Strategies to Remove Selectable Marker Genes from Transgenic Plants

11/29/14 1Dept of Plant Biotechnology


CONTENTSCONTENTS


INTRODUCTION• Transformation: “Uptake of foreign DNA by

cells.” (Lycett and Grierson).

• The transferred gene is known as a transgenetransgene and the organism that develop after a successful gene transfer is known as a transgenictransgenic.

• The expression of the transgene in transgenic may be ‘transient’ ‘transient’ or ‘stable’. ‘stable’.



Some of the marker geneS and Selective agentS uSed for Some of the marker geneS and Selective agentS uSed for plant tranSformation (# can be uSed for chloroplaSt plant tranSformation (# can be uSed for chloroplaSt

tranSformation)tranSformation)

6

Source: Sundar et al., 2008


Contd…

Need for using SMGs in Genetic transformation

• To differentiate transformed from untransformed cells.• Transformation without use of selectable markers

reduced the transformation frequency to approx. half in Potato (De Vetten et al., 2003) and up to four fold in Barley (Holme et al., 2006).

• Help in substantial reduction in the number of untransformed cells.

• It saves selection time taken to select the transformed cells when the gene that is transformed confers insect resistance, disease resistance etc.

• Sometimes use of SMG solely is enough to produce transgenic (herbicide resistant crops).


Need for SMG removal• Potential impact on transgenic crops for both food safety

and environment.• Horizontal gene transfer from GM plants back to

pathogenic micro-organisms resulting in antibiotic resistant pathogen.

• Risk of SMG transfer from GM crops to another crop or wild relative.

• Regulating sequences of SMGs can influence the expression of other transgene and endogenous gene.

• Continuous expression of marker may interfere with normal plant growth and development.

• SMG removal lead to decrease in the insert size, which in turn increases transformation frequency.


• SMGs will serve no purpose after the selection of transformants.

• Risk of transformed plants behaving as ‘voluntary weeds’ in next season.

In addition to the above mentioned purposes, SMG removal will provide additional advantages like, Same SMG can be recycled for re-use in

subsequent re-transformation events.Removal of metabolic burden from unwanted

SMG expression.


Contd…

Alternatives for the use of SMGs

Non-selectable marker gene systems (Reporter genes)Non-selectable marker gene systems (Reporter genes)

• They are important as the alternatives for use of SMGs.

• They have been used in co-transformation experiments to confirm the transgenic events where escapes are more common.

• Ex: lacZ, uidA, Luc, GFP, etc.


NoN-selectable marker geNes or reporter geNes demoNstrated iN NoN-selectable marker geNes or reporter geNes demoNstrated iN traNsgeNic plaNtstraNsgeNic plaNts


Source: Miki et al., 2004

Strategies to remove

Strategies to remove

SMGs from transgenics.

SMGs from transgenics.


Although it is Although it is possible to obtain possible to obtain transgenic plants transgenic plants without selection, without selection,

the majority of the majority of screened plants screened plants

would be would be untransformed.untransformed.


Avoiding the use of SMG• Without use of SMG, transformed plants were

distinguished from non-transformed plants using PCR.• The efficiency of the method has been tested in several

crops like Potato, Barley, Tobacco, etc.• In PotatoIn Potato, two A.tumefaciens strains (LBA4404 and

AGL0) were used for transformation without the use of any SMGs.

• Transformation frequency for putative transgenic shoots by PCR. LBA4404- 0.2% and AGL0-4.5%. Here stable transformation frequency reduced approx. by half in this system (de vetten et al., 2003).


11/29/14 Dept of Plant Biotechnology 16

Tobacco- nptII Barley

So, it was revealed that, even though it is possible to produce transgenic without using selection, it is very laborious and useful for just a few plant species.

3.4%

2.8%

90%

0.8%

Li et al., 2009Holme et al., 2006

TE= No. of successful transformants/ unit amount of DNA usedTF= No. of stable transformants/ Total no. of cells used for trnsfmtn

Co-transformation

• “Simultaneous transformation of two or more genes.”

• GoI and SMGSMG are co-transformed using different types of binary vectors. After segregation, transgenic plants with only GOI are selected.

• TYPES: 1)Two T-DNAs on two binary vectors.

2)Two T-DNAs on one binary vector.


Two T-DNAs on two binary vectors

Mixture methodMixture method: Two different Two different Agrobacterium Agrobacterium strains, one with SMG, strains, one with SMG,

and the other with GOI.and the other with GOI.

One-strain methodOne-strain method: A single : A single Agrobacterium Agrobacterium with two expression with two expression

vectors, one with SMG, and other one vectors, one with SMG, and other one with GOI.with GOI.


Two T-DNAs on one binary vector

Super binary vector methodSuper binary vector method: Two T-Two T-DNA constructs were produced on a DNA constructs were produced on a single binary vector and both T-DNA single binary vector and both T-DNA

separated by an intervening DNA separated by an intervening DNA fragmentfragment.

Twin T-DNA methodTwin T-DNA method: A small T-DNA A small T-DNA insert called ‘LB’ and ‘RB’ were used in insert called ‘LB’ and ‘RB’ were used in

the place of polylinker between GOI the place of polylinker between GOI and SMG which were already in and SMG which were already in

another set of ‘LB’ and ‘RB’.another set of ‘LB’ and ‘RB’.

Variants of ‘Two T-DNA on one binary vector’ method

DRB binary vector methodDRB binary vector method:RB1-SMG-RB2-GOI-LB.RB1-SMG-RB2-GOI-LB.

(Lu (Lu et al., et al., 2001).2001).

One T-DNA on one binary vectorOne T-DNA on one binary vector: SMG will be outside the borderSMG will be outside the border

RB-GOI-LB-SMG.RB-GOI-LB-SMG.(Huang (Huang et al., et al., 2004).2004).

Improved co-transformation strategies Improved co-transformation strategies for SMG removalfor SMG removal

Positive-negative selection with co-transformation system.

Transient positive selection of co-transformed plants. Most of the co-transformation methods require two steps:

One, co-transformation of GOI and SMG to obtain T0 transgenic plants. TwoTwo, removal of SMG by genetic segregation in T1 generation.

Not all the transgene transferred are stably integrated. In rare cases, an un-integrated T-DNA (with SMG) un-integrated T-DNA (with SMG) and a stably integrated GOI may co-exist in some cells.

Survive in selective medium for short selection time- Transient expression of SMG.

• This strategy was used to get SMG-free tobacco plants. Approx. 4.4% of SMG-free plants were recovered in T0 generation with transient expression of positive selection for a period of 2-4 weeks (Ramana and Veluthambi., 2010).


Transposition based SMG removal

• Transposons are mobile genetic elements that can jump around in the genome of an organism.

• Best characterized and widely used transposons are those of the Ac/Ds family transposons.

• The encoded transposase recognize the inverted repeat signals at both ends of the target gene (SMG or GOI)and catalyze relocation of the gene.

• GOI or SMG can be placed within the jumping sequence and eventually being excised and re-insert into other locations in the genome.


• GOI transposed GOI transposed and relocated. Can be evaluated for position effects (Cotsaftis et al., 2002).

• 10% of the Ac elements that are excised do not re-insert and therefore disappear from the genome or re-insert into a sister chromatid that is subsequently lost during somatic segregation.

• So, SMGs are transposed SMGs are transposed by placing them in between the inverted repeat signals, instead of GOI (Ebinuma et al., 2007).


Site-specific recombination mediated Site-specific recombination mediated SMG removalSMG removal

• Recombinase protein catalyzes recombination of DNA between two recognition sites.

• The outcome of recombination may be,– Site-specific excision (deletion).– Integration (addition).– Inversion.– Translocation.

Depending on the Depending on the position and position and relative relative orientation of two orientation of two recognition sites recognition sites on the DNA on the DNA molecule.molecule.

Cre-lox Cre-lox site-specific site-specific recombination recombination was first to be used for

SMG removal in Tobacco (1991).



Serine recombinase family have much longer recognition sites than tyrosine recombinase family. Based on amino acid sequence homology and mechanistic relatedness •The name derived from the conserved nucleophilic amino acid residue that use to attack the DNA.

Types of Site-specific SMG removalConstitutive expression of recombinase geneConstitutive expression of recombinase gene Transient recombinase expressionTransient recombinase expression

1)Restricted to sexually propagated 1)Restricted to sexually propagated crops.crops.2)Over-expression of recombinase gene 2)Over-expression of recombinase gene might cause abnormalities.might cause abnormalities.

High-level transient expression of recombinase proteins, without High-level transient expression of recombinase proteins, without the integration of recombinase genes into the genome. For this, the integration of recombinase genes into the genome. For this, Plant virus vectors and Agrobacterium T-DNA vectors can serve Plant virus vectors and Agrobacterium T-DNA vectors can serve as vehicles.as vehicles.Marker free Tobacco plants were derived by using Potato Virus Marker free Tobacco plants were derived by using Potato Virus X (PVX)-based vector to transiently express the X (PVX)-based vector to transiently express the CreCre recombinase recombinase gene (Kopertekh gene (Kopertekh et al., 2004et al., 2004).).Agro infiltration technique have also been used to perform Agro infiltration technique have also been used to perform Cre-Cre-mediated SMG removal in Tobacco (Kopertekh and Schiemann., mediated SMG removal in Tobacco (Kopertekh and Schiemann., 2007).2007).


Induced recombinase expressionInduced recombinase expression

Under the control of inducible promoter (Heat shock or Chemical).Under the control of inducible promoter (Heat shock or Chemical).The recombinase gene can also be designed for auto-excision.The recombinase gene can also be designed for auto-excision.This method used inThis method used in•Rice Rice (Cre-lox)- (Cre-lox)- Sreekala Sreekala et al., 2005.et al., 2005.•Tomato Tomato (Cre-lox)(Cre-lox)- Zhang - Zhang et al., et al., 2009.2009.•Tobacco Tobacco (FLP-FRT)(FLP-FRT)- Woo - Woo et al., et al., 2009.2009.

Under the control of inducible promoter (Heat shock or Chemical).Under the control of inducible promoter (Heat shock or Chemical).The recombinase gene can also be designed for auto-excision.The recombinase gene can also be designed for auto-excision.This method used inThis method used in•Rice Rice (Cre-lox)- (Cre-lox)- Sreekala Sreekala et al., 2005.et al., 2005.•Tomato Tomato (Cre-lox)(Cre-lox)- Zhang - Zhang et al., et al., 2009.2009.•Tobacco Tobacco (FLP-FRT)(FLP-FRT)- Woo - Woo et al., et al., 2009.2009.

Chemically inducedChemically induced

Auto-excision inducedAuto-excision induced

Use of ICR (IntraChromosomal Homologous Use of ICR (IntraChromosomal Homologous recombination) for SMG removalrecombination) for SMG removal


• In nature, integration of In nature, integration of bacteriophage bacteriophage λλ into into E.coli E.coli genome genome occurs by recombination between occurs by recombination between phage attachment (attP) and phage attachment (attP) and bacterial attachmant (attB) regions.bacterial attachmant (attB) regions.•Bacterial IHF and virally encoded Bacterial IHF and virally encoded Int Int are required for integration.are required for integration.•A transformation vector, pattP-A transformation vector, pattP-ICR designed containing two attP ICR designed containing two attP regions flanking regions flanking nptII, GFP nptII, GFP and and a a tms2 tms2 gene.gene.•Next to attP site, a TBS Next to attP site, a TBS (Transformation Booster (Transformation Booster Sequence) , which enhances Sequence) , which enhances homologous and illegitimate homologous and illegitimate recombination was positioned.recombination was positioned.

• In nature, integration of In nature, integration of bacteriophage bacteriophage λλ into into E.coli E.coli genome genome occurs by recombination between occurs by recombination between phage attachment (attP) and phage attachment (attP) and bacterial attachmant (attB) regions.bacterial attachmant (attB) regions.•Bacterial IHF and virally encoded Bacterial IHF and virally encoded Int Int are required for integration.are required for integration.•A transformation vector, pattP-A transformation vector, pattP-ICR designed containing two attP ICR designed containing two attP regions flanking regions flanking nptII, GFP nptII, GFP and and a a tms2 tms2 gene.gene.•Next to attP site, a TBS Next to attP site, a TBS (Transformation Booster (Transformation Booster Sequence) , which enhances Sequence) , which enhances homologous and illegitimate homologous and illegitimate recombination was positioned.recombination was positioned.

Use of NUclease(Meganulease, ZFN, TALENs)


Name of the Nuclease

Source/Components Recognition site Comments

Meganuclease Bacteria, Archae- bacteria, Yeast, etc

12-40 bp Presence of recognition sequence is a concern.

ZFNs Zinc finger (recognition domain), RENs (cleavage domain)

3-6 Zn fingers each with 9-18 bp specificity

Off-target cleavage may occur

TALENs Transcription activator like-effector (binding domain), RENs (cleavage domain)

33-34 amino acid sequences (highly conserved). Two of them highly variable (correlate with specific nd recognition)

There are chances of Off-target recognition


Introduction• Brassica juncea (Indian mustard) is an important

sources of edible oil, accounts for 12% of the total edible oil throughout the world.

• One of the major causes for significant yield loss is infestation of sap sucking hemipteran pest- Lypaphis erysimi (mustard aphid) during time of flowering and silique formation.

• Also carriers of pathogenic viruses.• Among several plant defense chemicals (α-

amylase inhibitor, lectins, RIPs, enzyme inhibitors, chitinases), Lectins play an important role in managing several important pests.


• A homodimeric mannose binding Allium sativum leaf agglutinin (ASAL) exhibits insecticidal activity against sap sucking hemipterans namely, mustard aphid, rice brown plant hopper, green leafhopper and chickpea aphid.

• Consequently transgenic expression of ASAL had been accomplished in mustard, rice and chickpea.

• Among several strategies, Cre/lox P method has been widely used for recombination mediated SMG removal.


Material and methodsMaterial and methods

• Plant material: Brassica juncea cv. B-85.

• Plant transformation vector and transformation:

pBKhgASAL- pBKhgASAL- ~ 7.0 kb ~ 7.0 kb pBK16.2-pBK16.2- ~10.6 kb ~10.6 kb

pBKhgASALpBKhgASAL

pBK16.2pBK16.2



Lane 1: Lane 1: ASAL ASAL positive controlpositive controlLane 2: Lane 2: ASAL ASAL negative controlnegative controlLane 3-8: Lane 3-8: HindIII HindIII digested digested genomic DNA from six genomic DNA from six ASAL ASAL lineslinesLane 9: Lane 9: Cre Cre positive controlpositive controlLane 10: Lane 10: Cre Cre negative controlnegative controlLane11-15: Lane11-15: HindIII HindIII digested digested genomic DNA from five genomic DNA from five Cre Cre lines.lines.

Lane 1: Lane 1: ASAL ASAL positive controlpositive controlLane 2: Lane 2: ASAL ASAL negative controlnegative controlLane 3-8: Lane 3-8: HindIII HindIII digested digested genomic DNA from six genomic DNA from six ASAL ASAL lineslinesLane 9: Lane 9: Cre Cre positive controlpositive controlLane 10: Lane 10: Cre Cre negative controlnegative controlLane11-15: Lane11-15: HindIII HindIII digested digested genomic DNA from five genomic DNA from five Cre Cre lines.lines.

Lane 1: Purified native Lane 1: Purified native ASAL ASAL protein as positive controlprotein as positive controlLane 2: negative controlLane 2: negative controlLane 3-6: Crude protein from four Lane 3-6: Crude protein from four transgenic mustard plantstransgenic mustard plants

Lane 1: Purified native Lane 1: Purified native ASAL ASAL protein as positive controlprotein as positive controlLane 2: negative controlLane 2: negative controlLane 3-6: Crude protein from four Lane 3-6: Crude protein from four transgenic mustard plantstransgenic mustard plants

Transformation and regeneration was done. Selection media containing either 30 mg/l hygromycin or 5

mg/l bialaphos were used to select calli and shoots transformed by pBKhgASAL or pBK16.2. Followed by regeneration.

• Crossing of T1 transgenic lines: T0 plants were selfed to

get T1 seeds.

Four T1 [ASAL-lox-hpt-lox] lines were crossed with four T1 [cre-bar] lines reciprocally.

F1 hybrid seeds were harvested, surface-sterilized, germinated and transferred to green house for further analyses.

Screening of transgenic plants by PCR: The analysis of T0, T1, F1 hybrid and F2 plants were carried out with the DNA extracted from mature leaf tissue of putative transformed plants as well as control plants using ASAL, hpt, Cre specific forward and reverse primers.

Southern blot analysis of Southern blot analysis of HindIII HindIII digested genome DNA from nine digested genome DNA from nine hpt hpt negative Fnegative F11 hybrid lines hybrid lines

Southern blot analysis of Southern blot analysis of HindIII HindIII digested genome DNA from nine digested genome DNA from nine hpt hpt negative Fnegative F11 hybrid lines hybrid lines

A: 362 bp ASAL sequence B: 1.1 kb Cre sequence C: 980 bp hpt sequencesA: 362 bp ASAL sequence B: 1.1 kb Cre sequence C: 980 bp hpt sequences

Expression of Expression of ASAL ASAL in Fin F11 hybrid lines hybrid lines

A: Western blot analysis of nine hpt –ve F1 hybrid mustard plants.B: ELISA analysis for expression of ASAL in total soluble protein.

A: Western blot analysis of nine hpt –ve F1 hybrid mustard plants.B: ELISA analysis for expression of ASAL in total soluble protein.

• Hemagglutination and thermal stability assay:

• Total soluble protein extracted from F1 hybrid lines containing ASAL gene.

• Blood was collected from the rabbit and dispensed into the wells of the plate.

• Stability of the protein was assessed by its ability to agglutinate rabbit erythrocytes upon incubation at different temperatures.


A: Rabbit erythrocytes were incubated with Panel I-V. Well no.1- No protein control, Well no.2- Protein from untransformed plant, Well no.3-7- TSP from ASAL expressing F1 hybrid plants.B: Thermal stability assay- Well no.1 (I-V)- No protein control, Rabbit erythrocyte incubated with ASAL positive F1 hybrid plants (Panel I-V., Well no.2-7) pre-incubated at different temperatures.

A: Rabbit erythrocytes were incubated with Panel I-V. Well no.1- No protein control, Well no.2- Protein from untransformed plant, Well no.3-7- TSP from ASAL expressing F1 hybrid plants.B: Thermal stability assay- Well no.1 (I-V)- No protein control, Rabbit erythrocyte incubated with ASAL positive F1 hybrid plants (Panel I-V., Well no.2-7) pre-incubated at different temperatures.


•In planta insect bioassay: •F1 plants bearing ASAL gene and control plants were subjected to bioassay with nymphs of L.erysimi in cages with one end open. •Survival of insects was monitored at the interval of 24 hrs for 9 days.•ANOVA followed by Duncun’s multiple range tests were conducted to compare the significance of differences among all the transgenic and control plants for the insect bioassay experiment.

Graphical representation of variation in number of aphids on each ASAL expressing progenies over a period of 9 days

Graphical representation of variation in number of aphids on each ASAL expressing progenies over a period of 9 days

Graphical representation of fecundity pattern of aphids on each ASAL expressing progenies over a period of 9 days

Graphical representation of fecundity pattern of aphids on each ASAL expressing progenies over a period of 9 days

Establishment of marker free FEstablishment of marker free F22 plants plants


SummarySummary• The average frequency of marker gene elimination-

39.5%.• Earlier studies- 26% and 29.1% in Rice (Hoa et al. and

Sreekala et al. respectively).• Transgenically expressed ASAL protein- stable at 370C• Loses its functionality at 55-750C which eliminated all the

possibility of native ASAL protein to be an allergen.• Insect mortality in the ASAL expressing transgenic

plants was found to be 70% (control- 35%).• The level of ASAL expression -vely correlated with the

fecundity of the insects.• Cre-lox system have been successfully used to establish

ASAL positive and both hpt and Cre negative F2 mustard plants.


ConclusionConclusion• Despite the large number of marker genes that exist for

plants, only a few marker genes are used for most plant research and crop development.

• However, to date, no experiment has provided any evidence that the antibiotic markers presently in use pose risks to human or animal health (ISAAA, 2014 Editorial publication).

• Besides minimizing public concerns, the absence of resistance genes in transgenic plants could also reduce the costs for developing GM products and lessen the need for time-consuming safety evaluations, thereby speeding up the commercial release of new products.



Strategies to Remove Selectable Marker Genes from Transgenic Plants

Science

plant transformation

normal plant growth

use of smgwithout use

transgenic plants

transgenic crops

transformation frequency

transgenic events

risk of smg transfer