Progress 03/01/04 to 09/30/06
Outputs
THis project was to study the possibility of artificially inducing tracheray element (TE) formation from undifferentiated cells of Douglas fir, a tissue culture system has been developed. Strips of xylem along with cambium obtained from young Douglas fir trees (4-10 year) were inoculated on solidified Murashige and Skoog (MS) medium supplemented with 2, 4-dichlorophenoxy acetic acid (2,4-D) and 6-benzylaminopurine (BA) and incubated in at 24C. This treatment gave 100 % callusing on all the explants inoculated. The callus obtained was subcultured in MS suspension culture media. Trials were carried out to differentiate the calli to TE's by modifying the media components (2,4-D and activated charcoal) and environmental conditions and reached yields of approximately 65% TEs. The presence of lignified cell walls was easily detected by polarized light, as secondary thickened cell walls and phloroglucinol-HCl staining specific for lignin. The presence of lignin was further
confirmed by pyrolysis GC-MS analysis, Infra-red spectroscopy and the amount of lignin was quantified by the thioglycolic acid assay. Carbohydrate analysis of the TE revealed the presence of cellulose and hemicelluloses and this finding is consistent with the composition of Douglas fir wood. The outcomes of this project have been the development of a model in-vitro system to induce cell differentiation which can be applied to examine wood formation from fundamental chemical/biochemical perspectives.
Impacts
Wood is one of the most important sustainable raw materials. The supply of wood from forest plantations can substitute for wood from old growth natural forest resources. Wood quality related traits (end use attributes) that improve wood value are of immense importance to the forest products industry. Therefore by understanding how wood is formed in the tree gives us a mechanism on how to grow trees for added value based on its properties. The developed model in-vitro system is an invaluable tool to rapidly assess targeted genes that regulate wood properties in Douglas fir for specific end uses.
Publications
- No publications reported this period
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Progress 10/01/04 to 09/30/05
Outputs
To study the induction of tracheray element(TE)formation from undifferentiated cells Douglas fir, a tissue culture system is being investigated. Strips of xylem (cambium) obtained from young trees (4-10 y) were inoculated on solidified Murashige and Skoog(MS)medium supplemented with 2mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 2mg/l 6-benzylaminopurine(BA) and incubated in the dark. This treatment gave 100% callusing on all the explants inoculated. The callus obtained were sustained by subsequent subcultures. Trials were carried out to differentiate the calli obtained to TEs by modifying the media components and various growth hormones levels. These attempts have so far not met with any success. Incubation in light caused the greening of callus, whereas culturing in alternate light and dark periods did not cause any effect. When callus was inoculated in MS media supplemented with 2g/l activated carbon (AC) the presence of lignin was detected by phloroglucinol-HCl
staining. The presence of lignified cell wall is easily detected by plain polarized light, as secondary thickened cell walls are strongly birefringent. However the presence of lignin in the callus was confirmed when the cell wall preparations were analyzed using Fourier transform infrared spectroscopy (FTIR) and pyrolysis gas chromatography-mass spectrometry (Pyro-GC/MS). FTIR spectra showed strong absorption peak at 1626 cm-1 characteristic of lignin fractions. Pyro-GC/MS suported the presence of lignin. Lignin quantification was done by lignoithioglycolysis acid assay at a 3.74% content in cell wall preparation obtained from callus cultured in MS supplemented with 2 g/l AC. This trend leads to the conclusion that although lignin is being synthesized by the cells its not undergoing the process of secondary cell wall formation and programmed cell death. However recently when the media was supplemented with acid washed AC, lignin formation with stray occurrence of a few secondary
thickened cells were seen as observed by the strong birefringence with plain polarized light and auto fluorescence caused by lignin. More repetitions and sampling will be done to establish if this finding is merely a chance occurrence or a definite result of this particular treatment. A separate experiment was carried out to understand how the production of callus is affected by age, position of the explant and type of plant growth hormone used on a 30 year old tree. The results showed that, maximum number explants produced callus when the explants were taken from the base of the tree (within 0.5 m from the base) and inoculated on MS supplemented with 2 mg/l 2,4-D and 2 mg/l BA (77.3% callusing). The worst performance was noted when the explants collected from the extreme top of the tree were inoculated on MS supplemented with 2mg/l indole-3-acetic acid (IAA) and 2mg/l BA (14.7% callusing). Thus it can be concluded that younger trees (4-10 year old) are better than mature trees for
inducing callusing and tissue used from upper portion of a mature tree is less responsive in tissue culture than the one used from the base.
Impacts
Wood is still the worlds most important raw materials and world-wide demand will continue to increase. The supply of wood from forest plantations can substitute for wood from old growth natural forest and reduce the pressure on primary forests. But in order to achieve performance related traits that improve wood quality and wood value is of immense importance for the forest products industries of the future. The outcome of is to understand the relationships between tree physiology, wood science and tree breeding for desirable wood quality traits.
Publications
- No publications reported this period
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Progress 10/01/03 to 09/30/04
Outputs
The primary challenge in this study was to induce callus from xylem strips and hypocotyl explants of Douglas fir and further, to trans-differentiate the callus cells into xylem tracheids. The first stage of this project was to develop protocols to grow callus tissue from various sources of tissue (cambium and hypocotyls (from germinated seeds)) from Douglas fir. The base culture media used was Murashige and Skoog. Variations on the base media included the addition of phytohormones (6-Benzylaminopurine and 2,4-D 2,4-Dichlorophenoxyacetic acid). Callus was clearly formed after 3 weeks incubation as observed by light microscopy. Current work is focused on inducing differentiation of the callus to form secondary cell walls.
Impacts
Wood is one of the most important raw materials. The supply of wood from forest plantations can substitute for wood from old growth natural forest resources. Wood quality related traits that improve wood quality and wood value would be of immense importance for the forest products industries. Therefore by understanding how wood is formed in the tree gives us a mechanism on how to grow trees for added value based on its properties.
Publications
- No publications reported this period
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