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Artificial chromosome platforms are described in plants. Because the function of centromeres is largely epigenetic, attempts to produce artificial chromosomes with plant centromere DNA have failed. The removal of the centromeric sequences from the cell strips off the centromeric histone that is the apparent biochemical marker of centromere activity. Thus, engineered minichromosomes have been produced by telomere mediated chromosomal truncation. The introduction of telomere repeats will cleave the chromosome at the site of insertion and attach the accompanying transgenes in the process. Such truncation events have been documented in maize, Arabidopsis, barley, rice, Brassica and wheat. Truncation of the nonvital supernumerary B chromosome of maize is a favorite target but engineered minichromosomes derived from the normal A chromosomes have also been recovered. Transmission through mitosis of small chromosomes is apparently normal but there is loss during meiosis. Potential solutions to address this issue are discussed. With procedures now well established to produce the foundation for artificial chromosomes in plants, current efforts are directed at building them up to specification using gene stacking methods and editing techniques., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Crop improvement is a never ending process. With a transgenesis approach, it is not inconceivable to envision a continuous addition of new transgenes to existing cultivars. Previously, we described a recombinase-directed gene stacking method in tobacco (Hou et al., Mol Plant 7:1756-1765, 2014). Being able to stack DNA to a previous location ensures that the number of genetic loci does not increase with each new round of transgene addition. Whereas the previous demonstration was conducted through polyethylene glycol to mediate uptake of DNA into tobacco protoplasts, we now describe protocols for using biolistic transformation to stack DNA in tobacco and rice.

The rapidly advancing field of plant synthetic biology requires transforming plants with multiple genes. This has sparked a growing interest in flexible plant transformation vectors, which can be used for multi-gene transformations. We have developed a novel binary vector series, named the PC-GW series (GenBank: KP826769-KP826773), for Agrobacterium-mediated plant transformation. The PC-GW vectors use the pCAMBIA vector backbone, and contain NPTII, hpt, bar, mCherry or egfp genes as selectable markers for plant transformation. In a modified multiple cloning site (MCS) of the T-DNA region, we have placed the attR1, attR2 and ccdB sequences for rapid cloning of one to four genes by Gateway™-assisted recombination. In addition, we have introduced four meganuclease sites, and other restriction sites for multi-gene vector construction. Finally, we have placed a CaMV 35S promoter and a 35S terminator on the 5' and 3' ends of the MCS. The CaMV 35S promoter is flanked by PstI restriction sites that can be used to replace it with another promoter sequence if needed. The PC-GW vectors provide choices for selectable markers, cloning methods, and can accommodate up to eight gene constructs in a single T-DNA, thereby significantly reducing the number of transformations or crosses needed to generate multi-transgene expressing plants., (Copyright © 2015. Published by Elsevier Inc.)

Engineered minichromosomes are small chromosomes that contain a transgene and selectable marker, as well as all of the necessary components required for maintenance in an organism separately from the standard chromosome set. The separation from endogenous chromosomes makes engineered minichromosomes useful in the production of transgenic plants. Introducing transgenes to minichromosomes does not have the risk of insertion within a native gene; additionally, transgenes on minichromosomes can be transferred between lines without the movement of linked genes. Of the two methods proposed for creating engineered minichromosomes, telomere-mediated truncation is more reliable in plant systems. Additionally, many plants contain a supernumerary, or B chromosome, which is an excellent starting material for minichromosome creation. The use of site-specific recombination systems in minichromosomes can increase their utility, allowing for the addition or subtraction of transgenes in vivo. The creation of minichromosomes with binary bacterial artificial chromosome vectors provides the ability to introduce many transgenes at one time. Furthermore, coupling minichromosomes with haploid induction systems can facilitate transfer between lines. Minichromosomes can be introduced to a haploid-inducing line and crossed to target lines. Haploids of the target line that then contain a minichromosome can then be doubled. These homozygous lines will contain the transgene without the need for repeated introgressions., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Species in genus Nannochloropsis, especially N. oceanica and N. gaditana, have been evolving as the model microalgae for both application and theory studies. The position effect of genome integration, the carrying capability limitation of integrative vectors and the instability of non-integrative vectors have hindered Nannochloropsis genetic modification with concatenate genes and extremely long DNA fragments. The molecular tools including genetic transformation, homologous recombination, gene edition, gene stacking and episome vectors for transient gene expression and diverse reporters and selection markers have been rapidly developing in Nannochloropsis species. The construction of animal and plant artificial chromosomes with "top down" strategy has set fine examples for the construction of Nannochloropsis artificial chromosomes (NannoACs). It seems that the methods and materials to set the foundation for constructing NannoACs are at hands. In this review, we outlined the current status of transgenes in Nannochloropsis species, summarized the limitations of both integrative and non-integrative vectors, and proposed a tentative approach to construct NannoACs by doubling and stabilizing the genome first, and then truncating the natural chromosomes. NannoACs once constructed will facilitate transferring the desired traits and concatenate genes into Nannochloropsis genetic backgrounds, thus contributing towards its genetic improvement and synthetic biological studies. [ABSTRACT FROM AUTHOR]

Transgene integration typically takes place in an easy-to-transform laboratory variety before the transformation event is introgressed through backcrosses to elite cultivars. As new traits are added to existing transgenic lines, site-specific integration can stack new transgenes into a previously created transgenic locus. In planta site-specific integration minimizes the number of segregating loci to assemble into a breeding line, but cannot break genetic linkage between the transgenic locus and nearby undesirable traits. In this study, we describe an additional feature of an in planta gene-stacking scheme, in which the Cre (control of recombination) recombinase not only deletes transgenic DNA no longer needed after transformation but also mediates recombination between homologous or non-homologous chromosomes. Although the target site must first be introgressed through conventional breeding, subsequent transgenes inserted into the same locus would be able to use Cre-mediated translocation to expedite a linkage drag-free introgression to field cultivars. [ABSTRACT FROM AUTHOR]

Genetic engineering of rice provides a means for improving rice grain quality and yield, and the introduction and expression of multiple genes can produce new traits that would otherwise be difficult to obtain through conventional breeding. GAANTRY (Gene Assembly in Agrobacterium by Nucleic acid Transfer using Recombinase technologY) was previously shown to be a precise and robust system to stably stack ten genes (28 kilobases (kb)) within an Agrobacterium virulence plasmid Transfer-DNA (T-DNA) and obtain high-quality Arabidopsis and potato transgenic events. To determine whether the GAANTRY system can be used to engineer a monocotyledonous crop, two new T-DNA constructs, carrying five (16.9 kb) or eleven (37.4 kb) cargo sequences were assembled and transformed into rice. Characterization of 53 independent transgenic events demonstrated that more than 50% of the plants carried all of the desired cargo sequences and exhibited the introduced traits. Additionally, more than 18% of the lines were high-quality events containing a single copy of the introduced transgenes and were free of sequences from outside of the T-DNA. Therefore, GAANTRY provides a simple, precise and versatile tool for transgene stacking in rice and potentially other cereal grain crops. [ABSTRACT FROM AUTHOR]

• Nanoparticle-based genetic transformation can transiently overexpress transgenes in intact tissues of Cannabis sativa. • Nanoparticle-based genetic transformation can simultaneously overexpress multiple transgenes. • Cationic polymer-modified silicon-coated gold nanoparticles are an effective carrier to introduce foreign DNA in Cannabis tissues. Cannabis sativa (Cannabis) is a multipurpose plant species consisting of specific lineages that for centuries has either been artificially selected for the production of fiber or the psychoactive drug Δ9-tetrahydrocannabinol (THC). With the recent lifting of previous legal restrictions on consuming Cannabis, there has been a resurgence of interest in understanding and manipulating Cannabis genetics to enhance its compositions. Yet, recently developed approaches are not amenable to high-throughput gene stacking to study multi-genic traits. Here, we demonstrate an efficient nanoparticle-based transient gene transformation protocol where multiple gene plasmids can be expressed simultaneously in intact Cannabis leaf cells in a very short time (5 days). Constructs encoding two soybean transcription factors were co-grafted onto poly-ethylenimine cationic polymer-modified silicon dioxide-coated gold nanoparticles (PEI-Au@SiO 2). Infiltration of the DNA-PEI-Au@SiO 2 into Cannabis leaf tissues resulted in the transcription of both soybean genes and the localization of fluorescent-tagged transcription factor proteins in the nuclei of Cannabis leaf cells including the trichomes, which are the cell types that biosynthesize valuable cannabinoid and terpene metabolites. Our study exemplifies a rapid transient gene transformation approach that will be useful to study the effects of gene stacking in Cannabis. [ABSTRACT FROM AUTHOR]

Transgenic crops that produce insecticidal toxins from the bacterium Bacillus thuringiensis Berliner ( Bt) were first commercialised in 1996. The risk that pests have the potential to evolve resistance to Bt toxins is one of the most serious challenges to this technology. Gene stacking, pyramiding two Bt genes into one variety, is considered to be an effective insect resistance management (IRM) strategy. In this study, insect-resistant rice expressing two Bt genes was developed by sexual crossing, and then characterised. Homozygous rice lines of two pyramided Bt genes were obtained in the F generation. Quantification of Bt toxin showed that protein concentrations of Cry1Ab, Cry1Ac and Cry2A in the two-gene lines were comparable with their single-gene parents, while the expression of cry1C gene decreased after gene stacking. Four two-gene lines showed higher activity to striped stem borer ( Chilo suppressalis Walker) than parental lines in the laboratory bioassay. All pyramided lines and their hybrids exhibited excellent efficacy against stemborers and leaffolders in field evaluation, while most pyramided lines had no significant differences from original variety in yield under spraying of insecticide. These results demonstrate that the two-gene lines have commercial potential and could serve as a valuable IRM strategy. Copyright © 2011 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Abstract: Gene flow between coexisting oilseed rape varieties and rape volunteers cannot be solely studied on field experiments. Previously, a model (GeneSys: [Colbach, N., Clermont-Dauphin, C., Meynard, J.M., 2001. GeneSys: A model of the inluence of croping system on gene escape from herbicide-tolerant rapeseed crops to rape volunteers. I. Temporal evolution of a population of rapeseed volunteers in a field. Agric. Ecosyst. Environ. 83, 235–253; Colbach, N., Clermont-Dauphin, C., Meynard, J.M., 2001. GeneSys: A model of the inluence of croping system on gene escape from herbicide-tolerant rapeseed crops to rape volunteers. II. Genetic exchanges among volunteer and cropped populations in a small region. Agric. Ecosyst. Environ. 83, 255–270]) was therefore developed to quantify the effects of cropping systems on gene flow from rapeseed varieties to rape volunteers in time and in space. In the present work, the model was improved to simulate simultaneously the spread of three different genes. Four different regions were then surveyed from 1995 to 2001: one region comprised only farmers’ fields with frequent rape crops in the rotations; the three remaining regions comprised the French experimental GMO platforms with 2–3 GM and one non-GM rape variety as well as neighbour farmers’ fields. For all these fields, cropping history was recorded during the study and for at least three previous years. In the first region, rapeseed volunteers were assessed in crops as well as on road and field margins in 1 year. In the three remaining regions, volunteer densities and genotypes were assessed each year after harvest and rape harvest genotypes determined during 2 years. The comparison of simulated and observed data showed that the model usually satisfactorily predicts volunteer densities and genotype proportions as well as harvest genotype proportions. Several problem areas were identified: volunteers in road and field margins were usually badly predicted; the model frequently underestimated volunteer densities in spring crops; gene flow via pollen dispersal was underestimated when the distance from the pollen source increased. The model was then used to simulate the spread of herbicide-tolerant rape volunteers in a region grown with one or several GM herbicide-tolerant varieties and more particularly the stacking of several resistance genes in individuals. The simulations also showed the contamination of the harvest of non-GM rape varieties by GM seeds resulting from gene flow. [Copyright &y& Elsevier]