A. M. Vieitez

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.

In vitro plantlet regeneration of mature chestnut

SummaryShoot formation and plantlet regeneration have been induced from shoot tip expiants of mature chestnut. Stump sprouts were collected in winter from three clones of Castanea sativa χ B. crenata hybrids selected for their resistance to root rot disease. After cold storage at 4°C they were planted in perlite in the glasshouse and the new shoots used to establish initial expiant cultures on Murashige and Skoog’s medium with half strength nitrates containing BAP. The effects of nine macroelement formulae on the multiplication rate and elongation of the cultures were studied. The optimal concentration of BAP for shoot multiplication was 0.2 mg l-1. The addition of 0.01-1.0 mg 1_1 of NAA did not improve the multiplication rate nor the general appearance of the cultures. Rooting was achieved by dipping the basal ends of shoots regenerated in vitro in a 0.5 g l-1 solution of IBA for 15 min or for 2 min at 1.0g l-1.

Somatic Embryogenesis in Mature Quercus Robur Trees

Somatic embryo induction and plant regeneration have been obtained in tissues from mature Quercus robur L. trees. Epicormic shoots were forced to flush in branch segments collected from the crown of trees growing in selected stands on different collection dates. Expanding leaves from five genotypes, cultured following a multistage treatment procedure, produced somatic embryos at frequencies ranging from 0.3 to 3.6% of leaf explants, depending on genotype and collection date. After being induced, somatic embryos started a recurrent process by secondary embryogenesis which amplified the 15 embryogenic lines established. Plant recovery was achieved in 60% and 17% of matured embryos from two genotypes.

Cryopreservation of embryogenic cultures of Quercus robur using desiccation and vitrification procedures

Oak embryogenic cultures are generally maintained by repetitive embryogenesis. To facilitate management of embryogenic lines and limit the risks of somaclonal variation and contamination a cryopreservation protocol should be developed. In this work we investigated the ability of several pre-treatments to enable 4-6mg clumps (1.0-1.5mm) of globular-heart stage somatic embryos of Quercus robur to withstand freezing in liquid nitrogen. In the best of the two embryogenic culture lines used, 56% of clumps resumed embryogenesis after cooling when they had been pre-treated by successive pre-culture on 0.3 and 0.7M sucrose supplemented media followed by desiccation in the air flow of a laminar flow cabinet to water contents of 24-34%. In both lines, embryogenesis resumption rates of about 70% were achieved by pre-culture on 0.3M sucrose medium followed by application of a vitrification solution (PVS2) for 60-90min prior to rapid plunging in liquid nitrogen.

In vitro adventitious bud regeneration from internode segments of beech

Internode explants collected from in vitro grown shoots of two clones of Fagus sylvatica L. (European beech) and five clones of F. orientalis Lipski (Oriental beech) were used to evaluate their bud regeneration capacity. Adventitious shoot-buds formed on callus, which developed from internode segments cultured in a Woody Plant Medium supplemented with different concentrations of either thidiazuron (TDZ) or benzyladenine (BA). After 4 weeks of culture on induction media, the explants were transferred to a proliferation medium supplemented with 2.2 μM BA, 9.1 μM zeatin and 2.9 μM indole-3-acetic acid (IAA) for another 8 weeks. Medium containing TDZ was much more efficient than medium containing BA in inducing adventitious buds, the optimal TDZ concentration being 4.5 μM and the optimal BA concentration 17.8 μM. Genotypic variation in shoot regeneration capacity was observed among the two Fagus species and between clones within each species, with a significant interaction between TDZ concentration and genotype regarding mean bud number. Thidiazuron induction medium supplemented with a range of individual auxins was investigated, and it was found that IAA or indole-3-butyric acid at 2.9 μM enhanced the bud forming capacity of explants. Morphogenic response varied significantly with the position of the internode along the stem. The highest regeneration potential was obtained from apical internodes, while those distal to the apex were the least productive. Elongated shoots of adventitious origin can be readily proliferated by axillary branching.

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.

Protocol for Micropropagation of Castanea Sativa

En Protocols for Micropropagation of Woody Trees and Fruits (eds. S. M. Jain and H. HAGGMAN , pags. 299-312.)

Somatic embryogenesis and plantlet regeneration from cell suspension cultures of Fagus sylvatica L.

SummaryEmbryogenic cell suspension cultures and somatic embryos of five genotypes of beech, were obtained from aged cultures derived from immature zygotic embryos cultured on solid medium containing both 2, 4-dichlorophenoxyacetic acid and N6-benzyladenine. The origin of somatic embryos was traced from single cells. Embryos remained arrested at the globular stage on liquid media, further development was achieved after plating embryogenic aggregates on Murashige and Skoogs medium with half strength major salts supplemented with glutamine and low levels of growth regulators. Cultures of different genotypes showed significant differences in maturation frequency which was not affected by the hormone treatments assayed. The frequency of conversion of embryos into plantlets was low. This frequency increased after cold storage of embryos for up to 7 months.

Somatic Embryogenesis in Cultured Immature Zygotic Embryos in Chestnut

Summary Mature and immature zygotic embryos from two specimens of different Castanea sativa x C. crenata clones were cultured in vitro to induce the production of somatic embryos. After 2 months of culture, nodular embryogenic callus developed on a small proportion of the explants obtained from immature zygotic embryos and cultured on Murashige and Skoog medium supplemented with 0.1-1 mg - L -1 2,4dichlorophenoxyacetic acid (2,4-D) with or without 1-2 mg - L -1 zeatin or 6-benzylaminopurine (BAP). Globular and heart-shaped embryoid structures differentiated following transfer to medium with no 2,4-D. Mature somatic embryos were achieved after transfer to Woody Plant Medium. Germination of mature embryos was incomplete. Embryogenic competence was maintained throughout 10 months of regular subculturing.

Induction of somatic embryogenesis in explants of shoot cultures established from adult Eucalyptus globulus and E. saligna × E. maidenii trees

A reproducible procedure for induction of somatic embryogenesis (SE) from adult trees of Eucalyptus globulus Labill. and the hybrid E. saligna Smith × E. maidenii has been developed for the first time. Somatic embryos were obtained from both shoot apex and leaf explants of all three genotypes evaluated, although embryogenic frequencies were significantly influenced by the species/genotype, auxin and explant type. Picloram was more efficient for somatic embryo induction than naphthaleneacetic acid (NAA), with the highest frequency of induction being obtained in Murashige and Skoog medium containing 40 µM picloram and 40 mg l(-1) gum Arabic, in which 64% of the shoot apex explants and 68.8% of the leaf explants yielded somatic embryos. The embryogenic response of the hybrid was higher than that of the E. globulus, especially when NAA was used. The cultures initiated on picloram-containing medium consisted of nodular embryogenic structures surrounded by a mucilaginous coating layer that emerged from a watery callus developed from the initial explants. Cotyledonary somatic embryos were differentiated after subculture of these nodular embryogenic structures on a medium lacking plant growth regulators. Histological analysis confirmed the bipolar organization of the somatic embryos, with shoot and root meristems and closed procambial tissue that bifurcated into small cotyledons. The root pole was more differentiated than the shoot pole, which appeared to be formed by a few meristematic layers. Maintenance of the embryogenic lines by secondary SE was attained by subculturing individual cotyledonary embryos or small clusters of globular and torpedo embryos on medium with 16.11 µM NAA at 4- to 5-week intervals. Somatic embryos converted into plantlets after being transferred to liquid germination medium although plant regeneration remained poor.