Department of Forest Sciences, Department of Agricultural Sciences Faculty of Agriculture and Forestry and Viikki Graduate School in Molecular Biosciences University of Helsinki The effects of abiotic and biotic factors on somatic embryogenesis and seedlings of Pinus sylvestris (L.) Jinrong Lu ACADEMIC DISSERTATION To be presented, with the permission of the Faculty of Agriculture and Forestry of the University of Helsinki, for public examination in lecture room B6, B-building (Latokartanonkaari 7), on April1 5 th , 2011, at 12 noon. Helsinki 2011 2 Supervisors: Professor Fred O. Asiegbu Department of Forest Sciences University of Helsinki, Helsinki Professor Ari Pappinen Faculty of Science and Forestry University of Eastern Finland, Joensuu Pre-examiners: Docent Karoliina Niemi The Central Union of Agricultural Producers and Forest Owners (MTK) Helsinki, Finland Docent Malin Elfstrand Department of Forest Mycology & Pathology Swedish University of Agricultural Sciences Opponent: Adjunct professor Yill Sung Park Candian Wood Fibre Centre PO Box 4000, Fredericton, New Brunswick E3B 5P7 CANADA Cover: Somatic embryos of Socts pine at the prematuring, maturing and germinating stage. ISSN 1795-7079 ISBN 978-952-10-4291-1 (paperback) ISBN 978-952-10-4292-8 (pdf) http://ethesis.helsinki.fi Helsinki University Printing House Helsinki 2011 3 4 TABLE OF CONTENTS ABBREVIATIONS USED ..................................................................................................................... 5 LIST OF PUBLICATIONS ..................................................................................................................... 7 ABSTRACT ............................................................................................................................................ 8 1. INTRODUCTION ............................................................................................................................... 9 1.1. Somatic embryogenesis-a potential in vitro propagation method for conifers ............... 9 1.1.1. Development of somatic embryogenesis in conifers ........................................................... 9 1.1.2. Four main phases of the somatic embryogenesis and conditions ...................................... 10 1.1.3. Biochemical and molecular aspects of conifer somatic embryogenesis ............................ 13 1.1.4. Somatic embryogenesis in pine ......................................................................................... 18 1.2. Ethylene in somatic embryogenesis ............................................................................. 21 1.2.1. Ethylene biosynthesis ........................................................................................................ 21 1.2.2. ACC Synthaseg650the key enzyme in ethylene biosynthesis pathway ................................. 22 1.2.3. Regulation of ethylene biosynthesis .................................................................................. 23 1.2.4. Ethylene in somatic embryogenesis ................................................................................... 25 1.3. Interactions between rhizosphere microbes and development of P. sylvestris roots ... 27 1.3.1. Ectomycorrhizal fungi and its interaction with host trees .................................................. 27 1.3.2. Pathogenic fungus - host interaction .................................................................................. 28 1.3.3. Fungal elicitors, effectors and host-microbes recognition ................................................. 29 3. MATERIALS AND METHODS ...................................................................................................... 33 3.1. Plant material and microbes used ................................................................................. 33 3.2. Initiation of embryogenic tissue cultures (I) ................................................................ 33 3.3. Maintenance and production of mature somatic embryos ............................................ 33 3.4. Induction treatment of somatic tissues (II) ................................................................... 34 3.5. Determination of ethylene production (II) ................................................................... 34 3.6. Elicitor preparations and sample treatment (III) .......................................................... 34 3.7. RNA isolation, reverse transcription PCR and quantitative PCR ................................ 35 3.8. Sequences and phylogenetic analyses of PsACS1 and PsACS2 ................................... 35 3.9. Induction of P. sylvestris seedlings by different microbes (IV) ................................... 35 3.10. Statistical analyses ........................................................................................................ 35 4. RESULTS AND DISCUSSION ........................................................................................................ 37 4.1. Parental effects on the somatic embryogenesis of P. sylvestris (Paper I) ................... 37 4.2. ACC synthesis genes in somatic embryogenesis of P. sylvestris (Paper II) ................ 38 4.3. Response of somatic embryos to fungal cell wall elicitors (paper III) ......................... 39 4.4. Induced defence responses in P. sylvestris seedlings (paper IV) ................................. 41 6. ACKNOWLEDGEMENTS .............................................................................................................. 44 7. REFERENCES .................................................................................................................................. 46 5 ABBREVIATIONS USED 2,4-D 2,4-dichlorophenoxyacetic acid ABA abscisic acid ACC 1-Aminocyclopropane-1-carboxylic acid ACS ACC synthase AdoMet S-adenosylmethionine AFAP actin filament-associated protein AOA amino-oxyacetic acid AspRS aspartyl-tRNA synthetase BA 6-benzylaminopurine BLAST basic logical alignment search tool cDNA complementary deoxyribonucleic acid DCR Douglas-fir cotyledon revised medium ECM ectomycorrhiza / ectomycorrhizal ECs embryogenic cultures EF-1A elongation factor 1 alpha EF-Tu elongation factor thermo unstable HBK3 homeobox of knox3 JA jasmonic acid LEA late embryogenesis abundant protein LP von Arnold and Eriksson medium MAMP microbe-associated molecular patterns MS Murashige and Skoog medium NAA g302-naphthaleneacetic acid NCBI national center for biotechnology P6 Pinus taeda basal medium PAMP pathogen-associated molecular patterns PAs polyamines PDT partial drying treatment PEG polyethylene glycol (4000) PGR plant growth regulator PRRs pattern-recognition receptors 6 RNA ribonucleic acid ROS reactive oxygen species RT-PCR reverse transcription polymerase chain reaction SBH1 Suillus bovinus isolate1 in Helsinki SE somatic embryogenesis tRNA transfer RNA 7 LIST OF PUBLICATIONS The thesis is based on the following publications, which will be referred to in the text with their Roman numerals (I-IV). I. Niskanen AM, Lu J, Seitz S, Keinonen K, Pappinen A, von Weissenberg K (2004). Effect of parent genotype on somatic embryogenesis in Scots pine (Pinus sylvestris L.). Tree Physiology 24:1259–1265. II. Lu J, Vahala J, Pappinen A (2011). Involvement of ethylene in somatic embryogenesis in Scots pine (Pinus sylvestris L.) (Plant Cell, Tissue and Organ Culture; Manuscript, Accepted). III. Lu J, Aronen T, Pappinen A, Asiegbu FO (2010). Response of somatic embryos of Scots pine to fungal cell wall elicitors. Forest Pathology. doi: 10.1111/j.1439- 0329.2010.00641.x IV. Lu J, Pappinen A, Timonen S (2011). Scots pine seedling-microbe interactions in the absence of physical contact (Manuscript, Submitted). 8 ABSTRACT Somatic embryogenesis (SE) is an asexual form of plant propagation that occurs in nature and mimics many of the events of sexual reproduction. Pinus sylvestris (L.) is an important source of timber in Northern Eurasia but it is recalcitrant to somatic embryogenesis. Several factors important for the success of the P. sylvestris embryogenic cultures have not been thoroughly investigated. In this study, we examined the effects of parental genotypes on the SE in P. sylvestris, the involvement of the gaseous plant growth regulator, ethylene in SE, and also biotic effects on somatic embryos as well as on seedlings. We tested parental effects on immature embryo initiation for different media, storage periods, and on the maturation process. Maternal effects were found to be crucial for SE in the absence of paternal effects. No maternal-paternal interaction was observed at any stage of somatic embryo production. Additionally the role of ethylene at different developmental stages of SE was investigated. Two ACC synthase genes, PsACS1 and PsACS2, were isolated and characterized. PsACS1 was expressed during the proliferation stage in all tested genotypes, whereas PsACS2 was only expressed in somatic embryos of each genotype. Ethylene production in embryos at stage 3 was significantly higher than the other stages. In a parallel study, the response of somatic embryos to fungal elicitors was investigated. Three fungi, a mutualistic ectomycorrhizal (ECM) fungus (Suillus bovinus), a weak Scots pine pathogen (Heterobasidion parviporum) and a strong pathogen (H. annosum) were used. The gene expression patterns for embryos exposed to the H. parviporum elicitor were found to be similar to that documented for S. bovinus among the tested genes. By contrast somatic embryos exposed to the H. annosum elicitor had a different pattern of regulation which was marked by a delayed response, and in some cases death of the embryos. Furthermore, interaction without direct contact between P. sylvestris seedlings and microbes (mutualistic and pathogenic fungus, cyanobacterium) were investigated. Several novel genes expressed in seedlings treated with ECM fungus were isolated which suggested that physical contact is not necessary for elicitation of host responses. The results suggest that somatic embryos and seedlings of P. sylvestris are genetically well equipped to respond to fungal elicitor/exudates and could serve as a suitable model for reproducible molecular studies in conifer tree patho- and symbiotic systems. 9 1. INTRODUCTION 1.1. Somatic embryogenesis-a potential in vitro propagation method for conifers 1.1.1. Development of somatic embryogenesis in conifers Clonal propagation is a technique used for producing large amount of genetically identical individuals by means of organ, tissue or cell culture (Nehra et al., 2005, Bonga et al., 2010). Clonal propagation methods include somatic embryogenesis (SE), micropropagation and macropropagation. The main difference is in the tissue explants used. Macropropagation involves mainly the rooting of cuttings, whereas micropropagation is based on the induction of either pre-existing meristems (auxiliary buds) or adventitious buds (Nehra et al., 2005). However, the age of donor plant has been the obstacle preventing its wide application in conifers (Park 2002). This is a serious limitation for forest trees because, by the time the genetic superiority of a clone has been genetically tested and determined, the donor plant would have been too old to be used for mass propagation (Park 2002). Somatic embryogenesis is an asexual form of plant propagation, which mimics many of the events of sexual reproduction in nature (Santacruz-Ruvalcaba et al., 1998). The application of SE technology makes it possible to circumvent the age problem, at least for some conifer species (Klimaszewska & Cyr 2002, Nehra et al., 2005, Bonga et al., 2010). Since the early descriptions of the process of SE in carrot (Steward et al., 1958) and the ‘totipotency’ theory by Schwann and Schleiden (Gautheret 1983), somatic embryo formation has been achieved for a variety of plant species, including angiosperms and gymnosperms (Breton et al., 2004, Park et al, 2006, Szczygiel et al. 2007, Aronen et al. 2009). The theoretical basis is that cells are capable of regeneration to give a whole plant. Plant tissue culture techniques developed rapidly after the discovery of plant growth regulators (PGRs). Unlike flowering plants, regeneration through SE in conifers is a more difficult process as many species are recalcitrant to in vitro conditions (Bonga et al., 2010). The first hints of SE in gymnosperms occurred in the late 1970s and early 1980s with the descriptions of embryolike structure in Pinus banksiana (Durzan & Chalupa 1976). These structures were unable to undergo further development. However, it was only a few years later that the mature somatic embryos were converted into plantlets from immature embryos of Picea abies (Chalupa 1985, Hakman et al., 1985). In the last two decades, the technology of conifer somatic embryogenesis has developed quickly due to its potential impact on the forest 10 industry. Somatic embryogenesis has been successfully induced in many coniferous species (Attree & Fowke, 1993, Stasolla et al., 2002, Stasolla & Yeung 2003, Park et al., 2006, Szczygiel et al., 2007, Aronen et al., 2009, Pullman et al., 2009, Bonga et al., 2010). Since SE has a vast potential for propagating large-scale plant production, a number of conifer species such as lobloly pine (Pinus taeda) Monterrey pine (Pinus radiata) and Norway spruce (P. abies) have been commercialized to provide the plant materials with desired characteristics and breeding goals (Bozhkov & von Arnold 1998, Pullman et al., 2003b, Aquea et al., 2008). Besides, SE also could be used for conserving endangered plant species (Maruyama et al., 2007). The most important advantage of conifer propagation by SE is that the embryogenic tissue can be cryopreserved, in liquid nitrogen indefinitely without changing its genetic characters (Ford et al., 2000a, Ford et al., 2000b, Häggman et al., 2000), while the corresponding trees are tested in the field. This advantage offers great flexibility in genetic studies to propagate desired genotypes consistently at any time (Häggman et al., 2000). 1.1.2. Four main phases of the somatic embryogenesis and conditions The establishment and regeneration of somatic embryos is successfully achieved through a continuous series of developmental steps including: the initiation and proliferation of embryogenic tissue, embryo maturation and germination. Initiation and proliferation of embryogenic cultures In coniferous species, asymmetric cell divisions within the embryogenic tissue mark the initiation of somatic embryo formation. The two cells originating from such divisions are morphologically distinct; one is small and densely cytoplasmic, whereas the other is larger and vacuolated. The densely cytoplasmic cell will develop into the embryo proper, whereas the vacuolated cell will form the suspensor (Nagmani et al., 1995). Initiation of embryogenic tissues in coniferous species is generally achieved from juvenile tissues, such as immature or mature zygotic embryos, cotyledons or the entire female gametophyte.The most successful explants used for pine are precotyledonary immature and cotyledonary mature embryos (Gupta & Durzan, 1986b, Keinonen-Mettälä et al., 1996, Pullman et al., 2009). In addition, megagametophytes, young seedlings, and re-induced cotyledon somatic embryos have been 11 used to initiate somatic embryogenesis in different coniferous species (Becwar et al., 1990, Lelu & Bornman 1990, Malabadi &VanStaden, 2005). In SE of pine, several basal media are often used (modified Murashige and Skoog medium (MS), LP [AE], Douglas-fir cotyledon revised medium (DCR), and P. taeda basal medium (P6)) (Murashige & Skoog 1962, von Arnold & Eriksson 1981, Chalupa 1985, Gupta & Grob 1995) with low concentrations of carbohydrates, auxin (2,4-dichlorophenoxyacetic acid (2,4- D) or a-naphthaleneacetic acid (NAA)) and cytokinin (BA). Other factors including pH, nitrogen level and light regime can also affect the induction process (Pullman et al., 2002). Moreover, different genotypes of the same species may require different culture conditions (Becwar & Pullman 1995, Lipavska & Konradova 2004). Somatic embryogenesis begins with a plant embryo excised from a seed and incubated on the initiation medium in darkness. Embryogenic cultures began to grow from the micropylar end of the megagametophyte with a unique white, fluffy and translucent appearance (Becwar & Pullman 1995). This is the initiation phase of SE and as long as the embryogenic tissue is maintained on the initiation medium, it will continue to proliferate indefinitely. During the early filamentous stage, embryo proper cells and suspensor cells develop continuously on the proliferation medium (Keinonen-Mettälä et al., 1996). Maintenance of embryogenic tissues can occur in a liquid or on solid medium with the same composition as the induction medium (Gupta & Durzan 1987, Tautorus et al., 1991). At this stage, ECs could be cryopreserved in liquid nitrogen (Ford et al., 2000a, Häggman et al., 2000) and also could be used to continue the somatic embryogenesis process - maturation. Somatic embryo maturation Upon transfer onto the maturation medium, the embryo increases in size with a more globular head, and attains an elongated filamentous shape. Maturation stage could be divided into five according to the differentiation of cotyledon. Stage 1 refers to small embryos consisting of an embryonic region of small, densely cytoplasmic cells subtended by a suspensor. Stage 2, embryos have a prominent embryonic region that becomes more opaque. Stage 3 represents somatic embryos in which the cotyledons are visible and characterized by distinct shoot and root apical meristems. Stage 4 is characteristic of germinating embryos, and at stage 5 the embryos are fully mature (Hakman & Fowke 1987). 12 It is well documented that a PGR, abscisic acid (ABA), has a major role in stimulation of somatic embryo maturation stage in many conifer species (Stasolla et al., 2002, Pullman et al., 2003a, Lipavska & konradova 2004, Aronen et al. 2009). ABA consumption differs from species to prevent precocious germination. For example, in Picea likiangensis, higher amount of ABA was used than that in P. sylvestris to give the best result (Aronen et al., 2009, Chen et al., 2010). The function of ABA on embryo maturation at molecular level will be discussed later. Besides ABA, polyethylene glycol (PEG4000) is often used during the maturation stage to maintain a high osmolarity in the medium. Effect of PEG mimics the naturally occurring water stress on seeds during late stages of maturation and improves the conversion of proembryonic masses into torpedo-shaped embryos (Attree et al., 1991, Ramarosandratana et al., 2001, Stasolla et al., 2003). The combination of ABA and PEG is the most commonly used factor for promoting maturation of somatic embryos of several genera of conifers (Li et al., 1997, Klimaszewska & Smith, 1997, Pullman et al., 2003a, Aronen et al., 2009). However, it was also found that PEG promoted maturation but inhibited further development of somatic embryos in P. abies (Bozhkov & Von Arnold, 1998) and in Picea glauca (Stasolla et al., 2003). The use of carbohydrate and desiccation treatment has been examined for conifer somatic embryos maturation (Ramarosandratana et al., 2001, Bomal et al., 2002, Malabadi et al., 2004, Pullman & Buchana, 2008). In addition to being an important energy source and osmotic agent, sucrose may serve a developmental signaling or regulatory role in conifer SE development (Lipavska & Konradova 2004, Iraqi et al., 2005, Pullman & Buchana, 2008). It has also been shown that reduced amounts of amino acids and peptides such as tripeptide glutathione (Stasolla et al., 2004) or higher percentages (to 1%) of the gelling agent Gelrite may decrease the water content of the somatic embryos. Consequently, the embryos do not need further drying treatment (Klimaszewska & Smith 1997, Klimaszewska et al., 2000). Such effect is similar to that caused by non-plasmolyzing osmotica, such as PEG4000. Germination of somatic embryos Individual mature somatic embryos are picked from the callus and placed onto germination medium. In general, the structure of the shoot apical meristems of cotyledonary somatic 13 embryos appears less organized than those observed in the zygotic counterparts (Yeung et al., 1998). Numbers of cotyledons varied from 5 to 8 even within the same genotype. Efficient germination of somatic embryos is dependent on the quality of embryos. Only those mature embryos that have accumulated enough storage materials and acquired desiccation tolerance at the end of maturation develop into plantlets (Konradova et al., 2003, Lipavska & Konradova 2004). Partial drying treatment (PDT) together with high percentage of gellan gum has been shown to produce more high quality embryos (Klimaszewska & Smith 1997, Malabadi et al., 2004) as well as improve the germination of somatic embryos in conifers (Bomal & Tremblay 1999, Bomal et al., 2002). However, PDT was not found to be essnetial for germination in some species if the embryos were picked up at the cotyledon stage (Choudhury et al., 2008, Aronen et al., 2009). Another factor reported to affect germination and conversion of conifer somatic embryos is light quality. A discrete, light-sensitive pre-germination stage and a later germination (radicle emergence) stage were identified by the differential response of somatic embryos to light of different wavelengths (Merkle et al., 2006). Kvaalen and Appelgren (1999) reported that red wavelengths improved the germination frequencies of P. abies somatic embryos and enhanced hypocotyl and taproot lengths. While blue or ultraviolet has no direct effect on hypocotyls growth (Fernbach & Mohr 1990) but affects glutamine synthetase during seed germination of P. sylvestris (Canovas et al., 1998). 1.1.3. Biochemical and molecular aspects of conifer somatic embryogenesis Physiological and biochemical study during somatic embryogenesis Most studies have focused on the morphological and morphogenic aspects of conifer SE, as described above. However, few have dealt with the physiology of the process. The reason is partly due to the fact that physiological responses often differ among genotypes within the same species (Stasolla et al., 2002). The success of SE depends on the quality of somatic embryos. Knowledge of the physiological and biochemical environments associated with zygotic embryo development will improve somatic embryogenesis (Silvera et al., 2004, Tereso et al., 2007, Klimaszewska et al., 2004, 2009). 14 Spermine 2,4-D+BA (PDT) - ABA -2,4-D -- BA + ABA Proliferation 1st stage of embryo 2nd stage 3rd stage ger m i n at i on Satge Histodiffe- rentiation Apical meristem differentiation Cotyledon development Storage product deposition Lipids and proteins Starch Glutamine, glytamic acid, arginine Polyamines Ethylene, ABA Endogenois hormones Ascorbate / Dehydroascorbate Ascorbic acid metabolism Spermidine Fig. 1. Summary of the major physiological events during somatic embryo maturation of pine from the early-filamentous stage to germination stage. steady values; increasing values; decreasing values. During maturation the embryos undergo structural development and also accumulate compounds that are necessary for further embryo development (Brownfield et al., 2007, Tereso et al., 2007). Such compounds include storage compounds that provide carbon and energy sources for the maturation phase and for germination (Lipavska &Konradova 2004, Brownfield et al., 2007). From proliferation to maturation of somatic embryos, morphological changes, storage products, PGRs, as well as antioxidants changes have been demonstrated (Fig. 1). Storage product accumulation during somatic embryogenesis has been studied in pine (Brownfield et al., 2007, Tereso et al., 2007). Somatic embryo accumulates starch first, followed by lipids and proteins (Silveira et al., 2004, Brownfield et al., 2007) (Fig.1.). The 15 differences between somatic embryo and zygotic embryo in storage starch and protein showed that storage protein might be a marker for embryo quality (Tereso et al., 2007). Changes in PGR levels, especially those of ABA, have been observed during embryogenesis of both angiosperms (Sghaier et al., 2009, 2010) and gymnosperms (Silveira et al., 2004, Vales et al., 2007, Wang et al., 2009, Chen et al., 2010). Unlike somatic embryos of many angiosperm crops, it has been shown that early-stage somatic embryos of most conifers require high concentration of exogenous ABA in order to develop into cotyledon-stage embryos (Stasolla et al., 2002, Lelu-Walter et al., 2008). In a developing zygotic embryo, endogenous ABA is relatively low during the initial stages of development. It reaches maximum level at stage 3 during maturation and declines again during the late stages, as the seed dries (Silveira et al., 2004). The dynamic expression of ABA-responsive gene during embryo maturation in P. taeda was a further proof that high concentration of ABA was crucial for embryo maturation (Vales et al. 2007). The ABA- responsive LEA-like (late embryogenesis abundant protein) gene expression pattern in zygotic embryos provides reasonable evidence for producing high quality somatic embryos (Vales et al., 2007). High levels of ABA in maturing embryos have been associated with increased storage deposition and inhibition of precocious germination (Hay & Charest 1999, Lipavska & Konradova 2004). Storage proteins of coniferous species have also been found to be high in arginine and glutamine (Rodriguez et al., 2006). Some studies on glutamine, glutamic acid, and arginine revealed that the changes in amino acid metabolism were associated with the storage protein deposition during somatic embryo maturation (Garin et al., 2000, Klimaszewska et al., 2004, Malabadi & Van Staden 2005). Glutathione redox state improved somatic embryo quality (Belmonte et al., 2005, 2007). Application of D buthionine- sulfoximine, which inhibits the biosynthesis of reduced glutathione, could improve somatic embryo yield and quality (Belmonte et al., 2007). The polyamines (PAs) spermidine and spermine and their precursor diamine putrescine involved in modulating many plant metabolisms are considered as plant hormones (Silveira et al., 2004, El Meskaoui & Trembaly, 2009, Noceda, et al. 2009). It was found that PA activities steadily increased during the development of somatic embryos, from embryogenic suspensor mass until early cotyledonary stages (Monocha et al., 1999, Niemi et al., 2002a, 16 Noceda et al., 2009) (Fig. 1). Polyamines played an important quantitative and qualitative role during the maturation of P. taeda somatic embryos (Minocha et al., 1999, Silveira et al., 2004) and P. sylvestris (Niemi et al., 2002b, 2007). It was well known that polyamines and ethylene derive from the same precursor, S-adenosylmethionine and polyamines regulate plant metabolism by influencing the biosynthesis of ethylene (Adams & Yang, 1979). However, few studies were done on the function of ethylene in somatic embryogenesis of Pinus. Changes in ascorbic acid (vitamin C) metabolism were also investigated during the maturation process (Malabadi & Van Staden 2005). Application of ascorbic acids together with other vitamins and organic acids produced statistically significant increases in early- stage embryo growth (Pullman et al., 2006). The study showed that the ascorbate (reduced form), dehydroascorbate (oxidized form) ratio increased sharply at the beginning of the maturation period in embryogenic tissues, whereas it remained unaltered in non embryogenic tissue (Pullman et al., 2006). Partial drying treatment of somatic embryos is needed in some conifers for the physiological and biochemical changes before germination (Stasolla et al., 2001, 2002, Jones & Staden 2001). The initial water loss during desiccation is thought to be a signal setting up the events by which desiccation tolerance is established (Bomal et al., 2002, Konrádová et al., 2003). It has been suggested that the desiccation reduces endogenous ABA content (Vales et al., 2007) and ethylene level (Kong & Yeung 1994, Stasolla et al., 2002). Molecular markers during induction and maturation of somatic embryos Somatic embryogenesis has been studied at the molecular level in conifers. Many processes of plant development and differentiation, including embryogenesis, are directly or indirectly regulated by alterations of gene expression patterns. Transcript profile in somatic embryo development of P. taeda has provided a basis for manipulation of embryo development in tissue culture (Cairney et al., 2000). When compared with Arabidopsis thaliana, many developmental pathways and signaling pathway were found to be conserved between angiosperms and gymnosperms (Cairney & Pullman 2007). During in vitro embryogenic process, two major processes that require the reprogramming of gene expression pattern are the induction and the maturation stages. Identification of embryo- 17 specific and embryo-stage-specific molecular markers during embryogenesis would provide a new approach for improvement of somatic embryo production (Fischerova et al., 2008, Wang et al., 2009). Many genes involved in the early stages of somatic embryo development have been studied by comparing embryogenic and non-embryogenic cultures in P. radiata (Bishop- Hurley et al., 2003, Aquea & Arce-Johnson 2008). OTUBAINS family participating in the ubiquitin pathway was also detected during early somatic embryo development in P. radiata (Aquea & Arce-Johnson 2008). The meristem regulation gene family, WOX (Wuschel related homeobox), its transcription factors were expressed in earliest somatic embryo stage and can be used as a possible marker for embryo quality in P. abies (Palovaara & Hakman 2008). PgAGO, isolated from P. glauca, is required for shoot and root apical meristem differentiation. Suppression of PgAGO will result in the abnormal embryo development (Tahir et al., 2006). ABA sensing geneVIVIPAROUS 1 in maize (Wang et al., 2009), ABI 3 in A. thaliana (Fischerova et al., 2008) and PaVP1 in P. abies are transcriptional factors expressed in early embryo maturation stage (Fischerova et al., 2008, Wang et al., 2009). Several efforts have been made to obtain high quality somatic embryos at the maturation stage (Rodriguez et al., 2006, Pullman et al., 2005, Belmonte & Stasolla 2009). ABA-mediated late embryogenesis genes, for example, LEA-like gene were isolated and studied (Vales et al., 2007, Wang et al., 2009). HBK3 (homeobox of Knox 3) gene, which play an important role in plant development, was also found to be involved in somatic embryo maturation through alteration in glutathione and ascorbate metabolism (Belmonte et al., 2007, Belmonte & Stasolla 2009). Embryo-specific patterns of glutamine synthetase genes could be useful as a molecular marker for early stages of vascular differentiation and the quality of somatic embryos in pine (Rodriguez et al., 2006). Late embryo development marker gene homeodomain-leucine zipper (PgHZ1) was isolated from P. glauca, which was related to storage product accumulation in embryos. Other genes have also been found to be developmentally regulated during somatic embryogenesis. These include genes encoding pathogenesis related proteins encoding a basic class IV chitinase and a beta-1,3-glucanase (Dong & Dunstan 1997), heat-shock proteins (Dong & Dunstan 1996) and stress related genes (Frederico et al., 2009, Mathieu, et al., 2009). Transcript profiling studies are expected to provide insight into somatic embryo development in conifers as well as make mass propagation applicable for the forest industry (Cairney et al., 2000, 2006). 18 1.1.4. Somatic embryogenesis in pine Somatic embryogenesis of conifers for clonal propagation has become an integral component of tree improvement strategies. With its capacity for long-term preservation and technology method employed, it is seen to be the preferred technology to accelerate selection and operational deployment of value-added genotypes. However, pine species are often more recalcitrant toward somatic embryogenesis tissue culture methods due to their specific physiological and biochemical properties (Bonga et al., 2010). At present, SE has been applied to 23 species of Pinus. The whole developmental process has been successful in production of plantlets in several species. These species include Pinus banksiana (von Aderkas et al., 2005, Park et al., 2006) P. elliottii (Merkle et al., 2006), P. kesiya (Malabady et al., 2004), P. monticola (Percy et al., 2000), P. patula (Malabadi & Van Staden 2005), P. pinaster (Lelu et al., 1999, Miguel et al., 2004), P. radiata (Aquea et al., 2008), P. strobus (Park et al., 2006), P. sylvestris (Keinonen-Mettälä et al., 1996, Häggman et al., 1999, Lelu et al., 1999, Aronen et al., 2009) and P. taeda (Pullman et al., 2006, Pullman & Buchanan 2008). The culture medium composition is important for the success of SE. It should provide all the nutrients and PGRs for each stage. Information from analyses of zygotic embryogenesis may help improve SE protocols for initiation, maintenance, and maturation (Brownfield et al., 2007, Pullman & Buchanan 2008, Pullman et al., 2009). Recently, some stage-specific condition has been analyzed by comparison with zygotic embryos, such as carbohydrates (Pullman et al., 2008). During initiation stage, adding D-xylose and D-chiro-inositol could increase initiation rate (Pullman et al., 2009). And sucrose, raffinose, melibiose, stachyose and fructose were the major contributors to osmotic potential at late seed development (Pullman & Buchanan 2008). These data suggest stage-specific media composition for each step in the somatic embryogenesis protocol. Additionally, adjusting ABA concentration according to embryo development is equally necessary (Vales et al., 2007). Current state of knowledge of somatic embryogenesis in P. sylvestris Pinus sylvestris (L.) is an important source of timber in Northern Europe and is often used in the pulp and paper industry. Clonal forestry in P. sylvestris has generated keen interest in Finland and Sweden (Keinonen-Mettälä et al., 1996, Häggman et al., 1999). However, study of SE in P. sylvestris lags far behind parallel study in P. taeda, which has been used for 19 industry application. Embryogenic cultures have been established both from precotyledonary embryos isolated from immature seeds (Keinonen-Mettälä et al., 1996, Sarjala et al., 1997, Häggman et al., 1999) and from mature embryos (Hohtola 1995). Several factors affect the success of SE in P. sylvestris, which include seed collection time (KeinonenMettala et al., 1996), explant storage time and temperature (Häggman et al., 1999), culture medium condition include auxin and cytokinin concentration, ABA level and polyamines (Niemi et al., 2007, Lelu-Walter et al., 2008), and the genotype of the parent trees (Lelu-Walter et al., 2008). These factors have to be modulated specifically for initiation and embryo maturation. A successful protocol for SE of Scots pine has recently been developed (Lelu et al., 2008, Aronen et al., 2009). The age of immature zygotic embryo as explants was found to be two weeks after fertilization, which gave the best ratio for initiation of somatic embryogenic cultures (Keinonen-Mettälä et al., 1996). The media used for the induction and maturation of somatic embryogenesis include: DCR basic medium (Gupta & Durzan 1986a), MSG (Becwar et al., 1990) and W-pat (Pullman & Gupta 1991). For induction and proliferation, DCR medium can be supplemented with casein hydrolysate, glutamine, 2,4-D, BA and maltose (Keinonen-Mettälä et al., 1996, Häggman et al., 1999, Lelu et al., 1999, Aronen et al., 2009). Embryogenic cultures could be cryopreserved at the proliferation stage for long-term storage (Häggman et al., 1998, 2000). For the somatic embryo maturation, high molecular weight PEG 4000 along with a high ABA and low water content in the maturation medium have been reported to yield high quality mature somatic embryos (Lelu-Walter et al., 2008, Aronen et al., 2009). However, recalcitrant genotypes still exist throughout the processes (Aronen et al., 2009). Apart from those factors caused by the medium, donor genotype has significant effects on SE initiation in Scots pine (Häggman et al., 1999, Lelu-Walter et al., 2008, Aronen et al., 2009). The genotype phenomena have been investigated in cotton (Michel et al., 2008). The differences between clonal genotypes were caused by the genetic integrity of megagametophytes. In pines, megagametophytes commonly contain multiple archegonia, which are capable of producing multiple genotypes within a megagametophyte. Differences were also observed for the ECs of the dissected immature seeds but without the megagametophyte effect (Yildirim 2005, Özkurt et al., 2008). Microsatellite results have shown that genotypic instability causes mutation in SE of P. sylvestris (Burg et al., 2007). The reasons for genotypic instability are partially due to the immature zygotic embryo, which 20 carry not fully developed genomic information. Stress from PGRs or unsuitable medium conditions might be another reason for genotypic instability (Lopes et al., 2006, Burg et al., 2007). In this case, genotype screening still should be done to achieve successful somatic embryos production (Burg et al., 2007, Marum et al., 2009). Plant hormonal factors have been studied in P. sylvestris. Apart from 2,4-D and BA in the proliferation medium (Lelu et al., 1999), Pas and ethylene have been tested for the growth of ECs (Sarjala et al., 1997, Niemi et al., 2002a, Pullman et al., 2003b, Niemi et al., 2007). A general decrease in the endogenous PA pool occurred towards the later stages of embryo development. The changes in PA transcription level at early stage of zygotic embryos (Vuosku et al., 2006) were a prerequisite for the success of somatic embryogenesis. Ethylene also has been reported to affect the processes of SE in pine (Pullman et al., 2003b, Kumar et al., 2009). More information of ethylene on SE will be discussed separately. Integrated studies in P. sylvestris with an emphasis on biochemical and physiological aspects of each stage of somatic embryo development are needed. Understanding the variability between zygotic and somatic embryos should provide a feasible basis to develop SE culture protocols that normalize response among genotypes at each step of the process (Brownfield et al., 2007, Pullman & Buchanan 2008). Applications of somatic embryogenesis in P. sylvestris Genetically identical study material of an individual tree has great value for many scientific studies (Sutton 2002). Genetic improvement of forest trees is at an early stage especially in SE (Häggman et al., 2006). So far most of the research on pine species has focused on the development of genetically improved genotypes through DNA-transformation protocols. There are reports on studies of genes involved in the improvement of embryo development in conifers, such as germin-like gene and PgAGO gene (Bishop-Hurley et al., 2001, Mathieu et al., 2006, Tahir et al., 2006, Klimaszewska et al., 2010). A range of genes that are of commercial interest have been studied by genetic transformation, for example, alteration of cellulose and hemicelluloses composition by manipulation of lignin biosynthesis (Diouf 2003, Henderson et al., 2006), as well as genes to enhance resistance to herbicide and insects (Brukhin et al., 2000, Bishop-Hurley et al., 2001, Wang et al., 2010). In P. sylvestris, cotyledons and embryogenic cultures have successfully been used as targets for genetic transformation (Häggman & Aronen 1998). Later on a transgenic line was achieved by particle bombardment of pollen before fertilization (Aronen et al., 2003). The frequency of 21 transgenic progenies was very low by this method. In this case, combining recently improved SE techniques with transgenic method would be an option to obtain more transgenic progenies (Aronen et al., 2003). However, the cumbersome nature of DNA transformation in SE of conifer, together with costs and public opinion toward transgenic plant make the technique less attractive for breeding purposes (Häggman et al., 2006). Embryogenic suspension cells usually give uniform growth condition. The suspension cells could respond more rapidly to pathogens or their elicitors (Schwacke & Hager 1992). Utilization of this system to study the response reaction has been carried out on agricultural and horticultural plants, but very few on conifers (Kvaalen & Solheim 2000, Elfstrand et al., 2001). A defensin-like gene spi 1 with enhanced CaMV 35S promoter has been transformed to embryogenic cultures in P. abies (Elfstrand et al., 2001). The transformed plantlet had reduced fungal growth in the sapwood after inoculation of H. annosum (Elfstrand et al., 2001). Interactions between embryogenic cultures and plant pathogen in P. sylvestris (Terho et al., 2000) and in P. abies (Kvaalen & Solheim 2000) have also been tested in order to select the resistance genotypes. Some limitations should be considered in studies of host-parasite interactions. The effects of the medium, the lack of tissue structures, unnatural environments, somaclonal variations and genetic instabilities are important factors to be considered when the results of experiments with tissue cultures are evaluated (Cry & Klimaszewska 2002, Marum et al., 2009). However, together with gene cloning and genomics technology applied in SE of conifers, the discovery and introduction of value-added traits, such as high wood quality and resistance to biotic and abiotic stresses, will facilitate wider application of SE propagation in forests plantation (Nehra et al., 2005). 1.2. Ethylene in somatic embryogenesis 1.2.1. Ethylene biosynthesis Gaseous ethylene has been recognized as a plant hormone for almost a century (Neljubov 1901; Crocker & Knight 1908). It influences a diverse array of plant growth and developmental processes, such as germination, flower senescence, cell elongation, fruit 22 ripening and the response to a wide variety of biotic and abiotic stresses (Yang & Hoffman 1984, Kende1993, Bleecker & Kende 2000, Klee & Clark 2004). Figure 2. The ethylene biosynthetic pathway The enzymes that catalyze each step are shown above the arrows. AdoMet: S-adenosyl- methionine; Met: methionine; ACC: 1-aminocyclopropane-1-carboxylic acid; MTA: methylthioadenine. Inputs that regulate the enzymes are also shown above the pathway, either via a transcriptional or post-transcriptional mechanism. See text for further details (Argueso et al., 2007). The biosynthesis of ethylene occurs through a relatively simple metabolic pathway (Fig. 2) that has been extensively studied and well documented in plants as reviewed by Yang & Hoffman (1984), Kende (1993) and Zarembinski & Theologis (1994). Ethylene is derived from the amino acid methionine, which is converted to AdoMet by AdoMet synthetase. AdoMet is then converted to 1-aminocyclopropane-1-carboxylic acid (ACC) and 5´-deoxy- 5´methylthioadenosine (MTA) by the enzyme ACS (Adams & Yang 1979, Argueso et al., 2007), which is the rate-limiting step in ethylene biosynthesis. Methylthioadenine is recycled to methionine through the Yang cycle, which allows high rates of ethylene production without depletion of the endogenous methionine pool (Miyazaki & Yang 1987). ACC is converted to ethylene by ACC oxidase (ACO). ACO has a small gene family and is found as a rate limiting enzyme in ethylene biosynthesis (Peck & Kende 1995, Argueso et al., 2007). 1.2.2. ACC Synthaseg650the key enzyme in ethylene biosynthesis pathway In most plant species, ACS is encoded by multigene families, which are differentially regulated by various environmental and developmental factors. The genes that encode ACS were first isolated from zucchini (Cucurbita pepo) (Sato & Theologis 1989) and numerous 23 ACS genes have since been identified and characterized in various plant species since then (Barry et al., 1996, Arteca & Arteca 1999, Yamagami et al., 2003, Chae & Kieber 2005). In A. thaliana, 12 putative genes that encode eight functional proteins have been analyzed. They can be divided into three main groups based on their C-terminal sequences and act as homo- or hetero-dimeric proteins (Yamagami et al., 2003). The form of heterodimers might act to increase the versatility of ethylene responses, enhance the capacity to regulate ethylene production during A. thaliana growth, development and response to different stresses (Tsuchisaka & Theologis 2004a, Argueso et al., 2007). Each member of this protein family has the unique and overlapping expression in response to different conditions (Tsuchisaka & Theologis 2004b, Tsuchisaka et al., 2009). Another well-studied case of transcriptional regulation of ACS is that of fruit development in the Lycopersicon esculentum. The L. esculentum ACS family consists of at least eight genes, and these are differentially regulated by various biotic and abiotic factors (Barry et al., 2000, Alexander & Grierson 2002, Bartley & Ishida 2007). Apart from herbal plants, eight ACSs have also been obtained from poplar (Populus trichocarpa) and deposited in the GenBank (POPTR05028, POPTR12462, POPTR16947, POPTR20081, POPTR31859 POPTR32359, POPTR39736 and POPTR40659). However, a few ACSs have been reported for gymnosperms. Two ACS genes related to somatic embryogenesis of Scots pine were first isolated and registered with the GenBank in 2005 (accession number: AY366205 and AY366206). Several ACSs associated to wound, insect infestation and wood formation were reported for other conifer species (Ralph et al., 2007, Barnes et al., 2008). 1.2.3. Regulation of ethylene biosynthesis Abiotic Stress Almost all plant tissues have the capacity to synthesize ethylene, although in most cases the amount of ethylene produced is very low. Expression of the ACS family in higher plants is induced by a diverse group of abiotic stresses, such as: wounding, anaerobiosis, chilling, drought, auxin, ripening and senescence processes (Chae & Kieber 2005), and also by multi stimuli including: LiCl, CuCl 2 , auxin, cycloheximide, aminooxyacetic acid (AOA) and ethylene (Arteca & Arteca 1999, Chae & Kieber 2005, Wi et al., 2010). One of the most studied abiotic stimulus involving stress-ethylene responses is wounding. In A. thaliana, the expression of multiple ACS genes increases after wounding (Tsuchisaka & 24 Theologis 2004b, Ralph et al., 2007). Both jasmonic acid (JA) and ethylene are increased synergistically when plants are wounded by pests or pathogen (Turner et al., 2002, Onkokesung et al., 2010). However, ethylene has been shown to be antagonist to JA induced defence reaction (Shoji et al., 2000, Ankala et al., 2009). In L. esculentum, JA is a major regulator of wound responses, and ethylene seems to act together with JA to regulate wound- responsive genes encoding proteinase inhibitor (pin) proteins (Wasternack et al., 2006, Onkokesung et al., 2010). Ankala et al., (2009) pointed out that JA functions upstream of ethylene in maize insect resistance 1-cysteine protease (Mir1-CP) pathway after herbivory wounding. The balance of these two pathways is critical depending on the plant species and type of stress (Ankala et al., 2009, Wi et al., 2010). Biotic factors The involvement of ethylene in response to pathogen attack has long been recognized (Boller 1991). Plant-derived ethylene is associated with a resistance response when plants are attacked by pathogens (van Loon et al., 2006). Ethylene was also observed in the roots of Medicago sativa inoculated with Rhizobium, and this is associated with defence responses (Ligero et al., 1987). Some micro-organisms, such as plant-growth promoting rhizobacteria, are also able to modulate ethylene responses by altering the levels of ACC produced by plants (Naveed et al., 2008, Govindasamy et al., 2009). Through this reaction, which is catalyzed by the microbial- encoded enzyme ACC deaminase, ACC is hydrolyzed to g302-ketobutyrate and ammonia. This in turn decreases the levels of ACC that are available for ethylene production and improves growth and yield of the plant (Naveed et al., 2008). Transcriptional and post transcriptional regulation of ACC synthase Transcripts of ACS genes could be regulated during the course of development and in response to various external cues. As discussed above, various biotic and abiotic stresses can influence the transcription of different ACS genes. In A. thaliana, diverse patterns of ACS transcripts have been detected in roots, leaves, flowers, siliques, stems, and etiolated seedlings (Yamagami et al., 2003, Tsuchisaka & Theologis 2004b, Wang et al., 2005). The genes ACS2, 4, 5, 6, 7, 8 which are expressed in the flower are also observed in other tissues. A well- studied case is the transcriptional regulation of ACS in L. esculentum fruit development. Two 25 systems of ethylene production have been proposed (Barry et al., 2000, Klee & Clark 2004). System 1 is functional during normal vegetative growth; it is ethylene auto-inhibitory and responsible for producing the basal levels of ethylene detectable in all of the tissues including non-ripening fruit. System 2 operates during the ripening of climacteric fruit and during petal senescence when ethylene is autostimulatory and requires the induction of both of the ACS and ACO. By this theory, when the fruit ripening process commenced, the ACS family becomes integrated in it and responds to different conditions (Barry et al., 2000). ACS protein turnover is also important for regulating the production of ethylene. Various studies are consistent with a model in which the C-terminal region of ACS proteins plays a crucial role in regulating their turnover (Chae & Kieber 2005) (Fig. 3), for example, the phosphorylation of AtACS2 and AtACS6 (Type 1) by MPK6 leads to the accumulation of ACS protein (Kim et al., 2003). This, in turn, causes elevated levels of cellular ACS activity, which leads to increased ethylene production (Kim et al., 2003, Liu & Zhang 2004). Induction of ACS activity through a phosphorylation-dependent mechanism was observed in L. esculentum, which involves increased turnover of the ACS protein (Spanu et al., 1990). Figure 3. Cartoon representing type 1, 2, and 3 ACS proteins ACS proteins in A. thaliana can be divided into three main groups, based on their C-terminal sequences. The conserved catalytic core, present in all ACS proteins, is shaded, whereas putative CDPK (calcium-dependent protein kinase) (Sebastia et al., 2004, Tsuchisaka & Theologis 2004a) and MAPK (mitogen-activated protein kinase) phosphorylation sites (Liu & Zhang 2004) are represented by “S” in Type 1 and 2 ACS proteins. 1.2.4. Ethylene in somatic embryogenesis Effect of ethylene on the growth of ECs The gaseous PGR, ethylene, regulates many aspects of plant morphogenesis. Modification of ethylene composition and its interaction in the nutrient medium has been the primary strategy to manipulate morphogenesis in vitro (Kumar et al., 1998, Yoo et al., 2009). In the model 26 plant of Alfalfa, the requirement for ethylene is optional during the induction of somatic embryogenesis; too much ethylene production might be inhibitory for the initiation stage (Huang et al., 2001). The similar function was also observed in conifers (Pullman et al., 2003b). At the proliferation stage, auxin is often required for cell differentiation (Find et al., 2002). However, increased ethylene production at this stage was also detected (Kvaalen 1994). High ethylene production in the ECs will decrease the initiation rate and embryo maturation (El Meskaoui & Tremblay 2001, Pullman et al., 2003b). A decrease in ethylene production during proliferation, for example, by silver nitrate (AgNO 3 ), was found to increase the induction rate and growth in lobloly pine (Pullman et al., 2003b, Kumar et al., 2009). Differences in ethylene production were observed among ECs with varying maturation capacities (El Meskaoui & Tremblay 2001). Regulation of ethylene biosynthesis is considered necessary for embryo production. Effect of ethylene biosynthesis on somatic embryo maturation At the maturation stage, several studies have revealed that high ethylene production inhibit somatic embryo development (Stasolla et al., 2002, Minocha et al., 2004, Kumar et al., 2009). Non-embryogenic lines, which will not produce somatic embryos, accumulate more ethylene than its corresponding embryogenic lines (Kumar et al., 1989). Higher accumulation of ethylene has been found to reduce the number of cotyledonary embryos and increase morphological abnormal embryo production (Kong & Yeung 1994, Kong et al., 1999). Since the function of endogenous ethylene is crucial in somatic embryo development, optimal ethylene level should be determined for in vitro culture (Tsuchisaka et al., 2009). ECs grown on artificial medium, might contain components that can increase ethylene production, control of ethylene production level during the maturation stage will help to improve somatic embryo formation. Regulation of ethylene biosynthesis is well studied in A. thaliana and in L. esculentum (Yamagami et al., 2003, Bartley & Ishida 2007). Inhibiting ethylene production by 1-Methylcyclopropene, aminoethyoxyvinyl glycine, and NAA could prolong the fruit storage time (Martinez-Romero et al., 2007, Yuan & Li 2008). In somatic embryogenesis of conifers, the use of AgNO 3 , an ethylene antagonist was found to increase embryos production in black spruce (El Meskaoui & Tremblay 2001, Huang et al., 2001, Pullman et al., 2003a, Jimenez 2005, Kumar et al., 2009). Modifying the concentration of ACC, the immediate precursor of ethylene, was reported to increase endogenous ACC content and in the process improve the embryo production in the genotype lines with low ethylene production (El 27 Meskaoui & Tremblay 2001). AOA, an inhibitor of ethylene synthesis have been used to decrease ethylene production during maturation stage to obtain high embryo production (El Meskaoui & Tremblay 2001, Huang et al., 2001). However, the effect of ethylene on somatic embryo maturation in P. sylvestris has not been thoroughly studied. 1.3. Interactions between rhizosphere microbes and development of P. sylvestris roots In nature, plants including forest trees are exposed to a diverse array of microbes, fungi, bacteria and viruses at every stage of their life time (Pearce 1996). The relationship between P. sylvestris and their microbial partners could range from latent entophytic to mutualistic associations and fatal infections. 1.3.1. Ectomycorrhizal fungi and its interaction with host trees Ectomycorrhizal fungi are group fungi that form a mutualistic association with plant roots (Smith & Read, 2008). ECM fungi form symbiotic association with trees, which include species in the Pinaceae, Betulaceae, and Fagaceae genera (Smith & Read, 2008, van der Heijden et al., 2008). During ECM symbiosis, the fungal partner and host plant could establish an efficient mechanism for water and nutrient uptake (Nehls 2008). An ECM root is characterized by the presence of three structural components: a sheath or mantle of fungal tissues around the root, a Hartig net is formed by ECM hyphae between the epidermal and cortical cells and external hyphal elements which form essential connections with soil and sporocarps of the fungi forming the ectomycorrhizas (Smith & Read, 2008). The ECM increases the contact area of plant roots in soil. The symbiosis facilitates the uptake of nutrients such as N and P to the plant root efficiently while the ECM fungi obtain carbohydrates from the tree. During the symbiotic interaction, the ECM fungi partner releases PGRs including auxins, cytokinins, ethylene and PAs (Niemi et al., 2003, Felten et al., 2009, Splivallo et al., 2009, Sarjala et al., 2010). ECM fungus has been used to induce root formation of cuttings and somatic embryo germination (Niemi & Häggman 2002, Niemi et al., 2004). Use of ECM fungus P. tinctorius in somatic embryogenesis of P. sylvestris has been reported to increase somatic embryos germination (Niemi & Häggman 2002). 28 Cyanobacteria are aquatic, photosynthetic and often called “blue green algae” (Whitton & Potts 2000). They form a wide variety of symbiotic associations with eukaryotic hosts such as plants, sponges and fungi (Adam & Duggan 2008). It is found that cycads are the only group among gymnosperms that have been shown to form symbiosis with cyanobacteria (Lindblad et al., 1987; Osborne 2007). Plant cyanobionts normally belong to the genus Nostoc, which is commonly found free- living in nature (Rai et al., 2002). Nostoc cyanobacteria fix nitrogen in special cells called heterocyst (Zhang et al., 2006). During symbiosis, cyanobacteria provide organic nitrogen to the plant and in return get water, minerals and protection from ultraviolet (light) damage (Honegger 1991, Kneip et al., 2007). Unlike other nitrogen-fixing Rhizobia or Frankia sp. that has specific host preference, cyanobacteria show a broad host range. Another remarkable difference is plant cyanobionts are always endosymbionts, form symbiotic cavities in the plant (Rai et al., 2002, Kneip et al., 2007). The biological and molecular nitrogen-fixation mechanism in Nostoc cyanobacteria has been studied (Fay 1992, Singh et al., 1994, Berman-Frank et al., 2003). Since nitrogenase enzyme complex is very sensitive to oxygen, nitrogen fixation is restricted to the dark period in unicellular cyanobacteria, when oxygen levels are low (Berman-Frank et al., 2003). Some organism such as the oceanic picoplanktion has been found to lack photosysten II of photosynthesis, which enables it to perform nitrogen fixation during the day (Bothe et al., 2010). In addition to this protection, concentration of oxygen can be decreased via biochemical pathway such as cyanoglobin, an oxygen scavenging molecules (Hill et al., 1996). In forest soil, the biological nitrogen is always a limiting factor in forest tree growth. The exogenous source of nitrogen into boreal forest is important. The largest natural input of nitrogen to the forest is through cyanobacteria that form symbionts with moss (Adams & Duggan 2008). However, litter chemical components and moisture are often the limitations to nitrogen fixation through cyanobacteria association (Gundale et al., 2009). 1.3.2. Pathogenic fungus - host interaction In the forest, many fungi cause diseases to trees. In tree-pathogen interaction, the host tree could be extremely susceptible or completely resistant to pathogenic fungi. One of the major pathogens of forest trees is the root and butt rot fungus, Heterobasidion spp. which causes significant decay to conifer trees in Europe and North America (Korhonen & Stenlid 1998, 29 Asiegbu et al., 2005). According to their host preferences and physiological and biochemical characters, Heterobasidion spp. has been devided to three different species in Europe (Karlsson and Stenlid 1991, Korhonen & Stenlid 1998). H. annosum s.s. (P-type) which mainly attacks Pinus species, although it has a broad host range including species of Picea, Larix, Juniperus and sometimes even deciduous trees like Betula and Alnus. H. parviporum (S-type) attacks mainly Norway spruce and has only occasionally been found on other native trees in most parts of Europe. And H. abietinum (F group) infects firs (Korhonen & Stenlid 1998, Maijala et al., 2003). The pathogen is spread through root contacts. The tree cell wall is the first barrier against infection by a pathogen. Heterbasidion annosum can secret a number of cell wall degrading enzymes to degrade the root cell walls (Korhonen & Stenlid 1998, Maijala et al., 2003, Asiegbu et al., 2004); whereas H. parviporum can kill young growing close to infected Picea stumps, but obviously its pathogenicity towards pine is relatively weak. Plant resistance mechanisms induced by microbes include basal resistance (Jones & Dangl 2006, Kiraly et al., 2007) and acquired or induced resistance (Shoresh et al., 2005, Jones & Dangl 2006). Several signalling molecules are known to regulate the plant defence responses. Normally plant reacts to biotrophic pathogen infection by inducing cell death, which is regulated by a SA dependent pathway (Glazebrook 2005). In contrast, necrotrophic pathogens benefit from host cell death which implies that SA regulated cell death does not limit their growth. Plants respond to necrotrophic pathogens by different defence pathways activated by JA/ethylene molecules (Glazebrook 2005, Jones & Dangl 2006). 1.3.3. Fungal elicitors, effectors and host-microbes recognition The term “elicitor” was originally used to refer to molecules and other stimuli that induce the synthesis and accumulation of antimicrobial compounds (phytoalexins) in plant cells (Keen 1975). It is now commonly used to describe molecules that stimulate any plant defence mechanism (Dixon 1986, Dixon & Lamb 1990). Many of these molecules have the ability to induce plant defence responses and are referred to as elicitors (Leitner et al., 2008). Microbe associated molecular patterns (MAMPs) are released in plants as complex mixtures of elicitors (Nürnberger & Brunner 2002, Jones & Dangl 2006). The skeleton of fungal cell walls contains 80-90% polysaccharides including chitin, cellulose, non-cellulosic g533-glucans and g302-glucans (Bartnick 1968, Mazan et al., 2006). Many fungal elicitors, such as chitin, g302-1, 30 3-Glucan, a polysaccharide oligochitin and glycoprotein from the cell wall, elicit the innate immunity for the defence of the plant (Hahn 1996, Wolski et al., 2007). The use of cell-wall components from the ECM fungi Amanita muscaria, Hebeloma crustuliniforme and from the conifer pathogen H. annosum have been described by Schwacke & Hager (1992). Usually, when a fungus comes into contact with a plant, certain molecular and chemical components of the fungal cell wall play key roles in the recognition process. Effectors are microbe-derived molecules whose intrinsic activities are to interact with and thus affect the host (de Wit et al., 2009). Plant pathogens secrete a suite of effectors that collectively promote their virulence (Lee & Schneewind 2001). In resistant plants, effectors are directly or indirectly recognized by cognate resistance proteins that reside either inside the plant cell or on plasma membranes (Jones & Dangl 2006, Ellis et al., 2009). To date, 10 fungal effectors, four avr genes and six extracellular proteins (ECPs) have been isolated from Cladosporium fulvum. All avrs and ECPs proved to be virulent factors in C. fulvum - L. esculentum pathosystem (Bolton et al., 2008, de Wit et al., 2009). However, necrotrophic fungi for which no gene-for-gene relationship has been established produce several necrogenic host-specific peptide effectors. ToxA was the first peptide toxin produced by P. tritici-repentis and caused necrosis in particular genotypes of wheat (Ciuffetti et al., 1997). Similar to the effectors of biotrophic fungal pathogens, toxic peptides from necrotrophic pathogens represent effectors that interact with host targets. Little is known about effectors from ECM fungi, it is hoped that the availability of genome sequences of fungal symbionts will facilitate identification of potential effectors (Martin & Selosse 2008). During the process of plant interaction with microbes, recognition plays a central role in the contacts between plants and their pathogens. Pathogens must be able to recognize the presence of their host plants in their environment, detect their host features in order to colonize/invade the plant and cause infection (Jones & Dangl 2006, Boller & Felix 2009). Successful pathogens should also be able to overcome plant defence responses (Jones & Dangl 2006). The plant partners release beneficial and also harmful compounds to the rhizosphere, such as amino acids, organic acids, sugar, phenolics, polysaccharides and proteins (Bais et al., 2006). These compounds will either stimulate or inhibit the surrounding microbes (Bais et al., 2006, Hartmann et al., 2009). The host plant also release some molecules, such as flavonoids or related compounds derived from plant phenylpropanoid metabolism, which are fundamental 31 molecules involved in plant defence (Eckardt 2006). On the other hand, pathogenic fungi secrete some chemical compounds or a large array of enzymes to suppress the host defence responses and cause desease (Jones & Dangl 2006, de Wit et al., 2009). Pattern recognition emerged as a fundamental process in the immune response of plants during the past 10 years (Boller & Felix 2009). Podila et al. (2002) showed that early symbiotic interactions occurred during pre-infection stage. Others reported that plants possessed pattern recognition receptors (PRRs) that enable it to perceive molecular signatures for easy of identification of different classes of microbes (Jones & Dangl 2006, Boller & Felix 2009). For example, one identified g533-glucan binding protein in legumes was proved as the specific binding site for 1,6-g533-linked and 1,3-g533-branched heptaglucoside (HG) in the cell wall of the oomycete Phytophthora sojae (Fliegmann et al., 2004). Very few studies have described the response of gymnosperm species to fungal elicitors (Schwacke & Hager 1992, Salzer et al., 1997). In cyanobacteria plant-microbe interaction, the MAMPs recognized by PRRs are typically associated with a specific class of microbes regardless of pathogenicity (He et al., 2007, Boller & Felix 2009). 32 2. AIMS OF THE STUDY Somatic embryogenesis is a potential vegetative propagation method for conifers, including P. sylvestris. The aim of this study was to improve SE propagation method of P. sylvestris through biotic and abiotic methods and to study the plant-microbe interaction using somatic embryos and seedlings. The objectives of the present study were as follows: 1) Investigate the effect of genotypes and initiation media on somatic embryogenesis of Scots pine (Paper I). 2) Isolate and characterize ACS genes from somatic embryogenic tissues of Scots pine and study the role of ethylene in embryo maturation (Paper II). 3) Investigate effects of fungal cell wall elicitors secreted from pathogenic (Heterobasidion spp.) and ECM (S. bovinus) fungus on somatic embryogenesis on P. sylvestris (Paper III). 4) Study the interactions between P. sylvestris seedling roots and microbes without direct physical contact (Paper IV). 33 3. MATERIALS AND METHODS 3.1. Plant material and microbes used Immature female cones from controlled crosses of seven Scots pine elite trees were collected and stored at 4 °C for different periods of cold treatment (I). All the initiated lines were used for the study of genotype effects on the success of somatic embryo production (I). The selected lines from these sets of ECs were subsequently used for ACS isolation and ethylene regulation treatments (II). Newly sprouted needles, roots or shoots from four-year-old Scots pine emblings were collected for ribonucleic acid (RNA) extraction for the study of ACS expression (II). Six embryogenic lines with abundant embryo production provided by Finnish Forest Research Institute, Punkaharju Research Forests were used for the study of elicitor effects (paper III). Scots pine seeds (E2866 × E2350, Asikkala × Anjalankoski, Punkaharju Research Unit, Finnish Forest Research Institute) were surface sterilized, germinated and used for the study of indirect interaction with microbes (IV). Heterobasidion annosum (Fr.) Bref. sensu stricto (isolate 03012) (III, IV) and H. parviporum Niemelä & Korhonen (isolate 03014) (III) were cultured on 2% malt extract medium. Suillus bovinus (L.: Fr.) O. Kuntze (isolate SBH1) (III, IV) grew on a Melin-Norkrans medium (Molina and Palmer 1982). Cyanobacteria Nostoc spp. (JR 990770) (IV) was cultured on Z8X medium (Kotai 1972). 3.2. Initiation of embryogenic tissue cultures (I) The immature female cones from 7x7 cross lines were surface sterilized. Seed coats were removed and the whole megagametophytes with immature embryos were placed horizontally on the initiation medium in 90-mm diameter Petri dishes. The following media were used: Modified MSG, Modified W-pat and Modified DCR20 (I). One to three cones per family were used for each cold-storage treatment (I). The plates were left to grow in the dark at 23°C. The total number of immature zygotic mega-gametophytes used in the experiment was 5264. 3.3. Maintenance and production of mature somatic embryos After initiation, cultures were transferred to DCR proliferation medium supplemented with 500 mg l –1 casein hydrolysate, 13.6 g541M 2,4-D, 2.2 g541M BA and 2% maltose (I, II, III). The somatic embryogenic tissues were sub-cultured at two week intervals, with nine of the 34 embryogenic tissues sub-cultured on a 9-cm-diameter Petri dish with 20 ml of culture medium. The cultures were proliferated for two months. To mature the embryo cultures, the tissues were first cultured on pre-maturation medium for two weeks. The cultures were moved to 0.1% active charcoal medium without growth regulators for a week. Thereafter they were transferred to maturation medium, DCR with 8% PEG 3500 and 15.1 g541M ABA (I, II) or with 80 g541M of ABA (III). 3.4. Induction treatment of somatic tissues (II) The ECs of the cell line BG8 (I) grown on DCR proliferation medium was used for induction treatment (II). Six pieces (0.2 g each) of ECs were randomly sampled, dipped into 100 ml of one of the following: 500 g80M CuCl 2 , 100 g80M 2,4-D, 100 g80M BA) or 100 g80M ABA in DCR medium for 6 h. ECs in DCR medium without treatment were used as the control. After each treatment, three pieces of the ECs were collected and frozen in liquid nitrogen and stored at - 70 °C for RNA extraction. The remaining three pieces were used for the measurement of ethylene production. 3.5. Determination of ethylene production (II) The different ECs of the cell line BG8 and somatic embryos at different developmental stages of the cell line BG8 and BC15 were incubated in a 5 ml gas-tight vial for 16 h for ethylene production measurement. Ethylene was measured by gas chromatograph (Varian 3700) equipped with porapak Q column (80-100 mesh, 1 m × 3.2 mm) according to Vahala et al. (1998). Oven, injector and detector temperatures were set at 40 °C, 150 °C and 200 °C, respectively. 3.6. Elicitor preparations and sample treatment (III) Fungal mycelia collected from fresh cultures of S. bovinus, H. parviporum and H. annosum were used for crude cell wall elicitor preparation (Ayers et al., 1976, Schwacke and Hager 1992). The final products were autoclaved, lyophilised and stored at -20°C. Six mature somatic embryos were treated with fungal cell wall elicitors for one month to investigate their effects on the root germination rate. The embryos of cell line L76 were subsequently chosen for detailed molecular biological investigation at 1, 7 and 15 days after elicitor treatment. The germinated roots were collected for RNA extraction and quantitative real time PCR. 35 3.7. RNA isolation, reverse transcription PCR and quantitative PCR Total RNA was extracted according to Chang et al. (1993) (II, III, IV). Reverse transcription polymerase chain reaction (RT-PCR) was used for gene expression study. Complementary deoxyribonucleic acid (cDNA) was reverse transcribed with avian myeloblastosis virus (Promega, Madison, WI, USA) (II, IV) or with Superscript III (Invitrogen) reverse transcriptase and with OLE4 reverse primer (II) or anchor T (5g397-(T) 20 VN-3g397) as primers (III, IV). Normal PCR was performed according to each primers melting temperature (II, IV). Quantitative real time PCR was made with SYBR Green I Master Mix (Roche, Germany) according to the manufacturer's recommendations (III). 3.8. Sequences and phylogenetic analyses of PsACS1 and PsACS2 The cloned sequences were analyzed by NCBI BLAST (national center for Biotechnology, basic logical alignment search tool) (II, IV). Amino acid sequence of PsACS1 and PsACS2 alignments were made using ClustalW (II). The evolutionary history of 29 taxa from A. thaliana, L. esculentum, P. glauca, Picea engelmannii × P. glauca, Pseudotsuga menziesii and P. taeda was inferred using the Neighbour-Joining method. Phylogenetic analyses were conducted with MEGA4 (II). 3.9. Induction of P. sylvestris seedlings by different microbes (IV) The pine seeds were surface sterilized and then germinated on water agar for two weeks at room temperature in the dark until the radicles were 0.5-2 cm long. The germinating seedlings were transferred to their respective 100 ml test tubes in which the medium had been set diagonally sloping against the side of the tube. A sterile membrane tube which had one end sealed was placed in the test tube and filled with the sterile light gravel. The tubes were sealed with cotton wool. The seedlings were incubated in the growth chamber for four weeks. Then two agar cubes were inoculated inside the membrane tube which was placed adjacent to the roots of the seedling. Seedlings without inoculation were used as control. After six weeks post inoculation, the weight of the root and number of root tips were determined. Total RNA was isolated from the root tips and used for RT-PCR. 3.10. Statistical analyses 36 Data obtained for the survival of the cell lines, ethylene production and embryo production was statistically analyzed using analysis of variance (ANOVA) (I, II). The difference in root tip numbers, germination between fungal cell wall elicitor treatments, cell lines and treatment times were analyzed using independent sample one-way ANOVA and Tukey’s test (III, IV)). All the tests were performed using SPSS for Windows release 15.0 (SPSS, Chicago, IL, USA). 37 4. RESULTS AND DISCUSSION 4.1. Parental effects on the success of the somatic embryogenesis of P. sylvestris (Paper I) The effect of parental genotypes on the cultures was evaluated at the initiation of the tissue culture period, after six months incubation on maintenance culture and at embryo maturation. The maternal effect was more significant than the paternal effect at the initiation stage of P. sylvestris (I). Lelu et al. (1999) observed a significant maternal tree effect in both P. sylvestris and P. pinaster at the initiation stage. Different initiation rates were found among different species (Park et al., 2006). The overall percentage of culture initiation in this study was 13% and the variation between families was 1 to 42%. The initiation success was about three times higher for cross-pollinated (14%) families than for self-pollinated (4%) families. Four of the trees in our experiment (P451, K801, K828 and K1005) were included in earlier somatic embryo production experiments (Keinonen-Mettälä et al., 1996, Häggman et al., 1999) and were the most responsive of 138 tested trees. This pre-selection of material may explain the high initiation percentage in the study of Aronen et al., (2009). After six months culturing in maintenance culture, the genotype of both maternal and paternal lines had a significant effect on the survival of the offspring (paper I, Table 4). The significance of the maternal genotype diminished and that of the paternal genotype correspondingly became detectable among the surviving lines. Klimaszewska et al. (2001) reported that two thirds of the initiated P. strobus lines survived the proliferation stage. Similar result was also observed in P. sylvestris, 67 % of initiated lines survived for at least one year in maintenance culture (Häggman et al., 1999). A small percentage of the lines were eliminated during the proliferation stage (Häggman et al., 1999). Survival of embryogenic cultures may partly depend on factors related to tissue culture techniques. For example, the two-week subculture period and culture medium may not have been optimal for all the genotypes selected. Chandler & Young (1995) studied P. radiata whereas Lelu et al. (1999) investigated P. sylvestris, both groups reported successful proliferation on hormone free culture medium. The uses of different carbohydrates at proliferation and maturation stages as well as adjustment of ABA concentration have also been tested (Aronen et al., 2009). 38 Only the maternal genotype significantly influenced the production of mature somatic embryos (paper I, Table 5). The mature somatic embryos production rate was 69% (paper I, Fig. 3), which indicated that fewer lines were eliminated at the maturation stage than at either the initiation stage or the proliferation stage. The differences in somatic embryo production varied among cell lines within families. The most and least productive lines were scattered within different families. Large variation in mature embryo production among lines was observed recently in self- and cross- pollinated seed families (Lelu et al., 2008). The percentage in producing mature somatic embryo is rather low in P. sylvestris (Häggman et al., 1999). However, recalcitrant lines did not produce or produced very limited amount of embryos, which have low germination rate (Aronen et al., 2009). In a word, selection of elite maternal genotypes is crucial for initiation, proliferation and embryo maturation in SE of P. sylvestris. 4.2. ACC synthesis genes in somatic embryogenesis of P. sylvestris (Paper II) The involvement of plant growth regulator ethylene in somatic embryo maturation of P. sylvestris was investigated. Genes encoding ACC, the rate-limiting enzyme in ethylene biosynthesis pathway, were isolated and characterized. PsACS1 and PsACS2, were isolated, cloned and sequenced from the ECs. These two ACS genes contain seven conserved boxes, comparable with the known A. thaliana AtACS7 and one coniferous PgACS1 (II, Fig. 1 and S1). The ACS genes are known to belong to a multi-gene family. In A. thaliana, 12 putative ACS genes encoding eight functional proteins have been analysed (Yamagami et al., 2003). However, only few studies have been carried out on Gymnosperms. PgACS1 and PgACS4 were cloned from P. glauca in response to wounding and insect infestation damages which induced expression (Ralph et al., 2007) and one PtaACS1 gene was isolated from P. taeda in response to bending stress (Barnes et al., 2008). In this study, PsACS1 was found to be expressed in the ECs at the proliferation and the maturation stages. At the proliferation stage, both 2,4-D and BA could induce PsACS1 expression but only 2,4-D treated samples induced high ethylene production (paper II, Fig. 2A and 2B); whereas 2,4-D decreased the PsACS2 transcript level thereby supporting results from earlier observations (Lelu et al., 1999). Wochok and Wetherell (1971) showed that 2,4- D inhibited carrot embryos maturation by inducing the production of ethylene. At the 39 maturation stage, differences in expression of PsACS1 were not detectable within the tissues and also within the cell lines (paper II, Fig. 3 A). Thus it probably functions like a house keeping gene to keep ECs differentiation under stress conditions. The PsACS2 transcript was expressed in embryos at the maturation stage. The high PsACS2 transcript abundance was correlated with the higher ratio of cotyledonary stage embryos, and the production of ethylene (II, Fig. 3A and B, Fig. 4). Since ACS is the rate limiting enzyme to ethylene production, it was presumed that the expression of PsACS2 is coincident with ethylene production during somatic embryo maturation (Fig. 4). Hence, the expression of PsACS2 could serve as a genetic marker for the early maturation stage. Cytokinin and Cu + could induce the expression of PsACS2 in plant regulation free medium. A previous identified PgACS1 from white spruce, shared 93% similarity at the amino acid level with PsACS2, was reported to be expressed at the embryo stage and was not induced by wound or insect infestation (Ralph et al., 2007). Investigating expression of ACC synthase genes further enhanced our understanding of the role of ethylene in embryo development and maturation, and improved the efficiency of somatic embryogenesis of P. sylvestris. It would be useful to identify additional ACSs in P. sylvestris for a better understanding of the involvement of ethylene in development and in responses to stresses. 4.3. Response of somatic embryos to fungal cell wall elicitors (paper III) Germination was tested among six cell lines with elicitor treatment. Two cell lines had the highest rooting ability. Under the elicitor treatment, the ability of germination in two cell lines appeared to be favoured by the H. parviporum or the S. bovinus elicitor. The elicitor from H. annosum decreased the germination and subsequent root development in all the genotypes (III, Fig. 2). It was interesting to observe that H. parviporum elicitor increased the germination in some genotypes. Generally, when an ECM fungus interacts with the host plant, it secretes different PGRs including auxins, cytokinins, ethylene and PAs to favour colonization and also nutrient intake from the host plant (Felten et al., 2009, Splivallo et al., 2009). The differences between the two pathogens H. annosum and H. parviporum in the induction of germination / root growth could lie on the apparent differences in their host preferences (Korhonen & Stenlid 1998) or their cell wall components (Schwacke & Hager 1992). However, only one isolate from each fungus was tested with somatic embryos. This 40 study proved that fungal cell wall elicitors could elicite similar defense responses in the emblings as well as living fungal hyphae. The effects of the fungal cell wall elicitors on a subset of genes that encode proteins involved in cell wall synthesis and modification, and the role in mediating stress responses were further examined. Gene transcripts that encoded actin (Act-1) and peroxidase (Pero-1) were up- regulated at 7-day after exposure of the emblings to S. bovinus or to H. parviporum cell wall elicitors. (Paper III, Fig. 3). Actin cytoskeleton forms a dynamic network that rapidly reorganizes in response to a variety of stimuli and regulated by JA mediated plant defense responses (Staiger 2000, Turner et al., 2002, Shimada et al., 2006). In this study, the results also indicated that the weak fungal pathogens had similar defence responses to ECM fungus, but a little higher expression for each tested genes. The weak pathogenic fungus may act as non pathogenic fungus to P. sylvestris since the ethylene synthase gene was not accumulated as high as that induced by H. annosum. Hence we concluded that in P. sylvestris–H. parviporum pathosystem, JA signaling pathway but not ethylene regulates the defense responses. This reaction is similar to the wound induced defence responses in maize as described by Ankala et al. (2009). Similar responses were also detected in genes involved in cell replication, hormone, signal transduction and metabolism, such as EF-1A (elongation factor-1 alpha) and Ps-ACS1 (ethylene synthase) (II) (III, Fig. 3). EF-1A will be discussed in paper IV since we also isolated a similar gene. The significantly increased transcripts of PsACS1 indicated that ethylene was involved in the defence against necrotrophic fungi. This result was in line with the observation that necrotrophic fungus elicits defense response regulated by ethylene and JA signaling pathway (Glazebrook 2005). The fungi used in this study also appear to have possessed some characteristic features in their cell walls that activated plant innate immunity. Recently, the term ‘MAMPs’ and ‘PAMPs’ (microbe- or pathogen-associated molecular patterns) have been used to describe fungal cell wall elicitors such as oligosaccharide (Leitner et al., 2008) and g302-glucan (Wolski et al., 2007). The defense responses in somatic embryos to fungal elicitors indicated that the young emblings are already well equipped with innate immunity to respond to stress. 41 4.4. Induced defence responses in P. sylvestris seedlings (paper IV) Ectomycorrhizal fungus S. bovinus (SBHI) (III, IV), cyanobacteris Nostoc spp. and pathogenic fungus H. annosum (III, IV) were used to study the plant-microbe interaction (IV, Fig.1). After one month incubation, S. bovinus treated seedling grew as well as the uninoculated control. The number of root tip was similar as that of the control, but with higher percentage of dichotomous roots (IV, Fig. 2 & 3). Similar results have previously been observed by Laajanen et al. (2007) that ECM fungi did not induce lateral root formation. The numbers of dichotomous roots obtained in this study were similar to that observed by direct fungus inoculation in pine (Niini & Raudaskoski 1998, Sarjala et al., 2010). The genetic change may be the reason for the morphological change. We have observed that act1 increased (paper III) during treatment with S. bovinus cell wall extracts. The host plant manipulated cell wall regulation gene, such as act1, against ECM infection. Chitinase genes, required for root cell wall reorganization, are known to be expressed when host plants encounter ECM infection (Martins et al., 1999, Tagu et al., 2002 Frettinger et al., 2007). Herrman et al. (2007) reported that chitinase was only expressed in the lateral roots, where dichotomous roots form. The host plant often responds to ECM fungi by changing root morphological and genetical state to sustain the ECM symbiosis. Increased number of the root tips and weight of the seedlings were observed in cyanobacteria treated seedlings. Cyanobacteria provide biological nitrogen to seedling during the incubation period. However, the nodule-like structure needs to be investigating further since Nostoc cyanobacteria usually form the cyanbiont cavities inside the plant (Adams 2008). The H. annosum treated seedlings were smaller than the control and the needle tips turned brown and necrotic (Fig. 3). Six gene fragments were cloned and sequenced from the SBH1 treated samples in addition to the control. The isolated gene products encoded five different proteins (paper IV, Table1.). Ps-Con1 and Ps-Con2 were isolated from the control and were found to encode aspartyl- tRNA synthetase (AspRS). AspRS, a class IIb aminoacyl tRNA synthetase (AARS), may be involved in large complexes of aaRS, the EF-1A elongation factor, and ribosomes (Stapulionis & Deutscher 1995). The gene encoding elongation factor-1A (EF-1A) is functionally homologous to the prokaryotic elongation factor EF-Tu (Kidou & Ejiri 1998), one of the well known PAMPs that trigger the defence response in plant (Hahn 1996, Zipfel et 42 al., 2006, Schwessinger & Zipfel 2008). The ECM fungus and weak pathogenic fungus could induce expression of EF-1A in emblings (Paper III) while downregulated by H. annosum. Another gene, Ps-Sbe1, encodes the GTP-binding protein, which shares homology to domain II of the elongation factors (EFs) EF-G and EF-Tu. EF-Tu is the most abundant bacterial proteins and is actively used as a PAMP for recognition by A. thaliana plants (Kunze et al., 2004). The Ps-Sbe2 was also found to have a 99% similarity to a human gene that encodes rag C protein, NACA (nascent-poly-peptide-associated complex g302-polypeptide) pseudogene. Several lines of evidence suggested that the g302-NAC could function as a transcriptional coactivator (Akhouayri et al. 2005) and may play a role in proliferation and differentiation of the cells (Al-Shanti & Aldahoodi 2006). The Ps-Sbe3 fragment contains putative conserved domain, SGA1 (Glucoamylase and related glycosyl hydrolases), which function in carbohydrate transport and the related metabolism. Ps-Sbe3 also contains a small part encoding actin filament-associated protein (AFAP) with 86% identity. AFAP acts as an adaptor protein that links signalling molecules to actin (III) filaments (Baisden et al., 2001). Fragment Ps-Sbe4 (Table1.) was similar to bilirubin oxidase / multicopper oxidase (MCOs). MCOs such as CueO, bilirubin oxidase, and laccase contain four Cu sites, type (I) Cu, type (II) Cu, and a pair of type (III) Cu's in the protein molecule. Cu (III) oxidase converts the final electron acceptor O 2 to H 2 O without releasing activated oxygen species (ROS) (Sakurai & Kataoka 2007). It is found that increased ROS also increased ethylene production during plant–fungus interaction (Wi et al., 2010). The expression of Ps-Sbe4 probably indicates that fewer oxygen species and less amount of ethylene will be released during ECM fungus-plant interaction. Possibly this could explain how the system regulate ethylene, an antagonist of JA signalling pathway, during plant-ECM fungus interaction. 43 5. CONCLUSIONS Many factors including medium conditions, cold storage time, paternal and maternal effects on the success of SE in P. sylvestris were investigated. We demonstrated that maternal effects occurred in the absence of paternal effects. Moreover, the absence of no maternal × paternal interaction suggests that selecting elite maternal genotypes were crucial for the success of SE. Effects of plant growth regulator ethylene on embryo maturation from various genotypes were investigated. The study of ACC synthase genes further enhanced our understanding of the role of ethylene in embryo development and maturation and improved the efficiency of somatic embryogenesis of P. sylvestris. In parallel to the above studies, the effects of biotic factors (fungal cell wall elicitors) on somatic embryos were also studied. Fungal cell wall elicitors from S. bovinus or H. parviprum induced different plant responses by compared to that from pathogenic H. annosum. The study demonstrates that mature somatic embryos of P. sylvestris are already well equipped with innate immune system to respond to defend the environmental/biological stress and could serve as suitable model plants to study tree-fungal interaction at molecular level. Finally, the results of the interaction experiments with seedlings indicate that elicitation of conifer host defences could be provoked without direct physical contact with the associating microbe. 44 6. ACKNOWLEDGEMENTS The present study was carried out at the University of Helsinki, Faculty of Agriculture, Department of Agricultural Sciences (former Department of Applied Biology) and Department of Forest Sciences. The project was supported by Helsinki University Research Foundation, Project No. 2108012. European Community project No. QLK5-CK-1999-00679, forest breeding foundation in 2007 and in 2008 and Suomen Metsätieteellinen Seura 2010. I wish to express my sincere gratitude to emeritus Prof. Kim von Weissenberg, former supervisor, who accepted me as a PhD student. I am also grateful to my advisor Dr. Ari Pappinen for leading me to my PhD thesis topic ‘somatic embryogenesis of Scots pine’ and patiently arranging funding for me. I would like to give my thanks to Prof. Fred O. Asiegbu, head of the forest pathology division and the most recent supervisor of this work, for his encouragement, helpful supervision and valuable comments. I wish to thank members of my thesis follow-up group (Prof. Teemu Teeri and Dr. Tuija Aronen) for their support and encouragement over all these years. My sincere thanks go to all the co-authors, especially Dr. Jorma Vahala, who led me to the “Ethylene” work in addition to his contribution in the lab, his editing and critical remarks on the manuscript. Thanks also go to Anna-Maija Niskanen, my coworker and dear friend, for her support, in editing and commenting on my manuscripts. Special thanks also for your kindness, willingness to help during this journey. I am indebted to Dr. Hanna Pasonen for her thoughtful comments on my manuscripts and friendly support. I learned a lot from your attitude towards life. You are not only a colleague to me, but more than that, a dear friend, and mentor. I am also grateful to my former colleague, Dr. Sanna Seppänen for your friendship, sharing laughter and tears of life. I wish to gratefully acknowledge the useful comments I received from Dr. Kari Korhonen, you are my hero as a scientist. I wish to express my appreciation to all my colleagues and friends in KPAT and MPAT, especially to Marjo Kilpinen, Dr. Anna Kärkönen, Dr Laura Mattinen, Dr. Timo Hytönen, Lahja Pesonen, Terttu Parkkari and Jouko Närhi for your assistance in addition to creating a 45 friendly environment at work. Docent Risto Kasanen, Dr. Yeshtila Degefu, Dr. Nora Garcia, Hui Sun and all the other former and present members of Forest pathology group are heartily thanked for being such good friends and colleagues, and sharing the ups and downs of both professional and private life. I also wish to thank my dear friends, inside and outside of the University, especially to Huang Hua (Xu), Lu Wei, Yu Liying, Chang Wei, Wang Limei, Liu Changli, Katariina Mustakallio and Oili Lähelmä. Without you, my life in Finland would have been lonely. It has been a long and hard trip to this point. I wish to dedicate this thesis to my husband Xing, without you, I wouldn’t have found my lost files in the new computer system. Thanks for your patience, encouragement and sometimes criticism to keep me going in life. My dear honeys, Huan and Ying, you both are my heart and my all. I feel grateful that Ying understands that I have to work late. My sincere gratitude also goes to my father, mother-in- law, brother and sisters back in China. Espoo, May, 2010 46 7. REFERENCES Adams DG, Duggan PS, 2008. Cyanobacteria-bryophyte symbioses. Journal of Experimental Botany 59, 1047-58. Adams DO, Yang SF, 1979. 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