Co-transformation Efficiency Research Articles

Characterization of Agrobacterium-mediated co-transformation events in rice using green and red fluorescent proteins.

Biotechnologists seeking to develop marker-free transgenic plants have established co-transformation methods. For co-transformation using mixed Agrobacterium strains, the mix ratio of Agrobacterium strains and selection scheme may influence co-transformation frequency. This study used fluorescent GFP and RFP markers to compose different selection schemes for observation of the selective dynamics of transformed rice cells and to investigate the factors affecting co-transformation efficiency. We utilized GFP and RFP markers in co-transformation and tested the combinations of an antibiotic-selectable vector (pGFP-HPT) and a single RFP vector (pRFP) and of two antibiotic-selectable vectors (pGFP-HPT and pRFP-HPT) in rice. The pGFP-HPT/pRFP combination resulted in 70.9% to 81.2% of co-transformation frequencies while lower frequencies (56.6% on average) were obtained with the pGFP-HPT/pRFP-HPT combination. Based on GFP/RFP segregation patterns, 55% of the pGFP-HPT/pRFP co-transformants contained unlinked T-DNAs and segregated single RFP progeny, which simulated the selection process of marker-free transgenic plants that carry an actual gene of interest. Transgene expression levels in the rice lines varied as revealed by RT-PCR, and tandem-linked T-DNAs were detected in co-transformants, suggesting that transgene expression might be affected by duplicated T-DNA structures. Co-transformation via mixed Agrobacterium strains is feasible, and approximately 55% of the pGFP-HPT/pRFP co-transformants contained unlinked T-DNAs and segregated single RFP progeny. The pGFP-HPT/pRFP and the pGFP-HPT/pRFP-HPT vector combinations showed distinctive selective dynamics of transformed rice cells, suggesting that co-transformation efficiency depends on both vector system and selection scheme.

Agrobacterium rhizogenes-mediated hairy root transformation as an efficient system for gene function analysis in Litchi chinensis

BackgroundLitchi chinensis Sonn. is an economically important fruit tree in tropical and subtropical regions. However, litchi functional genomics is severely hindered due to its recalcitrance to regeneration and stable transformation. Agrobacterium rhizogenes-mediated hairy root transgenic system provide an alternative to study functional genomics in woody plants. However, the hairy root transgenic system has not been established in litchi.ResultsIn this study, we report a rapid and highly efficient A. rhizogenes-mediated co-transformation system in L. chinensis using Green Fluorescent Protein (GFP) gene as a marker. Both leaf discs and stem segments of L. chinensis cv. ‘Fenhongguiwei’ seedlings were able to induce transgenic hairy roots. The optimal procedure involved the use of stem segments as explants, infection by A. rhizogenes strain MSU440 at optical density (OD600) of 0.7 for 10 min and co-cultivation for 3 days, with a co-transformation efficiency of 9.33%. Furthermore, the hairy root transgenic system was successfully used to validate the function of the key anthocyanin regulatory gene LcMYB1 in litchi. Over-expression of LcMYB1 produced red hairy roots, which accumulated higher contents of anthocyanins, proanthocyanins, and flavonols. Additionally, the genes involving in the flavonoid pathway were strongly activated in the red hairy roots.ConclusionWe first established a rapid and efficient transformation system for the study of gene function in hairy roots of litchi using A. rhizogenes strain MSU440 by optimizing parameters. This hairy root transgenic system was effective for gene function analysis in litchi using the key anthocyanin regulator gene LcMYB1 as an example.

A Cisgenic Approach in the Transformation of Bread Wheat cv. Saratovskaya 29 with Class I Chitinase Gene

Background: Bread wheat is one of the major crops grown worldwide, showing high demand for new varieties with traits such as pathogen resistance. As the public acceptance of transgenic plants remains low, a novel approach – cisgenesis – is being developed to introduce the genes from the same or closely related species. Objective: This study presents a cisgenic approach used for the transformation of wheat with class I chitinase gene derived from T. aestivum cv. Stepnaya 15, co-transformed with acetohydroxyacid synthase gene that provides tolerance to imidazolinone herbicides. Methods: Calli from immature embryos of spring bread wheat Triticum aestivum cv. Saratovskaya 29 were used for co-transformation with two independent Minimal Expression Units (MEUs): class I chitinase and Acetohydroxyacid Synthase (AHAS) gene. For identification of cisgenic plants, genomic DNA was extracted from the leaves of imazethapyr-resistant regenerant plants at the plantlets stage and screened by polymerase chain reaction. The efficiency of transformation was calculated as the relation of regenerated plants with chitinase gene insert to the total number of calli in the experiment. Results: The average transformation efficiency in four series of experiments (total number of calli - 2299) was found to be 1.84% (ranging from 0.3% to 3.4%), while total co-transformation efficiency reached 87.93%. Conclusion: The high efficiency of co-transformation in the experiment promotes it as a very useful technique for the production of wheat lines, free of the selectable marker gene. To our knowledge, this is the first report of cisgenic bread wheat, where both target and selectable genes are derived from wheat.

Comparison of three Agrobacterium-mediated co-transformation methods for generating marker-free transgenic Brassica napus plants

BackgroundGeneration of marker-free transgenic plants is very important to the regulatory permission and commercial release of transgenic crops. Co-transformation methods that enable the removal of selectable marker genes have been extensively used because they are simple and clean. Few comparisons are currently available between different strain/plasmid co-transformation systems, and also data are related to variation in co-transformation frequencies caused by other details of the vector design.ResultsIn this study, we constructed three vector systems for the co-transformation of allotetraploid Brassica napus (B. napus) mediated by Agrobacterium tumefaciens and compared these co-transformation methods. We tested a mixed-strain system, in which a single T-DNA is harbored in two plasmids, as well as two “double T-DNA” vector systems, in which two independent T-DNAs are harbored in one plasmid in a tandem orientation or in an inverted orientation. As confirmed by the use of PCR analysis, test strips, and Southern blot, the average co-transformation frequencies from these systems ranged from 24 to 81% in T0 plants, with the highest frequency of 81% for 1:1 treatment of the mixed-strain system. These vector systems are valuable for generating marker-free transgenic B. napus plants, and marker-free plants were successfully obtained in the T1 generation from 50 to 77% of T0 transgenic lines using these systems, with the highest frequency of 77% for “double T-DNA” vector systems of pBID RT Enhanced. We further found that marker-free B. napus plants were more frequently encountered in the progeny of transgenic lines which has only one or two marker gene copies in the T0 generation. Two types of herbicide resistant transgenic B. napus plants, Bar+ with phosphinothricin resistance and Bar+EPSPS+GOX+ with phosphinothricin and glyphosate resistance, were obtained.ConclusionWe were successful in removing selectable marker genes in transgenic B. napus plants using all three co-transformation systems developed in this study. It was proved that if a appropriate mole ratio was designed for the specific length ratio of the twin T-DNAs for the mixed-strain method, high unlinked co-insertion frequency and overall success frequency could be achieved. Our study provides useful information for the construction of efficient co-transformation system for marker-free transgenic crop production and developed transgenic B. napus with various types of herbicide resistance.

Enhancing fructooligosaccharides production by genetic improvement of the industrial fungus Aspergillus niger ATCC 20611

Aspergillus niger ATCC20611 is one of the most potent filamentous fungi used commercially for production of fructooligosaccharides (FOS), which are prospective components of functional food by stimulating probiotic bacteria in the human gut. However, current strategies for improving FOS yield still rely on production process development. The genetic engineering approach hasn’t been applied in industrial strains to increase FOS production level. Here, an optimized polyethylene glycol (PEG)-mediated protoplast transformation system was established in A. niger ATCC 20611 and used for further strain improvement. The pyrithiamine resistance gene (ptrA) was selected as a dominant marker and protoplasts were prepared with high concentration (up to 108g−1 wet weight mycelium) by using mixed cell wall-lysing enzymes. The transformation frequency with ptrA can reach 30–50 transformants per μg of DNA. In addition, the efficiency of co-transformation with the EGFP reporter gene (egfp) was high (approx. 82%). Furthermore, an activity-improved variant of β-fructofuranosidase, FopA(A178P), was successfully overexpressed in A. niger ATCC 20611 by using the transformation system. The transformant, CM6, exhibited a 58% increase in specific β-fructofuranosidase activity (up to 507U/g), compared to the parental strain (320U/g), and effectively reduced the time needed for completion of FOS synthesis. These results illustrate the feasibility of strain improvement through genetic engineering for further enhancement of FOS production level.

Generation of Marker- and/or Backbone-Free Transgenic Wheat Plants via Agrobacterium-Mediated Transformation.

Horizontal transfer of antibiotic resistance genes to animals and vertical transfer of herbicide resistance genes to the weedy relatives are perceived as major biosafety concerns in genetically modified (GM) crops. In this study, five novel vectors which used gusA and bar as a reporter gene and a selection marker gene, respectively, were constructed based on the pCLEAN dual binary vector system. Among these vectors, 1G7B and 5G7B carried two T-DNAs located on two respective plasmids with 5G7B possessing an additional virGwt gene. 5LBTG154 and 5TGTB154 carried two T-DNAs in the target plasmid with either one or double right borders, and 5BTG154 carried the selectable marker gene on the backbone outside of the T-DNA left border in the target plasmid. In addition, 5BTG154, 5LBTG154, and 5TGTB154 used pAL154 as a helper plasmid which contains Komari fragment to facilitate transformation. These five dual binary vector combinations were transformed into Agrobacterium strain AGL1 and used to transform durum wheat cv Stewart 63. Evaluation of the co-transformation efficiencies, the frequencies of marker-free transgenic plants, and integration of backbone sequences in the obtained transgenic lines indicated that two vectors (5G7B and 5TGTB154) were more efficient in generating marker-free transgenic wheat plants with no or minimal integration of backbone sequences in the wheat genome. The vector series developed in this study for generation of marker- and/or backbone-free transgenic wheat plants via Agrobacterium-mediated transformation will be useful to facilitate the creation of “clean” GM wheat containing only the foreign genes of agronomic importance.

Stacking resistance to crown gall and nematodes in walnut rootstocks

BackgroundCrown gall (CG) (Agrobacterium tumefaciens) and the root lesion nematodes (RLNs) (Pratylenchus vulnus) are major challenges faced by the California walnut industry, reducing productivity and increasing the cost of establishing and maintaining orchards. Current nematode control strategies include nematicides, crop rotation, and tolerant cultivars, but these methods have limits. Developing genetic resistance through novel approaches like RNA interference (RNAi) can address these problems. RNAi-mediated silencing of CG disease in walnut (Juglans regia L.) has been achieved previously. We sought to place both CG and nematode resistance into a single walnut rootstock genotype using co-transformation to stack the resistance genes. A. tumefaciens, carrying self-complimentary iaaM and ipt transgenes, and Agrobacterium rhizogenes, carrying a self-complimentary Pv010 gene from P. vulnus, were used as co-transformation vectors. RolABC genes were introduced by the resident T-DNA in the A. rhizogenes Ri-plasmid used as a vector for plant transformation. Pv010 and Pv194 (transgenic control) genes were also transferred separately using A. tumefaciens. To test for resistance, transformed walnut roots were challenged with P. vulnus and microshoots were challenged with a virulent strain of A. tumefaciens.ResultsCombining the two bacterial strains at a 1:1 rather than 1:3 ratio increased the co-transformation efficiency. Although complete immunity to nematode infection was not observed, transgenic lines yielded up to 79% fewer nematodes per root following in vitro co-culture than untransformed controls. Transgenic line 33-3-1 exhibited complete crown gall control and 32% fewer nematodes. The transgenic plants had thicker, longer roots than untransformed controls possibly due to insertion of rolABC genes. When the Pv010 gene was present in roots with or without rolABC genes there was partial or complete control of RLNs. Transformation using only one vector showed 100% control in some lines.ConclusionsCG and nematode resistance gene stacking controlled CG and RLNs simultaneously in walnuts. Silencing genes encoding iaaM, ipt, and Pv010 decrease CG formation and RLNs populations in walnut. Beneficial plant genotype and phenotype changes are caused by co-transformation using A. tumefaciens and A. rhizogenes strains. Viable resistance against root lesion nematodes in walnut plants may be accomplished in the future using this gene stacking technology.

A Novel Co-immunoprecipitation Protocol Based on Protoplast Transient Gene Expression for Studying Protein–protein Interactions in Rice

Protein–protein interactions constitute the regulatory network that coordinates diverse cellular functions. Co-immunoprecipitation (co-IP) is a widely used and effective technique to study protein–protein interactions in living cells. However, the time and cost for the preparation of a highly specific antibody is the major disadvantage associated with this technique. In the present study, a co-IP system was developed to detect protein–protein interactions based on an improved protoplast transient expression system by using commercially available antibodies. This co-IP system eliminates the need for specific antibody preparation and transgenic plant production. Leaf sheaths of rice green seedlings were used for the protoplast transient expression system which demonstrated high transformation and co-transformation efficiencies of plasmids. The transient expression system developed by this study is suitable for subcellular localization and protein detection. This work provides a rapid, reliable, and cost-effective system to study transient gene expression, protein subcellular localization, and characterization of protein–protein interactions in vivo.

A comparison of strategies for multiple-gene co-transformation via hairy root induction

Hairy root is a transformed root tissue in which transfer DNA (T-DNA) is inserted in the genome by Agrobacterium rhizogenes. To establish a system for multiple-gene co-transformation in hairy roots, we evaluated four different strategies using A. rhizogenes. The genes gusA and mgfp5 were located in separate plasmids, which were transformed into two different batches of A. rhizogenes (strategy 2AR) or a single batch (strategy 2BV). The two reporter genes were also inserted in one T-DNA (strategy 1TD) or two different T-DNAs (strategy 2TD) in a binary vector. Over 90% of infected Nicotiana tabacum leaf discs formed hairy roots in all four groups, which was not significantly different from the infection efficiency of wild-type A. rhizogenes. Proportions of co-transformed hairy roots with strategies 2AR, 2BV, 1TD, and 2TD were 65.4, 40.0, 78.6, and 82.1%, respectively, which indicated that all of the strategies were suitable for co-transformation of multiple genes. High variation in growth rate and heterologous protein expression indicated that further screening is required to identify the clone with the highest productivity. Our results indicated that strategies 1TD and 2TD achieved the highest co-transformation efficiency. Combination with strategy 2AR or 2BV provides additional options for co-transformation of multiple transgenes.

Use of the Cre-loxP recombination system as an estimate for Agrobacterium-mediated co-transformation of tobacco leaves

Agrobacterium tumefaciens-mediated transformation of tobacco leaves (Nicotiana tabacum) is used to study gene expression in a heterologous genetic background. Here, the Cre-loxP recombination system was used to detect T-DNA transfer by two A. tumefaciens cells harboring different binary vectors. Cre, under the control of the CaMV 35S promoter, was cloned into one vector, and a loxP cassette into another vector. A mixture of A. tumefaciens, in which each cell contained either a Cre- or loxP-vector, was co-infiltrated into tobacco leaves. After two days, excision of loxP-flanked DNA was detected by PCR and used as an estimate for co-transformation events. Strongest excision (> 50%) was observed when the loxP cassette was cloned into vector pPZP112 and Cre into pISV2678. This fast and easy technique can be used to assess the co-transformation efficiency of tobacco cells in future studies.

Cisgenic barley with improved phytase activity

The cisgenesis concept implies that plants are transformed only with their own genetic materials or genetic materials from closely related species capable of sexual hybridization. Furthermore, foreign sequences such as selection genes and vector-backbone sequences should be absent. We used a barley phytase gene (HvPAPhy_a) expressed during grain filling to evaluate the cisgenesis concept in barley. The marker gene elimination method was used to obtain marker-free plant lines. Here, the gene of interest and the selection gene are flanked by their own T-DNA borders to allow unlinked integration of the two genes. We analysed the transformants for co-transformation efficiency, increased phytase activities in the grain, integration of the kanamycin resistance gene of the vector-backbone and segregation between the HvPAPhy_a insert and the hygromycin resistance gene. The frequencies of the four parameters imply that it should be possible to select 11 potentially cisgenic T(1) -lines out of the 72 T(0) -lines obtained, indicating that the generation of cisgenic barley is possible at reasonable frequencies with present methods. We selected two potential cisgenic lines with a single extra copy of the HvPAPhy_a insert for further analysis. Seeds from plants homozygous for the insert showed 2.6- and 2.8-fold increases in phytase activities and the activity levels were stable over the three generations analysed. In one of the selected lines, the flanking sequences from both the left and right T-DNA borders were analysed. These sequences confirmed the absence of truncated vector-backbone sequences linked to the borders. The described line should therefore be classified as cisgenic.

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

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

Selectable marker elimination in the T0 generation by Agrobacterium-mediated co-transformation involving Mungbean yellow mosaic virus TrAP as a non-conditional negative selectable marker and bar for transient positive selection

Transient selection involving the bar gene and non-conditional negative selection against stable T-DNA integration through the use of the Mungbean yellow mosaic virus (MYMV) transcriptional activator protein gene (TrAP) were used in a novel co-transformation strategy to generate selectable marker gene (SMG)-eliminated transgenic tobacco plants in the T(0) generation itself. Two compatible binary plasmids, pCam-bar-TrAP-gus harbouring bar as an SMG and the MYMV TrAP gene as a non-conditional negative selectable marker, and pGA472 with the nptII gene as an unselected experimental gene of interest (GOI) were placed in the Agrobacterium tumefaciens strain EHA105 and used for co-transformation. Transient selection with 5 mg l(-1) phosphinothricin (PPT) for 2-4 weeks and subsequent establishment in a PPT-minus medium yielded 114 plants from 200 leaf discs. The unselected nptII gene was detected by Southern blot analysis in 13 plants, revealing a co-transformation efficiency of 11.5%. Five of these plants harboured only the nptII gene (GOI) and not the bar gene (SMG). Thus, SMG elimination was achieved in the T(0) generation itself in 4.4% (5/114) of plants, which were transiently selected for 2-4 weeks on PPT. MYMV TrAP, a non-conditional negative selectable marker, effectively reduced the recovery of plants with stable integration of the SMG (bar).

Highly efficient transformation of intact yeast-like conidium cells of Tremella fuciformis by electroporation

Tremella fuciformis is one of higher basidiomycetes. Its basidiospore can reproduce yeast-like conidia, also called the blastospore by budding. The yeast-like conidia of T. fuciformis is monokaryotic and easy to culture by submerged fermentation similar to yeast. So it is a good recipient cell for exogenous gene expression. In this study, two expression vectors pGlg-gfp containing gpd-Gl promoter and gfp gene and pGlg-hph containing gpd-Gl promoter and hph gene were constructed. The lowest sensitive concentration of hygromycin for the blastospore was determined on three types of media. Our experiments showed that the lowest sensitive concentration of hygromycin for the blastospore was 5 microg/mL on MA medium. The intact blastospores were transformed with the expression vector pGlg-hph by electroporation. The putative transformants were obtained by the MA selective medium. Experimental results showed that the most effective parameters for the electroporation of intact blastospores were obtained by using STM buffer, 1.0x10(8) cells/mL of blastospores, 200 microL in transformation volume, 6 microg plasmid, 2.0 kV/cm of electric pulse voltage, stillness culturing on MB liquid medium for 48 h after electroporation. In these transformation conditions, the efficiency reached 277 colonies/microg DNA. Co-transformation of plasmid pGlg-gfp and pGlg-hph with ratio of 1:1 was performed by electroporation with the optimal parameters. The putative co-transformants were obtained by the MA selective medium. Eight randomly selected colonies from the vast putative co-transformants were analyzed by PCR detection and Southern blotting. The experiments showed that the gfp was integrated into the genomes of three transformants. The co-transformation efficiency was 37.5%. Green fluorescence was observed under laser scanning confocal microscope in these gfp positive transformants. This indicates that the exogenous gfp can be expressed effectively in the yeast-like conidia of T. fuciformis.

Agrobacterium mediated genetic transformation of summer squash (Cucurbita pepo L. cv. Australian green) with cbf-1 using a two vector system

Efficient plant regeneration via shoot tip provided a basis for the optimization of the genetic transformation protocol. Therefore, experiments were conducted to establish an efficient in vitro regeneration protocol in summer squash for genetic co-transformation. 6-benzylaminopurine at 0.05 mg l−l was found to be optimum concentration of direct regeneration from shoot tip. Effective root system was induced in shootlets in indole-3-aceticacid 0.5 mg l−l. Two vectors namely pCAMBIA 2200 harboring marker gene nptII and pCAMBIA 0390 harboring gene, encoding C-repeat binding factor (cbf1) were used for co-transformation taking shoot tips as explants from in vitro germinated seeds. Explants were selected after co-cultivation on kanamycin supplemented medium and shoots and roots were induced. The transgenic plants were confirmed by polymerase chain reaction (PCR) and further southern blot analysis confirmed the integration of nptII and cbf1 genes in genome of summer squash with co-transformation efficiency of 0.7 percent.

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

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

Obtaining marker-free transgenic soybean plants with optimal frequency by constructing a three T-DNA binary vector

Obtaining marker-free plants with high efficiency will benefit the environmental release of transgenic crops. To achieve this point, a binary vector pNB35SVIP1 with three T-DNAs was constructed by using several mediate plasmids, in which one copy of bar gene expression cassette and two copies of VIP1 gene expression cassette were included. EHA101 Agrobacterium strain harboring the final construct was applied to transform soybean (Glycine max) cotyledon nodes. Through 2 - 3 months regeneration and selection on 3 - 5mg/L glufosinate containing medium, transgenic soybean plants were confirmed to be obtained at 0.83% - 3.16%, and co-transformation efficiency of both gene in the same individual reached up to 86.4%, based on southern blot test. By the analysis of PCR, southern blot and northern blot combining with leaf painting of herbicide in T1 progenies, 41 plants were confirmed to be eliminated of bar gene with the frequency of 7.6% . Among the T1 populations tested, the loss of the alien genes happened in 22.7% lines, the silence of bar gene took place in 27.3% lines, and VIP1 gene silence existed in 37.1% marker-free plants. The result also suggested that the plasmid with three T-DNAs might be an ideal vector to generate maker-free genetic modified organism.

Efficient transformation and expression of gfp gene in the edible mushroom Pleurotus nebrodensis

An efficient transformation method mediated by PEG-protoplasts was developed for the newly commercial edible mushroom Pleurotus nebrodensis. Two plasmids were used to co-transform protoplasts of P. nebrodensis. One plasmid is pAN7-1 containing a positive selectable marker gene hph conferring hygromycin B resistance. Another plasmid is pBlue-GFP containing a reporter gene gfp conferring green fluorescent protein. PCR and Southern blot analysis showed that hph gene or/and gfp gene were integrated into the genome of P. nebrodensis transformants. The transformation efficiency of the positive selectable marker gene hph was 3 transformants per microgram of plasmid pAN7-1 DNA, which was about 30 times higher than that previously reported in thoroughly studied Pleurotus species such as Pleurotus ostreatus. The transformation efficiency of the reporter gene gfp was 9 transformants per microgram of plasmid pBlu-GFP DNA. The co-transformation efficiency was 23.68%. This is the first report that a “reporter” gene, green fluorescent protein gene can be successfully stably expressed in this Pleurotus species.

Resistance to Anticarsia gemmatalis Hübner (Lepidoptera, Noctuidae) in transgenic soybean (Glycine max (L.) Merrill Fabales, Fabaceae) cultivar IAS5 expressing a modified Cry1Ac endotoxin

Somatic embryos of the commercial soybean (Glycine max) cultivar IAS5 were co-transformed using particle bombardment with a synthetic form of the Bacillus thuringiensis delta-endotoxin crystal protein gene cry1Ac, the β-glucuronidase reporter gene gusA and the hygromycin resistance gene hpt. Hygromycin-resistant tissues were proliferated individually to give rise to nine sets of clones corresponding to independent transformation events. The co-bombardment resulted in a co-transformation efficiency of 44%. Many histodifferentiated embryos and 30 well-developed plants were obtained. Twenty of these plants flowered and fourteen set seeds. The integration and expression of the cry1Ac, gusA and hpt transgenes into the genomes of a sample of transformed embryos and all T0, T1 ,T 2 and T3 plants were confirmed by Gus activity, PCR, Southern and western blot, and ELISA techniques. Two T0 plants out of the seven co-transformed plants produced seeds and were analyzed for patterns of integration and inheritance until the T3 generation. Bioassays indicated that the transgenic plants were highly toxic to the velvetbean caterpillar Anticarsia gemmatalis, thus offering a potential for effective insect resistance in soybean.

Optimization of wheat co-transformation procedure with gene cassettes resulted in an improvement in transformation frequency

Genetic manipulation using gene cassettes was applied to the elite wheat variety EM12 via particle bombardment, which allows an improvement in transformation frequency. We simultaneously transferred to wheat immature embryos with two non-linked genes, gus and bar, on either separate gene cassettes or one plasmid. The linear gene cassettes were excised and purified by restriction digestion of the plasmid, and consisted of promoters, open reading frames and terminators. No difference was observed in GUS transient expression of between gene cassettes and single whole plasmid. However, the stable transformation frequency was significantly increased to 1.1% using gene cassettes, compared with 0.4% when using single plasmid. Procedures of the efficient co-transformation with gene cassettes were developed. Factors influencing on the transformation frequency were also studied in order to optimize the procedure. These were acceleration pressure, target distance, gold particle size, the quantity ratio of gene cassettes and the age of target explants. Based on the transient and stable expression of the gus gene cassettes, optimization of transformation parameters improved the reproducibility of transformation in the elite wheat variety.