Protocols for Micropropagation of Woody Trees and Fruits

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B. PINTOS ET AL.
Efficient protocols for microspore embryogenesis induction in culture have been
developed in recent years for many herbaceous crops (reviewed in Maluszynski et al.,
2003), and in a lesser extent for some woody plants (Bueno et al., 1997, 2005;
Germana & Chiancone, 2003; Höfer, 2004). In trees, with a long reproductive cycle,
high levels of heterozygosity and sometimes self-incompatibility, methods for obtainning
homozygous plants are of strong interest, as their production through conventional
methods requires several generations which is difficult to realize in woody
plants. In contrast, gametic embryogenesis by isolated microspore culture allows the
single step development of complete homozygous lines from heterozygous parents.
With the aim of developing a gametic embryogenesis system for olive, a protocol
for efficient in vitro microspore culture and proembryo production has been developed
(Bueno et al., 2004, 2005). Different pollen developmental stages and stress
conditions (heat or cold shock) were evaluated. The correlation of inflorescence and
anther morphology and the suitable stage of microspore development were analysed.
The morphology of responsive buds was identified, which corresponded with microspores
from the late uninucleate to early binucleate pollen stages. Symmetrical divisions
of microspores as well as resulting multinucleate structures and pro-embryos
were observed. Microspore reprogramming, the induction of embryogenic divisions
and the formation of multicellular proembryos have been achieved as the basis for
developing gametic doubled-haploid plant production in olive.
2.1. Induction of Gametic Embryogenesis
2. EXPERIMENTAL PROTOCOL
The induction of gametic embryogenesis in Olea europaea L. was obtained from
microspore culture. During the process, several factors which may affect pollen
embryogenesis in vitro were considered.
2.1.1. Physiological Condition of the Donor Plants
Although the physiological status of donor plants may influence dramatically the
androgenic process, this parameter has not been investigated in depth in woody
plants. In herbaceous crops, growth conditions (temperature, photoperiod, light
irradiance, soil traits and culture techniques) have an influence on the endogenous
levels of plant growth regulators and on the nutritional status of anther tissues.
However, these factors are difficult to control in woody plants in the field.
2.1.2. Plant Genotype
The genotype of the donor plant affects the frequency of pollen plant induction and
therefore it is important to analyse the response of various genotypes to achieve a
high frequency in haploid plant production. The genotype determines the number of
embryogenic pollen grains, playing a decisive role on pollen embryogenesis. This
genetic ability controls the development of multicellular structures with symmetric
divisions in the microspores. (Barro & Martin, 1999). In this work, floral buds were
taken from trees of the cultivar ‘Arbequina’ (from the Olive World Germplasm Bank
in Córdoba, Spain).