Mining and validation of pyrosequenced simple sequence repeats (SSRs) from American cranberry (Vaccinium macrocarpon Ait.).

The American cranberry (Vaccinium macrocarpon Ait.) is a major commercial fruit crop in North America, but limited genetic resources have been developed for the species. Furthermore, the paucity of codominant DNA markers has hampered the advance of genetic research in cranberry and the Ericaceae family in general. Therefore, we used Roche 454 sequencing technology to perform low-coverage whole genome shotgun sequencing of the cranberry cultivar 'HyRed'. After de novo assembly, the obtained sequence covered 266.3 Mb of the estimated 540-590 Mb in cranberry genome. A total of 107,244 SSR loci were detected with an overall density across the genome of 403 SSR/Mb. The AG repeat was the most frequent motif in cranberry accounting for 35% of all SSRs and together with AAG and AAAT accounted for 46% of all loci discovered. To validate the SSR loci, we designed 96 primer-pairs using contig sequence data containing perfect SSR repeats, and studied the genetic diversity of 25 cranberry genotypes. We identified 48 polymorphic SSR loci with 2-15 alleles per locus for a total of 323 alleles in the 25 cranberry genotypes. Genetic clustering by principal coordinates and genetic structure analyzes confirmed the heterogeneous nature of cranberries. The parentage composition of several hybrid cultivars was evident from the structure analyzes. Whole genome shotgun 454 sequencing was a cost-effective and efficient way to identify numerous SSR repeats in the cranberry sequence for marker development.

Environment. Sustainable development of the agricultural bio-economy.

A U.S. farm policy shift to joint production of commodities and ecological services will advance sustainable agriculture.

Inhibition of growth of microcultured Hancornia speciosa shoots by 3beta-hydroxylated gibberellins and one of their C-3 deoxy precursors.

Gibberellins (GAs) A(1), A(3), A(4) and A(7), all 3beta-hydroxylated, growth-active GAs, significantly inhibited shoot elongation and the formation of nodes in in vitro-grown Hancornia speciosa, as did GA(20), a 3-deoxy precursor of GA(1). Ancymidol, an early-stage inhibitor of GA biosynthesis, significantly retarded shoot elongation without affecting the formation of nodes. Co-application of ancymidol and GA(1 )did not overcome the ancymidol-induced growth retardation. Trinexapac-ethyl, which can inhibit 3beta-hydroxylation (GA activation) and 2beta-hydroxylation (GA inactivation), gave no significant response on either shoot elongation or node formation, while two isomers of 16,17-dihydro GA(5), also inhibitors of GA 3beta-hydroxylation, significantly inhibited both shoot growth and the formation of nodes. These unusual results may indicate a unique metabolism for GAs in microcultured shoots of H. speciosa.

Physiological significance of anthocyanins during autumnal leaf senescence.

The light screen hypothesis states that foliar anthocyanins shade the photosynthetic apparatus from excess light. In this paper we extend the light screen hypothesis, postulating that plant species at risk of photoinhibitory conditions during autumnal leaf senescence often utilize anthocyanins to protect the photosynthetic apparatus during the period of nutrient resorption. When senescence-related photosynthetic instabilities are compounded by other environmental stresses, particularly low temperature, severe photoinhibition may result in reduced resorption of critical foliar nutrients, which can significantly affect plant fitness. There is evidence that environments where low and often freezing temperatures are common in autumn selectively favor the production of anthocyanins in senescing foliage. The stimuli for, and the timing and location of, autumnal anthocyanin production are all consistent with a photoprotective role for these pigments in senescing leaves. Furthermore, differences in nitrogen allocation strategies between early and late successional species appear to affect photosynthetic stability during leaf senescence, resulting in a reduced need for foliar autumnal anthocyanins in many early successional plants. The ecological and physiological evidence presented in this paper suggest that, for many deciduous species, the production of anthocyanins provides effective photoprotection during the critical period of foliar nutrient resorption.

Resistance to the birch leafminer Fenusa pusilla (Hymenoptera: Tenthredinidae) within the genus Betula.

Thirteen Betula species were tested for resistance to the birch leafminer, Femusa pusilla (Lepeletier), using no-choice assays. Birch leafminers were able to oviposit into expanding leaves of all Betula individuals tested. Larvae did not survive within any of the tested individuals of three species, B. alleghaniensis (Britt.), B. grossa (S. & Z.), and B. lenta (L.). Leafminer eggs deposited into the leaves of these species hatched, and larvae fed for a short period before dying. These three species were classified as highly resistant to birch leafminer, based on very low percent of mines (0.6-3.1%) with a diameter >3 mm. Eight species, B. papyrifera (Marsh), B. pendula (Roth), B. turkestanica (Litvin), B. glandulifira (Regal), B. ermanii (Cham.), B. platyphylla variety japonica [(Miq.) Hara], B. populifolia (Marsh) and B. maximowicziana (Regal) were classified as susceptible, with percent of mines >3 mm diameter of 87-94%. Two species, B. costata (Trautv.) and B. davurica (Pall.), displayed intermediate and variable resistance. B. davurica exhibited a mechanism of resistance not observed in the other species, Eggs oviposited into the leaves of resistant B. davurica individuals became surrounded by an area of discolored and necrotic tissue, and died. This response resembles the programmed cell death associated with a hypersensitive response.

Sucrose utilization during potato microtuber growth in bioreactors.

Potato microtubers are used as pathogen-tested in vitro stocks for certified seed potato production. Microtubers grown in a rotating bioreactor grew at a faster rate when the medium was replaced frequently. Although the total microtuber number was not affected, the number of microtubers over 1 g quadrupled when 75% of the medium was replaced every 2 weeks when compared with no medium refreshment. Significantly slower microtuber growth rates resulted when a lower sugar concentration (40 g 1-1 instead of 80 g 1-1) was used or when a mixture of glucose and fructose replaced sucrose. Although high sucrose levels are necessary for optimal microtuber production, the sucrose supplied was rapidly hydrolyzed into glucose and fructose, making the long-term maintenance of desirable sucrose levels difficult. These results indicate that successful strategies to reduce sucrose hydrolysis without inhibiting microtuber growth will improve the efficiency of sucrose utilization in potato microtuber bioreactors.

Taxol production in nodule cultures of Taxus.

The in vitro synthesis of secondary compounds from plants is one source of scarce and valuable phytopharmaceuticals. Often, some level of cellular or tissue differentiation is needed for the biosynthesis of many of these important compounds. Nodule cultures, consisting of cohesive multicellular units displaying a high degree of differentiation, were initiated from cultured needles of seven Taxus cultivars (Taxus cuspidata, Taxus x media 'Hicksii', Taxus x hunnewelliana 'Richard Horsey', Taxus x media 'Dark Green Spreader', Taxus x media 'L. C. Bobbick', and Taxus brevifolia). Under normal semicontinuous perfusion culture conditions (bimonthly refreshments to yield 0.2% sucrose), only trace amounts of taxol were detected from Taxus nodule cultures. However, with an elevated sucrose level (0.5% or 1.0%), taxol production was enhanced in T. cuspidata nodules to approximately 12 micrograms taxol/g nodule dry weight (dw). Stimulation of taxol production by elevated sucrose levels occurred even in the absence of other nutrients. The effect of increased sucrose on taxol induction does not appear to be due to an osmotic effect in the medium, suggesting that the increase in taxol production may be correlated with a metabolic process within the nodules. Although sucrose had a significant effect on taxol production, taxane precursors or elicitors of terpenes, as well as other plant secondary metabolites, had no effect on the production of taxol from these cultures. In addition to taxol, the higher sucrose levels also induced the production of 7-epi-10-deacetyltaxol, cephalomannine, and 7-epi-10-deacetylcephalomannine, so that total content of these taxanes equaled approximately 39 micrograms taxane/g dw nodules.

Expression of inducible angiosperm promoters in a gymnosperm, Picea glauca (white spruce).

Electrical discharge particle acceleration was used to test the transient expression of numerous inducible angiosperm promoters in a gymnosperm Picea glauca (white spruce). Promoter expression was assayed in three different tissues capable of in vitro regeneration, zygotic embryos, seedlings and embryogenic callus. The promoters tested include the light-inducible Arabidopsis and soybean ribulose-1,5-bisphosphate small subunit promoters and a maize phosphoenolpyruvate carboxylase promoter; a soybean heat-shock-inducible promoter, a soybean auxin inducible promoter and a maize alcohol dehydrogenase promoter. Promoters were cloned into a promoter-less expression vector to form a promoter-beta-glucuronidase-nopaline synthase 3' fusion. A similar construct was made using the cauliflower mosaic virus 35S (CaMV 35S) promoter as a control. All promoters were expressed in white spruce embryos, yet at levels lower than CaMV 35S. In addition, in the embryos the heat-shock and the alcohol dehydrogenase promoters showed inducible expression when given the proper induction stimulus. In seedlings, expression of all promoters was lower than in the embryos and expression was only inducible with the heat-shock promoter in the cotyledons. Of the tissues tested, the expression level of all promoters was lowest in embryogenic callus. Interestingly, the expression of the beta-glucuronidase gene in embryogenic callus was restricted to the proembryonal head cells regardless of the promoter used. These results clearly demonstrate the use of particle bombardment to test the transient expression of heterologous promoters in organized tissue and the expression of angiosperm promoters in a gymnosperm.

Stable transformation of Populus and incorporation of pest resistance by electric discharge particle acceleration.

Three different target tissues (protoplast-derived cells, nodules, and stems) and two unrelated hybrid genotypes of Populus (P. alba x P. grandidentata 'Crandon' and P. nigra 'Betulifolia' x P. trichocarpa) have been stably transformed by electric discharge particle acceleration using a 18.7 kb plasmid containing NOS-NPT, CaMV 35S-GUS, and CaMV 35S-BT. Four transformed plants of one hybrid genotype, NC5339, containing all 3 genes were recovered and analyzed. Two expressed GUS and one was highly resistant to feeding by 2 lepidopteran pests (the forest tent caterpillar, Malacosoma disstria, and the gypsy moth, Lymantria dispar.) Pretreatment of the target tissues, fine-tuning of the bombardment parameters, and the use of a selection technique employing flooding of the target tissues were important for reliable recovery of transformed plants.

Shoot culture dynamics of six Populus clones.

Shoot tips of five genotypically diverse Populus clones, P. alba x P. grandidentata 'Crandon,' P. nigra 'Betulifolia' x P. trichocarpa, P. nigra x P. laurifolia 'Strathglass,' P. maximowiczii x P. trichocarpa 'Androscoggin' and P. deltoides x P. nigra 'Eugenei,' were collected from hardwood cuttings, sterilized,and established in vitro. Stable shoot cultures were obtained from all clones except P. deltoides x P. nigra 'Eugenei'. The four poplar clones that formed stable shoot cultures together with a previously established P. tremula 'Erecta' clone were placed as two-node explants on either Murashige and Skoog medium or Woody Plant Medium containing benzyladenine to determine the rate of shoot multiplication, shoot growth and other responses of the clones. All five poplar clones showed rapid shoot multiplication when cultured in the presence of 0.4-1.0 microM benzyladenine on Murashige and Skoog medium, although P. tremula 'Erecta' produced a greater number of healthy shoots when grown on Woody Plant Medium. Individual shoot growth of all clones was more vigorous when the medium contained 0-0.1 microM benzyladenine, and 100% of such shoots rooted ex vitro.

Recovery of plants from leaf protoplasts of hybrid-poplar and aspen clones.

Leaf protoplasts were isolated from shoot cultures of two hybrid poplar clones (Populus alba × P. grandidentata 'Crandon', NC-5339 and P. nigra 'Betulifolia' × P. trichocarpa, NC-5331) and the Upright European Aspen (P. tremula 'Erecta') and were cultured in contact with screen discs floated in liquid medium. Protoplast culture was influenced by the growth medium of the source shoot cultures, the protoplast purification procedure, the plating density, and the presence or absence of a coconut water and casein hydrolysate supplement added to the culture medium. The protoplast-derived cells divided more quickly and with higher incidence than previously reported for hybrid poplars. Shoots were regenerated from the protoplast-derived calli and were maintained as shoot cultures. Plants were developed from microcuttings rooted ex vitro and were grown-on in the greenhouse and field.

Techniques for enhanced release of leaf protoplasts in Populus.

Microscopic examination of Populus leaf tissue following enzyme treatment revealed two factors contributing to low protoplast yields: (1) poor penetration of the enzymes into the tissue, and (2) entrapment of protoplasts in leaf debris during protoplast purification procedures. A simple combination of rapid grinding of the tissue in an Omni-mixer prior to enzyme treatment and forceful washing of leaf-debris after digestion provided high exposure of the cells, uniform digestion, and high yields of protoplasts of two Populus clones. Protoplasts exhibited cell wall regeneration and long-term viability in culture. The relative yield advantages of the techniques varied with the inherent digestibility of each clone but could produce up to 600 percent greater protoplast yields in a woody plant species in which protoplast isolation was previously limited. The techniques were also applicable to an herbaceous species, Solanum etuberosum.

Plant Leaf and Stem Proteins. II. Isozymes and Environmental Cabbage.

The activity of 10 enzymes separated by acrylamide disc gel electrophoresis of leaf and stem extracts from Dianthus grown under summer and winter conditions was studied. While banding was constant and highly reproducible under each environment, differences between the 3 cultivars and between the tissues were evident. No significant differences in the isozyme patterns of glutamate dehydrogenase, 6-phosphogluconate dehydrogenase, glucose-6-phosphate dehydrogenase, malate dehydrogenase, and catalase were observed between the 2 environments. Loss of activity was observed under winter conditions with amylase and lactate dehydrogenase and loss of certain isozymic components was evident with acid phosphatase and esterase. Prominent changes were observed in peroxidase isozymes, the hardy cultivars developing additional isozymic components under winter conditions. Only minor changes in the total protein banding were seen. The enzymes showed considerable stability in those tissues killed by the freezing conditions.

Plant leaf and stem proteins. I. Extraction and electrophoretic separation of the basic, water-soluble fraction.

Simplified but highly reproducible extraction and electrophoretic procedures have been developed for plant stem and leaf proteins using recent chemical and technical advances. The method is applied to the separation of the basic, water-soluble proteins found in stem and leaf tissues of 3 Dianthus clones. While most of the highly reproducible protein bands appear in all 3 selections, and many are also common to both leaf and stem tissue, others are characteristic for the variety or tissue.