0221-B2
Yill-Sung Park[1] and Krystyna Klimaszewska
Somatic embryogenesis (SE) in conifers is a recently developed cloning technique, where an unlimited number of genetically identical copies of trees can be produced from a single seed. The most important practical application of SE is in tree improvement and clonal forestry. Genetically superior and high-value trees may be developed through breeding, and the use of tested and cloned trees of the improved trees, i.e. clonal forestry, can improve forest productivity much more than conventional breeding techniques. With the expectation that more natural forests will be designated as ecological reserves or as recreational forests, wood production on remaining commercial sites must be increased. Clonal forestry can offset the deficit by deploying the best available genetic stock to commercial sites. SE and cryopreservation are the key technologies that make the development of genetically superior tree lines and the practice of clonal forestry possible. Successful implementation of clonal forestry requires that SE technology be sufficiently refined. The current state of SE technology in the context of clonal forestry is reviewed. Some issues concerning the practice of clonal forestry, including management of plantation diversity, are discussed.
Somatic embryogenesis (SE) is a cloning technique based on tissue culture, where an unlimited number of identical embryos can be produced. Since its first success with forest conifer trees, this technology has been developed for several conifer genera and species. Plants derived from somatic embryos are now routinely produced, especially for spruce, Douglas-fir, and some pine species. Somatic embryogenesis is becoming an efficient method for cloning genetically improved trees. Some applications of SE include provision of cell lines for genetic engineering, ex situ conservation of rare species or populations, and use in research to gain better understanding of genetics and embryo development. However, the most important application of SE is in tree improvement because the use of cloned trees in commercial plantation forestry can have a major impact on forest productivity. Through controlled breeding, genetically superior and productive clonal lines may be developed, and careful deployment of these clonal lines could drastically improve forest productivity. For example, clonal testing of white spruce at age 10 showed a genetic gain of five-to-six fold compared with conventional seed orchards. In this paper, we review the development of SE in conifer species in the context of tree improvement and plantation forestry. The specific purposes of this paper are to review current achievements in SE technology, to examine technical requirements of SE for use in clonal forestry, and to discuss issues relating to clonal forestry. Discussion is focused primarily on coniferous species, in which SE is well developed and commercially important.
To date, in the family Pinaceae, SE has been achieved in 41 species and hybrids belonging to five genera, i.e., Abies, Larix, Picea, Pinus, and Pseudotsuga. By far, the largest body of work has been directed towards SE in spruce (Picea) species, particularly P. abies, P. glauca, P. glauca x engelmannii, and P. mariana. This was due, in part, to the relative ease of SE initiation and regeneration of somatic seedlings from somatic embryos in these species compared with the other genera in the Pinaceae family, as well as their economic importance to the forest industry. Somatic embryogenesis in conifers is a multistage process, and each stage represents different challenges. In general, plant regeneration by SE is divided into five stages.
Initiation of embryogenic tissue - The initiation of embryogenic tissue (ET) begins with culturing of either immature or mature zygotic embryos on semi-solid nutrient media with plant growth regulators. The frequency of SE initiation is often influenced by the developmental stage of the zygotic embryos, the level of growth regulators, and the genetic makeup of explants.
Proliferation or maintenance - This stage involves the establishment of embryogenic culture and continuous growth of ET that produces an increase in fresh mass upon periodical subculture onto a fresh semi-solid or liquid medium. At this stage, the vigorously growing ET may be cryopreserved for long-term storage. The ET will proliferate continuously as long as it is subcultured onto fresh medium every 12 to 21 days and, thus, unlimited quantities of ET may be obtained.
Maturation of somatic embryos - This stage marks the appearance of the cotyledonary somatic embryos from ET through histodifferentiation. This stage may last 6 to 12 weeks. Some protocols apply a pre-maturation step, which involves a brief (3 to 7 d) culture of embryogenic tissue on a medium devoid of plant growth regulators and containing activated charcoal prior to transfer onto a maturation medium. The medium can be either liquid or semi-solid.
Germination of somatic embryos - Somatic embryos are usually germinated in vitro on a semi-solid nutrient medium that contains sucrose. This stage is completed after the elongation of an epicotyl and the development of needles, which occur most frequently after 12 to 16 weeks. Before germination, mature somatic embryos can be either partially desiccated at high relative humidity or dried to a low water content at low relative humidity.
Early growth ex vitrum - This stage involves the establishment of in vitro-grown somatic seedlings in a substrate under greenhouse conditions. Typically, during the first 2 to 3 weeks of growth, a high relative humidity is provided to facilitate the plants’ acclimatization to ambient conditions. Then, normal greenhouse practice is followed.
Somatic embryogenesis has many advantages over other in vitro vegetative propagation techniques, such as organogenesis. However, the most important advantage of SE is that the ET can be maintained in liquid nitrogen (cryopreservation) at a temperature ranging from -130 to -196°C without loss of viability or change in genetic makeup. The general approach is to facilitate the gradual removal of free water from the embryogenic cells and to minimize the formation of intracellular ice by using slow cooling. Most current protocols entail the incubation of a tissue suspension in a proliferation medium of decreased osmotic potential, treated with sorbitol and dimethylsulphoxide for a period of 24 to 48 hours. Then, the tissue suspension is cooled to -35 to -45°C, and the cultures are stored in liquid nitrogen. For regeneration, the frozen tissue is thawed rapidly for 1 to 2 minutes at 37°C, the storage solution is removed via draining, and the cultures are transferred onto a fresh semi-solid medium. Growth of cultures typically occurs within 1 to 2 weeks after thawing.
The ability to cryopreserve ET without change in genetic makeup or loss of regenerative capacity offers an opportunity to develop genetically improved clonal lines. This is accomplished by dividing each ET mass into two portions, one of which is cryopreserved while the other is propagated to produce clonal plants for testing in the field. Once field testing has shown which are the best performing clones, the corresponding ET can be retrieved from liquid nitrogen, thawed and used for propagating plants for commercial plantations.
The actual development of superior clonal varieties would involve controlled crossbreeding of proven superior trees, followed by selection of SE clones developed from seed obtained by this crossbreeding. The selection of SE clonal lines is based on genetic testing. The clonal varieties developed in this manner will utilize indigenous genes in the most favorable combinations without introducing foreign genes.
Clonal forestry may be defined broadly as any use of cloned trees in forestry, including the use of bulk-propagated families. However, more restrictively, it refers to the use of tested clonal lines in plantation forestry. There are many advantages to clonal forestry, particularly for deployment of tested clones; it offers (1) genetic improvement much greater than possible through conventional tree breeding; (2) the flexibility to rapidly introduce suitable clones to meet changing breeding goals or environments; and (3) the ability to carefully manage diversity in clonal plantations.
Implementation of clonal forestry requires three critical steps. First, a sufficiently refined cloning technique must be developed. In conjunction with cryopreservation, SE is the only technology that makes implementation of clonal forestry possible in conifers. The second step is the development of superior clonal lines and this involves the establishment of long-term genetic testing of clonal lines developed by breeding. The final step is large-scale production and deployment of tested clonal lines in plantation forestry. The following are some technical issues to consider for successful implementation of clonal forestry.
Initiation and plant conversion rates - Sufficiently high SE initiation and subsequent plant conversion rates are important to maintain genetic diversity of clonal plantations while achieving a high level of genetic gain. Therefore, improving SE initiation rate has been a major area of SE research and is influenced by several factors, such as composition of culture media, stage of maturity of a zygotic embryo (ZE), and genetic factors. The current SE initiation rate in spruce species, including Picea glauca, P. mariana, and P. abies, is sufficiently high, at over 65%, when immature ZE explants are used. Furthermore, about 80% of these embryogenic clones produce normal plants. But the initiation rate in pine species, with the exception of Pinus strobus (Klimaszewska et al. 2001), has been low. Intensive research is being carried out to improve this step of SE, particularly for pine plantation species such as Pinus taeda and P. radiata. Somatic embryogenesis process is also influenced by genetic factors, and understanding genetic control is an important element in improving the SE initiation (Park et al. 1993).
Genetic stability of cryopreserved clones - Technically, cryopreservation of embryogenic lines is easily achieved; however, it is important to demonstrate that embryogenic clones are maintained without change in genetic makeup or loss of viability during cryogenic storage. Comparisons of clones that were retrieved from cryopreservation and propagated through the SE process, grown in a greenhouse, and planted in a nursery test produced highly consistent results for in vitro and ex vitrum characters, indicating that genetic integrity is maintained during cryogenic storage (Park et al. 1998). Also, genetic stability of clones was evaluated using randomly amplified polymorphic DNA (RAPD) fingerprints (DeVerno et al. 1999). Variant banding patterns were detected in embryogenic tissue that had been subcultured for 2 and 12 months and in trees regenerated from aberrant somatic embryos. There was no banding pattern variation among plants regenerated from somatic embryos that were normal in appearance. These results suggest that it is important to avoid prolonged subculture and to select somatic embryos of normal morphology when propagating for clonal deployment.
Genetic integrity of clonal lines - In conifer species that require immature seed to initiate SE, whole megagametophytes are routinely used as explants. The megagametophytes in Pinaceae commonly contain multiple archegonia (egg cells), which are capable of producing multiple genotypes within a megagametophyte because multiple fertilizations can occur. Thus, there is a possibility that an embryogenic clone may contain more than one genotype. It is necessary that a clonal line be consisting of a single genotype.
Genetic testing of clonal lines - The primary reason for conducting clonal tests is to identify suitable clones for deployment in clonal forestry. Clonal testing generally involves a number of candidate clones evaluated over a range of common test sites with respect to traits of interest. Normally, testing a large number of clones will result in larger genetic gain than a small number. However, clonal testing is constrained by limitations in logistics and resources. Therefore, it is important to establish replicated common garden clonal tests containing a large but manageable number of clones. The genetic testing is a long-term field experiment that may last to rotation age and beyond, and the tests will be evaluated at regular intervals. The data obtained at a given time may be used to select candidate clones for clonal deployment.
The majority of clonal testing and selection programs are managed within the private sector, represented by forestry companies and third-party providers. Forestry companies known to be active in this area include Bioforest (Chile), Carter Holt Harvey (New Zealand), International Paper (USA), JD Irving Limited (Canada), Rayonier (New Zealand and USA), MeadWestvaco (USA) and Weyerhauser (USA). Other private or related organizations include AFOCEL (France), Arbogen (USA), CellFor (Canada), GenFor (Chile) and Rubicon (New Zealand). Governmental and academic entities include the Canadian Forest Service (Canada) and Swedish University of Agricultural Sciences (Sweden). Comprehensive publications with respect to clonal selection are few in number, with benchmark reports available for P. glauca (Park et al. 1998), Picea abies (Högberg et al. 2001), and P. glauca x engelmannii (Cyr et al. 2001). With respect to other commercial conifer species, clonal selection efforts occur primarily in the private sector and information is available at best from conference proceedings.
Commercial production and clonal deployment - Historically, conifer SE has been developed using Petri-dish based systems and in vitro germination. Although these approaches are suitable for the establishment of clonal trials, they are viewed as inadequate for commercial production. Promising developments in the automation of the SE process include liquid maintenance culture, bioreactor maturation, and embryo purification and desiccation (Timmis 1998, Cyr et al. 2001). Liquid culture methods, which facilitate rapid bulk-up, uniformity, and improved embryo yield, have been developed for Picea spp., Pseudotsuga menziesii, and Pinus taeda.
The development of artificial seeds has received significant attention. An approach, entailing direct sowing of somatic embryos, has been developed (Cyr et al. 2001). This proprietary system, demonstrated on a research scale for Picea spp., Pseudotsuga menziesii, Pinus radiata, and Pinus taeda, was described as suitable for use in commercial greenhouses using conventional sowing practices.
As an alternative, mass propagation of superior clonal lines can be done by serial rooting of cuttings taken from a number of donor (stock) somatic plants. This is easily achieved, particularly for spruce species, which can produce rooted cuttings for approximately 5 years. The cost of producing stecklings from stock plants is about twice the cost of seedlings (G. Adams, JD Irving, Ltd., personal communication).
The main concern about clonal forestry is that a narrow genetic base may make clonal plantations more vulnerable to diseases and insects and that this may lead to plantation failure. In general, it is assumed that the more clonal lines deployed in a plantation, the lower the risk. But increasing the number of clones in a plantation will reduce genetic gain. Thus, it is necessary to balance genetic gain and plantation diversity. Using various quantitative approaches, scientists generally agree that planting 10 - 30 clones mixed in a plantation should be sufficient to offer protection yet still confer the benefits of clonal forestry (Hühn 1987; Libby 1982; Roberds and Bisher 1997; Zobel 1993). After deciding the appropriate number of clones, a clonal deployment strategy should consider the configuration of deployed clones, which can be clones in a random mixture or monoclonal blocks in a mosaic structure (Libby 1982). Alternatively, a ‘mixture of clones and seedlings (MOCAS)’ is proposed for New Brunswick, Canada (Park 2002). For example, in a plantation, 60% of the plants can be a mixture of the best clones identified from genetic tests and the remaining 40% of the plants can be propagated from low-cost seed-orchard seed. MOCAS will increase the initial plantation diversity. The reason for proposing MOCAS is that, by one-half rotation age, about 40% of the plantation is commercially thinned leaving superior quality trees for final harvesting.
Furthermore, evaluation of genetic tests at regular intervals until the rotation age will lead to continually revised clonal compositions that are available for each clonal plantation establishment. Thus, the diversity of clonal plantations can also be managed through time. The up-to-date data from clonal test are also keys to the flexibility of clonal forestry to rapidly deploy suitable clones with changing breeding goals or other objectives.
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| [1] Natural Resources Canada,
Canadian Forest Service, Atlantic Forestry Centre, PO Box 4000, Fredericton,
New Brunswick E3B 5P7, Canada. Email: [email protected] |