Sucrose requirements and lipid utilization during germination of interior spruce (Picea glauca engelmannii complex) somatic embryos

Both somatic and excised zygotic embryos of interior spruce (Picea glauca engelmannii complex) required exogenous sucrose in the medium for germination in vitro. Over a period of 29 days on sucrose-containing medium germinants with roots and epicotyls developed from both kinds of embryo, and their content of linolenic acid (9,12,15-18:3) increased about six- to eightfold. Without added sucrose, embryos showed retarded growth or were necrotic, and the content of linolenic acid was barely detectable in their fatty acid profiles. Through¹⁴C-sucrose uptake studies, it was determined that germinants consumed only 25% of the sucrose available in a 1% (wt/vol) sucrose-containing medium. Since no radiolabelled fatty acids were detected, it appears that externally supplied sucrose was not used in the synthesis of lipids. Although sucrose was present during plantlet development, 72% of the initial lipids were consumed. To some extent, the plantlets appeared to be obligate storage lipid utilizers.Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid - FAMEs Fatty acid methyl esters - HPLC High-performance liquid chromatography     

Bioreactor culture of Picea mariana Mill. (black spruce) and the species complex Picea glauca-engelmannii (interior spruce) somatic embryos. Growth parameters

Somatic embryo cultures of Picea mariana and the species complex P. glauca-engelmannii were each grown in 7.5-l-capacity mechanically-stirred bioreactors containing 61 medium (LP, von Arnold and Eriksson) with 30 mm sucrose. Growth of both species occurred with no observable signs of shear stress due to mechanical agitation. Growth kinetics were analysed using an array of parameters (settled culture volume, packed culture volume, osmolarity, conductivity, pH). These were compared with fresh weight, dry weight, and somatic embryo number in order to determine what parameters were highly correlated with growth and embryo number. Increasing the sucrose concentration from 30 mm to 60 mm resulted in an increase in biomass and total number of somatic embryos. For P. mariana a maximum dry weight of 6.3 gl^–1 and 3076 embryos ml^–1 occurred in LP medium with 60 mm sucrose after 10–12 days of culture. For P. glauca-engelmannii a maximum dry weight of 4.3 gl^–1 and 2278 embryos ml^–1 occurred in LP medium with 60 mm sucrose after 6–8 days culture. For all sucrose concentrations, fresh weight, dry weight and embryo number were closely correlated with packed culture volume and conductivity for P. mariana, and settled culture volume, packed culture volume and conductivity for P. glauca-engelmannii.Correspondence to: D. I. Dunstan     

Polyamine biosynthesis during somatic embryogenesis in interior spruce (Picea glauca x Picea engelmannii complex)

Putrescine, spermidine, and spermine levels during somatic embryogenesis of interior spruce (Picea glauca x Picea engelmannii complex) were quantified On abscisic acid supplemented growth medium putrescine and spermidine levels increased two-fold coinciding with maturation of the early somatic embryos to globular embryos. Polyclonal antibodies raised against Escherichia coli arginine decarboxylase (ADC) and ornithine decarboxylase (ODC), following affinity purification specifically recognized spruce ADC and ODC, which corresponded to 85kD and 65kD bands on western blots of total protein extracts from embryogenic masses, Immunoassays using these antibodies showed increased ADC levels corresponding to embryo maturation while ODC levels remained the same. From these results it is concluded that polyamines are involved in the maturation of somatic embryos of interior spruce.Abbreviations ADC arginine decarboxylase - BSA bovine serum albumin - ODC ornithine decarboxylase - PBS phosphate buffered saline - PCA perchloric acid - SDS-PAGE sodium dodecyl sulfateporyacrylamide gel electrophoresis     

The role of sucrose during maturation of black spruce (Picea mariana) and white spruce (Picea glauca) somatic embryos

The present study was conducted to understand the role of sucrose in the medium on the maturation of black spruce and white spruce somatic embryos. A maturation medium containing 6% sucrose, which hydrolyzed into glucose and fructose, gave significantly more embryos than a medium containing 3.16% of each glucose and fructose. Preventing the complete sucrose hydrolysis by a daily transfer of the tissues onto fresh medium significantly decreased the yield of somatic embryos compared to when sucrose was allowed to complete its hydrolysis. This reduction was not due to the manipulation of the tissues during the transfer, since a daily in situ transfer did not affect embryo production. To verify if the better embryo production observed on a medium containing 6% sucrose was due to the increasing osmotic pressure of the medium, this increasing osmotic pressure was simulated with a sequence of media containing different concentrations of glucose and fructose. Unexpectedly and for both species, this simulation did not improve somatic embryo production, which stayed similar to the one obtained on constant osmotic pressure. To understand these results, embryos produced on the different treatments were analyzed in terms of sucrose, glucose, fructose and starch levels and protein contents. The embryo carbohydrate content was independent from the carbohydrate used in the maturation medium. However, embryos matured on 6% sucrose allowed to hydrolyze during the maturation period contained significantly more soluble and insoluble proteins than embryos matured on any other treatment. Furthermore, embryos with a higher protein content also exhibited a higher epicotyl appearance frequency. The role of sucrose as a regulatory factor during the maturation of spruce somatic embryos is discussed.     

Cryopreservation of interior spruce (Picea glauca engelmanni complex) embryogenic cultures

Summary Embryogenic cultures of interior spruce derived from 12 full-sib families were subjected to cryopreservation, with a 97 % success rate for 357 genotypes. Analyses suggested that cryotolerance was not related to family ranking (height increment), embryogenic potential or culture dispersability in suspension, and long-term storage in or above liquid nitrogen did not affect regenerative potential. By contrast, differences in cryotolerance among cell lines appeared to be prevalent in certain families. Analysis with a DNA fingerprinting probe used for clonal identification demonstrated no evidence of somaclonal variation as a result of cryopreservation. The results of this work indicate the applicability of cryopreservation as a long-term storage strategy for spruce embryogenic cultures from a wide genetic background.Abbreviations ABA ± abscisic acid - BA N⁶-benzyladenine - BSA bovine serum albumin - CTAB cetyldimethylethyl-ammonium bromide - DMSO dimethylsulfoxide - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid disodium salt - IBA indole-3-butyric acid - LN liquid nitrogen - PEG polyethylene glycol - SLS N-lauroyl sarcosine - Tris tris[hydroxymethylamino] methane - 2,4-D 2,4-dichlorophenoxyacetic acid     

Understanding changes in black (Picea mariana) and white spruce (Picea glauca) foliage biomass and leaf area characteristics

Key message

Growth conditions related to inter-tree competition greatly influence black and white spruce foliage biomass and projected leaf area characteristics.

Abstract

Foliage characteristics such as biomass and area are important among other reasons because they can be related to tree growth. Despite their economic and ecologic importance, equations to characterize foliage biomass and projected area of black (Picea mariana (Miller) BSP) and white (Picea glauca (Moench) Voss) spruces are sparse. Total foliage biomass and projected leaf area, foliage biomass and leaf area density, and relative vertical distribution of black and white spruces foliage biomass and leaf area were modelled with linear and nonlinear mixed effect models. A total of 65 white spruces and 57 black spruces were destructively sampled at four different locations in Alberta, Québec, and Ontario, Canada. Our results show that for each species, total tree foliage biomass and projected leaf area is proportional to stem diameter, total height, and crown length. The addition of crown length in the equations improved the precision of the predictions of total foliage biomass for both species and diminishes greatly the site level random effect. An increase in DBH for black spruce and in the DBH to total height ratio for white spruce skewed the relative vertical foliage biomass distribution toward the base of the living crown. According to our results, growth conditions or tree development stage influence both foliage biomass and leaf area characteristics of black and white spruces. Our results emphasize the importance of inter-tree competition on foliage biomass characteristics.     

Evaluation of somaclonal variation during somatic embryogenesis of interior spruce (Picea glauca engelmannii complex) using culture morphology and isozyme analysis

Somaclonal variation during interior spruce (Picea glauca engelmannii complex) somatic embryogenesis was evaluated using culture morphology and isozyme analysis. Genotype-specific abscisic acid-dependent developmental profiles and isozyme patterns were similar for subclone and parent line embryogenic cultures and cotyledonary somatic embryos. Extensive analysis of fifteen hundred subclone embryos of one genotype revealed no isozyme pattern variation. Initiation of embryogenic cultures was dependent on the developmental stage of the explant although cultures derived from different stages were morphologically similar. The embryogenic cultures initiated from interior spruce embryos show a high degree of genetic stability in that the morphological behavior and isozyme phenotype were always consistent with that of the explant genotype. These results support the conclusion that this culture system is appropriate for clonal propagation of interior spruce.     

Carbohydrate requirements for the development of black spruce (Picea mariana (Mill.) B.S.P.) and red spruce (P. rubens Sarg.) somatic embryos

Different carbohydrates were investigated for somatic embryo development of black spruce and red spruce. They were tested in a basal maturation medium consisting of Litvay's salts at half-strength containing 1 g l^-1 glutamine, 1 g l^-1 casein hydrolysate, 7.5 M abscisic acid, and 0.9% Difco Bacto-agar. A comparison of different sucrose concentrations showed that 6% was optimal for embryo development. Among the nine carbohydrates tested, sucrose, fructose, glucose, maltose, and cellobiose supported embryo development while arabinose, mannitol, myo-inositol, and sorbitol did not. A comparison of sucrose, glucose, and fructose at three concentrations showed that the general pattern of response for both species followed concentration expressed as a percentage, independent of the molarity of carbohydrate in the medium. Interspecific differences were observed concerning carbohydrate requirements. For red spruce, 6% fructose was found best for embryo development, while no such preference was observed for black spruce. No significant difference was observed in the number of embryos produced with 6% sucrose or 3% sucrose plus an equimolar concentration of either mannitol, sorbitol, or myo-inositol in the maturation medium, suggesting that the effect of the carbohydrate on the maturation was partly osmotic.     

Susceptibility of burned black spruce (Picea mariana) forests to non-native plant invasions in interior Alaska

As climate rapidly warms at high-latitudes, the boreal forest faces the simultaneous threats of increasing invasive plant abundances and increasing area burned by wildfire. Highly flammable and widespread black spruce (Picea mariana) forest represents a boreal habitat that may be increasingly susceptible to non-native plant invasion. This study assess the role of burn severity, site moisture and time elapsed since burning in determining the invisibility of black spruce forests. We conducted field surveys for presence of non-native plants at 99 burned black spruce forest sites burned in 2004 in three regions of interior Alaska that spanned a gradient of burn severities and site moisture levels, and a chronosequence of sites in a single region that had burned in 1987, 1994, and 1999. We also conducted a greenhouse experiment where we grew invasive plants in vegetation and soil cores taken from a subset of these sites. In both our field survey and the greenhouse experiment, regional differences in soils and vegetation between burn complexes outweighed local burn severity or site moisture in determining the invasibility of burned black spruce sites. In the greenhouse experiments using cores from the 2004 burns, we found that the invasive focal species grew better in cores with soil and vegetation properties characteristic of low severity burns. Invasive plant growth in the greenhouse was greater in cores from the chronosequence burns with higher soil water holding capacity or lower native vascular biomass. We concluded that there are differences in susceptibility to non-native plant invasions between different regions of boreal Alaska based on native species regeneration. Re-establishment of native ground cover vegetation, including rapidly colonizing bryophytes, appear to offer burned areas a level of resistance to invasive plant establishment.     

Effects of NaCl on responses of ectomycorrhizal black spruce (Picea mariana), white spruce (Picea glauca) and jack pine (Pinus banksiana) to fluoride

Black spruce ( Picea mariana ), white spruce ( Picea glauca ) and jack pine ( Pinus banksiana ) were inoculated with Suillus tomentosus and subjected to potassium fluoride (1 m M KF and 5 m M KF) in the presence and absence of 60 m M NaCl. The NaCl and KF treatments reduced total dry weights in jack pine and black spruce seedlings, but they did not affect total dry weights in white spruce seedlings. The addition of 60 m M NaCl to KF treatment solutions alleviated fluoride-induced needle injury in ectomycorrhizal (ECM) black spruce and white spruce, but had little effect in jack pine seedlings. Both KF and 60 m M NaCl treatments reduced E values compared with non-treated control seedlings. However, with the exception of small reductions of K_r by NaCl treatments in black spruce, the applied KF and NaCl treatments had little effect on K_r in ECM plants. Chloride tissue concentrations in NaCl-treated plants were not affected by the presence of KF in treatment solutions. However, shoot F concentrations in ECM black spruce and white spruce treated with 5 m M KF + 60 m M NaCl were significantly reduced compared with the 5 m M KF treatment. The results point to a possible competitive inhibition of F transport by Cl. We also suggest that the possibility that aquaporins may be involved in the transmembrane transport of F should be further investigated.     

Winter hardening in first-year black spruce (Picea mariana) seedlings

Winter hardening of first-year black spruce [ Picea mariana (Mill.) B.S.P.] seedlings was studied by assessing a number of morphological and physiological changes under three hardening regimes: 1) early removal (ER), in which seedlings were exposed to natural daylengths and low ambient temperatures outside. 2) extended greenhouse culture (EG), in which seedlings were exposed to natural daylengths and warm temperatures, and 3) short day (SD), in which seedlings were exposed to short daylengths and low ambient temperatures outside. Measurements included needle primordia initiation, embryonic shoot volume, terminal bud mitotic index, embryonic shoot average cell volume, and shoot tip frost hardiness. EG seedlings formed buds containing 4 times as many needle primordia as ER stock. Embryonic shoot volume increased with number of needle primordia initiated, until late in the hardening period, when significant reductions in meristem volumes of SD and EG stock were observed. Frost hardiness increased sooner in seedlings which set bud in response to short days, but SD treatment did not result in significantly greater frost hardiness at the end of the trial. Frost hardiness was correlated with mitotic index of the embryonic shoot. Cell size in the embryonic shoot declined in seedlings of all treatments during hardening, however, EG seedlings had significantly lower cell volumes by the end of the trial in comparison to ER and SD seedlings.     

Thermal acclimation of photosynthesis in black spruce [Picea mariana (Mill.) B.S.P.

We investigated the thermal acclimation of photosynthesis and respiration in black spruce seedlings [ Picea mariana (Mill.) B.S.P.] grown at 22/14 °C [low temperature (LT)] or 30/22 °C [high temperature (HT)] day/night temperatures. Net CO₂ assimilation rates ( A _net) were greater in LT than in HT seedlings below 30 °C, but were greater in HT seedlings above 30 °C. Dark and day respiration rates were similar between treatments at the respective growth temperatures. When respiration was factored out of the photosynthesis response to temperature, the resulting gross CO₂ assimilation rates ( A _gross) was lower in HT than in LT seedlings below 30 °C, but was similar above 30 °C. The reduced A _gross of HT seedlings was associated with lower needle nitrogen content, lower ribulose 1·5-bisphosphate carboxylase/oxygenase (Rubisco) maximum carboxylation rates ( V _cmax) and lower maximum electron transport rates ( J _max). Growth treatment did not affect V _cmax :  J _max. Modelling of the CO₂ response of photosynthesis indicated that LT seedlings at 40 °C might have been limited by heat lability of Rubisco activase, but that in HT seedlings, Rubisco capacity was limiting. In sum, thermal acclimation of A _net was largely caused by reduced respiration and lower nitrogen investments in needles from HT seedlings. At 40 °C, photosynthesis in LT seedlings might be limited by Rubisco activase capacity, while in HT seedlings, acclimation removed this limitation.     

Transformation of white spruce (Picea glauca) somatic embryos by microprojectile bombardment

Cotyledonary somatic embryos of white spruce [Picea glauca (Moench) Voss] were subjected to microprojectile bombardment with a gene construct containing a gus::nptll fusion gene. Somatic embryos were used to re-induce the embryogenic tissue after bombardments. Histochemical assay using X-gluc as a substrate showed that all the embryos (100%) were GUS positive 48 h after bombardment. However, only thirteen out of 605 embryos (2.2%) remained GUS positive after two months in culture. Three of those thirteen (23%) embryo-derived tissues consistently showed GUS activity for eight months in culture. These putatively transfomed embryogenic tissues were subjected to Southern blot analysis and the results suggested integration of the gus::nptll gene expression cassette in the white spruce genome.Abbreviations ABA (±)abscisic acid - BA benzyladenine - bp base pair - 2,4-D 2,4-dichlorophenoxyacetic acid - kb kilobase - gus E. coli gene uid A for -glucuronidase - nptll neomycin phosphotransferase II - X-gluc 5-bromo-4-chloro-3-indolyl--D-glucuronic acid     

Elevated growth temperatures reduce the carbon gain of black spruce [Picea mariana (Mill.) B.S.P.]

We explored the effect of high‐growth temperatures on a dominant North American boreal tree, black spruce [Picea mariana (Mill.) B.S.P.]. In 2004 and 2005, we grew black spruce at either 22 °C/16 °C day/night temperatures [low temperature (LT)] or 30°/24 °C [high temperature (HT)] and determined how temperature affected growth, leaf morphology, photosynthesis, respiration and thermotolerance. HT spruce were 20% shorter, 58% lighter, and had a 58% lower root : shoot ratio than LT trees. Mortality was negligible in the LT treatment, but up to 14% of HT seedlings died by the end of the growing season. HT seedlings had a higher photosynthetic temperature optimum, but net photosynthesis at growth temperatures was 19–35% lower in HT than LT trees. HT seedlings had both a lower apparent maximum ribulose‐1,5‐bisphosphate carboxylation capacity (V_cmax) and a lower apparent maximum electron transport rate (J_max) than LT trees, indicating reduced allocation to photosynthetic components. Consistently, HT needles had 26% lower leaf nitrogen content than LT needles. At each measurement temperature, HT seedlings had 20–25% lower respiration rates than LT trees; however, this did not compensate for reduced photosynthetic rates at growth temperature, leading to a greater ratio of dark respiration to net carbon dioxide assimilation rate in HT trees. HT needles had 16% lower concentrations of soluble sugars than LT needles, but similar starch content. Growth at high temperatures increased the thermotolerance of black spruce. HT trees showed less PSII inhibition than LT seedlings and no increase in electrolyte leakage when briefly exposed to 40–57 °C. While trees that develop at high temperatures have enhanced tolerance for brief, extreme heat events, the reduction in root allocation indicates that seedlings will be more susceptible to episodic soil drying and less competitive for belowground resources in future climates of the boreal region.     

Nutrient status of black spruce (Picea mariana [Mill.] BSP) forest soils dominated by Kalmia angustifolia L.

A study was conducted to determine soil chemistry in an uncut black spruce (Picea mariana) forest with and without the ericaceous understory shrub Kalmia angustifolia, as well as on a cut black spruce forest currently dominated by Kalmia. The organic (humus) and mineral (Ae, upper and lower B horizons) soils associated with Kalmia from uncut and cut forests, and non-Kalmia soils from uncut forest, were analyzed for selected soil properties. In general, mineral soils (B horizon) associated with Kalmia in uncut forest have lower values for organic matter (3.25%), organic nitrogen (0.66 mg·g⁻¹), Fe³⁺ (95.4 μg·g⁻¹) and Mn²⁺ (9 μg·g⁻¹), and higher values for pH (4.12) and Ca²⁺ (27 μg·g⁻¹) compared to non-Kalmia (organic matter, 3.43%; organic-N, 1.15 mg·g⁻¹; Fe³⁺, 431 μg·g⁻¹; Mn²⁺, 23.2 μg·g⁻¹; pH, 3.14; Ca²⁺, 15.6 μg·g⁻¹) and cut black spruce-Kalmia (organic matter, 3.74%; organic-N, 0.94 mg·g⁻¹; Fe³⁺, 379 μg·g⁻¹; Mn²⁺, 27 μg·g⁻¹; pH, 2.87; Ca²⁺, 25.2 μg·g⁻¹) forest. The high C:N ratio in Kalmia mineral soil from upper B (29.73) and lower B (identified as B+) (33.08) in uncut black spruce forest was recorded compared to non-Kalmia soils in B (18.17) and B+ (17.05) horizons in uncut black spruce forest. Phenolics leached out from Kalmia litter were lower in Kalmia associated soils than the non-Kalmia soils from the uncut forest, and Kalmia associated soils from the cut forest area. Results indicate that soils associated with Kalmia were nutrient poor particularly for nitrogen, phosphorus, iron and manganese, and provide some basis for the hypothesis that Kalmia has dominated microsites that were nutrient poor prior to Kalmia colonization.     

Actin distribution in somatic embryos and embryogenic protoplasts of white spruce (Picea glauca)

Summary Examination of unfixed immature somatic embryos of white spruce (Picea glauca) with fluorescent rhodamine-labeled phalloidin revealed an extensive network of fine actin microfilaments (MFs) in the embryonal region which were not detected in specimens fixed with formaldehyde. Transition cells linking the embryonal region and suspensor cells contained fine MFs as well as bundles of MFs. The large, highly vacuolated suspensor cells were characterized by actin MF cables only. Treatment of embryos with cytochalasin B (CB) removed the fine MFs from the embryonal region and transition cells, but many MF cables in suspensor cells were resistant. Full recovery from CB treatment was observed in most somatic embryos. Embryogenic protoplasts capable of regenerating to somatic embryos in culture were released from only the embryonal region of somatic embryos. Both uninucleate and multinucleate embryogenic protoplasts retained the extensive network of fine actin MFs. In contrast, protoplasts derived from vacuolated suspensor cells and vacuolated free-floating cells contained thick MF bundles and were not embryogenic. Distinct MF cages enclosed nuclei in multinucleate protoplasts and may be responsible for preventing nuclear fusion. Microspectrophotometric analyses showed that the DNA contents of embryonal cells in the embryo and embryogenic protoplasts were similar and characteristic of rapidly dividing cell populations. However, transition and suspensor cells which released nonembryogenic protoplasts appeared to be arrested in G₁, and suspensor cells showed signs of DNA degradation.     

Vicilin-like seed storage proteins in the gymnosperm interior spruce (Picea glauca/engelmanii)

A seed storage protein cDNA was characterized from a library of interior spruce (Picea glauca/engelmanii complex) cotyledonary stage somatic embryos. The deduced amino acid sequence predicts a 448 amino acid (50 kDa) polypeptide with 28–38% identity with angiosperm vicilin-like 7S globulins. XXC/G codon usage is low (47%) relative to monocot angiosperms while pairwise comparisons show that spruce, monocot, and dicot vicilins are approximately equal in amino acid divergence. Although small by comparison, the spruce vicilin contains an N terminal hydrophilic region characteristic of angiosperm large vicilins. Genomic Southern blotting predicts that the cDNA is encoded by a gene family.     

High-resolution analysis of stem radius variations in black spruce [Picea mariana (Mill.) BSP] subjected to rain exclusion for three summers

Key message

A rain exclusion repeated for 3 years resulted in larger summer stem contractions in three of the sites in the third year of the experiment and in larger winter contractions in the northern sites. However, there was no pronounced stress reaction in the stem radius variations of mature black spruce since total stem expansion was not reduced.

Abstract

Future climate warming is expected to produce more severe and frequent periods of drought with consequent water stresses for boreal species. In this paper, we present a high-resolution analysis of stem radius variations in black spruce under rain exclusion. All summer long rain exclusions were applied for three consecutive summers to mature trees on four sites along a latitudinal gradient. The stem radius variations of control and treated trees were monitored year-round at an hourly resolution with automatic point dendrometers. The seasonal patterns of shrinking and swelling were analyzed using a sequential analysis technique and the daily patterns of contraction and expansion were extracted. Overall, the treated trees followed their diurnal cycles of contraction and expansion during the rain exclusions and no significant cumulative difference in stem expansion between control and treated trees was observed over the 3 years. In the third year trees subjected to rain exclusion showed larger stem contractions in summer on three out of four sites and larger winter contractions were observed on the northern sites. This study shows that repeated summer rain exclusion does not necessarily lead to a direct evident stress reaction, showing the resilience of the boreal forest.     

A fungal endophyte of black spruce (Picea mariana) needles is also an aquatic hyphomycete

An aquatic hyphomycete, Dwayaangam sp., was isolated from superficially sterilized black spruce (Picea mariana) needles submerged in aerated water in a small glass chamber (microcosm). The internal transcribed spacer (ITS) sequence of this fungus and of a commonly encountered foliar endophyte isolated from P. mariana showed a high degree of similarity. When sporulation was induced in the microcosm, both the aquatic hyphomycete and the endophyte isolate produced similar aquatic conidia after 30 days, which is longer than previously documented in similar studies. Without the use of molecular tools, the link between the aquatic and endophytic phases of the fungus would have gone unnoticed. This is the first time that a fungal endophyte of conifer needles has been shown to have an aquatic phase. Its presence both as a foliar endophyte and a sporulating aquatic fungus suggests an alternating life cycle between the two environments.     

Hygromycin resistance is an effective selectable marker for biolistic transformation of black spruce (Picea mariana)

 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. Received: 1 December 1997 / Revision received: 2 January 1999 / Accepted: 15 June 1999