Journal of Experimental Botany, Vol. 54, No. 383, pp. 835-844,
February 1, 2003
© 2003 Oxford University Press
Transgenic loblolly pine (Pinus taeda L.) plants expressing a modified 
-endotoxin gene of Bacillus thuringiensis with enhanced resistance to Dendrolimus punctatus Walker and Crypyothelea formosicola Staud
Received 4 April 2002; Accepted 30 September 2002
1 North Carolina State University, Forest Biotechnology Group, Centennial Campus, PO Box 7247, Raleigh, NC 27695-7247, USA
2 Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, PR China
3 To whom correspondence should be addressed. Fax: +1 919 515 7801. E-mail: wei_tang{at}ncsu.edu
Abbreviations: BA, benzyladenine; B.t., Bacillus thuringiensis; CaMV, cauliflower mosaic virus; 2,4-D, 2,4-dichlorophenoxyacetic acid; IBA, indole-3-butyric acid; NOS, nopaline synthase; NPTII, neomycin phosphotransferase II gene; PCR, polymerase chain reactions.
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A synthetic version of the CRY1Ac gene of Bacillus thuringiensis has been used for the transformation of loblolly pine (Pinus taeda L.) using particle bombardment. Mature zygotic embryos were used to be bombarded and to generate organogenic callus and transgenic regenerated plants. Expression vector pB48.215 DNA contained a synthetic Bacillus thuringiensis (B.t.) CRY1Ac coding sequence flanked by the double cauliflower mosaic virus (CaMV) 35S promoter and nopaline synthase (NOS) terminator sequences, and the neomycin phosphotransferase II (NPTII) gene controlled by the promoter of the nopaline synthase gene was introduced into loblolly pine tissues by particle bombardment. The transformed tissues were proliferated and selected on media with kanamycin. Shoot regeneration was induced from the kanamycin-resistant calli, and transgenic plantlets were then produced. More than 60 transformed plants from independent transformation events were obtained for each loblolly pine genotype tested. The integration and expression of the introduced genes in the transgenic loblolly pine plants was confirmed by polymerase chain reactions (PCR) analysis, by Southern hybridization, by Northern blot analysis, and by Western blot analysis. Effective resistance of transgenic plants against Dendrolimus punctatus Walker and Crypyothelea formosicola Staud was verified in feeding bioassays with the insects. The transgenic plants recovered could represent a good opportunity to analyse the impact of genetic engineering of pine for sustainable resistance to pests using a B. thuringiensis insecticidal protein. This protocol enabled the routine transformation of loblolly pine plants that were previously difficult to transform.
Key words: Bacillus thuringiensis (B.t.) CRY1Ac, biolistic transformation, insect feeding bioassay, Pinus taeda L.
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Introduction |
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Loblolly pine (Pinus taeda L.) is one of the most widely planted forest species in both tropical and subtropical regions and an important tree in commercial forestry worldwide. Dendrolimus punctatus Walker and Crypyothelea formosicola Staud are two of the major pests threatening the wood production of loblolly pine. Systemic chemical pesticides for these insect pests are usually harmful, and the implementation of an environmentally friendly way of controlling this pest would be a preferable option (Huang et al., 2002). Compared to chemicals, insecticidal proteins do not work as contact or external poisons, but instead as internal poisons. Therefore, the transgenic approach would be a valuable strategy for the control of these insect pests because the insecticidal protein was eaten by the pests (Sardana et al., 1996; Cho et al., 2001). As the use of Bacillus thuringiensis genes to transform plants for protection against insect pests is currently considered to be the most reliable strategy (Estruch et al., 1997; Schuler et al., 1998), investigations were initiated to determine the susceptibility of insect pests to B. thuringiensis insecticidal proteins and to identify the candidate genes for the transformation of plants (Guerreiro et al., 1998). To date, six different endotoxins of B. thuringiensis have been tested against pests (Guerreiro et al., 1998), and the toxin expressed by the cry1Ac gene, widely used to confer resistance to lepidopterae (Dandekar et al., 1998), has been demonstrated to be the most effective.
Conventional breeding of woody species is a low-efficiency and time-consuming process due to their long life cycles. Genetic engineering could alleviate these problems by incorporating known genes into elite genetic backgrounds. Genetic modification of pine for insect, disease, and stress resistance, lignin content, and the improvement of wood quality is thus of major commercial importance. Pine improvement by conventional plant breeding methods has been limited, mainly due to the prohibitively long reproduction cycles and slow seed maturation of these plants. Therefore, the application of genetic engineering techniques to pine improvement appears to be an attractive alternative. Since transgenic plants from microprojectile-mediated gene transfer have been reported for rice (Alam et al., 1998; Christou et al., 1991; Datta et al., 1998), soybean (Christou et al., 1989), papaya (Fitch et al., 1990), potato (Perlak et al., 1993), tobacco (Iida et al., 1991), and poplar (Han et al., 1997; McCown et al., 1991), potential progress has been made in plant genetic transformation (Franche et al., 1997; Schuler et al., 1998). At present, the regeneration of transgenic conifers via Agrobacterium-mediated transformation has been reported for Larix decidua (Huang et al., 1991), larch (Shin et al., 1994), hybrid larch (Larix kaempferixL. decidua) (Levee et al., 1997), white pine (Pinus strobus L.) (Levee et al., 1999), Norway spruce (Picea abies L.) (Wenck et al., 1999), white spruce (Picea glauca) (Le et al., 2001), and loblolly pine (Tang et al., 2001). Stable transformation and regeneration of transgenic conifers using biolistics has been achieved for Picea abies (Walter et al., 1999), Larix laricina (Klimaszewska et al., 1997), Picea glauca (Ellis et al., 1993), Picea mairana (Charest et al., 1996), and Pinus radiata (Walter et al., 1998). Although transformation protocols are available for pine (Birch, 1997; Humara et al., 1999; Tzfira et al., 1996), there is no report available on the stable genetic transformation of loblolly pine by particle bombardment.
In this article, the first transformation of loblolly pine using particle bombardment for expression of an agronomic trait is reported. In the investigation reported here, a synthetic CRY1Ac gene was introduced into three loblolly pine genotypes. The data revealed the correct integration and expression of the CRY1Ac gene within the loblolly pine genome. Furthermore, there was a good correlation between CRY1Ac gene expression and insect bioassays performed on transformed plants. In this investigation, a reproducible method for the gene transfer and regeneration of transformed plants from the loblolly pine mature zygotic embryos using particle bombardment was established. A key to develop this optional genetic transformation protocol is to use mature zygotic embryos of different genotypes of loblolly pine as targeting tissues. In this study, transgenic loblolly pine plants with enhanced resistance to Dendrolimus punctatus Walker and Crypyothelea formosicola Staud were evaluated.
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Materials and methods |
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Plant materials and plasmid construction
Mature seeds of three loblolly pine genotypes were used for transformation experiments, one was collected from Zhangjiajie Loblolly Pine Seed Orchard (genotype J-29, provided by Professor Dongxiang Xu, Hunan Province, China), and two were from Yingde Loblolly Pine Orchard (genotypes E-11 and E-44, provided by Professor Weihua Zhong) in October 1996. Seeds were stored in plastic bags at 4 °C before they were used for tissue culture. Seeds were disinfected by immersion in 70% ethyl alcohol for 30 s and in 0.1% mercuric chloride for 10–15 min, followed by five rinses in sterile distilled water. Mature zygotic embryos were aseptically removed from the megagametophytes and placed horizontally on a solidified callus induction medium in 125 ml Erlenmeyer flasks or 15x100 mm Petri dishes. Mature zygotic embryo explants were used for transformation experiments after being cultured on a pretreatment medium that consisted of TE medium (Tang et al., 2001) supplemented with 10 mg l–1 2,4-dichlorophenoxyacetic acid (2,4-D), 4 mg l–1 benzyladenine (BA), and 4 mg l–1 kinetin for 3 d.
T-DNA of plasmid pB48.215 (Fig. 1) contained two genes, one is a synthetic Bacillus thuringiensis (B.t.) CRY1Ac coding sequence flanked by the double cauliflower mosaic virus (CaMV) 35S promoter (Kay et al., 1987) and nopaline synthase (NOS) terminator sequences, and the other is a selectable marker gene, neomycin phosphotransferase II (NPTII) controlled by the promoter of the nopaline synthase gene (Li et al., 1994). Plasmid construction was produced following the procedure reported by Li et al. (1994). The double CaMV 35S promoter and translation enhancer fragments (Gallie and Kado, 1989) cut by HindIII and BamHI from pD511 were cloned to pBin437 to produce pBin438 (Li et al., 1994). The Bacillus thuringiensis (B.t.) CRY1Ac gene fragments cut by BamHI and SaII from pB48.103 (Li et al., 1994) were inserted in pBin438 to produce pB48.215 (Li et al., 1994). Plasmid pB48.215 were transferred into E. coli strain DH5
and bacteria were cultured on LB medium (Sambrook et al., 1989) with 100 mg l–1 carbenicillin and 50 mg l–1 kanamycin. Plasmid pB48.215 were largely isolated from bacterium cultures grown overnight at 37 °C in liquid LB medium (Sambrook et al., 1989) supplemented with 100 mg l–1 carbenicillin and 50 mg l–1 kanamycin and purified by CsCl ethidium bromide density gradient centrifugation (Sambrook et al., 1989), and then used for biolistic transformation.
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translation enhancer, and T-DNA borders (LB left border, and RB right border). The arrows indicate the translation orientation of the genes. The probe used in Southern blot analysis of transgenic plants is the restriction enzymes BamHI and SalI fragment of B.t. gene, HindIII was used to digest genomic DNA isolated from transgenic plants, and their positions are indicated immediately above. Binding sites of PCR primers nrp and nfp are shown as black rectangles.Transformation and regeneration of transgenic plants
Microprojectile bombardment was carried out using the Biolistic PDS 1000/He System (Bio-Rad, Hercules, Calif.) at 1100 psi following the manufacturer’s instructions and protocol. All accessible parts of this equipment were surface-sterilized using 70% ethyl alcohol 15 min before use. Microcarriers were prepared as follows: 3 mg gold (0.6, 1.0, and 1.1 µm) particles, suspended in 50 µl distilled water, were mixed with 20 µg DNA of plasmid, followed by the addition of 175 µl CaCl2 (2.5M) and 70 µl spermidine (0.1 M, Sigma). The mixture was gently vortexed for 10 mix at room temperature and then centrifuged briefly. The gold particles were washed twice with 100% ethanol and resuspended in 100% ethanol (50 µl). Fifteen microlitres microcarrier suspension was used for each bombardment. Explants were placed on the TE callus induction medium (Tang et al., 2001) for 3 d before bombardment. Bombardment was performed with a rupture disc pressure of 1100, 1350, and 1550 psi, respectively, a gap distance of 1.5 cm, a macrocarrier travel distance of 8 mm, and target distance of 12 cm. After bombardment, embryos of all three genotypes were transferred to TE callus induction medium (Tang et al., 2001). After a period of 21–28 d, they were transferred to a selective TE medium supplemented with 15 mg l–1 of kanamycin. Embryos were transferred to new selective medium every 3 weeks. After calli were formed from embryos, calli were subcultured every 14 d. Kanamycin-resistant calli were maintained on selection medium for longer than was practically required for regeneration, to ensure that a true frequency for kanamycin resistance was established. Approximately 1 µg DNA was introduced per bombardment. Similar samples of all three families that were not bombarded and bombarded with gold particles without DNA served as the controls. Treatments were replicated three times.
Organogenic calli were initiated from transformed mature zygotic embryos cultured on TE induction medium (Tang et al., 2001) with 15 mg l–1 kanamycin. Plant regeneration was carried out on differentiation medium supplemented with selection pressure according to a procedure previously described (Tang and Ouyang, 1999), i.e. precise composition of the medium. All media were supplemented with 3% sucrose and 0.3% Phytagel (Sigma). Media were adjusted to pH 5.8 prior to autoclaving 20 min at 121 °C. All cultures were cultured at 25 °C in a culture room. Adventitious shoot induction was conducted in the dark and adventitious shoot differentiation, proliferation and rooting were conducted at 25 °C under a 16 h photoperiod with cool fluorescent light (100 µmol m–2 s–1). Differentiation was evaluated by the percentage of calli forming transgenic adventitious shoots. The acclimatization of regenerated plantlets was conducted by decreasing the relative humidity to ambient conditions over a period of 1 week, and then the plantlets were established in soil. The frequency of calli forming transgenic shoots and the frequency of transgenic shoots forming roots were determined in the 9th week of culture. The data were analysed by the Analysis of Variance (ANOVA).
Polymerase chain reaction (PCR) analysis
Loblolly pine genomic DNA was extracted from 300–500 mg fresh needle tissue of control and putative transgenic plants using a Genomic DNA Isolation Kit (Sigma) following the manufacturer’s protocol. These transgenic plants were 8–10 cm in height and established in soil for one year in a greenhouse, respectively. The primer set utilized for amplification of insert DNA was the nptII forward primer (nfp) 5'-ACAAACAGACAATCGGCTGC-3' and reverse primer (nrp) 5'-AAGAACTCGTCAAGAAGGCG-3'. A total of 100–300 ng of genomic DNA was used as the template in a 50 µl PCR reaction mix containing 200 µM each of dATP, dCTP, dGTP, and dTTP, 35 pmol of each primer, 2.5 U Taq DNA polymerase (Promega), 1.5 mM MgCl2, and 5 µl 10x buffer [500 mM KCl, 100 mM Tris-HCl (pH 9.0 at 25 °C), 1% Triton X-100, 15 mM MgCl2]. The reaction proceeded in a programmable MJ MiniCycler (MJ Research, Watertown, Mass., USA). The PCR conditions were 95 °C for 5 min followed by 30 cycles of 95 °C for 30 s, 52 °C for 1 min, and 72 °C for 2 min. Cycling was followed with a final incubation of 72 °C for 10 min. PCR products were observed under UV after electrophoresis on a 1.0% agarose gel with 0.1% ethidium bromide. Molecular marker is 1-kb DNA marker (Gibco-BRL).
Southern hybridization
Total genomic DNA was isolated from transgenic plants by using a Genomic DNA Isolation Kit (Sigma) following the manufacturer’s protocol. Twenty µg of total genomic DNA from putative transgenic and non-transformed control plants was digested overnight with the restriction enzyme HindIII (Boehringer Mannheim) at 37 °C. Digested DNA of each line were separated through a 1% agarose gel prepared in 1x TBE (Sambrook et al., 1989) and fragments were transferred from the agarose gels to a nylon membrane (Hybond-N; Amersham) and cross-linked to the membrane by UV using Stratalinker (1200 Joules 100; Stratagene). The DNA fixed on membranes was hybridized (at 65 °C) with the Bacillus thuringiensis (B.t.) CRY1Ac gene probe (BamHI and SacI (Boehringer Mannheim) fragment of B.t. gene), which was labelled with ß-[32P]dCTP (Ready to Go Labeling Beads (Pharmacia)). Hybridization was performed according to Sambrook et al. (1989) in 5x SSPE, 5x Denhardt’s solution, 0.1% SDS, 0.1 mg ml–1 denatured fish sperm, 0.1 g ml–1 dextran sulphate for 20 h before the membranes were washed with 0.1x SSPE, 0.1% SDS solution at 65 °C. Hybridizing bands were detected by exposure to Kodak X-OMAT autoradiography films at –80 °C for 3 d.
Isolation of RNA and Northern blot analysis
Ribonucleic acid was extracted from frozen leaf tissue as previously described (Chang et al., 1993). Needle samples were collected from PCR and Southern-positive transgenic loblolly pine plants and immediately frozen in liquid nitrogen and stored at –80 °C. Samples of RNA were extracted from 1.5 g of needle tissue using the procedure of Chang et al. (1993). The isolated RNA was precipitated twice with 4 M LiCl for 60 min at 0 °C to remove traces of DNA and small RNA species. Denatured total RNA samples were fractionated by 1.2% formaldehyde agarose gel electrophoresis (1.5% [w/v] agarose) and then capillary blotted onto ‘Zeta Probe’ blotting membranes (Bio-Rad). The blot was probed using a B.t. probe fragment labeled with ß-[32P]dCTP. Conditions for hybridization and washing of blots were those recommended for use (Sambrook et al., 1989). Hybridization of the probe DNAs to the blot was recorded on blue-sensitive X-ray® film. The integrity and the amount of RNA applied to each lane were verified by ethidium bromide staining.
Western blot analysis
Plant tissues (1 g fresh weight) were ground with a microfuge pestle in 2 vols of ice-cold 50 mM Hepes buffer (pH 7.0). The slurry was centrifuged for 10 min (13 000 g) in a microcentrifuge at 4 °C and the supernatant was collected. The protein concentration was determined using a protein dye-binding assay kit (Bio-Rad). The supernatant solutions were heat-denatured at 100 °C for 5 min after addition of an equal volume of 2x SDS sample buffer [100 mM Tris-Cl (pH 6.8), 200 mM dithiothreitol, 4% SDS, 0.01% bromophenol blue, and 20% glycerol]. Analysis by SDS-PAGE was carried out on 10% SDS-polyacrylamide gels according to Sambrook et al. (1989). Prestained molecular-weight standards (broad range; Bio-Rad) were used as markers. The separate proteins were transferred to nitrocellulose®filters (Bio-Rad) by semi-dry electroblotting for 25 min at 25 V with transfer buffer [48 mM TRIS-Cl (pH 9.2), 39 mM glycine, 10% SDS, and 20% methanol]. Immunodetection was carried out using anti-B.t. antiserum (Clontech) at a 1:2000 dilution in TBS [100 mM Tris-Cl (pH 7.4) and 0.9% NaCl]. Alkaline phosphatase-conjugated anti-rabbit IgG (Sigma Chemicals) was used as the secondary antibody at 1:5000 dilution in TBS and this was detected using nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate (Bio-Rad).
Insect bioassays
The eggs of Dendrolimus punctatus Walker and Crypyothelea formosicola Staud were collected in 1998 from loblolly pine plants at the Zhangjiajie Loblolly Pine Orchard, Hunan province, China and then were kept in an environmental chamber at 2% relative humidity, and under a photoperiod of 16:8 h (light:dark) (Shelton et al., 1991; Metz et al., 1995; Cao et al., 1999). For tests of transgenic calli, a needle containing 15–20 eggs was placed on transgenic calli maintained in containers of 1% water agar under a 16:8 h (light:dark) regime at 25 °C. In some cases, transgenic calli were inoculated with freshly hatched larvae instead of eggs. Larval mortality was scored after 7 d. For assays of transgenic plants, all positive transgenic plants expressing the CRY1Ac protein were tested indiviually for lethality and growth retardation of Dendrolimus punctatus Walker and Crypyothelea formosicola Staud larvaes using a whole plant-feeding assay. The test plants were individually infested with larvaes of 3–5 mm in length for 7 d at 25 °C under a 16/8 h light/dark regime. Transgenic plants were placed on a piece of moistened filter paper in a Petri dish (100x200 mm). Larvae were placed on the shoot, and the Petri dish was sealed with masking tape. After 7 d, the number of alive and dead larvae was determined for each Petri dish. A similar infestation was also done for control plants. The control plants are untransformed regenerants of the same age as the transformed ones. The bioassay was replicated three times for each plant. The mortality rate was expressed as a percentage (the number of dead larvae/number of larvae applied) x 100%. The data were analysed by the Analysis of Variance (ANOVA).
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Transformation, selection, and regeneration of transgenic plants
Bombarded embryos were first induced to form calli by being incubated in a medium without kanamycin for 28 d. The use of the preculture on TE callus induction medium (Tang et al., 2001) for 28 d produced a significantly higher number of callus than did the control. The initial 28 d of culture without kanamycin selection was intended to promote active proliferation of calli on the surface of the bombarded embryos. The optimal concentration for selecting transformed cells and tissues was determined by culturing mature zygotic embryos on callus induction medium containing different concentration of kanamycin. According to the preliminary experiments, 15 mg l–1 kanamycin was used to identify transformed cells and tissues. Two to three weeks after bombarded mature zygotic embryos were transferred onto callus induction medium supplemented with 15 mg l–1 kanamycin, kanamycin-resistant calli were observed. Callus was formed on cotyledons, hypocotyls, and radicles of embryos. Proliferation of transgenic calli was achieved by subcultured kanamycin-resistant calli on fresh callus induction medium with kanamycin. This sequential manipulation was critical for transformation success as prior attempts to obtain transformants from embryos transferred directly to TE medium containing kanamycin after bombardment failed.
Experiments were completed involving between 1000 and 9000 zygotic embryos for each genotype. On a selective medium with 15 mg l–1 of kanamycin, calli appeared on the surface of the necrotic embryos. After about two months of culture, such calli occurred on approximately 5% of the zygotic embryos. For the genotype J-29, small groups of calli appeared on 2–5% of the zygotic embryos, with considerable variability. On the genotype E-11, less than 1% of the explants produced calli. While the transformation process has been improved with further experimentation, it remained difficult to obtain transformed calli for each experiment; most of the results were obtained from 5–10 transformation assays for the E-11 and E-44 genotypes. For all three genotypes, isolated calli were then cultivated on the same selective medium. After 9 weeks of culture on differentiation medium (Tang et al., 2001) with 15 mg l–1 kanamycin, adventitious shoots were observed (Fig. 2A). For the J-29 genotype, groups of between 1 and 10 transformed adventitious shoots grew directly on primary explants. About 40% of these adventitious shoots were converted into plantlets.
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Both frequency of zygotic embryos forming transgenic calli and frequency of calli forming transgenic shoots was influenced by the genotypes of loblolly pine. The frequency of putative transgenic calli ranged from 3.2% (genotype E-11) to 28.7% (genotype E-44) (data not shown). Putative transgenic adventitious shoots (Fig. 2A) were formed on the surface of kanamycin-resistant calli, 9 weeks after kanamycin-resistant calli were transferred to differentiation medium. The frequency of adventitious shoots ranged from 12.7±1.6% (genotype E-11) to 24.9±2.0% (genotype J-29) on the differentiation medium supplemented with BA and IBA in the 9th week of culture (Table 1). Root primordia were usually formed at the base of shoots. Elongation and rooting of transgenic shoots (Fig. 2B), and acclimatization of regenerated plantlets were carried out according to a procedure previously described (Tang et al., 2001). Rooting frequency 34.3±3.9% (genotype E-11) to 47.9±4.3% (genotype J-29) (Table 1) was observed and both growth and phenotype of regenerated plantlets appeared similar to the untreated control. Regenerated plantlets from transgenic organogenic calli of loblolly pine were transferred from 125 ml Erlenmeyer flasks into a perlite:peatmoss:vermiculite (1:1:1 by vol) soil mixture, and acclimatized plantlets were successfully established in the soil in greenhouse (Fig. 2C).
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According to the protocol described, more than 300 transgenic calli were obtained with J-29, more than 200 with E-44 and only 60 with the E-11 genotype. Between 5 and 20 shoots were generated from each of the 300 calli obtained from the J-29. For E-44, 3–10 shoots were obtained from each group of embryos from the 200 transgenic calli. Only 60 regenerated shoots were obtained for the E-11 due to a low frequency of regeneration from transformed calli. Molecular characterization of transformed plants were performed on selected transgenic plants from independent events obtained with all three families.
Confirmation of T-DNA integration and gene expression
Polymerase chain reaction (PCR) was used for the primary analysis of transformants. Loblolly pine genomic DNA was extracted from fresh tissues of control and putative transgenic plants, respectively. The primer set used for amplification of insert DNA was the nptII forward primer (nfp) and reverse primer (nrp). PCR analysis was carried out as a rapid identification for the insert DNA in putative transgenic plantlets derived from kanamycin-resistant calli from three genotypes (E-11, E-44, and J-29) of loblolly pine. The expected 717 bp band (for nptII) was amplified in the transformed plantlets, but not in the non-transformed plant control (Fig. 3). These PCR results confirm that the regenerated plantlets from kanamycin-resistant calli contain the transgene derived from the pB48.215 plasmid. Southern hybridization of genomic DNA was then carried out to confirm that the T-DNA was integrated into the plant genome.
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Southern blotting was used to evaluate the number of T-DNA copies integrated into the plant genome. Restriction enzyme HindIII and probe (CRY1Ac) were chosen in order to detect fragments of the T-DNA (Fig. 1). It was postulated that the number of copies of the CRY1Ac gene, located on the right side of the T-DNA, is in fact representative of the copy number of the whole T-DNA. Among the 50 plants studied, 35 plants (70%) presented one T-DNA copy (Fig. 4), 8 (16%) two copies, 5 (10%) three copies, but 1 (2%) plant also showed four copies and 1 (2%) five copies (data not shown). These results are consistent with observations made in various dicotyledonous plants including tobacco, petunia, tomato, and sunflower (Tinland, 1996; Zambryski, 1988). The integration of foreign genes in regenerated transgenic plants was also observed in Pinus radiata via particle bombardment (Walter et al., 1998). In this investigation, only transgenic plants with one to three copies of the CRY1Ac were selected for Northern hybridization, Western blot, and insect bioassays.
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Transgenic plantlets from independent transformation events were analysed by Northern hybridization. Total RNA was isolated from control and putative transgenic plants using the procedure reported by Chang et al. (1993). No bands were detected in non-transformed control plants, whereas bands were observed in transgenic plants (Fig. 5). These results confirm that the foreign genes have been integrated into the Pinus taeda genome and expressed at the transcription level in transgenic plants.
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Western blot hybridization was used to evaluate the expression of gene at the protein level. Proteins were extracted from about 1 g of fresh needles collected from transgenic plants with positive Northern hybridization, respectively, and the detection of the insecticidal protein was performed by Western blotting (Sambrook et al., 1989) using a rabbit polyclonal antiserum, raised against the CRY1Ac protein previously purified in the authors’ laboratories (dilution of 1/2000 v/v). A secondary goat anti-rabbit antiserum alkaline phosphatase conjugate (Sigma), was then used for final detection, at a dilution of 1/5000 (v/v). Expression of the CRY1Ac gene was tested by Western analyses on proteins extracted from 48 transformed plantlets of the 50 independent transgenic calli analysed in this investigation. In 42 of them, the polyclonal antibody detected protein (Fig. 6). As shown in Fig. 6, an expected signal of 68 kDa molecular weight was observed with purified CRY1Ac protein and with needle extracts from transformed plants. However, in the other six transformed plants, although the CRY1Ac gene was detected by Southern blotting, the protein was not detected by Western analyses. The absence of an immunosignal in the six transformed plants was related to high copy numbers and possible co-suppression. These six plants were not resistant to the larvae.
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Insect bioassay
Bioassays were performed using transgenic calli and plants derived from all three loblolly pine genotypes. Insect resistance of transgenic plants with introduced synthetic Bacillus thuringiensis CRYIAc genes have been confirmed by insect bioassay (Cheng et al., 1998; Stewart et al., 1996; Xiang et al., 2000). In this investigation, the transgenic calli and regenerated plants confirmed by Southern, Northern, and Western blot analysis were selected for insect bioassay. Insecticidal activity was evaluated by placing larvae of Dendrolimus punctatus Walker and Crypyothelea formosicola Staud on the transgenic calli (Table 2) and transgenic plants to be tested (Table 3). The eggs of Dendrolimus punctatus Walker and Crypyothelea formosicola Staud were collected from loblolly pine trees (Fig. 3D) at Zhangjiajie Loblolly Pine Orchard. The eggs (Fig. 3E) were incubated according to the method described in the Materials and methods. It was observed that the larvae often bore small curves on the first or second day of feeding on the regenerated plants (Fig. 3F) positively identified by Southern, Northern, and Western blot analysis. Two days later, some larvae died and the others ceased feeding and became stunted, depending on the age of the larvae. However, the larvae on the non-transformed control plants of the same age as the transformed ones and susceptible plants continued to feed and grew well to more than 0.7 cm in length after 7 d of feeding. Eventually these larvae pupated later without mortality, leaving behind severely damaged plants.
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Discussion |
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Through extensive evaluations of factors involved in the particle bombardment-mediated transformation, the present protocol was developed with which a large number of stably transformed loblolly pine plants were generated. This protocol, with slight modification as necessary, appears applicable to all loblolly pine families and other coniferous species that have been recalcitrant to transformation. This protocol could be useful to genetic engineering of the commercially important loblolly pine clones with genes conferring virus resistance, disease resistance, stress tolerance, wood property, lignin contents, and other economical qualities.
Mature zygotic embryos of loblolly pine were used as target tissues for particle bombardment. Actively dividing cells in embryos where the dividing state is established during the 3 d pretreatment provided a window of competence for transformation. Tobacco cells bombarded at the M- and G2-phases have 4–6 times higher transformation efficiencies than those at the S- and G1-phases (Iida et al., 1991). T-DNA integrates preferentially into sequences that can potentially be transcribed (Perlak et al., 1991). Studies on the integration of T-DNA into the plant genome strongly suggest that host DNA synthesis is required (Gheysen et al., 1987). Thus, the greater the number of actively dividing cells in the tissue being bombarded, the higher probability of obtaining stable expression in the bombarded tissue. In the present protocol it was critical that the embryos were bombarded and were cultured in TE medium for a time at least sufficient to induce cell division on the target tissue. Cells hit by the DNA-coated particles at this stage were likely to be the cells from which proliferating calli are derived. Ellis et al. (1993) observed that the percentage of spruce somatic embryos expressing the GUS gene after bombardment increased progressively with advanced developmental stages. The transformed calli developed on the surface of the embryos. Among three different sizes of gold particles (0.6, 1.0, and 1.1 mm), the smallest particles were most efficient in delivering the genes. Different levels of helium gas pressures (1,100, 1,350, and 1,550 pounds per square inch) did not affect transformation efficiency (data not shown).
The CRY1Ac protein was not detected by Western analysis in six of the 48 transgenic plants where the CRY1Ac gene was detected by Southern blotting. This may be due either to a level of protein below the detection threshold of the antibody, which is about 0.1% of total protein in this assay, or to a lack of expression of the protein. It could also be explained by a recombination event within the T-DNA that has altered the structure of the CRY1Ac gene. As previously foreseen (Gould, 1998), the CRY1Ac gene conferred resistance to Dendrolimus punctatus Walker and Crypyothelea formosicola Staud, as demonstrated by the bioassays. A correlation between the detection of the insecticidal protein in needle extracts from transformed plants and resistance to Dendrolimus punctatus Walker and Crypyothelea formosicola Staud was observed in most cases. However, the observations suggested that, in some cases, the level of resistance and development stage could be related. This aspect will be checked carefully when the plants are transferred into soil for a field trial. Suitable insect management will be implemented in parallel with monitoring the expression of the CRY1Ac gene throughout the five year experiment. Up to now, all the results dealing with the evaluation of the efficiency of a B. thuringiensis (B.t.) gene in agricultural conditions have been obtained on annual crops (Huang et al., 2002). Thus, the observations to be carried out on loblolly pine will be very valuable for the future development of a B.t. strategy on perennial trees. Besides the stability of gene expression, it must be borne in mind that a targeted insect can develop resistance to toxins from B. thuringiensis (Rousch, 1997; Gould, 1998). Therefore, the use of genes with different resistance mechanisms is ideally required to maintain a B.t. strategy. The CRY1B gene could be used in order to obtain a cumulative effect with CRY1Ac (Guerreiro et al., 1998), since the gut receptors are different for the two proteins. Furthermore, integrated pest management, including trapping, using parasitoids, nematodes or entomophagous fungi, and adapted agricultural practices (optimizing of herbicides and other pesticide uses), also have to be considered. This is particularly relevant for a perennial tree like loblolly pine.
The transformation of loblolly pine for resistance to Dendrolimus punctatus Walker and Crypyothelea formosicola Staud is a first step in the process of creating insect-resistant transgenic loblolly pine plants. Although economically important, the Dendrolimus punctatus Walker and Crypyothelea formosicola Staud is not restricted to China and is currently present worldwide and considered to be the most devastating and economically important insect pest of loblolly pine. The practicality of this work will benefit not only the loblolly pine producers, but also the environment worldwide. The next step could therefore be the large-scale development of loblolly pine plants resistant to Dendrolimus punctatus Walker and Crypyothelea formosicola Staud in the field. The same approach could be applied to other loblolly pine families and conifers. Genetically modified plants from other conifers could also be produced. The results presented in this work open the way to new opportunities to improve the loblolly pine species, not only for other agronomic traits but also for those of technological interest.
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The authors are grateful to Professor Zhigang Pan of the Chinese Academy of Forestry Sciences for his constant support of this work and excellent technical assistance, to Professor Weihua Zhong of South China Agricultural University and Professor Dongxiang Xu of Wulin University for the collection of seeds, to Professor Feng Guo of Institute of Zoology, Chinese Academy of Sciences for technical advice on insect breeding assays, to members of the Plant Biotechnology Laboratory and the Biochemical Engineering Laboratory of the Chinese Academy of Sciences for their helpful collaboration during the bioassays. Part of this work was presented in a laboratory meeting of the Forest Biotechnology Group, North Carolina State University. We thank Professor Fan Ouyang, Dr Ron Sederoff, and Dr Ross Whetten for their discussion and encouragement and corrections of the text. This work was partially supported by China High Technology Program ‘863’ Project.
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