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

The goal of the following experiment is to transform flex using an agrobacterium mediated plant transformation floral dip method. This is achieved by growing the flex plants to an appropriate flowering stage with selecting primary in fluorescent buds. Next, the flowers are dipped in the infiltration medium composed of agrobacterium cells carrying the appropriate plasmid.

The treated plants are then maintained normally until they set seeds. The seeds are germinated and direct. PCR is performed from their leaves, DNA, in order to identify and select positive transformants to grow to maturity.

The results show that by using agrobacterium and floral dipping on flax, followed by direct PCR testing, transformation and selection of positive transformants is both easy and deficient. There are several advantages for this technique over many existing methods. Floral dipping via agrobacterium is simple, inexpensive, efficient, and most importantly, gives a higher transformation rate than the currently available methods for flax transformation.

Demonstrating the procedure will be nsma baki, a PhD student from my laboratory, Six weeks in advance of floral dipping. Start growing the plants. Prepare five inch pots with soil, and so the flax seeds a quarter of an inch deep.

Bury four in each pot. Ensure that the soil over the seeds is firmly packed. Then water the pots regularly and grow the plants under a 14 hour day diurnal cycle.

Mainten maintain the temperature between 23 and 27 degrees Celsius. Check regularly for primary in fluoresces cutoff leaves as needed to search for these buds when the buds have just formed. The in fluoresces are ready for transformation.

Transforming at this stage is very critical to this protocol. For the main treatment, use the main branch for other treatments and un dipped controls. Use the side branches which produce fewer flowers.

Be sure to label the branches and flowers with the date and type of treatment they receive before the floral dipping. Prepare agrobacterium cells carrying the plasmid of interest in this case, BRI 9 0 9. These preparation steps beginning with cloning are detailed in the text protocol.

Two days before floral dipping, thaw out the transformed agrobacterium stored at negative 80 degrees Celsius in 50%Glycerol grow these cells up to the stationary phase in five to 20 milliliter vials with LB containing the appropriate antibiotics. This step is usually done overnight. An OD between 0.5 and one is acceptable from the stationary phase.

Inoculate one to 100 dilutions by pouring five milliliters of the previously grown cells into 500 milliliters of LB and grow them overnight. A 28 to 30 degrees Celsius was shaking at 150 RPM. When the OD of the culture is between 0.5 and one, ideally 0.8, collect the cells with a five minute 5, 000 G spin at room temperature.

Then re suspend the cells in infiltration medium, composed of 500 milliliters of autoclave water with 5%sucrose. When the cells are fully resuspended, add 0.05%silhouette 77 to the mixture and mix. Then pour the mixture into a dish such as an eight inch by eight inch Pyrex glass dish.

In later dipping rounds, reduce the silhouette 77 concentration to 0.03%Using the best flower stage and silhouette. 77 concentration are both very critical factors for a successful yield of positive transformers. Now perform the floral dip.

First, lay the plant on its side. Then dip the visible buds into the infiltration medium for one to two minutes. After the dip, leave the plant sideways, overnight, and cover it with plastic to keep the plant humid.

The next day, return the plant to its normal conditions. After 10 to 14 days, the bud should grow larger. Then repeat the floral dipping on the same branches with a lowered concentration of silhouette.

77, using the branch labels to maintain precision. Later, evaluate the plant when the seeds are mature and ready to be collected. Take one seed from each flower to begin testing for positive transformants.

The seed should be grown as previously described. Planting three to six seeds in each five inch pot. The seeds will germinate in four to six days after being sowed.

When grown under 14 hours of light in approximately 10 to 14 days after germination, true leaves will develop, which can be used to test the plant. Using direct PCR elect five to 10 milligrams of leaf from each seedling into a weighing dish. When the weight is checked, put the leaves in a centrifuge tube.

Add 180 microliters of 50 millimolar sodium hydroxide to each tube. Then incubate the tubes for 10 minutes at 95 degrees Celsius. After the heat incubation, neutralize each reaction with 20 microliters of one molars hydrochloride at pH 8.0.

TD NA primers or insert primers should be used to generate a specific replicon from transformant plants. Then use one microliter from each extract as the template for the direct PCR reaction. After evaluating the PCR results, grow the seedlings that are positive to maturity.

If no positive transformants were obtained, pick new seeds from the same flowers of the dipped plants and repeat the screen. In some cases, not all seeds from a flower will be transformed. T one flowers were collected from the main and side shoots of a single T zero plant.

Several primer pairs were used for direct PCR. The positions of these primers are indicated with blue arrows. In a direct PCR eight of 12 T one plants tested positive.

The first two gels show primers that make amplicons from regions between TDNA and the gene of interest. Overall, there was no significant difference in the transformation of flowers collected from the main and side branches indicated As m and S, this gel shows use of a primer from within the TD NA region only. When attempting this procedure, it is important to remember to wear gloves as you're preparing the infiltration medium and dimming the plants as solid 77 is toxic and can harm your skin.

After watching this video, you should have a good understanding of how to transform flax or any other plant with similar branching anatomy to flax, using agrobacterium and floral dipping. This branching anatomy allows simple flower tracking during the treatment, thus making the selection and testing of positive transformers easier.