The overall goal of this method is to characterize the pattern formation of embryos during plant embryogenesis using the model organism. This method can help answer key questions in the plant matter field, such as embryogenesis in Arabidopsis. The main advantage of this technique is that embryo pattern formation in the model plant Arabidopsis can be characterized by the recommended operation.
Demonstrating the procedure is Jinlin Feng, a graduate student from my laboratory. To begin this procedure, add 100 seeds to a 1.5 milliliter tube. Add one milliliter of sodium hypochlorite to sterilize the seeds.
Invert the tube several times to ensure that the seeds and solution are well mixed. After five minutes, centrifuge the tube at 1200 Gs for 30 seconds. Remove the supernatant and wash the seeds with sterile water four times to remove any residual sodium hypochlorite.
Next, add 4.4 grams of MS basal salt mixture and 10 grams of sucrose to one liter of ultra pure water. Stir to dissolve the salt and sucrose. Using potassium hydroxide at one mol, adjust the solution's pH to 5.8.
Then, add three grams of gelling agent and sterilize the solution at 121 degrees Celsius for 15 minutes. Pour 30 milliliters of the resulting MS medium into a 12-centimeter culture dish. Place the sterilized seeds on the MS plate.
Transfer the plate to a refrigerator, and keep it four degrees centigrade in the dark for three days to stratify the seeds. Next, keep the plate in a growth chamber under long-day conditions for eight days. Then, transfer the plants to soil in a tray.
Cover the tray with a transparent white lid to prevent water loss. Leaving a small gap to avoid vitrification of the plants. After five days, remove the lid and cultivate the plants in a walk-in chamber under long-day conditions.
After about 20 days of growth, use a thread to mark 10 to 15 flower buds at the position of the seed stalks and the inflorescence at 8:00 PM.Define the beginning of the ovule's pollination when the marked buds open to flower as 8:00 AM the next morning. To begin preparing the Hoyer's solution, add 24 grams of chlorhydrate to a 50 milliliter tube. Wrap the tube completely in aluminum foil, making sure the foil seals out all light.
Add nine milliliters of ultra pure water, and three milliliters of glycerol. Shake the tube to dissolve the chlorhydrate. Then, let the prepared Hoyer's solution stand overnight at room temperature to eliminate any bubbles.
To begin, attach a piece of double sided adhesive tape to a microscope glass slide. Place a silique, grown for the desired number of days after pollination on the side with the pseudoseptum perpendicular to the surface. Using a syringe needle, split the carpals on both sides of the pseudoseptum.
Next, attach the two dissected carpals to the slide so that the ovules and the silique are visible under a stereoscope. Dispense 70 microliters of Hoyer's solution onto the glass slide. Then dip a pair of fine-tipped tweezers into the solution to moisten the tip.
Using the stereoscope and tweezers, move the ovules into the Hoyer's solution to clean the embryos. After this, gently apply a cover slip to the slide, making sure not to generate any bubbles. Placed the finished slide into a box in a dark room for two to 12 hours at room temperature.
After the necessary time has passed, use the microscope's DIC function to examine the cleared ovules. Locate the embryos under a low powered field, and then change to a high-power field to observe the cellular pattern in the developed embryos. Then, examine the embryos at different developmental stages.
To begin, prepare the DR5 GFP plasmid and introduce it into the wild-type plants by agrobacterium to methassians mediated transformation as outlined in the text protocol. Select the T1 plants using MS medium plates containing hydrolysin. Then, transfer and cultivate the plants as outlined in the text protocol to obtain T3 plants.
Using a thread, mark the flower buds of the homozygous T3 plant at the position of the seed stalks at the inflorescence at 8:00 PM.After this, mix three milliliters of glycerol with 47 milliliters of ultra pure water to generate a 6%glycerol solution. Then attach a piece of double-sided adhesive tape to a microscope glass slide. Place a T3 plant's silique on the slide with the pseudoseptum perpendicular to the surface.
Using a syringe needle, split the carpals on both sides of the pseudoseptum. Attach the two dissected carpals to the slide so that the ovules and the silique are visible under a stereoscope. Next, dispense 30 microliters of 6%glycerol onto the slide.
Dip a pair of fine tipped tweezers into the solution to moisten the tip. Using the stereoscope and tweezers, move the ovules into the glycerol solution. Gently place a cover slip onto the slide.
Then, tap the slide quickly and gently to extrude the embryo from the ovule. After this, use confocal laser scanning microscopy to examine the slide for green fluorescent protein fluorescence. Find the isolated embryos under a low-powered field, and record their location.
Change to a high powered field and set the excitation light source to 488 nanometers to observe GR5 GFP expression in the embryos. Then, detect the GFP expression, and characterize the embryo pattern formation as outlined in the text protocol. In this study, embryogenesis in Arabidopsis is observed via ovule clearance, as can be seen.
After ovule clearance, the embryos at different developmental stages are distinguishable using differential interference contrast microscopy. The expression pattern of GR5 GFP during embryogenesis is then recorded under confocal laser scanning microscopy. DR5 is seen to be expressed in the root pole of the wild type globular and heart shaped embryos.
DR5 is seen expressed in the embryonic cotyledon tips, and in the vasculature of mature wild-type embryos. After this, the role of naa10 in Arabidopsis during embryogenesis is examining it. The hypothesis observed to be abnormally divided, with askew and vertical division in naa10 compared to transverse asymmetric division in wild type plants.
Ass seen here, oxen has nearly uniform distribution in most naa10 embryos at the globular stage, and retains a broader signal in the hypotheses compared to the wild-type embryos. These results indicate that naa10 is required for embryogenesis, and that it may be invoved via the oxen signaling pathway in Arabidopsis. While attempting this procedure, it's important to remember how to grow Arabidopsis as plant material.
Following this procedure, other measures like in-situ hybridization can be performed in order to answer additional questions like expression of cell specific genes. After its development, this technique paves the way for researchers in the field of embryogenesis to explore the molecular mechanism by which specific genes mediate embryogenesis in Arabidopsis. After watching this video, you should have a good understanding of how to characterize embryogenesis in Arabidopsis.
