Cisgenic Plants Research Articles

Intellectual property rights and plants made by new genomic techniques: Access to technology and gene-edited traits in plant breeding

Gene editing has the potential to make new crop varieties faster and more efficiently. New and more suitable crop varieties can increase sustainable agriculture, for instance, in the form of disease-resistant varieties that facilitate integrated pest management. The European Commission's proposal on the regulation of gene-edited and cisgenic plants produced with New Genomic Techniques (including CRISPR/Cas) has re-opened the discussion on Intellectual Property Rights on plants in Europe. We provide an overview of the possible impact of patent rights and Plant Variety Rights on the availability of technology and gene-edited alleles to breeders on an European level. We highlight potential problems with the two Intellectual Property Right systems and indicate potential avenues to solutions.

Genetic Transformation of Potato without Antibiotic-Assisted Selection

The genetic engineering of plants often relies on the use of antibiotic or herbicide resistance genes for the initial selection of primary transgenic events. Nevertheless, the commercial release of genetically modified crops containing any marker gene encounters several challenges stemming from the lack of consumer acceptance. The development of strategies enabling the generation of marker-free transgenic plants presents an alternative to address public concerns regarding the safety of biotech crops. This study examined the capabilities of highly regenerative potato cultivars to develop transgenic plants without the presence of selective substances in their media. Internodal segments of in vitro potato plants were inoculated with the Agrobacterium strain AGL0 carrying plasmids, which contained the GFP or RFP gene driven by the CaMV 35S promoter to monitor the transformation process by observing in vivo green or red fluorescence. Despite the absence of selective pressure, inoculated explants demonstrated comparable or even higher transient expression compared to experiments based on antibiotic assistant selection. Consequently, under non-selective conditions, non-transgenic, chimeric, and fully fluorescent potato plantlets were concurrently developed. Among the five tested cultivars, the regeneration efficiency of non-chimeric transgenic plants varied from 0.9 (‘Chicago’) to 2.7 (#12-36-42) plants per 100 detached plantlets. Depending on the regenerative characteristics of potato varieties (early, intermediate, or late), a specific time interval can be determined when a blind collection of transgenic plantlets is more successful, streamlining the transformation procedure. The results indicate that the outlined procedure is simple and reproducible, consistently achieving the transformation efficiency of 7.3–12.0% (per 100 inoculated explants) in potato cultivars without selective pressure. The described transformation procedure holds the potential for obtaining cisgenic or intragenic potato plants with new valuable traits that do not carry marker genes.

Bioengineering of horticultural crops in Russia and in the world

Modern bioengineering technologies make it possible to create fruit and berry crops with signs that are unattainable by traditional breeding methods, while significantly reducing the time of breeding work. A review of more than 90 scientific publications in the period from 1989 to 2020 with research work on the creation of transgenic fruit and berry crops using various bioengineering technologies, which showed a wide variety of methods for modifying and identifying the resulting plants.
 For example, the modification of the genome of an apple tree using genes that accelerate flowering, the so-called “Fast breeding” technology, allows you to speed up breeding work to create valuable varieties. The developed new varieties of transgenic apple trees are resistant to the fungi, bacteria, apple moth, and also reduced ability to browning fruits, with an increased content of sucrose, etc.
 Modern methods of genetic engineering make it possible to significantly accelerate the processes of creating highly productive varieties of fruit crops with increased or complete resistance to viruses. Transgenic papaya, expressing the gene for the envelope protein of the virus, made it possible to save plantations in Hawaii. The plum varieties created with the help of bioengineering were resistant to the plum pox virus (PPV), which poses a greater danger to perennial fruit crops.
 In Russia, clonal rootstocks of apple and pear trees resistant to herbicides, strawberries and pears with improved fruit taste, rootstocks and varieties of plums and cherries resistant to the Plum Pox Virus have been created and field tested, a technology for obtaining cisgenic plants has been developed and applied using the example of apple and tomato trees that do not contain viral and bacterial sequences. Methods of genomic editing and accelerated selection of fruit crops are being developed.

Cisgenics and intragenics: boon or bane for crop improvement

Recent advances in molecular biology and genetic engineering have made it possible to increase agricultural yields when compared to conventional methods. However, lots of challenges are to be addressed due to changing climatic conditions. Although genetically modified organisms (GMOs) have proven their potential in a few crops, but needs to be explored in majority of the field/vegetable crops to overcome food and nutritional security in view of alarming population explosion. In spite of advantages from GMO crops due to the presence of foreign DNA, queries regarding their safety, environmental dangers and health effects needs to be addressed. One of the major environmental issues concerning transgenic crops is the mixing of genetic components across species that cannot hybridize naturally. Due to these limitations, new revolutionary technologies have been developed, such as intragenesis and cisgenesis for the transformation and development of superior plants. While cisgenesis entails genetic modification employing a complete copy of natural genes with their native regulatory components that only belong to sexually compatible species, intragenesis refers to the transfer of unique combinations of genes and regulatory sequence inside the same species. In cisgenesis, the donor genes are the same genes employed in conventional breeding. The two benefits of cisgenics are avoiding linkage drag and making greater use of existing gene alleles. This method significantly shortens the time it takes to breed plants by combining conventional methods with cutting-edge biotechnological tools. Because of this, plant genomes can be altered without causing drastic changes to the whole plant population and the environmental effects of cisgenic plants cannot be compared to those of transgenics. Transgenesis and cisgenesis share the same transformation methods; hence, cisgenic, intragenic and transgenic plants produced through random insertion do not pose any distinct risks with regard to host genome modifications. In contrast, using new genome techniques lessens the dangers related to potential unintentional changes to the host DNA. The use of cisgenesis and intragenesis as alternatives to transgenesis has been restricted to a small number of species due to incomplete understanding of the required regulatory sequences.

The study of pangenomes and super-pangenomes is a necessary requirement for the creation of promising intragenic and cisgenic plants, as well as the disclosure of the nature of heterosis

The study of pangenomes and super-pangenomes is a necessary requirement for the creation of promising intragenic and cisgenic plants, as well as the disclosure of the nature of heterosis

Updated scientific opinion on plants developed through cisgenesis and intragenesis.

In 2012, EFSA issued an opinion on plants developed through cisgenesis and intragenesis. With the development of New Genomic Techniques (NGTs) in the last decade, cisgenic and intragenic plants can now be obtained with the insertion of a desired sequence in a precise location of the genome. EFSA has been requested by European Commission to provide an updated scientific opinion on the safety and the risk assessment of plants developed through cisgenesis and intragenesis, in order to (i) identify potential risks, comparing them with those posed by plants obtained by conventional breeding and Established Genomic Techniques (EGTs) and (ii) to determine the applicability of current guidelines for the risk assessment of cisgenic and intragenic plants. The conclusions of the previous EFSA opinion were reviewed, taking into consideration the new guidelines and the recent literature. The GMO panel concludes that no new risks are identified in cisgenic and intragenic plants obtained with NGTs, as compared with those already considered for plants obtained with conventional breeding and EGTs. There are no new data since the publication of the 2012 EFSA opinion that would challenge the conclusions raised in that document. The conclusions of the EFSA 2012 Scientific Opinion remain valid. The EFSA GMO Panel reiterates from these conclusions that with respect to the source of DNA and the safety of the gene product, the hazards arising from the use of a related plant‐derived gene by cisgenesis are similar to those from conventional plant breeding, whereas additional hazards may arise for intragenic plants. Furthermore, the EFSA GMO Panel considers that cisgenesis and intragenesis make use of the same transformation techniques as transgenesis, and therefore, with respect to the alterations to the host genome, cisgenic, intragenic and transgenic plants obtained by random insertion do not cause different hazards. Compared to that, the use of NGTs reduces the risks associated with potential unintended modifications of the host genome. Thus, fewer requirements may be needed for the assessment of cisgenic and intragenic plants obtained through NGTs, due to site‐directed integration of the added genetic material. Moreover, the GMO panel concludes that the current guidelines are partially applicable and sufficient. On a case‐by‐case basis, a lesser amount of data might be needed for the risk assessment of cisgenic or intragenic plants obtained through NGTs.

Criteria for risk assessment of plants produced by targeted mutagenesis, cisgenesis and intragenesis.

EFSA was asked by the european Commission to develop criteria as advice for consideration for the risk assessment of plants produced by targeted mutagenesis, cisgenesis and intragenesis. EFSA proposes in this statement six main criteria to assist the risk assessment of these plants. The first four criteria are related to the molecular characterisation of the genetic modification introduced in the recipient plant. The four criteria evaluate whether any exogenous DNA sequence(s) is/are present (Criterion 1), whether such sequence derives from the breeders' gene pool (Criterion 2), the type of integration (Criterion 3) and whether any endogenous plant gene is interrupted (Criterion 4). Depending on the evaluation of the above criteria, the product can be a genome edited plant where no exogenous DNA sequence is present, or a cisgenic or intragenic plant where the cisgenic and intragenic sequence are introduced by targeted insertion and no plant endogenous genes are interrupted. In these cases, two more criteria are assessed to evaluate the history of safe use (Criterion 5) and the structure and function of the new allele (Criterion 6). If cisgenic and intragenic sequence are introduced by random integration without interruption of an endogenous gene, or when no risk is identified when an endogenous gene is interrupted, the criteria 5 and 6 will also be assessed. Evaluating the history of safe use is an important part of the proportionate risk assessment of cisgenic, intragenic and genome‐edited plants since the newly introduced allele may already be present in nature. However, when the history of safe use cannot be sufficiently demonstrated, the function and structure of the introduced allele should be carefully assessed. Recommendations are also included on the aspects that need further elaboration for full applicability of the criteria proposed herein are also included.

A CRISPR/Cas9-Based System with Controllable Auto-Excision Feature Serving Cisgenic Plant Breeding and Beyond.

Transgenic or genetically modified crops have great potential in modern agriculture but still suffer from heavy regulations worldwide due to biosafety concerns. As a promising alternative route, cisgenic crops have received higher public acceptance and better reviews by governing authorities. To serve the purpose of cisgenic plant breeding, we have developed a CRISPR/Cas9-based vector system, which is capable of delivering target gene-of-interest (GOI) into recipient plants while removing undesired genetic traces in the plants. The new system features a controllable auto-excision feature, which is realized by a core design of embedded multi-clonal sequence and the use of inducible promoters controlling the expression of Cas9 nuclease. In the current proof-of-concept study in Arabidopsis thaliana (L.) Heynh., we have successfully incorporated a GOI into the plant and removed the selection marker and CRISPR/Cas9 components from the final product. Following the designed workflow, we have demonstrated that novel cisgenic plant germplasms with desired traits could be developed within one to two generations. Further characterizations of the vector system have shown that heat treatment at 37 °C could significantly improve the editing efficiency (up to 100%), and no off-target mutations were identified in the Arabidopsis background. This novel vector system is the first CRISPR/Cas9-based genome editing tool for cisgenic plant breeding and should prove powerful for other similar applications in the bright future of precision molecular breeding.

Genome-Wide Analysis of the Apple CBL Family Reveals That Mdcbl10.1 Functions Positively in Modulating Apple Salt Tolerance.

Abiotic stresses are increasingly harmful to crop yield and quality. Calcium and its signaling pathway play an important role in modulating plant stress tolerance. As specific Ca2+ sensors, calcineurin B-like (CBL) proteins play vital roles in plant stress response and calcium signaling. The CBL family has been identified in many plant species; however, the characterization of the CBL family and the functional study of apple MdCBL proteins in salt response have yet to be conducted in apple. In this study, 11 MdCBL genes were identified from the apple genome. The coding sequences of these MdCBL genes were cloned, and the gene structure and conserved motifs were analyzed in detail. The phylogenetic analysis indicated that these MdCBL proteins could be divided into four groups. The functional identification in Na+-sensitive yeast mutant showed that the overexpression of seven MdCBL genes could confer enhanced salt stress resistance in transgenic yeast. The function of MdCBL10.1 in regulating salt tolerance was also verified in cisgenic apple calli and apple plants. These results provided valuable insights for future research examining the function and mechanism of CBL proteins in regulating apple salt tolerance.

Selection of transgenic citrus plants based on glyphosate tolerance conferred by a citrus 5-enolpyruvylshikimate-3-phosphate synthase variant.

We have defined the conditions for citrus transformations using glyphosate as selection agent. This protocol results in high transformation rate and low incidence of chimeric shoots. Glyphosate, the most widely used herbicide in the world, specifically inhibits 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), an essential enzyme of the shikimate pathway. Various laboratory-generated or naturally evolved glyphosate-resistant EPSPS variants have been used to produce glyphosate-tolerant transgenic crops, enabling highly effective weed control in agriculture. In this study, we explored the potential of using a citrus EPSPS variant that mimics the previously reported Eleusine indica glyphosate-resistant TIPS (T102I + P106S) mutant for selection of transgenic citrus plants in the presence of glyphosate. We found that glyphosate did not suppress bud formation on 'Duncan' grapefruit seedling explants, but inhibited non-transgenic bud outgrowth to produce shoots in a concentration-dependent manner. At certain concentrations, glyphosate had dramatic effect on the transformation rate and the percentage of non-chimeric transgenic shoots in this newly developed selection system. Specifically, at 0, 10, 20, and 50 μM of glyphosate, the citrus TIPS EPSPS-based selection resulted in transformation rates of 4.02, 5.04, 14.46, and 40.78%, respectively, and 6.41, 23.96, 42.94, and 40.17% of non-chimeric transgenic shoots, respectively. These results indicate that the citrus TIPS EPSPS-glyphosate selection system is highly efficient and can be used as an alternative to antibiotic-based selection methods in citrus genetic transformation. Furthermore, the selection conditions defined in this study are expected to greatly facilitate the production of genetically modified, market-friendly citrus plants, such as cisgenic and intragenic plants.

A Cisgenic Approach in the Transformation of Bread Wheat cv. Saratovskaya 29 with Class I Chitinase Gene

Background: Bread wheat is one of the major crops grown worldwide, showing high demand for new varieties with traits such as pathogen resistance. As the public acceptance of transgenic plants remains low, a novel approach – cisgenesis – is being developed to introduce the genes from the same or closely related species. Objective: This study presents a cisgenic approach used for the transformation of wheat with class I chitinase gene derived from T. aestivum cv. Stepnaya 15, co-transformed with acetohydroxyacid synthase gene that provides tolerance to imidazolinone herbicides. Methods: Calli from immature embryos of spring bread wheat Triticum aestivum cv. Saratovskaya 29 were used for co-transformation with two independent Minimal Expression Units (MEUs): class I chitinase and Acetohydroxyacid Synthase (AHAS) gene. For identification of cisgenic plants, genomic DNA was extracted from the leaves of imazethapyr-resistant regenerant plants at the plantlets stage and screened by polymerase chain reaction. The efficiency of transformation was calculated as the relation of regenerated plants with chitinase gene insert to the total number of calli in the experiment. Results: The average transformation efficiency in four series of experiments (total number of calli - 2299) was found to be 1.84% (ranging from 0.3% to 3.4%), while total co-transformation efficiency reached 87.93%. Conclusion: The high efficiency of co-transformation in the experiment promotes it as a very useful technique for the production of wheat lines, free of the selectable marker gene. To our knowledge, this is the first report of cisgenic bread wheat, where both target and selectable genes are derived from wheat.

Horizontal Stacking of PAPhy_a Cisgenes in Barley Is a Potent Strategy for Increasing Mature Grain Phytase Activity.

Mature grain phytase activity (MGPA) in the Triticea tribe cereals has evolved through gene duplications and neo-functionalization of the purple acid phosphatase phytase gene (PAPhy) in a common ancestor. Increased gene copy number of the PAPhy_a gene expressed during seed development has augmented the MGPA in cereals like rye and wheat. PAPhy_a phytase is highly stable and a potent enzyme in feed. However, barley only contains one HvPAPhy_a gene and the MGPA levels needs to be increased to substitute for the addition of microbial phytases to the feed. A substantial increase in MGPA for cisgenic barley was achieved with one extra homozygous HvPAPhy_a insert when the plants were grown in the greenhouse. In the current study, the stability of increased MGPA was confirmed in open field grown cisgenic barley. Furthermore, the gene dose response of phytase cisgenes from three different cisgenic barley plants were horizontally stacked. Cisgenic barley with 0, 1, 2, 3, 4, and 6 extra HvPAPhy_a inserts demonstrated a perfect positive linear correlation with the level of MGPA. The current study provides new insight into the potential of stacking of cisgenes in crops and suggests cisgene stacking as a versatile strategy for crop improvement.

Development of a Taqman real-time PCR method to quantify nptII in apple lines obtained with ‘established’ or ‘new breeding’ techniques of genetic modification

Cisgenic plants must be free of exogenous genetic elements such as antibiotic or herbicide resistance genes commonly used in the selection phase of a gene transfer protocol. However, the use of a selection marker is essential for the transformation of many fruit crops, including apple (Malus × domestica), for which the efficiency of DNA integration is very low. Currently, the approach with the highest chances of success relies on the removal of undesired exogenous genes by means of an inducible site-specific recombinase enzyme and its recognition sites. We developed a quantitative, rapid and cost-effective method based on real-time PCR to quantify the copy number of nptII marker gene in apple lines and to evaluate its elimination after the activation of the recombinase system. MdTOPO6 gene was chosen as endogenous reference gene for apple due to the single-copy presence in the haploid genome and to the species-specificity. A recombinant plasmid harboring specific sequences of both the reference gene and the target gene nptII was used as calibrator to build the standard curves. The limit of quantification of the method was evaluated, and precision and trueness of the quantification performances proved to be valid according to international reference criteria. Finally, this method was applied to characterize transgenic and cisgenic apple lines, and to investigate the possibility of removing an entire T-DNA cassette from the genome of edited apple plants.

Development of a Taqman real-time PCR method to quantify nptII in apple lines obtained with ‘established’ or ‘new breeding’ techniques of genetic modification

Cisgenic plants must be free of exogenous genetic elements such as antibiotic or herbicide resistance genes commonly used in the selection phase of a gene transfer protocol. However, the use of a selection marker is essential for the transformation of many fruit crops, including apple (Malus × domestica), for which the efficiency of DNA integration is very low. Currently, the approach with the highest chances of success relies on the removal of undesired exogenous genes by means of an inducible site-specific recombinase enzyme and its recognition sites. We developed a quantitative, rapid and cost-effective method based on real-time PCR to quantify the copy number of nptII marker gene in apple lines and to evaluate its elimination after the activation of the recombinase system. MdTOPO6 gene was chosen as endogenous reference gene for apple due to the single-copy presence in the haploid genome and to the species-specificity. A recombinant plasmid harboring specific sequences of both the reference gene and the target gene nptII was used as calibrator to build the standard curves. The limit of quantification of the method was evaluated, and precision and trueness of the quantification performances proved to be valid according to international reference criteria. Finally, this method was applied to characterize transgenic and cisgenic apple lines, and to investigate the possibility of removing an entire T-DNA cassette from the genome of edited apple plants.

Is it only the regulatory status? Broadening the debate on cisgenic plants

In current debates on emerging technologies for plant breeding in Europe, much attention has been given to the regulatory status of these techniques and their public acceptance. At present, both genetically modified plants with cisgenic approaches—using genes from crossable species—as well as transgenic approaches—using genes from different species—fall under GMO regulation in the EU and both are mandatorily labelled as GMOs. Researchers involved in the early development of cisgenic GM plants convey the message that the potential use and acceptance of cisgenic approaches will be seriously hindered if GMO regulations are not adjusted. Although the similar treatment and labelling of transgenic and cisgenic plants may be a legitimate concern for the marketability of a cisgenic GM plant, there are concerns around their commercialization that reach beyond the current focus on (de)regulation. In this paper, we will use the development of the cisgenic GM potato that aims to overcome ‘late blight’—the most devastating potato disease worldwide—as a case to argue that it is important to recognize, reflect and respond to broader concerns than the dominant focus on the regulatory ‘burden’ and consumer acceptance. Based on insights we gained from discussing this case with diverse stakeholders within the agricultural sector and potato production in Norway during a series of workshops, we elaborate on additional issues such as the (technical) solution offered; different understandings of the late blight problem; the durability of the potato plant resistance; and patenting and ownership. Hence, this paper contributes to empirical knowledge on stakeholder perspectives on emerging plant breeding technologies, underscoring the importance to broaden the scope of the debate on the opportunities and challenges of agricultural biotechnologies, such as cisgenic GM plants. The paper offers policy-relevant input to ongoing efforts to broaden the scope of risk assessments of agricultural biotechnologies. We aim to contribute to the recognition of the complex socio-ecological, legal and political dimensions in which these technological developments are entangled as a means to acknowledge, discuss and respond to these concerns and thereby contribute to more comprehensive and responsible developments within agricultural biotechnology.

Why Organic Farming Should Embrace Co-Existence with Cisgenic Late Blight–Resistant Potato

The EU regulation on organic farming does not allow the use of genetically modified organisms (GMOs) which are subject to Directive 2001/18/EC. Mutagenesis using irradiation or chemicals is genetic modification, but the organisms obtained through these techniques are not subject to the provisions of the GMO directive. Such mutants can therefore be used in organic agriculture. Derived from its basic principles, organic farming can only use natural substances to control disease and crops should be resilient, which, in the case of disease resistance, means that durable (horizontal) resistance is preferred to vertical (single gene) resistance. Cisgenesis can achieve such a durable resistance by introducing multiple resistance genes in one step. These multiple-resistant plants only contain natural genes that can also be introduced by breeding. In case cisgenic plants are not subject to the provisions of the GMO legislation, they can even be legally used in organic agriculture. In case they are not exempted from the GMO regulation, the question is: why obstruct a cisgenic potato crop that can hardly be distinguished from a potato crop that is the result of conventional breeding? Among the reasons why organic agriculture does not allow the use of GMOs it is mentioned that genetic engineering is unpredictable, it causes genome disruption and it is unnatural. However, our knowledge of plant genome evolution and breeding has increased dramatically. We now know that breeding is more unpredictable and causes more genome disruption than genetic engineering. Recent field trials have shown the efficacy of cisgenic late blight–resistant potatoes carrying multiple resistance genes. Large-scale growing of such durably resistant potatoes would not only be environmentally beneficial by it would strongly reducing the need for fungicide sprays in conventional potato cultivation and it would also reduce the disease pressure in organic potato cultivation.

Durable Late Blight Resistance in Potato Through Dynamic Varieties Obtained by Cisgenesis: Scientific and Societal Advances in the DuRPh Project

Abstract From 2006 through 2015, a research project on Durable Resistance in potato against Phytophthora (DuRPh) was carried out at Wageningen University and Research Centre. Its objective was to develop a proof of principle for durable resistance against late blight by cisgenesis. This public-funded project aimed at stimulating research on genetic modification and public debate on innovative genetic techniques. It was decided to clone and transfer late blight resistance (R) genes of crossable wild potato species (cisgenes) by Agrobacterium tumefaciens-mediated transformation without non-potato genes. A stack of multiple R genes were planned to be inserted into established varieties, thereby creating a dynamic variety in which the composition of the stacks may vary over space and time. Cisgenic plants were selected based on the expression of all inserted R genes and trueness-to-type. Within the project, 13 R genes from wild potato species were genetically mapped and three of them were cloned. Four varieties were transformed with one to three R genes. This was initially done using kanamycin resistance provided by a selectable marker gene of synthetic origin in order to quickly test the performance and stability of the introduced R genes and stacked R gene combinations. Once the functioning thereof was confirmed, marker-free transformations were conducted; thus, true cisgenic events were selected. The results about the different R genes, their chromosomal location, their specificity, the background dependence, the maximum size of a stack, its regeneration time and associated somaclonal variation frequency and its stability were studied. After selection and characterisation in the laboratory, the best cisgenic events were assessed in field trials for late blight resistance. This showed that inserted R genes were capable of turning a susceptible variety into a resistant one. Maximising longevity of the resistance was assured through resistance management research. It was shown that stacking of multiple R genes and monitoring how to deploy these stacks spatially and temporally could reduce fungicide use by over 80%. Communications through media and field demonstrations were manifold to allow public and policymakers to decide if cisgenesis is an acceptable tool to make potato farming more sustainable. Future deployment of the DuRPh strategy will depend largely on its status as a genetically modified crop or its exemption thereof. Worldwide near eradication of late blight would increase global annual potato production by close to 80 million tons, thereby contributing considerably to the needed additional global future food supply.

Efficient heat-shock removal of the selectable marker gene in genetically modified grapevine

Cisgenesis is one of the new plant breeding technologies emerging as a promising tool for the future, more publicly accepted than the traditional transgenic approach. One of the requirements for a cisgenic plant is the absence of selectable marker genes in the genome. In this study, a system for marker gene removal after selection of transgenic plants has been tested in grapevine. This is based on a binary vector containing a heat-shock-inducible promoter which, upon induction, activates a recombinase to produce the excision of a FRT-flanked box. After the removal of this cassette hosting both the marker gene, nptII, and the recombinase itself, the reporter gene gus may be expressed. Gene transfer experiments on grapevine embryogenic callus were carried out via Agrobacterium tumefaciens. Different heat-shock treatments with variable temperatures and heat incubations times were tested on a selected line and the optimal conditions for a complete removal of nptII with the subsequent gus transcription were found. Plants were analysed by means of qPCR on genomic DNA, to quantify nptII removal, and by a fluorimetric assay to measure gus activity. Our study is conceived as a proof-of-concept to investigate the feasibility of this method in grapevine in view of developing an efficient cisgenic approach in this valuable fruit crop.

Revisiting GMOs: Are There Differences in European Consumers' Acceptance and Valuation for Cisgenically vs Transgenically Bred Rice?

Both cisgenesis and transgenesis are plant breeding techniques that can be used to introduce new genes into plant genomes. However, transgenesis uses gene(s) from a non-plant organism or from a donor plant that is sexually incompatible with the recipient plant while cisgenesis involves the introduction of gene(s) from a crossable—sexually compatible—plant. Traditional breeding techniques could possibly achieve the same results as those from cisgenesis, but would require a much larger timeframe. Cisgenesis allows plant breeders to enhance an existing cultivar more quickly and with little to no genetic drag. The current regulation in the European Union (EU) on genetically modified organisms (GMOs) treats cisgenic plants the same as transgenic plants and both are mandatorily labeled as GMOs. This study estimates European consumers’ willingness-to-pay (WTP) for rice labeled as GM, cisgenic, with environmental benefits (which cisgenesis could provide), or any combination of these three attributes. Data were collected from 3,002 participants through an online survey administered in Belgium, France, the Netherlands, Spain and the United Kingdom in 2013. Censored regression models were used to model consumers’ WTP in each country. Model estimates highlight significant differences in WTP across countries. In all five countries, consumers are willing-to-pay a premium to avoid purchasing rice labeled as GM. In all countries except Spain, consumers have a significantly higher WTP to avoid consuming rice labeled as GM compared to rice labeled as cisgenic, suggesting that inserting genes from the plant’s own gene pool is more acceptable to consumers. Additionally, French consumers are willing-to-pay a premium for rice labeled as having environmental benefits compared to conventional rice. These findings suggest that not all GMOs are the same in consumers’ eyes and thus, from a consumer preference perspective, the differences between transgenic and cisgenic products are recommended to be reflected in GMO labeling and trade policies.

Improving resistance of different apple cultivars using the Rvi6 scab resistance gene in a cisgenic approach based on the Flp/FRT recombinase system

Cisgenic apple plants of two different cultivars were developed by transferring the Rvi6 scab resistance gene of Malus floribunda 821, using a new transformation vector based on the Flp/FRT recombinase system. Transformation experiments on seven different cultivars resulted in 22 transgenic lines for the cultivars ‘Brookfield Baigent’, ‘Mitchgla’, ‘Novajo’, and ‘Pinova’, whereby 16 lines thereof were resistant to Venturia inaequalis strain 104 (race 1). Analysis of the transgenic lines revealed Rvi6 mRNA expression levels comparable to several traditional bred Rvi6 containing cultivars and identified four transgenic lines, harboring a single T-DNA insertion, as suitable for the production of cisgenic lines. The T-DNA insertion site of these lines was determined, and lines were subject to induction of the recombinase system. Two cisgenic lines originating from the cultivars ‘Brookfield Baigent’ and ‘Pinova’ were obtained for which the exact excision of the recombinase cassette was confirmed by sequencing the previously determined T-DNA integration site. Further investigations revealed both cisgenic lines as fully resistant to V. inaequalis race 1. Rvi6 mRNA expression of the cisgenic lines and traditionally bred Rvi6 harboring cultivars was still comparable. The transformation vector developed is useable for the production of cisgenic apple plants to a certain extent.