Bermuda grass (Cynodon dactylon) is notoriously difficult to control with some commonly used herbicides. We cloned a cytochrome P450 gene from Bermuda grass, named P450-N-Z1, which was found to confer tolerance to multiple herbicides in transgenic Arabidopsis. These herbicides include: (1) acetolactate synthase (ALS) inhibitor herbicides nicosulfuron and penoxsulam; (2) p-hydroxyphenylpyruvate dioxygenase (HPPD)-inhibiting herbicide mesotrione; (3) synthetic auxin herbicide dicamba; (4) photosynthesis inhibitor bentazon. We further generated transgenic soybean plants expressing P450-N-Z1, and found that these transgenic soybean plants gained robust tolerance to nicosulfuron, flazasulfuron, and 2,4-dichlorophenoxyacetic acid (2,4-D) in greenhouse assays. A field trial demonstrated that transgenic soybean is tolerant to flazasulfuron and 2,4-D at 4-fold and 2-fold the recommended rates, respectively. Furthermore, we also demonstrated that flazasulfuron and dicamba are much more rapidly degraded in vivo in the transgenic soybean than in non-transgenic soybean. Therefore, P450-N-Z1 may be utilized for engineering transgenic crops for herbicide tolerance.

Key message Transgenic soybean event CAL-16 expressing fusion Bt protein Cry1Ab-Vip3A was developed for conferring broad-spectrum resistance to lepidopteran pests Abstract Lepidopteran insect species are important soybean pests causing significant yield loss and quality degradation worldwide. Transgenic soybeans expressing crystal (Cry) insecticidal proteins have been utilized for insect pest management. However, the efficacy of currently adopted insect-resistant soybean is challenged by insect resistance evolution. Vegetative insecticidal proteins (Vips) are highly active against a broad spectrum of lepidopteran insects. They differ from Cry in modes of action, and show great potential for lepidopteran pest management. Here, we report the creation and characterization of a transgenic soybean event CAL-16 which expresses a fusion protein of Cry1Ab and Vip3A. CAL-16 is a single copy T-DNA insertion transgenic event highly resistant to a broad-spectrum of lepidopteran insects. Insect bioassays demonstrated that CAL-16 caused 100% mortality to neonates of Helicoverpa armigera, Spodoptera litura, Agrotis ipsilon, Spodoptera exigua and Spodoptera frugiperda. Field trial also demonstrated its excellent resistance to Leguminivora glycinivorella, a severe pest feeding on soybean seeds. The expression of the fusion protein was found to be constitutively high in CAL-16 throughout developmental stages, and highly stable over 12 generations. Moreover, there was no statistical difference in agronomic traits between CAL-16 and its non-transgenic recipient control plants in field trial. In conclusion, CAL-16 is an elite soybean event with high efficacy toward major lepidopteran pests. It is expected to be released for commercial cultivation in the near future in China as it has been deregulated in China in 2023.

Alpha-amylases are crucial hydrolase enzymes which have been widely used in food, feed, fermentation, and pharmaceutical industries. Methods for low-cost production of α-amylases are highly desirable. Soybean seed, functioning as a bioreactor, offers an excellent platform for the mass production of recombinant proteins for its ability to synthesize substantial quantities of proteins. In this study, we generated and characterized transgenic soybeans expressing the α-amylase AmyS from Bacillus stearothermophilus. The α-amylase expression cassettes were constructed for seed specific expression by utilizing the promoters of three different soybean storage peptides and transformed into soybean via Agrobacterium-mediated transformation. The event with the highest amylase activity reached 601 U/mg of seed flour (one unit is defined as the amount of enzyme that generates 1 micromole reducing ends per min from starch at 65 °C in pH 5.5 sodium acetate buffer). The optimum pH, optimum temperature, and the enzymatic kinetics of the soybean expressed enzyme are similar to that of the E. coli expressed enzyme. However, the soybean expressed α-amylase is glycosylated, exhibiting enhanced thermostability and storage stability. Soybean AmyS retains over 80% activity after 100 min at 75 °C, and the transgenic seeds exhibit no significant activity loss after one year of storage at room temperature. The accumulated AmyS in the transgenic seeds represents approximately 15% of the total seed protein, or about 4% of the dry seed weight. The specific activity of the transgenic soybean seed flour is comparable to many commercial α-amylase enzyme products in current markets, suggesting that the soybean flour may be directly used for various applications without the need for extraction and purification.