Development of transgenic finger millet (<Emphasis Type="Italic">Eleusine coracana</Emphasis> (L.) Gaertn.) resistant to leaf blast disease

Finger millet plants conferring resistance to leaf blast disease have been developed by inserting a rice chitinase (chi11) gene through Agrobacterium-mediated transformation. Plasmid pHyg-Chi.11 harbouring the rice chitinase gene under the control of maize ubiquitin promoter was introduced into finger millet using Agrobacterium strain LBA4404 (pSB1). Transformed plants were selected and regenerated on hygromycin-supplemented medium. Transient expression of transgene was confirmed by GUS histochemical staining. The incorporation of rice chitinase gene in R₀ and R₁ progenies was confirmed by PCR and Southern blot analyses. Expression of chitinase gene in finger millet was confirmed by Western blot analysis with a barley chitinase antibody. A leaf blast assay was also performed by challenging the transgenic plants with spores of Pyricularia grisea. The frequency of transient expression was 16.3% to 19.3%. Stable frequency was 3.5% to 3.9%. Southern blot analysis confirmed the integration of 3.1 kb chitinase gene. Western blot analysis detected the presence of 35 kDa chitinase enzyme. Chitinase activity ranged from 19.4 to 24.8. In segregation analysis, the transgenic R₁ lines produced three resistant and one sensitive for hygromycin, confirming the normal Mendelian pattern of transgene segregation. Transgenic plants showed high level of resistance to leaf blast disease compared to control plants. This is the first study reporting the introduction of rice chitinase gene into finger millet for leaf blast 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.     

Novel insect resistance in Brassica napus developed by transformation of chitinase and scorpion toxin genes

Transgenic plants with introduced pest-resistant gene offer an efficient alternative insect control. The novel insect-resistant gene combination, chitinase(chi) and BmkIT(Bmk), containing an insect-specific chitinase gene and a scorpion insect toxin gene was introduced into Brassica napus cultivar via Agrobacterium-mediated transformation. Fifty-seven regenerated plantlets with kanamycin-resistance were obtained. Transgenic plants were verified by Southern blot analysis. Enzyme-linked immunosorbent assay (ELISA) and bioassay of artificial inoculation with diamondback moth (Plutella maculipenis) (DBM) larvae indicated that some of the transgenic plants were high-level expression for both chitinase and scorpion toxin proteins and performed high resistance against the tested pest infestation. The genetic analysis of T₁ progeny confirmed that the inheritance of introduced genes followed the Mendelian rules.     

Agrobacterium-mediated transformation of kabocha squash (Cucurbita moschata Duch) induced by wounding with aluminum borate whiskers

An efficient genetic transformation method for kabocha squash (Cucurbita moschata Duch cv. Heiankogiku) was established by wounding cotyledonary node explants with aluminum borate whiskers prior to inoculation with Agrobacterium. Adventitious shoots were induced from only the proximal regions of the cotyledonary nodes and were most efficiently induced on Murashige–Skoog agar medium with 1 mg/L benzyladenine. Vortexing with 1% (w/v) aluminum borate whiskers significantly increased Agrobacterium infection efficiency in the proximal region of the explants. Transgenic plants were screened at the T₀ generation by sGFP fluorescence, genomic PCR, and Southern blot analyses. These transgenic plants grew normally and T₁ seeds were obtained. We confirmed stable integration of the transgene and its inheritance in T₁ generation plants by sGFP fluorescence and genomic PCR analyses. The average transgenic efficiency for producing kabocha squashes with our method was about 2.7%, a value sufficient for practical use.     

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 T₀ plants (67%). Segregation of hph from ap24 was accomplished in the T₁ 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 T₁ 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 T₂ 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.     

The effect of co-cultivation and selection parameters on <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of Chinese soybean varieties

In the present study, an efficient Agrobacterium-mediated gene transformation system was developed for soybean [Glycine max (L.) Merrill] based on the examinations of several factors affecting plant transformation efficiency. Increased transformation efficiencies were obtained when the soybean cotyledonary node were inoculated with the Agrobacterium inoculum added with 0.02% (v/v) surfactant (Silwet L-77). The applications of Silwet L-77 (0.02%) during infection and l-cysteine (600 mg l⁻¹) during co-cultivation resulted in more significantly improved transformation efficiency than each of the two factors alone. The optimized temperature for infected explant co-cultivation was 22°C. Regenerated transgenic shoots were selected and produced more efficiently with the modified selection scheme (initiation on shoot induction medium without hygromycin for 7 days, with 3 mg l⁻¹ hygromycin for 10 days, 5 mg l⁻¹ hygromycin for another 10 days, and elongation on shoot elongation medium with 8 mg l⁻¹ hygromycin). Using the optimized system, we obtained 145 morphologically normal and fertile independent transgenic plants in five important Chinese soybean varieties. The transformation efficacies ranged from 3.8 to 11.7%. Stable integration, expression and inheritance of the transgenes were confirmed by molecular and genetic analysis. T₁ plants were analyzed and transmission of transgenes to the T₁ generation in a Mendelian fashion was verified. This optimized transformation system should be employed for efficient Agrobacterium-mediated soybean gene transformation.     

OBPC Symposium: Maize 2004 &; beyond—Plant regeneration,gene discovery,and genetic engineering of plants for crop improvement

Summary The development of robust plant regeneration technology in cereals, dicots and ornamentals that is in turn coupled to a high-frequency DNA transfer technology is reported. Transgenic cereals that include maize, Tripsacum, sorghum, Festuca and Lolium, in addition to dicots that include soybean, cotton and various ornamentals such as petunia, begonia, and geranium have been produced following either somatic embryogenesis or direct organogenesis independent of genotype. Coupled with these regeneration protocols, we have also identified several interesting genes and promoters for incorporation into various crops and ornamentals. In addition, the phenomenon of direct in vitro flowering from cotyledonary nodes in soybean is described. In in vitro flowering, the formation of a plant body is suppressed and the cells of the cotyledonary node produce complete flowers from which fertile seed is recovered. This in vitro flowering technology serves as a complementary tool to chloroplast transformation for developing a new transgenic pollen containment strategy for crop species. Recently, the center has undertaken to screen the expression response of the 24 000 Arabidopsis genes to nitric oxide. This signaling molecule upregulated 342 genes and downregulated 80 genes. The object here was to identify a population of promoters that can be manipulated by using a signaling molecule. In addition, in keeping with the mission of enhancing greenhouse profitability for North West Ohio growers, we cloned a number of genes responsive for disease resistance from ornamentals that play an important role in disease management and abiotic stress. We have constructed a plant transformation vector with CBF3 gene under the rd29A promoter for engineering cold and freezing tolerance in petunia. Leaf dises of Petunia×hybrida v26 were used for Agrobacterium-mediated transformation, and 44 hygromycin-resistant T0 plants were obtained. The presence of CBF3 gene was confirmed in all the transgenic plants by PCR and Southern analyses.     

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.     

Susceptibility to Agrobacterium tumefaciens and cotyledonary node transformation in short-season soybean

 Short-season adapted soybean [Glycine max (L.) Merrill] genotypes (maturity group 0 and 00) were susceptible to Agrobacterium tumefaciens in tumor-formation assays with A. tumefaciens strains A281, C58 and ACH5. The response was bacterial-strain and plant-cultivar dependent. In vitro Agrobacterium-mediated transformation of cotyledonary node explants of these genotypes with A. tumefaciens EHA105/pBI121 was inefficient but resulted in a transgenic AC Colibri plant carrying a linked insertion of the neomycin phosphotransferase and β-glucuronidase (gus) transgenes. The transgenes were transmitted to the progeny and stable gus expression was detected in the T₇ generation. The low rate of recovery of transgenic plants from the co-cultured cotyledonary explants was attributed to inefficient transformation of regenerable cells, and/or poor selection or survival of such cells and not to poor susceptibility to Agrobacterium, since, depending on the cultivar, explants were transformed at a rate of 27–92%, but transformation events were usually restricted to non-regenerable callus. Received: 8 January 1998 / Revision received: 30 June 1999 / Accepted: 12 July 1999     

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

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

Introduction and constitutive expression of a tobacco hornworm (<Emphasis Type="Italic">Manduca sexta</Emphasis>) chitinase gene in soybean

Summary Embryogenic soybean [Glycine max (L.) Merrill] cultures were transformed with a Manduca sexta chitinase (msc) gene using microprojectile bombardment. A 1.7 kb DNA fragment encoding a tobacco hornworm chitinase was cloned into the rice transformation vector pGL2, under the control of the maize ubiquitin promoter and linked to the hpt gene as a selectable marker. After bombardment, hygromycin-resistant tissues were isolated and cultured to give rise to clones of transgenic material. Four hygromycin-resistant clones were converted into plants. Two clones were positive for the msc gene via polymerase chain reaction (PCR) and Southern blot analysis. The integration inheritance, and expression of transgenes were confirmed by molecular analysis of transgenic soybean plants. Progeny analysis showed that the introduced genes were inherited and segregated in a 3:1 Mendelian fashion. DNA blot experiments and progeny inheritance analysis indicated that the plants contained several copies of the msc gene and that the insertion occurred at a single locus. Northern blotting analysis confirmed the expression of the transgenes. Western blot analysis of transgenic plants and their progeny revealed the presence of a protein with a molecular weight of 48kDa that reacted with the Manduca sexta antibody. Progeny from the chitinase-positive plants were tested for their resistance to the soybean cyst nematode. Plants expressing the insect chitinase did not manifest enhanced resistance to the soybean cyst nematode.     

Production of transgenic soybean lines expressing the bean pod mottle virus coat protein precursor gene

Soybean (Glycine max. Merrill. cv. Fayette) cotyledonary nodes were transformed with bean pod mottle virus (BPMV) coat protein precursor (CP-P) gene via Agrobacterium-mediated transformation. The transformation rate was low, and only five primary transformants derived from five different cotyledons were obtained from 400 original cotyledons. Southern blot hybridization verified the integration of the BPMV CP-P gene. Inheritance and expression of this gene in R₁ plants were also demonstrated. About 30% of R₂ plants derived from one transgenic line showed complete resistance to BPMV infection, as assessed by symptomatology and ELISA, suggesting that homozygous, but not hemizygous, plants exhibit the resistant phenotype.Abbreviations BAP 6-benzyladenine phosphate - BPMV bean pod mottle virus - CP-P coat protein-precursor - CTAB hexadecyltrimethylammonium bromide - DAS-ELISA double antibody sandwich-enzyme-linked immunosorbent assay - IBA indole-butyric acid - kbp kilobase pairs - MES 2-(N-Morpholino)ethanesulfonic acid - NOS nopaline synthase - NPTII neomycin phosphotransferase II - NTP nucleoside triphosphate - PBS phosphate-buffered saline - PCR polymerase chain reaction - PVP polyvinyl pyrrolidone - VPg viral genome-linked protein     

Co-transformation of canola by chimeric chitinase and <Emphasis Type="Italic">tlp</Emphasis> genes towards improving resistance to <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis>

Canola (Brassica napus) plants were co-transformed with two pathogenesis-related protein genes expressing a Trichoderma atroviride chitinase with a chitin-binding domain (chimeric chitinase) and a thaumatin-like protein (tlp) from Oryza sativa conferring resistance to phytopatogenic fungi by Agrobacterium-mediated transformation. The putative transgenic plants were confirmed by PCR. After measuring the specific activity of the chimeric chitinase and glucanase activity for tlp genes, transgenic plants with high specific activity were selected for southern blot analysis to confirm the copy number of the genes. In vitro assays, the antifungal activity of crude extracted protein against Sclerotinia sclerotiorum showed that the inhibition percentage in double transgenic plants was between 55 and 62, whereas the inhibition percentage in single-gene transformants (chimeric chitinase) ranged from 35 to 45 percent. Importantly, in greenhouse conditions, the double transgenic plants showed significant resistance than the single-gene transformant and wild type plants. The results in T₂ generation using the intact leaf inoculation method showed that the average lesion diameters were 10, 14.7 and 29 mm for the double transformant, single-gene transformant and non-transgenic plants, respectively. Combined expression of chimeric chitinase and tlp in transgenic plants showed significantly enhanced resistance against S. sclerotiorum than the one that express single-gene transformant plants. These results suggest that the co-expression of chimeric chitinase and tlp can confer enhanced disease resistance in canola plant.     

Genetic transformation of peanut (Arachis hypogaea L.) using cotyledonary node as explant and a promoterlessgus::nptII fusion gene based vector

We have generated putative promoter tagged transgenic lines inArachis hypogaea cv JL-24 using cotyledonary node (CN) as an explant and a promoterless gus::nptII bifunctional fusion gene mediated byAgrobacterium transformation. MS medium fortified with 6-benzylaminopurine (BAP) at 4 mg/l in combination with 0.1 mg/l α-napthaleneacetic acid (NAA) was the most effective out of the various BAP and NAA combinations tested in multiple shoot bud formation. Parameters enhancing genetic transformation viz. seedling age,Agrobacterium genetic background and co-cultivation periods were studied by using the binary vector p35SGUSINT. Genetic transformation with CN explants from 6-day-old seedlings co-cultivated withAgrobacterium GV2260 strain for 3 days resulted in high kanamycin resistant shoot induction percentage (45%); approximately 31% transformation frequency was achieved with p35S GUSINT in Β-glucuronidase (GUS) assays. Among thein vivo GUS fusions studied with promoterless gus::nptII construct, GUS-positive sectors occupied 38% of the total transient GUS percentage. We have generated over 141 putative T₀ plants by using the promoterless construct and transferred them to the field. Among these, 82 plants survived well in the green house and 5 plants corresponding to 3.54% showed stable integration of the fusion gene as evidenced by GUS, polymerase chain reaction (PCR) and Southern blot analyses. Twenty-four plants were positive for GUS showing either tissue-specific expression or blue spots in at least one plant part. The progeny of 15 T₀ plants indicated Mendelian inheritance pattern of segregation for single-copy integration. The tissue-specific GUS expression patterns were more or less similar in both T₀ and corresponding T₁ progeny plants. We present the differential patterns of GUS expression identified in the putative promoter-tagged transgenic lines in the present communication.     

Combined expression of a barley class II chitinase and type I ribosome inactivating protein in transgenic <Emphasis Type="Italic">Brassica juncea</Emphasis> provides protection against <Emphasis Type="Italic">Alternaria brassicae</Emphasis>

Alternaria leaf spot caused by Alternaria brassicae, or A. brassicola, is one of the major fungal diseases of Brassica juncea (Indian mustard). To develop resistance against this fungal disease, the barley antifungal genes class II chitinase (AAA56786) and type I ribosome inactivating protein (RIP; AAA32951) were coexpressed in Indian mustard via Agrobacterium-mediated transformation. The stable integration and expression of transgenes in T₀ plants were confirmed by Southern blot and Western analysis. The transgenic lines showing inheritance in Mendalian fashion (3:1) were further evaluated by in vitro studies and under greenhouse conditions for resistance to the A. brassicae fungal pathogen. The transgenic plants showed up to 44% reduction in A. brassicae hyphal growth in in vitro antifungal assays. In green house screening, the transgenic plants sprayed with A. brassicae spores showed resistance through delayed onset of the disease and restricted number, size, and expansion of lesions as compared to wild type plants. These results indicate that the expression of chitinase and RIP from a heterologous source in B. juncea provide subsequent protection against Alternaria leaf spot disease and can be helpful in increasing the production of Indian mustard.