PCR amplification with degenerate primers targeted to highly conserved amino acid motifs within the MYB domain was used to demonstrate that black spruce (Picea mariana) possesses a diverse MYB gene family. Amino acid sequence comparisons revealed three broad MYB subfamilies, one of which shares extensive similarity with maize C1, a central regulator of anthocyanin biosynthesis. A cDNA clone encoding a MYBR2R3 protein from P. mariana with high levels of sequence homology to maize C1 was shown to transactivate the Bz2 promoter in combination with maize R in embryonal tissues of both black spruce and larch. Functional dependence on the maize R protein, and the presence of a conserved C-terminal GIDPxTH motif, support the conservation of MYBR2R3 function in conifers, and demonstrate that the basic components of MYBR2R3-dependent transcriptional regulation have been conserved between angiosperms and gymnosperms.

Black spruce (Picea mariana) is one of about 40 species of the genus Picea A. Dietr. (family Pinaceae) scattered throughout the cooler parts of the north temperate zone and higher elevations in the south (Farrar 1995). Alone or with white spruce and tamarack [Larix laricina (Du Roi) K. Koch], black spruce forms the northern boundary of tree-form growth.

Using particle bombardment of mature somatic embryos followed by the induction of secondary embryogenesis in the presence of hygromycin, we produced over 90 lines of transgenic embryonal masses expressing β-glucuronidase from two genotypes of black spruce. Transformation efficiencies of up to 7% (1 transgenic line per 14 embryos bombarded) were achieved by extending the period of selection from 8 to 12 weeks. Proliferation of transformed embryonal masses in the presence of hygromycin had no effect on either embryogenicity or embryo maturation. Southern blot hybridization and PCR amplification confirmed the presence of the hygromycin phosphotransferase gene in genomic DNA. The expression of the β-glucuronidase gene in the needles of regenerated seedlings support the potential for long-term transgene expression in spruce.

The toxicities of four candidate peptides, which have potential for engineering disease resistance into poplars and conifers, were tested and compared in vitro. Cecropin B, (Ala8,13,18)-magainin II amide, and the two synthetic membrane interactive molecules (Peptidyl MIMs(tm)) D2A21 and D4E1 inhibited germination of spores of the fungal pathogens Cronartium ribicola J.C. Fisch., Gremmeniella abietina (Lagerberg) Morelet, Melampsora medusae Thuem., Nectria galligena Bres. in Strass., Ophiostoma ulmi (Buisman) Nannf., andSeptoria musiva Peck. Minimal inhibitory concentrations (MICs) of peptides required to achieve >95% inhibition of germination of conidia and urediniospores ranged from 0.3 to >5 µM. Permutation analyses on MICs confirmed that peptides significantly reduced germination of fungus spores (P = 0.0038) and that MICs of the two synthetic peptides were, on average, lower than those of the two natural peptides (P = 0.0012). In contrast, peptides had much less of an effect on the pollen of Picea, Pinus, and Populus species and seeds of white pine; MICs from 10 µM to >50 µM of peptide were needed to arrest germination. Peptidyl MIM(tm) D2A21 combined high antifungal activity with low pollen toxicity. Similar to some antimicrobial peptides from plants, Peptidyl MIM(tm) D2A21 could not prevent germination of fungal spores in the presence of potato dextrose broth or chloride salts with divalent cations. This may explain why the peptide had only a limited effect on fungal mycelium grown in potato dextrose broth medium.Key words: antimicrobial peptides (AMP), white pine blister rust, scleroderris canker, poplar leaf rust, Dutch elm disease, septoria canker.

The gene coding for green fluorescent protein (GFP) from the jellyfish Aequorea victoria was successfully used as a vital marker for the transformation of three woody plant species, black spruce (Picea mariana (Mill.) BSP), white pine (Pinus strobus L.) and poplar (Populus spp.). The gfp gene and the gene conferring resistance to kanamycin (nptII) were introduced by microprojectile bombardment or Agrobacterium tumefaciens-mediated technology. Screening by fluorescence microscopy of the transformed plant material, under the selection of kanamycin, identified five to eight cell lines from each tree species that clearly expressed GFP. Expression of GFP was observed in somatic embryonal cells of the coniferous species and in stem sections of poplar. For all species, GFP transgene expression was stable over multiple subcultures. Stable integration of the gfp gene into plant genomes was confirmed by Southern hybridization or polymerase chain reaction (PCR) analysis. We conclude that GFP can be used as a vital marker and reporter protein in transformation experiments with gymnosperms and deciduous trees.

Embryonal-suspensor tissue (EST) of Mediterranean cypress (Cupressus sempervirens L.) was tested for microprojectile-DNA delivery (by the PDS-1000/He device) for different subculture periods (9, 15, and 21 days) using the plasmid vectors pRT99GUS [containing the β-glucuronidase (GUS) and neomycin phosphotransferase (NPT II) genes, and the CaMV 35S promoter], pBI426 (with a GUS::NPT II fusion gene under the control of a duplicated 35S RNA promoter), and pCGUδ0 (containing the GUS gene with the ubiquitin intron, under the control of the sunflower ubiquitin promoter). The relative strengths of the promoters as determined by GUS assays were sunflower ubiquitin>35S-35S-AMVE>35S. The highest expression level was observed when 15-day-subcultured EST was bombarded with the pCGUδ0 gene construct, which also showed high activity of the chloramphenicol acetyltransferase and NPT II genes. Green fluorescent areas were observed on EST when bombarded with the p35S-GFP plasmid, carrying the gene for the green fluorescent protein from the bioluminescent jellyfish Aequorea victoria.

Excised, immature zygotic embryos of Larix laricina (Du Roi) K. Koch (tamarack) gave rise to embryogenic cultures on modified Murashige and Skoog (MSG) medium supplemented with growth regulators. Three lines of embryonal masses were maintained for 1 year by biweekly subcultures prior to the maturation experiments. All of these lines showed the ability to produce mature somatic embryos. Both elevated medium osmolality (315.0n543.6 mmol·kg n1 ) and presence of abscisic acid (ABA) at 40 μM stimulated the maturation process when applied simultaneously. Sucrose was most effective at 0.4 M, and polyethylene glycol (PEG) at 5 or 10% was effective only in combination with 0.2 or 0.4 M sucrose. The germination frequency of somatic embryos depended on both osmolality and ABA concentration in the maturation medium. Over 90% of mature somatic embryos were capable of secondary somatic embryogenesis when placed on the induction medium. This particular ability was exploited in order to achieve genetic transformation. Four vectors were delivered to the embryonal masses and somatic embryo cells via bombardment of DNA-coated gold particles. The vectors pBI426 and pRT99gus carried a gene encoding resistance to kanamycin, pRT66gus to hygromycin, and pRT55gus to methotrexate. All vectors carried the gene coding for β-glucuronidase (GUS) and were over 6 kilobases in size. Assays for both transient and stable transformation were carried out. The only vector that yielded two transgenic lines was pBI426. These lines of embryonal masses, designated as 2D1 and 2D2, were analysed by hybridization of the plasmid to the genomic Southern blots and revealed several insertions of the vector. Line 2D1 gave rise to young germinants that expressed the GUS gene uniformly throughout the root, hypocotyl, and cotyledons but failed to develop further. Line 2D2 gave rise to transgenic plants that displayed random and ipatchyi expression of the GUS gene. Polymerase chain reaction (PCR) amplification of the GUS insert in 6-month-old 2D2 transgenic plants showed the presence of diagnostic fragment in all parts of the plants. Resume : Des embryons zygotiques immatures de Larix laricina (Du Roi) K. Koch (mOlze laricin) ont donnO du tissu embryogne sur un milieu Murashige et Skoog modifiO (MSG) par liajout de rOgulateurs de croissance. Trois lignOes embryognes ont OtO maintenues pendant 1 an par sous-cultures bihebdomadaires avant les expOriences de maturation. Ces trois lignOes ont dOmontrO leur aptitude ‡ produire des embryons somatiques matures. Une osmolalitO OlevOe (315,0n

The tobacco rbcS (ribulose bisphosphate carboxylase small subunit) promoter, fused to the β-glucuronidase (GUS) reporter gene, was delivered to black spruce (Piceamariana (Mill.) BSP) tissues via microprojectile DNA bombardment, and its regulation was studied. The expression of the tobacco rbcS promoter–GUS chimeric gene was dependent on the presence of chloroplasts in black spruce tissues, as demonstrated in two ways: (i) there was no GUS activity expressed in zygotic embryos where no chloroplasts were observed, whereas it was expressed in light- and dark-grown seedlings that contained mature or immature chloroplasts; (ii) a herbicide, Norflurazon, destroyed chloroplast structure in seedlings and inhibited the expression of the tobacco rbcS promoter–GUS chimeric gene. A control chimeric gene, the cauliflower mosaic virus (CaMV) 35S promoter–GUS fusion gene was not inhibited by Norflurazon. Unlike in angiosperms, light had no effect on the expression of tobacco rbcS promoter–GUS chimeric gene. Both light- and dark-grown seedlings showed GUS activity, and expression in dark-grown seedlings was not enhanced by light. These results suggest that the tissue-specific regulation of the rbcS promoter may be conserved between angiosperms and conifers, but that the light regulation of this promoter may not be conserved.

Stable genetic transformation of black spruce (Picea mariana) and tamarack (Larix laricina) was obtained via microprojectile bombardment of tissues from two stages of somatic embryogenesis (mature cotyledonary somatic embryos and suspension from embryonal masses) using the Biolistic PDS-1000/He device. A total of 14 transgenic lines were obtained with the two species with the vectors pRT99GUS and pBI426 using low level kanamycin selection. The integration of the foreign genes was confirmed by Southern hybridization and complex patterns were observed. Transgenic seedlings were regenerated for both species and expression of the GUS gene observed in needles. Two chimeric genes containing components from black spruce genes were used for transient gene expression into conifer tissues; a cDNA (MBF1) coding for a myb C1 related gene and a chalcone synthase promoter. Both components were proven functional when reintroduced into their original genome and in L. laricina tissues. Furthermore, the chimeric MBF1 gene was functional when introduced into corn cell suspension. These results indicate the evolutionary conservation of the genes involved in the anthocyanin pathways in gymnosperms and angiosperms. Additionally, the Green Fluorescent Protein gene from a jellyfish (Aequorea victoria) was evaluated as a marker for genetic transformation in conifer tissues.

Both Larix and Picea are widely distributed conifers in temperate climates and are economically valuable to the pulp and paper and lumber industries. These genera include species that are biologically interesting because of their various growth habits and their ability to produce interspecific hybrids (Boyle et al. 1989; Burns and Honkala 1990). The distribution and biology of these species have been dealt with by Burns and Honkala (1990). Larix and Picea include species that were successfully propagated through organogenesis and somatic embryogenesis (Diner et al. 1986; Klimaszewska 1989; von Aderkas et al. 1990; Attree and Fowke 1991; Bonga and Pond 1991; Tautorus et al. 1991; Bonga and von Aderkas 1992; Lelu et al. 1993). The germplasms of these species have traditionally been stored at 4 °C in the form of seeds. These trees are not threatened with extinction per se, but germplasms from certain provenances need to be preserved.

Mature pollen of lodgepole pine (Pinusconcorta Dougl.), yellow cypress (Chamaecyparisnootkatensis (D. Don) Spach), western hemlock (Tsugaheterophylla (Raf.) Sarg.), jack pine (Pinusbanksiana Lamb.), and black spruce (Piceamariana (Mill.) B.S.P.) was bombarded with gold particles coated with four different plasmid constructions, pRT99GUS, pBM113Kp, pAct1-D, and pGA984, using the biolistic PDS-1000/He device. A protocol was devised for efficient gene transfer and gene expression assay in pollen. False positive results for expression of the β-glucuronidase (GUS) gene assayed with the substrate X-glucuronide were observed with pollen of yellow cypress, western hemlock, and lodgepole pine. The highest levels of transient GUS gene expression were obtained with plasmid pBM113Kp, which carried the GUS gene under the control of the wheat abscisic acid inducible early methionine promoter. The plasmids pRT99GUS (35S promoter) and pAct1-D (rice actin promoter) yielded similar intermediate levels of transient GUS gene expression. The pollen-specific promoter of the α-tubulin gene from Arabidopsisthaliana (pGA984) yielded the lowest levels of gene expression in pollen. Of the four species, yellow cypress showed the lowest levels of transient GUS gene expression and black spruce yielded the highest levels. The neomycin phosphotransferase II (NPT II) gene was also tested as a reporter gene for pollen transformation and was easily assayed via ELISA. The fusion gene between NPT II and GUS genes was detected at a lower level than the nonfused NPT II gene when under the control of the same 35S promoter. The method devised here could be used for the study of tissue-specific gene expression in conifer pollen.

The somatic embryogenesis process was evaluated as a potential tool for operational vegetative propagation using individuals from families currently used in the J.D. Irving, Ltd. black spruce tree improvement program. Most families were responsive although the number of individuals within families capable of producing embryogenic tissue (ET) varied greatly (1–70%). Seventy-four percent of the ET clones produced mature embryos and most of these germinated. Greenhouse survival was initially low (11%) but improved in subsequent experiments to 45% as growing regimes were refined. Demonstration plantings of the resulting somatic plants were established at two sites in New Brunswick. A total of 206 clones were cryopreserved. The potential for integrating somatic embryogenesis techniques into tree improvement and stock production programs is discussed. Key words: tree improvement, somatic embryogenesis, clonal propagation, black spruce, biotechnology

The genomic RNA of potyviruses has a characteristic 5' non-translated region (5'NTR) to which a viral protein, VPg, is covalently attached. This suggests that the viral RNA lacks a conventional cap structure and thus its translation may not proceed in the same way as most cellular mRNAs. To investigate the role of the 5'NTR during translation, various derivatives of the turnip mosaic potyvirus (TuMV) leader were fused to the reporter gene beta-glucuronidase (GUS). These constructs were used to monitor the efficiency of translation in vitro in a rabbit reticulocyte lysate and in planta following microprojectile DNA delivery into tobacco cell suspensions. GUS transcripts fused with the TuMV 5'NTR, whether they were capped or not, were efficiently translated, whereas GUS transcripts without the viral leader needed to be capped for expression. When transcripts of the viral leader were supplied in excess over functional transcripts, translation was inhibited in a dose-dependent manner. Similarly, transcripts synthesized from the reverse complement of the 5'NTR inhibited translation to the same extent as the wild-type sequence, indicating that cap independence was not conferred by a specific sequence within the viral leader. A stable hairpin loop was placed in front or after the viral sequence. This hairpin loop normally prevented translation of control GUS transcripts but when the viral leader was positioned after it a significant level of GUS activity was measured, whether the transcripts were capped or not. On the other hand, when the hairpin loop was positioned after the viral leader, no GUS activity was measured. These results suggested that ribosomes bound to an internal site within the TuMV 5'NTR and then presumably scanned the sequence for the initiator AUG.

Germination and plantlet development in somatic embryos of Larix x leptoeuropaea were affected by the duration of the maturation treatment and the concentrations of sucrose and abscisic acid in the maturation media. Extension of the maturation period from 3 weeks to 4 weeks resulted in a significant decrease in germination and plantlet development frequencies. There was no significant effect of abscisic acid concentration on either the number of somatic embryos germinated or the number of plantlets obtained, but it affected the rapidity of the epicotyl development. Sucrose at 0.2 M, applied during maturation, was significantly more beneficial in attaining high germination rates than at 0.1 M. High germination rates (92 and 93%) and plantlet development rates (74 and 80%) were achieved when somatic embryos were matured for a 3-week period on media with either 40 or 60 μM abscisic acid, respectively, and 0.2 M sucrose prior to transfer to the growth regulator-free germination medium. Two acclimatization methods were applied: the first required 10 to 12 weeks and ensured 97% plantlet survival under greenhouse conditions; the second required 2–3 weeks and ensured 86% plantlet survival. This represents the first detailed study of the effects of maturation regimes on the recovery of somatic embryo-derived plants of Larix.