Volume 5 Supplement 7
Transgenic aspen and birch trees for Russian plantation forests
© Shestibratov et al; licensee BioMed Central Ltd. 2011
Published: 13 September 2011
Aspen(Populus tremula) and birch (Betula pubescens) are the fast-growing trees successfully used for the aim of plantation forestry. These species have the great potential in Russia to meeting the need for paper, timber and other wood-based products. However, enhanced growth rate, decreased lignin content and herbicide resistance are the required properties of new trees for plantation forestry. Breeding of forest trees is a slow process due to the long generation intervals typical of most forest trees and because many traits can only be properly assessed at rotation age. Genetic modification is an alternative method that can be used for new trees creation.
Genetic transformation experiments were carried out using vectors pBI-4CL, pBI-Xeg, pGS and pBIBar and supervirulent Agrobacterium tumefaciens strain CBE21. pBI-4CL plasmid contains the expression cassette harboring inverted fragments of 4-coumarat-CoA-ligase gene (GeneBank AY043494). pBI-Xeg vector was constructed using cDNA of xyloglucanase gene cloned from Penicillium canescens. pGS and pBIBar vectors contains pine glutamine synthetase gene (GS) and phosphinothricin acetyltransferase gene (bar). For transcription control of expression 35S promoter used in all cassettes harboring the gene of interest.
Internodes from in vitro aspen plants (Populus tremula) and leaves from in vitro birch plants (Betula pubescens) cultivating on WPM medium were used as an explants for Agrobacterium-mediated transformation [1, 2]. Transformed shoots selected on the modified MS medium with 25 (aspen) or 50 (birch) mg/l kanamycin.
To detect DNA fragments from binary vectors transformants were analysed by PCR. Expression of recombinant genes was detected by RT-PCR. Lignin content determination performed by using the Klason method.
Results and conclusions
Most of GS lines have resistance to subletal dose of phosphinotricin as well as in the in vitro and greenhouse conditions. Comparative biometric analysis of GS plants in a greenhouse makes it possible to select 7 and 3 superior lines of birch and aspen respectively. Growth rate enhancement of selected lines was 20-25 %.
Lignin content of extract-free wood from control and transgenic aspen plants.
Acid soluble lignin, %
Acid insoluble lignin, %
Total lignin, %
Lignin reduction, %
Aspen and birch transgenic plants with bar gene coding resistance against herbicide phosphinotricin were also created. Transformation experiments were carried out using vector pBIBar. 19 aspen and 9 birch lines were selected as a result of transformation. Presence of bar gene was confirmed in all lines. Herbicide resistance was observed at 16 and 7 lines of aspen and birch respectively. Lines tested in a greenhouse demonstrated normal phenotype without somaclonal variations and may be recommended for the field trials.
To combine several new properties in one plant the co-transformation method was developed. This method allows transferring recombinant DNA fragments from three T-DNAs of two plasmids independently . Efficiency of co-transformation was up to 100 %. Currently we attempt to apply this method for the production of superior aspen and birch plants with enhanced growth rate, decreased lignin content and herbicide resistance at the same time.
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- Shadrina TE, Schestibratov KA: Efficient shoot regeneration from leaf explants of Betula pendula, B. pubescens and B. nigra. Acta Hort (ISHS). 2010, 885: 349-353.View ArticleGoogle Scholar
- Lebedev VG, Schestibratov KA, Shadrina TE, Bulatova IV, Abramochkin DG, Miroshnikov AI: Cotransformation of aspen and birch with three T-DNA regions from two different replicons in one Agrobacterium tumefaciens strain. Russ. J. Genet. 2010, 46 (11): 1282-1289. 10.1134/S1022795410110025.View ArticleGoogle Scholar
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