Protocols for Obtaining Zygotic and Somatic Embryos for Studying the Regulation of Early Embryo Development in the Model Legume Medicago truncatula

The overall goal of the following procedures is to demonstrate how many cargo tula embryos can be obtained at different developmental stages to investigate the genetic regulation of embryogenesis in plants, this can be accomplished by linking pod morphology with the different zygotic embryo developmental stages, and by culturing leaf explan to obtain somatic embryos at the different stages. Ultimately, quantitative PCR and beta glucuronidase can be used to assess the expression of genes during embryogenesis at the specific developmental stages of interest. The advantages of these techniques are the tagging of individual flowers is not required for the in vivo experiments, and are optimized in vitro procedures, provide complimentary experimental approaches.

Visual demonstration of the culture procedure is important because it allows the details of the manipulations to be more closely followed. Demonstrating the in vivo procedure will be ing Wang while Kim Nolan will be demonstrating the in vitro procedure. To facilitate zygotic embryo development, pierce the surface of the seed coats of seeds from a Monte cargo trinocular plant and soak the seeds overnight, fully covered in water.

The next day, sow three seeds in each of 10 15 centimeter diameter pots in potting mixture and transfer the pots to a glass house. Grow the plants under a 14 hour photo period with a 23 to 19 degrees Celsius day night temperature, retaining the healthiest seedlings after germination so that there is one plant per pot and surrounding the seedlings by trellis for the plant stems to climb when pods appear. Collect at the appropriate stages outlined in table one and figure one to obtain the desired early embryo development stage.

Next, use forceps to isolate the les from the pods. Alternatively, for more refined microscopy, fully clear in Hoyer solution, the embryo stages can be confirmed by imaging the embryos under a standard compound microscope. Then collect the number of UL required for the experiment from the central region of the pod and store them at the appropriate temperature for downstream analysis for somatic embryo development.

In vitro harvest leaflets from the latest, nearly fully expanded tate leaf of an elongating stem of an tran atula cultivar that is able to readily form embryos in culture. Keep the tate leaves on moist paper toweling in a culture pot to avoid dehydration. Then working in a UV sterilized biosafety cabinet, placed the leaves into the mesh ball of an autoclave spring tea infuser.

An immersed infuser in 70%Ethanol in an autoclave 250 milliliter screw cap. Polycarbonate culture pot after 30 seconds, drain the leaf tissue and then immerse the infuser in a one to eight diluted bleach solution for 10 minutes. With occasional gentle swirling, gently swirl the pot and drain the excess water.

Then rinse the infuser. One more time in fresh deionized water in a new pot. Now use sterile forceps to transfer the leaves from the tea infuser into a fresh culture, pot of sterile deionized distilled water screw on the sterile cap.

Then rinse the leaves by inversion and swirling. Then allow the leaflets to float on the sterile water to plate the implants. Trim the edges of the individual sterilized leaflets using sterile technique.

Then cut the remaining rectangle into two or three, eight to 10 by three to five millimeter rectangular explan with the mid vein in the center of each X explan plate. Six x explants per agar solidified culture medium in nine centimeter diameter. Petri dishes with the ABA seal side down to maintain the usual leaf orientation.

Then seal the Petri dishes with perfil and incubate the plates in the dark at 28 degrees Celsius in a controlled temperature room. After three weeks, use a sterile stainless steel spatula and forceps to subculture the differentiating hole explants onto fresh medium. The first embryos should be observed in about four weeks as callousing occurs, and the callus exceeds the size of the largest explan shown here.

Transfer only 50%of the individual callus when subculture to fresh medium. Finally use a stereo microscope to collect the different embryo stages over a four to eight week incubation period processing the explants according to the experimental objectives. After harvesting zygotic embryos as just demonstrated downstream in C two hybridization or other appropriate strategies can be employed of particular interest as demonstrated in these images, the distinctiveness of the SSIS and Spencer can be observed by following the plated X plans.

Through weeks four to eight somatic embryos at discreet stages can be located and picked for immediate analysis. For example, these data illustrate how embryos at the early Olein stage exhibit the expression of one type of medicago Tula eoin gene, but not another. A particular advantage of the somatic embryo system is that transformation of the callus can be carried out without regenerating a whole plant.

Indeed, as demonstrated here. Beta glucuronidase can be used to visualize gene expression at the different stages of embryogenesis. After watching the video, you should have a good understanding of how to select and dissector pod to obtain Andres.

You should also have a good understanding of how to prepare and plate sterile explan for somatic embryogenesis After their development. These techniques have enabled the investigation of the transcription regulation of the early stages of embryogenesis in relation to embryo growth development and nutrient storage.