Protoplast is a cell without a cell wall. It is the living part of the plant cell and consists of the cytoplasm and nucleus. Protoplasts can be isolated from various plant tissues, usually released by tissue digestion with cellulase and pectinase enzymes purified from wood-decaying fungi. The best and largest number of protoplasts can be isolated from the leaf pulp tissue. Cultivation of protoplasts in a sterile liquid medium is called protoplast culture. The basic principle of protoplast culture is the aseptic isolation of many living protoplasts and their culture in a nutritive medium suitable for their growth and development.

What We Offer

As a leading provider of plant science and technology research, Lifeasible offers comprehensive protoplast isolation and culture services, including:

Protoplast Donors Selection

All plant organs, such as leaves, petioles, shoot tips, roots, fruits, embryonic sheaths, embryonic axes, stems, embryos, pollen grains, healing tissues, and cell suspension cultures, can be used as materials for protoplast isolation. To obtain high-quality protoplasts, we use the most vigorous tissues for you.

Protoplasts Isolation

We offer the following two methods to isolate protoplasts, which mainly involve using cellulase and pectinase to digest the tissues.

• Mechanical isolation method: This is accomplished by cutting plasmodesmally separated tissues with a knife and releasing protoplasts by de-plasmodesmosis. The number of protoplasts released in this method is minimal.
• Enzymatic separation method: Enzymes that can digest cell walls (e.g., cellulase, pectinase, etc.) are used to remove cell walls by treatment and release protoplasts. By immersing the plant tissue in a hypertonic solution, we ensure that the protoplasts maintain their integrity and avoid the risk of rupture.

Protoplasts Purification

The protoplast solution may also include subcellular debris, such as chloroplasts, vascular components, undigested cells, and broken protoplasts. We offer the following methods to purify protoplasts.

• Sedimentation: The protoplast mixture is microfiltered and centrifuged at low speed for 5 min, and the precipitate is washed 3-4 times with the same sugar concentration as the enzyme solution and then washed with culture medium.
• Floatation method: If the protoplasts purified by centrifugation contain more fragments and a small number of old cells, centrifugation can be performed with 20% sucrose, and the protoplasts will float on the surface of the liquid. Fragments and old cells settle down and can be purified with high purity but low yield.
• Discontinuous gradient method: The crude extract of protoplasm is placed in a solution with a discontinuous concentration gradient, and the protoplasts with different specific gravity are separated after centrifugation.

Protoplast Viability Detection

To ensure the viability of protoplasts, we provide the following methods for comprehensive testing:

• Visual inspection: Observe under a microscope and determine the viability of protoplasts based on cell morphology and fluidity.
• Fluorescein diacetate (FDA) method: FDA is a non-polar substance that can freely cross the plasma membrane of the cell, in living cells, FDA is cleaved by esterase and fluoresces (fluorescein). In living cells, FDA is cleaved by esterase and fluoresces (fluorescein). Since luciferin cannot freely pass through the plasma membrane, cell activity can be determined under a fluorescence microscope by observation of fluorescent cells.
• Evan blue method: Evan blue cannot pass through the plasma membrane; only when the plasma membrane is severely damaged the cells can be stained, so the activity can be determined by whether the cells are stained or not.
• Oxygen uptake is measured by oxygen electrode.
• Photosynthetic activity.

Protoplasts Culture

We offer a variety of methods for culturing protoplasts. Depending on the needs of your project, we will select an effective and economical culture method for you.

Applications of Protoplast Culture

• Basic theory research
• Somatic cell hybridization
• Genetic transformation
• Saprobic variation and mutant screening
• Isolation and introduction of organelles
• Germplasm conservation

We Do Better

Lifeasible has a strict control system for factors affecting the isolation and culture of protoplasts, such as the physiological state of the isolated material, enzymatic conditions (enzyme mass, concentration, enzymatic temperature, enzymatic time, osmotic pressure of the enzyme solution), the separation conditions (number of centrifugations, centrifugation speed, purification method, duration of separation), and environmental conditions (temperature of the operating environment, influence of separation paraphernalia). We are based in the plant technology industry with professional and fast technical services, and our strict system and mature platform guarantee the success of your project.

If your organization requires the signing of a confidentiality agreement, please contact us by email.

Reference

1. Eriksson, Tage R. "Protoplast isolation and culture." Plant protoplasts. Crc Press, 2018. 1-20.

*If your organization requires the signing of a confidentiality agreement, please contact us by email.

For research use only, not intended for any clinical use.

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