Protocols for Micropropagation of Woody Trees and Fruits

MICROPROPAGATION OF MEDITERRANEAN CYPRESS
Rooting. The rooting of adventitious shoots was carried out as described with the
axillary shoots. From 15 to 60% of rooted shoots are obtained after 8 months.
2.2.4. Somatic Embryogenesis
Somatic embryogenesis is a powerful system of in vitro plant regeneration. When all
the steps of a protocol are optimised (i.e., induction and proliferation of embryogenic
masses, somatic embryo development and maturation, embryo conversion to plantlets),
it is possible to obtain high numbers of plantlets genetically uniform and identic to
the tissue (explant) from which the embryogenic line originated. Differently from
the broad-leaf woody species in which different tissues (e.g., leaf explants, portions
of embryos, flower organs and root tips) have been reported to show embryogenic
potential, the elective embryogenic tissue of the conifers is the immature embryo. In
time, effective protocols of somatic embryogenesis have been developed for a large
number of conifers (Ahuja, 2000) and always using immature embryos as the original
explants. In particular, embryogenic masses arise from the suspensor cells, i.e., a small
group of elongated cells which are present in basal portion of the embryonic axis.
In cypress, the embryogenic suspensor masses (ESMs) are white, translucent and
mucilaginous, with a high percentage of filamentous pro-embryos (Figure 1D). Differently,
non-embryogenic callus is white to yellow, never translucent or mucilaginous,
and with low organogenic potential. Proliferating ESMs of cypress consist primarily
of clusters of somatic pro-embryos, very similar to the late pro-embryo stage of zygotic
embryos (Attree & Fowke, 1991). The clusters are polarised structures, initially organised
into an embryonic region subtended by multiple, closed and short suspensors.
By cleavage polyembryogenesis, these structures continually initiate embryos which
generally develop simultaneously to the filamentous stage. We report here a protocol
of somatic embryogenesis from immature embryos as developed by Lambardi (2000).
In vitro induction and maintainance of embryogenic suspensor masses (ESMs). The
zygotic embryo of C. sempervirens reaches the full maturity in the autumn of the
second year after fertilisation. In the spring of the second year, the female cone turns
brown and the embryo starts to acquire firmness. At this point the seed coat can be
removed and the immature embryo safely excised from the megagametophyte.
The first step of the procedure starts with the mechanical isolation of immature
seeds in sterile conditions. Thereafter, the isolated seeds will be decontaminated for
1 min with 70% ethanol and 10 min in 0.1% HgCl2, followed by multiple rinses with
sterile distilled water. The disinfected seeds will be stored in the dark at 4°C for 5–7
days in a small amount of sterile distilled water to soften the seed coat before the
embryo excision. The immature embryos are then dissected from the megagametophyte
working with a stereoscope under the sterile air of a laminar-flow hood.
Culture conditions for the induction and the maintenance of ESMs. To induce EST
formation, excised embryos are plated on the EIM (embryogenic induction medium)
described in Table 1, supplemented with 500 mg/l casein hydrolysate and10 µM 2,4dichlorophenoxyacetic
acid (2,4-D). The embryos are then incubated at 23 ± 1°C in
the dark, and subcultured every 21 days. In these conditions, ESMs start to appear
101