The soil bacterium B. thuringiensis produces a wide range of proteins (8-endotoxins) that are included in crystals formed during sporulation and characterised by distinct insecticidal spectra (de Maagd et al. 2001). Bt spores contain high levels of 8-endotoxins harmful to specific insects that belong to the Lepidopteran, Dipteran and Coleopteran orders and are known as the major pests of annual crops and perennial tree species. The Bt spores and the crystal (Cry) proteins are ingested by the insect and solubilized within the alkaline midgut. The protoxins are then activated by proteinases and finally the active Bt toxin binds to specific molecular receptors causing the irreversible damage of the midgut epithelium by colloid osmotic lysis (Knowles and Dow 1993). B. thuringiensis has been used as a commercial insecticide for more than 50 years and to date an extensive number of reports have demonstrated that Bt proteins have negligible potential adverse effects against humans, animals and non-target invertebrates (Shelton et al. 2002). More than 130 Bt genes encoding different 8-endotoxins have been isolated and, among this extremely large gene array, those coding for the CryIA(a) and CryIA(c) proteins have been used to develop transgenic trees resistant to Lepidoptera (Table 12.1). In addition, the CryIIIA(a) protein has been chosen by different research groups to specifically target Coleopteran pests. The first generation of Bt trees expressing wild-type genes was characterised by extremely low levels of insect pest resistance due to the inefficiency of the bacterial codon usage in plants. The ability to withstand dangerous insects was subsequently increased using synthetic Bt genes containing coding sequences that were adapted to the plant transcriptional and translational machinery. It is worth noting that all the different Bt genes used in these studies were placed under the control of the enhanced 35S Cauliflower Mosaic Virus (CaMV) promoter.
12.2.1 Transfer of Bt Genes into Forest Tree Species
The genus Populus is considered a model system in forest tree biotechnology due to the availability of effective transformation and regeneration protocols developed for an increasing number of species and hybrids and to other favorable features (Taylor 2002; Confalonieri et al. 2003). The first Bt poplar was obtained in 1991 by McCown and coworkers who used particle bombardment to introduce a partially modified CryIA(a) gene in a Populus alba x Populus grandidentata genotype. One of the regenerated Bt lines was able to significantly affect in greenhouse the growth of two main pests, the forest tent caterpillar Malacosoma disstria Hübner (Lepidoptera, Lasiocampidae) and the gypsy moth Lymantria dispar L. (Lepidoptera, Lymantriidae), which are responsible for severe damage to poplars (Robison et al. 1994). The same transgenic line, subsequently evaluated during the field-growing season, maintained high expression levels of the CryIA(a) 8-endotoxin after winter dormancy (Kleiner et al. 1995). The wild-type CryIAc646 sequence was transferred to Populus nigra, P. alba and Populus tremula x P. alba. However, insect bioassays performed on transgenic P. nigra lines with larvae of Hyphantria cunea Drury (Lepidoptera, Arctiidae) and L. dispar did not show significant differences in larval mortality (Balestrazzi et al. 1994). When the bacterial CryIIIA gene was expressed in a P. tremula x Populus tremuloides genotype, the resulting GM plants were able to induce mortality of the leaf beetle larvae Chrysomela tremulae Fabricius (Coleoptera, Chrysomelidae) although transgene expression was detected only by reverse transcriptase-polymerase chain reaction (RT-PCR) (Cornu et al. 1996). A partially modified CryIB gene, encoding a protein toxic to the cottonwood leaf beetle Chrysomela scripta Fabricius (Coleoptera, Chrysomelidae) and forest tent caterpillar, was transferred to the P. nigra x Populus maximowiczii hybrid clone 'NM6' and feeding assays revealed a decrease of larval feeding in one of the tested transgenic lines (Francis 1996).
Poplar plantations in China are constantly threatened by defoliators such as the poplar lopper Apocheima cinerarius Erschoff (Lepidoptera, Geometridae) and the gypsy moth. Insect-resistant transgenic P. nigra plants were obtained by Agrobacterium-mediated genetic transformation using the CryIA(c) gene (Tian et al. 1993; Wang et al. 1996) and field evaluation was subsequently carried out on fourteen transgenic P. nigra lines in Manas (China). Results from this research showed that Bt poplars were protected against the damage caused by the larvae of the two main defoliators A. cinerarius and Orthosia incerta Hufnagel (Lepidoptera, Noctuidae)
(Hu et al. 2001). Interestingly, cross-protection of non-transgenic trees located in the same plantation was also observed. Several transgenic lines of Populus trichocarpa x Populus deltoides and Populus x euramericana hybrids carrying a CryIIIA gene were produced by Meilan et al. (2000). Transgenic plants showed very low feeding damage when infested by C. scripta larvae under natural conditions. More recently, Genissel et al. (2003) reported the expression of a synthetic CryIIIA(a) gene, specifically targeted to Coleoptera, in the hybrid poplar (P. tremula x P. tremuloides) clone INRA 353-38. In this construct the Bt gene was controlled by a hybrid promoter containing elements from both the 35SCaMV and the nopaline synthase promoters. The Bt toxin, found in mature leaves at a level corresponding to 0.05-0.0025% of the total soluble proteins, was lethal to C. tremulae at all developmental stages.
In contrast to poplar, Eucalyptus species are recalcitrant to in vitro propagation and genetic transformation. For these reasons, a limited number of transgenic studies is currently available (Campbell et al. 2003). Eucalyptus trees represent a valuable source of hardwood timber and pulp for paper. Plantations in Australia can be rapidly defoliated by insect pests such as the Tasmanian eucalypt leaf beetle Chrysophtharta bimaculata Olivier (Coleoptera, Chrysomelidae). The CryIIIA gene was introduced into Eucalyptus camaldulensis under the control of the pea plastocyanin gene promoter in order to direct accumulation of the protein to the young expanding leaves, tissues usually attacked by insect pests (Harcourt et al. 2000). One of the regenerated transgenic lines was able to confer resistance to early instars of C. bimaculata and Chrysophtharta agricola (Chapuis) and to the native chrysomelid beetle Chrysophtharta variicollis (Chapuis). This was the first useful trait introduced into a commercially relevant eucalypt species.
An emerging field in forest tree biotechnology is certainly represented by the genetic transformation of conifers with genes improving productivity (Tang and Newton 2003). The use of particle bombardment to transform conifers and the regeneration of genetically modified trees expressing genes for insect pest resistance was first reported by Ellis et al. (1993). In this study, the transgenic Bt white spruce (Picea glauca) plants were resistant to the spruce budworm Choristoneura fumiferana Clemens (Lepidoptera, Tortricidae). A Bt gene was subsequently transferred into the European larch (Larix decidua) by Agrobacterium-mediated genetic transformation and the regenerated plants were able to withstand attacks from the larch casebearer Coleophora laricella Hubner (Lepidoptera, Coleophoridae) (Shin et al. 1994). More recently, a synthetic CryIA(c) gene was introduced into the loblolly pine (Pinus taeda L.) by direct gene transfer (biolistics) to mature zygotic embryos (Tang and Tian 2003). Three different genotypes (J-29, E-11 and E-44, respectively) were used in this study. Feeding bioassays demonstrated that the transgenic plants were resistant to Dendrolimus punctatus Walker (Lepidoptera, Lasiocampidae) and Cryptothelea formosicola Strand (Lepidoptera, Psychidae), considered among the major pests threatening this forest species. A positive correlation between the presence of the 8-endotoxin in needle extracts from the Bt plants and insect pest resistance was also observed. Transgenic plants of Pinus radiata expressing the CryIA(c) gene were obtained by biolistic transformation of embryogenic tissue (Grace et al. 2005). Bioassays carried out using larvae of the painted apple moth (Teia anartoides Walker) revealed variable levels of resistance.
The major role played by poplars in the research carried out to engineer insect pest resistance into forest trees is clearly evidenced in Table 12.1.
Genetic engineering of the woody fruit plants has been hampered by the necessity to optimize transformation/regeneration systems suitable for the most recalcitrant species. Notwithstanding these difficulties, improved transgenic fruit species expressing agronomically relevant traits have been described as in the case of the major fruit tree crop Citrus (Gomez-Lim and Litz 2004). Unfortunately, only a few reports are currently available on insect pest resistance as shown in Table 12.1 where only apple and walnut trees are listed. James et al. (1993) reported the production of transgenic apple (Malus x domestica) plants with a CryIA(c) gene; however, results from feeding assays are not available. The codling moth Laspeyresia pomonella L., known as a major insect pest of apple and pear, causes severe damage to walnut (Juglans regia) production. The wild type CryIA(c) gene inserted in walnut somatic embryos resulted in inadeguate expression levels (Dandekar et al. 1994), while the use of a synthetic CryIA(c) gene allowed expression levels adequate to control L. pomonella (Dandekar et al. 1998). Sixty-one GM embryo lines were obtained and tested with first instar codling moth larvae. In 34% of these lines, 80-100% mortality was observed in the presence of detectable amounts of the S-endotoxin. The Bt protein was toxic to codling moth larvae at a very low concentration, corresponding to 0.01% of the total cellular proteins.
The increasing availability of protocols for gene transfer widely applicable to different genotypes and species will hasten the production of new varieties of insect-resistant fruit trees.
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