Table 1.

Troubleshooting table.

Step	Problem	Possible reason	Solution
13	Poor/bad quality protoplast yield	Poor plant growth conditions	Check that the pH of the MS media is between 5.6-5.8. Check and optimize growth conditions, such as light intensity, temperature, and photoperiod.
		Ineffective enzymatic digestion	Optimize digestion time for each transgenic line, accession, and plant tissue.
25	The protoplasts do not adhere properly	Insufficient glow discharging	Verify and adapt glow discharge conditions.
29	Non-vitrified ice in the samples	Many cells were not properly plunge frozen, producing a thick sample	Adjust the number of protoplasts when plunge freezing.
		Poor blotting conditions	Adjust the blotting parameters adequately, depending on the type of filter paper to be used, as well as the blotting force and time.