Protocols for Micropropagation of Woody Trees and Fruits

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A. GIOVANELLI AND A. DE CARLO
at the radicle region of the embryo after 4–5 week of culture. However, it should be
underlined that, in the Mediterranean cypress, the ability to initiate ESMs from
immature embryos is strongly influenced by the genotype and by the developmental
stage of the zygotic embryo. This means that contrasting results can be obtained when
collecting the seeds from different trees, or even from the same tree but in different
years.
Similarly to other conifers (Becwar et al., 1990; David et al., 1995), the developmental
stage of the zygotic embryo is particularly critical for the induction of
embryogenic tissue in cypress. As reported by Lambardi (2000), ESMs was mainly
found in immature embryos isolated from cones collected in an elapse of time
ranging from late-April to late-June. Differently, non-embryogenic calli originated
on explants collected throughout almost the entire sampling period. However, it was
observed that ESMs always originated from embryos that were morphologically
at the same stage of maturity (early-cotyledonary stage), characterised by the two
cotyledons just differentiated and still tightly joined. Hence, it is presumable that the
time difference among the trees as for the occurrence of the “embryogenic window”
(over a 2 week period) reflected a lack of coincidence in the course of their embryo
maturation processes.
As for the maintenance and the proliferation of the ESMs, in order to avoid
the appearance of extensive browning and necrosis, after the third subculture the
embryogenic lines are transferred onto the same medium, but with a lower auxin
concentration. In particular, either 2,4-D alone at a 5-µM concentration, or a combination
of α-naphtaleneacetic acid (NAA) and BA (5 µM each) proved to be effective
for the maintenance of the embryogenic lines (Lambardi, 2000). By the regular
subculturing every 1–2 weeks in one of the above hormone combination, the embryogenic
lines remain prolific (the culture volume doubles approximately every 2 weeks),
maintaining high concentrations of filamentous somatic embryos (Figure 1E). Following
this procedure, the embryogenic lines can be maintained for several years,
although occasionally sudden turning of the lines to a non-embryogenic condition
can occur.
Sallandrouze et al. (1999) reported the initiation of a single C. sempervirens
embryogenic line following a slightly different procedure. The immature embryos
were collected in mid-February and cultured on a hormone-free MS (Murashige
& Skoog, 1962) medium, supplemented with 15 g/L of both fructose and glucose,
4 g/L charcoal, 10 mL/L coconut water and 7 g/L Bacto-agar.
Embryo development and maturation. For maturation of somatic embryos, the ESMs
are transferred on hormone-free DCR medium (embryo maturation medium, EMM),
supplemented with 0.5 g/l activated charcoal, and they are cultivated in the darkness.
In these conditions, the somatic embryos differentiate until the cotyledonary stage,
but the hypocotyls and radicle regions remain poorly developed. To complete the
conversion of somatic embryos to plantlets (Figure 1F), the cotyledonary somatic
embryos should individually be transferred on filter-paper bridges, soaked with liquid
hormone-free DCR medium, within the tissue culture tubes. Differently, Sallandrouze
et al. (1999) reported the addition of bovine serum albumin (BSA) into the culture
medium to obtain the maturation of cypress somatic embryos to the cotyledonary