Eucalyptus
Abstract
The genus Eucalyptus is currently an important source of timber, cellulose pulp, and energy in a world that is becoming increasingly more conscious of the need to use renewable resources. This chapter initially explains the importance of hybridization and cloning techniques in intensive breeding programs that occurred in Brazil during the last few decades. The strength of these programs is based on the selection of the genetic material and the evaluation of the factors that can influence the expected genetic gains in forestry breeding programs using seeds. Factors such as endogamy, floral development, pollen viability, and pollination are discussed together with seed vigor and the influence of environmental factors on correct evaluation of candidate material. The recent application of molecular markers in eucalypt breeding programs is also mentioned with particular emphasis on genetic diversity, genotyping, and their association with disease resistance. An introduction into Eucalyptus tissue culture is also given as a prelude to the current status of genetic transformation using biolistics and Agrobacterium tumefaciens in this genus. The final sections describe the current challenges, disease resistance, and wood quality for large-scale cultivation and utilization of eucalyptus wood as prime material.
1 Introduction
In the last century, the genus Eucalypts became an important multipurpose source of timber in many industrial applications for production of pulp and paper, charcoal, energy, furniture, and housing. The genus Eucalyptus is a member of the Myrtaceae family, mainly originated from Australia, comprising of more than 700 species (Brooker, 2000). The trees form tall open forests, woodlands, and occur in environments ranging from areas of high rainfall to semi-arid regions, and from sea level to subalpine altitudes. A few species have been described as occurring outside the Australian territory (Eldridge et al., 1994); Eucalyptus deglupta is endemic to the Indonesian islands of Sulawesi and Ceram, Mindanao in the southern Philippines, northern New Guinea, and New Britain; Eucalyptus urophylla, E. orophila, and E. wetarensis occur in Timor and adjoining islands of the Lesser Sunda group (Pryor et al., 1995). Several factors contributed to the success of Eucalypts; the fast growth rate and large biomass production, the ability to grow in a wide range of environments and soils, good wood quality for solid wood products and short cellulose fiber, suitable for pulp production, particularly for paper and tissue. A considerable number of Eucalyptus plantations were established in the early 1900s in Brazil and South Africa along the railway tracks providing charcoal for the locomotives. Today, several countries in Asia, South America, southern Europe, and Africa, have an estimated area of planted forest around 16–19 million hectares (FAO, 2000). The predominance species in commercial plantations (around 80%) are Eucalyptus grandis, E. globulus, E. camaldulensis, and their hybrids (Potts, 2004). E. grandis, E. urophylla, and their hybrids are mainly planted in tropical and subtropical regions, while E. globulus is preferred in temperate climates (Potts, 2004).
One important factor determining the expansion of large areas of commercial Eucalyptus plantations was the development of cloning techniques, initiated in 1975 in the Popular Republic of Congo (Delwaulle et al., 1983). Outstanding genotypes, usually hybrids, having large and straight trunk with good wood properties were selected to make rooted cuttings and planted in large extensions of land. Vegetative propagation has been intensively used by the pulp and paper industry, producing highly uniform timber and allowing further gains in productivity in the pulping process. In the mid 1970s the development of tissue culture techniques and in vitro propagation of Eucalyptus spp. provided new opportunities for mass propagation, on a commercial scale, of selected genotypes. At the same time, the growing importance of Eucalyptus for the pulp and paper industry, particularly in South America and Africa, led to the establishment of breeding programs selecting better hybrids for cloning and to improve the basic populations introduced from Australia.
In the last 10 years, the advances in tissue culture techniques, plant regeneration, and genetic transformation, using mainly the Agrobacterium system, allowed the development of the first transgenic Eucalyptus trees. Also, advances in the use of molecular markers have played an important role in helping breeders to select Eucalyptus trees with better wood quality, disease resistance, and stress tolerance. More recently, the development of genomic approaches, such as major expressed sequence tag (EST) sequencing programs, have increased the interest in the application of biotechnological tools in order to produce better Eucalyptus trees.
Our chapter provides an overview of the main topics involved in Eucalyptus biotechnology, from cloning and hybrids production, breeding, application of molecular markers, tissue culture and plant regeneration, genetic transformation, main diseases affecting the Eucalyptus plantations, wood quality for the pulp and paper industry, energy production and biomaterials, and also the identification of genes involved in disease resistance and wood formation through functional genomics.
1.1 Hybridization, Cloning, and Breeding
1.1.1 Hybridization and cloning of Eucalyptus spp.: evolution and its importance for the forest sector in Brazil
The development of forestry plantations for industrial purposes must be oriented to increasing industrial competitiveness in the distinct market segments. In such a scenario, forestry-based companies must take into account the influence that forestry raw material can have on their competitive capacity. The modern concept of competitiveness includes generating products to meet the customer's requirements at low costs, in a sustainable manner and with minimum environmental impact. Therefore, the development of tree breeding programs to obtain quick gains, and a well-established cloning system have become important. Vegetative propagation methods should rapidly transform the genetic gains, obtained through breeding, into benefits for the industry. One of the most efficient tools to acquire these objectives is the combination of interspecific hybridization and establishment of clonal forestry derived from superior hybrid individuals (Assis, 2000).
In this context, hybridization can have a great impact on tree breeding programs, combining superior wood characteristics with tolerance to biotic and abiotic stresses, thus representing a significant source of superior individuals, capable of introducing genetic gains into forest productivity and wood properties. Crossing species with different characteristics allows the production of complementary wood properties in trees to meet special industrial requirements.
The effective and quick integration of genetic gains obtained with hybrids into the industrial process depends basically on the existence of functional large-scale cloning systems. Mass vegetative propagation perfectly complements hybridization for producing clonal forestry and has some advantages over the sexual methods of mass reproduction of selected families, besides being the best way to commercially exploit the heterosis found in several Eucalyptus hybrid crosses. By capturing the total genetic variance (Zobel, 1992), vegetative propagation allows for maximum benefits from wood properties and productivity, besides allowing for the production of more uniform raw material, which from an industrial point of view is highly beneficial to the industrial process and product quality. Therefore, tree breeding programs that focus on these aspects of the forest industry will have a great impact on the three important components of the competitive process: productivity, product quality, and production costs (Assis, 2001).
Recognizing the importance of hybridization and cloning in the context of industrial production has led to a rapid evolution of the techniques and processes that constitute these activities. As a result, methods to produce hybrid seeds and commercial cloning are now well understood and highly effective from the technical and operational perspective in Eucalyptus species. Developing the concept of indoor breeding orchards and the creation of artificially induced protogyny (AIP) has made controlled crosses operationally feasible. Hybrid production by controlled pollination is thus a simple and functional process. Indoor breeding orchards do not require flower isolation, saving a lot of time. On the other hand, AIP enables pollination without the need to emasculate the flowers. These two technologies allow large-scale controlled crossing, which is technically difficult to carry out and also economically unfeasible using traditional methods.
In the cloning systems for commercial-scale propagation, the development of microcutting and mini-cutting to replace rooting stem cuttings led to the development of super-intensive systems to produce vegetative propagules, which can now be done in much more controlled environments, allowing greater physiological and phytosanitary control. The use of more appropriate propagation facilities, with better environmental control, has contributed to improve rooting rates. The development of these technologies for Eucalyptus marked the beginning of a new cycle in the commercial cloning of vegetatively propagated plants, especially for woody species (Assis, 2001).
In the last two decades, cloning Eucalyptus spp. has produced relevant progress for the forestry companies, especially solving problems associated with diseases such as canker (Cryphonectria cubensis) and productivity improvement (Campinhos and Ikemori, 1983). Currently, the focus on cloning has shifted to industrial requirements, rather than remaining limited to disease resistance and increased volume. Wood properties that positively influence industrial processes and product quality are considered, especially where cloning has an important role to play.
1.1.1.1 Historical and technical evolution of Eucalyptus hybridization and cloning in Brazil
1.1.1.1.1 Hybridization
Considering the length of forest production cycles, the commercial use of Eucalyptus hybrids is relatively recent in Brazil. Spontaneous hybrids randomly formed at the tree farm in Rio Claro, São Paulo, at the Companhia Paulista de Estradas de Ferro, provided the base for the first plantations for industrial purposes. These hybrids highlighted aspects of resistance to Eucalypt canker (Ferreira, 1997; Alfenas et al., 2004) and to the presence of heterosis at the individual level during the 1970s. Therefore, these hybrids were also the starting point for the introduction of the concept of clonal forestry in Brazil, during the same decade (Campinhos and Ikemori, 1980). In the 1980s, when the importance of hybrids for industry was recognized, hybridization became an essential tool, helping to create a strong and competitive forestry industry (Assis and Mafia, 2007).
The first synthetic Eucalyptus hybrids were produced in the 1970s by open-pollination between compatible species (Assis, 1985; Gomes, 1987; Martins and Ikemori, 1987). Since this technique was limited by crosses between species that flowered at the same time, in the 1980s a controlled crossing method was developed in South Africa (Hodgson, 1967; Van Wick, 1977). Although it was difficult to perform, it enabled the expansion of hybrids produced artificially in Brazil (Brigatti et al., 1983; Assis, 1985; Martins and Ikemori, 1987). This method is based on the exploitation of protandry where pollen reaches physiological maturity in anthesis, but the stigma is not receptive for another few days. To make controlled crosses using this technique, the flowers must be emasculated, isolated, and pollinated after 5–7 days. Therefore, the method has a low operational yield, mainly because of the number of visits needed to make the crosses (Harbard et al., 2000). Moreover, relatively high losses are caused by injury to the flower buds during emasculation and isolation, contributing to reduce the efficiency of the crossings.
In 1996, a new technique for controlled crossing was developed, called AIP. This new technique was initially developed at Riocell S.A. (Assis and Jardim, unpublished results) and its great advantage is higher operational yield and better use of the flower buds. This technique was conceived based on two discoveries made in Chile and in Portugal. OSP (one stop pollination) was developed in Chile (Harbard et al., 2000), allowing receptiveness induced by cutting lengthwise or cutting off the upper third of the pistil of emasculated flowers. In Portugal, it was found that this receptiveness could be achieved before anthesis (Trindade et al., 2001). AIP was developed by combining these two discoveries. This technique consists of the artificial transformation of protandry into protogyny, by cutting the top of the floral bud operculum. At the same time, the upper third of the pistil is cut during the preanthesis stage, i.e., when the flower is still closed, and pollen is applied immediately after induction. Thus, there is no need to emasculate the flowers to make crosses.
Later, this new technique was tested simultaneously in Brazil, at Aracruz Guaíba, and at Commonwealth Scientific and Industrial Research Organisation (CSIRO) in Australia (Assis et al., 2005) with the same success rate observed at both sites. Besides reducing the number of visits, AIP does away with the need for emasculation and made it possible to obtain more seeds in Eucalyptus-controlled crosses. Operational productivity rose from 35 to 400 flowers per man/hour using this technique.
At the same time as AIP was created, the concept of indoor breeding orchard was developed, in screened houses, doing away with the need to isolate individual flowers, umbels or branches, enabling the isolation of whole plants in a collective way (Assis et al., 2005). When some type of contamination is acceptable, this technique can be implemented in open areas that are not isolated. Currently it is the most widely used method for Eucalyptus-controlled crosses. The development of indoor breeding orchards was enabled by the development of technologies to induce early flowering in potted plants. Flower production in small plants is necessary to cultivate them within greenhouses and also to allow easier access to the floral buds. In order to obtain flowered plants in pots, grafting of physiologically adult branches is used combined with application of the flowering inductor paclobutrazol.
The combined use of these technologies (AIP and indoor breeding orchards) allows large-scale controlled crosses and enables the multiplication of highly superior full-sib families, a task formerly considered technically and economically impossible. This is very important to improve difficult-to-root species, since the superiority of pure or hybrid full-sib families can be captured and multiplied on a large scale. This technique is widely disseminated in forestry companies and now accounts for most controlled crosses performed (Assis and Mafia, 2007).
1.1.1.1.2 Cloning techniques
The first rooted eucalyptus cutting was obtained in Australia in the 1940s (Eldridge et al., 1993). During the next decade, studies carried out by French researchers in the Congo, Tunisia, and Morocco played an important role in understanding physiological phenomena involved in the rooting of Eucalyptus, such as juvenility and maturation, as well as aspects of rejuvenation in Eucalyptus and their importance in cloning adult trees. This knowledge was important to establish the concept of Eucalyptus clonal forests, which occurred in the 1970s in Tunisia (Chaperon, 1987) and in Aracruz in Brazil (Campinhos and Ikemori, 1980). However, the first large-scale cloning system was established in Brazil, by Aracruz, and it served as a model for the development of clonal forests, whose use was extended to other Brazilian companies and to several parts of the world. After the success achieved by Aracruz, it was quickly disseminated, and now it is the main system for the reproduction of Eucalyptus species and clones.
Until the 1990s, the development of cloning techniques for Eucalyptus species evolved relatively slowly. For over a decade, the commercial production of vegetative propagules continued to be done by the original system, in clonal banks, before being replaced by the clonal hedges system. On the other hand, the techniques initially used for the stem cutting rooting of Eucalyptus, as well as the rooting facilities, remained practically the same until the early 1990s. In 1992, the first great change in the rooting technique occurred when microcutting (Assis et al., 1992; Xavier and Comério, 1996) and later mini-cutting were created (Assis, 1996; Higashi et al., 2000).
Microcutting is a rooting method where the propagules are obtained from shoot apices originating from micropropagated plants and in mini-cutting originating from auxiliary sprouts of plants cloned by stem cuttings. After rooting of the first shoot, the two techniques are identical, varying only in the origin of the initial propagule source. In some clones using mini-cutting, some propagation cycles (serial propagation) are required to reactivate before acquiring full rooting capacity potential. In microcuttings such propagation cycles come naturally by monthly in vitro subculturing of explants. Micropropagation is unnecessary for easy-to-root species because high levels of juvenility can be obtained easily by inducing basal shoots, therefore in such cases, mini-cutting is technically and economically feasible.
When plants are established in the mini-clonal hedges to form the mini-stumps by whatever means, multiplication is performed using the mini-cutting technique (Assis and Mafia, 2007). Microcutting and mini-cutting are the most modern concepts for cloning Eucalyptus species in large scale and they are currently the most widely used techniques in Brazil. The creation of these technologies was the watershed in the evolution of Eucalyptus cloning systems and they resulted in profound changes in the main technical segments that constitute this activity. It marked the beginning of a new cycle in the propagation of woody species.
A major consequence of creating microcutting and mini-cutting was the great change in the methods to produce vegetative propagules in large scale. The clonal hedges that had replaced the original clonal banks system at the beginning of the 1980s were replaced by the mini-clonal hedges. Propagule production began to be performed in a super-intensive manner, supported by hydroponic systems in controlled environments (indoor mini-clonal hedges). Currently, two hydroponic systems are used: sand bed with drip irrigation (Higashi et al., 2000) and intermittent flooding (Campinhos et al., 2000).
The development of these technologies also influenced changes in the conception of rooting facilities that were important for their evolution and improvement. When Eucalyptus cloning attained industrial proportions, most of the physical structures used to root stem cuttings were extremely simple, consisting of partial shading and a misting system. When the mini-cutting technology began to be used, greenhouses had to be used to achieve greater control over the rooting environment. Their importance in rooting became clear and led to their widespread use. Therefore, at the same time as rooting methods were developed, a major evolution occurred in the propagation structures. On the other hand, the development of super-intensive vegetative propagule production systems in hydroponic mini-clonal hedges, with better nutritional and phytosanitary control of the mother plants, is technically and economically advantageous in the commercial production of Eucalyptus plantlets. Currently, mini-cutting is the cloning technique most widely used by the large Brazilian forestry companies.
In easy-to-root hybrid clones, micropropagation is not commonly used as a rejuvenation method or simply as a method to rapidly broaden the clonal base of recently selected clones. Except in the systems that use microcutting, where it is a mandatory phase, mini-cutting from sprouts of rooted stem cuttings has worked well for these species and the tendency is that micropropagation will no longer be used for this purpose, especially due to its high costs. Nevertheless, there are technical advantages in using micropropagated plants to form mini-clonal hedges, even in easy-to-root clones. In clones that are difficult to root, micropropagation is still a technique that can potentially increase predisposition to rooting. This class includes E. globulus hybrid clones that are increasingly used in plantations for pulp production.
1.1.1.2 Importance of hybridization and cloning in Brazil
In recent years, the growing integration between the different segments of industrial production based on planting forests has been a major factor in the development of technologies dealing with forest production for industrial purposes. The greater proximity between forestry, industry, and commerce has enabled an integrated view of the segments: forest × industrial process × product quality, which is the base for acquiring competitive differentials in industries. In this way, new prospects arose in the development of technologies that could promote a positive impact on the industrial production chain and, consequently, on industry performance. On the other hand, the perception of the great economic gains by industry as a result of the increased quality of raw materials has significantly enhanced the genetic improvement programs, above all due to their capacity to promote quantitative and qualitative changes in the raw material.
In this context, increasing forest productivity and improvement of wood properties for industrial use are important demands on Eucalyptus breeding programs in Brazil. The main objectives of these demands are the reduction of operational costs, improving raw material performance in industrial processing, and rendering it appropriate for the manufacture of high quality products for different market segments (Assis, 2000). The main contribution of genetic improvement for the forestry-based industry is the generation of superior individuals that can lead to gains in forest productivity, industrial process, and product quality. The greatest challenges for tree breeders have been to use effective strategies to obtain individuals that are superior in both growth and wood quality. The main obstacles to overcome these challenges are that the species traditionally planted in Brazil present certain limitations concerning the wood quality, compared with other Eucalyptus species. In this context, interspecific hybridization is a very useful tool, especially since it allows transfer of genes from species with high wood quality but slow growth, to species that have an inferior wood quality but are well adapted to the planting sites and with excellent growth. The main advantage of hybridization, besides the capacity to allow the combination of differentiated characteristics in distinct species, is the possibility of producing trees with superior growth as a result of heterosis or hybrid vigor, a common phenomenon in Eucalyptus (Martin, 1989; Denison and Kietzka, 1992; Nikles, 1992).
The differences between the various species of Eucalyptus adapted to this country, with respect to wood properties, tolerance to biotic and abiotic stresses, as well as to the manifestation of heterosis has been the main factor to produce individuals with superior growth, adaptation, and wood quality, through hybridization. Thus, the shortest path between industrial demands and their fulfillment by genetic improvement programs is to seek complementarity among species, by increasing the number of desirable characteristics of the wood, combined with the commercial utilization of heterosis. Enhancement of the forest quality by the commercial use of heterosis, through the vegetative propagation of Eucalyptus hybrids in Brazil is clear and there is evidence that it is a very useful phenomenon for a faster gain in forest productivity. In this sense, the contribution of Eucalyptus hybridization to forest development in Brazil is very significant. Most benefits from hybrid use, especially in forest productivity, are accredited to the manifestation of heterosis for growth and to the complementarity of certain species concerning characteristics that combine to produce more appropriate genotypes for the different planting environments (Assis, 2000).
Currently, the vast majority of Eucalyptus improvement programs in Brazil are based on hybrids. In 2005, 84.5% of the clonal plantings of Eucalyptus in Brazil were hybrids, where 66.5% of which were hybrids from controlled crosses. The hybrid E. grandis × E. urophylla is most often used, with 65% of the planted area. Fifteen percent of the remainder is still natural and spontaneous E. urophylla hybrids, 3% Rio Claro hybrids, 0.5% E. urophylla × E. camaldulensis, 0.4% E. urophylla × E. globulus and 0.6% E. camaldulensis × E. grandis (Assis, 2004).
Wood properties are very important for industry because they have an impact on processing costs, production gains, and the technological qualification of the products and are therefore essential in production processes of raw material for industrial use. In this sense, hybridization tends to play an increasingly important role in the Eucalyptus planting programs in Brazil. Because of the success achieved in southern Brazil concerning the incorporation of E. globulus genes into site-adapted species and clones, mainly due to the significant enhancement of the wood quality to make pulp, there is a tendency to seek the source of wood quality in this crossing, by pulp companies. A major breakthrough is expected in wood quality to make short-fiber pulp using E. globulus hybrids.
From the perspective of industrial processes, E. globulus is the most appreciated Eucalyptus species by the pulp mills, because its wood presents technological properties that are especially important for the pulping process. Either because it provides greater production gains, or due to the lower manufacturing costs, its wood tends to be a major competitive differential point for the companies. Evaluations performed on E. globulus hybrids show that it is possible to maintain the same forest productivity levels obtained in E. grandis, E. saligna, E. dunnii, E. urophylla and several of their hybrids, but with positive changes in wood and pulp quality. Compared to the traditional genetic materials, these hybrids allow increasing wood density; reduction of 4% in lignin content; a 3% increase in pulp yield; 3% increase in hemicellulose content; 1m3 reduction of wood per ton of pulp in specific consumption and a 5kg reduction of the chlorine dioxide per ton of cellulose in bleaching, among others.
The forests that are being planted using the new genetic materials from this new type of crossing will provide major gains for the industries, significantly improving their competitive capacity on the short-fiber pulp market. Together with the gains in production and cost reduction, the variability found in the selected individuals also allows the obtention of fibers with anatomical characteristics that are appropriate for different market segments, allowing the qualification of pulp for each specific product.
The efficiency of hybridization to produce genetic gains is fully acknowledged, being used by most forestry companies in Brazil. However, since the hybrid offspring are heterogeneous, transforming these gains quickly and effectively into benefits for the industry depends on functional, large-scale cloning methods. Thus, if on the one hand, interspecific hybridization in Eucalyptus has been the fastest way to achieve genetic gains, cloning on the other hand is the most efficient way of incorporate these gains into the industrial production processes. Cloning as a tool for the implementation of clonal forests derived from high-quality hybrids, is still the ideal technical complement to maximize the benefits of hybridization in the context of forest production for industrial purposes. Currently its maximum potential has been achieved in establishing clonal forests derived from controlled interspecific hybrids, which produce better quality wood, greater volume, growth, and higher resistance to biotic and abiotic factors. In this way, cloning plays a major role and for this reason it has received much attention in forestry research programs in Brazil. The consolidation of cloning as a method for the commercial reproduction of superior Eucalyptus trees had a positive effect on productivity, costs of industrial processes, and product quality. Cloning Eucalyptus in large scale is currently one of the most important factors in promoting increased productivity, improvement, and homogenization of wood technological properties as an industrial raw material (Assis et al., 2004).
Almost 30 years after the introduction of the concept of clonal forests in Brazil, cloning is definitely a part of the raw material production processes used in various sectors of industrial activity, above all pulp and paper and charcoal, which account for 78% and 21% of the area planted with clones, respectively. In 2004, the area planted with clones surpassed 1000000ha. The current annual rate of new clonal planting is over 250000ha, with a tendency for further growth.
Brazilian clonal forestry was initially developed from spontaneous hybrids and natural hybrids presenting heterosis for growth, besides resistance to diseases, above all Eucalyptus canker. Although these hybrids were produced empirically, they were responsible for the great breakthrough in forest productivity that occurred in recent years. Forest productivity was increased threefold by cloning them. Currently the clonal forests are derived from individuals generated in genetic improvement programs, whose main strategy is based on the production of controlled interspecific hybrids. The cellulose increment (AMI cell) was around 6tha−1year−1 in the 1970s. This productivity is now around 12tha−1year−1 for the leading companies in this industry. Using E. globulus in the composition of hybrids, it is expected that trees planted from 2010 onward will achieve productivities of 16tha−1year−1.
1.1.1.3 Future perspectives
Due to the current importance of hybridization and cloning for industrial forests, their use has been consolidated and they are increasingly becoming part of the forest-based industrial production processes. The progress achieved in the different techniques involving these activities will ensure new levels of quality and constitute new platforms for the development of Brazilian forestry.
The advances that have occurred in controlled pollination techniques, for instance, will allow the intense use of hybrids in breeding programs of the industries that use Eucalyptus as raw material. On the other hand, hybrid production will be increasingly oriented to crosses with E. globulus and its subspecies, mainly because of the quality of this wood for pulp production. The use of adapted species and clones, combined with this species, has allowed a significant improvement in the wood quality, in environments where the pure species is not well adapted. This will allow the use of E. globulus hybrids both in subtropical and tropical areas, with significant gains in wood quality.
Although there has been significant progress in techniques and Eucalyptus rooting methods culminating in a robust, functional, well-established system, several studies on cloning are being performed to develop new systems that could be technically and economically more appropriate than the current ones. Propagule production methods evolved from an extensive system, with low propagule production per square meter, passing through an intensive system in clonal gardens until they reached the present super-intensive systems, with high propagule productivity per square meter in the mini-clonal hedges supported by hydroponics. The search for new advances in this field is directly related to establishing production systems for mini-propagules in bioreactors, using minimum space and with a significantly higher propagule production per square meter. A further advantage of these technologies is that they use highly rejuvenated tissues, which may provide better technical results both for plant production and for the quality of the clonal forests.
1.2 Factors Affecting Expected Genetic Gains in Forest Breeding Programs Using Seeds
Genetic improvement has the main objective of obtaining genotypes, through selection, that present adaptations to environmental conditions, resistance to disease and insects, characteristics desirable for productivity, and the multiple uses of wood. Many species of the genera Eucalyptus and Pinus have been intensely used in reforestation programs in Brazil and many other countries. The importance of Eucalyptus seeds of the best quality, from the best origins and the establishment of test species, seeds and progeny allowed the selection of genetic material with the potential for adaptation and productivity under the edaphoclimatic conditions found in different regions of Brazil.
The beginning of any breeding program is the selection of the species and populations to be used, by species tests and the origin of the desired characteristics (Resende et al., 2005). The strategies for genetic improvement of exotic forest species has been based on the selection of species/origins and the selection of individuals from the base population, exploring the natural genetic variability existing amongst populations and individuals (Ferreira, 1992). Vencovsky and Barriga 1992 concluded that the knowledge of the type of genetic action that predominates in the genetic basis of a character is an important ingredient in an efficient breeding program. Gene effects are classified into two basic types, allelic interactions (dominance interactions between alleles at an interlocus level) and nonallelic (epistatic actions that arise when alleles or genotypes are influenced by the genetic constitution of other loci).
According to Resende et al. 2005, based on experimental evaluation, the selection should be based on means and variance, recommending that the selected material should have an elevated mean and ample genetic variability, allowing continuous gains with selection through various generations. A way to estimate the progress of the selection process is very important when using quantitative genetics in plant improvement (Vencovsky and Barriga, 1992). By the general expression of gain (ΔG) “ΔG = ds × h2”, it can be observed that the progress is a direct function of the magnitude of the differential selection (ds = difference between the mean of the selected group and the mean of the original population) and of the heritability coefficient (h2) (Wright, 1976; Zobel and Talbert, 1984). The expected progress varies in accordance with the selection scheme and is detailed by Shelbourne 1969, Vencovsky 1978, Vencovsky and Barriga 1992, and Cruz and Regazzi 2001.
Ferreira 1985 stated that the gains obtained depend on the standard used for comparison (selected material or not), the effect of age on the evaluation, and environmental variation at the sites where the progeny were tested. Sometimes the expected genetic gain at a determined phase of the improvement program cannot be achieved. Factors related to the management of the improved populations, factors related to the production of seeds and saplings, genotype × environment interaction and others, can contribute negatively and these factors are discussed individually below.
1.2.1 Selection of genetic material
The selection based on indices has been used in forest improvement, offering larger gains when compared to other selection methods (Cotterill, 1986). The utilization of selection indices in Eucalyptus spp. improvement requires the estimation of a series of parameters, indices, and progress with selection, to permit the breeder to choose an index adequate for each situation (Resende et al., 1990). Resende et al. 2005 states that selection methods applicable to the perennial species can be classified in accordance with the selection units and the procedure used to predict the genetic values. According to the author, the procedure currently used to estimate the variation components and predict the genetic values is AM-BLUP (animal model, evaluation of genetic variation) associated with DFREML (derivative free maximum likelihood).
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Relationship between the flowering period, the typical qualities of the trees, and the behavior of the respective progenies (material from Coff's Harbour). It was observed that the same trees that originated from inferior progenies presented early flowering and atypical fruits, demonstrating the occurrence of hybrids in the selected material and a loss in productivity due to the heterogeneity in the trees.
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Correlation of the clonal behavior with the behavior of the progenies using diameter at-breast-height (DBH) and height data and comparisons with the inheritance estimates of the progenies (material from Zimbabwe). A clone-progeny correlation for DBH (r = 0.020) was very weak (Figure 1) and low for height (r = 0.33) (Figure 2), this shows that clones with good performance do not always present good progenies. Gene dominance could favor the selection of heterozygotic trees and heterogenic descendents with low productivity.
Correlation between the DBH values (cm) for progenies (2 years) × clones (3.5 years)
[Source: Oda et al., 1989]
This study shows that intense selection can reduce the effective size of the population, leading to the presence of different degrees of endogamy, demonstrating the loss of vigor in the saplings originating for endogamous crosses. In relation to the concept of the effective population size, Vencovsky 1987 discussed the genetic representativity contained within a sample in relation to immediately previous generation. Thus, the effective size is related to the genetic size of the population and not the number of individuals within the population (Resende and Vencovsky, 1990).
Correlation between the height (m) for progenies (2 years) × clones (3.5 years)
[Source: Oda et al., 1989]
1.2.2 Correlations and correlated responses between characteristics
The study of the correlations between characteristics is important in genetic improvement, because the general preoccupation is to improve the material, not only for isolated characteristics but simultaneously for a group of characteristics (Vencovsky, 1978). According to the author, the expected alteration in the mean of a determined characteristic Y, when the selection is for characteristic X is a response correlated with the system of selection considered. The correlation visualized directly in an experiment is phenotypic in nature, provoked by two factors: genetic and environmental (Vencovsky and Barriga, 1992). In genetic improvement it is important to know the association between characteristics, especially when the selection of one of them presents difficulties, such as low heritability or in the measurement or identification (Cruz and Regazzi, 2001). According to the authors, in genetic studies it is important to differentiate and quantify the degree of genetic and environmental association between the characteristics, such that the genetic correlations are of a heritable nature and as such usable in breeding programs.
Cruz and Regazzi 2001 discussing simultaneous selection of characteristics stated that the use of the index theory of selection is an efficient alternative, permitting the combination of multiple information contained within the experimental unit and making possible selection based on a number of variables that unite various attributes of economic importance. The authors reviewed various proposals to obtain selection indices (classic index proposed by Smith, 1936 and Hazel, 1943; index based on desired gains; base index; index based on the sum the ranks; “weight free” and “parameter free” index).
In Pinus radiata, Dean et al. 1983 studied 30 families of half-siblings and detected negative correlations between volume and wood basic density. The gain estimates for these characteristics, combined into one index, revealed that it is not possible to improve wood density and volume simultaneously. Moraes 1987 detected a low negative genetic correlation between growth characteristics and wood basic density in open-pollinated E. grandis progeny. However, this correlation was not sufficient to seriously weaken the possibility of gains in wood basic density.
Reviewing other genetic and phenotypic correlations between wood basic density and growth characteristics, Moraes 1987 observed the existence of high correlations between height, DBH, and volume, however a correlation between these characteristics and basic wood density was of different magnitudes, both positive and negative. For Sturion 1993 these contrasting results demonstrate that generalization could provoke losses in a selection process, depending on the adopted strategy.
1.2.3 Endogamy
According to Wright 1976, endogamy signifies the crossing of related individuals or descendents of the same individual. The most extreme case of endogamy is self-fertilization; however, there are various degrees of endogamy before this, such as crossing between siblings and members of small isolated populations. The author further states that endogamy does not always cause a reduction in vigor or depression by endogamy, which is caused by the accumulation of recessive deleterious genes and not solely because of self-fertilization.
Eldridge 1978, working with the proportion of self-fertilization and open-crossing in Eucalypts, confirmed predominance for open-crosses, although this proportion differed between species. Reddy and Rockwood 1989 reported that the quantity of self-fertilization in Eucalypts is higher than 7%. Estimates for the degree of natural self-fertilization vary among the species of Eucalyptus: 24% in E. obliqua (Brown et al., 1975), 18% in E. stoatei (Hopper and Moran, 1981) and 10–38% in E. grandis (Hodgson, 1976a).
Hodgson 1976b studied some aspects of flowering and the reproductive system in E. grandis and observed that progenies from self-fertilization were subject to various forms of endogamic depression, including deformation of the leaves and stem, reduction in vigor in comparison with progenies from open-crosses. The author concluded that the product of a seed producing plantation suffers a degree of reduction because of the quantity of self-fertilization, estimated at 30% (Van Wyk, 1981).
Various studies were carried out in order to verify the effects of different degrees of endogamy in Eucalypts, such as Van Wyk 1981, Eldridge and Griffin 1983, Maêda 1987, Griffin and Cotterill 1988, and Hardner and Potts 1995. Eldridge 1976 stated that endogamic depression in Eucalyptus is expressed as low growth of the saplings in the field and low viability of their seeds. Eldridge and Griffin 1983 working with Eucalyptus regnans, observed that the number of seeds varied from 42 per 100 in self-fertilized to 71 seeds in open-pollinated and 90 seeds in cross-pollinated flowers.
Irregular flowering and a deficiency in pollination are factors that can provoke endogamy. The presence of endogamy causes various prejudicial effects in Eucalypts, including deformed leaves and stems, reduction in vigor of the saplings, reduced height, reduced survival of saplings in the field, and reduction in the production of viable seeds.
1.2.4 Progeny tests
Individuals selected using an estimation of genetic parameters are then analyzed using progeny tests to ascertain their reproductive value as seed producers and later used to form seed producing plantations (Kageyama, 1980). Shimizu et al. 1982 presented the procedures and recommendations for the installation of progeny tests.
The best way to evaluate the genotypic value of the selected trees is through their progenies, permitting the estimation of their value as progenitors (Weir, 1977). This procedure permits the separation of trees with phenotypic superiority caused by the good planting conditions from those that present genotypic superiority (Allard, 1971; Weir, 1977). Hodge et al. 1996 compared the genetic parameters of progenies by open-pollination and controlled pollination, using E. globulus and E. nitens, and evaluated using two types of populations resulting from open-pollination (native population and a seed producing plantation) and two types of populations resulting from controlled pollination (using families of siblings from self-pollination and pollination between individuals). For E. nitens two populations were evaluated, families from the seed producing plantation using open-pollination and self-pollinated sibling families. The progeny tests were established at various sites and after 2 years height, DBH, and cylindrical volume were measured. The authors observed that early estimates of heritability for the open-pollinated progenies derived from native populations could be exaggerated, probably due to the high degree of endogamous depression. It was concluded that genetic gain estimates of open-pollinated populations can be overestimated because of (1) inflated heritability estimates and (2) flaws in the evaluation genotype × environment interaction.
One point to be discussed during the installation of the progeny test is sapling preparation. Generally the selection of saplings is made in order to make the forest more productive and homogenous. The appearance of abnormal saplings could be linked to endogamous effects; however Nogueira 2005 observed that a controlled cross carried out using two unrelated E. grandis trees, resulted in progeny with a 3:1 proportion of normal and abnormal individuals, respectively. The study identified two random amplified polymorphic DNA (RAPD) markers linked to the principal gene that causes the abnormal phenotype.
The preparation of saplings for the installation of progeny tests for E. grandis was studied by Mello et al. 1993. The authors studied the influence of two systems for the utilization of seeds on the behavior of 14 progenies and three controls, using two methods of sapling preparation: (a) seeded directly into tubes, thinned and selected and (b) seeded into a germinator, planted and utilize all the seedlings. At 2.7 years of age, the results indicated that significant differences exist between the two systems. The progenies presented superior DBH, basal area, and cylindrical volume when submitted to sapling selection in the nursery. It was also observed that there was a significant interaction between the progenies and the sapling production system.
1.2.5 Seed production
When the seed production plantation is established, it is expected that the seeds produced represent the desired crosses, and it is important that the pollination is effective between the genetic material present in the plantation. Mora et al. 1981 reviewed the aspects of seed production in forest species, citing various authors who emphasize that abundant flowering and the production of good quality seeds depend on various factors related to the processes of pollination, pollen germination, pollen tube growth, fertilization, and embryo development. Thus, the success of the plantation depends, among other things, on the quality of the pollen produced and germination efficiency. Contrasting differences in the germination efficiency of the pollen between the selected genetic materials in the plantation, would suggest that not all the materials will participate equally as the masculine progenitor. Thus, it is important to evaluate pollen viability in the trees selected as part of the breeding matrices and to detect male sterility. It is also important to study tree management techniques that influence pollen quality.
Weir and Zobel 1975 discussed seed production plantations for selected material. According to the authors, the strategy to develop and test material adequate for future generations should be elaborated early, during the first generation of the improvement program. If the program is of long duration, the consequences of a restricted genetic base and associated risk of endogamy should be avoided by the continuous inclusion of new genetic material within the population. Thus, the ideal way to select the maximum number of unrelated individuals for advanced generations is diallelic or partial diallelic, both give good estimates of the parental value of the clones, and general and specific combining abilities of the parent trees.
Kellison 1969 stated that due to the fact that seed productivity is dependent on environmental variables, the geographic location, plantation area, and clone dispersal should be considered before a seed producing plantation is installed. For the installation of plantations for the production of hybrid seeds by open-pollination, the following points should be considered: (a) that the species have coincident flowering; (b) the ratio of pollen receptors to pollen donors should be adequate to favor hybridization; (c) the pollen receptor species should have a shorter period of flowering in relation to the pollen donor species; (d) the pollen receptor species should be relatively or completely self-incompatible; and (e) the selection of hybrid saplings in the nursery is essential and should be preceded by studies on the architecture and inheritance of the characteristics of interest to assess its operational viability. The hybrid seeds produced by controlled pollination and the progeny tests are the base of this study.
Basic studies such as the phenology of the selected clones, determination of the general and specific combining abilities and self-compatibility studies are necessary to conduct a prudent improvement program in order to achieve the pre-established objectives.
1.2.6 Pollen viability
Studies on Eucalyptus pollen, in general, aim at developing techniques adequate for pollen management, including genetic conservation, controlled production of hybrids, and the determination of pollen quality/viability. Sousa and Pinto Jr. 1993 reported the existence of intra- and interspecific differences in pollen germination; these could be genetic or physiological in origin, suggesting the need to develop specific culture media that imitate natural conditions. Studies on the in vitro germination of Eucalyptus pollen were carried out by Boden 1958, Gabrielli et al. 1965, Borges et al. 1973, Griffin et al. 1982, Cangiani 1988, Sousa 1988, Menck et al. 1990, and Sousa and Pinto Jr. 1993.
Sousa 1988 stated that the nutritional state of the plant during pollen development could affect longevity. Due to the general opinion that boron is necessary and germination efficiency is poor in media with only sucrose, Sousa 1988 studied the effect of boron and calcium on E. urophylla and E. tereticornis pollen germination. The authors concluded that boron was necessary although they were unable to define the exact concentration. Therefore, the nutritional status of the seed producing trees, with particular respect to boron, is very important. On the other hand, calcium doses of 220ppm improved the germination efficiency of the two species tested. It would be interesting to observe the effect of boron supplements in the seed production plantations with respect to pollen viability and seed quality.
Suzano Company evaluated 55 E. grandis matrices of a second-generation clonal seed production plantation, from different origins, that are part of Suzano's breeding program. Branches containing flowers were collected, the open flowers and the green buds were removed, and the branch was placed in a recipient with water and left in the greenhouse. The buds in anthesis and the open flowers were cut from the branches and the stamens were removed for pollen extraction. A large variation in the efficiency of pollen germination was observed between the trees analyzed, the amplitude of the variation was from 0.15 to 93.96%, with a mean of 44.77%. This variation in the efficiency of pollen germination was also observed by Boden 1958. As these trees are part of the Suzano's clonal seed orchard, second generation, this data is important to evaluate the pollination among the selected genetic material. Thus, variations in pollen germination efficiency will influence the pollination within the plantation and as such influence the genetic quality of the seed. To investigate pollen germination efficiency between locations, the variation in five different locations was determined: Location 1 = 2.56–92.45%, Location 2 = 10.78–69.92%, Location 3 = 25.03–79.17%, Location 4 = 0.15–82.74%, and Location 5 = 1.22–93.96%. From these data, it can be observed that there is an ample variation in germination efficiency within the progenies used, with the range of efficiencies varying with location probably due to physiological/environmental factors. Thus, any variation detected at the same location should be due to genetic effect.
According to Eldridge 1976 there are only a few examples of male sterility in Eucalypts. The detection of these individuals is important since these trees will not participate as pollen donors.
1.2.7 Pollination
In natural populations, Eucalypts exhibit a mixed crossing system, but predominantly allogamy (Griffin, 1989). The pollination of the genus Eucalyptus is mainly entomophilic with the principal pollinator being the bee, Apis mellifera L. (Pacheco, 1982). Novelli et al. 1982 investigated the influence of bee pollination on seed production in a clonal plantation of Eucalyptus citriodora and demonstrated an increase in the number of viable seeds per kilogram and the number of capsules per panicle. Pacheco 1982 studied the effect of bee hives (A. mellifera L.) in a seed producing plantation of E. saligna on production and quality of the seeds. The pollen from the trees in the center of the plantation were labeled using P32 and it was shown that the number of flowers containing labeled pollen decreased with the increase in distance from the source up to a distance of 300m. Other results demonstrated a gradual reduction in the number of seeds per fruit with an increase in distance from the hive. At 0–50m, the number of seeds was 40% higher than that from 300 to 350m from the hive, with the authors demonstrating pollination activity of the bees up to a distance of 350m from the hive.
Maêda 1987 estimated for an E. grandis seed producing plantation, the fertilization rate for the characteristic that produces albino plantlets. The observed rate was 5.57, which represented a coefficient of endogamy of 0.03. The author considered this rate to be low and the variability of the plantation was maintained by efficient insect pollination. Although there appeared to be a large number of insects present on the plants, only a few effectively participated in the movement of pollen between flowers. When the quantity of pollen transported, visiting frequency and movement among the flowers was observed, A. mellifera was confirmed as the most important insect in E. grandis pollination.
According to Griffin 1989, pollen is frequently transported to larger distances than those inferred by direct observation of the insect flight behavior. The pollen collected from one flower is not necessarily deposited in the next to be visited. The biological aspects of the Eucalyptus flower suggests that (a) feeding behavior is not strongly conditioned by flower structure, therefore sticky pollen could be distributed over the whole insect, not solely the part that enters into contact with the stigmas and (b) pollen can maintain its viability for approximately 8 days on the insect's body, indicating that the transported pollen remains functional during this time.
Barbour et al. 2006 evaluated the phenology of flowering in species of the subgenus Symphyomyrtus native to Tasmania and E. nitens, introduced species. The difference in the flowering period of the native and an introduced species was highlighted as the principal barrier to gene flow by pollen. The authors state that the results show the importance of knowing the factors that affect gene flow, with the objective of identifying species and populations, native and exotic, with the highest risk of gene flow.
Griffin and Hand 1979 stated that knowledge about the receptiveness of the stigma, besides the processes of floral development and pollen release, is fundamental to the study of reproductive biology. Sousa and Pinto Jr. 1993 studied stigma receptiveness in E. dunnii in order to suggest ways to improve the efficiency of controlled pollination. The authors observed that for maximum efficiency, consequently better seed production, pollination should be carried out on the 6th day after anthesis. The receptive period for Eucalyptus stigma can vary within the subgenus (Pryor, 1951), between species of the same subgenus (Griffin and Hand, 1979) and within the same species. Griffin and Hand 1979 highlighted that there could be differences in floral receptiveness within the same tree. The similarity in floral development rates of different species within the same subgenus Symphyomyrtus, but growing in different hemispheres, suggests to the authors that this process is under strong genetic control.
According to Eldridge 1976, the flowers of Eucalyptus are hermaphrodite (male and female in the one flower), because protandry has been observed in some species, with examples of self-incompatibility and male sterility; however, allogamy can be considered as predominant in the genus. Although protandry occurs, this does not eliminate the possibility of self-fertilization, since flowering in the canopy lasts longer than the receptive period of a flower.
Kageyama 1979 studied the isolation of seed production areas, trying to minimize undesirable pollination problems because of the facility in intercrossing between large numbers of Eucalyptus species. The fact that pollination is predominantly entomophilic within the genus highlights the need to take care in the minimal distance between plantations to prevent possible contamination. A minimum distance of 200m between seed production areas is recommended. The authors also suggested the use of barriers consisting of other genera or unimportant Eucalyptus species to effectively isolate the seed production areas.
1.2.8 Floral development, phenology of flowering, and fruit formation in Eucalypts
The period and intensity of flowering varies within and between species, especially where they grow as exotic plants (Griffin, 1989). This could cause two sorts of problems: firstly, when the populations selected for improvement contain material from different origins they could function as a series of subpopulations partially isolated because of the different flowering periods, decreasing the effective size of the population and increasing the risk of endogamy; secondly, variations in the interaction between the species and the insect vector could affect the pollination efficiency between members of the population.
Studies on floral development and flowering/fruiting intensities in Eucalyptus populations have been carried out by Moggi 1959 with E. camaldulensis, E. resinifera, E. rudis, E. tereticornis, E. botryoides, and E. gomphocephala; Hodgson (1976a, 1976b) with E. grandis; Ashton 1975 with E. regnans; Ferreira 1977 with E. grandis; Mora and Ferreira 1978 with E. urophylla; Aguiar and Kageyama 1987 with E. grandis; Graça 1987 with E. dunnii; and Souza 1996 with E. camaldulensis. Mora and Ferreira 1978 studied the flowering of E. urophylla clones in Piracicaba (SP, Brazil). For each clone a branch was chosen to represent flowering in order to follow floral development in accordance with the following stages: (a) inflorescence formation, without individualization of the floral buds until the first operculum falls; (b) from the fall of the first operculum to the fall of the second; (c) flowering (opening of the floral bud); (d) fruit development; and (e) natural seed release. Proposing a vision more quantitative, percentages were calculated related to the production and development of the flower buds, flowers, and fruits. The authors observed that, in general, from the initial development of the buds to flowering takes 4–5 months and fruit formation to natural seed release 7–8 months. The natural loss of floral buds during flowering, fertilization, and fruit formation was 54.85%. The flowering period varied among the clones and could be put into the following categories: clones that did not flower during the experimental period; clones with an extremely short flowering period; clones that flowered almost the whole year; clones with a flowering peak from November to February; clones that flower from April to August; and clones with incompatible flowering characteristics.
Mori et al. 1988b suggested that to obtain maximum production, it is necessary to know the factors that affect productivity and use management techniques to alleviate these problems. Thus, basic knowledge about the biological process involved in seed production is important to predict and study the factors that could reduce productivity (Mora et al., 1981).
The variations in the number of viable seeds per kilogram could be related to the pollination efficiency, frequently weakened by the high variability in flowering or restricted genetic bases are shown in Table 1.
Degree of genetic improvement |
Number of viable seeds (kg−1) |
|---|---|
SCA (seed collection area)—Austrália |
400000 to 800000 |
SCA—Brasil |
800000 to 1000000 |
SSO (seed orchard by seeds) |
80000 to 400000 |
CSo (clonal seed orchard) |
500000 to 1000000 |
1.2.9 Seed vigor and size, germination and initial development of the seedlings
The level of physiological quality of the seeds should be evaluated through their capacity to germinate and seedling vigor (Popinigis, 1977; Aguiar, 1984). Popinigis 1977 suggested an index for the speed of emergence (SEI) in the determination of relative vigor between lots of seeds and an index for the speed of germination (SGI).
Valeri et al. 1984 commented that previously published papers demonstrate that the size of the seed is one of the factors that affect germination, with larger seeds producing more vigorous plantlets that develop faster. Thus, a classification by size would reduce losses associated with the less vigorous plantlets produced by small seeds that are quickly dominated by the plantlets form larger seeds (Aguiar et al., 1979). The authors studied the influence of E. saligna seed size on germination and initial plantlet development. The results demonstrated that plantlets from larger seeds reached the size for thinning and transplanting quicker than those from smaller seeds. Similar results were obtained by Pereira and Garrido 1975 with E. grandis, observing that larger seeds presented a higher SEI than the smaller seeds.
The occurrence of self-pollination contributes to the loss of seed quality, provoking the appearance of albino and abnormal plantlets (Kageyama, 1981; Maêda, 1987). Oda et al. 1991 studied the influence of the number of viable seeds per kilogram and the size of the seeds on the percentage of albino and abnormal plantlets. This study used E. grandis seeds collected at different times of the year and the seeds were classified into three groups (<0.50, 0.59–0.71, >0.71mm). The results demonstrated that larger seeds were less likely to produce albino or abnormal plantlets and suggested that the production of small seeds could be linked to self-pollination.
Suzano 1994a evaluated the behavior of different progenies of E. grandis with respect to seed vigor and initial plantlet growth, looking for possible links between these variables and the quality of the sapling and the future forest. The seeds of six progenies from the clonal seed orchard (second generation) were divided into two groups, >0.59mm but smaller than 0.71mm and >0.71mm. The germination efficiency, SEI and the height of the plantlets at 32 and 40 days after seeding were evaluated. Differences between the progenies were demonstrated, thus during thinning the plantlets that have higher SEIs and faster initial growth will be preferentially represented in the final product from the nursery. In this way, when breeding populations and commercial plantations are formed, not all of the progenies are equally represented, thus resulting in lower genetic variability, due to the reduction in the effective population number, and failure to achieve the expected results.
1.2.10 Quality of the seedling for planting
The selection of seedlings in the nursery by height is one of the parameters used to assess quality. The advantage of this selection is to form more uniform lots with a better use of the seedlings in the field (Coelho, 1984). According to the author, the practice of sapling selection in the nursery is a controversial question. Some authors state that a certain time after planting, the forest presented a more homogenous development even though the seedlings from the nursery were not selected. However, other studies report that the selection of taller seedlings give rise to taller trees. The removal of defective seedlings or seedlings with low vigor is, however, necessary. For example, of the total number of seedlings produced in the nursery by Companhia Suzano de Papel e Celulose, 20% were considered as abnormal, those presenting defects such as bifurcations and loss of vigor. Part of these abnormalities could be explained by different degrees of endogamy in the seed production plantation.
Coelho 1984 evaluated the silvicultural behavior of E. grandis seedlings up to 1-year-old, based on seed, plantlet, and sapling size. The author observed that selection by size of the seedling had highly significant effects on height and diameter, suggesting that the selection for this species by seed or seedling should be encouraged in order to obtain trees with better growth qualities. Various studies demonstrate that the selection of seedlings can enable an initial gain in height, but with time this difference is reduced or disappears (Donald, 1976; Balloni et al., 1978; Morais and Brune, 1983; Suzano, 1994b).
In this way, the selection of seedlings has an effect on the formation of commercial plantations where all the progenies do not participate in equal quantities, thus the genetic variability could be reduced due to the reduction in the size of the effective population.
1.2.11 Interaction genotype × environment (G × E)
Zobel and Talbert 1984 stated that when the tests are carried out at the same location, any effect is limited only by the genotype. This fact has two consequences: (a) the heritability could be overestimated, resulting in overestimation of the expected gains when the material is planted in an untested environment and (b) the genotypes selected in one environment may not be the best in other environments resulting in a reduction in the genetic gain. The authors also suggested that the G × E interaction affects the strategy in forestry breeding programs as seen by tree mortality and/or reductions in growth.
Various studies have reported that stability is genetically controlled (Barriga, 1980; Torres, 1988). Kageyama 1980 evaluated the genetic variation in E. grandis (origin Coff's Harbour, Australia) generated by open-pollination in five locations: Agudos, Anhembi, Brotas, Lençois Paulista, and Resende (SP, Brazil). The evaluation characteristics were as follows: form of the tree trunk, DBH, and height at 2 years old. The effect of the location, significant on all of the characteristics, reflected in the reduction in heritability (at the level of the location, individual plants, mean of the families, and within the families) and, therefore, in the selection for the locations as a group. The same tendency was observed by Pinto Jr. 1984 in progenies of E. urophylla S.T. Blake from Island of Flowers (Indonésia), using conjugated origin testes and locations in four Brazilian states, Aracruz (ES), Anhembi (SP), Bom Despacho (MG), and Planaltina (DF).
Mori et al. 1986 estimated the genetic parameter for E. saligna from three locations (Itatinga (ACS); Areia Branca (PSC); and Salto (PS)) and planted at three test sites: General Câmara (RS), Brotas (SP), and Bom Despacho (MG). The characteristics evaluated were DBH, height, and cylindrical volume at 3-years-old. The coefficient of heritability when analyzed together was inferior to the majority of the coefficients obtained by test location, demonstrating losses in heritability because of the progeny × location interaction. The losses in the selection due to this interaction were estimates at up to 88.3% for cylindrical volume.
Mori et al. 1988a studied the progeny × location interaction in 30 seven-years-old progenies of E. urophylla generated by open-pollination from the Island of Flowers (Indonesia) at four locations: Aracruz (ES), Belo Oriente (MG), Bom Despacho (MG), and Grão Mogol (MG). The values of the mean heritability (heritability coefficients at the plant level, progeny mean, and within the progenies) for the four locations were superior to the values obtained when the data was analyzed together, demonstrating the great effect of progeny × location interaction. The losses in genetic gain due to the progenies × location interaction for the cylindrical volume, DBH, and height, were 26.73%, 15.74%, and 8.14%, respectively. The authors compared the genetic gains (%), using the progeny mean of the cylindrical tree volume and three improvement strategies: multiple populations with selection of progeny for each location; the selection of individuals with a greater capacity to adapt to the study location, stable selection; traditional selection not considering environmental difference and the progenies × location interaction. For this comparison, a selection proportion of 1:6 between progenies was used, which made possible the selection of progenies predictably more productive (selection of 5 progenies from 30). The genetic gains in percentage are shown in Table 2.
Strategy |
Aracruz (ES) |
Belo oriente (MG) |
Bom despacho (MG) |
Grão mogol (MG) |
Mean |
|---|---|---|---|---|---|
Multipopulation |
18.81 |
10.18 |
14.18 |
26.10 |
17.32 |
Stable |
5.09 |
0.79 |
10.72 |
13.25 |
7.46 |
Traditional |
1.15 |
1.93 |
1.09 |
1.94 |
1.53 |
Carvalho 1989 evaluated the G × E interaction in 50 clones of E. grandis, in the region of São Mateus (ES) in three traditionally reforested areas, using the following genetic parameters: heritability, standard deviation from the estimate of heritability, coefficient of genetic variation, and the quotient between genotypic variation and the coefficient of experimental variation, calculated using the measurable parameters DBH, height, cylindrical volume, and wood basic density. It was observed that the estimated heritability values were relatively high for all the measured characteristics when considered at each location. When the data was analyzed together, there was a general decrease for all the characteristics involved in growth (DBH, height, and volume), and less pronounced for basic density. Van Wyk et al. 1989 tested the stability of 31 hybrid Eucalyptus clones at 10 locations in South Africa, observing that many clones were stable for tree height. According to the authors, the results indicated that the selection of clones should be cautious since the behavior of each clone is location specific.
Cruz and Regazzi (1994, 2001) discussed the effect of the G × E interaction on the prediction of selection gains obtained using a particular improvement strategy. The authors presented situations in which it had been desirable to make gain predictions. These studies demonstrated that the effects of the G × E interaction should be considered in breeding programs, since it is possible that losses in the expected gains could occur in the selection of genotypes. According to Mori et al. 1988a the G × E interaction, when badly administered, could result in a reduction of expected gains making it difficult for the breeding program to achieve its goals. The authors suggested, in order to minimize losses, a more adequate multipopulation strategy is necessary that considers the specificity of the genetic material for particular environmental conditions, through the selection of individuals more adapted and productive for each ecological region.
A genetic improvement program should have well-defined strategies, planned for short- and the long-term objectives, in order to obtain genetic material improved for the desirable characteristics. This material should guarantee the sustainability of commercial plantations and the continuity of the improvement program through the selection of advanced generations of trees and superior clones. The breeder should be acquainted with the factors that could reduce the expected genetic gains and how to intervene in the improvement strategies to minimize these losses.
1.3 Application of Molecular Markers in Eucalypt Breeding
The success of a breeding program depends on the genetic resources, which are being explored, the selection procedures, and the strategy used. There are numerous examples of successful Eucalypt breeding programs throughout the world. The average Eucalypt productivity has been improved in three decades from 12m3ha−1year−1 in the 1960s, to a current value of 60m3ha−1year−1. The current performance has been reached because of applied silvicultural research and especially the breeding programs using high performance clones with fast growth, excellent wood quality, rusticity on poor soil, and resistance to diseases and pests.
The success of the breeding programs depends on the ability to distinguish heritable and environmental factors. Genetic markers are heritable when associated with the characters of interest and can increase the efficiency of selection. The segregation analysis of molecular markers permits the construction of genomic or linkage maps, to estimate gene frequency, especially useful to characterize the population gene diversity and germplasm in breeding programs for the genetic conservation of Eucalypts (Mori, 1993). Paternity tests and the study of mating system of different Eucalypt species have contributed to obtain superior clones in breeding programs (Camargo, 2001).
A genetic marker is a character capable of detecting differences between two or more individuals or organisms, be able to distinguish progenitors and progenies, and should present a series of attributes such as high level of polymorphism, stability in different environments, detection of high numbers of unlinked loci and of simple inheritance. In the following sections will be present the main types of molecular markers as well as their main applications in Eucalypt breeding programs.
1.3.1 Application of molecular markers in breeding programs
The selection of Eucalypt phenotypic characters for commercial purposes is time consuming because of the time taken to make field evaluations. One way to shorten this time is the use of molecular markers. Various types are appropriate for use in Eucalypt breeding programs and some examples are given below.
1.3.1.1 Characterization of genetic diversity
The characterization of gene diversity in breeding programs is very important for researchers. Molecular markers are very useful to study the structure of populations, families, clones, cultivars, and also to discriminate gene pools from different populations.
Polymorphism based upon isoenzyme loci was studied in populations of E. urophylla, E. grandis, and E. saligna to develop tests for discrimination of interspecific hybrids from contaminants. Mori et al. 1996 concluded that the Est-1, Est-2, and Idh-1 loci were useful for the purpose because some alleles were present in a population and absent in others and vice versa. Clones and progenies of five E. grandis subpopulations were analyzed by isoenzyme loci observing that the average inbreeding (f) was 0.08 and outcrossing rate (t) was 0.88. Much of the genetic diversity was within the subpopulations and there was practically no diversity between subpopulations (FST = 0.011) (Mori and Kageyama, 2001).
Sixty-nine progenies were analyzed representing one open-pollinated family of E. urophylla trees. RAPD markers allowed the identification of 72 loci that were analyzed using Jaccard's coefficient, generating matrices of genetic distances. The genetic distances between individuals were 0.40 through 12 groups. The progenies also showed different bark patterns, allowing the establishment of distinct groups. However, the groups based on genetic distances using DNA analysis did not correspond of those based on bark pattern (Pigato and Lopes, 2001).
A total of 44 natural hybrids of Eucalyptus, cultivated in central Brazil were analyzed. The RAPD markers presented efficient discriminating power, determining a mean genetic distance of 54% among them and genetic divergence from 24% to 73%. This demonstrated that there is a wide genetic base among individuals, which is desirable in breeding programs. Clustering analysis established by UPGMA (Unweighted Pair Group Method with Arithmetic mean) method, using 80% as a cut off criterion for the total genetic distance, established nine distinct groups with average genetic divergence above 60% (Caixeta et al., 2003).
The study of genetic diversity among trees is an important stage of any breeding program that targets the exploration of heterozygosis. The prediction of heterozygosis has been extensively used in Eucalyptus to obtain better hybrid combinations. In a study using 40 E. grandis and E. urophylla trees (two 10 × 10 circulate partial diallel crosses) were analyzed for their genetic diversity. The crosses were produced in a previous study based on the tree diversity using RAPD markers. Seventeen microsatellites amplified 75 allelic forms and gave a heterozygosis value of 26.1%. The genetic distance varied from 10% to 100%, whereas the mean genetic distance was 64.6%. The cluster analysis for the 40 trees showed high diversity and formed two groups (one for each species), although some genotypes were outside their species-specific group. In comparison with groups formed by the RAPD markers, microsatellite markers were more efficient in discriminating species. However, the correlation values among RAPD and microsatellite markers were low and negative. Microsatellite markers were efficient to discriminate trees of E. grandis and E. urophylla (Muro-Abad et al., 2005).
1.3.1.2 Fingerprinting
The characterization and identification of individuals are important procedures in plant breeding. Rocha et al. 2002 used RAPD and simple sequence repeat (SSR) markers to obtain an exclusive fingerprint for 15 genotypes of Eucalypt hybrids with high potential for vegetative propagation used in breeding programs. Those two molecular techniques produced a set of markers that allowed an accurate identification of all genotypes. The RAPD procedures were also used by Pimenta et al. 2001 to obtain information on origin, genetic distance, and relationship among clones.
Hybridization between three species of Eucalyptus in the Series Curviptera, E. macrocarpa, E. pyriformis, and E. youngiana was investigated using RAPD markers. The dendrogram based on genetic similarities showed the relative proximity and distance among the individuals. Two clusters were identified by the UPGMA dendrogram. One of them included all of the E. macrocarpa genotypes and also one of E. macrocarpa hybrid. The other included all of the E. youngiana and E. pyriformis genotypes and their hybrids (Neaylon et al., 2001).
1.3.1.3 Quantitative trait loci (QTL) mapping
Most Eucalyptus breeding programs have used interspecific hybridization, making it possible to capture nonadditive genetic variance. Propagating genotypes via vegetative propagation greatly enhanced the possibility to use genetic linkage maps for accelerating breeding programs by marker-assisted selection (MAS) and recombination (Grattapaglia and Sederoff, 1994).
Linkage maps were constructed mainly by RAPD dominant markers (Verhaegen et al., 1997), amplified fragment length polymorphism (AFLP) dominant markers (Gaiotto and Grattapaglia, 1997), and SSR co-dominant markers (Brondani et al., 1998). The maps could identify genomic regions in eucalypts with significant effects on expression of economically important characters. Various characters were studied by QTL analysis, such as, characters associated to eucalypt rooting (Marques et al., 2002), insect resistance (Shepherd et al., 1995), plant growth (Squilassi and Grattapaglia, 1998), and Puccinia psidii rust resistance in E. grandis (Junghans et al., 2001). There are also studies for different eucalypt species and cultures in which few loci with major effects controlling a relatively large proportion (from 10% to 40%) of total phenotypic variation for quantitative characters of silvicultural importance, such as, wood volume and wood density were detected.
Squilassi and Grattapaglia 1998 mapped QTLs in eucalypts for wood volume by RAPD markers, using linkage disequilibrium on selected progenies. The study showed that the MAS was efficient to select through the family mean level; however, it was not efficient at the individual level.
Gion et al. 2001 studied 201 full-sib families from interspecific hybrids between E. urophylla and E. grandis for eight genes involved in the lignin biosynthesis (PAL (phenylalanine ammonia-lyase), COMT1 (caffeate/5-hydroxyconiferaldehyde O-methyltransferase 1), COMT2, 4CL (4-coumarate:coenzyme A ligase), C4H (cinnamate 4-hydroxylase), CCoAOMT (caffeoyl coenzyme A O-methyltransferase), CAD2 (cinnamyl alcohol dehydrogenase 2), and CCR (cinnamoyl-coenzyme A reductase) and three Myb transcription factors. The genes were mapped using the single strand conformation polymorphism (SSCP) technique. These genes were located on the two parental genetic maps constructed with polymerase chain reaction (PCR)-based markers. The lignin monomeric compositions (S/G ratio) were obtained using the thioaciolysis method and the QTL analysis for S/G ratio was performed using the interval mapping procedure. Several regions controlling part of the variation were identified, showing that multipoint estimates of the total variation explained through the QTL were 38.0% and 18.5% for E. urophylla and E. grandis, respectively. The study shows that it should be possible to follow the manipulation of lignin quality in breeding programs using molecular markers.
1.3.1.4 MAS
Some studies were carried out using isoenzyme markers to allow earlier selection of superior genotypes. Menck 1996 studying the acid phosphatase isoenzyme in clones of E. grandis cultivated in vitro found a correlation between superior genotypes for growth characters and those for efficiency by the use of phosphorous. The author also observed correlations between superior genotypes on the field and higher activities of acid phosphatase enzyme.
The transfer of disease resistance alleles in plants can be expedited by the use of DNA markers. If the markers are tightly linked to the resistance alleles they can be used for MAS. One effective use of MAS is found in the process of pyramiding resistance alleles. The use of MAS is based upon the principle that a gene or a block of genes is associated with a molecular marker of easier identification, making the selection for that marker more efficient than the selection for the phenotypic character. MAS in eucalypt has focused mainly on wood growth, which is an important commercial character, easily measured but of low heritability.
Experimental full-sib families of E. grandis and E. urophylla were used to evaluate the comparative efficiency of selection based on molecular markers associated with a favorable QTL and the regular phenotypic selection for diameter at breast height (Squilassi and Grattapaglia, 1998). The authors did not study all possible crosses, evaluating only the maternal QTL allelic contribution, because an effective assessment of MAS in eucalypt depends on experiments where the phenotypic data are gathered at adequate ages and QTL information is preferentially available for all progenitors involved in the pedigree (Squilassi and Grattapaglia, 1998).
Using the BSA (bulked segregant analysis) procedure, Bortoloto 2006 developed a SCAR (sequenced characterized amplified region) marker to detect the flowering time region on E. grandis map. The marker presented linkage to the EMBRA 7 (Brondani et al., 1998) microsatellite marker on a paternal map. The author also detected linkage of the EgLFY flowering gene to the EMBRA 6 (Brondani et al., 1998) microsatellite marker indicating the region for flowering control.
1.3.1.5 Recombination of genotypes and seed orchard
Eucalyptus species are widely used for planting through tropical and subtropical regions of the world and they are predominantly outcrossing, highly heterogeneous, and genetically diverse (Moran and Bell, 1983). Using microsatellites and AFLP markers, Zelener et al. 2005 studied six provenances and 37 selected half-sib families of E. dunnii to make a selection to establish a seed orchard. The estimated genetic differentiation showed low values among provenances (θP = 0.026) and high values among families (θS = 0.174). A high proportion of the total variation observed within families suggested that the orchard design should be based upon individual or family selection rather than provenance selection.
A E. dunnii breeding population of 46 provenances from Australia and selected for fitness through subtropical and cold environments was screened by AFLP and microsatellite markers to estimate the genetic diversity. The markers presented no significant differences between the original breeding population and the selected genotypes of seed orchard confirming that the seed orchard can be established with a limited number of individuals without problems of inbreeding (Poltri et al., 2005).
Microsatellite markers were used to genotype potential pollen donor and maternal progenies from a E. globulus seed orchard in Chile and an E. grandis seed orchard in Uruguay. The resulting data were used to infer the most likely pollen parents for the seed from each seed orchard. The estimated distances from maternal tree to the most likely male parent served as the data set basis to model the pattern of gene flow in both orchards. The results indicate that the pollen dispersal may reach distances as far as 300m or more (Russell et al., 2001).
1.3.1.6 Mating system
Eucalypts present hermaphrodite flowers making possible selfing and outcrossing rates at different levels. Different authors have observed that eucalypts present a model of mixed mating, predominantly by outcrossing, however inbreeding can occur by selfing and crossing amongst relatives.
Moran and Brown 1980 studying a population of Eucalyptus delegatensis by isoenzymes, observed an average outcrossing rate of 0.77, with differences occurring between young (r = 0.66) and adult (r = 0.85) plants. Through time, the populations change and usually there is a tendency for endogamous individuals to die, increasing the level of heterozygosity in the population. Moran et al. 1989 studying a seed orchard of E. regnans observed an outcrossing rate (r = 0.91) higher than that of the natural population (r = 0.74). The lower rate of outcrossing in natural populations could be due to inbreeding between physically close relatives. In a seed orchard there are no relatives and the inbreeding occurs via selfing. Mori 1993 investigating a seed orchard of E. grandis, also using isoenzymes found an outcrossing rate of 0.88.
Patterson et al. 2001 determined the outcrossing rates at two positions on the tree: the top and the bottom of the canopy in E. globulus, in a remnant of a native stand in southern Tasmania using isoenzyme markers. In trees previously determined to have high levels of self-incompatibility, outcrossing rates were high at both, the top and bottom of the canopy (from 0.87 to 0.99). In contrast, the outcrossing rates in self-compatible trees were significantly different in both positions of the canopy: lower at the bottom (from 0.27 to 0.66) than the top (from 0.74 to 0.90). These results were of particular significance to collection of open-pollinated seed for breeding or deployment, where unfortunately, the most accessible seed may also be the most inbred.
Mori et al. 2004 studying E. grandis progenies by microsatellite markers observed an outcrossing rate of 0.67 indicating that the species shows crossing among relatives and selfing.
1.3.1.7 Diagnosis of disease resistance
Eucalypt rust, caused by P. psidii, is one of the most serious diseases in Brazil and considered to be the most serious threat to eucalypt plantations worldwide. Junghans et al. 2001 analyzed the number of sorus and pustule size to characterize rust severity on E. grandis seedlings which were further screened with RAPD markers. One of the markers (AT/917) showed complete co-segregation with Ppr-1 (P. psidii resistance, gene 1). All the resistant plants showed the marker, while no susceptible plants had this marker. The AT9/917 sequence was used to obtain specific primers (SCAR), which will be used for screening clones in an E. grandis genomic library.
Using microsatellite markers, Mori et al. 2004 also studied rust resistance character in progenies of E. grandis. Nei's genetic distance varied up to 0.400 and the index of gene diversity varied from 0.383 to 0.713, having an average of 0.587.
1.3.1.8 Analysis of paternity
It is very common to use molecular markers to check the paternity of a superior elite tree obtained by open-pollination. The analysis of paternity is especially important to determine the other parent contributing to the genotype with superior gene combination.
Estimates of the level of multiple paternities correlated with outcrossing within and between fruits in a pre outbred population of the bird-pollinated mallee, Eucalyptus rameliana, were made by Sampson 1998 using six isoenzyme loci. The correlation of outcrossing paternity (rp) was positive and significant within fruits (0.26) and the effective number of mates for a single fruit was estimated to be 3.85. The specialization of floral structure and phenology in E. rameliana for bird pollination has probably contributed to correlation of paternity within fruits because there are fewer male parents available at any one time when compared to the mass-flowering species.
Paternity analysis using microsatellite markers was conducted on a E. nitens seed orchard in Victoria, Australia. An average of 40 seeds per clone were germinated and screened for paternity using the four loci. Paternal contribution varied among clones, suggesting that panmitic pollination was not occurring, probably due to differences in flowering time and flower numbers between the clones such that each clone was subjected to a different pollen pool. Distance between clones was another important factor influencing paternal contribution (Grosser et al., 2001).
1.3.1.9 Comparison of breeding generations of Eucalyptus
Pigato and Lopes 2001 studied the diversity and the genetic distances in four generations of E. urophylla, which provided data to help guide the breeding program. The initial base population was introduced by seeds collected in Indonesia (P0 generation). In the subsequent segregating generations originated by open-pollination, recombinations were designated as P1, P2, and P3. One hundred and seventy four individual trees representing the four generations were analyzed. The RAPD technique allowed the identification of 86 positions analyzed using the Jaccard Coefficient. The genetic distance from P0 generation was 0.33, 0.34 from P1, 0.40 from P2, and 0.38 from the P3 generation. The genetic distances between individuals increased in relation to the base population, being 0.15% from P1 generation, 18.93% from P2, and 13.31% from P3, showing an increase in genetic diversity in the advanced generations, despite selective processes. Genetic diversity of 14 populations of E. grandis were studied by Mezzena 2003 utilizing microsatellite markers. The populations were at different levels and breeding generations. The inbreeding coefficients within the populations were very similar (f = 0.16). The author observed a little loss of heterozygosity from F1 (Ho = 0.64) to F2 (Ho = 0.60) breeding generations.
2 Development of Transgenic Eucalypts
2.1 In Vitro Culture in Eucalypts as a Prelude to Genetic Transformation
From the agribusiness point of view, eucalypts are important in many aspects. Essential oils can be extracted from the leaves; tannins from the bark; and wood, methanol, charcoal, and cellulose for paper production can be extracted from the trunk. Hence, it is not strange that millions of hectares are planted in the world. Currently, Brazil has the biggest collections of Eucalyptus spp., even bigger than Australia and Indonesia (Ferreira, 1992). In addition, this genus has species with various interesting attributes such as fast growth, adaptation to poor soils, fertile hybrid formation, and capacity to produce roots from stump sprouts (coppiced).
Commercially, Eucalyptus propagation has been traditionally done by seed, but, the cellulose and paper industry has stimulated research to attempt clonal multiplication (Xavier and Comério, 1996; Rosse et al., 1996), to optimize field production, reduce costs, and to achieve better genetic gain (Thorpe and Harry, 1990). Ikemori et al. 1994 showed that pulp and cellulose production was significantly higher in cutting cloned forests (10.9tha–1year–1) than in unimproved seedling forest (5.9tha–1year–1). However, the propagation via rooted cuttings offers difficulties such as loss of rooting capacity in the adult trees (Brune, 1982; Burger and Lee, 1987) and plagiotropic, a bushlike growth pattern (Durand-Cresswell et al., 1985; Flynn et al., 1990). Eucalyptus spp., despite the fact that many species can propagate by cutting due to the presence of an epicormic bud at the base of adult trees others, such as E. regnans, E. nitens, E. fraxinoides, and E. deglupta, are recalcitrant (Hartney, 1980).
In cloning, tissue culture has been used for several years as a tool to propagate uniform and selected material with major commercial value (Karnosky, 1981; Haissig et al., 1987; Hartmann et al., 1990; Kozai and Kubota, 2001). On the other hand, the development of breeding programs with woody plants for additive or nonadditive genetic variation has been too slow. Therefore, tissue culture has been effective in these programs, aiming to diminish the time to obtain intra-and interspecific hybrids. Recently, in vitro culture has contributed efficaciously to the research on biolistic or Agrobacterium tumefaciens transformation with the objective to reach new genetic commercial arrangement for forestry at short, medium, and long terms (Rochange et al., 1995; Machado et al., 1997; González et al., 2002).
There are many reviews reporting in vitro techniques showing morphogenesis either via organogenesis or embryogenesis to obtain whole plants in Eucalyptus spp. (Hartney, 1982; Durand-Cresswell et al., 1985; LeRoux and van Staden, 1991a; Sita, 1993). In this context, problems with genotype, contamination, and phenol oxidation have been reported as serious challenges to be overcome. For the time being, problems with somaclonal variation and genetic stability (Tibok et al., 1995; Azmi et al., 1997; Rani and Raina, 1998) or vitrification (Bunn, 2005; LeRoux and van Staden, 1991b) have not been cited as important drawbacks.
Tissue culture is a strong biotechnological tool for micropropagation with potential large-scale propagation of Eucalyptus. Having elite material with determined degrees of genetic gain so as to replace seeds by cloned propagation, with low cost and high quality products for wood and cellulose and competitiveness is the basic premise of any company. Here we attempt to review the use of in vitro tissue and organ cultures to produce Eucalyptus clonal forestry, showing different experimental procedures according to the literature available by the author.
Species |
Explant |
Basal medium |
Growth regulators (μM) |
Organogenesis (via) |
Rooting (μM) |
References |
|---|---|---|---|---|---|---|
Eucalyptus torelliana |
Nodal explants from tree |
MS |
BAP: 2.2 + KIN: 0.93 |
Shoot induction |
NAA: 11 |
Gupta et al., 1983 |
E. sideroxylon |
Nodal explants from coppice regrowth of tree |
MS |
BAP: 2–4 + NAA: 0.5–1 |
Shoot induction |
IBA: 10 |
Burger, 1987 |
E. citriodora |
Mature seed |
B5; B5M B5R |
NAA: 16.0 NAA: 27 — |
Directly somatic embryogenesis |
Muralidharan et al., 1989 |
|
E. macarthurii; E. smithii Hybrid MG25 E. saligna |
Nodal explants from 1-year-old plant |
MS |
BAP: 0.88 + NAA: 0.05 |
Shoot induction |
IBA: 10 |
LeRoux and van Staden, 1991a |
E. globulus |
Nodal explants from coppice stumps of tree |
MS |
BAP: 2.5 + NAA: 1.25 |
Shoot induction |
IBA: 10 |
Bennett et al., 1994 |
E. grandis |
Leaf explants from in vitro plants |
KG, G22, R5, GBA |
BAP: 0.2; BAP: 2.0 + NAA: 2.5; Zeatin: 8.0 + NAA: 0.5; BAP: 5.0 + NAA: 0.5; |
Callus |
IBA: 1.25 |
Lainé and David, 1994 |
E. urophylla |
Hypocotyl explants from 14-day-old |
MS |
BAP: 0.9 +NAA: 1.1; Zeatin: 4.6 |
Callus |
NAA: 5.4 BAP: 0.04 |
Tibok et al., 1995 |
E. dunnii |
Seedlings—3 days old |
B5 |
NAA: 16.5 |
Callus—somatic embryogenesis |
Termignoni et al., 1996 |
|
E. grandis × E. urophyla |
Hypocotyls, cotyledonary-node, from 14-day-old seedlings Primary leaves with different ages from in vitro plants |
SP |
TDZ: 2; BA/NAA: 2.5/0.5, 5/0.5, 10/0.5; Zeatin/NAA: 2.5/0.5, 5/0.5, 10/0.5 |
Callus |
IBA: 2.5 |
Barrueto et al., 1999 |
E. impensa |
Shoot segments from trees |
MS |
BAP: 0.25 + Kin: 2.5 |
Shoot induction |
IBA: 5 + NAA: 0.5 |
Bunn, 2005 |
2.1.1 The explants
Table 3 shows that different explants that have been utilized to initiate Eucalyptus tissue culture and that the use of nodal segments from elite adult plants is more frequent. Although contamination has been recorded (Fossard et al., 1977; Durand-Cresswell et al., 1985), this has not been described as a problem in Eucalyptus, even if explants from field or simply surface-sterilized by HgCl2 or NaOCl was used (Figure 3) (Rao and Venkateswara, 1985; Gupta and Mascarenhas, 1987; Bennett et al., 1994). However, it seems that this cannot be generalized since 70% of in vitro-inoculated nodal explants from forest trees show contamination, while greenhouse nodal fragments remained virtually contamination-free (Fossard et al., 1977). On the other hand, with respect to surface sterilization of explants, most reports show use of NaOCl or HgCl2 with varying periods from 10–20min for NaOCl, and from 5–15min for HgCl2. Concentrations varied from 0.12% to 9.0% of NaOCl and from 0.02% to 0.1 % for HgCl2. Prior to the treatments, fungicide has sometimes been used (LeRoux and van Staden, 1991a, 1991b). Also, the material sometimes was thoroughly rinsed with tap water and detergent (Gupta and Mascarenhas, 1987; Das and Mitra, 1990), before sterilizing with HgCl2 or commercial bleach.
Seedlings of E. grandis × E. urophylla approximately 14-day-old in SP medium, seeds surface disinfected with NaOCl (9%) for 10min, showing hypocotyls, cotyledons, roots, and contamination free. Growth conditions: 24 ± 2°C, 16h photoperiod, and photosynthetic photon flux density at 50μmm−2s−1
Ca(OCl)2 is an energetic oxidant, with low phytotoxicity, similar to NaOCl. However, due to its powder formulation and the need to be filtered, few studies have used it as a surface sterilizer for the inoculation material. In these cases, concentrations varied from 5% to 7% for 5–20min (Cresswell and Nitsch, 1975; Fossard et al., 1977; Trindade and Pais, 1997).
Other chemical substances against bacteria and fungi have rarely been mentioned in Eucalyptus studies, besides ethanol and H2O2. Alkydimethylbenzalkonium chloride, ADBAC for example, was a product rarely cited (Bunn, 2005), and biocide-type isothiazolones such as PPM (Niedz and Bausher, 2002) were not used in Eucalyptus micropropagation. The later is heat stable, with a broad spectrum against plant contaminants (Compton and Koch, 2001). Physical methods such as the use of hot water and UV light have practically not been mentioned in explant sterilization procedures for Eucalyptus.
In general, phenolic oxidation is mostly inconvenient in in vitro cultures (Prieto et al., 2005) often demanding the use of various alternative substances to overcome it (Ziv and Halevy, 1983). Changes in peroxidase activity and level of phenolic compounds were showed by Arezki et al. 2001 in E. camaldulensis.
In Eucalyptus, browning has hindered the establishment of some protocols such as those related to protoplasts (Teulières and Boudet, 1991), cell suspension (Teulières et al., 1989; Barrueto et al., 1997), nodal explants from adult trees (Rao, 1988; Das and Mitra, 1990), or floral origin (Warrag et al., 1991), being necessary the use of antioxidant substances such as polyvinylpyrolidone and ascorbic acid in several cases, or adsorbents of toxic substances such as activated charcoal. Other methods reported the use of periodic subcultures, initial dark period, or using explants from established shoot cultures (LeRoux and van Staden, 1991a, 1991b).
2.1.2 Shoot multiplication and hormone treatment
Once the explants have been established in a basal medium, multiplication follows via organogenesis or embryogenesis. Usually the basal medium used in this phase is an extension of the medium used previously, frequently a Murashige and Skoog (MS) medium (Table 3). Salt and vitamins from the MS nutrient medium have been used in their original form (Rao, 1988; MacRae and van Staden, 1990; Bennett et al., 1994; Yang et al., 1995) or with modifications (Hartney, 1982; Barrueto et al., 1999) depending on the purpose (induction of organogenesis or somatic embryogenesis) and the genotype used.
Auxin and cytokinin have been useful to obtain in vitro plants from basal medium at different levels. Calli have been obtained with 2μM thidiazuron (TDZ) using juvenile material from a E. grandis × E. urograndis hybrid (using cotyledon, hypocotyls, and cotyledonary node), and plants were obtained when these calli were submitted to different concentrations of 6-benzylaminopurine/α-naphthalene acetic acid (BAP/NAA) or Zeatin/NAA. The best results were obtained with Zeatin: 5.0μM and NAA 0.5μM, respectively, using cotyledonary node explant (Barrueto et al., 1999).
Calli were also obtained with hypocotyl juvenile material from E. grandis using a combination of 21.5μM NAA and 4.6μM kinetin (Kin) and organogenesis was observed when the material was transferred to a hormone-free medium (Warrag et al., 1991). Callus induction was also observed in E. citriodora, from cotyledons and hypocotyls submitted to different auxins: IAA (indole-3-acetic acid), NAA, 2,4-D (2,4-dichlorophenoxyacetic acid) (range: 0.1–5.0mgl−1) plus coconut milk (15%); however, organogenesis occurred when calli were placed in 2.7μM Zeatin and 1.1μM IAA (Sita, 1979). According to the same author, calli from leaves originated from mature plants induced with 27μM NAA and 28μM IAA, but no organogenesis was observed. Organogenesis was observed in E. urophylla, from hypocotyl explants when MS was supplemented with several NAA/BAP concentrations, showing better results with NAA at 1.1μM and BAP at 0.88μM (Tibok et al., 1995); however, shoot production of E. grandis × urophyla was also effective, at 13.7 per explant with NAA/BAP at 0.54μM and 0.44μM on apical shoots and auxiliary bud, respectively (Yang et al., 1995).
At low concentrations (NAA 0.54μM and BAP 0.9μM), shoot multiplication from nodal explants of juvenile Eucalyptus of many species (E. smithii, E. saligna, etc.) showed good results (LeRoux and van Staden, 1991a, 1991b). Using auxiliary meristems, shoots were also obtained with different genotypes of E. tereticornis, inoculated onto MS basal medium. Among the growth regulators tested, NAA/BAP/IAA at 5.3, 4.4, 1.1 (μM), respectively, was more effective giving 32–45 shoots per explant. Repeated subculture in MS plus IAA 0.1μM and BAP 0.44μM, produced around 200 shoots per explant (Rao, 1988). With nodal segments from an 8–10-year-old E. tereticornis, 18–22 shoots per explant were achieved with modified MS supplemented with NAA 0.54μM and BAP 4.44μM (Das and Mitra, 1990). Low concentrations of growth regulators were also used with Eucalyptus impensa to obtain shoot multiplication in half-strength MS medium plus BAP 2.5μM and Kin 2.5μM from shoot segments as explants (Bunn, 2005).
Regeneration competency from auxiliary and apical buds was observed in 20-year-old E. citriodora. This was achieved by incubating the explants at 15°C for 72h, and then incubating them in a liquid MS-2 media (120revmin−1). Five to eight shoots per bud developed. After this, individual shoots were transferred to solid MS-2. In this new condition, 10–15 shoots were obtained per explant (Gupta et al., 1981). The MS-2 medium was supplemented with Kin 0.93μM, BAP 1.33μM, calcium pantothenate, and biotin (0.1mgl−1 of each). According to the authors, it could be possible to get 100000 viable plants per year from a single bud using this protocol.
Somatic embryogenesis was obtained with E. citriodora using NAA 16.13μM and zygotic embryos as a starting point. The basal medium (B5) was supplemented with sucrose 50g (Muralidharan et al., 1989). In a peculiar protocol, somatic embryogenesis was also described for E. grandis. This was obtained with leaves from in vitro seedlings using 2,4-D (2.3μM) for callus induction. Callus proliferation was reached in MS medium with 4gl−1 of activated charcoal containing (μM): 0.05 NAA, 0.44 BAP, and 0.27 gibberellin. However, additional study was mentioned for implanting large-scale production (Watt et al., 1991). In conclusion, low concentrations of BAP and NAA seem to be more effective for Eucalyptus shoot multiplication under a wide range of experimental conditions (Table 3); however, these wide experimental conditions, basically, included Eucalyptus solid cultures and not liquid cultures, probably because liquid medium can cause vitrification—hyperhydricity (Monsalud et al., 1995).
2.1.3 Shoot elongation
Usually, shoot elongation has been obtained using a half-strength medium. In E. camaldulensis, regenerated shoots were transferred to quarter strength B5 basal medium plus NAA 0.05μM (Kawazu et al., 1996). In E. grandis × E. urophylla shoot elongation was more efficiently obtained with MS medium containing half-strength potassium nitrate and sucrose (Yang et al., 1995). On the other hand, the elongation of shoot clusters may be mediated with gelling agents. In E. grandis, the MS medium containing Gelrite plus a combination of NAA/BAP/GA (μM): 0.05, 0.44, and 0.27, respectively, improved shoot-cluster elongation (MacRae and van Staden, 1990). In contrast, optimum shoot elongation of Eucalyptus torelliana and E. camaldulensis was achieved with solid and liquid MS-2 media, respectively, using Kin/BAP: 0.23μM and 0.44μM, respectively (Gupta et al., 1983). In addition, E. grandis shoot elongation was promoted in MS plus gibberellic acid 2.7μM prior to rooting (Sita and Rani, 1985).
2.1.4 Rooting
The formation of roots from excised shoots in one-fourth strength MS containing IAA 10μM with no cytokinin was reported by Hartney 1982. In addition, shoots of Eucalyptus sideroxylon from modified MS (half-strength salts, full-strength organic constituents) rooted with 10μM IBA (indole-3-butyric acid). IBA was more effective than NAA in adult or coppice tissues (Burger, 1987). Differences in rooting between juvenile and old tree material were also noted in E. citriodora (Gupta et al., 1981). In general, this is a physiological characteristic of woody species that in vitro regeneration and rooting ability decrease with age. For E. tereticornis, the presence of IBA in the basal medium was essential for rooting, as well as the use of a dark period before the transfer of shoots to light (Das and Mitra, 1990). A period of 72h in darkness, prior to a 16h photoperiod along with hormonal treatment, was used to root four Eucalyptus genotypes, giving different rooting ability, inclusive, deficient rooting was observed in Eucalyptus macarthurii and E. smithii. In addition, these species also showed poor rooting from coppice cuttings (LeRoux and van Staden, 1991a). Eucalyptus globules showed better results when low concentrations of IBA (1.0–2.5μM) were used and when NH4NO3 was removed from the root-induction medium (Bennett et al., 2003).
Rooting of different clones of E. globulus was verified after different periods of rejuvenation using subculturing routines (Trindade and Pais, 1997). Root induction improved when riboflavin and choline chloride were included in the medium and boron was removed from the rooting medium. The authors also reported that IBA dipping before transfer to the rooting medium improved the appearance of adventitious roots. Experiments with E. grandis × E. urophylla using modified SP supplemented with IBA 2.5μM for 5 days and transfer of shoots to a similar medium with activated charcoal (1gl−1) greatly improved rooting but resulted in little callus formation at the base of some shoots (Barrueto et al., 1999).
Despite auxin being an important factor in root induction, care must be taken to avoid callus induction at the base of the shoots (Mehra-Palta, 1982; Rao, 1988; Cheng et al., 1992). On the other hand, cytokinin is a critical component of the multiplication media, but inhibitory for the rooting media. However, subsequent adventitious root production in rooting medium was better when Kin was used during the last multiplication (Bennett et al., 1994). As seen above, rooting is a highly clone-dependent characteristic, hence, one must be careful when testing or improving rooting conditions, especially with shoots from a mature tree, older the tree, smaller the rooting ability.
2.1.5 Temperature and light
In general, the culture conditions for in vitro Eucalyptus plant establishment were a constant temperature of 25 ± 2°C and a 16h photoperiod, involving different ranges of photosynthetic photon flux density (PPFD) from cool fluorescent light bulbs (Rao and Venkateswara, 1985; Subbaiah and Minocha, 1990; Termignoni et al., 1996, Sartoretto et al., 2002; Bennett et al., 2003).
2.1.6 Hardening plantlets
The transfer from tube to soil under greenhouse condition is an important step in the micropropagation process. Usually, intermittent mist or a plastic cover is required as well as plastic pots with sterile vermiculite and adequate natural or artificial light and temperature. When established, plantlets are transferred to polyethylene bags with a mixture of soil and sand. Survival is variable, but, usually high. Generally, a hardening protocol contains steps similar to those previously described with variation in the composition of the rooting substrate (inclusion of fertilizer, fungicide, etc).
Finally, the plants are taken to the field and their performance is evaluated in terms of survival, growth rates, and biomass production. On the whole, micropropagated Eucalyptus plants have the same appearance as their sources (Mehra-Palta, 1982; Gupta and Mascarenhas, 1987; Warrag et al., 1989; LeRoux and van Staden, 1991a). Consequently, tissue culture techniques have been observed with great interest in agronomy, forestry, and ornamental plant production, due to the economic benefits of clonal propagation on a commercial level; however, the productivity of commercial clones can be reduced by viral infections, but culture of meristem tips is a good way to combat this (Rutledge and Douglas, 1988).
2.2 Genetic Transformation of Eucalypts
To maintain and sustain forest vegetation, conventional approaches have been exploited for propagation and improvement, but tree breeding efforts are restricted to the most valuable and fast growing species. However, such methods are limited with several inherent bottlenecks because trees are generally slow growing, long-lived, sexually self-incompatible, and highly heterozygous plants. Due to the prevalence of high heterozygosity in these species, a number of recessive deleterious alleles are retained within populations, resulting in high genetic load and inbreeding depression. This limits the use of traditional breeding methods such as selfing and backcrossing, and makes it difficult to fix desirable alleles in a particular genetic background (Williams and Savolainen, 1996). Thus, conventional breeding is rather slow and less productive and cannot be used efficiently for the genetic improvement of trees. To circumvent these impediments clonal or vegetative propagation has been deployed for recovering dominant, additive, and epistatic genetic effects to select superior genotypes. Plant tissue culture and genetic transformation methods offer an important option for effective multiplication and improvement of trees within a limited time frame. Biotechnological approaches for in vitro regeneration, mass micropropagation techniques, and gene transfer studies in tree species have been encouraging, particularly in the last decade. With these techniques, genetic engineering assumes additional significance, allowing introduction of desired gene(s) in a simple step, for precision breeding of forest trees (Giri et al., 2004).
Until recently, trees were considered to be recalcitrant for genetic transformation studies involving molecular techniques. The main obstacle for genetic transformation of trees is the regeneration of transformed plantlets. Agrobacterium-based genetic transformation is normally the main method used for developing transgenic trees. Further regeneration of plants from single cells is a requisite for Agrobacterium-mediated gene transfer to achieve homogenetically transformed plants (Giri et al., 2004).
Choice of explants having competence for transformation and regeneration is a crucial factor. At this point in time efficient tissue culture techniques become the foundation for genetic transformation studies. In addition to the regeneration through organogenesis, somatic embryogenesis definitely offers the advantage of single cell regeneration and currently appears to be the most promising approach to introduce new genes into woody tree species (Giri et al., 2004).
Despite the progress in recent years, challenges remain for the transformation of hardwood trees. Although elite individual of hardwoods such as poplar, eucalypts, and sweet gum can be maintained by vegetative propagation methods, genotypic variation in regeneration prohibits the inclusion of many genotypes in tree improvement programs. Even for the elite genotypes that are propagated for clonal deployment, transformation can be limited by Agrobacterium susceptibility or in vitro regeneration. At the moment hardwood transformation is largely limited to either the clones that are easy to transform and regenerate or juvenile materials that have higher regeneration and transformation potential. Maturation is another common problem. While seedling or juvenile tree explants are relatively easy to regenerate and transform, explants of mature trees tend to lose regeneration potential. Unfortunately, elite clones are selected from older trees with years of field performance data (Nehra et al., 2005).
A limitation to the use of this technique lies in the general recalcitrance of eucalypts to transformation and poor regeneration capacity (Poke et al., 2005). At this time, the stable transformation has only been successful in a small number of species including E. camaldulensis, E. globulus, E. urophylla, E. grandis, and E. urophylla × E. grandis hybrids (Mullins et al., 1997; Ho et al., 1998; Moralejo et al., 1998; González et al., 2002; Tournier et al., 2003; Valerio et al., 2003). These are predominantly laboratory studies. Field studies of transgenic eucalypts have been reported in the United Kingdom and Spain in 1995, in Portugal and South Africa in 1997, and Uruguay and Chile in 1997/1998 (Potts et al., 2001) including field testing of glyphosate-resistant transgenic E. grandis (Llewellyn, 2000).
Despite the clear potential of genetic engineering for improving woody plants during the last 10 years, progress has been slow on Eucalyptus, which is still considered recalcitrant. In particular, regeneration and tissue culture is often very poor, probably due to the high concentration of phenolic compounds in the cells (Tournier et al., 2003) or to low endogenous cytokinin content as shown by Azmi 1999. Regeneration capacity is even lower on a selective antibiotic-containing medium making it often impossible to recover transgenic shoots even when stable transformation is achieved (Serrano et al., 1996; Sartoretto et al., 2002).
2.2.1 Gene transfer
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Availability of the trait to be transferred as cloned-DNA;
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Availability of a powerful transfer system; and
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Availability of a reliable regeneration system predominantly from a single transformed cell.
The last two points are solved in principle, although regeneration is still a problem. Regeneration is more an art than a science, particularly in transformation of Eucalyptus clones. However, recipes are available and, with sufficient trials and the use of a range of different genotypes, success should be achievable. Transfer studies are predominantly via Agrobacterium, as such transformations are more stable than microbombardment techniques during subsequent meiosis. Microbombardment has, however, the advantage that it can transform meristematic cells, therefore the regeneration of plants is relatively easy to achieve. The most critical problem is still gene isolation. Most of these isolated genes are used in transformation experiments for basic research to elucidate biochemical pathways and add knowledge particularly in the field of metabolomics (Wenzel, 2006). Most of the patented genes available in the market, which are expected to give a determinant trait, have not been sufficiently tested in Eucalyptus. However, patented genes for specific traits in engineered plants have not generated improved commercial Eucalyptus clones, yet.
2.2.2 The use of biolistics in the genetic transformation of Eucalyptus
Microprojectile bombardment, which bypasses the problems associated with host specificity of Agrobacterium and regeneration systems, offers an alternative approach for the delivery of DNA into cells that are also competent for regeneration. Because the particle gun concept involves physical introduction of DNA into the cell, it potentially allows the transformation of any living cell, independent of its type or localization in the tissue (Sartoretto et al., 2002).
Serrano et al. 1996 obtained stable transformation of E. globulus cells for the first time, using biolistic DNA delivery in zygotic embryos as the target material; however, no transgenic plants were regenerated. Sartoretto et al. 2002 reported a procedure for genetic transformation of calli from a E. grandis × E. urophylla hybrid using particle bombardment of calli derived from cotyledon and hypocotyl. However, the calli were unable to regenerate transgenic shoots, suggesting that the conditions suitable for regeneration are unsuitable for transformation and vice versa. In spite of some interesting advances using the particle gun delivery system, no transgenic plants of any Eucalyptus species have been reported using the biolistic strategy.
2.2.3 Eucalyptus transformation using A. tumefaciens
Mullins et al. 1997 obtained stable transformation of one clone of E. camaldulensis using leaf explants inoculated with A. tumefaciens. In this work, the reporter gene β-glucuronidase and the NPTII gene were used for selecting transgenic tissues. Ho et al. 1998 reported an efficient system for transformation of seedling material from E. camaldulensis by the inoculation of hypocotyls segments with A. tumefaciens. The work used the Ti-plasmid vector harboring chimeric neomycin phosphotransferase and β-glucuronidase genes.
Moralejo et al. 1998 regenerated transgenic E. globulus plants using hypocotyls and cotyledons from young seedlings. The explants were inoculated with A. tumefaciens and the genes introduced were GUS and NPTII. Due to confidentiality problems, no precise transformation protocol has been published for most of the eucalypt species. Maunders et al. 1997, even though the protocol is not described, reported a reliable transformation system for several species of Eucalyptus including commercially important E. grandis and E. globulus, starting from either mature elite clones or improved seed material.
Transformation of forest tree species remains difficult, particularly within the genus Eucalyptus. Harcourt et al. 2000 produced transgenic plants of E. camaldulensis containing both the insecticidal cryA3 gene and the bar gene by A. tumefaciens-mediated transformation of seedling explants. The transgenic lines exhibited tolerance to the broad-spectrum herbicide and resistance to the first instars of chrysomelid beetles. These plants are likely to provide better insect and weed control options in plantations, particularly during the vulnerable establishment phase, mainly in elite genetic background.
Chen et al. 2001 regenerated transgenic E. camaldulensis plants of elite clones. The genes introduced were cinnamate 4-hydroxylase (C4H) and NPTII. The main objective of this work was to alter the lignin content of the wood. The transgenic plants were obtained but the wood modification was not evaluated. For Eucalyptus species, transformation and plantlet regeneration are generally more efficient with juvenile materials, such as hypocotyls, cotyledons, and leaf disks from in vitro-germinated seedlings, than with clonally derived material from field-grown trees that have poor regenerability in tissue culture (MacRae and van Staden, 1999). For forest tree species, the true value of genetic engineering lies in its integration into conventional breeding programs to improve economically important traits that cannot be modified by conventional means within a reasonable time frame. González et al. 2002 obtained transgenic E. grandis × E. urophylla plants using seedling material and A. tumefaciens. The chimeric construct contained the nptII gene and the Lhcb1*2 gene, coding the 28kDa chlorophyll a/b binding pea protein from LHCII antenna. The transformation system used the inoculation of seeds with A. tumefaciens followed by sonication for a few seconds. The objective of the work was to develop a transformation protocol and evaluate the alteration in the process of photosynthesis.
Tournier et al. 2003 regenerated E. grandis × E. urophylla juvenile clones with the antisense CAD gene and NPTII like selective gene. The transgene expression was demonstrated with high inhibition of endogenous CAD gene, but physiological and biochemical wood modifications were not evaluated.
Valerio et al. 2003 developed a procedure for A. tumefaciens-mediated genetic transformation of a juvenile E. camaldulensis clone using antisense CAD gene. Some transgenic lines exhibited a strong inhibition of CAD activity, associated with a decrease in transcription. The wood chemical analysis showed no differences in lignin quantity, composition or pulp yield, compared to control trees. These results underline the problems of extrapolating genetic engineering results from a model to a genetically distant target plant species. Other techniques such as RNA interference-type procedures may be used for more complete gene suppression.
Recent developments in transgenic trees can have multidirectional benefits. The benefits range from manipulating generation time, plant protection, wood quality, production of compounds of pharmaceutical value, and recovery of polluted soils.
These successes have opened up avenues to include agronomically more useful genes for transfer into tree species as has been demonstrated in crop plants (Giri and Vijaya, 2000). Following the global trend in forestry biotechnology there are now 24 genetically modified tree species that have been approved for field trials. Recently, it has been emphasized that genetically modified trees can be excellent tools for physiological research (Herschbach and Kopriva, 2002). The research completed so far demonstrates the potential of these techniques in the improvement of forest tree species. One of the most important aspects of transgenic trees is integration of the introduced gene into the genome and its expression. For long-lived tree species, new questions arise regarding the stability of the integration and expression of foreign genes. Biosafety considerations, including the impact of transgene dispersion through pollen and unexpected effects on nontargeted organisms, are now receiving attention. With recent research developments, molecular genetics provide tools that may allow genetic improvement to make up lost ground. If current progress in tissue culture and genetic transformation combined with biotechnological applications continues, the future may witness super tree species tailored for special agronomic and economic characteristics (Giri et al., 2004).
Private companies claim to possess routine transformation protocols for different Eucalyptus species and hybrids; however, because of the confidentiality issues, these methods are not widely available (MacRae and van Staden, 1999).
3 Important areas for improvement using genetic transformation
3.1 Eucalyptus Diseases, Control Strategies, and Genomic Approaches to Identify Genetic Resistance
3.1.1 Eucalyptus diseases and control strategies
Eucalyptus cultivation in Brazil was limited only to a few thousand hectares until the 1970s, when eucalypts were considered to be practically immune to diseases. The increasing demand for forest products and the need to conserve the native forests pushed the expansion of Eucalyptus plantations to about 3.4 million hectares. About 1 million hectares of this are planted with 362 different clones, of both pure species and hybrids, distributed in areas varying from 10 to 34000ha/clone. It is expected that within this year an additional 250000ha of eucalypt forests comprising 55 clones will be planted, occupying areas varying from 10 to 9000ha/clone/company. The expansion of plantations to warmer and more humid regions conducive to infection by plant pathogens, the regional peculiarities of climate and soil, combined with the possible introduction of diseases from other countries and adaptation of some local pathogens from native Myrtaceae to Eucalyptus species have resulted in the incidence of various fungal and bacterial diseases that cause significant losses in some highly susceptible clones. In spite of the risks, cloning has proved to be an excellent tool for disease control. The knowledge of Eucalyptus species and their interactions with different potential diseases is important to establish strategies to ensure stability of wood production. This review summarizes the main Eucalyptus field diseases and their control.
3.1.1.1 Rust
The symptoms are the appearance of yellow-colored powdery urediniosporic sporulation over the affected organs is the typical feature for rust diagnosis (Figures 4a–c). In highly susceptible materials, the infection causes deformation, necrosis, hypertrophy, mini-cankers, and death of the apical meristems (Figure 4d). Although the uredinial phase is more common and is the main form of pathogen dispersal, less frequently, teliospores can be produced, during the warmer periods, on fully expanded leaves.
Eucalyptus rust (Puccinia psidii). (a) Infected apical shoots of Eucalyptus urophylla; (b) pathogen esporulation on E. globulus leaf; (c) rust incidence on stem and leaves of Syzygium jambos; and (d) necrosis and death of apical shoots of E. grandis
Causal agent: Puccinia psidii Winter.
Control: The existence of high inter- and intraspecific genetic variability for resistance to rust allows for disease control by planting resistant clones, progenies, or species. Corymbia citriodora, C. torelliana, E. camaldulensis, E. microcorys, E. pellita, E. pilularis, E. propinqua, E. resinifera, E. robusta, E. saligna, E. tereticornis, and E. urophylla are important sources of resistance. In regions, favorable to rust infection, planting of E. grandis seeds (provenances: South Africa and Coff's Harbour 9583), E. phaeotricha, E. cloeziana, E. globulus, and E. nitens should be avoided. There is, however, ample intraspecific variability, which permits selection and cloning of resistant genotypes for planting. In E. grandis, resistance might be dominant and controlled by a major gene (Ppr-1) (Junghans et al., 2003). Thus, rust can be controlled through use of resistant progenies, whose seeds are harvested from resistant homozygous mother plants, as practiced by some Brazilian forest industries in São Paulo and southern Bahia. It is also possible to select trees with rapid growth characteristics, which rapidly escape the disease by virtue of the microclimate in the upper canopy being unfavorable for disease development. Similarly, selection can be made from clones of E. globulus and related species that rapidly pass through the susceptible juvenile leaf stage to produce resistant adult leaves. In the case of highly susceptible genetic materials of high commercial value, the disease can be controlled by systemic fungicide spray such as triadimenol (Bayfidan 25 PM ou 25 CE) (0.5gi.a.l−1) and azoxystrobin (Amistar 500 WG) (0.1gi.a.l−1) at 2 weeks intervals. In nurseries, especially in clonal hedges and mini hedges, the disease is controlled by fortnightly spraying of triadimenol or azoxystrobin, using the above concentrations.
3.1.1.2 Cylindrocladium leaf blight
The lesions can develop at the base, at the apex, or at the leaf margins, and can cover a large portion of the leaf area, inducing severe premature defoliation of the lower, middle, and apical thirds of the tree canopy during the 1st and 2nd years after planting (Figure 5a). It is believed that, when only the basal or middle third of the canopy is affected, the trees tend to recover. However, under disease favorable conditions, the apical portion is also defoliated, especially in highly susceptible materials, leading to reduced plant growth. Defoliation also allows for high light intensity penetration into the stand leading to growth of competing weeds (Figure 5a). Disease symptoms can vary depending upon the species of Cylindrocladium and Eucalyptus. The leaf spots of C. pteridis are smaller, circular, or elongated and light gray progressing to light brown in color (Ferreira et al., 1995) (Figure 5b), while those of C. candelabrum, C. floridanum, C. ilicicola, C. parasiticum, and C. scoparium are larger, light to dark brown with a gray green halo (Alfenas et al., 2004) (Figure 5c). In most Eucalyptus species, the lesions are light or pale brown, but in E. cloeziana they are dark brown.
Cylindrocladium leaf blight. (a) Defoliation in commercial plantation of eucalyptus hybrid (E. grandis × E. urophylla), the arrows indicate growth of weeds due to light penetration increase; (b) smaller leaf spot, typical of C. pteridis; and (c) larger leaf spot, typical of Cylindroclaium spp.
Causal agents: C. candelabrum, C. floridanum, C. ilicicola, C. ovatum, C. parasiticum, C. pteridis, and C. scoparium.
Control: Considering the natural recovery of infected plants during subsequent periods, climatically unfavorable to the disease, no control measure is currently used. However, because of high reduction of the photosynthetic area, a significant loss in volume is expected, which may justify adoption of control measures to reduce potential losses. In this case, planting blight-resistant progenies, provenances, species, and clones is the best control strategy. The determination of inheritance model and genetic base is essential for a breeding program to obtain resistant materials.
3.1.1.3 Rhizoctonia leaf blight
In field plantations and clonal hedges, the infection starts on the leaves of the lower branches and progresses to the plant apex, causing intense leaf blight and defoliation (Silveira et al., 2000) (Figure 6a). The disease is characterized by large and irregular leaf spots (Figures 6b and c). Depending on the disease intensity, whitish-mycelium-covered branches and leaves can be observed (Figure 6b), with the possible presence of whitish or light to dark-brown sclerotia (Figure 6d). Initially, the affected leaves show irregular light-gray to light-brown lesions of different sizes leading to blight of almost all leaves that become pale in color. Initially, infected leaves remain attached to the plant, but tend to fall with time. Other marked characteristics of the disease are hanging leaves attached by the fungal hyphae, adhering to one another, and connected by hyphae resembling a web (web-blight) (Figure 6c). The pathogen survives in soil, from which it disseminates by water splash to the surface of lower leaves or by growing epiphytically up the trunk reaching the higher portions of the canopy. In general, the fungus does not sporulate, and the most important features are sclerotia formation along the infected organs, right angle branching of hyphae, and presence of a constriction at the first septum in the branched hyphae when observed under the microscope. The sexual phase of some isolates of Rhizoctonia solani (Thanatephorus cucumeris) can be produced under controlled conditions, but is rarely found in natural infections (Silveira et al., 2000).
Rhizoctonia leaf blight. (a) High defoliation; (b) leaves with lesions and pathogen mycelium (arrows); (c) leaves with blight symptoms stuck each other, with the fungal mycelia holding the dead leaf; and (d) dark-brown sclerotia over infected leaves
Causal agents: Rhizoctonia solani (AG1-1B) and binucleate species of Rhizoctonia not yet identified.
Control: Although there are no studies about the genetic variability for resistance, it is unlikely that resistant genotypes will be found within eucalypt species. However, artificial inoculations should be conducted to examine this hypothesis.
3.1.1.4 Bacterial leaf blight
At the initial phases of infection, the disease is characterized by water-soaked translucent leaf spots (Figure 7a), resulting from water leakage into the intercellular spaces, and is followed by intense defoliation, girdling, and mortality of the apical portion of highly susceptible materials (Figure 7b). As the disease progresses, the lesions become necrotic and dry with perforations and deformation of the leaf blade (Figure 7c). Its precise diagnosis requires laboratory examination, using exudation tests in which bacterial slime emanates from newly formed lesions.
Causal agents: Species of Xanthomonas axonopodis, X. campestris, Pseudomonas cichorii, and others (Gonçalves, 2003).
Control: Selection and planting of resistant genotypes.
3.1.1.5 Phaeophleospora leaf blight
Angular purple brown spots, distributed on both sides of mature leaves (Figures 8a–c), result from exudation of conidial masses (cirri) (Figure 8d), black sporulation resembling black mold. The disease is sometimes confused with the leaf spots caused by Cylindrocladium, plant bacterial infection or phosphorus deficiency, especially if observed on the upper leaf surface. However, typical sporulation of the pathogen is the major characteristic for diagnosing the disease. It generally occurs on old leaves of plants in the field or in the nursery.
Causal agent: Kirramyces epicoccoides (Phaeoseptoria epicoccoides = Phaeophleospora eucalypti), teleomorph Mycosphaerella suttoniae (Crous, 1998).
Control: No specific control measure has been used, but selection and planting of resistant genotypes may be effective.
Bacterial leaf blight. (a) Water-soaked translucent leaf spot on Eucalyptus cloeziana plants; (b) death of apical portion of E. urophylla; and (c) differences in the symptoms
Phaeophleospora leaf spot. (a) Angular purple brown spot with fungi sporulation; (b) healthy leaf and leaves with symptoms; (c) detail of angular spots; and (d) exudation of conidial masses (cirri)
3.1.1.6 Pilidiella leaf spot
Large, light brown to pale leaf spots, with dark concentric halos (Figure 9) formed by the exudation of spore mass. Typical conidiophores and conidia of the pathogen can be observed by microscopic examination of histological sections through the pycnidium. This fungus penetrates host tissues through wounds (leaf friction by the strong winds), insect (thrips, larval, and aphids), or mite injuries and also through the lesions caused by other leaf pathogens as P. psidii and Cylindrocladium spp. The photosynthetic area is reduced in case of high disease severity, but defoliation is not usually observed.
Pilidiella leaf spot. Leaf spot showing pathogen penetration by wounds or other pathogens lesions
Causal agent: Pilidiella eucaliptorum (= Coniella fragariae).
Control: No specific control measures are employed, but planting of resistant clones is the best strategy of control.
3.1.1.7 Aulographina leaf spot
Circular or elongated dark-brown corky spots occur over the main vein, petiole, and twigs (Ferreira, 1989) (Figures 10a and b). Superficial dark-brown to black, elongated, curved, or branched fruiting bodies with a longitudinal slit (hysterothecia) are formed over the lesions (Ferreira, 1989) (Figure 10c). Asci and the ascospores of the fungus can be observed by microscopic examination of histological sections of the ascomata.
Aulographina leaf spot. (a) Typical symptoms; (b) detail of corky spots over the main vein; and (c) typical hysterothecia
Causal agent: Aulographina eucalypti.
Control: The disease does not cause important damage, therefore, no specific control measures are being used.
3.1.1.8 Mycosphaerella leaf spot
The fungus infects young leaves of E. globulus, E. nitens, and E. dunnii and mature leaves of many other species, including E. grandis, E. saligna, E. urophylla, and their hybrids. The spots vary from circular to irregular circular shape and are light to pale brown, being darker on the lower leaf surface (Figures 11a–d). Dark ascomata, asci, and ascospores are formed on the lesions.
Mycosphaerella leaf spot. (a) Typical spot of Mycosphaerella suberosa; (b) typical spot of M. parkii; (c) lesions caused by M. marksii; and (d) typical spot of M. juvenis
Causal agents: The disease is caused by various species of Mycosphaerella. Although it is not well known, it is believed that M. parkii (anamorph = Stenella parkii), M. suberosa (anamorph = not determined), and M. suttoniae (anamorph = Phaeophleospora epicoccoides) are the most common species on Eucalyptus in Brazil.
Control: No control measures have been adopted in Brazil, but for species such as E. globulus, E. maidenii, E. dunnii, and E. nitens it is possible to select faster growing clones, which rapidly pass to the less susceptible adult stage and, thus escape the disease.
3.1.1.9 Cryptosporiopsis leaf spot
The pathogen infects leaves on the basal third branches without causing significant defoliation. The lesions are light to grayish-brown of varying sizes, semicircular or circular in shape (Figure 12a). They are encircled by a dark halo and the center has a dark rust colored spot of up to 6mm diameter (Ferreira et al., 1998) (Figure 12b). Typical conidiophores and conidia can be observed microscopically in the histological sections of conidiomata. Like Pilidiella eucaliptorum and Hainesia lythri, the fungus penetrates the host through wounds.
Cryptosporiopsis leaf spot. (a) Lesions on the lower (left) and upper side (right) of leaf and (b) detail of spot lesions
Causal agent: Cryptosporiopsis eucalypti.
Control: Since the disease does not cause significant economical losses, no specific control measures have been adopted. However, it is believed that planting of resistant genotypes may be the best strategy.
3.1.1.10 Ralstonia wilt
The first symptoms appear on 4- to 8-month-old plants. Initially, the leaves show wilting, becoming reddish, yellowish (Figure 13a), and latter pale to dark-brown in recently dead plants (Figure 13b). Stem section of wilted plants, exudates bacterial pus as cream-colored drops (Figures 13c and d).
Ralstonia wilt. (a) Plant with wilt symptoms and basal defoliation; (b) dead plants; (c) symptoms in the stem section immediately after cut; and (d) exudates bacterial pus (arrows)
Causal agent: Ralstonia solanacearum (= Pseudomonas solanacearum).
Control: Production of plantlets exempt of contamination with the pathogen.
3.1.1.11 Ceratocystis wilt, dieback, and canker
Initially are observed epicormic shoots along the trunk, and depression in the bark, then dieback, canker, wood discoloration, and wilt, leading to plant death (Figures 14a–d, Figures 15e–g).
Dieback, canker, and wilt caused by Ceratocystis fimbriata. (a) Development of basal shoots due to veins colonization by the fungus; (b) natural infection showing dieback; (c) dead plant; and (d) canker
Continue from Figure 14. Transversal (e) and radial (f) xylem discoloration symptoms; and (g) inoculated plant with wilt symptoms (left), and detail of stem discoloration (right)
Causal agents: Ceratocystis fimbriata.
Control: Planting of resistant genotypes.
3.1.1.12 Eucalyptus canker
Infection may be observed on 6-month-old plants (Figure 16a). When it occurs on young or adult plants of small stem diameter or on mini-stumps in clonal hedges, usually causes death by stem girdling. The canker can occur at any height of the stem, but usually occurs at the tree base (basal canker), causing superficial or deep lesions on the bark surrounded by callus (Figures 16b–d). A typical canker is formed if the lesion is deep and localized at a specific point of the trunk, while in superficial lesions, not reaching the cambium, the plant responds by producing new tissues resulting in the trunk swelling and bark cracking at the infection point (Figure 16e). The weakened trunk at this point can break (Figure 16f), especially in regions of strong winds. Dark pycnidia and or perithecia produced on dead bark are the signs of the disease, essential for unequivocal diagnosis.
Eucalyptus canker caused by Chrysophorte cubensis. (a) Scattered death of Eucalyptus saligna plants in the field; (b) typical canker without bark removal; (c) typical canker (deep lesion flanked by callus); (d) basal canker; (e) trunk swelling, and bark cracking on the infection point; and (f) plant with canker, broken by wind at the infection point
Causal agent: Chrysophorte cubensis (= Cryphonectria cubensis = Diaporthe cubensis = Endothia eugeniae).
Control: Planting of resistant species, provenances, families, or clones. C. citriodora, C. torelliana, E. cloeziana, E. pilularis, E. paniculata, E. pellita, E. urophylla, E. robusta, E. resinifera, and E. microcorys are the more resistant species, while provenances of E. grandis and E. saligna are the most susceptible. However, there is a high intraspecific variability as to resistance, which allows for selection and cloning of resistant genotypes for planting. In clonal hedges, canker can be controlled by selective shoot harvesting that reduces the stress on the mini-stumps, avoiding predisposition to the disease.
3.1.1.13 Pink disease or rubelosis
Lesions and girdling are observed on the stem and branches of 1- to 3-year-old plants. A pink colored mycelium grows on young lesions (Figures 17a and b). Epicormic shoots emerge from below the girdled portion of the stem (Figure 17a). Later, the lesions dry out and lose the typical color (Figure 17c), leaving behind cankers (Figure 17d) on the thicker nongirdled stem and branches. The stem may break and lose apical dominance (Figures 17e and f). Pink to salmon colored mycelium, containing basidia and basidospores may be observed on the lesions.
Pink disease or Eucalyptus rubelosis, caused by Erythricium salmonicolor. (a) Pink mycelium and epicormic shoots below infected area; (b) and (c) stem lesion showing mycelium growth and bark cracking; (d) typical canker; (e) trunk breaks on the infection point; and (f) trunk break and loss of apical dominance
Causal agent: Erythricium salmonicolor (= Corticium salmonicolor).
Control: Selection and cloning of resistant genotypes.
3.1.1.14 Coniothyrium canker
Small discrete necrotic lesions with a strong depression in the bark are formed along the trunk (Figures 18a–c). The infection occurs through the younger tissues of the green stem. In highly susceptible genotypes, the lesions coalesce and cause extensive necrosis, generally followed by kinopocket formation (gummosis) (Figure 18d) and reduced plant growth, dieback, and emission of epicormic shoots along the trunk, due to partial cambium death and stem girdling. The fungus produces globose and substomatal pycnidia, containing conidiospores and conidia.
Coniothyrium canker. (a), (b), and (c) Necrotic lesions with a depression in the bark; and (d) details of the xylem infection with kinopocket formation
Causal agent: The disease, first described in South Africa, was attributed to Coniothyrium zuluense (Wingfield et al., 1997). In Brazil, the disease was found on E. grandis and attributed to Coniothyrium sp. (Ferreira, 1977).
Control: In Brazil, it has not caused serious damage to the affected plants, thus no control measures have been adopted.
3.1.1.15 Botryosphaeria canker
The infection occurs in young tissues, resulting in breakage with the forking of the main stem at the infection point, gum exudation, darkening, depression, and bark cracking (Figures 19a–f). Generally, the lesions are superficial and confined to the bark region, showing fructification of the pathogen. Pseudothecia containing asci and ascospores are formed on the lesions.
Botryosphaeria canker. (a) Canker; (b) depression and bark cracking; (c) gum exudation; (d) break of apical trunk; (e) forking of main stem in the infection point; and (f) details of superficial infection in the bark region
Causal agent: Botryosphaeria ribis.
Control: No specific control measures have been adopted.
3.1.2 Approaches to identify genetic resistance
3.1.2.1 Sources of disease resistance and breeding strategies
Considering that a relatively small number of clones, possibly with narrow genetic base, are planted in some regions of Brazil, it is imperative to establish an interspecies breeding program to obtain new resistant genotypes. Predicting the eventual dry periods and predominance of high temperatures, drought resistant species, such as E. camaldulensis, should be considered for crossings. Other species, such as E. pellita (Papua New Guinea provenance) and E. urophylla, are excellent source of resistance to canker. The former is also an important source of resistance to leaf blights. On the other hand, E. globulus can be used as a gene source for reducing the lignin and extractive contents, increasing wood density, improving pulp yield, and fiber quality, while E. grandis (Atherton or Coff's Harbour) has high adaptability and is suitable for high cellulose yield. Introgression of genes of E. globulus should be carried out using pollen obtained from elite mother plants from the southern part of Brazil, Uruguay, Spain, Chile, Portugal, or Australia, where it is possible to cultivate these plants as a pure species. In the final composition, hybrids should contain 50–75% genes of E. grandis, obtained by a series of crossing and back crossings, and selfings if necessary. However, E. deglupta, E. resinifera, and E. robusta can also be tested, although little is known about their resistance to diseases, fiber quality, and adaptability. Since E. globulus and E. pellita have contrasting characteristics, for example, disease resistance, site adaptability and extractives, and lignin content, they should contribute a maximum of 12.5% of genes in constituting interspecific hybrids. Since wood lignin and extractives have essential functions in defense mechanisms, reduction of their concentrations can potentially result in higher susceptibility. Thus, in each generation of crosses, the resistant genotypes should be selected through artificial inoculation with specific pathogens of interest for each region. It is also essential to determine the resistance of commercial clones and its genetic basis to different diseases such as stem canker, rust, leaf blights (fungal and bacterial), ceratocystis wilt, and bacterial wilt through artificial inoculations of controlled crossing progenies. When resistance is dominant, resistant homozygous mother plants should be used for seed harvesting, since irrespective of the pollen origin, the progeny should be resistant.
3.1.2.2 Genomic approaches to identify genes for rust resistance
Currently, one of the biggest threats to Eucalyptus plantations is neotropical rust caused by the biotrophic fungus P. psidii. This disease attacks young trees, normally younger than 2 years old, thus it principally affects nurseries and recently planted areas, and depending on the severity of the infestation can result in a significant to almost total loss of production. In order to investigate the genes differentially expressed during P. psidii infection, Moon et al. 2007 constructed two SAGE (serial analysis of gene expression, Velculescu et al., 1995) libraries representing susceptible and resistant material. Susceptible and resistant individuals were selected from a segregating population of half-siblings of E. grandis (Suzano Papel e Celulose), after being naturally infected with P. psidii Winter under field conditions. Bulks representing each phenotype were formed using ten susceptible and ten resistant individuals selected from an E. grandis segregating population of half-siblings (Suzano Papel e Celulose). 31645 and 39964 tags, representing a cumulative gene count of 4095 and 5213, were generated from the susceptible and resistant bulks, respectively. The Z-test indicated that 239 were preferentially expressed in the susceptible library and 232 in the resistant. Using the National Center for Biotechnology Information (NCBI) public database (http://www.ncbi.nlm.nih.gov), which contains approximately 15000 Eucalyptus ESTs and complementary DNAs (cDNAs), the authors were able to associate nucleotide sequences to 40 and 72 tags, susceptible and resistant, respectively.
Table 4 shows the differences in the number of transcripts within each functional category for the differentially expressed genes for the susceptible and resistant libraries. It can be seen that in the susceptible library the expression of genes involved in metabolism and the production of energy is considerably reduced showing about 25% of the level observed in the resistant material, probably reflecting the generally debilitated state of the infected susceptible plants. Genes preferentially expressed in the resistant library in this category were associated with the metabolism of sugar nucleotides and carbohydrates, indicating a higher biosynthetic activity directed toward the production of structural components.
Category |
Susceptible library (%) |
Resistant library (%) |
|---|---|---|
Metabolism and energy |
4.4 |
20.1 |
Processes |
32.3 |
31.4 |
Transport |
1.1 |
2.3 |
Structural and organization of structure |
4.8 |
16.5 |
Information pathways |
19.3 |
1.7 |
Unknown function |
38.1 |
28.0 |
Another interesting category was structural and organization of structure, which represents the genes involved in cell wall synthesis and cytoskeletal organization. This category was reduced in the susceptible material, approximately 33% of the level observed in the resistant library. Genes involved in the synthesis of the cell wall were preferentially expressed in the resistant library, including those associated with cellulose and lignin biosynthesis. We also observed genes involved in the formation of the cytoskeleton, the tubulins, preferentially expressed in the resistant material.
Although the number of transcripts produced for the processes category was almost the same in the two libraries, the genes that they represent show differing pictures in the presence of the pathogen. In the susceptible library, the preferentially expressed genes demonstrated a response to oxidative stress and general stress response proteins. However, the resistant material showed the induction of genes more specifically for defense responses, including a catalase and a kinase.
The category representing the information pathways was almost ten times more expressed in the susceptible than the resistant material. These genes were associated with ubiquitin regulated protein turnover and proteases were mainly expressed in the susceptible library indicating a very active protein degradation system.
From these preliminary results Moon et al. 2007 suggest that two completely different processes are occurring in the susceptible and resistant plants. Firstly, from the generalized lesions observed in the susceptible material, it is obvious that plant is fighting a loosing battle against the pathogen and is trying to limit the oxidative damage within the infected leaves and active proteolysis is occurring within the areas surrounding the necrotic lesions. Secondly, in the resistant plants various distinct processes are occurring simultaneously contributing to the expression of the resistant phenotype. Primary metabolism shows an increase in the expression of genes involved in the production of the raw materials for cell wall formation, sugar nucleotides, and carbohydrates. Coupled to this is the preferential expression of the genes associated with cellulose biosynthesis and the principal genes involved in lignin biosynthesis and polymerization, most probably re-enforcing the cell wall structure making fungal penetration more difficult. The preferential expression of genes involved in the formation of the cytoskeleton and vesicular transport factors would also indicate that the process of cellular polarization is occurring (reviewed by Schmelzer, 2002). Cellular polarization is defined as the process of cytoskeletal organization that allows the translocation of the cell nucleus and transport of vesicles containing cell wall matrix material or specific defense related proteins directly to the site of infection. This process is documented as being an important process during the host cell response to pathogens. The authors also observed catalase and a kinase, preferentially expressed in the resistant material, probably involved in signal transduction during the initial infection and later in the process of systemic acquired resistance. Unfortunately, they were unable to identify any gene specifically involved in the destruction of the fungal pathogen, such as chitinases, thaumatins, or R genes, probably due to the lack of publicly available Eucalyptus ESTs that represent these genes. The authors suggest that sustainable resistance to P. psidii in E. grandis is the result of more than one predominant gene.
3.2 Wood Quality and the Transcriptome Involved in Eucalyptus Wood Formation
3.2.1 Wood quality requirements for pulp and paper, charcoal biomass fuel end-uses
Eucalyptus wood has gained a respectable position as raw material for several utilizations. Thanks to an excellent forestry technology, which was developed in countries like Brazil, Portugal, South Africa, Spain, Chile, Uruguay, and Australia, Eucalyptus has reached the status of “super-tree”. In a historical first moment, the efforts coming from tree breeding and silvicultural techniques were directed to the production of volume and/or weight in the trees. The acquired productivity would provide the desired competitiveness to the wood-based businesses. Wood-specific unit costs and forest operation costs were dramatically reduced by these technologies. Thanks to the high productivity of the planted forests and to the low production costs, Eucalyptus wood soon gained importance with several industries, such as pulp and paper, charcoal for steel manufacturing, lumber, and furniture. This raw material could easily supply fibers and biomass fuel, in unbeatable conditions. In a short period of time, the world quickly became surprised and enchanted with Eucalyptus for its fast growth rates and wood quality. The quality targets are becoming more and more sophisticated for each of the wood utilizations. In the beginning, the selection of the raw materials was based on productivity (volume or dry weight), yields in the manufacturing conversion processes, and low wood costs. Today, the needs for product differentiation and for adding value in the production chain have oriented wood and trees to new requirements. Besides the traditional needs for productivity, yields, and costs, there are two new and important wood requirements: tree and wood uniformities. The control of forest variability may be seen as a simple task, but it is tremendously difficult. The anticipation using the cloning techniques was very high with the aim of controlling variability. However, the dream of having very similar trees has not been realized. Although a lot more uniform, the cloned trees show a considerable level of variability. This happens in the tree characteristics and in the wood quality (anatomical, physical, and chemical properties).
Currently, the foresters have placed attention on the production of trees having good shape, straight trunks, higher shape factors, and low percentage of bark. Additionally, the foresters also consider the resistance of the trees to pests and diseases, and tolerance to detrimental weather conditions (frost, winds, water deficits in the soil, flooding, etc). All these issues are demanding a lot of attention and research. The target for high quality Eucalyptus planted forests is not that simple. Even being healthy, with good shape and fast growth, the trees may not be suitable to some end-uses. For example, E. grandis trees, with fantastic growth rates, and tree shapes, may not be well suitable to the production of charcoal. The wood density may be too low for this purpose, the logs become very cracked when dried and the charcoal is poor in density, in calorific value per volume, very bulky, and generates more fines during manufacturing and handling. On the other hand, trees of Eucalyptus robusta may give low pulp yield in the conversion to kraft pulp due to the excess of extractives and/or lignin.
In short, now it is just the right time not only to have forests and trees, but also with end-use oriented qualities. A very good forest oriented for the production of charcoal will not be so desirable for kraft pulping, since both utilizations have opposite requirements in terms of wood lignin content. Whereas for charcoal, the best wood quality is the one with very high lignin content and wood density but for kraft pulping, lower the lignin content the better. The wood density for pulping cannot be very high due to the problems with liquor impregnation.
Although the knowledge for the required wood characteristics is well understood by most researchers, the truth is that we are still finding many mistakes in the way breeding programs are being performed. The major mistake is the precarious sampling procedure being used in the majority of the tree and wood quality improvement programs. As far as the variability remains high for many qualitative parameters, even for cloned forests, the size of sampling should be larger than is generally used. Additionally, the sampling should be more representative, collecting samples from all segments of the population according to their frequency. Currently, there is a trend to take few trees with the average volume. Average sized trees are not synonymous of trees with average basic density, or average lignin content, etc. The first recommendation has to be made related to trees and wood sampling. Definitively, the size and the representativeness of these samples need to be improved. The second recommendation is to increase the number of analyses, the number of replications for each required quality parameters being evaluated. In general, the analyses are made with a single measurement and the mean value is used in the tree and wood improvement program. Depending on the statistical error that the forest breeder is willing to allow, the numbers of replications have to be considerably higher. There is a chance that the breeder is accepting what is accepted as a good genome but is definitively not good for a given property of the tree or wood. This is something that needs to be immediately reevaluated.
3.2.1.1 Eucalyptus wood quality requirements for the production of kraft pulp
Independent of the pulp mill, the pulp sector has fundamental issues including high productivity, high operational efficiency (no losses, no problems, no breaks, and no stops), low production costs, and uniform quality of the process and products. For achieving these targets, the raw material must be as uniform as possible so as not to cause strong impacts on the pulping process and pulp qualities. To reduce this variability, the pulp mill engineers blend woods. Blending very different wood is acceptable if the result has a reasonable average quality. This is not an ideal situation, but it is the most usual.
When the pulp maker asks for uniform wood he is not only referring to wood basic density, but also to a series of wood quality parameters that are very important in the conversion process: proportion of bark in the wood chips, wood chip dimensions, decay level of the wood, wood moisture, wood chip bulk density, etc. The objective is to have a cooking and bleaching operation with the minimum variability, without undesirable surprises. The final quality must be uniform and within the specification limits and the process losses should be minimal. When the mill manager standardizes the wood intake, the first thing is to guarantee an appropriate quantity of dry wood being fed to the digesters. It is important to keep chip bulk density as uniform as possible for continuous addition of the same dry weight of wood to the digesters. The mill manager is carrying out “the management of the quantity of dry wood taken into the mill.” When a uniform flow of wood dry weight is guaranteed, the liquor, steam, and chemical flows do not sharply change and the process runs smoothly, and the process and product quality is more easily achieved.
The second type of management is “management of wood variability” that tries to guarantee a low variability of wood parameter such as wood basic density, lignin content, extractives content, and active alkali consumption during kraft cooking, bleaching chemical consumption, yields in the conversion of the wood to bleached pulp, etc. There are other associated goals, such as to minimize the overload of dry solids to the recovery boiler turnover; reduce the specific wood consumption in cubic meters of wood by air-drying metric tons of pulp; guarantee stable quality and to reduce the production costs.
Management of dry quantities and management of wood variability are the basic requirements in any pulp mill. Having fulfilled these needs, the next type of management is “management for product differentiation” or “tailor-made orientation in the manufacture of products.” This type of management requires substantial changes and offers important challenges to the mill personnel. The changes may happen in wood quality (for example, low and high basic density woods), process conditions (for example, ECF or ECF-Light bleaching sequences), or even other recently added qualities (certified or noncertified wood). Differentiation of products is more easily achieved in mills with more than one fiberline. This means that the mill may run each fiberline with a differentiated product, without experiencing the usual troubles in making the transition from one product to another using a single fiberline. Anyhow, the tailor-made concept will only be a winner when the pulp maker has guaranteed the two previous types of management: dry quantity and variability. It is very simple to say, but very difficult to understand and to implement. Conflicts and misunderstandings are frequent between commercial, production, and product innovation areas in a pulp mill. Each of these areas has their own needs, product uniformity, product uniqueness, and product differentiation. As a result, few Eucalyptus pulp mills have products that may be said to be completely differentiated in their products portfolio. Most of the pulp manufacturers aim to have a single product, as uniform as possible, with the minimum cost, and maximum in productivity and in operational efficiency.
It is relatively difficult to say what is the single most important wood characteristic for a given pulp mill. The reason is that there is not a universal wood property to be managed. Depending on the pulp mill bottleneck, wood quality is defined to guarantee the maximum performance of a particular mill. The most common bottlenecks are the capacities of digester, recovery boiler, drying machine, lime kiln and alkalinization, pulp washing, pulp bleaching chemicals. As a conclusion, it may be said that the type of mill bottlenecks will define the most desirable wood quality. This is the case for existing mills. For new greenfield mills, the quality may be previously built at the forest. However, soon the mill starts up, and the bottlenecks will appear to define the new wood quality standards. This is the reality, no doubt about it. This is also the cause of domestic conflicts within the company.
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Wood cleanliness. This is an item not clearly understood by many forest managers, who try to mechanize their operations to reduce costs and do not realize the impact on wood cleanliness, which in turn affects pulp mill efficiency. The wood must be as clean as possible in terms of bark, soil, leaves, stones, decayed wood, etc.
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Wood basic density. To the pulp maker, the uniformity of wood density is very important. The more uniform is the wood, the better and simpler is the management of the quantity and variability. In many mills the design capacity allows the use of light woods, which are easier to be impregnated with cooking liquor and demand a lower alkali charge. However, when the digester is the bottleneck, denser wood is used to raise mill production in the digester. Again, the kind of bottleneck is the source of specifications for the wood quality. When the mill solves the bottleneck using wood quality, normally it is the forest breeders who need to produce wood with the desired specification, sometimes simply by changing clones or searching for new clones that fit the specifications. Pulp mills are short-term oriented and forest breeders are long term. The conclusions and behaviors are very different in both cases. Depending on the bottleneck experienced, some mills prefer to use low-density wood (0.40–0.45gcm–3), other medium (0.50–0.55gcm–3), and others high density (0.55–0.60gcm–3).
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Active alkali consumption and pulp yield. These variables are a consequence of the wood characteristics such as lignin, extractives, ash, density, Eucalyptus species, tree age, etc. The pulp maker wants to manufacture more and better, without overloading the production process. For this reason, the wood quality improvement programs need to be oriented by processing qualities in pulp manufacturing.
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Specific wood consumption (m3/adt = cubic meters of wood by air-dry ton of pulp). This consumption is the result of many interconnected wood and kraft process variables: wood basic density, pulp yield, decay of wood, process losses of fibers, wood chipping operations, etc. The specific consumption of the wood is responsible for important fraction of the pulp cost. Wood is the main component in the pulp production cost, even in low-cost wood countries. For this reason, it is one of the most vital indicators to the mill manager. All qualitative wood characteristics that may impact the specific wood consumption should be optimized.
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Lignin content and lignin type. Lignin is abundant in Eucalyptus wood, especially those planted in Brazil. The total lignin content in Brazilian Eucalyptus wood varies from 24% to 32%, relatively high for hardwoods. This affects the pulp yield in the conversion to kraft pulp, as well as the consumption of active alkali and the generation of dry by the recovery boiler. There are Eucalyptus species with lower lignin contents and as such are more appropriate for pulping operations. E. globulus and E. dunnii when compared to E. urograndis, E. urophylla, E. grandis, and E. saligna, offer woods of 2–8% less total lignin (based on dry wood). For this reason, tree breeders are trying to combine in hybrids for cloning these wood characteristics in association with the fast growth rates from other species. Thus, gains in wood-specific consumption, alkali charges, and pulp yield should be significant. As a rule, for 1.2–1.5% reduction in lignin, the kraft pulp yield increases 1% based on dry wood. The active alkali consumption reduces around 0.2–0.3% for the same lignin reduction. Therefore, the production of low lignin clones is a new challenge for hybridization and cloning, without the need for genetic modification to insert genes for low lignin in the wood. At the same time, in Portugal, the need to maintain the purity of E. globulus and to improve the forest productivity should be possible through genetics and silviculture. With this in mind, the Portuguese foresters are willing to improve the productivity of E. globulus in equivalent tons of pulp per hectare per year. In Brazil, Eucalyptus commercial forests produce from 9 to 15adtha–1year–1, while in Portugal the E. globulus forests produce 6–8adtha–1year–1. Even with lower growth rates, the E. globulus trees are offering a competitive raw material thanks to the better wood basic density, lignin content, and pulp yield. However, not only the quantity of lignin in the wood is important, but also its quality. Lignin with high syringyl/guaiacyl ratio offers easy cooking and pulp bleaching because guaiacyl type lignin is more difficult to be removed during the chemical reactions. This ratio in Brazilian Eucalyptus varies from 2 to 3, but in Portuguese E. globulus it may vary from 2.5 to 6.
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Extractives content. Extractives are undesirable in the pulping process because they have an impact on pulp yield and contaminate the process and the final product with pitch. The total extractives content in the Eucalyptus wood varies as a function of species, age, silvicultural stresses, pests, and diseases, etc. Values are variable from 1.5% to 6%. Extractive can be removed in several ways, normally extracting them with a solvent (water, caustic soda, ethanol, toluene, dichloromethane, etc.). In all cases, the lower the extractives, the better is the pulping, bleaching, and cleanliness of the final product.
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Ash content. Wood is rich in mineral elements and these are absorbed by the trees as nutrients. When the wood is harvested, the minerals are exported from the soil through the trees to the mills. Minerals are measured in the wood as ash content, after burning the sawdust of the sampled wood. Ash content in Eucalyptus wood varies from 0.3% to 1.0%, the most important minerals being calcium, potassium, and magnesium. With the trend to use closed water cycles in the pulp mills, these minerals are able to build up in these systems, bringing enormous problems with incrustations, pitch formation, formation of “stones” in the recovery boiler, etc. Ash content is very variable among the Eucalyptus species and for this reason it is an important parameter for tree breeding. When low ash woods are selected, the exportation of nutrients from the soil is minimized. The trees are more efficient in wood formation using or immobilizing fewer minerals in their tissues. These minerals, also known as nonprocess elements, leave the pulp mills as pollution (solid residues, air pollution particulate or dust, and dissolved ions in liquid effluents). This means that a fantastic natural resource is transformed into pollution, in mills not oriented to prevent or to develop clean production techniques for controlling and recycling these minerals. For this reason, the emphasis today is to prevent the intake of minerals into the mills, both those in the wood composition, or as contaminants (soil, stones, etc.). Bark is also very rich in ash (about 5–10 times richer in ash content than wood), thus the contamination of logs and chips with bark brings additional amounts of minerals into the milling process affecting pulp making.
3.2.1.2 Eucalyptus wood quality requirements for the production of paper
All these wood characteristics and related consequences mentioned until now favor the pulp mills in their targets for productivity, costs, and efficiency. However, they are only part of the wood features to be evaluated. Eucalyptus pulp is a raw material for the manufacture of several grades of papers. For each paper grade and for each paper mill design, there are different wood and pulp quality requirements. It is important to mention that, independent of the type of paper to be manufactured, all the paper makers have what are known as basic physiological needs. These needs are similar to those for the pulp manufacturer: productivity, operational efficiency, quality, and costs.
Productivity requires a high-speed paper machine, fast drainage at the wet end, high consistency after the wet presses, and minimum number of paper sheet breaks along the machine. Quality implies the maximum percentage of paper in the specification range and minimum generation of broken paper lines. Machine operation efficiency is the dream of any paper manufacturer. He wants his machine working smoothly, at the maximum speed as possible, no breaks, and achieving the required quality in the manufactured products. The consequence of all this is that the specific unit cost is also optimized here. No doubt that a good pulp is the one able to provide good paper machine runnability and appropriate quality in the end product.
Some of the desired pulp properties are closely related to these performances and directly related to wood quality, others depend on the conversion of wood to pulp (cooking, bleaching), and many are a combination of these two factors influencing the pulp quality. For example, some properties that are related to pulping and bleaching are viscosity and degradation of cellulose chains, fiber deformations and individual fiber strengths, surface charges in fibers, etc. One very important pulp property that is related to wood quality and pulp conversion is the hemicellulose content in the pulp. This parameter depends on the wood content and the ability of the pulping process to preserve it in the fibers.
There are many pulp properties that are dependent both on wood quality and on pulping/bleaching processes. There are also many cases where the exigencies are placed a lot on the wood quality, when the wood is not the only factor to determine the pulp quality for paper. Wood quality affects properties such as WRV—water retention value, WWS—wet web strength, fiber bonding, and individual fiber strength. However, other pulp properties are directly related to pulp manipulation processes, for example, the fine content of the pulp (parenchyma cells and fiber fragments) is generated in operations such as wood chipping, pulp pumping, pulp dynamic mixers, pulp dewatering presses, etc.
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Drainage at the wet end section in the paper machine. This behavior is very much affected by the fiber population (number of fibers per gram of pulp), by the initial or refined pulp freeness (drainability of the pulp measured as Canadian Standard Freeness or Schopper Riegler degree), by the Water Retention Value (hydration and swelling ability of the pulp furnish), and by the fine content in the pulp furnish.
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Paper sheet strength along the paper machine, mainly at the wet end and press section. Sheet behavior is very much dependent on the individual fiber strength, fiber bonding, furnish contaminants (shives, sand, solid debris, etc.) and consolidation of the paper web. Individual fiber strength is related to fiber wall thickness, fibril angle, fiber deformations, and micro-fractures and the Eucalyptus species used.
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Fiber population or the number of fibers per gram of pulp. The fiber population is related to the weight of each individual fiber, to fiber coarseness and to the percentage of fiber wall in the fiber volume. There are a number of fiber properties associated to fiber population and fiber coarseness: fiber wall fraction (ratio between cell wall thickness and fiber ray), Runkel index, fiber flexibility index (ratio between the lumen diameter and fiber diameter), index of fiber collapsibility, ratio fiber wall thickness and fiber perimeter, wood basic density, and fiber length. Pulps with lower fiber population show better drainage in the wet end and the paper sheets are more porous, bulkier, more permeable, and absorbent. They are very much appreciated by paper makers because they allow faster machine speeds, if they furnish enough strength to the wet paper sheet.
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Individual fiber strength. This fiber characteristic is very difficult to be measured in short fibers such as those from Eucalyptus. There are tests correlated to this parameter such as the zero spans, very useful for predicting pulp quality and behavior in the paper machines.
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Fiber bonding ability. This test is measured by the wet/dry zero span technique or by other equipment for bonding tests, such as the Scott bond tester. It is also related to the hemicellulose content of the fibers, fiber population, fiber drainability (CSF or SR), fiber fines content, and fiber collapsibility.
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Fiber swelling. This fiber property is affected by the pulping and bleaching operations during pulp manufacture and by the pulp hemicellulose content. Several properties are associated to the swelling of fibers: water retention value, fiber charges, fiber wall microporosity, and fiber wall microfractures.
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Fiber deformations. The deformations in the fibers are measured as curl index, fiber kinks, fiber latency, and fiber microfractures in the cell wall. They affect fiber strength, but they provide substantial improvements in the paper sheet porosity, bulk, smoothness, and water absorption.
Eucalyptus pulps are special products in the manufacture of bulky and/or opaque papers. Today, Eucalyptus pulps are preferred raw materials in the manufacture of tissue, printing and writing, carton boards, industrial filter, impregnation based, cigarette, and many other papers. Eucalyptus fibers may be the sole fiber in the pulp furnish or to be part of a blend with other short and/or long fibers.
Tissue papers demand softness, smoothness, absorption, bulk, and the exact strength to provide machine runnability and very fast drainage in the wet end. The fibers cannot collapse because this will flatten the paper surface, the paper becomes stronger in tensile, but all the tactile properties are lost. Pulp fines are also undesirable for two reasons: fiber bonding and building up in the paper machine white water system, reflecting in drainability losses. The most indicated Eucalyptus fibers for tissue manufacture are those showing low fiber population and consequently high coarseness, low fine content, low bonding ability, low hemicellulose content, high bulk, and water absorption in the manufactured paper sheets. Fiber deformations are also important, since these deformations improve the bulk, porosity and absorption of the paper. It is important to remember that fiber deformations may be artificially created in the pulp mills. The manufacture of industrial filter papers and impregnation-based papers demand the same properties, but at different levels. This means, to go to these specialty paper markets, the differentiation must be even more pronounced. Thus the simplest way to move between very specialty markets is to work towards very high coarseness (low fiber population, high wood basic density), low hemicellulose content, and to intensify fiber deformations (by high consistency presses, fiber shredding, or pulp flash drying).
For printing and writing papers, the desirable paper properties are formation, paper strength, porosity, dimensional stability, and opacity. A higher fiber population provides improved opacity associated with lower fiber coarseness. Fiber bonding and hemicellulose and pulp fine content is important to improve strength. However, there are limits depending on each paper machine system and operation. A very high fiber population may improve opacity and formation, but drainage at the wet end and consistency after wet presses may deteriorate and machine speed is reduced. Fiber deformation may not be so important, but may help to balance the pulp properties, since it may be created by machines. A higher hemicellulose content favors refining, bonding, consolidation of the paper web and strength properties (tensile, burst, tear, folding). An ideal pulp should have high strength at the low levels of refining. However pulp refining raises energy costs, reduces the life of refiner discs, reduces machine drainage and machine speed, increases steam consumption and a very important paper property that is dimensional stability. Definitively, the best pulps are those showing good strength at low levels of refining. Paper maker is very sensitive to this. Besides these properties, there is another wood anatomical characteristic that is very important to printing grade paper: vessel element content and vessel dimensions (especially the diameter). Large, wide, and numerous vessels are undesirable for P&W (printing and writing) papers giving a defect known as vessel picking. The paper maker needs to have special conditions to combat the vessel-picking tendency in the paper. Thus, wood with smaller and less numerous vessels is preferred.
There are many other grades of papers manufactured with Eucalyptus pulps, but generally Eucalyptus fibers are used to improve paper formation, opacity, smoothness, dimensional stability, bulk, and porosity. The Eucalyptus fiber population in the pulps is rigid and difficult to collapse an important property for paper making. There is another key driver to paper makers for using fibers: the market pulp prices of this fiber. Thanks to the low production costs, high pulping yield, and lower chemical and wood consumption, Eucalyptus pulps are generally less expensive than softwood pulps. No doubt that the production costs are also key issues for paper makers. The same are to the entire Eucalyptus pulp and paper production chain.
Eucalyptus pulps have today gained the status of the most admired fiber supply. They are growing in an unbeatable rate in the paper business. Eucalyptus pulps are versatile and may be used as the single fiber or blended with others, such as hardwoods, softwoods or recycled fibers. Wood and fiber quality improvements due to genetic and silvicultural operations and of the conversion process may contribute to further worldwide appreciation of Eucalyptus.
3.2.1.3 Eucalyptus wood quality requirements for wood charcoal manufacturing and biomass fuel
Brazil has had enormous success in the utilization of Eucalyptus biomass as firewood and for charcoal production. The first biomass fuel-oriented forest plantations were based on high wood density Eucalyptus species, such as E. paniculata, E. camaldulensis, E. tereticornis, C. citriodora, and C. maculata. Wood basic density is a fundamental wood characteristic because it leads to better quality charcoal and a higher calorific value per volume of wood or charcoal. However, tree growth rate is also important in order to reach the maximum possible production of dry biomass per hectare (trunk, branches and bark). Thus, if an Eucalyptus species leads to high-density wood, but its growth rate is poor, the production of total biomass may not economically attractive. For this reason, the wood biomass segment has directed its efforts to the diversification of species, and hybridization. Currently, more species are being explored in the forest breeding: E. cloeziana, E. pellita, E. urophylla, and hybrids, such as E. urograndis. These species are adapted to grow in tropical regions of Brazil, where the attacks of pests and diseases are more frequent, due to higher temperature and humidity. In case that charcoal production could migrate to temperate regions, where there are other species very suitable such as E. dunnii, E. viminalis, E. benthamii, E. saligna, E. globulus, and hybrids.
It is important to mention that wood density is a key property, but there are other wood characteristics that are important when wood is destined for fuel purposes. The trees should be as straight as possible to favor the feeding of the charcoal ovens or biomass furnaces or chippers. The bark content is required to be as low as possible, since the wood for energy is not debarked and Eucalyptus bark has a higher percentage of minerals, lower carbon content, basic density and calorific value. Furthermore, the high phosphorous content in the bark may make difficult the utilization of this material in the production of some grades of charcoal.
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Basic Wood density and forest growth rate. It is essential to optimize these two key parameters at once. Faster growth and higher wood density are desirable with the goal of maximizing dry biomass production per hectare.
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Lignin content, lignin composition, and carbon content. The lignin calorific value is higher than that obtained from the cellulose and hemicelluloses carbohydrates. The carbon content in lignin is somewhat higher than in the wood carbohydrates. For this reason, the higher the lignin content, the higher the carbon content, and the better the wood is for fuel. The ratio syringyl/guaiacyl is another point to be considered since guaiacyl lignin is richer in carbon content than syringyl. This means that, contrary to the wood for pulping, the wood for energy the syringyl/guaiacyl ratio should be as low as possible.
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Ash content. Wood minerals do not generate heat, but they consume heat during combustion and they also reduce the carbon content of the wood and bark based on dry weight content. Both wood and bark should have minimal ash content.
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Volatile extractives. Charcoal production implies substantial dry weight losses and the usual yields when manufacturing charcoal with Eucalyptus wood varies from 30% to 40%. Many types of wood extractives are very volatile and they are lost in the exhaust flue gases during controlled combustion. Depending on the manufacturing process, the extractive content of wood and bark may affect the charcoal yield.
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Wood moisture. High-density woods have lower moisture content due to the fact that they have less open spaces or porosity to hold water. For this reason, they are easily dried for consumption as fuel raw material. High speed drying is a good quality parameter for wood selection, thus the lower the wood and bark moisture, the better is the raw material is for biomass fuel.
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Anatomical composition of the wood. It has been proven that wood that is rich in vessel elements and parenchyma cells is less recommended for charcoal manufacturing. In general, the lower the density and the quantity of these anatomical elements, the more friable and poorer quality charcoal is produced.
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Fissured and cracked logs. It is very important that the logs have good dimensional stability, with minimal cracks and fissures. These wood defects give a higher fine content in the charcoal and a lower yield as consequence, since fines have to be screened out from the charcoal to improve its quality.
Wood may be engineered and improved by selection, genetics and silvicultural operations, therefore for maximization of results, it is important to know and to understand the parameters that need to be improved. They need to show good heritability and to add value to the conversion processes and to the end-use products. Sound planning, representative sampling, high quality evaluation, and data interpretation are vital to the success of any wood quality improvement program. Many of the current successes can be attributed to the excellent opportunities offered by hybridization and cloning techniques. Wood basic density has been the most important quality parameter to predict wood quality but it is not the unique wood characteristic. Another point is that wood basic density is a very good parameter to compare wood from the same species, or with similar behavior for a given utilization. For example, even with the same wood density, two very different species of Eucalyptus may show very different behavior in pulping and papermaking. E. saligna wood with 0.5gcm–3 usually shows a completely different performance than E. robusta wood with exactly the same wood basic density.
Definitively, there are many roads to walk in the direction to the future. Thus, future improvement programs need to be efficient and efficacious if they are to maintain continuous competitiveness of Eucalyptus as source of industrial raw material. These programs must not only face economic and quality issues, but also environmental and social performances. These new roads are demanding new additional challenges, and because of this, science, technology, knowledge, and goodwill are to be soundly matched.
3.2.2 Characterization of the transcriptome involved in the process of wood formation in Eucalyptus
Wood is formed from the vascular cambium, which is the secondary meristem, responsible for the diametric growth of the stem. When the cambial initials divide, one cell remains at meristematic status while the other is destined to become a xylem or a phloem mother cell. During differentiation, the xylem mother cells undergo an ordered series of developmental steps that include cell division, cellular expansion, and deposition of secondary cell wall, giving rise to supporting, storage, and conducting cells (Larson, 1994). The xylem transport function is carried out by a specific cell type, the vessel elements, which undergo a differentiation process characterized by successive deposition of secondary cell wall layers, culminating in PCD (programmed cell death) and autolysis.
The genetic factors controlling wood formation in Eucalyptus are still not fully understood. In recent years, many genes involved in wood formation have been identified by large-scale genomic approaches in Poplars, Pines, and Eucalyptus (Hertzberg et al., 2001; Lorenz and Dean, 2002; Paux et al., 2004). Hertzberg et al. 2001 established a hierarchical pattern of gene expression through different zones of developing xylem in Populus by isolating cells at different stages of xylogenesis. By microarray analysis, the authors showed that genes encoding enzymes involved in cellulose and lignin biosynthesis, as well as a large number of transcription factors and potential xylogenesis regulators are under strict control at each xylem differentiation stage. Paux et al. 2004 developed a targeted approach of functional genomics by the construction of a xylem-leaves subtractive library to identify genes involved in the control of Eucalyptus wood formation. The two main classes of ESTs preferentially expressed in xylem were related to auxin signaling through ubiquitin proteolysis, cell wall biosynthesis, and remodeling. More recently, the induction of tension in wood has been used as a model to study wood formation due to its higher cellulose content and poor lignification (Paux et al., 2005; Andersson-Gunnerås et al., 2006).
In the attempt to investigate the genes expressed during juvenile wood formation in E. grandis, when growth is maximized, Carvalho et al. 2008 produced two SAGE libraries (Velculescu et al., 1995) from the cambial region of 3- and 6-year-old trees representing juvenile wood. Overall, the authors identified 444 genes involved in cellulose biosynthesis, nucleotide sugar metabolism, and lignin biosynthesis, as well other physiological processes related to wood formation. The tissue samples were collected from a half-sibling population of E. grandis originated from a single mother tree of a second generation of clonal seeds orchard, introduced from Coff's Harbour, Australia. A total of 40 3-year-old and 40 6-year-old trees were sampled from stands located in Itapetininga, State of São Paulo, Brazil (23°35′20″ S and 48°03′11″ W) at an altitude of 656m. The 3- and 6-year-old trees were spaced at 3 × 1.5m and had an average height of 18 and 25m, respectively. The cambial region of the trees were harvested in the same morning during the summer of 2003, by opening a window (20 × 15cm) in the bark at breast height, scraping the stem exposed tissue and the inner surface of the bark, and immediately freezing the sample in liquid nitrogen (Figures 20a and b). The stem tissue was scraped until the fibrous material below the differentiating cells was reached. The inner side of the bark was also scraped to guarantee that all the meristematic material would be represented in the SAGE libraries. This procedure was firstly used by Foucart et al. 2006 who showed that the cambial cells get adhered to the removed bark. Figures 20(c) and (d) also shows two transversal sections of Eucalyptus wood (xylem, cambial region, and phloem) where it is possible to observe that the meristematic cells tend to follow the phloem cells when the bark is removed. Two bulks, one representing the 3-year-old trees and the other the 6-year-old trees, were made by grinding and mixing all the samples material.
Biological material sampling. (a) Removal of the tree bark. (b) sampling the cambial region by scraping the tissue. (c) transversal section of Eucalyptus wood before removal of the bark. (d) transversal section of the Eucalyptus wood after removal of the bark. Bars = 100μm
The 3- and 6-year-old SAGE libraries were produced from the sequencing of 737 and 703 clones generating 22660 and 22024 tags, respectively. As the number of tags produced by the two libraries was very similar, the authors combined the sequencing reads from both and analyzed the data as one library representing the cambial region of juvenile E. grandis trees. The SAGE 2000 software extracted 43304 tags from the sequencing data file and produced 26958 tags with three or more copies representing 3066 unique tags or genes. From this total the authors have assigned ESTs to 657 unique tags (data not shown), 444 and produced 26958 tags with three or more copies representing 3066 unique tags or genes. From this total the authors have as signed ESTs to 657 unique tags (data not shown), 444 with a defined function, and 213 with unknown functions. For most of the unique tags (79%) it was not possible to associate an EST or a complete cDNA due to the small number of Eucalyptus sequences currently available in public databases (Figure 21a).
Distribution of the 3066 genes analyzed. (a) Tags mismatches: tags with no homology to the Eucalyptus public EST sequences, Expressed sequences: tags associated to an EST with unknown function in the GenBank, Identified genes: tags associated to an EST with a probable function defined in the GenBank. (b) Functional classification of the 444 identified genes. Functional Categories: metabolism and energy, cellular processes, transport, structure and organization of structure, and information pathways
The 444 tags that presented a tag–gene association were annotated and assigned to functional categories as defined by Rison et al. 2000. The most expressed categories were metabolism and energy and structure and organization of structure, followed by information pathways and processes (Figure 21b).
It is important to note that among the tags in the subcategory autotrophic metabolism, there are genes associated with glycolysis, TCA cycle, alcoholic fermentation, and ATP synthesis. In nonphotosynthetic organs, carbohydrates are consumed through respiration to produce energy and carbon skeletons for cellular metabolism and biosynthesis of structural molecules, including cell wall polymers. However, the quantity of free O2 in the cambial region might be limited by the physical barrier imposed by the bark and, also due to the O2 consumption during respiration. Thus, a proportion of the necessary energy to secondary growth could be provided by alcoholic fermentation (Kimmerer and Stringer, 1988). The possible role of alcoholic fermentation as an alternative or principal supply of energy in juvenile E. grandis trees is also suggested by the presence of alcohol dehydrogenase and pyruvate decarboxylase transcripts. Other recent studies have reported the presence of transcripts or proteins associated with anaerobic respiration during xylem formation and secondary growth (Gion et al., 2005; Juan et al., 2006; Ranik et al., 2006).
Another interesting observation in the work of Carvalho et al. 2008 was the existence of tags representing light induced proteins and components of the photosystems I and II in wood forming tissues (Carvalho et al., 2008). The presence of functional chloroplasts with active photosystems in the outer peridermal layers (chlorenchyma) and also in deeper stem tissues such as ray cells and pith, have been reported in many woody species (Pfanz et al., 2002), what could explain the presence of transcripts related to photosynthesis in Carvalho's data. Supporting this result Celedon et al. 2007 also found rubisco proteins using 2D-LC-MS/MS in the same samples used in the previous study. The outer bark of the stems have a rather low permeability to gaseous diffusion, leading to accumulation of CO2 in the intercellular air spaces, around 500–800 times higher ambient air (Pfanz et al., 2002). Thus, stem photosynthesis may be an important mechanism to partially reduce the internal anaerobiosis, avoiding a further restriction to dark respiration and ATP production (Pfanz et al., 2002).
3.2.2.1 Sugar-nucleotide metabolism and cell wall biosynthesis
Sugar-nucleotide metabolism provides the precursors for biosynthesis of hemicelluloses and pectins during wood formation. These precursors represent 65% and 26–36% of the primary and secondary cell walls, respectively (Mellerowicz et al., 2001). The UDP-glucose dehydrogenase (UGDH) and UDP-glucuronate decarboxylase (UXS), responsible for UDP-glucuronate and UDP-xylose production, respectively, were represented by highly frequent tags (Figure 22a). However, only one UGDH tag with 22 copies was observed, while the total expression of the three UXS tags accounted for 47 copies. This expression pattern is consistent with previously reported findings pointing to a low activity of UGDH when compared to other enzymes in subsequent reaction steps in the same pathway, suggesting its function as rate limiting in the synthesis of matrix polysaccharides (Dalessandro and Northcote, 1977).
Transcript profiling of genes related to the main biosynthetic routes of cell wall components. For each enzyme the corresponding tag(s), as well as its expression level are presented. *Tags identified as potential alternative transcripts. Pathways related to (a) Sugar-nucleotide interconversions, pectin, hemicellulose, and cellulose biosynthesis. (b) Lignin biosynthesis. GT8D, glycosyl transferase family 8D; UXS, UDP-xylose synthase; UGDH, UDP-glucose dehydrogenase; PAL, phenylalanine ammonia-lyase; C4H, cinnamate 4-hydroxylase; 4CL, 4-coumarate: coenzyme A ligase; CCR, cinnamoyl-coenzyme A reductase; CAD, cinnamoyl alcohol dehydrogenase; C3H, coumarate 3-hydroxylase; HCT, hydroxycinnamoyltransferase; COMT, caffeate/5-hydroxyconiferaldehyde O-methyltransferase; CCoAOMT, caffeoyl-coenzyme A O methyltransferase; F5H, ferulic acid/coniferaldehyde 5-hydroxylase; SAMS, S-adenosyl-methionine-synthetase; GS1, cytosolic glutamine synthetase
Transcript profiling of genes related to the main biosynthetic routes of cell wall components. For each enzyme the corresponding tag(s), as well as its expression level are presented. *Tags identified as potential alternative transcripts. Pathways related to (a) Sugar-nucleotide interconversions, pectin, hemicellulose, and cellulose biosynthesis. (b) Lignin biosynthesis. GT8D, glycosyl transferase family 8D; UXS, UDP-xylose synthase; UGDH, UDP-glucose dehydrogenase; PAL, phenylalanine ammonia-lyase; C4H, cinnamate 4-hydroxylase; 4CL, 4-coumarate: coenzyme A ligase; CCR, cinnamoyl-coenzyme A reductase; CAD, cinnamoyl alcohol dehydrogenase; C3H, coumarate 3-hydroxylase; HCT, hydroxycinnamoyltransferase; COMT, caffeate/5-hydroxyconiferaldehyde O-methyltransferase; CCoAOMT, caffeoyl-coenzyme A O methyltransferase; F5H, ferulic acid/coniferaldehyde 5-hydroxylase; SAMS, S-adenosyl-methionine-synthetase; GS1, cytosolic glutamine synthetase
Among the UXS tags, one (TACTCGGTTG) with 27 copies is associated with the Arabidopsis AtUXS3 soluble form. The other two, occurred at a frequency of 20 copies and are associated with the Arabidopsis AtUX4 Golgi membrane form (Harper and Bar-Peled, 2002). A more detailed analysis of the ESTs associated with the AtUXS4 tags reveled that the tag (TCATTATCAA) was present in both ESTs sequences distant 27 and 146bp from the poly-A, suggesting that probably two alternative transcripts of AtUXS4 are expressed in E. grandis wood forming tissues.
Besides UDP-xylose production, another important UDP-glucuronate-derived branch leads to pectin metabolism. The pectic polymers are mainly important in the primary cell wall where they comprise approximately 47% of the polysaccharides. Despite its importance in cell wall structure, only a few genes responsible for pectin biosynthesis have been identified to date.
In dicotyledonous plants, the primary cell wall consists basically of a cellulose microfibrils framework embedded in a polysaccharide matrix of pectin and cross-linked glycans (Carpita and Gibeaut, 1993). During cell extension occur modifications in the structure and composition of the cross-linked pectin-xyloglucans (Bourquin et al., 2002). For example, xyloglucan endotransglycosylases (XETs) are responsible for cell wall remodeling during primary cell wall biosynthesis by cutting and rejoining the xyloglucan chains. XETs probably act during secondary cell wall deposition, as well as, by creating and reinforcing the connections between primary and secondary wall layers (Bourquin et al., 2002). Despite its well-known importance in cell wall remodeling, only one XET tag with 19 copies was observed, although this result could be due to the lack of Eucalyptus XET ESTs publicly available. Moreover, an increased number of transcripts for pectinesterases and pectate lyases were observed during increased secondary growth in poplar tension wood (Andersson-Gunnerås et al., 2006). However, under normal growth conditions experienced in our study, two pectinesterases tags at a frequency of 35 and 11 copies were observed in the cambial region library, and only one low expressed tag for a polygalacturonase (Figure 22a) in contrast to the 11 highly expressed genes identified in active poplar cambium (Geisler-Lee et al., 2006).
3.2.2.2 Cellulose biosynthesis
Current models of cellulose biosynthesis involve not only CESA proteins but also membrane-associated proteins like KORRIGAN (endo-1,4-β-glucanase) and SUSY (sucrose synthase) (Joshi et al., 2004). Five tags corresponding to Eucalyptus grandis cellulose synthase (EgCesA) genes were identified, one showing high similarity to the primary cell wall related cellulose synthase gene EgCesA4, and the other four showing high similarity to the secondary cell wall related genes EgCesA1, EgCesA2, and EgCesA3. Two tags (AATTGATATG and GAATCAAAAT) represented the EgCesA1 gene, the first occurred in both ESTs sequences at a distance of 151 and 30bp from the poly-A tail, indicating a possible alternative transcript (Figure 22a).
According to Ranik and Myburg 2006, some genes implicated in Eucalyptus secondary wall formation (EgCesA1, EgCesA2, and EgCesA3) have higher expression levels than those involved in primary cell wall formation (EgCesA4, EgCesA5, and EgCesA6) in xylem. Carvalho et al. 2008 observed the same transcriptional profiles for primary cell wall EgCesA genes. The EgCesA4 tag shows low expression (Figure 22a) and the EgCesA5 and EgCesA6 tags are represented only as single-copy transcripts (Carvalho et al., 2008). The higher expression of the secondary cell wall associated CesA genes was expected since the sample contained more cells from the xylem side of the cambial region. Ranik and Myburg 2006 reported the EgCesA3 gene as the highest-expressed CesA gene in secondary xylem. In contrast, the results of Carvalho et al. 2008 pointed out the EgCesA1 as the most expressed (71 copies) cellulose synthase gene in the E. grandis juvenile wood forming tissue (Figure 22a).
The pool of UDP-glucose destined to cellulose synthesis can be produced either by UDP-glucose pyrophosphorylase or sucrose synthase. In Carvalho's study, the higher frequency of SUSY transcripts compared to UDP-glucose pyrophosphorylase transcripts strongly suggests that SUSY activity is probably the main source of UDP-glucose for cellulose synthesis in E. grandis differentiating xylem (Figure 22a). SUSY transcripts were the most abundant carbohydrate-active enzymes (CAZyme) transcripts in poplar and have been shown to be highly expressed during secondary cell wall biosynthesis in tension wood formation (Andersson-Gunnerås et al., 2006). The membrane-associated SUS (SUSY) was detected in developing cotton fibers giving rise to a functional model for cellulose biosynthesis where this enzyme directly channels UDP-glucose to the membrane-bound cellulose synthesis complex avoiding competition from the cellular metabolic pool of UDP-glucose, and providing a more efficient cellulose synthesis (Amor et al., 1995), especially important during active secondary growth.
3.2.2.3 Lignin biosynthesis
The phenylpropanoid pathway starts with the deamination of phenylalanine to cinnamic acid by PAL. Cinnamic acid is then converted to coumaric acid by C4H and diverted to monolignol synthesis (Figure 22b). Both enzymes from the early steps of phenylpropanoid biosynthesis were represented by highly frequent tags (Figure 22b). Both PAL genes (AtPAL1 and AtPAL2) are believed to be the most important players in lignin synthesis during vascular lignification among the four Arabidopsis PAL genes (Raes et al., 2003). The C4H tag was observed at a frequency of 36 copies and is associated with the poplar PtreC4H2 gene, which is more xylem specific than the PtreC4H1 and more weakly expressed in phloem (Lu et al., 2006).
Only one tag representing the E. camaldulensis 4CL1 gene (Figure 22b) was observed. The enzyme 4CL produces CoA thioesters of hydroxycinnamic acids, which are the precursors for H (p-coumaryl alcohol), G (coniferyl alcohol), and S (sinapyl alcohol) lignin subunits. One poplar 4CL gene was reported to be up-regulated in the cambial zone undergoing lignification (Hertzberg et al., 2001) confirming its important role in the initial steps of lignin synthesis. Lignin polymers in angiosperm wood are composed by great amounts of G and S units while only small amounts of H units are added. This is because H units are predominantly deposited into the middle lamella and cell corners followed by G units, which are mainly laid down in the secondary wall, and S units that are deposited at the late stages of lignification (Lewis, 1999). Thus, it is expected that the key enzymes in the biosynthesis of the monolignols G and S show higher expression levels during secondary growth. The tags for COMT, CCoAOMT, and S-adenosylmethionine synthase (SAMS) showed a general higher expression level than those for 4CL and CCR (Figure 22b). These results agree with Paux et al. 2005, who suggested that the expression of 4CL, CCR, and CAD is under a common transcriptional control while COMT and CCoAOMT form another co-regulated transcriptional cluster. Similar expression profiles for these two enzymes was also observed by Hertzberg et al. 2001 supporting this hypothesis.
The determining step for the diversion of p-coumaryl-CoA for G and S monolignols synthesis is its conversion into p-coumaroyl shikimic acid/quinic acid by hydroxycinnamoyltransferase (HCT), since p-coumarate 3-hydroxylase (C3H) is not able to use p-coumaryl-CoA as substrate (Schoch et al., 2001). It has been shown that p-coumaroyl shikimate and p-coumaroyl quinate are important intermediates in the phenylpropanoid pathway with HCT acting both upstream and downstream of C3H, producing caffeoyl CoA (Hoffmann et al., 2004). Interestingly, the C3H tag, similar to the Arabidopsis CYP98A3 class of P450 gene whose expression is more evident in lignifying vascular cells (Nair et al., 2002), was almost four times more expressed than the HCT tag (Figure 22b). Alternatively, C3H can also act on p-coumaric acid precursors producing caffeic acid, which in turns can be diverted to ferulate, by COMT, or to caffeoyl CoA, by 4CL (Figure 22b).
Although COMT was first believed to convert caffeic acid into ferulate (Dixon, 2001), it was subsequently shown that COMT preferentially catalyzes the conversion of 5-hydroxyferulate, 5-hydroxyconiferaldehyde, and 5-hydroxyconiferyl alcohol into sinapic acid, sinapaldehyde, and sinapyl alcohol, respectively, and thus acts preferentially on ferulic acid/coniferaldehyde 5-hydroxylase (F5H) derived products (Parvathi et al., 2001). A differential regulation for F5H and COMT genes is supported by recent findings obtained through a proteomic approach where F5H proteins were not found among the expressed proteins during poplar cambial regeneration, while COMT isoforms were detected at all stages (Juan et al., 2006). Consistent with this previous result, our data showed that COMT transcripts expression was almost twice the F5H expression level (Figure 22b).
According to Carvalho's data, the CCoAOMT transcripts had the highest expression level of all lignin biosynthetic enzymes observed in E. grandis wood forming tissue (Figure 22b). This is in agreement with the results of Paux et al. 2004 that showed a preferential expression of this gene during Eucalyptus wood formation. CCoAOMT adds a methyl radical to caffeoyl CoA, producing feruloyl CoA in an alternative route for monolignol production (Zhong et al., 1998). The importance of this alternative route was demonstrated by the down-regulation of CCoAOMT in transgenic tobacco and poplar plants leading to an altered S/G ratio and significantly decreased lignin content (Zhong et al., 1998, 2000). As the number of CCoAOMT transcripts is much higher than the number for COMT, we suggest that the synthesis of S and G units is being preferentially carried out directly from caffeoyl CoA in E. grandis.
It is interesting to note that CCoAOMT and SAMS tags have similar high expression levels (Figure 22b), even though the presence of a common transcriptional regulatory system for both genes is still unknown. Although SAMS is a housekeeping enzyme involved in methionine metabolism, its activity occurs in xylem tissue undergoing secondary growth (Juan et al., 2006). According to Cantón et al. 2005, SAMS might be functioning in a local providing of methyl groups consumed by CCoAOMT and COMT thus ensuring high rates of lignification.
The lignification processes also requires a large nitrogen input to support the first step in the phenylpropanoid biosynthesis catalyzed by PAL. Extensive amounts of ammonium are liberated through phenylalanine deamination and an efficient system to recycle nitrogen is needed to prevent a severe N deficiency in plants during active lignification (Cantón et al., 2005). Therefore, the authors proposed a mechanism in which the liberated nitrogen is locally recycled and re-incorporated into glutamine by GS1 (glutamine synthetase). Carvalho's results also suggest a possible role for GS1 during lignification as the GS1 tag showed high expression (101 copies), occupying the 17° position among the 50 most expressed tags in E. grandis juvenile wood forming library (Table 4; Figure 22b).
The final step in lignin biosynthesis is the polymerization of monolignols that is conducted by peroxidases and laccases (Baucher et al., 2003). Although the role of laccases in lignin polymerization is still a matter of debate, their importance during wood formation has been reported (Paux et al., 2004). The results observed by Carvalho et al. 2008 corroborate a greater role for laccases in lignin polymerization during Eucalyptus wood formation due to the lack of tags representing peroxidases (Figure 22b). Supporting this idea, no significant decrease in peroxidase expression was observed in poplar tension wood (characterized by decreased lignin content), while the laccase gene lac3 is co-regulated with the lignin biosynthesis genes (Andersson-Gunnerås et al., 2006). However, Hertzberg et al. 2001 identified two peroxidase genes up-regulated in poplar lignifying tissues, as well as, two laccases with an overlapping expression pattern, indicating specific but different roles in the polymerization process. The authors also demonstrated the induction of a dirigentlike protein coincident with lignification. Dirigent proteins can mediate the monolignols coupling mainly in the S (1) sublayer and middle lamella of lignifying secondary xylem cell walls (Burlat et al., 2001). Carvalho et al. 2008 found one low expressed tag similar to the Forsythia × intermedia dirigent protein and a highly expressed tag similar to the Picea glauca dirigent protein (Figure 22b). The authors suggested that probably there are similar roles for laccases and dirigent proteins in monolignol polymerization in E. grandis.
The data obtained by Carvalho et al. 2008 offers an insight into the expression of functionally related genes involved in cell wall biosynthesis during the first years of development of Eucalyptus, and this information is just beginning to be combined to produce an overview of the important genes for wood formation. In the near future, the information generated by functional genomics, molecular markers, transgenesis, will provide the breeders with important information to decide in the selection of the best genotypes to improve productivity, resistant to biotic and abiotic stresses, and better wood quality.
- 4CL
- 4-coumarate:coenzyme A ligase
- AFLP
- amplified fragment length polymorphism
- AIP
- artificially induced protogyny
- BAP/NAA
- 6-benzylaminopurine/α-naphthalene acetic acid
- BSA
- bulked segregant analysis
- C3H
- p-coumarate 3-hydroxylase
- C4H
- cinnamate 4-hydroxylase
- CAD2
- cinnamyl alcohol dehydrogenase 2
- CAZyme
- carbohydrate-active enzymes
- CCR
- cinnamoyl-coenzyme A reductase
- CCoAOMT
- caffeoyl coenzyme A O-methyltransferase
- COMT1
- caffeate/5-hydroxyconiferaldehyde O-methyltransferase 1
- CSIRO
- Commonwealth Scientific and Industrial Research Organisation
- DBH
- diameter at-breast-height
- EST
- expressed sequence tag
- F5H
- ferulic acid/coniferaldehyde 5-hydroxylase
- G × E
- genotype × environment
- HCT
- hydroxycinnamoyltransferase
- IAA
- indole-3-acetic acid
- IBA
- indole-3-butyric acid
- Kin
- kinetin
- MAS
- marker-assisted selection
- MS
- Murashige and Skoog
- NCBI
- National Center for Biotechnology Information
- OSP
- one stop pollination
- PAL
- phenylalanine ammonia-lyase
- PCD
- programmed cell death
- PCR
- polymerase chain reaction
- PPFD
- photosynthetic photon flux density
- QTL
- Quantitative trait loci
- RAPD
- random amplified polymorphic DNA
- SAGE
- serial analysis of gene expression
- SAMS
- S-adenosylmethionine synthase
- SCAR
- sequenced characterized amplified region
- SEI
- speed of emergence
- SGI
- speed of germination
- SSCP
- single strand conformation polymorphism
- SSR
- simple sequence repeat
- SUSY
- sucrose synthase
- TDZ
- thidiazuron
- UGDH
- UDP-glucose dehydrogenase
- UXS
- UDP-glucuronate decarboxylase
- XETs
- xyloglucan endotransglycosylases
- cDNAs
- complementary DNAs
