Genetic transformation of Vitis vinifera via organogenesis

Background  

Efficient transformation and regeneration methods are a priority for successful application of genetic engineering to vegetative propagated plants such as grape. The current methods for the production of transgenic grape plants are based on Agrobacterium-mediated transformation followed by regeneration from embryogenic callus. However, grape embryogenic calli are laborious to establish and the phenotype of the regenerated plants can be altered.     

多年生黑麦草成熟胚再生体系的建立及基因枪转化

目的:建立以多年生黑麦草成熟胚为起始材料的再生体系,用于基因枪转化。方法:多年生黑麦草成熟种子在附加 5mg L 2,4 D的MS培养基上诱导愈伤组织,转至新继代培养基上产生胚性愈伤组织。分化培养基为无激素MS培养基。再生植株在培养基成分减半的无激素MS培养基生根,之后移栽至土壤。基于这一再生体系,用含有水稻几丁质酶基因RC2 4的质粒pARN6和含有草丁膦乙酰转移酶基因Bar的质粒pDB1,通过基因枪轰击胚性愈伤组织。用附加PPT的继代培养基进行转化植株的抗性筛选。结果:共获得 2 4 3株再生植株。通过PCR进行检测,获得1 8株整合有RC2 4基因的植株,1 5株整合有Bar基因的植株,同时转入 2个基因的植株 2株。     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of meadow fescue (<Emphasis Type="Italic">Festuca pratensis</Emphasis> Huds.)

Meadow fescue (Festuca pratensis Huds.) is an important cool-season forage grass in Europe and Asia. We developed a protocol for producing meadow fescue transgenic plants mediated by Agrobacterium tumefaciens transformation. Embryogenic calli derived from mature embryos were transformed with A. tumefaciens strain AGL1 carrying the binary vector pDM805, coding for the phosphinothricin acetyltransferase (bar) and β-glucuronidase (uidA) genes. Bialaphos was used as the selective agent throughout all phases of tissue culture. In total, 40 independent transgenic plants were recovered from 45 bialaphos-resistant callus lines and an average transformation efficiency of 2% was achieved. The time frame from infection of embryogenic calli with Agrobacterium to transferring the transgenic plants to the greenhouse was 18 weeks. In a study of 11 BASTA-resistant transgenic lines, the uidA gene was expressed in 82% of the transgenic lines. Southern blot analysis revealed that 82% of the tested lines integrated one or two copies of the uidA gene. C. Gao and J. Liu contributed equally to the work.     

Generation of large numbers of independently transformed fertile perennial ryegrass (Lolium perenne L.) plants of forage- and turf-type cultivars

Perennial ryegrass (Lolium perenne L.) is the most important grass species in areas with a temperate climate. Biolistic transfer of a ubiquitin promoter driven nptII expression cassette into mature or immature tissue derived calli of perennial ryegrass followed by paromomycin selection, resulted in the rapid and efficient production of fertile transgenic ryegrass plants. Transformation efficiencies after paromomycin selection in combination with the nptII selectable marker compared favourably with hygromycin selection in combination with the hph selectable marker. In total 83 independent nptII expressing plants were produced. Transformation frequency was highly affected by genotype, explant, selection regime and the duration of the callus induction period. The optimised transformation protocol for mature embryo derived calli of turf-type or forage-type cultivars resulted in an average transformation efficiency of 5.2% or 6.6% respectively. This converts into 1.7 or 2.2 independent transgenic plants per bombardment. Immature inflorescence- and immature embryo-derived calli were also successfully used as target for the gene transfer, resulting in transformation efficiencies of up to 3.7% or 11.42% respectively. Transgenic plants were transferred to soil 12 or 9 weeks after excision of mature and immature embryos or inflorescences respectively. Transgene integration and expression were confirmed by PCR and ELISA or western blot analysis. Southern blot analysis confirmed the independent nature of the transgenic lines. The majority of lines showed the integration of two to six transgene copies, while 21% of the analysed lines had a single copy insert. A short tissue culture period in comparison to recently published reports seems to be beneficial for the production of normal and fertile transgenic ryegrass plants. Consequently we report for the first time molecular evidence for sexual transgene transmission in fertile transgenic perennial ryegrass.     

Development of an efficient plant regeneration system for Russian wildrye (<Emphasis Type="Italic">Psathyrostachys juncea</Emphasis>)

Russian wildrye [Psathyrostachys juncea (Fisch.) Nevski] is a cool-season forage grass with a broad adaptation to semi-arid regions of North America. In order to explore the potential of biotechnology for genetic improvement of this important forage species, we developed an efficient tissue culture system. Embryogenic calli were induced from mature embryos with an induction frequency in the range of 2-7%. The selected highly embryogenic calli allowed the regeneration of dozens of plants from a single callus. Individual embryogenic calli were then used to establish single genotype-derived suspension cultures. Eighteen embryogenic cell suspension lines were established from three cultivars (Bozoisky-Select, Sawki and Tetracan). A relatively high green plant regeneration frequency, up to 70%, was achieved from plated cell clusters of the established suspension cultures. The regenerated plants were fertile after two winters of vernalization in the field. This efficient plant regeneration system provides a solid basis for generating transgenic plants.     

Development of an efficient Agrobacterium-mediated transformation system and production of herbicide-resistant transgenic plants in garlic (Allium sativum L.)

The genetic improvement of garlic plants (Allium sativum L.) with agronomical beneficial traits is rarely achieved due to the lack of an applicable transformation system. Here, we developed an efficient Agrobacterium-mediated transformation procedure with Danyang, an elite Korean garlic cultivar. Examination of sGFP (synthetic green fluorescence protein) expression revealed that treatment with 2-(N-morpholino) ethanesulfonic acid (MES), L-cysteine and/or dithiothreitol (DTT) gives the highest efficiency in transient gene transfer during Agrobacterium co-cultivation with calli derived from the roots of in vitro plantlets. To increase stable transformation efficiency, a two-step selection was employed on the basis of hygromycin resistance and sGFP expression. Of the hygromycin-resistant calli initially produced, only sGFP-expressing calli were subcultured for selection of transgenic calli. Transgenic plantlets produced from these calli were grown to maturity. The transformation efficiency increased up to 10.6% via our optimized procedure. DNA and RNA gel-blot analysis indicated that transgenic garlic plants stably integrated and expressed the phosphinothricin acetyltransferase (PAT) gene. A herbicide spraying assay demonstrated that transgenic plants of garlic conferred herbicide resistance, whilst nontransgenic plants and weeds died. These results indicate that our transformation system can be efficiently utilized to produce transgenic garlic plants with agronomic benefits.     

A simple and rapid Agrobacterium-mediated transformation protocol for cotton (Gossypium hirsutum L.): Embryogenic calli as a source to generate large numbers of transgenic plants

A protocol is presented for efficient transformation and regeneration of cotton. Embryogenic calli co-cultivated with Agrobacterium carrying cry1Ia5 gene were cultured under dehydration stress and antibiotic selection for 3–6 weeks to generate several transgenic embryos. An average of 75 globular embryo clusters were observed on selection plates and these embryos were cultured on multiplication medium followed by development of cotyledonary embryos on embryo maturation medium to obtain an average of 12 plants per Petri plate of co-cultivated callus. About 83% of these plants have been confirmed to be transgenic by Southern blot analysis. An efficiency of ten kanamycin-resistant plants per Petri plate of co-cultivated embryogenic callus was obtained. The simplicity of the procedure and the efficiency of the initial material allow transformation of any variety where a single regenerating embryogenic callus line can be obtained. In addition, multiple transformations can be performed either simultaneously or sequentially. The method is extremely simple, reliable, efficient, and much less laborious than any other existing method for cotton transformation.V.G. Sunnichan and R. Kumria contributed equally to this investigation     

Particle bombardment-mediated transformation of barley with an Arabidopsis NDPK2 cDNA

As barley is recalcitrant to transformation with current methods, a new improved system is required to apply genetic transformation in breeding programs. In a previous study, we defined optimal conditions for plant regeneration (PR) using mature embryos. This study was conducted to establish an improved transformation system employing the previously adjusted regeneration conditions. Optimal DNA delivery condition for the embryogenic calli developed from mature embryos was bombardment pressure of 1,100 psi at the target distance of 6 cm. The feasibility of the regeneration and DNA delivery conditions was confirmed by developing transgenic barley plants transformed with the Arabidopsis nucleoside diphosphate kinase 2 (AtNDPK2) cDNA via particle bombardment of embryogenic calli from mature embryos. Stable integration of AtNDPK2 cDNA into barley genome was confirmed by PCR and Southern blot analysis of AtNDPK2 transgene. Transgenic plants showed about 10% reduction in membrane damage caused by methyl viologen, indicating the expression of AtNDPK2 transgene. The results demonstrated that the transformation system developed in this study employing the PR from mature embryo-derived embryonic callus is applicable in transgenic barley production.     

Development of highly efficient genetic transformation protocols for table grape Sugraone and Crimson Seedless at low <Emphasis Type="Italic">Agrobacterium</Emphasis> density

Highly efficient genetic transformation protocols and the regeneration of transgenic plants of Sugraone and Crimson Seedless grapevines (Vitis vinifera L.) were achieved from embryogenic calli co-cultured with low Agrobacterium tumefaciens densities. The sensitivity of embryogenic cultures to kanamycin, as well as the effect of Agrobacterium strains, C58(pMP90) or EHA105, and the bacterial concentration (0.06 or 0.2 at Optical Density OD₆₀₀) on transformation efficiency were studied. Embryogenic cultures showed different kanamycin sensitivities and the total suppression of embryo differentiation at 20 and 50 mg/l kanamycin for Crimson Seedless and Sugraone, respectively. sgfp gene expression was evaluated in callus co-cultured with each bacterial strain. Although GFP transient expression was higher with A. tumefaciens EHA105 in both cultivars at the beginning of the culture, there were no significant differences 28 days post-inoculation. However, the concentration of Agrobacterium did affected transformation efficiency: 0.06 OD₆₀₀ being more effective for the transformation of Crimson Seedless and 0.2 OD₆₀₀ for Sugraone. By following the optimised procedure, 21 and 26 independent transgenic plants were generated from Sugraone and Crimson Seedless respectively, three to five months post-infection. PCR analyses were carried out to verify the integration of the sgfp and nptII genes into grapevine genome and the stable integration of the sgfp gene was confirmed by Southern blot.     

Production of herbicide-resistant sweet potato plants transformed with the <Emphasis Type="Italic">bar</Emphasis> gene

Herbicide-resistant sweet potato plants were produced through biolistics of embryogenic calli derived from shoot apical meristems. Plant materials were bombarded with the vectors containing the β-glucuronidase gene (gusA) and the herbicide-resistant gene (bar). Selection was carried out using phosphinothricin (PPT). Transformants were screened by the histochemical GUS and Chlorophenol Red assays. PCR and Southern-blot analyses indicated the presence of introduced bar gene in the genomic DNA of the transgenic plants. When sprayed with Basta, the transgenic sweet potato plants was tolerant to the herbicide. Hence, we report successful transformation of the bar gene conferring herbicide resistance to sweet potato.     

The development of herbicide-resistant maize: stable Agrobacterium-mediated transformation of maize using explants of type II embryogenic calli

One of the limitations to conducting maize Agrobacterium-mediated transformation using explants of immature zygotic embryos routinely is the availability of the explants. To produce immature embryos routinely and continuously requires a well-equipped greenhouse and laborious artificial pollination. To overcome this limitation, an Agrobacterium-mediated transformation system using explants of type II embryogenic calli was developed. Once the type II embryogenic calli are produced, they can be subcultured and/or proliferated conveniently. The objectives of this study were to demonstrate a stable Agrobacterium-mediated transformation of maize using explants of type II embryonic calli and to evaluate the efficiency of the protocol in order to develop herbicide-resistant maize. The type II embryogenic calli were inoculated with Agrobacterium tumefaciens strain C58C1 carrying binary vector pTF102, and then were subsequently cultured on the following media: co-cultivation medium for 1 day, delay medium for 7 days, selection medium for 4 × 14 days, regeneration medium, and finally on germination medium. The T-DNA of the vector carried two cassettes (Ubi promoter-EPSPs ORF-nos and 35S promoter–bar ORF-nos). The EPSPs conferred resistance to glyphosate and bar conferred resistance to phosphinothricin. The confirmation of stable transformation and the efficiency of transformation was based on the resistance to phosphinothricin indicated by the growth of putative transgenic calli on selection medium amended with 4 mg l^?1 phosphinothricin, northern blot analysis of bar gene, and leaf painting assay for detection of bar gene-based herbicide resistance. Northern blot analysis and leaf painting assay confirmed the expression of bar transgenes in the R₁ generation. The average transformation efficiency was 0.60%. Based on northern blot analysis and leaf painting assay, line 31 was selected as an elite line of maize resistant to herbicide.     

Efficient regeneration and transformation of plantain cv. “Gonja manjaya” (Musa spp. AAB) using embryogenic cell suspensions

In banana and plantain research, it is essential to establish embryogenic cell suspensions together with a highly efficient regeneration and transformation system. This article describes the development of an embryogenic cell suspension (ECS), regeneration, and transformation for plantain cv. “Gonja manjaya”. ECS was established using highly proliferative multiple buds. The frequency of embryogenic friable callus formation was 56.8% of the cultured explants. Friable embryogenic calli with many translucent proembryos were transferred to liquid medium and homogenous cell suspensions were established within 3–4 mo. Approximately 25,000 to 30,000 plants per 1.0 ml of settled cell volume were regenerated in approximately 13–14 mo. ECSs were transformed using Agrobacterium strain EHA 105 harboring the binary vector pBI121. About 50–60 transgenic plants per 0.5 ml settled cell volume were regenerated on selective medium containing 100 mg l⁻¹ kanamycin. Histochemical GUS assays using different tissues of putatively transformed plants demonstrated stable expression of uidA gene. The presence and integration of the uidA gene were confirmed by PCR and Southern blot analysis, respectively. This is the first report showing establishment of embryogenic cell suspension cultures and Agrobacterium-mediated transformation of an important plantain cultivar, “Gonja manjaya”. This study shows the huge potential for genetic transformation of plantains for disease or pest resistance, as well as tolerance to abiotic factors such as drought stress using this robust regeneration and transformation protocol.     

An efficient particle bombardment system for the genetic transformation of asparagus (Asparagus officinalis L.)

The microprojectile bombardment method was used to transfer DNA into embryogenic callus of asparagus (Asparagus officcinalis L.) and to produce stably transformed asparagus plants. Embryogenic callus, derived from UC 157 and UC72 asparagus cultivars, was bombarded with tungsten particles coated with plasmid DNA that contained genes encoding hygromycin phosphotransferase, phosphinothricin acetyl transferase and -glucuronidase. Putatively transformed calli were identified from the bombarded tissue after 4 months selection on 25 mg/L hygromycin B plus 4 mg/L phosphinothricin (PPT). By selecting embryogenic callus on hygromycin plus PPT the overall transformation and selection efficiencies were substantially improved over selection with hygromycin or PPT alone, where no transgenic clones were recovered. The transgenic nature of the selected material was demonstrated by GUS histochemical assays and Southern blot hybridization analysis. Transgenic asparagus plants were found to withstand the prescribed levels of the PPT-based herbicide BASTATM for weed control.Abbreviations GUS -glucuronidase - HPT hygromycin phosphotransferase - bar phosphinothricin acetyl transferase gene - PPT phosphophinothricin - NAA naphthalene acetic acid - 2iP 2-isopentenyl adenine     

Transformation of chickpea: effect of genotype,explant, <Emphasis Type="Italic">Agrobacterium</Emphasis>-strain and composition of culture medium

Reproducible and high-frequency transgenic plant regeneration from callus and embryo axes of four different genotypes of chickpea (Cicer arietinum) was achieved after Agrobacterium-mediated transformation. Three different strains of Agrobacterium (EHA105, AGL1 and LBA4404) harboring the binary vector pCAMBIA1301 containing β-glucuronidase (GUS) and hygromycin phosphotransferase (hpt) genes under the control of a CaMV35S promoter were used. The highest number of transgenic plants was obtained from cotyledonary node-derived calli of genotype Pusa-256. A highly efficient rooting was achieved on Murashige and Skoog medium supplemented with indole-3-butyric acid. The stable integration of the gene was confirmed by molecular analyses of the transformed plants. Inheritance of GUS and hpt gene was followed through two generations and they showed the expected 3:1 inheritance.     

Expression of the B subunit of <Emphasis Type="Italic">E. coli</Emphasis> heat-labile enterotoxin in tobacco using a herbicide resistance gene as a selection marker

The B subunit of Escherichia coli heat-labile enterotoxin (LTB) has been transformed to plants for use as an edible vaccine. We have developed a simple and reliable Agrobacterium-mediated transformation method to express synthetic LTB gene in N. tabacum using a phosphinothricin acetyltransferase (bar) gene as a selectable marker. The synthetic LTB gene adapted to the coding sequence of tobacco plants was cloned to a plant expression vector under the control of the ubiquitin promoter and transformed to tobacco by Agrobacterium-mediated transformation. Transgenic plants were selected in the medium supplemented with 5 mg l^-1 phosphinothricin (PPT). The amount of LTB protein detected in the transgenic tobacco was approximately 3.3% of the total soluble protein, approximately 300-fold higher than in the plants generated using the native LTB gene under the control of the CaMV 35S promoter. The transgenic plants that were transferred to a greenhouse had harvested seeds that proved to be resistant to herbicide. Thus, the described protocol could provide a useful tool for the transformation of tobacco plants.     

High-frequency plant regeneration through callus initiation from mature embryos of maize (<Emphasis Type="Italic">Zea Mays</Emphasis> L.)

An efficient maize regeneration system was developed using mature embryos. Embryos were removed from surface-sterilized mature seeds and sliced into halves. They were used as explants to initiate callus on induction medium supplemented with 4.0 mg l^–1 2,4-dichlorophenoxyacetic acid (2,4-D). The induction frequency of primary calli was over 90% for all inbred lines tested. The primary calli were then transferred onto subculture medium supplemented with 2.0 mg l^–1 2,4-D. Following two biweekly subcultures, embryogenic calli were formed. Inclusion of a low concentration (0.2 mg l^–1) of 6-benzylaminopurine (BA) in the subculture medium significantly promoted the formation of embryogenic callus. The addition of silver nitrate (10 mg l^–1) also supported an increased frequency of embryogenesis. The embryogenic callus readily formed plantlets on regeneration medium supplemented with 0.5 mg l^–1 BA. The regenerated plantlets were transferred to half-strength Murashige and Skoog medium supplemented with 0.6 mg l^–1 indole-3-butyric acid to develop healthy roots. The regenerated plantlets were successful on transfer to soil and set seed. Using this system, plantlets were regenerated from seven elite maize inbred lines. The frequency of forming green shoots ranged from 19.8% to 32.4%. This efficient regeneration system provides a solid basis for genetic transformation of maize.Abbreviations BA 6-Benzylaminopurine - 2,4-D 2,4-Dichlorophenoxyacetic acid - IBA Indole-3-butyric acid - KT KinetinCommunicated by M.C. Jordan     

Development of an embryogenic callus induction method for centipede grass (<Emphasis Type="Italic">Eremochloa ophiuroides</Emphasis> Munro) and subsequent plant regeneration

The present study demonstrates the establishment of embryogenic tissue from seeds and (seedling-derived hypocotyls) shoot base explants derived from seedlings of Eremochloa ophiuroides. The highest percentage of callus induction obtained from seed and young shoot base explants was 52.0% and 66.6% on Murashige and Skoog (MS) basal media supplemented with 9.0 μM and 18.1 μM 2,4-dichlorophenoxyacetic acid (2,4-D), respectively. The type of callus obtained from both types of explants was off-white to yellow in color and non-friable and shiny in texture. Excised callus from the explants was subcultured onto fresh media of the same recipe for further proliferation. After 10–12 d of subculture, a yellow, globular, friable embryogenic callus was obtained from the initial callus. The highest percentage of embryogenic calli obtained at 40.0% was observed on media containing 2.2 μM 2,4-D. The highest regeneration rate of 46.6% was observed on MS media supplemented with 0.4 μM 2,4-D and 2.2 μM benzylaminopurine (BA). Regenerated shoots were rooted in MS basal medium. Plants with well-developed roots were transferred to pots containing a soil mix and acclimatized in greenhouse conditions. Four weeks post-transfer, acclimatized plants showed 100% survival and remained healthy and green. This is the first report of a successful method for induction of somatic embryogenesis with subsequent plant regeneration in centipede grass and demonstrates the establishment of embryogenic callus and efficient plant regeneration with potential application in the development of genetic transformation systems for centipede grass.     

Optimization of a mature embryo-based in vitro culture system for high-frequency somatic embryogenic callus induction and plant regeneration from japonica rice cultivars

Optimization of the conditions for an efficient induction of somatic embryogenic calli and regeneration of plants from mature seeds of japonica rice cultivars was attempted. The number, color, size, shape, and appearance time of the induced embryogenic calli varied among the rice cultivars depending on the type of basal medium (LS, MS, N6). Presence of adequate amount of sucrose in the medium was an absolute requirement for embryogenic callus formation and shoot induction. Induction of the embryogenic calli, whose overall rates ranged from 30 to 56%, was most efficient in N6 medium supplemented with 3.0 mg l^–1 of 2,4-D and 30 g l^–1 of sucrose. Agar concentration in the regeneration medium was also critical for the shoot induction. Kinetin was found to be more effective for shoot regeneration compared with BA, while the highest shoot regeneration frequencies were observed when either cytokinin was combined with high concentration (2.0 mg l^–1) of NAA. The optimal concentration of kinetin for the highest shoot regeneration frequency (6777%) was different among the cultivars tested. The embryogenic calli-derived shoots rooted on a plant growth regulator-free MS medium were successfully established in soil, producing fertile seeds.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated high frequency transformation in dwarf recalcitrant rice cultivars

The Agrobacterium-mediated transformation was done in rice (Oryza sativa L. var. indica) cv. HKR126 and elite cross-bred cv. Pusa Basmati1 (PB1), using strain LBA4404 containing pCAMBIA1300 cloned with gene cassettes; potato proteinase inhibitor and Bacillus thuringiensis endotoxin (plasmid JDW53) or mannitol-1-phosphate dehydrogenase (plasmid RKJ108). Co-cultivation with scutellar-calli derived from mature seeds showed stable and highly efficient transformation. In cvs. HKR126 and PB1, 35 % and 41 % of hygromycin resistant calli were obtained. The transformation efficiency in PB1 (22.0 %) was much higher than in HKR126 (12.5 %). Similarly, PB1 had higher plant regeneration efficiency than HKR126. The shoots regenerated per callus were, 3–4 in HKR126 and 5–6 in PB1. The transformation efficiency with pRKJ108 (18.6 %) was higher than pJDW53 (15.9 %). Polymerase chain reaction (PCR) analysis showed the presence of transgenes in regenerated transgenic plants of both cultivars.     

Production of herbicide-resistant transgenic sweet potato plants through <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> method

Transgenic herbicide-resistant sweet potato plants [Ipomoea batatas (L.) Lam.] were produced through Agrobacterium-mediated transformation system. Embryogenic calli derived from shoot apical meristems were infected with Agrobacterium tumefaciens strain EHA105 harboring the pCAMBIA3301 vector containing the bar gene encoding phosphinothricin N-acetyltransferase (PAT) and the gusA gene encoding β-glucuronidase (GUS). The PPT-resistant calli and plants were selected with 5 and 2.5 mg l⁻¹ PPT, respectively. Soil-grown plants were obtained 28–36 weeks after Agrobacterium-mediated transformation. Genetic transformation of the regenerated plants growing under selection was demonstrated by PCR, and Southern blot analysis revealed that one to three copies of the transgene were integrated into the plant genome of each transgenic plant. Expression of the bar gene in transgenic plants was confirmed by RT-PCR and application of herbicide. Transgenic plants sprayed with Basta containing 900 mg l⁻¹ of glufosinate ammonium remained green and healthy. The transformation frequency was 2.8% determined by herbicide application which was high when compared to our previous biolistic method. In addition, possible problems with multiple copies of transgene were also discussed. We therefore report here a successful and reliable Agrobacterium-mediated transformation of the bar gene conferring herbicide-resistance and this method may be useful for routine transformation and has the potential to develop new varieties of sweet potato with several important genes for value-added traits such as enhanced tolerance to the herbicide Basta.