Regeneration Of Transgenic Plants Research Articles

A two-step protocol for shoot regeneration from hypocotyl explants of oilseed rape and its application for Agrobacterium-mediated transformation

A two-step protocol for improving the frequency of shoot regeneration from oilseed rape (Brassica napus L.) hypocotyl explants was established. The protocol consists of a pre-culture on callus induction medium (CIM) and a subsequent shoot regeneration on shoot induction medium (SIM). The SIM was Murashige and Skoog medium supplemented with different concentrations of 6-benzylaminopurine (BA; 2–5 mg dm−3) and naphthaleneacetic acid (NAA; 0.05–0.15 mg dm−3). Maximum frequency of shoot regeneration (13 %) was on the SIM medium containing 4 mg dm−3 BA and 0.1 mg dm−3 NAA, but it increased to 24.45 % when 20 μM silver thiosulphate (STS) was added. Strikingly, an extremely high frequency of shoot regeneration up to 96.67 % was reached by a two-step protocol when hypocotyl explants had been pre-cultured for 7 d on a CIM medium containing 1.5 mg dm−3 2,4-dichlorophenoxyacetic acid. In addition, the shoot emergence was also 7 d earlier than that observed by use of the one-step protocol. The two-step protocol was also applied for regeneration of transgenic plants with cZR-3, a nematode resistance candidate gene. As a result, 43 plants were generated from 270 shoots and from these 6 plants proved to be transgenic.

Preliminary results for genetic transformation of shoot tip of Eucalyptus saligna Sm. via Agrobacterium tumefaciens

The regeneration of transgenic plants of Eucalypt is the largest difficulty for the genetic transformation of this genus; in addition a low rate of transformed plants is reached. The aim of this research was to evaluate acetosyringone (3',5'-Dimethoxy-4'-hydroxyacetophenone) on the co-culture medium during genetic transformation of shoot tips of Eucalyptus saligna via Agrobacterium tumefaciens and to promote the explants selection supposedly transformed. Shoot tip from multiple shoots was used as explants. These explants were pre-cultured during two days before the transformation. Strain EHA105 of A. tumefaciens harboring the plasmid pBI121 was used. The treatments were: 0 and 100 μM acetosyringone added to the co-culture, after co-culture the explants were cultured in multiplication medium supplemented with 250 mg.L-1 Cefotaxime® and each sub-culture the kanamycin levels were increased from 50 to 150 mg.L-1. The transient expression of the uidA gene in shoot tips was evaluated after the end of the co-culture (fifth day) and after seven days of culture on medium containing kanamycin. The presence of 100 µM acetosyringone at the co-culture of shoot tips of Eucalyptus saligna promoted higher transient expression of the uidA gene and retards toxic effects caused by kanamycin.

Regeneration of transgenic plants by Agrobacterium-mediated transformation of somatic embryos of juvenile and mature Quercus robur

A protocol was developed for genetic transformation of somatic embryos derived from juvenile and mature Quercus robur trees. Optimal transformation conditions were evaluated on the basis of the results of transient GUS expression assays with five oak embryogenic lines and a strain of Agrobacterium tumefaciens (EHA105) harbouring a p35SGUSINT plasmid containing a nptII and a uidA (GUS) genes. For stable transformation, embryo clumps at globular/torpedo stages (4-10 mg) were inoculated with EHA105:p35SGUSINT bacterial cultures, cocultivated for 4 days and selected in proliferation medium with 75 mg/l of kanamycin. Putatively transformed masses appeared after 20-30 weeks of serial transfers to selective medium. Histochemical and molecular analysis (PCR and Southern blot) confirmed the presence of nptII and uidA genes in the plant genomes. Transformation efficiencies ranged from up to 2% in an embryogenic line derived from a 300-year-old tree, to 6% in a juvenile genotype. Twelve independent transgenic lines were obtained from these oak genotypes, and transgenic plantlets were recovered and acclimatized into the soil. This is the first demonstration of the production of transformed somatic embryos and regenerated plants from juvenile and mature trees of Q. robur and suggests the possibility of introducing other genetic constructions to develop trees that are tolerant/resistant to pathogens and/or biotic stresses.

An embryogenic suspension cell culture system for Agrobacterium-mediated transformation of citrus

A method for the genetic transformation of several citrus cultivars is described, including cultivars observed to be recalcitrant to conventional epicotyl-mediated transformation. Embryogenic cell suspension cultures, established from unfertilized ovules were used as target tissues for Agrobacterium-mediated transformation. Several modifications were made to the culture environment to investigate factors required for efficient transfer of the T-DNA and the subsequent regeneration of transgenic citrus plants. It was determined that co-cultivation of citrus cells and Agrobacterium in EME medium supplemented with maltose (EME-M) and 100μM acetosyringone for 5days at 25°C was optimum for transformation of each of the citrus cultivars. Efficient selection was obtained and escapes were prevented when the antibiotic hygromycin B was used as a selection antibiotic following transformation with an Agrobacterium strain containing hptII in the T-DNA region. Transgenic embryo regeneration and development was enhanced in medium that contained a liquid overlay consisting of a 1:2 mixture of 0.6M BH3 and 0.15M EME-M media. PCR and Southern blot analyses confirmed the presence of the T-DNA and the stable integration into the genome of regenerated plants, while RT-PCR demonstrated variable amounts of RNA being transcribed in different transgenic lines. This protocol can create an avenue for insertion of useful traits into any polyembryonic citrus cultivar that can be established as embryogenic cell suspension cultures, including popular specialty mandarins and seedless cultivars.

Generation of transgenic plants expressing plasma membrane-bound antibodies to the environmental pollutant microcystin-LR

In this paper we describe the engineering and regeneration of transgenic tobacco plants expressing a recombinant plasma membrane-retained antibody specific to microcystin-LR (MC-LR), the environmental toxin pollutant produced by cyanobacteria. The antibody was created by a genetic fusion of the antigen binding regions of the microcystin-specific single chain antibody, 3A8, with the constant regions from the murine IgG1κ, Guy's 13, including a membrane retention sequence at the C-terminal end of the antibody heavy chain. The antibody produced in the leaves was shown to be functional by binding to MC-LR in an ELISA with antibody yields in transgenic plant leaves reaching a maximum of 1.2 μg g(-1) leaf f.wt (0.005% total soluble protein). Antibody-MC-LR complexes formed in leaves after addition of MC-LR to hydroponic medium around the roots of transgenic plant cultures.

High-efficiency and stable genetic transformation of pear (Pyrus communis L.) leaf segments and regeneration of transgenic plants

Pear (Pyrus communis L.) is a nutrient-dense fruit with strong consumer demand and high commercial value. However, most cultivated pear varieties are often susceptible to diseases caused by fungi, bacteria, and viruses. The purpose of the present study was to establish an efficient genetic transformation and regeneration protocol, paving the way for genetic engineering of pear cultivars with enhanced disease resistance. Major factors that influence transformation and regeneration were examined and optimal conditions were established for efficient transformation from leaf explants of ‘Old Home’, a valuable pear interstem and rootstock. High transformation efficiency was achieved largely due to an improved infection/transformation induction strategy. Co-cultivation of Agrobacterium cells and leaf segments on a liquid induction medium yielded a fivefold increase in transformation frequency. Southern hybridization analysis revealed presence of reporter gene uidA in the genomic DNA samples from independent transgenic plants, confirming the integration of the transgene in recipient pear genomes. The stability of T-DNA integration was evaluated by the consistent presence of the Km selectable marker and the expression pattern of the introduced reporter gene uidA was analyzed by GUS histochemical assay.

Robust circadian rhythms of gene expression in Brassica rapa tissue culture.

Circadian clocks provide temporal coordination by synchronizing internal biological processes with daily environmental cycles. To date, study of the plant circadian clock has emphasized Arabidopsis (Arabidopsis thaliana) as a model, but it is important to determine the extent to which this model applies in other species. Accordingly, we have investigated circadian clock function in Brassica rapa. In Arabidopsis, analysis of gene expression in transgenic plants in which luciferase activity is expressed from clock-regulated promoters has proven a useful tool, although technical challenges associated with the regeneration of transgenic plants has hindered the implementation of this powerful tool in B. rapa. The circadian clock is cell autonomous, and rhythmicity has been shown to persist in tissue culture from a number of species. We have established a transgenic B. rapa tissue culture system to allow the facile measurement and manipulation of clock function. We demonstrate circadian rhythms in the expression of several promoter:LUC reporters in explant-induced tissue culture of B. rapa. These rhythms are temperature compensated and are reset by light and temperature pulses. We observe a strong positive correlation in period length between the tissue culture rhythm in gene expression and the seedling rhythm in cotyledon movement, indicating that the circadian clock in B. rapa tissue culture provides a good model for the clock in planta.

Biolistic Transformation of Citrullus vulgaris Schrad (Watermelon)

In this study, DNA-coated gold particles were used to transform cotyledon sections including axillary meristem of Citrullus vulgaris using the biolistic method for optimizing the transformation parameters and eventually regeneration of putative transgenic watermelon plants. Initially, five biolistic parameters namely helium pressure, macrocarrier to target tissue distance, explant ages, DNA concentrations and osmotic treatment were optimized using plasmid pRQ6 (carrying both gusA and hpt genes). The optimized biolistic parameters for cotyledon sections of Citrullus vulgaris explants were determined as follow: 1100 psi helium pressure, 6 cm target distance, 4-day-old explants, concentration of DNA at 1.2 µg/bombardment and osmotic treatment for 24 h on medium supplemented with 0.6 M mannitol prior to bombardment. Using the optimized parameters, transformation of watermelon cotyledons was carried out using pAHG11, pCAMBIA 1301, co-transformation (pAHG11+pCAMBIA 1301) and pCambar plasmids. Bombarded tissue’s were selected on either 5 mg L-1 hygromycin or 2 mg L-1 PPT. Finally, the transformants produced were subjected to GUS histochemical assay. Integration of the transgenes into transgenic watermelon genome was confirmed by PCR analysis.

Agrobacterium Mediated Transformation of Maize (Zea mays L.)

Agrobacterium tumefaciens mediated transformation may offer a better alternative than the biolistic gun for genetic transformation of maize plants. This gene delivery system results in a greater proportion of stable, low-copy number transgenic events than does the biolistic gun, and is highly efficient. In the present work, we studied maize transformation using A. tumefaciens by identifying some important factors that affect transformation efficiency subsequent tissue culture and regeneration of transgenic plants. Although, all genotypes produced embryogenic calli, S61 and A188 lines had higher percent (75% and 71% respectively) of regeneration than the other ones. Transformant events obtained when immature embryos (1.5-2 mm) were inoculated with A. tumefaciens LBA4404 harboring a standard binary vector pCAMBIA3301 after 72 h pretreatment culture of the embryos. Polymerase chain reaction (PCR) confirmed the presence of the gus and bar genes in the genome of regenerated plants. The transformation frequency (the number of independent, PCR-positive transgenic plants per 100 embryos infected) was 6.45% for S61 genotype. Therefore, our results identified suitable genotype (S61), embryo size (1.5-2 mm), A. tumefaciens strain (LBA4404), pretreatment culture, and appropriate antibiotic (Timentin) for Agrobacterium mediated transformation of Z. mays.

Genetic transformation of mature embryos of bread (T. aestivum) and pasta (T. durum) wheat genotypes

The objective of the present study is to develop an efficient protocol for regeneration of transgenic wheat plants using Agrobacterium- mediated transformation of mature embryos of hexaploid bread wheat (Triticum aestivum) and tetraploid pasta wheat (Triticum durum). The data indicated that embryogenic calli were formed within 7 days in the presence of 2 mgl-1 2,4-D. Adventitious shoots emerged from the embryonic calli in the presence of 2 mgl-1 BA. Shoot regeneration frequency varied between wheat cultivars according to their genetic background differences. Regeneration frequency was higher in the cultivar Gemmiza 10 (95 %) compared with the other cultivars tested. Mature embryos derived callus of the cultivars Gemmiza 10 and Gemmiza 9 were co-cultivated with A. tumefaciens strain LBA4404 harboring a binary vector pBI-121 containing the neomycin phosphotransferase-II gene (npt-II). The resulted putative transgenic plantlets were able to grow on kanamycin containing medium. A successful integration of the transgene was confirmed by analyzing the T0 plantlets using Southern hybridization and PCR amplification. The gus gene expression can be detected only in the transgenic plants. The reported protocol is reproducible and can be used to regenerate transgenic wheat plants expressing the genes present in A. tumifaciens binary vectors.

Transformation of chickpea: effect of genotype, explant, Agrobacterium-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.

Selection system and co-cultivation medium are important determinants of Agrobacterium-mediated transformation of sugarcane

A reproducible method for transformation of sugarcane using various strains of Agrobacterium tumefaciens (A. tumefaciens) (AGL0, AGL1, EHA105 and LBA4404) has been developed. The selection system and co-cultivation medium were the most important factors determining the success of transformation and transgenic plant regeneration. Plant regeneration at a frequency of 0.8-4.8% occurred only when callus was transformed with A. tumefaciens carrying a newly constructed superbinary plasmid containing neomycin phosphotransferase (nptII) and beta-glucuronidase (gusA) genes, both driven by the maize ubiquitin (ubi-1) promoter. Regeneration was successful in plants carrying the nptII gene but not the hygromycin phosphotransferase (hph) gene. NptII gene selection was imposed at a concentration of 150 mg/l paromomycin sulphate and applied either immediately or 4 days after the co-cultivation period. Co-cultivation on Murashige and Skoog (MS)-based medium for a period of 4 days produced the highest number of transgenic plants. Over 200 independent transgenic lines were created using this protocol. Regenerated plants appeared phenotypically normal and contained both gusA and nptII genes. Southern blot analysis revealed 1-3 transgene insertion events that were randomly integrated in the majority of the plants produced.

Regeneration and characterization of transgenic kumquat plants containing the Arabidopsis APETALA1 gene

‘Meiwa’ kumquat (Fortunella crassifolia Swingle.) is famous for its relatively short juvenility, delicious flavor, human health benefits and high resistance to citrus canker. To establish kumquat transformation system and to further shorten its juvenility, Agrobacterium-mediated epicotyledon segment transformation of APETALA1 (AP1 from Arabidopsis) gene was conducted. Transformation efficiency ranged from 1.00 to 4.08% depending on seedling age, and 20 day age seedlings proved to be the best explants for transformation. Five stable transgenic plants were obtained as revealed by GUS assay, and further confirmed by specific PCR and Southern blot analyses. After transfer to the greenhouse, one transgenic line (J3) flowered at the 11th month and continued to flower in the next years, till the third year when all non-transformed and transgenic plants but J66 flowered. Gene expression analysis of AP1 and four endogenous flowering genes CiAP1, CiFT, CiLFY, and CiTFL1 by real-time RT-PCR suggested that CiFT and CiTFL1 played important roles in the regulation of flowering in transgenic AP1 kumquat. Factors influencing kumquat transformation efficiency and the relationship between flowering time in transgenic AP1 kumquat and expression levels of endogenous FT and TFL1 genes were discussed.

Pre- and post-agroinfection strategies for efficient leaf disk transformation and regeneration of transgenic strawberry plants

Following previously described Agrobacterium tumefaciens-mediated transformation procedures for Fragaria x ananassa Duch. 'Chandler', we undertook several experiments to establish the importance of some parameters affecting transformation. The most important factor that increased the percent recovery of transformants was the introduction of a pre-selection phase, in-between co-cultivation and selection, in which leaf disks were cultured on pre-selection regeneration medium containing validamycin A, timentin, and cefotaxime. The average percentage of leaf disks forming shoots on selection medium containing cefotaxime (250 mg l(-1)) + timentin (250 mg l(-1)) was 5.4% and about three shoots per regenerating leaf disk. Maximum transformation percentage, based on polymerase chain reaction, was 31.25%. Transgene integration and copy number were assessed by Southern hybridization confirming single copy as well as multiple copies of transgene integration in shoots as well as roots separately. This confirmed the non-chimeric nature of these transgenic plants. The system is very promising for the regeneration of genetically transformed cells and obtaining transgenic strawberry plants at high efficiency.

Agrobacterium mediated transformation of sugarcane for borer resistance using Cry 1Aa3 gene and one-step regeneration of transgenic plants

Sugarcane borers are the major biotic pest in sugarcane that accounts nearly 25-30% yield loses. Most of the present sugarcane varieties are not resistant to borers. Development of borer resistance in sugarcane through transgenic technology could be the best approach. Cry series of genes are known for development of insect pest resistance in plants in particular for the most damaging order 'Lepidoptera'. The sugarcane (Saccharum hybrids) genotype CoC671 was transformed by Agrobacterium mediated method. Cry 1Aa3 gene driven by CaMV35S promoter and Ocs terminator with selectable marker nptII driven by Nos promoter and terminator harboring in binary vector pBinAR was transformed into Agrobacterium tumefaciens strain EHA 105 was used in sugarcane transformation. Young leaf roll discs were infected and regenerated plants were obtained in one step after infection with minimal callus phase. Three to four shoots per explant were developed after 7-10 days incubation. Regenerated shoots were allowed to grow further. Two months old putative transgenic plants were taken for molecular characterization. Out of 65 plants tested by PCR, 24 plants were positive for Cry1Aa3 gene. This represents nearly 28% of the transformation efficiency. All these plants were analyzed by Southern blot hybridization for integration of the inserted gene. Four plants showed single copy number and another two showed more than two integrations. All those 24 plants that are positive by PCR were subjected to ELISA test for expression of foreign protein. It was observed that four transgenic plants showed around ten-fold high level expression and another two plants showed with moderate levels, 3-5 fold expression.

Generation of transgenic plants expressing antibodies to the environmental pollutant microcystin‐LR

We describe the engineering, regeneration, and characterization of transgenic tobacco plants expressing a recombinant antibody specific to microcystin-LR (MC-LR), the environmental toxin pollutant produced by species of cyanobacteria. The antibody was created by a genetic fusion of the antigen-binding regions of the microcystin-specific single-chain antibody, 3A8, with constant regions from the murine IgG1kappa, Guy's 13. IgG transgenes were controlled by a leader peptide that targets the transgene products to the secretory pathway and also allows for rhizosecretion. The antibody, extracted from the leaves or rhizosecreted into hydroponic medium by transgenic plants, was shown to have functional binding to MC-LR. Antibody yields in transgenic plant leaves reached a maximum of 64 microg/g leaf fresh weight (0.6% total soluble protein), and the rate of antibody rhizosecretion reached a maximum of 5 microg/g root dry weight/24 h. Rhizosecreted antibody bound to MC-LR in hydroponic medium, and transgenic plants grew more efficiently on medium containing MC-LR compared to wild-type controls. This proof of concept paves the way for applications to produce diagnostic antibodies to microcystin-LR, remove it from the environment by phytoremediation, or enhance yields in crops exposed to MC-LR.-Drake, P. M. W., Barbi, T., Drever, M. R., van Dolleweerd, C. J., Porter, A. J. R., Ma, J. K.-C. Generation of transgenic plants expressing antibodies to the environmental pollutant microcystin-LR.

Regeneration of transgenic citrus plants from the trimmed shoot/root region of etiolated seedlings

Transformation and high efficient regeneration of transgenic plants from the trimmed etiolated shoot/root region (TESRR) of Anliucheng sweet orange [Citrus sinensis (L.) Osb.] seedling was reported. A visual green fluorescent protein (GFP) marker gene was introduced to evaluate transformation efficiency by using the explants from TESRR and epicotyls. The transformation protocol was: infection 20 min, co-culture 3 d, selection culture 30 d, and rooting 15 d. Out of a total of 288 sprouted shoots obtained from TESRR, 34 shoots (11.8 %) yielded GFP expression. In contrast, only 2 (3.0 %) of the 67 sprouted shoots from epicotyl transformation yielded GFP expression. In all plants showing the green fluorescence an expected 500 bp GFP fragment was proved by PCR analysis. Southern blot analysis further confirmed the integration of GFP gene into citrus genome. Transgenic plantlets were obtained within 80 d using the TESRR, compared within 150 d by using epicotyls.

Genetic transformation of blue grama grass with the rolA gene from Agrobacterium rhizogenes: regeneration of transgenic plants involves a “hairy embryo” stage

Until recently, information about the effects of transforming plants with the rolA gene of Agrobacterium rhizogenes has been restricted mainly to dicots in which a severely wrinkled phenotype, reduced internode distances, and abnormal reproductive development were commonly observed. In this work, we analyzed the effects associated with the expression of this gene in a new genetic context: the forage grass genome. Transgenic P35S•rolA plants of blue grama grass (Bouteloua gracilis) were obtained by a biolistic approach employing embryogenic chlorophyllic cells as the target material. Four independent transgenic lines with regeneration capacity were recovered, which showed stable integration of this transgene as demonstrated by polymerase chain reaction and Southern blot hybridization. Growth of the rolA-transformed lines under greenhouse conditions provided evidence for a new biotechnological application for the rolA gene in plants, namely, the improvement of biomass production in forage grasses. Additionally, we described here a new phenotypic marker (referred to here as the “hairy embryo” syndrome) that can be instrumental for the early identification of transformation events when transforming grasses with this gene.

Direct gene transfer study and transgenic plant regeneration after electroporation into mesophyll protoplasts of Pelargonium × hortorum, ‘Panaché Sud’

Direct genetic transformation of mesophyll protoplasts was studied in Pelargonium x hortorum. Calcein and green-fluorescent protein (GFP) gene were used to set up the process. Electroporation (three electric pulses from a 33-microF capacitor in a 250-V cm(-1) electric field) was more efficient than PEG 6000 for membrane permeation, protoplast survival and cell division. Transient expression of GFP was detected in 33-36% of electroporated protoplasts after 2 days and further in colonies. A protoplast suspension conductivity of >1,500 microS cm(-1) allowed high colony formation and plant regeneration. Stable transformation was obtained using the plasmid FAJ3000 containing uidA and nptII genes. When selection (50 mg l(-1) kanamycin) was achieved 6 weeks after electroporation, regenerated shoots were able to grow and root on 100 mg l(-1) kanamycin. The maximum transformation efficiency was 4.5%, based on the number of colonies producing kanamycin-resistant rooted plants or 0.7% based on the number of cultured protoplasts. Polymerase chain reaction (PCR) analysis on in vitro micropropagated plants showed that 18 clones out of 20 contained the nptII gene, while the uidA gene was absent. These results were confirmed after PCR analyses of five glasshouse-acclimatized clones.

Optimizing Initiation and Maintenance of Vitis Embryogenic Cultures

Stamens and pistils from mature grapevines and leaves from in vitro micropropagation cultures were used to optimize parameters influencing somatic embryogenesis in Vitis. Embryogenic competence was dependent on species/variety, explant type and developmental stage, medium composition, and growth regulator concentration. Of varieties evaluated, a greater number produced embryogenic cultures from stamens and pistils (26) compared with leaves (six). Among the different stamen and pistil stages, Stage II and III explants produced the maximum embryogenic response regardless of genotype and medium composition. Of seven culture media tested, the highest embryogenic response was recorded from varieties cultured on MSI (18) and PIV (16) media. Experiments annually repeated over 3 to 10 years demonstrated reproducible results. Highly reliable protocols for somatic embryogenesis were obtained for 29 Vitis species and varieties, including 18 Vitis vinifera varieties, Vitis riparia, Vitis rupestris, Vitis champinii, and eight Vitis hybrids. Embryogenic cultures were maintained on X6 medium for a period of 6 months to 2 years depending on the variety and used in studies involving genetic transformation and transgenic plant regeneration.