Factors Affecting Transformation Efficiency Research Articles

Cotyledon peeling method for passion fruit protoplasts: a versatile cell system for transient gene expression in passion fruit (Passiflora edulis).

Passion fruit (Passiflora edulis) is a perennial evergreen vine that grows mainly in tropical and subtropical regions due to its nutritional, medicinal and ornamental values. However, the molecular biology study of passion fruit is extremely hindered by the lack of an easy and efficient method for transformation. The protoplast transformation system plays a vital role in plant regeneration, gene function analysis and genome editing. Here, we present a new method ('Cotyledon Peeling Method') for simple and efficient passion fruit protoplast isolation using cotyledon as the source tissue. A high yield (2.3 × 107 protoplasts per gram of fresh tissues) and viability (76%) of protoplasts were obtained upon incubation in the enzyme solution [1% (w/v) cellulase R10, 0.25% (w/v) macerozyme R10, 0.4 M mannitol, 10 mM CaCl2, 20 mM KCl, 20 mM MES and 0.1% (w/v) BSA, pH 5.7] for 2 hours. In addition, we achieved high transfection efficiency of 83% via the polyethylene glycol (PEG)-mediated transformation with a green fluorescent protein (GFP)-tagged plasmid upon optimization. The crucial factors affecting transformation efficiency were optimized as follows: 3 μg of plasmid DNA, 5 min transfection time, PEG concentration at 40% and protoplast density of 100 × 104 cells/ml. Furthermore, the established protoplast system was successfully applied for subcellular localization analysis of multiple fluorescent organelle markers and protein-protein interaction study. Taken together, we report a simple and efficient passion fruit protoplast isolation and transformation system, and demonstrate its usage in transient gene expression for the first time in passion fruit. The protoplast system would provide essential support for various passion fruit biology studies, including genome editing, gene function analysis and whole plant regeneration.

Optimization of cabbage (Brassica oleracea var. capitata L.) protoplast transformation for genome editing using CRISPR/Cas9.

Genome editing techniques, such as Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated systems (CRISPR/Cas9) are undoubtedly becoming an indispensable tool for improving food crops and tackling agricultural challenges. In the present study, key factors affecting transformation efficiency, such as PEG4000 concentration, incubation time, and plasmid amount were evaluated to achieve efficient delivery of CRISPR/Cas9 vector into cabbage protoplasts. Using amplicon sequencing, we confirmed a significant effect of PEG4000 concentration and incubation time on the induced target mutations. By optimizing the transformation protocol, editing efficiency of 26.4% was achieved with 40 µg of plasmid and 15 minutes incubation with 50% PEG4000. While these factors strongly affected the mutation rate, the viability of the transformed protoplasts remained high. Our findings would be useful for successful genome editing in cabbage and other brassicas, as well as in research areas such as gene function analysis and subcellular localization that rely on transient transformation methods in protoplasts.

Overexpression of TcCHS Increases Pyrethrin Content When Using a Genotype-Independent Transformation System in Pyrethrum (Tanacetum cinerariifolium).

Pyrethrum (Tanacetum cinerariifolium) is one of the most important industrial crops for the extraction of pyrethrins, which are natural insecticidal compounds. Progress in pyrethrum molecular breeding with the objective of increasing pyrethrin content has been slow for lack of a suitable gene transfer system. Regeneration recalcitrance is a crucial barrier to establishing a genetic transformation system in pyrethrum. Therefore, in this study, an Agrobacterium-mediated transformation system in pyrethrum was developed using shoot apical meristems from germinated seedlings. Factors affecting transformation efficiency were optimized. Optimal conditions included explants at the “no true leaf” stage with a half apical meristem, an Agrobacterium tumefaciens cell density of OD600 = 0.5, two days of cocultivation, and the incorporation of 1.5 mg L−1 6-BA and 30 mg L−1 kanamycin into the selection medium. Under the optimized conditions, two expression cassettes (proTcCHS-GUS and proRbcS-TcCHS) were successfully transformed into pyrethrum. Polymerase chain reaction (PCR), Southern blotting, reverse-transcription quantitative PCR (RT-qPCR), and histochemical staining confirmed the identity of proTcCHS-GUS transgenic plants. PCR and RT-qPCR analyses confirmed the identity of proRbcS-TcCHS transgenic plants. The transformation efficiency was 0.83% (5 transgenic lines/600 infected explants). The relative concentration of pyrethrins in proRbcS-TcCHS transformants (OX T0-1: 1.50% or OX T0-2: 1.24%) was higher than that in nontransformed plants (WT: 0.76%). Thus, the genetic transformation system overcame the low regeneration efficiency and integrated a foreign gene into the pyrethrum genome. The new system is a suitable and effective tool for creating high-yielding cultivars of pyrethrum.

Development of Efficient, Reproducible and Stable Agrobacterium-Mediated Genetic Transformation of Five Potato Cultivars

SUMMARYThe developments in transformation technology have enabled the scientists to incorporate, mutate or substitute gene(s) leading to a particular trait; advancing it to a point where only few technical limitations remain. Genotype dependency and explant types are important factors affecting transformation efficiency in potato. In the present study, a rapid, reproducible and stable Agrobacterium-mediated transformation procedure in potato was developed by a combination of different plant growth regulators. Leaf discs and internodal explants of five cultivars of potato, i.e. Lady Olympia, Granola, Agria, Désirée and Innovator were infected with Agrobacterium tumefaciens strain LBA4404 containing pBIN19 expression vector with β-glucuronidase gusA gene under the control of 35S CaMV promoter. Kanamycin was used as plant selectable marker for screening of primary transformants at concentration of 100 mg/L. Both explants responded positively; internode being more suitable explant for better transformation efficiency. Based on GUS histochemical assay, the transformation efficiency was 22, 20, 18.6, 15 and 10% using the internodal explant, and 15, 12, 17, 8 and 6% using leaf discs as explant in Lady Olympia, Granola, Agria, Désirée and Innovator respectively. Furthermore, PCR assays confirmed the presence of gusA and nptII genes in regenerated plants. The molecular analysis in succeeding progeny showed proper integration and expression of both genes. The results suggest Lady Olympia as the best cultivar for future transformation procedures. Overall, the short duration, rapidity and reproducibility makes this protocol suitable for wider application of transgenic potato plants.

A high-throughput transient expression system for rice.

Rice is an important global crop and represents a vital source of calories for many food insecure regions. Efforts to improve this crop by improving yield, nutritional content, stress tolerance, or resilience to climate change are certain to include biotechnological approaches, which rely on the expression of transgenes in planta. The throughput and cost of currently available transgenic expression systems is frequently incompatible with modern, high‐throughput molecular cloning methods. Here, we present a protocol for isolating high yields of green rice protoplasts and for PEG‐mediated transformation of isolated protoplasts. Factors affecting transformation efficiency were investigated, and the resulting protocol is fast, cheap, robust, high‐throughput, and does not require specialist equipment. When coupled to a high‐throughput modular cloning system such as Golden Gate, this transient expression system provides a valuable resource to help break the “design‐build‐test” bottleneck by permitting the rapid screening of large numbers of transgenic expression cassettes prior to stable plant transformation. We used this system to rapidly assess the expression level, subcellular localisation, and protein aggregation pattern of nine single‐gene expression cassettes, which represent the essential component parts of the β‐cyanobacterial carboxysome.

Study of different factors affecting Agrobacterium- mediated genetic transformation in lentil (Lens culinaris Medik.)

The present work was aimed to study the various factors affecting Agrobacterium tumefaciens mediated genetic transformation of three BARI masur variety of lentil (Lens culinaris Medik.), namely, BM-4, BM-5 and BM-6. Agrobacterium strain LBA4404 containing plasmid pBI121 which carrying gus and nptII as screenable and selectable marker genes, respectively, was used for transformation. Factors affecting transformation efficiency, such as, type of explants, bacterial density, incubation time and co-cultivation period were studied. The result showed that decapitated embryo (DE) explants of BM-6 variety of lentil inoculated in Agrobacterium suspensions at density of 1.0 (OD600) for 45 minutes and co-cultivated on MS medium with hormones in the dark for 3 days gave the highest transient expression of gus gene (86.90%). The lowest percentage (15.00%) of GUS +ve explants observed in cotyledonary node (CN) of BM-5 variety of lentil when they were inoculated in Agrobacterium suspensions at density of 0.5 (OD600) for 15 minutes. Using this protocol agronomical and economically important trait could be transfer to the locally grown lentil varieties.

Genetic Transformation of Wheat: State of the Art

The review provides the latest achievements in the field of wheat transformation and analysis of the factors affecting transformation efficiency. A comparative analysis of the most commonly used methods of wheat transformation, i.e., direct gene transfer by biolistic transformation and by Agrobacterium tumefaciens in vitro and in planta, is carried out. The stages and components of methods that affect transformation efficiency are examined in detail. Since the first successful biolistic transformation of wheat in 1992 and Agrobacterium- mediated transformation in 1997, 25 to 20 years have passed. Since then, all physical and biological parameters for the heterologous DNA delivery to the wheat genome and regeneration of plant transformants in vitro have been investigated and described in detail. Information on the influence of key parameters and factors on increasing transformation efficiency of highly productive wheat varieties is presented.

Identification of novel genes in the carotenogenic and oleaginous yeast Rhodotorula toruloides through genome-wide insertional mutagenesis

BackgroundRhodotorula toruloides is an outstanding producer of lipids and carotenoids. Currently, information on the key metabolic pathways and their molecular basis of regulation remains scarce, severely limiting efforts to engineer it as an industrial host.ResultsWe have adapted Agrobacterium tumefaciens-mediated transformation (ATMT) as a gene-tagging tool for the identification of novel genes in R. toruloides. Multiple factors affecting transformation efficiency in several species in the Pucciniomycotina subphylum were optimized. The Agrobacterium transfer DNA (T-DNA) showed predominantly single-copy chromosomal integrations in R. toruloides, which were trackable by high efficiency thermal asymmetric interlaced PCR (hiTAIL-PCR). To demonstrate the application of random T-DNA insertions for strain improvement and gene hunting, 3 T-DNA insertional libraries were screened against cerulenin, nile red and tetrazolium violet respectively, resulting in the identification of 22 mutants with obvious phenotypes in fatty acid or lipid metabolism. Similarly, 5 carotenoid biosynthetic mutants were obtained through visual screening of the transformants. To further validate the gene tagging strategy, one of the carotenoid production mutants, RAM5, was analyzed in detail. The mutant had a T-DNA inserted at the putative phytoene desaturase gene CAR1. Deletion of CAR1 by homologous recombination led to a phenotype similar to RAM5 and it could be genetically complemented by re-introduction of the wild-type CAR1 genome sequence.ConclusionsT-DNA insertional mutagenesis is an efficient forward genetic tool for gene discovery in R. toruloides and related oleaginous yeast species. It is also valuable for metabolic engineering in these hosts. Further analysis of the 27 mutants identified in this study should augment our knowledge of the lipid and carotenoid biosynthesis, which may be exploited for oil and isoprenoid metabolic engineering.

A simple and efficient Agrobacterium tumefaciens-mediated plant transformation of Brassica rapa ssp. pekinensis.

The present study aims to investigate the numerous factors influencing Agrobacterium tumefaciens-mediated genetic transformation of Chinese cabbage (Brassica rapa ssp. pekinensis). Factors affecting transformation efficiency, such as age of explants, Agrobacterium concentration, and effect of acetosyringone, pre-cultivation, infection and co-cultivation time of Agrobacterium were examined. The pre-cultured hypocotyls from young seedlings prior to exposure to Agrobacterium showed higher shoot regeneration. The plant transformation with the modest A. tumefaciens concentrations (0.8 OD) and the 3 days co-cultivation periods increased transformation efficiency. Plant growth hormones [1-naphthyl acetic acid (NAA) and 6-benzyl amino purine (BAP)] were essential for callus and shoot formation. Root formation was effective in half strength MS medium without supplementation of root-inducing hormones. To maintain selection pressure, plant subculture was carried out every 2 weeks with selective antibiotics. The putative transgenic plants were acclimatized in the greenhouse. Polymerase chain reaction was performed to confirm the integration of T-DNA into the genome of transgenic plants. A transformation efficiency of 15 % was obtained. This protocol allows effective transformation and indirect regeneration of Brassica rapa.

Genotype independent and efficient Agrobacterium-mediated genetic transformation of the medicinal plant Withania somnifera Dunal

Withania somnifera one of the most reputed Indian medicinal plant has been extensively used in traditional and modern medicines as active constituents. A high frequency genotype and chemotype independent Agrobacterium-mediated transformation protocol has been developed for W. somnifera by optimizing several factors which influence T-DNA delivery. Leaf and node explants of Withania chemotype was transformed with A. tumefaciens strain GV3101 harboring pIG121Hm plasmid containing the gusA gene encoding β-glucuronidase (GUS) as a reporter gene and the hptII and the nptII gene as selection markers. Various factors affecting transformation efficiency were optimized; as 2 days preconditioning of explants on MS basal supplemented with TDZ 1 μM, Agrobacterium density at OD600 0.4 with inclusion of 100 μM acetosyringone (As) for 20 min co-inoculation duration with 48 h of co-cultivation period at 22 °C using node explants was found optimal to improved the number of GUS foci per responding explant from 36 ± 13.2 to 277.6 ± 22.0, as determined by histochemical GUS assay. The PCR and Southern blot results showed the genomic integration of transgene in Withania genome. On average basis 11 T0 transgenic plants were generated from 100 co-cultivated node explants, representing 10.6 % transformation frequency. Our results demonstrate high frequency, efficient and rapid transformation system for further genetic manipulation in Withania for producing engineered transgenic Withania shoots within very short duration of 3 months.

Improved dominant selection markers and co-culturing conditions for efficient Agrobacterium tumefaciens-mediated transformation of Ustilago scitaminea

Ustilago scitaminea is the causal agent of sugar-cane smut disease. There is, however, no genetic transformation method for it. Here we report the development of an efficient mutagenesis method based on Agrobacterium tumefaciens-mediated transformation. To improve transformation efficiency, a range of conditions, including the codon-usage preference of the selection marker gene, promoters and the culture conditions for transformation were optimized. A strong promoter to drive marker gene expression, optimized codon usage of selection marker gene, controlled water content and pH of co-culture medium were critical factors affecting transformation efficiency. Our findings provide a useful tool for genetic analysis of this important plant pathogen.

Retraction Note: High-efficiency Agrobacterium-mediated transformation of chickpea (Cicer arietinum L.) and regeneration of insect-resistant transgenic plants

To develop an efficient genetic transformation system of chickpea (Cicer arietinum L.), callus derived from mature embryonic axes of variety P-362 was transformed with Agrobacteriumtumefaciens strain LBA4404 harboring p35SGUS-INT plasmid containing the uidA gene encoding β-glucuronidase (GUS) and the nptII gene for kanamycin selection. Various factors affecting transformation efficiency were optimized; as Agrobacterium suspension at OD600 0.3 with 48 h of co-cultivation period at 20°C was found optimal for transforming 10-day-old MEA-derived callus. Inclusion of 200 μM acetosyringone, sonication for 4 s with vacuum infiltration for 6 min improved the number of GUS foci per responding explant from 1.0 to 38.6, as determined by histochemical GUS assay. For introducing the insect-resistant trait into chickpea, binary vector pRD400-cry1Ac was also transformed under optimized conditions and 18 T0 transgenic plants were generated, representing 3.6% transformation frequency. T0 transgenic plants reflected Mendelian inheritance pattern of transgene segregation in T1 progeny. PCR, RT-PCR, and Southern hybridization analysis of T0 and T1 transgenic plants confirmed stable integration of transgenes into the chickpea genome. The expression level of Bt-Cry protein in T0 and T1 transgenic chickpea plants was achieved maximum up to 116 ng mg−1 of soluble protein, which efficiently causes 100% mortality to second instar larvae of Helicoverpa armigera as analyzed by an insect mortality bioassay. Our results demonstrate an efficient and rapid transformation system of chickpea for producing non-chimeric transgenic plants with high frequency. These findings will certainly accelerate the development of chickpea plants with novel traits.

Agrobacterium tumefaciens-mediated genetic transformation of Salix matsudana Koidz. using mature seeds

An Agrobacterium tumefaciens-mediated transformation method was developed for Salix matsudana Koidz. using mature seeds as starting material. Multiple shoots were induced directly from embryonic shoot apices of germinating seeds. Although thidiazuron, 6-benzylaminopurine and zeatin induced multiple shoot induction with high frequency, zeatin (4.5 μM) was more effective for elongation of shoots and roots. The binary vector pCAMBIA1303, which contained neomycin phosphotransferase as a selectable marker gene and β-glucuronidase as a reporter gene, was used for transformation. Factors affecting transformation efficiency were examined for optimization of the procedure. Up to 35 of 180 seeds regenerated kanamycin-resistant shoots under optimal transformation conditions as follows: seeds were precultured for 4 days, apices of embryonic shoots were removed and infected with A. tumefaciens strain LBA4404 grown to a cell density equivalent (OD600) of 0.6, and then the infected explants were cultivated at 21 °C for 4 days. Storage of seeds at -20 °C for as long as 3 years had no significant effect on the induction of kanamycin-resistant shoots. Using this method, transgenic plants were obtained within ∼5 months with a transformation frequency of 7.2%. Analysis by polymerase chain reaction (PCR) showed that 36.4-93.8% of plants from all 13 tested kanamycin-resistant lines were PCR positive. Several 'escapes' were eliminated by a second round of selection. PCR, Southern blot and reverse transcriptase-PCR analyses of selected transgenic individuals 2 years after cutting propagation confirmed the successful generation of stable transformants. Our method, which minimizes the duration of axenic culture, may provide an alternative procedure for transformation of other recalcitrant Salix species.

Agrobacterium tumefaciens-mediated genetic transformation of the Taxol-producing endophytic fungus Ozonium sp EFY21

An efficient Agrobacterium tumefaciens-mediated genetic transformation method was successfully established for a newly isolated Taxol-producing fungus, Ozonium sp EFY21. A specific hygromycin B resistance expression vector, pCAMBIA1304'AN7-1, was constructed for fungal transformation. Key factors affecting transformation efficiency were thoroughly investigated and optimized. PCR amplification and Southern hybridization were used to verify the transformation events. This study should pave the way for future genetic modification studies of Ozonium sp EFY21.

RETRACTED ARTICLE: High-efficiency Agrobacterium-mediated transformation of chickpea (Cicer arietinum L.) and regeneration of insect-resistant transgenic plants

To develop an efficient genetic transformation system of chickpea (Cicer arietinum L.), callus derived from mature embryonic axes of variety P-362 was transformed with Agrobacterium tumefaciens strain LBA4404 harboring p35SGUS-INT plasmid containing the uidA gene encoding β-glucuronidase (GUS) and the nptII gene for kanamycin selection. Various factors affecting transformation efficiency were optimized; as Agrobacterium suspension at OD(600) 0.3 with 48h of co-cultivation period at 20°C was found optimal for transforming 10-day-old MEA-derived callus. Inclusion of 200μM acetosyringone, sonication for 4s with vacuum infiltration for 6min improved the number of GUS foci per responding explant from 1.0 to 38.6, as determined by histochemical GUS assay. For introducing the insect-resistant trait into chickpea, binary vector pRD400-cry1Ac was also transformed under optimized conditions and 18 T(0) transgenic plants were generated, representing 3.6% transformation frequency. T(0) transgenic plants reflected Mendelian inheritance pattern of transgene segregation in T(1) progeny. PCR, RT-PCR, and Southern hybridization analysis of T(0) and T(1) transgenic plants confirmed stable integration of transgenes into the chickpea genome. The expression level of Bt-Cry protein in T(0) and T(1) transgenic chickpea plants was achieved maximum up to 116ngmg(-1) of soluble protein, which efficiently causes 100% mortality to second instar larvae of Helicoverpa armigera as analyzed by an insect mortality bioassay. Our results demonstrate an efficient and rapid transformation system of chickpea for producing non-chimeric transgenic plants with high frequency. These findings will certainly accelerate the development of chickpea plants with novel traits.

Development of genotype-independent regeneration system for transformation of rice (Oryza sativa ssp. indica)

Rice (Oryza sativa ssp. indica) is an important economic crop in many countries. Although a variety of conventional methods have been developed to improve this plant, manipulation by genetic engineering is still complicated. We have established a system of multiple shoot regeneration from rice shoot apical meristem. By use of MS medium containing 4 mg L(-1) thidiazuron (TDZ) multiple shoots were successfully developed directly from the meristem without an intervening callus stage. All rice cultivars tested responded well on the medium and regenerated to plantlets that were readily transferred to soil within 5-8 weeks. The tissue culture system was suitable for Agrobacterium-mediated transformation and different factors affecting transformation efficiency were investigated. Agrobacterium strain EHA105 containing the plasmid pCAMBIA1301 was used. The lowest concentration of hygromycin B in combined with either 250 mg L(-1) carbenicillin or 250 mg L(-1) cefotaxime to kill the rice shoot apical meristem was 50 mg L(-1) and carbenicillin was more effective than cefotaxime. Two-hundred micromolar acetosyringone had no effect on the efficiency of transient expression. Sonication of rice shoot apical meristem for 10 s during bacterial immersion increased transient GUS expression in three-day co-cultivated seedlings. The gus gene was found to be integrated into the genome of the T(0) transformant plantlets.

Progress on Agrobacterium-mediated Transformation of Banana

The development of improved Musa by conventional breeding remains to be a difficult endeavor because of the long generation times, various levels of ploidy, sterility of most edible cultivars and limited genetic variability. Therefore, genetic engineering may offer an alternative method for crop enhancement. The transformation system mediated by Agrobacterium tumefaciens has been used successfully to transform banana during the past 10 years. This article reviews recent progresses on transformation of banana mediated by Agrobacterium tumefaciens and summarizes the factors affecting transformation efficiency. Existing problems and its prospects are briefly discussed.

Salt-tolerant transgenic perennial ryegrass ( Lolium perenne L.) obtained by Agrobacterium tumefaciens-mediated transformation of the vacuolar Na +/H + antiporter gene

The objective of this study was to obtain a salt-tolerant perennial ryegrass ( Lolium perenne L.) by transforming it with a rice vacuolar membrane Na +/H + antiporter gene via the Agrobacterium-mediated method. To optimize the transformation conditions, two Agrobacterium tumefaciens strains, LBA4404 and EHA105, carrying plasmid pCAMBIA3301, were used to transform embryogenic calli of perennial ryegrass; two factors affecting transformation efficiency, acetosyringone and mannitol, were then determined. Addition of acetosyringone during the incubation of bacteria and the co-cultivation process increased the transformation rate. Transformation efficiency could be further increased by osmotic treatment with 0.4 mol/l mannitol before infection. Using this optimized transformation system, the rice vacuolar Na +/H + antiporter gene, OsNHX1, was transferred into perennial ryegrass by Agrobacterium-mediated method. The transformants were confirmed by both PCR and Southern blot analysis. The resultant transgenic ryegrass had better salt-tolerance. After stress treatment for 10 weeks with 350 mmol/l NaCl, transgenic plants survived, while wild-type plants did not. The leaves of transgenic plants accumulated higher concentrations of Na +, K + and proline than those of the control plants. These results demonstrate the OsNHX1 gene was successfully transferred into perennial ryegrass by Agrobacterium-mediated method, and the salt-tolerance of perennial ryegrass was improved by overexpression of the OsNHX1 gene.

Infection by Agrobacterium tumefaciens increased the resistance of leaf explants to selective agents in carnation ( Dianthus caryophyllus L. and D. chinensis)

Selection is critical in plant transformation and the selection pressure is one of the most important factors affecting transformation efficiency. In some cases, a suitable concentration for selective agents is chosen based on regeneration tests and the potential influence of bacterial infection on the resistance of explants to selective agents has been probably neglected. In order to evaluate the effect of agrobacterial infection on the resistance of explants to selective agents, we performed several experiments on two carnation cultivars infected by two strains (EHA101 and C58C1), carrying only a helper plasmid, of Agrobacterium tumefaciens. The selective agents were kanamycin, mannose and phosphinothricin (PPT). The results have showed that the bacterial infection indeed increases the tolerance of leaf explants to the selective agents and this increase is genotype-dependent. In general, without the bacterial infection, the highest concentrations for shoot formation were 25–50 mg l −1 for kanamycin, 3–4 g l −1 for mannose and 0 mg l −1 for PPT depending on genotype. After the bacterial infection, shoot formation was observed at either 100 mg l −1 kanamycin, or 4–6 g l −1 mannose or 1–2 mg l −1 PPT depending on cultivar. It seems that there is an interaction between the bacterium strains and the genotypes in relation to shoot formation. The results in this study have provided evidence that bacterial infection can increase the plant resistance to selective agents, which should be considered when establishing a critical concentration threshold of selective agents for transformation.

Regeneration of herbicide-tolerant black locust transgenic plants by SAAT.

A protocol based on SAAT (sonication-assisted Agrobacterium-mediated transformation) has been developed to obtain herbicide-resistant transgenic black locust (Robinia pseudoacacia L.) plants. Cotyledon explants were co-cultivated with Agrobacterium AGL1 strain carrying the pTAB16 plasmid (bar and gusA genes). The effects of bacterial concentration (OD550 of 0.3, 0.6, 0.8) and method of infection (sonication vs immersion) on bacterial delivery were determined by assaying cotyledons for transient beta-glucuronidase expression 3 days after infection. SAAT increases transient expression efficiency especially at an OD550 of 0.6. After determining bacterial concentration and infection method, other factors affecting transformation efficiency, such as explant preconditioning and period of time before applying selection, were tested. From these experiments, the preferred protocol for black locust cotyledon transformation should include sonication of preconditioned cotyledons in AGL1 suspension, coculture for 3 days with 100 microM acetosyringone and transfer to selection medium with 4 mg/l phosphinothricin and 150 mg/l timentin. Of the initial explants, 2% produced at least one transgenic shoot. Genetic transformation was confirmed by Southern hybridization, chlorophenol red assay and herbicide tolerance of the regenerated plants.