Generating Transgenic Plants with Single-copy Insertions Using BIBAC-GW Binary Vector

The overall goal of this methodology is to generate transgenic plants carrying intact, single-copy insertions of a transfer DNA, or T-DNA, in an efficient manner. The BIBAC-Gateway vector is a valuable tool for generating transgenic plants that carry intact, single-copy insertions of sequences of interest that show stable gene expression. With the BIBAC-Gateway vector, one can use Gateway recombination technology to insert sequences of interest with minimal effort.

All BIBAC derivatives, including the BIBAC-Gateway vectors, are suitable for transferring large, transgenic DNA fragments into plant genomes. The BIBAC-GW vectors can be used to insert sequences of interest into many plant systems, such as maize, rice, and tomato. There are two BIBAC-GW plasmids available.

The BIBAC-RFP-GW plasmid contains a DsRed gene that is expressed in seed coats. The BIBAC-BAR-GW plasmid contains a gene providing resistance to glufosinate. Both vectors contain a kanamycin-resistant gene as a selection marker in bacteria.

To insert the sequences of interest into the binary vector, first prepare the Gateway Entry and binary vectors, as described in the text protocol. To carry out a Gateway reaction, prepare the LR recombination reaction according to the supplier's suggestions in a 1.5-milliliter microcentrifuge tube at room temperature. Mix 100 to 300 nanograms of the supercoiled Entry clone and 300 nanograms of the BIBAC-Gateway vector.

Adjust the volume of the mixture to 16 microliters with TE.Finally, add four microliters of LR Clonase enzyme mix, and mix by vortexing. Incubate the mixture at 25 degrees Celsius for one hour. Terminate the LR reaction by adding two microliters of Proteinase K solution to the mixture.

Mix and incubate at 37 degrees Celsius for 10 minutes before proceeding with transformation into E.coli, as described in the text protocol. To prepare the Arabidopsis plants for transformation, grow Arabidopsis plants in a greenhouse or climate-controlled growth chamber until they are flowering. Clip the first bolts to allow more secondary bolts to emerge.

Plants are ready for dipping four to six days after clipping, when the plants have many immature flower heads and not many fertilized siliques. To perform floral dipping, dip inflorescences for five to 10 seconds in Agrobacterium suspension. Use gentle agitation.

Wrap the above-ground parts of the plants in cling film to keep the humidity high, and cover the plant pots with a box to keep the plants in the dark. Incubate the plants for two days in a greenhouse or growth chamber. After two days, remove the box and the cling film and grow the plants to maturity in a greenhouse or growth chamber.

To increase the efficiency of transformation, the same plants can be re-dipped seven days after the first dipping. Next, when the transformed plants are mature and dry, harvest the seeds. Pool and analyze the seeds of plants transformed with the same construct as a single set.

In case plants are transformed with a BIBAC-RFP-Gateway plasmid derivative, identify transgenic plants by analyzing the seeds using fluorescence microscopy. To detect DsRed expression in seed coats, image the seeds at an excitation of 560 nanometers and emission of 600 to 650 nanometers. Separate the fluorescent seeds from non-fluorescent counterparts using forceps.

To screen for transgenic plants transformed with a BIBAC-BAR-Gateway plasmid derivative, sow the seeds in trays filled with soil. Ensure an even spreading of seeds over trays by suspending seeds in 0.1%agar in 0.5x MS medium, and spread the seeds using a one-milliliter pipette. Stimulate the seeds to germinate in a synchronous manner by incubating the seeds for at least two days at four degrees Celsius.

This can be done before or after sowing the seeds. Two and three weeks after sowing the seeds, spray the seedlings with 0.5%glufosinate-ammonium solution. Use 500 milliliters of glufosinate-ammonium solution per one square meter.

Transfer surviving seedlings to pots. Analyze the glufosinate-ammonium-resistant plants by PCR for the presence of the construct of interest. Determine the number of T-DNA integrations and their integrity by DNA blotting using restriction enzymes.

This method allows identification of single as well as repeated integrations at the same or different loci in the genome. Use a series of restriction digestions to identify the different integration patterns possible. Select an enzyme that cuts once in the middle of the T-DNA, to be able to independently probe sequences upstream and downstream of the restriction site.

Also, select an enzyme or combination of enzymes cutting out the entire sequence of interest at once. Take care to use only restriction enzymes that are not sensitive to cytosine methylation. After performing DNA blotting, as detailed in the text protocol, perform blot analysis.

The analysis depends on the restriction strategy used. When analyzing the blot using the strategy with one restriction site in the middle of the T-DNA, count the number of the fragments detected. In this strategy, the number of hybridized fragments suggest the number of T-DNA integrations.

Compare the number of the fragments detected with a probe for the left and the right side of the T-DNA. If a different number of hybridizing fragments is detected with the two probes, then either multiple T-DNA integrations in an inverted repeat or incompletely integrated T-DNAs are present. To identify tandem and inverted arrangements of the T-DNAs, estimate the sizes of the hybridizing fragments on the blot based on the size of the marker bands, and compare the estimated sizes with the expected fragment sizes calculated based on the restriction strategy.

To examine if the transgenic sequence of interest is intact, analyze the blot prepared according to the strategy with restriction sites at the extremities of the sequence of interest. Estimate the size of the hybridizing fragments on the blot based on the size of the marker bands, and compare it with the expected size. An intact insertion yields a single fragment with a defined length.

Any deviation from the expected length indicates incomplete integration. The DNA restriction and blotting strategy to determine the number and intactness of the T-DNA integrations is shown. The ScaI restriction site divides the reporter sequence into the left border proximal parts that are detected when using a probe for bar and the right border proximal parts that are detected when using a probe for eYFP.

The BglII and PciI restriction sites are positioned within the T-DNA, outside the reporter construct. A double digestion results in a fragment with a defined size, and any deviation is an indication for incomplete insertions. The number of hybridizing fragments identified reflects the number of T-DNA insertions.

One blot containing genomic DNA of nine transgenics digested with ScaI was hybridized with probes for bar and eYFP. Several lanes indicate transgenics with single T-DNA integrations. Lane four displays two fragments with bar and one with eYFP, indicating either a truncation of the eYFP sequence or inverted repeat around the right border.

Lane six displays two fragments with bar and eYFP. One fragment has a weak intensity with the bar probe. The other fragment has a high intensity, indicating two T-DNA insertions, one of which is truncated.

After watching this video, you should have a good understanding of how to incorporate your DNA sequence of interest into a BIBAC-GW binary vector using Gateway recombination, followed by Arabidopsis transformation, screening, and assessment of the number of T-DNA integrations and their integrity. Generating and characterizing transgenic plants is time-consuming. When using BIBAC-Gateway as a binary vector, the process of identifying transgenics with intact, single-copy integrations can be shortened.

About half of the transgenics generated with BIBAC derivatives carry intact, single-copy integrations. Generally, individuals do not realize that many transgenic plants generated using common binary vectors carry truncated copies of a transgene or transgenes that show gene silencing. With BIBAC derivatives, most of the transgenic plants generated carry intact, single integrations of a transgene that show seldom gene silencing.

When using BIBAC-GW for generating transgenics, it is important to transform a sufficient number of plant materials, as the overall transformation efficiency of BIBAC derivatives is lower than that of commonly used binary vectors. The transformation efficiency of BIBAC-GW is generally between 0.2%and 0.5%After identifying transgenic plants carrying intact, single-copy T-DNA integrations, if needed, further analysis can be performed to define the precise genomic location of the T-DNA insertion. Transgenics with intact, single-copy integrations show comparable expression levels of the transgenes.

This indicates that integration position of the T-DNA in the genome has little effect on the expression level of the transgene.