Charles H. Michler

Pathogen challenge, salicylic acid, and jasmonic acid regulate expression of chitinase gene homologs in pine.

To better understand the molecular regulation of defense responses in members of the genus Pinus, we tested the expression of various chitinase homologs in response to pathogen-associated signals. PSCHI4, a putative extracellular class II chitinase, was secreted into liquid medium by pine cells and was also secreted by transgenic tobacco cells that ectopically expressed pschi4. Extracellular proteins of pine were separated by isoelectric focusing; PSCHI4 was not associated with fractions containing detectable beta-N-acetylglucosaminidase or lysozyme activities. However, other fractions contained enzyme activities that increased markedly after elicitor treatment. The pschi4 transcript and protein accumulated in pine seedlings challenged with the necrotrophic pathogen Fusarium subglutinans f. sp. pini, with the protein reaching detectable levels in susceptible seedlings concomitant with the onset of visible disease symptoms. Additional chitinase transcripts, assigned to classes I and IV based on primary sequence analysis, were also induced by pathogen challenge. Jasmonic acid induced class I and class IV but not class II chitinase, whereas salicylic acid induced all three classes of chitinase. These results show that multiple chitinase homologs are induced after challenge by a necrotrophic pathogen and by potential signaling molecules identified in angiosperms. This suggests the potential importance of de novo pathogenesis-related (PR) gene expression in pathogen defense responses of pine trees.

Epicormic buds in trees: a review of bud establishment, development and dormancy release

The formation of epicormic sprouts on the boles of trees is a phenomenon that has, until recently, been poorly understood. Renewed interest in the topic in the last two decades has led to significant advances in our knowledge of the subject, especially in regard to bud anatomy, morphology and ontogeny. There exists, however, no comprehensive synthesis of results from different disciplines across genera and geographical areas; this review seeks to fill that void and provide a comprehensive framework capable of guiding future research. A trees potential for producing epicormic branches is dependent on the number of buds that are produced on a growing shoot, the development of those buds and associated meristems over time and the factors that promote sprout formation or bud death. Based on the descriptions of a limited number of researched species, we were able to describe four different developmental strategies for epicormics based on characteristics of meristem development. Control over epicormic bud dormancy is complex, but it is clear that the traditional view of auxin-mediated dormancy release is incomplete. Genetic control over epicormic development is yet to be empirically proven. Future research should focus on clarifying these physiological and genetic controls of epicormic bud development as well as developing more robust methods for tracking epicormics in ecological and silvicultural studies.

High frequency somatic embryogenesis from leaf tissue of Populus spp.

Abstract A high frequency somatic embryogenesis protocol was developed for biotechnological manipulations of hybrid poplar in which embryogenic callus was derived from non-embryonic explants. Somatic embryos of Populus alba × P. grandidentata (NC-5339) were differentiated directly on leaf explants and indirectly from embryogenic cell suspension callus initiated from leaf tissue. Leaf explants were induced to form embryogenic callus in darkness on Murashige and Skoog (MS) medium with 5 mg/1 2,4-dichlorophenoxyacetic acid (2,4-D), 0.05 mg/l benzyladenine (BA), and 30 gg/l sucrose. The auxin to cytokinin ratio (5 mg/l 2,4-D to 0.05 mg/l zeatin) was optimized for globular-shaped embryo initiation from cell suspension callus. Heart-shaped embryo development occurred after removal of 2,4-D from the culture medium, and development progressed to embryo maturity on the same medium. Late stage torpedo-shaped embryos germinated following a 1-week subculture on MS medium containing 5 mg/l IAA and 0.05 mg/l BA, and germinated embryos were successfully transferred ex vitro to greenhouse growth conditions.

Influence of regeneration method and tissue source on the frequency of somatic variation in Populus to infection by Septoria musiva

Septoria leaf spot and canker are serious diseases of many hybrid poplar clones in plantations established for biomass production. Developing resistant clones through breeding is the best long-term strategy to minimize tree damage caused by this disease. Tissue culture and somaclonal selection techniques may reduce the time needed to develop disease resistance in poplars. We used a single source clone of a hybrid Populus to determine the influences of explant tissue source and regeneration method on the frequency of somatic variation in disease resistance. Plants were regenerated adventitiously via shoots and somatic embryos from callus derived from several tissue sources and from axillary buds. The resulting plants expressed somatic variation in disease resistance in different frequencies, except for the plants regenerated from hardwood cuttings. Several regenerated plants from various tissue sources exhibited variant morphological phenotypes, providing further evidence of the instability of this clone when cultured in vitro. Although mutagenic effects of the culture regimes alone cannot be ruled out, results of this study suggest that the explant source and culture method influenced how frequently variant plants were recovered.

Biotechnological efforts for preserving and enhancing temperate hardwood tree biodiversity, health, and productivity

Hardwood tree species in forest, plantation, and urban environments (temperate regions of the world) are important biological resources that play a significant role in the economy and the ecology of terrestrial ecosystems, and they have aesthetic and spiritual value. Because of these many values of hardwood tree species, preserving forest tree biodiversity through the use of biotechnological approaches should be an integral component in any forestry program in addition to large-scale ecologically sustainable forest management and preservation of the urban forest environment. Biotechnological tools are available for conserving tree species as well as genetic characterization that will be needed for deployment of germplasm through restoration activities. This review concentrates on the biotechnological tools available for conserving, characterizing, evaluating, and enhancing hardwood forest tree biodiversity. We focus mainly on species grown for lumber and wood products, not species grown mainly for fiber (pulp and paper production). We also present a brief summary of the importance of non-wood forest products from temperate hardwood tree species (a research area that needs further development using biotechnological techniques) and a few case studies for preserving forest tree biodiversity.

Technological advances in temperate hardwood tree improvement including breeding and molecular marker applications

Hardwood forests and plantations are an important economic resource for the forest products industry worldwide and to the international trade of lumber and logs. Hardwood trees are also planted for ecological reasons, for example, wildlife habitat, native woodland restoration, and riparian buffers. The demand for quality hardwood from tree plantations will continue to rise as the worldwide consumption of forest products increases. Tree improvement of temperate hardwoods has lagged behind that of coniferous species and hardwoods of the genera Populus and Eucalyptus. The development of marker systems has become an almost necessary complement to the classical breeding and improvement of hardwood tree populations for superior growth, form, and timber characteristics. Molecular markers are especially valuable for determining the reproductive biology and population structure of natural forests and plantations, and the identity of genes affecting quantitative traits. Clonal reproduction of commercially important hardwood tree species provides improved planting stock for use in progeny testing and production forestry. Development of in vitro and conventional vegetative propagation methods allows mass production of clones of mature, elite genotypes or genetically improved genotypes. Genetic modification of hardwood tree species could potentially produce trees with herbicide tolerance, disease and pest resistance, improved wood quality, and reproductive manipulations for commercial plantations. This review concentrates on recent advances in conventional breeding and selection, molecular marker application, in vitro culture, and genetic transformation, and discusses the future challenges and opportunities for valuable temperate (or “fine”) hardwood tree improvement.

Chloroplast DNA variation of northern red oak (Quercus rubra L.) in Indiana

Chloroplast DNA (cpDNA) variation was examined in 48 northern red oaks at 14 sites representing contrasting glacial histories and age structures within the state of Indiana in the United States. PCR-RFLP of three intergenic regions revealed five haplotypes. Haplotype I was common to seven sites and was the most frequent (17 trees). Haplotype II was common to five sites and was nearly as frequent as haplotype I (16 trees). Haplotypes III, IV and V were equally infrequent and did not occur together. Genetic diversity resided among rather than within populations (GST = 0.73 ± 0.14). This preliminary survey shows that cpDNA diversity will be a useful tool for the investigation of ancient seed dispersal patterns in northern red oak.

Using molecular methods to improve the genetic management of captive breeding programs for threatened species

Captive breeding programs are powerful tools for the conservation of our natural resources. Both animal and plant biodiversity are in global decline (International Union for Conservation of Nature [IUCN] 2008), and the trend is for levels and rates of endangerment to continue increasing (Butchart et al. 2005; Chapter 1 by Honeycutt and colleagues). When in-situ conservation efforts are insufficient at stopping or reversing the decline of a species, captive populations often represent the only alternative for forestalling extinction. For example, it is currently estimated that hundreds of amphibian species will soon be extinct if emergency measures are not taken to protect populations in captivity until the threats to wild populations can be halted or overcome (Gascon et al. 2007). The success of numerous captive breeding programs has been well documented. As just a few of many examples, captive breeding programs have saved the black-footed ferret (Mustela nigripes), Przewalski’s horse (Equus caballus przewalskii), and the California condor (Gymnogyps californianus) from final extinction (after the last wild populations were extirpated) and new wild populations of the golden lion tamarin (Leontopithecus rosalia), Arabian oryx (Oryx leucoryx), and whooping crane (Grus americana) have been successfully re-established from captive stocks. Conservation breeding programs aim to maintain populations that are representative of their wild counterparts, to provide a reservoir for future reintroductions and recovery efforts (see Chapter 12 by Rhodes and Latch). Thus, the genetic goals of captive population management are to minimize genetic drift, retain genetic diversity, restrict inbreeding, and limit adaptation to captivity (Lacy 1994). The foundations of most captive breeding programs are pedigree analyses, which are used to manage both the demography and genetics of captive populations (Ballou & Foose 1996). Accurate pedigrees provide information on inbreeding, the kinships among individuals, and the distributions of individual founder contributions to a population. Collectively, this information is used to produce regular breeding recommendations intended to meet demographic and genetic goals. Although pedigree analyses are quite effective, they do have limitations. The most basic limitation of pedigree-based management is that it requires complete and accurate pedigrees to be effective. Thus, the genetic and demographic management of captive populations are hampered when missing or inaccurate parentage records produce incomplete pedigrees. When a captive pedigree is inaccurate or incomplete, molecular data have the potential to improve breeding program management. Molecular markers can

Proceedings. 14th Central Hardwood Forest Conference

Proceedings of the 14th Central Hardwood Forest conference held 16-19 March in Wooster Ohio. Includes 102 papers and abstracts dealing with silviculture, wildlife, human dimensions, harvesting and utilization, physiology, genetics, soils, nutrient cycling, and biometrics.

The pine Pschi4 promoter directs wound-induced transcription

Abstract Mechanical wounding stimulates the accumulation of Pschi4 transcripts (encoding a putative extracellular chitinase) in pine trees. To gain insight into the transcriptional regulatory region(s) in this gymnosperm defense gene, the 5′-flanking region of Pschi4 was fused to the uid A reporter gene encoding β-glucuronidase (GUS), and the construct was functionally tested for transient expression in onion and pine cells. As little as 200 bp of the upstream sequence was sufficient to promote activated transcription and nearly maximum levels of transient expression in onion cells. Similar results were obtained in bombarded pine cells, although maximum expression appeared to require the entire 4.5 kb of upstream sequence. Having defined potential regulatory regions in the 5′ flanking region of Pschi4 in transient assays, the pine promoter- uid A fusions were functionally tested in stably transformed tobacco plants. As little as 200 bp of the upstream sequence was sufficient to direct wound-inducible transcription. Furthermore, particle bombardment was sufficient to stimulate transgene expression in the transgenic tobacco, indicating that bombardment per se leads to wound responses in vivo. Taken as a whole, our data demonstrate that a gymnosperm promoter can recruit a wound-regulated signal transduction pathway in angiosperms. This suggests that the downstream components of wound signaling pathways are functionally conserved across broad taxonomic groups.