Visualization of Leaf and Bracteal Nectaries of Cotton using Digital Microscopy to Improve Scoring Accuracy and Data Preservation

The current study aims on the method that accurately scores the expression of the nectary trait. To illustrate the nectary trait expression, both cotton leaf and bracteal nectaries were used as model tissues. This method overcomes the limitation of traditional scoring, which is unreliable and inaccurate for nectary trade scoring.

To begin, transport a cooler and labeled Ziploc bags to the greenhouse for sample collection, collect young leaf tissue from two to three month old greenhouse grown cotton plants and place it into the labeled sample bags. Place at least two leaves per plant sample into the respective labeled sample bag and seal the bag securely after collection. Place each sealed sample bag containing leaf tissue into the cooler.

Collect healthy flowers from the top branches when the plants are at the mid flowering stage. Place at least two flowers into a labeled sample bag and seal the bag. Transfer the sealed sample bags containing flowers into the cooler for transport to the laboratory.

Turn on a microscope and complete the initial startup steps. Place a half folded A4 white sheet cut to match the size of the microscope stage onto the stage. Use the small light knobs on the console controller to adjust the light on the microscope stage to the maximum intensity.

Then rotate the large brightness knob three quarters of the way to set it to medium high. On the computer, select 10 x magnification in the microscope software. Place the leaf tissue onto the precut white sheet on the microscope stage with the abaxial side facing up and center it on the stage.

Gently rotate the course and fine adjustment knobs to focus on the mid rib of the leaf. Continue adjusting until the image appears clear and without blur. Position the lower end of the mid rib where the nectary is located at the center of the field of view while ensuring clear visibility of the diverging lateral veins.

Using the course and fine adjustment knobs further refine the focus to improve digital image clarity. Capture the nectary image. When prompted by the software, save the image with the sample identification number at the required location, then assign a score from one to four based on the observed leaf nectary phenotype.

Use forceps or hand to remove the bracts from the flower. Place the flower onto a clean cutting board. Using a sterile blade, cut the stalk of the flower, then make a straight incision along the edge of the removed bracts.

Place the prepared flower tissue upside down onto the pre-cut white sheet on the microscope stage. Use the course and fine adjustment knobs to improve the clarity of the image displayed on the screen. Save the image with the appropriate sample identification number.

Digital image scoring of the leaf abaxial surface on the lower side of the mid rib showed two phenotypes with score one with no nectaries on the mid rib. And score four with a fully developed nectary with nectar. When flower samples were analyzed for bracteal nectaries, two phenotypes were observed with score one showing no nectary and score four showing a fully formed nectary producing nectar.

Digital images of nectary plants clearly showed both leaf and bracteal nectar structures at 10 x, 20 x, and 40 x magnification. Selected leaf digital images corresponding to the standard scoring format showed score one with absence of nectary, score two with an underdeveloped nectary, score three with a smaller nectar, and score four with a fully developed nectary. Accurate scoring of nectary less plants from the segregating populations of nectary and nectary less crosses is essential for the selection of nectary less plants that can reduce plant insect damage as well as reduce yield losses.

So selection of this trait is important for downstream applications like DNA markers associated with the nectary less trait that are valuable tools for marker assisted selection.