Development Of Marker Free Transgenic Plants: Biosafety Concern

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However, the methods to introduce foreign DNA in a plant cell, either by Agrobacterium, microinjection, particle gun, or protoplast transformation, are relatively inefficient. The additions of gene to the desired trait of the plant also require selection marker genes to select the transformed cells and tissues. Approximately fifty different selection systems have been developed over the past several years. Despite the large number of systems, marker genes that confer resistance to the antibiotics kanamycin (nptII) and hygromycin (hpt) or the herbicide phosphinothricin (bar) have been used in most plant research and crop development techniques. Selection markers are not required in mature plants, especially when they are grown in fields. The European Union suggests avoiding the use of selectable markers in genetically engineered crops, and the ultimate goal is to introduce as few foreign sequences, in addition to the gene of interest, as possible. Moreover, the generation of marker-free transgenic plants responds not only to public concerns over the safety of genetically engineered (GE) crops, but supports multiple transformation cycles for transgenic pyramiding.

In addition, the existence of marker genes in transgenic crops could evoke additional, lengthy risk assessments for release of crops that contain useful novel traits.

Markers that promote plant regeneration have been recently described. However, continuous expression of these markers may interfere with normal plant growth and development. Removal of this type of marker from plant tissues is necessary unless expression is under good control. Furthermore, current transformation technologies permit only the introduction of a very limited number of genes into plants. Retransformation of the same line is needed for multiple trait modifications. New selectable markers are thus needed with each transformation to pyramid the same crop variety with different desirable traits. The number of selectable marker genes that are suitable for each crop species is usually very limited. This is especially true for transformation of recalcitrant species. Marker excision can allow reuse of a marker after each transformation step. Marker elimination will not only appease some potential environmental and consumer concerns, it will also remove technical barriers for plant genetic transformation (Herrera-Estrella et al., 1983 and Bevan et al., 1983). In the recent years, concerns have been raised that the presence of such genes might be an unpredictable hazard to the ecosystem as well as to human health. For example some of the genes like Bt. Genes and herbicide resistant gene might be transferred by out- crossing into weeds; and the presence of resistance genes against antibiotic in food products might theoretically lead to the spread of these resistances via gut bacteria in humans- although there is as yet no scientific evident to support the listed statement.

The very success of antibiotics in medicine has now become a problem. Many bacteria, including pathogens of infectious diseases, are already resistant and can no longer be controlled with the particular antibiotic. These concerns have been taken seriously and various governments have initiated studies in which such scenarios are now under investigation. How ever the most elegant way to over-come all the concerns is just to remove the cause of concern - the selectable marker gene itself.

The drawbacks of traditional markers are becoming apparent even in practical research.

Two different marker gene systems are required if plants which have already been genetically modified are to be transformed again. But there are only a few available for each crop species.

If several marker genes left over from various developmental phases accumulate in a plant, the stability of the genetically engineered trait can be impaired.

The more genes and marker genes are transferred, the greater the probability of unforeseen effects occurring in the plant (Pleiotropic effect); the role of one gene is effected by the other gene..

In the next generation of transgenic plants, antibiotic-resistance markers will be the exception rather than the rule. But there is still a long way to go before sufficient new procedures and strategies. . In this updated review article the current technologies has been discussed to elimated the selection marker gene from the plant genome and this review article is very useful in terms for developing marker free transgenic keeping in mind the environmental and human health concerns.

Methods to Eliminate Marker genes from nuclear genome

Co- transformation

The co-transformation method is very simple method to eliminate marker gene from the nuclear genome. Co-transformation involves transformation with two plasmids that target insertion at two different plant genome loci. One plasmid carries a selective marker gene and the other carries gene of interest.

Basic three methods are used in the co-transformation system

two different vector carried by different Agrobacterium strains(McKnigh et al., 1987; Deblock and Debrouwer, 1991; De Neve et al., 1997). And biolistic introduction of two plasmids in the same tissue.

two different vectors in the same Agrobacterium cell (De Framond et al., 1986; Daley et al., 1998; Sripriya et al., 2008,)

The two T-DNAs can be borne by a single binary vector (2 T-DNA system) (Komari et al., 1996; Xing et al., 2000; Mathews et al., 2001; McCormac et al., 2001; Miller et al., 2002).

In all the three methods selectable marker gene can be eliminated from the plant genome at the time of segregation and recombination that occurs during sexual reproduction by selecting on the transgene of interest and not the SMG in progeny. Inspite of all these there are several limitations which is inevitable. The methods describe above is very time consuming and compatible only for fertile plants. The second is; the tight linkage between co-integrated DNAs limits the efficiency of co-transformation. Indeed integration of SMG and the transgene is at indiscriminate event, both the SMG and transgene may integrate in the same loci and that is not feasible for co-transformation. However, the overall advantages of these methods remain unclear. Most of the research paper documented the limitations of the Co-transformation methods that are limited and useful only for flowering plants but Nick de Vetten et al., 2003 developed a silencing construct (pKGBA50mf-IR 1.1) and transformed in to Potato (karnico) via. Highly virulent LBA4404 or AGL0 Agrobacterium mediated transformation without the use of selection marker gene. They have developed PCR based detection method and > 2% of the recovered shoots showed a complete gene silencing of the GBSS1 gene resulting in an amylose free phenotype. They have successfully developed a protocol which is useful for vegetative plants and also for flowering plants.

Site Specific Recombination

Recombination is very clear phenomenon in Biological system, recombination take place between the two homologous DNA molecules. In Bacteriophage site specific recombination take place between defined excision sites in the phage and in the bacterial chromosome. In site specific recombination, DNA strand exchange take place between segments possessing only a limited degree of sequence homology (Kolbe 2002; Coates et al., 2005).

Basically three site specific recombination system is well known and described for the elimination of selection marker gene.

Cre/lox site specific recombination system

FLP/FRT recombination system from Saccharomyces cerevisiae.

R/RS recombination system from Zygosaccharomyces rouxii,

The recombination sites are typically between 30 and 200 nucleotide in length and consist of two motifs with a partial inverted repeat symmetry, to which the recombinase binds and which flank a central crossover sequence at which the recombination take place (Fig. ). The unique ability of Cre to catalyze a crossover between directly repeated lox sites flanking any fragment of DNA has been exploited to remove selectable marker genes from transgenic plants. The pairs of sites between which the recombination occurs are usually identical, but there are exception e.g. attP and attB of λ integrase (Landy 1989). The simplest approach is to generate plants that express the cre gene and to cross them with plants in which the selectable marker gene is flanked by lox sites. The marker gene is excised in the F1 generation and the cre gene is segregated away in the subsequent generation. Selection marker gene can be eliminated either by re-transformation (Odell et al., 1990; Dale and Ow 1991; Russell et al., 1992) or by Crossing over (Bayley et al., 1992; Russell et al., 1992; Chakraborti et al., 2008 ). The retransformation and crossing over strategy was very labour intensive and time consuming and in both the approaches the selection marker gene is eliminated at F1 generation. The answer to the above problem was an auto excision system controlled by inducible promoter and with this system the F1 progengy is free of selection marker gene. This is very well studied in most of the agronomical important crops and successfully marker free transgenic plants were generated in Arabiodopsis, maize tobacco and rice (Hoff et al, 2001, Zuo et al, 2001, Zhang et al, 2003, Yuan et al, 2004, Sreekala et al., 2005 ). Recently B.G.Ma et al., 2009 have developed transgenic tomato using salicyclic acid inducible Cre/loxP recombination system. Through this system they have developed 41% transgenic tomato that ar marker free ( nptII gene) in the F1 generation.

Transposon-based marker methods

Two transposon mediatd strategy has been developed to generate marker free transgenic plants. This strategy involves Agrobacterium mediated transformation followed by intragenomic relocation of transgene of interest, and its subsequent segregation form the selectable marker in the progeny (Goldsbrough et al., 1993) or excision of marker gene from the genome (Ebinuma el al., 1997). Both strategy were developed using the maize Ac/Ds transposable element but could be adapted to use similar autonomous transposable element.

Ebinuma et al., 1997 proves the feasibility of this strategy by eliminating the isopentyl transferase (ipt) marker gene from transgenic tobacco plants. Transgenic plants constitutively expressing the ipt gene have elvated sytokinin to auxin ratios resulting in a loss of apical dominace, suppression of root formation and what is referred to as shooty phenotype. Transformed tobacco leaf disc with a T-DNA containing nptII and gus gene and a chimeric Ac element which included a 35S-ipt gene, two thirds of this differentiated adventitious shoots showed and extremely shooty phenotype. Upon subculturing this phenotypic distinct shoots normal shoots were developed which indicated the removal of ipt gene expression.

The basic advantage of this strategy is; Marker free transgenic can easily be screened at T0 generation, avoiding the need of sexual cross plants and thereby making the strategy applicable to the vegetative propogated crops like banana, potato, grapes and so on.

Inspite of all the advantageous the limitations for this strategy is reflected some inevitable like the frequency for generation of marker free transgenic is very less as expected. The genomic instability of transgenic plants because of the continuous presence of heterologous trasnsposons (Scutt et al., 2002)

Chemically inducible system

From past several years recombination system is very often used in plant transformation to eliminate selection marker gene. Cre/loxrecombination system of Bacteiophage P1 is one of the system developed in the context of marker removal in transgenic plants ( Dale and Ow 1991). And in order to remove the cre gene from the transgenic plants, retransformation and out-crossing approaches have been used that enables the loss of cre gene in subsequent generation although it is very laborious and time constraint job (Dale and Ow 1991). Now several approaches were used to over come these shortcomings by using some chemical inducers (Yuang et al., 2004, Zhang et al., 2006) or by Heat shock (Wang et al., 2005; Cuellar et al., 2006).

The CLX vector system benefits also from a particularly regulated system of chemical induction [D. W. Ow, 2001]. The procedure could be used for vegetatively propagated species and may be particularly well adapted to crop species requiring transformation by the regeneration of embryo cultures.

Marker free transgenic tomato expressing cry1AC were obtained by using chemically regulated Cre/lox mediated site specific recombination system. The marker gene nptII was eliminated by two directly oriented loxP sites was located between the CaMV35S promoter and a promoterless cry1AC. Upon induction by 2 μM β-estradiol, sequence encoded the selectable marker and cre sandwiched by two loxP sites were excised from the tomato gemone (Yuyang et al., 2006). Using the Cre/loxP recomnbination system the expression of Cre recombinase was under the control of estrogen receptor based transactivator XVE. Upon induced by β- estradiaol, the slection marker gene fused with Cre recombinase, flanked by two lox sites were auto excised from the Arbidopsis genome the chemical inducible system was reliable method for generating marker free transgenic. (Jianru Zuo et al., 2001). Recently Chaoyang Lin, et al., 2008 have reported a chemical induce method for creating selectively terminable transgenic rice. In that they have used benzothiadiazole herbicide (Bentazon) which has been used for weed control of several major crops, such as rice, corn wheat, cotton and soyabean. These crops express cytochrome P450 as detoxifying the herbicide benzaton. They generate benzaton sensitive rice plants by suppressing the expression of this detoxification gene through Antisense RNA or benzaton sensitive transgenic rice with high glyphosate tolerance.

Heat inducible system

The site specific recombination system is used widely in the applied biotechnology for generating marker free transgenic plants. Cre/loxP, FLP/frt recombination system and the knowledge of promoters is keeping the researchers upper hand for generating marker free transgenics. ShangX Y et al ., 2006 have developed the transgenic tobacco using FLP/frt recombinase system in which the expression of FLP was tightly under the control of hsp (heat shock protein). Two different construct were used in this approach(frt containing vector pCAMBIA1300-betA-frt-als-frt and the FLP expression vector pCAMBIA1300-hsp-FLP-hpt) and through the process of retransformation The FLP recombinase gene was introduced into transgenic (betA-frt-als-frt) tobacco.In re-transgenic plants, after heat shock treatment, the marker gene als flanked by two identical orientation frt sites could be excised by the inducible expression of FLP recombinase under the control of hsp promoter. Excision of the all gene was found in 41% re-transgenic tobacco plants. Heat inducible strategy for the elimination of selection marker gene was used also in vegetative propogated plants like potato (Cuellar W et al., 2006) and seed producing plants like Tobacco (Wamg Y et al., 2005) In this strategy HSP70 was used as heat inducible promoter in Cre/lox recombination system. A new binary expression vector based on the 'genetically modified (GM)-gene-deletor' system was constructed. In this vector, the gene coding for FLP site-specific recombinase under the control of a heat shock-inducible promoter HSP18.2 from Arabidopsis thaliana and isopentenyltransferase gene (ipt), as a selectable marker gene under the control of the cauliflower mosaic virus 35S (CaMV 35S) promoter, were flanked by two loxP/FRT fusion sequences as recombination sites in direct orientation. Further characterization fot eh transgenic tobacco plants confirmas the elimination of the ipt gene along with gusA in the primary stage. Heat inducible approach provides a reliable strategy for auto-excising a selectable marker gene from calli, shoots or other tissues of transgenic plants after transformation and producing marker-free transgenic plants. The disadvantage of this method is not negotiable. When auto-excision constructs are used, the recombinase can be activated by a chemical compound or by a heat shock in the shoots and seeds or during a subculture step and an extra regeneration step. The latter possibility lengthens the time to obtain marker-free transgenic plants and can introduce (additional) somaclonal variation.

Positive selection system to eliminate marker gene

Some marker genes for positive selection enable the identification and selection of genetically modified cells without injury or death of the non-transformed cell population (negative selection). In this case, the selection marker genes should give the transformed cell the capacity to metabolize some compounds that are not usually metabolized. This fact will give the transformed cells an advantage over the non-transformed ones. The addition of this new compound in the culture medium, as nutrient source during the regeneration process, allows normal growth and differentiation of transformed cells, while non-transformed cells will not be able to grow and regenerate de novo plants.

The gus gene: The gene gus codes for the β-glucuronidase enzyme (GUS; EC and was isolated from Escherichia coli. This gene is widely used as a reporter gene in transgenic plants. In this system, the selective agent is a glucuronide derivative of benzyladenine (benzyladenine N-3-glucuronide), an inactive form of the plant hormone cytokinin. This glucuronide present in the selection medium can be hydrolyzed by the GUS enzyme produced in the transformed cells, releasing active cytokinin (benzyladenine) in the medium. This cytokinin will be a stimulator for transformed cell regeneration while non-transformed cell development is arrested.

The selective agent (benzyladenine N-3-glucuronide) does not have any effect on the non-transformed cells because the cytokinin is in its inactive form.

There is only one report concerning the successful use of this system in the effective recovery of transgenic plants (Joersbo and Okkels, 1996; Okkels et al., 1997).

The manA gene: The man gene codes for the phosphomannose isomerase enzyme (PMI; EC isolated from Escherichia coli. In the presence of mannose, the PMI converts mannose-6-phosphate into fructose-6-phosphate in transformed cells that can be immediately incorporated in the plant metabolic pathway. Thus, mannose can be used as the sole carbohydrate source for the transformed cells. This selection system is immediate and extremely efficient (Joersbo et al., 1998).

Mannose cannot usually be metabolized by non-transformed cells and is converted into mannose-6-phosphate by endogenous hexokinase. Therefore, when mannose is added to the culture medium, plant growth may be minimized due to mannose-6-phosphate accumulation. The mannose-6-phosphate toxicity in plant cells was shown to be responsible for apoptosis, or programmed cellular death, through induction of an endonuclease, responsible for DNA laddering (Stein and Hansen, 1999). Mannose-6-phosphate accumulation also causes phosphate and ATP starvation that deplete cell of energy for critical functions such as cell division and elongation, giving rise to growth inhibition. Therefore, mannose is a hexose that fills the desirable requirements for a good selection agent: it is (a) soluble in plant culture media; (b) absorbed by plant cells; (c) cheap; (d) easily available and (e) safe.

Although most plant species are sensitive to mannose, some species, especially dicotyledonous, have shown a considerable insensitivity to this sugar, including carrot, tobacco, sweet potato and legumes. Other species are extremely sensitive and have been successfully transformed using mannose as selective agent, such as sugar beet, maize, wheat, oat, barley, tomato, potato, sunflower, oilseed rape and pea (Joersbo et al., 1998; 1999; 2000; Negrotto et al., 2000; Wang et al., 2000).

Some plant transformation protocols that use the positive selection system with PMI were at least 10 times more efficient than the traditional protocols based on the use of kanamycin as selection agent (Wright et al., 2001).

The xylA and DOGR1 genes: A similar positive selection system has been developed using the xylose isomerase gene (xylA) isolated from Thermoanaerobacterium thermosulfurogenes or from Streptomyces rubiginosus, as selection marker gene (Haldrup et al., 1998a; 1998b). Transgenic plants of potato, tobacco and tomato were successfully selected in xylose-containing media.

Recently, the DOGR1 gene encoding 2-deoxyglucose-6-phosphate phosphatase (2-DOG-6-P) was used to develop a positive selection system for tobacco and potato plants (Kunze et al., 2001). DOGR1 gene, which has been isolated from yeast, gives resistance to 2-deoxyglucose (2-DOG) when over-expressed in transgenic plants.

Negative selection

An alternative and potentially more efficient strategy is based on the incorporation of a negative selection step. Finally, the combination of using a mixture of mechanisms, transient selection, sequential transformation, negative marker genes, P-DNA and a mutated virD2 gene together should be capable of producing high frequency marker-free transgenic plants by co-transformation methods. Recently, a novel marker gene has been characterized, dao1, encoding D-amino acid oxidase that it can be used as for either positive or negative marker, depending on the substrate Erikson et al., 2004. Therefore, it is possible to apply the negative selection after a positive selection using one marker gene, dao1, via changing D-alanine or D-serine to D-isoleucine or D-valine for the substrates.Conversion

of an externally provided specific substrate into its phytotoxic derivative by the marker gene encoded enzyme enables this counter selection. The tms2 gene was the first conditional selective marker gene to be used in tobacco (Depicker et al., 1988) and in Arabidopsis (Karlin-Neuman et al., 1991). Indoleacetic acid hydrolase (IAAH) encoded by the tms2 gene confers sensitivity of plants to naphthaleneacetamide (NAM) because IAAH converts NAM to the potent auxin naphthaleneacetic acid (NAA) which inhibits seedling growth. Other conditional markers proven to be effective in dicots are aux2 in cabbage (Beclin et al., 1993), the HSV-tk gene in tobacco (Czako and Marton, 1994), a bacterial cytochrome P450 mono-oxygenase gene in tobacco (O'Keefe et al., 1994) and Arabidopsis (Tissier et al., 1999), and codA in Arabidopsis (Kobayashi et al., 1995) and tobacco (Schlaman and Hooykaas, 1997). So far, the cytochrome P450 (the product of which catalyses the dealkylation of a sulfonylurea compound, R7402 into its cytotoxic metabolite) and codA (whose product cytosine deaminase converts the non-toxic 5-fluorocytosine into phytotoxic 5-fluorouracil) are the only genes to have been used as conditional negative selectors in monocots. Both have been proven to be effective in barley (Koprek et al., 1999). The only gene used in rice so far is the cytochrome P450 (Chin et al., 1999).

Auto Excision strategy to eliminate marker gene

As most of the methods to eliminate the gene selection marker form the plant genome is known. The earlier methods of auto excision like heat inducible system and chemical inducible system is time consuming and also marker gene is eliminated in the nest generation after segregation. in terms of money wise and for development of transgenic plant without maker gene.

. Now from last few years scientist have crated a very novel and ideal method to eliminate the gene selection marker in single generation. This method is mainly called as "Auto excision strategy" in which marker is easily eliminated in the T1 seeds of the transgenic plants ( the seeds which is collected from the T0 plants). The next generation of the transgenic plants will be marker free.

Auto excision strategy is very recently introduced and used in the plant biological system to eliminate gene selection marker from the plant genome.

Auto excision system is controlled by pollen -and /or seed specific promotes, it was reported that the highly efficient auto-excision of selective markers is successfully achieved in tobacco (Mlynarova et al., 2006; Luo et al 2007). Auto excision strategy relies on floral specific promoters to regulate the expression of cre recombinase to generate marker free transgenic plants.

The functionally characterized promoters were used in the strategy and the system is successfully demonstrated in rice (Xianquan et al., 2008) the novel marker free approach mediated by the Cre/loxp recombination system and the Cre gene was under the control of floral specific promoter OsMADS45. The marker gene nptII was completely removed from the T1 progenny of the rice with 37.5% efficiency.

Dimitri Verweire et al., 2007 have developed homologous marker free transgenic plants of Arabidopsis thaliana introducing a germline-specific auto-excision vector containing a cre recombinase gene under the control of a germline-specific promoter (APETALA1 and SOLO DANCERS genes from Arabidopsis (Arabidopsis thaliana) Columbia-0)transgenic plants become genetically programmed to lose the marker when its presence is no longer required. Usnign this method the frequency of regerating marker free transgenic lines in Arabidopsis is 83%-100%. Isnpite of all the above auto excision strategy is having its limitations like it is successful only in flowering plants. It will not be useful for the vegetative propogated plants like grapes, potato and banana.

Abiotic stress related Gene as selection marker

Co- transformation, Site specific integration, Chemical induced and Heat induced marker gene elimination method in which the marker gene is eliminated in second generation, except the autoexcision strategy where the selection marker gene is eliminated in F1 generation. Here we have discussed the novel approach for the development of Marker free transgenic. It is well known fact that various genes encode proteins which protect the plants at the time of several environmental stresses like drought stress, salt stress and at the time of Oxidative stress. Till date so many gene which is well characterized in Arabidopsis or in several agronomical important crop can be used for the development of marker free transgenic plants. Incorporation of such well characterized gene in to those plants which are salt sensitive plants, include rice (Oryza sativa), maize (Zea mays), soybean (Glycine max) beans (Phaseolus vulgaris) and Tomato(Lycopersicum esculantum) is one hand contribution to the Agriculture sector for developing transgenic plants.

The basic ideas behind this strategy is, Plant tissue or plant senses high Na+ concentration in the soil/media and initiates signal transduction to activate a set of stress responsive genes for salt tolerance. The gene is incorporated into the plant tissue or explants without the selection marker gene. After transformation the tissue will grow under the pressure of salt stress and explants which grows well without any deformities is selected and grown further in the salt stress medium till vegetative proliferation of the explants. In the whole experiment there is no need to use the selection marker. The gene itself can be used as selection marker to select the transformed tissue.

ESKIMO1 gene involved in plant water economy as well as cold acclimation and salt tolerance (Bouchabke-Coussa O, et al., 2008). Yoshida et al.2000 attempted to make the yeast Na'-ATPase function in plant cells. The ENAl gene that encodes the S. cerevisiae Na+-ATPase was placed under the control of the CaMV35S promoter and introduced into BY2 cells. Transgenie BY2 cells which produced Enal protein were able to grow in modified LS medium containing 120 mM of LiCl, conditions which markedly inhibited the growth of untransformed cells. Sanan-Mishra N, et al., 2005 Recently explored the potential role of PDH45 (pea DNA helicase45) in overcoming salinity stress. PDH45 mRNA is induced in pea seedlings in response to high salt, and its overexpression driven by a constitutive cauliflower mosaic virus-(35)S promoter in tobacco plants confers salinity tolerance. The over-expression of barley group 3 LEA gene HVA1 in leaves and roots of rice and wheat lead to improved tolerance against osmotic stress as well as improved recovery after drought and salinity stress

In case of unknown gene it will be difficult to use the system.

Screening will tedious and boredom because there will be a chances of escaped of untransformed calli.

Conclusion and Future prospective

The field of marker gene removal continues to produce new innovations. For example, the possibilities of increasing the number of different heterologous recombinase systems

available by molecular evolution approaches have been discussed, and new marker gene and marker-free strategies are under development Schubbert et al., 1998. The removal of marker gene and backbone from the transgenic plants supports multiple transformation cycles for transgene pyramiding. Though research continues, it is clear that several viable methods for the removal of unwanted marker genes already exist. It seems highly likely that continued work in this area will soon remove the question of unwanted marker genes from the debate concerning the public acceptability of transgenic crop plants. The techniques for marker gene removal under development will also facilitate the more precise and subtle engineering of the plant genome, with widespread applications in both fundamental research and biotechnology.