Fate of introduced genetic markers in transformed root clones and regenerated plants of monohaploid and diploid potato genotypes

Summary Agrobacterium transformation of stem internodes of four monohaploid (839-79, 849-7, 851-23, 855-1) and two diploid (M9 and HH260) potato genotypes using hairy root-inducing single (LBA 1020, LBA 9365, LBA 9402) and binary (LBA 1060KG) vectors is reported. Various media and successive culture steps were tested for plant regeneration from different transformed root clones. The fate of introduced genetic markers in root clones and regenerated plants (hairy root phenotype, hormone autotrophy, opine production, kanamycin resistance, -glucuronidase activity), the ploidy stability and protoplast yield were analysed. The transformation efficiency of stem internodes (hairy root production) and the regeneration capacity of the transformed root clones greatly differed within and between the various potato genotypes. The regenerated plants obtained after transformation with both types of vectors often showed the absence of one or more genetic markers. However, transformation with the binary Agrobacterium vector generally resulted in the stable presence of the opines in all transformed root clones and most regenerated plants. In HH260, transformation efficiency, plant regeneration of transformed root clones, protoplast yield and ploidy stability were the highest as compared to the other genotypes. The application of these transformed plants as marker lines in gene mapping and gene expression studies is indicated.     

Efficient Method of Agrobacterium-mediated Transformation for Triticale (x Triticosecale Wittmack)

Transgenic plants of triticale cv. Wanad were obtained after transformation using three combinations of strain/vectors. Two of them were hypervirulent Agrobacterium tumefaciens strains (AGL1 and EHA101) with vectors containing bar under maize ubiquitin 1 promoter (pDM805), and both hpt under p35S and nptII under pnos (pGAH). The third one was a regular LBA4404 strain containing super-binary plasmid pTOK233 with selection genes the same as in pGAH. The efficiency of transformation was from 0 to 16% and it was dependent on the selection factor, auxin pretreatment, and the strain/vector combination. The highest number of transgenic plants was obtained after transformation with LBA4404(pTOK233) and kanamycin selection. Pretreatment of explants with picloram led to the highest number of plants obtained after transformation with both Agrobacterium/vector systems LBA4404(pTOK233) and EHA101(pGAH) and selected with kanamycin. Transgenic character of selected plants was examined by PCR using specific primers for bar, gus, nptII, and hpt and confirmed by Southern blot hybridization analysis. There was no GUS expression in T₀ transgenic plants transformed with gus under p35S. However the GUS expression was detectable in the progeny of some lines. Only 30% of 46 transgenic lines showed Mendelian segregation of GUS expressing to GUS not expressing plants. In the remaining 70% the segregation was non-Mendelian and the rate was much lower than 3:1. Factors that might effect expression of transgenes in allohexaploid monocot species are discussed.     

Green fluorescent protein as an efficient selection marker for Agrobacterium rhizogenes mediated carrot transformation

Agrobacterium rhizogenes mediated transformation combined with a visual selection for green fluorescent protein (GFP) has been applied effectively in carrot (Daucus carota L.) transformation. Carrot root discs were inoculated with A4, A4T, LBA1334 and LBA9402 strains, all bearing gfp gene in pBIN-m-gfp5-ER. The results indicate that transformed adventitious roots can be visually selected solely based on GFP fluorescence with a very high accuracy. The method requires no selection agents like antibiotics or herbicides and enables a reduction of labour and time necessary for tissue culture. Moreover, individual transformants can be easily excised from the host tissue and cultured separately. All of the 12 used carrot cultivars produced transformed adventitious roots and the frequency of discs producing GFP expressing adventitious roots varied from 13 to 85%. The highest transformation rate was found for A4T and LBA1334 strains possessing chromosomal background of A. tumefaciens C58. The results encourage that visual selection of transformed, fluorescing adventitious roots can be highly effective and applied routinely for the production of carrot transgenic plants.     

Study of the factors influencing Agrobacterium-mediated transformation of pea (Pisum sativum L.)

Factors influencing the efficiency of Agrobacterium-mediated transformation of pea were tested using highly efficient, direct regeneration system. The virulence of three Agrobacterium strains (octopine LBA 4404, nopaline C58C1 and succinamopine, hypervirulent EHA 105) clearly varied giving 1 transgenic plant per 100 explants for LBA 4404, 2.2 for C58C1 and 8.2 for EHA 105. To test the efficacy of selection agents we used the hypervirulent EHA 105 strain carrying pGPTV binary vector with one of four different selection genes: nptII, hpt, dhfr or bar. The mean number of transgenic, kanamycin-resistant plants for two cultivars tested was 4.2 per 100 explants and was slightly higher than the number of phosphinothricin-resistant plants (3.6 plants per 100 explants). The proportion of transgenics among kanamycin-selected plants was also higher than among phosphinothricin-resistant plants (35% and 28% respectively). There was no regeneration on hygromycin or methotrexate media (transformation with hpt and dhfr genes). Acetosyringone had no apparent influence on efficiency of transformation with hypervirulent EHA 105 strain, however it did affect the rate of transformation when moderately virulent C58C1 was used. Recovery of transgenic plants was enhanced after application of 5-azacytidine. The presence of integrated T-DNA was checked by PCR and confirmed by Southern hybridization. T-DNA was stably transmitted to the next generation.     

Transformation of protoplasts and intact cells from slowly growing embryogenic callus of wheat (Triticum aestivum L.)

A procedure for culturing protoplasts from slowly growing embryogenic calli of wheat was developed. The procedure was dependent on the ability to isolate large numbers of culturable protoplasts from slowly growing embryogenic callus. Approximately 68% of the isolated protoplasts divided, and 22% formed colonies; of the latter, 67% continued to proliferate. Plating efficiency was reduced when protoplasts were transformed by polythylene glycol, electroporation, and/or Agrobacterium. Intact cells were also directly transformed by electroporation. Direct electroporation of the Agrobacterium binary vector into intact cells resulted in a significant increase of GUS activity over the control.     

Mannose selection system used for cucumber transformation

The selectable marker system, which utilizes the pmi gene encoding for phosphomannose-isomerase that converts mannose-6-phosphate to fructose-6-phosphate, was adapted for Agrobacterium-mediated transformation of cucumber (Cucumis sativus L.). Only transformed cells were capable of utilizing mannose as a carbon source. The highest transformation frequency of 23% was obtained with 10 g/l mannose and 10 g/l sucrose in the medium. Molecular, genetic analysis, and PMI activity assay showed that the regenerated shoots contained the pmi gene and the gene was transmitted to the progeny in a Mendelian fashion. The results indicated that the mannose selection system, which is devoid of the disadvantages of antibiotic or herbicide selection, could be used for cucumber Agrobacterium-mediated transformation.     

Novel recombinant binary vectors harbouring Basta (bar) gene as a plant selectable marker for genetic transformation of plants

Genetic transformation is one of the most widely used technique in crop improvement. However, most of the binary vectors used in this technique, especially cloning based, contain antibiotic genes as selection marker that raise serious consumer and environmental concerns; moreover, they could be transferred to non-target hosts with deleterious effects. Therefore, the goal of this study was reconstruction of the widely used pBI121 binary vector by substituting the harmful antibiotic selection marker gene with a less-harmful selection marker, Basta (herbicide resistance gene). The generated vectors were designated as pBI121NB and pBI121CB, in which Basta gene was expressed under the control of Nos or CaMV 35S promoter, respectively. The successful integration of the new inserts into both the vectors was confirmed by PCR, restriction digestion and sequencing. Both these vectors were used in transforming Arabidopsis, Egyptian wheat and barley varieties using LBA4404 and GV3101 Agrobacterium strains. The surfactant Tween-20 resulted in an efficient transformation and the number of Arabidopsis transformants was about 6–9 %. Soaked seeds of wheat and barley were transformed with Agrobacterium to introduce the bacteria to the growing shoot apices. The percentage of transgenic lines was around 16–17 and 14–15 % for wheat and barley, respectively. The quantitative studies presented in this work showed that both LBA4404 and GV3101 strains were suitable for transforming Egyptian wheat and barley.     

The maize Knotted1 gene is an effective positive selectable marker gene for Agrobacterium-mediated tobacco transformation

We have assessed the use of a homeobox gene knotted1 (kn1) from maize as a selectable marker gene for plant transformation. The kn1 gene under the control of cauliflower mosaic virus 35S promoter (35S::kn1) was introduced into Nicotiana tabacum cv. Xanthi via Agrobacterium-mediated transformation. Under nonselective conditions (without antibiotic selection) on a hormone-free medium (MS), a large number of transgenic calli and shoots were obtained from explants that were infected with Agrobacterium tumefaciens LBA4404 harboring the 35S::kn1 gene. On the other hand, no calli or shoots were produced from explants that were infected with an Agrobacterium strain harboring pBI121 (nptII selection) or from uninfected controls cultured under identical conditions. Relative to kanamycin selection conferred by nptII, the use of kn1 resulted in a 3-fold increase in transformation efficiency. The transgenic status of shoots obtained was confirmed by both histochemical detection of GUS activity and molecular analysis. The results presented here suggest that kn1 gene could be used as an effective alternative selection marker with a potential to enhance plant transformation efficiency in many plant species. With kn1 gene as a selection marker gene, no antibiotic-resistance or herbicide-resistance genes are needed so that potential risks associated with the use of these traditional selection marker genes can be eliminated.     

Polymerase chain reaction analysis of transgenic plants contaminated byAgrobacterium

Agrobacterium-mediated genetic transformation is a method of choice for the development of transgenic plants. The presence of latentAgrobacterium that multiplies in the plant tissue in spite of antibiotic application confounds the results obtained by polymerase chain reaction (PCR) analysis of putative transgenic plants. The presence ofAgrobacterium can be confirmed by amplification of eitherAgrobacterium chromosomal genes or genes present out of transfer DNA (T-DNA) in the binary vector. However, the transgenic nature ofAgrobacterium-contaminated transgenic plants cannot be confirmed by PCR. Here we report a simple protocol for PCR analysis ofAgrobacterium-contaminated transgenic plants. This protocol is based on denaturation and renaturation of DNA. The contaminating plasmid vector becomes double-stranded after renaturation and is cut by a restriction enzyme having site(s) within the PCR amplicon. As a result, amplification by PCR is not possible. The genomic DNA with a few copies of the transgene remains single-stranded and unaffected by the restriction enzyme, leading to amplification by PCR. This protocol has been successfully tested with 4 different binary vectors and 3Agrobacterium tumefaciens strains: EHA105, LBA4404, and GV3101.     

High frequency <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation and plant regeneration via direct shoot formation from leaf explants in <Emphasis Type="Italic">Beta vulgaris</Emphasis> and <Emphasis Type="Italic">Beta maritima</Emphasis>

We have developed a new procedure for Agrobacterium-mediated transformation of plants in the genus Beta using shoot-base as the material for Agrobacterium infection. The frequency of regeneration from shoot bases was analyzed in seven accessions of sugarbeet (Beta vulgaris) and two accessions of B. maritima to select materials suitable for obtaining transformed plants. The frequency of transformation of the chosen accessions using Agrobacterium strain LBA4404 and selection on 150-mg/l kanamycin was found to be higher than that in previously published methods. Genomic DNA analysis and -glucuronidase reporter assays showed that the transgene was inherited and expressed in subsequent generations. In our method, shoot bases are prepared by a simple procedure, and transformation does not involve the callus phase, thus minimizing the occurrence of somaclonal variations.     

Analysis of conditions forAgrobacterium-mediated transformation of tobacco cells in suspension

We have developed anAgrobacterium-mediated transformation system, using tobacco cell suspensions, that permits evaluation of factors affecting transformation within seven days of co-cultivation. Tobacco cell transformation was determined by monitoring -glucuronidase (GUS) activity detected in plant cell extracts. The use of a chimeric gene construct, 35S-GUS/INT, containing a portable intron in theuidA reading frame, assured only plant-specific GUS expression. During the co-cultivation period, induction of the bacterialvir-region was monitored using a heterologous gene construct composed of avirB promoter fragment from pTiC58 fused to the chloramphenicol acetyltranferase (CAT) gene ofTn9. Tobacco cell transformants were confirmed by antibiotic selection of transformed plant cells and by X-Gluc staining. Maximum transformation was obtained when plant suspension cultures were growing rapidly which also was coincidental with elevated levels of bacterialvir-region expression. One week after co-cultivation, the transformed cultures exhibited a stable pattern of GUS activity which remained constant without antibiotic selection. The system was used to compare the virulence of a number ofAgrobacterium strains. GUS activity of plant cells co-cultivated with a strain containing a cointegrate plasmid was 3-fold higher than that of one with a binary configuration of the T-DNA. When the co-cultivatingAgrobacterium strain also carried the plasmid used to monitorvir induction, the frequency of transformation was reduced by as much, as 97%.     

Improved <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated genetic transformation of GNA transgenic sugarcane

Six plasmids carrying a snowdrop lectin (Galanthus nivalis agglutinin, GNA) and one of three selection markers were successfully transferred into two sugarcane cultivars (FN81–745 and Badila) via Agrobacterium-mediated transformation. Agrobacterium strains LBA4404, EHA105 and A281 that harboured a super-binary vector were used for sugarcane transformation. The use of the hygromycin (Hyg) resistance gene (hpt II), phosphinothrincin (PPT) resistance gene (bar) or G418 resistance gene (npt II) as a screenable marker facilitated the initial selection of GNA transgenic sugarcane callus with different efficiencies and helped the rapid segregation of individual transformation events. All the three selective marker genes were controlled by CaMV 35S promoter, while GNA gene was controlled by promoter of RSs-1 (rice sucrose synthase-1) or Ubi (maize ubiquitin). Factors important to successful transformation mediated by Agrobacterium tumefaciens were optimized, which included concentration of A. tumefaciens, medium composition, co-cultivated methods with plant tissue, strain virulence and different selective marker genes. An efficient protocol for sugarcane transformation mediated by A. tumefaciens was established. The GNA gene has been integrated into sugarcane genome as demonstrated by PCR and Southern dot blotting detections. The preliminary results from bioassay demonstrated a significant resistance of the transgenic sugarcane plants to woolly aphid (Ceratovacuna lanigera Zehnther) indicating thus the possibility for obtaining a transgenic sugarcane cultivar with resistance to woolly aphid.     

Recent advances in barley transformation

Barley, an important member of the cereals, has been successfully transformed through various methods such as particle bombardment, Agrobacterium tumefaciens, DNA uptake, and electroporation. Initially, the transformation in barley concentrated on developing protocols using marker genes such as gus, bar, and hpt. Immature embryos and callus derived from immature embryos were targeted for transformation. Subsequently, genes of agronomic and malting importance have been deployed in barley. Particle bombardment appears to be the preferred choice for barley transformation in the majority of the reports, although Agrobacterium-mediated transformation is being used more often. The current review focuses on the challenges encountered in barley transformation such as somaclonal variation, development of transformation systems for commercial cultivars, gene expression, stability and inheritance, and gene flow. Newer markers such as the green fluorescent protein (gfp), firefly luciferase, and phosphomannose isomerase were found to be useful in the selection of transgenic plants. Tissue-specific promoters such as those for B1-hordein and D-hordein genes, and spike-specific promoters, are increasingly used to drive gene expression. The review also describes recent research on gene-tagging through transformation, insertion of disease resistance, and abiotic stress resistance genes, transformation with genes for improved malting quality, nutrient content, feed quality, and the production of feed enzymes and pharmaceutical compounds.     

Efficient transformation of Arabidopsis thaliana: comparison of the efficiencies with various organs,plant ecotypes and Agrobacterium strains

Summary The efficiency of Agrobacterium-mediated transformation of Arabidopsis thaliana was compared with different organs, Arabidopsis ecotypes, and Agrobacterium strains. Efficiency of shoot regeneration was examined using hypocotyl, cotyledon and root explants prepared from young seedlings. Hypocotyl expiants had the highest regeneration efficiency in all of the four Arabidopsis ecotypes tested, when based on a tissue culture system of callus-inducing medium (CIM: Valvekens et al. 1988) and shoot-inducing medium (SIM: Feldmann and Marks 1986). Histochemical analysis using the ß-glucuronidase (GUS) reporter gene showed that the gusA gene expression increased as the period of preincubation on CIM was extended, suggesting that dividing cells are susceptible to Agrobacterium infection. In order to obtain transgenic shoots, hypocotyl explants preincubated for 7 or 8 days on CIM were infected with Agrobacterium containing a binary vector which carries two drug-resistant genes as selection markers, and transferred to SIM for selection of transformed shoots. Of four Arabidopsis ecotypes and of three Agrobacterium strains examined, Wassilewskija ecotype and EHA101 strain showed the highest efficiency of regeneration of transformed shoots. By combining the most efficient factors of preincubation period, Arabidopsis ecotype, tissue, and bacterial strain, we obtained a transformation efficiency of about 80–90%. Southern analysis of 124 transgenic plants showed that 44% had one copy of inserted T-DNA while the others had more than one copy.Abbreviations AIM Agrobacterium infection medium - CIM callus-inducing medium - CTAB cetyltrimethylammonium bromide - 2,4-D 2,4-dichlorophenoxy-acetic acid - GUS ß-glucuronidase - hph hygromycin phosphotransferase - IAA indole-3-acetic acid - IBA indole-3-butyric acid - 2ip N -(2-isopentenyl) adenine - NPTII neomycin phosphotransferase II - RIM root-inducing medium - 35S cauliflower mosaic virus 35S promoter - SIM shoot-inducing medium     

Genetic transformation of strawberry: Stable integration of a gene to control biosynthesis of ethylene

Summary Efficient methods ofAgrobacterium-mediated transformation are described for two Pacific Northwest cultivars of strawberry (Fragaria &#x00D7;ananassa), Tristar and Totem. We report stable incorporation of a gene for control of ethylene biosynthesis, into strawberry (cultivar Totem) for the first time. Cultivar Tristar was transformed with disarmed strains ofAgrobacterium tumefaciens (A. tumefaciens), LBA4404 or EHA101, containing a binary vector with marker genesuidA andnptII. Cultivar Totem was transformed withA. tumefaciens strains EHA101 or EHA105 harboring binary vectors with selectable marker genesnptII orhpt and with a gene for S-adenosylmethionine hydrolase (SAMase) for control of ethylene biosynthesis. The frequency of transgenic shoots ranged from 12.5% to 58.8% of the original treated explants when using plasmids containing the gene encoding SAMase. Primary shoot regenerants obtained on selection medium were subjected to several iterations of tissue isolation and reculture on higher stringency selection medium for recovering uniformly transformed plantlets. Transgenic plants were confirmed by their ability to undergo rooting on medium with selection at 60 mg/liter kanamycin or 10 mg/liter hygromycin. About 95&#x2013;100% of the transformation events from different experiments were capable of profuse rooting in the presence of selection. Insertion of the SAMase gene and its integration into the strawberry genome were confirmed by Southern hybridization. About 500 plants from 250 independent transgenic events have been successfully transferred to soil for further evaluation.     

Agrobacterium-mediated sorghum transformation

Agrobacterium tumefaciens was used to genetically transform sorghum. Immature embryos of a public (P898012) and a commercial line (PHI391) of sorghum were used as the target explants. The Agrobacterium strain used was LBA4404 carrying a `Super-binary' vector with a bar gene as a selectable marker for herbicide resistance in the plant cells. A series of parameter tests was used to establish a baseline for conditions to be used in stable transformation experiments. A number of different transformation conditions were tested and a total of 131 stably transformed events were produced from 6175 embryos in these two sorghum lines. Statistical analysis showed that the source of the embryos had a very significant impact on transformation efficiency, with field-grown embryos producing a higher transformation frequency than greenhouse-grown embryos. Southern blot analysis of DNA from leaf tissues of T₀ plants confirmed the integration of the T-DNA into the sorghum genome. Mendelian segregation in the T₁ generation was confirmed by herbicide resistance screening. This is the first report of successful use of Agrobacterium for production of stably transformed sorghum plants. The Agrobacterium method we used yields a higher frequency of stable transformation that other methods reported previously.     

Factors affecting transformation efficiency of embryogenic callus of Upland cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis>) with <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

A reliable and high-efficiency system of transforming embryogenic callus (EC) mediated by Agrobacterium tumefaciens was developed in cotton. Various aspects of transformation were examined in efforts to improve the efficiency of producing transformants. LBA4404 and C58C3, harboring the pgusBin19 plasmid containing neomycin phosphortransferase II (npt-II) gene as a selection marker, were used for transformation. The effects of Agrobacterium strains, acetosyringone (AS), co-cultivation temperature, co-cultivation duration, Agrobacterium concentration and physiological status of EC on transformation efficiency were evaluated. Strain LBA4404 proved significantly better than C58C3. Agrobacterium at a concentration of 0.5 × 10⁸ cells ml^–1 (OD₆₀₀=0.5) improved the efficiency of transformation. Relatively low co-cultivation temperature (19 °C) and short co-cultivation duration (48 h) were optimal for developing a highly efficient method of transforming EC. Concentration of AS at 50 mg l^–1 during co-cultivation significantly increased transformation efficiency. EC growing 15 days after subculture was the best physiological status for transformation. An overall scheme for producing transgenic cotton is presented, through which an average transformation rate of 15% was obtained.     

A study of some factors affectingAgrobacterium transformation and plant regeneration ofDendranthema grandiflora Tzvelev (syn.Chrysanthemum morifolium Ramat.)

In an attempt to develop a system for producing transformed plants from explants ofDendranthema grandiflora, the susceptibility of the cultivar Super White to various wild-type strains ofAgrobacterium tumefaciens andA. rhizogenes was investigated. Tumour formation was not a reliable indicator of the ability of a related disarmed strain to mediate transformation. Following inoculation of explants with disarmedAgrobacterium strains, a number of shoots developed on selective media. However, none of these shoots were transformed. By co-cultivating stem internode explants with a mixed inoculum of wild-type and disarmed strains, it was possible to obtain a callus stably transformed withAgrobacterium carrying a disarmed T-DNA. Histological analysis of explants revealed that shoot regeneration initially occurred from the cells of the epidermis and subsequently from the cortex. However, the cells which were susceptible to T-DNA transfer were confined to the vascular tissue.     

Agrobacterium tumefaciens-mediated transformation of Rhipsalidopsis gaertneri

A protocol for Agrobacterium tumefaciens-mediated genetic transformation of Rhipsalidopsis cv. CB5 was developed. Calluses derived from phylloclade explants and sub-cultured onto fresh callus induction medium over a period of 9–12 months were co-cultivated with A. tumefaciens LBA4404. Plasmid constructs carrying the nptII gene, as a selectable marker, and the reporter uidA gene were used. Transformed Rhipsalidopsis calluses with a vigorous growth phenotype were obtained by extended culture on media containing 600 mg l⁻¹ kanamycin. After 9 months of a stringent selection pressure, the removal of kanamycin from the final medium together with the culture of the transformed calluses under nutritional stress led to the formation of several transgenic adventitious shoots. Transformation was confirmed by GUS staining (for uidA gene), ELISA analysis and Southern blot hybridization (for the nptII gene). With this approach, a transformation efficiency of 22.7% was achieved. Overall results described in this study demonstrate that Agrobacterium-mediated transformation is a promising approach for this cactus species.