Simple and versatile selection of Arabidopsis transformants

Abstract

The selection of large amounts of seeds of Arabidopsis thaliana obtained after in planta transformation under sterile conditions and in tissue culture is relatively labour-intensive, time-consuming and expensive. We describe here an alternative selection procedure that uses regular greenhouse conditions and functions with a wide variety of selective agents. The selection system is based on the inert growth substrate Grodan and was intensively tested for the selection of transgenic Arabidopsis seeds with kanamycin, hygromycin, BASTA, sulfadiazine and methotrexate. However, the system has the capacity to be applicable for more selective agents and to a wider range of plant species. For Arabidopsis, the effectiveness of selective agents is comparable to or better than that obtained with tissue culture procedures.

Novel sul I binary vectors enable an inexpensive foliar selection method in Arabidopsis

“…To determine how effectively the pCS- and pSUN-derived vectors confer resistance, the initially isolated lines were subjected to a concentration gradient of 0 - 200 mg/L sulfadiazine. As expected from previous results [12], the pCS-derived plants exhibited resistance and grew well in media containing up to 200 mg/L sulfadiazine. The pSUN-derived plants however were less tolerant, exhibiting good growth at levels up to 50 mg/L, with a gradual loss of fitness at higher levels of selection (data not shown).…”

“…A lesser known and less frequently utilized plant selectable marker is the sulfonamide resistance gene ( sul I), which confers resistance to sulfadiazine and other sulfonamide chemicals [9]. Since the first demonstration of its utility in tobacco, others have utilized sul I for selection in potato [10,11] as well as Arabidopsis [12,13]. Arguably, sul I is used less often than npt II, hpt II, and bar simply due to its infrequent inclusion as a marker in the commonly used plant transformation vectors.…”

“…Sulfonamides also exhibit substantial potency as a plant selection agent; e.g. 5 mg/L sulfadiazine is sufficient for Arabidopsis selection [12]. …”

Plant Biosystems Design Research Roadmap 1.0

“…Since plant biosystems design may require multiple iterations of Design-Build-Test-Learn (DBTL) cycles (for details see Section 3 ), it is essential that the genetically modified plants or de novo plant systems can be easily upgraded for improving performance or adding new functions. In general, upgrading the plant genome requires consecutive stable plant transformation processes, which is constrained by a limited number of selectable marker genes available for plant transformation, including widely used selectable marker genes conferring antibiotic (e.g., kanamycin and hygromycin) or herbicide (e.g., BASTA) resistance [ 65 ], along with some nonantibiotic and nonherbicide markers such as plant phosphomannose isomerase [ 66 ], broad-specificity amino acid racemase [ 67 ], and fluorescent proteins [ 68 , 69 ]. For enabling upgradability of plant biosystems design, it would be desirable to consider marker-free plant transformation systems, in which the selectable marker gene can be excised from the plant genome after transformation (Figure 4 (e)).…”