6 Apr 2022

Protoplast Culture of Potato

Anjali Singh, MS

As a content and community manager, I leverage my expertise in plant biotechnology, passion for tissue culture, and writing skills to create compelling articles, simplifying intricate scientific concepts, and address your inquiries. As a dedicated science communicator, I strive to spark curiosity and foster a love for science in my audience.

Table of Contents

Potato (Solanum tuberosum L.) is one of the important crops of people’s interest. It’s grown in around 180 countries worldwide with the largest parts in Asia and then in Europe and South and Central America.

Introduction to Tissue Culture of Potato

Potato (Solanum tuberosum L.) is one of the important crops of people’s interest. It’s grown in around 180 countries worldwide with the largest parts in Asia and then in Europe and South and Central America.

Because of its constant skyrocketing demands in the market, the plant is also of scientists’ interest for research studies, including increasing productivity of the plants, germplasm conservation, and getting rid of infectious diseases. In all these studies, tissue culture has a major role to play because it’s the technique that allows researchers to grow plants in lab conditions and achieve the goals they are looking for.

The technique is also used by some culturists and industries to produce disease-free plants in mass with increased productivity. The first report of potato culture establishment goes a half-century ago.

The in vitro propagation of potatoes is done by using meristem tips, nodal cuttings, and micro tubers to maintain the genetic integrity of the multiplied clones. Techniques like differentiation and organogenesis and embryogenesis have been reported to cause some genetic changes in potato plants.

In this article, we’ll talk about the protoplast culture technique to propagate potato plants.

What is Protoplast Culture?

Protoplast culture is a tissue culture technique in which protoplasts of plants are isolated using enzymatic or mechanical methods and cultured in a suitable tissue culture media for lant regeneration.

The factors that influence the liberation of protoplast from leaves depend on:

• the physiological state of the material leaves
• the kinds of degrading enzymes
• the composition of the reaction solution
• the concentration and type of osmotic stabilizers

Procedure to Potato Protoplast Culture

The given below procedure is taken from the study of Kikuta Yoshio, Fujino Kaien, Saito Wataru, Masuda Kiyoshi, and Okazawa Yoz. Protoplast Culture Of Potato: An Improved Procedure For Isolating Viable Protoplasts. Journal of the Faculty of Agriculture, Hokkaido University, 62(4), 429-439.

Plant Material and surface sterilization

• Collect the expanded leaves of the potato plants.
• Sterilize the leaves with 70% ethanol for 30 sec and immersed them in 10 % Sodium hypochlorite solution (effective chlorine was 0.5%) with a drop of Tween ~20 for 5 min.
• Wash the leaves several times using sterilized water.
• Blot dry the leaves using sterile filter paper.
• Remove the midrib of leaves and cut the epidermis into 1.0 mm strips.
• Preincubate the explants in 0.44 M mannitol solution for 2 hr in order to induce plasmolysis in mesophyll cells.

Enzyme purification

• Use a cell wall degradation enzyme mixture containing 1% Cellulase Onozuka R-10 and 0.03% Macerozyme to liberate the protoplasts of the leaves
• Though commercially available enzymes contain some purity, so you can either use them as they are or purify them by using chromatography technique on Sephadex G50 column.

Incubation Procedure

• Incubate the cut segment of potato leaves immersed in 90 mm diameter plastic Petri dishes with 8 ml of enzyme preparations, 0.44 M mannitol, and 5 mM MES buffer, pH 5.4.
• Keep the dished under gentle agitation at 25°C for more than 3 hr.
• For long-term incubation, replace the incubation medium with a fresh medium, after 5 hours of complete incubation, and place the dishes in an incubator at 20°C.

Potato Protoplast Purification

• Filter the incubation mixture in an incubator at 20°C and collect it by centrifugation at 95 xg for 3 min.
• Resuspend the obtained protoplast in 2 ml of 0.44 M mannitol and 5 mM MES buffer, pH 6.0.
• It will form a four-part discontinuous Percoll/0.44 M mannitol density gradient centrifugation system. The system should be consist of 2 ml each of 6 %/25 % Percoll in 0.44 M mannitol with 5 mM MES buffer, pH 6.0.
• Centrifuge the system at 400 xg for 5 min to separate cell types and cell debris.

Measure Protoplast Population

• Use a 20 microliter droplet counting method to estimate protoplast densities and distributions by using the mean of 6 protoplast determinations present in 20 microliter aliquots.
• Multiply the counted numbers with 50 to obtain the protoplast density in ml.

Assess Protoplast Viability

• Assess the viability by testing the ability of the protoplast to accumulate (a) the vital dye stain neutral red (0.4% w/v) (b) fluorescein from a 0.01% v/v solution of fluorescein diacetate or to exclude the mortal stain Evans Blue (0.0025% w/v).

Protoplast Regeneration

• Culture protoplasts at 104/ml cell density in 6 mm plastic dishes containing 4 ml of protoplast culture medium.
• Prepare the culture medium using (Mineral salts in mg/I) and adjust pH to 5.8.

• Incubate the cultures at 20°C in darkness and observe the divided cells on day 7 and cell colonies on day 14.
• Dilute the cultures with cell colonies in a fresh culture medium containing 0.22 M mannitol at 14 days intervals.
• After 6 weeks, transfer the cultures from liquid medium to solid agar medium for callus formation and plant regeneration.

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So, visit plantcelltechnology.com today and find out more about our product and services and how they help you to excel in your tissue culture processes.

Happy Culturing!!