<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of the medicinal plant <Emphasis Type="Italic">Centaurea montana</Emphasis>

An efficient transformation system was developed for Centaurea montana by co-cultivation of leaf explants with Agrobacterium tumefaciens strain AGL1 that contained a plasmid harboring the isopentenyl transferase gene under the control of the developmentally regulated Atmyb32 promoter of Arabidopsis thaliana and the gene encoding for hygromycin resistance under the control of the Cauliflower Mosaic Virus 35S (CaMV35S) promoter. A total of 990 explants were infected with Agrobacterium, and 18 shoots were regenerated resulting in an overall transformation efficiency of 1.8%. Molecular analyses, including PCR, Southern blotting and RT-PCR, were performed on T₀ and T₁ plants to confirm chromosomal integration and expression of the transgene in the phenotypically normal transformed plants. Transformation of C. montana was also performed using A. tumefaciens supervirulent strain EHA105 harboring the β-glucuronidase (GUS) reporter gene. Expression of the GUS gene in the putative transgenics was confirmed using a histochemical GUS assay.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of Perilla (<Emphasis Type="Italic">Perilla frutescens</Emphasis>)

Agrobacterium tumefaciens-mediated transformation system for perilla (Perilla frutescens Britt) was developed. Agrobacterium strain EHA105 harboring binary vector pBK I containing bar and γ-tmt cassettes or pIG121Hm containing nptII, hpt, and gusA cassettes were used for transformation. Three different types of explant, hypocotyl, cotyledon and leaf, were evaluated for transformation and hypocotyl explants resulted in the highest transformation efficiency with an average of 3.1 and 2.2%, with pBK I and pIG121Hm, respectively. The Perilla spp. displayed genotype-response for transformation. The effective concentrations of selective agents were 2 mg l⁻¹ phosphinothricin (PPT) and 150 mg l⁻¹ kanamycin, respectively, for shoot induction and 1 mg l⁻¹ PPT and 125 mg l⁻¹ kanamycin, respectively, for shoot elongation. The transformation events were confirmed by herbicide Basta spray or histochemical GUS staining of T₀ and T₁ plants. The T-DNA integration and transgene inheritance were confirmed by PCR and Southern blot analysis of random samples of T₀ and T₁ transgenic plants.     

The transformation of pea (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.): applicable methods of <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated gene transfer

Three methods of transformation of pea (Pisum sativum ssp. sativum L. var. medullare) were tested. The most efficient Agrobacterium tumefaciens-mediated T-DNA transfer was obtained using embryonic segments from mature pea seeds as initial explants. The transformation procedure was based on the transfer of the T-DNA region with the reporter gene uidA and selection gene bar. The expression of β-glucuronidase (GUS) in the regenerated shoots was tested using the histochemical method and the shoots were selected on a medium containing phosphinothricin (PPT). The shoots of putative transformants were rooted and transferred to non-sterile conditions. Transient expression of the uidA gene in the tissues after co-cultivation and in the course of short-term shoot cultivation (confirmed by histochemical analysis of GUS and by RT-PCR of mRNA) was achieved; however, we have not yet succeeded in proving stable incorporation of the transgene in the analysed plants.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis> L.) via vacuum infiltration

AnAgrobacterium-mediated gene transfer system with recovery of putative transformants was developed for cotton (Gossypium hirsutum L.) cv. Cocker-312. Two-month-old hypocotyl-derived embryogenic calli were infected through agroinfiltration for 10 min at 27 psi in a suspension ofAgrobacterium tumefaciens strain GV3101 carrying tDNA with theGUS gene, encoding β-glucuronidase (GUS), and the neomycin phosphotransferase II (nptII) gene as a kanamycin-resistant plant-selectable marker. Six days after the histochemicalGUS assay was done, 46.6% and 20%GUS activity was noted with the vacuum-infiltration and commonAgrobacterium-mediated transformation methods, respectively. The transformed embryogenic calli were cultured on selection medium (100 mg/L and 50 mg/L kanamycin for 2 wk and 10 wk, respectively) for 3 mo. The putative transgenic plants were developed via somatic embryogenesis (25 mg/L kanamycin). In 4 independent experiments, up to 28.23% transformation efficiency was achieved. PCR amplification and Southern blot analysis fo the transformants were used to confirm the integration of the transgenes. Thus far, this is the only procedure available for cotton that can successfully be used to generate cotton transformants.     

Isolation and characterization of <Emphasis Type="Italic">Brittle2</Emphasis> promoter from <Emphasis Type="Italic">Zea Mays</Emphasis> and its comparison with <Emphasis Type="Italic">Ze19</Emphasis> promoter in transgenic tobacco plants

ADP-glucose pyrophosphorylase (AGPase) represents a key regulatory step in starch synthesis. A 0.9 kb of 5′ flanking region preceding Brittle2 gene, encoding the small subunit of maize endosperm AGPase, was cloned from maize genome and its expression pattern was studied via the expression of β-glucuronidase (GUS) gene in transgenic tobacco. Analysis of GUS activities showed that the 0.9 kb fragment flanking Brittle2 gene was sufficient for driving the seed-preferred expression of the reporter gene. The activity of the 0.9 kb 5′ flanking fragment was compared with that of the tandem promoter region from a zein gene (zE19, encoding a maize 19 kDa zein protein). The results indicated that both promoters were seed-preferred in a dicotyledonous system as tobacco and the activity of zE19 promoter was three to fourfold higher than that of the 0.9 kb fragment flanking Brittle2 gene in transgenic tobacco seeds. At the same time, zE19-driven GUS gene expressed earlier than Brittle2 promoter during seed development. Histochemical location of GUS activity indicated that both promoters showed high expression in embryos, which is different from similar promoters tested in maize.     

Enhanced resistance against bacterial wilt in transgenic tomato (<Emphasis Type="Italic">Lycopersicon esculentum</Emphasis>) lines expressing the <Emphasis Type="Italic">Xa21</Emphasis> gene

To enhance bacterial wilt resistance in tomato plants and simplify the protocol of Agrobacterium tumefaciens mediated gene transfer, parameters affecting transformation efficiency in tomato have been optimized. A. tumefaciens strain EHA101, harboring a recombinant binary expression vector pTCL5 containing the Xa21 gene under the control of the CaMV 35S promoter was used for transformation. Five cultivars of tomato (Rio Grande, Roma, Pusa Ruby Pant Bahr and Avinash) were tested for transformation. Transformation efficiency was highly dependent on preculture of the explants with acetosyringone, acetosyringone in co-cultivation media, shoot regeneration medium and pre-selection after co-cultivation without selective agent. One week of pre-selection following selection along with 400 μM acetosyringone resulted in 92.3% transient GUS expression efficiency in Rio Grande followed by 90.3% in Avinash. The presence and integration of the Xa21 gene in putative transgenic plants was confirmed by polymerase chain reaction (PCR) and Southern blot analyses with 4.5–42.12% PCR-positive shoots were obtained for Xa21 and hygromycin genes, respectively. Transgenic plants of the all lines showed resistance to bacterial wilt. T₁ plants (resulting from self-pollination of transgenic plants) tested against Pseudomonas solanacearum inoculation in glasshouse, showed Mendelian segregation.     

Effects of plant genotype and bacterial strain on <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of <Emphasis Type="Italic">Brassica oleracea</Emphasis> L. var. <Emphasis Type="Italic">capitata</Emphasis>

Two inbred lines of Brassica oleracea L. var. capitata were transformed with two Agrobacterium tumefaciens strains harboring resistance to herbicide Basta: AGL1/pDM805 and LBA4404/pGKB5 (LB5-1). Inoculated cotyledons and hypocotyls provided equally good explants and manifested a high percentage of shoot regeneration on MS medium supplemented with 1 mg/l benzyladenine and 0.5 mg/l indole-3-butyric acid. The P₃₄I₅ genotype was superior to P₂₂I₅ in shoot regeneration (48.1 vs. 26.9%), multiplication, and acclimation in the greenhouse (76 vs. 40%). A. tumefaciens AGL1/pDM805 provided more regenerated shoots per explant, especially in the case of cotyledon explants, and the higher transformation rate (up to 35% vs. up to 12%) as compared to LB5-1. Putative transformants survived spraying with 10–30 mg/l phosphinothricin. Transformation was confirmed by GUS assay and PCR analysis in T0 and T1 generations. Published in Russian in Fiziologiya Rastenii, 2007, vol. 54, No. 5, pp. 738–743. The text was submitted by the authors in English.     

<Emphasis Type="Italic">Agrobacterium</Emphasis> -mediated transformation of cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis>) using a heterologous bean chitinase gene

Cotton (Gossypium hirsutum L., var. Coker 312) hypocotyl explants were transformed with three strains of Agrobacterium tumefaciens, LBA4404, EHA101 and C58, each harboring the recombinant binary vector pBI121 containing the chi gene insert and neomycin phosphotransferase (nptII) gene, as selectable marker. Inoculated tissue sections were placed onto cotton co-cultivation medium. Transformed calli were selected on MS medium containing 50 mg l⁻¹ kanamycin and 200 mg l⁻¹ cepotaxime. Putative calli were subsequently regenerated into cotton plantlets expressing both the kanamycin resistance gene and βglucuronidase (gus) as a reporter gene. Polymerase chain reaction was used to confirm the integration of chi and nptII transgenes in the T₁ plants genome. Integration of chi gene into the genome of putative transgenic was further confirmed by Southern blot analysis. ‘Western’ immunoblot analysis of leaves isolated from T₀ transformants and progeny plants (T₁) revealed the presence of an immunoreactive band with MW of approximately 31 kDa in transgenic cotton lines using anti-chitinase-I polyclonal anti-serum. Untransformed control and one transgenic line did not show such an immunoreactive band. Chitinase specific activity in leaf tissues of transgenic lines was several folds greater than that of untransformed cotton. Crude leaf extracts from transgenic lines showed in vitro inhibitory activity against Verticillium dahliae.Transgenic plants currently growing in a greenhouse and will be bioassayed for improved resistance against V. dahlia the causal against of verticilliosis in cotton.     

Two seed coat-specific promoters are functionally conserved between <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> and <Emphasis Type="Italic">Brassica napus</Emphasis>

Canola is one of the most important cash crops in Canada, and a national project named “Designing Oilseeds for Tomorrow’s Market” was undertaken to improve seed meal quality of this strategically important crop. As a part of this project, our group is focusing on identifying seed coat-specific promoters for canola (Brassica napus). These promoters will be used to genetically modify canola seed coat to reduce or eliminate anti-nutritional components from the meal. The Arabidopsis thaliana BAN promoter (AtBANpro) and δVPE promoter (AtδVPEpro) were isolated and fused to GUS reporter gene to generate transgenic canola plants. These plants were analyzed by GUS staining and microtome sectioning which showed that both promoters are seed coat-specific in canola: AtBANpro in inner seed coat layer and AtδVPEpro in outer seed coat layer. Therefore, the two Arabidopsis promoters can be used to modify genes in seed coat of canola for further improving its seed qualities.     

<Emphasis Type="Italic">Organogenic callus</Emphasis> as the target for plant regeneration and transformation via <Emphasis Type="Italic">Agrobacterium</Emphasis> in soybean (<Emphasis Type="Italic">Glycine max</Emphasis> (L.) Merr.)

A regeneration and transformation system has been developed using organogenic calluses derived from soybean axillary nodes as the starting explants. Leaf-node or cotyledonary-node explants were prepared from 7 to 8-d-old seedlings. Callus was induced on medium containing either Murashige and Skoog (MS) salts or modified Finer and Nagasawa (FNL) salts and B5 vitamins with various concentrations of benzylamino purine (BA) and thidiazuron (TDZ). The combination of BA and TDZ had a synergistic effect on callus induction. Shoot differentiation from the callus occurred once the callus was transferred to medium containing a low concentration of BA. Subsequently, shoots were elongated on medium containing indole-3-acetic acid (IAA), zeatin riboside, and gibberellic acid (GA). Plant regeneration from callus occurred 90 ∼ 120 d after the callus was cultured on shoot induction medium. Both the primary callus and the proliferated callus were used as explants for Agrobacterium-mediated transformation. The calluses were inoculated with A. tumefaciens harboring a binary vector with the bar gene as the selectable marker gene and the gusINT gene for GUS expression. Usually 60–100% of the callus showed transient GUS expression 5 d after inoculation. Infected calluses were then selected on media amended with various concentrations of glufosinate. Transgenic soybean plants have been regenerated and established in the greenhouse. GUS expression was exhibited in various tissues and plant organs, including leaf, stem, and roots. Southern and T₁ plant segregation analysis of transgenic events showed that transgenes were integrated into the soybean genome with a copy number ranging from 1–5 copies.     

Genetic transformation of NERICA,interspecific hybrid rice between <Emphasis Type="Italic">Oryza glaberrima</Emphasis> and <Emphasis Type="Italic">O. sativa</Emphasis>, mediated by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

We developed an efficient gene transfer method mediated by Agrobacterium tumefaciens for introgression of new rice for Africa (NERICA) cultivars, which are derivatives of interspecific hybrids between Oryza glaberrima Steud. and O. sativa L. Freshly isolated immature embryos were inoculated with A. tumefaciens LBA4404 that harbored binary vector pBIG-ubi::GUS or pIG121Hm, which each carried a hygromycin-resistance gene and a GUS gene. Growth medium supplemented with 500 mg/l cefotaxime and 20 mg/l hygromycin was suitable for elimination of bacteria and selection of transformed cells. Shoots regenerated from the selected cells on MS medium containing 20 g/l sucrose, 30 g/l sorbitol, 2 g/l casamino acids, 0.25 mg/l naphthaleneacetic acid, 2.5 mg/l kinetin, 250 mg/l cefotaxime, and 20 mg/l hygromycin. The shoots developed roots on hormone-free MS medium containing 30 mg/l hygromycin. Integration and expression of the transgenes were confirmed by PCR, Southern blot analysis, and histochemical GUS assay. Stable integration, expression, inheritance, and segregation of the transgenes were demonstrated by molecular and genetic analyses in the T₀ and T₁ generations. Most plants were normal in morphology and fertile. The transformation protocol produced stable transformants from 16 NERICA cultivars. We also obtained transformed plants by inoculation of calluses derived from mature seeds, but the frequency of transformation was lower and sterility was more frequent.     

A rapid<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated<Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> transient assay system

Stable transformation ofArabidopsis thaliana is a lengthy process that involves up to 3 mo of plant growth and seed selection. We have developed a rapid, 3-wk transient assay system to test the functionality ofcis-regulatory regions controlling expression of a reporter gene in plants before undertaking stable transformation. Two-week-oldArabidopsis seedlings were vacuum-infiltrated withAgrobacterium tumefaciens cultures carrying various upstream regulatory regions controllinguidA (β-glucuronidase [GUS]) expression. Seedlings were fixed and stained for GUS activity 3–5 d following infiltration. Regulatory regions tested in this system include the cauliflower mosaic virus (CaMV)35S promoter, the upstream regulatory region of ribosomal protein geneL23A-1, and a temperature-inducible regulatory region (HSP101B) also fromArabidopsis. The percentage of seedlings positive for GUS activity varied depending on the construct used, with the CaMV35S promoter producing the highest number of GUS-positive seedlings. Temperature induction treatments elicited increased GUS expression in seedlings transformed with theHSP101B regulatory region. Regardless of construct, GUS expression levels were higher in seedlings collected 5 d followingAgrobacterium infiltration than those collected 3–4 d postinfiltration.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated genetic transformation of <Emphasis Type="Italic">Perilla frutescens</Emphasis>

A reproducible plant regeneration and an Agrobacterium tumefaciens-mediated genetic transformation protocol were developed for Perilla frutescens (perilla). The largest number of adventitious shoots were induced directly without an intervening callus phase from hypocotyl explants on MS medium supplemented with 3.0 mg/l 6-benzylaminopurine (BA). The effects of preculture and extent of cocultivation were examined by assaying -glucuronidase (GUS) activity in explants infected with A. tumefaciens strain EHA105 harboring the plasmid pIG121-Hm. The highest number of GUS-positive explants were obtained from hypocotyl explants cocultured for 3 days with Agrobacterium without precultivation. Transgenic perilla plants were regenerated and selected on MS basal medium supplemented with 3.0 mg/l BA, 125 mg/l kanamycin, and 500 mg/l carbenicillin. The transformants were confirmed by PCR of the neomycin phosphotransferase II gene and genomic Southern hybridization analysis of the hygromycin phosphotransferase gene. The frequency of transformation from hypocotyls was about 1.4%, and the transformants showed normal growth and sexual compatibility by producing progenies.     

Optimization of factors influencing microprojectile bombardment-mediated genetic transformation of seed-derived callus and regeneration of transgenic plants in <Emphasis Type="Italic">Eleusine coracana</Emphasis> (L.) Gaertn

Microprojectile bombardment mediated genetic transformation parameters have been standardized for seed derived callus of Eleusine coracana. Plasmid pCAMBIA 1381 harboring hygromycin phosphotransferase (hptII) as selectable marker gene and β-glucuronidase (gus A) as reporter gene, was used for the optimization of gene transfer conditions. The transient GUS expression and survival of putative transformants were taken into consideration for the assessment of parameters. Optimum conditions for the microprojectile bombardment mediated genetic transformation of finger millet were 1,100 psi rupture disk pressure with 3 cm distance from rupture disk to macrocarrier and 12 cm microprojectile travel distance. Double bombardment with gold particles of 1.0 μm size provided maximum transient GUS expression and transformation efficiency. Osmotic treatment of callus with 0.4 M sorbitol enhanced efficiency of particle bombardment mediated genetic transformation. Regenerative calli were bombarded at optimum conditions of bombardment and placed on regeneration medium with hygromycin to obtain transformed plants. The integration of hptII and gus A genes was confirmed with PCR amplification of 684 and 634 bp sizes of the bands respectively from putative transformants and Southern blot hybridization using PCR amplified DIG labeled hptII gene as probe. PCR analysis with hptII gene specific primers indicated the presence of transgene in T₁ generation plants. Thus a successful genetic transformation system was developed using particle bombardment in E. coracana with 45.3% transformation efficiency. The protocol will be helpful for the introgression of desired genes into E. coracana.     

Functional analysis of the <Emphasis Type="Italic">egl3</Emphasis> upstream region in filamentous fungus <Emphasis Type="Italic">Trichoderma reesei</Emphasis>

In the filamentous fungus Trichoderma reesei, endoglucanase III (EGIII) is coordinately expressed with other cellulases during growth on cellulose, its derivatives, and L-sorbose. To elucidate EGIII induction mechanism, we cloned and sequenced the upstream region of egl3 encoding EGIII. Two GGCTAA motifs, a putative binding site for ACEII and xylanase regulator Xyr1, were found on the template strand of the egl3 upstream region. Deletion analysis of the egl3 upstream region using the beta-glucuronidase (GUS) reporter system revealed that removal of regions containing the GGCTAA motifs and the region between −1,045 and −1,002 bp containing GGCTAT motif severely affected GUS inducibility. Furthermore, mutation of the two GGCTAA motifs and the GGCTAT motif of this region led to a significant decrease in GUS activity. These data indicate that both GGCTAA and GGCTAT are key motifs for egl3 expression, and that egl3 induction may also be controlled by Xyr1. This hypothesis was supported by in vitro electrophoretic mobility shift assay, in which heterologously expressed Xyr1 specifically bound not only GGCTAA but also GGCTAT motif.     

Pollen-Specific Expression of <Emphasis Type="Italic">Oryza sativa Indica</Emphasis> Pollen Allergen Gene (<Emphasis Type="Italic">OSIPA</Emphasis>) Promoter in Rice and <Emphasis Type="Italic">Arabidopsis</Emphasis> Transgenic Systems

Earlier, a pollen-specific Oryza sativa indica pollen allergen gene (OSIPA), coding for expansins/pollen allergens, was isolated from rice, and its promoter—upon expression in tobacco and Arabidopsis—was found active during the late stages of pollen development. In this investigation, to analyze the effects of different putative regulatory motifs of OSIPA promoter, a series of 5′ deletions were fused to β-glucuronidase gene (GUS) which were stably introduced into rice and Arabidopsis. Histochemical GUS analysis of the transgenic plants revealed that a 1631 bp promoter fragment mediates maximum GUS expression at different stages of anther/pollen development. Promoter deletions to −1272, −966, −617, and −199 bp did not change the expression profile of the pollen specificity. However, the activity of promoter was reduced as the length of promoter decreased. The region between −1567 and −199 bp was found adequate to confer pollen-specific expression in both rice and Arabidopsis systems. An approximate 4-fold increase in the GUS activity was observed in the pollen of rice when compared to that of Arabidopsis. As such, the OSIPA promoter seems promising for generation of stable male-sterile lines required for the production of hybrids in rice and other crop plants.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of <Emphasis Type="Italic">indica</Emphasis> rice cv. ADT 43

A reproducible and highly efficient protocol for Agrobacterium tumefaciens-mediated transformation of indica rice (Oryza sativa L. subsp. indica cv. ADT 43) was established. Prior to transformation, embryogenic callus were induced from mature seeds incubated on Linsmaier and Skoog (LS) medium supplemented with 2.5 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.0 mg l⁻¹ thiamine-HCl. Callus, intact mature seeds, and other in vitro derived explants (leaf bases, leaf blades, coleoptiles, and root-tips) were immersed in a bacterial suspension culture of A. tumefaciens strain EHA 105, OD600 of 0.8, and co-cultivated on LS medium for 2 days in the dark at 25 ± 2°C. Based on GUS expression analysis, 10 min incubation time of explants on a co-cultivation medium containing 100 μM acetosyringone was optimum. Following β-glucuronidase (GUS) assay and polymerase chain reaction (PCR) analysis, transformants were identified. Stable integration of the transgene was confirmed in four putatively transformed T₀ plants by Southern blot analysis. The copy number of the transgene in these lines, one to two, was then determined. Among the observations made, necrosis of co-cultivated explants was a problem, as well as sensitivity of callus to Agrobacterium infection. Levels of necrosis could be minimized following co-cultivation of explants in a medium consisting of 30% LS and containing 10 g l⁻¹ (14), polyvinyl pyrrolidone, 10% coconut water, and 250 mg l⁻¹ timentin (15:1). This latter medium also increased the final transformation efficiency to 15.33%.     

Efficient regeneration of <Emphasis Type="Italic">Brassica napus</Emphasis> L. hypocotyls and genetic transformation by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

An efficient system for shoot regeneration and Agrobacterium-mediated gene transfer into Brassica napus was developed through the modification of the culture conditions. Different concentrations of benzyladenine (1.5, 3.0 and 4.5 mg dm^–3) and thidiazuron (0.0, 0.15 and 0.30 mg dm^–3) were evaluated for shoot regeneration of 7, 14 and 21-d-old hypocotyl explants. Maximum shoot regeneration frequency was obtained in 21-d-old explants using 4.5 mg dm^–3 benzyladenine and 0.3 mg dm^–3 thidiazuron. Under above culture condition, the highest percentage of shoot regeneration frequency was 200 %. Agrobacterium-infected explants grown on the selection medium gave rise to transgenic shoots at a frequency of 11.8 %. Transformed shoots rooted when cultured on a medium supplemented with 2 mg dm^–3 of indolebutyric acid and 10 mg dm^–3 kanamycin. The rooted plantlets were successfully established in the soil and developed fertile flowers and viable seeds. Evidences for transformation were confirmed by GUS assay and PCR analysis.