Insertion and Characterization of the cry1Ia1 Gene in the Potato Cultivar Spunta for Resistance to Potato Tuber Moth

Potato tuber moth (Phthorimaea operculella) is a serious pest of potatoes in tropical and subtropical regions of the world, including South Africa. The cry1Ia1 gene (from Bacillus thuringiensis) under the control of the 35S cauliflower mosaic virus promoter was transformed into the potato (Solanum tuberosum) cultivar Spunta to develop a cultivar with resistance to potato tuber moth for release in South Africa. Two transformation events, ‘SpuntaG2’ and ‘SpuntaG3’, were selected and subjected to extensive molecular analyses as required by the regulatory agencies of South Africa. Southern hybridization experiments indicated that ‘SpuntaG2’ and ‘SpuntaG3’ had one and three copies of the cry1Ia1 gene, respectively, and that the gene insertion was stable through multiple clonal generations. Furthermore, the sequence of the cry1Ia1 gene in ‘SpuntaG2’ was compared with the known sequence of the cry1Ia1 gene and found to be identical. Polymerase chain reaction (PCR) amplification using primers for plasmid ‘‘backbone’’ genes demonstrated that ‘SpuntaG2’ contained no backbone plasmid genes, whereas ‘SpuntaG3’ contained several backbone plasmid genes. Therefore, further analyses were limited to ‘SpuntaG2’, and event-specific primers were developed for this cultivar. Analysis of the left and right border regions in ‘SpuntaG2’ demonstrated that the insertion of the cry1Ia1 gene did not disrupt any functional genes nor did it create new open reading frames that encoded proteins with a significant match to the non-redundant sequence database queried by the BLASTP program. Enzymelinked immunoabsorbent assays (ELISA) tests indicate that the cry1Ia1 gene was expressed at a mean concentration of 2.24 mg g fresh weight in leaf tissue and 0.12 mg g fresh weight in tubers. This study demonstrates the extensive molecular characterization that is necessary to apply for deregulation of a genetically modified crop and these data have been used in a regulatory package for the general release of ‘SpuntaG2’. Potato tuber moth is a serious pest of potatoes in South Africa, causing losses as much as $5.4 million per annum to the South African potato industry (Visser and Schoeman, 2004). Larval mining on the plant results in the loss of leaf tissue, death of growing points, and weakening or breakage of stems (Raman, 1980), all of which can reduce yield. Tuber mining results in tubers that are not marketable or consumable and increases tuber susceptibility to infection by potato pathogens in the field and in storage (Visser, 2004). Host plant resistance to potato tuber moth is highly desirable, and components of resistance such as glandular trichomes and acetylated glycoalkaloids (leptines) have been identified in wild potato species such as Solanum berthaultii, S. polyadenium, S. tarijense, and S. chacoense. However, attempts to introduce Received for publication 19 Jan. 2010. Accepted for publication 15 Apr. 2010. This project was supported by the U.S. Agency for International Development (USAID) and the Michigan Agricultural Experiment Station (MAES). We thank the employees of Envirologix (Portland, ME), including Bruce Ferguson, Terry Goddard, Whitney King-Buker, Karen Larkin, and Joan Lawton, for their antibody production and ELISA development expertise. Corresponding author. E-mail: [email protected]. J. AMER. SOC. HORT. SCI. 135(4):317–324. 2010. 317 these resistance components into cultivated potato have been difficult due to linkage with undesirable traits (Kalazich and Plaisted, 1991). At present, there is no potato germplasm produced from host plant resistance breeding that has appreciable levels of resistance to potato tuber moth (Lagnaoui et al., 2000). Therefore, commercial producers rely on insecticide application, generally applied at weekly intervals, for potato tuber moth control. Control is not always satisfactory, and damage levels vary between seasons and years, depending largely on the survival of over-wintering moths and their reinfestation of newly planted fields (Visser, 2004). Most small-scale farmers cannot afford or do not have access to insecticides. Furthermore, there are no insecticides registered for use in South Africa to control potato tuber moth under storage conditions, including Bacillus thuringiensis (Bt) sprays (Nel et al., 2002). This is problematic for small-scale farmers who store their potatoes in rustic buildings or diffused light storages, which are accessible by the potato tuber moths. Thus, the only insect pest management strategy that gives consistently complete control against the potato tuber moth in the field as well as storage is the use of genetically modified (GM), insect-resistant potatoes containing the cry1Ia1 gene (Visser, 2004). Bacillus thuringiensis is an aerobic, gram-positive soil bacterium that accumulates high levels of target-specific, insecticidal crystal proteins during sporulation. These proteins have no known detrimental effects on beneficial insects, mammals, or birds (Barton and Miller, 1993; McGaughey and Whalon, 1992). The introduction of the Bt toxin gene via genetic engineering produces host plant resistance for management of target insects. The major advantages of this delivery system are increased efficacy, reduced application costs, and minimal scouting needs compared with a conventional insecticide spray strategy (Lambert and Peferoen, 1992). Previous studies have shown that potatoes transformed with a codon-modified Btcry1Ia1 gene (previously classified as Bt-cryV ) have high levels of Bt expression, with 80% to 100% potato tuber moth mortality in detached leaf tests (Douches et al., 1998; Li et al., 1999). In an effort to develop a potato tuber moth resistant potato cultivar for humanitarian release in South Africa, a cry1Ia1 vector was developed and transformed into the potato cultivar Spunta. The public release of any GM crop is a highly regulated event and requires the rigorous molecular characterization of the transgenic event, detailed data on protein safety, and multiple years of agronomic trials (Codex Alimentarius, 2003; Koenig et al., 2004). At the molecular level, it is first necessary to characterize the organization of the inserted genetic material, including copy number and sequence data for the inserted material and the surrounding region (Codex Alimentarius, 2003). The inserted DNA must be sequenced to verify that it has not been modified from the known sequence in the vector and for future protein safety evaluations. The surrounding genome must be sequenced to determine if an existing open reading frame (ORF) has been altered or if a new ORF has been introduced by the insertion event. This sequencing also allows for the development of event-specific polymerase chain reaction (PCR) assays that are used to monitor the presence of GM crops in the food and animal feed supply once the GM crop is released for public use. Because each insertion event must be sequenced and evaluated and because multiple integrations events can adversely affect the transgene function (Jorgensen et al., 1996), it is preferable to identify and select a GM plant with only one copy of the transgene. T-DNA vector backbone sequences are frequently incorporated into the plant genome during Agrobacterium tumefaciens-mediated transformation (De Buck et al., 2000; Oltmanns et al., 2009). Therefore, it is necessary to determine if a GM plant carries backbone sequences and to select those that do not. Once a transgenic event has been selected, one must demonstrate that the transgene and its effect are inherited in a stable manner throughout several generations. Because of the cost involved in gaining regulatory approval for release of a GM crop, the private sector is responsible for the deregulation of nearly all of GM crops being grown worldwide. It is often necessary for public institutions/researchers to team with the private sector to pursue regulatory approval for release of a GM crop. A good example of this is the release of herbicide-tolerant soybeans (Glycine max) that were jointly developed by the Brazilian Agricultural Research Corp. (Brası́lia, Brazil) and the chemical company BASF , (Ludwigshafen, Germany). They were recently given approval for release by the Brazilian Biosafety Technical Commission (BASF, 2010). Golden rice (Oryza sativa) is another example; however, it has not yet been released (Golden Rice Humanitarian Board, 2009). Currently, the only example of a publicly released GM crop is papaya ringspot virus-resistant papaya (Carica papaya) that was developed and released for the Hawaiian industry (Gonsalves, 1998). The research presented here is the first in a series of three articles including Quemada et al. (2010) and Douches et al. (2010) documenting the extensive research necessary to apply for the release of a GM potato (‘SpuntaG2’) in South Africa. This project was funded by the U.S. Agency for International Development (Washington, DC) and is a collaborative effort between Michigan State University Potato Breeding and Genetics Program (East Lansing) and the Agricultural Research Council, Vegetable and Ornamental Plant Institute [(ARCVOPI), Pretoria, South Africa]. Materials and Methods CONSTRUCT DETAILS. The vector used for transformation of the potato cultivar Spunta was pSPUD5 [previously published as pBIML5 (Douches et al., 2002)], which contains a codonmodified version of the cry1Ia1 gene [previously called cryV in older literature (Douches et al., 1998)] obtained from Syngenta (Basel, Switzerland). In this vector, the cry1Ia1 gene is under the control of a 35S cauliflower mosaic virus (CaMV) promoter (Fig. 1) and the nptII gene is present as a selectable marker. The pSPUD5 vector was transformed into A. tumefaciens LBA4404. PLANT MATERIAL AND TRANSFORMATION. The potato cultivar Spunta was used for all transformations and the method of transformation was as reported in Douches et al. (1998). Rooted plantlets were transplanted into pots in the greenhouse. The leaves and tubers from these clones were used for molecular analyses and bioassays. DETERMINATION OF COPY NUMBER. Genomic DNA of selected ‘Spunta cry1Ia1’ and non-transgenic ‘Spunta’ plants was extracted from leaf tissue of greenhouse-grown plants according to Saghai-Maroof et al. (1984). The DNA was digested with the restriction enzyme XbaI (which recognizes a single restriction site between the T-borders) to assess T-DNA insert number. The resulting fragments were electrophoretically separated through a 1% (w/v) agarose gel and transferred to a nylon membrane (Hybond N; Amersham, Little Chalfont, 318 J. AMER. SOC. HORT. SCI. 135(4):317–324. 2010. England) for Southern analysis as described by Douches et al. (1998). A digoxygenin (DIG)-labeled random-primed probe was made from a commercial kit, using DIG-11dUTP as the label, and the entire cry1Ia1 gene as the template. Labeling was done according to the manufacturer’s instructions (Roche Applied Science, Indianapolis). For chemiluminescence detection, disodium 2-chloro-5-(4-methoxyspiro {1,2-dioxetane-3,2#-(5#-chloro) tricyclo[3.3.1.13,7 ] decan}-4-yl)-1phenyl phosphate CDP-Star (Roche) was used according to manufacturer’s instructions. The hybridized membrane was then exposed to X-Ray film (Hyperfilm, Amersham) to generate autoradiographs. STABILITY OF TRANSFORMATION EVENT. Further experiments were conducted to determine if the transformation event was stable from one clonal generation to another. The ‘SpuntaG2’ and ‘SpuntaG3’ lines used for the initial Southern analysis were maintained in tissue culture and went through 12 cycles of vegetative propagation before being exported to South Africa. The tubers imported into South Africa (SA Generation 1) were planted in the greenhouse at the ARC-VOPI and leaves (SA Generation 2) were collected for Southern analysis. Two further generations of vegetative propagation were conducted in South Africa and the leaves of SA Generation 4 were collected for Southern analysis as well. Untransformed ‘Spunta’ potato genomic DNA from two plants was included in the Southern blots to serve as a negative control. Genomic DNA extraction, probe preparation, and Southern analysis were conducted as indicated previously in this article. For each Southern, DNA was digested with XbaI (cuts once in the T-DNA region upstream of the cry1Ia1 gene) or NdeI (does not cut in the T-DNA region). SEQUENCE COMPARISON OF INSERTED DNA AND PSPUD5 TDNA. Plant genomic DNA was extracted from leaf tissue using the DNeasy Plant Mini Kit (69104; Qiagen, Valencia, CA) according to manufacturer’s instructions. The oligonucleotides used for amplification were prepared by Michigan State University’s Research Technology Support Facility (East Lansing) (Table 1). PCR amplifications were conducted using Taq DNA polymerase (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. A thermal cycler (model 9600; PerkinElmer, Wellesley, MA) was programmed for a hot start (95 C, 5 min) and 30 cycles of 95 C for 1 min, then placed at the appropriate temperature for each primer pair for 1 min, and 72 C for a 1 min 30 s extension, with a final extension of 72 C for 5 min. For clear sequencing, Table 1. Primer sequences used in the polymerase chain reaction (PCR) for amplification of the T-DNA region in the potato cultivar SpuntaG2. Primer sets Primer sequence Annealing