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Efforts to Initiate Construction of a Disease Resistance Package on a Designer Chromosome in Tobacco.

THE DOMINANT GENE N localizes TMV infection by a hypersensitive response, and was originally transferred to cultivated tobacco from N. glutinosa via interspecific crossing and chromosome substitution by Holmes (1938). Gerstel (1948) later showed that recombination between the H chromosome of tobacco and the substitution chromosome could occur. TMV-resistant flue-cured varieties using N have since been developed, but are not widely grown because of reduced yields and quality associated with the presence of the gene (Chaplin et al., 1961, 1966; Chaplin and Mann, 1978). This negative association has been difficult to break using multiple cycles of backcrossing, and is probably due to linkage drag effects caused by undesirable N. glutinosa genes linked to N. Similar difficulties have been described after other conventional interspecific gene transfers (Legg et al., 1981; Zeven et al., 1983; Koebner and Shepherd, 1988). Traditional backcrossing to the recurrent parent can be ineffective in reducing the size of linked alien chromosomal segments surrounding gene(s) of interest (Young and Tanksley, 1989). For some resistance genes of interspecific origin, isolation of the sequence per se by gene cloning followed by reintroduction through transformation may increase opportunities for successfully deploying the genes.

Crop varieties of the future are expected to possess a large number of transgenes. In addition to backcrossing transgenes into existing elite lines, plant breeders must also continue work to improve agronomic characteristics using conventional methods. If separate transgenes are inserted into different breeding lines, then multiple breeding crosses coupled with arduous selection during breeding generations would be required to combine all of them into an elite line. This process could also be problematic in that sexual intermating could break beneficial linkage blocks that were very difficult to fix within the lines. In addition, it could be difficult to remove a group of transgenes from an elite line if it became necessary. These concerns are in addition to problems that might be associated with insertional mutagenesis caused by transgene integration into transcribed regions. Such interruption can occur frequently, and many transformants can exhibit mutant phenotypes (Heberle-Bors et al., 1988; Koncz et al., 1989; Feldmann, 1991; Lindsey et al., 1993; Birch, 1997). Since most mutations are of an unfavorable nature, the pyramiding of multiple transgenes in elite lines could theoretically act to significantly reduce yields and quality, as compared with nontransformed lines.

Plant breeders have noted the importance of considering linkage between transferred genes (Campbell et al., 2000). By linking beneficial genes, a block would be created that would segregate as a single unit. Linkages between a few transgenes can be created by simply placing them on the same construct. This approach becomes increasingly difficult as the number of transgenes increases because of technical hurdles associated with building constructs with a large number of genes. It might be ideal if transgenes could be located on a single linkage group physically separated from the crop genome. This would simplify selection during breeding, avoid problems caused by insertional mutagenesis, and allow rapid transfer of multiple transgenes to elite lines.

In yeast and human systems, artificial chromosomes have been constructed (Murray and Szostak, 1983; Harrington et al., 1997). Additional chromosomes can be accessed in plants through the use of interspecific crosses. In tobacco (N. tabacum, 2n = 48), chromosome addition line NC152 (2n = 50) has been developed by adding a single pair of chromosomes from N. africana (Wernsman, 1992). Campbell et al. (1994) proposed to use the addition chromosome of NC152 as a "designer" chromosome into which numerous transgenes could be targeted and inherited as a single linkage block. The designer chromosome could be used as a "gene shuttle" to rapidly and efficiently transfer a transgene linkage block from genotype to genotype or to remove a set of transgenes if it ever became necessary. This chromosome is mitotically stable and inherited in a predictable fashion. Campbell et al. (1994) expected the integrity of the linkage group to be preserved because recombination between the N. africana chromosome and the tobacco genome was not detected. Carlson (1995) showed that high-yielding genotypes of flue-cured tobacco containing the alien chromosome of NC152 could be produced, and that quality characteristics may be enhanced in lines possessing the extra chromosome.

The first objective of this work was to generate multiple independently-transformed lines of NC152 possessing the cloned TMV resistance gene N. These lines may be valuable as parental sources of TMV resistance, as they do not possess any accompanying deleterious N. glutinosa chromatin. The second objective was to identify transformants in which N had been inserted into the addition chromosome. For each independent transformant, [BC.sub.1][F.sub.1] families which segregated for the presence of N and the addition chromosome were produced. Cosegregation analyses and examination of rates of transmission of TMV resistance to egg nuclei were used in an attempt to identify lines in which N had been inserted in the addition chromosome. The goal was to initiate construction of a disease resistance package on the addition chromosome by linking the TMV resistance gene with a gene native to the chromosome that confers reduced susceptibility to some isolates of potato virus Y (PVY) and tobacco etch virus (TEV).

MATERIALS AND METHODS

Transformation

The transgenic doubled haploid chromosome addition line NC152-dhfr-996 (2n = 50) produced by Campbell et al. (1994) was used as an explant source for transformation by Agrobacterium tumefaciens, according to the procedure of An et al. (1986). The addition chromosome in this line was previously tagged with a mutant dhfr transgene conferring resistance to the antibiotic methotrexate (Mtx). The Agrobacterium strain possessed plasmid pTG34, bearing the selectable marker nptII and a 13.0 kb XhoI fragment from a genomic DNA clone containing N and its cis regulatory factors (Whitham et al., 1994). Two-hundred forty inoculated 1-[cm.sup.2] leaf disks were placed on solid MS culture medium (Murashige and Skoog, 1962). Twenty uninoculated leaf disks were also maintained as a regeneration control.

After 2 d of cocultivation with the vector, inoculated leaf disks were transferred to shoot regeneration medium comprised of MS inorganic salts supplemented with 4.0 mg [L.sup.-1] indole acetic acid, 2.5 mg [L.sup.-1] kinetin, 30 g [L.sup.-1] sucrose, and 7 g [L.sup.-1] agar. Also added were 250 mg [L.sup.-1] cefotaxime, 0.5 mg [L.sup.-1] Mtx, and 100 mg [L.sup.-1] kanamycin to eliminate contaminating bacteria and select for transformed cells. Uninoculated control disks were transferred to MS medium without antibiotics. Disks were transferred to fresh medium every 14 to 21 d. Regenerated shoots were transferred to rooting medium consisting of MS inorganic salts plus 30 g [L.sup.-1] sucrose and 7 g [L.sup.-1] agar. Rooted plants were transferred to soil-filled pots in a growth room and were designated as [R.sub.0] transformants.

Resistance to TMV

Response of [R.sub.0] transformants to infection by strain U1 of TMV was evaluated using the detached leaf test of Rufty et al. (1987). Plants whose leaves exhibited the localized lesions of a hypersensitive response 4- to 5-d postinoculation were classified as TMV-resistant ([TMV.sup.R]). Leaves that did not produce a hypersensitive response were classified as coming from TMV-susceptible ([TMV.sup.S]) plants.

Development of Advanced Generations

Selected [TMV.sup.R] [R.sub.0] transformants and 4 [TMV.sup.S] regeneration control [R.sub.0] regenerates were transplanted into pots in a greenhouse. Only one or two transformants per leaf disc were selected in order to minimize the number of duplicate insertion events evaluated. [BC.sub.1][F.sub.1] families were developed for each selected [R.sub.0] individual by first crossing them as females with [TMV.sup.S]/[Mtx.sup.S]/[PVY.sup.S] cultivar Petite Havana. [TMV.sup.R] [F.sub.1] plants were then backcrossed as females to Petite Havana. Single [TMV.sup.R] [F.sub.1] plants used to produce the [BC.sub.1][F.sub.1] generations were obtained by screening segregating [F.sub.1] families of 4 to 16 plants for TMV resistance.

Cosegregation Analysis for N and dhfr

[BC.sub.1][F.sub.1] families segregating for the designer chromosome ([Mtx.sup.R]) and TMV resistance (N) were derived from selected [TMV.sup.R] [R.sub.0] transformants and were used to evaluate linkage between N and dhfr. For each [BC.sub.1][F.sub.1] family, [Mtx.sup.R] seedlings were isolated by seeding surface-sterilized seeds onto medium consisting of MS inorganic salts supplemented with 1 mg [L.sup.-1] Mtx and 7 g [L.sup.-1] agar in 100 x 15 mm petri plates. After 14 d, [Mtx.sup.R] seedlings were removed from the agar and transplanted to soil-filled pots in a growth room. For initial evaluation, only 6 [Mtx.sup.R] plants from each family were tested for TMV resistance using the previously-described detached leaf test. For families exhibiting five or six [TMV.sup.R] individuals per six [Mtx.sup.R] plants, 6 to 39 additional [Mtx.sup.R] plants were tested for resistance to TMV.

Transmission of TMV Resistance in Selected [BC.sub.1][F.sub.1] Families

Additional testing was done on selected families to determine the rates of transmission of TMV resistance through the egg without selection for Mtx resistance. Previous data indicated that the addition chromosome was transmitted to female gametes of individuals monosomic for the chromosome at a rate of [approximately equals] 7 to 10%. [BC.sub.1][F.sub.1] families were selected for transmission analysis based on lack of fit to a model of a single N locus segregating on a N. tabacum chromosome. Lack of fit was determined using a chi-square goodness-of-fit test for a 1:1 ratio of [Mtx.sup.R]/[TMV.sup.R]:[Mtx.sup.R]/[TMV.sup.S] plants. The test was applied to each family, and the model was rejected if [chi square] [is greater than or equal to] 3.84 (P [is less than] 0.05, 1df).

For transmission analysis, 99 to 100 whole, unselected [BC.sub.1][F.sub.1] plants from each selected family were inoculated with TMV. Two leaves per 30-d-old plant were inoculated using a cotton-tipped applicator, and inoculum was prepared as previously described. Plants were evaluated for response to TMV 5 to 6 d postinoculation. Ratios of [TMV.sup.R]:[TMV.sup.S] plants were tested against a model of a single N locus being transmitted on the addition chromosome. Chi-square analysis was performed to test if genetic segregation of TMV resistance fit a 7.67 [TMV.sup.R]:92.33 [TMV.sup.S] ratio. This ratio was based on the 7.67% transmission rate of the alien chromosome observed in this experiment. The model hypothesizing insertion on the designer chromosome was accepted if [chi square] [is less than] 3.84 (P [is greater than] 0.05, 1df).

Molecular Analysis

Southern gel-blot hybridizations and polymerase chain reactions (PCRs) were conducted for individuals from a single family (GH96-3700 [BC.sub.1][F.sub.1]) in which cosegregation and transmission analyses suggested insertion of N in the addition chromosome. Plants were those used in the cosegregation analysis. For Southern blots, genomic DNA was extracted according to Rogers and Bendich (1985) and 5 mg DNA/sample was digested with XbaI according to manufacturer's recommendations. XbaI cuts at a single site within the T-DNA borders of pTG34 and within the N open reading frame. Digested DNA was electrophoresed and transferred to nylon membranes according to Sambrook et al. (1989). Hybridization of a 545 bp N-specific radiolabeled probe to membranes and preparation of autoradiograms were performed according to Sambrook et al. (1989). The probe was created by PCR amplification using primers (5'-ACCAGAATGATATGTTCCAC-3') and (5'-GGACTCAACGTTAATTCTCTG-3'), and was double labeled with [Alpha]-[sup.32]P-dCTP and [Alpha]-[sup.32]P-dATP using a random primed labeling kit according to manufacturer's instructions.

Reaction mixes for PCR assays were constructed according to Taq DNA Polymerase manufacturer's recommendations (Boehringer Mannheim, Indianapolis, IN). Reaction parameters were 94 [degrees] C for 1 min, 55 [degrees]C for 1 min, and 72 [degrees] C for 1 min for 25 cycles, using 250 ng of genomic DNA and 250 ng of each of the above primers in 100 mL reaction volumes. The expected PCR product in plants carrying the N transgene was 545 bp.

Isolation of Disomic dhfr dhfr-N N Lines

Six [Mtx.sup.R]/[TMV.sup.R] plants from the family GH96-3700 [BC.sub.1][F.sub.1] were crossed as females with N. africana to produce an array of gynogenic haploid plants according to Burk et al. (1979). The detached leaf test described above was used to identify [TMV.sup.R] plants. [TMV.sup.R] plants were then screened for resistance to Mtx by culturing three 0.7-em diameter leaf disks/plant on the regeneration medium described above containing 0.5 mg [L.sup.-1] Mtx. [TMV.sup.R]/[Mtx.sup.R] haploid plants were then chromosome doubled according to Kasperbauer and Collins (1972).

RESULTS

Production of Transgenic Material

More than 400 plants were regenerated from culture of leaf discs inoculated with A. tumefaciens. The detached leaf test is a highly reliable assay for identifying plants possessing the N-gene (Rufty et al., 1987). Inoculated leaves from transgenic [TMV.sup.R] plants exhibited a clear hypersensitive response comparable to that observed for leaves from non-transgenic [TMV.sup.R] materials. One-hundred forty-six [TMV.sup.R] [R.sub.0] plants from 121 different leaf disks were selected for generation of [BC.sub.1][F.sub.1] families. This ensured that at least 121 of the 146 transgenic plants were independent transformants. Some plants would undoubtedly have contained multiple transgene insertions, and some insertions would have been lost during generation of [BC.sub.1][F.sub.1] families due to segregation. However, a minimum of 121 different N loci were being evaluated for linkage to dhfr on the addition chromosome. Using the binomial probability distribution formula (Steel et al., 1997), it was estimated that 74 independent transformants would have been required to have a 95% probability of obtaining at least one transgene insertion in the designer chromosome (assuming comparable chromosome sizes, random integration, and lack of excessive heterochromatic regions in the addition chromosome). Thus, we felt that the population of 146 [R.sub.0] transformants was sufficient to isolate at least one individual with an insertion in the designer chromosome.

Generation of Segregating Families

After crossing each selected [R.sub.0] individual to [TMV.sup.S] Petite Havana, [TMV.sup.R] [F.sub.1] plants were selected from 137 different [R.sub.0] transformants. Nine [TMV.sup.R] [R.sub.0] plants did not yield any [TMV.sup.R] [F.sub.1] plants. This could be due to transgene inactivation phenomena (Matzke and Matzke, 1995; Park et al., 1996) or loss of the gene due to somatic segregation in chimeric [R.sub.0] plants. After pollination of single [F.sub.1] individuals with Petite Havana, [BC.sub.1][F.sub.1] seed was harvested, and 121 to 968 seeds per family were plated onto growth medium containing Mtx. Methotrexate-resistant plants were easily identified, as they formed roots and green leaves, while susceptible plants germinated but died within 8 d. Because [F.sub.1] plants would have been monosomic for the designer chromosome, the percentage of surviving [BC.sub.1][F.sub.1] plants was indicative of the rate of transmission of the addition chromosome to female gametes. This data is summarized in Fig. 1, which illustrates that seven [BC.sub.1][F.sub.1] families appeared to exhibit obviously different rates of transmission than the majority of the families. For one family, it was not possible to isolate any [Mtx.sup.R] individuals from a large number of progeny, probably due to a loss of the addition chromosome. Six [BC.sub.1][F.sub.1] families exhibited high percentages of [Mtx.sup.R] individuals (40.5-49.6%). These six percentages were interpreted as possibly being caused by translocations of the N. africana chromosome segment containing dhfr to a chromosome of the N. tabacum genome. Data from subsequent testing of these six families has supported the translocation hypothesis by showing transfer of dhfr and the N. africana PVY resistance gene to the N. tabacum genome (subject of future publication). Excluding the seven anomalous families, the mean percent dhfr transmission rate was determined to be 7.67% (range: 1.65-16.53%).

[GRAPH OMITTED]

Linkage Analysis

Six to forty-five [Mtx.sup.R] plants from each of the 136 [BC.sub.1][F.sub.1] families derived from [TMV.sup.R] [R.sub.0] transformants were tested for TMV resistance (Table 1). If an N insertion had occurred on the addition chromosome, it was predicted that 100% of [Mtx.sup.R] [BC.sub.1][F.sub.1] individuals would also exhibit resistance to TMV. Out of 136 families, the number exhibiting this association was an unexpected zero. There were, however, several very high ratios of [Mtx.sup.R]/[TMV.sup.R]: [Mtx.sup.R]/[TMV.sup.S] plants (e.g., 41:4 and 21:2). These high ratios might be explained by three possibilities: (i) presence of two or more independently segregating N loci in [BC.sub.1][F.sub.1] progenies, (ii) insertion on the addition chromosome with occasional inactivation or loss of the N transgene, or (iii) insertion on the addition chromosome with rare recombinatory events between the addition chromosome and the N. tabacum genome. Transmission analysis and molecular assays were used to gain insight on these possibilities.
Table 1. Segregation of tobacco mosaic virus (TMV) resistance
in [Mtx.sup.R] individuals from derived [BC.sub.1][F.sub.1]
families.([dagger])

                                 #              [BC.sub.1][F.sub.1]
# [TMV.sup.R] Plants:   [BC.sub.1][F.sub.1]      Family Reference
# [TMV.sup.S] Plants         Families            ([double dagger])

 0:6(*)                          5                      --
 1:5                            13                      --
 2:4                            17                      --
 3:3                            46                      --
 4:2                            28                      --
 5:1                             1                      --
 7:5                             4                      --
 8:4                             6                      --
 9:3                             3                      --
13:11                            1                      --
15:8                             1                      --
20:5(*)                          1                   GH96-3558
20:7(*)                          1                   GH97-0459
14:3(*)                          1                   GH96-3856
10:2(*)                          2            GH97-0024 and GH97-0321
18:3(*)                          1                   GH96-3878
17:2(*)                          1                   GH96-3853
19:2(*)                          1                   GH97-0062
20:3(*)                          1                   GH97-0110
21:2(*)                          1                   GH97-0209
41:4(*)                          1                   GH96-3700

(*) Indicates that the ratio of [Mtx.sup.R]/:[Mtx.sup.R]/[TMV.sup.S]
plants did not fit a model of 1:1 segregation at the 0.05 significance
level.

([dagger]) Six initial [Mtx.sup.R] plants/family were tested for TMV
resistance. For families exhibiting 5-6 [TMV.sup.R] individuals, 6-39
additional [Mtx.sup.R] plants were tested for TMV resistance.

([double dagger]) Family references are provided only for those
families for which the ratio of [Mtx.sup.R]/[TMV.sup.R]:[Mtx.sup.R]/
[TMV.sup.S] plants was significantly higher than that expected under
a model of 1:1 segregation. These families were selected for
transmission analysis.


Transmission of TMV Resistance in [BC.sub.1][F.sub.1] Progenies

Given the aforementioned possibilities that might have been complicating our efforts to identify desired insertion events, eleven [BC.sub.1][F.sub.1] families exhibiting high ratios of [Mtx.sup.R]/[TMV.sup.R]:[Mtx.sup.R]/[TMV.sup.S] plants were tested for transmission of TMV resistance to female gametes without selection for [Mtx.sup.R]. Results from inoculations of 99 to 100 plants from eleven [BC.sub.1][F.sub.1] families are shown in Table 2. A model of a single N insertion being transmitted on the addition chromosome (7.67% [TMV.sup.R]) was tested using a Chi-square test. Very high ratios of [TMV.sup.R]:[TMV.sup.S] plants in 10 families supported the possibility of multiple independently segregating N insertions. In one family, GH96-3700 [BC.sub.1][F.sub.1], TMV resistance was transmitted to female gametes in 7 out of 100 events (Table 2). This ratio was highly suggestive of an insertion of N in the addition chromosome in this family.
Table 2. Transmission of tobacco mosaic virus (TMV) resistance
to female gametes in selected [BC.sub.1][F.sub.1] families.

                                     Resistance to TMV

                                 Observed           Expected([dagger])

[BC.sub.1][F.sub.1]   Res.([double       Sus.                [chi
Family                  dagger])     ([sections])   Res.    square]

GH96-3558                  88             12        7.67    911.21(*)
GH96-3700                   7             93        7.67      0.06
GH96-3853                  92              8        7.67   1004.21(*)
GH96-3856                  80             19        7.59    748.16(*)
GH96-3878                  78             22        7.67    698.46(*)
GH97-0024                  65             35        7.67    464.12(*)
GH97-0062                  96              4        7.67   1101.74(*)
GH97-0110                  77             23        7.67    678.74(*)
GH97-0209                  88             12        7.67    911.21(*)
GH97-0321                  72             28        7.67    584.37(*)
GH97-0459                  76             24        7.67    659.30(*)

(*) Significantly different from the expected ratio at the 0.05
probability level ([Chi] [is greater than or equal] 3.84;
P < 0.05, 1 df).

([dagger]) A model of a single N locus being transmitted on the
addition chromosome was tested. Expected segregation ratios were
based upon 7.67% transmission of the chromosome to female gametes.

([double dagger]) Res. = resistant.

([sections]) Sus. = susceptible.


Molecular Analysis (GH96-3700 [BC.sub.1][F.sub.1] Family)

A PCR assay was conducted on GH96-3700 [BC.sub.1][F.sub.1] individuals from the cosegregation analysis in order to gain insight on the possibilities that transgene inactivation, transgene loss, or interchromosomal recombination might have been confounding efforts to demonstrate linkage between dhfr and N on the addition chromosome. A PCR was conducted on 24 [Mtx.sup.R] individuals from the cosegregation analysis (Fig. 2). Selected individuals included all 4 observed [Mtx.sup.R]/[TMV.sup.S] plants. The PCR detected the presence of N, as expected, in each [TMV.sup.R] plant. PCR did not indicate the presence of the N transgene in any of the 4 [Mtx.sup.R]/[TMV.sup.S] plants. Southern blot testing of individuals from this analysis (Fig. 3) indicated the presence of a single N locus in resistant plants and verified further that N was not present in the few observed [Mtx.sup.R]/[TMV.sup.S] plants. The possibility that transgene silencing was occurring in these plants was therefore not supported.

[ILLUSTRATIONS OMITTED]

Collectively, the cosegregation analysis, transmission data, and molecular evidence suggest that a single insertion of the N-gene was present in the addition chromosome in family GH96-3700 [BC.sub.1][F.sub.1]. It appeared that the N transgene was occasionally being lost. This study does not reveal the precise mechanism of loss of the N transgene. There are numerous reports describing mechanisms of transgene inactivation (Matzke and Matzke, 1995; Park et al., 1996), but few describing mechanisms of transgene loss. Intra-transgenic recombination between clustered transgene insertions may cause elimination of transgene sequences (Pawlowski and Somers, 1996). This possibility seems unlikely in our case as only a single N insert was apparent in [TMV.sup.R] GH96-3700 [BC.sub.1][F.sub.1] individuals. We feel that the inability to show perfect cosegregation between dhfr and N may have been due to low frequency, and previously undetected, interchromosomal recombinatory events.

Isolation of Disomic dhfr dhfr-N N Lines

An array of 129 maternally-derived haploid plants was generated from six [Mtx.sup.R]/[TMV.sup.R] plants of the GH96-3700 [BC.sub.1][F.sub.1] family. Ten of these plants (7.75%) were found to be [TMV.sup.R]. Each [TMV.sup.R] haploid plant was also found to be [Mtx.sup.R]. These haploid plants have been chromosome doubled to produce doubled haploid plants (2n = 50) that are homozygous for dhfr, N, and the N. africana potyvirus resistance factor(s). Maternal transmission of TMV resistance was, again, entirely consistent with placement of the N-gene on the alien chromosome in this family. Cosegregation of TMV resistance and Mtx resistance during this procedure also provided additional evidence that N was linked with dhfr on the addition chromosome. If there was a single N locus segregating on a N. tabacum chromosome in this [BC.sub.1][F.sub.1] family, the observed results would be expected only 0.098% of the time.

DISCUSSION

The study and use of N. glutinosa N-mediated TMV resistance in tobacco breeding has a history of events that exemplify the successes and challenges associated with the introgression of disease resistance factors from related species. This work describes the generation of more than 100 transgenic TMV-resistant lines of flue-cured tobacco. The cloning of N by Whitham et al. (1994) followed by reintroduction through transformation may increase the potential for deploying TMV resistance in commercial flue-cured tobacco cultivars. This system eliminates the problem of flanking N. glutinosa chromatin and accompanying negative effects on yield and quality.

With some effort, as shown in this experiment, transgene insertion events can be identified which can be advantageous to plant breeders. Coupling phase linkages can greatly simplify selection and allow rapid transfer of beneficial linkage blocks between genotypes. Evidence presented in this paper shows that we obtained one insertion of the N-gene in the proposed designer chromosome. This demonstrates the feasibility of transferring a gene of agronomic importance to the addition chromosome. The work initiated construction of a disease resistance gene linkage block by linking TMV resistance with a potyvirus resistance gene(s) native to the chromosome. One disturbing problem was the occasional loss of the N transgene, possibly due to recombination between the addition chromosome and the N. tabacum genome. An important characteristic of a designer chromosome system would be that recombination between the alien chromosome and the crop genome would not occur so that any established linkage package would be preserved during breeding procedures. Previous work did not indicate evidence of recombination (Witherspoon, 1987; Wernsman, 1992; Campbell et al., 1994). Additional investigations are underway to better understand this possible recombination. It is expected that recombination would be extremely infrequent when in the disomic condition because of the presence of a homologue with which to pair.

One might question the practicality of a designer chromosome approach because of the low frequency at which desired insertions can be obtained. The authors would agree that a large "up-front" investment of effort is required to isolate desired transgene insertions in the addition chromosome. The advantages of the system would increase, however, as the number of transgenes added to the chromosome increases. The benefits would be in terms of a greatly simplified approach to transferring a large set of transgenes from genotype to genotype during breeding procedures. An additional concern relates to a possible necessity for introducing a different selectable marker with each new transgene introduction. With strategic construct design, this can be avoided. For example, the dhfr transgene in NC152 is positioned between the borders of a maize Ds element. When exposed to the Ac transposase, dhfr can be excised from the designer chromosome.

We used the approach of evaluating a large number of random T-DNA insertion events to find the desired linkage. A significant amount of research has been published on site-specific transgene integration mechanisms. Targeted transgene insertion into genomic DNA based on sequence homology has been achieved at high frequencies in yeast and other fungi (Timberlake and Marshall, 1989), but at extremely low frequencies in mammalian cells (Baker et al., 1988; Jasin et al., 1996). Targeted transgene insertion based on homologous recombination has been demonstrated for plant nuclear genomes (Paszkowski et al., 1988; Lee et al., 1990; Offringa et al., 1990; Halfter et al., 1992) and chloroplast genomes (Zoubenko et al., 1994; Carrer and Maliga, 1995). Except for plastid genomes, however, site-specific integration based on homologous recombination is not yet practical in plants. Several recombinase-mediated site-specific transgene insertion strategies (e.g., Cre-lox, FLP-FRT) have also been evaluated in plants (reviewed by Ow and Medberry, 1995). While interesting, this technology does not yet seem practical for constructing transgene linkage blocks for a number of reasons (Ow and Medberry, 1995). It is therefore not clear that these mechanisms offer any advantages over the approach used in the research presented here.

Carlson (1995) transferred the N. africana addition chromosome to N. glutinosa (2n = 24). This related species possesses one-half the chromosome number of cultivated tobacco and, therefore, the probability of random insertion into the designer chromosome would theoretically be greatly enhanced. After transformation of N. glutinosa and isolation of desired individuals through linkage analysis, the chromosome could be readily transferred back to N. tabacum using interspecific hybridization and backcrossing to N. tabacum with selection for Mtx resistance. This system was not used in the work described here because N. glutinosa is the source of N-mediated TMV resistance.

ACKNOWLEDGMENTS

The authors would like to thank Dr. Barbara Baker, USDAARS, for her cooperation in providing us with the N construct pTG34 used in this work. This research was supported in part by Philip Morris USA.

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Abbreviations: bp, base pairs; Mtx, methotrexate; PCR, polymerase chain reaction; PVY, potato virus Y; TEV, tobacco etch virus; TMV, tobacco mosaic virus.

Ramsey S. Lewis and Earl A. Wernsman(*)

Department of Crop Science, North Carolina State University, Raleigh, NC 27695-7620. Received 31 Jan. 2000. (*) Corresponding author (ewernsma@cropserv1.cropsci.ncsu.edu).
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