Factors influencing axillary shoot proliferation and ... - Tree Physiology

484 RENAU-MORATA, OLLERO, ARRILLAGA AND SEGURA
exclude a possible ABA influence in our experiments. The use
of defoliated microcuttings makes it difficult to compare our
results with those reported by Piola and Rohr (1996) for juvenile
C. libani microcuttings; nevertheless, we found that an incubation
temperature of 30 °C enhanced shoot yield in juvenile
microcuttings of both C. atlantica and C. libani. Piola and
Rohr (1996) found that a 3-h pulse with 0.1 mM BA induced
the highest proliferation rate (4.5 buds per explant) in C. libani
microcuttings. We observed similar results for C. libani microcuttings
grown in the presence of 2.2 µM BA; however, BA
negatively affected bud sprouting in C. atlantica microcuttings.
Nested ANOVA attributed most of the variability in shoot
yield from cultured microcuttings of juvenile origin to single
seedlings (77 and 65% for C. atlantica and C. libani, respectively).
This strong genotypic effect was even more evident in
cultured explants of bicentennial C. libani trees, where only
three out of the six genotypes were successfully established in
vitro. A similar genotypic effect on the bud-forming capacity
of explants was observed by Tang et al. (2001). In some conifers,
variation in morphogenic responses was evident not only
among embryos from different seed lots but also among embryos
obtained from controlled crossings (von Arnold and
Eriksson 1982, 1986, Shen and von Arnold 1982). We observed
differences in the initial in vitro performance of the
three genotypes, but these differences decreased with increasing
number of subcultures.
Usually, in vitro manipulation of woody plants is more difficult
with mature explants than with juvenile explants, and
Cedrus was no exception. A comparison between cedar explants
of juvenile origin and those of adult origin showed that
microcuttings were the preferred explant for axillary shoot
proliferation in juvenile and adult cultures. Although this response
did not require the presence of cytokinin in the culture
medium, Z increased axillary shoot yield in explants of mature
origin. Hormone-free nutrient media have been successfully
employed for culture establishment of other mature Pinaceae
including Pseudotsuga menziesii (Mirb.) Franco and Pinus
lambertiana Dougl. (Gupta and Durzan 1985), although the
presence of growth regulators favored axillary bud sprouting
in shoot apices and microcuttings isolated from 20-year-old
Larix occidentalis Nutt. trees (Chesick et al. 1990).
Shoot proliferation rates in cultures of adult C. libani were
generally greater on basal MSBN/2 medium than on basal
WPM. Although the influence of nutrient medium on in vitro
morphogenesis is well documented, its effect remains one of
the most empirical aspects of plant cell culture (Preece 1995).
Compared with basal MSBN/2 medium, basal WPM has
higher concentrations of calcium, sucrose, micronutrients and
organic supplements, and contains biotine and folic acid (both
absent in MSBN/2 medium). A reduced amount of nutrients
stimulates axillary branching in some plants (Karhu 1997) and
low calcium availability can affect apical meristem integrity
(McCown and Sellmer 1987), thus favoring axillary bud development;
however, we are unable to offer an explanation for
the differential effect of MSBN/2 and WPM media on the
basis of our experimental data.
TREE PHYSIOLOGY VOLUME 25, 2005
Adventitious organogenesis offers higher potential for
shoot production than axillary bud proliferation, and is the preferred
method for coniferous micropropagation because coniferous
buds are generally induced directly on the explant
(Thorpe et al. 1991). With a few exceptions, however, techniques
for adventitious budding of coniferous explants obtained
from trees in the adult growth phase are still limited, because
the caulogenic potential of isolated organs is affected by
the ontogenic age of the tissues (von Aderkas and Bonga 2000,
Giri et al. 2004). In our experiments, callus derived from shoot
apices differentiated adventitious buds in the presence of cytokinin,
but only when explants of juvenile origin were used and
we were unable to promote their elongation. In contrast,
Hosseyni et al. (1999) reported the induction of adventitious
organogenesis (buds and needles) from winter buds of 10–15year-old
C. libani cultured on WPM with BA or kinetin. This
difference between studies may be associated with tree age,
because our trees were older.
We successfully generated adventitious shoots from C. atlantica
embryos, which opens up the possibility of mass propagation
of this species. Our regeneration system included: (1)
adventitious budding in the presence of 9.0 µM Z; (2) bud development
on medium containing 4.4 µM Z; and (3) shoot
elongation on cytokinin-free medium. Zeatine or benzyladenine
alone was enough to stimulate both direct and indirect
adventitious budding, which is in agreement with previous
findings in other conifer species (Thorpe et al. 1991); however,
the presence of Z improved the bud-forming capacity of the
explants, and the process of bud development required transfer
of C. atlantica embryos to a medium with cytokinin. In contrast,
in other conifers, bud development depends on transfer
of explants to hormone-free medium (Gómez and Segura
1994, Mata et al. 2001, Villalobos-Amador et al. 2002, Schestibratov
et al. 2003).
Successful micropropagation of many woody species is frequently
limited by their reluctance to form adventitious roots,
and C. libani and C. atlantica were no exception. In many species,
rooting can be achieved by adding auxins to the culture
medium or by using different support substrates (Hartmann et
al. 1997). Activated charcoal, a high sucrose concentration,
variations in environmental temperature conditions and light
reduction in the rooting zone improve auxin-induced rooting
in many species, including conifers (George 1993). A variety
of ancillary compounds such as paclobutrazol, phosphoric
acid, calcium, triacontanol, oligosaccharins and coumarin
(George 1993, Haissig and Davis 1994, Tantos et al. 2001,
Nandi et al. 2002) can improve rooting capacity in recalcitrant
species. In some conifer species, infection with A. rhizogenes
enhances rhizogenesis (McAfee et al. 1993, Tzfira et al. 1996,
Mihaljevic et al. 1998, Villalobos-Amador et al. 2002). In our
experiments, all of these variables failed to induce rooting in
shoots of axillary or adventitious origin of C. libani and C. atlantica.
Nicholson (1984) and Nandi et al. (2002) were able to
induce rooting in C. deodara (D. Don) G. Don and rooting has
been reported in C. libani and C. atlantica (Dirr and Heuser
1987) cuttings, although in the last two species, rooting percentages
were minimal and rooting was observed only when
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