Genetic Transformation of Torenia fournieri L. with the Bacillus thuringiensis Cry1Ab Gene Confers Resistance to Mythimna separata (Walker)

Abstract

Torenia fournieri L. is a popular ornamental plant in the genus Torenia, widely used in commercial landscaping, especially during the summer. Additionally, Torenia has served as a model ornamental plant in many studies exploring ornamental characteristics and pest control through genetic engineering. To date, no research has been reported on developing insect-resistant Torenia expressing genes from Bacillus thuringiensis (Bt). In this study, a recombinant vector carrying the Cry1Ab gene from Bt, pBI121-Cry1Ab, was constructed and transferred into T. fournieri via Agrobacterium tumefaciens-mediated transformation. A total of 13 shoots survived on the kanamycin selection medium, among which four putative transgenic lines, designated L1, L2, L7, and L11, were molecularly confirmed by PCR and Southern blot analysis, indicating successful integration of the Cry1Ab gene into the genomes of these lines. Quantitative real-time PCR and ELISA results further verified the successful expression of the Cry1Ab gene in the leaves of all four transgenic lines. Insect bioassay results demonstrated that all four transgenic lines showed strong resistance to the insect pest, Mythimna separata, with mortality rates ranging from 59.9% to 100.0%, in contrast to a larval mortality rate of 16.2% in the wild-type Torenia. Additionally, these transgenic lines significantly decreased in larval survival rates compared to those fed on wild-type plants. Furthermore, these transgenic lines activated superoxide dismutase (SOD) activity at 12 and 24 h, and catalase (CAT) activity at 72 h, while suppressing SOD activity at 72 h, and peroxidase (POD) activity over time. Our findings indicate that these transgenic lines exhibit high resistance to the insect pest and provide new insights into controlling insect pests in ornamental plants through genetic approaches.

Keywords: Cry1Ab; insect resistance; oriental armyworm; protective enzymes; transgenic Torenia.

Figure 1

Construction of recombinant vector pBI121-Cry1Ab used for Torenia transformation. (A) Schematic diagram of the recombinant construction of pBI121-Cry1Ab harboring the kanamycin resistance gene nptII driven by the NOS promoter. RB and LB, right and left borders of T-DNA reg; NOS-Pro, nopaline synthase promoter; NOS-Ter, nopaline synthase terminator; CaMV 35S Pro, cauliflower mosaic virus 35S promoter. (B) The recombinant construction of pBI121-Cry1Ab. (B) Restriction analysis of recombinant plasmid pBI121-Cry1Ab with endonucleases BamHI and SalI. Lane M, DNA marker DL 15,000 (TaKaRa Biotechnology (Dalian) Co., Ltd., Dalian, China); lane 1, digested pBI121-Cry1Ab plasmid.

Figure 2

The generation of Cry1Ab-transgenic Torenia plants. (A) The 4-week-old in vitro Torenia plants; (B) Agrobacterium-infected leaf discs on the selection medium; (C) elongation of kanamycin-resistant shoots on the shoot elongation medium; (D) putative transgenic Torenia plants in a greenhouse chamber.

Figure 3

Molecular analysis of Cry1Ab-transgenic Torenia lines. (A) PCR-amplified fragments of putative transgenic lines and wild-type plants using Cry1Ab-specific primers. Lane M, DL2000 marker (TaKaRa Biotechnology (Dalian) Co., Ltd.); lane 1, plasmid pBI121-Cry1Ab (positive control); lane 2, wild-type Torenia (negative control); lane 3-6, transgenic lines L1, L2, L7, and L11. (B) Southern blot analysis of HindIII-digested genomic DNA isolated from PCR-positive transgenic lines. W, wild-type Torenia; lanes 1-4, transgenic lines L1, L2, L7, and L11.

Figure 4

Levels of Cry1Ab transcripts in the leaves of four transgenic Torenia lines and wild-type plants. WT, wild-type plants; L1, L2, L7, and L11, transgenic lines expressing the Cry1Ab gene. Data are presented as means + standard error. The β-Actin gene was used as an internal standard to normalize Cry1Ab expression. Different lowercase letters above the bars indicate significant differences among the plant lines (one-way ANOVA followed by Tukey’s honestly significant difference (HSD) post hoc test, p < 0.05).

Figure 5

Levels of Cry1Ab protein content in the leaves of transgenic Torenia and wild-type plants. WT, wild-type plants; L1, L2, L7, and L11, transgenic lines expressing the Cry1Ab gene. Data are presented as means + standard error. Different lowercase letters above the bars indicate significant differences among the plant lines (one-way ANOVA followed by Tukey’s HSD post hoc test, p < 0.05).

Figure 6

Resistance of transgenic plants expressing the Cry1Ab gene to Mythimna separata. The mortality rate (A) and survival rate (B) of Mythimna separata larvae fed on leaves from transgenic and wild-type plants. WT, wild-type plants; L1, L2, L7, and L11, transgenic lines expressing the Cry1Ab gene. Data are presented as means + standard error. Different lowercase letters above the bars and asterisks indicate significant differences between transgenic lines and wild-type plants (one-way ANOVA followed by Tukey’s HSD post hoc test, p < 0.05; *, p < 0.05).

Figure 7

Effect of transgenic plants expressing the Cry1Ab gene on the activities of protective enzymes in Mythimna separata. Activities of SOD (A), POD (B), and CAT (C) in Mythimna separata larvae fed on leaves from transgenic and wild-type plants. WT, wild-type plants; L1, L2, L7, and L11, transgenic lines expressing the Cry1Ab gene. Data are presented as means + standard error. Different lowercase letters above bars indicate significant differences between transgenic lines and wild-type plants (one-way ANOVA followed by Tukey’s HSD post hoc test, p < 0.05).