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