Plant protoplasts provide a universal cell-based experimental system. Biological macromolecules such as DNA, RNA and proteins can be introduced into protoplasts by methods such as PEG calcium fusion, electroporation and microinjection. Through transient manipulation of signal transduction and metabolic pathways, transcription and translation, the study of cell-autonomous regulation and response. Combined with model organisms with abundant genetic resources, such as rice, Arabidopsis, etc., this system has been widely used in functional genomics analysis. In this series of experiments, the extraction of protoplasts is the basis, and the concentration of the protoplast suspension needs to be adjusted in subsequent transformation experiments. The protoplast extraction kit can help to obtain relatively pure protoplasts quickly and efficiently, and the JIMBIO CL counter can accurately count the suspension accurately.

1. Materials and methods

1.1 Materials and instruments

Rice seed; nutrient soil; vermiculite; light incubator; centrifuge; JIMBIO CL counter or

Jimbio CCplus counter, etc.

Protoplast extraction kit (Jiangsu Berryli Biotechnology Co., Ltd.).

1.2 Experimental method

1.2.1 Rice planting: dry seed sowing, 28℃ short-day soil cultivation for 10 days.

1.2.2 Cut the leaf sheath (young stem) of the rice seedling into slices with a thickness of 0.5-1mm, take 3-5g, and extract the protoplasts with the kit.

1.2.3 After obtaining the protoplast suspension, use the JIMBIO CL counter to count and determine the concentration.

1. Results and analysis

Figure 1. Picture of protoplasts extracted under the hemocytometer

Figure 2. Counting result of the instrument

Figure 3. Histogram result of instrument count

It can be seen from Figure 1 that the protoplasts extracted by the kit are relatively pure and have no obvious impurities. The counting results shown in Figures 2 and 3 show that the particle size is equivalent to the result observed by the hemocytometer, and the particle size distribution is normal. Table 1 compares the counting results of the instrument and the counting plate. It can be seen that the CV value of the instrument is smaller than that of the counting plate, and the counting is more stable. The counting result of the instrument is slightly higher than that of the counting plate. This is because it is impossible to remove impurities during the extraction process. Completely, there will be some small impurity particles, which have been subjectively eliminated when counting on the counting board. This part is not large, and the user can also select the area to count.

Recommended model: Jimbio CL or Jimbio CC plus