Immunochemical analysis of PAI content in embryogenic and non-embryogenic calli of cultivar S29, grown on callus-initiation and regeneration media, showed the absence of PAI in the non-embryogenic callus for both types of nutrient medium used (Fig. 2 a, b). However, the embryogenic callus contained PAI in either case. PAI content on regeneration medium was twofold greater than that on callus-initiation medium (Fig. 2 c, d).
Thus, PAI content possibly reflects the embryogenic capacity of the wheat-callus tissue, which depends on culture-medium composition as well as on genotype (Karabaev and Dzhardemaliyev 1994). We assume that the PAI-expressing cells of the embryogenic sites of callus-tissue origin behave similarly to the apical meristematic cells of an adult wheat plant.
Analysis of the time-course of PAI content in wheat embryos and callus tissue during callus formation and the subsequent regeneration is described in Fig. 3. PAI content was the same in the embryos of all three genotypes studied, possibly due to the presence of initial cells in developing embryonal apices. There was a synchronous decrease in PAI content (to as low as 50%) on days 8 and 15 of callus formation. This decrease reflects de-differentiation and callus-tissue formation. From day 22 on, the callus PAI content was increasing, with significant PAI- content differences observed among the genotypes under study on day 30 of callus formation. The callus PAI content of LA was significantly greater than those of its sister line LB and cultivar S29 (Fig. 3). The number of embryogenic calli in, LA was 51% greater than that in LB over the same period of culture (Fig. 4).
The significant PAI-content differences among the genotypes under study were confirmed by additional statistical processing of the results by the classification trees method. Comparing the mean values of callus PAI content on day 30 of culture led us to group the genotypes as follows; (i) cultivar S29 and the tall line LB (mean value: 91.67) and (ii) the dwarf line LA (mean value: 283.3).
After the embryogenic calli had been transferred to regeneration medium, PAI content continued increasing in all the genotypes. This increase, however, was much more rapid and reached higher values in LA than in the other genotypes. On day 39 of regeneration (day 39 of culture), the genotypic differences smoothed out a little, possibly corresponding to a shift to whole-plant regeneration (Fig. 3).
On the basis of our estimates of the time-course of PAI content in wheat embryos during callus formation and the subsequent regeneration, we assume that this marker is an "embryonal antigen" (Moiseeva 1991), and its content is associated with the intensity of embryogenic processes occurring in wheat embryos. Because callus initiation and plant regeneration are different processes and are likely to be regulated by different genetic mechanisms (Karabaev and Dzhardemaliyev 1994), one can assume that the embryonal antigen used by us is associated, to a degree,with the Rht genetic system. This follows from the fact that the dwarf line, which has a greater embryogenic potential, had much more PAI than did the tall sister line and the parent cultivar.
Thus, the immunochemical determination of PAI is fully suited for breeding and bioengineering programs to estimate the embryogenic capacity of the newly raised wheat, lines in the in vitro culture.
Acknowledgments
We thank Dmitry N. Tychinin (IBPPM RAS) for the English translation of the manuscript.