17. Place on the magnetic separator.
18. Collect the cleared supernatant, which contains the purified
PCR amplicon (see Note 46).
19. Conduct Sanger sequencing (see Note 48).
20. Identify hairy root or plant lines containing gene knockout
mutation(s) using ICE to analyze obtained Sanger sequencing
data (see Notes 49 and 50).
3.5.4 Ploidy Level
Analysis
Regeneration of polyploid primary transformant (T0) plants is not
unusual in plant tissue-culture dependent A. tumefaciens-mediated
transformation. As an increase in tomato ploidy level can alter the
development of vegetative and/or reproductive organs, it is necessary to select diploid primary transformant (T0) individuals [9].
1. Chop 1.0 cm
2 of leaf material from a primary transformant
(T0) plant in 200 μL of chilled nuclei extraction buffer for
2 min using a razor blade.
2. Filter the suspension through a 50-μm nylon filter to remove
cell fragments and large debris.
3. Add 800 μL of chilled staining buffer to the isolated nuclei.
4. Mix by pipetting.
5. Measure the DNA content of about 5000 to 10,000 nuclei per
leaf sample using a flow cytometer (see Note 51).
6. Analyze with FloMax software to identify diploid primary
transformant (T0) plant lines.
3.6 Crossing Gene
Knockout Plant Lines
to Wild-Type
Here, we describe a method to cross an obtained gene knockout
plant line to the wild-type. This allows the exclusion of somaclonal
variation and the generation of single-gene knockout plant lines
from multiple-gene knockout plant lines. Alternatively, it can be
used to develop heterozygous gene knockout plant lines from
homozygous gene knockout plant lines that are low in seed set or
sterile (see Note 52).
1. Select an immature bud (see Note 53) as female parent, which
shows a little yellowing of the petals (see Note 54), from a
T-DNA free gene knockout plant line (see Note 55).
2. Remove sepals from the female parent using ultrafine point
forceps.
3. Emasculate the female parent (see Note 56) using ultrafine
point forceps to carefully pinch the side of the petals and anther
cone and pull them off thereafter (see Note 57).
4. Remove all other flowers present on the inflorescence to which
the female parent belongs (see Note 58).
5. Select an open flower as male parent from a wild-type plant.
CRISPR-Cas-Mediated Gene Knockout
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18. Collect the cleared supernatant, which contains the purified
PCR amplicon (see Note 46).
19. Conduct Sanger sequencing (see Note 48).
20. Identify hairy root or plant lines containing gene knockout
mutation(s) using ICE to analyze obtained Sanger sequencing
data (see Notes 49 and 50).
3.5.4 Ploidy Level
Analysis
Regeneration of polyploid primary transformant (T0) plants is not
unusual in plant tissue-culture dependent A. tumefaciens-mediated
transformation. As an increase in tomato ploidy level can alter the
development of vegetative and/or reproductive organs, it is necessary to select diploid primary transformant (T0) individuals [9].
1. Chop 1.0 cm
2 of leaf material from a primary transformant
(T0) plant in 200 μL of chilled nuclei extraction buffer for
2 min using a razor blade.
2. Filter the suspension through a 50-μm nylon filter to remove
cell fragments and large debris.
3. Add 800 μL of chilled staining buffer to the isolated nuclei.
4. Mix by pipetting.
5. Measure the DNA content of about 5000 to 10,000 nuclei per
leaf sample using a flow cytometer (see Note 51).
6. Analyze with FloMax software to identify diploid primary
transformant (T0) plant lines.
3.6 Crossing Gene
Knockout Plant Lines
to Wild-Type
Here, we describe a method to cross an obtained gene knockout
plant line to the wild-type. This allows the exclusion of somaclonal
variation and the generation of single-gene knockout plant lines
from multiple-gene knockout plant lines. Alternatively, it can be
used to develop heterozygous gene knockout plant lines from
homozygous gene knockout plant lines that are low in seed set or
sterile (see Note 52).
1. Select an immature bud (see Note 53) as female parent, which
shows a little yellowing of the petals (see Note 54), from a
T-DNA free gene knockout plant line (see Note 55).
2. Remove sepals from the female parent using ultrafine point
forceps.
3. Emasculate the female parent (see Note 56) using ultrafine
point forceps to carefully pinch the side of the petals and anther
cone and pull them off thereafter (see Note 57).
4. Remove all other flowers present on the inflorescence to which
the female parent belongs (see Note 58).
5. Select an open flower as male parent from a wild-type plant.
CRISPR-Cas-Mediated Gene Knockout
335