Cisgenics, Intragenics and Site-specific Mutagenesis

Transcription

1 Cisgenics, Intragenics and Site-specific Mutagenesis K. Veluthambi School of Biotechnology Madurai Kamaraj University South Asia Biosafety Conference September 18-19,

2 An important concern of Genetically Modified (GM, Transgenic) crops Mixing of genetic materials between unrelated plants which can not hybridize by natural means Respect to Nature Perceived health risk Spreading of new gene combinations in Nature

3 Alternatives to Transgenics? CISGENESIS & INTRAGENESIS Plants must be transformed with genetic material derived from the species itself or from closely related species capable of sexual hybridization Foreign sequences such as Selectable marker genes and Vector backbone sequences should be absent

4 CISGENESIS (Schouten et al., 2006) Full CDS including introns of a gene originating from the sexually compatible gene pool of the recipient plant along with gene s own promoter and terminator are used for transformation INTRAGENESIS (Rommens et al., 2004) The full or partial CDS of genes originating from the sexually compatible gene pool of the recipient plant can be used in sense or antisense orientation. The promoter and terminator could originate from sexually compatible gene pool of the recipient plant (not necessarily from the cisgene itself).

5 CISGENESIS AND INTRAGENESIS Holme et al. (2013) Plant Biotechnol. J. 11:

6 Advantage of Cisgenesis over Conventional Breeding No Linkage Drag

7 ADVANTAGES OF CISGENESIS AND INTRAGENESIS Great potential to overcome the limitations of classical breeding The transfer of genes between sexually compatible plants can be speeded up. Linkage drag associated with conventional breeding is avoided. Tightly linked inferior traits can be completely eliminated. The exchange of genetic material is precise. In intragenesis, higher expression is achieved by using a more efficient promoter and lower expression is achieved through gene silencing.

8 Scope for exemption from Regulatory Requirements The gene pool exploited by intragenesis and cisgenesis is identical to the gene pool available for traditional breeding. Therefore, cisgenesis and intragenesis have good scope to be treated on par with conventional breeding and exempted from regulations that govern transgenics.

9 CHALLENGES AND LIMITATIONS OF CISGENESIS AND INTRAGENESIS Only those of the traits in sexually compatible gene pool can be transferred to a crop. Genome sequences responsible for the trait including the endogenous promoters and terminators should be cloned. Development of marker and vector backbone free plants.

10 Examples of Application of Cisgenesis/Intragenesis High amylopectin potato de Vetten et al. (2003) Down regulation of granule bound starch synthase gene expression by RNA silencing P GBSS GBSS nos t Has been changed to GBSS terminator in 2007 Intragenesis Profound reduction in amylose content

11 Prevention of browning of cut potato tubers Rummens et al. (2004) P DNA border P DNA border P GBSS Ubi3 t PPO (Plastidial polyphenol oxidase) Intragenesis

12 Apple Scab Resistance Fungus Venturia inaequalis Resistance gene (HcrVf2) from the wild apple Malus floribundus Vanblaere et al. (2011) Cisgenesis P CDS term Genomic clone of HcrVf2 Intragenesis Joshi et al. (2011) P SSU CDS SSU t HcrVf2

13 Grey mould resistance in strawberry Fungus : Botrytis cinerea Gene: Strawberry polygalacturonase inhibiting protein (PGIP) Schaart et al. (2004) Intragenesis P Exp2 PGIP Exp2 t Expansin fruit specific

14 Selectable marker free strategies 1. No selectable marker used for transformation (High amylopectin potato de Vetten et al., 2003) 2. Transient expression of the selectable marker gene (Browning-free potato Rommens et al., 2004) 3. Recombinase/Recombination site (Apple scab resistance Joshi et al., 2011) 4. Co-transformation (Seed expression of barley phytase gene Holme et al., 2012)

15 Co transformation and Marker Elimination Selectable marker Gene of interest T DNA border Ebinuma et al. (2001) Plant cell reports 20:

16 STRATEGY FOR SELECTABLE MARKER ELIMINATION Co transformation with single strain of Agrobacterium harbouring a cointegrate plasmid with the selectable marker (hph ) and reporter (gus) genes and a binary plasmid with the gene of interest (chi11) (pgv2260::pssj1, pcam chi11, psb1) Selection on Hyg medium (50 mg/l) 21-day-old PB callus

17 Maps of the Binary Plasmid and Cointegrate Plasmid T DNAs used for co transformation pgv2260::pssj1 ( A Cointegrate vector with selection marker and reporter genes) EcoRI LB P35S Int-gus nos 3 tml3 hph P35S RB hph probe 1.0-kb >2.7-kb pcam chi11 (A Binary vector with a Gene of Interest Rice chi11) HindIII PstI EcoRI PstI EcoRI HindIII LB chi11 PUbi1 RB chi11 probe PUbi1 probe >2.0-kb 1.1-kb 1.0-kb

18 STRATEGY FOR MARKER ELIMINATION IN THE T 1 GENERATION BY GENETIC SEPARATION Genotype of the Co transformed plant C H C chitinase gene H hygromycin resistance / gus genes

19 Genotype of Cotransformed parent C H Selfing CcHh CcHh X Gametes CH Ch ch ch CH Ch ch ch CH Ch ch ch CH CCHH CCHh CcHH CcHh T 1 progenies Ch ch CCHh CcHH CChh CcHh CcHh cchh Cchh cchh ch CcHh Cchh cchh cchh

20 Southern blot analysis of T 0 rice plants transformed with A. tumefaciens C58C1 (pgv2260::pssj1, pcam chi11, psb1) C U 1 2* * C U * * kb kb Probe: hph gene EcoRI LB P35S Int-gus nos 3 tml3 hph P35S RB hph probe 1.0-kb >2.7-kb Sripriya et al. (2008) Plant Cell Rep. 27:

21 Southern blot analysis of T 0 rice plants transformed with A. tumefaciens C58C1 (pgv2260::pssj1, pcam chi11, psb1) Probe: chi11 LB HindIII chi11 PstI EcoRI PUbi1 PstI EcoRI HindIII CoT2, 6, 20 & 23 are co transformed chi11 probe 1.0-kb 3.1-kb Sripriya et al. (2008) Plant Cell Rep. 27:

22 Segregation analysis of co transformed plants obtained by transformation with Agrobacterium tumefaciens C58C1 (pgv2260::pssj1, psb1, pcam chi11) T 0 lines Total number of T 1 plants Number of T 1 plants a GUS + a GUS - b T-DNA copy (hph) Segregation ratio χ 2 value Cointegrate T-DNA loci CoT : CoT : CoT : CoT : a Segregation based on GUS staining b T DNA copy numbers were determined by junction fragment analysis with hph probe

23 Southern blot analysis of T 1 plants of the co transformed plant line CoT23 GUS ve GUS + ve GUS ve GUS + ve kb C U kb C U hph probe kb GUS ve P C U kb GUS ve P E C chi11 probe Sripriya et al. (2008) Plant Cell Rep. 27:

24 Junction fragment analysis of T 1 plants of the line CoT23 to determine copy number of the chitinase gene C U T kb HindIII PstI EcoRI PstI EcoRI HindIII LB chi11 PUbi1 1.0-kb chi11 probe >2.0-kb PUbi1 probe Sripriya et al. (2008) Plant Cell Rep. 27:

25 Efficiency of Marker Elimination in Rice by Co-transformation Total number of independent transgenic lines Number of cotransformed lines 20 4 (CoT2, CoT6, CoT20, CoT23) Co-transformed lines with unlinked integrations (Marker-free plants) 2 (CoT6, CoT23)

26 Beyond cisgenesis and intragenesis What? Cisgenesis and intragenesis are stilll generating new risks because the insertions at random locations (ectopic) in the genome could have unpredictable pleiotropic effects The endogenous gene in the recipient plant is not altered Focus is thus shifting to Gene Targeting to achieve site-specific mutagenesis

27 Gene Targeting by Positive/Negative Selection Strategy LB RB DT A GE hph NE DT A T DNA Homologous recombination GENE Native gene locus GE hph NE Targeted gene locus Positive selection hph Negative selection diphtheria toxin A chain (DT A) Terada et al. (2002)

28 Gene Targeting of the Rice OsMADS1 gene by Positive/Negative Selection

29 Schematic diagram of Gene targeting binary vector construction for OsMADS1 gene Binary vector used for gene targeting of OsMADS1

30 a) Genomic structure of OsMADS1 in rice HindIII SII B SI B 5 3 SII SI H 5 region, 3 region Intron Exon b) Genomic structure of knock out OsMADS1 locus in rice SacII SacI BamHI 13.7kb 6.5 kb 4.5 kb BamHI SacI SacII HindIII OsMADS1 (upstream) tml3 hph p35s OsMADS1 (3 UTR) HindIII 11.5 kb 14.7 kb 11.0 kb HindIII EcoRV 4.55 kb EcoRV PstI PstI

31 Putative gene targeted (GT) rice plant Putative GT 2 callus in regeneration medium Putative GT 2 plant in rooting medium Putative GT 2 plant in greenhouse Rice transformation yielded three independent putative GT plants (Total number of calli used for infection 2643)

32 PCR analysis of putative GT plants to exclude ectopic T DNA integration hph primers DT A primers 1 kb + H20 Control ( ) Control GT 1 plant GT 1 plant (+) Control 1 kb + H20 Control ( ) Control GT 1 plant GT 1 plant (+) Control Kb Kb bp bp GT plants have the hph gene but not the DT A gene

33 Southern blot analysis with the hph probe to confirm the OsMADS1 gene disruption GT 1 HindIII C/HindIII HindIII SmaIII EcoRI EcoRV SacI SacII B.v B.v SII,SI H,EV SI,SII EV H Kb Probe hph The hph gene was integrated as a single copy in the OsMADS1 locus and no ectopic T DNA integration was observed. Enzyme used HindIII SmaI EcoRI EcoRV SacI SacII Expected fragment size (Kb)

34 Southern blot analysis of GT 1 plant with the DT A gene probe DT 5 BamHI + EcoRI c E E BamHI+ PstI B.v B.v B P B P DT A DT A Probe DT A Internal fragment size 650 bp No ectopic integration DT A gene is not found in the transgenic plants

35 Southern blot analysis to confirm the OsMADS1 gene disruption SacI SacII BamHI HindIII Probe 3 part of the OsMADS1 gene C E C E C E C E HSII SI B H B SI SII Enzymes used SacI SacII BamHI HindIII C E C E C E C E Expected fragment size (s) (Kb)

36 Southern blot analysis to confirm the OsMADS1 gene disruption HindIII EcoRI PstI EcoRV Probe 5 part of the OsMADS1 gene C E C E C E C E E H EV E P P H EV C 23.1 E H E Base change in the vector homologous sequence is efficiently transferred to the target locus. E. g. EcoRI digestion Enzymes used HindIII EcoRI PstI EcoRV C E C E C E C E Expected fragment size (s) (Kb)

37 Southern blot analysis of T 1 GT 1 plants to identify homozygous mutants BamHI Bv/ HindIII kb 23.1 C C +C +C kb kb B B Expected banding pattern: Control plant 4.95 kb Hemizygous 4.95 kb kb Homozygous 4.5 kb Plant No: 3, 5, 7, 9 and 11 were homozygous B B BamHI 4.5 kb BamHI OsMADS1 (upstream) tml3 hph p35s OsMADS1 (3 UTR) Probe 3 Part of the OsMADS1 gene

38 Future Goal Cisgenesis and intragenesis have created an opportunity to initiate a new dialogue among scientists, breeders and consumers to discuss about a new group of genetically modified crops which are consumer friendly. An essential component of the dialogue is to communicate that cisgenic/intragenic modification is restricted to a modulation of existing traits using genes from the sexually compatible gene pool and further that the plants are devoid of any DNA from other gene pools. This dialogue should help in addressing many consumer concerns and help in securing public acceptance.

39 Thank you Research support from DBT, Govt. of India is acknowledged 39