Cointegrate Vector Research Articles

Improved Agrobacterium-mediated co-transformation and selectable marker elimination in transgenic rice by using a high copy number pBin19-derived binary vector

A high copy number, selectable marker gene (SMG)-free Agrobacterium binary vector pBin19Δ nptII was constructed by deleting the nptII gene from pBin19. The binary vectors with the RK2 and pVS replication origins exist in 12 and 3 copies, respectively, in Agrobacterium. The tobacco osmotin gene ( ap24) was cloned in pBin19Δ nptII and the resultant plasmid pBin19Δ nptII- ap24 was mobilized into the Agrobacterium tumefaciens strain C58C1 Rif r harbouring the single-copy cointegrate vector pGV2260::pSSJ1. The T-DNA of the cointegrate vector harboured the hph (SMG) and gus genes. Transformation of Oryza sativa L. var. Pusa Basmati1 with Agrobacterium tumefaciens (pGV2260::pSSJ1, pBin19Δ nptII- ap24) yielded 14 independent hyg +/GUS + transgenic plants. Southern blot analysis with hph and ap24 probes revealed that 12 out of the 14 transgenic plants were co-transformed and harboured hph, gus and ap24 genes. The new multi-copy binary vector yielded 86% co-transformation efficiency. SMG elimination by genetic separation of the cointegrate T-DNA with the hph/ gus genes and binary vector T-DNA with the ap24 gene was accomplished in four out of ten primary co-transformants that were forwarded to the T 1 generation.

Transgene stacking and marker elimination in transgenic rice by sequential Agrobacterium-mediated co-transformation with the same selectable marker gene

Rice chitinase (chi11) and tobacco osmotin (ap24) genes, which cause disruption of fungal cell wall and cell membrane, respectively, were stacked in transgenic rice to develop resistance against the sheath blight disease. The homozygous marker-free transgenic rice line CoT23 which harboured the rice chi11 transgene was sequentially re-transformed with a second transgene ap24 by co-transformation using an Agrobacterium tumefaciens strain harbouring a single-copy cointegrate vector pGV2260::pSSJ1 and a multi-copy binary vector pBin19∆nptII-ap24 in the same cell. pGV2260::pSSJ1 T-DNA carried the hygromycin phosphotransferase (hph) and β-glucuronidase (gus) genes. pBin19∆nptII-ap24 T-DNA harboured the tobacco osmotin (ap24) gene. Co-transformation of the gene of interest (ap24) with the selectable marker gene (SMG, hph) occurred in 12 out of 18 T(0) plants (67%). Segregation of hph from ap24 was accomplished in the T(1) generation in one (line 11) of the four analysed co-transformed plants. The presence of ap24 and chi11 transgenes and the absence of the hph gene in the SMG-eliminated T(1) plants of the line 11 were confirmed by DNA blot analyses. The SMG-free transgenic plants of the line 11 harboured a single copy of the ap24 gene. Homozygous, SMG-free T(2) plants of the transgenic line 11 harboured stacked transgenes, chi11 and ap24. Northern blot analysis of the SMG-free plants revealed constitutive expression of chi11 and ap24. The transgenic plants with stacked transgenes displayed high levels of resistance against Rhizoctonia solani. Thus, we demonstrate the development of transgene-stacked and marker-free transgenic rice by sequential Agrobacterium-mediated co-transformation with the same SMG.

Generation of selectable marker-free sheath blight resistant transgenic rice plants by efficient co-transformation of a cointegrate vector T-DNA and a binary vector T-DNA in one Agrobacterium tumefaciens strain

Co-transformation of Oryza sativa L. var. Pusa Basmati1 was done using an Agrobacterium tumefaciens strain harbouring a single-copy cointegrate vector and a multi-copy binary vector in the same cell. The T-DNA of the cointegrate vector pGV2260::pSSJ1 carried the hygromycin phosphotransferase (hph) and beta-glucuronidase (gus) genes. The binary vector pCam-chi11, without a plant selectable marker gene, harboured the rice chitinase (chi11) gene under maize ubiquitin promoter. Co-transformation of the gene of interest (chi11) with the selectable marker gene (hph) occurred in 4 out of 20 T(0) plants (20%). Segregation of hph from chi11 was accomplished in two (CoT6 and CoT23) of the four co-transformed plants in the T(1) generation. The selectable marker-free (SMF) lines CoT6 and CoT23 harboured single copies of chi11. Homozygous SMF T(2) plants were established in the lines CoT6 and CoT23. Northern and Western blot analysis of the homozygous SMF lines showed high level of transgene expression. In comparison to untransformed controls, chitinase specific activity was 66- and 22-fold higher in the homozygous SMF T(2) plants of lines CoT6 and CoT23, respectively. The lines CoT6 and CoT23 exhibited 38 and 40% reduction in sheath blight disease, respectively.

Development of Indica Basmati rice harboring two insecticidal genes for sustainable resistance against lepidopteran insects

Rice variety Basmati 370 was transformed with a co-integrate vector pSM6 carrying cry1Ac and cry2A genes. The hygromycin resistance gene on a separate vector, pROB5 was used as a selectable marker. One to two week old scutellum derived calli were transformed with a biolistic gun. Transgenic status of different transformed lines was evaluated by molecular analysis (PCR, Dot blot, Southern blot, Western blot and ELISA) and insect feeding assays with rice leaf folder (RLF) and yellow stem borer (YSB). An average transformation efficiency of 1.98% was observed in a set of experiments. Plants from five different transgenic lines exhibited 100% mortality against rice leaf folder and segregated in Mendelian fashion. Insect bioassays revealed that there is no synergistic or antagonistic effect of cry1Ac and cry2A genes on the development of resistance against RFL and YSB.

Mungbean yellow mosaic virus-Vi Agroinfection by Codelivery of DNA A and DNA B From One Agrobacterium Strain.

Agroinfection of bipartite geminiviruses is routinely done by mixing two Agrobacterium strains that independently harbor partial tandem repeats of DNA A and DNA B. We report here an improved agroinfection method for bipartite geminiviruses that utilizes one strain of Agrobacterium that harbors DNA A and DNA B partial tandem repeats on two compatible replicons. A cointegrate vector, pGV2260∷pGV1.3A, with the partial tandem repeat of Mungbean yellow mosaic virus-Vi (MYMV-Vi) DNA A and a binary vector, pGA1.9B, with the partial tandem repeat of MYMV-Vi DNA B gave an agroinfection efficiency of 24% when harbored in two Agrobacterium strains and an efficiency of 61% when harbored in one Agrobacterium strain. A combination of binary vectors, pGA1.9A with MYMV-Vi DNA A partial tandem repeat and pGA1.9B with DNA B partial tandem repeat, gave an agroinfection efficiency of 74% when harbored in two strains. But pGA1.9A and pPZP1.9B (a partial tandem repeat of DNA B), when present in the same Agrobacterium strain, gave 100% agroinfection. Accumulation of viral DNA was shown by Southern blotting. The single-strain method using two compatible replicons consistently gave 100% agroinfection efficiency.

A Cointegrate Ti Plasmid Vector for Agrobacterium tumefaciens - mediated Transformation of indica Rice cv Pusa Basmati 1

An intermediate vector pSSJ1 was constructed by cloning a hph gene and a gus gene with catalase intron in pGV1500. pSSJ1 was cointegrated into a disarmed receptor Ti plasmid pGV2260 harboured in Agrobacterium tumefaciens strain C58C1RifR. The resulting A. tumefaciens strain C58C1RifR (pGV2260::pSSJ1) stably transformed Oryza sativa L. cv Pusa Basmati 1 scutellum-derived calli at 26% frequency. Introduction of the plasmid pSSJ3 (3′virB, virG and virC of pTiB0542) into A. tumefaciens C58C1RifR (pGV2260::pSSJ1) resulted in the elevation of acetosyringone-induced T -strand accumulation. Rice transformation efficiency of the cointegrate plasmid pGV2260::pSSJ1 increased from 26% to 33% in the presence of pSSJ3 and from 26% to 35% in the presence of pToK47 (complete virB, virG and virC). T-DNA integration in To plants was confirmed by Southern hybridization analysis. Inheritance analysis of the T0 plants with single-copy T-DNA insertions revealed segregation of hygromycin resistance in 3:1 ratio. The feasibility of rice transformation with a cointegrate Ti plasmid vector is clearly established.

Generation of selection marker-free transgenic plants by cotransformation of a cointegrate vector T-DNA and a binary vector T-DNA in one Agrobacterium tumefaciens strain

Cotransformation of Nicotiana tabacum was done using a single Agrobacterium tumefaciens strain harbouring a cointegrate vector (one copy per cell) and a binary vector (ten to 15 copies per cell). The T-DNAs of the cointegrate plasmid, pGV2260::pSSJ1 and the binary plasmid, pGA472 carried hph and nptII as plant selection markers, respectively. When the binary T-DNA marker ( nptII) was used for selection, the non-selected cointegrate T-DNA with hph cotransformed at 36% frequency. However, upon using the cointegrate T-DNA with hph for selection, cotransformation of binary T-DNA with nptII was much higher (56–74%). Segregation of the T-DNA markers hph and nptII was followed in the T 1 generation to screen for the elimination of the selection marker. The elimination of nptII of the multicopy binary vector was found in the progeny of only one of the three T 0 plants and at a low frequency of 3%. However, elimination of hph of the single-copy cointegrate vector was found at 16–18% frequency in the progeny of both the T 0 plants analysed. The use of the T-DNA of low copy number cointegrate vector for initial selection improves the cotransformation frequency of non-selected T-DNA of the multicopy binary vector. This strategy also increases the frequency of generation of selection marker-free transgenic plants in the T 1 generation.

Transgenic rice as a system to study the stability of transgene expression: multiple heterologous transgenes show similar behaviour in diverse genetic backgrounds

The success of contemporary breeding programmes involving genetic engineering depends on the stability of transgene expression over many generations. We studied the stability of transgene expression in 40 independent rice plant lines representing 11 diverse cultivated varieties. Each line contained three or four different transgenes delivered by particle bombardment, either by cotransformation or in the form of a cointegrate vector. Approximately 75% of the lines (29/40) demonstrated Mendelian inheritance of all transgenes, suggesting integration at a single locus. We found that levels of transgene expression varied among different lines, but primary transformants showing high-level expression of the gna, gusA, hpt and bar transgenes faithfully transmitted these traits to progeny. Furthermore, we found that cry1Ac and cry2A transgene expression was stably inherited when primary transformants showed moderate or low-level expression. Our results show that six transgenes (three markers and three insect-resistance genes) were stably expressed over four generations of transgenic rice plants. We showed that transgene expression was stable in lines of all the rice genotypes we analysed. Our data represent a step forward in the transfer of rice genetic engineering technology from model varieties to elite breeding lines grown in different parts of the world.

Particle-bombardment-mediated co-transformation of elite Chinese rice cultivars with genes conferring resistance to bacterial blight and sap-sucking insect pests

Transgenic rice plants were generated using particle bombardment to simultaneously introduce the rice Xa21 gene eAective against bacterial blight and the Galanthus nivalis agglutinin (snowdrop lectin; gna) gene eAective against sap-sucking insect pests, specifically the brown plant hopper. Using three plasmids, we co- transformed 5- to 10-d-old, mature seed-derived rice (Oryza sativa L.) callus of two elite Chinese rice cultivars, Eyi 105 and Ewan 5. The plasmids carried a total of four genes. The gna and Xa21 genes were carried on separate plasmids. The selectable marker hygromycin phosphotransferase (hpt) and the reporter gene b-glucu- ronidase (gusA) were linked on the same, co-integrate vector. We recovered over 160 independently derived transgenic rice plants. Over 70% of the transgenic plants carried all four genes, as confirmed by polymerase chain reaction and/or Southern blot analysis. Furthermore, 70% of transgenic plants carrying all four genes also co- expressed all four genes, as confirmed by growth on selection media (hpt), GUS histochemical assays (gusA), western blotting (gna) and reverse transcriptase-poly- merase chain reaction (Xa21) analysis. The co-expres- sion eAciency reported for the four transgenes in our study is the highest ever found in any transgenic plant population generated through co-transformation. The linked genes (hpt and gusA) co-integrated with a frequency of near 100%, and we observed a co-integra- tion frequency greater than 70% for the genes carried on separate plasmids. We observed no preferential integra- tion of any particular gene(s). Genetic analysis con- firmed Mendelian segregation of the transgenes in subsequent generations. We report, for the first time, generation and analysis of transgenic rice lines carrying genes eAective against more than one taxa of pathogen or pest, substantiating that particle bombardment rep- resents an eAective way to introduce unlinked complex multiple traits into plants.

Multiple traits of agronomic importance in transgenic indica rice plants: analysis of transgene integration patterns, expression levels and stability

We cotransformed indica rice (Oryza sativa L. cvs. Basmati 370 and M7) with three plasmids, carrying a total of four genes, by particle bombardment. The Bacillus thuringiensis (Bt) δ-endotoxin genes cry1Ac and cry2A were carried on separate vectors, while the gna (snowdrop lectin) and hpt (hygromycin phosphotransferase) genes were linked on the same, cointegrate vector. We evaluated the molecular and expression profiles of 29 independently derived transgenic lines over two generations. The gna and hpt genes cointegrated with a frequency of 100% as expected. Furthermore, 60% of the transgenic plants carried all four genes. Southern blot analysis showed that transgene copy number ranged from 1 to 15. We used western blots to determine protein expression levels in R0 and R1 plants. We observed wide variation in the expression levels of the nonselected transgenes among independently-derived lines, but expression levels within lines derived from the same clone were similar. Consistent with previous reports, we observed no correlation between transgene copy number and the level or stability of protein expression. We show that the introduction of multiple agronomically valuable genes into the rice genome by cotransformation is a practical strategy for engineering elite rice varieties.

Molecular Characteristics of Transgenic Wheat and the Effect on Transgene Expression

A population of R0 transgenic wheat plants, generated by particle bombardment, was analyzed to define molecular, genetic and phenotypic properties resulting from transformation with a cointegrate vector, or cotransformation with two separate plasmids. By evaluating the progeny of 70 independently-derived transgenic plants, we also identified rare events such as chimerism and transgene elimination, which provide valuable information concerning the development of transgenic cereal plants following bombardment experiments. The frequency of chimerism in our transgenic wheat plants was very low. Furthermore, while transgene elimination did occur, this was also a very rare event. We determined the copy numbers of integrated transgenes and the levels of transgene expression. Comparisons to transgenic rice plants generated in the same manner demonstrated some similarities, but also important differences in transgene behavior. Whereas in rice there is no evidence for any direct relationship between transgene copy number and transgene expression or stability, multicopy populations in wheat demonstrated a bias towards higher levels of expression for the two genes and the maize ubiquitin promoter evaluated in the present study.

Agrobacterium-mediated transformation of five wild Solanum species using in vitro microtubers

A genetic transformation method, using in vitro microtubers and Agrobacterium-mediated transformation has been developed for five wild Solanum species: S. verrucosum, S. hjertingii, S. papita, S. stoloniferum, S. demissum, which range in ploidy from diploid to hexaploid. A disarmed A. tumefaciens strain, C58 harbouring the co-integrate vector pGV3580::pKU2 with the genes of neomycin phosphotransferase (NPTII) and hygromycin phosphotransferase (HPTII) as selectable markers, was used. Selection of putative transformants was based on their ability to grow and produce roots on a medium containing 150 mg/l kanamycin. The transgenic nature of the putative transformants was confirmed by Polymerase Chain Reaction analysis and by NPTII dotblot assay to show the expression of the NPTII gene. Additionally, the transmission of transgenes, NPTII and HPTII in selfed-sexual progeny of some transgenic plants was also determined.

Transformation of passionfruit (Passiflora edulis fv flavicarpa Degener.) using Agrobacterium tumefaciens

Leaf and stem explants of passionfruit (Passiflora eadulis fv flavicarpa) were co-cultivated with a disarmed strain of Agrobacterium tunefaciens harbouring the co-integrate vector pMON200. Four plants of passionfruit were regenerated from leaf explants on agar-solidified Murashige and Skoog (1962) based medium containing 4.43 μM 6-benzyl-aminopurine and supplemented with 86 μM kanamycin sulphate. The four plants were rooted by transfer to MS based medium with 14.7 μM 3-indolebutyric acid and 2.68 μM α-naphthyleneacetic acid for 7 d, followed by MS based medium lacking growth regulators. Both media used for rooting contained 172 μM kanamycin sulphate. Rooted plants were potted and grown to maturity. Three of the plants synthesised nopaline and expressed neomycin phosphotransferase activity; DNA dot blot and polymerase chain reaction analyses confirmed the presence of the neomycin phosphotransferase gene in three plants.

Celery transformation by Agrobacterium tumefaciens: cytological and genetic analysis of transgenic plants

Transgenic celery plants were obtained following co-cultivation of petiole explants with Agrobacterlum tumefaciens containing pMON200, a cointegrate vector carrying genes for kanamycin resistance and nopaline synthase. Transformants were selected by ability of callus to grow in the presence of 50mg/l kanamycin. Transformation was confirmed either by the presence of nopaline or by Southern blots. Cytological analysis of 14 transformed plants revealed chromosomal aberrations, both in structure and number. Only 20% of the regenerated plants had the normal karyotype. Kanamycin resistance behaved as a monogenic, dominant trait, segregating in a 3:1 ratio in three families derived from plants with normal karyotypes.