Transgene stacking in potato using the GAANTRY system

Objective 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). We examined the ability of the GAANTRY Agrobacterium rhizogenes ArPORT1 ‘10-stack’ strain to generate transgenic potato plants. Results The 28.5 kilobase 10-stack T-DNA, was introduced into Lenape potato plants with a 32% transformation efficiency. Molecular and phenotypic characterization confirmed that six of the seven tested independent transgenic lines carried the entire desired construct, demonstrating that the GAANTRY 10-stack strain can be used can be used in a tissue culture-based callus transformation method to efficiently generate transgenic potato plants. Analysis using droplet digital PCR showed that most of the characterized events carry one or two copies of the 10-stack transgenes and that ‘backbone’ DNA from outside of the T-DNA was absent in the transgenic plants. These results demonstrate that the GAANTRY system efficiently generates high quality transgenic potato plants with a large construct of stacked transgenes. Electronic supplementary material The online version of this article (10.1186/s13104-019-4493-8) contains supplementary material, which is available to authorized users.


Introduction
Agrobacterium are soil microbes that have been harnessed for their ability to transfer DNA into plant cells [1,2]. This technique has revolutionized agriculture, providing a way to identify and test gene functions and to transfer superior trait genes into crops without the added and often unwanted genes that come along during breeding schemes. An important aspect associated with the transfer of DNA into plants is the stability of the insertion event. Agrobacterium T-DNAs can sometimes be incomplete or concatenated repeats [3], exhibit genetic instability or gene silencing [4]. Agrobacterium-mediated transformation of plants with one or a few genes is relatively routine, but the assembly and transformation of large constructs carrying multiple genes and their efficient use to generate high-quality transgenic plants has been a challenge.
Previous research developed the GAANTRY (Gene Assembly in Agrobacterium by Nucleic acid Transfer using Recombinase technologY) system for transgene stacking [5]. The system is based on the combined use of unidirectional integration and excision controlled by three site-specific serine recombinases and has been shown to be an effective and stable system for stacking multiple genes within an Agrobacterium virulence plasmid T-DNA [5,6]. The gene stacking system utilizes easy-to-handle 'P Donor' and 'B Donor' cloning vectors for the insertion of sequences of interest. The P and B Donor vectors contain either attP or attB recognition sites enabling precise integration into the GAANTRY ArPORT1 strain. The resulting Agrobacterium strain can then be directly used for plant transformation. The gene stacking strategy is efficient, precise, modular, and allows control over of the orientation and order in which genes are stacked within the T-DNA. The stacking process was previously demonstrated to successfully assemble a 28.5 kb 10-stack T-DNA construct containing ten cargo sequences, including eight transgenes that confer
We wanted to examine the ability of the GAANTRY ArPORT1 10-stack strain to produce stable clean single copy transgenic events in the important crop species of potato. Potato is a member of the Solanaceae family, which contains multiple other crop species and utilizes a tissue culture-based callus transformation method, which is distinctly different than the floral dip transformation method used for Arabidopsis [5].
We present the utilization of the GAANTRY transgene stacking system to produce genetically engineered Lenape potato plants carrying the 28.5 kb '10-stack' T-DNA. Multiple independent transgenic events were produced, phenotypically and molecularly characterized. Our analysis evaluated both the fidelity and completeness of T-DNA integration, and the copy number of the inserted sequences within the potato genome.

Results and discussion
The GAANTRY ArPORT1 10-stack strain was previously assembled and validated [5]. Details and the GenBank accessions for the donor plasmids carrying the promoter and gene sequences assembled within the 10-stack assembly are described in Additional file 2: Table S1. A simplified diagram of the 28.5 kb 10-stack T-DNA is shown in Fig. 1a.
The 10-stack GAANTRY strain was used to transform potato B5141-6 (cv. Lenape) in two independent transformation experiments. A total of 86 internodes were co-cultivated with the ArPORT1 10-stack strain. A total of 28 shoots regenerated and rooted under kanamycin selection. Resistance to kanamycin is conferred by the nptII transgene, which is located adjacent to the right border (RB) of the T-DNA (Fig. 1a). Nodal cuttings from the rooted kanamycin resistant plants were excised and rooted on media containing the sulfadiazine selection agent (resistance is conferred by sul1, located adjacent to the Left Border (LB) of the T-DNA). Twenty-five plants survived and rooted in the presence of sulfadiazine, indicating the presence and expression of the sul1 resistance gene in 89% of the transgenic plants that were recovered.
The overall transformation rate based on kanamycin selection was 32.6% and it was 29.1% if both kanamycin and sulfadiazine resistance was required. Ten randomly selected kanamycin-resistant independent T 0 transgenic events were transferred to soil and grown in the greenhouse. Seven of the plants successfully grew to maturity and were further characterized.
The selection marker and reporter gene phenotypes of these seven transgenic potato plants were further examined. All seven plants exhibited resistance to kanamycin in tissue culture media (Fig. 1b) and produced leaves with detectable red fluorescence (Fig. 1c). Six of the events (85%) were positive for β-glucuronidase histochemical staining of leaves (Fig. 1d) and these same six events exhibited resistance to sulfadiazine by successfully rooting on sulfadiazine containing tissue culture media (Fig. 1e), but line 3 was sensitive to sulfadiazine and did not root. Luciferase enzyme activity was detected in in leaf protein extracts from six of the events, but line 3 lacked detectable activity (Fig. 1f ).
The presence of other portions of the 10-stack T-DNA was examined using PCR amplification of the transgene sequences using genomic DNA isolated from each transgenic event as template. The analysis illustrates that the mybA and uidA transgenes were detected in all of the seven tested lines, while the bar and eGPF transgenes were detected in six of the tested events, but not within line 3 (Fig. 2).
The nptII transgene copy number was measured for the seven selected transgenic events using droplet digital PCR [16]. Five of these events (71%) carried a single copy of the nptII transgene, while two events (29%) carried two copies (Additional file 1: Fig. S1). The transgene copy number for the sul1 gene from these seven events was also determined, and the results show that four of the plants (57%) were single copy, two carried two copies (29%) and one event (line #3; 14%) lacked the sul1 transgene (Additional file 1: Fig. S1).
The presence of sequences from outside of the T-DNA construct was also examined using genomic PCR screening. The analysis did not detect the presence of 'backbone' sequence from outside of the left border region in any of the seven transgenic lines, despite the fact that two of these lines contained two copies of sul1 gene located adjacent to the Left Border of the T-DNA (Fig. 2e and Additional file 1: Fig. S1).
Taken together, these results suggest that four of the seven lines likely carried a single complete copy of the 28.5 kb 10-stack T-DNA construct. Two of the independent events appeared to carry two complete T-DNA copies, and a single event (line #3) contains only a partial T-DNA integration which lacks the sul1, luciferase, eGFP, and bar transgenes.
Together these results indicate that 4 out of 7 (57%) of the transgenic lines likely carry an intact, backbone free, single copy T-DNA integration demonstrating that the GAANTRY system can be used in tissue culture-based transformation method for the production of transgenic potato with stacked gene constructs.
Rates of potato transformation for this study appeared to be somewhat lower than what we typically observe for potato transformation. This may be due to the larger size of the 10-stack T-DNA and/or characteristics of the ArPORT1 strain in potato transformation. Both the high rate of single copy transgenic production (57% of the tested lines) and the lower rates of transformation may be because the GAANTRY T-DNA is launched directly from the low copy virulence plasmid, rather than a higher copy binary vector. The study by Oltmanns et al. [17] launched the T-DNA from the picA locus of the Agrobacterium tumefaciens chromosome and also recovered a lower transformation rate than observed for a binary vector. The lower transformation rate of the GAANTRY system is not a significant problem, since multiple highquality transgenic events were recovered within the small population of seven transgenic events that were analyzed.
Genetic engineering offers a powerful way to introduce or modify complex traits within crop plants. The GAANTRY system enables the assembly and maintenance of multi-gene stacked constructs and can produce high quality transgenic events with low copy number Luciferase Units Potato transgenic event number transgene integrations that are free of sequences outside of the T-DNA in both Arabidopsis [5], and potato. Thus, the ArPORT1 10-stack GAANTRY strain provides an effective technique to generate and efficiently introduce multiple genes into crop plants like potato and suggests that the GAANTRY system will likely be useful in other Solanaceous plants as well.

Methodology
Potato (Solanum tuberosum) B5141-6, the variety formerly known as Lenape [18] were maintained in a tissue culture chamber at 23 °C, 16 h light and grown in a greenhouse in Sunshine Mix #1 (Sungro Horticulture). Plant material was micropropagated in tissue culture on Shoot Media (Table 1) from excised 1 or 2 node segments (1 cm in size) when plantlets reached approximately 10 cm in height, or every 6-8 weeks.