Frans A. Krens

Cisgenic plants are similar to traditionally bred plants: international regulations for genetically modified organisms should be altered to exempt cisgenesis.

![][1] ![][2] ![][3] The testing and release of genetically modified organisms (GMOs)—in particular GM plants—is tightly regulated internationally to prevent any negative effects on the environment or human health. However, these regulations are based on transgenic organisms and do not discriminate between transgenic plants and cisgenic plants, although we believe that they are fundamentally different (see sidebarNow, cisgenic plants fall under regulations designed for transgenic organisms, possibly because there have not yet been any applications for the approval of the deliberate release of cisgenic plants into the environment. Definitions of key terms in relation to plants Cisgenesis is the genetic modification of a recipient plant with a natural gene from a crossable—sexually compatible—plant. Such a gene includes its introns and is flanked by its native promoter and terminator in the normalsense orientation.Cisgenic plants can harbour one or more cisgenes, but they do not contain any transgenes. Transgenesis is the genetic modification of a recipient plant with one or more genes from any non‐plant organism, or from a donor plant that is sexually incompatible with the recipient plant. This includes gene sequences of any origin in the anti‐sense orientation, any artificial combination of a coding sequence and a regulatory sequence, such as a promoter from another gene, or a synthetic gene. Traditional breeding encompasses all plant breeding methods that do not fall under current GMO regulations.As the European legal framework defines GMOs and specifies various breeding techniques that are excluded from the GMO regulations,we use this framework as a starting point, particularly the European Directive 2001/18/EC on the deliberate release of GMOs into the environment (European Parliament, 2001). Excluded from this GMO Directive are longstanding cross breeding, in vitro fertilization, polyploidy induction,mutagenesis and fusion of protoplasts from sexually compatible plants (European Parliament, 2001). Although transgenesis and cisgenesis both use the same genetic … [1]: /embed/graphic-1.gif [2]: /embed/graphic-2.gif [3]: /embed/graphic-3.gif

Do cisgenic plants warrant less stringent oversight

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A high efficiency technique for the generation of transgenic sugar beets from stomatal guard cells

An optimized protocol has been developed for the efficient and rapid genetic modification of sugar beet (Beta vulgaris L.). A polyethylene glycol-mediated DNA transformation technique could be applied to protoplast populations enriched specifically for a single totipotent cell type derived from stomatal guard cells, to achieve high transformation frequencies. Bialaphos resistance, conferred by the pat gene, produced a highly efficient selection system. The majority of plants were obtained within 8 to 9 weeks and were appropriate for plant breeding purposes. All were resistant to glufosinate-ammonium–based herbicides. Detailed genomic characterization has verified transgene integration, and progeny analysis showed Mendelian inheritance.

Functional analysis and expression profiling of HcrVf1 and HcrVf2 for development of scab resistant cisgenic and intragenic apples

Apple scab resistance genes, HcrVf1 and HcrVf2, were isolated including their native promoter, coding and terminator sequences. Two fragment lengths (short and long) of the native gene promoters and the strong apple rubisco gene promoter (PMdRbc) were used for both HcrVf genes to test their effect on expression and phenotype. The scab susceptible cultivar ‘Gala’ was used for plant transformations and after selection of transformants, they were micrografted onto apple seedling rootstocks for scab disease tests. Apple transformants were also tested for HcrVf expression by quantitative RT-PCR (qRT-PCR). For HcrVf1 the long native promoter gave significantly higher expression that the short one; in case of HcrVf2 the difference between the two was not significant. The apple rubisco gene promoter proved to give the highest expression of both HcrVf1 and HcrVf2. The top four expanding leaves were used initially for inoculation with monoconidial isolate EU-B05 which belongs to race 1 of V. inaequalis. Later six other V. inaequalis isolates were used to study the resistance spectra of the individual HcrVf genes. The scab disease assays showed that HcrVf1 did not give resistance against any of the isolates tested regardless of the expression level. The HcrVf2 gene appeared to be the only functional gene for resistance against Vf avirulent isolates of V. inaequalis. HcrVf2 did not provide any resistance to Vf virulent strains, even not in case of overexpression. In conclusion, transformants carrying the apple-derived HcrVf2 gene in a cisgenic as well as in an intragenic configuration were able to reach scab resistance levels comparable to the Vf resistant control cultivar obtained by classical breeding, cv. ‘Santana’.

Transfer of cytoplasm from new Beta CMS sources to sugar beet by asymmetric fusion. 1. Shoot regeneration from mesophyll protoplasts and characterization of regenerated plants.

SummaryFor our program on the transfer of cytoplasmic male sterility (CMS) by cybridization inBeta vulgaris L. (sugar beet), we have developed a procedure for the isolation and culture of mesophyll protoplasts of sugar beet followed by shoot regeneration. A prerequisite proved to be the presence in the media of n-propylgallate (nPG), a lipoxygenase inhibitor. Sustained divisions were found in all accessions that were tested. Plating efficiencies and regeneration ability varied greatly from one experiment to the other and appeared to be accession-dependent. Shoots could be easily transferred to soil. A majority of the regenerants (72%) retained the diploid chromosome number. Somaclonar variation in phenotype was low (4.9%). Mitochondrial DNA probes, capable of discriminating different cytoplasms ofBeta spp. showed no rearrangements due to the protoplast and in vitro culture phase, indicating that these probes can be used to identify cybrids after asymmetric fusions. The data presented here open up possibilities for genetic engineering using protoplasts in one of the worlds most important arable crops.

Factors influencing induction, propagation and regeneration of mature zygotic embryo-derived callus from Allium cepa

A systematic study on the effects of subspecies, cultivar, basal medium, sucrose concentration and 2,4-dichlorophenoxyacetic acid concentration on callus induction, propagation and subsequent plant regeneration in Allium cepa has been carried out. Mature zygotic embryos from two onion (cvs. Sturon and Hyton) and two shallot (cvs. Tropix and Atlas) varieties were used as explants. After callus initiation and growth on both Murashige and Skoog (MS) and Gamborgs B5 modified by Dunstan and Short (BDS) basal media with different 2,4-dichlorophenoxyacetic acid and sucrose concentrations for eight weeks, lines were identified on which compact or friable callus was induced. Callus induction and propagation were largely determined by the concentration of 2,4-dichlorophenoxyacetic acid whereas subspecies, cultivar, sucrose concentration and basal media were of less importance. After callus propagation for twelve weeks, 315 lines from a total of 3348 embryos initially subcultured were selected to test their regeneration capacity on growth regulator-free medium. It was found that shallot formed more shoots and roots than onion. The MS basal medium proved to be more beneficial for shoot regeneration and root formation than the BDS basal medium. There were no differences in plant regeneration among selected calli which had been previously subcultured on different concentrations of 2,4-dichlorophenoxyacetic acid and sucrose. The results show that plant regeneration strongly depended on the line: 45.4% from 315 tested lines could produce shoots while 93.0% formed roots.

Tissue-specific expression of the β-glucuronidase reporter gene in transgenic strawberry (Fragaria × ananassa) plants

Abstract. The strawberry (Fragaria spp) is regarded as a false fruit because it originates from the receptacle, which is a non-ovarian tissue. For this reason, fruit-specific promoters isolated from plant species in which the fruit is derived from the ovary wall might not be suited to control gene expression in a fruit-specific way in strawberry. In order to achieve (false) fruit-specific expression in strawberry, we tested the petunia FBP7 (floral binding protein7) promoter, which proved to be active in the receptacles of petunia flowers, in transgenic strawberry fruits. In strawberry plants containing the FBP7 promoter fused to the β-glucuronidase (GUS) reporter gene (gus), GUS activity was found in floral and fruit tissues of all developmental stages tested but not in leaf, petiole and root tissue. Surprisingly, Northern blot analysis showed the presence of gus-derived mRNAs in root (strong) and petiole (weak) tissue of fbp7-gus plants in addition to the floral and fruit tissues. Therefore, it is concluded that the histological GUS phenotype does not necessarily correspond with expression at the mRNA level. mRNA quantification using the TaqMan polymerase chain reaction technology confirmed the Northern results and showed that in red strawberry tissue the cauliflower mosaic virus 35S promoter is at least sixfold stronger than the FBP7 promoter.

Agrobacterium tumefaciens-mediated transformation of Allium cepa L.: the production of transgenic onions and shallots

This paper describes the development of a reliable transformation protocol for onion and shallot (Allium cepa L.) which can be used year-round. It is based on Agrobacterium tumefaciens as a vector, with three-week old callus, induced from mature zygotic embryos, as target tissue. For the development of the protocol a large number of parameters were studied. The expression of the uidA gene coding for β-glucuronidase was used as an indicator in the optimization of the protocol. Subspecies (onion and shallot) and cultivar were important factors for a successful transformation: shallot was better than onion and for shallot cv. Kuning the best results were obtained. Also, it was found that constantly reducing the size of the calli during subculturing and selection by chopping, thus enhancing exposure to the selective agent hygromycin, improved the selection efficiency significantly. Furthermore, callus induction medium and co-cultivation period showed a significant effect on successful stable transformation. The usage of different Agrobacterium strains, callus ages, callus sources and osmotic treatments during co-cultivation did not influence transformation efficiency. The highest transformation frequency (1.95%), was obtained with shallot cv. Kuning. A total of 11 independent transformed callus lines derived from zygotic embryos were produced: seven lines from shallot and four lines from onion. Large differences in plantlet production were observed among these lines. The best line produced over 90 plantlets. Via PCR the presence of the uidA and hpt (hygromycin phosphotransferase) genes could be demonstrated in these putative transformed plants. Southern hybridization showed that most lines originated from one transformation event. However, in one line plants were obtained indicating the occurrence and rescue of at least three independent transformation events. This suggested that T-DNA integration occurred in different cells within the callus. Most transgenic plants only had one copy of T-DNA integrated into their genomes. FISH performed on 12 plants from two different lines representing two integration events showed that original T-DNA integration had taken place on the distal end of chromosomes 1 or 5. A total of 83 transgenic plants were transferred to the greenhouse and these plants appeared to be diploid and normal in morphology.

The effect of exogenously-applied phytohormones on gene transfer efficiency in sugarbeet (Beta vulgaris L.)

Abstract Cellular competence for regeneration and transformation has been monitored in a recalcitrant crop, i.e. sugarbeet (Beta vulgaris L.). Explants for Agrobacterium inoculation were taken from the transition zone between hypocotyl and cotyledonary petioles. De novo generation of adventitious shoots was very efficient (60%–70%) from these cotyledonary node explants. Regeneration could not be further stimulated by manipulation of cytokinin type or concentration. The mode of regeneration was direct, without a callus phase. Dedifferentiation, as induced by a preculture period on 2,4-D, gave significantly higher gene transfer frequencies, but regeneration was irreversibly impaired. NAA mimicked the effect of 2,4-D on gene transfer. However, explants retained their regenerative capacity upon pretreatment with NAA. Using the procedure described here, transgenic sugarbeet plants were obtained after selecting on 250 mg/l kanamycin at a frequency of 1%.

Intergeneric transfer of a partial genome and direct production of monosomic addition plants by microprotoplast fusion.

Results are reported on the transfer of single, specific chromosomes carrying kanamycin resistance (KanR) and β-glucuronidase (GUS) traits from a transformed donor line of potato (Solanum tuberosum) to a recipient line of the tomato species Lycopersicon peruvianum through microprotoplast fusion. Polyethylene glycol-induced mass fusion between donor potato microprotoplasts containing one or a few chromosomes and normal recipient diploid L. peruvianum protoplasts gave several KanR calli. A high frequency of plants regenerated from KanR calli expressed both KanR and GUS, and contained one or two copies of npt-II and a single copy of gus. Genomic in situ hybridization showed that several microprotoplast hybrid plants had one single potato donor chromosome carrying npt-II and gus genes and the complete chromosome complement of the recipient L. peruvianum (monosomic additions). Several monosomic-addition hybrid plants could be regenerated within the short time of 3 months and they were phenotypically normal, resembling the recipient line. These results suggest that the transfer of single chromosomes is tolerated better than is the transfer of the whole donor genome. The unique advantages of microprotoplast fusion are discussed: these include the direct production of monosomic addition lines for the transfer and introgression of economically important traits in sexually-incongruent species, the construction of chromosome-specific DNA libaries, high-resolution physical mapping and the identification of alien chromosome domains related to gene expression.