Agrobacterium-mediated transformation of finger millet (Eleusine coracana (L.) Gaertn.) using shoot apex explants

A new Agrobacterium-mediated transformation system was developed for finger millet using shoot apex explants. The Agrobacterium strain LBA4404 harboring binary vector pCAMBIA1301, which contained hygromycin phosphotransferase (hptII) as selectable marker gene and β-glucuronidase (GUS) as reporter gene, was used for optimization of transformation conditions. Two finger millet genotypes, GPU 45 and CO 14, were used in this study. The optimal conditions for the Agrobacterium-mediated transformation of finger millet were found to be the co-cultivation of explants obtained on the 16th day after callus induction (DACI), exposure of explants for 30 min to agrobacterial inoculum and 3 days of co-cultivation on filter paper placed on medium supplemented with 100 μM acetosyringone (AS). Addition of 100 μM l-cysteine in the selection medium enhanced the frequency of transformation and transgenic plant recovery. Both finger millet genotypes were transformed by Agrobacterium. A frequency of 19% transient expression with 3.8% stable transformation was achieved in genotype GPU 45 using optimal conditions. Five stably transformed plants were fully characterized by Southern blot analysis. A segregation analysis was also performed in four R₁ progenies, which showed normal Mendelian pattern of transgene segregation. The inheritance of transgenes in R₁ progenies was also confirmed by Southern blot analysis. This is the first report on Agrobacterium-mediated transformation of finger millet. This study underpins the introduction of numerous agronomically important genes into the genome of finger millet in the future.     

Factors influencing <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated genetic transformation of <Emphasis Type="Italic">Eleusine coracana</Emphasis> (L.) Gaertn

Agrobacterium-mediated transformation protocol has been developed for Eleusine coracana (var. PR-202) by varying several factors which influence T-DNA delivery. Green nodular regenerative calli with meristematic nodules of seed origin were used as the target tissue for Agrobacterium tumefaciens-mediated gene transfer. The highest frequency of transformation (44.4%) was observed when callus was infected, co-cultivated and incubated at 22°C. Incorporation of higher level of CuSO₄ in the regeneration medium had significantly positive effect on the recovery of transformed plants. PCR analysis of T ₀ and T ₁ generation plants with nptII-specific primers revealed the amplification of nptII gene. Southern blot analysis of six regenerated plants confirmed selectable marker gene integration in three plants. This is a first report on Agrobacterium-mediated genetic transformation of finger millet and will pave the way for further studies in this and other millet crops.     

Enhanced resistance to blast (Magnaporthe grisea) in transgenic Japonica rice by constitutive expression of rice chitinase

Rice blast is the most devastating plant disease in Japan. Our goal is to create new rice varieties which show enhanced resistance against blast, regardless of the race of blast. By an Agrobacterium-mediated transformation method, we reintroduced a rice class-I chitinase gene, Cht-2 or Cht-3, under the control of the enhanced CaMV 35S promoter and a hygromycin phosphotransferase gene, as a selection marker into the Japonica rice varieties Nipponbare and Koshihikari, which have retained the best popularity over a long period in Japan. In regenerated plants (R₀), the Cht-2 product was found to accumulate intracellularly whereas the Cht-3 product was found to be targeted extracellularly. The transgenic rice plants which constitutively expressed either chitinase gene showed significantly higher resistance against the rice blast pathogen Magnaporthe grisea races 007.0 and 333. Both high-level expression of the chitinase and blast-resistance were stably inherited by the next generation in several lines. Received: 16 November 1998 / Accepted: 30 January 1999     

Improved <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation and direct plant regeneration in four cultivars of finger millet (<Emphasis Type="Italic">Eleusine coracana</Emphasis> (L.) Gaertn.)

We have developed an improved Agrobacterium-mediated transformation and rapid regeneration system for four cultivars (‘CO(Ra)-14’, ‘PR-202’, ‘Try-1’ and ‘Paiyur-2’) of finger millet using optimized transformation and direct plant regeneration conditions. The shoot apical meristems (SAMs) were used as explants in this study. Agrobacterium strain EHA105 carrying binary vector pCAMBIA1301 was used to optimize the transformation conditions. Concentration of hygromycin, the optical density of the culture, infection time, age of the explants, co-cultivation period, the concentrations of acetosyringone and antibiotics were optimized to improve the transformation frequency. The highest frequency of mean transient gus expression (85.1%) was achieved in cultivar ‘CO(Ra)-14’. The entire transformation procedure, from initiating SAMs to planting putative transgenic plantlets in the greenhouse, was completed within 45 days with the highest stable transformation frequency of 11.8% for ‘CO(Ra)-14’. PCR, gus staining and Southern blot analyses were performed in T₀ and T₁ generations to confirm the gene integration. Six events from T₀ had a single copy of the transgene and showed a normal Mendelian pattern of segregation. To our knowledge, this is the first report on the high frequency transformation of finger millet by Agrobacterium and subsequent recovery of transgenic plants via direct plant regeneration without a callus phase, in short duration (45 days). The proposed protocol could be supportive in breaking through the bottleneck in transformation and regeneration of finger millet cultivars.     

Over-expression of a cacao class I chitinase gene in Theobroma cacao L. enhances resistance against the pathogen, Colletotrichum gloeosporioides

Theobroma cacao L. plants over-expressing a cacao class I chitinase gene (TcChi1) under the control of a modified CaMV-35S promoter were obtained by Agrobacterium-mediated transformation of somatic embryo cotyledons. Southern blot analysis confirmed insertion of the transgene in eight independent lines. High levels of TcChi1 transgene expression in the transgenic lines were confirmed by northern blot analysis. Chitinase activity levels were measured using an in vitro fluorometric assay. The transgene was expressed at varying levels in the different transgenic lines with up to a sixfold increase of endochitinase activity compared to non-transgenic and transgenic control plants. The in vivo antifungal activity of the transgene against the foliar pathogen Colletotrichum gloeosporioides was evaluated using a cacao leaf disk bioassay. The assay demonstrated that the TcChi1 transgenic cacao leaves significantly inhibited the growth of the fungus and the development of leaf necrosis compared to controls when leaves were wound inoculated with 5,000 spores. These results demonstrate for the first time the utility of the cacao transformation system as a tool for gene functional analysis and the potential utility of the cacao chitinase gene for increasing fungal pathogen resistance in cacao.     

Obtaining the transgenic lines of finger millet Eleusine coracana (L.). with dinitroaniline resistance

The current data is dedicated to the study of bioballistic and Agrobacterium-mediated transformation of finger millet with the constructs carrying the mutant α-tubulin gene (TUAml), isolated from R-biotype goosegrass (Eleusine indica L.), for the decision of problem of dinitroaniline-resistance. It was found that 10 μM of trifluralin is optimal for the selection of transgene plants of finger millet. PCR analysis of transformed lines confirmed the transgenic nature of plants. The analysis of seed of T₁ of transgenic lines confirmed heterozygous character of inheritance of trifluralin resistance.     

Combined expression of chitinase and β-1,3-glucanase genes in indica rice (Oryza sativa L.) enhances resistance against Rhizoctonia solani

Agrobacterium-mediated transformation of rice was done using the binary vector pNSP3, harbouring the rice chitinase (chi11) gene under maize ubiquitin promoter and the tobacco β-1,3-glucanase gene under CaMV 35S promoter in the same T-DNA. Four of the six T₀ plants had single copies of complete T-DNAs, while the other two had complex integration patterns. Three of the four single-copy lines showed a 3:1 segregation ratio in the T₁ generation. Northern and western blot analyses of T₁ plants revealed constitutive expression of chitinase and β-1,3-glucanase genes. Homozygous T₂ plants of the single-copy lines CG20, CG27 and CG53 showed 62-, 9.6- and 11-fold higher chitinase activity over the control plants. β-1,3-Glucanase activity was 1.1- to 2.5-fold higher in the transgenic plants. Bioassay of homozygous T₂ plants of the three single-copy transgenic lines against Rhizoctonia solani revealed a 60% reduction in sheath blight Disease Index in the first week. The Disease Index increased from 61.8 in the first week to 90.6 in the third week in control plants, while it remained low (26.8–34.2) in the transgenic T₃ plants in the corresponding period, reflecting the persistence of sheath blight resistance for a longer period.     

Introduction and constitutive expression of a rice chitinase gene in bread wheat using biolistic bombardment and the bar gene as a selectable marker

 Our long-term goal is to control wheat diseases through the enhancement of host plant resistance. The constitutive expression of plant defense genes to control fungal diseases can be engineered by genetic transformation. Our experimental strategy was to biolistically transform wheat with a vector DNA containing a rice chitinase gene under the control of the CaMV 35 S promoter and the bar gene under control of the ubiquitin promoter as a selectable marker. Immature embryos of wheat cv ‘Bobwhite’ were bombarded with plasmid pAHG11 containing the rice chitinase gene chi11 and the bar gene. The embryos were subcultured on MS2 medium containing the herbicide bialaphos. Calli were then transferred to a regeneration medium, also containing bialaphos. Seventeen herbicide-resistant putative transformants (T₀) were selected after spraying with 0.2% Liberty, of which 16 showed bar gene expression as determined by the phosphinothricin acetyltransferase (PAT) assay. Of the 17 plants, 12 showed the expected 35-kDa rice chitinase as revealed by Western blot analysis. The majority of transgenic plants were morphologically normal and self-fertile. The integration, inheritance and expression of the chi11 and bar genes were confirmed by Southern hybridization, PAT and Western blot analysis of T₀ and T₁ transgenic plants. Mendelian segregation of herbicide resistance was observed in some T₁ progenies. Interestingly, a majority of the T₁ progeny had very little or no chitinase expression even though the chitinase transgene was intact. Because PAT gene expression under control of the ubiquitin promoter was unaffected, we conclude that the CaMV 35 S promoter is selectively inactivated in T₁ transgenic wheat plants. Received: 12 May 1998 / Accepted: 15 May 1998     

Transgenic indica Rice Variety Pusa Basmati 1 Constitutively Expressing a Rice Chitinase Gene Exhibits Enhanced Resistance to Rhizoctonia solani

Agrobacterium-mediated transformation of an elite indica rice variety, Pusa Basmati 1, was performed using LBA4404 (pSB1, pMKU-RF2) that harbours a rice chitinase gene (chi11) under the control of the maize ubiquitin (Ubi1) promoter-intron. Right border (gus) and left border (hph) flanking sequences and the transgene (chi11) in the middle of the T-DNA were used as probes in Southern analysis. Out of eleven independent T0 plants regenerated, three had single copy T-DNA insertions and eight had multiple T-DNA insertions. Nine T₀ plants carried the complete T-DNA with the chitinase transgene. Two T₀ plants did not carry chi11, though they had other T-DNA portions. Three plants harbouring single copy insertions and one plant harbouring two inserted copies were analyzed in detail. A segregation ratio of 3:1, reflecting T-DNA insertion at a single locus, was observed in the progeny of all the four T₀ plants. Northern and western blot analyses of T₁ plants revealed constitutive expression of chitinase at high levels. Bioassays of T₁ plants indicated enhanced resistance to the sheath blight pathogen, Rhizoctonia solani, in comparison to control plants. A homozygous transgenic line was established from one T₀ line, which exhibited the maximum resistance to R. solani.     

Agrobacterium-mediated transformation of indica rice with chitinase gene for enhanced sheath blight resistance

Four rice indica genotypes of local importance were transformed with RC7, rice chitinase cDNA clone through Agrobacterium-mediated gene transfer method using mature seed derived calli as explants. The putative hygromycin resistant calli showed varied level of regeneration efficiency ranging from 2.0 to 7.6 %. The stable integration and expression of RC7 was confirmed through polymerase chain reaction (PCR) and Western analysis. Transformation efficiency ranged from 0.9 to 5.2 %. The expression of RC7 (35 kDa chitinase) in different tissues of transgenic plant (root, sheath and leaf) was proved through Western analysis and in terms of increased chitinase activity. The inheritance of transgene was studied through PCR and Western analysis in transgenic plants of Pusa Basmati 1. Bioassays with transgenic plants of local cultivars exhibited enhanced resistance up to 33.3 % to rice sheath blight pathogen Rhizoctonia solani under glasshouse conditions. Enhanced expression or 3-to 4-fold increased activity of chitinase in transgenic plants was correlated with sheath blight resistance.     

Functional markers based molecular characterization and cloning of resistance gene analogs encoding NBS-LRR disease resistance proteins in finger millet (<Emphasis Type="Italic">Eleusine coracana)</Emphasis>

Magnaporthe grisea, the blast fungus is one of the main pathological threats to finger millet crop worldwide. A systematic search for the blast resistance gene analogs was carried out, using functional molecular markers. Three-fourths of the recognition-dependent disease resistance genes (R-genes) identified in plants encodes nucleotide binding site (NBS) leucine-rich repeat (LRR) proteins. NBS-LRR homologs have only been isolated on a limited scale from Eleusine coracana. Genomic DNA sequences sharing homology with NBS region of resistance gene analogs were isolated and characterized from resistant genotypes of finger millet using PCR based approach with primers designed from conserved regions of NBS domain. Attempts were made to identify molecular markers linked to the resistance gene and to differentiate the resistant bulk from the susceptible bulk. A total of 9 NBS-LRR and 11 EST-SSR markers generated 75.6 and 73.5% polymorphism respectively amongst 73 finger millet genotypes. NBS-5, NBS-9, NBS-3 and EST-SSR-04 markers showed a clear polymorphism which differentiated resistant genotypes from susceptible genotypes. By comparing the banding pattern of different resistant and susceptible genotypes, five DNA amplifications of NBS and EST-SSR primers (NBS-05_504, NBS-09₇₁₁, NBS-07₆₈₈, NBS-03₅₀₉ and EST-SSR-04₂₄₁) were identified as markers for the blast resistance in resistant genotypes. Principal coordinate plot and UPGMA analysis formed similar groups of the genotypes and placed most of the resistant genotypes together showing a high level of genetic relatedness and the susceptible genotypes were placed in different groups on the basis of differential disease score. Our results provided a clue for the cloning of finger millet blast resistance gene analogs which not only facilitate the process of plant breeding but also molecular characterization of blast resistance gene analogs from Eleusine coracana.     

Over Expression of Rice chitinase Gene in Transgenic Peanut (Arachis hypogaea L.) Improves Resistance Against Leaf Spot

A Rice chitinase-3 under enhance version of CaMV 35S was introduced into peanut (Arachis hypogaea L.) through Agrobacterium mediation. Agrobacterium tumefaciens strain LB4404 was used harboring the binary vector (pB1333-EN4-RCG3) containing the chitinase (chit) and hygromycin resistance (hpt) gene as selectable marker. Putative transgenic shoots were regenerated and grown on MS medium supplemented with 5 mg/l BAP, 1 mg/l kinetin, and 30 mg/l hygromycin. Elongated shoots were examined for the presence of the integrated rice chitinase gene along with hygromycin gene as selectable. The integration pattern of transgene in the nuclear genome of the putative transformed plants (T₀) was confirmed through Southern hybridization analysis of the genomic DNA. Survival rate of the in vitro regenerated plantlets was over 60% while healthy putatively transgenic (T₀) plants with over 42% transformation frequency were produced through Agrobacterium mediated gene transfer of the rice chitinase gene and all the plants flowered and set seed normally. T1 plants were tested for resistance against Cercospora arachidicola by infection with the microspores. Transgenic strains exhibited a higher resistance than the control (non-transgenic plants). chitinase gene expression in highly resistant transgenic strains was compared to that of a susceptible control. A good correlation was observed between chitinase activity and fungal pathogen resistance.     

Development of transgenic rice plants expressing maize anthocyanin genes and increased blast resistance

The functional association of flavonoids with plant stress responses, though widely reported in the literature, remains to be documented in rice. Towards this end we chose a transgenic approach with well characterized regulatory and structural genes from maize involved in flavonoid biosynthesis. Activation of anthocyanin pathway in rice was investigated with the maize genes. Production of purple anthocyanin pigments were observed in transformed Tp309 (a japonica rice variety) calluses upon the introduction of the maize regulatory genes C1 (coloured-1), R (red) and the structural gene C2 (coloured-2, encoding chalcone synthase). In addition, stable transgenic plants carrying the maize C2 gene under the control of the maize Ubiquitin promoter were generated. A localized appearance of purple/red pigment in the leaf blade and leaf sheath of R₀ C2 transgenic seedlings was observed. Such a patchy pattern of the transgene expression appears to be conditioned by the genetic background of Tp309, which is homozygous for dominant color inhibitor gene(s) whose presence was unravelled by appropriate genetic crosses. Southern blot analysis of the transgenic plants demonstrated that c2 cDNA was integrated into the genome. Western blot analysis of these primary transgenics revealed the CHS protein while it was not detected in the control untransformed Tp3O9, suggesting that Tp309 might have a mutation at the corresponding C2 locus or that the expression of this gene is suppressed in Tp309. Further analysis of C2 transgenics revealed CHS protein only in three out of sixteen plants that were western-positive in the R₀ generation, suggesting gene silencing. Preliminary screening of these R₁ plants against the rice blast fungus Magnaporthe grisea revealed an increase in resistance.     

Self-fertile transgenic wheat plants regenerated from isolated scutellar tissues following microprojectile bombardment with two distinct gene constructs†

A system for enhanced induction of somatic embryo-genesis and regeneration of plants from isolated scutellar tissue of wheat has been developed. This system has been successfully used in the development of a simple and reproducible protocol for the production of self-fertile transgenic wheat plants. The procedure is rapid resulting in the production of transgenic plantlets within 12 weeks from initiation of cultures and it avoids the need for establishing long-term callus, cell suspension or protoplast cultures. Somatic embryos regenerated from scutella bombarded with plasmid pBARGUS were selected on L-phosphinothricin (L-PPT) to obtain herbicide-resistant self-fertile transgenic plants. Phosphinothricin acetyltransferase (PAT) activity was observed at varying levels in 50% of the plants selected on L-PPT whereas none of the plants showed β-glucuronidase (GUS) activity. Molecular analysis of PAT-positive plants confirmed stable integration of both bar and gus genes in R₀ and R₁ progeny plants. Segregation of the PAT activity and herbicide resistance in R₁ progeny plants confirmed the Mendelian inheritance of the bar gene. Additionally, isolated scutella bombarded with plasmid DNA containing a gus::nptII fusion gene driven by a rice actin promoter and its first intron were selected in the presence of geneticin to obtain fully fertile transgenic plants. Functional expression of the fusion gene was demonstrated in transgenic plants by GUS and neomycin phospho-transferase (NPTII) enzyme assays. Southern blot analysis confirmed the integration of transgenes into the wheat genome. Histochemical GUS staining showed transmission of the fusion gene to floral organs of primary transformants and confirmed Mendelian segregation of the transgene in R₁ progeny.     

Effective selection and regeneration of transgenic rice plants with mannose as selective agent

A new method for the selection of transgenic rice plants without the use of antibiotics or herbicides has been developed. The phosphomannose isomerase (PMI) gene from Escherichia coli has been cloned and consitutively expressed in japonica rice variety TP 309. The PMI gene was transferred to immature rice embryos by Agrobacterium-mediated transformation, which allowed the selection of transgenic plants with mannose as selective agent. The integration and expression of the transgene was confirmed by Southern and northern blot analysis and the activity of PMI indirectly proved with the chlorophenol red assay. The results of genetic analysis showed that the transgenes were segregated in a Mendelian fashion in the T₁ generation. The establishment of this selection system in rice provides an efficient way for producing transgenic plants without using antibiotics or herbicides with a transformation frequency of up to 41%.     

Transgenic Lines of Oryza Sativa (Basmati 2000) Expressing Xa21 Demonstrate Enhanced Resistance to Bacterial Blight

An efficient transformation system for rice was established by co-cultivating calli, derived from 21-day-old scutellum, with Agrobacterium tumefaciens cells (OD600 = 0.04), maintained on filter paper, moistened with 4 mL of a medium, and supplemented with 400 μM acetosyringone, for a period of 2 days. Presence of the transgene was confirmed by polymerase chain reaction, and stable integration and copy number of the transgene were determined by Southern blot analysis. Among seven plants analyzed, six possessed single T-DNA integration events, while one plant was found to have two integrated copies of the T-DNA. A total of 45 T₀ plants were grown in the greenhouse to obtain the T₁ generation. T₁ plants evaluated for presence of the transgene and for response to inoculation with the bacterial leaf blight pathogen, Xanthomonas oryzae pv. Oryzae, exhibited Mendelian segregation (3:1) for the transgene as well as enhanced resistance to bacterial blight     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated creeping bentgrass (<Emphasis Type="Italic">Agrostis stolonifera</Emphasis> L.) transformation using phosphinothricin selection results in a high frequency of single-copy transgene integration

Genetic transformation of creeping bentgrass mediated by Agrobacterium tumefaciens has been achieved. Embryogenic callus initiated from seeds (cv. Penn-A-4) was infected with an A. tumefaciens strain (LBA4404) harboring a super-binary vector that contained an herbicide-resistant bar gene driven either by the CaMV 35S promoter or a rice ubiquitin promoter. Plants were regenerated from 219 independent transformation events. The overall stable transformation efficiency ranged from 18% to 45%. Southern blot and genetic analysis confirmed transgene integration in the creeping bentgrass genome and normal transmission and stable expression of the transgene in the T₁ generation. All independent transformation events carried one to three copies of the transgene, and a majority (60–65%) contained only a single copy of the foreign gene with no apparent rearrangements. We report here the successful use of Agrobacterium for the large-scale production of transgenic creeping bentgrass plants with a high frequency of a single-copy transgene insertion that exhibit stable inheritance patterns.Abbreviations 2,4-D: 2,4-Dichlorophenoxyacetic acid - bar: Bialaphos resistance gene - GUS: -Glucuronidase - PPT: Phosphinothricin - ubi: Ubiquitin Communicated by J.M. Widholm     

Transgenic rice plants expressing the snowdrop lectin gene (<Emphasis Type="Italic">gna</Emphasis>) exhibit high-level resistance to the whitebacked planthopper (<Emphasis Type="Italic">Sogatella furcifera</Emphasis>)

Transgenic rice plants, expressing snowdrop lectin [Galanthus nivalis agglutinin (GNA)], obtained by Agrobacterium-mediated genetic transformation, were evaluated for resistance against the insect, the whitebacked planthopper (WBPH). The transgene gna was driven by the phloem-specific, rice-sucrose synthase promoter RSs1, and the bar was driven by the CaMV 35S promoter. In our previous study, the transgenic status of these lines was confirmed by Southern, Northern and Western blot analyses. Both the transgenes, gna and bar, were stably inherited and co-segregated into progenies in T₁ to T₅ generations. Insect bioassays on transgenic plants revealed the potent entomotoxic effects of GNA on the WBPH. Also, significant decreases were observed in the survival, development and fecundity of the insects fed on transgenic plants. Furthermore, intact GNA was detected in the total proteins of WBPHs fed on these plants. Western blot analysis revealed stable and consistent expression of GNA throughout the growth and development of transgenic plants. Transgenic lines expressing GNA exhibited high-level resistance against the WBPH. As reported earlier, these transgenics also showed substantial resistance against the brown planthopper and green leafhopper .     

Transgenic tomato plants expressing a wheat endochitinase gene demonstrate enhanced resistance to <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">lycopersici</Emphasis>

Various chitinases have been shown to inhibit the growth of fungal pathogens in in vitro as well as in planta conditions. chi194, a wheat chitinases gene encoding a 33-kDa chitinase protein, was overexpressed in tomato plants (cv. Pusa Ruby) under the control of maize ubiquitin 1 promoter. The integration of transgene in tomato plants was confirmed with polymerase chain reaction (PCR) and Southern blot analysis. The inheritance of the transgene in T₁ and T₂ generations were shown by molecular analysis and the hygromycin sensitivity test. The broad range of chitinase activity was observed among the transgenic lines in T₀ and a similar range was retained in the T₁ and T₂ generations. Most importantly, the transgenic tomato lines with high chitinase activity were found to be highly resistant to the fungal pathogen Fusarium oxysporum f. sp. lycopersici. Thus, the results demonstrated that the expression of the wheat endochitinase chi194 in tomato plants confers resistance against Fusarium wilt disease caused by the fungal pathogen Fusarium oxysporum f. sp. lycopersici.