Selection Of Transgenic Shoots Research Articles

Kanamycin selection in temporary immersion bioreactors allows visual selection of transgenic citrus shoots

In mature citrus transformation, the nptII gene is most commonly used for selection and it is confounded by the high number of non-transformed, escaped shoots that develop on semi-solid kanamycin selection medium, even at high concentrations. Selection in liquid medium with kanamycin in temporary immersion bioreactors might provide a better means of distinguishing between transformed and non-transformed shoots. A dose-response curve was constructed for wild-type Carrizo rootstock in liquid medium to evaluate the effects of kanamycin concentration on the number and the length of microshoots. Kanamycin at 200 mg/l was chosen as the optimal concentration for selection of transgenic mature citrus shoots in bioreactors. At this dose, most non-transgenic microshoots turned yellow and their lengths and numbers were significantly reduced in comparison to the no kanamycin controls. Selection of transgenic shoots in bioreactors was tested after Agrobacterium transformations of mature Carrizo and Valencia using three different binary vectors containing nptII as the selectable marker. Shoots developed on semi-solid medium and were transferred to temporary immersion bioreactors containing liquid MS medium with 200 mg/l kanamycin. After two weeks of culture in bioreactors, 21 dark green shoots were visually selected on the basis of color from a total of 6882 microshoots, and 17 of them (81%) were confirmed as transgenic with either the GUS histochemical assay, GFP fluorescence or PCR. Yellow shoots (5675) to be discarded from pTLAB21 and pCAMBIA2301 transformations were also tested for GUS or GFP expression and only one (0.01%) was positive. Kanamycin selection of mature transgenic shoots in temporary immersion bioreactors permitted transgenics to be visually distinguished on the basis of color, and reduced labor and consumable costs for PCR screening, particularly when reporter genes were not used.

Agrobacterium-mediated transformation efficiency of carrot embryogenic callus using nptII and hpt selective genes

Experiments were carried out to compare the efficiency of embryogenic carrot calli transformation using selective genes nptII and hpt. Our data showed that it is more preferable to use kanamycin concentration 100 mg/L and hygromycin 20 mg/L for the selection of transgenic shoots. Under the same conditions, the use of kanamycin yielded a greater number of transgenic shoots. It is, therefore, more preferable to use vector constructs containing gene nptII for genetic transformation of the Nantskaya 4 carrot cultivar.

Cyanamide hydratase as selectable marker in potato

The selectable marker cyanamide hydratase/cyanamide was successfully used to generate transgenic potato plants from the cultivars Russet Burbank, Ranger Russet, Bintje, Desiree, Kardal and Pentland Dell. Up to 3,000 transgenics per person per year were produced. The efficiency of transgenic production varied among cultivars, and was in general 2–3 times lower than the transformation efficiency (TE) using the selectable marker kanamycin. Differences between cultivars in sensitivity to cyanamide selection were observed, but in general a concentration of 30 mg/l was applied for selection of transgenic shoots. A stepwise increase of cyanamide concentration during the transformation procedure for Russet Burbank resulted in an improved TE via a reduction of the escape efficiency from 69 to 29 %. The cultivars differed in the hormone concentration and duration (2,4-D and Zeatin riboside) required for the production of transgenics, predominantly during the phase of shoot initiation. Only for cultivars Bintje and Pentland Dell, adaptations in the hormone scheme are required during the transformation procedure with cyanamide selection compared to selection on kanamycin. Upon application of a transformation protocol for Russet Burbank, only in 1 % of the plants (in a population of 241) Agrobacterium could be detected, but these bacteria did not contain the vector for transgene transfer anymore.

Stable Agrobacterium-mediated transformation of the halophytic Leymus chinensis (Trin.)

In this study, an efficient procedure for stable Agrobacterium -mediated transformation of Leymus chinensis (Trin.) was established. Agrobacterium tumefaciens strain EHA105, harboring a binary vector pCAMBIA2300, was used for transformation, along with a sweet potato 2-cysteine peroxiredoxin ( 2-Cys Prx ) gene under the control of the stress-inducible sweet potato anionic peroxidase 2 ( SWPA2 ) promoter and the neomycin phosphotransferase ( nptII ) gene under the control of the cauliflower mosaic virus (CaMV) 35 S promoter. We found that a one-month-old callus derived from mature seeds could be efficiently transformed. Seven-day preculture followed by inoculation with the addition of 100 μmolL -1 acetosyringone (AS) and then a 3 day co-cultivation were performed before selection. Selection of transgenic shoots was done in the presence of 150 mgL -1 kanamycin (KM). An optical density at a wavelength of 600 nm (OD 600 ) of approximately 0.4 for A. tumefaciens infection solution and 20 min of infection time gave the highest transformation efficiency. Polymerase chain reaction (PCR) analysis of KM-resistant plants and newly regenerated rhizomes revealed stable transformation of the 2- Cys Prx gene and the nptII gene, with the highest transformation frequency of 4.93%. RT-RCR analysis was conducted using salt stressed transgenic plants, and the results suggested that 2-Cys Prx had low transcription levels under non-stressed conditions, and increased transcription after 6 h of 200 mM NaCl stress. This gene continued to demonstrate high levels of transcription until 6 h after withdrawal of stress, with a slow recovery. The method reported herein provides a direct opportunity for improvement of the quality traits of L. chinensis via genetic transformation. Keywords: Leymus chinensis , Agrobacterium -mediated transformation, 2-Cys peroxiredoxin, gene transformation

Agrobacterium tumefaciens-mediated transformation protocol of Jatropha curcas L. using leaf and hypocotyl segments

In the present study, Agrobacterium tumefaciens-mediated transformation protocol has been optimized for Jatropha curcas—an important biofuel plant using leaf segments (LS) and hypocotyl segments (HS) from in vitro-seedlings. Among different accessions used, ‘GJ-Ran-C2’ was selected as the best for transformation. A. tumefaciens strain EHA101, containing the binary vector pIG121Hm was used for transformation, along with the GUS reporter gene containing intron and CaMV 35S as promoter. Various parameters were optimized to get the highest transient transformation expression (TTE). Augmentin® was used in the medium to control A. tumefaciens. Selection of transgenic shoots was done in the presence of kanamycin. Integration of GUS gene was confirmed by PCR analysis, whereas the expression of GUS transgene was confirmed by RT-PCR, using actin gene as internal control. GUS histochemical analysis of explants just after cocultivation showed blue color in approximately all the LS and HS but stable transformation efficiency was 5% in case of LS, whereas 4% in HS.

Transformation of potato via Agrobacterium coated microparticle bombardment

The transformation of potato (Solanum tuberosum L. cv. Desiree) was extended by the Agrobacterium-mediated biolistic method. Using this approach transgenic shoots could be obtained at a similar frequency to that achieved through conventional biolistics. Leaves from shoot cultures were bombarded with gold particles coated in Agrobacterium tumefaciens cells harboring a binary plasmid encoding three genes of interest in the T-DNA. Nine shoots were obtained from 20 shots, with selection of transgenic shoots on a series of media containing progressively increasing concentrations of hygromycin from 5 to 20 mg dm−3.

Agrobacterium tumefaciens-mediated transformation of Withania somnifera (L.) Dunal: an important medicinal plant

This report describes Agrobacterium tumefaciens-mediated transformation of Withania somnifera--an important Indian medicinal plant. A. tumefaciens strain LBA4404, containing the binary vector pIG121Hm was used for transformation, along with the gusA reporter gene with intron under the transcriptional control of the Cauliflower Mosaic Virus (CaMV) 35S promoter. The leaf segments from two-and-a-half-month-old green house-grown seedlings were more efficient in transformation, as compared to those from the in vitro-grown shoots. Second expanded leaf from the shoot tip gave the highest transient transformation efficiency. Selection of transgenic shoots was done in the presence of 50 mg l(-1) kanamycin. Polymerase chain reaction analysis of T(0) transgenic plants showed the presence of gusA and nptII genes. The expression of these transgenes in T(1) progeny was confirmed by RT-PCR. The integration of gusA gene was confirmed by Southern blot analysis. The transformation efficiency was found to be 1.67%.

Green fluorescent protein as a screenable marker to increase the efficiency of generating transgenic woody fruit plants

The green fluorescent protein (GFP) from Aequorea victoria has been introduced into three different citrus genotypes [Citrus aurantium L., C. aurantifolia (Christm.) Swing. and C. sinensis L. Osbeck×Poncirus trifoliata (L.) Raf.] which are considered recalcitrant to transformation, mainly due to low transformation frequencies and to the regeneration of escape shoots at high frequencies from the Agrobacterium-inoculated explants. High-level GFP expression was detected in transgenic cells, tissues and plants. Using GFP as a vital marker has allowed us to localize the sites of transgene expression in specific cells, always occurring in callus tissue formed from the cambium of the cut ends of explants. Whereas green fluorescent shoots regenerated in all cases from this callus, most escapes regenerated directly from explants with almost no callus formation. Thus, development of callus from cambium is a prerequisite for citrus transformation. Furthermore, in vivo monitoring of GFP expression permitted a rapid and easy discrimination of transgenic and escape shoots. The selection of transgenic shoots could be easily favored by eliminating the escapes and/or by performing shoot-tip grafting of the transgenic buds soon after their origin. GFP-expressing shoots have also been observed in citrus explants co-cultivated with Agrobacterium but cultured in a medium without the selective agent kanamycin. This opens the possibility to rescue the transgenic sectors and to regenerate transgenic plants without using selectable marker genes conferring antibiotic or herbicide resistance, which is currently a topic of much discussion for the commercialization of transgenic plants.

A wound-induced promoter driving npt-II expression limited to dedifferentiated cells at wound sites is sufficient to allow selection of transgenic shoots.

There is much data to indicate that only a small number of cells in plant explants are competent for stable transformation by Agrobacterium. Circumstantial evidence suggests that certain cells reentering cell division at wound sites are competent for transformation by Agrobacterium. We have discovered a member of the intracellular PR gene family from asparagus (AoPR1) which is strongly expressed upon wounding and during the reactivation of the cell cycle in cultured asparagus cells, but which shows very little expression in intact plant tissues. The promoter from the AoPR1 gene was fused to an intron-containing GUS reporter gene and shown to be more strongly expressed than the commonly used CaMV 35S constitutive promoter in target cells for plant transformation. A transcriptional fusion of the AoPR1 promoter with an NPT-II gene was found to be a very efficient marker for the selection of transgenic tobacco callus. Expression of the AoPR1-NPT-II gene allowed efficient shoot formation on transgenic callus and efficient adventitious root formation on transgenic shoots. These latter observations provided firm evidence that transformation selection marker gene expression is most crucial at the early stages of the transformation process, during the establishment of transformed micro-calli.