Heartwood is a determining factor of wood quality and understanding the biology of heartwood may allow us to control its formation. Heartwood formation is a form of senescence that is accompanied by a variety of metabolic alterations in ray parenchyma cells at the sapwood-heartwood transition zone. Although senescence has been studied at the molecular level with respect to primary growth, the cell maturation and death events occurring during heartwood formation have been difficult to study because of their location and timing. Analysis of global gene expression patterns during the transition from sapwood to heartwood may offer a powerful means of identifying the mechanisms controlling heartwood formation. Previously, we developed cDNA microarrays carrying 2567 unigenes derived from the bark/cambium region, sapwood and transition zone of a mature black locust tree. Here, we describe the use of these microarrays to characterize seasonal changes in the expression patterns of 1873 genes from the transition zone of mature black locust trees. When samples collected in summer and fall were compared, 569 genes showed differential expression patterns: 293 genes were up-regulated (> twofold) in summer (July 5) and 276 genes were up-regulated in fall (November 27). More than 50% of the secondary and hormone metabolism-related genes on the microarrays were up-regulated in summer. Twenty-nine out of 55 genes involved in signal transduction were differentially regulated, suggesting that the ray parenchyma cells located in the innermost part of the trunk wood react to seasonal changes. We established the expression patterns of 349 novel genes (previously unknown or no-hit), of which 154 were up-regulated in summer and 195 were up-regulated in the fall.

Summary We developed an effective detection method for wood-decaying fungi by hybridization of immobilized Sequence-Specific Oligonucleotide Probes with florescent-labeled PCR-amplified fungal rDNA internal transcribed spacer sequences. This method takes advantage of both the sequence specificity of Southern blot hybridization and the sensitivity of the previously reported PCR-based fungal species identification methods. Both in vitro cultured fungal strains and naturally decaying wood samples were used to demonstrate that this method is robust and practical for detection of incipient wood-decaying fungi. It can be a useful tool for microbial ecology, plant pathology, protection of wood products in service, preservation efforts for high-value furniture and wood-based art and DNA fingerprinting for tracking the source of contamination of wood decay fungi.

We studied the in vitro responses of cambial tissue and dormant vegetative buds obtained from top and epicormic branches of three mature black locust (Robinia pseudoacacia L.) trees. Cambial tissues isolated from epicormic branches produced more callus than cambial tissues isolated from top branches, whereas in vitro shoot cultures derived from buds excised from top branches grew faster than those derived from buds excised from epicormic branches. There were no significant differences between the two branch sources in in vitro bud break or shoot multiplication from bud explants or cambial-derived callus tissue, respectively. Furthermore, the top branches, generally considered to be the most mature in a tree, were not recalcitrant in terms of morphogenic capacity compared to epicormic branches.

A repeatable and fast transformation-regeneration system has been developed for a woody legume, black locust (Robinia pseudoacacia L.). Hairy roots were produced from hypocotyl segments inoculated with Agrobacterium rhizogenes R1601, a super-virulent strain with eukaryotic kanamycin resistance gene, in less than 1 week and resulted in subsequent shoot regeneration within 4 weeks. Transgenic black locust trees were established in soil 12 weeks after inoculation with the A. rhizogenes strain. The hairy root phenotype found in regenerated plants, which has been characterized as having wrinkled leaves and abundant root development, became evident after 3 months of growth. Southern blot analysis clearly showed multiple integration of T-DNA (transferred DNA) and the presence of neomycin phosphotransferase (NPTII) gene sequences in the transformed black locust genome.

The most commonly planted species in the genus Robinia is R. pseudoacacia L. (black locust, common locust, yellow locust). Black locust is a member of the family Fabaceae, and forms root nodules in association with nitrogen-fixing Rhizobium (Allen and Allen 1981). This allows the species to grow in relatively poor soil. Black locust is a medium-sized tree that has deciduous, compound leaves and shows rapid, indeterminate growth. The twigs usually have stipular spines that flank the leaf scar, beneath which lie naked, often superposed buds (Harlow et al. 1979). The flowers of black locust are insect-pollinated, perfect, papilionate, very fragrant, and are borne in large, white, pendulous racemes (Fig. 1). Most seedlings are produced from outcrossing (Surles et al. 1990). The fruit is a many-seeded legume 5–10 cm in length. Black locust has a diploid chromosome number of 20 (Darlington and Wylie 1955), and cambial cells were reported to contain 2.4 pg of DNA (Singh and Siminovitch 1976).

Chloroplast DNA (cpDNA) was purified from blue spruce (Picea pungens Engelm.) and Engelmann spruce (P. engelmannii Parry ex Engelm.), and was digested with several restriction endonucleases. Restriction fragment length polymorphisms (RFLPs) were identified that differentiated both species. Intraspecific conservation of the RFLPs that distinguished each species was confirmed by examining eight trees from the natural range of blue spruce and six of Engelmann spruce. Four Engelmann spruce x blue spruce F1 hybrids had cpDNA with the banding pattern of blue spruce, indicating paternal transmission of the chloroplast markers. These data were then used to assess the parentage of three naturally occurring putative hybrids. The presence of typical Engelmann spruce banding patterns indicates that Engelmann spruce was the pollen parent, yet interspecific crosses with blue spruce as the female parent are known to be incompatible. Thus, the cpDNA data suggest these trees are not F1 hybrids.

Callus cultures were established from internodal segments of shoot cultures from two mature black locust Robinia pseudoacacia L. trees. Callus of both trees produced shoots on Murashige and Skoog (MS) medium supplemented with 10 microM 6-benzylaminopurine alone or in combination with 1 microM naphthaleneacetic acid. Regenerated shoots were successfully multiplied on MS medium containing 0.32 microM 6-benzylaminopurine, and produced roots on 0.1 strength MS medium containing 1 microM indole-3-butyric acid. One clone consistently outperformed the other with respect to shoot proliferation and proportion of shoots that produced roots. This distinction had previously been observed in shoots produced from bud explants obtained from the mature trees.

In a partial diallel mating design among 20 blue and 20 Engelmann spruce parents, the interspecific crosses were successful only with Engelmann spruce as the female parent. No viable seed were obtained from the reciprocal cross among the 60 full-sib families attempted. Under the conditions of artificial pollination and a controlled germination environment, an average of 0.3% of the seed germinated on a total seed basis across all 20 Engelmann spruce females. Many abnormalities were observed among the germinating hybrid seed, suggesting hybrid inviability also contributes to the low crossability between these two species. Isozyme analysis was used to confirm the interspecific hybrids between blue and Engelmann spruce based on the unique genotypic compositions of the hybrids relative to the two species. No natural F1 hybrids between blue and Engelmann spruce were observed in this study based on isozyme analysis of mature individuals or their seedling progeny. Analyses included samples of open-pollinated seed from blue and Engelmann spruce females located in an area where both species are present in close proximity, often side-by-side, and where pollen shed and female strobilus receptivity in the two species are coincident. In addition, there was evidence of possible gametic selection or hybrid inviability among the full-sib progeny based on deviations of observed from expected segregation ratios for progeny isozyme genotypes. Deviations tended to favor the allele more common to both species rather than the allele unique or more common to only one species. Also, the elevationally allopatric blue and Engelmann spruce subpopulations were less divergent genetically than the sympatric subpopulations.

Five Agrobacteriumtumefaciens strains, and one A. rhizogenes strain, incited tumors on Robiniapseudoacacia L. (black locust) tissues that were inoculated invitro. Four phytohormone-independent tumors, incited by A. tumefaciens strains A6, A348, A274, and A208, were examined using Southern analysis. T-DNA sequences were detected in, and appeared to be integrated into, the DNA from all the tumors. Some tumors tested negative for the presence of octopine or nopaline. All of these tumors had T-DNAs that were truncated on the right ends, i.e., missing the region in which those particular opine synthetic loci are present on the Ti plasmid. Kanamycin-resistant callus was also obtained after inoculation of cotyledons with A. tumefaciens strain A281 carrying the binary vector pGA472, and Southern analysis indicated that nptII sequences were integrated into the R. pseudoacacia genome.

Chloroplast DNA (cpDNA) was purified from blue spruce (Picea pungens Engelm.) and white spruce [P. glauca (Moench) Voss], and was digested with several different restriction endonucleases. Restriction fragment length polymorphisms (RFLPs) were identified that differentiated the cpDNA of both species. Intraspecific conservation of the RFLPs that differentiated each species was confirmed by examining trees from across the natural range of each species. Ten F1 hybrids were examined, and the cpDNA from each showed the banding pattern of the paternal species. Cloned Petunia cpDNA containing part of the rbcL gene hybridized to polymorphic bands, while a cloned maize mtDNA probe of the coxII gene failed to hybridize to any band.

Determinisme genetique des capacites de croisements intraspecifiques chez l'epicea bleu et de Engelmann etudie par croisements dialleliques partiels, realises en champs en 1983

Buds excised from the stems of five dormant, mature (20- to 30-year old) black locust (Robinia pseudoacacia L.) trees were placed on MS basal medium with various levels of 6-benzylaminopurine. In all treatments, bud explants from two of the trees produced shoots which could be subcultured. Whole plants were obtained from cultures of these two trees. Explants from two other trees became vitrified or produced callus, respectively, when cultured on medium containing between 0.032 and 1.0 μM 6-benzylaminopurine; subculturable shoots were only obtained when the buds from these trees were cultured on medium containing 3.2 μM 6-benzylaminopurine. No shoot cultures which could be subcultured were obtained from the fifth tree used in these experiments. The whole plants produced in these experiments were transferred to a greenhouse, and were phenotypically normal five months after culture initiation (three months after transfer to the greenhouse).

Thirteen loci from 11 enzyme systems were identified among full-sib and half-sib progeny of blue and Engelmann spruce. Eleven of the loci were expressed in bud, embryo, and megagametophyte tissue; the remaining two loci were expressed only in embryo and megagametophyte tissue. There were no mobility differences observed between loci expressed in seed and bud tissues. The mode of inheritance for 10 of the loci was confirmed based on progeny genotypic distributions. For the two loci not expressed in bud tissue, acid phosphatase (Acp-2) and diaphorase (Dia-2), inheritance was inferred from pooled segregation ratios of megagametophytes from open-pollinated seed from heterozygous females. The inheritance of glutamate oxaloacetate transaminase (Got-3) was also inferred from segregation ratios and diploid embryo phenotypes of open-pollinated progeny owing to a lack of variability at this locus among the 40 parents in the mating design. Two loci, aldolase (Ald) and malate dehydrogenase (Mdh-2), were monomorphic among the 20 parents of both species. Key words: isozymes, Engelmann spruce, blue spruce, Picea.

The extent and causes of variation of chromosome number in anther cultures of Arabidopsis thaliana (L.) Heynh. were studied. Comparisons of the chromosome number heterogeneity of different tissue types were conducted. Primary callus induced from anthers developed substantial aneuploidy and polyploidy. Shoots regenerated from anther callus, including diploids, dihaploids and parentally derived diploids, displayed no greater variability of chromosome number than did normal plants.

Induction of Arabidopsis thaliana (L.) Heynh. anther cultures on liquid and agar-solidified media was attempted. Anthers floated on stationary liquid medium produced greater numbers of calluses as compared with medium containing agar. Mean chromosome numbers were lower for liquid-grown tissue and maximum shoot regeneration rates were higher.

The effects of activated charocal in the culture medium on regenerant shoots of Arabidopsis thaliana (L.) Heynh. were examined. In these experiments, shoots generated from anther-derived callus were observed to root more frequently on charcoal-containing medium than on medium lacking activated charcoal. Further, concurrent shoot growth and development was greater on charcoal medium than on a rooting medium previously used.