Plant Physiology

Table 1: Viability of Red Cabbage Protoplasts determined by Evan's blue

# stained (non-viable)

# unstained (non-viable)

Plant Physiology (Biology 327) - Dr. Stephen G. Saupe; College of St. Benedict/ St. John's University; Biology Department; Collegeville, MN 56321; (320) 363 - 2782; (320) 363 - 3202, fax; ssaupe@csbsju.edu

Monitoring Protoplast Viability with Evan's Blue and Neutral Red

Question(s): Can Evan's blue and Neutral red be used to examine the protoplasts for viability? What percent of the protoplasts are viable?

Hypotheses: Viable protoplasts will exclude Evan's blue appearing clear or yellowish against a blue background. Intact viable protoplasts will accumulate neutral red and turn red. Most of the protoplasts will be viable.

Protocol:

Add a drop of neutral red (0.1% in 0.7 M sorbitol) to a drop of the protoplast suspension. Neutral red readily passes through many membranes and accumulates in the vacuole. It is best to use a geranium or other unpigmented leaf protoplast (i.e., unpigmented red cabbage protoplast) for this exercise. Describe and sketch (or photograph) the results. Include a caption and plate magnification.
Add a drop of Evan's blue (0.5% in 0.7 M sorbitol) to another drop of protoplasts (on a regular slide). Viable protoplasts will appear white or yellowish against a blue background. Describe and sketch (or photograph) the results. Include a caption and plate magnification.
Gently vortex the protoplast sample. Prepare another slide with Evan's blue and protoplasts. Determine the viability (percent of the protoplasts that are viable) of your sample by randomly counting 50 protoplasts and scoring them for Evan's blue staining. Record these data in Table 1. Note, count only round, intact protoplasts - ignore malformed ones. Do your Evan's blue data correlate with your observations of cyclosis?

Data, Analysis & Conclusions:

Describe the effect of adding Neutral red to the protoplast suspension. Offer an explanation for your observations. Do the results support our hypothesis?
Describe the effect of adding Evan's Blue to the protoplast suspension. Offer an explanation for your observations. Do the results support our hypothesis?
What do you conclude about the viability of the protoplasts? How do the vital stain data correlate with your observations of cyclosis? Do the results support our predictions?