An Efficient Clearing Protocol for the Study of Seed Development in Tomato (Solanum lycopersicum L.)

This method can help researches quickly and effortlessly find abnormal periods of tomato embryo development. The main advantage of this protocol is a treatment of tomato seeds with sodium hypochlorite which provides a basis for the observation of tomato embryos. Begin with the chemical preparation and harvesting of the Solanum lycopersicum fruits three to 23 days after flowering or DAF.

Immediately put the fruits on ice and divide them into early, middle and late fruits, based on the days after flowering. Break the early fruit, put it on a slide and collect fresh seeds carefully with precision forceps under 1x to 4x magnification of the stereo microscope. For middle and late fruits, break the fruit and directly collect fresh seeds using precision forceps.

In the conventional method, transfer the freshly collected seeds immediately into a two milliliter centrifuge tube containing 1.5 milliliters FAA fixative and place the tube on the orbital shaker at 5 rpm for four hours at room temperature. Then dehydrate the seeds in 70%95%and 100%ethanol for one hour each on an orbital shaker at 5 rpm. Once done, place the seed in the three to five drops of clearing solution present on the slide, and gently cover it with a cover slip.

Use a single concave slide for middle and late seeds. Depending on the developmental stages of the seeds, incubate them at room temperature for clearing. After incubation, observe the samples with a differential interference contrast, or DIC microscope equipped with a digital camera at 10x, 20x and 40x magnification.

Adjust and optimize the transmitted light brightness, DIC slider and condenser aperture in real time for each sample and capture images. Transfer the fresh seeds collected from the broken fruits into a two milliliter centrifuge tube containing 1.5 milliliters of disinfectant solution. Incubate the samples on an orbital shaker at 30 rpm for three to 50 minutes at room temperature.

Once the innermost seed coat outline is clearly visible, discard the disinfectant solution. For middle and late seeds, transfer the seeds onto a slide. Use forceps and a dissecting needle to remove the seed mucilage under 1x to 4x magnification of the stereo microscope.

Transfer the seeds back into the original centrifuge tube using forceps. Wash them five times in 1.5 milliliters of deionized water for 10 seconds each, and discard the deionized water. Next, add the double volume of the clearing solution to the seeds.

Depending on the seed stage, give the intermittent vacuum treatment for zero to 50 minutes, with 10 minute intervals after each vacuum treatment of 10 minutes. After vacuum treatment, replace the clearing solution. To facilitate the clearing of seeds, place the centrifuge tube protected from light for 30 minutes to seven days at room temperature.

In the case of late embryos, replace the solution daily with a fresh clearing solution, followed by subjecting the seeds to vacuum treatment for 10 minutes. Place the cleared seeds on the slide or single concave slide. Using the DIC microscope equipped with a digital camera, adjust and optimize the transmitted light brightness, DIC slider and condenser aperture in real time for each sample and capture the image.

In the conventional method of clearing S.lycopersicum seeds, the dense endosperm cells blocked the visualization of early embryos at 3 and 6 DAF. The decreased permeability during the seed growth resulted in fuzzy images after 9 DAF. The seed mucilage and seed coat gradually became denser, preventing the penetration of clearing agents from 13 DAF onward.

The outline of the embryo inside 14 to 19 DAF seeds was extremely blurred, Despite the extended treatment time of seven days. The internal structure in 22 DAF seeds was completely invisible. The stripped seed coat mucilage produced by the seed coat epidermal cells was prominent from 13 DAF onward.

The sodium hypochlorite treatment enabled a clear identification of the inner seed coat and detachment of most of the adherent mucilage without seed damage. In the optimized clearing protocol the seeds showed satisfactory transparency in all tested developmental stages. The distinct cell layers of the seed coat at 3 DAF, distinguishable endosperm cells at 5 DAF, the stick shaped embryo at seven DAF, the globular stage at 9 DAF, and the heart stage at 11 DAF were observed.

In the optimized protocol, the degree of the curl of cotyledons and shoot apical meristem was very easily captured at various stages of embryonic development. Sterilization removes seed mucilage and seed coat pigment, which enhance seed penetration and visualization. Vacuum treatment can accelerate clearing solution penetration.