In this work we investigate the hypothetical association of PAI
with the embryogenic capacity of the wheat- callus tissue in an in
vitro culture, with the example cultivar Saratovskaya 29 (S29)
and its near-isogenic lines differing in the Rht-B1c
alleles.
Materials and methods
The tall common-wheat (Triticum aestivum L.)
cultivar S29 (tall wheat cultivar Saratovskaya 29) and its
near-isogenic lines LA (dwarf near-isogenic Saratovakaya 29 lines
carrying Rht-B1c alleles) and LB (tall sister near-isogenic
Saratovskaya 29 lines carrying Rht-B1a alleles) were used. LA
and LB were raised in a backcross by Lobachev (2000). The dwarf
common-wheat line ANK11 (All-Russia Institute of Plant Industry's
collection, St. Petersburg, Russia) was a donor and S29 a recipient
of the Rht-B1c allele. The allele originates in the Tom Thumb
form (McIntosh et al. 1998). The sister near-isogenic lines LA and LB
are theoretically 99.2% identical in genotype to the recipient
cultivar, S29. Phenotypically, the lines are identical except
for the plant-height trait: LA is a dwarf line carrying the
Rht-B1c allele, and LB is a tall line carrying the Rht-B1a
allele.
For callus initiation, immature, (14-day-old) wheat embryos were cultured on Linsmaier-Skoog medium (2,4 D: 2 mg ml-1; kinetin: 0.5 mg l-1). The embryogenic calli were regenerated on Blaydes medium (IAA: 0.5 mg ml-1; kinetin: 0.5 mg l-1).
Experiments to determine PAI (proliferative antigen of initials) content and to assess its time course changes had six replicated samples. Each sample consisted of three calli chosen from each genotype at the start of the experiment (immature embryo); on days 8, 15, 22 and 30 (sampling from callus-initiation medium); and on days 32,35, and 39 (sampling from regeneration medium). The relative quantitative assay of PAI content was done with an immunochemical test-system using monospecific anti-PAI antibodies raised by us as described earlier (Volodarsky et al. 1979; Sumaroka et al. 2000).
The antigen concentration was estimated semiquantitatively as described in Clausen (1988). The concentration titer of PAI (maximum dilution that makes precipitation with antibodies visible to the unaided eye) was used as a measure of the antigen concentration. The concentration titer of 1:2 was taken as 100%. The total water-soluble proteins obtained from the stem apices of four-day-old wheat seedlings were used as the initial test-system. This was because PAI is localized not only in callus but also (and primarily) in wheat-stem apex (Sumaroka et al. 2000). By way of example, Fig. 1 shows the concentration titer of PAI present in the embryogenic callus of LA on callus-initiation medium (day 30 of culture). Here the concentration titer was 1:4 (Fig. 1, well no. 5), because at a 1:8 dilution no precipitation band was observed (Fig. 1, well no. 6). To this titer corresponds the relative PAI-concentration C = 200%. C was ultimately estimated by averaging over the data gathered from six replicated samples, which sometimes yielded C values non-divisible by 2.
The number of embryogenic calli of the genotypes under study was determined visually (Nabors 1982). Each experiment had three replicated samples, each sample consisting of 40 calli. The results were processed statistically by a one-factorial analysis of variance with determination of the mean square deviation (Maksimov 1984). The gene effect (%) was determined by comparing the mean values of the trait under study among LA, LB, and S29 only when there was a significant difference. In assessing the gene effect, we took as 100% the magnitude of yield of the LB embryogenic-callus on day 30 of culture (Lobachev 2000). In addition, the mean values of PAI content were compared among the calli of LA, LB, and S29 by the classification trees method described in Breiman et al.(1984).