Efficient Gene Stacking in Rice Using the GAANTRY System

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.

Keywords: Agrobacterium; Gene stacking; Genetic engineering; Oryza sativa; Site-specific recombinase.

Fig. 1

Composition of the 5-stack T -DNA. Diagram of an Agrobacterium rhizogenes virulence plasmid containing the 5-stack T-DNA. Cargo sequences, including the promoter, gene coding sequence and transcription terminator, are displayed as a colored arrow showing the orientation of transcription or a dark blue rectangle (TBS insulator). The T-DNA right border (RB) and left border (LB) locations are shown in green. Abbreviations are as follows: hptII, hygromycin phosphotransferase 2, Rluc (Renilla luciferase), TBS: Transformation Booster Sequence, Fluc (Firefly luciferase) and GUSPlus (β-glucuronidase encoding reporter gene). The gentamicin bacterial resistance marker (GmR) is shown outside the RB region of the T-DNA, within the native Agrobacterium virulence plasmid (pRi)

Fig. 2

Composition of the 11-stack T -DNA and PCR validation of cargo sequences. a Diagram of the GAANTRY 11-stack 37.4 kb T-DNA. See Fig. 1 legend for a description of the diagram. PCR products that bridge junctions between each cargo sequence are indicated by the numbered rectangles below the T-DNA. b Gel electrophoresis image of the PCR amplicons spanning each junction. Abbreviations are as follows: hptII (hygromycin phosphotransferase 2), Rluc (Renilla luciferase), TBS (Transformation Booster Sequence), Fluc (Firefly luciferase), GUSPlus (β-glucuronidase encoding reporter gene), bar (bialaphos resistance), eGFP (enhanced Green Fluorescent Protein), tdTom (tdTomato red fluorescent protein), EPSPS (5-enol-pyruvylshikimate-3-phospate synthase), and nptII (neomycin phosphotransferase 2)

Fig. 3

Representative phenotypes observed in the GAANTRY 11-stack T₁ rice plants. a Hygromycin sensitive wildtype Nipponbare (WT, left) and resistant (11-stack right) seedlings 7 days post germination. b Measured Renilla luciferase activity for 11-stack leaf samples. c Firefly luciferase activity for 11-stack leaf samples. The average background levels of activity detectable in wildtype leaf extracts is shown with the dashed line at 1.0 × 10³ units. d β-glucuronidase activity detected in green tissues and not in roots in a histochemically stained 7-day old 11-stack seedling. e Finale herbicide sensitivity in wildtype (left) and tolerance in 11-stack (right) leaves. f Observed green fluorescence localized in the root tips of a 11-stack rice plant. g The phenotypes of a wildtype (left) and 11-stack (right) seedlings germinated on media containing hygromycin and paromomycin antibiotics as well as glufosinate and glyphosate herbicides

Fig. 4

The GAANTRY 5-stack T-DNA and summary of the phenotypes and genotypes of the independent events. The structure of the 16.9 kb 5-stack GAANTRY T-DNA (top). PCR amplicons used to determine the presence of the T-DNA cargo sequences and the LB backbone sequence are indicated by the numbered rectangles below the T-DNA diagram. Amplicon 5* is fully within the GUSPlus cargo sequence, while the other amplicons span the junction between cargo sequences. The distance of each complete cargo sequence is from the right border (in kb) is shown below the diagram. Colored rectangles indicate the presence of the associated phenotype and/or genotype for each independent transgenic event. The asterisks (*) mark the events with a single copy of the hptII and GUSPlus transgenes. The plus sign (+) marks the event that contained sequence outside of the T-DNA beyond the left border

Fig. 5

The GAANTRY 11-stack T-DNA and a summary of the phenotypes and genotypes of the independent events. The GAANTRY 37.4 kb 11-stack T-DNA (top). PCR amplicons used to determine the presence of the T-DNA cargo sequences and the LB backbone sequence are indicated by the numbered rectangles below the T-DNA diagram. Amplicons marked with an asterisk are within a single cargo sequence, while those without, span junctions between cargo sequences. The distance of each complete cargo sequence is from the right border (in kb) is shown below the T-DNA diagram. Colored rectangles indicate the presence of the associated phenotype and/or genotype for each independent transgenic event. The asterisks (*) mark the events with a single copy of the hptII, GUSPlus and nptII cargo sequences

Fig. 6

Transgene copy number in the GAANTRY 5- and 11-stack events. The percent of total tested events with zero, one, two, three to four or more than four copies of the indicated transgenes (hptII, GUSPlus or nptII) is shown