Pinaceae Research Articles

Rapid assessment of wood density of live trees using the Resistograph for selection in tree improvement programs

Wood density is traditionally determined by a volumetric method that is accurate but expensive for large-scale sampling. A new device called the Resistograph was investigated for rapid assessment of relative wood density of live trees in progeny trials. Fourteen full-sib families of loblolly pine (Pinus taeda L.) produced by a six-parent half-diallel mating design were tested at four sites. For each family, wood density was measured with the traditional volumetric method and then compared with the Resistograph readings (amplitude). Amplitude had weak (0.29) to moderate (0.65) phenotypic correlations with wood density on an individual-tree basis over the four sites. The family mean correlation between the two measurements, however, was much stronger (0.92). The additive genetic correlation between the two measures was also high (0.95). Individual-tree breeding values of amplitude yielded more accurate rankings than phenotypic values. The rankings of the parental, general-combining abilities were identical for the two measures. Both wood density and amplitude were under strong genetic control at the family level (full-sib family heritability (h2fs) = 0.95 for wood density and h2fs = 0.85 for amplitude). The efficiency of using the Resistograph as a means of indirect selection for improvement of wood density was 87% at the family level. Results from this study suggest that the Resistograph could be used reliably and efficiently to assess relative wood density of live trees for selection in tree improvement programs. The method is rapid, nondestructive, and much cheaper than the traditional volumetric method.

Histology of White Pine Blister Rust in Needles of Resistant and Susceptible Eastern White Pine.

White pine blister rust, Cronartium ribicola, has plagued the forests of North America for almost a century. Over past decades, eastern white pine (Pinus strobus) that appear to tolerate the disease have been selected and incorporated into breeding programs. Seeds from P. strobus with putative resistance were collected from Oconto River Seed Orchard, Nicolet National Forest, WI. Seedlings were grown for 5 months and artificially inoculated with basidiospores of C. ribicola in two replicated greenhouse experiments. Needles from infected seedlings were fixed, sectioned, and stained with a variety of histological reagents, and rate of mortality for the remaining seedlings was monitored. The most susceptible families suffered 50% mortality in approximately half the time of the more resistant families. Extensive inter- and intracellular hyphae were observed in needles from seedlings of susceptible families, whereas hyphal proliferation was restricted in needles of resistant seedlings. Needles from resistant families had pronounced responses to infection. Phenolics, observed with phloroglucinol-HCl staining, were deposited around infection sites where dense mycelial masses were present. Abnormal host cell growth and rapid cell death in the immediate area of infection were also observed in some eastern white pine families.

Patterns of branch permeability with crown depth among loblolly pine families differing in growth rate and crown size

Patterns in branch permeability with crown depth and permeability at the top of the main stem were analyzed for loblolly pine (Pinus taeda L.) trees from families selected on the basis of growth rate (fast, slow) and crown size (large, small). Analysis of variance with levels of crown size nested within levels of growth rate was used to test for differences in main stem permeability. Permeability at the top of the stem averaged 2.0×10−12 m2. There were no significant differences in permeability between families selected for fast and slow growth, but permeability was significantly lower for families selected for large crowns than for families selected for small crowns. Branch permeability averaged 0.74×10−12 m2 and decreased significantly with crown depth. Large-crown families had higher overall branch permeability than small-crown families. Average permeability in branches did not differ significantly between fast- and slow-growing families. Large crown-families had significantly larger current leaf area: total leaf area ratios in the lower two-thirds of the crown, and a weak but significant association was found between permeability and current leaf area: total leaf area ratios for a given relative crown depth. Our results suggest that ecotypic and adaptive processes simultaneously affect the overall patterns of stem and branch permeability in loblolly pine families.

Genetic variation in branchlet carbon and nitrogen isotope composition and nutrient concentration of 11-year-old hoop pine families in relation to tree growth in subtropical Australia

Genetic variation in tree growth and branchlet carbon isotope composition (δ 13C), nitrogen (N) isotope composition (δ 15N) and nutrient (N, P, K, Ca, Mg, Mn, Fe, Cu and Zn) concentration of 11-year-old hoop pine ( Araucaria cunninghamii Ait. ex D. Don) half-sib families were examined at two contrasting sites (31 families with 8 blocks sampled at a wet site and 27 families with 9 blocks at a dry site) in south-east Queensland, Australia. Genetic correlations were determined between the tree growth traits, between the physiological traits, and between the physiological and growth traits. The potential of using these tree physiological traits was evaluated for assisting in selection of elite hoop pine families with improved water-use efficiency (WUE) as reflected in branchlet δ 13C, nutrient (particularly N)-use efficiency (NUE) and tree growth. At the wet site there was significant genetic variation in tree growth, branchlet δ 13C, nutrient concentration and mineral concentration among the 11-year-old hoop pine families, with heritability estimates ranging from 0.26 to 0.72. At the dry site, there was also significant genetic variation in some of the corresponding variables with heritability estimates ranging from 0.26 to 0.54. There seemed to be significant family variation in branchlet δ 15N at the dry site ( P=0.068), with a moderate heritability estimate of 0.21, but not at the wet site. Some strong genetic correlations were also found between the tree growth traits, between the physiological traits, and between the tree growth and the physiological traits. Tree WUE and branchlet nutrient concentrations were significantly influenced by rainfall and soil fertility. Branchlet δ 15N at the wet site was significantly lower than that at the dry site, suggesting that soil microbial–plant interactions might play a role in discriminating soil available 15N in favor of 14N for tree uptake (lower δ 15N in plant tissues) at the wet site. Branchlet δ 13C δ 15N and nutrient (particularly N) concentration show promise as physiological and nutritional traits for assisting in the selection of elite hoop pine families with improved tree WUE, NUE and growth for the more water- and nutrient-limited environments in subtropical Australia.

Brassinolide improves embryogenic tissue initiation in conifers and rice.

Somatic embryogenesis (SE), the most promising technology for the large-scale production of high-value coniferous trees from advanced breeding and genetic engineering programs, is expected to play an important role in increasing productivity, sustainability, and the uniformity of future U.S. forests. To be successful for commercial use, SE technology must work with a variety of genetically diverse trees. Initiation in loblolly pine ( Pinus taeda L.), our main focus species, is often recalcitrant for desirable genotypes. Initiation percentages of loblolly pine, Douglas-fir [ Pseudotsuga menziesii (Mirb.) Franco], and Norway spruce ( Picea abies L., Karst.) were improved through the use of brassinolide. Brassinosteroids, which include brassinolide, are a relatively new group of natural plant growth regulators that are found in many plant species. They have been shown to have diverse, tissue-specific, and species-specific effects, including the stimulation of cell elongation and ethylene production and increasing resistance to abiotic stress. In our media, brassinolide was effective at concentrations ranging from 0.005-0.25 micro M. Using control medium (no brassinolide) and brassinolide-supplemented (0.1 micro M) medium, we achieved improved initiation percentages in loblolly pine, Douglas-fir, Norway spruce, and rice-15.0% to 30.1%, 16.1% to 36.3%, 34.6% to 47.4%, and 10%, respectively. Brassinolide increased the weight of loblolly pine embryogenic tissue by 66% and stimulated initiation in the more recalcitrant families of loblolly pine and Douglas-fir, thus compensating somewhat for genotypic differences in initiation. Initiation percentages in loblolly pine were improved through the combination of modified 1/2-P6 salts, 50 mg/l activated carbon (AC), adjusted levels of Cu and Zn (to compensate for adsorption by AC), 1.5% maltose, 2% myo-inositol (to raise the osmotic level, partially simulating the megagametophyte environment), 500 mg/l casamino acids, 450 mg/l glutamine, 2 mg/l alpha-naphthaleneacetic acid, 0.63 mg/l 6-benzylaminopurine, 0.61 mg/l kinetin, 3.4 mg/l silver nitrate, 10 micro M cGMP, 0.1 micro M brassinolide, and 2 g/l Gelrite. Across 12 open-pollinated families of loblolly pine, initiation percentages ranged from 2.5% to 50.7%, averaging 22.5%.

Families of loblolly pine that are the most stable for resistance to fusiform rust are the least predictable

In an extensive series of trials with open-pollinated families of loblolly pine (Pinus taeda L.), resistance to fusiform rust disease (caused by Cronartium quercuum (Berk.) Miyabe ex Shirai f. sp. fusiforme) at individual test sites was relatively unpredictable for the families deemed most resistant. The most resistant families were also the most stable for performance across test sites, with stability defined as the slope of the regression of family means for rust infection versus site means for rust infection. A family's R-50 value (its predicted rust infection level when the site mean infection is 50%) was correlated to its stability parameter or slope (r = 0.78). On average, any one family's level of infection (% galled) was reasonably predictable for any given infection level at a given site; the average coefficient of determination (r2) was 0.78 for the regression of family means for rust infection versus site means for rust infection. However, the six most stable families for resistance had the lowest r2 values (average r2 = 0.58). We speculated that the lower predictability for the most resistant families was due to interactions of specific resistance genes in these families and corresponding avirulence and (or) virulence levels in the pathogen populations that may differ among sites. Although the predictability of the individual resistant families was relatively low, if these families were bulked into a resistant seed lot, they performed in a more predictable manner with r2 = 0.74 for the regression of the bulk mean versus site means. Bulks of four to six highly resistant families appeared to be a good solution to obtain stable and predictable performance across a range of sites.

Branchlet nutrient concentration in hoop pine (Araucaria cunninghamii) relative to family, stable carbon and oxygen isotope ratios and growth rate in contrasting environments.

Genetic variation in branchlet nutrient (N, P, K, Na, Ca, Mg, Mn and Fe) concentrations and mineral concentration (sum of branchlet P, K, Na, Ca, Mg, Mn and Fe concentrations) of 8-9-year-old hoop pine (Araucaria cunninghamii Ait. ex D. Don) half-sib families was assessed for four canopy positions at a wet site (23 families) and two canopy positions at an N- and water-limiting dry site (22 families) in relation to tree growth and associated branchlet carbon (delta13C) and oxygen (delta18O) isotope composition in southeast Queensland, Australia. Branchlet nutrient and mineral concentrations varied significantly among families and with canopy position and site. Depending on the canopy position sampled, the hoop pine family effect accounted for 0 to 13.8% of the total variation in branchlet N concentration, and for 0 to 30.3% of the total variation in branchlet mineral concentration at the wet site. The corresponding values for the family effect at the dry site were 0-13.3% for branchlet N concentration and 0-25.7% for branchlet mineral concentration. There were significant variations in branchlet P, K, Ca and Mg concentrations at both sites, and these variations differed with canopy position. Relationships between family means of branchlet N concentration and tree growth or delta13C or delta18O varied with canopy position at both sites. At the wet site, there were significant positive correlations between branchlet mineral concentration in the upper-outer or upper-inner canopy and tree height (r = 0.26 and 0.37, P < 0.01) and between branchlet mineral concentration and delta13C (r = 0.24, P < 0.01) in the upper-inner canopy, and a significant negative correlation between branchlet mineral concentration and delta13C (r = -0.21, P < 0.05) in the upper-outer canopy. At the dry site, branchlet mineral concentrations in the upper-inner and upper-outer canopy were significantly correlated with branchlet delta13C (r = -0.28 and -0.51, P < 0.01), and branchlet N concentration in the upper-inner canopy was significantly correlated with tree growth (r = 0.29, P < 0.01). A significant correlation between branchlet delta18O (an index of stomatal conductance) and branchlet mineral concentration at the dry site (r = 0.39, P = 0.020) indicated that stomatal conductance might be a factor regulating the variation in branchlet mineral concentration of the hoop pine families. Both branchlet N concentration and mineral concentration at particular canopy positions assist in selecting hoop pine families with improved tree growth and N- and water-use efficiency in environments where both N deficiency and a limited water supply are major factors affecting plantation productivity.

Growth and leaf nutrient responses of loblolly and slash pine families to intensive silvicultural management

Intra-specific variation in foliar nutrient response to fertilization treatments and associated growth performance is largely unknown in southern pines. Sixteen open-pollinated families from each of three pine taxa—genetically improved loblolly pine (PTI; Pinus taeda), improved slash pine (PEI; Pinus elliottii var. elliottii), and unimproved slash pine (PEU)—were planted in two locations under a common split–split plot experimental design in north Florida. At each site, two levels of silvicultural treatments (low intensity—no fertilization; high intensity—fertilization and weed control) were implemented. Vector analysis suggested that N, P, and K were nutrients mostly commonly limiting growth, while Mg and Ca were generally not deficient for the three taxa over four different sampling periods during the growing season. The period of highest nutrient demand occurred during the growing season from June to September, with deficiencies easing throughout the remainder of the year. Differential family responses in foliar nutrient levels (especially N and P) were closely associated with current-year foliage biomass production and annual height growth for the three taxa. PTI and PEI families with constant, but minor, foliar N and P deficiencies during the growing season had higher foliage biomass production and height increments than those exhibiting excess, dilution, or severe deficiency. PEU families generally had lower nutrient requirements than PTI and PEI families. Differences in nutrient demand for growth existed between PEU and PEI. Differences between PEI and PEU in foliar nutrient response to intensive management from this study, along with findings in growth performance and crown structure, suggested that PEI had growth strategies more closely aligned with PTA than with PEU. Significant intra-specific differences in growth and nutritional characteristics also indicated that family forestry could be a viable option suited for intensive plantation management because family-specific fertilizer prescriptions may be needed to further improve stand growth.

Variation among slash pine families in chlorophyll fluorescence traits

Photochemical quenching, nonphotochemical quenching, and yield of photosystem II were measured on seedlings of full-sibling, open-, and self-pollinated slash pine (Pinus elliottii Engelm. var. elliottii) families. Our results reveal that genetic variation in photochemical quenching and yield of photosystem II exists within this species. The pattern of variation found in these traits is consistent with the variance profile expected to occur as a result of segregation among nuclear genes. Variation among families accounted for 17% of the total variation observed in photochemical quenching, whereas the component for trees within families made up slightly more than 25% of the total. Less variation, both among families as well as among trees within families, was found for yield of photosystem II. A strikingly different pattern was observed for nonphotochemical quenching. Other than the error term, only pretreatment effects contributed significantly to the variation observed. This suggests that nonphotochemical quenching is largely influenced by environmental factors. With regard to associations between fluorescence and growth traits, both height and diameter growth were found to be positively correlated with photochemical quenching (0.36 and 0.33, respectively) when selfed and open-pollinated families were analyzed along with control-pollinated families.

Evaluating Growth and Resistance of Eastern and Western White Pine to White Pine Weevil and Blister Rust in the Northeast

Abstract Eastern white pine is highly susceptible to damage from white pine weevil, and to some extent white pine blister rust, in the northeastern United States. Western white pine has shown resistance to the weevil, but is highly susceptible to blister rust in the West. Objectives of this study were to compare the growth and resistance of eastern and western white pine to damage from the weevil and blister rust and to identify families of western white pine suitable for planting in the East. A field trial containing 76 half-sib families of western white pine and two half-sib families of eastern white pine was established in 1983 at two sites, one each in New York and Maine. After 14 yr, tree height and diameters were measured, and resistance to weevil and rust assessed. Tree height and diameters were significantly greater for eastern white pine than western white pine. Western white pine sustained approximately fourfold less weevil damage at both sites, but was more impacted by rust at one site. Cluster and discriminant analyses were used to group families using three variables: height growth, resistance to weevil, and resistance to blister rust. One single group combined superior height growth with moderate resistance to both pests. Selections made from within these families have the most promise for planting western white pine in the East. North. J. Appl. For. 20(1):19–26.

Avaliação e seleção precoce para crescimento de Pinus taeda

O objetivo deste trabalho foi avaliar a eficiência de diferentes métodos de seleção quanto aos caracteres ligados ao crescimento do fuste e o potencial da seleção precoce de genitores. Em 48 progênies de meios-irmãos de Pinus taeda L., foi medido o diâmetro do fuste a 30 cm e a 1,30 m do solo, aos 16 e 84 meses de idade, além da altura total. As progênies foram cultivadas em espaçamento tradicional e reduzido. Os componentes de variância, parâmetros e valores genéticos aditivos foram obtidos a partir de um modelo de análise individual. A seleção indireta foi de eficiência similar à direta quanto ao diâmetro (93%) e menor quanto à altura (78%). A seleção seqüencial foi 16% (altura) e 28% (diâmetro) mais eficiente que a seleção única. A eficiência da seleção combinada foi maior em relação a direta para diâmetro. Vinte genitores selecionados por valores genéticos aditivos quanto ao diâmetro, aos 16 e 84 meses, aumentaram a média em 7,6% e 8,1%, respectivamente. A alta magnitude da correlação genética no que diz respeito ao diâmetro entre idades revelou que esse caráter, avaliado aos 16 meses de idade, prediz o crescimento aos 84 meses. Isso reduz custos de avaliação de progênies, antecipa o desbaste de genitores e a oferta de sementes melhoradas.

Additional virulence genes and sonication enhance Agrobacterium tumefaciens-mediated loblolly pine transformation.

Additional virulence (vir) genes in Agrobacterium tumefaciens and sonication were investigated for their impact on transformation efficiency in loblolly pine (Pinus taeda L.). Mature zygotic embryos of loblolly pine were co-cultivated with disarmed A. tumefaciens strain EHA105 containing either plasmid vector pCAMBIA1301 or vector pCAMBIA1301 with an additional 15.8-kb fragment carrying extra copies of the Vir B, Vir C, and Vir G regions from the supervirulent plasmid pTOK47. pCAMBIA1301 contains hygromycin resistance and the beta-glucuronidase (GUS) reporter gene. Expression of GUS was observed after 3-6 days of co-cultivation, with peak expression at approximately 21 days. The highest numbers of GUS-expressing areas were visible up to 21 days after co-cultivation, declining rapidly thereafter. Both transient and stable transformation efficiencies increased when the explants were sonicated before co-cultivation and/or the additional virB, virC, and virG genes were included with the pCAMBIA1301 plasmid T-DNA. Use of the plasmid with additional vir genes and sonication dramatically enhanced the efficiency of Agrobacterium-mediated gene transfer not only in transient expression but also in the recovery of hygromycin-resistant lines. Stably transformed cultures and transgenic plants were produced from embryos transformed with A. tumefaciens EHA105 carrying pCAMBIA1301 or pCAMBIA1301+pTOK47 in the three families of loblolly pine. The presence of the introduced GUS and hygromycin phosphotransferase genes in the transgenic plants was confirmed by polymerase chain reaction and Southern hybridization analyses.

Effects of Nursery Characteristics on Field Survival and Growth of Loblolly Pine Rooted Cuttings

Abstract Morphological characteristics of rooted cuttings and seedlings of four open-pollinated loblolly pine families cultured at the G.H.W. Weyerhaeuser Nursery near Washington, North Carolina were assessed after lifting. Cuttings were visually assigned to nine grading classes and six putative cull classes. The nine grading classes represented a factorial of three root collar diameter classes (small [4–6 mm], medium [6–8 mm] and large [8–10 mm]) and three root quality classes (poor, fair, and good). Putative cull classes included runts, poor foliage coverage, jumbos, “dog-legged” (one horizontal root at a right angle to the stem), multiple leaders, and excessive sweep.After assessment, the rooted cuttings and seedlings were established in a randomized block design on a coastal site in southern Beaufort County, North Carolina. Five years after establishment in the field, survival, height, diameter, presence or absence of fusiform rust incidence, number of leaders, and sweep were assessed. Analyses of variance were conducted to detect differences among the putative cull classes. Nursery rooting cutting morphological traits were regressed on fifth year field traits. Iterative logistic regression was performed between the nursery traits (continuous variables) and survival (binary variable).Overall, fifth year survival of the rooted cuttings was higher than that of the seedlings (77 versus 42%, respectively), and their stem volume was not significantly different from seedlings (0.259 versus 0.285 m3). For the four open-pollinated families studied, these results indicate that no culling standards are needed for rooted cutting planting stock to perform at least as well as seedling planting stock. However, eliminating cuttings with poor foliage coverage (less than 2.5 cm) and culling or pruning rooted cuttings with multiple leaders is recommended to reduce the number of forked trees in the field. South. J. Appl. For. 26(4):207–213.

Genetic trends in wood density and radial growth with cambial age in a radiata pine progeny test

The main objective of this study was to describe trends in genetic parameters for wood density and radial growth through cambial age in a radiata pine progeny test established in the south of Chile. Wood samples from 31 half-sib families of radiata pine were obtained and submit- ted to an X-ray densitometry procedure. The analyzed traits were total ring width (TRW), ring area (RA), and average ring density (ARD). Statis- tical analyses were conducted to estimate the heritability of individual traits at the ring level and the ring-to-ring genetic correlation between ARD and radial growth. The pattern of change of genetic parameters with cambial age is especially affected between rings 6 to 10, which can be related with the transition from juvenile wood to adult wood. The genetic control of ring density was strong at cambial ages 2 and 3 and dropped to zero within the transition zone (rings 6 and 8). After ring 10, the genetic control of ARD varied from low to moderate. From cambial ages 3 to 9, the genetic correlation between ring density and radial growth was positive. From rings 5 to 9, the phenotypic correlation was also positive but low. At rings 8 and 9, the relationship between radial growth and density changed and strong within-plot competition effects possibly affected the phenotypic correlation between ring density and radial growth. After ring 9, the genetic correlation was negative but weak. The phenotypic correlation between ring density and radial growth increased its negative magnitude towards cambial age 14, which may have been the result of local micro site influences, such as competition for light and nutrients.

Relationship between early family-selection traits and natural blister rust cankering in western white pine families

Blister rust (Cronartium ribicola) cankering incidence was compared among half-sib families of both artificially and naturally inoculated western white pine (Pinus monticola). In the first test, artificially inoculated seedlings of six families had their individual infection spots tallied and any infected needles were removed before cankers developed. These healthy seedlings were classified as high or low spotters and were planted in row plots with four high spotters paired with four low spotters. After 7 years, 2 pairs had equal cankers, 12 had more rust on the high spotters, and 12 had more rust on the low spotters. The mean number of cankers per tree was 22 and 23 for high and low spotters, respectively. In the second test, 203 artificially inoculated western white pine families were ranked for their relative susceptibility to blister rust based on (i) family mean counts of individual infection spots per seedling (spotting) and (ii) the presence of only small cankers (slow canker growth resistance). These results were compared with the natural cankering incidence of the same families in eight plantations. There were few significant differences in spotting incidence among inoculated families. The range in field cankering in the test plantations (15–63 families each) was 22–88%. Seven plantations, including two with families significantly different in infection-spotting incidence, lacked a significant correlation (Spearman, P < 0.05) between percentage field cankering and spotting incidence from inoculations. Although one plantation had a significant correlation (r s = 0.64; P < 0.005) between spotting incidence from inoculation and cankering in a plantation, there was no overall trend across plantations. Incidence of slow canker growth resistance from artificial inoculation ranged from 0 to 18%. There was a significant positive correlation between field cankering and slow canker growth resistance, as determined by inoculation only in three of eight plantations. However, when the families were placed into slow canker growth classes, there was a trend of reduced cankering with increasing class of slow canker growth resistance (mean Spearman's r s value = 0.87 (P < 0.01) across all plantations). These studies indicate that selection on the basis of slow canker growth resistance resulted in less cankering in plantations than selection on the basis of reduced infection spotting.

Diurnal changes in gas exchange and carbon partitioning in needles of fast- and slow-growing families of loblolly pine (Pinus taeda).

We investigated diurnal and seasonal changes in carbon acquisition and partitioning of recently assimilated carbon in fast- and slow-growing families of loblolly pine (Pinus taeda L.) to determine whether fast-growing families exhibited greater carbon gain at the leaf level. Since planting on a xeric infertile site in Scotland County, NC, USA in 1993, five Atlantic Coastal Plain (ACP) and five "Lost Pines" Texas (TX) families have been grown with either optimal nutrition or without fertilization (control). In 1998 and 1999, gas exchange parameters were monitored bimonthly in four families and needles were analyzed bimonthly for starch and soluble sugar concentrations. Although diurnal and seasonal effects on net photosynthesis (A(net)) and maximum rate of light-saturated photosynthesis (A(max)) were significant, few family or treatment differences in gas exchange characteristics were observed. The A(net) peaked at different times during the day over the season, and A(max) was generally highest in May. Instantaneous water-use efficiency (WUE(i)), derived from gas exchange parameters, did not differ among families, whereas foliage stable isotope composition (delta(13)C) values suggested that TX families exhibited lower WUE than more mesic ACP families. Although there were no diurnal effects on foliar starch concentrations, needles exhibited pronounced seasonal changes in absolute concentrations of total nonstructural carbohydrates (TNC), starch and soluble sugars, and in partitioning of TNC to starch and sugars, mirroring seasonal changes in photosynthesis and shoot and root growth. In all families, foliar starch concentrations peaked in May and decreased to a minimum in winter, whereas reducing sugar concentrations were highest in winter. Some family and treatment differences in partitioning of recently assimilated carbon in needles were observed, with the two TX families exhibiting higher concentrations of TNC and starch and enhanced starch partitioning compared with the ACP families. We conclude that growth differences among the four families are not a function of differences in carbon acquisition or partitioning at the leaf level.

Growth of ponderosa pine seedlings as affected by air pollution

The effect of air pollution on seedling survival and competitive ability is important to natural and artificial regeneration of forest trees. Although biochemical and physiological processes are sensitive indicators of pollution stress, the cumulative effects of air pollutants on seedling vigor and competitive ability may be assessed directly from whole-plant growth characteristics such as diameter, height, and photosynthetic area. A few studies that have examined intraspecific variation in seedling response to air pollution indicate that genotypic differences are important in assessing potential effects of air pollution on forest regeneration. Here, we studied the effects of acid rain (no-rain, pH 5.1 rain, pH 3.0 rain) and ozone (filtered, ambient, twice-ambient) in the field on height, diameter, volume, the height:diameter ratio, maximum needle length, and time to reach maximum needle length in seedlings of three families of ponderosa pine ( Pinus ponderosa Dougl. ex Laws). Seedling diameter, height, volume, and height:diameter ratio related significantly to their pre-treatment values. Twice-ambient ozone decreased seedling diameter compared with ozone-filtered air. A significant family-by-ozone interaction was detected for seedling height, as the height of only one of the three families was decreased by twice-ambient ozone compared with the ambient level. Seedling diameter was larger and the height:diameter ratio was smaller under pH 3.0 rain compared to either the no-rain or the pH 5.1-rain treatment. This suggests greater seedling vigor, perhaps due to a foliar fertilization effect of the pH 3.0 rain.

Regeneration of transgenic loblolly pine (Pinus taeda L.) from zygotic embryos transformed with Agrobacterium tumefaciens.

Embryos of 24 open-pollinated families of loblolly pine (Pinus teade L.) were used as explants to conduct in vitro regeneration. Then, Agrobacterium tumefaciens strain GV3101 harboring the plasmid pPCV6NFHygGUSINT was used to transform mature zygotic embryos of seven families of loblolly pine. The frequency of transformation varied among families infected with A. tumefaciens. The highest frequency (100%) of transient beta-glucuronidase (GUS)-expressing embryos was obtained from family 11-1029 with over 300 blue spots per embryo. Expression of the GUS reporter gene was observed in cotyledons, hypocotyls, and radicles of co-cultivated mature zygotic embryos, as well as in callus and shoots derived from co-cultivated mature zygotic embryos. Ninety transgenic plants were regenerated from hygromycin-resistant callus derived from families W03. 8-1082 and 11-1029. and 19 transgenic plantlets were established in soil. The presence of the GUS gene in the plant genome was confirmed by polymerase chain reaction. Southern blot, and plant DNA/T-DNA junction analysis. These results suggest that an efficient A. tumefaciens-mediated transformation protocol for stable integration of foreign genes into loblolly pine has been developed and that this transformation system could be useful for future studies on transferring economically important genes to loblolly pine.

Agrobacterium-mediated transformation and assessment of factors influencing transgene expression in loblolly pine (Pinus taeda L.).

This investigation reports a protocol for transfer and expression of foreign chimeric genes in loblolly pine (Pinus taeda L.). Transformation was achieved by co-cultivation of mature zygotic embryos with Agrobacterium tumefaciens strain LBA4404 which harbored a binary vector (pBI121) including genes for beta-glucuronidase (GUS) and neomycin phosphotransferase (NPTII). Factors influencing transgene expression including seed sources of loblolly pine, concentration of bacteria, and the wounding procedures of target explants were investigated. The expression of foreign gene was confirmed by the ability of mature zygotic embryos to produce calli in the presence of kanamycin, by histochemical assays of GUS activity, by PCR analysis, and by Southern blot. The successful expression of the GUS gene in different families of loblolly pine suggests that this transformation system is probably useful for the production of the genetically modified conifers.

Plant regeneration from embryogenic cultures initiated from mature loblolly pine zygotic embryos

Mature zygotic embryos of eight (open-pollinated) families of loblolly pine (Pinus taeda L.) were cultured on eight different basal salt formulations, each supplemented with 36.2 muM 2,4-dichlorophenoxyacetic acid, 17.8 muM 6-benzyladenine, 18.6 muM kinetin, 500 mg l(-1) casein hydrolysate, and 500 mg l(-1) L-glutamine for 9 wk; embryogenic tissue was formed on cotyledons, hypocotyls, and radicles of mature zygotic embryos. Callus was subcultured on the callus, proliferation, medium, the same as the induction medium, bat with one-fifth concentration of auxin and cytokinin for 9 wk. On this medium a white to translucent, glossy, mucilaginous embryogenic callus containing embryogenic suspensor masses (ESMs) was obtained. The highest frequency of explants forming embryogenic tissue, 17%, occurred on a modified Murashige and Skoog salts basal medium containing the concentration of KNO3, Ca(NO3)(2)-4H(2)O, NH4NO3, KCI, ZnSO4-7H(2)O(2), and MnSO4-H2O, 720, 1900, 00 250, 25.8, and 25.35 mg l(-1), respectively. Embryogenic suspension cultures were established by culturing embryogenic callus in liquid callus proliferation medium. Liquid cultures containing ESMs were transferred to medium containing abscisic acid, polyethylene glycols, and activated charcoal for stimulating ther production of cotyledonary somatic embryos. Mature somatic embryos germinated for 4-12 wk on medium containing indole-butyric acid, gibberellic acid, 6-benzyladenine, activated charcoal, and reduced sucrose concentration (15 g l(-1)). Two hundred and ninety-one regenerated plantlets were transferred to a perlite:peatmoss:vermiculite (1:1:1) mixture, then the plants were transplanted to soil in the earth, and 73 plantlets survived in the field.