Cross Arabidopsis for complementation (A).

Aim: Cross two transgenic Arabidopsis lines and generate hybrids. Each of the two parental
lines express a part of the biosynthetic pathway of the tyrosine cyanogenic glucoside (a
natural product). The resulting hybrid will express the complete dhurrin pathway, and
accumulate dhurrin.

Theory: Arabidopsis is autogamous, which means that it self fertilizes before the flowers
open. This is a trait important for generation of homozygous lines. However, Arabidopsis
lines can be crossed in the laboratory to produce hybrids. To accomplish this, the immature
anthers are removed and the stigma is allowed to mature to a state where it will accept
pollen and be fertilized. Then pollen is introduced from the other parental line. Arabidopsis
will produce 3-6 inflorescences depending on the daylight length. Often crosses work best
using the primary inflorescence and avoiding the first 2-3 flowers (counting from below).
As the plants get older, the flowers tend to become smaller and the failure rate for crosses
increases.

You will be given two transgenic lines denoted UGT and 2x, respectively. Cross the two
lines together and obtain a hybrid. If the cross is successful, the resulting hybrids will
produce a new metabolite called dhurrin which can be readily scored by HPLC (high
pressure liquid chromatography). The UGT line expresses the UGT85B1 gene from
sorghum (AAF17077) under control of the 35S cauliflower mosaic virus promoter;
35S::UGT85B1. The UGT line contains the aacC marker conferring resistance to the
antibiotic gentamycin. The 2x lines expresses two sorghum genes, CYP79A1 (Q43135) and
CYP71E1 (AF029858), both under control of a 35S cauliflower mosaic virus promoter. The
two genes have been introduced with the NptII marker conferring resistance to the antibiotic
kanamycin, in the same construct: 35S::CYP79A1/35S::CYP71E1. Accordingly, CYP79A1
and CYP71E1 are located next to each other in the Arabidopsis chromosome. The resulting
hybrids should express all three sorghum genes.

CYP79A1 and CYP71E1 catalyze the conversion of the aromatic amino acid tyrosine to p-
hydroxymandelonitrile, the aglycone of the cyanogenic glucoside dhurrin (figure 1). p-
Hydroxymandelonitrile is an unstable compound that will decompose and release toxic
hydrogen cyanide (HCN) and p-hydroxybenzaldehyde, which will be oxidized to p-
hydroxybenzoic acid. In Arabidopsis, p-hydroxybenzoic acid will be detoxified by addition
of a glucose molecule giving rise to high levels of p-hydroxybenzoic acid glucosides
derivatives, which can be readily detected by HPLC. The 2x plants have a stressed and
stunted phenotype due to the accumulation of the introduced xenobiotics (foreign
metabolites) (figure 2). The UGT line expresses the sorghum glucosyltransferase,
UGT85B1 that will glucosylate p-hydroxymandelonitrile to the cyanogenic glucoside
dhurrin (figure 1). If UGT85B1 is expressed in a Arabidopsis line that does not express
CYP79A1 and CYP71E1, no new metabolites will accumulate as the UGT85B1 substrate is
absent. However, if introduced into the 2x line by e.g. crossing, the hybrid line will a)

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accumulate dhurrin and b) complement the stressed and stunted phenotype of the 2x line
(figure 2).

Week 35: August 29th. Prepare Arabidopsis for crossing (A1)

Work in groups of two. It is important that you stay in the same group through out the
exercise.

From the UGT line (female parent) remove the sepals,

petals and anthers with the tweezers from 3-4 flowers
(figure 3). The tweezers are expensive, sharp and
pointed and will not work if bended – please take
good care of the tweezers! The flowers has to be at
the stage just before they open (the white petals are
becoming to become visiable) (figure 3), if the
flowers are too immature the stigma will not mature,
if the flowers are too open the flower is most likely
already fertilized. Carefully “brush” over the flower
with the side of the tweezer to open op the flower.
The anthers should be below the stigma; at this stage
they are not mature and the pollen is not shed. Be
very careful that you do not damage the stigma, style,
and the vascular tissue; if you do, the cross will not be
successful as the inflorescence and/or stigma will wilt.
Place the inflorescence/flower under the
stereomicroscope to see what you are removing.
Healthy plants usually have three flowers on the main shoot suitable for crossing. Prepare 2-
5 plants (3 flowers on each) to ensure that you have enough stigma that will mature and are
undamaged. It is very important that all anthers are removed. Have one of the teachers
examine your plants, to ensure that the plants have been prepared correctly, as the rest of the
exercise depends on the quality of the plants. Se figure 3 for how the flowers should look
before and after preparation.

When you are done preparing the flowers, carefully remove the rest of the florescence, so
that siliques only will develop on the flowers prepared.

Week 35 September 1st. Cross Arabidopsis (A2).

Examine the flowers you prepared Monday under the microscope and ensure that the stigma
has matured and all anthers are removed.

Select a flower from the 2x line (male parent) - the flower should be open and under the
stereomicroscope you should be able to see that the anthers are shedding pollen (figure 4).
Remove the flower with the tweezers and at the same time squeeze it near the base with the
tweezers so that the flower “opens up”. Examine the flower under the stereomicroscope,

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pollen should be dispersed from the anthers. Pollinate the stigmas from the UGT line with
pollen from flowers from the 2x line. To avoid cross contamination with pollen from other
lines, carefully wipe the tweezers with moisturized tissue paper.

If you have more than 6 flowers ready for pollination, do not pollinate some of them. Label
the stigma you have NOT pollinated with e.g tape. The un-pollinated stigma should not
develop into siliques and serves negative controls.

Week 36 September 5th Check crosses (A3).

If the pollination has been successful, the style will have elongated and a silique will start to
develop. Examine the stigma under the stereo-microscope and make a note of what you see.

In 2-3 weeks the seeds should be ready for collection, and the siliques can be collected
when they begin to turn yellow. If the siliques dry on the plant, it may spit open, and the
seeds will be lost. Dry the siliques at room temperature for ~2 weeks before planting. Damp
seeds will have poor germination rates. The teachers will collect your mature siliques and
dry them due to the time limits of this course.

Week 40 October 6th. Sow hybrids (A4).

Carefully collect the siliques and sow 5-10 seeds in each of 2 9 cm pots filled with soil.
Label each pot with a yellow sticker. (The yellow label is used to label transgenic lines,
wild type plants are labeled with stickers). Sow 10 seeds of each of the parental lines, UGT
and 2x and, in individual pots. The latter two pots are negative controls. Finally sow ~10
seeds of a dhurrin producing line as positive control. The seeds for the negative and positive
control will be provided by one of the teachers. Carefully water the 5 pots. The pots will be
placed in a growth chamber.

Obtain hybrid seeds from one of the teacher. These seeds are offspring (F2) from a similar
cross that we made earlier; the F1 line has been selfed.

Work in a sterile bench. Sterilize the seeds by first washing with 500 µl 5% NaOCl and
0,02 % Triton X-100, followed by 3 washes with 500 µl sterile water. Place ~1/3 of the
seeds on MS plates with kanamycin, ~1/3 of the seeds on MS plates with gentamycin, and
~1/3 of the seeds on MS plates with both kanamycin and gentamycin. These plates will be
placed in a growth chamber.

Week 41 Oktober 13th. Score the Arabidopsis seedlings on selective plates (A5).
The F2 seedlings that are green are resistant to the antibiotics. Count the number of white
and green seedlings. What does the numbers tell you?

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Week 42. HPLC analysis of Arabidopsis hybrids. The teachers will do this during the
autumn holiday.

Week 43. Oktober 24th. Evaluate results from HPLC analysis (A6).

Week 43: Upload your report by 27-10 at the latest.

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Figure 1 COOH

NH2
HO

tyrosine

CYP79A1
H N
OH

HO

p-hydroxyphenylacetaldoxime

CYP71E1
CN

OH

HO

p-hydroxymandelonitrile

Sorghum UGT85B1
CN

O
Glc

HO

dhurrin

Figure 2

wt UGT 2x f1
Female parent Male parent

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Figure 3

Figure 4