BACKGROUND Gene editing using the CRISPR/Cas9 system has become a routinely applied method in several plant species. The most convenient gene delivery system is Agrobacterium-mediated gene transfer with antibiotic selection and stable genomic integration of transgenes, including Cas9. For elimination of transgenes in the segregating progeny, selfing is applied in many plant species. This approach, however, cannot be widely employed in potato because most of the commercial potato cultivars are self-incompatible. RESULTS In this study, the efficiency of a transient Cas9 expression system with positive/negative selection based on codA-nptII fusion was tested. The PHYTOENE DESATURASE (PDS) gene involved in carotenoid biosynthesis was targeted. A new vector designated PROGED::gPDS carrying only the right border of T-DNA was constructed. Using only the positive selection function of PROGED::gPDS and the restriction enzyme site loss method in PCR of genomic DNA after digestion with the appropriate restriction enzyme, it was demonstrated that the new vector is as efficient in gene editing as a traditional binary vector with right- and left-border sequences. Nevertheless, 2 weeks of positive selection followed by negative selection did not result in the isolation of PDS mutants. In contrast, we found that with 3-day positive selection, PDS mutants appear in the regenerating population with a minimum frequency of 2-10%. Interestingly, while large deletions (> 100 bp) were generated by continuous positive selection, the 3-day selection resulted in deletions and substitutions of only a few bp. Two albinos and three chimaeras with white and green leaf areas were found among the PDS mutants, while all the other PDS mutant plants were green. Based on DNA sequence analysis some of the green plants were also chimaeras. Upon vegetative propagation from stem segments in vitro, the phenotype of the plants obtained even by positive selection did not change, suggesting that the expression of Cas9 and gPDS is silenced or that the DNA repair system is highly active during the vegetative growth phase in potato. CONCLUSIONS Gene-edited plants can be obtained from potatoes by Agrobacterium-mediated transformation with 3-day antibiotic selection with a frequency high enough to identify the mutants in the regenerating plant population using PCR.

中文翻译：

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通过农杆菌介导的转化在马铃薯中产生无转基因的PDS突变体。

背景技术使用CRISPR / Cas9系统进行基因编辑已成为几种植物中的常规应用方法。最方便的基因传递系统是农杆菌介导的基因转移，可进行抗生素选择和包括Cas9在内的转基因的稳定基因组整合。为了消除分离后代中的转基因，自交被应用于许多植物物种中。然而，由于大多数商业马铃薯品种是自交不亲和的，因此该方法不能广泛用于马铃薯中。结果在这项研究中，测试了基于codA-nptII融合的具有正/负选择的瞬时Cas9表达系统的效率。靶向类胡萝卜素生物合成的PHYTOENE DESATURASE（PDS）基因。构建仅携带T-DNA右边界的命名为PROGED :: gPDS的新载体。使用适当的限制酶消化后，仅使用PROGED :: gPDS的正选择功能和基因组DNA PCR中的限制性酶切位点丢失方法，证明新载体在基因编辑方面与传统的二元载体一样有效。右边界和左边界序列。但是，经过2周的阳性选择和阴性选择后，并未分离出PDS突变体。相反，我们发现，经过3天的阳性选择，PDS突变体以至少2-10％的最小频率出现在再生群体中。有趣的是，尽管通过连续的阳性选择产生了大的缺失（> 100 bp），但3天的选择仅导致了几个bp的缺失和取代。在PDS突变体中发现了2个白化病和3个带有白色和绿色叶区的chimaeras，而所有其他PDS突变体植物都是绿色的。根据DNA序列分析，一些绿色植物也属于嵌合体。在从茎段进行无性繁殖后，即使通过正向选择获得的植株的表型也没有改变，这表明Cas9和gPDS的表达被沉默了，或者DNA修复系统在马铃薯的营养生长阶段具有很高的活性。结论基因编辑的植物可以通过农杆菌介导的转化和3天的抗生素选择而从马铃薯获得，其频率足够高以使用PCR鉴定再生植物群体中的突变体。根据DNA序列分析，一些绿色植物也属于嵌合体。在从茎段进行无性繁殖后，即使通过正向选择获得的植株的表型也没有改变，这表明Cas9和gPDS的表达被沉默了，或者DNA修复系统在马铃薯的营养生长阶段具有很高的活性。结论基因编辑的植物可以通过农杆菌介导的转化和3天的抗生素选择而从马铃薯获得，其频率足够高以使用PCR鉴定再生植物群体中的突变体。根据DNA序列分析，一些绿色植物也属于嵌合体。在从茎段进行无性繁殖后，即使通过正向选择获得的植株的表型也没有改变，这表明Cas9和gPDS的表达被沉默了，或者DNA修复系统在马铃薯的营养生长阶段具有很高的活性。结论基因编辑的植物可以通过农杆菌介导的转化和3天的抗生素选择而从马铃薯获得，其频率足够高以使用PCR鉴定再生植物群体中的突变体。提示Cas9和gPDS的表达被沉默或DNA修复系统在马铃薯的营养生长阶段非常活跃。结论基因编辑的植物可以通过农杆菌介导的转化和3天的抗生素选择而从马铃薯获得，其频率足够高以使用PCR鉴定再生植物群体中的突变体。提示Cas9和gPDS的表达被沉默或DNA修复系统在马铃薯的营养生长阶段非常活跃。结论基因编辑的植物可以通过农杆菌介导的转化和3天的抗生素选择而从马铃薯获得，其频率足够高以使用PCR鉴定再生植物群体中的突变体。