The preparation of plant protoplasts can be used for: (1) due to its ability to regenerate cell walls, carry out successive cell divisions and regenerate complete plants; (2) with the ability to ingest exogenous macromolecules and organelles, as well as bacteria and viruses, so it is a good material for genetic manipulation and gene transfer; (3) through the fusion of protoplasts of homo- and hetero-species of plants, heterokaryotic organisms can be produced, realizing the realization of somatic cell hybridization that breed new varieties.

Operation method

Preparation of plant protoplasts

Principle

Removal of plant cell walls can be done either mechanically by hand or by utilizing enzymatic methods. The first person to utilize mechanical methods to prepare protoplasts was Klercker (1892), and it was not until 1960 that Cocking, a British scientist, first prepared protoplasts in large quantities by enzymatic methods. In this experiment, cellulase and pectinase were used to digest the cell walls and separate the cells using the plant's leaf pulp tissue as the material.

Materials and Instruments

Oilseed rape or spinach or tobacco
Calcium chloride Distilled water 2 Sucrose Enzymatic solution
Microscope Centrifuge Centrifuge tubes Scissors Tweezers Straws Petri dishes Slides Coverslips

Move

1、Taking materials Select different materials according to the purpose and conditions of the experiment

2、Separation of protoplasts

(1) Tear the lower epidermis of leaves

(2) Enzymatic digestion Cut the torn leaves into small pieces, put them in a small petri dish with 5mL of enzyme solution, let the side with the torn leaves contact with the enzyme solution, and let the side with the torn leaves contact with the enzyme solution, and let the side with the torn leaves contact with the enzyme solution, and let the side with the torn leaves contact with the enzyme solution, and then enzymatically digest the leaves for 60-90 minutes at 25-28℃.

3、Collection and purification

(1) Pipette the enzyme solution into a 5mL centrifuge tube and centrifuge at 600 rpm for more than 5 minutes to settle the protoplasts.

(2) Recover the supernatant (digest), leaving the protoplasts and residue at the bottom of the tube.

(3) Add about 2mL of calcium chloride solution and blow gently to homogenize.

(4) Slowly inject about 2-3mL of sucrose into the bottom of the centrifuge tube with a syringe, resulting in a lower sucrose solution and an upper protoplasmic suspension.

(5) Centrifuge at 600rpm for 10 minutes, a green band appears between the two liquid phases, which is pure protoplasts.

4. Observation or Cultivation

Take a small amount of protoplasts on a slide, cover the slide to observe its morphology, or inoculate the protoplasts on the medium for culture to regenerate the plant.

Caveat

It takes a long time to cultivate suspended cell lines. Therefore, we should focus on improving the culture conditions, including the selection of suitable medium, including the type of medium, additives, hormone types and levels, and the culture method, and changing the medium according to the state of the cultures, selecting suitable exosomes and the induction period.

Common Problems

Protoplasm is a bare, active cell, the body wrapped with the nucleus, organelles, cytoplasm, etc. Therefore, protoplasm has the ability to regenerate the cell wall, continuous division and generate a complete plant, i.e., it has cellular totipotency. Protoplasts can ingest DNA, plasmids, viruses, bacteria, organelles and other exogenous materials, which are good materials for genetic manipulation and gene transfer in plant cell engineering.

Source from "Advances in the preparation and viability of plant protoplasts" Biotechnology Bulletin.

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