Expression of the <Emphasis Type="Italic">rol</Emphasis>B gene enhances adventitious root formation in hardwood cuttings of aspen

Summary An elite aspen hybrid (Populus × canescens × P. grandidentata) was transformed with Agrobacterium tumefaciens strain EHA105 that harbored a binary vector (pBI121) carrying the nptII gene under the nos promoter and tandem rolB-uidA (GUS) genes with the CaMV 35S or heat shock promoter. Among 32 independent kanamycin-resistant plants, 25 plants were confirmed by polymerase chain reaction and Southern blot analyses to contain all three genes, whereas five plants contained only nptII or/and uidA genes and two plants had both the rolB and nptII or uidA genes. Integration of the rolB gene significantly increased rooting ability of hardwood cuttings. Heat shock-rolB-transformed plants rooted at significantly higher percentage than the CaMV 35S-rolB-transformed plants. Heat shock treatment further enhanced rooting of heat shock-rolB-transformed plants. Exposure to exogenous auxin did not significantly increase the rooting percentage of transgenic hardwood cuttings, but increased the number of roots induced. This research shows great potential to improve rooting of hardwood cuttings of difficult-to-root woody plants which are commercially important to the horticultural and forestry industry. The transgenic plants with gain-of-function in hardwood-cutting rooting can facilitate research in the understanding of adventitious rooting from hardwood cuttings of recalcitrant woody plants.     

Protocol for transformation of the apple rootstock Jork 9 with the rolB gene and its influence on rooting

A suitable protocol for transformation has been developed for the apple rootstock Jork 9 using Agrobacterium tumefaciens strain EHA101A(pEHA101A)(pSCV1.6). Root formation was increased by transforming the rootstock with A. tumefaciens strain C58C1(pGV3850)(pB-B:GUS), which contains the nptII, rolB and gus genes on the T-DNA. Transformation for all of the introduced genes was confirmed by polymerase chain reaction and Southern blot analyses. Of the 18 independent shoot lines obtained after transformation only ten contained at least one copy of intact T-DNA, while six lines were missing the gus gene and two lines were missing both the gus and rolB genes. The rooting experiments showed that introduction of the rolB gene increased root percentage and root number, giving 13.8 roots per shoot compared to 2.3 for untransformed shoots. More than two copies of the rolB gene decreased the number of roots and percentage of rooted shoots.     

Agrobacterium-mediated DNA transfer in sugar pine

DNA transfer using Agrobacterium tumefaciens has been demonstrated in sugar pine, Pinus lambertiana Dougl. Shoots derived from cytokinin-treated cotyledons formed galls after inoculation with A. tumefaciens strains containing the plasmid pTiBo542. A selectable marker, neomycin phosphotransferase II, conferring resistance to kanamycin, was transferred into sugar pine using a binary armed vector system. Callus proliferated from the galls grew without hormones and in some cases, kanamycin-resistant callus could be cultured. Southern blots provided evidence of physical transfer of T-DNA and the nptII gene. Expression of the nptII gene under control of the nos promoter was demonstrated by neomycin phosphotransferase assays. Several aspects of DNA transfer were similar to those previously observed in angiosperms transformed by A. tumefaciens. This is the first evidence for DNA transfer by Agrobacterium in this species and the first physical evidence for transfer in any pine. These results bring us closer to genetic engineering in this commercially important genus of forest trees.     

Expression of a reporter gene is reduced by a ribozyme in transgenic plants

A chimeric gene encoding a ribozyme under the control of the cauliflower mosaic virus (CaMV) 35S promoter was introduced into transgenic tobacco plants. In vivo activity of this ribozyme, which was designed to cleave npt mRNA, was previously demonstrated by transient expression assays in plant protoplasts. The ribozyme gene was transferred into transgenic tobacco plants expressing an rbcS-npt chimeric gene as an indicator. Five double transformants out of sixteen exhibited a reduction in the amount of active NPT enzyme. To measure the amount of ribozyme produced, in the absence of its target, the ribozyme and target genes were separated by genetic segregation. The steady-state concentrations of ribozyme and target RNA were shown to be similar in the resulting single transformants. Direct evidence for a correlation between reduced npt gene expression and ribozyme expression was provided by crossing a plant containing only the ribozyme gene with a transgenic plant expressing the npt gene under control of the 35S promoter, i.e. the same promoter used to direct ribozyme expression. The expression of npt was reduced in all progeny containing both transgenes. Both steady-state levels of npt mRNA and amounts of active NPT enzyme are decreased. In addition, our data indicate that, at least in stable transformants, a large excess of ribozyme over target is not a prerequisite for achieving a significant reduction in target gene expression.     

pBECKS

A series of binary T-DNA vectors (pBECKS) has been created for use in theAgrobacterium-mediated genetic transformation of plants. The pBECKS series has corrected the undesirable features of the popular pBIN19 vector; the deleterious mutation within the coding sequence ofnptII has been amended and the cloning sites are now adjacent to the right border repeat in order to reduce the possibility of producing truncated sequences of novel genes within transformants. One set of vectors incorporates various combiantions of the marker genesgusA,C1/Lc,nptII,hph, andbar, for pursuit of early and stable transformation events. A set of constructs which contain deleted T-DNA borders in various combinations and display predictably altered efficacies for gene transfer has also been created. A modular set of vectors has been designed to facilitate the insertion and transfer of novel gene sequences by providing anptII-linked plant expression cassette orlacZ-multiple cloning site. A range of antibiotic resistance genes has been incorporated into the non-T-DNA part of the vectors in order to facilitate their selection across the range ofAgrobacterium virulence strains.     

Expression of <Emphasis Type="Italic">rol</Emphasis> genes in transgenic soybean (<Emphasis Type="Italic">Glycine max</Emphasis> L.) leads to changes in plant phenotype,leaf morphology,and flowering time

Soybean (Glycine max L.) cultivar NARC-4 was transformed with constructs carrying rolA, rolB, or rolC genes, each under the control of the Cauliflower Mosaic Virus 70S promoter. Cotyledonary nodes of soybean seeds were infected with Agrobacterium tumefaciens strain LBA4404 carrying one of the three rol genes, along with nptII in the plasmid pLBR. The efficiency of the transformation varied slightly among the three constructs, with frequencies of 6, 7, and 5% for the rolA, rolB, and rolC genes, respectively, being observed. Southern blot analysis confirmed the integration of rol genes in the soybean genome with varying numbers of copies of the transgene. All transformed plants showed enhanced rooting, but the number of adventitious roots was higher for transformants carrying the rolB transgene. rolA and rolC transformants showed dwarf phenotypes, clustered branching, and variations in leaf morphology. Furthermore, these plants flowered within a short period of time and produced lower numbers of flowers. rolB transformants showed variations in phenotype, including dwarf to semi-dwarf and shrubby growth, abnormal stem growth, short internodes, variations in leaf morphology, and greenish to yellowish-green colored leaves. These plants also flowered early, but dwarf plants produced low numbers of flowers, while shrubby plants produced high numbers of flowers, but these were mostly infertile.     

Characterization of hairy root-phenotype in transgenic <Emphasis Type="Italic">Hypericum perforatum</Emphasis> L. clones

Hairy root-regenerated clones of Hypericum perforatum L. grown in vitro similarly to those successfully adapted to ex vitro conditions showed phenotype features typical for plants transformed with Agrobacterium rhizogenes T-DNA. These included reduced apical dominance, increased branching, dwarfing and reduced fertility. Transgenic clones differed in ability to develop root system as a necessary condition for transfer to the soil. One of the profiling characters, capability of hypericin biosynthesis was altered as well. Dark glands as the sites of hypericin accumulation and/or synthesis exhibited significantly higher densities on both, leaves and petals of transgenic clones comparing to controls. In the genome of transgenic clones, rolABC genes were detected. Both clones harboured similar copy number of individual rol genes. However, copy numbers descended from rolA to rolC gene in both clones.     

Phenotypic Effects of Isolated pRiA4 TL-DNA rol Genes in the Presence of Intact TR-DNA in Transgenic Plants of Solanum dulcamara L.

The effect of the rol genes, together with the TR-DNA of pRiA4on the phenotype of Solanum dulcamara plants, was analysed.Plants transformed by Agrobacterium strain BN1010: :rolA (rolA^7plus;TR⁺)exhibited severe leaf wrinkling, whereas plants transformedby strain BN1010: :rolC (ro/C⁺TR⁺) had a typical ‘hairyroot’ phenotype. Leaf discs excised from these latterplants produced roots on hormone-free medium. BN1010: :rolABC(rolABC⁺TR⁺) transformed plants had an exaggerated transformedphenotype. Some of the BN1010: :rolABC transformants had positivelygeotropic root growth which correlated with the presence ofmultiple copies of the TR-DNA. S. dulcamara plants, transformedby the TR-DNA region only, exhibited epinasty. Scanning electronmicroscopy of plants containing various regions of agropineRi T-DNA revealed that transformation causes changes in basicplant siructure.     

Characterization of transgenic plants derived from hairy roots ofHyoscyamus muticus

Mature plants were regenerated via protoplasts fromAgrobacterium rhizogenes-transformed root cultures ofHyoscyamus muticus L., and chemical analyses were performed on 34 individual plants. The regenerated plants showed strong phenotypic differences from clone to clone as well as from the control plants. Polymerase chain reaction studies revealed that the plants exhibiting the strongest phenotypic alterations contained therol (A, B and C) genes, whereas the plants with fewer alterations had lost them. The plants produced hyoscyamine, scopolamine and a range of different calystegins, and considerable somaclonal variation was observed. Alkaloid production in the plants transgenic for therol genes was clearly reduced. The pattern of calystegins was similar within all the regenerated plants lackingrol genes. Among the plants withrol genes, the calystegin B₁ was not detectable. It seems clear that the presence ofrol genes is detrimental to the alkaloid accumulation in the transgenic plants in contrast to hairy root cultures.Abbreviation PCR Polymerase chain reaction     

Stable transformation and direct regeneration in Coffea canephora P ex. Fr. by Agrobacterium rhizogenes mediated transformation without hairy-root phenotype

A system for genetic transformation of Coffea canephora by co-cultivation with Agrobacterium rhizogenes harbouring a binary vector has been developed. The objective of the present study was the genetic transformation and direct regeneration of transformants through secondary embryos bypassing an intervening hairy root stage. Transformants were obtained with a transformation efficiency up to 3% depending on the medium adjuvant used. A. rhizogenes strain A4 harbouring plasmid pCAMBIA 1301 with an intron uidA reporter and hygromycin phosphotransferase (hptII) marker gene was used for sonication-assisted transformation of Coffea canephora. The use of hygromycin in the secondary embryo induction medium allowed the selection of transgenic secondary embryos having Ri T-DNA along with the T-DNA from the pCAMBIA 1301 binary vector. In addition transgenic secondary embryos devoid of Ri-T-DNA but with stable integration of the T-DNA from the binary vector were obtained. The putative transformants were positive for the expression of the uidA gene. PCR and Southern blot analysis confirmed the independent, transgenic nature of the analysed plants and indicated single and multiple locus integrations. The study clearly demonstrates that A. rhizogenes can be used for delivering transgenes into tree species like Coffea using binary vectors with Agrobacterium tumefaciens T-DNA borders.     

Transformation of rapid cycling cabbage (Brassica oleracea var. capitata) with Agrobacterium rhizogenes

Summary Genetically transformed cabbage (Brassica oleracea var. capitata) roots were obtained after inoculation with two engineered Agrobacterium rhizogenes strains, each harbouring a plant selectable marker gene in their T-DNA. Axenic root clones resistant to kanamycin or hygromycin B were established, most of which did not exhibit the phenotypic characteristics of Ri-transformed roots. Shoot regeneration was induced from roots after treatment with 2,4-dichlorophenoxyacetic acid (2,4-D). The resulting plants exhibited various phenotypes: some looked normal, while others showed the transformed phenotype observed in other species. Direct evidence for genetic transformation was obtained by molecular hybridization. The trait was transmitted to the progeny. Transformed cabbage plants can be obtained within 6 months using this approach.     

Transformation of the monocot Alstroemeria by Agrobacterium rhizogenes

An efficient procedure is described for transformation of calli of the monocotyledonous plant Alstroemeria by Agrobacterium rhizogenes. Calli were co-cultivated with A. rhizogenes strain A13 that harbored both a wild-type Ri-plasmid and the binary vector plasmid pIG121Hm, which included a gene for neomycin phosphotransferase II (NPTII) under the control of the nopaline synthase (NOS) promoter, a gene for hygromycin phosphotransferase (HPT) under the control of the cauliflower mosaic virus (CaMV) 35S promoter, and a gene for -glucuronidase (GUS) with an intron fused to the CaMV 35S promoter. Inoculated calli were plated on medium that contained cefotaxime to eliminate bacteria. Four weeks later, transformed cells were selected on medium that contained 20 mg L^–1 hygromycin. A histochemical assay for GUS activity revealed that selection by hygromycin was complete after eight weeks. The integration of the T-DNA of the Ri-plasmid and pIG121Hm into the plant genome was confirmed by PCR. Plants derived from transformed calli were produced on half-strength MS medium supplemented with 0.1 mg L^–1 GA₃ after about 5 months of culture. The presence of the gusA, nptII, and rol genes in the genomic DNA of regenerated plants was detected by PCR and Southern hybridization, and the expression of these transgenes was verified by RT-PCR.     

Agrobacterium-mediated transformation of the apple rootstock Malus micromalus Makino with the RolC gene

Summary Malus micromalus Makino was genetically engineered with the rolC gene via Agrobacterium-mediated transformation. The antibiotics carbenicillin, hygromycin, and kanamycin (Km) inhibited shoot regeneration from in vitro leaf explants. The leaf segments were infected with Agrobacterium tumefaciens strain LBA4404 harboring one of the three different plasmids. Shoots were regenerated only from leaf segments infected with LBA4404 harboring the plasmid with the introncontaining neomycin phosphotransferase II gene as the selectable marker. Preculture of leaf segments on regeneration medium for 2 d before Agrobacterium treatment reduced formation of Km-resistant calluses. Transformation was confirmed by a histochemical β-glucuronidase (GUS) assay, amplification of the rolC gene by polymerase chain reaction (PCR), and by Southern blot analysis. The rolC-transformed M. micromalus shoots showed an increased rooting ability without auxin treatment, and reduced height, internode length and leaf areas. This research shows the potential application of using the rolC gene for developing dwarf apple rootstocks.     

Expression of a Novel Antiporter Gene from Brassica napus Resulted in Enhanced Salt Tolerance in Transgenic Tobacco Plants

Tobacco leaf discs were transformed with a plasmid pBIBnNHX1, containing the selectable marker neomycin phosphotransferase gene (nptII) and Na⁺/H⁺ vacuolar antiporter gene from Brassica napus (BnNHX1), via Agrobacterium tumefaciens-mediated transformation. Thirty-two independent transgenic plants were regenerated. Polymerase chain reaction (PCR) and Southern blot analyses confirmed that the BnNHX1 gene had integrated into plant genome and Northern blot analysis revealed the transgene expression at various levels in transgenic plants. Transgenic plants expressing BnNHX1 had enhanced salt tolerance and could grow and produce seeds normally in the presence of 200 mM NaCl. Analysis for the T₁ progenies derived from seven independent transgenic primary transformants expressing BnNHX1 showed that the transgenes in most tested independent T₁ lines were inherited at Mendelian 3:1 segregation ratios. Transgenic T₁ progenies could express _BnNHX1 and had salt tolerance at levels comparable to their T₀ parental lines. This study implicates that the BnNHX1 gene represents a promising candidate in the development of crops for enhanced salt tolerance by genetic engineering.     

Locations and stability of Agrobacterium-mediated T-DNA insertions in the Lycopersicon genome

Summary The genomic distribution and genetic behavior of DNA sequences introduced into the tomato genome by Agrobacterium tumefaciens were investigated in the backcross progeny of 10 transformed Lycopersicon esculentum x L. pennellii hybrids. All transformants were found to represent single locus insertions based on the co-segregation of restriction fragments corresponding to the T-DNA left and right border sequences in the backcross progeny. Isozyme and restriction fragment length polymorphism (RFLP) markers were used to test linkage relationships of the insertion in each backcross family. The T-DNA inserts in 9 of the 10 transformants were mapped in relation to one or more of these markers, and each mapped to a different chromosomal location. Because only one insertion did not show linkage with the markers employed, it must be located somewhere other than the genomic regions covered by the markers assayed. We conclude that Agrobacterium-mediated insertion in the Lycopersicon genome appears to be random at the chromosomal level. No discrepancies were found between the T-DNA genotype and the nopaline phenotype in the 322 backcross progeny of the nopaline positive transformants. Backcross progeny of two nopaline negative transformants showed incomplete correspondence between the T-DNA genotype and the kanamycin resistance phenotype. No alteration of T-DNA was observed in progeny showing a discrepancy between T-DNA and kanamycin resistance. However, two kanamycin resistant progeny plants of one of these two transformants possessed altered T-DNA restriction patterns, indicating genetic instability of the T-DNA in this transformant.Journal article no. 1223 of the New Mexico Agricultural Experiment Station     

Agrobacterium-mediated transformation of the ectomycorrhizal symbiont Laccaria bicolor S238N

The development of an efficient transformation system is required to alter the expression of symbiosis-regulated genes and to develop insertional mutagenesis in the ectomycorrhizal basidiomycete Laccaria bicolor S238N. Vegetative mycelium of this fungus was transformed by Agrobacterium tumefaciens-mediated gene transfer. The selection marker was the hygromycin resistance gene of Escherichia coli (hph) under the control of the gpd promoter from Agaricus bisporus and the CaMV 35S terminator as part of the T-DNA. PCR amplification of hph and Southern blot analyses showed that the genome of the hygromycin-resistant transformants contained the cassette. The latter proved mostly single copy and random integration of part of the transgene into the fungal genome. A. tumefaciens-mediated gene transfer should facilitate future development of insertional mutagenesis, targeted gene disruption and RNA interference technology in L. bicolor.     

Genetic transformation of oilseed rape (Brassica napus) by the Ri T-DNA of Agrobacterium rhizogenes and analysis of inheritance of the transformed phenotype

Summary Genetically transformed repeseed (Brassica napus) roots were obtained by in vitro inoculation of excised stem segments with Agrobacterium rhizogenes. Axenic root organ clones were established and they exhibited a phenotype characteristic of transformed roots: rapid growth, reduced apical dominance and root plagiotropism. Stem regeneration was induced by exposing root fragments to 2,4-dichloroacetic acid (2,4-D) in liquid medium, followed by transfer to solid regeneration medium. The resulting plants exhibited the transformed phenotype observed in other species where similar experiments have been performed. Direct evidence for genetic transformation was obtained from opine assays and molecular hybridization. Sexual transmission of the transformed phenotype was Mendelian, and a probable case of T-DNA insertion into two independent loci within the same plant was detected. The estimated optimal time necessary to obtain transformed oilseed rape plants using this approach is 2 months.