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​Protoplast Culture: Isolation and Culture Methods (Part-2)
​Protoplast Culture: Isolation and Culture Methods (Part-2)

​Protoplast Culture: Isolation and Culture Methods (Part-2)

Anjali Singh

29th Apr 2022

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Tissue culture is an advanced biotech technique used in labs to grow disease-free plants on a commercial scale. And, among the many tissue culture techniques, such as callus culture, organ culture, and embryo culture, one is the protoplast culture technique.

Introduction

Tissue culture is an advanced biotech technique used in labs to grow disease-free plants on a commercial scale. And, among the many tissue culture techniques, such as callus culture, organ culture, and embryo culture, one is the protoplast culture technique.

In protoplast culture, protoplasts isolated from any plant part, including root, shoot, leaves, or embryo, is cultured in an artificial media under artificial conditions favoring cell division and plant regeneration.

In the previous part of this article, you’ve learned what is protoplast culture, how protoplasts are isolated from the plant parts, and how their viability is checked for their use in tissue culture procedures.

In this article, we will review the types of culture media types used to grow protoplasts in a lab environment and the techniques that are used by scientists to culture protoplasts.

Protoplast Culture Media Types

The culture medium contains all the nutrients and vitamins required for the growth and development of plants in lab conditions. Mostly MS media is used for such purposes. However, often a modified MS media or B5 media is found to be suitable for the regeneration of plants. It means the formulation of the culture media depends on the species of the plants and their specific growth requirements.

Some special feature of protoplast culture media are given below:

  • There should be less iron and zinc and no ammonia in the culture medium.
  • For the membrane stability, the calcium concentration should be 2-4 times more compared to that used in normal cell culture media.
  • Mainly glucose is the preferred source of carbon, however, a combination of glucose and sucrose can also be used.
  • A high auxin/kinetin ratio is required to induce cell divisions, however, a high kinetin/auxin ratio is needed for regeneration.
  • Some vitamins can be used as they are used in standard tissue culture media.

Other than the nutritional components, the osmoticum (the chemical which increases the osmotic pressure of the solution) is another factor that needs to be taken care of while preparing the tissue culture media.

For plant regeneration from protoplasts, first, they are required to develop cell walls to proceed to the stage of cell division. Protoplast is cultured either on semi-solid media or liquid media. However, there are also instances where protoplasts are first cultured on liquid media and then transferred to solid media for further development.

  • Agar Culture

Agar is the most widely used solidifying agent in labs. As much concentration of agar should only be used during the experiments that it forms a soft layer of the gel after being mixed with protoplast suspension.

The protoplasts are cultured on the plate using Bergmann’s cell plating technique. Here, the protoplasts are fixed in a position for cell division and further growth. The agar technique is efficient to prevent the formation of clumps in the cultures.

  • Liquid culture

It’s the most preferred method for the protoplast culture. Using this technique, the density of the protoplasts can be manipulated and they are easy to dilute and transfer. Moreover, the osmotic pressure of the cultures can also be diluted.

Protoplast Culture Methods

The protoplasts are cultured in labs using the following techniques:

  • Feed Layer Technique

In this technique protoplast suspension is exposed to X-rays and then plated on agar plates. It’s used to culture protoplasts at low density.

Moreover, the feel layer technique is also suitable for the selection of hybrid cells and specific mutants on plates.

  • Co-culture Protoplasts

This technique is used to culture together protoplasts of two different species. And, you must also note that it can only be used if the protoplasts of the two plant species are morphologically distinct.

  • Micro-Drop Culture

In this technique, protoplasts are cultures in a special dish, known as Cuprak dishes. The dish has outer and inner chambers. The inner chamber has several walls, in which individual protoplast droplets in nutrient media can be added. The outer chamber contains water to maintain the humidity for the growth of the cultures.

How Plant Cell Technology Is Helping Culturists Worldwide In Their Tissue Culture Application?

Plant Cell Technology is helping tissue culturists around the world by providing unique and world-class products and services that smoothen their process. It has MS media, agar, gellan gum, Plant Preservative Mixture (PPM), culture vessels, Biocoupler (TM), and masks in its store to facilitate your processes.

And, that’s not it! Plant Cell Technology also offers consultation services to culturists of all sizes that help to get instant solutions to your tissue culture problems.

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!!

Anjali

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