Comparative analysis of transgenic rice plants obtained by Agrobacterium-mediated transformation and particle bombardment

We compared rice transgenic plants obtained by Agrobacterium-mediated and particle bombardment transformation by carrying out molecular analyses of the T₀, T₁ and T₂ transgenic plants. Oryza sativa japonica rice (c.v. Taipei 309) was transformed with a construct (pWNHG) that carried genes coding for neomycin phosphotransferase (nptII), hygromycin phosphotransferase (Hyg^r), and -glucuronidase (GUS). Thirteen and fourteen transgenic lines produced via either method were selected and subjected to molecular analysis. Based on our data, we could draw the following conclusions. Average gene copy numbers of the three transgenes were 1.8 and 2.7 for transgenic plants obtained by Agrobacterium and by particle bombardment, respectively. The percentage of transgenic plants containing intact copies of foreign genes, especially non-selection genes, was higher for Agrobacterium-mediated transformation. GUS gene expression level in transgenic plants obtained from Agrobacterium-mediated transformation was more stable overall the transgenic plant lines obtained by particle bombardment. Most of the transgenic plants obtained from the two transformation systems gave a Mendelian segregation pattern of foreign genes in T₁ and T₂ generations. Co-segregation was observed for lines obtained from particle bombardment, however, that was not always the case for T₁ lines obtained from Agrobacterium-mediated transformation. Fertility of transgenic plants obtained from Agrobacterium-mediated transformation was better. In summary, the Agrobacterium-mediated transformation is a good system to obtain transgenic plants with lower copy number, intact foreign gene and stable gene expression, while particle bombardment is a high efficiency system to produce large number of transgenic plants with a wide range of gene expression.     

Conifer genetic engineering: transgenic Pinus radiata (D. Don) and Picea abies (Karst) plants are resistant to the herbicide Buster

A biolistic transformation procedure was applied to co-transform embryogenic tissue of Pinus radiata and Picea abies with two plasmid DNAs. The first vector contained the bar gene, specifying resistance to the herbicide glufosinate, under the control of the maize ubiquitin promoter. This plasmid also contained the Pinus radiata germin cDNA sequence, in either sense or antisense orientation, driven by the ubiquitin promoter. The second vector contained both the nptII gene under control of the CaMV 35S promoter for selection of transgenic tissue on geneticin and the uidA reporter gene under control of the double CaMV 35 promoter. Polymerase chain reaction analysis of selected geneticin-resistant tissue showed that the transformation rates for the co-bombarded plasmid were high in both Pinus radiata (75%) and Picea abies (86%). A combination of phenotypic analysis and Northern hybridisation demonstrated that a number of the transgenic lines expressed all four transgenes. Regenerated plantlets from Pinus radiata and Picea abies transgenic lines were spray-tested with commercial rates of Buster (glufosinate at 0.5, 1.0, 2.0 and 4.0 kg active ingredient per hectare). Transgenic plants survived and continued to grow with minor or no damage to their needles, whereas non-transgenic plants regenerated from the same cell lines died within 8 weeks of spraying. To our knowledge, this is the first report on genetically engineered herbicide resistance in conifers, and the results demonstrate that this trait is a feasible option for plantation forestry.     

A comparison of transgenic barley lines produced by particle bombardment and Agrobacterium-mediated techniques

Two barley transformation systems, Agrobacterium-mediated and particle bombardment, were compared in terms of transformation efficiency, transgene copy number, expression, inheritance and physical structure of the transgenic loci using fluorescence in situ hybridisation (FISH). The efficiency of Agrobacterium-mediated transformation was double that obtained with particle bombardment. While 100% of the Agrobacterium-derived lines integrated between one and three copies of the transgene, 60% of the transgenic lines derived by particle bombardment integrated more than eight copies of the transgene. In most of the Agrobacterium-derived lines, the integrated T-DNA was stable and inherited as a simple Mendelian trait. Transgene silencing was frequently observed in the T₁ populations of the bombardment-derived lines. The FISH technique was able to reveal additional details of the transgene integration site. For the efficient production of transgenic barley plants, with stable transgene expression and reduced silencing, the Agrobacterium-mediated method appears to offer significant advantages over particle bombardment.     

Seasonal variation of growth and development of the roots of five second year conifer species in the nursery

Every other week over their second growing season, stem height, collar diameter, shoot and root dry masses, number of lateral roots and length of the tap root were measured on nursery grown seedlings ofAbies balsamea L. Mill.,Pinus banksiana Lamb.,Pinus resinosa Ait.,Picea mariana Mill. BSP andPicea glauca Moench Voss. Root elongation, branching and mycorrhizal development were also recorded.Given species showed distinct seasonal growth patterns. The rate and timing of maximum root growth (mg/dry weight/week) differed markedly between species.Except for the increase in height ofPinus banksiana, root and shoot growth were not negatively correlated.The results are discussed in relation to the performance of tree seedlings in the nursery.     

Chromosome analysis and DNA homology in threePicea species,P. mariana,P. rubens,andP. glauca (Pinaceae)

A detailed karyotype analysis was made on the somatic complement ofPicea rubens andP. glauca. B-chromosomes were observed in someP. glauca populations. The karyotypes are generally asymmetrical with most of the chromosomes having median to median-submedian centromeres.Picea glauca chromosomes 2, 3, 7, and 8 have secondary constriction on their short arm and chromosome 10 has a secondary constriction on the long arm. Chromosome 3 was the most easily identifiable, as it has two secondary constrictions located on the short arm. InP. rubens, all the chromosomes but chromosomes 8 and 9 have one to four distinctive secondary constrictions. In general, the diagrammatic comparisons show a high degree of similarity amongP. mariana, P. rubens, andP. glauca. GenomicP. mariana probe strongly hybridized to dots of genomic DNA fromP. rubens andP. glauca indicating that there is a high sequence homology among these three species. The synchronizing agent, hydroxyurea was used at different concentrations to enhance the mitotic index of cell suspensions derived from embryogenic cultures. Hydroxyurea at 1.25 mM increased significantly the mitotic index. An increase of hydroxyurea from 1.25 mM to 5 mM and 10 mM resulted in a steady decrease of mitotic index.     

Hydraulic acclimation to shading in boreal conifers of varying shade tolerance

The purpose of this study was to determine how shading affects the hydraulic and wood‐anatomical characteristics of four boreal conifers (Pinus banksiana, Pinus contorta, Picea glauca and Picea mariana) that differ in shade tolerance. Plants were grown in an open field and under a deciduous‐dominated overstory for 6 years. Sapwood‐ and leaf‐area specific conductivity, vulnerability curves, and anatomical measurements (light and scanning electron microscopy) were made on leading shoots from six to nine trees of each treatment combination. There was no difference in sapwood‐area specific conductivity between open‐grown and understory conifers, although two of four species had larger tracheid diameters in the open. Shaded conifers appeared to compensate for small diameter tracheids by changes in pit membrane structure. Scanning electron microscopy revealed that understory conifers had thinner margo strands, greater maximum pore size in the margo, and more torus extensions. All of these trends may contribute to inadequate sealing of the torus. This is supported by the fact that all species showed increased vulnerability to cavitation when grown in the understory. Although evaporative demand in an understory environment is low, a rapid change into fully exposed conditions could be detrimental for shaded conifers.     

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     

RAPD variations among pure and hybrid populations ofPicea mariana, P. rubens, andP. glauca (Pinaceae), and cytogenetic stability ofPicea hybrids: Identification of species-specific RAPD markers

Random amplified polymorphic DNA (RAPD) analysis was used to determine genetic relationships amongP. mariana (black spruce),P. rubens (red spruce), andP. glauca (white spruce) and to assess the degree of polymorphism within populations from different provenances and among spruce hybrids. Eleven arbitrary decamer primers were used to amplify genomic DNAs extracted from embryogenic cultures and seedlings. Species-specific RAPD markers were identified.Picea mariana andP. rubens showed similar RAPD profiles confirming their close genetic relationship. Species-specific RAPD markers were identified and were useful in distinguishing white spruce from black and red spruces. RAPD differentiation between populations within each species was small. The level of polymorphism was much higher in spruce hybrid populations than in the pure species. Cytological analysis ofP. mariana ×P. rubens hybrids showed normal mitotic behaviour at prophase, metaphase, anaphase, and telophase. All the hybrids analyzed from different cross combinations were euploids.     

Transformation and regeneration of loblolly pine: shoot apex inoculation with Agrobacterium

Loblolly pine (Pinus taeda L.) is the mostimportant tree species in US commerce and has much to gain through geneticengineering. This species can be transformed using particle bombardment andAgrobacterium; however, the regeneration of plants fromtransgenic tissues has been difficult and the recovery of transgenic plants hasbeen rare. A shoot-based and genotype-independent transformation methodemploying Agrobacterium tumefaciens was used to facilitaterecovery of plants and permit the transformation of elite germplasm. Shootsfrom4–6 week old seedlings and adventitious shoots from culture wereinoculated with A. tumefaciens EHA101 (pGUS3), or EHA105(pSSLa.3), subjected to selection and regenerated. Shoots that survivedexhibited expression of the uidA gene (GUS) in a patterncharacteristic of the either the CaMV35S promoter (pGUS3), or the larch RbcSpromoter (pSSLa.3) transferred. Recovered plants were screened using PCRamplification. Southern DNA analyses and amplification of the T-DNA borderjunction confirmed genomic integration of both transferreduidA and nptII genes. In this proofofconcept study, the overall recovery of P. taeda shoots wasfair (10–20%), while recovery of intact rooted plants was poor (>1%)due to difficulty in rooting. Recovery of intact rooted plants from inoculatedshoots of P. eldarica and P. radiatawas more efficient (10–30%). The addition of a shoot multiplication stepand effective rooting protocols will improve the efficiency of this genotypeindependent transformation method in P. taeda, and inotherPinus spp.     

A simple and effective method for protein subcellular localization using <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of onion epidermal cells

A modified Agrobacterium-mediated transformation protocol has been successfully used for transient expression of the intrinsically fluorescent proteins and their fusion proteins in onion epidermis. The mean of the transformed cells rate per peel is about 10.5±0.9%, while that of the particle bombardment method is at the range 2.0±0.4%. To compare with the prevailing method of micro-projectile bombardment, the modified Agrobacterium-mediated transformation may provide with higher efficiency and even more simplified manipulability on a lower budget.     

Inheritance of mitochondrial DNA in the conifer Larix

Summary Restriction fragment length polymorphisms between Larix leptolepis and Larix decidua were identified in heterologous hybridization experiments, using wheat mitochondrial DNA probes specific for atp9, coxI, nad3/rps12, and orf25. Analysis of eight individuals of each reciprocal hybrid of these two species revealed that mitochondrial DNA was maternally inherited. Furthermore, sequences homologous to wheat orf25 were also identified in Larix gmelini, Larix siberica, Larix olgensis, and Larix laricina, as well as Ginkgo biloba, Picea mariana, Picea glauca and Pinus contorta.     

A comparison of the composite and repeat pressurization methods of pressure–volume analysis for shoots of four North American conifer species

Significantly different water relations attributes were derived for temperate conifers measured using the repeat pressurization (RP) and composite (CM) pressure–volume (PV) procedures. In the RP method, single shoots were measured 10–20 times for xylem water potential and mass during air-drying to produce each PV curve. In contrast, for CM PV curves 25–30 shoots were air-dried to relative water contents (R) ranging from 1.0 to 0.5 before being pressurized once. Aggregation of these 25–30 paired values produced single PV curves. Pinus banksiana, P. resinosa and Picea mariana, but not Pinus strobus, had lower full turgor osmotic potential, shallower slope of the linear segment of the PV curves and higher symplast fraction with the CM method. Data points along the linear segment of PV curves were obtained to lower R using the CM method. Reanalysis using similar R ranges eliminated differences between PV methods for Picea mariana but not Pinus banksiana and P. resinosa.     

Mapped DNA probes from loblolly pine can be used for restriction fragment length polymorphism mapping in other conifers

A high-density genetic map based on restriction fragment length polymorphisms (RFLPs) is being constructed for loblolly pine (Pinus taeda L.). Consequently, a large number of DNA probes from loblolly pine are potentially available for use in other species. We have used some of these DNA probes to detect RFLPs in 12 conifers and an angiosperm. Thirty complementary DNA and two genomic DNA probes from loblolly pine were hybridized to Southern blots containing DNA from five species of Pinus (P. elliottii, P. lambertiana, P. radiata, P. sylvestris, and P. taeda), one species from each of four other genera of Pinaceae (Abies concolor, Larix laricina, Picea abies, and Pseudotsuga menziesii), one species from each of three other families of Coniferales [Sequoia sempervirens (Taxodiaceae), Torreya californica (Taxaceae) and Calocedrus decurrens (Cupressaceae)], and to one angiosperm species (Populus nigra). Results showed that mapped DNA probes from lobolly pine will cross-hybridize to genomic DNA of other species of Pinus and some other genera of the Pinaceae. Only a small proportion of the probes hybridized to genomic DNA from three other families of the Coniferales and the one angiosperm examined. This study demonstrates that mapped DNA probes from loblolly pine can be used to construct RFLP maps for related species, thus enabling the opportunity for comparative genome mapping in conifers.     

Efficient screening for expressed sequence tag polymorphisms (ESTPs) by DNA pool sequencing and denaturing gradient gel electrophoresis (DGGE) in spruces

There is an urgent need to accelerate the development of informative codominant markers of coding regions such as ESTPs (expressed sequence tag polymorphisms) to estimate map synteny within and among taxa. A set of primer pairs for 207 ESTs or cDNAs from Picea and Pinus taxa was screened on three distantly-related taxa in the genus Picea, P. mariana (Mill.) B.S.P., P. glauca (Moench) Voss and P. abies (L.) Karst. Of these, 118 (57%) resulted in positive amplification of single-locus gene products in the first two species. To detect polymorphism, these 118 markers were further screened on a panel of 10 pedigree parents for each of P. mariana and P. glauca, either by agarose gel electrophoresis (AGE) or by parallel denaturing gradient gel electrophoresis (DGGE) with standard conditions of 15-45% urea-formamide. Of these, 87 and 74 were found polymorphic in P. mariana and P. glauca, respectively, and 65 were polymorphic in both species. DNA pool sequencing has been explored as a possible strategy to increase economically the detection throughput of SNPs and small indels, and to characterize the types of DNA polymorphism detected by DGGE. Different DNA samples of known sequences were pooled in different ratio mixtures before and after PCR amplifications to determine their minimum relative abundance for detection of DNA polymorphisms by sequencing. For detection of a polymorphism in the DNA pools, the minimum level of relative abundance was 10%. Pooling DNA samples before or after PCR amplification had no effect on the detection of polymorphism by sequencing. For each species panel, the DNAs were pooled and then amplified and sequenced for the 118 primer pairs. With this strategy, the number of ESTPs increased to 107 in P. mariana and 106 in P. glauca, and the number of ESTPs shared by both species increased to 99. About half of the ESTP markers displayed both SNP and indel polymorphisms while the other half displayed only SNPs. Most of the additional ESTPs were amenable to detection by DGGE or CAPS (Cleaved Amplified Polymorphic Sequence) for mapping purposes.     

Microprojectile bombardment of plant tissues increases transformation frequency by Agrobacterium tumefaciens

Bombardment of plant tissues with microprojectiles in an effective method of wounding to promote Agrobacterium-mediated transformation. Tobacco cv. Xanthi leaves and sunflower apical meristems were wounded by microprojectile bombardment prior to application of Agrobacterium tumefaciens strains containing genes within the T-DNA encoding GUS or NPTII. Stable kanamycin-resistant tobacco transformants were obtained using an NPTII construct from particle/plasmid, particle-wounded/Agrobacterium-treated or scalpel-wounded/Agrobacterium-treated potato leaves. Those leaves bombarded with particles suspended in TE buffer prior to Agrobacterium treatment produced at least 100 times more kanamycin-resistant colonies than leaves treated by the standard particle gun transformation protocol. In addition, large sectors of GUS expression, indicative of meristem cell transformation, were observed in plants recovered from sunflower apical explants only when the meristems were wounded first by particle bombardment prior to Agrobacterium treatment. Similar results in two different tissue types suggest that (1) particles may be used as a wounding mechanism to enhance Agrobacterium transformation frequencies, and (2) Agrobacterium mediation of stable transformation is more efficient than the analogous particle/plasmid protocol.     

A set of primers for amplification of mitochondrial DNA in Picea abies and other conifer species

Thirteen primer pairs generating intraspecific length and/or presence‐absence polymorphism in Picea abies have been obtained from a P. abies mtDNA library, using different methodologies (agarose gel electrophoresis, polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP), single‐strand conformation (SSCP). Poly‐morphism tests were extended successfully to other Picea species (P. omorika, P. engelmanii and P. glauca) and species belonging to other conifer genera (Abies alba, Larix laricina and Pinus pinaster). This set of PCR‐based mitochondrial markers can provide promising tools for studying phylogeography or phylogeny in P. abies and other conifer species in which the mt genome is generally the only one to be transmitted via the female gamete.     

Organogenesis in vitro of tissues from mature conifers

Summary Shoot and embryolike structures were obtained in cultures of various tissues of mature conifer trees. Morphogenesis occurred in haploid and diploid tissues of female cones ofPinus mugo andPicea abies, although only rarely. Vegetative shoots dissected from early spring buds ofAbies balsamea, Picea glauca, andPseudotsuga menziesii were more responsive, with most of the organized growth occurring at the base of young needles. The following sequence of treatments was most effective: (a) collect twigs with buds shortly before budbreak, (b) cold store for at least 1 month, (c) force buds to break before excision of the vegetative shoots (keep submerged in water during excision), and (d) soak explant in malonic acid, 1 g/l, for 15 min before transfer of the shoots to the nutrient medium.     

Postglacial vegetational and fire history: pollen, plant macrofossil and charcoal records from two Alaskan lakes

Pollen, plant macrofossil and charcoal analyses of sediments from two Alaskan lakes provide new data for inferring Lateglacial and Holocene environmental change. The records span the past 14,700 years at Lost Lake, 240 m a.s.l., central Alaska, north of the Alaska Range and 9600 years at Grizzly Lake, 720 m a.s.l., Copper River Plateau, south of the Alaska Range. Salix shrubs expanded in the herb tundra about 14,400 cal b.p., and Betula shrub tundra became established at ca. 13,200 cal b.p. Diminished Betula shrub cover in association with the increased abundance of herbaceous taxa occurred at 12,500–11,600 cal b.p., although the timing of these changes is not well constrained. Populus expanded at 11,200 cal b.p. and formed dense stands until 9600–9400 cal b.p. when Picea glauca forests or woodlands became established at both sites. The abundance of Alnus viridis increased markedly around 8500 cal b.p. at both sites, marking the development of alder shrub thickets around the lakes and on mountain slopes in these areas. Boreal forests dominated by Picea mariana became established around 7200 cal b.p. at Grizzly Lake and 5700 cal b.p. at Lost Lake. At Grizzly Lake, marked vegetational oscillations occurred within the past 8500 years; for example, A. viridis expanded at 2750 cal b.p. and 450 cal b.p. and declined at 150 cal b.p. Some of these oscillations coincide with large-scale climatic events, such as the Little Ice Age cooling (LIA), and they probably reflect vegetational sensitivity to climatic change at this high site. Microscopic charcoal at Lost Lake suggests that fire was important in the lateglacial birch tundra, probably because of severe moisture deficits of the regional climate and/or high abundance of fine fuels. On the basis of the Grizzly Lake microscopic charcoal record, regional fires were common between 8500 and 6800 cal b.p. and between 450 and 150 cal b.p. Around Grizzly Lake, the mean return intervals of local fires estimated from macroscopic charcoal were ∼386 years between 6800 and 5500 cal b.p. when Picea glauca dominated over P. mariana, ∼254 years between 5500 and 3900 cal b.p. when P. mariana was more abundant than P. glauca, and ∼200 years after 3900 cal b.p. in both P. glauca and P. mariana dominated forests. Correlation analysis of pollen and microscopic charcoal at Grizzly Lake reveals that increased fire activity led to the reductions of P. glauca, P. mariana, and tree Betula in association with the expansions of A. viridis, Epilobium, Lycopodium clavatum, and L. annotinum.     

Biolistic mediated site-specific integration in rice

Cre-lox mediated site-specific integration in tobacco or Arabidopsis used polyethylene glycol or Agrobacterium, respectively, to deliver the integrating DNA. The polyethylene glycol method is inconvenient since it requires the use of protoplasts. The Agrobacterium method is inefficient as the single-stranded T-DNA is not a substrate for Cre-lox recombination. In this study, we tested the biolistic method for the site-specific insertion of DNA into the rice genome. Two target callus lines, each harboring a single genomic lox target, were generated by Agrobacterium-mediated transformation. The target callus lines were subjected to a second round of transformation by particle bombardment with a construct designed to excise the plasmid backbone from the integrating DNA, followed by the recombination of the integrating DNA into the genomic lox target. Site-specific integration was obtained from both target callus lines. Three integrant plants were regenerated from one target line and were found to have a precise copy of the integrating DNA at the target site, although only one plant has the integrating DNA as the sole copy in the genome. Site-specific integration through the biolistic delivery of DNA can be considered for other plants that are transformable via particle bombardment.