Highly efficient generation of bacterial leaf blight-resistant and transgene-free rice using a genome editing and multiplexed selection system

Background Rice leaf blight, which is a devastating disease worldwide, is caused by the bacterium Xanthomonas oryzae pv. oryzae (Xoo). The upregulated by transcription activator-like 1 (UPT) effector box in the promoter region of the rice Xa13 gene plays a key role in Xoo pathogenicity. Mutation of a key bacterial protein-binding site in the UPT box of Xa13 to abolish PXO99-induced Xa13 expression is a way to improve rice resistance to bacteria. Highly efficient generation and selection of transgene-free edited plants are helpful to shorten and simplify the gene editing-based breeding process. Selective elimination of transgenic pollen of T0 plants can enrich the proportion of T1 transgene-free offspring, and expression of a color marker gene in seeds makes the selection of T2 plants very convenient and efficient. In this study, a genome editing and multiplexed selection system was used to generate bacterial leaf blight-resistant and transgene-free rice plants. Results We introduced site-specific mutations into the UPT box using CRISPR/Cas12a technology to hamper with transcription-activator-like effector (TAL) protein binding and gene activation and generated genome-edited rice with improved bacterial blight resistance. Transgenic pollen of T0 plants was eliminated by pollen-specific expression of the α-amylase gene Zmaa1, and the proportion of transgene-free plants increased from 25 to 50% among single T-DNA insertion events in the T1 generation. Transgenic seeds were visually identified and discarded by specific aleuronic expression of DsRed, which reduced the cost by 50% and led to up to 98.64% accuracy for the selection of transgene-free edited plants. Conclusion We demonstrated that core nucleotide deletion in the UPT box of the Xa13 promoter conferred resistance to rice blight, and selection of transgene-free plants was boosted by introducing multiplexed selection. The combination of genome editing and transgene-free selection is an efficient strategy to accelerate functional genomic research and plant breeding.

Pollen Flow of Winter Triticale (x Triticosecale Wittmack) Investigated with Transgenic Line Expressing β-Glucuronidase Gene

A transgenic winter triticale line expressing the uidA gene, encoding β-glucuronidase, was used to assess the pollen flow in field experiments over two consecutive vegetation seasons in central Poland. The experimental design included two variants of mixed transgenic and non-transgenic lines. Pollen grains were collected using passive traps located at 0, 10, 30, 60 and 85 meters from the transgenic line. GM pollen grains were detected histochemically by staining with x-Gluc. A positive effect of temperature increase, as well as the strength and direction of the wind on the number and spread of pollen grains was observed. Regardless of the experiment year and variant, only few pollen grains were observed at a distance of 85 m. In the first year of the study the amount of pollen grains at 85 m was 300-fold lower than at the source and 140-fold lower in the second year. The number of transgenic pollen grains was two times lower when the field with the transgenic triticale was surrounded by a non-transgenic line, compared to an empty field. On the basis of the obtained results, we suggest 100 m as the distance for triticale pollen migration, although longer flight incidents are possible in extreme atmospheric conditions.

A Lévy-flight diffusion model to predict transgenic pollen dispersal

The containment of genetically modified (GM) pollen is an issue of significant concern for many countries. For crops that are bee-pollinated, model predictions of outcrossing rates depend on the movement hypothesis used for the pollinators. Previous work studying pollen spread by honeybees, the most important pollinator worldwide, was based on the assumption that honeybee movement can be well approximated by Brownian motion. A number of recent studies, however, suggest that pollinating insects such as bees perform Lévy flights in their search for food. Such flight patterns yield much larger rates of spread, and so the Brownian motion assumption might significantly underestimate the risk associated with GM pollen outcrossing in conventional crops. In this work, we propose a mechanistic model for pollen dispersal in which the bees perform truncated Lévy flights. This assumption leads to a fractional-order diffusion model for pollen that can be tuned to model motion ranging from pure Brownian to pure Lévy. We parametrize our new model by taking the same pollen dispersal dataset used in Brownian motion modelling studies. By numerically solving the model equations, we show that the isolation distances required to keep outcrossing levels below a certain threshold are substantially increased by comparison with the original predictions, suggesting that isolation distances may need to be much larger than originally thought.

Effectiveness of border areas in confining the spread of transgenic Brassica napus pollen

The development of transgenic Brassica napus L. cultivars requires field trials in agricultural settings. For field testing of transgenic constructs that have not been granted full environmental release, current Canadian Government regulations require either large isolation zones (200 m) or 10 m wide borders of synchronously flowering, non-transgenic B. napus to contain transgenic pollen. To investigate the effectiveness of border areas in containing transgenic B. napus pollen, border areas 15 to 30 m wide were planted around 30 m × 60 m central plots of bromoxynil-herbicide-resistant transgenic B. napus strains. Four field trials were conducted at Carman and Winnipeg, Manitoba, Canada in 1994 and 1995. Seed samples were harvested from the border area at 0, 2.5, 5, 10, and 15 m for the four cardinal directions and additionally at 20, 25, and 30 m for two cardinal directions. These seed samples were planted in the field in 1995 and 1996 and the seedlings screened for the presence of bromoxynil-resistant plants (i.e., from outcrossed seeds). Distance from the central plot significantly affected outcrossing rates while environment (site–year) and direction effects were non-significant. Outcrossing rates averaged 0.70% at 0 m and declined exponentially to 0.02% at 30 m. More than four-fifths of the total outcross events detected occurred in the first 10 m of border area indicating that border areas effectively reduce pollen-mediated gene flow in B. napus but cannot completely eliminate it. Key words: Transgenic canola; Brassica napus; pollen containment