54. The stigma of the selected female parent should be receptive to
pollen.
55. Alternatively, select an immature bud as female parent from a
primary transformant (T0) plant line that has difficulty
setting seed.
56. Removal of the anthers is needed because tomato is a selfpollinating species.
57. Pull the petals and anther cone straight off and away from the
flower axis to avoid (a) damaging the stigma and style and
(b) pollen transfer.
58. Prevent pollen transfer from these flowers to the female parent.
59. Collected pollen should be visible to the naked eye on the tip of
the ultrafine point forceps.
60. Provide the stigma with a visible amount of pollen to help
assure fertilization.
61. Indicate both parents and the date of crossing.
62. This will increase the likelihood that fertilization of the female
parent will occur.
63. It is recommended to perform multiple crosses, since not all of
them might succeed.
64. To obtain single-gene knockout lines, identified heterozygous
F1 lines can be selfed and their progeny can be analyzed as
described in Subheading 3.5.
References
1. Rothan C, Bres C, Garcia V, Just D (2016)
Tomato resources for functional genomics.
In: Causse M, Giovannoni J, Bouzayen M,
Zouine M (eds) The tomato genome. Springer,
Berlin, Germany, pp 75–94
2. Van Eck J (2018) Genome editing and plant
transformation of solanaceous food crops. Curr
Opin Biotechnol 49:35–41
3. Ron M, Kajala K, Pauluzzi G, Wang D, Reynoso MA, Zumstein K et al (2014) Hairy root
transformation using Agrobacterium rhizogenes
as a tool for exploring cell type-specific gene
expression and function using tomato as a
model. Plant Physiol 166:455–469
4. Van Eck J, Keen P, Tjahjadi M (2019) Agrobacterium tumefaciens-mediated transformation of tomato. In: Kumar S, Barone P and
Smith M (Eds) Transgenic plants (methods in
molecular biology 1864). Humana Press,
New York, NY, pp 225–234
5. Shikata M, Ezura H (2016) Micro-tom tomato
as an alternative plant model system: mutant
collection and efficient transformation. In:
Botella J, Botella M (eds) Plant signal transduction (methods in molecular biology 1363).
Humana Press, New York, NY, pp 47–55
6. Garcia D, Narva ´ez-Va ´squez J, Orozco-Ca ´rdenas ML (2015) Tomato (Solanum lycopersicum). In: Wang K (ed) Agrobacterium
protocols (methods in molecular biology
1223). Springer, New York, NY, pp 349–361
7. Lampropoulos A, Sutikovic Z, Wenzl C,
Maegele I, Lohmann JU, Forner J (2013)
GreenGate—a novel, versatile, and efficient
cloning system for plant transgenesis. PLoS
One 8:e83043
8. Schiml S, Fauser F, Puchta H (2017)
CRISPR/Cas-mediated in planta gene targeting. In: Busch W (ed) Plant genomics (methods in molecular biology 1610). Humana
Press, New York, NY, pp 3–11
9. Ellul P, Garcia-Sogo B, Pineda B, Rı ´os G,
Roig L, Moreno V (2003) The ploidy level of
transgenic plants in Agrobacterium-mediated
340
Gwen Swinnen et al.
pollen.
55. Alternatively, select an immature bud as female parent from a
primary transformant (T0) plant line that has difficulty
setting seed.
56. Removal of the anthers is needed because tomato is a selfpollinating species.
57. Pull the petals and anther cone straight off and away from the
flower axis to avoid (a) damaging the stigma and style and
(b) pollen transfer.
58. Prevent pollen transfer from these flowers to the female parent.
59. Collected pollen should be visible to the naked eye on the tip of
the ultrafine point forceps.
60. Provide the stigma with a visible amount of pollen to help
assure fertilization.
61. Indicate both parents and the date of crossing.
62. This will increase the likelihood that fertilization of the female
parent will occur.
63. It is recommended to perform multiple crosses, since not all of
them might succeed.
64. To obtain single-gene knockout lines, identified heterozygous
F1 lines can be selfed and their progeny can be analyzed as
described in Subheading 3.5.
References
1. Rothan C, Bres C, Garcia V, Just D (2016)
Tomato resources for functional genomics.
In: Causse M, Giovannoni J, Bouzayen M,
Zouine M (eds) The tomato genome. Springer,
Berlin, Germany, pp 75–94
2. Van Eck J (2018) Genome editing and plant
transformation of solanaceous food crops. Curr
Opin Biotechnol 49:35–41
3. Ron M, Kajala K, Pauluzzi G, Wang D, Reynoso MA, Zumstein K et al (2014) Hairy root
transformation using Agrobacterium rhizogenes
as a tool for exploring cell type-specific gene
expression and function using tomato as a
model. Plant Physiol 166:455–469
4. Van Eck J, Keen P, Tjahjadi M (2019) Agrobacterium tumefaciens-mediated transformation of tomato. In: Kumar S, Barone P and
Smith M (Eds) Transgenic plants (methods in
molecular biology 1864). Humana Press,
New York, NY, pp 225–234
5. Shikata M, Ezura H (2016) Micro-tom tomato
as an alternative plant model system: mutant
collection and efficient transformation. In:
Botella J, Botella M (eds) Plant signal transduction (methods in molecular biology 1363).
Humana Press, New York, NY, pp 47–55
6. Garcia D, Narva ´ez-Va ´squez J, Orozco-Ca ´rdenas ML (2015) Tomato (Solanum lycopersicum). In: Wang K (ed) Agrobacterium
protocols (methods in molecular biology
1223). Springer, New York, NY, pp 349–361
7. Lampropoulos A, Sutikovic Z, Wenzl C,
Maegele I, Lohmann JU, Forner J (2013)
GreenGate—a novel, versatile, and efficient
cloning system for plant transgenesis. PLoS
One 8:e83043
8. Schiml S, Fauser F, Puchta H (2017)
CRISPR/Cas-mediated in planta gene targeting. In: Busch W (ed) Plant genomics (methods in molecular biology 1610). Humana
Press, New York, NY, pp 3–11
9. Ellul P, Garcia-Sogo B, Pineda B, Rı ´os G,
Roig L, Moreno V (2003) The ploidy level of
transgenic plants in Agrobacterium-mediated
340
Gwen Swinnen et al.