The overall goal of Induced Somatic Sector Analysis is to provide a medium to high throughput technique for the functional characterization of gene and promoter constructs in tree secondary stem tissue within comparatively short time frames. This method can help answer key questions around where and when danger expressed as well as their role in biologically important processes such as wood formation and secondary stem development. The main advantage of this technique is that it is efficient, easy and relatively a fast procedure that is applicable to a wide range of tree species.
To begin, prepare a clean, 50 milliliter screw cap tube with 19 milliliters of fresh, 28 degree Celsius LB medium with the appropriate antibiotics for bacterial selection. Add one milliliter of A.tumefaciens culture and check the optical density, or OD, at 600 nanometres. If the OD is above 0.1, dilute further with warm LB.Place the A.tumefaciens culture into a shaking incubator until the OD 600 is between 0.4 and 0.6.
Once this is achieved, centrifuge the cells for 15 minutes at four degrees Celsius and 1, 000 x g. Decant the liquid from the tube and immediately re-suspend the cell pellet with one milliliter of cooled MS medium. Transfer the suspension to a clean, two milliliter microtube and store on ice until required.
Begin the experiment during the spring or early summer and select plants with an active and fast growing cambium. This is confirmed if the phloem can be easily peeled from the xylem. Identify a straight section of stem near the base of the plant and remove any leaves or branches.
Using a sharp scalpel, cut a one square centimeter cambial window by creating two parallel, 20 millimeter incisions through the phloem at five millimeters apart and then a single, horizontal cut that bisects the two vertical cuts at the basal end. Heal the phloem strip upwards exposing the developing xylem tissue. And then, using a pipette, add sufficient inoculation medium to fully wet the surface of the exposed xylem.
Immediately, reinsert the phloem strip into the stem and bind it into place tightly with parafilm. Repeat these inoculation steps for all genes or promoters of interest creating new cambial windows at least one centimeter above or below existing windows and at a 90 degree offset. Additionally, inoculate windows with the positive and negative control vectors on the same stem or stems, as the experimental inoculates.
Monitor stem growth periodically and once radial growth of at least five millimeters has been observed, harvest the tissue for a beta-glucuronidase, or GUS assay. To harvest the test material, first excise the cambial window from the stem removing any tissue that is not part of new growth. For studies relating to mature xylem and phloem cell or tissue morphology, peel the phloem from the xylem.
Alternatively, for investigations requiring that the cambial zone remain intact, or promoter expression patterns to be assessed, slice the cambial window transversely using a razor blade to create discs of between 0.5 and one millimeter thickness. Place the harvested cambial window tissues into individual, 14 milliliter round bottom tubes and rinse twice in 0.1 molar phosphate buffer at pH 7 for five minutes each time ensuring that the tissue is completely submerged. At the end of the second rinse, take care to remove all excess solution.
Next, add five milliliters of GUS reagent to each tube. If this does not completely cover the tissue sample, add additional reagent. Incubate the tubes for ten minutes at 55 degrees Celsius in the dark.
Following this, incubate the tubes for a further 12 to 16 hours on a shaker at 37 degrees Celsius in the dark using gentle agitation. Upon removal from the incubator, take a random subset of the tubes and check the pH of the GUS reagent using litmus paper with pH range of zero to seven. If any of them have a pH lower than six, label them and check the remainder of the tubes.
Label and exclude any tubes with a pH lower than six. Decant the GUS reagent from the tubes and replace it with sufficient 70 percent ethanol to cover the tissue. Store the samples at four degrees Celsius until needed.
Using a clean pair of forceps, remove all the cambial window tissue from a sample tube and place it onto a small tray or petri dish for microscopic visualization. Place the sample under the lens of a dissecting microscope between 1X and 4X magnification and identify and tally the number of cells or tissues displaying bright blue staining. In examples where the phloem was removed, count the number of sectors in cambial tissue found on the surface of the developing xylem.
For samples cut into discs, count the number of sectors found in the different cell or tissue types. Ensure that both sides of the disc are viewed and that sectors that occur across two discs are matched to ensure that numbers are not overestimated. Place only the tissue containing sectors back into the tube containing 70 percent ethanol and store until needed.
Repeat the analysis for any additional cambial windows harvested including the positive and negative controls. To aid in the accurate identification in the classification of sectors, we suggest you become familiar with secondary stem anatomy and development including stem responses to wounding. This figure shows several successful transformations seen here as regions of blue staining.
An example phloem peel window displays multiple transformed sectors and discs harvested from two tree species show sectors of transformation and subsequent radial growth. Transformation in a range of tissue types can also be observed. Here stem sectors can be seen in the periderm, phloem, wound parenchyma, and several other tissue regions of white poplar.
This image shows the results of gene promoter expression in cambial derivatives. In this example, we see the proportion of staining in each tissue type in a trial involving a range of CesA promoters from eucalyptus grandis transformed into stems of eucalypt hybrids. Expression in the phloem, developing and developed xylems is represented as a percentage.
Average number of transformation events per centimeter of cambium inoculated can also be calculated for each promoter or gene of interest. This table shows the data for typical sectors identified in eucalypt and poplar subjects transformed with a positive control or promoter of interest analyzed using the disc harvesting method. Once mastered, this technique can provide a fast, efficient and widely applicable method for dissecting the complex nature of cambial differentiation with formation in secondary stem development.
After watching this video, you should have a good understanding of how to prepare plants and bacteria, how to transform, grow and harvest stems as well as how to identify and classify transgenic tissue for phenotypic assessment.
