M. C. San-José

Somatic embryogenesis from stem and leaf explants of Quercus robur L.

Abstract Internodal and leaf segments from pedunculate oak (Quercus robur L.) seedlings were used as explant source to induce somatic embryogenesis. Auxin treatment influenced embryogenic response, which only occurred in explants initially cultured on media containing 4 mg/l naphthaleneacetic acid (NAA) and different benzyladenine (BA) concentrations. After 6 weeks of culture on induction medium, the explants were transferred to medium supplemented with 0.1 mg/l BA and 0.1 mg/l NAA, and 4 weeks later, they were subcultured in a growth-regulator-free medium, in which somatic embryos arose through indirect regeneration on the surface of a nodular callus. Somatic embryos were induced in explants of two out of four seedling provenances. The induction frequency ranged from 16% in leaf explants to 4% in internodal explants. Somatic embryos developed two cotyledons, which were translucent or opaque-white in appearance, but anomalous morphologies were also observed. Different embryogenic lines were established and maintained by repetitive embryogenesis in multiplication medium containing 0.1 mg/l BA plus 0.05 mg/l NAA. These results indicate that tissues from explants other than Q. robur zygotic embryos are able to produce embryogenic cultures.

Improving genetic transformation of European chestnut and cryopreservation of transgenic lines

The aim of the present work was to study the effect of the developmental stage of the somatic embryos and of the genotype on the genetic transformation of embryogenic lines of European chestnut (Castanea sativa Mill.) and the cryopreservation of the embryogenic lines that are generated. As an initial source of explants in the transformation experiments, it was found that the use of somatic embryos isolated in the globular stage or clumps of 2–3 embryos in globular/heart-shaped stages was more effective (30%) than when embryos at the cotyledonary stage were used (6.7%). All of the seven genotypes tested were transformed, and transformation efficiency was clearly genotype dependent. Three transgenic lines were successfully cryopreserved using the vitrification procedure, and the stable integration of the uidA gene into the transgenic chestnut plants that were regenerated subsequent to cryopreservation was demonstrated.

Progress towards clonal propagation of Camellia japonica cv. Alba Plena by tissue culture techniques

SummaryOptimal rooting conditions have been determined for shoot cultures of Camellia japonica cv. Alba Plena derived from a 50 year old tree: dipping the base of shoots in 1 g l−l solution for 15 min, followed by 12 days’ darkness, induced 87% rooting in shoots cultured with Woody Plant Medium (WPM) macronutrients. Halving the auxin concentration or exposure time considerably reduced this rate, and root formation was severely inhibited if an initial dark period for the entire shoot was not used. The type of support (agar or paper bridges) did not significantly affect the rooting percentage or number of roots per rooted shoot, but liquid media induced greater root elongation. No significant differences were observed between the use of WPM and a modified Heller’s medium as regards the rooting percentages achieved, but the number of roots formed was considerably greater with WPM, as was the survival rate after transfer to soil (70% for transfer as against 35% with the modified Heller’s medium). Best surviva...

Development of rejuvenation methods for in vitro establishment, multiplication and rooting of mature trees.

The utility of the micropropagation of forest trees is limited by the efficiency with which selected trees can be reproduced vegetatively. As is well known, there are several woodland species for which satisfactorily successfull establishment in vitro has still not been achieved, at least for adult trees. Juvenile trees are in general readily cloned by conventional techniques, but the ease of propagation of many trees tends to diminish as they approach a size sufficient to allow reliable evaluation of their crop potential (Bonga, 1987).

MICROPROPAGATION OF Fagus spp.

The genus Fagus,a member of the Fagaceae family, comprises ten species of moncecious trees native to the Northern Hemisphere Temperate Zone regions of Eurasia and Eastern North America. The name Fagus (related to Greek phagein, to eat) is a reference to the distinctive triangular nuts of these species, which are eaten by both wildlife and humans. The chief members of this genus are Fagus sylvatica L. (European beech), which is one of the economically most important deciduous trees of Central Europe and together with oaks defines the climax vegetation of this region; F. orientalis Lipski (Oriental beech), a native of the temperate regions of Eastern Europe, the Balkan peninsula, the Caucasus and Asia Minor; F. grandiflora Ehrh. (American beech), a native of Eastern North America that is both of great

Adventitious shoot regeneration from in vitro leaves of adult Camellia reticulata

Experiments were conducted with Camellia reticulata cv. Captain Rawes to develop an adventitious regeneration system. Leaves were taken from axillary shoot proliferation cultures in WPM medium that had been established from mature trees. They were sectioned, and then plated in Petri dishes on media containing various combinations of cyto- kinins and auxins; the best response was induced by 2 mg I“1 BA + 1 mg I-1 IBA. The shoots obtained were multiplied by axillary branching on WPM + 2 mg I-1 BA + 2 mg I-1 Z + 2 mg I-12iP + 0.01 mg I-1 IBA. There was no significant difference in multiplication coefficient (the product of the proportion of explants forming axillary shoots and the mean number of new segments per explant) between shoots of adventitious and axillary origin, but there was between the various types of explant used in the multiplication stage: shoot tips (ST1) and nodal segments (ns) of harvested shoots longer than 14- 15 mm, and whole harvested shoots 7-10 mm long (ST2). The best results were ac...

Conservation of Hardwood Forest Species

Conservation of forest genetic resources is essential for meeting the demand for future wood products. Genotypes with improved characteristics are nowadays widely produced through conventional breeding programmes, by selection of mature superior trees, by genetic transformation procedures, etc., which are important for increasing the productivity of forestry clonal plantations. Strategies for forest biodiversity conservation today are well defined, among which cryopreservation is viewed as a complementary storage method, important for plant tissues with specific characteristics (vegetatively propagated species). In addition, many hardwood forest trees produce recalcitrant seeds (seeds that cannot be stored for long periods under conventional conditions) that only would be stored on a long-term basis through cryopreservation. The availability of simple, reliable and cost-effective strategies for conservation of hardwood forest species (with special attention to recalcitrant species of the Fagaceae family) will be highlighted in this review. Specifically, emphasis will be addressed to the following topics: (i) medium-term conservation through slow growth storage; (ii) cryopreservation techniques; (iii) selection of explants for cryopreservation: in vivo collection of embryonic axes and dormant buds and in vitro collection of shoot tips and embryogenic cultures; and (iv) genetic stability of cryopreserved material. The limited application of cryopreservation to the development of large cryobanks of hardwood forest species also will be mentioned.

Improved efficiency of in vitro propagation of Camellia reticulata cv. Captain Rawes

SummaryEffects of various factors were studied on the in vitro multiplication and rooting of a clone of Camellia reticulata cv. Captain Rawes established in vitro from shoots of an adult tree. Multiplication rates were increased by repeated harvesting of cultures in which shoots were subcultured in a horizontal rather than a vertical position. Best rooting responses were obtained with 175.3 mM sucrose in the rooting medium using 8 week old shoots cultured in 500 ml glass jars; these shoots contained more anthocyanin than those from test-tube cultures. The orientation and repeated harvesting of the mother shoots had no effect on rooting response.