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Anther culture
Spikes were collected when the microspores were estimated to be at
the mid- to lateuninucleate stages, and were subjected to cold
pretreatment for about 1 week at 4C. Anthers of the primary and
secondary florets from the middle of each spike were inoculated on
the C17 medium (Wang and Chen 1986) with 0.9% sucrose and 0.7% agar
(pH 5.6). Spikes were sterilized with 70% ethanol, and 12 anthers
were placed in each phi 18 x105 - mm test tube containing 5 ml medium
under aseptic condition. All cultures were maintained at 29C in the
dark. The number of induced calli and embryoid were surveyed after
about 40 days of incubation.
For the statistical analysis, all data were transformed by aresine.
Analysis of variance (ANOVA) woo performed to evaluate effects of
genotypes and environments.
Results
Experiment 1
Effect of the absence of 4B and 41) chromosome arms is given in
Table 1. The number of cultured anther per
line was about one thousand, expect for DT 4BS and DT4DS. In these
two DT lines, the removal of the long arms resulted in poor growth
and small number of tillers. Embryoid induction frequency in
Haruyutaka was 87.8% in the first replicate and 14.8% in the second
replicate. CS euploid showed the induction frequency of 6.1% and
4.8%, in the first and second replicates, respectively. DT 4DL
produced 7.6% embryonic structures which was the highest among the
lines. On the contrary, DT 4DS had a low induction response with the
mean of below 8%. However, the ANOVA indicated that all DT lines were
not different from CS and there was a significant difference between
replications (p<0. 01), because all lines in the first replicate
showed higher induction frequency than in the second replicate.
Consequently, it was found that the absence of chromosome arms of 4B
and 4D had no significant effect on embryoid induction from anther
culture.
Experiment 2
Effect of the Rht1 and Rht2 genes on embryoid induction
in the TD set is shown in Table 2. The
number of cultured anther of each line is about one thousand. In the
TD set, the control TD carrying the rht1 + rht2 alleles
had the mean induction frequency of 8.3%. There was no line that
inferior induction response to the control, while the Rht1
+ rht2 and rht1 + Rht2 lines had slightly
high induction frequencies. The first replicate promoted a markedly
high induction response over 20% in all lines. The ANOVA shows no
significant differences among lines
but a significant. difference among replications (p<0.01).
In the Itana set shown in Table
3, the control
Itana carrying the rht1 + rht2 alleles and the
semidwarf Rht1 + Rht2 line had similar induction
frequency around 4.5%. The Rht1 + rht2 line had a
slightly high response. However, differences. between genotypes were
not larger than the variation due to genotype x
replication
interaction.
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