Yill-Sung Park

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.

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.

Factors influencing somatic embryogenesis induction in Eucalyptus globulus Labill.: basal medium and anti-browning agents

The low induction rates of somatic embryogenesis (SE) in Eucalyptusglobulus hamper scaling up the process for commercialization. We analyzed the effectiveness of several media (MS, 1/2MS, B5, WPM, DKW and JADS) during SE induction and expression. MS and B5 were the best media for SE induction and embling regeneration. In general, MS was the best medium for expression, independently of the medium previously used during induction. Several anti-browning compounds (ascorbic acid, charcoal, DTE, DTT, PVP, PVPP and silver nitrate) were added to the expression medium (MS), but all decreased SE potential and only DTE, charcoal and silver nitrate reduced explant browning. When added only during the induction period, anti-browning agents reduced accumulation of phenolics but also severely reduced SE potential. Continuous exposure completely inhibited the SE response. The negative impact of anti-browning agents on SE potential raises a question about the role of production/accumulation of phenolics in the SE process.

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.

Realized genetic gains observed in a first generation seedling seed orchard for jack pine in New Brunswick, Canada

Jack pine seedlots collected from a seedling seed orchard owned by J.D. Irving Limited (JDISSO) were compared in a block-plot genetic gain test on four sites in New Brunswick, Canada. Three seedlots representing different genetic quality of the orchard were included: before rogueing (UNR), following the first rogueing (1STR), and following the second rogueing (2NDR). Two stand seedlots were included to represent an average unimproved commercial seedlot (UC) for comparison. Individual tree growth and stem straightness were recorded up to age 15 (one-third rotation age). For individual tree growth, there was no strong evidence of better performance of the UNR over the UC, but the 1STR and 2NDR grew much faster. For volume per hectare, all the JDISSO seedlots outperformed the UC. The realized gains per hectare were higher than those on individual tree basis, but both increased with rogueing time and decreased with age. Improvement in stem straightness was also significant for all the orchard seedlots. The realized genetic gains were generally comparable to the respective predicted ones from the family tests. Analyses of individual sites indicated that the realized gain varied greatly with site, especially for growth traits. Deploying good seedlots to better sites would result in more volume gains. Overall, results of this study indicate the efficiency of the seed orchard procedure for jack pine.

Allocation of varietal testing efforts for implementing conifer multi-varietal forestry using white spruce as a model species

Abstract• IntroductionMulti-varietal forestry (MVF) is the deployment of tested tree varieties in plantation forestry. Computer simulation using POPSIM Simulator identified optimal combination of numbers of families, varieties per family and ramets per variety (nf, nc and nr, respectively) yielding the largest genetic gain for a specific status number (NS) in a varietal test (VT) intended for MVF of conifers.• Results and discussionTesting 40 to 80 full-sib families and 20 to 30 varieties per family would be optimal for a VT. This nf interval was insensitive to the number of candidate varieties planted, ratio of genetic variances and selection restriction. It was influenced somewhat by individual narrow-sense heritability (h2), required NS and mating design. Lower h2, lower NS and designs with fewer matings per parent tree favoured the lower range of the nf interval. The optimal nr was 6. It was not markedly affected by the required NS, family size or selection restriction but was strongly influenced by h2 and the ratio of genetic variances. Larger h2 or an introduction of non-additive genetic variance allowed planting fewer ramets per variety.