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CELL BIOLOGY & MOLECULAR GENETICS

Inheritance of Multiple Transgenes in Wheat

^a Dep. of Agronomy, Univ. of Nebraska, Lincoln, NE 68583 USA
^b Dep. of Plant Pathology, Univ. of Nebraska, Lincoln, NE 68583 USA
^c Center for Biotechnology, Univ. of Nebraska, Lincoln, NE 68583 USA
^d Center for Biotechnology/Dep. of Agronomy, Univ. of Nebraska, Lincoln, NE 68583 USA

pbaenziger1{at}unl.edu

	ABSTRACT

TOP NOTES ABSTRACT INTRODUCTION Materials and methods Results and discussion Conclusions REFERENCES

 
Understanding and predicting transgene linkage relationships is required to utilize efficiently transformation technologies in breeding programs. This study was conducted to determine the co-transformation frequency of three gene sequences by means of a three-plasmid co-transformation system and to determine the mode of inheritance for two genes of interest. Twenty-five independently transformed wheat (Triticum aestivum L.) plants were produced by microprojectile bombardment of 1080 immature embryos with a three-plasmid system. Polymerase chain reaction (PCR) analyses of T₁ progeny from each of the 25 T₀ plants indicated co-transformation of all three plasmids at a frequency of 36%. Two of nine transgenic families containing all three plasmids were characterized for the segregation of the two genes of interest in T₁, T₂, and T₂ testcross generations by PCR. These data suggest that the two genes of interest are linked in both families studied. On the basis of the inheritance of the two genes of interest, one transgenic family would be desirable for use in a breeding program because it contained tightly linked genes, whereas the other family studied would not be desirable for use in a breeding program because the two genes segregated aberrantly.

Abbreviations: LBA, leaf bleach assay • MS, Murashige and Skoog • NPT II, neomycin phosphotransferase II • PCR, polymerase chain reaction • RL, RNase L • 2-5A, 2-5A synthetase

	INTRODUCTION

TOP NOTES ABSTRACT INTRODUCTION Materials and methods Results and discussion Conclusions REFERENCES

 
PLANT BREEDERS have the ability to broaden the gene pool in their breeding programs via transformation technologies. The use of the tools of biotechnology, including transformation, can provide a strong complement to traditional breeding methods. Wheat and other monocotyledonous plants are often transformed via microprojectile bombardment (Vasil et al., 1992; Weeks et al., 1993), although Agrobacterium tumefaciens mediated transformation of wheat has been achieved (Cheng et al., 1997).

For many traits, it is often desirable to transfer multiple genes or to pyramid several novel genes in one line. Transferring multiple genes can be achieved through transformation using one gene at a time, but this would likely prove to be impractical and inefficient (Chen et al., 1998). By means of microprojectile bombardment, a number of plasmids containing genes of interest and a selectable marker can be mixed before bombardment and used for co-transformation (Chen et al., 1998). The co-transformation of multiple transgenes present on separate plasmids has been employed successfully via microprojectile bombardment in a number of plant species, including common bean (Phaseolus vulgaris L.; Aragão et al., 1996), maize (Zea mays L.; Gordon-Kamm et al., 1990; Spencer et al., 1990, 1992; Brettschneider et al., 1997; Zhang et al., 1996), oat (Avena sativa L.; Pawlowski et al., 1998), soybean [Glycine max (L.) Merr.]; (Hadi et al., 1996), rice (Oryza sativa L.; Kohli et al., 1998; Chen et al., 1998; Maqbool and Christou, 1999), and wheat (Triticum aestivum L.; Barro et al., 1998). Aragão et al. (1996), Brettschneider et al. (1997), Chen et al. (1998), and Maqbool and Christou (1999) reported co-transformation frequencies ranging from 20 to 80% determined on the basis of presence of the DNA sequence. In addition, Gordon-Kamm et al. (1990) and Spencer et al. (1990, 1992) reported co-transformation frequencies of 18, 50, and 63% determined on the basis of co-expression of two transgenes. To our knowledge, the largest number of plasmids transferred via microprojectile bombardment is 12 and 13 in soybean and rice, respectively (Hadi et al., 1996; Chen et al., 1998).

Transgenes must be integrated into the chromosome and inherited in a stable manner to be of use in a breeding program. Traits controlled by multiple genes or pyramided genes transferred via transformation are most useful to plant breeders if the transgenes are inherited as a tightly linked unit. Little is known about the fate of transgenes once integrated into the plant chromosome. Commonly, transgene segregation is based on expression patterns, which may lead to distorted segregation ratios (Pawlowski and Somers, 1996) due to gene silencing and reduced transgene expression (Finnegan and McElroy, 1994; Matzke and Matzke, 1995; Taylor, 1997; Depicker and Van Montagu, 1997; Stam et al., 1997). Transgene segregation can also be characterized by determining transgene DNA segregation by Southern hybridization and polymerase chain reaction (PCR). However, characterizing transgene segregation at the DNA level does not provide information about the expression of the transgene, which must be considered before using the transgenic material in a breeding program. Transgene inheritance characterized at the DNA level avoids expression-related distortions.

Transgenes are generally expected to behave as dominant genes and segregate in a 3:1 ratio for transgenic to wild-type (non-transgenic) progeny when the plant is self-pollinated, because the transgene locus is considered to be hemizygous in the primary transformant (Pawlowski and Somers, 1996). Transformation experiments in several different crops report that transgenes are often, but not always, inherited in a simple, Mendelian fashion (Srivastava et al., 1996; Barro et al., 1998; Gallo-Meagher and Irvine, 1996; Register et al., 1994, Armstrong et al., 1995; Christou, 1997; Pawlowski et al., 1998; Ulian et al., 1994, 1996; Yao et al., 1997; Christou et al., 1989; Tomes et al., 1990). In addition, co-transformation experiments utilizing genes originating on separate plasmids indicate that those genes are often, but not always, inherited together and act as linked genes (Chen et al., 1998; Walters et al., 1992; Kohli et al., 1998; Pawlowski and Somers, 1998). Little of the previous research was done with wheat or with multiple plasmids in microprojectile bombardment. Hence, additional research is needed before transgenic wheat lines involving multiple genes of interest can be used in a breeding program.

We produced and analyzed 25 independently transformed wheat families co-bombarded with a three-plasmid system. The three-plasmid system consisted of two genes of interest and a selectable marker gene. Our goal was to determine the co-transformation frequency of the three plasmids, and the genotypic segregation patterns associated with the two genes of interest in two generations of self-pollination and one testcross generation.

	Materials and methods

TOP NOTES ABSTRACT INTRODUCTION Materials and methods Results and discussion Conclusions REFERENCES

 
Gene Constructs
A three-plasmid system (Fig. 1) consisting of a selectable marker plasmid and the two parts of the 2-5A system was used for wheat transformation. Construction of plasmids RL and 2-5A are described by Mitra et al. (1996). The selectable marker plasmid (pUbiNPTII-I) contained neomycin phosphotransferase II (NPT II), driven by the maize ubiquitin promoter (Christensen et al., 1992). In addition to RNase L and 2-5A synthetase genes, the RL and 2-5A plasmids contain NPT II genes driven by the T-DNA nos (nopaline synthase) promoter. The nos promoter, however, is a very weak promoter in wheat compared with the ubi promoter, which drives the NPT II gene present on pUbiNPTII-I (Mitra, 1999, unpublished data).

View larger version (16K): [in this window] [in a new window]   Fig. 1 A partial map of plasmids (a) pRL, (b) p2-5A, and (c) pUbiNPTII-I used in the wheat transformation, including restriction sites used for Southern hybridization

A 1 : 0.5 : 0.5 molar ratio of pUbiNPTII-I, pRL, and p2-5A plasmid DNA totaling 25 µg, was precipitated onto 1-µm gold particles following a modification of Sanford et al. (1987). Immature embryos were bombarded two times at a distance of 5 cm below the microcarrier assembly shelf using a Biolistic PDS-1000/He Particle Delivery System (Bio-Rad Lab., Hercules, CA). The helium pressure of the bombardment was 9.3 MPa (1350 psi). The gap distance between the rupture disk and macrocarrier was 0.6 cm, and the macrocarrier travel distance was 11 mm. The bombarded embryos remained on the high osmotic medium for 16 to 24 h post-bombardment.

Sixteen to twenty-four hours post-bombardment, embryos were placed on callus induction medium containing the pre-culture medium supplemented with 25 mg L^-1 geneticin (G418; Sigma, St. Louis, MO) for 2 wk in the dark at 24°C. G418, the selective agent for NPT II, was added to the medium to select for transformed tissues. Following the 2-wk induction phase, tissue was sectioned into approximately 2-mm pieces. Care was taken to label callus pieces derived from the same embryo lineage. The induced calli were transferred to shoot regeneration medium composed of MS salts and vitamins supplemented with 0.25 mg L^-1 2,4 D and 40 mg L^-1 maltose, pH 5.7 at 24°C, under a 16-h light (intensity of 180 µmol m^-2 s^-1) and 8-h dark regime for 2 wk. Selection pressure of 25 mg L^-1 G418 was maintained during shoot regeneration. Differentiating calli were transferred to root elongation medium consisting of MS salts and vitamins and 40 mg L^-1 maltose. The selection pressure of 25 mg L^-1 G418 was maintained during elongation, and rooted plants were acclimated to soil and grown to maturity in the greenhouse.

NPT II Expression Analysis
T₀ plants were assayed for the expression of NPT II to distinguish transformed plants from non-transformed plants by two NPT II expression assays. A modification of a leaf bleach assay (LBA) described by Cheng et al. (1997), was conducted on young leaf tissue from plants at approximately the three-leaf stage. In addition, total protein was extracted from leaf tissue and used in an enzyme-linked immunosorbant assay (ELISA) according to the specifications of a NPT II ELISA kit (5 Prime3 Prime, Inc., Boulder, CO). T₀ plants were determined to be positively transformed if NPT II protein was detected by ELISA. The ELISA and LBA were both performed on 80 T₁ plants to verify the accuracy of the LBA.

Molecular Characterization
Total genomic DNA was isolated from wheat plants at the three-leaf stage following the procedure of Dellaporta et al. (1983). PCR reactions were performed on all T₁, T₂, and T₂ testcross plants by amplifying the coding regions of transgenes present on pRL and p2-5A using the following sets of primers: RNase L (forward) 5'-GCAGGGATCATAACAACCC-3' and (reverse) 5'-GGGCGGTATATTTACTGTGG-3'; 2-5A synthetase (forward) 5'-TTCCTCAGTCCTCTCACCAC-3' and (reverse) 5'-GAGCTGCCTTCTCAGGTACT-3'. T₁ plants were also subjected to PCR amplification of the ubi I promoter region specific to pUbiNPTII-I, utilizing ubi I primers (forward) 5'-TTGACAACAGGACTCTACAG-3' and (reverse) 5'-GTGTGGAGGGGGTGTCTATT-3'. The expected PCR products for each primer pair were 1059, 633, and 658 bp for RNase L, 2-5A synthetase, and ubi I promoter region, respectively.

Cycling parameters began with an initial template denaturation at 94°C for 5 min followed by 30 cycles of 1) 94°C for 30 s, 2) 54°C (RNase L), 58°C (2-5A synthetase), 56°C (ubi I) for 30 s, and 3) 72°C for 1 min. Thirty cycles were followed by a final extension time of 5 min at 72°C. Following PCR amplification, 10-µL PCR product was loaded into a 0.8% (w/v) agarose gel and subjected to electrophoresis for 1 h at 85 V followed by visualization under UV light.

Southern hybridization was carried out on a subset of 20 progeny to confirm the accuracy of the PCR. Fifteen micrograms of genomic DNA was digested overnight with HindIII and separated on a 0.8% agarose gel at 25 V overnight and alkali blotted onto a Zeta-probe GT genomic tested blotting membrane according to the manufacturer's specifications (Bio-Rad, Lab., Hercules, CA). Twenty-five nanograms probe DNA was radiolabeled with -³²P-labeled dCTP by means of the Prime-It II random primer labeling kit according to the manufacturer's protocol (Stratagene, La Jolla, CA). PCR products consisting of the coding regions for RNase L and 2-5A synthetase were used as probes to detect RNase L and 2-5A synthetase, respectively. Pre-hybridization and hybridization were carried out at 65°C in the presence of high salt buffer following the manufacturer specifications. Membranes were analyzed by autoradiography with X-Omat AR5 film (Eastman Kodak, Rochester, NY).

Population Construction and Analysis
Twenty T₁ seeds (unless fewer seeds were available), harvested from each of 25 independently transformed wheat plants (T₀), were planted in the greenhouse and evaluated for segregation of RNase L (RL) and 2-5A synthetase (2-5A) to estimate the pattern of inheritance of each transgene. The genomic DNA collected from each T₁ plant was used for PCR amplification of RL and 2-5A to determine if each of the transgene sequences was present or absent. An aliquot of each T₁ DNA sample from each T₀ family was pooled together by T₀ family and subjected to PCR amplification of the ubi I promoter to indicate whether the selectable marker plasmid was present. Amplification of the ubi I promoter region allowed an appropriate co-transformation frequency of all three plasmids to be calculated.

The PCR data for RL and 2-5A segregation were subjected to goodness of fit testing for independently assorting gene ratios of 9:3:3:1 (3:1 RL x 3:1 2-5A), 225:15:15:1 (15:1 RL x 15:1 2-5A), 45:3:15:1 (15:1 RL x 3:1 2-5A), 45:15:3:1 (3:1 RL x 15:1 2-5A) and two independently inserted linked transgene ratios of 12:0:3:1 (3:1 RL and 2-5A x 3:1 2-5A alone) and 12:3:0:1 (3:1 RL and 2-5A x 3:1 for RL alone) by the ² statistic (P < 0.05). On the basis of the T₁ segregation patterns observed, self-pollinated T₂ seeds were harvested from T₁ plants from T₀ Families 20 and 26 for further transgene segregation analysis. The families were chosen because T₁ analysis of Family 20 indicated linked insertions of RL and 2-5A, whereas T₁ data from Family 26 indicated insertions of RL and 2-5A may not be linked.

At least 10 T₂ progeny from each of five T₁ plants (RL positive/ 2-5A positive) from Family 20 and six T₁ plants (RL positive/ 2-5A positive) from Family 26 were scored for the presence or absence of RL and 2-5A using PCR. Progeny scoring was subjected to ² goodness of fit tests for T₀ Families 20 and 26. T₂ progeny from Family 20 were tested against genetic ratios of 3:1 for linked genes from a hemizygous parent, and 15:1 for two linked insertions of RNase L and 2-5A synthetase, and for linked or unlinked genes from a homozygous parent (1:0). T₂ progeny from Family 26 were also tested against genetic ratios of 3:1 and 15:1, as well as a 9:3:3:1 ratio, indicative of independent insertions of RNase L and 2-5A synthetase from a hemizygous parent. Chi-square statistics in single gene ratios were calculated using Yates' correction factor for small data sets (Steel et al., 1980).

T₂ testcrosses were made by crossing selected T₂ plants from T₀ Families 20 and 26 as a male by the approach method (Rosenquist, 1927). The female plant was a non-transgenic spring wheat cv. Oxen. Testcross seed was harvested and planted in the greenhouse. Genomic DNA from each of the testcross progeny was used for PCR amplification of RNase L and 2-5A synthetase. PCR data were tested against expected ratios of 1:1:1:1 (independently segregating transgenes), 1:1 (linked transgene segregation), and 1:0 (homozygous for both transgenes) by ² goodness of fit tests (P < 0.05).

	Results and discussion

TOP NOTES ABSTRACT INTRODUCTION Materials and methods Results and discussion Conclusions REFERENCES

 
Wheat Transformation
Twenty-five independently transformed wheat plants were produced from co-bombardment of 1080 immature embryos at a transformation frequency of 2.3%. Leaf bleach assay (LBA) analyses of all 25 T₀ plants confirmed the expression of NPT II. A comparison of the LBA and ELISA expression assays conducted on 80 T₁ plants found total agreement between the two assays and indicated that a leaf bleach assay was a highly effective and reliable assay to determine the expression of NPT II in transgenic wheat plants (data not shown).

Co-Transformation Efficiency
PCR amplification of sequences specific to each plasmid in T₁ progeny allowed the co-transformation frequency to be calculated (Fig. 2) . Results from southern blotting confirmed the accuracy of our PCR detection system in a subset of progeny (Fig. 3) . This provides evidence that gene fragmentation did not influence the PCR by producing false negatives, as was reported by Aragão et al. (1996) and Register et al. (1994) in common bean and maize experiments, respectively.

View larger version (48K): [in this window] [in a new window]   Fig. 2 (a) PCR amplification of a 1059 bp RNase L fragment in 14 T1 progeny segregating for RNase L: lanes: 1, 1 kb ladder; 2-15, T1 progeny; 16, skip; 17, pRL, positive control; 18, skip; 19, non-transformed plant, negative control. (b) PCR amplification of a 633 bp 2-5A synthetase fragment in 16 T1 progeny segregating for 2-5A synthetase: Lanes: 1, 1-kb ladder; 2-17, T1 progeny; 18, p2-5A, positive control; 19, non-transformed plant, negative control

View larger version (95K): [in this window] [in a new window]   Fig. 3 Southern hybridization of RNase L in six segregating T2 testcross progeny. DNA was restricted with HindIII and hybridized with RNase L probe. The positions of DNA size markers are given to the left of the panel. Lanes: 1 through 6, T2 testcross progeny; 7 non-transformed plant, negative control; 8, pRL, positive control. Comparative results of the PCR amplification of RNase L are given at the top of the panel

T₀ plants would be expected to contain pRL and p2-5A at nearly the same frequency, because an equal molar (1:1) concentration of the two plasmids was used in the bombardment and no selection pressure was placed on either gene. pRL (64%) was present in T₀ plants more often than p2-5A (48%). This result was likely the result of chance, due to the small number of transgenic families studied. Assuming that RL and 2-5A have an equal chance of integration, our data fit the expected ratio of 1:1 when tested at the 95% level. It is possible that there is an "expression threshold" of 2-5A synthetase in the plant that may have been responsible for p2-5A being present at a lower frequency than pRL, because 2-5A synthetase is known to be extremely toxic to plants (Mitra et al., 1996).

RNase L and 2-5A synthetase Segregation in T₁ Progeny
Three of the nine T₀ families (19, 25, 26), containing the two transgenes of interest fit a 9:3:3:1 ratio for the segregation of RL and 2-5A in T₁ progeny (Table 2) . A 9:3:3:1 ratio indicates that RL and 2-5A behave as two independent loci, thus suggesting integration occurred at independent locations in the plant chromosome. Family 6 fits a 12:3:0:1 ratio indicating that integration occurred at two locations, with one locus containing linked RL and 2-5A and the other locus containing RL alone. Family 25 also fits ratios of 45:15:3:1 and 45:3:15:1 in addition to a 9:3:3:1, making it difficult to decipher the segregation pattern without a larger sample size. T₁ progeny from Family 20 fit a 3:1 ratio and a 15:1 ratio for RL and 2-5A, providing evidence that the two transgenes were linked at one or possibly two integration sites in this event. The remaining four families do not fit any of the tested ratios and contain a large number of progeny in the RL negative/2-5A negative category, which would not be characteristic of any simple Mendelian ratio. Larger sample sizes are needed to better determine the inheritance of RL and 2-5A relative to one another in the T₁ generation.

View this table: [in this window] [in a new window]   Table 2 Chi-square analysis of 2-5A synthetase (2-5A) and RNase L (RL) in T1 progeny derived from six independent events using segregation ratios of 9:3:3:1, 12:0:3:1, 12:3:0:1, 225:15:15:1, 45:15:3:1, 45:3:15:1, 3:1, and/or 15:1 based on PCR

Family 20 Characterization
T₂ and T₂ testcross progeny in Family 20 confirmed our hypothesis from T₁ data that RL and 2-5A act as tightly linked genes in this family. T₂ progeny from each of five T₁ plants (RL positive/ 2-5A positive) fit a 3:1 ratio for a linked RL and 2-5A. In addition, T₂ progeny from two of the five T₁ plants also fit a 15:1 ratio, though not as well as a 3:1 ratio (Table 3) . The sum total of T₂ progeny from the five T₁ plants fit the 3:1 ratio and segregated 65 RL, 2-5A positive to 22 RL, 2-5A negative. None of the five T₁ plants selected to produce T₂ progeny were homozygous for both transgenes, which would be expected to occur in one out of every three T₁ plants positive for both RL and 2-5A.

Family 26 Characterization
T₂ and T₂ testcross progeny in Family 26 did not confirm our hypothesis from T₁ data that RL and 2-5A act as independently assorting genes. In the T₁ generation, offspring from Family 26 were present in all four possible categories (RL positive/2-5A positive., RL positive/2-5A negative, RL negative/2-5A positive, RL negative/2-5A negative), and fit the 9:3:3:1 ratio expected if both transgenes segregated independently. After selecting RL positive/2-5A positive T₁ individuals to self-pollinate and produce T₂ progeny, we observed only three individuals out of 128 in the RL negative/2-5A positive category and none in the RL positive/2-5A negative category, indicating that RL and 2-5A were not independently assorting (Table 3).

T₂ progeny from T₁ plants 97-216-1 and 97-217-3 fit a 3:1 and 15:1 ratio for linked RL and 2-5A, while progeny from 97-220-1 fit only the 3:1 ratio. This result would indicate a linked insert(s) of RL and 2-5A, which did not agree with the T₁ segregation patterns. T₂ progeny from 97-216-1 and 97-220-2 appear to fit a 12:0:3:1 ratio for two independent insertions, one with linked RL and 2-5A and one with 2-5A alone, but this ratio also contradicts the T₁ segregation.

Five of the six T₂ plants used as the male parent in the testcrosses were RL positive/2-5A positive, and the remaining T₂ parent (98-237-1) was deliberately chosen to be RL negative/ 2-5A positive. If our hypothesis of independently assorting RL and 2-5A were true, the two transgenes should segregate in a 1:1:1:1 ratio if the T₂ parent was heterozygous for both transgenes. None of the six testcross families fit a 1:1:1:1 ratio, confirming results of T₂ analysis that indicated RL and 2-5A were not independently assorting. Progeny from four of the five crosses using RL positive/2-5A positive parents (98-211-2, 98-213-1, 98-217-1, and 98-223-1) fit an expected ratio of 1:1 for linked DNA containing RL and 2-5A (Table 4), suggesting a single linked insertion. The fifth (98-215-1) was most likely homozygous or possibly fit a 15:1 ratio in the T₂ generation. The 12 testcross progeny evaluated from the RL negative/ 2-5A positive T₂ testcross parent (98-237-1) did not contain RL, confirming T₂ data that indicated RL and 2-5A were not always present together in Family 26. We have no explanation for these testcross results, as the parent plant could not have been homozygous or heterozygous at a single locus. Also, there is no genetic data from previous generations suggesting multiple, unlinked insertions for 2-5A in this family.

When examining all of the data generated in the T₁, T₂, and T₂ testcross generations, a stable pattern of inheritance could not be elucidated. In the T₁ generation, RL and 2-5A appeared to be independently assorting. The data generated in the T₂ and T₂ testcross generations provide evidence that RL and 2-5A are linked and not independently assorting. There is linkage present between RL and 2-5A in Family 26, but the stability of this linkage is clearly in question. The inheritance of RL and 2-5A in Family 26 suggested that the transgenes were not stably inherited resulting in aberrant segregation ratios.

A possible explanation for the unstable behavior of RL and 2-5A in Family 26 is reduced transgene transmission to progeny or transgene loss, which may be the result of partial transgene integration or transgene rearrangement or may be due to some sort of chromosomal aberration, perhaps a deletion induced by the transformation event.

Rearrangements can result from the site of transgene integration (Christou et al., 1989; Zhang et al., 1996; Gallo-Meagher and Irvine, 1996; Ulian et al., 1994; Spencer et al., 1992; Walters et al., 1992). Pawlowski and Somers (1996) described several different mechanisms of reduced transgene transmission or transgene loss, which include the transgene integration event being linked to a deleterious mutation transmitted to offspring at a low frequency or intra-transgenic recombination occurring between clustered homologous copies of the integrated plasmids.

Chromosome rearrangements and aberrations have been reported extensively in tissue culture experiments. Youssef et al. (1989) suggested that while chromosome rearrangements and aberrations occurred during the tissue culture process, self-pollination would restore euploidy in some progeny lines. Therefore, chromosome aberrations may be present in Family 26, but in subsequent generations of self-pollination those aberrations are eliminated.

	Conclusions

TOP NOTES ABSTRACT INTRODUCTION Materials and methods Results and discussion Conclusions REFERENCES

 
This experiment clearly shows that transferring two transgenes and a selectable marker present on three separate plasmids can result in the successful co-integration of both genes of interest in a desirable form amenable to wheat breeding programs. In this study, 25 transgenic plants were produced at a frequency of 2.3%, of which nine (36%) contained all three transgenes. Family 20 was the only transgenic line that did not contain T₁ progeny in the RL positive/2-5A negative or RL negative/2-5A positive categories, and was determined, by further genetic analyses, to contain tightly linked genes of interest.

Progeny analysis of Family 20 demonstrates that it is possible to produce transgenic wheat containing tightly linked copies of two genes of interest, which would be desirable for use in a breeding program. A simple backcrossing program could be employed to transfer both RL and 2-5A to existing cultivars or elite breeding lines.

Genetic analyses of Family 26 indicated that multiple transgenes do not always behave as a stable, tightly linked gene cluster. Family 26 is a good example of a transgenic line that would not be desirable for use in a breeding program. The ambiguous segregation ratios associated with Family 26 provide evidence that T₁ transgene analyses alone are not sufficient to properly understand genetic mechanisms involved in transgene integration and inheritance. Additional analyses are required to further understand and verify transgene inheritance.

	ACKNOWLEDGMENTS

 
We thank Dr. Robert H. Silverman of the Cleveland Clinic Foundation, Cleveland, OH, for providing the RNase L gene. This research was supported in part by the University of Nebraska Center for Biotechnology and the Nebraska Wheat Board. B.T.C. was supported by the Channing B. and Katherine W. Baker Endowment fund from the University of Nebraska-Lincoln.

	NOTES

TOP NOTES ABSTRACT INTRODUCTION Materials and methods Results and discussion Conclusions REFERENCES

 
Nebraska Agricultural Research Division, Journal Series No. 12806.

Received for publication October 26, 1999.

	REFERENCES

TOP NOTES ABSTRACT INTRODUCTION Materials and methods Results and discussion Conclusions REFERENCES

 

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