Isolation of L-glufosinate-tolerant embryogenic lines in various genotypes

--E. Lupotto, M. C. Lusardi, E. Nielsen* and G. Forlani*

*Dipartimento di Genetica e Microbiologia, Universita di Pavia The active principle of the herbicide BASTA (Hoechst AG) is L-glufosinate or L-phosphinotricine (L-PPT), an analogue of L-glutamic acid and two L-alanine residues. Upon removal of these residues by peptidases, PPT results in a potent inhibitor of GS (glutamine synthetase). This enzyme plays a central role in the assimilation of ammonia and in the regulation of nitrogen metabolism in plants (Miflin and Lea, Ann. Rev. Plant Physiol. 28:299, 1977). L-glufosinate belongs to the so-called "total herbicides" which represent, to date, the most important classes of compounds of different chemical nature, for chemical control of weeds. These compounds can be applied in low dosages, are stable, safe for animals, and completely degraded by the microorganisms in soil, and act on specific target sites in the biosynthetic pathways of the plant cell (Comai and Stalker, Oxford Surv. of Plant Mol. Cell. Biol. 3:166, 1986). Resistance to L-PPT has been reported in alfalfa cells, after a stepwise selection on growing levels of L-PPT, resulting in gene amplification (Donn et al., J. Mol. Appl. Genet. 2:621, 1984), and by introgression in tobacco, potato and tomato plants, via Agrobacterium-mediated transformation of the bar gene, encoding for phosphinotricine acetyltransferase (PAT), a detoxifying enzyme (De Block et al., EMBO J. 6:2513, 1987). In vitro selection for L-glufosinate in cultures may lead to the isolation of resistant cell lines differing from the control counterparts in different features. Resistance to L-glufosinate can be due either to a mechanism of gene amplification or to the presence of altered forms of the target enzyme glutamine synthetase as well as to the presence of naturally occurring detoxifying enzymes.

Maize cultures were established from immature embryos of different genotypes. They were chosen because the derived callus was typically of type II (Armstrong and Green, Planta 164:207, 1985) highly embryogenic and friable. This was done in order to enhance the homogeneity of the material grown in selective conditions, the cellular population to be subjected to selection, and the subsequent regeneration of the selected cell lines. A derivative of B79 culture was also considered (Lupotto et al., MNL 62:31, 1988) because of its high regenerability and friability compared to the direct callus culture obtained in B79. Also a cell line of type I callus was considered, LC10, derived from a F2 population of the cross W64AxA188, which was particularly suited to this work because it was established as long-term highly regenerable culture. The genotypes considered are listed in Table 1. The curve of sensitivity to L-glufosinate in the culture medium was drawn for each genotype.

Table 1.  Level of selection of different maize genotypes on L-glufosinate.

The effect of L-glufosinate varies depending on the genotype considered. The LD50 in the various cases lies between 0.05 and 0.2mM with the exception of LC10, which displayed a higher LD50 (around 0.15mM). Resistant clones were obtained in all cases at different levels of selection (0.05, 0.1 and 0.2mM for type II calli and 0.05, 0.2 and 0.3mM for type I LC10 culture) (Table 1). A total fresh weight of 4 grams embryogenic callus was considered for each genotype and spread over 30ml agar N6P medium (Lupotto et al., MNL 62:31, 1988) in 90mm petri dishes. Growing isolates were subcultured 8-9 times after the first passage before being classified as stable resistant clones, in the presence of L-glufosinate. At that time their GI on glufosinate was identical to the non-selected counterparts grown on control medium.

When a L-glufosinate tolerant isolate was considered stable, it was also embryogenic, at least at the histological level checked by observing the presence of well defined embryogenic structures. Regeneration was stimulated in all the resistant cell clones in each genotype by transferring callus portions on MS (Murashige and Skoog, 1962) hormone-free medium and subculturing them each week onto fresh medium in the light. Regeneration was easily obtained in B79 and Va85xB79, while a longer time in MS hormone-free medium was required for 154/LC12 and B73xA188. Regeneration was also obtained in LC10 type I callus culture at higher levels of L-glufosinate (0.2 and 0.3 mM). In this case, plants were easily established in soil. The only evident abnormality registered in them was the presence of tassel-seed plants, a very common abnormal feature of in vitro regenerated plants not connected with the selection.

Figure 1. A: in vitro sensitivity to L-glufosinate of the enzyme glutamine synthetase in the various selected cell clones. B: levels of the specific activity of GS in 5 isolates compared among each other.

Analysis of the level of specific activity and curve of sensitivity to L-glufosinate in vitro of glutamine synthetase in selected calli was performed on frozen and fresh callus tissues. Calli were extracted in Tris-HCl 100mM, b-mercaptoethanol 6mM pH 7.4 (l mg-1 FWT), mixed with PVPP and dialyzed overnight at 4 C. Two hundred ul of each extract were tested in the presence or absence of L-glufosinate in a reaction mixture consisting of Tris-HCl 100mM pH 7.4, b-mercaptoethanol 6mM, ATP 10mM, L-Glutamic acid 100mM, NH20H-HCl 100 mM, MgCl2 20mM. The reaction was run 3 hrs at 35 C, then stopped by addition of 750µl colorimetric dye: Fe(CNO3)3 10% + TCA 5% + HCl 6.7%. Each sample was read at 535nm (coeff. molar est. 761 M-1 cm-1). Total protein content was measured with the Lowry method. Specific activity of glutamine synthetase varies according to each genotype, with the highest value detected in 154/LC12 selected on 0.2mM L-glufosinate. The glutamine synthetase sensitivity to L-glufosinate reflects in all the cases the LD50 of the tissues, being around 10-4M (Fig. 1). However, because the tolerant calli do not show a real enhancement in the level of their glutamine synthetase activity in comparison to controls, further analyses are in progress to ascertain the nature of the tolerance obtained.

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