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Plant biotechnology provides a means for the rapid genetic improvement of crops including the enhancement of complex traits like yield and nutritional quality through the introduction and coordinated expression of multiple genes. GAANTRY (gene assembly in Agrobacterium by nucleic acid transfer using recombinase technology) is a flexible and effective system for stably stacking multiple genes within an Agrobacterium virulence plasmid transfer DNA (T-DNA) region. The system provides a simple and efficient method for assembling and stably maintaining large stacked constructs within the GAANTRY ArPORT1 Agrobacterium rhizogenes strain. The assembly process utilizes unidirectional site-specific recombinases in vivo and an alternating bacterial selection scheme to sequentially assemble multiple genes into a single transformation construct. A detailed description of the procedures used for bacterial transformation, selection, counter selection, and genomic PCR validation with the GAANTRY system are presented. The methods described facilitate the efficient assembly and validation of large GAANTRY T-DNA constructs. This powerful, yet simple to use, technology will be a convenient tool for transgene stacking and plant genetic engineering of rice and other crop plants.

Conventional methods of DNA sequence insertion into plants, using Agrobacterium-mediated transformation or microprojectile bombardment, result in the integration of the DNA at random sites in the genome. These plants may exhibit altered agronomic traits as a consequence of disruption or silencing of genes that serve a critical function. Also, genes of interest inserted at random sites are often not expressed at the desired level. For these reasons, targeted DNA insertion at suitable genomic sites in plants is a desirable alternative. In this paper we review approaches of targeted DNA insertion in plant genomes, discuss current technical challenges, and describe promising applications of targeted DNA insertion for crop genetic improvement.

Abstract 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.

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]

Summary: Biotechnology provides a means for the rapid genetic improvement of plants. Although single genes have been important in engineering herbicide and pest tolerance traits in crops, future improvements of complex traits like yield and nutritional quality will likely require the introduction of multiple genes. This research reports a system (GAANTRY; Gene Assembly in Agrobacterium by Nucleic acid Transfer using Recombinase technologY) for the flexible, in vivo stacking of multiple genes within an Agrobacterium virulence plasmid Transfer‐DNA (T‐DNA). The GAANTRY system utilizes in vivo transient expression of unidirectional site‐specific recombinases and an alternating selection scheme to sequentially assemble multiple genes into a single transformation construct. To demonstrate GAANTRY's capabilities, 10 cargo sequences were sequentially stacked together to produce a 28.5‐kbp T‐DNA, which was used to generate hundreds of transgenic events. Approximately 90% of the events identified using a dual antibiotic selection screen exhibited all of the introduced traits. A total of 68% of the tested lines carried a single copy of the selection marker transgene located near the T‐DNA left border, and only 8% contained sequence from outside the T‐DNA. The GAANTRY system can be modified to easily accommodate any method of DNA assembly and generate high‐quality transgenic plants, making it a powerful, yet simple to use tool for plant genetic engineering. [ABSTRACT FROM AUTHOR]

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 Pst I 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. [ABSTRACT FROM AUTHOR]

Hevea brasiliensis (Euphorbiaceae) is the major source of the world natural rubber. Susceptibility of Hevea brasiliensis to various biotic and abiotic stresses and incidence of tapping panel dryness are the major constraints in natural rubber production. Gene stacking permits the integration of multiple genes for complex traits simultaneously into the target plant. In the present study, gene stacking was achieved by repeated genetic transformation for the integration of two genes, manganese superoxide dismutase (MnSOD) for abiotic stress tolerance and 3-hydroxy-3-methyl-glutaryl-CoA reductase (hmgrI) for enhanced latex yield. Initially, using the embryogenic callus derived from immature zygotic embryo as the target tissue, Agrobacterium mediated transformation by vacuum infiltration was carried out with the binary vector harboring MnSOD gene and nptII as the selectable marker gene. High frequency (30%) transformation was obtained and was detected by GUS histochemical staining and PCR using MnSOD gene specific primer. This MnSOD transgenic callus was used as the target tissue for the integration of hmgrI gene containing hpt as the selectable marker gene. PCR using hpt gene specific primer was performed for the detection of hmgrI gene integration. Somatic embryo induction (32%) was achieved from the multiple gene integrated callus lines in modified MS medium with BA and Kin (0.3 mg l-1 each) and GA3 (0.5 mg l-1). Fifteen percent of the embryos were matured and were further cultured for plant regeneration. This is the first report on multiple gene integration in Hevea brasiliensis. [ABSTRACT FROM AUTHOR]

The ability to create artificial gene-clusters for genetic transformation could facilitate the development of crops with multiple engineered traist, or with traits which result from the expression of multiple genes. A simple method to assemble artificial gene-clusters was developed by designing a multiple cloning site consisting of an array of homing endonuclease cleavage sites into a single vector. These enzymes are also known as intron-or intein-encoded endonucleases, and have very long recognition sequences, which makes them very rare cutters. The resulting vectors are pUGA for microprojectile-mediated transformation, and pUGA2 for Agrobacterium-mediated transformation. In addition, a series of unidirectional shuttle vectors containing various combinations of homing endonuclease restriction sites was constructed. Gene cassettes can be cloned into individual shuttles, and then transferred to either pUGA or pUGA2 to construct artificial gene-clusters. To test the feasibility of this approach, a six-gene cluster was constructed and transformed into soybean via microprojectile bombardment and into tobacco via Agrobacterium. The genes were assayed for expression in both the T0 and T1 generations for three independent transgenics. Up to five of the six genes were expressed. Additional changes to the construction of individual gene cassettes may improve the frequency with which all genes in the cluster are expressed. [ABSTRACT FROM AUTHOR]