produced more entire plantlets. Besides, it appeared that
the addition of an elongation step after shoot initiation was
beneficial not only to produce normal-appearing, elongated
shoots, but also to increase their rooting ability.
Rooted plants were transferred to pots with substrate and
kept in a growth chamber at 25 C and a 16/8 photoperiod
(Fig. 2f). For acclimatization and hardening, a transparent
plastic cup was placed covering the whole plant, and humidity
was progressively reduced by drilling the cup. Once
plants were acclimated, their ploidy was checked by flow
cytometry. Small pieces of young leaves were chopped,
processed basically as described in Corral-Martı´nez and
Seguı´-Simarro (2012), and analyzed using a Partec PA-I
Ploidy Analyzer. Ploidy analysis of 88 microspore-derived
plants revealed that 5 (5.7 %) were haploid, while 61
(69.3 %) were diploid and 18 (20.5 %) showed higher
ploidies. The histograms of 4 plants presented multiple
peaks or excessive background noise that precluded an
unambiguous determination of their ploidy. Diploid plants
were analyzed with microsatellite (SSR) molecular markers
using the procedure and markers described in Corral-
Martı´nez and Seguı´-Simarro (2012). These markers were
proven heterozygous for the ‘Bandera’ donor plants. The
61 diploid plants analyzed were consistently found homozygous
for all the SSR makers used, as expected since
they came from isolated microspore cultures where anther
wall tissue is filtered out. These results are in agreement
with our previous studies with these SSR markers in