This work was supported by the Ogelbay fund.

1. Growing the Plants
<ol>
	<li>6 weeks prior to dipping, fill 5 inch pots with soil and sow flax seeds &#188; inch deep in the soil (4 seeds per pot). Be sure to firm the soil over the seeds. Water the plants regularly and maintain them in long daylight (14 hr light and 10 hr dark).</li>
	<li>Check plants regularly for primary inflorescences (clusters of flowers&#8217; organs). The plants are ready for transformation when the buds are visible and have just formed in the inflorescence (Figure 1 and 2). To see the buds, cut the leaves around it if necessary. 
	NOTE: Using the best flower stage is critical and is detailed in the Discussion.
	<ol>
		<li>Use the main branch of the plant for the experimental treatment as it produces more flowers than the side braches. Use the side branches either as controls (not to be dipped) or for other experimental treatments.</li>
		<li>Alternatively, use the main and side branches of the same plant for the experimental treatment and use another plant as a control or for other experimental treatments. Use labels to mark individual branches and individual flowers with the date and type of treatment.</li>
	</ol>
	</li>
</ol>
2. Cloning and Transformation in to Escherichia coli (E. coli) Cells
<ol>
	<li>Clone the fragment/gene of interest into the multiple cloning site of a plant binary vector harboring the T-DNA.
	<ol>
		<li>Perform cloning in one step or two steps.
		<ol>
			<li>Directly clone small inserts in this protocol (~500 bp) into the plant binary vector. In this protocol, use the plant binary vector (PRI909). Otherwise, use any other plant binary vectors in a similar strategy.</li>
			<li>Alternatively, clone large inserts (&#8805;6.5 kb) in two steps: first using a general commercial cloning kit (not plant specific), then subclone into the plant binary vector.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Set up a PCR reaction to amplify the gene of interest. Design the primers with restriction sites at the 5&#8217; end (according to the multiple cloning site of the plant binary vector in use).
	<ol>
		<li>Perform a standard PCR using genomic flax DNA with the following cycling conditions: an initial hold of 24 &#176;C, then 2 min at 94 &#176;C, followed by 30 cycles of 98 &#176;C for 5 sec, 60 &#176;C for 15 sec, and 72 &#176;C for 2 min. Perform a final extension step at 72 &#176;C for 5 min followed by an indefinite hold at 4 &#176;C.</li>
		<li>Separate PCR products on 1% Tris/Borate/EDTA (TBE) agarose gel, run the gel at 100 V for 1 hr. Purify the products from the gel and quantify via Nanodrop.</li>
	</ol>
	</li>
	<li>Set up the ligation mix to clone the PCR product in the commercial cloning vector. Follow manufacturer&#8217;s protocol.</li>
	<li>Transform the ligation mix into chemically competent E. coli cells as follows.
	<ol>
		<li>Add 2 &#181;l of the ligation mix into one vial of chemically competent E. coli cells. Incubate on ice for 30 min.</li>
		<li>Heat shock the cells for 30 sec at 42 &#176;C (Heat shock time and temperature depends on the type of cells used).</li>
		<li>Incubate on ice and add 250 &#181;l of RT super optimal broth with catabolite repression (S.O.C) medium.</li>
		<li>Cap the tube and incubate in an orbital shaker at 200 rpm at 37 &#176;C for one hr.</li>
		<li>Spread 10 - 50 &#181;l from each transformation on a prewarmed LB plate (prewarmed at 37 &#176;C) + appropriate selective antibiotic (determine the selective antibiotic based on the type of commercial cloning vector used). Incubate plates at 37 &#176;C O/N.</li>
	</ol>
	</li>
	<li>Pick ~10 colonies for mini prep plasmid purification, using a commercial kit. Mini prep plasmid purification is generally performed as following. 
	NOTE: Buffer names are specific to the commercial kit used but their general functions are similar.
	<ol>
		<li>Inoculate the single colonies in 2 - 5 ml LB medium containing the appropriate selective antibiotic (determined based on the type of commercial vector used).</li>
		<li>Incubate for approximately 8 hr at 37 &#176;C with vigorous shaking (~300 rpm).</li>
		<li>Harvest the cells by centrifugation at 6,000 x g for 10 min.</li>
		<li>Resuspend the pellet in the 250 &#956;l resuspension buffer. Mix and vortex to completely disperse the pellet.</li>
		<li>Lyse the bacterial cells by adding 250 &#181;l of the lysis buffer, mix thoroughly by inverting the tubes 4 - 6 times.</li>
		<li>Neutralize the lysate in the neutralization buffer and invert 4 - 6 times to mix. Centrifuge for 10 min at ~17,900 x g on a table-top microcentrifuge.</li>
		<li>Transfer the supernatants from previous step to a spin column. Centrifuge again at ~17,900 x g for 30 - 60 sec.</li>
		<li>Wash the spin column by adding 0.75 ml washing buffer and centrifuge for 30 - 60 sec. Discard the flow-through.</li>
		<li>Centrifuge for an additional 1 min to remove residual wash buffer. Place the spin column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 &#956;l water to the center of each column. Let stand for 1 min, and centrifuge for 1 min.</li>
	</ol>
	</li>
</ol>
3. Analyze the Purified Plasmids for the Presence of Insert
<ol>
	<li>Restriction Digest:
	<ol>
		<li>Set up a restriction digest to determine the presence of the insert by digesting the plasmid with the restriction enzymes used to clone the insert. A typical double restriction digest reaction as follows: 1 &#181;g of purified plasmid, 1 &#181;l of each restriction enzymes, 2 &#181;l 10x restriction digest buffer + 2 &#181;l BSA (if applicable), X &#181;l water (to a total of 20 &#181;l)</li>
		<li>Mix gently and incubate at recommended temperature (varies from one enzyme to another).</li>
		<li>Analyze the restriction digest reaction on 1% TBE agarose gel, run at 100 V for 1 hr and look for drop-out of appropriate size.</li>
	</ol>
	</li>
	<li>PCR analysis:
	<ol>
		<li>Set up a PCR with the purified plasmid, using primers from the commercial vector region to amplify inside the insert or the junction between the vector and the insert. Common primers are M13 forward and reverse and T3/T7 primers.</li>
		<li>In this protocol, use the following PCR cycling conditions: an initial hold of 24 &#176;C, then 2 min at 94 &#176;C, followed by 18 cycles at 98 &#176;C for 5 sec, 60 &#176;C for 15 sec, and 72 &#176;C for 2 min. Perform a final extension step at 72 &#176;C for 5 min followed by an indefinite hold at 4 &#176;C.</li>
		<li>Load PCR products on a 1% TBE agarose gel and run for 100 - 120 V for 1 hr.</li>
	</ol>
	</li>
	<li>Sequencing:
	<ol>
		<li>Send the purified plasmid to a commercial sequencing facility to analyze and confirm the presence of the insert.</li>
		<li>Once the correct construct is obtained, use it for the second step of cloning (into the plant binary vector). 
		NOTE: Steps 2.3 - 3.3 can be eliminated if the cloning is completed in one step.</li>
	</ol>
	</li>
</ol>
4. Cloning into the Plant Binary Vector (PRI909) and E. coli Transformation
<ol>
	<li>Set up a double restriction digest reactions to linearize the plant binary vector and isolate the previously cloned insert using the same restriction enzyme sites that were added to the primers (step 2.2).</li>
	<li>Analyze the restriction digests using gel electrophoresis, run at 100 V for 1 - 2 hr. Cut the insert and the linearized plant binary vector from the gel.</li>
	<li>Use a commercial kit to purify the gel products. Refer to manufacturer&#8217;s manual.</li>
	<li>Set up a ligation reaction to ligate the insert into the plant binary vector. The insert to vector ratio depends on the size of the insert and the plant binary vector. In this protocol the LIS1 insert was 6.5 kb and the plant binary vector was 9 kb. Hence, use a 1:2 insert to vector ratio.</li>
	<li>Repeat steps 2.4 - 3 for the bacterial transformation, plasmid purification and analysis. Once the correct construct (insert + plant binary vector) is obtained, proceed to the electroporation in step 5.</li>
</ol>
5. Electroporation into Agrobacterium tumefaciens Electrically Competent Cells
<ol>
	<li>Thaw a vial of Agrobacterium tumefaciens electrically competent cells on ice.</li>
	<li>Add 1 ng of binary vector plasmid DNA to 20 &#181;l of competent cells, on ice, and mix gently.</li>
	<li>Chill a 0.1 cm electroporation cuvette on ice.</li>
	<li>Transfer the competent cell/DNA mix to the electroporation cuvettes, and tap to collect the mixture at the bottom. Put the cuvette in the electroporator machine and pulse (voltage and time conditions depend on the size of cuvette and the electroporator used).</li>
	<li>Add 1 ml of SOC media and transfer cells to a 15 ml tube.</li>
	<li>Incubate for 1 hr at 28 - 30 &#176;C, shaking at 100 rpm. Plate 50 - 100 &#956;l of cells on LB agar + appropriate selective antibiotic (depend on the plant binary plasmid and the strain of Agrobacterium used. In this protocol use 50 &#956;g/ml kanamycin and 100 &#956;g/ml streptomycin).</li>
	<li>Incubate the plates up to 48 hr at 28 - 30 &#176;C.</li>
	<li>Repeat step 2.5 for colony selection and plasmid purification.</li>
	<li>Repeat step 3 to analyze the plasmid and to check the integrity of the insert and T-DNA before using the construct for floral-dipping. Repeat step 3.2, using multiple PCR primers across the entire insert region and across the T-DNA regions and by sequencing as in step 3.3. 
	NOTE: In this protocol 4 different primers were designed across the T-DNA and 10 primers across the insert.</li>
	<li>Once the presence of the plant binary plasmid in the selected Agrobacterium colony is confirmed, place cells in 50% glycerol and store at -80 &#176;C. Store remaining colonies on the plate at 4 &#176;C if they are to be used within one week7.</li>
</ol>
6. Floral-Dipping
NOTE: 2 days prior to floral-dipping:
<ol>
	<li>Grow Agrobacterium cells to the stationary phase (OD600 between 0.5 - 1 is acceptable) in LB + appropriate antibiotics in liquid media. 
	NOTE: These are the same antibiotic as in step 5.6, based on the plant binary vector and the type of Agrobacterium strain used.</li>
	<li>Start the culture with 1:100 dilutions of a saturated (5 ml) O/N culture and grow for 24 - 48 hr at 28-30 &#176;C while shaking at 150 rpm. The culture should have reached the midlogarithmic phase and more likely will be approaching or at stationary phase1. OD of approximately 0.8 ( again OD between 0.5 to 1 are all acceptable)8. Collect the cells by centrifuging at 5,000 x g at RT.</li>
	<li>Resuspend the cells in infiltration medium (5.0% sucrose + 0.05 - 0.003% Silwet L-77). For the first round of dipping, use 0.05% Silwet-77. For the second and third rounds, reduce the concentration to 0.03% (detailed in Discussion).</li>
	<li>Proceed with the floral-dip step. Lay the plant on its side and dip only the visible buds in the infiltration medium for 1-2 min. Leave the plant on its side and cover it with plastic wrap to maintain high humidity in the dome (Figure 3).</li>
	<li>On the next day, place the plant in an upright position and maintain normally.</li>
	<li>When the bud grows larger (usually after about 10 - 14 days), repeat steps 6.1 - 6.4. Reduce the dipping time to 30 - 60 sec and Silwet-77 concentration to 0.003%.</li>
	<li>Maintain the plants normally until their seeds are mature and ready to be collected (Figure 4).</li>
</ol>
7. Selection of Positive Transformants with Direct PCR
<ol>
	<li>Sow the T1 seeds on soil as in step 1.1.
	<ol>
		<li>Alternatively, make Murashige and Skoog basal salt medium (MS medium) by adding 2.2 g of MS medium + 4 g of agar in 500 ml water. Autoclave and pour into small plant pots. Keep in the 4 &#176;C until used.</li>
		<li>Sow the seed by placing them on the solidified MS medium. Keep under long daylight (14 hr light and 10 hr of dark). Seeds will germinate in about 4 - 6 days (Figure 5).</li>
		<li>Use one seed from each flower as a starting point. If no positive transformant is obtained, repeat this step by picking another seed from the flower. 
		NOTE: Test different seeds from the same flowers, because in some cases not all seeds from one flower will be transformed. Selecting additional seeds from experimental flowers is sometimes necessary.</li>
	</ol>
	</li>
	<li>Check on the seedlings regularly. In approximately 10 - 14 days after germination, when true leaves develop, test the plants with direct PCR.</li>
	<li>Prepare the leaf DNA extract by cutting 2 - 3 leaves (5 - 10 mg in weight) from each seedling and place them in a microcentrifuge tube.</li>
	<li>Add 180 &#181;l of 50 mM NaOH to each tube, and incubate for 10 min at 95 &#176;C.</li>
	<li>Neutralize the extract by adding 20 &#181;l of 1 M Tris-HCl (pH 8.0).</li>
	<li>Use 1 &#181;l of the extract in the direct PCR reaction, using primers designed across the T-DNA or the insert (Figure 7), to select for positive transformants.
	<ol>
		<li>In this protocol, use direct PCR with an initial hold of 24 &#176;C, then 2 min at 98 &#176;C, followed by 40 cycles of (98 &#176;C for 10 sec, annealing step for 15 sec, and an extension step at 68 &#176;C). Perform a final extension step at 68 &#176;C for 5 min followed by an indefinite hold at 4 &#176;C. (Refer to Table 1 for details on primers, annealing temperatures and extension times for the cycles).</li>
	</ol>
	</li>
	<li>Identify positive transformants and grow them to maturity. If the seeds were germinated in MS medium, transplant to soil in a bigger pot (Figure 5).</li>
</ol>

<ol>
	<li>Bent, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arabidopsis+thaliana+floral+dip+transformation+method.">Arabidopsis thaliana floral dip transformation method.</a> Methods Mol Biol. 343, 87-103 (2006).</li><li>Beranova, M., Rakousky, S., Vavrova, Z., Skalicky, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sonication+assisted+Agrobacterium-mediated+transformation+enhances+the+transformation+efficiency+in+flax+(Linum+usitatissimum+L.).">Sonication assisted Agrobacterium-mediated transformation enhances the transformation efficiency in flax (Linum usitatissimum L.).</a> Plant Cell Tiss Organ Cult. 94, 253-259 (2008).</li><li>Chen, Y., Schneeberger, R. G., Cullis, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+site-specific+insertion+sequence+in+flax+genotrophs+induced+by+environment.">A site-specific insertion sequence in flax genotrophs induced by environment.</a> New Phytol. 167, 171-180 (2005).</li><li>Clough, S. J., Bent, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Floral+dip:+a+simplified+method+for+Agrobacterium-mediated+transformation+of+Arabidopsis+thaliana.">Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana.</a> Plant J. 16 (6), 735-743 (1998).</li><li>Cullis, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+and+Control+of+Rapid+Genomic+Changes+in+Flax.">Mechanisms and Control of Rapid Genomic Changes in Flax.</a> Ann Bot. 95, 201-206 (2005).</li><li>Dong, J. Z., McHughen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+procedure+for+production+of+transgenic+flax+plants+using+Agrobacterium+tumefaciens.">An improved procedure for production of transgenic flax plants using Agrobacterium tumefaciens.</a> Plant Sci. 88, 61-71 (1993).</li><li>Logemann, E., Birkenbihl, R. P., Ulker, B., Somssich, I. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+method+for+preparing+Agrobacterium+cells+that+simplifies+the+Arabidopsis+transformation+protocol.">An improved method for preparing Agrobacterium cells that simplifies the Arabidopsis transformation protocol.</a> Plant Methods. 2 (16), (2006).</li><li>Mara, C., Grigorova, B., Liu, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Floral-dip+transformation+of+Arabidopsis+thaliana+to+examine+pTSO2::&#946;-glucuronidase+reporter+gene+expression.">Floral-dip transformation of Arabidopsis thaliana to examine pTSO2::&#946;-glucuronidase reporter gene expression.</a> J Vis Exp. (40), (2010).</li><li>Mlynarova, L., Bauer, M., Nap, J. -. P., Pretova, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+efficiency+Agrobacterium-mediated+gene+transfer+to+flax.">High efficiency Agrobacterium-mediated gene transfer to flax.</a> Plant Cell Rep. 13, 282-285 (1994).</li><li>Mu, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+transformation+of+maize+female+inflorescence+following+floral+dip+method+mediated+by+agrobacterium.">Genetic transformation of maize female inflorescence following floral dip method mediated by agrobacterium.</a> Biotechnology. 11 (3), 178-183 (2012).</li><li>Trieu, A. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformation+of+Medicago+truncatula+via+infiltration+of+seedlings+or+flowering+plants+with+Agrobacterium.">Transformation of Medicago truncatula via infiltration of seedlings or flowering plants with Agrobacterium.</a> Plant J. 22 (6), 531-541 (2000).</li><li>Yasmeen, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Planta+transformation+of+tomato.">In Planta transformation of tomato.</a> Plant Mol Biol Rep. 27, 20-28 (2009).</li><li>Zale, J. M., Agarwal, S., Loar, S., Steber, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+stable+transformation+of+wheat+by+floral+dip+in+Agrobacterium+tumefaciens.">Evidence for stable transformation of wheat by floral dip in Agrobacterium tumefaciens.</a> Plant Cell Rep. 28, 903-913 (2009).</li><li>Zhan, X. C., Jones, D. A., Kerr, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+of+flax+plants+transformed+by+Agrobacterium+rhizogenes.">Regeneration of flax plants transformed by Agrobacterium rhizogenes.</a> Plant Mol Biol. 11, 551-559 (1988).</li></ol>

The authors have nothing to disclose.

In some plant species, such as flax (Linum usitatissimum), successful plant transformation has been limited. Previously, transformation in flax has required an Agrobacterium infection by wounding and co-cultivation, applying biolistic particles or using ultrasound sonication, followed by regeneration; a process that is both long and prone to being accompanied by many mutational events. Moreover, the selection process of these techniques requires the use of antibiotic selectable markers such as kanamycin. However, it has been noted in the literature that this method of selection produces many false positives, as flax tends to escape high concentrations of antibiotics6,9,14. Another disadvantage of the previous techniques in flax transformation has been the low transformation rates2,6.
In the protocol described here, Agrobacterium-mediated plant transformation via floral-dipping was shown to result in a high transformation rate for flax (50 - 60%). Transformants were obtained from flowers dipped and collected from main and side branches. Selection of positive transformants was simply done by growing T1 plants on soil and screening their leaves soon after they germinated, by-passing the use of antibiotic selection, a step previously used as a norm in floral-dip for other plant species. By performing direct PCR testing of leaves, and using the appropriate T-DNA primers, positive transformants can be rapidly selected. This technique is simple, inexpensive and easy to perform, yet results in a much higher transformation rate than those previously reported for Arabidopsis and other plant species using this method1,10,12. It is also the highest reported transformation rate for flax.
However, there are critical steps in the procedures including the selection of the best flower stage and the best surfactant concentration, so that the Agrobacterium can penetrate into the plant cells without killing the flower organs. If an early bud stage is used (Figure 2A) with high Silwet-77 concentration of more than 0.05%, the flower will not develop nor set seeds. If late bud stage is used (Figure 2C), although the transformation might work, it will occur at a much lower rate. Similar results were obtained with Arabidopsis floral dip transformation1,4. For this protocol, all the floral stages were tested with different Silwet-77 concentrations and the best stage was determined to be the middle bud stage (Figure 2C) with Silwet-77 at 0.05% for the first dipping, followed by a second dipping at the late bud stage (Figure 2C) with a slightly reduced Silwet-77 concentration of 0.03%. The transformation also worked well using the early bud stage (Figure 2A) with a low silwet-77 concentration of 0.003%, followed by a second dipping with middle bud stage (Figure 2B) at higher Silwet-77 concentration of 0.05%.
In this protocol, some other parameters were attempted to optimize the transformation rate, but found to have no effects on the final outcome. Examples include extending the time after dipping that the plants lay on their side and covered in plastic from one day to two days; using an OD of more than 1 for Agrobacterium culture, instead of 0.5 - 1; increasing the dipping time to 5 - 15 min instead of 1 - 2 min. Again we have not noticed any effect on the transformation rate using these strategies. The most effective factors, however, were found to be using healthy plants at the correct flower stages, and using the best Silwet-77 concentration. We noticed that two dipping intervals, works somehow better than one time, even though one time dipping also works.
Modification to this protocol can be achieved by reducing the Silwet-77 concentration to as little as 0.003% in the second or third dipping. Since Silwet-77 is toxic, too high a concentration results in the flowers developing poorly, resulting in no seed yield. The dipping frequency can be reduced to one, with the second or third events eliminated if the plants are not looking healthy and the buds are not developing well.
A major limitation of this technique is the low number of flowers produced by the flax, the limited number of seeds obtained from each flower, and the long life cycle of flax. It takes 6 - 8 weeks from seed sowing to have the primary buds ready for the first dipping and an additional 8 - 10 weeks post-dipping to get to the T1 generation. In total, a range of 5 - 6 months is needed to obtain the T1 generation. Unlike other plant species, which flower anytime of the year, some flax varieties flower better at specific times the year. So thoughtful planning for this technique is important.
In summary, our results of floral dip with two different flax varieties: the fiber flax, Stormont Cirrus (responsive and plastic), and the oil flax, Bethune (stable and non-responsive), show that Agrobacterium-mediated plant transformation via floral-dip is an applicable and efficient method for flax transformation and can be used to replace the previously used techniques for flax transformation. The modifications of the floral-dip method in this protocol will be applicable for use with any other plant species and not limited to flax.

Flax (Linum usitatissimum) is an important crop grown widely for its fibers and oils. Transformation of the flax genome is possible with techniques such as wounding, Agrobacterium infection, and co-cultivation in tissue culture, applying biolistic particles or ultrasound sonication followed by regeneration. However, these techniques have many disadvantages, including proclivity to many mutational events and an extended time to obtain the transgenic lines. Some of these methods can also be expensive and require skilled and efficient manipulation of the instruments, resulting in low recovery seedlings. Most importantly, these technique often result in low transformation rates2,6.
Agrobacterium-mediated plant transformation via floral-dip is a simple and efficient approach to generate transgenic plants. It has been routinely and successfully used for many plant species such as Arabidopsis thaliana1,4, Medicago truncatula11, tomato12, wheat13 and maize10. However, it has not been thought of as a viable technique for flax transformation due to several factors, such as the low numbers of flowers produced by flax, limited number of seeds obtained from each flower, the large seed size, and the thick coat, which could also be problematic for such genetic transformation process. Additionally, the selection segment of the floral-dip technique requires germinating transformed seeds on a plant media containing an antibiotic, with transformed progenies distinguished based on their ability to germinate and stay green, while non-transformed progenies either do not germinate or germinate but bleach out quickly and die. In the current literature, it has been noted that wild-type flax tends to escape high concentration of antibiotic selections, producing false positive results, and making the selection of T1 progenies based on antibiotic resistance more difficult6,14. Also, when a high concentration of antibiotic was added to the selection medium, the rate of observed transformation dropped dramatically9.
In this protocol, we used Agrobacterium and the floral-dip method to transform a line of fiber flax, Stormont cirrus (responsive and plastic), which has been shown to respond to stresses in the environment by altering its genome3,5. To overcome the antibiotic escape problem, we have chosen to do direct PCR testing of DNA from T1 leaves, instead of selection by adding the antibiotic to the plant media. We took advantage of the simple anatomy of flax to track specific flowers at the time of treatment. This tracking system allowed selection of seeds from specific flowers and germination on soil without adding antibiotic. Positive transformants were simply identified by testing DNA obtained from leaves using the fast and efficient method of direct PCR. Our results demonstrate that the floral-dip method worked very well in this line of flax and surprisingly resulted in a very high transformation rate (50 - 60%), higher than those previously observed for Arabidopsis thaliana, which was reported to be 0.1 - 1%1, and also higher than other plant species10,12. We also tested another variety of linseed (oil flax), Bethune (stable and non-responsive), and our preliminary data indicates that floral-dip also works for this variety of flax.
The goal of this protocol is to show that Agrobacterium and floral-dip can be used to generate transgenic flax. We show that this technique is simple, inexpensive, and quicker than other methods of flax transformation. More importantly it results in a much higher transformation rate than the other methods of flax transformation2,6. The anatomy of Arabidopsis thaliana, which has many branches and flowers, makes it difficult to distinguish dipped and non-dipped flowers on the same plant. Therefore, large numbers of seeds, approximately 20,000 seeds per plant, need to be screened to identify positive transformants8. Flax, on the other hand, has fewer branches (one main branch and a few side branches) and fewer flowers, producing approximately 100 seeds per plants, which makes it possible to track individual flowers and to select specific seeds during screening process.
We propose that floral-dip is an applicable method to transform any related species of flax, a genus of approximately 200 species. This method gives much higher transformation rate than other methods of flax transformation. We are also proposing that the direct PCR screening of T1 leaf DNA is an efficient way to overcome the problem of antibiotic resistance escape that often produces many false positives. Direct PCR screening can be applied to any other plant species and is not limited to flax. The simple seed tracking technique employed in this protocol can be applied to any other plant species with branching anatomy similar to flax.

<table><tbody><tr><td>Flax seeds of the original Stormont Cirrus variety (PL)</td><td></td><td></td><td></td></tr><tr><td>5&quot; pots</td><td></td><td></td><td></td></tr><tr><td>Potting soil</td><td></td><td></td><td></td></tr><tr><td>Greenhouse with appropriate light setting</td><td></td><td></td><td></td></tr><tr><td>Thermocycler</td><td></td><td></td><td></td></tr><tr><td>Agarose gel electropheresis equipment</td><td></td><td></td><td></td></tr><tr><td>Digital imaging setup</td><td></td><td></td><td></td></tr><tr><td>Silwet-77</td><td>LEHLE SEEDS</td><td>VIS-01</td><td>Toxic, wear gloves</td></tr><tr><td>GoTaq Green Master Mix</td><td>Promega</td><td>Part# 9PIM712</td><td></td></tr><tr><td>Terra PCR Direct Polymerase Mix</td><td>Clontech</td><td>639270</td><td></td></tr><tr><td>Binary vector PRI 909</td><td>Takara</td><td>3260</td><td></td></tr><tr><td>Agrobacterium tumefaciens LBA4404 E</td><td>Takara</td><td>9115</td><td></td></tr><tr><td>TOPO TA cloning kit</td><td>Invitrogen</td><td>K4595-01</td><td></td></tr><tr><td>Sucrose</td><td>Fisher Scientific</td><td></td><td></td></tr><tr><td>Electroporator and cuvettes</td><td>Bio-Rad</td><td>165-2092</td><td></td></tr><tr><td>Shaker</td><td></td><td></td><td></td></tr><tr><td>Spinner</td><td></td><td></td><td></td></tr><tr><td>Plastic wrap and aluminum foil wrap</td><td></td><td></td><td></td></tr><tr><td>speedSTAR DNA polymerase</td><td>Takara</td><td>RR070A/B</td><td></td></tr><tr><td>QlAquick gel extraction kit</td><td>Qiagen</td><td>28704</td><td></td></tr><tr><td>QIAGEN plasmid mini kit</td><td>Qiagen</td><td>12123</td><td></td></tr><tr><td>SalI-HF enzyme</td><td>NEB</td><td>R3138S</td><td></td></tr><tr><td>SacI-HF enzyme</td><td>NEB</td><td>R3156S</td><td></td></tr><tr><td>T4 DNA ligation kit</td><td>NEB</td><td>M0202</td><td></td></tr><tr><td>Murashige Skoog</td><td>Sigma</td><td>M5524</td><td></td></tr><tr><td>Agar</td><td>Fisher Scientific</td><td>A360-500</td><td></td></tr></tbody></table>

floral-dip transformation of flax (linum usitatissimum) to generate transgenic progenies with a high transformation rate

Agrobacterium-mediated plant transformation via floral-dip is a widely used technique in the field of plant transformation and has been reported to be successful for many plant species. However, flax (Linum usitatissimum) transformation by floral-dip has not been reported. The goal of this protocol is to establish that Agrobacterium and the floral-dip method can be used to generate transgenic flax. We show that this technique is simple, inexpensive, efficient, and more importantly, gives a higher transformation rate than the current available methods of flax transformation.
In summary, inflorescences of flax were dipped in a solution of Agrobacterium carrying a binary vector plasmid (T-DNA fragment plus the Linum Insertion Sequence, LIS-1) for 1 - 2 min. The plants were laid flat on their side for 24 hr. Then, plants were maintained under normal growth conditions until the next treatment. The process of dipping was repeated 2 - 3 times, with approximately 10 - 14 day intervals between dipping. The T1 seeds were collected and germinated on soil. After approximately two weeks, treated progenies were tested by direct PCR; 2 - 3 leaves were used per plant plus the appropriate T-DNA primers. Positive transformants were selected and grown to maturity. The transformation rate was unexpectedly high, with 50 - 60% of the seeds from treated plants being positive transformants. This is a higher transformation rate than those reported for Arabidopsis thaliana and other plant species, using floral-dip transformation. It is also the highest, which has been reported so far, for flax transformation using other methods for transformation.

Figure 1 - 4 illustrate some of the steps within the protocol. In Figures 1 and 2, the leaves around the inflorescence buds are cut to expose them to the Agrobacterium cells and the different bud stages that were used to develop the protocol. Figure 3 shows the process of flax floral-dip. Figure 4, shows an example of how the main and side branches can be labeled and how individual flowers can be tracked and identified. Figure 5 shows how the T1 progenies can be germinated on the MS plant media and then transplanted to soil for maturity. Figure 6 illustrates how wild-type flax can escape high concentrations of kanamycin, confirming previous findings in the literature6,9,14.
Figure 7 shows an example of direct PCR amplification from positive T1 transformants. The T1 flowers were collected from the main and side shoots of a single T0 plant. As can be seen from the direct PCR, 8/12 T1 plants tested positive by PCR and have amplified the different regions across the T-DNA. Our primers were also designed between the LIS-1 insert and the multiple cloning sites (Figure 7B and C). We used additional primers from the plant binary vector to amplify different segments of the T-DNA, such as the left border and the NOS terminator (data not shown) or the right border and the multiple cloning site (Figure 7D). Primers specific to the LIS-1 insert were also used in this protocol (data not shown). A list of primers is provided in Table 1. However, the sequences of these primers depends on the sequence of the T-DNA plant binary vector and the insert used for the floral-dip. We also noted that there was no significant difference in the transformation rate between flowers collected from the main and side branches.
<img alt="Figure 1" src="/files/ftp_upload/52189/52189fig1highres.jpg" /> 
Figure 1. Cutting the leaves around the primary inflorescence buds to expose them to the Agrobacterim cells. (A) The buds are covered by leaves. (B) Leaves have been cut around the buds to expose them. (C) Magnified image from plant in (A) after cutting to expose buds. <a href="https://www.jove.com/files/ftp_upload/52189/52189fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52189/52189fig2highres.jpg" /> 
Figure 2. The different bud stages that were used in this protocol to determine the best stage to use for the floral dip. (A) The early stage bud is approximately 2 mm. (B) The middle stage bud is approximately 5 mm. (C) The Late stage bud is approximately about 1 cm. <a href="https://www.jove.com/files/ftp_upload/52189/52189fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/52189/52189fig3highres.jpg" /> 
Figure 3. The process of flax floral-dipping.&#160; (A) The primary inflorescences are dipped in the infiltration media containing the Agrobacterium cells. (B) Magnified from (A). (C) The dipped plants are laid flat until the next day, and the dipped branches are covered with plastic to maintain high humidity. <a href="https://www.jove.com/files/ftp_upload/52189/52189fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/52189/52189fig4highres.jpg" /> 
Figure 4. The process of flower tracking and seed collections, from the T0 treatedplants. (A) An example of the whole plant with the main branch (the tallest branch in the center) and the side branches. (B - D) An example of the flowers from the different branches. (E) An example of the seeds collected from individual flowers (labeled a - k) from the main branch. <a href="https://www.jove.com/files/ftp_upload/52189/52189fig4highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/52189/52189fig5highres.jpg" /> 
Figure 5. The T1 seedlings are grown without antibiotic selection. (A) T1 seeds are germinated on the MS plant media. (B) Positive transformants, as determined by direct PCR, are transplanted to soil and grown to maturity. <a href="https://www.jove.com/files/ftp_upload/52189/52189fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/52189/52189fig6highres.jpg" /> 
Figure 6. Antibiotic escape, a problem for T1 selection, is overcome by direct PCR screening. (A) Wild-type flax seeds germinated on MS plant media without antibiotic. (B) Wild-type flax seeds germinated on MS plant media+ increasing concentrations of kanamycin (200 &#181;g/ml, 600 &#181;g/ml, 1 mg/ml). (C) Wild-type flax seeds germinated on MS plant media with 2 mg/ml kanamycin. (D) PCR from wild-type flax and T1 seedling using kanamycin primers, all amplified the kanamycin gene (legends: EZ1: DNA marker, FlaxS, wild-type flaxS, C: control non-dipped branch, Ma: T1 progeny from flower &#8220;a&#8221; collected from the main branch, Sc,Sd,Se,Sf: T1 progenies of different flowers &#8220;c,d,e,f&#8221; collected from the side branch, W: no-DNA). <a href="https://www.jove.com/files/ftp_upload/52189/52189fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" src="/files/ftp_upload/52189/52189fig7highres.jpg" /> 
Figure 7. An example of successful PCR amplifications of T1 progenies using the direct PCR method. (A) Diagram of the Plant binary vector+ the cloned LIS-1 insert. Blue arrows indicate the position of PCR primers used in the direct PCR screening (modified from Takara). (B) PCR with primers M13F+3&#8217; (C) PCR with primers M13R+18a (D) PCR with primers right border (RB) and multiple cloning site (MCS) **Each lane represents T1 from individual flowers collected from C: control branch (non-dipped), M: main branch flower a-g, S: side branch flower b-c&#8217;. <a href="https://www.jove.com/files/ftp_upload/52189/52189fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Forward primer sequence source</td>
			<td>sequence 5&#39;-3&#39;</td>
			<td>Reverse primer sequence source</td>
			<td>Sequence 5&#39;-3&#39;</td>
			<td>Annealing Temprature (&#176;C)</td>
			<td>Extension Time (Sec)</td>
			<td>Expected size (bp)</td>
		</tr>
		<tr>
			<td>M13F (T-DNA)</td>
			<td>CTGCAAGGCG 
			ATTAAGTTGG</td>
			<td>3&#39; (LIS-1 insert)</td>
			<td>GAGGATGGAA 
			GATGAAGAAGG</td>
			<td>57</td>
			<td>40</td>
			<td>450</td>
		</tr>
		<tr>
			<td>18a (LIS-1 insert)</td>
			<td>TATTTTAACCC 
			TATCTCCCAACAC</td>
			<td>M13R (T-DNA)</td>
			<td>ATTAGGCACC 
			CCAGGCTTTA</td>
			<td>57</td>
			<td>40</td>
			<td>520</td>
		</tr>
		<tr>
			<td>MCS (T-DNA)</td>
			<td>TGGTCATAGC 
			TGTTTCCTGTG</td>
			<td>RB (T-DNA)</td>
			<td>TTTAAACTGA 
			AGGCGGGAAA</td>
			<td>60</td>
			<td>20</td>
			<td>200</td>
		</tr>
		<tr>
			<td>LB (T-DNA)</td>
			<td>TTTGATGGTG 
			GTTCCGAAAT</td>
			<td>NOS (T-DNA)</td>
			<td>GAATCCTGTT 
			GCCGGTCTT</td>
			<td>60</td>
			<td>30</td>
			<td>380</td>
		</tr>
		<tr>
			<td>NPTII (T-DNA)</td>
			<td>GCGATACCGT 
			AAAGCACGAG</td>
			<td>NTPII (T-DNA)</td>
			<td>GCTCGACGTT 
			GTCACTGAAG</td>
			<td>65</td>
			<td>45</td>
			<td>502</td>
		</tr>
	</tbody>
</table>
Table 1. Some of the primers used for the direct PCR testing.

Watch this Scientific Journal Video about Floral-Dip Transformation of Flax Linum usitatissimum to Generate Transgenic Progenies with a High Transformation Rate at JoVE.com

Floral-Dip Transformation of Flax Linum usitatissimum to Generate Transgenic Progenies with a High Transformation Rate

Here, we present a protocol to transform flax using Agrobacterium-mediated plant transformation via floral-dip. This protocol is simple to perform and inexpensive, yet yields a higher transformation rate than the current available methods for flax transformation.

Floral-Dip Transformation of Flax (Linum usitatissimum) to Generate Transgenic Progenies with a High Transformation Rate

Agrobacterium-mediated plant transformation via floral-dip is a widely used technique in the field of plant transformation and has been reported to be ...

floral-dip-transformation-flax-linum-usitatissimum-to-generate

Research

JoVE Journal

Biology

29.2K Views.  Case Western Reserve University. Here, we present a protocol to transform flax using Agrobacterium-mediated plant transformation via floral-dip. This protocol is simple to perform and inexpensive, yet yields a higher transformation rate than the current available methods for flax transformation.

Video: Floral-Dip Transformation of Flax Linum usitatissimum to Generate Transgenic Progenies with a High Transformation Rate

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

When generating transgenic plants, generally the objective is to have stable expression of a transgene. This requires a single, intact integration of the transgene, as multi-copy integrations are often subjected to gene silencing. The Gateway-compatible binary vector based on bacterial artificial chromosomes (pBIBAC-GW), like other pBIBAC derivatives, allows the insertion of single-copy transgenes with high efficiency. As an improvement to the original pBIBAC, a Gateway cassette has been cloned into pBIBAC-GW, so that the sequences of interest can now be easily incorporated into the vector transfer DNA (T-DNA) by Gateway cloning. Commonly, the transformation with pBIBAC-GW results in an efficiency of 0.2&#8211;0.5%, whereby half of the transgenics carry an intact single-copy integration of the T-DNA. The pBIBAC-GW vectors are available with resistance to Glufosinate-ammonium or DsRed fluorescence in seed coats for selection in plants, and with resistance to kanamycin as a selection in bacteria. Here, a series of protocols is presented that guide the reader through the process of generating transgenic plants using pBIBAC-GW: starting from recombining the sequences of interest into the pBIBAC-GW vector of choice, to plant transformation with Agrobacterium, selection of the transgenics, and testing the plants for intactness and copy number of the inserts using DNA blotting. Attention is given to designing a DNA blotting strategy to recognize single- and multi-copy integrations at single and multiple loci.

Using a pBIBAC-GW binary vector makes generating transgenic plants with intact single-copy insertions, an easy process. Here, a series of protocols is presented that guide the reader through the process of generating transgenic Arabidopsis plants, and testing the plants for intactness and copy number of the inserts.

When generating transgenic plants, generally the objective is to have stable expression of a transgene. This requires a single, intact integration of the ...

Genetics

Plant Growth and Agrobacterium-mediated Floral-dip Transformation of the Extremophyte Schrenkiella parvula

Schrenkiella parvula is an extremophyte adapted to various abiotic stresses, including multiple ion toxicity stresses. Despite high-quality genomic resources available to study how plants adapt to environmental stresses, its value as a functional genomics model and tool has been limited by the lack of a feasible transformation system. In this protocol, we report how to generate stable transgenic S. parvula lines using an Agrobacterium-mediated floral-dip method. We modified the transformation protocol used for A. thaliana to account for unique traits of S. parvula, such as an indeterminate flowering habit and a high epicuticular wax content on leaves. Briefly, S. parvula seeds were stratified at 4 &#176;C for five days before planting. Plants were grown at a photoperiod of a 14 h light and 10 h dark and a 130 &#181;mol m-2s-1 light intensity, at 22 &#176;C to 24 &#176;C. Eight to nine week-old plants with multiple inflorescences were selected for transformation. These inflorescences were dipped in an infiltration solution of Agrobacterium tumefaciens GV3101 carrying the pMP90RK plasmid. We performed two rounds of flower dipping with an interval of three to four weeks to increase the transformation efficiency. The T1 seeds were collected and dried for four weeks in a container with desiccants before germination to screen for candidate transformed lines. Resistance to BASTA was used to screen T1 plants. We sprayed the BASTA solution three times with an interval of three days starting at two week-old plants to reduce false positives. A BASTA drop test was performed on surviving individual plants to identify true positive transformants. The transformation efficiency was 0.033%, yielding 3&#8211;4 transgenic plants per 10,000 T1 seeds propagated.

Plant Growth and Agrobacterium-mediated Floral-dip Transformation of the Extremophyte Schrenkiella parvula

Agrobacterium-mediated transformation using a floral-dip method can be successfully employed to create stable transgenic lines of the extremophyte model Schrenkiella parvula. We present a protocol modified from that for Arabidopsis thaliana, considering different growth habits and physiological characteristics of the extremophyte.

Schrenkiella parvula is an extremophyte adapted to various abiotic stresses, including multiple ion toxicity stresses. Despite high-quality genomic ...

TransFLP &#x2014; A Method to Genetically Modify Vibrio cholerae Based on Natural Transformation and FLP-recombination

Several methods are available to manipulate bacterial chromosomes1-3. Most of these protocols rely on the insertion of conditionally replicative plasmids (e.g. harboring pir-dependent or temperature-sensitive replicons1,2). These plasmids are integrated into bacterial chromosomes based on homology-mediated recombination. Such insertional mutants are often directly used in experimental settings. Alternatively, selection for plasmid excision followed by its loss can be performed, which for Gram-negative bacteria often relies on the counter-selectable levan sucrase enzyme encoded by the sacB gene4. The excision can either restore the pre-insertion genotype or result in an exchange between the chromosome and the plasmid-encoded copy of the modified gene. A disadvantage of this technique is that it is time-consuming. The plasmid has to be cloned first; it requires horizontal transfer into V. cholerae (most notably by mating with an E. coli donor strain) or artificial transformation of the latter; and the excision of the plasmid is random and can either restore the initial genotype or create the desired modification if no positive selection is exerted. Here, we present a method for rapid manipulation of the V. cholerae chromosome(s)5 (Figure 1). This TransFLP method is based on the recently discovered chitin-mediated induction of natural competence in this organism6 and other representative of the genus Vibrio such as V. fischeri7. Natural competence allows the uptake of free DNA including PCR-generated DNA fragments. Once taken up, the DNA recombines with the chromosome given the presence of a minimum of 250-500 bp of flanking homologous region8. Including a selection marker in-between these flanking regions allows easy detection of frequently occurring transformants.
This method can be used for different genetic manipulations of V. cholerae and potentially also other naturally competent bacteria. We provide three novel examples on what can be accomplished by this method in addition to our previously published study on single gene deletions and the addition of affinity-tag sequences5. Several optimization steps concerning the initial protocol of chitin-induced natural transformation6 are incorporated in this TransFLP protocol. These include among others the replacement of crab shell fragments by commercially available chitin flakes8, the donation of PCR-derived DNA as transforming material9, and the addition of FLP-recombination target sites (FRT)5. FRT sites allow site-directed excision of the selection marker mediated by the Flp recombinase10.

A quick method to modify the genome of V. cholerae is described. These modifications include the deletion of single genes, gene clusters and genomic islands as well as the integration of short sequences (e.g. promoter elements or affinity-tag sequences). The method is based on the natural transformation and FLP-recombination.

Several methods are available to manipulate bacterial chromosomes1-3. Most of these protocols rely on the insertion of conditionally replicative plasmids ...

Immunology and Infection

Inducible, Cell Type-Specific Expression in Arabidopsis thaliana Through LhGR-Mediated Trans-Activation

Inducible, tissue-specific expression is an important and powerful tool to study the spatio-temporal dynamics of genetic perturbation. Combining the flexible and efficient GreenGate cloning system with the proven and benchmarked LhGR system (here termed GR-LhG4) for the inducible expression, we have generated a set of transgenic Arabidopsis lines that can drive the expression of an effector cassette in a range of specific cell types in the three main plant meristems. To this end, we chose the previously developed GR-LhG4 system based on a chimeric transcription factor and a cognate pOp-type promoter ensuring tight control over a wide range of expression levels. In addition, to visualize the expression domain where the synthetic transcription factor is active, an ER-localized mTurquoise2 fluorescent reporter under control of the pOp4 or pOp6 promoter is encoded in driver lines. Here, we describe the steps necessary to generate a driver or effector line and demonstrate how cell type specific expression can be induced and followed in the shoot apical meristem, the root apical meristem and the cambium of Arabidopsis. By using several or all driver lines, the context specific effect of expressing one or multiple factors (effectors) under control of the synthetic pOp promoter can be assessed rapidly, for example in F1 plants of a cross between one effector and multiple driver lines. This approach is exemplified by the ectopic expression of VND7, a NAC transcription factor capable of inducing ectopic secondary cell wall deposition in a cell autonomous manner.

Inducible, Cell Type-Specific Expression in Arabidopsis thaliana Through LhGR-Mediated Trans-Activation

Here, we describe the generation and application of a set of transgenic Arabidopsis thaliana lines enabling inducible, tissue-specific expression in the three main meristems, the shoot apical meristem, the root apical meristem, and the cambium.

Inducible, tissue-specific expression is an important and powerful tool to study the spatio-temporal dynamics of genetic perturbation. Combining the ...

Co-expression of Multiple Chimeric Fluorescent Fusion Proteins in an Efficient Way in Plants

Information about the spatiotemporal subcellular localization(s) of a protein is critical to understand its physiological functions in cells. Fluorescent proteins and generation of fluorescent fusion proteins have been wildly used as an effective tool to directly visualize the protein localization and dynamics in cells. It is especially useful to compare them with well-known organelle markers after co-expression with the protein of interest. Nevertheless, classical approaches for protein co-expression in plants usually involve multiple independent expression plasmids, and therefore have drawbacks that include low co-expression efficiency, expression-level variation, and high time expenditure in genetic crossing and screening. In this study, we describe a robust and novel method for co-expression of multiple chimeric fluorescent proteins in plants. It overcomes the limitations of the conventional methods by using a single expression vector that is composed of multiple semi-independent expressing cassettes. Each protein expression cassette contains its own functional protein expression elements, and therefore it can be flexibly adjusted to meet diverse expression demand. Also, it is easy and convenient to perform the assembly and manipulation of DNA fragments in the expression plasmid by using an optimized one-step reaction without additional digestion and ligation steps. Furthermore, it is fully compatible with current fluorescent protein derived bio-imaging technologies and applications, such as FRET and BiFC. As a validation of the method, we employed this new system to co-express fluorescently fused vacuolar sorting receptor and secretory carrier membrane proteins. The results show that their perspective subcellular localizations are the same as in previous studies by both transient expression and genetic transformation in plants.

We have developed a novel method for co-expressing multiple chimeric fluorescent fusion proteins in plants to overcome the difficulties of conventional methods. It takes advantage of using a single expression plasmid that contains multiple functionally independent protein expressing cassettes to achieve protein co-expression.

Information about the spatiotemporal subcellular localization(s) of a protein is critical to understand its physiological functions in cells. Fluorescent ...

Chemistry

CcCIPK14 Gene Function Analysis to Illuminate the Efficient Root Transgenic System

An efficient and stable transformation system is fundamental for gene function study and molecular breeding of plants. Here, we describe the use of an Agrobacterium rhizogenes mediated transformation system on pigeon pea. The stem is infected with A. rhizogenes carrying a binary vector, which induced callus after 7 days and adventitious roots 14 days later. The generated transgenic hairy root was identified by morphological analysis and a GFP reporter gene.To further illustrate the application range of this system, CcCIPK14 (Calcineurin B-like protein-interacting protein kinases) was transformed into pigeon pea using this transformation method. The transgenic plants were treated with jasmonic acid (JA) and abscisic acid (ABA), respectively, for the purpose of testing whether CcCIPK14 responds to those hormones. The results demonstrated that (1) exogenous hormones could significantly upregulate the expression levelof CcCIPK14, especially in CcCIPK14 over-expression (OE) plants; (2) the content of Genistein in CcCIPK14-OE lines was significantly higher than the control; (3) the expression level of two downstream key flavonoid synthase genes, CcHIDH1 and CcHIDH2, were up-regulated in the CcCIPK14-OE lines; and (4) the hairy root transgenic system can be used to study metabolically functional genes in non-model plants.

CcCIPK14 Gene Function Analysis to Illuminate the Efficient Root Transgenic System

Here we present an efficient and stable transformation system for the functional analysis of the CcCIPK14 gene as an example, providing a technical basis for studying the metabolism of non-model plants.

An efficient and stable transformation system is fundamental for gene function study and molecular breeding of plants. Here, we describe the use of an ...

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&beta;-glucuronidase Reporter Gene Expression

The ability to introduce foreign genes into an organism is the foundation for modern biology and biotechnology. In the model flowering plant Arabidopsis thaliana, the floral-dip transformation method1-2 has replaced all previous methods because of its simplicity, efficiency, and low cost. Specifically, shoots of young flowering Arabidopsis plants are dipped in a solution of Agrobacterium tumefaciens carrying specific plasmid constructs. After dipping, the plants are returned to normal growth and yield seeds, a small percentage of which are transformed with the foreign gene and can be selected for on medium containing antibiotics. This floral-dip method significantly facilitated Arabidopsis research and contributed greatly to our understanding of plant gene function. In this study, we use the floral-dip method to transform a reporter gene, &beta;-glucuronidase (GUS), under the control of TSO2 promoter. TSO2, coding for the Ribonucleotide Reductase (RNR) small subunit3, is a cell cycle regulated gene essential for dNDP biosynthesis in the S-phase of the cell cycle. Examination of GUS expression in transgenic Arabidopsis seedlings shows that TSO2 is expressed in actively dividing tissues. The reported experimental method and materials can be easily adapted not only for research but also for education at high school and college levels.

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&#946;-glucuronidase Reporter Gene Expression

This article illustrates the floral-dip method of Agrobacterium tumefaciens -mediated transformation of Arabidopsis thaliana. By introducing a cell-cycle regulated promoter-reporter, pTSO2::&beta;-glucuronidase (GUS), into Arabidopsis, we illustrates how one detects GUS reporter expression in transgenic seedlings.

The ability to introduce foreign genes into an organism is the foundation for modern biology and biotechnology. In the model flowering plant Arabidopsis ...

Environmentally Induced Heritable Changes in Flax

Some flax varieties respond to nutrient stress by modifying their genome and these modifications can be inherited through many generations. Also associated with these genomic changes are heritable phenotypic variations 1,2. The flax variety Stormont Cirrus (Pl) when grown under three different nutrient conditions can either remain inducible (under the control conditions), or become stably modified to either the large or small genotroph by growth under high or low nutrient conditions respectively. The lines resulting from the initial growth under each of these conditions appear to grow better when grown under the same conditions in subsequent generations, notably the Pl line grows best under the control treatment indicating that the plants growing under both the high and low nutrients are under stress. One of the genomic changes that are associated with the induction of heritable changes is the appearance of an insertion element (LIS-1) 3, 4 while the plants are growing under the nutrient stress. With respect to this insertion event, the flax variety Stormont Cirrus (Pl) when grown under three different nutrient conditions can either remain unchanged (under the control conditions), have the insertion appear in all the plants (under low nutrients) and have this transmitted to the next generation, or have the insertion (or parts of it) appear but not be transmitted through generations (under high nutrients) 4. The frequency of the appearance of this insertion indicates that it is under positive selection, which is also consistent with the growth response in subsequent generations. Leaves or meristems harvested at various stages of growth are used for DNA and RNA isolation. The RNA is used to identify variation in expression associated with the various growth environments and/or t he presence/absence of LIS-1. The isolated DNA is used to identify those plants in which the insertion has occurred.

Growing some flax varieties under nutrient stress results in genomic variation within a subset of the genome and phenotypic variation. A complex insertion at a specific site is associated with growth under various nutrient regimes and with changes in gene expression around this site.

Some flax varieties respond to nutrient stress by modifying their genome and these modifications can be inherited through many generations. Also ...

Hyperactive piggyBac Transposase-mediated Germline Transformation in the Fall Armyworm, Spodoptera frugiperda

Stable insertion of genetic cargo into insect genomes using transposable elements is a powerful tool for functional genomic studies and developing genetic pest management strategies. The most used transposable element in insect transformation is piggyBac, and piggyBac-based germline transformation has been successfully conducted in model insects. However, it is still challenging to employ this technology in non-model insects that include agricultural pests. This paper reports on germline transformation of a global agricultural pest, the fall armyworm (FAW), Spodoptera frugiperda, using the hyperactive piggyBac transposase (hyPBase).
In this work, the hyPBase mRNA was produced and used in place of helper plasmid in embryo microinjections. This change led to the successful generation of transgenic FAW. Furthermore, the methods of screening transgenic animals, PCR-based rapid detection of transgene insertion, and thermal asymmetric interlaced PCR (TAIL-PCR)-based determination of the integration site, are also described. Thus, this paper presents a protocol to produce transgenic FAW, which will facilitate piggyBac-based transgenesis in FAW and other lepidopteran insects.

Hyperactive piggyBac Transposase-mediated Germline Transformation in the Fall Armyworm, Spodoptera frugiperda

Successful germline transformation in the fall armyworm, Spodoptera frugiperda, was achieved using mRNA of hyperactive piggyBac transposase.

Stable insertion of genetic cargo into insect genomes using transposable elements is a powerful tool for functional genomic studies and developing genetic ...

Agrobacterium-Mediated Hairy Root Induction and Regeneration in Plants

Hairy Root Transformation and Regeneration in Arabidopsis thaliana and Brassica napus

Hairy root transformation represents a versatile tool for plant biotechnology in various species. Infection by an Agrobacterium strain carrying a Root-inducing (Ri) plasmid induces the formation of hairy roots at the wounding site after the transfer of T-DNA from the Ri plasmid into the plant genome. The protocol describes in detail the procedure of the injection-based hairy root induction in Brassica napus DH12075 and Arabidopsis thaliana Col-0. The hairy roots may be used to analyze a transgene of interest or processed for the generation of transgenic plants. Regeneration medium containing cytokinin 6-benzylaminopurine (5 mg/L) and auxin 1-naphthaleneacetic acid (8 mg/L) successfully elicits shoot formation in both species. The protocol covers the genotyping and selection of regenerants and T1 plants to obtain plants carrying a transgene of interest and free of T-DNA from the Ri plasmid. An alternative process leading to the formation of a composite plant is also depicted. In this case, hairy roots are kept on the shoot (instead of the natural roots), which enables the study of a transgene in hairy root cultures in the context of the whole plant.

Author Spotlight: Optimizing Hairy Root-Based Transformation Protocols for Enhanced Efficiency in Brassicaceae

The protocol describes hairy root induction using Arabidopsis primary inflorescence stems and Brassica napus hypocotyls. The hairy roots can be cultured and used as explants to regenerate transgenic plants.

Floral-Dip Transformation of Flax (Linum usitatissimum) to Generate Transgenic Progenies with a High Transformation Rate

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Hyperactive piggyBac Transposase-mediated Germline Transformation in the Fall Armyworm, Spodoptera frugiperda

Hairy Root Transformation and Regeneration in Arabidopsis thaliana and Brassica napus