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High-Stearic and High-Oleic Cottonseed Oils Produced by
Hairpin RNA-Mediated Post-Transcriptional
Gene Silencing
1
Qing Liu, Surinder P. Singh, and Allan G. Green*
Commonwealth Scientific and Industrial Research Organization Plant Industry, P.O. Box 1600, Canberra,
Australian Capitol Territory 2601, Australia
We have genetically modified the fatty acid composition of cottonseed oil using the recently developed technique of hairpin
RNA-mediated gene silencing to down-regulate the seed expression of two key fatty acid desaturase genes, ghSAD-1-
encoding stearoyl-acyl-carrier protein ⌬9-desaturase and ghFAD2-1-encoding oleoyl-phosphatidylcholine
6-desaturase.
Hairpin RNA-encoding gene constructs (HP) targeted against either ghSAD-1 or ghFAD2-1 were transformed into cotton
(Gossypium hirsutum cv Coker 315). The resulting down-regulation of the ghSAD-1 gene substantially increased stearic acid
from the normal levels of 2% to 3% up to as high as 40%, and silencing of the ghFAD2-1 gene resulted in greatly elevated
oleic acid content, up to 77% compared with about 15% in seeds of untransformed plants. In addition, palmitic acid was
significantly lowered in both high-stearic and high-oleic lines. Similar fatty acid composition phenotypes were also achieved
by transformation with conventional antisense constructs targeted against the same genes, but at much lower frequencies
than were achieved with the HP constructs. By intercrossing the high-stearic and high-oleic genotypes, it was possible to
simultaneously down-regulate both ghSAD-1 and ghFAD2-1 to the same degree as observed in the individually silenced
parental lines, demonstrating for the first time, to our knowledge, that duplex RNA-induced posttranslational gene silencing
in independent genes can be stacked without any diminution in the degree of silencing. The silencing of ghSAD-1 and/or
ghFAD2-1 to various degrees enables the development of cottonseed oils having novel combinations of palmitic, stearic,
oleic, and linoleic contents that can be used in margarines and deep frying without hydrogenation and also potentially in
high-value confectionery applications.
Although cotton (Gossypium hirsutum) is primarily
grown for fiber production, it is also the world’s sixth
largest source of vegetable oil. Cottonseed oil is typ-
ically composed of about 26% palmitic acid (C16:0),
15% oleic acid (C18:1), and 58% linoleic acid (C18:2).
The relatively high level of palmitic acid provides a
degree of stability to the oil that makes it suitable for
high-temperature frying applications, but is nutri-
tionally undesirable because of the low-density li-
poprotein cholesterol-raising properties of this satu-
rated fatty acid (Cox et al., 1995). Although
cottonseed oil has recently been shown to lower total
serum cholesterol compared with corn (Zea mays) oil
(Radcliffe et al., 2001), it did so by lowering the level
of the desirable high-density lipoprotein cholesterol
without reducing the level of the undesirable low-
density lipoprotein cholesterol, presumably because
of its significant content of palmitic acid. Further-
more, cottonseed oil is sometimes hydrogenated to
achieve the very high stability required in deep-
frying food service applications or to provide the
solidity required for margarine hard stock. Unfortu-
nately, the hydrogenation process results in the pro-
duction of trans-fatty acids, which are now recog-
nized as having cholesterol-raising properties
equivalent to those of saturated fatty acids (Ascherio
and Willett, 1997).
As a result of these factors, there is a growing trend
away from the use of oils that are rich in palmitic acid
and hydrogenated oils in favor of those that are both
nutritionally beneficial and can provide the required
functionality without hydrogenation. Oils that are
low in palmitic acid and rich in either oleic acid or
stearic acid (C18:0) meet these requirements, and
such fatty acid profiles have now been developed in
several oilseed species through genetic modification
of fatty acid synthesis. Selective breeding utilizing
natural variants or induced mutations has been used
to develop a range of improved oils in the major
temperate oilseed crops, including high-stearic (HS)
soybean (Glycine max; Graef et al., 1985), high-oleic
(HO) rapeseed (Brassica napus; Auld et al., 1992), HO
peanut (Arachis hypogaea; Norden et al., 1987), and HS
(Osorio et al., 1995) and HO (Soldatov, 1976) sun-
flower (Helianthus annuus). However, due to a lack of
any significant genetic variation for fatty acid com-
position in cottonseed oil and the allotetraploid na-
ture of cultivated cotton, classical breeding tech-
niques and induced mutagenesis have so far been
unsuccessful in developing improved cottonseed oil.
1
This work was supported by the Australian Cotton Research
and Development Corporation (grant no. CSP–78C).
* Corresponding author; e-mail allan.green@csiro.au; fax
61–2–62465000.
Article, publication date, and citation information can be found
at www.plantphysiol.org/cgi/doi/10.1104/pp.001933.
1732 Plant Physiology, August 2002, Vol. 129, pp. 1732–1743, www.plantphysiol.org © 2002 American Society of Plant Biologists
To overcome the limitations of conventional breed-
ing approaches, genetic engineering techniques have
now been successfully employed to modify the fatty
acid composition in a number of oilseed crops. In
particular, posttranslational gene silencing (PTGS) has
been used to down-regulate the activity of the desatu-
rase enzymes that control the synthesis of the major
seed oil fatty acids, principally stearoyl-acyl-carrier
protein (ACP) ⌬9-desaturase, which converts stearic
acid into oleic acid, and oleoyl-phosphatidylcholine
(PC)
6-desaturase, which converts oleic acid into li-
noleic acid. For example, stearic acid was raised to
around 40% in rapeseed oil through antisense-
mediated down-regulation of the stearoyl-ACP ⌬9-
desaturase activity (Knutzon et al., 1992), and very
high levels of oleic acid have been achieved in both
soybean and rapeseed through seed-specific cosup-
pression of oleoyl-PC
6-desaturase (Kinney, 1996).
However, the antisense and cosuppression strategies
used in these cases have variable and unpredictable
effectiveness and generally require the production of
large populations of transgenic plants to obtain an
acceptable number of lines exhibiting sufficient de-
grees of target gene suppression (Knutzon et al., 1992;
Kinney, 1996; Hamilton et al., 1998). This presents a
particular problem for their application in cottonseed
oil improvement because cotton is still relatively dif-
ficult to transform and requires long periods of time in
tissue culture for regeneration (Cousins et al., 1991;
Murray et al., 1999).
Recently, the discovery that RNA interference oc-
curs in plants and is mediated by sequence-specific
degradation of dsRNA has led to the development of
highly efficient methods of silencing plant genes
(Smith et al., 2000). Specifically designed genetic con-
structs, such as inverted repeats that encode RNA
having regions of self-complementarity, can reliably
generate hairpin RNA (hpRNA) transcripts that in-
voke sequence-specific RNA degradation targeted to
the double-stranded region of the hpRNA and to
homologous endogenous mRNA molecules. By uti-
lizing a partial sequence of an endogenous gene in
the inverted repeat regions of the silencing construct,
high-level silencing of the target gene expression can
be achieved. We have applied hpRNA-mediated
gene-silencing techniques to modify the expression
of the ⌬12-desaturase gene (FAD2) in Arabidopsis
seeds and have demonstrated that they result in much
higher efficiency and efficacy of gene silencing than
either antisense or cosuppression (Stoutjesdijk et al.,
2002). Such high-efficiency gene-silencing techniques
have now made it practicable to attempt genetic mod-
ification of fatty acid composition of cottonseed oil.
The ghSAD-1 and ghFAD2-1 genes in cotton, respec-
tively, encode stearoyl-ACP ⌬9-desaturase (Liu et al.,
1996) and microsomal
6-desaturase (Liu et al.,
1999b), also referred to as ⌬12-desaturase, which are
the key enzymes determining the fatty acid composi-
tion of cottonseed oil (Fig. 1). Here, we report the use
of hpRNA gene-silencing constructs to achieve seed-
specific silencing of both ghSAD-1 and ghFAD2-1 re-
sulting in the development of HS and HO cottonseed
oils, including molecular analysis of target gene ex-
pression, and comparisons of phenotypic patterns ob-
served in a range of independently derived transgenic
lines. Furthermore, we demonstrate the stable inheri-
tance of these phenotypes in progeny derived by ei-
ther selfing or intercrossing, and the generation of
further novel fatty acid compositions in hybrid prog-
eny expressing both HO and HS traits.
RESULTS
Identification of Transgenic Plants
Cotton cv Coker 315 was transformed with gene-
silencing constructs consisting of the ghSAD-1 or
ghFAD2-1 cDNA clone in either inverted repeat or
antisense configurations driven seed specifically by a
soybean lectin promoter (Fig. 2). Thirty-four and 25
Figure 1. Diagramatic representation of fatty acid biosynthetic path-
way in cottonseed.
Hairpin RNA-Mediated Silencing of Fatty Acid Desaturases in Cotton
Plant Physiol. Vol. 129, 2002 1733
fertile plants were established from kanamycin-
resistant calli for the ⌬9-HP and ⌬9-AS constructs,
respectively. Thirty-six and 27 fertile plants were
obtained by transformation with constructs ⌬12-HP
and ⌬ 12-AS, respectively. No obvious difference was
observed in terms of callus induction, somatic em-
bryogenesis, and establishment of fertile plants
among the four transformations. Transgenic status of
the regenerants was confirmed by PCR amplification
of the soybean lectin 3⬘ terminator DNA fragment
from genomic DNA.
Southern-blot analysis of genomic DNA was used
to confirm the integration of the transgene into the
cotton genome and to estimate the transgene copy
number. The Southern blots were performed with
HindIII-digested genomic DNA from the leaf tissue
of transgenic plants, and probed with a radiolabeled
DNA fragment consisting of the promoter region of
the soybean lectin gene. HindIII cuts once within the
T-DNA of the ⌬12 constructs and twice within the ⌬9
constructs, in each case downstream of the lectin
promoter. Probing of the Southern blots with the
lectin promoter identified uniquely sized fragments
that spanned the junction of the T-DNA to the adja-
cent genomic DNA in each transformation event,
verifying that the individual transgenic lines each
originated from independent transformation events.
A number of representative transgenic lines are
shown in Figure 3. Bands of different sizes were
interpreted to represent different transgene inser-
tions and demonstrated that several transgenic lines
contained multiple numbers of insertions (Fig. 3). In
the case of transgenic lines with multiple 5⬘ lectin
bands, different signal intensities were frequently
observed among these bands on the same lane. The
high-intensity bands may harbor multiple fragments
of similar sizes. Although highly variable among the
four individual transformations, in each case it is
clear that there are a significantly high proportion of
transgenic lines containing only a single insertion
locus (Table I).
Silencing of ⌬12-Desaturase Results in HO
Cottonseed Oil
The parental variety Coker 315 has a consistently
HO desaturation proportion (ODP) value ranging
from 0.80 to 0.85 (Fig. 4A), meaning that over 80% of
oleic acid produced in the developing seed is subse-
quently converted to linoleic acid by the action of the
oleoyl-PC ⌬12-desaturase enzyme. Many of the T
1
plants carrying the ⌬12-HP and ⌬12-AS constructs
showed a considerable reduction in ODP value,
down to as low as 0.04, indicating a profound (95%)
down-regulation of ⌬12-desaturase activity. The HP
construct was more effective than the AS construct in
achieving the down-regulation of ⌬ 12-desaturase ac-
tivity, with 18 of 34 (53%) of the ⌬12-HP T
1
plants
showing a reduction in ODP, compared with 10 of 27
(37%) for the ⌬12-AS T
1
plants.
The pattern of segregation for the ⌬12-silencing
trait was examined by analyzing fatty acid composi-
tion of individual selfed T
2
seeds borne on 15 of the
T
1
plants. An average of 35 T
2
seeds were analyzed
from each of the selected T
1
plants carrying the
⌬12-HP construct. Although Southern-blot analysis
(Fig. 3) showed the presence of single transgene in-
sertions in some lines (for example, ⌬12-HP*23 and
⌬12-HP*124) and multiple insertions in others (for
example, ⌬12-HP*83 and ⌬12-HP*128), in all cases
the ratio of low ODP (⌬12 silenced) to normal ODP
fitted a 3:1 ratio when tested using the Chi-squared
goodness of fit parameter, indicating a single gene
inheritance. However, two distinct segregation pat-
terns were evident and are typified by the examples
shown in Figure 4, B and C. In 11 of 15 lines exam-
ined, typified by line ⌬12-HP*23 (Fig. 4B), there was
a very high level of silencing of the ⌬12-desaturase in
Figure 2. Schematic diagram of the chimeric
silencing constructs transformed into cotton.
The ghSAD-1 and ghFAD2-1 genes in either
inverted repeat (HP) or antisense (AS) orienta-
tion were placed under the control of the seed-
specific soybean lectin promoter (Lec-P) and
terminator (Lec-T). The neomycin phosphotrans-
ferase selectable marker gene (NptII) was driven
by the Nos promoter (Nos-P). LB and RB corre-
spond to the T-DNA left and right borders, re-
spectively. The positions of EcoRI and HindIII
restriction enzyme sites are indicated.
Liu et al.
1734 Plant Physiol. Vol. 129, 2002
all T
2
seeds carrying the transgene, indicating that
the low ODP phenotype was inherited as a com-
pletely dominant trait. In contrast to this pattern, the
distribution for ODP in the T
2
seeds from the other
four T
1
plants, as typified by line ⌬12-HP*72 (Fig.
4C), revealed a more intermediate level of silencing
and a continuous distribution between the wild-type
value and that of the most silenced seed. The very
wide spread of ODP values in the silenced class
suggests that either the expression of this silencing
was highly variable, or that there were phenotypic
differences between transgene hemizygotes and ho-
mozygotes. Progeny testing would be necessary to
resolve these possibilities.
T
2
seeds were also analyzed from three self-
fertilized ⌬12-AS lines that showed distinct patterns
of ⌬12-desaturase down-regulation. For line ⌬12-
AS*132 (Fig. 4D), about one-quarter of the T
2
seeds
showed very high-level silencing of ⌬12-desaturase,
equivalent to the best silencing from the ⌬12-HP
lines, with the remaining seeds displaying the wild-
type phenotype. Such a 1:3 ratio suggests that silenc-
ing was only effective when the transgene was in the
homozygous state, with hemizygotes showing little
or no silencing. This type of segregation pattern was
also evident in line ⌬12-AS*126 (Fig. 4E), but the
degree of silencing was much lower in this case. In
the third line, ⌬12-AS*86 (Fig. 4F), there was moder-
ate to high silencing in all 30 T
2
seeds with no wild-
type phenotypes being observed. This pattern would
be consistent with the control of silencing by multiple
independent copies of the transgene, where the pop-
ulation size may have been insufficient to recover the
expected low frequency of null genotypes.
As expected, the pronounced silencing of ⌬12-
desaturase resulted in large reductions in linoleic
acid and concomitant increases in oleic acid (Table
II), regardless of whether the transgene was ⌬12-HP
or ⌬12-AS. In the most extreme case of silencing
using the ⌬12-HP construct (line ⌬12-HP*23), oleic
acid was increased from the normal level of 13% up
to 78%, and correspondingly the level of linoleic acid
reduced from normally 59% down to 4%. Interest-
ingly, palmitic acid was also significantly reduced,
down from 26% to 15%. Stearic acid is only present in
very small amounts and was unchanged in the ⌬12-
desaturase-silenced lines.
Silencing of ⌬9-Desaturase Results in HS
Cottonseed Oil
⌬9-desaturation is extremely active in developing
cotton seeds, with around 96% of stearic acid formed
during seed lipid synthesis being desaturated, ini-
tially to oleic acid, in cotton cv Coker 315 and only
2% to 3% of stearic acid remaining in the seed oil at
maturity. As was the case for ⌬12-desaturase, the
⌬9-HP construct was more effective than the ⌬9-AS
construct in silencing the target ⌬9-desaturase gene.
For the ⌬9-HP construct, 18 of 29 T
1
plants (62%)
showed reductions in stearic desaturation proportion
(SDP), whereas only 6 of 25 (24%) of the ⌬9-AS lines
were reduced. However, the degree of silencing
achieved was considerably less for ⌬ 9-desaturase
than was the case for ⌬12-desaturase. SDP was re-
duced from 0.96 in cotton cv Coker 315 down to 0.52
Figure 3. Southern hybridization analysis of nine representative T
1
transgenic lines harboring silencing transgenes. From left: lane 1,
⌬9-HP*6; lane 2, ⌬9-HP*51; lane 3, ⌬9-HP*62; lane 4, ⌬9-HP*150;
lane 5, ⌬9-AS*118; lane 6, ⌬12-HP*23; lane 7, ⌬12-HP*83; lane 8,
⌬12-HP*128; and lane 9, ⌬12-HP*124. Genomic DNA from each
line was digested with HindIII and probed with the whole promoter
fragment of the soybean lectin gene. The bands represent the trans-
gene insertions. The migration of DNA size markers is shown on the
left in kb.
Table I. Nos. of transgenic plants produced using gene-silencing
constructs and estimated nos. of transgene insertions
Transgene
Construct
No. of Confirmed
Transgenic
No. of Transgenic Plants
Having Transgene Copy No.
12⬎2
⌬9-HP 29 13 3 13
⌬9-AS 25 9 4 12
⌬12-HP 34 16 7 11
⌬12-AS 27 10 4 13
Hairpin RNA-Mediated Silencing of Fatty Acid Desaturases in Cotton
Plant Physiol. Vol. 129, 2002 1735
in the most silenced individual seed, representing
only a halving of ⌬9-desaturase activity compared
with the over 95% reduction that was achieved for
⌬12-desaturase using the same hpRNA strategy.
The pattern of segregation for the modified ⌬9-
desaturation trait was examined by analyzing fatty
acid composition of individual T
2
seeds borne on a
range of self-fertilized T
1
plants. An average of 48 T
2
seeds were analyzed from each of 10 T
1
plants car-
rying the ⌬9-HP construct. Segregation patterns for
five of these lines that typify the overall results are
shown in Figure 5. Three of the T
1
plants had indi-
vidual T
2
seeds that showed relatively high degrees
of silencing. In each of these lines, the class of seed
having reduced ⌬9-desaturation (mid-SDP) was dis-
tinct from that having the wild-type (high-SDP) phe-
notype. In the case of line ⌬9-HP*150 (Fig. 5B), the
ratio of mid-SDP:high-SDP fitted a 3:1 ratio expected
for the segregation of a single locus trait, even though
Southern-blot analysis revealed the presence of at
least six copies of the transgene (Fig. 3, lane 4). The
distribution of SDP for line ⌬9-HP*62 (Fig. 5C)
closely fitted a 15:1 ratio, suggesting the segregation
of two independent silencing loci, with Southern-blot
analysis indicating three to four copies of the trans-
gene (Fig. 3, lane 3). In contrast, the distribution of
SDP in line ⌬9-HP*37 (Fig. 5D) did not fit any pre-
dicted ratio, there being an excess of wild-type indi-
viduals compared with that expected with even a
single locus segregation. The distribution within the
mid-SDP classes was continuous in each of these
three lines and did not enable any putative discrim-
ination of transgene hemizygotes and homozygotes.
In the six other T
1
lines, exemplified by ⌬9-HP*6
(Fig. 5E) and ⌬9-HP*72 (Fig. 5F), the degree of ⌬9-
desaturase silencing was relatively minor. Although
in each case the distribution of SDP was continuous,
cleavage at an SDP value of 0.95, based on the cotton
cv Coker 315 wild type having a consistently higher
SDP value, resulted in the segregation of silenced to
wild-type individuals that fitted a 3:1 ratio in five of
the six lines. For the remaining line, the distribution
of SDP was continuous with only very minor reduc-
tions in SDP evident. None of the ⌬9-AS lines were
examined for segregation of the trait among T
2
seeds.
The partial silencing of ⌬9-desaturase resulted in
significant increases in stearic acid and concomitant
decreases in both oleic and linoleic acids (Table II) in
the ⌬9-HP and ⌬9-AS lines. In line ⌬9-HP*150, one of
the most extreme cases of silencing using the ⌬9-HP
construct, stearic acid was raised from normal levels
of 2% up to 40%, with oleic acid being reduced from
Figure 4. Frequency distribution for ODP in individual seeds of
cotton cv Coker 315 (A), and T
2
seeds from ⌬12-desaturase-silenced
lines ⌬12-HP*23 (B), ⌬12-HP*72 (C), ⌬12-AS*132 (D), ⌬12-AS*126
(E), and ⌬12-AS*86 (F).
Table II. Fatty acid composition, SDP, and ODP values for Coker 315, the highest stearic acid seed
obtained by silencing ghSAD-1 (from line ⌬9-HP*150), the highest oleic acid seed obtained by si-
lencing ghFAD2-1 (from line ⌬12-HP*23), and putative homozygous recombinant F
2
seed from the
cross ⌬9-HP*150 ⫻⌬12-HP*23
Line
Fatty Acid Composition
SDP ODP
Palmitic
(16:0)
Stearic
(18:0)
Oleic
(18:1)
Linoleic
(18:2)
Linolenic
(18:3)
Arachidic
(20:0)
%
Coker 315 25.6 2.3 13.2 58.5 0.1 0.3 0.97 0.82
⌬9-HP*150 14.9 39.8 3.8 38.8 0.2 2.4 0.52 0.91
⌬12-HP*23 15.3 2.3 78.2 3.7 0.1 0.3 0.97 0.05
F
2
13.7 39.9 37.4 6.0 0.6 2.4 0.52 0.15
Liu et al.
1736 Plant Physiol. Vol. 129, 2002
13% to 4% and linoleic acid down from 59% to 39%.
As was the case with ⌬12-desaturase silencing,
palmitic acid was also significantly reduced, down
from 26% to 15%.
Reduced ODP and SDP Is Seed Specific and
Associated with Reduced Desaturase mRNA Levels
Expression levels of the targeted ghSAD-1 and
ghFAD2-1 genes were examined by northern-blot
analysis of RNA isolated from developing seeds. We
have previously demonstrated that ghSAD-1 (Liu et
al., 1996) and ghFAD2-1 (Liu et al., 1999b) are highly
expressed in the developing cotton embryos, con-
comitant to the accumulation of storage lipids, with
the highest expression at mid-maturation stages.
RNAs originating from pooled samples of develop-
ing T
2
seeds from ⌬9-HP*62 and ⌬12-HP*23 (Fig. 6
,
lanes 2 and 3, respectively) were probed with the
ghSAD-1 or ghFAD2-1 cDNA clones at high strin-
gency. The ⌬9- and ⌬12-silenced lines showed drastic
reductions in ghSAD-1 and ghFAD2-1 mRNA levels,
respectively, compared with the Coker 315 control
(Fig. 6, lane 1), but each line had normal levels of
mRNA for the desaturase that was not targeted by
the silencing transgene. The sharp contrast in expres-
sion levels of the respective targeted desaturase
genes compared with those in the untransformed
cotton cv Coker 315 clearly indicates that the drastic
reduction in SDP and ODP levels in the transgenic
lines is associated with substantially lowered levels
of each transcript. Furthermore, analysis of leaf lipids
in a number of ⌬9-HP and ⌬12-HP lines (data not
shown) demonstrated that they were identical to
those of Coker 315, suggesting that the gene silencing
was restricted to the developing seeds as expected
from reporter gene studies using the soybean lectin
promoter (Townsend and Llewellyn, 2002).
Combination of HO and HS Traits by Crossing
⌬12-HP and ⌬9-HP Lines
The possibility of combining the silencing of the
⌬9-desaturase and ⌬12-desaturase genes into a single
line to generate further variability for fatty acid com-
position was examined initially by intercrossing the
⌬9-HP*150 and ⌬12-HP*23 T
1
plants. Based on
Southern-blot analysis and within-line trait segrega-
tion, the ⌬12-HP*23 parent contains only one copy of
the ⌬12-silencing transgene, whereas the ⌬9-HP*150
parent appears to contain at least six copies of the
⌬9-silencing transgene (Fig. 3), of which only one
copy seems to be contributing to the ⌬9-desaturase
silencing. Because the hybridization was performed
using hemizygous T
1
plants as parents, the F
1
seeds
are expected to segregate for the transgenes. Among
36 F
1
seeds analyzed nondestructively for fatty acid
composition, 12 seeds had levels of stearic and oleic
acids similar to wild type, 10 seeds had elevated
Figure 5. Frequency distribution for SDP in individual seeds of cot-
ton cv Coker 315 (A), and T
2
seeds from ⌬9-desaturase-silenced lines
⌬9-HP*150 (B), ⌬9-HP*62 (C), ⌬9-HP*37 (D), ⌬9-HP*6 (E), and
⌬9-HP*72 (F).
Figure 6. Northern-blot analyses of ghSAD-1- (A), ghFAD2-1- (B),
and ghKASII- (C) specific RNAs in developing embryos of transgenic
cotton and control. Samples were extracted from mid-maturation
embryos (approximately 30 d after fertilization) from untransformed
cotton cv Coker 315 (lane 1), T
2
seeds of ⌬9-HP*62 (lane 2) and
⌬12-HP*23 (lane 3), and F
2
seeds of the cross ⌬9-HP*150 ⫻⌬12-
HP*23 (lane 4).
Hairpin RNA-Mediated Silencing of Fatty Acid Desaturases in Cotton
Plant Physiol. Vol. 129, 2002 1737
stearic acid alone, seven seeds had elevated oleic acid
alone, and seven seeds had elevated levels of both
stearic and oleic acids, indicating that they carried
both ⌬9- and ⌬12-silencing transgenes. One F
1
plant
was established from this latter class and 199 indi-
vidual F
2
seeds borne on this plant were analyzed for
fatty acid composition to determine the phenotypes
of the transgene recombinants. The plots of SDP
against ODP (Fig. 7A) and stearic acid against oleic
acid content (Fig. 7B) clearly show the presence of
four classes representing the combinations of wild-
type and silenced phenotypes for each of the two
targeted desaturases. The ratio of silenced to wild
type for the ⌬12-desaturase (140:59) and for the ⌬9-
desaturase (152:47) in each case fitted a 3:1 ratio
expected for the segregation of a single dominant
gene (
2
1
⫽ 2.29 and 0.20, respectively). The joint
segregation of the two traits also closely fitted the
pattern expected for two independently segregating
genes (
2
1
⫽ 1.15).
Among the F
2
seeds, the pattern of silencing for the
individual ⌬9 and ⌬12 desaturases was similar to
that observed among the T
2
seeds of the parental
lines. Within the ⌬12-silenced genotypes, variation
for ODP was relatively narrow, ranging only be-
tween 0.04 and 0.13 in the ⌬9 wild-type class and
mainly between 0.07 and 0.24 in the ⌬9-silenced class.
In contrast, variation for SDP within the ⌬9-silenced
genotypes was very broad, ranging from 0.46 to 0.84
in the ⌬12 wild-type class and between 0.52 and 0.88
in the ⌬12-silenced class. This was consistent with the
relative variation observed for ODP and SDP, respec-
tively, in the T
2
seeds of the parental lines, ⌬12-HP*23
(Fig. 4B) and ⌬9-HP*150 (Fig. 5B). The combined
effects of the ⌬9- and ⌬12-desaturase silencing re-
sulted in a wide range of novel combinations of
stearic acid and oleic acid levels in the F
2
seeds
carrying both transgenes. There was a strong nega-
tive correlation between oleic acid and stearic acid
levels, with the extreme types having on the one
hand 10% stearic and 65% oleic, and on the other
hand 40% stearic and 38% oleic, reflecting the pre-
cursor/product relationship of these two fatty acids.
Because the two silencing transgenes are segregating
independently, it is expected that 1 of 16 of the F
2
seeds are homozygous for both the ⌬9- and ⌬12-
silencing transgenes. These are most likely to be the
seeds having around 30% to 35% stearic and 40% to
45% oleic acid, although progeny testing would be
necessary to confirm the genotypes of the individual
seeds. As was the case in both parental lines, palmitic
acid was around 10% lower in the lines carrying both
silencing constructs compared with the wild-type ge-
notypes, being as low as 12% of total fatty acids in the
putative group of double homozygous F
2
individu
-
als. Northern-blot analysis using the RNA extracted
from the developing F
2
embryos confirmed the si
-
multaneous silencing of ghSAD-1 and ghFAD2-1 (Fig.
6, A and B, lane 4).
In some of the initial transgenic lines and recombi-
nants expressing the HS trait, we observed that some
HS seeds, although germinating well at room tem-
perature on water-soaked filter paper, were slow to
establish and grow when transferred to soil. By ana-
lyzing fatty acid composition of a small proportion of
the seed and leaving the rest to germinate in soil, we
observed a correlation between high levels of stearic
acid and reduced survival ability of seedlings (data
not shown). However, germination and growth of HS
seeds was increased significantly when they were
germinated and grown at approximately 5°C higher
Figure 7. Joint segregation of ODP with SDP (A), and oleic acid with
stearic acid (B), in F
2
seed populations from the cross ⌬9-HP*150 ⫻
⌬12-HP*23. Seeds are classified as typical of the ⌬9-HP*150 HS
parent (‚), the ⌬12-HP*23 HO parent (F), recombinant HO and HS
(E), and wild-type cotton cv Coker 315 (Œ).
Liu et al.
1738 Plant Physiol. Vol. 129, 2002
temperature. Furthermore, all HS seeds gave rise to
viable plants when they were germinated and estab-
lished on Suc-supplemented tissue culture media. No
germination difficulties were observed with any of
the seeds expressing only the HO trait.
DISCUSSION
We have recently demonstrated in Arabidopsis
that inverted repeat gene-silencing constructs are
more efficient than either antisense or cosuppression
in down-regulating expression of the FAD2 gene en-
coding ⌬12-desaturase, both in terms of higher fre-
quency of transgenics showing silencing, and gener-
ally higher degrees of silencing, particularly in
heterozygotes (Stoutjesdijk et al., 2002). Those obser-
vations are now further supported by the current
results from cotton where inverted repeat constructs
targeted against either the FAD2-1 or the SAD-1 gene
resulted in substantially higher recovery of silenced
individuals than did the corresponding antisense
constructs, and by our recent demonstration that the
inclusion of an intron in the inverted repeat con-
structs targeted against the FAD2 gene results in
100% efficiency of silencing (Wesley et al., 2001). The
high efficiency of gene silencing obtainable through
the use of hpRNA-mediated PTGS makes this tech-
nique a valuable contribution to practical trait mod-
ification in agricultural plants. This is particularly
important for those plants, such as cotton, that have
relatively low transformation efficiency, or where it
is required to have larger transgenic populations to
obtain selectable marker free plants by segregation.
Our results demonstrate that hpRNA constructs
targeted specifically against the ghFAD2-1 gene can
almost completely silence ⌬12-desaturase activity in
developing cottonseed (ODP reduced to approxi-
mately 6% of normal levels), apparently without im-
pairing normal seed development or subsequent ger-
mination and plant growth. Because of its tetraploid
origin, two highly homologous copies (98% DNA
sequence similarity) of the ghFAD2-1 gene exist in
cotton, one in each of the A and D subgenomes, and
appear to contribute equally to the abundance of
ghFAD2-1 mRNA in the developing cottonseeds (Liu
et al., 1999b). Northern-blot analysis showed the
presence of only very low levels of ghFAD2-1 mRNA
during the period of rapid fatty acid synthesis in
developing seeds of the most highly silenced ⌬12-HP
transgenic line, confirming the expectation that
mRNA transcripts from both subgenomic copies of
FAD2-1 would be targeted for degradation. Interest-
ingly, the ghFAD2-1-silencing trait was inherited as a
single dominant gene in several of the highly si-
lenced inverted repeat lines, indicating that a single
copy of the silencing transgene can be sufficient to
achieve maximum suppression of the target
ghFAD2-1 gene. This feature was more pronounced
in the current cotton experiments than was the case
with FAD2 silencing in Arabidopsis (Stoutjesdijk et
al., 2002), where hemizygotes for highly effective
silencing transgenes approached, but generally did
not equal, the degree of silencing achieved in ho-
mozygotes. This difference may indicate that the tim-
ing and degree of expression of the lectin-driven
silencing transgene was a more effective fit to the
expression pattern of the targeted endogenous
FAD2-1 gene in cotton than was the case with the
napin-driven transgene in Arabidopsis. The pro-
found reduction in ⌬ 12-desaturase activity achieved
through hpRNA-mediated silencing of the ghFAD2-1
gene is considerably greater than that recently re-
ported in cottonseed by Chapman et al. (2001), where
transformation with a full-length, but nonfunctional,
rapeseed FAD2 sequence in sense orientation
achieved only a halving of ⌬12-desaturation, with
substantial amounts of linoleic acid (approximately
28%) remaining in the most extreme lines. In that
case, the introduced sequence has relatively low ho-
mology to the cotton FAD2 genes and it seems prob-
able that the reduction of ⌬12-desaturation is the
consequence of a phenomenon other than PTGS.
It is notable that even in the most highly ⌬12-
silenced lines, there is still a small amount of linoleic
acid (approximately 3%) that accumulates in the oil,
reflecting a low but significant residual level of ⌬12-
desaturase activity. This is almost identical to our
experience with silencing of the equivalent FAD2
gene in Arabidopsis using hpRNA-mediated PTGS
(Stoutjesdijk et al., 2002), where it seemed likely that
the residual ⌬ 12-desaturase activity is encoded by
other divergent ⌬12-desaturase genes that are not
effectively targeted by the silencing construct, pre-
sumably the FAD6 gene encoding a ⌬12-desaturase
that acts on oleic acid bound to glycerolipid sub-
strates in the plastid. It is possible that such FAD6-
mediated ⌬12-desaturation may also account for
some of the residual linoleic acid present in the
FAD2-silenced cotton lines. In addition, whereas
Arabidopsis has only a single FAD2 gene, cotton has
been shown by Southern-blot analysis to have at least
five copies (Liu et al., 1999b). Although the ghFAD2-1
member of this gene family appeared to be the major
contributor to the desaturation of oleic acid in the
seed oil, it is not known how much contribution the
other members make. We have isolated one other
member, ghFAD2-2 (Liu et al., 1999a), which appears
to be expressed only at low levels throughout the
plant, including in the seeds. Similar to the situation
in soybean (Heppard et al., 1996), this constitutively
expressed ghFAD2-2 sequence has only about 70%
identity with ghFAD2-1, which is probably a suffi-
cient mismatch for ghFAD2-2 mRNA to escape sig-
nificant homology-dependent silencing in the
ghFAD2-1-targeted lines and to perhaps contribute to
the residual ⌬12-desaturase activity in their seeds.
In contrast to the very high-level silencing of ⌬12-
desaturase, only intermediate down-regulation of
Hairpin RNA-Mediated Silencing of Fatty Acid Desaturases in Cotton
Plant Physiol. Vol. 129, 2002 1739
⌬9-desaturase was achieved in the current study. The
most highly silenced ⌬9-HP line recovered still accu-
mulated about 43% of C18 unsaturated fatty acids,
indicating that ⌬9-desaturation had only been halved
by the ghSAD-1-silencing transgene. The absence of
high-level reductions in ⌬9-desaturase activity in cot-
tonseed, even with inverted repeat gene-silencing
techniques, accords with previous attempts to down-
regulate this enzyme in rapeseed using antisense
techniques (Knutzon et al., 1992), where stearic acid
was also raised only up to 40%. This common expe-
rience possibly reflects the essential role that ⌬9-
desaturase plays in cellular lipid synthesis. All C18
unsaturated fatty acids present in plant cells,
whether in plastidic or microsomal membranes or
deposited as triacylglycerols in oleosomes, originate
from the desaturation of stearoyl-ACP in the plastid
by the stearoyl-ACP ⌬9-desaturase. Complete re-
moval of this enzyme activity would leave cells with-
out the ability to synthesize any C18 unsaturated
fatty acids and impair their ability to appropriately
manipulate membrane fluidity (Lightner et al., 1994).
This essential nature of stearoyl-ACP ⌬9-desaturase
may have been a factor favoring the evolutionary
development of the multigene families that encode it
in many plant species. For example, even though the
small genome of Arabidopsis has only one FAD2
gene encoding ⌬ 12-desaturase, it contains at least
five genes encoding stearoyl-ACP ⌬9-desaturase
(Ohlrogge and Jaworski, 1997). Similarly, Southern-
blot analysis in cotton has shown that the ⌬9-
desaturase multigene family consists of six to eight
members per diploid genome (Liu et al., 1996; Liu,
1998) and we have recently isolated at least four
cDNA clones with unique 3⬘-untranslated regions
(UTRs) from a cottonseed cDNA library (Q. Liu, un-
published data).
At present, neither the degree of sequence diversity
between the individual SAD genes, nor their relative
contribution to the overall ⌬9-desaturation activity in
the developing cottonseed storage lipids, are known.
Therefore, it is not possible to predict how much the
expression of other SAD genes is likely to be affected
by the ghSAD-1-silencing construct. It might be the
case that the SAD gene family has high levels of se-
quence homology and is capable of being globally
silenced by a ghSAD-1-silencing construct. If so, highly
expressed ghSAD-1-silencing transgenics accumulat-
ing very high levels of stearic acid might not be recov-
ered due to lethality resulting from either inadequate
levels of C18 unsaturated fatty acids in their mem-
brane lipids or inability of transgenic embryoids to
mobilize the HS lipids to support germination. In such
a case, it might be expected that only those transgenics
with weakly expressing silencing transgenes, and
consequently intermediate levels of stearic acid and
essential C18 unsaturated fatty acids, might be recov-
ered. However, in the current study we did not ob-
serve any substantially lower recovery rates from
transformations involving the ⌬9-desaturase-silencing
constructs compared with those involving the ⌬12-
desaturase constructs. Furthermore, the highest stearic
line had a dramatic reduction in ghSAD-1 mRNA, in-
dicative of high-level silencing of that gene. Therefore,
it appears more likely that the significant remaining
⌬9-desaturation activity in the HS lines is due to other
SAD genes escaping silencing, rather than a global but
weak silencing of all members of this gene family.
Assuming that it is possible to use PTGS to achieve
greater reductions in ⌬9-desaturase activity in cot-
tonseed than were obtained in the current study,
there may still be physiological limitations to the
level of stearic acid that can ultimately be attained. In
the present study, seed germination and seedling
establishment were impaired in some of the HS cot-
ton lines, but not in any of the HO lines, even though
initial indications are that both types had apparently
normal oil content (data not shown). This problem
was readily overcome by germinating the HS seeds
on Suc-containing medium, suggesting that it was
caused by poor ability to mobilize the altered seed oil
as an energy source. Similar germination problems
have been reported in HS mutants of soybean (Rah-
man et al., 1997) and HS genotypes of Brassica spp.
produced by antisense-mediated down-regulation of
stearoyl-ACP ⌬9 desaturase (Knutzon et al., 1992).
The inability of cottonseeds to effectively utilize HS
seed oils during germination could operate through a
number of mechanisms. Future studies comparing
germination of cottonseeds having various fatty acid
modifications, and analyzing the fatty acid structure
of triglycerides and other cellular lipids in the HS
cottonseed lines, should provide further insight into
this issue.
As expected, the principal effect of silencing ⌬9-
and ⌬12-desaturase was to alter the relative propor-
tions of the C18 fatty acids—stearic, oleic, and lino-
leic acids—by decreasing the levels of the fatty acids
downstream of the relevant enzyme and increasing
the levels of the immediate fatty acid substrate. How-
ever, palmitic acid was also significantly reduced in
the HS and HO lines, as well as in the lines carrying
both traits. One possible explanation for this may be
that the accumulation of oleic acid in the ⌬12-
silenced lines and stearic acid in the ⌬9-silenced lines
results in increased levels of oleoyl-CoA and
stearoyl-CoA, respectively, in the cytoplasm and that
this alters the relative selectivity of acyl-transferases
responsible for the movement of the fatty acids into
triglycerides in a manner that reduces the incorpora-
tion of palmitate. The concentrations of minor fatty
acids were relatively unaltered in the ⌬9- and ⌬12-
silenced lines. In particular, the levels of the cyclo-
propenoid fatty acids, malvalic acid and sterculic
acid, remained low (⬍1%) in oil extracts taken from
bulked HS or HO lines (data not shown). Thus, de-
spite the large increase in oleic acid in the ⌬12-
silenced lines, there was no evidence of increased
Liu et al.
1740 Plant Physiol. Vol. 129, 2002
production of the cyclopropenoid fatty acids that are
synthesized from oleic acid. This observation is con-
sistent with the theory that cyclopropenoid fatty acid
synthesis occurs mainly in the embryo axis of cotton-
seed (Wood, 1986), where it would be spatially sep-
arated from the highly enriched cotyledonary oleic
acid pool in the ⌬12-silenced lines. Ultimately, it will
be interesting to examine the levels of cycloprope-
noid fatty acids in the embryo axis lipids of the very
HO lines to determine whether they are substantially
altered as a result of the increased level of oleic acid.
The alterations to fatty acid composition of cotton-
seed oil achieved using PTGS should enable the de-
velopment of a range of cottonseed oils that better
match current end-use requirements. In particular,
the HO cottonseed oil containing predominantly
oleic acid (75%) and palmitic acid (15%) is expected
to be even more stable than the HO forms of other
oilseeds such as soybean, rapeseed, and sunflower,
and should be usable in long-life deep-frying appli-
cations, such as in the food service and snack food
sectors, without the need for hydrogenation and the
associated production of nutritionally undesirable
trans-fatty acids. Similarly, the HS cottonseed oil
should prove suitable for solid fat applications, such
as margarines and shortenings, without hydrogena-
tion. Although it may be possible to further reduce
linoleic acid below the 4% present in the best HO
lines, such a change may not be advantageous be-
cause there is evidence from studies with midoleic
genotypes of rapeseed (Xu et al., 2000) that a modest
level of linoleic acid in the oil is desirable from a
flavor standpoint, and should not be sacrificed for
the minimal further improvement in stability that
would result from its complete removal. In fact, it
may even be desirable to select HO lines that have
slightly higher levels of linoleic acid to produce an
optimal cooking oil.
The relatively high level of palmitic acid naturally
present in cottonseed oil has been an important con-
tributor to the stability of the oil and to the solidity of
its hydrogenated derivatives, but is nutritionally un-
desirable. Because the increases in oleic acid or
stearic acid are able to impart the required functional
properties on the modified oils, it should now be
possible to dramatically lower palmitic acid in cot-
tonseed oil without compromising performance.
Both the HO and HS oils developed in the current
study already have a significant reduction in palmitic
acid, and thereby enhanced nutritional value. Further
reductions in palmitic acid should be possible in both
the HO and HS oils through genetic manipulation of
the enzymes controlling palmitic acid synthesis, in
particular palmitoyl-ACP thioesterase and keto-acyl
synthase II.
The HO and HS characteristics behaved as inde-
pendent traits that were able to be brought together
in recombinant genotypes having elevated levels of
both fatty acids. The degree of silencing of both ⌬9-
and ⌬12-desaturase in these recombinant genotypes
was equivalent in its magnitude to that observed in
the individually silenced parental lines, demonstrat-
ing for the first time, to our knowledge, that hpRNA-
induced PTGS in independent genes can be stacked
without any diminution in the degree of silencing.
Because stearic acid and oleic acid percentages are
negatively correlated due to their precursor/product
relationship, the recombinant genotypes show inter-
mediate levels of both fatty acids. Furthermore, be-
cause it was possible to obtain various degrees of
elevation of oleic acid and stearic acid in individually
silenced transgenic lines, the opportunity exists to
develop a wide range of alternative palmitic, stearic,
and oleic acid combinations through recombination
of appropriately chosen parental lines. This should
enable the development of cottonseed fats and oils
that satisfy different application requirements, in
particular lines with fatty acid profiles matching
those of valuable specialty confectionery fats such as
cocoa (Theobroma cocoa) butter.
MATERIALS AND METHODS
Gene-Silencing Constructs
Gene-silencing constructs designed to target the endogenous cotton (Gos-
sypium hirsutum) genes encoding either stearoyl-ACP ⌬9-desaturase or mi-
crosomal ⌬12-desaturase, using the ghSAD-1 or ghFAD2-1 cDNA sequences,
respectively, in either antisense or inverted repeat configurations, are shown
in Figure 2. The silencing constructs were each driven by the soybean
(Glycine max) lectin promoter, which has been shown to direct seed-specific
expression of a

-glucuronidase reporter gene in cotton (Townsend and
Llewellyn, 2002). A HindIII/EcoRI fragment containing the soybean lectin
promoter and terminator sequences was excised from the pGLe-10 plasmid
(Cho et al., 1995) and engineered into the same restriction sites of binary
vector pBI121 (CLONTECH) from which the cauliflower mosaic virus 35S-
Gus-Nos chimeric gene had been removed. This formed a pBI-Lec binary
vector that was subsequently used to carry all the gene-silencing constructs
described in this paper. The ⌬9-desaturase antisense construct (⌬9-AS)
consisted of the entire ghSAD-1 cDNA (Liu et al., 1996) cloned behind the
lectin promoter in an antisense orientation. For the ⌬9-desaturase inverted
repeat construct (⌬9-HP), a 514-bp fragment was PCR amplified from the 5⬘
end of ghSAD-1 using oligonucleotides ⌬9s1 (5⬘-TTTTAATGCCATCGCC-
TCG-3⬘) and ⌬9a1 (5⬘-CTTCAGCAGTCCAAGCCCTG-3⬘) and inserted at
the 3⬘ end of the ghSAD-1 sequence to form an inverted repeat. This
ghSAD-1 inverted repeat construct was then ligated behind the lectin pro-
moter in the sense orientation in relation to the full-length ghSAD-1 se-
quence. A 1,351-bp fragment of ghFAD2-1 (Liu et al., 1999b) was PCR
amplified using oligonucleotides ⌬12s1 (5⬘-CCTGGCGTTAAACTG CTTTC-
3⬘) and ⌬12a1 (5⬘-CCATATAGTTTATTAATATAACAC-3⬘) and consisted of
the entire ⌬12-desaturase coding region, the full 3⬘-UTR, and a partial
5⬘-UTR. This ghFAD2-1 fragment was cloned behind the lectin promoter in
an antisense orientation to make the antisense construct (⌬12-AS). For the
⌬12-desaturase inverted repeat construct (⌬12-HP), an 853-bp fragment was
PCR amplified from the 5⬘ end of the ghFAD2-1 with oligonucleotides ⌬12s1
and ⌬12a2 (5⬘-TATGTTGCCGTAGGTGATC-3⬘) and ligated at the 3⬘ end of
ghFAD2-1 to form an inverted repeat. The whole ghFAD2-1 inverted repeat
unit was then ligated behind the lectin promoter in pBI-Lec binary vector in
a sense orientation in relation to the full-length ghFAD2-1 sequence.
Cotton Transformation
Transgenic cotton cv Coker 315 plants were generated by Agrobacterium
tumefaciens-mediated transformation, and selection on medium containing
kanamycin sulfate, by a modification of the method described by Cousins et
al. (1991). Cotton seedlings were germinated aseptically on Murashige and
Hairpin RNA-Mediated Silencing of Fatty Acid Desaturases in Cotton
Plant Physiol. Vol. 129, 2002 1741
Skoog medium (Murashige and Skoog, 1962) solidified using phytagel
(Sigma, St. Louis). Seedlings were maintained under low-light conditions at
28°C. Cotyledon explants from 10- to 14-d-old seedlings were cocultivated
with A. tumefaciens strain AGL1 containing the relevant gene construct for
2 d on the medium containing Murashige and Skoog macro- and micro-
elements and B
5
vitamins (Gamborg et al., 1968), 100 mg L
⫺1
myo-inositol,
30gL
⫺1
Glc, 0.2 mg L
⫺1
2,4-dichlorophenoxyacetic acid, 0.1 mg L
⫺1
kinetin,
and 0.93 g L
⫺1
magnesium chloride, and solidified using2gL
⫺1
phytagel.
The callus was induced on the same medium but supplemented with 50 mg
L
⫺1
kanamycin sulfate and 250 mg L
⫺1
cefotaxime at 28°C for 6 weeks.
Healthy calli were then transferred to Murashige and Skoog medium con-
taining 5 mg L
⫺1
6-(
␥
,
␥
-dimethylallylamino)-purine, 0.1 mg L
⫺1
naphtha
-
lene acetic acid, 25 mg L
⫺1
kanamycin, and 250 mg L
⫺1
cefotaxime for a
second selection period of 6 weeks at 28°C. Somatic embryogenesis was
initiated on the solidified Murashige and Skoog medium, without added
phytohormone or antibiotic, and the embryoids formed were then germi-
nated on Stewart and Hsu medium (Stewart and Hsu, 1977) solidified with
phytagel to produce transgenic cotton plantlets. Primary transgenic cotton
plantlets (herein referred to as the T
1
generation) were transferred to soil
and maintained in a greenhouse once leaves and roots developed.
Identification of Transgenic Plants by PCR
The presence of the transgenes in each regenerated cotton plant was
initially determined by PCR, using cotton genomic DNA as a template, and
the following oligonucleotides: 3Lec-s1, 5⬘-CATGTGACAGATCGAAG-
GAA-3⬘; and 3Lec-a1, 5⬘-ATCTAATTATTCTATTCAGAC-3⬘.
This process amplifies an approximately 300-bp DNA fragment compris-
ing the transcriptional terminator of the soybean lectin gene. Accordingly,
amplification only occurs from plant DNA containing the introduced chi-
meric genes. Further confirmation of transgenic status of the regenerated
cotton plants was obtained by Southern-blot analysis as described below.
DNA Isolation and Southern-Blot Analysis
Cotton genomic DNA was isolated according to Paterson et al. (1993) and
further purified using the CsCl gradient method as described by Sambrook
et al. (1989). Approximately 10
g of DNA was digested by HindIII and the
restriction fragments were separated on a 0.7% (w/v) agarose gel by elec-
trophoresis and transferred onto a Hybond N
⫹
nylon membrane (Amer
-
sham, Buckinghamshire, UK) using 0.4 m NaCl for 4 h. Southern-blot
analyses were carried out by hybridizing with an [
␣
-
32
P]dCTP-labeled DNA
fragment consisting of the promoter region of the soybean lectin gene that
is specific for the transgene. The hybridization and subsequent washing
were carried out as previously described (Liu et al., 1999b).
RNA Isolation and Northern-Blot Analysis
Cotton embryos at mid-maturation, approximately 30 d after fertilization,
were harvested and RNA was isolated using the RNeasy Plant Mini Kit
(Qiagen USA, Valencia, CA). RNA was separated on a denaturing formal-
dehyde gel and transferred onto a Hybond N
⫹
nylon membrane according
to Sambrook et al. (1989). The entire coding regions of the ghSAD-1 and
ghFAD2-1 genes were used as probes. A cDNA fragment containing the
entire coding region of the cotton keto-acyl synthase II gene (ghKASII) was
obtained (Q. Liu, unpublished data) and used to probe the blot as a control,
indicating the level of expression of nontargeted lipid synthesis genes in
each line. The hybridization and the after washing was essentially the same
as previously described (Liu et al., 1999b).
Fatty Acid Analysis
Self-pollinated seeds were harvested from each primary transgenic (T
1
)
plant and analyzed for fatty acid composition. As an initial screen, the total
lipids were extracted from pooled three-seed samples from each T
1
plant by
the method of Bligh and Dyer (1959) and used for fatty acid analysis.
Subsequently, interesting lines were examined in more detail by performing
fatty acid analysis on a number of individual T
2
seeds borne on each T
1
plant. In addition, some of the T
1
plants carrying the ghSAD-1 or ghFAD2-
1-silencing constructs were intercrossed to combine the traits. F
1
seeds from
these crosses were analyzed nondestructively by cutting off approximately
a one-sixth portion of the seed distal to the embryonic axis and crushing this
onto filter paper discs to provide expressed oil for analysis. The remaining
larger portion of each seed, containing the embryonic axis, was planted
directly into soil to establish F
1
plants. F
2
seeds were analyzed individually
for fatty acids by methylation of oil expressed from individual seeds.
Fatty acid methyl esters were prepared by alkaline transmethylation.
Samples of solvent-extracted or -expressed oil were loaded onto filter paper
discs and methylated in 2 mL of 0.02 m sodium methoxide for1hat90°C,
followed by addition of 1.5 mL of hexane and 2 mL of water. After vortexing
and phase separation, the upper hexane layer containing the fatty acid
methyl esters was transferred to a microvial. Fatty acid methyl esters were
analyzed by gas-liquid chromatography as previously described (Stoutjes-
dijk et al., 2002). Cyclopropenoid fatty acids were not routinely determined
on all lines. Relative fatty acid compositions were calculated as the percent-
age that each fatty acid represented of the total measured fatty acids. An
indirect method of assessing the cumulative effects of ⌬9-desaturase and
⌬12-desaturase activity during seed fatty acid synthesis is through the SDP
and ODP parameters, derived by the following formulae: SDP ⫽ (% oleic ⫹
% linoleic)/(% stearic ⫹ % oleic ⫹ % linoleic) and ODP ⫽ (% linoleic)/(%
oleic ⫹ % linoleic), respectively. These parameters represent the ratio of the
total fatty acid products of desaturation to the amount of fatty acid substrate
that was available, and are useful in illustrating the effects of gene silencing
on the activities of the target enzymes. Cottonseed oil typically has an SDP
value of around 0.97 and an ODP value of around 0.80, indicating that about
97% of stearic acid formed during fatty acid synthesis is subsequently
desaturated to oleic acid and about 80% of this is further desaturated to
linoleic acid. Phenotypic distributions for fatty acid composition, SDP, and
ODP in T
2
seed populations were compared with expected segregation
ratios using the Chi-squared goodness of fit test at P ⫽ 0.05.
ACKNOWLEDGMENTS
We are grateful to Clive Hurlstone and Rhenzong Liu for technical
assistance and to Dr. Danny Llewellyn and Dr. Belinda Townsend for advice
on cotton transformation. We also thank Dr. Lorraine Tonnet, Lorraine
Mason, and Richard Philips for analyzing fatty acid composition.
Received December 28, 2001; returned for revision January 23, 2002; ac-
cepted March 15, 2002.
LITERATURE CITED
Ascherio A, Willett WC (1997) Health effects on trans fatty acids. Am J Clin
Nutr 66: 1006S–1010S
Auld DL, Heikkinen MK, Erickson DA, Sernyk JL, Romero JE (1992)
Rapeseed mutants with reduced levels of polyunsaturated fatty acids and
increased levels of oleic acid. Crop Sci 32: 657–662
Bligh EG, Dyer WJ (1959) A rapid method of total lipid extraction and
purification. Can J Biochem Physiol 37: 911–917
Chapman KD, Austin-Brown S, Sparace SA, Kinney AJ, Ripp KG, Pirtle
IL, Pirtle RM (2001) Transgenic cotton plants with increased seed oleic
acid content. J Am Oil Chem Soc 78: 941–947
Cho MJ, Widholm JM, Vodkin LO (1995) Cassettes for seed-specific ex-
pression tested in transformed embryogenic cultures of soybean. Plant
Mol Biol Rep 13: 255–269
Cousins YL, Lyon BR, Llewellyn DJ (1991) Transformation of an Australian
cotton cultivar: prospects for cotton improvement through genetic engi-
neering. Aust J Plant Physiol 18: 481–494
Cox C, Mann J, Sutherland W, Chisholm A, Skeaff M (1995) Effects of
coconut oil, and safflower oil on lipids and lipoproteins in persons with
moderately elevated cholesterol levels. J Lipid Res 36: 1787–1795
Gamborg OL, Miller RA, Ojima K (1968) Nutrient requirements of suspen-
sion cultures of soybean root cells. Exp Cell Res 50: 151–158
Graef GL, Miller LA, Fehr WR, Hammond EG (1985) Fatty acid develop-
ment in a soybean mutant with high stearic acid. J Am Oil Chem Soc 62:
773–775
Hamilton AJ, Brown S, Yuanhai H, Ishizuka M, Lowe A, Solis A-GA,
Gierson D (1998) A transgene with repeated DNA causes high frequency,
post-transcriptional suppression of ACC-oxidase gene expression in to-
mato. Plant J 15: 737–746
Liu et al.
1742 Plant Physiol. Vol. 129, 2002
Heppard EP, Kinney AJ, Stecca KL, Miao GH (1996) Developmental and
growth temperature relation of two different microsomal
-6 desaturase
genes in soybeans. Plant Physiol 110: 311–319
Kinney AJ (1996) Improving soybean seed quality. Nature Biotechnol 14: 946
Knutzon DS, Thompson GA, Radke SE, Johnson WB, Knauf VC, Kridl JC
(1992) Modification of Brassica seed oil by antisense expression of a
stearoyl-acyl carrier protein desaturase gene. Proc Natl Acad Sci USA 89:
2624–2628
Lightner J, Wu J, Browse J (1994) A mutant of Arabidopsis with increased
levels of stearic acid. Plant Physiol 106: 1443–1451
Liu Q (1998) The isolation and characterisation of fatty acid desaturase
genes in cotton. PhD thesis. Sydney University
Liu Q, Singh SP, Brubaker CL, Green AG (1999a) Cloning and sequence
analysis of a novel member (accession No. Y10112) of the microsomal
-6
fatty acid desaturase family from cotton (Gossypium hirsutum). Plant
Physiol 120: 339
Liu Q, Singh SP, Brubaker CL, Sharp PJ, Green AG, Marshall DR (1999b)
Molecular cloning and expression of a cDNA encoding a microsomal
-6
fatty acid desaturase in cotton (Gossypium hirsutum L.). Aust J Plant
Physiol 26: 101–106
Liu Q, Singh SP, Sharp PJ, Green AG, Marshall DR (1996) Nucleotide
sequence of a cDNA from Gossypium hirsutum encoding a stearoyl-acyl
carrier protein desaturase (accession No. X95988) (OGR 96-012). Plant
Physiol 110: 1435
Murashige T, Skoog F (1962) A revised medium for rapid growth and
bioassays with tobacco tissue cultures. Physiol Plant 15: 473–497
Murray F, Llewellyn D, McFadden H, Last D, Dennis ES, Peacock WJ
(1999) Expression of the Talaromyces flavus glucose oxidase gene in cotton
and tobacco reduced fungal infection, but is also phytotoxic. Mol Breed 5:
219–232
Norden AJ, Gorbet DW, Knauft DA, Young CT (1987) Variability in oil
quality among peanut genotypes on the Florida breeding program. Pea-
nut Sci 14: 7–11
Ohlrogge JB, Jaworski JG (1997) Regulation of fatty acid synthesis. Ann
Rev Plant Physiol Plant Mol Bio 48: 109–136
Osorio J, Fernandez-Martinez J, Mancha M, Garces R (1995) Mutant sun-
flower with high concentration of saturated fatty acids in their oil. Crop
Sci 35: 739–742
Paterson AH, Brubaker CL, Wendel JF (1993) A rapid method for extraction
of cotton (Gossypium spp.) genomic DNA suitable for RFLP or PCR
analysis. Plant Mol Biol Rep 11: 122–127
Radcliffe JD, King CC, Czajka-Narins DM, Imrhan V (2001) Serum
and liver lipids in rats fed diets containing corn oil, cottonseed oil, or
a mixture of corn and cottonseed oils. Plant Foods Human Nutr 56: 51–60
Rahman SM, Takagi Y, Knoshita T (1997) Genetic control of high stearic
acid content in seed oil of two soybean mutants. Theor Appl Genet 95:
772–776
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning: A Laboratory
Manual, Ed 2. Cold Spring Harbor Laboratory Press, Plainview, NY
Smith NA, Singh SP, Wang MB, Stoutjesdijk PA, Green AG, Waterhouse
PM (2000) Totally silencing by intron-spliced hairpin RNAs. Nature 407:
319–320
Soldatov KI (1976) Chemical mutagenesis in sunflower breeding. In Pro-
ceedings of the Seventh International Sunflower Association, Vlaardin-
gen, The Netherlands. International Sunflower Association, Toowoomba,
Australia, pp 352–357
Stewart JM, Hsu CL (1977) In ovulo embryo culture and seedling develop-
ment of cotton (Gossypium hirsutum L.). Planta 137: 113–117
Stoutjesdijk P, Singh SP, Liu Q, Hurlstone C, Waterhouse P, Green A
(2002) hpRNA-mediated targeting of the Arabidopsis FAD2 gene gives
highly efficient and stable silencing. Plant Physiol 129: 1723–1731
Townsend BJ, Llewellyn DJ (2002) Spatial and temporal regulation of a
soybean (Glycine max) lectin promoter in transgenic cotton (Gossypium
hirsutum). Funct Plant Biol (in press)
Wesley V, Helliwell C, Smith N, Wang MB, Rouse D, Liu Q, Gooding P,
Singh S, Abbott D, Stoutjesdijk P et al. (2001) Construct design for
efficient, effective and high throughput gene silencing in plants. Plant J
27: 581–590
Wood R (1996) Comparison of the cyclopropene fatty acid content of cot-
tonseed varieties, glanded and glandless seeds, and various seed struc-
tures. Biochem Arch 2: 73–80
Xu XQ, Tran VH, Palmer MV, White K, Salisbury P (2000) Chemical,
physical and sensory properties of Monola oil, palm olein and their
blends in deep frying trials. Food Aust 52: 77–82
Hairpin RNA-Mediated Silencing of Fatty Acid Desaturases in Cotton
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