System in the USA. Maize germplasm is also available from the
North Central Regional Plant Introduction Station (NCRPI;
https://www.ars.usda.gov/midwest-area/ames/plant-introduction-research/).
2. Do not use fertilizer, as the seed endosperm contains sufficient
nutrients to promote growth.
3. Buffer B is used to facilitate degradation of the cell wall and
release of the fragile protoplasts. Therefore, from this point
onward, care should be taken in using the gentlest of manipulations and avoiding hard shaking of the sample.
4. Purified plasmids for transformation should be of a concentration >1000 ng/μL in water or 10 mM Tris/HCl buffer
(pH 8.5). A preparation kit that allows large-scale purification
of plasmid DNA with the final concentration of at least 1 μg/μ
L should be used instead of manual preparation since impurities might affect efficiency of transformation.
5. Polystyrene or glass material is important, because during the
PEG-initiated transformation the protoplasts might stick to
polyethylene or other plastics. Use 1 mL tips for which the
ends are cut off for a wider opening to make sure the protoplasts are not destroyed by pipetting.
6. All of the materials needed for DNA transformations of the
isolated protoplasts should be preassembled so that the transformations can be done quickly. Place in front of you the
following items: Buffer C (PEG solution), Buffer D (Incubation buffer), purified plasmids on ice, pipette for 500 μL with
cut tips, pipette for 20 μL with tips, and a pipet for 5 mL with
the dispenser attached. If introducing two plasmids, prepare
either 10 μL of 1 mg/μL of each plasmid, or premix the two
plasmids and dispense a total of 20 μL to the protoplasts. To
work quickly, shake the sample with one hand, and add everything with the other hand, one by one. After Buffer D is added,
shaking stops and the PEG in Buffer C becomes diluted. The
protoplasts will die if you stop shaking after adding Buffer C
(PEG solution) and before adding Buffer D, or shaking after
adding Buffer C (PEG solution) takes too long before addition
of Buffer D. Therefore, the entire process for one protoplast
sample, including addition of plasmids, Buffer C and Buffer D,
should be completed within a couple of minutes. If you cannot
work with two hands, then a partner can help with gentle
shaking of the protoplast-containing tube while you add everything step by step. When testing multiple enzymes and controls
for localization or interactions, each protoplast sample must be
completely processed before going on to the next DNA
transformation.
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North Central Regional Plant Introduction Station (NCRPI;
https://www.ars.usda.gov/midwest-area/ames/plant-introduction-research/).
2. Do not use fertilizer, as the seed endosperm contains sufficient
nutrients to promote growth.
3. Buffer B is used to facilitate degradation of the cell wall and
release of the fragile protoplasts. Therefore, from this point
onward, care should be taken in using the gentlest of manipulations and avoiding hard shaking of the sample.
4. Purified plasmids for transformation should be of a concentration >1000 ng/μL in water or 10 mM Tris/HCl buffer
(pH 8.5). A preparation kit that allows large-scale purification
of plasmid DNA with the final concentration of at least 1 μg/μ
L should be used instead of manual preparation since impurities might affect efficiency of transformation.
5. Polystyrene or glass material is important, because during the
PEG-initiated transformation the protoplasts might stick to
polyethylene or other plastics. Use 1 mL tips for which the
ends are cut off for a wider opening to make sure the protoplasts are not destroyed by pipetting.
6. All of the materials needed for DNA transformations of the
isolated protoplasts should be preassembled so that the transformations can be done quickly. Place in front of you the
following items: Buffer C (PEG solution), Buffer D (Incubation buffer), purified plasmids on ice, pipette for 500 μL with
cut tips, pipette for 20 μL with tips, and a pipet for 5 mL with
the dispenser attached. If introducing two plasmids, prepare
either 10 μL of 1 mg/μL of each plasmid, or premix the two
plasmids and dispense a total of 20 μL to the protoplasts. To
work quickly, shake the sample with one hand, and add everything with the other hand, one by one. After Buffer D is added,
shaking stops and the PEG in Buffer C becomes diluted. The
protoplasts will die if you stop shaking after adding Buffer C
(PEG solution) and before adding Buffer D, or shaking after
adding Buffer C (PEG solution) takes too long before addition
of Buffer D. Therefore, the entire process for one protoplast
sample, including addition of plasmids, Buffer C and Buffer D,
should be completed within a couple of minutes. If you cannot
work with two hands, then a partner can help with gentle
shaking of the protoplast-containing tube while you add everything step by step. When testing multiple enzymes and controls
for localization or interactions, each protoplast sample must be
completely processed before going on to the next DNA
transformation.
232
Maria Shumskaya et al.