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. 2026 Jul 14;7(4):100078. doi: 10.1016/j.abiote.2026.100078

Engineering potato inbred lines expressing Cry1Ac and Cry3A for dual resistance to potato tuber moth and Colorado potato beetle

Doudou Yang a,1, Weichao Lin a,1, Zhongmin Yang b,1, Deshan Zhou a,c, Juan Liu a, Jie Cheng a, Yuqin Zheng a, Yulin Gao d, Zhengqian Luo f, Linli Xu f, Chunzhi Zhang a, Jinzhe Zhang e, Enliang Liu f,⁎, Suhua Li a,⁎⁎
PMCID: PMC13445969  PMID: 42565032

Abstract

Potato (Solanum tuberosum) is critical for global food security. However, potato production is severely constrained by two major pests, potato tuber moth (Phthorimaea operculella, PTM) and Colorado potato beetle (Leptinotarsa decemlineata, CPB), which can cause catastrophic yield losses of up to 90% under outbreak conditions. Developing durable, broad-spectrum resistance within genetically tractable breeding systems poses a major challenge to potato improvement. Here, we established an effective dual-resistance strategy centered on a diploid inbred platform by co-expressing codon-optimized Bacillus thuringiensis (Bt) genes cry1Ac and cry3A in the self-compatible diploid inbred line A6-56 (Solanum tuberosum Group Stenotomum), as well as various tetraploid cultivars, via Agrobacterium-mediated transformation. The engineered diploid line exhibited strong and stable resistance to both PTM and CPB, providing a simplified, genetically stable system for trait integration compared with using conventional tetraploid backgrounds. Crucially, reciprocal crosses between the transgenic diploid line and another inbred line, E4-63 (Solanum tuberosum Group Phureja), produced F1 progeny that retained strong insect resistance and consistent transgene expression. These results demonstrate the stable Mendelian inheritance and functional conservation of Cry1Ac and Cry3A, underscoring the unique advantage of using diploid inbred lines for precise trait transmission and rapid deployment in breeding programs. Our findings provide both a powerful breeding resource and a scalable strategy for achieving durable, broad-spectrum insect resistance, which should advance the development of efficient, predictable, sustainable potato improvement systems.

Keywords: Potato inbred lines, Plant resistance breeding, cry1Ac, cry3A, Potato pests

1. Introduction

Potato (Solanum tuberosum L.) is a staple food ranked as the third most valuable crop for direct human consumption worldwide after rice (Oryza sativa) and wheat (Triticum aestivum) [1]. In China, the world's largest producer and consumer of potatoes, this crop is cultivated across four major agro-ecological zones: the Northern Single Cropping Region, the Central Double Cropping Region, the Southern Winter Cropping Region, and the Southwestern Mixed Cropping Region (Fig. S1) [2]. The major tetraploid cultivars planted in these regions include Atlantic, Jizhangshu No. 12 (JZS12), Longshu No. 7 (LS7), and Qingshu No. 9 (Q9). Nevertheless, potato production faces substantial constraints from various biotic and abiotic stress factors, which can reduce yields by 18% to 32% [3]. Among these, insect pests pose a persistent threat, damaging both potato yield and quality through direct herbivory and the transmission of plant pathogens [4].

The potato tuber moth (Phthorimaea operculella Zeller, PTM), a lepidopteran pest with high fecundity and wide distribution, damages potato foliage and tunnels into tubers. Severe infestations lead to yield losses of 85% to 100%. Originally native to the tropical mountains of South America, PTM has now spread to all tropical and subtropical regions worldwide, becoming a major threat to potato production in warmer climates [4]. In China, PTM was first reported in Liuzhou, Guangxi Province in 1937. Since then, PTM has expanded to all major potato-growing areas across the country, with particularly severe impacts observed in Yunnan, Guizhou, and Sichuan provinces (Fig. S1) [5,6]. Another highly destructive insect pest of potato is Colorado potato beetle (Leptinotarsa decemlineata Say, CPB). Both adults and larvae can completely devour the leaves of an entire potato plant, typically causing yield reductions of 30%–50%; under heavy infestation, yield losses can exceed 90% [7]. CPB was first recorded in the western United States in 1811. CPB experienced an initial major outbreak in Nebraska in 1859 and has since established itself across North America, Europe, and Asia [4]. Since its initial invasion into China in 1993, CPB has expanded its distribution to 42 counties across Xinjiang Uygur Autonomous Region and Heilongjiang Province, with an estimated annual range expansion rate of 100–200 km/year (Fig. S1) [8,9]. Moreover, major potato-growing regions in China substantially overlap with climatically suitable habitats for CPB [10], and some regions are also heavily affected by PTM infestations (Fig. S1). Therefore, an integrated management strategy targeting both PTM (Lepidoptera) and CPB (Coleoptera) is necessary.

The use of chemical insecticides is currently the most common strategy for managing potato pests. However, the widespread application of these chemicals has led to significant environmental pollution, the rapid development of insect resistance, and potential risks to human health [11]. Bacillus thuringiensis (Bt), a widely distributed Gram-positive bacterium, produces insecticidal parasporal crystal proteins during sporulation [12,13]. These proteins, which are primarily classified into families such as Cry, Cyt, and Vip, exhibit activity against specific insect orders, establishing them as a cornerstone of biological pest control and a key component of agricultural pest management worldwide [14]. Bt Cry proteins such as Cry1Ac and Cry3A are toxic to lepidopteran and coleopteran insect pests, respectively [15]. Estrada et al. conducted detached-leaf bioassays and tuber bioassays using two potato lines (ND5873 and Spunta) transformed with cry1Ac and reported 97% to 99% mortality against PTM [16]. Similarly, Amiri and Bakhsh generated cry1Ac transgenic potato lines (cv. Marabel) and observed 50% to 100% mortality against PTM larvae [17]. In a recent study, transgenic potatoes expressing Cry1C and Cry2A showed almost 100% mortality against PTM larvae [18]. In addition, transgenic potatoes expressing cry3A provided effective control against CPB [[19], [20], [21]]. Cry3B, Cry3Ca, and Cry1Ba/Cry1Ia also exhibit insecticidal activity against CPB [22,23].

Most commercial potato cultivars are autotetraploid. Therefore, tetrasomic inheritance and clonal propagation via tubers are currently the most common approaches to potato breeding and cultivation. However, the complexity of tetrasomic inheritance and the reliance on vegetative propagation have long posed significant challenges for potato researchers and breeders [24]. The re-domestication of diploid potatoes using advanced technologies such as whole-genome design would represent a breakthrough in potato breeding [[25], [26], [27]]. However, most tuber-bearing potato species are self-incompatible, which has hampered the development of inbred lines. Significant progress has been made in reinventing potato as a seed-propagated diploid crop, largely by overcoming self-incompatibility and inbreeding depression [9,[27], [28], [29]]. However, whether diploid potato inbred lines expressing Bt proteins could be generated remains unexplored.

In this study, we systematically investigated the insecticidal efficacy of Cry1Ac (effective against PTM), Cry3A (effective against CPB), and their combination against PTM and CPB larvae at different stages of development by transforming both diploid inbred and cultivated tetraploid potato with genes encoding these proteins. Using A6-56, a diploid inbred line with high genetic transformation efficiency, we generated a transgenic line expressing cry1Ac and cry3A and crossed this line with inbred line E4-63 through seed propagation to generate F1 progeny. These plants exhibited strong resistance to CPB and PTM, demonstrating the heritability of insect resistance. Therefore, strategically combining distinct Bt proteins is an effective approach for enhancing insect resistance and broadening the range of pest control in crops. The use of a diploid potato breeding system could accelerate the development of potato with enhanced pest resistance. The Bt-transgenic potato lines generated in this study effectively control major potato pests, offering important genetic resources for potato breeding to help ensure successful potato production.

2. Results

2.1. Heterologous expression of cry1Ac and cry3A in cultivated potato confers lethal effects against PTM and CPB, respectively

To investigate the role of cry1Ac in insect resistance, we introduced the pCHF3 vector containing a codon-optimized cry1Ac gene driven by the constitutive CaMV 35S promoter into potato cultivar Atlantic. Of the 12 independent transformation events generated, six lines tested positive for both nptII and cry1Ac by PCR (Fig. S2A). We conducted reverse-transcription–quantitative PCR (RT-qPCR) to identify lines with relatively high cry1Ac expression (Fig. S2B). We selected the two lines with the highest cry1Ac transcript levels (Atlantic_1Ac#7 and Atlantic_1Ac#9) for further analysis. To quantify Cry1Ac protein accumulation, we performed ELISA. Cry1Ac protein levels reached 9.62 and 16.39 ng/g fresh leaf weight in Atlantic_1Ac#7 and Atlantic_1Ac#9, respectively, whereas no detectable protein was observed in wild-type Atlantic (Fig. 1A). To evaluate resistance to herbivory, first-instar PTM larvae were allowed to feed on fresh leaves from the transgenic plants for 10 days. The surviving larvae on Atlantic_1Ac lines #7 and #9 were severely stunted, with altered morphology compared to those feeding on control Atlantic plants (Fig. 1B), and the larval survival rate was reduced to nearly 10% on these lines (Fig. 1C). These results demonstrate that heterologous expression of cry1Ac in potato confers robust resistance against PTM by significantly increasing larval mortality and severely suppressing larval growth.

Fig. 1.

Fig. 1

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Identification of potato cultivars harboring Cry1Ac or Cry3A and bioassay of their activities against PTM and CPB. A Cry1Ac levels in the leaves of Atlantic_1Ac transgenic lines. B Phenotypes of PTM larvae after feeding on the leaves of Atlantic and Atlantic_1Ac plants for 10 days. Bar = 0.5 cm. C Survival rates of PTM larvae after feeding on the leaves of Atlantic and Atlantic_1Ac plants for 15 days. D Cry3A contents in two Atlantic_3A transgenic lines. E Phenotypes of CPB after feeding on Atlantic and Atlantic_3A plants for 4 days. Bar = 0.5 cm. F-G Survival curves of CPB larvae after feeding on the leaves of Atlantic (F), JZS12, LS7, QS9 (G), and the respective transgenic plants harboring Cry3A protein. All data are presented as mean ± standard error (SE), with n = 30 larvae per treatment. Statistical significance was determined using a two-tailed Student's t-test: ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.

To confer resistance against another notorious potato pest, CPB, we generated a separate set of transgenic lines overexpressing a codon-optimized cry3A gene in multiple cultivated potato varieties. For Atlantic_3A, we obtained 20 independent transformation events, 18 of which harbored both nptII and cry3A, as determined by PCR. We selected Atlantic_3A#12 and Atlantic_3A#17 for further analysis based on their relatively high cry3A transcript levels, with their Cry3A contents measured about 150 ng/g and 715 ng/g, respectively. (Fig. 1D, Fig. S2C and D). In a four-day feeding assay with second-instar larvae, these transgenic lines significantly reduced larval feeding and severely inhibited the growth of the surviving larvae (Fig. 1E, Fig. S3A), with 40–83% mortality by day 2 compared with the wild-type control (Fig. 1F). In addition to larval toxicity, cry3A expression strongly deterred adult CPB feeding. Adults displayed minimal feeding on Atlantic_3A#12 and Atlantic_3A#17 over four consecutive days (Fig. S3B). We also generated cry3A-expressing transgenic lines in three other tetraploid cultivar backgrounds: JZS12, LS7, and Q9 (Fig. S2C and D, Fig. S3C). Consistent with the results observed in Atlantic, these lines also significantly suppressed larval growth, increased larval mortality, and reduced adult feeding upon CPB attack (Fig. 1G, Fig. S3D). These results demonstrate that heterologous cry3A expression confers robust and consistent resistance to both larval and adult CPB in potato across diverse genetic backgrounds.

2.2. Co-expressing cry1Ac and cry3A in potato inbred lines enhances resistance to both lepidopteran and coleopteran pests

Based on the resistance of these transgenic lines against PTM and CPB, we constructed a binary vector harboring both cry1Ac and cry3A (Fig. 2A) and generated transgenic potato plants co-expressing both genes in the self-compatible diploid inbred line A6-56 (Solanum tuberosum group Stenotomum) background. This potato line is capable of self-pollination, allowing it to propagate through true seeds, and is amenable to genetic transformation. We obtained 10 independent transformation events for A6-56_1Ac+3A. Four lines tested positive for nptII, cry1Ac, and cry3A, as determined by PCR. We selected two of these lines (#1 and #2) for further analysis based on high transcript levels (Fig. S4A and B). We identified putative transformants (A6-56_1Ac+3A#1, #2) by detecting nptII, cry1Ac, and cry3A in genomic DNA and confirmed the expression of cry1Ac/cry3A in each transgenic line (Fig. S4A and B). Protein analyses verified the successful heterologous accumulation of both Cry1Ac and Cry3A in plants (Fig. 2B–E).

Fig. 2.

Fig. 2

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Identification of diploid inbred lines harboring Cry1Ac and Cry3A and bioassay of their activities against PTM and CPB. A Diagram of the multigene assembly vector. B Cry1Ac contents in the leaves of A6-56_1Ac+3A transgenic plants. C-D Survival rates of PTM larvae after feeding on the leaves of A6-56 and A6-56_1Ac+3A plants for 15 days (C), biomass and larval phenotypes of PTM after feeding for 10 days (D). Bar = 0.25 cm. E Cry3A contents in the leaves of A6-56_1Ac+3A transgenic lines. F-G Survival curve (F), biomass and larval phenotypes (G) of CPB after feeding on the leaves of A6-56_1Ac+3A transgenic lines for 3 days. Bar = 0.25 cm. All data are presented as mean ± standard error (SE), with n = 30 larvae per treatment. Statistical significance was determined using a two-tailed Student's t-test: ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.

Bioassays with newly hatched PTM larvae feeding on these transgenic lines showed that cry1Ac expression was associated with significantly increased larval mortality (Fig. 2C). Surviving larvae feeding on these transgenic lines exhibited severely inhibited growth and reduced biomass (Fig. 2D). To further evaluate the resistance of potato plants co-expressing cry1Ac and cry3A against CPB, we conducted feeding bioassays using second-instar larvae. The transgenic lines caused over 75% larval mortality (Fig. 2F). The surviving larvae exhibited complete growth arrest and a significant reduction in biomass compared with the control (Fig. 2G). These results demonstrate that co-expressing cry1Ac and cry3A conferred strong and consistent resistance against PTM and CPB.

Additionally, we generated transgenic Atlantic lines with stable co-expression of both cry1Ac and cry3A (Fig. S4C and D, Fig. S5A and B). For Atlantic_1Ac+3A, 68 independent transformation events were generated. Among these, 47 lines were positive for nptII, cry1Ac, and cry3A. Based on transcript levels (one high, one medium), we selected lines #3 and #40 for further characterization. Consistent with the results in the A6-56 background, these transgenic Atlantic lines also significantly suppressed larval growth and increased PTM and CPB larval mortality, with Cry1Ac contributing to the mortality of PTM and Cry3A contributing to the mortality of CPB (Fig. S5C and D). Moreover, there was no significant difference in plant vigor, tuber number per plant, individual tuber weight, single plant tuber weight, or protein content in tubers between the transgenic and control plants (Fig. S5E–J). These findings demonstrate that co-expressing cry1Ac and cry3A in potato enhanced resistance against both PTM and CPB without compromising tuber weight or tuber quality.

2.3. Diploid potato expressing cry1Ac and cry3A shows stable insect resistance

To assess the stable inheritance of insect resistance in diploid potato, we used the diploid transgenic lines A6-56_1Ac+3A#1 and A6-56_1Ac+3A#2 separately as pollen donors, with the diploid inbred line E4-63 (Solanum tuberosum group Phureja) used as the female parent (Fig. S6A). We harvested seeds from the progeny and screened F1 plants by performing PCR to identify transgenes (nptII, cry1Ac, and cry3A), RT-qPCR to quantify cry1Ac and cry3A expression (Fig. S6B and C), and ELISA to evaluate protein accumulation (Fig. 3A and B). For the cross E×A_1Ac+3A#1, a total of 7 F1 plants were screened, and two lines (#1 and #2) with comparable expression levels were selected. For the cross E×A_1Ac+3A#2, 26 F1 plants were screened, and lines #7 and #18 were selected. As a result, two individual crosses from two F1 lines (E×A_1Ac+3A#1 and E×A_1Ac+3A#2) accumulated Cry1Ac and Cry3A, respectively, at levels comparable to those of the parental lines. To rule out any potential effects of hybrid vigor on insect resistance, we also generated a control line by crossing A6-56 with E4-63 (E×A) for use in subsequent bioassays.

Fig. 3.

Fig. 3

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Bioassay of intercross E×A_1Ac+3A lines against PTM and CPB. A-B Cry1Ac (A) and Cry3A (B) contents in the leaves of diploid potato line E×A_1Ac+3A. C-D Biomass on day 10 (C) and survival rate (D) of PTM larvae on day 15 after feeding on the leaves of diploid potato line E×A_1Ac+3A. E Biomass of CPB larvae after feeding on the leaves of diploid potato line E×A_1Ac+3A on day 3. F Leaf morphology of diploid potato line E×A_1Ac+3A after 3 days of feeding by CPB larvae. Bar = 1 cm. G Leaf area consumed by adult CPB on diploid potato line E×A_1Ac+3A. All data are presented as mean ± standard error (SE), with n = 30 larvae per treatment. Statistical significance was determined using a two-tailed Student's t-test: ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.

In feeding assays with PTM larvae, compared to negative control plants, E×A_1Ac+3A plants strongly inhibited larval weight gain (Fig. 3C) and caused nearly complete mortality by day 15, whereas larvae fed on control plants had already begun to pupate (Fig. 3D). Consistent with observations in the parental diploid line (Fig. 2B and C), the accumulation of Cry1Ac protein in F1 plants enhanced resistance, as evidenced by suppressed larval growth and increasing mortality. In bioassays with first-instar CPB larvae, transgenic F1 plants strongly suppressed larval growth (Fig. 3E) and deterred feeding (Fig. 3F). Adult beetles also exhibited markedly reduced feeding on leaves (Fig. 3G).

Finally, to assess whether reciprocal crosses would affect the stable inheritance of the Bt proteins, we used E4-63 as the paternal parent in crosses with transgenic lines A6-56_1Ac+3A#1 and A6-56_1Ac+3A#2 (Fig. S6D). The resulting F1 lines, A×E_1Ac+3A#1 and A×E_1Ac+3A#2, retained high cry1Ac and cry3A expression (Fig. S6E and F), as well Bt protein accumulation (Fig. S7A and B), and maintained strong resistance against both PTM (Fig. S7C and D) and CPB (Fig. S7E–G). We also measured protein accumulation in the tubers of F1 plants (Fig. S7H and I). Although both Cry1Ac and Cry3A accumulated to lower levels in tubers than in leaves, in feeding assays on F1 tubers, PTM larval growth was significantly inhibited: larval weight decreased by nearly 80% relative to the control after 6 days of feeding (Fig. S7J), and only 4.76% to 15% of the larvae reached pupation (Fig. S7K). These results demonstrate that the Bt genes were stably inherited in the resulting F1 progeny, highlighting the potential use of these lines in diverse crossing combinations for pest resistance breeding.

3. Discussion

Due to the high specificity and efficacy of Bt crystal proteins, Bt-transgenic technology has become a cornerstone of modern agricultural pest management [30]. To date, Bt crops such as Bt cotton (Gossypium hirsutum), maize (Zea mays), and rice have been successfully commercialized on a large scale and are widely planted in multiple countries, significantly reducing the use of insecticides and providing substantial economic and environmental benefits [30,31]. To safeguard the long-term sustainability of this technology against inevitable pest adaptation, the strategic pyramiding of multiple insecticidal genes with independent modes of action into elite crop varieties represents a critical path forward for delaying resistance and ensuring durable field performance [32]. For example, both mcry1Ab and mvip3A were introduced into sorghum (Sorghum bicolor): both genes target lepidopteran pests but use different mechanisms [33]. Bollgard 3 XtendFlex® Cotton is the first commercial cotton product with full federal approval to that combines three Bt proteins (Cry1Ac, Cry2Ab, and Vip3A) for both lepidopteran insect control and herbicide tolerance (Monsanto Company 2016). Another example is the expression of the hybrid insecticidal protein SN19, which contains cry1Ba-domain I–III and cry1Ia-domain II, in transgenic potato plants, showing promising efficacy against CPB and Tuta absoluta [34]. Moreover, the recent reconstruction of ancestral sequences of Bt proteins revealed that resurrected ancient toxins can exhibit broad-spectrum insecticidal activity. This analysis identified key structural hotspots for protein engineering that could guide the design of novel variants with improved activity or alternative modes of action [35].

Here, using an innovative multigene assembly approach, we successfully engineered transgenic potato lines in various backgrounds, including the diploid inbred line A6-56 and the tetraploid cultivars Atlantic, Jizhangshu No.12, Longshu No.7, and Qingshu No.9, for the simultaneous expression of cry1Ac and/or cry3A. The transgenic lines exhibited strong and specific resistance against both PTM and CPB, with Cry1Ac conferring effective control of PTM (with no effect on CPB), and Cry3A providing potent protection against CPB (with no effect on PTM) (Fig. 1and 2, Fig. S5). Therefore, we established a platform for generating dual-resistant germplasm that leverages the complementary specificities of Bt proteins in order to broaden pest resistance in potato.

In this study, Cry3A exhibited substantially higher transcript and protein levels than Cry1Ac across most transgenic lines. This disparity could likely be attributed to multiple factors beyond promoter strength, including positional effects of the transgene insertion [36], differences in codon optimization efficiency [37], transcript stability [38], protein stability [39], and post-transcriptional gene silencing [40]. Despite its relatively low accumulation level, Cry1Ac effectively suppressed PTM growth and development, as reflected by the association between Cry1Ac content and larval mortality (Fig. 2B and C). Nevertheless, to further enhance the overall insecticidal efficacy of this technique, future studies should combine molecular optimizations (e.g., codon usage in different promoters, positional effects) with the large-scale screening of independent transformation events to identify lines with commercially relevant levels of both proteins.

The barriers of self-incompatibility and inbreeding depression in potato have been overcome for several diploid lines [29,[41], [42], [43]]. Advances in diploid inbred-based breeding now hold the potential to fundamentally reshape the future of potato improvement and production [27]. In this study, we engineered transgenic potato plants co-expressing cry1Ac and cry3A in the background of the highly homozygous diploid potato line A6-56 (Solanum tuberosum group Stenotomum) and used this line as both the male and female parent in a cross with another highly homozygous diploid line, E4-63 (Solanum tuberosum group Phureja), to produce F1 progeny. Regardless of whether the transgenic line served as the male or female parent, stable inheritance and functional conservation of cry1Ac and cry3A were consistently observed, with all F1 plants maintaining comparable Cry protein levels and potent insecticidal activity against both PTM and CPB (Fig. 3). This consistent inheritance underscores the value of this diploid dual-Bt line as a breeding parent, offering flexibility in parental line selection for future breeding programs. Furthermore, the seed-propagated nature of these lines, coupled with the presence of stacked Bt genes, provides a sustainable, scalable approach to pest management, reducing the need for chemical insecticides and providing dual-resistance to PTM and CPB.

In summary, we established a durable insect-resistance germplasm library in various backgrounds, including diverse tetraploid potato cultivars, diploid inbred lines, and F1 plants harboring cry1Ac and cry3A, which exhibited effective resistance against both PTM and CPB. By leveraging the diploid inbred-based breeding system, we established an efficient pipeline for the rapid development of insect-resistant commercial crop varieties. Unlike conventional tetraploid breeding, which is constrained by the need for clonal propagation and lengthy selection cycles, our approach enables the direct introgression of stable Bt-based resistance into elite diploid lines through sexual crossing, substantially accelerating the breeding timeline. Thus, this work provides both a sustainable genetic repository for global potato security and a foundational platform for next-generation potato breeding. Future efforts should focus on the field-level validation of the durability of PTM and CPB resistance, optimization of resistance management strategies, and integration of additional Bt genes or RNAi-based approaches to further broaden and strengthen pest resistance in potato breeding systems.

4. Materials and methods

4.1. Plant materials

Two inbred parental potato lines were used in this study, A6-56 (Solanum tuberosum group Stenotomum) and E4-63 (Solanum tuberosum group Phureja), which were kindly provided by the International Potato Center [43]. Four commercially cultivated potato varieties were used in this study: Atlantic, Longshu No.7 (LS7), Jizhangshu No.12 (JZ12), and Qingshu No.9 (QS9). These varieties are widely grown in the potato-growing regions of China.

4.2. Vector construction and transformation

The nucleotide sequences of cry1Ac and cry3A were codon-optimized for potato according to pooled sequences of potato genes, and gene fragments were synthesized. The binary vector pCHF3 was digested with Sal I and Pst I, and the synthesized cry1Ac and cry3A fragments were separately introduced into this vector by homologous recombination to construct the single-gene expression vectors pCHF3-cry1Ac and pCHF3-cry3A, respectively, which were used to transform the tetraploid potato cultivars.

For multigene stacking, the codon-optimized cry1Ac and cry3A genes were each fused with the 35S promoter and the rbcS terminator using overlap PCR [44]. Using chimeric primers (comprising a 15-nt unique nucleotide, an Nb.BtsI site, and a gene-specific sequence), the Cry1Ac and Cry3A cassettes were amplified, digested with Nb.BtsI, and assembled in a single reaction into the Nb.BtsI-digested pYLTAC380N-UNiE vector to generate the transformation construct Atlantic_1Ac+3A, which was then used to transform Atlantic and A6-56. The vectors were introduced into Agrobacterium tumefaciens strain GV3101 via chemical transformation. Agrobacterium-mediated transformation of potato internodes was carried out as previously described [42].

Genomic DNA was isolated from the leaves of potato plants using the CTAB method [45]. PCR of nptII, cry1Ac, and cry3A was performed using specific primers listed in Table S1 to investigate the presence of transgenes in putative transgenic plantlets.

4.3. RT-qPCR

To analyze the transcript levels of the exogenous genes, total RNA was extracted from leaves from transgenic and non-transgenic potato plants using a TransZol Up Kit (TransGen) according to the manufacturer's instructions and reverse-transcribed using an All-in-one 1st Strand cDNA Synthesis SuperMix Kit (NovoScript). Quantitative RT-PCR was performed using TB Green® Premix Ex Taq (Takara) on a 7500 Fast Real-Time PCR system (Applied Biosystems) according to standard protocols. EIF3e was used as the internal reference. The specific primers used are listed in Table S1.

4.4. ELISA

Cry1Ac protein levels in the leaves and tubers of transgenic and non-transgenic potatoes were quantified by ELISA using a QualiPlate™ Kit (EnviroLogix, Portland, ME, USA), and Cry3A protein levels were determined using a PathoScreen Kit (Agdia Inc., Elkhart, IN, USA). Absorbance values at 450 nm were measured, and the concentration of the target protein was calculated according to the manufacturer's protocol.

4.5. PTM rearing and bioassays

A laboratory colony of PTM, which has been reared for over 50 generations and shows stable phenotypes, was used in this study. This colony was established from field populations collected in Kunming, Yunnan, China, with the initial specimens kindly provided by Prof. Yulin Gao (Institute of Plant Protection, Chinese Academy of Agricultural Sciences).

For leaf feeding assays, neonate PTM larvae were fed freshly collected potato leaves from either control or transgenic plants; the leaf petioles were wrapped with water-moistened absorbent cotton to prevent desiccation. Larval biomass was recorded after 10 days, and larval survival rate was recorded after 15 days. For tuber feeding assays, second-instar PTM larvae were pre-fed on non-transgenic potato tubers for 5 days, followed by tubers from control or transgenic plants. Larval biomass was measured after 6 days of feeding, and survival rate was recorded after 16 days. In both assays, each treatment comprised 30 larvae divided into three replicates of 10 larvae each, and the entire experiment was independently repeated three times.

4.6. Rearing and leaf biotoxicity assay of CPB

L. decemlineata adults were collected and all CPB experiments were performed in Urumqi, Xinjiang, China. After collection, the insects were placed in an artificial climate box. CPB was fed with potato cultivar Atlantic tubers in insect rearing cages in a greenhouse compartment at 25/22 °C under a 16:8 h light/dark photoperiod with 65% ± 5% relative humidity.

In the feeding assay, CPB larvae were provided with excised young leaves from potato plants. The number of surviving larvae was monitored daily across all experimental groups. The assay lasted for 4 days, at which point surviving larvae were counted and their final mass measured.

4.7. Analysis of agronomic traits

The agronomic characteristics of Atlantic potato plants, including plant height (from ground level to the panicle apex) and tuber weight, were assessed in during the seedling stage and at harvest. Protein content was determined using the Kjeldahl method following the procedures outlined in the National Food Safety Standard of China (GB 5009.5-2016, Method 1). The data were statistically analyzed to assess the phenotypic differences between transgenic and non-transgenic plants.

4.8. Statistical analysis

The data were processed using Excel and graphed using GraphPad software, and one-way ANOVA was used to analyze the significance of differences. A value of P < 0.05 was considered to be significantly different. ImageJ software was used to measure the area of leaf consumption.

CRediT authorship contribution statement

Doudou Yang: Data curation, Formal analysis, Writing – original draft. Weichao Lin: Data curation, Writing – original draft. Zhongmin Yang: Data curation, Validation. Deshan Zhou: Methodology. Juan Liu: Methodology. Jie Cheng: Methodology. Yuqin Zheng: Methodology. Yulin Gao: Resources. Zhengqian Luo: Resources. Linli Xu: Resources. Chunzhi Zhang: Resources. Jinzhe Zhang: Resources. Enliang Liu: Conceptualization, Supervision. Suhua Li: Conceptualization, Funding acquisition, Supervision, Writing – review & editing.

Declaration of competing interest

The authors declare that they have no conflict of interest.

Acknowledgements

This work was supported by Guangdong Major Project of Basic and Applied Basic Research (2021B0301030004), the Agricultural Science and Technology Innovation Program (CAAS-ZDRW202404), and the National Natural Science Foundation of China (32302375).

Footnotes

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.abiote.2026.100078.

Contributor Information

Enliang Liu, Email: liuenliang513@163.com.

Suhua Li, Email: lisuhua01@caas.cn.

Appendix A. Supplementary data

The following are the Supplementary data to this article:

Multimedia component 1
mmc1.docx (22.8KB, docx)
Multimedia component 2
mmc2.docx (4.4MB, docx)

Data availability

All data supporting the findings of this study are available within the paper and its Supplementary Information.

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Data Availability Statement

All data supporting the findings of this study are available within the paper and its Supplementary Information.

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