce marker free cis/intragenic
plant
1. Transformation without marker gene
2. Co- transformation
3. Site-specific recombination
4. Transposon based expelling system
18
19. Transformation without marker gene
Without the use of a selectable agent.
Depending on the regeneration and gene transfer frequencies,
some plantlets will be transformed.
Potato and cassava – 1-5% ( via Agrobacterium mediated
transformation)
Tobacco – 18% (via protoplast fusion)
Limited to model species and a low number of specific crop
cultivars, e.g. potato.
19
20. Co-transformation
It involves co-integration of gene of interest and a selectable
marker delivered by two separate DNA molecules and thereafter,
segregation of both in the progeny
It is based on Agrobacterium/ biolistic mediated transformation.
20
24. Methods used to produce marker-free
intragenic/cisgenic plants
24
25. Intragenic vector development
Components of intragenic vector:
1.a plant derived T-DNA like region that should contain 2 or
at least 1 T-DNA border like sequences in the correct
orientation.
2.an origin of replication(ori)
3.a selectable antibiotic resistant gene
25
26. P-DNA Approach
Functional plant analogs of the Agrobacterium T-DNA
borders.
Putative transfer DNAs from pooled DNA’s of 66 potato
accessions were isolated and PCR is done using border
specific degenerate primers.
Amplified fragment were sequenced.
Inverse PCR with nested primers to confirm the sequence of
the border like regions.
Identified sequence similarity with Agrobacterium T-DNA.
(Rommens et al., 2005)
27. Putative P-DNA both the inverted repeats flanking a 2.6-kb rice DNA
fragment share homology with T-DNA borders, especially with the
highlighted left border of Agrobacterium nopaline strains and right
border of octopine strains (shaded).
27
28. Limitations of P-DNA
• Found in some species only.
• finding such features on a single relatively short fragment in a
plant genome is rare.
• Reduced frequencies of gene transfer.
(Rommens et al., 2005)
Left border
1-2 kb apart Restriction
sites
Right border
28
29. Combines benefits of traditional breeding and genetic
engineering.
Identifying and assembling T-DNA like fragments with
functional equivalents of T-DNA border sequences.
single step without linkage drag
Without the incorporation of “foreign DNA”
29
33. )
• Objective : Improve the existing variety (Gala) for apple scab resistance
• Material : ‘Gala’ cultivar (susceptible variety) for apple scab caused
by Venturia inaequalis
33
34. Source of natural resistance
Source of natural resistance to scab diseases is known
Classical breeding - Vf resistance gene from Malus floribunda 821
(Lespinasse, 1989; MacHardy, 1996).
In this study HcrVf2 gene (homologue of the Cladosporium
fulvum resistance genes of the Vf region) has taken from ‘Florina’
cultivar.
34
36. Methodology
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
36
37. Results
• 6 Independent transformation experiments were performed
resulting in regeneration of 10 transgenic lines out of 1635
explants.
• 2 experiments failed to regenerate transgenic shoots.
37
38. • Eight transgenic lines (T7.1, T7.2, T7.3, T7.4, T8.1, T8.2, T8.3,
T11.2) showed nptIII insertion, confirming backbone integration.
Backbone integration: PCR analysis using primers specific for nptIII.
38
41. Conclusion
Cisgenic ‘Gala’ lines - HcrVf2.
DEX treatment is not impairing apple explant regeneration.
Elimination of fusion marker gene and the R recombinase gene,
occurred as expected.
Delayed negative selection with 5-FC: better recovery of cisgenic
shoots.
Overall no negative effects on the growth of the shoots.
High degree of backbone integration (80%) pose considerable
problem.
41
43. Materials used:
• Potato varieties –Atlantic (America) , Bintje (Dutch) , potae9 (Korea)
• Tested with five Phytophthora infestans isolates with varied virulence
• IPO-C
90128
• EC1
Pic9189
• DHD11 Korean
European
American
43
44. Vector
• Resistance governing genes
i. Rpi-vnt 1.1- S . Venturii
ii. Rpi-sto 1 - S. Stoloniferum
• pBINPLUS – binary vector
• pBINPLUS:Rpi-vnt1.1
• pBINPLUS:Rpi-sto1
• pBINAW2- modified form of
pBINPLUS
44
constructs
45. Detached leaf assays of transgenic
potatoes with single R gene constructs
• Non transformed Atlantic or Bintje were
susceptible to four P. infestans isolates.
45
47. Results
• 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 47
48. Functional validation of cisgenic
transformants by agroinfiltration and
resistance assays
• A. Avrvnt1.1- and Avrsto1-induced hypersensitive responses in
cisgenic transformant H43-7.
• B. Detached leaf assays for cisgenic transformant H43-7.
48
50. 50
Conclusion
• 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.
• Cisgenic upgrades of local potato varieties might even ensure food
security.
51. • Objective :
• Improving the viral resistance using RNAi-mediated
transcriptional regulation in marker free intragenic potato
without affecting plant phenotype and productivity.
51
52. Materials and methods
• Potato variety-‘Pirol NN’
• Vector- pMF1
(T-DNA was removed by VspI sites, and resulted expression
cassette was cut at AscI and SbfI sites to insert the hairpin
sequence- pWS-E1-Lhca3).
52
53. Construction of the intron-spliced-hairpin
construct for RNAi–mediated silencing
• pWS-E1-Lhca3 : vector
• Lhca3 gene promoter from
cv“Manhattan”
• rbcs1-ribulose-(1.5)-bisphosphate
carboxylase gene
• ColE1- E. coli origin of replication
• oriV - A. tumefaciens ori site
• trfA- replication protein,
• nptIII- neomycin phosphotransferase III
53
54. Molecular characterization of intragenic plants
• PCR analysis- pws 4.6, pws 10.1
• Southern blot analysis
Southern blot analysis
of EcoRV-digested DNA
isolated from
PCR-positive potato lines
using the probes specific
for the sequence
of Lhca3 gene promoter
(a) and the sequence of
eIF4E1 translation
initiation factor (b)
54
60. BIOSAFETY AND REGULATIONS
• Genetically modified
organisms (GMOs) and
the products are
regulated under the
“Rules for the
manufacture, use,
import, export &
storage of hazardous
microorganisms,
genetically engineered
organisms or cells,
1989”.
60
61. • These Rules are implemented by the Ministry of Environment, Forest and
Climate Change, Department of Biotechnology and State Governments
through six competent authorities.
61
62. Definitions in Rules, 1989
“Genome Edited cells/organisms”(GEd) living cells and/or organisms with
targeted genetic change(s) in genome.
It is used to create a wide range of genome modifications that includes:
• production of ‘nature-identical’ traits,
• production of cisgenic and intragenic plants and animals, and
• introduction of exogenous genes with minimum change in the
cell’s/organism’s genome.
“Genetic engineering” means the technique by which heritable material, which
does not usually occur or will not occur naturally in the organism or cell
concerned, generated outside the organism or the cell is inserted into said cell or
organism.
62
63. Why we need biosafety assessment for GEd ?
Currently available nucleases used for
genome editing experiments are not
completely error-free.
Therefore, biosafety assessment of
GEd organisms/Cells takes into
account :
1) Modified/introduced trait
efficacy as well as
2) The off-target effects leading to
undesirable genetic changes in
the genome and/or phenotype.
63
Point
mutations
and
deletions
Long
sequences
inserted
64. Tiered approach for the risk assessment of GEd
products / organisms
Regulatory scrutiny will be determined by:
• the kind of the genome editing tools/process used like CRISPR/Cas9
• extent of the resulting genetic change (edit),
• the characteristics of GEd organism/cells,
• un-intended changes if any
Risk categories:
• Group I - chemical/radiation mutagenesis, targeted editing using SDN-1(Site
Targeted Nucleases),ODM (Oligo Directed Mutagenesis)
• Group II - targeted editing using SDN-2
• Group III - targeted editing using SDN-3
64
65. • Group I -trait
performance data
using greenhouse or
nethouse.
• Group II - the trait
efficacy data would
be assessed on a
case-by-case basis
depending on the
crop-trait
combination.
• Group III - besides
green house/ net
house data, trait
efficacy data from
limited confined
field trials would be
required.
Draft Document on Genome Edited Organisms,DBT, 2020
65
66. • Regarding regulation of genome engineering technologies, there is still a
debate in our country.
• The definition of GE technology is very broad based and include
“modification, deletion or removal of parts of heritable material”.
• This implies that all new technologies (cis/intragenesis) will be subject to
regulation under provision of Rules, 1989.
• It is still under examination of regulatory agencies and appropriate
Guidelines and Standard Operating Procedures(SOPs) to be drafted.
66
67. ITALY:
• On May 18, 2018, MIPAAF (Italian Ministry of Agricultural, Food, and
Forestry Policies) approved a three-year sustainable agriculture research plan
‘BIOTECH’.
• This research focuses on genome editing and cisgenesis for grapevine, olive,
apple, citrus fruit, apricot, peach, cherry, strawberry, kiwifruit, eggplant, tomato,
basil, artichoke, wheat, rice, and poplar trees.
• In 2021, after the completion of the project, a decision will be taken about the
plant generated by BIOTECH, based on the legislation on force . 67
68. CANADA:
• Follows unique approach based on “Novel traits”
• Canadian Food Inspection Agency (CFIA)
• Novelty is the major trigger for regulatory oversight in Canada and not the
method used to introduce it.
• A herbicide-tolerant variety of canola was developed based on genome
editing oligonucleotides by Cibus Global, a San Diego based company -
approved by the CFIA, is in market from 2016.
• Gen-1 potato with reduced bruising and black spots and reduced asparagine
produced by RNAi silencing has been approved by CFIA for release in
2016.
• Cisgenic plants with ‘Novel Traits’ have to demonstrate that they are
environmentally safe and do not cause any deleterious effect on human and
animal health.
68
69. Cisgenic cultivar registration:
• Establishment of the superiority of the cisgenic cultivar over
the non-cisgenic based on field trials for crop production
traits
• Its impact on food/feed in regard to health effects should be
tested.
• Results are then submitted to the Variety Registration
Office (VRO) of the CFIA.
Ajjamada et al. 2016
69
71. Current status of cis/intragenic crops
• In most countries, the release of cisgenic or intragenic crops
currently falls under the same regulatory guidelines as
transgenic crops.
• The greatest expression of interest for less stringent
regulations of these crops has been within the USA and New
Zealand.
71
Apple production highly impaired by apple scab disease caused by v.en .
Many researches have come up with many resistant varieties but the incorporated genes were exogenous coupled with marker genes.
Inorder to find a solution vanblaere and his coworkers came out with the development of cisgenic apple.
As apple cultivars are self incompatible and highly heterozygous – breeding produces genotypes with new and distict characters
Monoculturing- susceptibility
Established cultivars are susceptible and resistant cultivars developed could not meet up the quality characters of already established caultivars
Schematic representation of the pMF1 vector containing the HcrVf2 gene controlled by its native regulatory sequences.
The segment between the left (LB)and the right border (RB) is transferred into the plant cell, and
the segment between the recombination sites (RS) is then removed on recombinase-mediated deletion,
with the exception of one of the RS.
Rec LBD – LIGAND BINDING DOMAIN inactivates the R recombinase
The gene hcrvf2 will be isolated from florina cultivar and ligated into the vector at pacl and ascl sites of the binary vector pMF1
To verify the correct insertion the gene is isolated and sequenced
Top 4 youngest leaves from 4 week old invitro shoots from gala cv were cut and cocultivated with agrobacterium for 3 days.
Transferred into regeneration medium with kanamycin
For callus induction it was kept at 25 degree for 2 weeks in dark
Resulting transgenic lines were transferred into fresh media after every 4-5 week
Slide
By PCR results they confirmed the presence of hcrvf2 & fusion marker gene in all the 10 transgenic lines.
For marker free transformation all 10 transgenic lines were subjected to recombination and subsequent regeneration through an induction step of R recombinase activity by culturing explts on dexamethasone containing medium.
Later for identification of the marker free transformants 2 diff strategies were applied
1.Normal neg selection
2.Delayed neg selection – prevent the plants containing cod A gene
From the putative cisgenic shoots which was confirmed by neg selection 3 lines selected
Earlier breeding for late blight resistance was done by introgressing R genes from S.demissum – there was rapid breakdown of the resistance.
Therefore combination of R genes from diff spectra came into the thought of researchers
Sarpomira a variety with 4 R genes with durable resistance was released but it failed bcz of no market value as farmers prefer already established varieties.
Transgenic atlantic and bintje with rpivnt1.1 were suscept to EC1 While that of Potae9 was resist to EC1
Those with rpisto1 were sucptible to 99189 but they were resistant to the other strains
So these 2 genes were combined as a cisgenic R gene stack in selected varieties.
19 lines were transferred to greenhouse for phenotypic characterization of the disease
After 3 weeks 5 showed abnormalities
14 lines were tested for their response to Avrvnt1 and avrsto1 after agroinfiltration
AGROINFILTRATION: it’s a methid to induce transient expression of genes in the transformed plant/leaf in order to produce a desired protein
PVY is most harmful viral disease
Severe cases leads to 50-100%yield loss
Classical control practices for the vector control + use of certified high grade potatoes provided only some amount of resistance
Therefore Breeding for viral resistance at genetic level was a promising approach
100 nodal explants
224 plantlets – were collected
For simplifying the search 8plantlets were pooled into I culture vessel
DNA isolation from pooling the samples from each culture vessel
PCR Analysis-Analysis of samples through amplification of chimeric union of left arm of hairpin construct with rbcs intron
Presence of intragenic plts in 4th and 10th vessel
DNA isolation from each individual plants in 4th and 10t vessel and PCR
2 independent events pws4.6, pws10.1 found positive and confirmed the presence of hairpin construct in both the lines
To verify the expression level of the 2 genes
To assess whether the reduction in m RNA of these 2 genes was because of RNAi silencing , the accumulation of hairpin derived si RNA was analysed by Northern blotting
Genome editing is a precise molecular method of mutation leading to deletion or addition or substitution of target base pair(s) in the native genes/ nucleic acid sequences. On the contrary, GE organisms (also known as GMOs/LMOs) typically contain foreign genes or DNA (with/ without prior knowledge of genome structure and function) derived from related or unrelated organisms to modify an existing trait or introduce a new trait. In addition, genome editing also facilitates the introduction of a foreign gene(s) to introduce a new trait(s), which is similar to GE organisms, but the site of integration is predetermined in GEd organisms unlike in GE organisms where site of foreign gene integration in the genome is random.
