Crop Science Grow Your Career with CSSA
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Figures Only
Right arrow Full Text (PDF) Free
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Related articles in Crop Science
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (2)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Tang, J.
Right arrow Articles by McVetty, P. B. E.
Right arrow Search for Related Content
PubMed
Right arrow Articles by Tang, J.
Right arrow Articles by McVetty, P. B. E.
Agricola
Right arrow Articles by Tang, J.
Right arrow Articles by McVetty, P. B. E.
Related Collections
Right arrow Cell Biology & Molecular Genetics
Right arrow Crop Genetics
Right arrow Other Oil Crops
Published in Crop Sci. 44:732-740 (2004).
© 2004 Crop Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA

CROP BREEDING, GENETICS & CYTOLOGY

Stability of the Expression of Acyl-ACP Thioesterase Transgenes in Oilseed Rape Doubled Haploid Lines

Jihong Tang, Rachael Scarth* and Peter B. E. McVetty

Department of Plant Science, University of Manitoba, Winnipeg, MB, R3T 2N2, Canada

* Corresponding author (Rachael_Scarth{at}UManitoba.CA).


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Transgene stability in doubled haploid (DH) lines is an important consideration for transgenic cultivar development programs. The objective of this study was to assess the expression stability of acyl-acyl carrier protein (ACP) thioesterase (TE) transgenes in oilseed rape (Brassica napus L.) DH lines. The DH lines were developed from microspores of F1 plants from the crosses between transgenic parents carrying the bay-TE (Uc FatB1), elm-TE (Ua FatB1), nutmeg-TE (Mf FatB1), or cuphea-TE (Ch FatB1) transgenes and three nontransgenic cultivars having distinct seed oil fatty acid compositions. Of 333 DH1 plants developed from microspore-derived embryos that had undergone selection for kanamycin resistance (the selectable marker) with bay-TE F1 and cuphea-TE F1 plants as the microspore donors, 20 plants did not show TE transgene expression. Polymerase chain reaction (PCR) and Southern blotting analyses confirmed that the absence of TE transgene expression in these 20 DH1 plants was due to escape of embryos from kanamycin selection or existence of an incomplete T-DNA copy without the TE transgene. No DH1 plant with completely silenced TE transgenes was detected. Thirty of 34 transgenic DH lines showed a stable level of the target fatty acid, lauric acid (C12:0) for the bay-TE and palmitic acid (C16:0) for the other three TE transgenes, over the two or three consecutive generations tested (DH2, DH3, and/or DH4 plants). Target fatty acids were significantly affected by temperature during seed development. DH lines carrying the elm-TE or the cuphea-TE transgene grown under high temperature conditions (25/20°C, day/night) during seed development showed higher levels of palmitic acid than under lower temperatures (20/15°C). These results support the application of the DH procedure in breeding programs for transgenic cultivars and indicate the important influence of environment.

Abbreviations: ACP, acyl carrier protein • DH, doubled haploid • MT, microspore treatment • nptII, neomycin phosphotransferase II • PCR, polymerase chain reaction • RT, root treatment • TE, acyl-acyl carrier protein thioesterase


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
CONVENTIONAL OILSEED RAPE CULTIVARS have traces of lauric acid and approximately 40 µg mg–1 palmitic acid in the fatty acid composition of the seed oil. Agrobacterium-mediated transformations of oilseed rape plants with ACP TE genes cloned from several plant species have led to the development of transgenic lines accumulating lauric acid or enhanced levels of palmitic acid in the seed oil (Jones et al., 1995; Voelker et al., 1996, 1997). Oils rich in such fatty acids have a number of food and nonfood uses, such as manufacturing of margarine, shortening, laundry detergent, and shampoo.

For practical applications, it is important that transgenes are inherited and expressed in a predictable, consistent, and stable manner (Campbell et al., 2000; Conner and Christey, 1994; Conner et al., 1998). Voelker et al. (1996) reported that oilseed rape lines with multiple copies (5–15) of the bay-TE transgene (Uc FatB1) were stable for more than five generations, without any apparent genetic instability or loss of the transgenic phenotype. However, instability in the expression of other transgenes has been observed in some studies (Assaad et al., 1993; Scheid et al., 1991; Zhong et al., 1999). For example, in a population of Arabidopsis thaliana (L.) Heynh. plants transgenic for a hygromycin resistance gene (hpt), 50% of the plants failed to transmit the resistance trait to the progeny, although the complete transgene was detected in all the plants (Scheid et al., 1991).

Unstable expression of transgenes has been associated with gene silencing (Charrier et al., 2000; Meyer and Saedler, 1996; Scott et al., 1998). Gene silencing is due to somatically or meiotically heritable repression of gene expression that is potentially reversible and is not due to mutation (Kaeppler et al., 2000). Gene silencing could result from the blocking of transcription initiation, transcriptional gene silencing (TGS), or from the degradation of mRNA after transcription, posttranscriptional gene silencing (PTGS) (Chandler and Vaucheret, 2001; Matzke and Matzke, 1998; Wassenegger, 2000). Possible factors inducing gene silencing include growth conditions of the transgenic plants and in vitro tissue culture processes such as those involved in DH line development, in addition to multiple transgene copies, the structure of transgene inserts (such as tandem repeats and truncated copies), vector sequence, and the genomic site of insertion (Charrier et al., 2000; Dale et al., 1998; Maqbool and Christou, 1999). The procedure of DH line development in Brassica includes an in vitro culture period (Ferrie and Keller, 1995). It has been reported that during in vitro culture, DNA methylation may occur (Brown et al., 1990; Kaeppler and Phillips, 1993; Olhoft, 1996), which could alter chromatin structures, thus leading to variation in gene expression (Kaeppler et al., 2000). Gene silencing due to in vitro culture has been observed (Kaeppler et al., 2000).

The DH procedure is an increasingly important method of creating genetically homozygous lines in Brassica breeding because complete homozygosity can be realized in a single generation with the DH technology, thus avoiding repeated generations of inbreeding for development of pure breeding lines (Ferrie and Keller, 1995). Transgene expression stability in DH lines is therefore an important consideration for transgenic cultivar development programs. The objective of this study was to assess the stability of four different TE transgenes in oilseed rape DH lines developed by microspore culture.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Parental Genotypes
The original seeds of oilseed rape transgenic parental lines TL1, TL3, TL5, and TL6, as well as two nontransgenic oilseed rape breeding lines 212/86 and QO4, were kindly provided by Calgene Inc. (Davis, CA). TL1 was developed by Agrobacterium-mediated transformation with the bay-TE gene Uc FatB1 (GenBank accession no. M94159, Voelker et al., 1992, 1996), TL3 with the elm-TE gene Ua FatB1 (accession no. U65644, Voelker et al., 1997), TL5 with the nutmeg-TE gene Mf FatB1 (accession no. U65642, Voelker et al., 1997), and TL6 with the cuphea-TE gene Ch FatB1 (accession no. U17076, Jones et al., 1995). The breeding line 212/86 was the recipient genotype used in the transformation for TL1, TL3, and TL6. QO4 was the recipient genotype for TL5. Transgenic plants carrying the bay-TE gene showed accumulation of lauric acid in the seed oil, whereas plants carrying any of the other three TE genes accumulated enhanced levels of palmitic acid in the seed oil, compared with nontransgenic oilseed rape cultivars (<50 µg mg–1 palmitic acid). The selectable marker for the transformations of the four TE transgenes was neomycin phosphotransferase II (nptII), which confers kanamycin resistance. The three nontransgenic cultivars used as parents in this study have distinct seed oil fatty acid compositions, which included the canola cultivar AC Excel (Rakow, 1993), the low linolenic acid canola cultivar Apollo (Scarth et al., 1995a), and the high erucic acid (C22:1) cultivar Mercury (Scarth et al., 1995b). As with 212/86 and QO4, the three cultivars have less than 50 µg mg–1 palmitic acid and no accumulation of lauric acid in the seed oil.

DH Line Development
DH lines used in this study were developed from F1 plants through microspore culture and chromosome doubling based on the procedure described by Ferrie and Keller (1995). The F1 originated from crosses between the four transgenic parental lines and the three nontransgenic cultivars. Microspores were isolated from unopened flower buds of F1 plants. Embryogenesis was induced by culturing the microspores in induction media at 32.5°C for 3 d. When small embryos turned green in color, they were transferred to solid B5 media for regeneration.

A total of 333 DH lines were developed from embryos which had undergone kanamycin selection for the detection of TE transgene silenced plants. For the selection, small embryos were cultured on B5 media containing 50 mg L–1 kanamycin for 3 wk. Embryos that survived kanamycin selection remained green and were transferred to fresh B5 media without kanamycin for subsequent regeneration.

Doubling of the chromosome number was conducted by two alternative treatments: microspore treatment (MT) or root treatment (RT). For MT, microspores were cultured in induction media containing 10 mg L–1 colchicine in the first 48 h of the culture process. For RT, the roots of haploid plantlets were immersed in 0.34% (w/v) colchicine solution for 2 h after the plantlets had grown in soil for 3 to 4 wk. DH1 plants, defined by Stringam et al. (1995) as plants developed directly from microspore culture after chromosome doubling treatments, were grown in a greenhouse and selfed to produce DH2 seeds. The DH lines were advanced by single seed descent for the production of DH3 and DH4 seeds in the greenhouse. DH2, DH3, and DH4 seeds were used to grow the trials in this study.

Experimental Design
Four experiments were conducted under confined environmental conditions in a growth room or greenhouse with individual plants grown in 150 mm pots. Self-pollinated seeds of each plant were harvested at full maturity and tested for the fatty acid composition of the seed oil.

In Exp. 1, DH1 plants of the 333 DH lines, which were directly regenerated from selected microspore-derived embryos, were grown in a greenhouse to assess TE transgene expression in DH lines developed from embryos selected for kanamycin resistance. The 333 lines included 15 DH1 plants from the crosses of the bay-TE transgenic parent TL1 with the three nontransgenic cultivars and 318 DH1 plants from the crosses of the cuphea-TE transgenic parent TL6 with the same three cultivars. Self-pollinated DH2 seeds from DH1 plants were grown to the two-leaf seedling stage for extraction of genomic DNA for PCR and Southern blotting as describe below.

In Exp. 2, a total of 34 DH lines, including 11 bay-TE transgenic lines, six elm-TE transgenic lines, five nutmeg-TE transgenic lines, and 12 cuphea-TE transgenic lines, were tested for seed oil fatty acid composition in concurrently grown plants of two or three consecutive generations to assess the stability of TE transgene expression over generations. For the DH lines carrying the bay-TE or the cuphea-TE transgenes, two generations (DH2 and DH3 plants) were tested; for the DH lines carrying the elm-TE or the nutmeg-TE transgenes, three generations (DH2, DH3, and DH4) were tested. Three plants were grown for each generation of each line in a randomized complete block design with three replicates in a growth room with controlled growth conditions of a 16-h photoperiod, 580 µmol m–2 s–1 light intensity, and day/night temperatures of 20/15°C.

In Exp. 3, six DH3 plants for each of 10 DH lines carrying the bay-TE, elm-TE, nutmeg-TE, or cuphea-TE transgenes, were grown to the bolting stage in a controlled environment with a 16-h photoperiod, 580 µmol m–2 s–1 light intensity, and day/night temperatures of 20/15°C to assess the effect of elevated temperatures during seed development on TE transgene expression. At the onset of flowering, three plants of each line were transferred to a second controlled environment with the same photoperiod and light intensity as described above, and day/night temperatures of 25/20°C until the completion of seed development to maturity. The remaining three plants of each line remained in the first environment to maturity.

In Exp. 4, the DH3 generation of eight bay-TE and cuphea-TE DH lines, including four lines for which the chromosome number was doubled by RT and four lines by MT, were grown in a controlled environment with 20/15°C day/night temperatures, in a completely randomized design with 14 plants from each line to test plant to plant variation in the target fatty acid level within TE transgenic DH lines.

Characterization of Transgenic DH Lines
Plant genomic DNA for PCR and Southern blotting analyses was extracted from approximately 3 g of cotyledon and young leaves by the CTAB (cetyltrimethylammonium bromide) procedure (Kidwell and Osborn, 1992). DH lines developed from embryos that had undergone kanamycin selection but did not show the expected fatty acid composition were characterized by PCR (Foolad et al., 1995). The sequences of the forward and reverse primers for the amplification of an internal fragment of each transgene by PCR were as follows: the primers used for a 1.0-kb fragment of the bay-TE transgene Uc FatB1 being 5'-GAGCTTGAAAAGGTTGCCTG-3' and 5'-GGTTCTGCGGGTATCACACT-3'; the primers for a 1.1-kb fragment of the cuphea-TE being 5'- GAACTTTTATCAACCA-3' and 5'-ACCTGCCCTTCACTCAG-3'; the primers for a 0.7-kb fragment of the kanamycin resistance gene nptII being 5'-AGACAATCGGCTGCTCTGAT-3' and 5'-TCATTTCGAACCCCAGAGTC-3'. In addition to the primers corresponding to the TE transgene, primers P1/P2, designed on the basis of the sequence of the endogenous napin gene published by Kridl et al. (1991), were added in each PCR reaction, which resulted in a 0.5-kb internal control band for both transgenic and nontransgenic plants.

The probes used for Southern blotting analyses were prepared by a digoxigenin (DIG)-labeling procedure with the PCR DIG Probe Synthesis Kit (Roche Diagnostics GmbH, Mannheim, Germany). To prepare the cuphea-TE probe, the TE transgene was amplified by PCR from genomic DNA of the cuphea-TE transgenic parental line TL6 with the forward primer PT1, 5'-ATTAGAGCCTCGGCTTCACTC-3', and the reverse primer PT2, 5'-GGATCCCATTGGATGATCTTT-3', designed on the basis of the published sequences of the TE gene. The amplified DNA fragment was cloned with the pGEM-T Easy Vector and JM109 Competent Cells (Promega Corp., Madison, WI). A white colony was used to inoculate LB Broth media and plasmid DNA carrying the cuphea-TE gene was extracted (Ausubel et al., 1994). With the plasmid DNA having the TE gene as the template, a 1.1-kb probe for the TE was produced and DIG-labeled by PCR under the presence of DIG-dUTP. A 0.7-kb DIG-labeled probe for the kanamycin resistance gene nptII was also prepared with plasmid DNA containing the nptII gene as the template.

Genomic DNA (5–10 µg) of cuphea-TE transgenic lines was digested with the restriction enzyme NsiI, separated by electrophoresis on 0.8% (w/v) agarose gel, and blotted onto nylon hybridization transfer membrane Hybond-N+ (Amersham Pharmacia, Amersham, UK). NsiI has a unique site located in the napin promoter. The napin promoter was isolated from B. rapa and used for seed-specific expression of the TE (Jones et al., 1995; Kridl et al., 1991). Hybridization and detection was conducted following a DIG nonradioactive hybridization and chemiluminescent detection procedure as described in the instructions (Roche, Germany) as follows. Hybridization was done overnight at 50°C in the presence of the cuphea-TE transgene probe, with stringency washes at 68°C in a 2x wash solution [2x SSC—1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate, containing 0.1% (w/v) SDS] for 2x for 30 min. After washes and blocking, the membrane was incubated with a dilution of Anti-Digoxigenin-Fab fragment conjugated to alkaline phosphatase (AP). Followed by treatment with a 1:100 dilution of the AP substrate, CDP-Star, the luminescent signal was recorded on X-ray film. Reprobing of the membrane with a second probe, the DIG-labeled 0.7-kb nptII fragment, was conducted by the same procedure as described above for the cuphea-TE probe after removing the chemiluminescent substrate CPD-Star with distilled water and stripping the TE probe in an alkaline probe-striping solution (0.2 NaOH M, 0.1% SDS). The sizes of the bands on the films were estimated on the basis of the 1-kb Plus DNA Ladder (Gibco/BRL Life Technologies Inc., Bethesda, MD) running alongside the digested DNA samples on the agarose gel.

The fatty acid composition of seed oils was determined by gas chromatography of the methyl ester derivatives of the fatty acids (Hougen and Bodo, 1973). A sample of 10 seeds was picked randomly from the seeds of each plant to be tested. The seed oil was extracted overnight with 1 mL heptane. Then, 300 µL of 0.5 M sodium methoxide was added for methyl ester derivitization. The oven temperature was programmed to increase from 190 to 230°C. The level of each fatty acid is reported as the percentage of the total fatty acids in the seed oil. Multiple comparisons and correlation studies of fatty acid data were performed as described by Ott (1993).


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
CONCLUSIONS
 REFERENCES
 
Expression Stability of TE Transgenes in Kanamycin Selected DH Plants
A total of 333 DH1 plants were developed from embryos which had undergone kanamycin selection. DH2 seeds from the DH1 plants were tested for the fatty acid composition (Table 1). Since the embryos survived kanamycin selection, the DH1 plants developed from the embryos were expected to have the kanamycin resistance gene nptII and the TE transgene, thus accumulating the target fatty acid of the TE in the seed oil. On the basis of the fatty acid composition of the seed oil, however, some of the DH1 plants did not show the expected transgenic phenotype (Table 1). Of the 15 DH1 plants that originated from crosses between the bay-TE transgenic parental line TL1 and nontransgenic plants, three plants showed the same phenotype as the nontransgenic control plants with no accumulation of lauric acid. Among 318 DH1 plants originating from the crosses of the cuphea-TE transgenic parental line TL6 and nontransgenic plants, 17 plants had palmitic acid levels ranging from 33 to 68 µg mg–1, the same phenotype as the nontransgenic control plants, indicating no expression of the cuphea-TE transgene.


View this table:
[in this window]
[in a new window]
  		Table 1. Number of oilseed rape DH1 plants developed from microspore-derived embryos having undergone kanamycin selection which were produced by microspore culture from crosses of transgenic parental lines TL1 and TL6 with nontransgenic oilseed rape plants and the minimum and maximum levels of the target fatty acids in the seed oil of DH2 seeds.

 
A possible cause for the lack of the expected transgenic phenotype in these DH1 plants having undergone kanamycin selection was that the TE transgene in these plants was silenced. The other possibilities were that these plants might have originated from nontransgenic embryos that escaped from kanamycin selection, or the plants might carry incomplete T-DNA copies which contained only the nptII but not the TE gene. To distinguish between these possibilities, PCR and Southern blotting analyses were performed.

In PCR analyses, the three DH1 plants, represented by Lanes 1 to 3 (Fig. 1) , from crosses between the bay-TE transgenic parent TL1 and nontransgenic parents, exhibited only an internal control band identical to that of the nontransgenic breeding line 212/86 (Lane 5). In PCR analyses with primers for amplification of a 1.0-kb internal fragment of the bay-TE transgene, the transgenic parent TL1 showed the expected 1.0-kb band (Lane 4, Fig. 1a), but the three DH1 plants did not. Similarly, in PCR analysis with primers for a 0.7-kb fragment of the nptII gene, the three plants did not show any band at approximately 0.7 kb (Fig. 1b). Therefore, these three DH1 plants did not carry either the bay-TE transgene or the kanamycin resistance gene. These plants must have originated from nontransgenic embryos which escaped from kanamycin selection. Thus, the lack of lauric acid accumulation was not due to TE transgene silencing.



View larger version (54K):
[in this window]
[in a new window]
  		Fig. 1. PCR analyses for the three DH1 plants that were developed from embryos selected with kanamycin and did not accumulate lauric acid in the seed oil. a. PCR with the bay-TE gene primers for a 1.0-kb fragment and the napin promoter primers for a 0.5-kb control band. b. PCR with the nptII gene primers for a 0.7-kb fragment and the napin promoter primers. Lanes 1 to 3, the three DH1 plants; Lane 4, the bay-TE transgenic parental line TL1; Lane 5, nontransgenic control. Sizes of DNA molecular weight markers (L) are indicated in base pair.

 
Of the 17 DH1 plants developed from crosses between the cuphea-TE transgenic parental line TL6 and nontransgenic parents, eight of them did not carry the nptII gene (Lanes 2, 6, 7, 8, 10, 11, 14, 16 in Fig. 2) , indicating that these plants had also escaped from kanamycin selection. The remaining nine plants showed the 0.7-kb band representing the nptII gene (Lanes 1, 3, 4, 5, 9, 12, 13, 15, 17, Fig. 2). However, in PCR analysis with primers for amplification of an 1.1-kb fragment of the cuphea-TE transgene, these nine plants did not show the 1.1-kb band representing the cuphea-TE transgene (Lanes 1 to 17, Fig. 3) as seen in the transgenic control plants (Lane 18, Fig. 3), indicating that the T-DNA copy in these nine DH1 plants was incomplete, i.e., T-DNA contained the nptII gene but not the cuphea-TE gene. Thus, the lack of the transgenic phenotype in these nine DH1 plants was also not due to transgene silencing, but rather to the absence of the cuphea-TE transgene.



View larger version (31K):
[in this window]
[in a new window]
  		Fig. 2. PCR analyses using the nptII gene primers for a 0.7-kb fragment and the napin promoter primers for a 0.5-kb control band for the 17 oilseed rape DH1 plants that were developed from embryos selected with kanamycin and did not show enhanced levels of palmitic acid in the seed oil. Lanes 1 to 17, the 17 DH1 plants; Lane 18, the cuphea-TE transgenic parental line TL6; Lane 19, nontransgenic control plants. Sizes of DNA molecular weight markers (L) are indicated in base pair (bp).

 


View larger version (28K):
[in this window]
[in a new window]
  		Fig. 3. PCR analyses using the cuphea-TE primers for the amplification of a 1.1-kb fragment and the napin promoter primers for a 0.5-kb control band for the nine oilseed rape DH1 plants that showed the nptII gene. Lanes 1 to 17, the nine DH1 plants with the same lane number as in Fig. 2; Lane 18, the cuphea-TE transgenic oilseed rape parental line TL6; Lane 19, nontransgenic control. Sizes of DNA molecular weight markers (L) are indicated in base pair (bp).

 
The cause for the occurrence of DH1 plants with incomplete T-DNA copies was investigated by Southern blotting analyses. When an internal fragment of the cuphea-TE gene was used as a probe, the cuphea-TE transgenic parental line TL6 displayed five bands, which were approximately 11.5, 9.0, 8.5, 8.0, and 4.0 kb long, respectively (Fig. 4a) . By reprobing the same membrane with an internal fragment of the nptII gene as a probe, TL6 also showed five bands, with four of the bands having the same sizes as detected by the TE gene probe, i.e., the bands of 11.5, 9.0, 8.5, and 8.0 kb long, respectively (Fig. 4b). This indicated that the T-DNA copies represented by the four bands in the parental line TL6 had both the nptII and the cuphea-TE transgenes (referred to as compete T-DNA copies). Another band detected with the nptII probe was 6.0 kb long (Fig. 4b), whereas no band of this length was detected with the cuphea-TE probe (Fig. 4a), indicating an incomplete T-DNA copy without the TE transgene. The 4.0-kb band detected with the TE probe suggested an incomplete T-DNA copy without nptII.



View larger version (35K):
[in this window]
[in a new window]
  		Fig. 4. Southern blotting analyses of oilseed rape cuphea-TE transgenic parental line TL6 and DH2 seedlings originating from eight DH1 plants by probing with the cuphea-TE transgene probe (a) followed by re-probing with the nptII gene probe (b) and schematic representation of the position of the probes in the T-DNA construct, not drawn to scale (c). The estimated sizes of the bands are shown in kilobase pair (kb). Lanes 1 to 8 represent the eight DH1 plants that were developed from crosses of the cuphea-TE transgenic parental line TL6 with nontransgenic plants and showed cuphea-TE expression by enhanced levels of palmitic acid (18.7–35.5% palmitic acid) in DH2 seed oil; P, a plasmid carrying the cuphea-TE transgene; CK, nontransgenic control plants. LB, left T-DNA border; RB, right T-DNA border; 35S, CaMV 35S promoter; tml, tml 3' region; nptII, neomycin phosphotransferase gene; napin 5', a seed specific promoter of the napin gene from B. rapa; napin 3', 3' termination fragment of the napin gene; cuphea-TE, the cuphea-TE transgene. Hatched bars represent the probes for the nptII gene and the cuphea-TE transgenes. The NsiI site is approximately 1.9 kb from RB and 5.9 kb from LB (Jones et al.., 1995; Kridl et al.., 1991; McBride and Summerfelt, 1990).

 
Southern blotting analyses for eight DH1 plants that showed TE expression by high palmitic acid levels (187–355 µg mg–1 palmitic acid) in the seed oil and were also developed from crosses of TL6 and nontransgenic plants like the 17 DH1 plants without TE expression, provided confirmation about the four complete T-DNA copies (Lanes 1 to 8, Fig. 4a,b). Also, these DH1 plants showed the 4.0-kb band representing a T-DNA copy without nptII and the 6.0-kb band representing a T-DNA copy without TE. In addition, the incomplete T-DNA copy without TE (represented by the 6.0-kb band) segregated from the complete T-DNA copies and also from the incomplete copy without nptII. For example, some plants (Lanes 1, 3, 5, and 7, Fig. 4b) showed some or all the four complete T-DNA copies but not the 6.0-kb band, and other plants (Lanes 4 and 7, Fig. 4a,b) showed only the 6.0- or the 4.0-kb bands but not both. The existence of a T-DNA copy without TE in the transgenic parental line TL6 and segregation of this incomplete T-DNA copy from other T-DNA copies provided an explanation for the occurrence of DH1 plants with an incomplete T-DNA copy only.

In summary, no plants with silenced TE transgene were detected in more than 300 bay-TE and cuphea-TE transgenic DH1 plants having undergone kanamycin selection. Therefore, the in vitro culture process does not necessarily induce transgene silencing, influencing the expression of the target fatty acids of TE transgenes. Influence of tissue culture processes on the expression of the bay-TE transgene has been excluded in a previous study (Voelker et al., 1996).

Stability of TE Transgenic DH Lines over Generations
The mean lauric acid level in the DH3 seeds (320 µg mg–1) of the 11 bay-TE lines were not significantly different from the mean lauric acid level in the DH4 seeds (304 µg mg–1) harvested from concurrently grown plants of these lines (Table 2). Comparison within individual lines over generations showed no significant difference in the lauric acid level in eight of the 11 lines. All the plants of the other three lines accumulated over 260 µg mg–1 lauric acid; therefore, the difference between the generations in the three lines was not caused by transgene silencing. The six elm-TE transgenic lines from crosses of TL3 with different nontransgenic parents showed stable expression over the three generations DH3, DH4, and DH5 (Table 2). The five nutmeg-TE lines also showed stability over the same three generations. One line (line 25) had a lower palmitic acid level in the DH3 seeds, but the DH4 and DH5 seeds were not significantly different. The 12 cuphea-TE transgenic DH lines showed stability over the DH3 and DH4 generations for the mean palmitic acid levels and in the comparisons within each line over generations in each cross with the nontransgenic parents (Table 2). In general, the target fatty acid level of the TE transgenes was stable over generations. Stable expression over generations has been observed in studies with various transgenes (Fearing et al., 1997; McCabe et al., 1999; Scott et al., 1998), including the bay-TE transgene in oilseed rape plants (Voelker et al.,1996). For example, the expression level of a transgenic CryIA(b) gene in Bt maize (Zea mays L.) lines was stable over successive backcross generations, without significant differences between BC1, BC2, BC3, and BC4 populations planted concurrently (Fearing et al., 1997).


View this table:
[in this window]
[in a new window]
  		Table 2. Mean lauric acid (C12:0) level in DH3 and DH4 seeds of bay-TE transgenic oilseed rape DH lines developed from crosses of the transgenic parent TL1 with three nontransgenic cultivars Apollo, AC Excel, and Mercury and mean palmitic acid (C16:0) level in DH3, DH4, and DH5 seeds of elm-TE, nutmeg-TE, and cuphea-TE transgenic oilseed rape DH lines from crosses of TL3, TL5, and TL6 with the same three nontransgenic cultivars.

 
Influence of Temperature on the Fatty Acid Composition
Exposure to different temperatures during seed development did not result in different levels of the target fatty acids in the two bay-TE DH lines and the three nutmeg-TE DH lines tested in this study (Table 3). The lauric acid level in the seed oil of the two bay-TE DH lines and the palmitic acid level in the seed oil of the three nutmeg-TE DH lines were the same in the two environments: 25/20°C and 20/15°C day/night temperatures. However, the level of the target fatty acid palmitic acid in the seed oil of the three elm-TE DH lines and the three cuphea-TE DH lines was influenced by temperature during seed development (Table 3). The average palmitic acid level of the three elm-TE transgenic lines was 3.1% higher at 25/20°C than at 20/15°C. The higher temperatures resulted in a mean palmitic acid level 13.4% higher in the three cuphea-TE transgenic lines. The increase in the palmitic acid level under higher temperatures was accompanied by increases in the levels of myristic acid (C14:0) and stearic acid (C18:0), and decreases in oleic acid (C18:1) and linolenic acid (C18:3) for both the elm-TE and the cuphea-TE DH lines (Table 3). For example, the 3.1% increase in the palmitic acid level in the seed oil of the elm-TE transgenic lines under elevated temperatures was accompanied by a 1.9% increase in the myristic acid level and a 0.5% increase in the stearic acid level. Growth conditions have been shown to influence expression of other transgenes in previous studies (Matzke et al., 1994; McCabe et al., 1999; Senior, 1998). Increased temperature (Conner et al., 1998; Köhne et al., 1998; Matzke et al., 1994) and high light intensity (van der Krol et al., 1990) were reported to affect transgene expression. For example, kanamycin-sensitive progeny from self-pollination of homologous 1-locus tobacco (Nicotiana tabacum L.) transgenic lines occurred at a frequency of 0.5 to 5.9 x 10–4 under close-to-optimum environmental conditions, but the frequency became as high as 1.5 to 3.8 x 10–3 under heat and/or drought stress (Conner et al., 1998). Another study in tobacco has shown differences in heat stability of two sequences coding for the same enzyme, where one sequence showed reduced expression but the expression of the other GC-rich sequence was stable under the same heat stress (Köhne et al., 1998). In this study, elm-TE and cuphea-TE transgenic DH lines showed increased levels of the targeted fatty acid under higher temperatures but bay-TE and nutmeg-TE DH lines did not. The difference of these transgenic lines in the response to temperature could be due to the differences in the DNA sequences of these TE transgenes or in the TE enzymatic activities, among other possible explanations.


View this table:
[in this window]
[in a new window]
  		Table 3. Mean fatty acid composition of oilseed rape DH3 plants carrying the bay-TE, elm-TE, nutmeg-TE, or cuphea-TE transgenes grown under low temperatures (20/15°C, day/night) and high temperatures (25/20°C) during seed development.

 
Influence of temperature on the fatty acid composition of the seed oil of nontransgenic oilseed rape cultivars has been reported (Deng and Scarth, 1998; Wilmer et al., 1996; Pritchard et al., 2000). However, the mechanism underlying the response of TE transgenic plants to the temperature may be different from that of nontransgenic oilseed rape cultivars. Increased temperatures led to enhanced levels of the saturated fatty acids (palmitic acid and stearic acid) accompanied by an increase in the monunsaturated fatty acid (oleic acid) in the seed oil of nontransgenic cultivars (Deng and Scarth, 1998). However, high temperature conditions resulted in an increased palmitic acid accompanied by a decreased oleic acid level in the TE transgenic lines. It is possible that the higher temperatures increased the relative activity of the TE compared to the enzymes for the synthesis of the C18 fatty acids, e.g., KAS II, an enzyme responsible for the elongation of palmitic acid-ACP to stearic acid-ACP, thus resulting in an accumulation of the target fatty acids of the TE with a reduction in the percentage of the C18 fatty acids.

Variation within TE Transgenic DH Lines
The seed oil of individual DH3 plants within the same transgenic DH line exhibited variation in the level of the target fatty acids as shown by the scatter plot of the lauric acid and palmitic acid levels (Fig. 5) . The range of the lauric acid level in the seed oil of individual DH3 plants of bay-TE DH line 962 was 30.0 to 42.6%, a 1.4-fold difference between the maximum and the minimum levels. The other seven DH lines carrying the bay- or the cuphea-TE transgenes had an 1.2- to 1.8-fold difference in the level of the target fatty acids within the same lines. The coefficients of variation (C.V., standard deviation over mean in percentage) of the eight lines were from 5 to 16%.



View larger version (18K):
[in this window]
[in a new window]
  		Fig. 5. Lauric acid (C12:0) or palmitic acid (C16:0) level (%) in the seed oil of individual DH3 plants of eight oilseed rape DH lines carrying the bay-TE or the cuphea-TE transgenes. Each dot represents the average of two tests of seed samples from the same plant.

 
Individual plants of the same DH lines are expected to be genetically identical (Wenzel and Foroughi-Wehr, 1994). However, this study showed variation in TE expression among plants within each of the eight DH lines carrying the bay-TE or the cuphea-TE transgenes. Variation within genetically identical transgenic plants has also been reported with the expression of a ß-glucuronidase transgene in asexually propagated transgenic tobacco plants (N. tabacum), which had C.V. values ranging from 4 to 20% (Bhattacharyya et al., 1994). Such plant-to-plant variation among genetically identical individuals has been ascribed to environmental and experimental error (Bhattacharyya et al., 1994).

DH3 plants derived from the different chromosome number doubling treatments, MT and RT, confirmed that the plant-to-plant variation in TE expression within individual DH lines was not associated with the in vitro culture process. Because the magnitude of the standard deviation is in proportion to the mean (Bowman and Watson, 1997) and the mean levels of the target fatty acid of the DH lines showed significant differences (Table 4), comparison of the variations of the lines was based on C.V., not on standard deviations. The four DH lines for which the chromosome number was doubled by MT had similar C.V. (8.4% on average) to the four lines doubled by RT (9.5% on average), with some DH lines (e.g., lines 962 and 995) from MT having a C.V. value smaller than lines from RT (e.g., line 1345). RT is applied to the haploid plants after the in vitro culture process. Therefore, any mutated loci caused by the culture process would be homozygous. However, if mutations occurred during the in vitro culture after chromosome doubling by MT, the mutated loci would be in a heterozygous state. Segregation of the heterozygous loci would increase the variation among plants within the individual DH lines. Therefore, the similar magnitude of the variation of the DH lines from the two treatments, RT and MT, indicated that no mutations with a significant influence on the target fatty acids of the TE in the transgenic DH lines tested in this study had occurred during the in vitro culture process. This is consistent with studies on the genetic stability of DH progenies in cereals, in which about 90% of the DH lines were estimated to be genetically uniform (Hu and Kasha, 1997). These results support the use of MT for doubling the chromosome number since DH lines from MT and RT were shown to have a similar degree of homozygosity and MT is more convenient and more effective than RT (Mathias and Robbelen, 1991; Möllers et al., 1994).


View this table:
[in this window]
[in a new window]
  		Table 4. Plant to plant variation within bay-TE and cuphea-TE oilseed rape transgenic DH lines for which the chromosome number was doubled by colchicine treatment at the initiation of microspore culture (MT) or root treatment after in vitro culture (RT).

 

   CONCLUSIONS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
REFERENCES
 
Expression stability of TE transgenes was studied in oilseed rape DH lines developed by microspore culture. No TE transgene silenced DH plants were detected in DH plants developed by microspore culture and selected with kanamycin at the embryo stage. Tests of 34 TE transgenic DH lines in controlled environments showed that the target fatty acid level of seed oil was usually identical in concurrently grown TE transgenic plants of different generations (DH3, DH4, and DH5 seeds), indicating stable inheritance and expression of the TE transgenes in DH plants. Higher temperatures during seed development produced higher target fatty acid levels than lower temperatures in two types of TE transgenic DH lines. DH lines also showed variation from plant to plant, with C.V. ranging from 5 to 16% for the eight DH lines tested. On the basis of comparison of the eight DH lines, which originated from two different chromosome number doubling treatments, RT and MT, in vitro culture processes did not induce genetic mutations, which significantly influence the target fatty acid level. These results support the application of the DH technology in breeding for TE transgenic oilseed rape cultivars.

Received for publication November 11, 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 


Related articles in Crop Science:

THIS ISSUE IN CROP SCIENCE

Crop Science 2004 44: 707-710. [Full Text]  



This article has been cited by other articles:


Home page
 Crop Sci.Home page
R. Scarth and J. Tang
Modification of Brassica Oil Using Conventional and Transgenic Approaches
Crop Sci., April 25, 2006; 46(3): 1225 - 1236.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Figures Only
Right arrow Full Text (PDF) Free
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Related articles in Crop Science
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (2)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Tang, J.
Right arrow Articles by McVetty, P. B. E.
Right arrow Search for Related Content
PubMed
Right arrow Articles by Tang, J.
Right arrow Articles by McVetty, P. B. E.
Agricola
Right arrow Articles by Tang, J.
Right arrow Articles by McVetty, P. B. E.
Related Collections
Right arrow Cell Biology & Molecular Genetics
Right arrow Crop Genetics
Right arrow Other Oil Crops

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
The SCI Journals Agronomy Journal Vadose Zone Journal
Journal of Plant Registrations Soil Science Society of America Journal
Journal of Natural Resources
and Life Sciences Education		 Journal of
Environmental Quality