6. Make a slit in the anther cone of the male parent using ultrafine
point forceps.
7. Drag the ultrafine point forceps upward through the inside of
the anther cone to collect pollen (see Note 59).
8. Apply collected pollen from the male parent using the ultrafine
point forceps to the stigma of the female parent (see Note 60).
9. Tag the crossed flower (see Note 61).
10. Repollinate the female parent one or 2 days later by repeating
steps 5–8 (see Note 62).
11. Ovary enlargement of the cross-pollinated flower can be
observed within 4 to 5 days if fertilization was successful.
Otherwise, the flower will abort (see Note 63).
12. F1 lines can be analyzed as described in Subheading 3.5 (see
Note 64).
4 Notes
1. Because the modular system is based on GreenGate technology, it can easily be adapted to any new development (e.g., for
alternative Cas endonucleases) [10].
2. For instance, to transfer three gRNAs to a binary vector of
choice, you can combine three Golden Gate entry modules for
one gRNA (with Golden Gate overlaps AB, BC and CD)
together with the Golden Gate entry module pGG-D-linkerII-G. If a binary vector containing six gRNAs is desired,
you can assemble six Golden Gate entry modules for one
gRNA (with Golden Gate overlaps AB, BC, CD, DE, EF and
FG). To obtain a binary vector with seven gRNAs, you can
combine five Golden Gate entry modules for one gRNA (with
Golden Gate overlaps AB, BC, CD, DE, and EF) together with
one Golden Gate entry module for two gRNAs (with Golden
Gate overlap FG) (see Table 3).
3. With the MagWell™ Magnetic Separator 96 (EdgeBio), the
magnetic beads will be attracted to one side and up from the
bottom of PCR tubes, allowing for easy recovery of your
samples.
4. Ultrafine point forceps can be purchased from any supplier.
5. BpiI from Thermo Scientific and BbsI-HF from New England
Biolabs are stable at À20
C.
6. Store the T4 ligase buffer in aliquots of 10–20 μL to minimize
degradation of DTT (and of ATP in case it is included in the
buffer).
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Gwen Swinnen et al.
point forceps.
7. Drag the ultrafine point forceps upward through the inside of
the anther cone to collect pollen (see Note 59).
8. Apply collected pollen from the male parent using the ultrafine
point forceps to the stigma of the female parent (see Note 60).
9. Tag the crossed flower (see Note 61).
10. Repollinate the female parent one or 2 days later by repeating
steps 5–8 (see Note 62).
11. Ovary enlargement of the cross-pollinated flower can be
observed within 4 to 5 days if fertilization was successful.
Otherwise, the flower will abort (see Note 63).
12. F1 lines can be analyzed as described in Subheading 3.5 (see
Note 64).
4 Notes
1. Because the modular system is based on GreenGate technology, it can easily be adapted to any new development (e.g., for
alternative Cas endonucleases) [10].
2. For instance, to transfer three gRNAs to a binary vector of
choice, you can combine three Golden Gate entry modules for
one gRNA (with Golden Gate overlaps AB, BC and CD)
together with the Golden Gate entry module pGG-D-linkerII-G. If a binary vector containing six gRNAs is desired,
you can assemble six Golden Gate entry modules for one
gRNA (with Golden Gate overlaps AB, BC, CD, DE, EF and
FG). To obtain a binary vector with seven gRNAs, you can
combine five Golden Gate entry modules for one gRNA (with
Golden Gate overlaps AB, BC, CD, DE, and EF) together with
one Golden Gate entry module for two gRNAs (with Golden
Gate overlap FG) (see Table 3).
3. With the MagWell™ Magnetic Separator 96 (EdgeBio), the
magnetic beads will be attracted to one side and up from the
bottom of PCR tubes, allowing for easy recovery of your
samples.
4. Ultrafine point forceps can be purchased from any supplier.
5. BpiI from Thermo Scientific and BbsI-HF from New England
Biolabs are stable at À20
C.
6. Store the T4 ligase buffer in aliquots of 10–20 μL to minimize
degradation of DTT (and of ATP in case it is included in the
buffer).
336
Gwen Swinnen et al.