Protocols for Micropropagation of Woody Trees and Fruits

MICROPROPAGATION FOR MICROSPORE EMBRYOGENESIS IN OLIVE 365
hundred microspores with a minimum of twenty replicates for each treatment were
tested. The viability of microspores determined immediately after isolation was 50–
77%. Microspore viability after cold-shock was 30–68%, while heat-shock treatments
reduced viability to 6–43% (Figure 2).
Figure 2. A) Microspore viability was determined by FDA staining. B) Survival of in vitro
olive microspore subjected to different shock treatments: cold (3°C) for 96 h or heat (33°C) at
different duration (15, 24 and 72 h) for the induction of pollen embryogenesis.
2.1.8. Isolation of Microspores
The composition of culture medium is a very important factor for haploid plant
induction. Generally, the basal medium consists of salts, vitamins, sugars and a
gelling agent. Many species require plant growth regulators such as auxins, cytokinins
and ABA in the culture medium for the production of haploid plantlets. In
some species, haploid induction frequency was enhanced with the addition of
polyamines, amino acids and silver nitrate in the induction medium. Different
sugars like sucrose, maltose, glucose and fructose also enhance the frequency of
microspore embryogenesis in some species (Bueno et al., 2000). Furthermore, in
cultures of isolated microspores, cell density may be critical. Best results have
generally been obtained between 5 × 10 3 and 2 × 10 4 microspores per ml (Zheng et al.,
2002).
Olive microspores were isolated from anthers using the following protocol
(Bueno et al., 2005). A total of 200 anthers were placed in 2 ml medium B adjusted
at pH 7.0 (Table 1). Anthers were agitated on a magnetic stirrer at 250 rpm for 1
min. The released microspore population was filtered through a 30 µm sieve which
retained the anthers. Next, a further 2 ml of medium B was added to the remaining
anthers. This extraction process was repeated a total of four times. The pooled
filtrate containing microspores was centrifuged at room temperature at 1200 rpm for
5 min. The microspore pellet obtained was re-suspended in 5 ml medium B. This
process was also repeated for four times.