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Abstract
Two previously optimized procedures for genetic transformation of two economically important high oleic sunflower genotypes (H. annuus cv.
capella and SWSR2 inbred line) were used to generate stably transformed sunflower plants of these genotypes expressing gus reporter gene:
Agrobacterium tumefaciens-mediated gene transfer to juvenile split apical meristems was applied to cv. capella, and a biolistic gene transfer
method using the same target tissue was applied to SWSR2 inbred line. Out of 120 originally used explants of cv. capella, five transgenic
regenerants could be identified by biochemical and molecular analysis. Thus, transformation efficiency amounted to 4.1%. For SWSR2 inbred line,
125 explants were used and six transgenic regenerants could be identified in an identical manner, resulting in a transformation efficiency of 4.8%.
The highest expression level of the gus gene resulted in a yield of recombinant protein of approx. 0.2% total soluble protein. Additionally, T1
progeny was tested for gus gene inheritance in both genotypes. From approx. 75% of the T0 plants, the gus gene was stably transmitted to the T1
progeny and expressed in a detectable amount. To test the reproducibility of the transformation procedures, they were used to introduce another
reporter gene, mgf5, into both genotypes. The resulting transformation efficiencies were 3.3 for both genotypes. gfp proved to be a suitable reporter
gene to select transgenic regenerants in early developing stages. The data presented here demonstrate for the first time the reproducible
transformation of high oleic sunflower genotypes with high frequency.
# 2006 Elsevier Ireland Ltd. All rights reserved.
Keywords: Gus gene; Helianthus annuus L.; Meristem transformation; Mgfp5 gene; Particle bombardment; Vacuum infiltration
1. Introduction
Sunflower (Helianthus annuus L.) is one of three most
important annual oil-bearing crops world-wide following
soybean (Glycine max L.) and rapeseed (Brassica napus L.)
[1]. The oil of high oleic sunflower genotypes can be used as
food oil or deep-frying fat [2,3]. Moreover, the high oxidative
performance of oleic acid and its very low content of
polyunsaturated fatty acids combined with low content of
stearic acid make them suitable for industrial applications like
cosmetics, pharmaceuticals, detergents, lubricants, metal
working fluids, surfactants or for chemical synthesis. However,
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E. coli DH5a carrying the plasmid pBI121 or pCAMBIA1302 was used as plasmid source for particle bombardment
experiments and positive controls. The culture of the bacteria
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Table 1
Summary of stable transformation events of T0 plants of high oleic H. annuus L.
genotypes, cv. capella using gus gene with Agrobacterium infiltration and
SWSR2 using biolistic gene transfer method
Genotype
cv. capella
SWSR2
120
77
125
82
77
82
40
5
4.1
40
6
4.8
a
Transformation frequency was calculated on the basis of positive PCR
plants in relation to the total number of used explants. PCR was performed with
gus-specific primer 1416 weeks after co-cultivation or bombardment.
Fig. 1. Variation of GUS activity among gus expressing T0 plants of H. annuus (A) cv. capella and (B) SWSR2 inbred line over a period of 1214 weeks on different
development media. One leaf was ground each time with extraction buffer and the crude protein extract was used in fluorometric GUS activity assay. For details see
materials and methods. Data represent mean of three parallel measurements. The level of variation was <5%.
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Table 2
Analysis of gus gene transmission to the T1 progeny in high oleic H. annuus L. cv. capella ands SWSR2 inbred line. GUS expression was recorded by the
histochemical GUS activity assay using a random leaf of each T1 plant
Genotype and plant number
No. of seeds
Capella
Capella
Capella
Capella
Capella
1
2
3
4
5
3
5
7
10
9
SWSR2
SWSR2
SWSR2
SWSR2
SWSR2
SWSR2
1
2
3
4
5
6
11
5
9
13
7
10
GUS activity
3
4
5
6
2
+
+
+
+
1
4
4
2
0
1/3
4/4
4/5
2/6
0/2
9
1
6
10
7
7
+
+
+
3
0
2
5
3
0
3/9
0/1
2/6
5/10
3/7
0/7
PCR was performed with digested DNA of all plants using gus specific primer.
tissue did not exhibit any blue color under identical assay
conditions.
The presence of respective T-DNA in 40 randomly chosen
regenerants, including the hitherto identified GUS positive
plantlets, was tested by PCR 1416 weeks after cocultivation,
using gus-specific primer. An amplification of the expected
830 bp fragment corresponding to the gus gene was observed in
all GUS positive plants, whereas no amplification was detected
in the samples from non-transformed plants (data not shown).
The resulting transformation frequency of cv. capella was
calculated on the basis of positive PCR plants and recorded as a
percentage from the total number of co-cultivated or
bombarded explants (Table 1). The frequency amounted to
4.1% using Agrobacterium infiltration method whereas this was
4.8% in SWSR2 inbred line using direct gene transfer method.
Finally, southern blot analysis of seven randomly chosen T0
plants confirmed the presence and integration of the gus
fragment gene into the sunflower genome of both genotypes
(Fig. 2).
Eventually, single or two hybridizing bands were observed
in cv. capella plants transformed with Agrobacterium infiltra-
Fig. 2. Southern blot hybridization of random independent T0 transgenic plants of two high oleic H. annuus L. genotypes, cv. capella and SWSR2 inbred line.
Genomic DNA was digested with EcoRI and hybridized with 32P-labelled gus probe. Lanes 1, 3, 7, 9 transgenic cv. capella plants (plants 4, 3, 2, and 1 respectively,
from Fig. 1), lanes 2, 4, 8 transgenic SWSR2 inbred line plants (plants 1, 4 and 6, respectively, from Fig. 1), lanes 5 and 6 non-transformed sunflower and lane 10
marker DNA. Numbering of T0 plants is according to Fig. 1.
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Fig. 3. Southern blot hybridization of DNA from T1 plants from two randomly chosen T0 plants of genotype cv. capella and SWSR2 inbred line transformed with the
gus gene. Genomic DNA was digested with HindIII and hybridized with 32P-labelled gus probe. (A) Lanes 14 transgenic cv. capella T1 plants from T0 plant 2, lane 5
non-transformed cv. capella and lane 6 marker DNA. (B) Lanes 1, 3, 4 transgenic SWSR2 inbred line T1 plants from T0 plant 1 and lane 2 non-transformed SWSR2
inbred line. Numbering of T0 plants is according to Fig. 1.
Fig. 4. Relative fluorescence of crude protein extracts from leaves of positive gfp transgenic plants of high oleic H. annuus L. genotypes, cv. capella and SWSR2
inbred line, after background substraction. One leaf was ground each time with extraction buffer and the crude protein extract was used in fluorometric GFP activity
assay. For details see materials and methods. Data represent mean of three parallel measurements. The level of variation was <8%.
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Fig. 5. Southern blot analysis of two mgfp5 expressing T0 plant of two high
oleic H. annuus L. genotypes, cv. capella and SWSR2 inbred line. Genomic
DNA was digested with EcoRI and hybridized with 32P-labelled probe corresponding to mgfp5 gene. Lane 1: transgenic cv. capella plant, lane 2: transgenic
SWSR2 inbred line plant, lane 3: non-transformed sunflower, and lane 4:
marker DNA.
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