Genetic transformation ofLotus corniculatus withAgrobacterium tumefaciens and the analysis of the inheritance of transgenes in the T1 generation

The herbage legume,Lotus corniculatus (bird's-foot trefoil), was transformed using the disarmedAgrobacterium tumefaciens strain LBA4404 (pAL4404) carrying a binary construct, pJit73. This plasmid carries two antibiotic resistance genes,aphIV andnptII encoding resistance to hygromycin and kanamycin respectively, and the easily detectable reporter gene,uidA encoding the enzyme -glucuronidase (GUS). Transgenic plants were regenerated from two separate co-cultivations of leaves withA. tumefaciens either with or without an acetosyringone pretreatment. A total of 110 putative transformants were regenerated, 52 (47%) of which grew on selection media containing hygromycin. Twenty-five plants were analysed further for morphological variation and presence of transgenes and were used to study the inheritance of expression of the transgenes in the T₁ generation. Expression patterns of the transgenes in the T₁ progeny generated from these 25 plants differed. In the majority of plant linesaphIV anduidA transgenes segregated together, but the apparent number of copies of the transgenes varied. No expression of either transgene was detected in the progeny from three plants, while the progeny from six other plants were resistant to hygromycin but had no GUS expression. Progeny of all of the remaining 16 plants had GUS activity. For three plants, inheritance data were consistent with more than one dose ofuidA andaphIV; another two plants yielded data expected for exactly one dose of both transgenes. In the progeny of the remaining 11 plants, the percentage of seedlings expressing bothuidA andaphIV was lower than expected.     

Identification of the aph IV gene from protoplast-derived maize plants by a simple nonradioactive DNA hybridization method

Summary Protoplast-derived, transformed maize plants were evaluated by Southern analysis for the presence of the aph IV gene which codes for resistance to the antibiotic, hygromycin B. This gene was used as a selectable marker for the transformation of maize protoplasts. Southern analysis was performed with fluorescein-labeled probe DNA. A new method for labeling molecular weight markers with fluorescein-N⁶ is presented. The nonradioactive Southern analysis method is compared to the radioactive method and the results show that the nonradioactive method is as sensitive as the radioactive method.     

Pearl millet transformation system using the positive selectable marker gene phosphomannose isomerase

Fertile transgenic pearl millet plants expressing a phosphomannose isomerase (PMI) transgene under control of the maize ubiquitin constitutive promoter were obtained using the transformation system described here. Proliferating immature zygotic embryos were used as target tissue for bombardment using a particle inflow gun. Different culture and selection strategies were assessed in order to obtain an optimised mannose selection protocol. Stable integration of the manA gene into the genome of pearl millet was confirmed by PCR and Southern blot analysis. Stable integration of the manA transgene into the genome of pearl millet was demonstrated in T₁ and T₂ progeny of two independent transformation events with no more than four to ten copies of the transgene. Similar to results obtained from previous studies with maize and wheat, the manA gene was shown to be a superior selectable marker gene for improving transformation efficiencies when compared to antibiotic or herbicide selectable marker genes.Abbreviations 2,4-D: 2,4-Diclorophenoxyacetic acid - IAA: Indole acetic acid - ICRISAT: International Crops Research Institute for the Semi-Arid Tropics - IZEs Immature zygotic embryos Communicated by H. Lörz     

Resistance to hygromycin B

Summary A bacterial gene encoding hygromycin phosphotransferase has been modified for expression in tobacco cells. The aphIV gene from Escherichia coli was inserted between the 5 sequence of an octopine synthase gene and the 3 sequence from a nopaline synthase gene. The new gene was incorporated between T-DNA border fragments in the broad-host-range vector pKT210 to form a micro-Ti plasmid. Agrobacterium tumefaciens containing this plasmid and a Ti plasmid as helper was used to incite crown gall tumors on aseptic tobacco plants. Samples of these galls could grow in the presence of hygromycin B, provided that the aph gene had been fused with the ocs gene to maintain the sense of the coding sequences. When the genes had been fused in the reverse antisense orientation none of the gall samples could grow on hygromycin. Unlike wild-type galls the hygromycin-resistant tissue contained DNA sequences homologous to the aphIV gene. Thus the modified gene can be introduced into tobacco cells and confer on them the ability to grow in the presence of hygromycin B.     

Characterization of transgenic rice plants that express rgp1, the gene for a small GTP-binding protein from rice

 The rgp1 gene, which encodes a small GTP-binding protein from rice, was introduced into rice protoplasts by electroporation. Transformed protoplasts were cultured on liquid protoplast-culture medium for 1 month, and then cells that had proliferated were transferred to a selection medium that contained 50 mg/l hygromycin B. Among 50 colonies that were selected and transferred to regeneration medium, 3 colonies generated shoots. However, two of the three shoots failed to form roots and ceased growing. A single regenerated shoot that formed roots was planted in soil and transferred to a greenhouse. Southern hybridization showed that the regenerated plant harbored a single copy of the introduced gene. The transformant (T₀) plant was shorter than the controls, it developed three times as many tillers as controls, it developed three times as many tillers as control plants but it produced mostly sterile seeds. In a test of hygromycin resistances, viable seeds segregated into resistant and sensitive seedings at a ratio of approximately 1 : 3. The progeny (T₁) plants were short with many tillers, and some produced seeds normally. The T₂ seedlings grew more rapidly than control seedlings for the first 28 days after germination, but control plants subsequently outgrew the T₂ plants. Northern blotting analysis revealed that the rgp1 gene in T₂ plants was expressed consitutively throughout all developmental stages. The results suggest that the observed phenotypic changes were due to expression of the exogenous rgp1 gene. Received: 21 September 1997/Accepted: 31 March 1998     

Green fluorescent protein as a visual selection marker for coffee transformation

The green fluorescent protein (GFP) was used as a visual selectable marker to produce transgenic coffee (Coffea canephora) plants following Agrobacterium-mediated transformation. The binary vector pBECKS 2000.7 containing synthetic gene for GFP (sgfp) S65T and the hygromycin phosphotransferase gene hph both controlled by 35S cauliflower mosaic virus CaMV35S promoters was used for transformation. Embryogenic cultures were initiated from hypocotyls and cotyledon leaves of in vitro grown seedlings and used as target material. Selection of transformed tissue was carried out using GFP visual selection as the sole screen or in combination with a low level of antibiotics (hygromycin 10 mg/L), and the efficiency was compared with antibiotics selection alone (hygromycin 30 mg/L). GFP selection reduced the time for transformed somatic embryos formation from 18 weeks on a hygromycin (30 mg/L) antibiotics containing medium to 8 weeks. Moreover, visual selection of GFP combined with low level of antibiotics selection improved the transformation efficiency and increased the number of transformed coffee plants compared to selection in the presence of antibiotics. Molecular analysis confirmed the presence of the sgfp-S65T coding region in the regenerated plants. Visual screening of transformed cells using GFP by Agrobacterium-mediated transformation techniques was found to be efficient and therefore has the potential for development of selectable marker-free transgenic coffee plants.     

Fertile transgenic barley by particle bombardment of immature embryos

Transgenic, fertile barley (Hordeum vulgare L.) from the Finnish elite cultivar Kymppi was obtained by particle bombardment of immature embryos. Immature embryos were bombarded to the embryonic axis side and grown to plants without selection. Neomycin phosphotransferase II (NPTII) activity was screened in small plantlets. One out of a total of 227 plants expressed the transferred nptII gene. This plant has until now produced 98 fertile spikes (T₀), and four of the 90 T₀ spikes analyzed to date contained the nptII gene. These shoots were further analyzed and they expressed the transferred gene. From green grains, embryos were isolated and grown to plantlets (T₁). The four transgenic shoots of Toivo (the T₀ plant) produced 25 plantlets as T₁ progeny. Altogether fifteen of these T₁ plants carried the transferred nptII gene as detected with the PCR technique, fourteen of which expressed the nptII gene. The integration and inheritance of the transferred nptII gene was confirmed by Southern blot hybridization. Although present as several copies, the transferred gene was inherited as a single Mendelian locus into the T₂ progeny.     

Selectable antibiotic resistance marker gene-free transgenic rice harbouring the garlic leaf lectin gene exhibits resistance to sap-sucking planthoppers

Rice, the major food crop of world is severely affected by homopteran sucking pests. We introduced coding sequence of Allium sativum leaf agglutinin, ASAL, in rice cultivar IR64 to develop sustainable resistance against sap-sucking planthoppers as well as eliminated the selectable antibiotic-resistant marker gene hygromycin phosphotransferase (hpt) exploiting cre/lox site-specific recombination system. An expression vector was constructed containing the coding sequence of ASAL, a potent controlling agent against green leafhoppers (GLH, Nephotettix virescens) and brown planthopper (BPH, Nilaparvata lugens). The selectable marker (hpt) gene cassette was cloned within two lox sites of the same vector. Alongside, another vector was developed with chimeric cre recombinase gene cassette. Reciprocal crosses were performed between three single-copy T₀ plants with ASAL- lox-hpt-lox T-DNA and three single-copy T₀ plants with cre-bar T-DNA. Marker gene excisions were detected in T₁ hybrids through hygromycin sensitivity assay. Molecular analysis of T₁ plants exhibited 27.4% recombination efficiency. T₂ progenies of L03C04(1) hybrid parent showed 25% cre negative ASAL-expressing plants. Northern blot, western blot and ELISA showed significant level of ASAL expression in five marker-free T₂ progeny plants. In planta bioassay of GLH and BPH performed on these T₂ progenies exhibited radical reduction in survivability and fecundity compared with the untransformed control plants.     

Production of fertile transgenic barley (Hordeum vulgare L.) plant using the hygromycin-resistance marker

Fertile transgenic barley (Hordeum vulgare L.) plants were obtained by high velocity particle bombardment. The plasmid pBCl was used to deliver the selectable hph gene and reporter Gus gene into immature embryo. After the selection culture 18 hygromycin resistant plants were obtained. Samples for Southern hybridization and enzymatic Gus assay were obtained from 11 plants. Southern hybridization confirmed the presence of the hph gene in the 11 hygromycin resistant plants(T₀). Enzymatic assay indicated that all the t₀ plants that showed hph positive in Southern analysis possessed detectable amount of Gus activity. To date all the 11 t₀ plants reached maturity and mature seeds were obtained Transmission of the hph gene to progeny(T₁) of two independent t₀ plants was confirmed by Southern hybridization.Abbreviations Adh Alcohol Dehydrogenase - BA 6-Benzylaminopurine - cv cultivar - 2,4-D 2,4-Dichlorophenoxyacetic Acid - Gus -Glucuronidase - hph Hygromycin Phosphotransferase - 4MU 4-Methyl-umbelliferone     

Inheritance of tissue-specific expression of barley hordein promoter-uidA fusions in transgenic barley plants

 Barley (Hordeum vulgare L.) hordeins are alcohol-soluble redundant storage proteins that accumulate in protein bodies of the starchy endosperm during seed development. Strong endosperm-specific β-glucuronidase gene-(uidA; gus) expression driven by B₁- and D-hordein promoters was observed in stably transformed barley plants co-transformed with the selectable herbicide resistance gene, bar. PCR analysis using DNA from calli of 22 different lines transformed with B₁- or D-hordein promoter-uidA fusions showed the expected 1.8-kb uidA fragment after PCR amplification. DNA-blot analysis of genomic DNA from T₀ leaf tissue of 13 lines showed that 12 (11 independent) lines produced uidA fragments and that one line was uidA-negative. T₁ progeny from 6 out of 12 independent regenerable transgenic lines tested for uidA expression showed a 3 : 1 segregation pattern. Of the remaining six transgenic lines, one showed a segregation ratio of 15 : 1 for GUS, one expressed bar alone, one lacked transmission of either gene to T₁ progeny, and three were sterile. Stable GUS expression driven by the hordein promoters was observed in T₅ progeny in one line, T₄ progeny in one line, T₃ progeny in three lines and T₂ or T₁ progeny in the remaining two fertile lines tested; homozygous transgenic plants were obtained from three lines. In the homozygous lines the expression of the GUS protein, driven by either the B₁- or D-hordein promoters, was highly expressed in endosperm at early to mid-maturation stages. Expression of bar driven by the maize ubiquitin promoter was also stably transmitted to T₁ progeny in seven out of eight lines tested. However, in most lines PAT expression driven by the maize ubiquitin promoter was gradually lost in T₂ or later generations; one homozygous line was obtained. In contrast, six out of seven lines stably expressed GUS driven by the hordein promoters in T₂ or later generations. We conclude that the B₁- and D-hordein promoters can be used to engineer, and subsequently study, stable endosperm-specific gene expression in barley and potentially to modify barley seeds through genetic engineering. Received: 28 May 1998 / Accepted: 19 December 1998     

Stable transformation ofArabidopsis with thebar gene using particle bombardment

A plasmid pARK 22 harbouring thebar gene encoding phosphinothricin acetyltransferase (PAT) under the control of the cauliflower mosaic virus (CaMV) 35S promoter and nopaline synthase (NOS) terminator was constructed and introduced into root sections ofArabidopsis thaliana using the pneumatic particle gun. The root sections that had been bombarded with this plasmid gave four to eight times higher yield of drug-resistant calluses than those sections bombarded with pCaMVNEO or pCH, which respectively contain the neomycin phosphotransferase and hygromycin phosphotransferase genes. Among a number of primary transformant (T₀) plants obtained from independent bialaphos-resistant calluses, three were studied by Southern blot hybridization and PAT enzyme activity analyses, confirming the stable integration of the foreign gene into theArabidopsis genome and its expression in plants. The progeny analysis showed transmission of the foreign gene and its expression in up to the T₂ generation. Some of the T₁ progeny showed morphological abnormalities. Thus, thebar gene can be used effectively to allow selection of transgenicA. thalianna plants.     

Inheritance of gusA and neo genes in transgenic rice

Inheritance of foreign genes neo and gusA in rice (Oryza sativa L. cv. IR54 and Radon) has been investigated in three different primary (T₀) transformants and their progeny plants. T₀ plants were obtained by co-transforming protoplasts from two different rice suspension cultures with the neomycin phosphotransferase II gene [neo or aph (3) II] and the -glucuronidase gene (uidA or gusA) residing on separate chimeric plasmid constructs. The suspension cultures were derived from callus of immature embryos of indica variety IR54 and japonica variety Radon. One transgenic line of Radon (AR2) contained neo driven by the CaMV 35S promoter and gusA driven by the rice actin promoter. A second Radon line (R3) contained neo driven by the CaMV 35S promoter and gusA driven by a promoter of the rice tungro bacilliform virus. The third transgenic line, IR54-1, contained neo driven by the CaMV 35S promoter and gusA driven by the CaMV 35S.Inheritance of the transgenes in progeny of the transgenic rice was investigated by Southern blot analysis and enzyme assays. Southern blot analysis of genomic DNA showed that, regardless of copy numbers of the transgenes in the plant genome and the fact that the two transgenes resided on two different plasmids before transformation, the introduced gusA and neo genes were stably transmitted from one generation to another and co-inherited together in transgenic rice progeny plants derived from self-pollination. Analysis of GUS and NPT II activities in T₁ to T₂ plants provided evidence that inheritance of the gusA and neo genes was in a Mendelian fashion in one plant line (AR2), and in an irregular fashion in the two other plant lines (R3 and IR54-1). Homozygous progeny plants expressing the gusA and neo genes were obtained in the T₂ generation of AR2, but the homozygous state was not found in the other two lines of transgenic rice.     

Molecular analysis of transgene and vector backbone integration into the barley genome following Agrobacterium-mediated transformation

We report a large-scale study on the frequency of transgene and T-DNA backbone integration following Agrobacterium-mediated transformation of immature barley embryos. One hundred and ninety-one plant lines were regenerated after hygromycin selection and visual selection for GFP expression at the callus stage. Southern blotting performed on a subset of 53 lines that were PCR positive for the GFP gene documented the integration of the GFP gene in 27 of the lines. Twenty-three of these lines expressed GFP in T₁ plantlets. Southern blotting with a vector backbone probe revealed that 13 of the 27 lines possessed one or more vector backbone fragments illustrating the regular occurrence of vector backbone integration following Agrobacterium infection of barley immature embryos.     

Expression of GUS in primary transformants and segregation patterns of GUS,TL- and TR-DNA in the T1 generation of hairy root transformants ofLotus corniculatus

Agrobacterium rhizogenes was assessed as a vehicle for transformation ofLotus corniculatus. Plants were co-transformed usingA. rhizogenes strain LBA 9402 harbouring the bacterial plasmid pRi1855 and the binary transformation vector pJit 73. pRi 1855 transfers both T_L and T_R sequences, while pJit 73 encodes β-glucuronidase (GUS) and also two selectable marker genes giving resistance to the antibiotics kanamycin and hygromycin. Three primary transformants (lines 1,6 and 12) were subjected to detailed morphological and biochemical analysis and lines 6 and 12 were also analysed at the molecular level. Tissues of both lines 6 and 12 were resistant to hygromycin and expressed GUS. Analysis of various tissues of each line showed a significantly lower GUS activity in line 6 than in line 12. Genetical analysis of progeny produced between control plants and lines 6 and 12 indicated that line 6 had one dose of theuid gene while line 12 had two or more independently segregating doses of the gene. Both lines 6 and 12 contained multiple copies of T_L-DNA, while only line 6 was T_R positive. In the progeny of lines 6 and 12 there was no evidence for linkage of T_L-DNA withuid, while in the progeny of line 6, T_R-DNA was under-represented. GUS-positive progeny which were free of both T_L and T_R sequences were identified from both lines.     

RNA Interference-Based Transgenic Maize Resistant to Maize Dwarf Mosaic Virus

Maize dwarf mosaic virus (MDMV) is a widespread pathogenic virus that causes serious loss of yield in maize (Zea mays). RNA interference (RNAi) triggered by hairpin RNA (hpRNA) transcribed from a transgenic inverted-repeat sequence is an effective way to defend against viruses in plants. In this study, an hpRNA expression vector containing a sense arm and an antisense arm of 150 bp separated by an intron of the maize actin gene was constructed to target the P1 protein (protease) gene of MDMV and used to transform Agrobacterium tumefaciens strain EHA105. The transformed Agrobacterium strain was used to transform maize embryonic calli isolated from immature embryos by an improved culture technique. In all, 46 plants were regenerated after stringent hygromycin B selection, and 18 of them were certified to be positive by PCR amplification. Of these positive plants, 13 were grown to produce offspring, and nine were identified by Southern blotting to have the transgene integrated with one or two copies. The resistance of three T₂ lines was evaluated in a field trial of dual MDMV inoculation in two environments and was found to be improved compared with the non-transformed control. The disease indexes of the transgenic plant lines h2, 13, and h1 were not significantly different from the highly resistant control line H9-21. The viral titers of the inoculated plants were detected by double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), and the result was in accord with the resistance evaluated in the field trial. The addition of uniconazole S3307 (0.25 mg l⁻¹) and ABT root-promoting powder (0.5 mg l⁻¹) showed a significant improvement of hardening in regenerated plantlets, which were stronger and generated a better fibrous root system than the control. This improvement could facilitate the transgenic operation of maize.     

Inheritance of a co-transferred foreign gene in the progenies of transgenic rice plants

The integration pattern and the inheritance of exogenous DNA in transgenic rice plants were analysed. Plasmid pCH (4.8 kb), that contains chimaeric cauliflower mosaic virus 35S promoter-hygromycin phosphotransferase structural gene, and plasmid pGP400 (7.2 kb), possessing oat phytochrome promoter and structural gene of bacterial -glucuronidase, were co-transferred into protoplasts of rice (Oryza sativa L.) plants via electroporation. Primary transformants (T₀ generation) and their progenies (T₁, T₂ and T₃) were selected by hygromycin B. Southern blot analysis of inserted genes in transgenic rice plants suggests the integration of an intact hygromycin phosphotransferase gene and non-functional DNA fragments into host genome. Co-inheritance of the hygromycin phosphotransferase gene and -glucuronidase gene was also observed. There were no significant differences in terms of the morphology and size of seeds between untransformed and transgenic plants (T₃ generation).     

Efficient <Emphasis Type="Italic">Agrobacterium-</Emphasis>mediated genetic transformation of oilseed mustard [<Emphasis Type="Italic">Brassica juncea</Emphasis> (L.) Czern.] using leaf piece explants

Leaf piece explants of five Brassica juncea (L.) Czern. cultivars were transformed with an Agrobacterium tumefaciens strain EHA105 harboring the plasmid pCAMBIA1301, which contains the β-glucuronidase (uidA) and hygromycin phosphotransferase (hpt) genes under the control of cauliflower mosaic virus 35S (CaMV35S) promoter. Transgenic plants were regenerated on Murashige and Skoog (MS) medium fortified with 8.87 μM 6-benzylaminopurine, 0.22 μM 2,4-dichlorophenoxyacetic acid, and 20 μM silver nitrate in the presence of 30 mg/l hygromycin. When co-culture took place in the presence of 100 μM acetosyringone, the efficiency of stable transformation was found to be approximately 19% in the T ₀ generation, with the transgenic plants and their progeny showing constitutive GUS expression in different plant organs. Southern blot hybridization of uidA and hpt genes confirmed transgene integration within the genome of transformed plants of each cultivar. Inheritance of hpt gene for single copy T-DNA inserts showed a 3:1 pattern of Mendelian segregation in progeny plants through germination of T ₁ seeds on MS medium containing 30 mg/l hygromycin. The protocol described here reports superior transformation efficiency over previously published protocols and should contribute to enhanced biotechnology applications in B. juncea.     

Expression of the cry1EC gene in castor (Ricinus communis L.) confers field resistance to tobacco caterpillar (Spodoptera litura Fabr) and castor semilooper (Achoea janata L.)

Castor (cv. DCS-9) has been transformed through Agrobacterium-mediated and particle gun bombardment methods using appropriate vectors containing the Bt chimeric gene cry1EC driven by enhanced 35S promoter. About 81 and 12 putative transformants were regenerated following selection on hygromycin and kanamycin, respectively. Southern analysis of DNA extracted from T₀ plants confirmed integration of the introduced gene in castor genome. The integration and inheritance of the introduced genes was demonstrated up to T₄ generation by PCR and Southern analysis. Southern analysis of two events having single and two copies showed the same pattern of integration in the subsequent generations. Insect feeding experiments conducted in the laboratory by releasing neonate larvae of castor semilooper and S. litura on leaf tissues excised from transgenic and control plants showed varying degrees of larval mortality and slow growth in larvae fed on transgenic leaf tissue. Field bioassays against Spodoptera litura and castor semilooper conducted for eight events in T₁–T₄ generations under net confinement were more informative and events conferring resistance to the two major defoliators were identified.     

Vacuum infiltration of Petunia hybrida pollen with Agrobacterium tumefaciens to achieve plant transformation

 Genetic transformation of Petunia hybrida with a reporter gene and selectable marker gene (35S-bar) was achieved in similar frequencies by pollinating flowers with pollen vacuum-infiltrated with Agrobacterium tumefaciens or applying a drop of Agrobacterium suspension to the stigma immediately prior to pollination. Nine percent of the T₁, and 5% of the T₂ progeny germinated in nutrient medium with 3 mgl/l Basta^R. Polymerase chain reaction assays indicated that of the Basta^R-resistant plants, 66% of the T₁ plants, and 61% of the T₂ plants harboured the GUS gene. Histochemical assays showed that 10% of the putatively transformed T₁ plants and 5% of their progeny expressed GUS in leaf tissue, pistils and young anthers. Southern hybridization confirmed genomic integration of the bar gene in one to three places in selected T₁ and T₂ progeny. Received: 12 March 1999 / Revision received: 1 October 1999 / Accepted: 20 October 1999     

Comparison of three selectable marker genes for transformation of tall fescue (<Emphasis Type="Italic">Festuca arundinacea</Emphasis> Schreb.) plants by particle bombardment

A variety of selection systems have been developed for transformation of forage crops. To compare the most frequently used systems, we tested three selectable marker genes for their selection efficiency under four selection procedures for the production of transgenic tall fescue. Embryogenic calluses initiated from mature embryos were bombarded with three constructs containing either the phosphinothricin acetyltransferase (bar) gene, the hygromycin phosphotransferase (hpt) gene or the neomycin phosphotransferase II (nptII) gene. Transformation efficiency was strongly influenced by the selectable marker gene, selection procedure and genotype. The highest transformation efficiency was observed using the bar gene in combination with bialaphos. Average transformation efficiencies with bialaphos, phosphinothricin (glufosinate), hygromycin and paromomycin selection across the two callus lines used in the experiments were 9.4%, 4.4%, 5.2% and 1.6%, respectively. Southern blot analysis revealed the independent nature of the tested transgenic plants and a complex transgene integration pattern with multiple insertions.