3.1.2 Growth
of Arabidopsis Cells
in Culture
Cell suspension cultures represent a source of nearly uniform cell
material for genomic, biochemical, physiological, and metabolomic
studies that can be performed under tightly controlled environmental conditions. ABRC maintains several widely used Arabidopsis cell cultures, one of which is the T87 cell line. This line
originates from Columbia accession seedlings and can photosynthesize in light [2]. It has been used to analyze gene expression
changes under stress conditions, hormone signaling pathways, the
circadian clock, and plant cell wall biosynthesis [3–5]. Transient
and stable transformation protocols for this line have also been
established [3, 6]. Maintenance of T87 as a cell suspension culture
has been detailed previously [7]. This cell culture can also be
maintained as callus, and subsequently used to reestablish a cell
suspension culture. Due to limited space, only the protocol describing creation and maintenance of the T87 callus culture will be
described here.
1. Prepare 1 L of NT-1 media by adding 4.3 g of MS salt mixture,
30 g sucrose, 0.18 g KH 2 PO 4 , 100 μl of 10 mg/mL thiamine
stock solution, 220 μL of 2 mg/mL 2,4-D stock solution, and
100 mg myo-Inositol to a beaker containing 0.8 L of ddH 2 O
and stir to dissolve (see Note 10).
2. Adjust the pH to 5.8 using 5 M NaOH. Add ddH 2 O to final
volume of 1 L.
3. Distribute 90 mL of freshly prepared NT-1 media and add
0.72 g Bacto™ Agar per Magenta™ box.
4. Cover boxes with a double layer of domestic aluminum foil to
autoclave for 20 min. Allow the media to cool before closing
with lid and store at 4
C.
5. Start a new callus culture by pipetting 1–2 mL of one-week-old
T87 cell suspension culture upon solid media (see Note 11).
6. Grow the culture at 24
C under continuous light
(40–100 μmol/m
2 s).
7. Subculture callus biweekly by transferring a 1 mm piece to fresh
solid media with a disposable inoculating loop (see step 6 and
Notes 12 and 13).
3.1.3 Planting Seeds
of Arabidopsis and Other
Members
of the Brassicaceae on Soil
Diverse mixes and media can be used for growing various Brassicaceae species. The term “soil” will be used here for any mix or
medium utilized for non-sterile growth of plants in pots or similar
containers. Commercial potting mixes are popular with researchers
due to their convenience and reliability. Potting mixes often employ
peat moss for moisture retention and perlite for aeration. Mixes
such as Lambert
® LM-111 support healthy plant growth and
include a starter nutrient charge, so that fertilization is not necessary in the early growth phases. Seeds can be planted by various
8
Christopher Calhoun et al.
of Arabidopsis Cells
in Culture
Cell suspension cultures represent a source of nearly uniform cell
material for genomic, biochemical, physiological, and metabolomic
studies that can be performed under tightly controlled environmental conditions. ABRC maintains several widely used Arabidopsis cell cultures, one of which is the T87 cell line. This line
originates from Columbia accession seedlings and can photosynthesize in light [2]. It has been used to analyze gene expression
changes under stress conditions, hormone signaling pathways, the
circadian clock, and plant cell wall biosynthesis [3–5]. Transient
and stable transformation protocols for this line have also been
established [3, 6]. Maintenance of T87 as a cell suspension culture
has been detailed previously [7]. This cell culture can also be
maintained as callus, and subsequently used to reestablish a cell
suspension culture. Due to limited space, only the protocol describing creation and maintenance of the T87 callus culture will be
described here.
1. Prepare 1 L of NT-1 media by adding 4.3 g of MS salt mixture,
30 g sucrose, 0.18 g KH 2 PO 4 , 100 μl of 10 mg/mL thiamine
stock solution, 220 μL of 2 mg/mL 2,4-D stock solution, and
100 mg myo-Inositol to a beaker containing 0.8 L of ddH 2 O
and stir to dissolve (see Note 10).
2. Adjust the pH to 5.8 using 5 M NaOH. Add ddH 2 O to final
volume of 1 L.
3. Distribute 90 mL of freshly prepared NT-1 media and add
0.72 g Bacto™ Agar per Magenta™ box.
4. Cover boxes with a double layer of domestic aluminum foil to
autoclave for 20 min. Allow the media to cool before closing
with lid and store at 4
C.
5. Start a new callus culture by pipetting 1–2 mL of one-week-old
T87 cell suspension culture upon solid media (see Note 11).
6. Grow the culture at 24
C under continuous light
(40–100 μmol/m
2 s).
7. Subculture callus biweekly by transferring a 1 mm piece to fresh
solid media with a disposable inoculating loop (see step 6 and
Notes 12 and 13).
3.1.3 Planting Seeds
of Arabidopsis and Other
Members
of the Brassicaceae on Soil
Diverse mixes and media can be used for growing various Brassicaceae species. The term “soil” will be used here for any mix or
medium utilized for non-sterile growth of plants in pots or similar
containers. Commercial potting mixes are popular with researchers
due to their convenience and reliability. Potting mixes often employ
peat moss for moisture retention and perlite for aeration. Mixes
such as Lambert
® LM-111 support healthy plant growth and
include a starter nutrient charge, so that fertilization is not necessary in the early growth phases. Seeds can be planted by various
8
Christopher Calhoun et al.