J. M. Bonga

In vitro culture of trees.

1. Introduction. 2. Laboratory Organization and Equipment. 3. Media Preparation. 4. Collection, Sterilization, Excision and Culture. 5. Clonal Propagation. 6. Special Cultures. 7. Secondary Metabolite Production. 8. Pathology. 9. Nitrogen Fixation. 10. Storage of Cultures. 11. Genetic Modification. 12. Epilogue. 13. References. 14. Indices.

Application of somatic embryogenesis in high-value clonal forestry: deployment, genetic control, and stability of cryopreserved clones

SummaryThe most important advantage of cloning conifers by somatic embryogenesis (SE) is that the embryogenic tissue can be cryopreserved without changing its genetic make-up and without loss of juvenility. This offers an opportunity to develop high-value clonal varieties by defrosting and repropagating cryopreserved clones after genetic testing has shown which clones are the best performers. In the current absence of cost-effective automated embling handling systems or artificial seed technology, the deployment of the high-value clones in clonal forestry can be achieved inexpensively by mass serial rooting of cuttings from juvenile donor plants produced from cryopreserved embryogenic cultures. In a genetic analysis of the SE process in white sprucePicea glauca, we found that induction of SE was under strong genetic control. Although the dominance variance diminished rapidly as the zygotic embryos matured, the additive variance remained relatively large during the induction phase. The genetic effects in the subsequent maturation and germination phases were less strong. Furthermore, genetic variation at the different phases of SE was not correlated. Thus, it is the induction phase of SE that can be manipulated by breeding. Most of the embryogenic clones were cryopreserved easily, i.e., there was no apparent genotype effect. To determine stability of cryopreserved clones, a set of 12 clones was retrieved after 3 and 4 years, respectively, from cryopreservation and repropagated by SE. An assessment of morphologicalin vitro development andex vitro survival and growth characters demonstrated general stability of the cryopreserved clones of white spruce.

Initiation of somatic embryogenesis in white spruce (Picea glauca): genetic control, culture treatment effects, and implications for tree breeding.

The degree of genetic control and the effects of cultural treatments on somatic embryogenesis (SE) in white spruce were investigated with material derived from six-parent diallel crosses, including reciprocals. Thirty zygotic embryos from both immature and mature cones of each family were cultured in media with either 2,4-D or Picloram immediately after the collection of cones and after 2 months of cold storage. There were significant differences in SE initiation between immature and mature explants, and fresh and cold-stored seeds, but there was no significant differences with culture media effect. Significant variances due to families and to family x treatment interactions were found. The mean percentage of explants that initiated SE in each family ranged from 3.3% to 54.6%, with an overall average of 30.5%. The partitioning of family variance revealed that 21.7% was due to general combining ability effects, 3.5% was due to maternal effects, and 5.5% was due to reciprocal effects, but that the specific combining ability (SCA) was negligible. Variance due to interactions of family x treatments collectively accounted for 32.6%, while the remaining 37.8% of variation was accounted for by random error. However, when comparing the responses obtained with the treatment combinations, the SE response for freshly excised immature embryo explants showed comparatively large SCA variance, whereas the SCA variance was very small in the other treatment combinations.

Optimized somatic embryogenesis in Pinus Strobus L.

SummarySomatic embryogenesis (SE) initiation in Pinus strobus was optimized by the manipulation of plant growth regulator (PGR) concentrations in the culture medium. Modified Litvay medium (MLV) of Litvay et al. (1985) supplemented with lower than routinely used PGR concentration increased initiation of established embryogenic cultures from approximately 20 to 53%. The original developmental stage of zygotic embryos had a pronounced effect on the SE response. The optimum stage was the pre- to shortly post-cleavage stage. A substantial genetic influence on initiation of SE was indicated by a significant variance component due to families. Genotype X collection date and genotype X media interactions had large effects on initiation of SE. The PGR levels in the culture medium prior to maturation had a significant effect on subsequent production of mature somatic embryos. Embryogenic tissue initiated and proliferated on medium with a low level of PGR consistently produced a high number of somatic embryos, indicating that optimized initiation protocol also enhanced somatic embryo production. Somatic embryos of 93 embryogenic lines (representing five families) that were initiated on media with different PGR concentrations were converted to plants at an overall frequency of 76%, and grown in the greenhouse. With these improved protocols, application of P. strobus SE in commercial clonal forestry is feasible as an alternative to traditional breeding and reforestation.

Initiation of somatic embryogenesis in Pinus banksiana, P. strobus, P. pinaster, and P. sylvestris at three laboratories in Canada and France

During 2002–2004, three laboratories in Canada and France collaborated to improve initiation of somatic embryogenesis (SE) in jack pine (Pinus banksiana Lamb.), eastern white pine (P. strobus L.), maritime pine (P. pinaster Ait.), and Scots pine (P.␣sylvestris L.), giving particular attention to the effects of (1) N-(2-chloro-4-pyridyl)-N′-phenylurea (CPPU) versus various concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and benzyladenine (BA), (2) differences in basal nutrient media, i.e., macro- and microelements, and (3) gelling agent concentration. The work was carried out separately at␣each laboratory, but the details of media compositions were shared and tested on their respective species. Results indicate that the developmental stage of the zygotic embryo (ZE) and genotype effects had a large influence on SE initiation, and that genetic effects were consistent over time. Different species responded differently to PGR types and concentration, basal nutrient media, trace elements, and their combinations. Currently, our best initiation rates based on a selected group of genotypes, optimal development stage of ZE, and medium are 3.9% for jack pine, 54.6% for eastern white pine, 76.2% for maritime pine, and 19.7% for Scots pine.

Rejuvenation of Tissues from Mature Conifers and its Implications for Propagation in Vitro

Clonal propagation of trees old enough to have expressed their desirable characteristics is an effective means of rapidly obtaining improved planting stock. However, cloning of mature trees of conifer species is often difficult or impossible with present-day techniques. Therefore, specimens are often selected for clonal propagation when they are still too young for proper assesment. For many species such assesment is, at present, not possible until the trees have reached about half their rotation age (Namkoong et al. 1980). Because of this predicament, maximum benefit of clonal propagation cannot be expected until we find the appropriate means either to clone older trees or to determine at a young age what the trees will be like at the end of their rotation.

Somatic embryogenesis in white spruce (Picea glauca): genetic control in somatic embryos exposed to storage, maturation treatments, germination, and cryopreservation.

Genetic controls for growth of embryogenic cultures, storage, maturation treatments, germination and cryopreservation in white spruce somatic embryogenesis (SE) were examined. These SE processes were under genetic control but less strongly so than the initiation phase. For all the SE characters examined, variance due to clones within families was significant and often the largest genetic component of variance. This was further partitioned using an additive-dominance-epistasis model. A relatively-large proportion of the total genetic variance was due to epistatic variance in the maturation and germination of somatic embryos. Embryogenic lines were cryopreserved easily without a distinct genetic influence being noticed.

Formation of haploid embryoids of Larix decidua: early embryogenesis

Callus initiation of sections of megagametophytes of Larix decidua occurs just below the cut surface and is followed by the formation of one or more long protruding cells. The long cells then divide transversely at their tips to yield small cytoplasmically-dense terminal cells. The latter divide, forming loose aggregates of dense cells, microcalli, which develop long cells that radiate in all directions and divide terminally to produce aggregates of small cells. This long/short cell alternation is repeated a few times. Eventually the aggregates divide in a polarized manner producing files of long cells predominantly in one direction. These loosely bundled long cells form a suspensor-like structure. The meristematic small cells continue dividing forming a mass of embryonal cells. This early embryoid eventually turns green and produces both a root and shoot. Haploid embryoids are also derived from long cells in which karyokinesis but not cytokinesis occurs, resulting in a four-nucleate coenocyte. The nuclei migrate to one pole and become surrounded by cell walls. The cells thus formed are the originators of a new embryoid.

Plant regeneration in Stone pine (Pinus pinea L.) by somatic embryogenesis

Regeneration of plants by somatic embryogenesis (SE) was achieved in Stone pine (Pinus pinea), one of the most characteristic tree species of the Mediterranean ecosystem. The initial explants were megagametophytes containing zygotic embryos from five selected half-sib families collected at different dates over 2 consecutive years. Rates of extrusion and initiation of SE differed in both years. However, qualitative patterns were very similar: for most families, the responsive developmental window was from late cleavage polyembryony to early cotyledonary stage. The highest overall mean frequencies of extrusion and SE initiation (7 and 0.9%, respectively, for the five families and the eight 2006 collections) were obtained on a modified Litvay’s medium with 9xa0μM 2,4-D and 4.5xa0μM BAP, supplemented with L-glutamine and casein hydrolysate. Families showed large differences in frequencies of SE initiation from year to year. Only seven embryogenic lines were induced in 2005, representing three of the five families tested, whereas 34 lines from all the families were obtained in 2006. Proliferation of embryonal masses (EM) was significantly improved when they were subcultured after dispersing in liquid medium and collected on filter paper disks, instead of being subcultured as small clumps. This effect showed a significant interaction with genotype. Several preconditioning treatments and culture media combinations were tested for embryo development and maturation. The high proliferation rate of EM hampered somatic embryo development. However, up to 42 mature embryos from different lines of three of the five families were obtained, 23 of them germinated and seven converted into somatic seedlings.

Comparison of Larch Embryogeny In Vivo and In Vitro

Larch species have been induced to form embryoids both from both haploid and diploid expiants. The developmental steps in embryogenesis of diploid expiants of Larix leptolepis, L. decidua, L. occidentalis and L. x eurolepis are outlined and compared with the embryogeny of zygotic embryos. The various terms commonly found in the classical embryological descriptions are discussed in terms of their usefulness in describing events in vivo. A number of tissue culture terms, such as proembryo(id), embryonal suspensor mass, and callus are discussed as well. This comparative embryological study is extended to haploid embryoid development, which is initially different from both somatic and zygotic embryogenesis.