Accelerated Stem Growth Rates and Improved Fiber Properties of Loblolly Pine: Functional Analysis Of CyclinD from Pinus taeda
- (Scion Research.)
A sustained supply of low-cost, high quality raw materials is essential for the future success of the U.S. forest products industry. To maximize stem (trunk) growth, a fundamental understanding of the molecular mechanisms that regulate cell divisions within the cambial meristem is essential. We hypothesize that auxin levels within the cambial meristem regulate cyclin gene expression and this in turn controls cell cycle progression as occurs in all eukaryotic cells. Work with model plant species has shown that ectopic overexpression of cyclins promotes cell division thereby increasing root growth > five times. We intended to test whether ectopic overexpression of cambial cyclins in the cambial zone of loblolly pine also promotes cell division rates that enhance stem growth rates. Results generated in model annual angiosperm systems cannot be reliably extrapolated to perennial gymnosperms, thus while the generation and development of transgenic pine is time consuming, this is the necessary approach for meaningful data. We succeeded in isolating a cyclin D gene and Clustal analysis to the Arabidopsis cyclin D gene family indicates that it is more closely related to cyclin D2 than D1 or D3 Using this gene as a probe we observed a small stimulation of cyclin D expression in somatic embryo culture upon addition of auxin. We hypothesized that trees with more cells in the vascular cambial and expansion zones will have higher cyclin mRNA levels. We demonstrated that in trees under compressive stress where the rates of cambial divisions are increased on the underside of the stem relative to the top or opposite side, there was a 20 fold increase in the level of PtcyclinD1 mRNA on the compressed side of the stem relative to the opposite. This suggests that higher secondary growth rates correlate with PtcyclinD1 expression. We showed that larger diameter trees show more growth during each year and that the increased growth in loblolly pine trees correlates with more cell divisions in the cambial meristem as expected. We isolated a promoter from a cambial specific gene and commenced development of transformation protocols for loblolly pine. Since our results show that cyclin D expression correlates with increased growth we continued with experiments to demonstrate the effect of cyclin overexpression upon tree growth. Vectors which constitutively express the cyclin D cDNA were constructed and transformed into a transgenic pine system through the collaboration with Forest Research, New Zealand. The transformation system for Pinus radiata is well established and we hoped to gain phenotypic information in a closely related pine, rather than await development of a robust loblolly pine transformation method. Transformation experiments were conducted by a biolistic method developed at Forest Research, NZ. A total of 78 transgenic embryogenic lines were generated and bulked up with a good representation of transgenic lines per construct. Transformed calli were originally identified by resistance to the antibiotic Geneticin contained in the medium. The transgenic nature of the selected lines was subsequently confirmed using histochemical GUS staining. To date, 10 out of 13 selected transgenic lines have produced embryos and we are currently harvesting the first transgenic plantlets. At present time 22 of those plantlets have been moved to GMO facilities. We will soon develop a strategy for assessing potential phenotypic differences between the transclones and non-transformed controls. Transgenic plants are being grown to a stage (approx. 1 year) when meaningful phenotypic evaluation can be conducted. The recent availability of 10,000 element loblolly pine cDNA microarray will permit the evaluation of cyclinD overexpression upon gene expression in transgenic Pinus.
- Research Organization:
- Georgia Institute of Technology, Atlanta, GA (United States)
- Sponsoring Organization:
- USDOE Office of Science (SC)
- DOE Contract Number:
- FC36-00ID13876
- OSTI ID:
- 860826
- Country of Publication:
- United States
- Language:
- English
Similar Records
GENETIC MODIFICATION OF GIBBERELLIC ACID SIGNALING TO PROMOTE CARBON SEQUESTRATION IN TREE ROOTS AND STEMS
Exploiting Genetic Variation of Fiber Components and Morphology in Juvenile Loblolly Pine