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.

Genetic control of somatic embryogenesis induction in Eucalyptus globulus Labill.

A reproducible protocol for somatic embryogenesis (SE) induction in Eucalyptus globulus from mature zygotic embryos is available since 2002. However, for the use of SE in tree breeding programs, the frequency of SE initiation needs to be improved and controlled, and this was investigated in 13 open-pollinated (OP) families over three consecutive years. A diallel mating design with five parent trees was used to study genetic control of SE induction. Results showed that SE induction varies across E. globulus families and over the years of seed production tested. Somatic embryogenesis was initiated on explants from 84% of the OP families tested in 2002 and 100% of the families tested in 2003 and 2004. The year 2003 gave best results for percentage of induction and total number of somatic embryos produced. Results concerning genetic control showed that SE induction is under the control of additive genetic effects, as 22.0% of variation in SE initiation was due to general combining ability (GCA) effect, whereas 6.4% was due to maternal effects. Neither specific combining ability (SCA) nor reciprocal effects were significant.

Analysis of the genetic stability of Eucalyptus globulus Labill. somatic embryos by flow cytometry.

Flow cytometry was used to measure the nuclear DNA content of Eucalyptus globulus Labill. somatic and zygotic embryos and leaves in order to determine if somatic embryogenesis induces DNA content and ploidy changes in this species. Mature zygotic embryos derived from open-pollination orchard families were collected from a location in the centre of Portugal. One group was kept for nuclear DNA content and ploidy analysis, and the other group was used for establishing embryogenic cultures. Mature zygotic embryos were grown on Murashige and Skoog (MS) medium supplemented with 3% (w/v) sucrose and 3 mg l(-1) alpha-naphthaleneacetic acid (NAA) for 3 weeks and then transferred to MS medium without growth regulators. Globular somatic embryos from approximately 8-month-old embryogenic cultures were used in the assay. DNA ploidy levels and the nuclear DNA content of mature zygotic embryos, somatic embryos and leaves from the mother field tree were determined using flow cytometry combined with propidium iodide staining. Zygotic embryos had a nuclear DNA content of 1.32 pg/2C, somatic embryos had a nuclear DNA content of 1.39 pg/2C and leaves from the field tree had a nuclear DNA content of 1.40 pg/2C. The values estimated for the somatic embryos and mother plant did not differ statistically from each other (P < or = 0.05), but both differed from those of the zygotic embryos (P < or = 0.05). These results clearly indicate that no changes were induced during the embryogenic process. However, the differences found between the field plants and zygotic embryos did suggest that some aspects must be evaluated carefully, as propidium iodide fluorescence may potentially be influenced by the presence of secondary compounds (e.g. anthocyanins, tannins) in E. globulus somatic embryos and mature leaves. Therefore we believe that the somatic embryogenesis methodology used did not induce major genetic changes in the somatic embryos and that our primary goal of "true-to-type" propagation was assured.

Early ovule development following self- and cross-pollinations in Eucalyptus globulus Labill. ssp. globulus.

The study was conducted to identify the self-incompatibility mechanism in Eucalyptus globulus ssp. globulus. Controlled self- and cross-pollinations were conducted on individual flowers from three mature trees that had self-incompatibility levels of 76, 99.6 and 100%. Flowers were harvested at 4, 6 and 8 weeks after pollination. Embryology was investigated by bright field microscopy on material harvested at 4 and 6 weeks after pollination. Fertilization had taken place at 4 weeks after pollination with zygotes and free nuclear endosperm visible. There was a greater proportion of healthy, fertilized ovules in the cross- compared with the self-pollination treatment, and approx. half the ovules examined from both pollen treatments were not fertilized or were degenerating. By 6 weeks after pollination a few zygotes were starting to divide. The number of healthy, fertilized ovules was still greater in the cross-pollination treatment, but the number of healthy fertilized ovules was lower in both treatments compared with 4 weeks after pollination, and many ovules were degenerating. Fertilized ovules were significantly larger than non-fertilized or degenerating ovules and this difference was detectable by eye at 6 and 8 weeks after pollination. The mechanism of self-incompatibility appears to have both late pre- and post-zygotic components.