xi Part 1 THE SCIENCE OF GENETIC MODIFICATION IN FOREST TREES 3 1. Genetic modification as a component of forest biotechnology C. Walter and M. Menzies While the term “biotechnology” refers to a broad spectrum of modern tools and the application of those tools, it is frequently equated with genetic engineering by the lay public. FAO noted in their 2004 report The State of Food and Agriculture that “biotechnology is more than genetic engineering” (FAO, 2004a). In fact, 81% of all biotechnology activities in forestry over the past ten years were not related to genetic modification (Wheeler, 2004). There are many definitions of biotechnology and they differ in their scope. FAO (2001) defines the term biotechnology as “any technological application that uses biological systems, living organisms, or derivatives thereof, to make or modify products or processes for specific use”. This definition, although accurate for the specific purposes for which it was intended, may contribute to the confusion surrounding the term. A simpler definition might be “the application of biological knowledge to practical needs such as technologies for altering reproduction, or technologies for locating, identifying, comparing or otherwise manipulating genes”. In short, forest biotechnology is associated with a broad spectrum of modern methods applicable to agricultural and forest science, only some of which are related to genetic engineering. In forestry, the definition of biotechnology covers all aspects of tree breeding and plant cloning, DNA genotyping and gene manipulation, and gene transfer. Forest biotechnologies can be classified in many ways (Yanchuk, 2001; Wheeler, 2004), but here they are grouped under five major, though undoubtedly overlapping, categories (Henderson and Walter, 2006; Trontin et al., 2007; El-Kassaby, 2003, 2004): r QSPQBHBUJPO r NPMFDVMBSNBSLFST r NBSLFSBTTJTUFETFMFDUJPO ."4 BOENBSLFSBTTJTUFECSFFEJOH ."# r HFOPNJDT NFUBCPMPNJDTBOEQSPUFPNJDT r HFOFUJDNPEJGJDBUJPOPSHFOFUJDFOHJOFFSJOH This chapter provides a brief discussion of these technologies in the context of existing or proposed deployment in commercial forestry. However, this should be read only as an introduction, and the reader is referred to the vast literature available on those subjects. 4 Forests and genetically modified trees PROPAGATION Plant cloning has been used for centuries for tree breeding and propagation using grafts and cuttings. Chinese fir (Cunninghamia lanceolata) has been propagated by cuttings for clonal forestry in China for more than 800 years (Li and Ritchie, 1999) and Japanese cedar (Cryptomeria japonica) has been propagated clonally by cuttings in Japan for plantations since the beginning of the fifteenth century (Toda, 1974). Some tree species are easier than others to propagate by cuttings. Easy-to- root hardwood species, such as poplars (Populus spp.), willows (Salix spp.) and some eucalypt (Eucalyptus) species, and conifer species, such as spruces (Larix spp.), redwood (Sequoia sempervirens), and some pines (Pinus spp.), are widely planted as cuttings in family or clonal plantations (Ritchie, 1991; Ahuja and Libby, 1993; Assis, Fett-Neto and Alfenas, 2004; Menzies and Aimers-Halliday, 2004). In the future, the use of vegetatively propagated trees for intensively managed, high- yielding plantations is expected to increase in all regions of the world. While the main use of propagation technologies has been for forest establishment of genetically-improved families or clones, there is also a conservation use for those species that are at risk, rare, endangered or of special cultural, economic or ecological value (Benson, 2003). Integrating traditional methods such as in situ conservation and seed storage with biotechnologies such as micropropagation and cryopreservation can provide successful solutions. Micropropagation Micropropagation refers to the in vitro vegetative multiplication of selected plant genotypes, using organogenesis and/or somatic embryogenesis. Approximately 34% of all biotechnology activities reported in forestry over the past ten years related to propagation (Chaix and Monteuuis, 2004; Wheeler, 2004). Micropropagation is used to multiply (bulk-up) desirable genotypes or phenotypes to create large numbers of genetically identical individuals of clones or varieties. These techniques are gaining increased attention by foresters and tree breeders because vegetative propagation offers a unique opportunity to bypass the genetic mixing associated with sexual reproduction. Organogenesis While macropropagation methods, such as cuttings, involve comparatively large pieces of tissue, micropropagation by organogenesis involves in vitro culture of very small plant parts, tissues or cells, particularly meristems from germinating embryos or juvenile plant apices. There are a number of stages in organogenesis, involving sterilization and shoot initiation, shoot elongation and multiplication, rooting and acclimatization. Sterilization is typically done with a diluted bleach solution, followed by initiation of shoots on an appropriate tissue culture medium. Shoots can develop from existing axillary meristems or from meristems of adventitious origin. Adventitious meristems can be stimulated from plant tissue, such as cotyledons or leaves, by exposure to a pulse of the plant hormone, cytokinin. Plants arising from shoots of adventitious origin may show undesirable Genetic modification as a component of forest biotechnology 5 advanced maturation characteristics (Frampton and Isik, 1987). There have been many different media developed for organogenesis, depending on the species (McCown and Sellmer, 1987). Following shoot initiation, shoots are elongated on a medium without cytokinin. The addition of 0.5–1.0% activated charcoal may be beneficial. Once shoots have elongated sufficiently, they can be cut into nodal sections or topped to stimulate lateral side shoot or shoot clump development, which can then be separated and elongated. When sufficient multiplication has been achieved, the shoots can be stimulated to form roots by transferring them to a medium containing auxin. Rooting may be done in vitro or ex vitro, depending on the species. Venting of the culture container by using a hole in the container lid covered with a permeable membrane or cotton wool during the time in auxin medium may help acclimatization for transfer ex vitro. Similarly, the container lid may be left loosened or unwrapped to allow some gaseous exchange and exposure to ambient humidity. Once shoots are transferred ex vitro and have rooted, the humidity may be gradually reduced to ambient conditions in an acclimatization phase. There are a number of methods available for maintaining or storing of clones in tissue culture by organogenesis, including repeated subculture (serial propagation), minimal growth media, cool storage and cryopreservation. Radiata pine clones have been maintained as shoots for more than ten years with repeated subculture every 6–8 weeks (Horgan, Skudder and Holden, 1997). However, long-term success at halting ageing is uncertain and the costs are high because of the requirement for regular transfers and a controlled environment. Using diluted nutrient concentrations in the media does reduce the need for regular subculturing, and radiata pine shoots have been maintained successfully for four years at 20–22 °C with annual subculturing (Horgan, Skudder and Holden, 1997). Successful cryopreservation of organogenic material has proved to be more difficult. Cotyledons from radiata pine zygotic embryos have been successfully frozen and thawed (Hargreaves et al., 1999). Cryopreservation of axillary meristems is also being attempted (Hargreaves et al., 1997) and results are now very promising (Hargreaves and Menzies, 2007). Organogenesis methods have been developed for a large number of forestry species for large-scale production, including hardwoods such as poplars, willows and eucalypts, and for conifers such as coast redwoods, radiata pine (Pinus radiata), loblolly pine (Pinus taeda) and Douglas fir (Pseudotsuga menziesii). More detailed protocols for various hardwoods and conifers can be found in Bonga and Durzan (1987a, b) and Bajaj (1986, 1989, 1991). Embryogenesis Another micropropagation technology that has been more recently developed and has promising applications for clonal forestry is somatic embryogenesis. Successful embryogenesis was first reported for sweetgum (Liquidambar styraciflua) in 1980 (Sommer and Brown, 1980) and for spruce (Picea abies) in the mid-1980s (Hakman and von Arnold, 1985; Chalupa 1985). Since then, somatic embryogenesis has been 6 Forests and genetically modified trees investigated for many forestry species, including hardwoods such as poplars, willows and eucalypts, and conifers such as spruces, larch (Larix spp.), pines and Douglas fir. Embryogenesis differs from organogenesis in that somatic embryos are formed from embryogenically competent somatic cells in vitro, with both shoot and root axes, and these embryos will germinate, whereas with organogenesis shoots are developed, and these must be rooted as mini-cuttings. As in organogenesis, there are a number of stages for embryogenesis, involving initiation of embryogenic tissue, multiplication, development and maturation, germination and acclimatization. Typically, embryogenic tissue is established from immature seeds, just after fertilization, using either embryos within intact megagametophytes or excised embryos. Tissue can be maintained or multiplied in a relatively