7 Aug 2020

Methods For Isolating Single-cells

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

Isn’t it amazing that we can grow a whole plant from just a single cell!? Imagine the number of plants you can get from a small clump of tissue!

However, it is not as easy as it may sound. Isolating single cells from the tissue clump without any damage and with full accuracy needs a lot of practice, effort, and focus.

In 1902, Haberlandt was the first person who made pioneering attempts to isolate and culture a single cell from a flowering plant. He knew that the idea would help him to investigate the properties of plant cells and their complementary influences.

While he failed in his attempts to induce the division in the cell, his vision and his ideas were successful.

Later, several scientists picked up the idea and entered the line of this investigation. Since then, there has been spectacular progress and technological advancements that have made it possible to isolate, culture, and induce division in single cells to form a whole plant.

This article will present the techniques that have been used worldwide to isolate the single cells in various tissue-culture laboratories.

Methods of Single-cell Isolation

There are mainly four available and extensively used methods in plant tissue culture labs for the isolation of single cells. The technique is categorized into two groups, based on the source from which the single-cell is isolated. These are: intact plant organs and culture tissue.

(1) Single-cell isolation from intact plant organs

This includes two types of methods of isolation: (i) Mechanical method and (ii) the Enzymatic method.

(i) Mechanical Method

Not one method works with the single-cell isolation of all the plants. So, several processes were designed to overcome this limitation. Given below are different mechanical methods used for isolation purposes:

• Tear across the leaf and expose the mesophyll cells. Then, scrap the cells with a fine scalpel. The disadvantage of this method is that you cannot get viable cells. It can be used for Arachis hypogea.
• Gently grind the leaves and clean the cells with filtration and centrifugation.
• Grind 10g of leaves in 40 ml of grinding medium (which consists of 20 μmol sucrose, 10 μmol MgCl2, 20 μmol Tris-HCl buffer, and pH 7.8) using a mortar and pestle.
  • Filter the homogenate using two layers of muslin cloth.
  • Wash the released cells in a grinding medium by gentle centrifugation.
  • Work for dicot and monocot (mainly grasses) species of the plants.

(ii) The Enzymatic Method

• Excise the plant embryo.
• Treat the embryo with a 1% macerozyme for 2 hours at 32 ℃, on a shaker (50 revs/min).
• Force the treated embryo through a 5 mL hypodermic syringe. Do this several times to release the single-cells.
• It can be used for plants like cabbage, carrot, lettuce, etc.
• This method helps in the isolation of viable cells.
• Various studies have shown that the presence of potassium dextran sulfate in the maceration mixture increases the yield of free cells.
• Enzymatic methods are found to be difficult to isolate single cells from cereal plants. The reason for the problem is the interlocking structure of the leaves of the plants. It includes: Hordeum vulgare, Zea mays, Triticum vulgare, etc.

Advantages of the mechanical method over the enzymatic method:

• The mechanical method protects the cells from the harmful effects of the chemicals used in the enzymatic method.
• The mechanical method does not involve the plasmolyzation of the cells.
• The mechanical method does not require any extra protection. However, the enzymatic method requires osmotic protection to avoid the damage caused by the macerozyme.

Disadvantages of the mechanical method:

• It is difficult to obtain viable cells by using the mechanical method of single-cell isolation.

(2) Single-cell isolation from cultured tissues

• Transfer the undifferentiated and friable calli to a liquid medium in a flask.
• Continuously agitate the medium.
• This generates a mild pressure on the clump of tissue which will break it into smaller clumps and single cells.
• This is also called suspension cultures.
• The advantage of agitation of the medium is that it maintains the uniformity of cells and clumps in the medium.
• The movement of the medium also enables a gaseous exchange.

Apart from the mentioned technique, various other techniques are also available for the isolation of single-cell from a clump of plant tissue.

Advantages of single-cell culturing from cultured tissue:

Whenever we perform an experiment and use a method, it is important to understand its advantages and disadvantages. Below are a some reasons why should do single-cell culturing and the benefits it provides over culturing of the whole tissue:

• Independent analysis of the biological functioning of the individual cells.
• To reveal the unique cellular and molecular characteristics of the cell.
• To study the response of the plant in the stress environment.
• Study the dynamic changes in the plant during differentiation
• Large-scale production and synthesis of the commercially important phytochemicals.
• To study the effect of various substances on the cellular response.
• To study the differentiation and development of the plants.

Reference

1. Bhojwani S. S. and Razdan M. K. (1983). Plant Tissue Culture: Theory and Practice. Elsevier publications.