GENETI C TRANSFORMATI ON AND HYBRI DI ZATI ON

Generation of transgenic plants of a potential oilseed crop

Camelina sativa by Agrobacterium-mediated transformation
Chaofu Lu Jinling Kang
Received: 16 July 2007 / Revised: 1 September 2007 / Accepted: 10 September 2007 / Published online: 27 September 2007
Springer-Verlag 2007
Abstract Camelina sativa is an alternative oilseed crop
that can be used as a potential low-cost biofuel crop or a
source of health promoting omega-3 fatty acids. Currently,
the fatty acid composition of camelina does not uniquely t
any particular uses, thus limit its commercial value and
large-scale production. In order to improve oil quality and
other agronomic characters, we have developed an efcient
and simple in planta method to generate transgenic came-
lina plants. The method included Agrobacterium-mediated
inoculation of plants at early owering stage along with a
vacuum inltration procedure. We used a uorescent pro-
tein (DsRed) as a visual selection marker, which allowed us
to conveniently screen mature transgenic seeds from a
large number of untransformed seeds. Using this method,
over 1% of transgenic seeds can be obtained. Genetic
analysis revealed that most of transgenic plants contain a
single copy of transgene. In addition, we also demonstrated
that transgenic camelina seeds produced novel hydroxy
fatty acids by transforming a castor fatty acid hydroxylase.
In conclusion, our results provide a rapid means to genet-
ically improve agronomic characters of camelina,
including fatty acid proles of its seed oils. Camelina may
serve as a potential industrial crop to produce novel bio-
technology products.
Keywords Agrobacterium Camelina sativa
Transgenic plants Unusual fatty acids
Vacuum-inltration
Abbreviations
DsRed Discosoma sp. red uorescent protein
FAH12 D12-Fatty acid hydroxylase
FAMEs Fatty acyl methyl esters
GC Gas chromatography
MS Murashige and Skoog
PCR Polymerase Chain Reaction
YEP Yeast extract-peptone medium
Introduction
Camelina [Camelina sativa (L.) Crtz., popular names gold-
of-pleasure, false ax] is an ancient crop of the Brassica-
ceae family. It was cultivated for oil production since
prehistoric times, and it was extensively grown in Europe
in the ninteenth century (Knorzer 1978). However, for
some unknown reasons, camelina production gradually
declined and was almost vanished after World War II. The
recent interests in camelina are inspired by its unique oil
composition. About 90% of fatty acids in camelina oil is
unsaturated. The essential polyunsaturated fatty acids
(linoleic acid, 18:2n-6 and a-linolenic acid, 18:3n-3) con-
stitute over 50% of total fatty acids, and linolenic acid is
the most predominant fatty acid (3540%). Therefore
camelina oil is an excellent source of omega-3 fatty acids
(Leonard 1998), which has been recommended in the diet
to achieve essentiality and cardiovascular benets
(Gebauer et al. 2006). Non-food uses of camelina oil have
Communicated by R. Reski.
C. Lu (&) J. Kang
Department of Plant Sciences and Plant Pathology,
Montana State University, Bozeman, MT 59717, USA
e-mail: clu@montana.edu
1 3
Plant Cell Rep (2008) 27:273278
DOI 10.1007/s00299-007-0454-0
also been exploited for the production of soaps, varnishes
and cosmetics (Putnam et al. 1993; Zubr 1997). Since
production cost of camelina is relatively lower than many
other oil crops such as rapeseed, corn and soybean, it is an
attractive potential crop for biofuels and many other
industrial applications (Putnam et al. 1993).
Currently, the lack of a clear utilization pattern of
camelina oil limits its uses and large-scale production
despite its adaptation to a wide range of climates (Putnam
et al. 1993). Camelina oil contains about 15% of eicos-
enoic acid (20:1), which is unique among many other
vegetable oils. The usefulness or disadvantage of eicose-
noic acid is not clear, however, it may present a hurdle to
obtain food approval (Leonard 1998). The high percent-
age of polyunsaturated fatty acids makes camelina oil
more susceptible to oxidation and thus is undesirable for
fuel and other industrial applications. Therefore it is
necessary to modify camelina oils to nd a role of this
potential crop in the world oilseed market. Traditionally,
cruciferous oil crops are amenable to genetic improve-
ment through breeding (Downey and Craig 1964), and
biotechnology (Jaworski and Cahoon 2003; Kinney 1996;
Singh et al. 2005; Thelen and Ohlrogge 2002). Limited
research has also been done to improve camelina oil
quality or other agronomic characters. A trial of seed
mutagenesis has been conducted and variations of fatty
acid proles were observed (Buchsenschutz-Nothdurft
et al. 1998). In vitro studies include somatic hybridization
studies with other Brassica species (Narasimhulu et al.
1994; Sigareva and Earle 1999), and plant regeneration
from leaf explants (Tattersall and Millam 1998), which
led to the establishment of a tissue culture-based, Agro-
bacterium-mediated transformation method (United States
Patent No. 20040031076).
In this report, we describe a simple and efcient trans-
formation system, which will provide a useful tool for
genetic improvement of camelina, and biological studies
using this crop species. We also present an example of
successfully expressing a castor fatty acid hydroxylase in
camelina seeds, resulting in accumulation of unusual
hydroxy fatty acids. Our results demonstrate that camelina
is an excellent candidate crop species to produce many
biotechnology products including unusual fatty acids.
Materials and methods
Plant material
A Camelina sativa cultivar Celine was used for all
experiments. For transformation, ve seeds were directly
germinated in 9 cm pots lled with soil and Sunshine Mix
(Clinton, OK, USA) (1:1) in the greenhouse. Growth
conditions in the greenhouse were 20/16C (day/night)
with natural lighting. The relatively humidity was main-
tained at 60%.
Agrobacterium strain, plasmid and bacterial growth
A binary plasmid pGDP-FAH12 constructed previously
(Lu et al. 2006) was used in this study (Fig. 1). In this
construct, a castor fatty acid hydroxylase (FAH12) gene
was driven by a seed-specic phaseolin promoter (Seng-
upta-Gopalan et al. 1985). The selection marker DsRed
(Clontech, Mountain View, CA, USA) was driven by the
constitutive cassava vein mosaic virus promoter (Verda-
guer et al. 1996). The plasmid was introduced into
Agrobacterium strain GV3101 (pMP90) by electroporation
with 0.2 cm cuvettes (Bio-Rad, Hercules, CA, USA) at a
eld strength of 2.5 kv/cm, restorors of 600 X and a
capacitance of 25 lF. Selection of transformed bacteria
was carried out on LB plates containing 100 lg/ml spec-
tinomycin and 40 lg/ml gentamycin.
Bacterial growth was commenced 23 days prior to
plant transformation. A single colony was inoculated into a
culture tube containing 5 ml YEP medium (5 g/l yeast
extract, 10 g/l peptone, 5 g/l NaCl, pH6.8) supplemented
with 100 mg/L spectinomycin. Overnight culture of the
5-ml bacteria was then transferred into a 2-l ask con-
taining 500 ml of the same medium and continued growing
for 2448 h at 28C. Bacteria were harvested by centrifu-
gation at 6,000 rpm for 10 min and the pellet was
suspended in the inltration medium, consisting of half-
strength MS salts, 50 g/l sucrose and 0.05% (v/v) Silwet
L77 (Lehle Seeds, Round Rock, TX, USA).
Inoculation of plants with Agrobacterium
Camelina plants were inoculated with the Agrobacterium
suspension prepared as described above. The pots with
plants at the owering stage were placed inside a 310 mm-
height vacuum desiccator (Bel-Art, Pequannock, NJ, USA)
and the inorescences were immersed into the inoculum
contained in a 300-ml beaker. The vacuum was applied and
held for 5 min at a pressure of 85 kPa. The treated plants
were covered by plastic bags for 24 h before returning to
normal growth in greenhouse until maturity.
Screening of seeds recovered from inoculated plants
Seeds harvested from transformed plants were illumi-
nated by a green LED ashlight (Streamlight Flashlights,
http://www.opticsplanet.net). Fluorescent seeds were
274 Plant Cell Rep (2008) 27:273278
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visually detected using a pair of red-lens sunglasses (KDs
Dark Red, http://www.originalkds.com).
PCR analysis of putative transgenic plants
DNA was extracted from young leaves by a CTAB-based
method (Lukowitz et al. 2000). PCR were carried out
using the following primers to amplify coding regions of
DsRed: 5
0
-ATGGCCTCCTCCGAGAACGT-3
0
(forward)
and 5
0
-ACCCTGAGACTGTTGGACAGAG-3
0
(reverse);
and FAH12: 5
0
-ATGGGAGGTGGTGGTCGCATGTCT
ACTG-3
0
(forward) and 5
0
-TTAATACTTGTTCCGG
TAC-3
0
(reverse). The total volume of 50 lL reaction
mixture contains *200 ng genomic DNA, 5 ll of each
primer (5 lM), 5 ll of dNTP mix (2 lM each) 10 ll
green GoTaq reaction buffer (5) and 0.25 ll GoTaq
DNA polymerase (Promega, Madison, WI, USA). Cycling
parameters began with an initial 94C for 3 min, followed
by 30 cycles of 94C 30 s, 55C 30 s, 72C 90 s and a
nal extension of 72C10 min. PCRproducts were analyzed
by electrophoresis in 0.8% agarose/ethidium bromide gels.
Fatty acid analysis of seed oils
Fatty acid methyl esters (FAMEs) were prepared by heat-
ing 13 seeds at 80C in 1 ml 2.5% H
2
SO
4
(v/v) in
methanol for 90 min, and extracted with 200 ll hexane and
1.5 ml of 0.9% NaCl (w/v), and 100 ll of organic phase
were transferred to autoinjector vials. One microlitre of
sample was injected into a Shimadzu 2010 GC tted with a
10-M 0.18-mm narrowbore carbowax column (Agilent).
The GC was programmed for an initial temperature of
180C for 1.5 min followed by an increase of 30C/min to
210C and maintained for a further 3 min.
Results
A simple transformation procedure for Camelina sativa
The plasmid construct used in this study is illustrated in
Fig. 1a. In order to quickly identify transgenic plants, we
used a Discosoma sp. uorescent protein (DsRed) as a
selection marker. This marker was recently developed to
select transgenic seeds of Arabidopsis thaliana (Stuitje
et al. 2003), and was successfully used in our previous
study (Lu et al. 2006). A full-length cDNA of castor
hydroxylase gene FAH12 was cloned from a castor cDNA
library, and was inserted into a binary vector pGate-DsRed-
Phas behind a seed-specic Phaseolin promoter by Gate-
way LR reaction (Lu et al. 2006). The resulting construct
was introduced into the Agrobacterium strain GV3101 by
electroporation.
Camelina plants at the early owering stages (Fig. 1b)
were used for transformation. We initially attempted to
transform camelina by the oral dipping method (Clough
and Bent 1998), i.e., submerging ower buds for 13 min
in Agrobacterium-containing solutions, but without suc-
cess. Since camelina plants were only about 30 cm high at
the early owering stage (Fig. 1d), we were able to place
the pots inside a desiccation chamber, which was con-
nected to a vacuum pump. We then applied vacuum at
about 85 kPa while immersing owers for 5 min in the
solution. The treatment did not have signicant deleterious
effects on plant survival and seed set.
Screening of transgenic seeds
Transgenic seeds were screened from dry mature seeds
using the visible DsRed marker (Fig. 1c). A total of 76
uorescent seeds were identied from about 6,000 seeds
Fig. 1 Generation of transgenic plants of Camelina sativa. a
Schematic representation of the gene construct pGDP-FAH12. The
selection marker DsRed is driven by the cassava vein mosaic virus
promoter (pCVMV). pPHAS Phaseolin promoter; Spc spectinomycin
resistance; LB, RB left and right borders of the binary vector. b
Camelina owers ready for transformation. c Screening of red
transgenic seeds expressing DsRed. d Transgenic plants growing in
pots. A desiccation chamber is shown for size comparison
Plant Cell Rep (2008) 27:273278 275
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that were harvested from 22 T0 plants. The frequency of
transformation was about 1.3%.
To evaluate transgenic effects on changing oil quality of
camelina seeds, we have introduced a castor fatty acid
hydroxylase gene, FAH12, into the transgenic plants along
with the DsRed selection marker. We analyzed fatty acid
compositions of three single uorescent seeds by gas
chromatography (GC). Figure 2 indicated that all three red
seeds accumulated novel fatty acids in seed oil. The four
novel fatty acids had been previously identied in trans-
genic Arabidopsis as ricinoleic acid (18:1OH), which is the
major component of castor oil, and three other hydroxy
fatty acids densipolic acid (18:2OH), lesquerolic acid
(20:1OH) and auricolic acid (20:2OH) (Broun and Som-
erville 1997; Smith et al. 2003). This result clearly
conrmed that the red uorescent seeds have been suc-
cessfully transformed and expressed the castor FAH12
gene.
We also performed PCR analyses of DsRed positive
transgenic plants using DsRed and FAH12 specic primers.
The amplication results revealed the presence of expected
DsRed (708 bp) and FAH12 (1164 bp) bands in all trans-
genic plants tested (Fig. 3), indicating that selection was
effective.
Progeny analysis of transgenic plants
The red transgenic seeds were able to germinate in soil, and
transgenic plants displayed a normal morphology such as
leaf size and plant height compared to non-transgenic
plants grown side-by-side. Most plants produced enough
seeds for analysis. We chose ten transgenic plants to
examine the inheritance of DsRed and FAH12 genes by the
number of uorescent and normal brown seeds in the
progeny. As shown in Table 1, the chi-square goodness-of-
t tests indicated that eight lines showed the expected 3:1
ratio, suggesting a single insertion of the transgene at a
single locus in these lines. Line #32 may have multiple
copies of the transgene since most seeds were red.
Three lines that have single inserts were grown for
further investigations. Homozygous plants were obtained
in the T3 generation, as determined by the DsRed
expression. We analyzed fatty acid compositions of the T4
seeds. Table 2 showed that hydroxy fatty acids were pro-
duced in all the transgenic seeds, indicating that the FAH12
transgene was transmitted to the T4 generation.
Fig. 2 Gas chromatographic analysis of FAMEs prepared from
single mature seeds of untransformed camelina (a) and transformed
lines expressing the castor FAH12 gene (b)
Fig. 3 PCR analysis of selected transgenic plants. DsRed and FAH12
were amplied separately from plasmid, transgenic plants (corre-
sponding to lines listed in Table 1) and non-transgenic control plants.
PCR products of both DsRed and FAH12 genes from each transgenic
plant were loaded in the same wells
Table 1 Segregation of transgenic (red) and non-transgenic (brown)
seeds in the T2 lines
Line no. Red seeds Brown seeds v
2
value
a
1 123 43 0.073
2 115 45 0.833
3 111 46 1.55
4 16 6 ND
5 153 52 0.015
12 135 48 0.148
31 145 63 3.10
32 46 0 ND
b
33 88 56 14.8
b
34 143 57 1.31
ND Not determined
a
df = 1, P = 0.05 of v
2
= 3.84
b
The segregation does not t the expected 3:1 ratio
276 Plant Cell Rep (2008) 27:273278
1 3
Discussion
Camelina is an under exploited crop species but has a great
potential of economical importance. In this report, we
demonstrate that camelina can be efciently transformed
by a simple method that does not involve lengthy and
tedious tissue culture procedures. The in planta Agrobac-
terium-mediated transformation was rst developed in the
model plant Arabidopsis thaliana (Clough and Bent 1998),
and some successful attempts were achieved on other plant
species such as the oilseed crop Brassica napus (Wang
et al. 2003) and the vegetable crop radish (Curtis 2003).
These achievements provide useful means to produce
valuable germplasms for crop breeding. Our results indi-
cated that Camelina sativa is very susceptible to
Agrobacterium-mediated transformation by oral dipping
along with vacuum inltration. A camelina plant attains
height about 3090 cm tall, and has branches that may
produce several hundreds of seeds in the eld. The plants
we used were grown in the greenhouse during the summer
of 2006. These plants were only about 50 cm at maturity,
and produced limited number of branches, therefore we
could only harvest about 300 seeds from each plant.
Clipping primary inorescences to encourage branching
and produce more ower buds may increase transformation
efciency for each plant. However, it was not necessary to
pre-treat camelina plants in order to facilitate vacuum
inltration. In contrast, one has to reduce the plant height
of B. napus, and the transformation efciency was very low
(Wang et al. 2003).
The selection marker DsRed uorescent protein
remained active in desiccated mature seeds, and the light-
colored camelina seed coat does not obstruct excitation or
emission of the orescence. This marker allowed us to
effectively select transgenic seeds, and rapidly evaluate
transgenic effect on fatty acid accumulation. Our trans-
formation system would provide a useful tool to rapidly
improve many agronomic characters including seed fatty
acid proles. Also, Camelina sativa may serve as a model
(oilseed) plant for biological and biotechnological studies.
In this report, we demonstrated that novel hydroxy fatty
acids were produced in camelina oils by seed-specic
expression of a castor fatty acid hydroxylase. Our next goal
is to increase unusual hydroxy fatty acid accumulation in
camelina seeds in order to provide an economical alter-
native to castor oil for the oleochemical industry.
Acknowledgments This work was supported by the Concurrent
Technologies Corporation, and by the Biobased Products Institute of
Montana State University.
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Line No. Fatty acids (% mol)
16:0 18:0 18:1 18:2 18:3 20:0 20:1 22:1 18:1OH 18:2OH 20:1OH 20:2OH
Celine 7.3 4.5 14.4 16.8 37.8 1.6 14.3 3.3
1 8.8 9.3 20.2 10.3 13.3 5.3 15.6 2.2 7.4 5.6 1.3 0.7
3 8.5 7.3 21.6 8.5 18.9 2.8 17.3 2.4 6.0 5.6 0.9 0.4
34 8.5 6.5 21.7 14.2 16.2 2.5 16.7 1.7 6.3 5.0 0.7 ND
Data represent mean values from 36 individual plants for each line
ND Not detectable
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