Protocols for Micropropagation of Woody Trees and Fruits

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A. GIOVANELLI AND A. DE CARLO
Repeated subcultures of explants on a BA-containing medium often results in a
poor elongation of the de novo-formed axillary shoots. In order to stimulate shoot
elongation, the explants are transferred to the elongation medium (EM) containing
activated charcoal (Capuana & Giannini, 1997). This way, over 75% of the axillary
shoots elongate rapidly and, after 4 weeks shoots longer than 15 mm are excised and
transferred to fresh PM. The favourable effect of activated charcoal on axillary shoot
elongation has reported for other conifers, such as Sequoia sempervirens Lamb.
(Boulay, 1978), Pinus halepensis Mill. (Lambardi et al., 1993), Picea abies L. Karst
(Ewald & Suss, 1993). Similarly, the inclusion of fructose in the culture medium
alone or in combination with sucrose, showed to stimulate shoot elongation in
walnut (Leslie et al., 2005). After five successive proliferation-elongation cycles
(i.e., culturing the explants alternatively in the PM and the EM), the average number
of elongated shoots longer than 15 mm per explant becomes constant (from 3 to 5),
showing that the stabilisation of the culture has been achieved. Following this protocol,
shoot cultures of Mediterranean cypress have been maintained for over 3 years
in satisfactory conditions.
For root induction, axillary shoots longer than 20 mm are excised from explants
at the end of elongation period and placed on root induction medium consisting of
half-strength SH with 20 gl –1 sucrose and 0.1 mM IBA (Capuana & Giannini, 1997).
After 7 days on root induction medium in dark conditions, shoots are transferred to
75 ml jars filled with a solid medium (expression medium) composed of peat, sand
and perlite (3:1:1, v:v:v) and moistened with half-strength SH. Each jar with five
induced shoots, is incubated at 20°C in a 16-h photoperiod at an irradiance of 80 µE
m –2 s –1 . Under these conditions 82% of induced shoots show 2–3 adventitious roots
(longer than 10 mm) after 8 weeks on solid medium (Figure 1C). Axillary shoots,
20–30 mm length, were induced to root on MS ½ with 10 gl –1 sucrose and 0.5 mgl –1
IBA (Spanos et al., 1997). Adventitious roots differentiated from 95% of shoots
after 4 weeks on medium with auxin.
Adult material. Apical portions from the disinfected shoots (20–25 mm in length)
are used for in vitro establishment of cultures, and axillary bud induction and shoot
elongation have been obtained following the same procedure as described for juvenile
material. Following the introduction in vitro, initial explants from adult stock plants
release dark exudations (phenols) which browned the medium surrounding the
explants. The detrimental effects of the phenolic exudations are markedly reduced
by weekly transfer of explants to a fresh medium, or simply moving the explants in
the same test tube on a fresh portion of the medium. This way, after 4 weeks, from 2
to 4 axillary shoots, longer than 10 mm, were developed from the pre-existent buds
of each healthy explant (Figure 1A) whilst multiple buds were developed at the base
of the primary explants and they formed a cluster. The subsequent transfer of
proliferating shoots on EM stimulates the elongation of shoots from axillary buds.
At the same time, the adventitious buds at the base of the primary explant remain
short and rapidly turn brown.
It is important to note that the new developing axillary shoots display morphological
juvenile characters (i.e., juvenile leaf traits with verticillate phyllotaxy). The
appearance of juvenile traits can be considered an effect of re-invigoration, due to