9. The liquid MS medium should be at room temperature. This is
the optimal temperature for Agrobacterium transfection.
10. Resuspend the bacterial pellet with the 1 mL micropipette
carefully in order to avoid bubble formation that could damage
bacteria.
11. Blank can be distilled water or liquid MS.
12. The OD 600 of the culture should be around 2.0–2.6. Higher or
lower values depend on the growth of the 50 mL Agrobacterium culture. The efficiency of the transformation decreases
when the OD 600 is less than 2.0.
13. Make 5 mL of infiltration buffer per plate.
14. The surfactant Silwet L-77 acts improving the adhesion of the
Agrobacterium to the cotyledons and induce the stomata infiltration of the Agrobacterium suspension by decreasing the
surface tension.
15. The hormone acetosyringone improves transformation efficiency in plants. It has been suggested that the Agrobacterium
VirA/VirG system located on the Ti plasmid recognizes the
acetosyringone as a host-specific signal and activates the virulence gene expression [11].
16. It is recommended to have single-use frozen aliquots of 1 M
acetosyringone ready to thaw and add to the infiltration buffer.
17. For co-infiltration of two constructs, OD 600 of 1 should be
calculated per each construct to obtain a final OD 600 of 2.
18. If the efficiency of a construct is too high, it is recommended to
decrease the OD 600 and test which is the best concentration for
each construct. If the efficiency is low, it is unlikely that an
increase in OD 600 would enhance the transfection and consequently the expression of a construct since an OD 600 of 2 corresponds to a high concentrated Agrobacterium solution.
19. Usually 3–4 mL of infiltration buffer with the Agrobacterium
are required per plate.
20. Often some seedlings float after the infiltration buffer is added.
These seedlings are not useful for the transformation. They
should be removed after vacuum infiltration.
21. The plates can be placed on a rack and several plates can be
vacuum infiltrated simultaneously (Fig. 1). It is important to
apply the vacuum slowly with the help of the manometer in
order to avoid damaging and stressing of the seedlings as much
as possible.
22. Start 1 min countdown when you observed bubbles being
released from the seedlings.
Seedling Transformation
153
the optimal temperature for Agrobacterium transfection.
10. Resuspend the bacterial pellet with the 1 mL micropipette
carefully in order to avoid bubble formation that could damage
bacteria.
11. Blank can be distilled water or liquid MS.
12. The OD 600 of the culture should be around 2.0–2.6. Higher or
lower values depend on the growth of the 50 mL Agrobacterium culture. The efficiency of the transformation decreases
when the OD 600 is less than 2.0.
13. Make 5 mL of infiltration buffer per plate.
14. The surfactant Silwet L-77 acts improving the adhesion of the
Agrobacterium to the cotyledons and induce the stomata infiltration of the Agrobacterium suspension by decreasing the
surface tension.
15. The hormone acetosyringone improves transformation efficiency in plants. It has been suggested that the Agrobacterium
VirA/VirG system located on the Ti plasmid recognizes the
acetosyringone as a host-specific signal and activates the virulence gene expression [11].
16. It is recommended to have single-use frozen aliquots of 1 M
acetosyringone ready to thaw and add to the infiltration buffer.
17. For co-infiltration of two constructs, OD 600 of 1 should be
calculated per each construct to obtain a final OD 600 of 2.
18. If the efficiency of a construct is too high, it is recommended to
decrease the OD 600 and test which is the best concentration for
each construct. If the efficiency is low, it is unlikely that an
increase in OD 600 would enhance the transfection and consequently the expression of a construct since an OD 600 of 2 corresponds to a high concentrated Agrobacterium solution.
19. Usually 3–4 mL of infiltration buffer with the Agrobacterium
are required per plate.
20. Often some seedlings float after the infiltration buffer is added.
These seedlings are not useful for the transformation. They
should be removed after vacuum infiltration.
21. The plates can be placed on a rack and several plates can be
vacuum infiltrated simultaneously (Fig. 1). It is important to
apply the vacuum slowly with the help of the manometer in
order to avoid damaging and stressing of the seedlings as much
as possible.
22. Start 1 min countdown when you observed bubbles being
released from the seedlings.
Seedling Transformation
153