Protocols for Micropropagation of Woody Trees and Fruits

MICROPROPAGATION FOR MICROSPORE EMBRYOGENESIS IN OLIVE 367
Figure 3. Normal microspores a) and “swollen” microspores b) after cold treatment in the
dark at 3°C for 96 h.
2.1.10. Induction of Microspore Embryogenesis
After stress treatment, the microspore population in medium B was centrifuged for 5
min at 1200 rpm at room temperature. The microspore pellet was resuspended in
induction medium AT3 I (Höfer et al., 1999) (Table 2) in 4-well plates with the final
density of the microspores being determined using a haemocytometer (Neubauer
chamber) and adjusted to 4–8 × 10 4 or 1–2 × 10 5 microspores/ml. The microspores were
incubated in a culture chamber at 25 ± 1°C in the dark.
The frequencies of microspore division and multicellular structure formation
were scored by DAPI staining from the first week to the fourth week of culture using
an inverted microscope. About 1200 microspores were counted for each treatment. The
first sporophytic binucleate microspores (Figure 4) were observed after 1 week of
culture. Multinucleate structures are produced following the interruption of gametophytic
pollen grain development and at the beginning of embryogenetic pathway.
These microspores continued to divide and the number of nuclei per microspore
increased to three, four, six or more fold during the next weeks. After the 5 weeks in
culture, further development of these proembryos was limited. In Lupinus spp. the
development of multicellular structures in isolated microspore cultures appears to be
limited by the rigid outer exine layer (Bayliss et al., 2004) which could also be the
case in olive.
The induction of microspore embryogenesis is usually achieved by a stress
treatment, and it is suggested to be associated with a symmetric microspore division
producing two equal nuclei as opposed to the differentiated generative and vegetative
nuclei resulting from asymmetric division in the normal gametophyte pathway (Zaki
& Dickinson, 1991). The influence of heat and cold shock treatments on the initiation
of microspore division (induction of embryogenesis) and the development of multicellular
structures are illustrated on Table 3. The sporophytic development pathway
induced by cold treatment of isolated olive microspores was also seen in cultures
subjected to heat shock although with much lower efficiency. The optimal treatment
was 3°C for 96 h (32.4% multicellular structures per dividing microspore) while the
best heat treatment was at 33°C for 24 h (15.2% multicellular structures per dividing
microspore).