Gene targeting using the Agrobacterium tumefaciens-mediated CRISPR-Cas system in rice

Background

The type II clustered, regularly interspaced, short palindromic repeat (CRISPR)/ CRISPR-associated protein 9 (Cas9) system is a novel molecular tool for site-specific genome modification. The CRISPR-Cas9 system was recently introduced into plants by transient or stable transformation.

Findings

Here, we report gene targeting in rice via the Agrobacterium tumefaciens-mediated CRISPR-Cas9 system. Three 20-nt CRISPR RNAs were designed to pair with diverse sites followed by the protospacer adjacent motif (PAM) of the rice herbicide resistance gene BEL. After integrating the single-guide RNA (sgRNA) and Cas9 cassette in a single binary vector, transgenic rice plants harboring sgRNA:Cas9 were generated by A. tumefaciens-mediated stable transformation. By analyzing the targeting site on the genome of corresponding transgenic plants, the mutations were determined. The mutagenesis efficiency was varied from ~2% to ~16%. Furthermore, phenotypic analysis revealed that the biallelic mutated transgenic plant was sensitive to bentazon.

Conclusions

Our results indicate that the agricultural trait could be purposely modified by sgRNA:Cas9-induced gene targeting. CRISPR-Cas9 system could be exploited as a powerful tool for trait improvements in crop breeding.     

Modeling a farm population to estimate on-farm compliance costs and environmental effects of a grassland extensification scheme at the regional scale

We used a farm-level modeling approach to estimate on-farm compliance costs and environmental effects of a grassland extensification scheme in the district of Ostprignitz-Ruppin, Germany. The behavior of the regional farm population (n = 585) consisting of different farm types with different production orientations and grassland types was modeled under the presence and absence of the grassland extensification scheme using the bio-economic model MODAM. Farms were based on available accountancy data and surveyed production data, while information on farm location within the district was derived from a spatial allocation procedure. The reduction in total gross margin per unit area was used to measure on-farm compliance costs. A dimensionless environmental index was used to assess the suitability of the scheme to reduce the risk of nitrate-leaching.Calculated on-farm compliance costs and environmental effects were heterogeneous in space and farm types as a result of different agricultural production and site characteristics. On-farm costs ranged from zero up to almost 1500 Euro/ha. Such high costs occurred only in a very small part of the regional area, whereas the majority of the grassland had low on-farm costs below 50 Euro/ha. Environmental effects were moderate and greater on high-yield than on low-yield grassland. The low effectiveness combined with low on-farm costs in large parts of the region indicates that the scheme is not well targeted. The soft scheme design results from an attempt to achieve environmental and rural development objectives with only one scheme. Improving the efficiency of the scheme would require designing separate instruments for the two distinct objectives. This is in line with the Tinbergen rule, which states that consistent economic policy requires that the number of instruments equals the number of targets.     

基于行星轮系的玉米穴施肥自动对种系统设计与试验

玉米播种同步穴施肥是提高肥料利用率的有效手段，本文针对现有穴施肥装置用于精量播种作业时种肥相对位置无法实时检测和控制的难题，设计了一种玉米自动对种穴施肥装置。提出了基于行星轮系的自动对种方法，在确保种距和肥距相等的前提下实时调节排种盘和排肥盘相位差，实现播种作业过程中种肥相对位置的实时调控。通过分析机具前进速度、排种器排肥器间距、株距等对落种落肥位置的影响，得到种肥相对位置计算方法，设计了穴施肥自动对种控制算法。搭建了基于STM32F103的控制系统，并完成硬件系统和软件系统的设计，可完成落种落肥等信息的检测和种肥相对位置的实时调控。验证试验结果表明，当前进速度分别为3、4、5、6 km/h时，种肥纵向平均间距分别为8.6、6.1、11.7、8.5 mm,满足对种穴施肥需求。