Transient expression of the uidA gene in Pinus pinea cotyledons: A study of heterologous promoter sequences

Transfer and expression of the β-glucuronidase gene (uidA) in cultured cotyledons of stone pine (Pinus pinea L.) was obtained by microprojectile bombardment. Conditions for optimum transient expression were established by using plasmid pBI121 delivered by 1.0 μm-diameter gold particles, into 1-day-old cultured cotyledons. Helium pressure of 6.2 MPa, microcarrier travel distance of 6 cm, and 0.8 μg of plasmid DNA per bombardment, were the best parameters for high levels of transient uidA expression. By using these parameters, 98% of bombarded cotyledons showed β-glucuronidase activity, with a mean of 63 Gus foci per cotyledon. This system was used to study the expression of uidA gene driven by several heterologous promoters. The expression under the control of the sunflower polyubiquitin gene (UbB1) promoter (Δ1 deletion) was higher (99% of GUS positive cotyledons) than under the control of the CaMV35S promoter, whereas the rice actin and the maize alcohol dehydrogenase gene promoters gave lower uidA expression, as determined histochemically. These results were confirmed by using the GUS fluorometric assay. Use of a deletion of the sunflower polyubiquitin promoter resulted in GUS activity detectable 35 days after bombardment, and significant levels of GUS activity were confirmed at the end of that period. The results will be useful to design protocols for stable transformation and high levels of transgene expression in P. pinea. This revised version was published online in June 2006 with corrections to the Cover Date.     

Transient expression of a reporter gene in bulbscales and immature embryos of three Lilium species is affected by 5'upstream sequences and culture conditions

In Lilium , a transformation system has not yet been developed. For efficient selection of cells expressing transferred genes following particle bombardment, the effects of 5'upstream regions on the transient expression of the β-glucuronidase gene ( gusA ) were estimated in bulbscales and immature embryos of lily. When four plasmids having the gusA gene under the control of the cauliflower mosaic virus (CaMV) 35S, maize alcohol dehydrogenase gene and rice actin gene ( Actl ) promoters, and the castor bean catalase introm were introduced by particle bombardment, the patterns of transient expression in the bulbscales showed differences among three Lilium species, L. x formolongi, L. dauricum and L. japonicum . In immature embryos of L x formolongi , transient expression was significantly influenced by age of embryos after self-pollination, duration of culture before bombardment, and culture conditions. Moreover, the transient gusA expression driven by six different 5'upstream regions, including the maize ubiquitin gene promoter and a modified CaMV 35S promoter were compared in both bulbscales and immature embryos. Use of the Actl and modified CaMV 35S promoters resulted in the greatest number of cells that transiently expressed gusA in both types of tissue of L. x formolongi . These two promoters are efficient for use in lily transformation.     

Integration,expression and inheritance of transgenes in hexaploid oat (Avena sativa L.)

Two oat varieties, Melys (spring variety) and Bulwark (winter variety) were transformed by particle bombardment of primary embryogenic callus using either a ubi-bar-ubi-gus co-integration vector or co-transformed (Melys) with a ubi-bar plasmid together with one of three plasmids containing the beta-glucuronidase (gus) gene under the control of either a rice actin promoter, a CaMV35S promoter or a wheat high molecular weight glutenin promoter. Morphologically normal and fertile transgenic plants were regenerated following callus selection with glufosinate ammonium. Evidence for the integration and functioning of the selectable (bar) and reporter (gus) genes in T0 and T1 plants was confirmed by PCR, Southern hybridisation, fluorescence in situ hybridisation (FISH), histochemical assays, and by progeny analysis. Transformation rates varied from 0.2 to 5.0 lines/plate of callus bombarded, with co-transformation frequencies of 83 to 100%, and co-expression frequencies of 60 to 100%. Copy numbers for the bar and gus gene varied from 3 to 17 and from 2 to 20 respectively. Cell and tissue specific expression of the gus gene was evident from the different promoters, with the HMW glutenin promoter showing endosperm specific expression in T1 seed. No expression of the gus gene under the CaMV35S promoter was detected in any tissues. Progeny analysis provided evidence of Mendelian inheritance of the introduced genes suggesting either one or two unlinked integration sites. This was confirmed by fluorescence in situ hybridisation to chromosome spread preparations. No segregation of the gus gene from the bar gene was observed in any of the progeny derived from co-transformation.     

Stable transformation of Eustoma grandiflorum by particle bombardment

Explants (7.5±2.5 mm) cut from stems and roots of 3-week-old Eustoma grandiflorum Grise, (lisianthus) cv. Glory White seedlings were bombarded with plasmid pBI221, which harbors the uidA gene encoding β-glucuronidase (GUS) driven by the cauliflower mosaic virus (CaMV) 35S promoter. More than 800 blue spots of GUS-expressing cells were observed per 90 explants. Explants bombarded with pARK22 harboring the bar gene encoding phosphinothricin acetyltransferase driven by the CaMV 35S promoter were selected for bialaphos resistance. Putative transgenic plants were obtained about 3 months after bombardment. Southern blot analysis of putative transgenic plants revealed the presence of the bar gene in their genome. Received: 10 April 1996 / Revision received: 7 November 1997 / Accepted: 22 November 1997     

Development of an efficient transient transformation protocol for avocado (Persea americana Mill.) embryogenic callus

An efficient protocol for transient transformation of avocado embryogenic callus has been established, using the PDS-1000/He system and the reporter gus gene driven by the sunflower polyubiquitin promoter. Best physical parameters for transient transformation were 900 psi helium pressure and 6 cm target distance. The level of transient gus expression was slightly higher when the amount of DNA per shot was increased from 0.6 to 1.8 μg, but it was not significantly modified by the type of microprojectile used (tungsten vs. gold particles). The transient transformation assay developed in this research was used to test the strength of different promoters and the expression of fluorescent reporter genes. Four constitutive promoters, sunflower polyubiquitin, CaMV35S, CaMV35S with enhancer, and rice actin 1, as well as a trichome-specific promoter, ATP, were analyzed. Polyubiquitin and ATP promoters yielded the highest number of gus expressing foci, while no expression was detected with the Act1 promoter from rice. Embryogenic callus was also bombarded with plasmids pXK7S*NF2 and pXK7RNR2, harboring the enhanced green fluorescent gene, EGFP, and the red fluorescent gene DsRed, respectively. Both fluorescent proteins were detected 24 and 72 h after bombardment, but the observed transformation efficiency was slightly higher in GFP bombarded cells. The transient transformation system described here can be used as a fast way to select suitable promoters and/or fluorescent genes needed to undertake stable transformation studies in avocado using currently available protocols.     

Genetic transformation of sweet potato by particle bombardment

Transient and stable expression of foreign genes has been achieved in sweet potato using the particle bombardment system of gene delivery. Callus and root isolates of two genotypes (Jewel and TIS-70357) with positive signs of transformation have been recovered. Tungsten microcarriers coated with plasmid DNA (pBI 221 containing the gusA gene) were accelerated at high velocity using a biolistic device into sweet potato target tissues. Histochemical examination of bombarded leaf and petiole explants revealed that most had cells expressing the gusA gene. When explants were cultured, calli and roots developed in most bombarded tissues. Similar results but with a lower frequency of transformation were observed when the plasmid pBI 121 (with gusA and antibiotic resistance npt II genes) was employed and bombarded explants cultured on an antibiotic selection medium. Subcultured roots and calli were positive for gusA expression when tested even after one year of in vitro culture, and thus the expression of the foreign gene is fairly stable. The particle bombardment approach of gene delivery appears to have a potential for generating transgenic sweet potatoes with useful agronomic traits.Abbreviations BA 6-benzylaminopurine - CaMV cauliflower mosaic virus - 2,4-D 2, 4-dichlorophenoxyacetic acid - GUS ß glucuronidase - NAA naphthaleneaceticacid - nos nopaline synthase gene - NPT II neomycin phosphotransferase II - MS Murashige and Skoog (1962) - MS-CP MS cell proliferation medium     

Evaluation of microprojectile-mediated DNA delivery and reporter genes for genetic transformation of the Mediterranean cypress (Cupressus sempervirens L.)

Embryonal-suspensor tissue (EST) of Mediterranean cypress (Cupressus sempervirens L.) was tested for microprojectile-DNA delivery (by the PDS-1000/He device) for different subculture periods (9, 15, and 21 days) using the plasmid vectors pRT99GUS [containing the β-glucuronidase (GUS) and neomycin phosphotransferase (NPT II) genes, and the CaMV 35S promoter], pBI426 (with a GUS::NPT II fusion gene under the control of a duplicated 35S RNA promoter), and pCGUδ0 (containing the GUS gene with the ubiquitin intron, under the control of the sunflower ubiquitin promoter). The relative strengths of the promoters as determined by GUS assays were sunflower ubiquitin>35S-35S-AMVE>35S. The highest expression level was observed when 15-day-subcultured EST was bombarded with the pCGUδ0 gene construct, which also showed high activity of the chloramphenicol acetyltransferase and NPT II genes. Green fluorescent areas were observed on EST when bombarded with the p35S-GFP plasmid, carrying the gene for the green fluorescent protein from the bioluminescent jellyfish Aequorea victoria. Received: 18 November 1996/ Revision received: 19 February 1997/ Accepted: 20 November 1997     

Optimised DNA delivery into Coffea arabica suspension culture cells by particle bombardment

An optimised bombardment protocol to introduce DNA into Coffea arabica suspension culture cells was developed. Osmotic preconditioning of cells and physical bombardment parameters including Helium pressure, gap and target distances affecting DNA delivery were evaluated by monitoring transient expression of the uidA gene driven by the CaMV35S promoter. The highest transient GUS expression was obtained when cells were subjected to a 0.5 M mannitol–sorbitol pre-treatment 4 h prior to bombardment and a Helium pressure of 1550 psi, a 9-mm gap distance and 12 cm target distance as physical bombardment parameters. The optimised protocol was tested with two coffee promoters: -tubulin and arabicin, which presented similar activity to the CaMV35S promoter in suspension culture cells by fluorometric GUS assays. GUS expression was reduced in bombarded tissue culture leaves, and only the CaMV35S and arabicin promoters showed histochemical activity in coffee endosperms. This is the first report of optimization of particle bombardment on coffee suspension culture cells, equivalent CaMV35S activity for a coffee promoter and transient -glucoronidase expression in coffee endo-sperms.     

Comparison of promoters and selectable marker genes for use in Indica rice transformation

The effectiveness of different promoters for use in Indica rice transformation was compared. Plasmids encoding the Escherichia coli uidA (gus) gene under the control of CaMV 35S, Emu, Act1 or Ubi1 promoters were delivered into cell suspension cultures by particle bombardment. Transient gene expression, 48 h after delivery, was greatest from plasmids utilising the constitutive promoters, Act1 and Ubi1. Gene expression in stably transformed tissue was examined by bombarding embryogenic Indica rice calli with a pUbi1-gas plasmid and a plasmid containing either the selectable marker gene, hph, which confers hygromycin resistance, or bar, which confers resistance to the herbicide phosphinothricin (BASTA) each under the control of the CaMV 35S, Emu, Act1 or the Ubi1 promoters. The bombarded calli were placed on the appropriate selection media and stained for GUS activity at 1 day, 3 weeks and 5 weeks after shooting. Callus bombarded with the pUbi1-hph or the pEmu-hph constructs gave a dramatic increase in the size of the GUS staining areas with time. No such increase in the size of GUS staining areas was observed in calli co-bombarded with pUbi1-gus and any of the bar containing constructs.Co-bombardment of calli with either the pEmu-hph or pUbi1-hph construct and a virus minor coat protein (cp) gene construct resulted in many fertile transgenic Indica rice plants, containing one to eight copies of both the hph and cp genes. These genes were stably inherited by the T1 generation.     

Generation of low copy number and stably expressing transgenic creeping bentgrass plants using minimal gene cassette bombardment

A minimal gene cassette comprised of the ubiquitin (Ubi) promoter + green fluorescent protein (Gfp) gene + Nos terminator DNA sequences, derived from the plasmid vector pPZP201-Gfp was utilized for transformation of creeping bentgrass using particle bombardment. Bentgrass calli bombarded individually with equivalent amounts of the cassette or whole plasmid DNA were compared for Gfp expression and the GFP-positive calli were subsequently regenerated into plants. Percentage of GFP expressing calli and the number of GFP spots/calli were significantly higher in calli that were bombarded with the minimal gene cassette when compared to the whole plasmid. The Gfp expression was stable up to the T₂ generation in minimal gene cassette transformants and there was a lower degree of gene silencing. Southern blot analysis of transgenic plants derived from minimum gene cassette bombardment revealed the presence of single or few copy of the transgene and fairly simple integration patterns. In comparison, whole plasmid transformants had multiple copies and complex integration patterns of the transgene. These results illustrate the advantages of using simple gene cassette for stable plant transformation in bentgrass with possible applications to other plant species.     

Optimization of parameters for particle bombardment of embryogenic suspension cultures of cassava (Manihot esculenta Crantz) using computer image analysis

Tissue derived from embryogenic suspension cultures of cassava was bombarded with microparticles coated with a plasmid containing theuidA gene, which codes for-glucuronidase (GUS). After 3 days, the effect of different bombardment parameters was evaluated by comparing the numbers of blue spots that resulted from histological GUS assays. Counting of blue spots was performed using a system comprised of a black and white video camera, a stereoscope and a personal computer. A reproducible counting method was established by optimizing GUS assay conditions, preparation of tissue samples and acquisition of video images in view of attaining the highest possible contrast between the blue spots and the surrounding tissue. The effects of bombardment pressure, microparticle size, number of bombardments, and osmotic pretreatment on GUS expression were investigated. Optimal transient expression of theuidA gene was observed after bombardment at 1100 psi, with a particle size of 1 µm, an osmotic pretreatment and two bombardments per sample. The highest number of blue spots observed was 2400 per square centimeter of bombarded tissue.     

Studies on genetic transformation of olive (<Emphasis Type="Italic">Olea europaea</Emphasis> L.) somatic embryos: I. Evaluation of different aminoglycoside antibiotics for <Emphasis Type="Italic">nptII</Emphasis> selection; II. Transient transformation via particle bombardment

As a first step to the establishment of a genetic transformation protocol for olive somatic embryos obtained from the seeds of cv. ‘Picual’, the efficiencies of different aminoglycoside antibiotics as selective agents to be used with the nptII marker gene, and the particle bombardment technique for transient transformation have been evaluated. Among the three antibiotics tested, paromomycin and kanamycin showed a similar inhibitory effect and, at 200 mg l⁻¹, both of them impaired callus growth after 8 weeks of culture. However, when isolated embryos were cultured in the presence of these antibiotics, a 20% of the embryos still remained viable at 400 mg l⁻¹. Neomycin was discarded as a selective agent since it showed only a moderate toxic effect. Contrary to solid medium, when olive callus was cultured in liquid medium supplemented with different paromomycin concentrations for 3 weeks, the callus growth was impaired at the lowest antibiotic concentration, 3 mg l⁻¹. Best conditions for transient transformation of olive callus using PDS-1000/He system were a 6 cm target distance and a 900 psi bombardment pressure. pCGU∆1 plasmid, containing the gus gene under the control of sunflower ubiquitin promoter yielded a significantly higher number of gus expression areas per bombarded explant than pGUSINT or pJGUS5 plasmids, where the gus gene is driven by CaMV35S promoter or CaMV35S with enhancer, respectively. Almost 45% of bombarded explants showed gus expression 12 weeks after bombardment.     

A biolistic approach for the transfer and expression of a gusA. reporter gene in embryogenic cultures of Pinus radiata

Summary The biolistic® particle delivery system was used for the delivery of DNA into embryogenic tissue culture cells of Pinus radiata D. Don. Several experiments with varying parameters were performed to increase the delivery efficiency. Six different controlling elements were cloned upstream of the ß-glucuronidase coding sequence (gusA reporter gene) and transient expression of the gusA reporter gene was compared three days after bombardment. The results clearly indicate a decrease in transient expression as follows: pEmu-derivatives with the ocs-enhancer-element > 2x CaMV 35S (with Kozak consensus-sequence) > 2x CaMV 35S (without Kozak consensus sequence) > CaMV 35S (with Kozak consensus-sequence) > CaMV 35S (without Kozak consensus sequence). Time course experiments monitoring gusA expression showed a significant decrease in the number of blue spots 10–14 days after bombardment. A few blue clumps however, were still detected 35 days after shooting. Embryo initials expressing the gusA gene in all cells were also detected. The results suggest that it will be possible to develop a reliable biolistic protocol for stable integration of genes into Pinus radiata embryogenic cultures which are capable of plant regeneration.Abbreviations ccc covalently closed circular DNA - lin linearised DNA - E restriction enzyme Eco RI - Sph restriction enzyme SpH I - 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine     

DNA uptake by imbibition and expression of a foreign gene in rice

Uptake of DNA by imbibition of dry and viable rice ( Oryza sativa L.) embryos from a DNA solution and expression of a foreign gene were detected using two different vectors contaíning gusA (β-glucuronidase) and hpt (hygromycin phosphotransferase) as reporter genes. The frequency of transient expression of gusA and hpt genes using the CaMV35S promoter was about 30 to 50%. The main sites of gusA gene expression were meristems of roots and vascular bundles of leaves. Also, DNA uptake, integration and expression of the hpt gene in selected rice were investigated by various PCR methods and Southern blot analysis of genomic DNA. It was shown that the hygromycin phosphotransferase (HPT) DNA was present in the rice genome in an integrated form and not as a plasmid form.     

Particle-inflow-gun-mediated genetic transformation of buffel grass (Cenchrus ciliaris L.): optimizing biological and physical parameters

The present study was conducted to optimize various biological and physical parameters for developing an efficient and reproducible gene transfer method for genetic transformation of buffel grass. Transformation was carried out using a helium-driven particle inflow gun (PIG). Embryogenic calli produced from mature seeds of buffel grass cv. CC-119 were separately bombarded with four plasmids, containing Actin (pAct1DX), Ubiquitin (pAHC-25; pAHC-27) and CaMV-35S (pCaMVGUS) promoters, coated on tungsten and gold particles. The efficiency of transformation was monitored through transient GUS expression. Different parameters, viz., the type of promoter, type and size of microcarrier, helium gas pressure, distance and time of bombardment, were standardized for delivering DNA into embryogenic calli. Bombardment with plasmid DNA carrying the actin promoter coated on 1.6 micro gold particles, at a helium pressure of 4 bars, a distance of 10 cm for 10 micro sec and 28 mm Hg vacuum in the chamber, produced the best result in transient GUS expression. The Actin promoter was found to be more efficient in driving expression of the GUS gene in buffel grass, followed by Ubiquitin and CaMV-35S promoters. Lower helium pressure was found to be sub-optimal, while higher pressure produced a smaller number of blue spots, probably due to excessive damage to the cells. Maximum of 385 blue spots was observed with gold particles of 1.6 micro size, whereas only 213 blue spots were recorded for tungsten particles of 1.0 micro size. The optimized parameters can be employed for genetic transformation of buffel grass with genes of agronomic importance.     

Effects of tissue type and promoter strength on transient GUS expression in sugarcane following particle bombardment

Effects of tissue type and promoter strength on transient GUS expression in the sugarcane (Saccharum spp. hybrids) cultivar NCo 310 were evaluated following microprojectile bombardment of leaf explants. GUS expression was histochemically or fluorometrically measured 48 h after delivery of the uidA gene. High levels of GUS expression were obtained in leaf segments isolated from young, expanding sugarcane leaves cultured for 1, 3, or 6 d prior to bombardment. The promoter derived from the maize ubiquitin 1 gene (Ubi-1) produced significantly more GUS foci and higher GUS activity levels compared to the recombinant Emu, rice actin 1 (Act1), and CaMV 35S promoters. Our transient expression system should facilitate efforts to identify promoters and elements which will regulate desired gene expression patterns in sugarcane and aid in development of an efficient stable transformation system.Abbreviations Act1 rice actin 1 gene - CaMV cauliflower mosaic virus - GUS ß-glucuronidase - Ubi-1 maize ubiquitin 1 gene - uidA GUS gene - X-Glu 5-bromo-4-chloro-3-indoylglucuronide     

Transient expression of lacZ in particle bombarded Gracilaria changii (Gracilariales, Rhodophyta)

Using a Biolistic PDS 1000/He system, healthy thalli of Gracilaria changii were bombarded with gold particles coated with plasmid DNA containing the lacZ reporter gene. Transient expression of lacZ was observed in bombarded thalli under the rupture-disc pressures of 4482, 6206, 7584 and 8963 KPa, two days after bombardment. Although G. changii exhibits a slight blue background, positive expression and the background colour can be clearly differentiated. The results indicate that lacZ could be a useful reporter gene and that SV40 promoter could be an effective promoter for Gracilaria transformation.     

Promoters for pregenomic RNA of banana streak badnavirus are active for transgene expression in monocot and dicot plants

Two putative promoters from Australian banana streak badnavirus (BSV) isolates were analysed for activity in different plant species. In transient expression systems the My (2105 bp) and Cv (1322 bp) fragments were both shown to have promoter activity in a wide range of plant species including monocots (maize, barley, banana, millet, wheat, sorghum), dicots (tobacco, canola, sunflower, Nicotiana benthamiana, tipu tree), gymnosperm (Pinus radiata) and fern (Nephrolepis cordifolia). Evaluation of the My and Cv promoters in transgenic sugarcane, banana and tobacco plants demonstrated that these promoters could drive high-level expression of either the green fluorescent protein (GFP) or the -glucuronidase (GUS) reporter gene (uidA) in vegetative plant cells. In transgenic sugarcane plants harbouring the Cv promoter, GFP expression levels were comparable or higher (up to 1.06% of total soluble leaf protein as GFP) than those of plants containing the maize ubiquitin promoter (up to 0.34% of total soluble leaf protein). GUS activities in transgenic in vitro-grown banana plants containing the My promoter were up to seven-fold stronger in leaf tissue and up to four-fold stronger in root and corm tissue than in plants harbouring the maize ubiquitin promoter. The Cv promoter showed activities that were similar to the maize ubiquitin promoter in in vitro-grown banana plants, but was significantly reduced in larger glasshouse-grown plants. In transgenic in vitro-grown tobacco plants, the My promoter reached activities close to those of the 35S promoter of cauliflower mosaic virus (CaMV), while the Cv promoter was about half as active as the CaMV 35S promoter. The BSV promoters for pregenomic RNA represent useful tools for the high-level expression of foreign genes in transgenic monocots.     

Stable Transformation of the Intact Cells of Chlorella Kessleri with High Velocity Microprojectiles

A transgenic expression system of Chlorella kessleri using the gene for β-glucuronidase (GUS) was developed. Cells of this unicellular green alga were bombarded with the plasmid pBI 121, which bears β-glucuronidase under the control of CaMV 35S promoter and the kanamycin resistant gene. Maximum GUS activity was obtained after 48 h of bombardment using a helium pressure of 900 kPa; GUS activity was then assayed for many generations. The stable transformants were able to grow on kanamycin containing medium after repeated passages between selective and nonselective medium and exhibited GUS activity comparable to that of control cells. Stable transformed cells were confirmed by polymerase chain reaction (PCR) and Southern hybridization of GUS probe with the genomic DNA of C. kessleri. This revised version was published online in July 2006 with corrections to the Cover Date.     

Transgenic Pinus radiata from Agrobacterium tumefaciens–mediated transformation of cotyledons

A method for Agrobacterium tumefaciens-mediated transformation of Pinus radiata cotyledon explants was developed using commercially available open-pollinated seed. Pinus radiata is the most widely planted commercial conifer species in the Southern Hemisphere. Reports on transformation of this species have relied on particle bombardment of embryogenic callus derived from immature embryos. The main drawback to the method is the small number of genotypes that are amenable to transformation and regeneration. Since more than 80% of genotypes of radiata pine can be regenerated using cotyledons from mature seed, cotyledon explants were cocultivated with A. tumefaciens strain AGL1 containing a plasmid coding for the neomycin phosphotransferase II (nptII) gene and the -glucuronidase (GUS) gene (uidA). Transformed shoots were selected using either geneticin or kanamycin. Critical factors for successful transformation were survival of the cotyledons after cocultivation and selection parameters. Of the 105 putative transformants that were recovered from selection media, 70% were positive for integration of the nptII gene when analysed by PCR. GUS histochemical assay for uidA expression was unreliable because of reaction inhibition by unidentified compounds in the pine needles. Further, only 4 of the 26 independent transformants characterised by PCR and Southern analysis contained an intact copy of both genes. The remaining 22 transformants appeared to have a truncated or rearranged copy of the T-DNA. It is possible that the truncation/rearrangements are due to the Cauliflower mosaic virus (CaMV) 35S promoter. Analysis of the T-DNA junction sites and sequencing of the introduced DNA will help elucidate the nature of T-DNA insertion so that genetic modification of radiata pine can be targeted effectively.Communicated by P. Debergh