Summary We developed procedures for the micropropaga-tion of Juniperus oxycedrus L. using shoot apices or nodal segments from mature plants. Of the media and explants ex-amined, best culture establishment was obtained with shoot apices cultured on modified Schenk and Hildebrandt medium (SH medium) without growth regulators; however, shoot mul-tiplication was only achieved when shoot apices isolated from shoots grown on SH medium without growth regulators were subcultured on SH medium containing 0.5 µM benzyladenine. None of the auxins and methods tested for root induction provided satisfactory results.
Keywords: auxins, benzyladenine, cytokinins, growth regula-tors, indoleacetic acid, indolebutyric acid, kinetin, naphthale-neacetic acid, tissue culture.
Introduction
Micropropagation by axillary shoot proliferation has been reported for many hardwood species (Bonga and von Aderkas 1992). In contrast, axillary shoot proliferation in conifers still presents technical difficulties (Boulay 1987, Thorpe et al. 1991). However, there is much interest in developing tech-niques for axillary shoot proliferation in conifers as a means of facilitating micropropagation of trees that are old enough to have demonstrated their superior value. We have developed a system for micropropagating Juniperus oxycedrus L. (Cupres-saceae) from apical and nodal explants of mature trees. Junipe-rus oxycedJunipe-rus was chosen for this study because the species has potential for reforestation of degraded Mediterranean areas (Ceballos and Ruiz 1971).
Materials and methods
Plant material
Terminal shoots from lateral branches (5 cm length) were collected from 30-year-old J. oxycedrus trees growing in their natural habitat (La Eliana, Valencia, Spain) and kept at 4 to 6 °C for 1--2 days. The shoots were surface sterilized by immersion in 1% NaOCl containing 0.01% Tween-20 for 15 min, followed by four 5-min washes with sterile distilled water. Subsequently, shoots apices (2 mm length) or nodal
segments (with 2--3 axillary buds) were excised from the terminal shoots and cultured on different nutrient media.
Media and culture conditions
The basal media tested were: Medium A with MS macronutri-ents (Murashige and Skoog 1962), Medium B with SH macro-nutrients (Schenk and Hildebrandt 1972), Medium C with the macronutrients of Gressoff and Doy (1972), and Medium D with the macronutrients of Heller (1953) supplemented with 1 mM (NH4)2SO4. In some experiments, Medium A was used with MS macronutrients reduced to 1/3 of their original con-centration (A/3). All media included MS micronutrients, vita-mins as described by Vieitez et al. (1985), and 30 g l−1 sucrose. Solidified nutrient medium was prepared by adding 0.7% Difco Bacto agar. All media were adjusted to pH 5.8. Growth regulators were added to media before autoclaving for 20 min at 120 °C (1 × 105 Pa). Explants were cultured either in 150 × 25 mm glass tubes each containing 20 ml of agar-solidified nutrient medium, or in continuously agitated (50 rpm) 125-ml Erlenmeyer flasks each containing 30 ml of liquid medium. Vessels were closed with polypropylene closures (Bellco).
Unless otherwise specified, cultures were maintained for 60 days in a growth chamber at 26 ± 2 °C and a 16-h photope-riod with light supplied by Sylvania (GTE Gro-lux, F36W/GRO, Germany) fluorescent tubes (80 µmol m−2 s−1 irradiance at culture level). In all experiments, cultures were examined for sprouting percentage, number of shoots per ex-plant and shoot length. Twelve exex-plants were cultured per treatment, and all experiments were repeated at least twice.
Culture establishment
To study the effect of nutrient medium on culture estab-lishment, apical explants were cultured on agar-solidified Me-dium A, B, C or D supplemented with benzyladenine (BA; 0, 0.05 or 0.5 µM). The effect of 0.05 µM indoleacetic aicd (IAA) on culture establishment was examined in apical explants cultured on Medium B supplemented with 0, 0.05, 0.5, 5 or 10 µM BA.
To study the influence of harvest time on culture estab-lishment, apical explants from branches collected in spring, summer, autumn and winter were cultured on agar-solidified Medium B supplemented with 0, 0.05 or 0.5 µM BA.
Axillary shoot proliferation in cultures of explants from mature
Juniperus oxycedrus
trees
M. P. GÓMEZ and J. SEGURA
Department of Plant Biology, Faculty of Pharmacy, University of Valencia, Avinguda Vicent Andrés Estellés s/n, 46100-Burjasot (Valencia), Spain
Received October 17, 1994
Tree Physiology 15, 625--628
Shoot multiplication
Shoot apices (0.5--0.8 cm) isolated from shoots grown for 2 months on Medium B without growth regulators were trans-ferred to the same medium supplemented with BA (0, 0.05 or 0.5 µM) alone or in combination with 0.05 µM kinetin (KIN). In another experiment, explants were initially maintained for 1, 2, 6, 24 or 48 h in liquid Medium B supplemented with 50 µM BA. After each induction time, tissues were transferred to solid basal Medium B. Total culture period was 60 days.
Root induction
Shoots (1 cm) excised from clumps that had been cultured on agar-solidified Medium B without growth regulators were cul-tured for 30 days on solidified Medium B supplemented with naphthaleneacetic acid (NAA), IAA or indolebutyric acid (IBA) (0.0, 0.5, 2.5, 5.0, 10.0, 12.5 or 25.0 µM), or combina-tions of NAA + IAA, NAA + IBA or IAA + IBA (2.5 and 12.5 µM), then transferred to the same medium without auxins. In a second experiment, shoots were dipped in sterile aqueous solutions (pH 5.6) of IAA, NAA or IBA (50, 100, 250 or 500 µM) for 10 min and then cultured on solidified Medium B without growth regulators. Rooting percentages were recorded after 60 days.
Statistical analysis
Significance of the treatment effects was determined by analy-sis of variance (Statgraphics, Version 6, Manugistics, Rockville, MD, USA), employing a completely random de-sign. Percentage data were subjected to arcsin transformation before analysis. Variations among treatment means were ana-lyzed by Tukey’s (1953) procedure.
Results and discussion
Culture establishment
A comparison of apical and nodal explants cultured on Me-dium A/3 with different concentrations of BA (0, 5, 10, 20, 30 or 40 µM) indicated that apical explants were more responsive to BA than nodal explants. The presence of BA in the culture medium inhibited sprouting percentage, the mean number of shoots per explant and shoot length. Concentrations of BA higher than 10 µM caused necrosis of the explants. The use of BA concentrations less than 10 µM or the substitution of BA for KIN (0.5 or 5.0 µM) did not improve the morphogenetic capacity of the explants. None of these treatments was effec-tive in promoting axillary bud multiplication (data not shown). Neither nutrient media nor BA concentration significantly affected sprouting percentages (99--100%) of apical explants after 60 days. Although the type of nutrient medium signifi-cantly influenced the mean number of shoots formed per apical explant, the proliferation rates were low, ranging from 1.4 to 2.2 shoots per explant (Table 1). The length of the regenerated shoots depended on the nutrient medium and BA concentra-tion. Maximal shoot elongation occurred on Medium B with-out BA (Table 1).
The presence of 0.05 µM IAA, alone or in combination with BA, in the induction medium significantly reduced sprouting percentage, number of shoots per explant and shoot elongation of the explants (data not shown). In other conifers, the presence of auxins also reduces cytokinin-induced axillary bud prolif-eration (Lin et al. 1991), although a low concentration of auxin can promote growth of axillary shoots by counteracting the inhibitory effects of high cytokinin concentrations on shoot elongation (see Nehra and Kartha 1994). Bud cultures in coni-fers have also been successfully established on media without
Table 1. Effects of nutrient media and BA concentration on shoot proliferation from shoot tips of mature Juniperus oxycedrus. Culture time was
60 days. Data are mean values from 12 replications; A, B, C and D are the different culture media.
BA (µM) Sprouting percentage No. shoots/explant Shoot length (cm)1
A B C D A B C D A B C D
0 100 100 100 100 1.6 1.8 1.7 2.0 0.8 bcd 2.0 e 0.9 cd 1.0 d
0.05 98 100 100 100 1.6 2.0 1.8 1.7 0.4 ab 0.5 abc 0.4 ab 0.4 ab
0.5 98 100 100 100 1.4 2.2 2.0 1.6 0.3 a 0.4 ab 0.3 a 0.3 a
Mean2 1.5 a 2.0 b 1.8 ab 1.7 ab
Analysis of variance
Source of variation df Mean squares
Sprouting percentage No. shoots/explant Shoot length
Nutrient medium (A) 3 225.0 ns 3 1.4* 1.5*4
BA concentration (B) 2 56.2 ns 0.0 ns 10.4*
A × B 6 56.2 ns 0.4 ns 1.0*
Error 132 112.5 0.3 0.1
1 Interaction of nutrient medium with BA; values followed by the same letter are not significantly different according to Tukey’s test at P = 0.05. 2 Effect of nutrient medium; values followed by the same letter are not significantly different according to Tukey’s test at P = 0.05.
3 ns = Not significant. 4 * = Significant at P = 0.01.
growth regulators (Boulay 1979, Walker et al. 1985, Mon-teuuis 1991).
Because time of explant collection can influence in vitro propagation of some conifers (von Arnold 1981, Bonga 1987, Laliberté and Lalonde 1988, Chesick et al. 1990, Lin et al. 1991), we examined the proliferative capacity of shoot apices collected in spring, summer, autumn and winter, and cultured on agar-solidified Medium B with or without BA (0.05 and 0.5 µM). Sprouting percentages (100%) and the mean number of shoots per explant (1.6 to 2) were not significantly influenced by time of collection, but shoot length was greater (1.9 cm) on media without BA than on media containing BA (data not shown).
Shoot multiplication
Apical buds, isolated from shoots grown on solidified basal Medium B, were subcultured on the same medium supple-mented with BA, KIN or combinations of both cytokinins. After 60 days of culture, sprouting percentage was 100%. Furthermore, subculture favored axillary bud development and the formation of multiple shoot cultures. The highest number of shoots per explant was obtained when the medium was supplemented with 0.5 µM BA, and the greatest shoot length was obtained when the medium was supplemented with 0.5 µM BA and 0.05 µM KIN (Table 2).
Exposure to liquid Medium B containing 50.0 µM BA prior to culture on solidified medium without BA did not increase the morphogenetic response of the explants. Proliferation rates were less in explants cultured in liquid medium than on solidi-fied medium with BA (data not shown). There was also a progressive loss of morphogenetic potential and an increase in hyperhydricity of the explants with increasing exposure to BA. However, pulse treatments of cytokinins have improved caulo-genesis in other conifers (Bornman 1983, von Arnold and Tillberg 1987, Jang and Tainter 1991).
Rooting and plant development
None of the auxins or auxin combinations tested promoted satisfactory rooting. The frequency of rooted shoots (data not shown) ranged from 7 to 10%, and there were no significant differences between the auxins or auxin combinations tested. Some treatments, especially those including NAA, induced basal callus formation. In a previous study (Gómez and Segura 1994), we found high rooting percentages with adventitious shoots regenerated from leaves of mature J. oxycedrus suggest-ing that the method of micropropagation affects the rootsuggest-ing capability of the regenerated shoots. The high rhizogenic po-tential of adventitious shoots regenerated from leaves may be associated with some rejuvenation process, because an inverse relation between rooting capacity and age has been observed in many woody species (Németh 1986, Evers et al. 1988).
Micropropagation of adult Juniperus oxycedrus plants was accomplished by axillary shoot multiplication. The procedure may be useful for clonal propagation of elite genotypes, but only after improvements in the rooting of proliferating shoots have been achieved.
Acknowledgments
This work was supported by the Diputación de Valencia and the Conselleria de Cultura, Educación y Ciencia de la Generalidad Valen-ciana, grant to P.G.
References
Bonga, J.M. 1987. Clonal propagation of mature trees: problems and possible solutions. In Cell and Tissue Culture in Forestry, Vol. 1.
Eds. J.M. Bonga and D.J. Durzan. Martinus Nijhoff Publishers, Dordrecht, The Netherlands, pp 249--271.
Bonga, J.M. and P. von Aderkas. 1992. In vitro culture of trees. Kluwer Academic Publishers, Dordrecht, The Netherlands, 236 p. Bornman, C.H. 1983. Possibilities and constraints in the regeneration
of trees from cotyledonary needles of Picea abies in vitro. Physiol.
Plant. 57:5--16.
Boulay, M. 1979. Propagation in vitro du Douglas par
micropropaga-tion de germinamicropropaga-tion aseptique et culture de bourgeons dormants d’arbres forestiers. AFOCEL, Nangis, France, pp 67--75. Boulay, M. 1987. In vitro propagation of trees species. In Plant Tissue
and Cell Culture. Eds. G.C. Green, D.A. Sommers, W.P. Hackett and D.D. Biesboer. AR Liss, New York, pp 367--382.
Ceballos, L. and J. Ruiz. 1971. Arboles y arbustos de la España peninsular. Instituto Forestal de Investigaciones Agrarias y Escuela Técnica Superior de Ingenieros de Montes, Madrid, Spain, 512 p. Chesick, E.E., D.E. Bilderback and G.M. Blake. 1990. In vitro
multi-ple bud formation by 20-year-old western larch buds and stems. HortScience 25:114--116.
Evers, P.V., J. Donkers, A. Prat and E. Vermeer. 1988. Micropropaga-tion of forest trees through tissue culture. Pudoc, Wageningen, The Netherlands, 84 p.
Gómez, M.P. and J. Segura. 1994. Factors controlling adventitious bud induction and plant regeneration in mature Juniperus oxycedrus
leaves cultured in vitro. In Vitro Cell. Dev. Biol. 30P:210--218.
Gressoff, P.M. and C.H. Doy. 1972. Development and differentiation of haploid Lycopersicum esculentum. Planta 107:161--170. Heller, R. 1953. Recherches sur la nutrition minerale des tissus
vègètaux cultivés in vitro. Ann. Sci. Nat. Bot. Biol. Veg. 14:1--223. Table 2. Shoot multiplication from shoot tips of mature Juniperus
oxycedrus cultured on Medium B with cytokinins. Culture time was
60 days. Data are mean values from 12 replications.
Cytokinins (µM) No. shoots/explant1 Shoot length (cm)1
BA KIN
Source of variation df Mean squares
No. shoots/explant Shoot length
Cytokinins 4 59.9*2 0.9*
Error 55 6.8 0.1
1 For each parameter, values followed by the same letter are not significantly different according to Tukey’s test at P = 0.05. 2 * = Significant at P = 0.01.
Jang, J.C. and F.H. Tainter. 1991. Micropropagation of shortleaf, Virginia and loblolly × shortleaf pine hybrids via organogenesis. Plant Cell Tissue Organ Cult. 25:61--67.
Laliberté, S. and M. Lalonde. 1988. Sustained caulogenesis in callus cultures of Larix × eurolepsis initiated from short shoot buds of a
12-year-old tree. Am. J. Bot. 75:767--777.
Lin, Y., M.R. Wagner and L.J. Heildmann. 1991. In vitro formation of axillary buds by immature shoots of ponderosa pine. Plant Cell Tissue Organ Cult. 26:161--166.
Monteuuis, O. 1991. Rejuvenation of a 100-year-old Sequoiadendron giganteum through in vitro meristem culture. I. Organogenesis and morphological arguments. Physiol. Plant. 81:111--115.
Murashige, T. and F. Skoog. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15:473--497.
Nehra, N.S. and K.K. Kartha. 1994. Meristem and shoot tip culture: requirements and applications. In Plant Cell and Tissue Culture.
Eds. I.K. Vasil and T.A. Thorpe. Kluwer Academic Publishers, Dordrecht, The Netherlands, pp 37--70.
Németh, J. 1986. Induction of rooting. In Biotechnology in
Agricul-ture and Forestry, Vol. 1, Trees I. Ed. Y.P.S. Bajaj. Springer-Verlag, Berlin, Germany, pp 49--64.
Schenk, R.V. and A.C. Hildebrandt. 1972. Medium and techniques for induction and growth of monocotyledonous and dicotyledonous plant cell cultures. Can. J. Bot. 50:199--204.
Thorpe, T.A., I.S. Harry and P.P. Kumar. 1991. Application of micro-propagation to forestry. In Micropropagation, Technology and
Ap-plication. Eds. P.C. Deberg and R.H. Zimmerman. Kluwer Academic Publishers, Dordrecht, The Netherlands, pp 311--336. Tukey, J.W. 1953. Some selected quick and easy methods of statistical
analysis. Trans. N.Y. Acad. Sci. 16:88--97.
Vieitez, A.M., M. San-José and E. Vieitez. 1985. In vitro plantlet
regeneration from juvenile and mature Quercus robur L. J. Hortic. Sci. 60:99--106.
von Arnold, S. 1981. In vitro propagation of Norway spruce. In Coll.
Int. Cult. in vitro Essences Forestiers. IUFRO, Fontainebleau,
France, pp 87--100
von Arnold, S. and E. Tillberg. 1987. The influence of cytokinin pulse treatments on adventitious bud formation on vegetative buds of
Picea abies. Plant Cell Tissue Organ Cult. 9:253--261.
Walker, N., E. Dumas, A. Franclet and F. Bekkaoui. 1985. Techniques de culture in vitro des meristémes de Sequoia sempervirens et Pinus pinaster. Ann. AFOCEL, Nangis, France, pp 8--29.
