Biology Reference
In-Depth Information
splice site in the GFP mRNA resulting in a truncated, nonfluorescent protein
(
Haseloff, 1999
). Fluorescent reporters attached the N-terminus of alpha and beta
tubulin-6 (the most commonly used isoforms) are effective when imaging microtu-
bules in living
Arabidopsis
cells. Transgenic plants expressing alpha tubulin fusions
are prone to show abnormal growth phenotypes such as twisting (
Abe & Hashimoto,
2005; Ueda, Matsuyama, & Hashimoto, 1999
). When driven from the 35S promoter,
microtubules are not visible in root tissues (
Ueda et al., 1999
). However, microtu-
bules in root cells can be visualized when constructs are driven from the ubiquitin1
promoter (
Ambrose, Allard, Cytrynbaum, & Wasteneys, 2011
).
15.1.1.2
Plant transformation
A. thaliana
transformation can be accomplished by simply coating young flower
buds with a solution of
A. tumefaciens
, sugar, and a surfactant (Clough & Bent,
1998). This “floral dipping” protocol has been optimized and described in greater
detail in several articles (
Davis, Hall, Millar, Darrah, & Davis, 2009; Desfeux,
Clough, & Bent, 2000; Logemann, Birkenbihl, Ulker, & Somssich, 2006; Zhang,
Henriques, Lin, Niu, & Chua, 2006
). Here, a common protocol that works well in
our hands is outlined. One of the more important aspects of this procedure is to start
with healthy, robust plants with ample developing flower buds because transforma-
tion occurs in the ovule of the flower (
Desfeux et al., 2000; Ye et al., 1999
). To start,
sow nine seeds in each of ten 4
4 in. pots. Grow the plants till the inflorescence
stem (the flower stalk) is 6-8 in. tall, which can take 3-5 weeks depending on the
genotype and growth conditions (the light regime and temperature are the primary fac-
tors). Once 6-8 in. tall, the floral stem is removed by cutting it near the base with a
clean razor blade. The plants are watered with a balanced fertilizer after cutting.
The removal of the primary floral stem releases multiple axillary floral meristems from
apical dominance and results in the outgrowth of the axillary floral buds. This increases
the total number of flowers available for transformation several fold (at least 4
).
When the axillary floral stems reach the height of 5-7 in. and are actively pro-
ducing fruit, they are gently submerged (dipped) in a suspension of
A. tumefaciens
in 5% sucrose and 0.02% of the surfactant Silwet L-77 (Clough & Bent, 1998). The
Silwet concentration can be varied from 0.005% to 0.02%. The first step in producing
the
A. tumefaciens
suspension is to grow a 500-ml culture in Luria broth (LB) at
28
C for 48 h (
A. tumefaciens
grows slower than
E. coli
). This culture is then cen-
trifuged at 4000 rpm for 20 min at room temperate. The resulting pinkish pellet is
gently resuspended in the sucrose/Silwet solution by slowly pipetting up and down
with a large bore pipette. The young pliable floral stems are then gently submerged in
the bacterial suspension for 30-60 s. When dipping the plants, one should avoid wet-
ting the rosette leaves and breaking the floral stems. Placing the plants in a humid
low-light environment, like a covered bucket or tray, for 2-3 days after dipping
the plants increases the transformation success (Clough & Bent, 1998). After 48-
72 h, the plants are slowly transitioned to normal growth conditions over the period
of a day. The plants will continue to grow and flower after dipping.
A. thaliana
read-
ily self-pollinates, and generating large numbers of seeds to screen for antibiotic
