Selection by gradual enrichment for putative dominant mutations in Black Mexican Sweet (BMS) cell cultures

We have selected several variant subcultures of a fast-growing BMS cell line (BMS-M, generously supplied by D.A. Somers, University of Minnesota). Our goal is to obtain dominant markers which will be useful in protoplast hybridization experiments to test the dominance of new, previously untested mutants. We have so far obtained BMS variant lines which are chlorsulfuron resistant (CHSR), glyphosate resistant (GLYR), and lysine/threonine resistant (LTR). If hybridization tests, or gene transfer, should prove that one of these new marker phenotypes is dominant, then we intend to derive other, more experimental mutations in this cell line. In that case, for example, the dominance of the new mutations could always be ascertained by the following cell hybridization and analysis:

Genetic analyses of chlorsulfuron-resistant, glyphosate-resistant, and lysine/threonine-resistant mutants obtained in cell cultures of tobacco and Arabidopsis, petunia, and maize, respectively, have shown that the lesions obtained were dominant (R. Chaleff and Ray, Science 223:1148, 1984; Haughn and Somerville, Mol. Gen. Genet. 204:430, 1986; K.A. Hibberd et al., Planta 148:183, 1980). Although a variety of genetically based mechanisms might yield BMS-M cells which are resistant to each of these cytotoxic substances, these published experiments demonstrate that dominant mutations of the type we desire are possible to obtain.

Chlorsulfuron is very toxic to BMS-M suspension cultures, and all cell division is halted after 5 days treatment with 10-6, 10-7 and 10-8 M. Growth is significantly retarded by 10-9 M (Fig. 1). Normal population doubling time (Td = 32 to 36h) is obtained when 10-10 to 10-12 M chlorsulfuron is present in these cultures. Enrichment selections were begun by treating the cells with the partially toxic drug concentration, 10-9 M. Serial, 7 day passages (1:1, 1:3, or 1:5 splits) on 10-9 M chlorsulfuron continued for 8 weeks, and then rapid increases in drug concentration to 10-8, 10-7, 5 x 10-7, and 10-6 M were made at 8, 11, 12, and 14 weeks after initiation of the selection. A stable, variant BMS subline (CHSR-1) resistant to 10-6 M chlorsulfuron was isolated after 15 weeks. This variant line has a 36h Td at 10-6 M (Fig. 1) and is therefore more than 100-fold resistant to this drug. This level of drug resistance is similar to that obtained in Arabidopsis (Haughn and Somerville, 1986). CHSR-1 has been cloned by protoplasting and plating in agar, and subclones will be used in all further analysis.

A glyphosate-resistant subline (GLYR-1) was selected in a similar manner in 22 weeks. Gradual enrichment selection was begun with applications of 0.25 mM glyphosate; this concentration is mildly inhibitory (50% growth inhibition by 7 days). GLYR-1 is now tolerant to 1.OmM, and the population fresh weight increases 5-fold in 7 days in the presence of herbicide. Wild type BMS-M is completely restricted in growth by 0.5, 1.0, and 2.0 mM glyphosate, so the increase in glyphosate resistance obtained so far is 2- to 4-times background. The sensitivity of these corn cells to glyphosate is much greater than that of petunia cells. In the latter cultures, 1.0 mM is the lowest completely toxic concentration (Steinrucken et al., Arch Biochem. Biophys. 244:169, 1986). GLYR-1 is being tested for evidence of gene amplification (D.M. Shah et al., Science, 233:478, 1986).

Lysine/threonine-resistant variant cell lines are easy to obtain by gradual enrichment with incremental additions of 1.0 mM to 4.0 mM amino acid over a period of 3 months.

Figure 1. Growth of resistant (---) and sensitive (--) BMS cell lines in the presence of various concentrations of chlorsulfuron.

J. Stadler, M. Leonard and H.C. Huang

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