Metla Project 3389
Biotechnology in forest genetics and tree breeding
Keywords: applications in forest tree breeding, biotechnology, cryopreservation, genetic modification, lignin biosynthesis, tissue culture, vegetative propagation
Research project group: Distinct projects 1 - Structure and function of forest ecosystems
Objectives The general aim of the research project is to develop and apply biotechnological methods as a part of forest genetics and tree breeding research. Both perspectives, basic research orientated attempt to understand physiological and genetic phenomena as well as development of practical applications, are considered equally important. Individual research topics fall into different areas of tree biotechnology: vegetative propagation and cryopreservation, genetic modification, and applications of marker techniques. The importance of vegetative propagation and cryopreservation is emphasized, because in these areas practical applications are most urgently needed in tree breeding programmes and operational forestry. Genetic modification is used as a research tool, and experience accumulated for evaluating the benefits and risks of potential future applications of this technique in forestry. Marker techniques are used in integrative way within the other research topics, and especially for studying genetic fidelity of vegetatively propagated and cryostored tree individuals.
Within vegetative propagation and cryopreservation studies, the most important goal is to investigate the effects of the techniques used on the genomes of the target trees – well-conserved genetic fidelity is one prerequisite for any practical applications. Another research topic is physiological ageing of trees that limits potentials of vegetative propagation especially in conifers. At the same time, the propagation and preservation techniques developed are adjusted to better meet the demands of practical breeding.
Lignin biosynthesis, more specifically the function and regulation of some precursor genes, is studied in silver birch using genetic modification. The growth and wood characteristics of the transgenic birch lines are determined, and more over, the ecological interactions between lignin-modified trees and other species, such as insect herbivores and mycorrhizas, are studied in order to get information for general and scientific discussions on gm-trees.
Results Vitrification successfully used for the first time in cryopreservation of silver birch
Cryopreservation is a modern technique used for the conservation of forest genetic resources. No expensive equipment or programming of slow cooling is needed, because vitrification is based on cryoprotective chemical treatment of samples. The method has now been used successfully for the first time with silver birch (Betula pendula Roth). Vitrification, just like traditional cryopreservation, refers to immersion of samples in liquid nitrogen at -196°C. For birch, vitrification is applicable for cryopreservation of tissue cultured material. In order to keep the cells alive during vitrification, it is essential that the formation of ice crystals within the cells is prevented. In traditional cryopreservation methods, cells are dehydrated, which causes extracellular crystallization during the slow cooling process from 4°C to -38°C. When samples – in this case, axillary buds - were processed with vitrification, they survived the transition from 4°C directly to liquid nitrogen. The highly concentrated solutions of cryoprotective agents used in vitrification prevent the crystallization of ice in the extracellular domain and both intra- and extracellular liquids remain vitreous during the freezing and cryostorage. When the results were studied at the molecular level, no signs of genetic effects were observed when DNA-markers of the birch plants regenerated after vitrification were compared to their donor trees. Vitrification is an equally efficient cryopreservation method as the traditional cryopreservation: the average recovery of the studied samples was 71%. As for equipment requirements, vitrification facilitates cryopreservation of silver birch even in smaller tissue culture laboratories.
Cloning by tissue culture does not affect genome of silver birch
Clonal trueness of micropropagated or cryopreserved material is essential for any application, especially with long-living tree species. In this project, the growth rate and morphology of regenerated silver birch (Betula pendula Roth) plants growing in the nursery were evaluated after different treatments: short-term and long-term tissue culture periods, cryostorage of in vivo buds and cryopreservation of in vitro shoot apices using four different slow-cooling cryopreservation protocols with PGD as cryoprotectant. Genetic fidelity of the regenerated plants compared to the original donor trees was evaluated using RAPD assays together with chromosome analysis. The regenerated plants showed no genetic or phenotypic changes, and can thus be considered as reliable material for any research, breeding or silvicultural activities.
Cloning of leaf-variegated birches for landscaping
Two naturally-occurring forms of birch (Betula pendula Roth), “golden-veined” birch (GV) and “white-flecked” birch (WF) were studied for their suitability to vegetative propagation and cryopreservation. The forms are characterized by specific leaf colourations, and could provide good material for ornamental and landscaping purposes in severe northern climate. Cloning of the WF birch was proven to be easy by micropropagation even after cryopreservation. After two years from grafting, more than 90 percent of the grafts were alive. Correspondingly, the micropropagation success percentage of GV birch was less than ten and the survival percentage of the grafts was approximately 50. The leaf colouration, which had been assumed to be inheritable, was lacking in the micropropagated progenies. Only the leaves of the oldest branches of the WF birch grafts showed white-fleckedness. The reason was found in the donor trees. It is typical for a WF donor birch to have strong year-to-year variation in foliar colouration, and the fleckedness is not expressed in the newest leaves of the current year’s growth. The amount of white-fleckedness in the donor tree was shown to be associated with growth conditions, dry conditions increasing the fleckedness. White-fleckedness in the older tree parts is explained by chlorophyll deficiency. The results indicate that when the cloned WF birches, either grafts or micropropagated plants age, they express more flecked leaf colouration. They are well suitable for landscaping. The GV donor birch did not show corresponding year-to-year variation in leaf colouration. The reason for the gold-coloured veins is so far unknown. To facilitate the use of the GV birch for decorative purposes, further studies will be needed to find the factor controlling leaf colouration and establish its usefulness.
Lignin modified silver birches and their ecological interactions
Transgenic silver birch lines were produced in order to modify lignin biosynthesis. The 35S- and UbB1-PtCOMT genes were transferred by the biolistic bombardment into silver birch, Betula pendula Roth, and the effects on syringyl (S) lignin unit synthesis were investigated. The transgenes were stably integrated into the B. pendula genomes, and their variable expression was observed. In 35S-PtCOMT lines, a reduced syringyl/guaiacyl (S/G) ratio and incorporation of abnormal 5-OH-G units into lignin were found apparently due to the RNA interference (RNAi)-based suppression of the COMT gene leading to reduced S lignin content in stems, leaves and roots. This work supports the essential role of COMT for S unit synthesis and the current view on the lignin biosynthesis in woody angiosperms. The unchanged morphology and growth characteristics of the 35S-PtCOMT modified B. pendula lines also indicate that plants are able tolerate large variation in the lignin S/G ratio. PtCOMT-promoter-GUS (β-glucuronidase) modified B. pendula lines were produced in order to examine the expression pattern of the PtCOMT gene. The main activity during the growing season was present in the new xylem and lignified phloem fibers. Our results also suggest the role of COMT in tension wood formation but not in response to wounding.
The transgenic birch lines together with the control were used for studying potential effects of lignin-modification on the interactions with insect herbivores. In controlled feeding experiments herbivores common in the boreal environment, i.e. geometrid larvae and the adults of birch leaf-feeding beetles were used. The feeding preferences of these herbivores differed in some cases among the tested birch lines. These differences could not be directly associated to lignin composition, but they might be explained by other than lignin related leaf characteristics, either natural or caused by transgene site effects. Lignin modification did not affect significantly growth performance of lepidopteran larvae. We also investigated the potential effect of the PtCOMT modification on the ectomycorrhiza (ECM) development between Betula pendula and Paxillus involutus in vitro. According to our results, both the non-transgenic controls and PtCOMT modified silver birch lines were able to form ECM with Paxillus involutus. In the development of ECM we observed line-dependent differences, which may possibly be related to the line-specific growth characteristics or decreased S/G ratio of root lignin of individual transgenic line. The beneficial effect of the inoculation on the plant survival, shoot and root growth and the lateral root formation of B. pendula plants were demonstrated. This in vitro study is the first report on the ECM symbiosis between lignin modified trees and mycorrhizal fungus.
Lignin studies are done in collaboration with eg prof. Häggman (Univ. of Oulu), Doc. Niemi (Univ. of Helsinki), prof. Chiang (NCSU, USA) and prof. Tsai (MTU, USA). All the experimental work with genetically modified birches is done under in vitro or controlled greenhouse conditions.
The Finnish Forest Research Institute,
Finlandiantie 18, FI-58450 PUNKAHARJU, FINLAND
Phone: +358 29 532 4233
Pehkonen, Teresa (2005,2008), Ryynänen, Leena (2005-08)
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