In this study, we report a technique to obtain Cas9-transgene-free Arabidopsis mutants and its potential application in other crops such as wheat and canola. This method is based on the ability of some plants to recover from Basta selection. Basta herbicide has an active ingredient, glufosinate-ammonium (Hoerlein, 1994; Takano and Dayan, 2020) with a mode of action that inhibits the glutamine synthetase enzyme. Glutamine synthetase plays a role in nitrogen metabolism in plants, and the inactivation of this enzyme results in the disruption of multiple metabolic pathways, including photosynthesis (Dan Hess, 2000; Dayan et al., 2019; Krogmann et al., 1959; Wendler et al., 1990; Wild and Wendler, 1993). The inhibition of glutamine synthetase results in the accumulation of toxic amounts of ammonia in the chloroplast (Bernard and Habash, 2009; Takano and Dayan, 2020). Plants exposed to this herbicide stop growing, resulting in death approximately two weeks later. Our lab routinely uses the phosphinothricin acetyltransferase gene (blpR) as a positive selection marker in plasmids for selecting transgenic plants which have been exposed to Basta. The blpR gene in transgenic plants confers resistance to glufosinate-ammonium by acetylating it to create an inactive conjugate, which allows plants to grow on growth media with the Basta selection agent (Block et al., 1987).
During our studies with transgenics plants expressing the blpR gene, we observed the ability of the non-transgenic plants to recover their growth once the plants were moved to media without the Basta selection agent. Since Basta doesn’t directly disrupt the photosynthesis system, the absence of the herbicide allowed some plants to regain their normal growth. Non-transgenic Arabidopsis seedlings growing on Basta media display stunted growth with discolored leaves. We observed 100 percent growth recovery once the affected seedlings were moved to normal growth media (Fig. 1F). The recovered seedlings were then able to grow normally in soil under greenhouse conditions. In contrast, Arabidopsis seedlings grown on kanamycin or hygromycin selection media could not recover their growth after being transferred to non-selection growth media (Fig. 1D). Kanamycin and hygromycin differ from Basta in the mode of action as both antibiotics interfere with protein synthesis in the chloroplast, causing permanent damage to plants (Davies et al., 1965; Davis, 1987; Hoerr et al., 2016; Weisblum and Davies, 1968).
The recovery of Basta-sensitive seedlings from Basta-selection media provides an opportunity to easily identify non-transgenic siblings in plant transformation studies. The isolation of non-transgenic siblings not only allows us to select Cas9-transgene-free mutants in Arabidopsis, as reported in this study, it also offers researchers several advantages in experimental design and result interpretation. For example, comparing transgenic plants to closely related non-transgenic siblings is particularly important when studying traits that may be influenced by minor genetic variations caused by the transformation process. Another more labor-intensive approach for identifying non-transgenic siblings in plant transformation studies involves self-pollination of heterozygous transgenic plants or backcrossing to the wild type, which is then followed by PCR assays or transgene selection analysis in paired sibling populations.
While other methods isolating Cas9-free mutants have been described, such as fluorescent-based isolation or grafting, these systems may not be compatible with all plant species or varieties (Gao et al., 2016; Yang et al., 2023). For example, although DsRed fluorescence selection offers a simple alternative for selecting Cas9-free plants, its application is limited to seeds with translucent seed coats, such as Arabidopsis and camelina. Plant seeds with thick seed coats, such as canola and wheat, limit the detection of DsRed fluorescent signals. In addition, grafting presents a practical challenge with the need for special equipment along with skilled personnel but cannot be readily performed in monocots due to the lack of a cambium layer of cells.
Although our method offers a simple alternative to isolate Cas9-free Arabidopsis plants, the method only allows the isolation of T-DNAs that still contain a selection marker gene. It does not track unknown T-DNA pieces inserted into the plant genome, the isolation of which is necessary to make true non-transgenic plants. Thus, if true non-transgenic plants are the goal, a whole genome sequence will be necessary to determine the presence of any such foreign DNA elements in any of the methods used for transgenes selection such as DsRed.
In addition to Arabidopsis, we investigated the recovery of field crops like camelina, canola, and wheat from Basta selection media. Camelina exhibited delayed recovery on media, primarily in root growth, but failed to grow in soil possibly due to prolonged exposure to the selection media. Fortunately, the use DsRed is a reliable transgene selection method for camelina studies (Sharma Koirala and Neff, 2020). The response of non-transgenic canola to Basta selection involved growth slowdown followed by leaf yellowing, with varying recovery rates upon transplantation to normal growth media. Although canola seedlings had a lower recovery percentage in soil than Arabidopsis, more seedlings exhibited green hypocotyls, suggesting potential for tissue culture approaches. Conversely, wheat had a higher recovery percentage in both media and soil, most likely attributed to its tightly packed crown structure and higher regenerative ability compared to canola (Huang and Taylor, 1993; Shang et al., 2021). These findings underscore the versatility of our Basta recovery methods across different crops, each presenting unique challenges and advantages. Since our study did not involve transgenic material for these crops, further testing is required to fully understand the application of the Basta recovery method in eliminating trans-mutagenic CRISPR-Cas9. Additionally, we present our Basta recovery method as an additional tool for crop improvement, emphasizing that it complements other methods rather than claiming superiority. This approach recognizes the diverse requirements of specific crops and offers flexibility in addressing them.