Daniel E. Keathley

Regeneration of a transgenic woody legume (Robinia pseudoacacia L., black locust) and morphological alterations induced by Agrobacterium rhizogenes-mediated transformation

Abstract 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.

Inheritance of plastids in interspecific hybrids of blue spruce and white spruce

SummaryChloroplast 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.

Novel gene expression profiles define the metabolic and physiological processes characteristic of wood and its extractive formation in a hardwood tree species, Robinia pseudoacacia.

Wood is of critical importance to humans as a primary feedstock for biofuel, fiber, solid wood products, and various natural compounds including pharmaceuticals. The trunk wood of most tree species has two distinctly different regions: sapwood and heartwood. In addition to the major constituents, wood contains extraneous chemicals that can be removed by extraction with various solvents. The composition and the content of the extractives vary depending on such factors as, species, growth conditions, and time of year when the tree is cut. Despite the great commercial and keen scientific interest, little is known about the tree-specific biology of the formation of heartwood and its extractives. In order to gain insight on the molecular regulations of heartwood and its extractive formation, we carried out global examination of gene expression profiles across the trunk wood of black locust (Robinia pseudoacacia L.) trees. Of the 2,915 expressed sequenced tags (ESTs) that were generated and analyzed in the current study, 55.3% showed no match to known sequences. Cluster analysis of the ESTs identified a total of 2278 unigene sets, which were used to construct cDNA microarrays. Microarray hybridization analyses were then performed to survey the changes in gene expression profiles of trunk wood. The gene expression profiles of wood formation differ according to the region of trunk wood sampled, with highly expressed genes defining the metabolic and physiological processes characteristic of each region. For example, the gene encoding sugar transport had the highest expression in the sapwood, while the structural genes for flavonoid biosynthesis were up-regulated in the sapwood-heartwood transition zone. This analysis also established the expression patterns of 341 previously unknown genes.

Novel aspects of transcriptional regulation in the winter survival and maintenance mechanism of poplar

Temperate woody plants have developed sophisticated winter survival and maintenance mechanisms that enable them to adapt rapidly to the annual cycle of environmental changes. Here, we demonstrate notable aspects of the transcriptional regulation adopted by poplar in winter/dormancy, employing biochemical and whole transcriptome analysis, and showing high levels of transcriptional activity in a broad spectrum of genes during the dormancy period. A total of 3237 probe sets upregulated more than threefold in winter/dormancy stems over summer/active-growth stems were identified. As expected, genes related to cold hardiness and defense were over-represented. Carbohydrate biosynthesis and transport-related genes were also actively expressed in winter/dormancy stems. Further biochemical analyses verified the dormancy/winter transcription phenotype. More than 60% of the winter upregulated transcription factors (TFs) were related to either biotic or abiotic stress. This finding substantiates that the major transcriptional network of winter/dormancy stems is related to stress tolerance, such as dehydration, cold tolerance and defense. Furthermore, during winter/dormancy, preferential expression of genes involved in cell wall biosynthesis or modification, indirect transcriptional regulation (RNA metabolism) and chromatin modification/remodeling were observed. Taken together, these findings show that regulation of gene expression associated with winter survival and maintenance extends beyond control by promoter-binding TFs to include regulation at the post-transcriptional and chromatin levels.

A molecular framework for seasonal growth-dormancy regulation in perennial plants.

The timing of the onset and release of dormancy impacts the survival, productivity and spatial distribution of temperate horticultural and forestry perennials and is mediated by at least three main regulatory programs involving signal perception and processing by phytochromes (PHYs) and PHY-interacting transcription factors (PIFs). PIF4 functions as a key regulator of plant growth in response to both external and internal signals. In poplar, the expression of PIF4 and PIF3-LIKE1 is upregulated in response to short days, while PHYA and PHYB are not regulated at the transcriptional level. Integration of light and environmental signals is achieved by gating the expression and transcriptional activity of PIF4. During this annual cycle, auxin promotes the degradation of Aux/IAA transcriptional repressors through the SKP–Cullin-F–boxTIR1 complex, relieving the repression of auxin-responsive genes by allowing auxin response factors (ARFs) to activate the transcription of auxin-responsive genes involved in growth responses. Analyses of transcriptome changes during dormancy transitions have identified MADS-box transcription factors associated with endodormancy induction. Previous studies show that poplar dormancy-associated MADS-box (DAM) genes PtMADS7 and PtMADS21 are differentially regulated during the growth-dormancy cycle. Endodormancy may be regulated by internal factors, which are specifically localized in buds. PtMADS7/PtMADS21 may function as an internal regulator in poplar. The control of flowering time shares certain regulatory hierarchies with control of the dormancy/growth cycle. However, the particularities of different stages of the dormancy/growth cycle warrant comprehensive approaches to identify the causative genes for the entire cycle. A growing body of knowledge also indicates epigenetic regulation plays a role in these processes in perennial horticultural and forestry plants. The increased knowledge contributes to better understanding of the dormancy process and consequently to precise manipulation of dormancy-related horticultural traits, such as flowering time.

Differential responses to in vitro bud culture in mature Robinia pseudoacacia L. (black locust)

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).

Detection and Species Identification of Wood-Decaying Fungi by Hybridization of Immobilized Sequence-Specific Oligonucleotide Probes with PCR-Amplified Fungal Ribosomal DNA Internal Transcribed Spacers

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.

Cambial tissue culture and subsequent shoot regeneration from mature black locust (Robinia pseudoacacia L.)

Callus initiation and subsequent shoot regeneration were obtained from cambial tissue of mature black locust trees. Shoot regeneration was highly tree specific, and was only achieved when calli derived from a particular genotype (tree #1) were transferred to medium containing 6-benzylaminopurine. The studies described here show that the immediate use of a mature genotype as starting material for tissue cultural manipulation is possible by using cambial explants with the proper in vitro conditions. We also found that stems could be stored at 4°C for 8 months prior to culture initiation, with no decline in the viability of the cultures. Tissue sources can thus be continuously available.

Genetic Transformation of Black Locust (Robinia pseudoacacia L.)

Black locust (Robinia pseudoacacia L.), a nitrogen-fixing and multipurpose legume tree species, is planted globally. It has a very rapid rate of growth, 2–6 cm/day (Hanover et al. 1992), and often outcompetes weeds and other vegetation. Unlike many other, fast-growing tree species, which usually have lower wood density, black locust produces decay-resistant wood with a very high wood density, 690kg/m3and higher. Recently, these attributes, in concert with black locust’s tolerance of low fertility sites, drought stress, and capacity for nitrogen fixation, have drawn attention to its potential for use in environmental restoration. In addition, black locust’s relatively small genome size (2.4 pg), amenability to tissue culture, and the relative ease with which it can be manipulated through Agrobacterium-mediated transformation facilitate the use of biotechnological techniques to genetically improve this tree legume.