Protocols for Micropropagation of Woody Trees and Fruits

MICROPROPAGATION FOR MICROSPORE EMBRYOGENESIS IN OLIVE 363
2.1.3. Correlation of Floral and Male Gametophyte Development
In most species, embryogenic anthers contain either late uninucleate microspores or
early binucleated pollen grains. In some tree species, such as olive tree and cork oak,
the transition from the microspore to the pollen grain is greatly asynchronous. Floral
buds similar in morphologic traits such as colour or size may contain microspores at
very different stages, e.g. tetrads, uninucleate microspores or even mature pollen.
The appropriate microspore developmental stage is crucial in pollen embryogenesis.
In our study in olive, developmental stages from tetrad to mature pollen are
presented with the corresponding anther, bud and inflorescence (Figure 1). A weak
relationship between bud size and microspore stage was observed. Inflorescences
were selected at different phenological stages. Buds and anthers were dissected and
the respective microspores were stained with 4’-6-diamidino-2-phenylindole (DAPI)
for the determination of the developmental stage (Figure 1A–F). The anthers were
placed on a glass slide in a few drops of 1mg/l DAPI in PBS plus 1% Triton X-100,
and tapped softly through the coverglass. Microspores were examined under a Nikon
fluorescence microscope and photographed under ultraviolet light (λ = 360 nm) with
a digital Coolpix 4500 Nikon camera. Open floral buds with yellow-greenish anthers
that began to emerge from the flower were identified as those with higher proportion
of microspores from the late uninucleate to the binucleate pollen stage. About 100
microspores for each development stages and a total of 50 floral buds were tested.
2.1.4. Bud Selection in Olive
Bud and anther length are similar in olive. It appeared that for pollen embryogenesis,
the optimal microspore developmental stage is quite short-lived and microspores at
an inappropriate developmental stage will neither divide nor produce embryos. We
found that the selection of bud morphology was more consistent than bud (or anther)
size. This parameter is probably maintained with other cultivars since flower size is
influenced by the number of flowers in each peduncle.
The best stage for the induction of gametic embryogenesis in cultivar Arbequina
is when the buds are approximately 2 mm long. This size corresponds to half-opened
flowers with yellowish anthers containing microspores at late uninucleate – early
binucleate pollen stage (Figure 1D,E). These microspores are at the optimal stage for
the induction of divisions which lead to proembryo formation
2.1.5. Pretreatment of Olive Buds
Generally cold and heat pretreatments are applied to flower buds in order to achieve
high frequencies of gametic embryogenesis (Bueno & Manzanera, 2003). Therefore,
the effect of cold and heat pretreatments on pollen embryogenesis were also evaluated
in olive. Stress pretreatments may be applied either in vivo and/or in vitro,
to floral buds, isolated anthers or even to isolated microspores. Stress can be applied
in different ways, physical, physiological or chemical. Typically, heat or cold shocks,
starvation, sugar or nitrogen depletion etc., yield good results (Zheng, 2003). In the
present work, a pretreatment was applied in vivo to trigger microspore embryogenesis
in olive. Small branches of olive bearing flowers were harvested. The harvested olive
branches (50 cm long) were wrapped in aluminium foil, with damp cotton wool at
the base of the stem and maintained in darkness at 4ºC for 9 days.