Protocols for Micropropagation of Woody Trees and Fruits

MICROPROPAGATION OF MEDITERRANEAN CYPRESS
stage. When transferred onto a BSA-free medium, the cotyledonary somatic embryos
were converted into whole plantlets.
2.3. Hardening and Transfer to the Field
The hardening of plants from micropropagation or from somatic embryogenesis is
obtained in 2-liter plastic pots, filled with a peat-sand-perlite substrate (2:1:2, v:v:v)
added with osmocote. The potted plantlets are maintained in greenhouse at 20–24°C
under misting and daylight conditions for 1–2 months, during which humidity should
be gradually reduced from 98 to 65%. The best period to harden-off the plantlets
from in vitro culture is the early spring. Following this procedure, 100% of successfully
hardened plants can be obtained.
Spanos et al. (1997) reported that rooted shoots were weaned into 80-mm pots
filled with a loam-peat-sand (7:2:3, v:v:v). Plants were maintained for 14 days under
mist conditions at 18–23°C and then placed under glasshouse conditions to complete
the hardening for 28 days. This way, high rates of plantlet survival (over 80%) were
obtained.
With the above described procedure, also the hardening of the rooted shoots,
originally derived from adult material, is easily achieved. However, the plants can
show initially typical signs of plagiotrophic growth which, however, starts to disappear
after 6 to 8 months.
3. CONCLUSIONS
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In the last decades, many breeding programmes have been developed in order to
select cypress genotypes of both superior growth habit, and high tolerance to the
cypress canker disease. Hence the exploitation of tissue culture systems can offer a
valuable alternative to traditional propagation for large-scale clonal reproduction of
selected genotypes. Ex-vitro plants can be easily obtained from embryos, seedlings
or very young plants but it is very difficult with mature selected trees. Preconditioning
of adult plant material with re-invigorating treatments as micrografting and repeated
grafting onto juvenile rootstock, represents a key step to overcome difficulties of
in vitro propagation of selected mature trees.
To date, C. sempervirens is the only species inside the genus Cupressus for
which a procedure of somatic embryogenesis has been described and the technique,
as well as in many other conifer species, seems to be the most promising among the
in vitro regeneration systems, taking into consideration its superior multiplication
potential. Moreover, embryogenic tissue is an ideal material for genetic transformation,
which would be highly advantageous in cypress, considering the time required for
the production of new genotypes by sexual crossing.
Data from this report show that two factors – the genotype and the developmental
stage of the zygotic embryo – are particularly critical to initiate ESMs from immature
embryos. The period during which the embryo responds to inductive treatments can be
as short as few days during its course of maturation. In this context, further investigations
will be advisable to obtain more information about the detection of the exact