Generation Of Marker-free Transgenic Plants Research Articles

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.

Production of marker-free transgenic plants from mature tissues of navel orange using a Cre/loxP site-recombination system

Genetic transformation with mature material as the explants could shorten the transgenic period and avoid seed dependence compared with genetic transformation using the epicotyl seedling stem segments as the receptor. Here, we constructed an Agrobacterium tumefaciens-mediated transformation for generation of marker-free transgenic plants from navel orange (Citrus sinensis Osbeck) mature stems using a Cre-loxP recombination system. To efficiently recover the regenerated buds from mature tissues, five recovery methods were compared: in vitro micrografting of 0.1–0.5 (1–2 weeks), > 0.5 cm (3–4 weeks) and > 1 cm long lignified bud and in vitro micrografting of explants with a bud and rooting regenerated bud. The data showed that in vitro micrografting of > 1 cm long regenerated bud with expanded leaves after one month of continuous culture for lignification was the optimal solution for plant recovery from mature tissues. Transgenic plants without selectable marker genes were created from navel orange (Citrus sinensis Osbeck) tissue using a transformation vector PLI-35SPR1aCB containing a Cre/loxP system recombination together with genes encoding the selectable marker isopentenyl transferase (IPT) and an anti-bacterial peptide (PR1aCB). Using IPT positive selection, the transformation efficiency determined by PCR was 0.9%, and in total, 20 transgenic plants were obtained. Southern blotting confirmed further their transgenicity. PCR and sequencing analysis demonstrated that both the Cre and IPT genes had been successfully removed from the transgenic plants (deletion efficiency 100%). Over all, using Cre/loxP system recombination together with the IPT positive selection, marker-free transgenic plants can be recovered efficiently from mature tissues of navel orange (Citrus sinensis Osbeck), which provides a potential method for production of transgenic plants from citrus mature tissue.

Construction of chimeric viruses based on pepper mild mottle virus using a modified Cre/loxP system

Cre/loxP, a site-specific recombination system, has been widely used for various purposes, including chromosomal translocations, generation of marker-free transgenic plants, tissue-specific activation of a reporter gene and efficient heterologous gene expression in plants. However, stable or transient expression of Cre recombinase in plants can cause chlorosis or necrosis. Here, we describe a modified Cre/loxP recombination system using a DNA fragment flanked with loxP sites in the same orientation in which necrosis induced by Cre recombinase in Nicotiana benthamiana leaves was alleviated. The modified system was successfully used to create functional GFP-tagged pepper mild mottle virus (PMMoV) and a chimeric virus with coat protein (CP) substitution assembled from separate pro-vector modules. Our results provide a new strategy and flexible technique to construct chimeric virus and infectious clones for plant viruses with large genomes.

Generation of marker-free transgenic plants concurrently resistant to a DNA geminivirus and a RNA tospovirus.

Global threats of ssDNA geminivirus and ss(-)RNA tospovirus on crops necessitate the development of transgenic resistance. Here, we constructed a two-T DNA vector carrying a hairpin of the intergenic region (IGR) of Ageratum yellow vein virus (AYVV), residing in an intron inserted in an untranslatable nucleocapsid protein (NP) fragment of Melon yellow spot virus (MYSV). Transgenic tobacco lines highly resistant to AYVV and MYSV were generated. Accumulation of 24-nt siRNA, higher methylation levels on the IGR promoters of the transgene, and suppression of IGR promoter activity of invading AYVV indicate that AYVV resistance is mediated by transcriptional gene silencing. Lack of NP transcript and accumulation of corresponding siRNAs indicate that MYSV resistance is mediated through post-transcriptional gene silencing. Marker-free progenies with concurrent resistance to both AYVV and MYSV, stably inherited as dominant nuclear traits, were obtained. Hence, we provide a novel way for concurrent control of noxious DNA and RNA viruses with less biosafety concerns.

무선발 형질전환 Bt벼의 도입유전자 구조 분석

Abstract A less simple approach developed for generation of marker-free transgenic plants is to select transformants without the use of selective marker genes. Some results about development of marker-free transgenic plants were obtained using a non-selective approach in several crops such as rice, potato and tobacco. However, the study did not provide evidence on detailed characterization of introduced gene on genome, a critical step for confirming the stable integration and transmission of a foreign gene. In this study, we evaluated structure and integration sites of transgene (m Cry1Ac ) in the transgenic Bt rice plants which were made via conventional Agrobacterium -mediated transformation by non-selective method. Structure and integration sites of transgene in these transgenic plants had similar fashion as those recovered under selection. Keywords Non-selective, Marker-free transgenic rice, Agrobacterium -mediated transformation, Transgene, integration site 서 론 선발마커유전자(Selective marker gene)는 목표유전자와 연결하여, 도입유전자가 형질전환 된 식물체를 선택적으로 선발하는데 사용된다. 통상적으로

An efficient method for the production of marker-free transgenic plants of peanut (Arachis hypogaea L.)

Recombinant genes conferring resistance to antibiotics or herbicides are widely used as selectable markers in plant transformation for selecting the primary transgenic events. However, these become redundant once the transgenic plants have been developed and identified. Although, there is no evidence that the selectable marker genes are unsafe for consumers and the environment, it would be desirable if the marker genes can be eliminated from the final transgenic events. The availability of efficient transformation methods can enable the possibility of developing transgenic events that are devoid of the marker gene/s upfront. Taking advantage of the high and consistent transformation potential of peanut, we report a technique for developing its transgenics without the use of any selectable marker gene. Marker-free binary vectors harboring either the phytoene synthase gene from maize (Zmpsy1) or the chitinase gene from rice (Rchit) were constructed and used for Agrobacterium tumefaciens-mediated transformation of peanut. The putative transgenic events growing in vitro were initially identified by PCR and further confirmed for gene integration and expression by dot blots assays, Southern blots, and RT-PCR where they showed a transformation frequency of over 75%. This system is simple, efficient, rapid, and does not require the complex segregation steps and analysis for selection of the transgenic events. This approach for generation of marker-free transgenic plants minimizes the risk of introducing unwanted genetic changes, allows stacking of multiple genes and can be applicable to other plant species that have high shoot regeneration efficiencies.

Feasibility of the seed specific cruciferin C promoter in the self excision Cre/loxP strategy focused on generation of marker-free transgenic plants

This work is focused on the generation of selectable marker-free transgenic tobacco plants using the self excision Cre/loxP system that is controlled by a strong seed specific Arabidopsis cruciferin C (CRUC) promoter. It involves Agrobacterium-mediated transformation using a binary vector containing the gus reporter gene and one pair of the loxP sites flanking the cre recombinase and selectable nptII marker genes (floxed DNA). Surprisingly, an ectopic activation of CRUC resulting in partial excision of floxed DNA was observed during regeneration of transformed cells already in calli. The regenerated T(0) plants were chimeric, but no ongoing ectopic expression was observed in these one-year-long invitro maintained plants. The process of the nptII removal was expected in the seeds; however, none of the analysed T(0) transgenic lines generated whole progeny sensitive to kanamycin. Detailed analyses of progeny of selected T(0)-30 line showed that 10.2% GUS positive plants had completely removed nptII gene while the remaining 86.4% were still chimeras. Repeated activation of the cre gene in T(2) seeds resulted in increased rate of marker-free plants, whereas four out of ten analysed chimeric T(1) plants generated completely marker-free progenies. This work points out the feasibility as well as limits of the CRUC promoter in the Cre/loxP strategy.

Single-step transformation for generating marker-free transgenic rice using the ipt-type MAT vector system.

The ipt-type MAT vector uses the ipt gene for regeneration of marker-free transgenic plants. However, it was pointed out that this system was not suitable for most economically important crops that regenerated through auxin-dependent embryogenesis. We report a single-step transformation system of rice using MAT vector. When we transformed scutellum tissues of 5 days pre-cultured rice seeds, marker-free transgenic rice plants directly regenerated from 25.5% infected scutellum tissues without forming ipt-intermediates within 4 weeks after an infection. Excision of the ipt gene caused the regeneration of marker-free transgenic rice plants through embryogenic tissues. Therefore, this system needs no selective agent and no sexual crossing for identification of transgenic plants not containing a selectable marker gene. This system is highly effective for generation of marker-free transgenic plants in economically important crops.

Mat (Multi-Auto-Transformation) vector system. The oncogenes of Agrobacterium as positive markers for regeneration and selection of marker-free transgenic plants

We have developed a new transformation method called MATVS (Multi-Auto-Transformation Vector System). The oncogenes (ipt or rol genes) of Agrobacterium are used as selectable markers to regenerate transgenic cells and to select marker-free transgenic plants in the MATVS. The chimeric ipt gene or the rol genes are combined withthe site-specific recombination R/RS system to remove the oncogenes from the transgenic cells after transformation. We report here the application of MATVS to transformation of tobacco, aspen, rice and snapdragon. (I) The GST-MAT vector pMAT8 has the native ipt gene and the R gene with a chemical inducible promoter (GST-II-27). Use of the GST-MAT vector generated marker-free transgenic tobacco plants cotaining a single copy transgene at high frequency. (2) Use of the GST-MAT vector pRBI11 containing the rbcS 3B-ipt gene produced transgenic marker-free hybrid aspen plants without crossing. (3) Use of the ipt-type MAT vector, pNPI30GFP, containing the 35S-ipt and 35S-R, genes, resulted in the regeneration of marker-free transgenic reice plants directly from infected scutellum tisues at high frequency within 1 mo. (4) Use of the rol-type MAT vector pNPI702, containing the rol genes and the 35S-R gene, produced transgenic marker-free plants of tobacco and snapdragon at high frequency without crossing. Our results show that the promoter of the ipt gene, the preculture periods of plant tissues and the culture medium are important factors to improve the generation efficiency of marker-free transgenic plants. We can rapidly produce marker-free transgenic plants without the production of ipt-shooty intermediates. Therefore, it is a most promising method to save time and work for the generation of marker-free transgenic plants in crops.