Application of Ectomycorrhizal Fungi in Vegetative Propagation of Conifers

In forestry, vegetative propagation is important for the production of selected genotypes and shortening the selection cycles in genetic improvement programs. In vivo cutting production, in vitro organogenesis and somatic embryogenesis are applicable with conifers. However, with most coniferous species these methods are not yet suitable for commercial application. Large-scale production of clonal material using cuttings or organogenesis is hindered by rooting problems and difficulties in the maturation and conversion limit the use of somatic embryogenesis. Economically important conifers form symbiotic relationship mostly with ectomycorrhizal (ECM) fungi, which increase the fitness of the host tree. Several studies have shown the potential of using ECM fungi in conifer vegetative propagation. Inoculation with specific fungi can enhance root formation and/or subsequent root branching of in vivo cuttings and in vitro adventitious shoots. Germination of somatic embryos and subsequent root growth can also be improved by the use of ECM fungi. In addition, inoculation can increase the tree's ability to overcome the stress related to ex vitro transfer. A specific interaction between a fungal strain and tree clone occurs during root induction and germination of somatic embryos. Multiple rooting factors exist in this interaction that complicate the predictability of the response to inoculation. Fungal-specific factors that influence rooting responses to inoculation may include plant growth regulator production, modification of the rooting environment, and interactions with beneficial microbes. A combination of these factors may act synergistically to result in positive responses in tree genotypes that are compatible with the fungus.     

Somatic embryogenesis in oak (<Emphasis Type="Italic">Quercus</Emphasis> spp.)

Summary The present review summarizes the factors involved in controlling the process of oak somatic embryogenesis as a method for vegetative plant propagation and includes also data on artificial seed production, cryopreservation and transformation. One major limitation, the inability to initiate embryogenic cultures from mature trees, has been recently overcome. Leaves from selected cork oak trees with an age of 50 yr and more have been used to initiale somatic embryogenesis (SE) with a frequency of up to 20%. These findings offer encouraging prospects for cloning proven superior plant material and to integrate this propagation system into tree improvement programs. Once the process of SE has been initiated, the multiplication cycle proceeds via secondary embryogenesis, which can be maintained indefinitely. Problems are reported by the formation of anomalous embryos. The mutability of somatic embryogenic cell lines of various oak species has been monitored by flow cytometry and molecular markers. No somaclonal variation was detected applying random amplified polymorphic DNA (RAPD) or amplified fragment length polymorphism (AFLP) markers, whereas DNA-content measurements via flow cytometry revealed tetraploidy in some cell lines after several years of continuous subculture. Maturation and low germination frequencies are the main bottlenecks for a broader use of this technique. Recently attention has been on embryo quality and parameters for conversion capacity such as high endogenous cytokinin level and low abscisic acid (ABA) level. Although oak is probably the species that is the most well-developed system for a broadleaved forest tree, data on growth performances of somatic embryo-derived plants are rare.     

Forest biotechnology: Innovative methods, emerging opportunities

Summary The productivity of plantation forests is essential to meet the future world demand for wood and wood products in a sustainable fashion and in a manner that preserves natural stands and biodiversity. Plantation forestry has enormously benefited from development and implementation of improved silvicultural and forest management practices during the past century. A second wave of improvements has been brought about by the introduction of new germplasm developed through genetics and breeding efforts for both hardwood and conifer tree species. Coupled with the genetic gains achieved through tree breeding, the emergence of new biotechnological approaches that span the fields of plant developmental biology, genetic transformation, and discovery of genes associated with complex multigenic traits have added a new dimension to forest tree improvement programs. Significant progress has been made during the past five years in the area of plant regeneration via organogenesis and somatic embryogenesis (SE) for economically important tree species. These advances have not only helped the development of efficient gene transfer techniques, but also have opened up avenues for deployment of new high-performance clonally replicated planting stocks in forest plantations. One of the greatest challenges today is the ability to extend this technology to the most elite germplasm, such that it becomes an, economically feasible means for large-scale production and delivery of improved planting stock. Another challenge will be the ability of the forestry research community to capitalize rapidly on current and future genomics-based elucidation of the underlying mechanisms for important but complex phenotypes. Advancements in gene cloning and genomics technology in forest trees have enabled the discovery and introduction of value-added traits for wood quality and resistance to biotic and abiotic stresses into improved genotypes. With these technical advancements, it will be necessary for reliable regulatory infrastructures and processes to be in place worldwide for testing and release of trees improved through biotechnology. Commercialization of planting stocks, as new varieties generated through clonal propagation and advanced breeding programs or as transgenic trees with high-value traits, is expected in the near future, and these trees will enhance the quality and productivity of our plantation forests.     

栎树繁殖生物技术进展

栎树是重要的森林树种.由于常规营养繁殖困难、种子繁育开花结实迟等原因,栎树的繁育问题一直未得到很好的解决.综述栎树繁殖生物技术的历史与进展;分析栎树的体细胞胚胎从诱导到萌发的过程及机理;阐述欧洲栓皮栎等树种的小孢子胚胎发生技术、双单倍体培养技术、体胚遗传转化技术、人工种子技术以及相关的遗传稳定性与变异性检测技术.     

Forest tree biotechnology

The past year has seen the fruits of biotechnological manipulation of forest trees approach commercial application. Advances in somatic embryogenesis have brought mass clonal propagation of the top commercial trees closer to reality, and efficient gene transfer systems have been developed for a number of conifers and hardwoods. Radical alterations in the quantity and quality of lignin in wood have been shown to be possible in softwoods and hardwoods through identification of naturally occurring mutants, as well as by engineering the lignin biosynthetic pathway with transgenes. The potential environmental and social impacts of the release of transgenic trees have become an increasingly contentious issue that will require more attention if we are to use these technologies to their full advantage.     

Progress in the biotechnology of trees

An increasing world population and rise in demand for tree products, especially wood, has increased the need to produce more timber through planting more forest with improved quality stock. Superior trees are likely to arise from several sources. Firstly, forest trees can be selected from wild populations and cloned using macropropagation techniques already being investigated for fruit tree rootstocks. Alternatively, propagation might be brought aboutin vitro through micropropagation or sustained somatic embryogenesis, with encapsulation of the somatic embryos to form artificial seeds. Tree quality could be improved through increased plant breeding and it is likely that experienced gained, to date, in the breeding of fruit species will be useful in devising strategies for forest trees. Since the development of techniques to regenerate woody plants from explant tissues, cells and protoplasts, it is now feasible to test the use of tissue culture methods to bring about improvements in tree quality. Success has already been achieved for tree species in the generation of somaclonal and protoclonal variation, the formation of haploids, triploids and polyploids, somatic hybrids and cybrids and the introduction of foreign DNA through transformation. This review summarizes the advances made so far in tree biotechnology, and suggests some of the directions that it might take in the future.     

Technological advances in temperate hardwood tree improvement including breeding and molecular marker applications

Hardwood forests and plantations are an important economic resource for the forest products industry worldwide and to the international trade of lumber and logs. Hardwood trees are also planted for ecological reasons, for example, wildlife habitat, native woodland restoration, and riparian buffers. The demand for quality hardwood from tree plantations will continue to rise as the worldwide consumption of forest products increases. Tree improvement of temperate hardwoods has lagged behind that of coniferous species and hardwoods of the genera Populus and Eucalyptus. The development of marker systems has become an almost necessary complement to the classical breeding and improvement of hardwood tree populations for superior growth, form, and timber characteristics. Molecular markers are especially valuable for determining the reproductive biology and population structure of natural forests and plantations, and the identity of genes affecting quantitative traits. Clonal reproduction of commercially important hardwood tree species provides improved planting stock for use in progeny testing and production forestry. Development of in vitro and conventional vegetative propagation methods allows mass production of clones of mature, elite genotypes or genetically improved genotypes. Genetic modification of hardwood tree species could potentially produce trees with herbicide tolerance, disease and pest resistance, improved wood quality, and reproductive manipulations for commercial plantations. This review concentrates on recent advances in conventional breeding and selection, molecular marker application, in vitro culture, and genetic transformation, and discusses the future challenges and opportunities for valuable temperate (or “fine”) hardwood tree improvement.     

Enhancement of American chestnut somatic seedling production

Somatic embryogenesis holds promise for mass propagation of American chestnut trees bred or genetically engineered for resistance to chestnut blight. However, low germination frequency of chestnut somatic embryos has limited somatic seedling production for this forest tree. We tested the effects of culture regime (semi-solid versus liquid), cold treatment, AC and somatic embryo morphology (i.e., cotyledon number) on germination and conversion of the somatic embryos. Cold treatment for 12 weeks was critical for conversion of chestnut somatic embryos to somatic seedlings, raising conversion frequencies for one line to 47%, compared to 7% with no cold treatment. AC improved germination and conversion frequency for one line to 77% and 59%, respectively, and kept roots from darkening. For two lines that produced embryos with one, two or three-plus cotyledons, cotyledon number did not affect germination or conversion frequency. We also established embryogenic American chestnut suspension cultures and adapted a fractionation/plating system that allowed us to produce populations of relatively synchronous somatic embryos for multiple lines. Embryos derived from suspension cultures of two lines tested had higher conversion frequencies (46% and 48%) than those from cultures maintained on semi-solid medium (7% and 30%). The improvements in manipulation of American chestnut embryogenic cultures described in this study have allowed over a 100-fold increase in somatic seedling production efficiency over what we reported previously and thus constitute a substantial advance toward the application of somatic embryogenesis for mass clonal propagation of the tree.     

Vegetative reproduction of trees in some European natural forests

Various means of vegetative reproduction in unexploited forests in western Europe are illustrated with examples. Root suckers are sometimes almost the only method of forest regeneration near the limits of tree growth on the Wadden islands and they can play an important role in forest gaps and riverine forests. Trunk suckers finally replacing their parent trees occur in Alnus, Tilia and Ulmus. Partial uprooting of trees, favoured by special soil conditions, was shown to be an important condition for vegetative reproduction. Temporary survival after uprooting gives opportunity for development of reiterative sprouts, that can replace the vertical axis of a fallen tree. Contact of living branches or even whole stems with soil or mouldering logs favours the growth of adventitious roots. Thus vegetatively reproduced individual trees establish before the uprooted parent tree finally dies. Examples of the clonal spreading of trees are given and a special strategy of layering its branches in pasture woods has been described for beech. Modern forestry rigorously eliminates conditions suitable for the vegetative reproduction of forest trees so their abilities in this respect are often underestimated. Vegetative reproduction seems to be particularly important under circumstances where natural growth is difficult e.g. near the limits of tree growth, on dynamic sites and under heavy shade.     

Genetically engineered trees for plantation forests: key considerations for environmental risk assessment

Forests are vital to the world's ecological, social, cultural and economic well‐being yet sustainable provision of goods and services from forests is increasingly challenged by pressures such as growing demand for wood and other forest products, land conversion and degradation, and climate change. Intensively managed, highly productive forestry incorporating the most advanced methods for tree breeding, including the application of genetic engineering (GE), has tremendous potential for producing more wood on less land. However, the deployment of GE trees in plantation forests is a controversial topic and concerns have been particularly expressed about potential harms to the environment. This paper, prepared by an international group of experts in silviculture, forest tree breeding, forest biotechnology and environmental risk assessment (ERA) that met in April 2012, examines how the ERA paradigm used for GE crop plants may be applied to GE trees for use in plantation forests. It emphasizes the importance of differentiating between ERA for confined field trials of GE trees, and ERA for unconfined or commercial‐scale releases. In the case of the latter, particular attention is paid to characteristics of forest trees that distinguish them from shorter‐lived plant species, the temporal and spatial scale of forests, and the biodiversity of the plantation forest as a receiving environment.     

Somatic embryogenesis in conifers—A case study of induction and development of somatic embryos in Picea abies

Vegetatively propagated material offers many advantages over seed material in forest tree breeding research and in reforestation programmes. Evidence is accumulating to suggest that using somatic embryos in forestry is a viable option. However, before somatic embryos can be used optimally in forestry, basic research aimed at increasing the number of responsive genotypes as well as the age of the primary explant is needed. This in turn requires the establishment of a basic understanding of the physiological and molecular processes that underlie the development of somatic embryos. The functions of genes and their developmental and tissue specific regulation are studied using transient and stable transformation techniques.The process of somatic embryogenesis can be divided into different steps: (1) initiation of somatic embryos from the primary explant, (2) proliferation of somatic embryos, (3) maturation of somatic embryos and (4) plant regeneration. Cortical cells in the primary explant are stimulated to go through repeated divisions so that dense nodules are formed from which somatic embryos differentiate. The first formed somatic embryos continue to proliferate and give rise to embryogenic cell lines. Embryogenic cell lines of Picea abies can be divided into two main groups A and B, based on morphology, growth pattern and secretion of proteins. Our results suggest that extracellular proteins play a crucial role in embryogenesis of Picea abies. Somatic embryos from group A can be stimulated to go through a maturation process when treated with abscisic acid. Mature somatic embryos can develop into plants.Abbreviations ABA abscisic acid - BA N⁶-benzyladenine - 2,4-D dichlorophenoxy acetic acid     

Efficiency,genotypic variability,and cellular origin of primary and secondary somatic embryogenesis of <Emphasis Type="Italic">Theobroma cacao</Emphasis> L.

Summary The development of efficient tissue culture systems for cacao holds the potential to contribute to the improvement of this tropical erop by providing a rapid and efficient vegetative propagation system for multiplication of elite genotypes. It may also find application in facilitation of germplasm movement across quarantine borders, enhancement of germplasm conservation via cryo-preservation, and development of genetic transformation systems. Somatic embryogenesis using floral tissue explants was previously the only tissue culture procedure for regeneration of cacao. We report the development of a secondary embryogenesis system utilizing primary somatic embryo cotyledon explants, which results in up to a 30-fold increase in somatic embryo production compared to primary somatic embryogenesis. The influence of genotype on the efficiency of the system was evaluated. To understand the cellular origins and developmental pathways operative in this system, we investigated the morphological changes occurring over time using light and scanning electron microscopy. While primary embryos arise from clusters of cells forming embryonic nodules, secondary embryos arise predominantly from the division of single cells, in a pathway reminiscent of zygotic embryogenesis. These results have important significance to the application of tissue culture to cacao improvement programs.     

Analysis of genetic stability at SSR loci during somatic embryogenesis in maritime pine (Pinus pinaster)

Somatic embryogenesis (SE) is a propagation tool of particular interest for accelerating the deployment of new high-performance planting stock in multivarietal forestry. However, genetic conformity in in vitro propagated plants should be assessed as early as possible, especially in long-living trees such as conifers. The main objective of this work was to study such conformity based on genetic stability at simple sequence repeat (SSR) loci during somatic embryogenesis in maritime pine (Pinus pinaster Ait.). Embryogenic cell lines (ECLs) subjected to tissue proliferation during 6, 14 or 22 months, as well as emblings regenerated from several ECLs, were analyzed. Genetic variation at seven SSR loci was detected in ECLs under proliferation conditions for all time points, and in 5 out of 52 emblings recovered from somatic embryos. Three of these five emblings showed an abnormal phenotype consisting mainly of plagiotropism and loss of apical dominance. Despite the variation found in somatic embryogenesis-derived plant material, no correlation was established between genetic stability at the analyzed loci and abnormal embling phenotype, present in 64% of the emblings. The use of microsatellites in this work was efficient for monitoring mutation events during the somatic embryogenesis in P. pinaster. These molecular markers should be useful in the implementation of new breeding and deployment strategies for improved trees using SE.     

Hydraulic limits on tree performance: transpiration,carbon gain and growth of trees

The state of knowledge concerning the influences of tree size, xylem dysfunction, growth conditions and within-species and between species genetics is reviewed. It is concluded that high plant hydraulic conductance is necessary for high productivity in forest trees, and this being the case, the implications for commercial forestry require further study.     

Initiation, long-term cryopreservation, and recovery of Abies alba Mill. embryogenic cell lines

Somatic embryogenesis of Abies alba (Mill.) has significant potential to become an effective method for vegetative propagation of this species. To induce somatic embryogenesis in A. alba, the influence of the mother tree, sampling dates, and cold treatment storage of cones were examined. The initiation frequencies ranged from 1.7% to 16.6%. The sampling date and cone storage, but not the mother tree, had a significant effect on the initiation of embryogenic cultures. Storage of embryogenic cell lines was tested through cryopreservation for 6 yr. Four out of 12 cryostored embryogenic cell lines recovered, and the regeneration of cotyledonary embryos was obtained with two cell lines. The ability of embryogenic cell masses to produce somatic embryos and the mean number of cotyledonary embryos were higher when the maturation protocol was based on embryogenic suspensions dispersed on filter paper. The properly developed germinants were obtained only from maturation media where 32 μM abscisic acid was used, being 16.2% when polyethylene glycol (PEG) was not present and 1.8% when supplemented with 10% (w/v) PEG, respectively. The present study provides evidence that it is possible to cryopreserve A. alba embryogenic cultures while maintaining their maturing ability for the lengthy period (6 yr) needed for progeny testing of field-grown trees. Therefore, our findings are important for further studies and advanced breeding work of the species; however, the conversion of germinants into ex vitro conditions still remains a significant challenge.     

Somatic embryogenesis from mature <Emphasis Type="Italic">Bambusa balcooa</Emphasis> Roxburgh as basis for mass production of elite forestry bamboos

A reliable protocol for mass propagation via somatic embryogenesis in mature bamboos has been established using pseudospikelets of Bambusa balcooa. Fourty percent of the explants gave rise to multiple regenerants within 4 months. This conversion rate is sufficiently high to use the process in commercial mass production. Further, shoot apical meristems can also be used as primary explants without lost of efficiency. Regenerated plants were uniform and identical to the mother plant and to plants obtained by axillary branching with respect to growth characteristics and morphology. Furthermore, epigenetic changes could not be detected by Methylation Sensitive AFLP (MSAP). During the complete process no changes in ploidy level could be observed. The process allows for a cost reduction for this tropical bamboo for forestry of up to 57% compared to micropropagation via axillary branching. For the first time, a reliable process based on somatic embryogenesis has been developed that is well suited for commercial micropropagation of elite mature bamboos.     

Some effects of animal grazing and browsing on vegetation

Summary

This is a review of some problems posed by research on tree biology. First, phase change and in vitro culture are discussed as they affect vegetative propagation of woody plants. Then the breeding of timber trees, with an evident and important trend to clonal propagation is considered. Tropical timber tree breeding has enormous potential but has only just started (on very few species). The trend to making tropical woodlands into planted pure stands, of converting production forestry into, ‘tree farming’, an aspect of agriculture, so to speak, is apparent. ‘Ideotypes’ have fallen out of favour but some important ideas as to biomass and its allocation remain and well-defined economic objectives must become paramount. A broad review of the tropical agricultural context with regard to trees, suggests an acute need for far better understanding of them and for much introduction and breeding to generate new woody food-crops.     

Biotechnological advances in guava (Psidium guajava L.): recent developments and prospects for further research

Guava (Psidium guajava L.), an important fruit crop of several tropical and sub-tropical countries, is facing several agronomic and horticultural problems such as susceptibility to many pathogens, particularly guava wilting caused by Fusarium oxysporium psidii, low fruit growth, short shelf life of fruits, high seed content, and stress sensitivity. Conventional breeding techniques have limited scope in improvement of guava owing to long juvenile period, self incompatibility, and heterozygous nature. Conventional propagation methods, i.e., cutting, grafting or stool layering, for improvement of guava already exist, but the long juvenile period has made them time consuming and cumbersome. Several biotechnological approaches such as genetic transformation may be effective practical solutions for such problems and improvement of guava. The improvement of fruit trees through genetic transformation requires an efficient regeneration system. During the past 2–3 decades, different approaches have been made for in vitro propagation of guava. An overview on the in vitro regeneration of guava via organogenesis, somatic embryogenesis, and synthetic seeds is presented. Organogenesis in several different genotypes through various explant selection from mature tree and seedling plants has been achieved. Factors affecting somatic embryogenesis in guava have been reviewed. Production of synthetic seeds using embryogenic propagules, i.e., somatic embryos and non-embryogenic vegetative propagules, i.e., shoot tips and nodal segments have also been achieved. Development of synthetic seed in guava may be applicable for propagation, short-term storage, and germplasm exchange, and distribution. An initial attempt for genetic transformation has also been reported. The purpose of this review is to focus upon the current information on in vitro propagation and biotechnological advances made in guava.     

Conservation of the Cedrus libani populations in Lebanon: history, current status and experimental application of somatic embryogenesis

Cedrus libani, the cedar of Lebanon, is a threatened conifer native to the Levant. Over 4000 years of exploitation have resulted in the fragmentation and degradation of the Lebanese cedar populations. Continued urban and agricultural development in Lebanon adds to the difficulty of effective conservation. Two protected areas have recently been established which contain two of the more important forests: a cedar dominated forest in the Shouf region and a mixed forest at Ehden. A number of other populations are protected by ministerial decrees, and there is a need for rigorous management of all the remaining populations. The application of in vitro techniques such as somatic embryogenesis may assist in the conservation of this species. We have produced somatic pro-embryos using immature zygotic tissue as explants cultured on half-strength MS medium containing an auxin and a cytokinin (10 M 2,4-D and 5 M BAP). The application of somatic embryogenesis to the Lebanese cedar would be in the propagation and preservation of selected genotypes, either those from old growth provenance for use in restoration, or those with desirable commercial or horticultural characteristics.