Jean-François Trontin

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.

Molecular evidence of true-to-type propagation of a 3-year-old Norway spruce through somatic embryogenesis

Abstract. Some progress has recently been made in establishing a system enabling somatic embryos to be initiated from old elite trees. We report here the first results demonstrating the molecular conformity of somatic embryos initiated from increasingly old Norway spruce (Picea abies (L.) Karst.), as indicated by an analysis of six nuclear microsatellites that showed an extremely high tendency to mutate during in vitro culture. No allelic difference was detected at these loci among plants regenerated from somatic embryos or between the former and mother plants. Moreover, phenotypical data acquired on the same 3- to 9-year-old plants growing in the field sampled for molecular analyses were totally in accord with the results on molecular conformity.

Early molecular events involved in Pinus pinaster Ait. somatic embryo development under reduced water availability: transcriptomic and proteomic analyses.

Maritime pine somatic embryos (SEs) require a reduction in water availability (high gellan gum concentration in the maturation medium) to reach the cotyledonary stage. This key switch, reported specifically for pine species, is not yet well understood. To facilitate the use of somatic embryogenesis for mass propagation of conifers, we need a better understanding of embryo development. Comparison of both transcriptome (Illumina RNA sequencing) and proteome [two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis with mass spectrometry (MS) identification] of immature SEs, cultured on either high (9G) or low (4G) gellan gum concentration, was performed, together with analysis of water content, fresh and dry mass, endogenous abscisic acid (ABA; gas chromatography-MS), soluble sugars (high-pressure liquid chromatography), starch and confocal laser microscope observations. This multiscale, integrated analysis was used to unravel early molecular and physiological events involved in SE development. Under unfavorable conditions (4G), the glycolytic pathway was enhanced, possibly in relation to cell proliferation that may be antagonistic to SE development. Under favorable conditions (9G), SEs adapted to culture constraint by activating specific protective pathways, and ABA-mediated molecular and physiological responses promoting embryo development. Our results suggest that on 9G, germin-like protein and ubiquitin-protein ligase could be used as predictive markers of SE development, whereas protein phosphatase 2C could be a biomarker for culture adaptive responses. This is the first characterization of early molecular mechanisms involved in the development of pine SEs following an increase in gellan gum concentration in the maturation medium, and it is also the first report on somatic embryogenesis in conifers combining transcriptomic and proteomic datasets.

Advances in Conifer Somatic Embryogenesis Since Year 2000

This review compiles research results published over the last 14 years on conifer somatic embryogenesis (SE). Emphasis is placed on the newest findings that affect the response of seed embryos (typical explants) and shoot primordia (rare explants) to the induction of SE and long-term culture of early somatic embryos. Much research in recent years has focused on maturation of somatic embryos, with respect to both yield and quality, as an important stage for the production of a large number of vigorous somatic seedlings. Attempts to scale up somatic embryo production numbers and handling have resulted in a few bioreactor designs, the utility of which may prove beneficial for an industrial application. A few simplified cryopreservation methods for embryonal masses (EM) were developed as a means to ensure cost-efficient long-term storage of genotypes during clonal field testing. Finally, recent long-term studies on the growth of somatic trees in the field, including seed production yield and comparison of seed parameters produced by somatic versus seed-derived trees, are described.

High subculture frequency, maltose-based and hormone-free medium sustained early development of somatic embryos in maritime pine

SummaryEmbryonal-suspensor mass (ESM) lines characterized by a spiky, morphotype (i.e., developed, early embryos escaping from the ESM periphery) were recently shown to produce the best maturation yields in Maritime pine. How to select or preserve such a valuable morphotype during ESM maintenance (prior to maturation treatment) is still unknown. Several maintenance procedures were tested; 2400 ESM from 10 lines were subcultured each, 7 or 14 d on maltose- or sucrosecontaining medium without plant growth regulator (PGR) or supplemented with 2,4-dichlorophenoxyacetic acid and 6-benzylaminopurine or a low concentration of, abscisic acid. Multiple components analyses based on growth rate, macromorphology data, and micromorphology data, collected for 6 mo., allowed for the association, of the spiky morphotype with additional traits (defining the phenotype I) such as whitish aspect, high vigor, high growth rate, and complex cellular organization (resulting from high early embryogenic ability). Since a gradual increase in growth rate as well as a decrease in cellular organization were concomitantly, observed during the 6 mo, experiment, we concluded that ESM growth and early somatic embryo development were disconnected. In some lines, the progressive loss of early embryogenic, ability (aging process) could be decreased using maltose-based and PGR-free medium. For most lines, the aging effect was minimized using a weekly subculture. An improved procedure for ESM maintenance prior to the maturation step is thus proposed.

Simple and efficient protocols for the initiation and proliferation of embryogenic tissue of Douglas-fir

Key messageThis paper describes the micropropagation of Douglas-fir via somatic embryogenesis using media and methods available in the public domain.AbstractDouglas-fir is a conifer species with high and increasing interest for forest industries in both New Zealand and France. Delivery of the best trees to the forest from breeding programmes is currently constrained by an inability to effectively and reliably multiply selections through vegetative propagation. Somatic embryogenesis coupled with cryopreservation is essential biotechnology tool in conifers for scaling up variety design and production. The aim of this work was therefore to develop protocols for the initiation and proliferation of Douglas-fir embryogenic cell lines based on the modifications of previously published techniques and media available in the public domain, especially successful methods developed for P. radiata at Scion. Three years of initiation experiments have resulted in a simple and efficient protocol. Disinfection of whole cones (instead of seeds) was sufficient to prevent contamination. Immature zygotic embryos were excised from megagametophytes and placed onto a modified Litvay medium (Plant Cell Rep 4(6):325–328, 1985***). At optimal development stages of zygotic embryos, the average initiation percentage of embryogenic tissue was 69.3% when our most effective protocol was used. Proliferation of initiated cell lines on a Glitz formulation was challenging, with a high percentage of cell lines composed of a mixture of both embryonal masses and callus. 2,4-D at a lower concentration reduced the number of such mixed lines. Repeated liquid suspension and subculture of the embryogenic parts of the tissue was an efficient means to increase the number of embryogenic cell lines with sustained proliferation. Maltose added to the proliferation medium in place of sucrose improved fresh mass gain and consistently increased early somatic embryo patterning and growth. A sample of proliferating cell lines was successfully cryopreserved, thawed and somatic embryos were matured and germinated from these lines. The method may be of practical interest and provide new opportunities to realize increased genetic gain in this species through the clonal-assisted deployment of the best genetic material in both New Zealand and France.

The role of arginine metabolic pathway during embryogenesis and germination in maritime pine (Pinus pinaster Ait.)

Vegetative propagation through somatic embryogenesis is critical in conifer biotechnology towards multivarietal forestry that uses elite varieties to cope with environmental and socio-economic issues. An important and still sub-optimal process during in vitro maturation of somatic embryos (SE) is the biosynthesis and deposition of storage proteins, which are rich in amino acids with high nitrogen (N) content, such as arginine. Mobilization of these N-rich proteins is essential for the germination and production of vigorous somatic seedlings. Somatic embryos accumulate lower levels of N reserves than zygotic embryos (ZE) at a similar stage of development. To understand the molecular basis for this difference, the arginine metabolic pathway has been characterized in maritime pine (Pinus pinaster Ait.). The genes involved in arginine metabolism have been identified and GFP-fusion constructs were used to locate the enzymes in different cellular compartments and clarify their metabolic roles during embryogenesis and germination. Analysis of gene expression during somatic embryo maturation revealed high levels of transcripts for genes involved in the biosynthesis and metabolic utilization of arginine. By contrast, enhanced expression levels were only observed during the last stages of maturation and germination of ZE, consistent with the adequate accumulation and mobilization of protein reserves. These results suggest that arginine metabolism is unbalanced in SE (simultaneous biosynthesis and degradation of arginine) and could explain the lower accumulation of storage proteins observed during the late stages of somatic embryogenesis.