Annual Report 2006

cytochimera among the mulberries (
L.) consecutively irradiated since 1979 in the
Gamma Field. The chimera has both tetraploid
cells and original diploid cells. The tetraploid
cells were localized in the L1 layer of the shoot
apical meristem (SAM) and diploid ones were
identified in the L2 and L3 layers. Generally,
angiosperm, such as mulberry, has a SAM
constructed with three cell layers, commonly
called L1, L2 and L3 from the epidermal cells to
central cell layer. In addition, the L1 cells
develop into the epidermal cells of a plant body
while L2 and L3 develop into the internal plant
cells. The macro-morphological phenotypes of
the cytochimera were found to be similar to the
original diploid cultivars. The sizes of the
stomata guard cells, which are derived from the
L1, were larger than that of the original diploid.
The longitudinal section of the SAM indicated
tetraploid L1 and diploid L2 and L3 cell layers
(Fig. 2). We induced the regenerated plantlets
via direct adventitious bud formation
by culturing immature leaves of the cytochimera.
By applying a flow cytometric analysis, we
recognized that most of the regenerated plants
were tetraploid suggesting that they tended to
be derived from the L1 cells. Other regenerated
plantlets retained the original chimerism. There
was no plantlet derived from only the L2 or L3
cells among the regenerated plantlets. In this
case, we successfully obtained the complete
tetraploid from the cytochimera by the
culture method.
Similar results were obtained on a mutant
cultivar of Japanese pear (
Nakai), cv. Osa Gold.The cultivar is a blackspot-disease-resistant
mutant derived from cv.
Osa Nijisseikithat is known as a self-compatible
Fig. 2.
Longitudinal sections of SAMs of a diploid wild type
(left) and cytochimera with tetraploid cells in L1 layer
mutant derived from cv. Nijisseikiin which self
compatibility is caused by the complete deletion
of S4 gene. However, PCR analysis of cv. Osa
Nijisseikireported that the original wild-type
(self-incompatible) cells have been retained and
are maintained in a chimera state (Sassa, et al.
1997). In our studies, we found that cv. Osa
Goldis also a chimera similar to cv. Osa
Nijisseikibecause a weak amplified fragment of
S4 gene was identified by PCR analysis (Fig. 3).
Using culture methods, we successfully
obtained regenerated plantlets via adventitious
buds that were induced on the leaves of
-cultured winter buds. The PCR analysis
indicated that most of the plantlets were wild
type (Fig. 3). The other regenerated plantlets
were chimera similar to the cv. Osa Nijisseiki,
judging from the PCR fragment pattern. In this
case, we were not successful in obtaining the
non-chimera cell plantlets by the
culture method.
These results indicate that by
culture of a periclinal chimera, we can obtain
non-chimera plantlets at a high frequency or
notatall. culture techniques are not
always a veritable panacea. We need to
develop new and more robust techniques
having application to any types of chimeras.
Fig. 3.
PCR analysis of the original chimeric pear, cv. Osa Gold, and regenerated plantlets (1-4) after culture
Regenerated plant let 1: same as original cv. Osa Gold; 2-4: same as Nijisseiki, from which Osa Gold is derived
S2, 4, 5: Self incompatible gene alleles 2, 4, 5