Protocols for Micropropagation of Woody Trees and Fruits

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2.1. Induction of Gametic Embryogenesis
B. PINTOS ET AL.
2. EXPERIMENTAL PROTOCOL
The induction of gametic embryogenesis in Quercus suber L. was obtained from
anther cultures. During the process, the following relevant factors for the embryogenic
response were considered:
1. Correlation between the developmental stages of catkins, anthers and
microspores.
2. Influence of a chilling pre-treatment (4° C) on the anther response.
3. Heat shock stress treatments of the anthers.
4. Effect of the addition of activated charcoal to the induction medium for
gametic embryogenesis.
2.1.1. Correlation between the Developmental Stages of Catkins, Anthers
and Microspores
A key factor for the successful induction of embryogenesis is the adequate developmental
stage of the microspore or the pollen grain. Therefore the accurate selection
of anther and catkin stages containing a high proportion of embryogenic microspores
is crucial. For that purpose, catkins were selected at five different phenologic stages
(Figure 1A). Anthers from those stages were dissected and the respective microspores
were stained with 4’-6-diamidino-2-phenylindole (DAPI) for the determination of
their developmental stage (Figure 1B–E). 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. We observed a good relationship among the phenologic stage of the
cork oak catkin, the anther size and colour, and the microspore stage.
2.1.2. Catkin Selection in Quercus suber L.
At the best stage for the induction of gametic embryogenesis in Quercus suber,
catkins are approximately 20 mm long and 5 mm thick. Flowers of about 2 mm in
size start to separate. At this stage, anthers are green-yellowish, of about 1.2 by 1.2 mm
size, containing 91% of the microspores in the late uninucleated or vacuolated phase
(Figure 1D). The nucleus is moved towards a pole due to the presence of a big central
vacuole. These microspores are in the optimal stage for the induction of gametic
embryogenesis (Pintos et al., 2005).
2.1.3. Pre-treatment on the Anther Response
Branches bearing catkins (Figure 2B) were collected from selected cork oak trees,
transported to the laboratory and preserved in darkness with moist cotton wrapped at
the base and enveloped in aluminium foil at 4° C for one, two or three weeks. The
highest embryogenic rate was obtained after a pre-treatment at 4° C during one week.
Longer cold pre-treatments, e.g., two or three weeks, produce a lower frequency of
embryogenesis (0.76% and 0.49% respectively).