4 Notes
1. The normal pH of the solution should oscillate between 3.8
and 4.2.
2. You can accelerate the cooling of the media by introducing the
bottle inside an ice bucket with a little bit of ice and tap water.
By doing this, you should be very careful of not overcool the
media, or else the media at the bottom of the bottle will start to
solidify.
3. Different strains of Agrobacterium need different selection
agents: GV3101 strain needs gentamicin and rifampicin, alongside the selection agent that the vector confers; meanwhile,
EHA101 only needs gentamicin and the selection agent that
the vector confers.
4. Do not vortex the cells or the transformation efficiency will
drop dramatically.
5. Longer incubation times do not affect transformation
efficiency.
6. Avoid generating smaller explants. At early stages, the tissue is
difficult to manipulate as is very fragile and desiccates very
easily.
7. If the exposure time is shorter, it could affect the number of
transformant explants.
8. Avoid exposing the explant too long on the paper to prevent
desiccation of the tissue.
9. Depending on the incubator chamber, the media from the
plates could evaporate more rapidly in some cases.
References
1. Vaughan J, Geissler C (2009) The new Oxford
book of food plants. OUP, Oxford
2. Simpson K, Cerda A, Stange C (2016a) Carotenoid biosynthesis in Daucus carota. Subcell
Biochem 79:199–217
3. FAOSTAT Database on Agriculture (2014).
In: food and agriculture Organization of the
United Nations, Rome, Italy
4. Simon P, Pollak L, Clevidence B, Holden J,
Haytowitz D (2009) Plant breeding for
human nutritional quality. Plant Breeding Rev
31:325–392
5. Kreuger M, Van Holst G (1993) Arabinogalactan proteins are essential in somatic embryogenesis of Daucus carota L. Planta
189:243–248
6. Quiroz-Figueroa F, Rojas-Herrera R, GalazAvalos R, Loyola-Vargas V (2006) Embryo
production through somatic embryogenesis
can be used to study cell differentiation in
plants. Plant Cell Tissue Organ Cult
86:285–301
7. Shiota H, Ko S, Wada S, Otsu C, Tanaka I,
Kamada H (2008) A carrot G-box binding
factor-type basic region/leucine zipper factor
DcBZ1 is involved in abscisic acid signal transduction in somatic embryogenesis. Plant
Physiol Biochem 46:550–558
8. Guan Y, Ren H, Xie H, Ma Z, Chen F (2009)
Identification and characterization of bZIPtype transcription factors involved in carrot
(Daucus carota L.) somatic embryogenesis.
Plant J 60:207–217
Carrot Nuclear Transformation
319
1. The normal pH of the solution should oscillate between 3.8
and 4.2.
2. You can accelerate the cooling of the media by introducing the
bottle inside an ice bucket with a little bit of ice and tap water.
By doing this, you should be very careful of not overcool the
media, or else the media at the bottom of the bottle will start to
solidify.
3. Different strains of Agrobacterium need different selection
agents: GV3101 strain needs gentamicin and rifampicin, alongside the selection agent that the vector confers; meanwhile,
EHA101 only needs gentamicin and the selection agent that
the vector confers.
4. Do not vortex the cells or the transformation efficiency will
drop dramatically.
5. Longer incubation times do not affect transformation
efficiency.
6. Avoid generating smaller explants. At early stages, the tissue is
difficult to manipulate as is very fragile and desiccates very
easily.
7. If the exposure time is shorter, it could affect the number of
transformant explants.
8. Avoid exposing the explant too long on the paper to prevent
desiccation of the tissue.
9. Depending on the incubator chamber, the media from the
plates could evaporate more rapidly in some cases.
References
1. Vaughan J, Geissler C (2009) The new Oxford
book of food plants. OUP, Oxford
2. Simpson K, Cerda A, Stange C (2016a) Carotenoid biosynthesis in Daucus carota. Subcell
Biochem 79:199–217
3. FAOSTAT Database on Agriculture (2014).
In: food and agriculture Organization of the
United Nations, Rome, Italy
4. Simon P, Pollak L, Clevidence B, Holden J,
Haytowitz D (2009) Plant breeding for
human nutritional quality. Plant Breeding Rev
31:325–392
5. Kreuger M, Van Holst G (1993) Arabinogalactan proteins are essential in somatic embryogenesis of Daucus carota L. Planta
189:243–248
6. Quiroz-Figueroa F, Rojas-Herrera R, GalazAvalos R, Loyola-Vargas V (2006) Embryo
production through somatic embryogenesis
can be used to study cell differentiation in
plants. Plant Cell Tissue Organ Cult
86:285–301
7. Shiota H, Ko S, Wada S, Otsu C, Tanaka I,
Kamada H (2008) A carrot G-box binding
factor-type basic region/leucine zipper factor
DcBZ1 is involved in abscisic acid signal transduction in somatic embryogenesis. Plant
Physiol Biochem 46:550–558
8. Guan Y, Ren H, Xie H, Ma Z, Chen F (2009)
Identification and characterization of bZIPtype transcription factors involved in carrot
(Daucus carota L.) somatic embryogenesis.
Plant J 60:207–217
Carrot Nuclear Transformation
319