Tissue Culture (Somatic Embryogenesis)-Induced Tnt1 Retrotransposon-Based Mutagenesis in Brachypodium distachyon.

Brachypodium distachyon is a model grass species for economically important cereal crops. Efforts are in progress to develop useful functional genomic resources in Brachypodium. A tobacco retrotransposon, Tnt1, has been used successfully in recent past to generate insertional mutagenesis in several dicot plant species. Tnt1 retrotransposon replicates, transposes, and inserts at multiple random genomic locations in the plant genome. Transposition occurs only during somatic embryogenesis but not during seed transmission. We developed Brachypodium transgenic plants that can express the Tnt1 element. Here, we describe an efficient tissue culture-based approach to generate Tnt1 insertional mutant population using transgenic Brachypodium line expressing the Tnt1 retrotransposon.

Retarded germination of Nicotiana tabacum seeds following insertion of exogenous DNA mimics the seed persistent behavior.

Tobacco seeds show a coat-imposed dormancy in which the seed envelope tissues (testa and endosperm) impose a physical constraint on the radicle protrusion. The germination-limiting process is represented by the endosperm rupture which is induced by cell-wall weakening. Transgenic tobacco seeds, obtained by insertion of exogenous genes codifying for seed-based oral vaccines (F18 and VT2eB), showed retarded germination with respect to the wild type and modified the expression of endogenous proteins. Morphological and proteomic analyses of wild type and transgenic seeds revealed new insights into factors influencing seed germination. Our data showed that the interference of exogenous DNA influences the germination rather than the dormancy release, by modifying the maturation process. Dry seeds of F18 and VT2eB transgenic lines accumulated a higher amount of reserve and stress-related proteins with respect to the wild type. Moreover, the storage proteins accumulated in tobacco F18 and VT2eB dry seeds have structural properties that do not enable the early limited proteolysis observed in the wild type. Morphological observations by electron and light microscopy revealed a retarded mobilization of the storage material from protein and lipid bodies in transgenic seeds, thus impairing water imbibition and embryo elongation. In addition, both F18 and VT2eB dry seeds are more rounded than the wild type. Both the morphological and biochemical characteristics of transgenic seeds mimic the seed persistent profile, in which their roundness enables them to be buried in the soil, while the higher content of storage material enables the hypocotyl to elongate more and the cotyledons to emerge.

Genetic analyses of the inheritance and expressivity of autonomous endosperm formation in Hieracium with different modes of embryo sac and seed formation.

Background and Aims: Apomixis, or asexual seed formation, in polyploid Hieracium subgenus Pilosella species results in clonal progeny with a maternal genotype. An aposporous embryo sac forms mitotically from a somatic cell, without prior meiosis, while embryo and endosperm formation is fertilization independent (autonomous). The latter two developmental components are tightly linked in Hieracium . Recently, two plants, AutE196 and AutE24, were identified from two different crosses. Both form embryo sacs via the sexual route by undergoing meiosis, and embryo development requires fertilization; however, 18 % of embryo sacs can undergo autonomous endosperm (AutE) formation. This study investigated the qualitative and quantitative inheritance of the AutE trait and factors influencing phenotype expressivity. An additional focus was to identify the linkage group bearing the AutE locus in AutE196. Methods: Crosses and cytology were used to examine the inheritance of AutE from AutE24 and AutE196, and to reintroduce apomictic components into AutE plants, thereby changing the ploidy of developing embryo sacs and increasing the dosage of AutE loci. Markers from a Hieracium apomict linkage map were examined within a backcrossed AutE196 mapping population to identify the linkage group containing the AutE196 locus. Key Results: Qualitative autonomous endosperm in the AutE24 line was conferred by a single dominant locus, and the trait was transmitted through male and female gametes in AutE196 and AutE24. Expressivity of the trait did not significantly increase when AutE loci from AutE196 and AutE24 were both present in the progeny, within embryo sacs formed via apospory, or sexually derived embryo sacs with increased ploidy. It remains unclear if these are identical loci. Conclusions: The qualitative trait of autonomous endosperm formation is conferred by single dominant loci in AutE196 and AutE24. High expressivity of autonomous endosperm formation observed in apomicts requires additional genetic factors. Potential candidates may be signals arising from fertilization-independent embryo formation.

Expression and genomic integration of transgenes after Agrobacterium-mediated transformation of mature barley embryos.

Mature embryos in tissue cultures are advantageous because of their abundance and rapid germination, which reduces genomic instability problems. In this study, 2-day-old isolated mature barley embryos were infected with 2 Agrobacterium hypervirulent strains (AGL1 and EHA105), followed by a 3-day period of co-cultivation in the presence of L-cystein amino acid. Chimeric expression of the b-glucuronidase gene (gusA) directed by a viral promoter of strawberry vein banding virus was observed in coleoptile epidermal cells and seminal roots in 5-day-old germinated seedlings. In addition to varying infectivity patterns in different strains, there was a higher ratio of transient b-glucuronidase expression in developing coleoptiles than in embryonic roots, indicating the high competency of shoot apical meristem cells in the mature embryo. A total of 548 explants were transformed and 156 plants developed to maturity on G418 media after 18-25 days. We detected transgenes in 74% of the screened plant leaves by polymerase chain reaction, and 49% of these expressed neomycin phosphotransferase II gene following AGL1 transformation. Ten randomly selected T0 transformants were analyzed using thermal asymmetric interlaced polymerase chain reaction and 24 fragments ranged between 200-600 base pairs were sequenced. Three of the sequences flanked with transferred-DNA showed high similarity to coding regions of the barley genome, including alpha tubulin5, homeobox 1, and mitochondrial 16S genes. We observed 70-200-base pair filler sequences only in the coding regions of barley in this study.

Somatic embryogenesis, tetraploidy, and variant leaf morphology in transgenic diploid strawberry (Fragaria vesca subspecies vesca 'Hawaii 4').

BACKGROUND: The diploid (2n = 2x = 14) strawberry model plant Fragaria vesca ssp. vesca 'Hawaii 4' was employed for functional analysis of expressed DNA sequences initially identified as being unique to Fragaria and of unknown or poorly understood function. 'Hawaii 4' is prominent in strawberry research due to its ease of Agrobacterium-mediated transformation and regenerability, and its status as the source of the first complete strawberry genomic sequence. Our studies of a set of transformants have documented intriguing, construct-associated effects on leaf morphology, and provide important and unexpected insights into the performance of the 'Hawaii 4' transformation and regeneration system. RESULTS: Following Agrobacterium-mediated transformation of leaf explants with gene constructs carried by Gateway® vectors, plants were regenerated using a modified version of an established 'Hawaii 4' protocol. Expanding upon the findings of prior studies, we documented that plantlet regeneration was occurring via a somatic embryogenic rather than an organogenic developmental pathway. Among transformants, several variations in leaf morphology were observed. Unexpectedly, a particular leaf variant type, occurring in ~17% of all regenerants independent of construct type, was found to be attributable to tetraploidy. The tetraploidy-associated alteration in leaf morphology could be differentiated from the leaf morphology of diploid regenerants on the basis of a quantitative ratio of leaf dimensions: B/A, where B is the width of the central leaflet and A is the overall width of the trifoliate leaf. Variant effects on leaf morphology of four different transgenic constructs were also documented, and were in all cases distinguishable from the effects of tetraploidy. CONCLUSIONS: These results define opportunities to optimize the existing 'Hawaii 4' protocol by focusing on treatments that specifically promote somatic embryogenesis. The reported morphological metric and descriptions will guide future transgenic studies using the 'Hawaii 4' model system by alerting researchers to the potential occurrence of polyploid regenerants, and to differentiating the effects on leaf morphology due to polyploidy versus transgenic manipulations. Finally, an intriguing spectrum of leaf morphology alterations resulting from manipulation of expressed sequences of uncertain function is documented, providing a foundation for detailed studies of the respective genes and their functional roles.

Regulation of Arabidopsis embryo and endosperm development by the polypeptide signaling molecule CLE8.

The plant seed is a major nutritional source for humans as well as an essential embryo development and dispersal unit. To ensure proper seed formation, fine spatial and temporal coordination between the embryo, endosperm, and maternal seed components must be achieved. However, the intercellular signaling pathways that direct the synchronous development of these tissues are poorly understood. Here we show that the Arabidopsis thaliana peptide ligand CLAVATA3/embryo surrounding region-related8 (CLE8) is exclusively expressed in young embryos and endosperm, and that it acts cell and noncell autonomously to regulate basal embryo cell division patterns, endosperm proliferation, and the timing of endosperm differentiation. CLE8 positively regulates expression of the transcription factor gene Wuschel-like homeobox8 (WOX8), and together CLE8 and WOX8 form a signaling module that promotes seed growth and overall seed size. These results demonstrate that seed development is coordinated by a secreted peptide ligand that plays a key early role in orchestrating cell patterning and proliferation in the embryo and endosperm.

Loss of function of the HSFA9 seed longevity program.

Gain of function approaches that have been published by our laboratory determined that HSFA9 (Heat Shock Factor A9) activates a genetic program contributing to seed longevity and to desiccation tolerance in plant embryos. We now evaluate the role(s) of HSFA9 by loss of function using different modified forms of HaHSFA9 (sunflower HSFA9), which were specifically overexpressed in seeds of transgenic tobacco. We used two inactive forms (M1, M2) with deletion or mutation of the transcription activation domain of HaHSFA9, and a third form (M3) with HaHSFA9 converted to a potent active repressor by fusion of the SRDX motif. The three forms showed similar protein accumulation in transgenic seeds; however, only HaHSFA9-SRDX showed a highly significant reduction of seed longevity, as determined by controlled deterioration tests, a rapid seed ageing procedure. HaHSFA9-SRDX impaired the genetic program controlled by the tobacco HSFA9, with a drastic reduction in the accumulation of seed heat shock proteins (HSPs) including seed-specific small HSP (sHSP) belonging to cytosolic (CI, CII) classes. Despite such effects, the HaHSFA9-SRDX seeds could survive developmental desiccation during embryogenesis and their subsequent germination was not reduced. We infer that the HSFA9 genetic program contributes only partially to seed-desiccation tolerance and longevity.

DiSUMO-like DSUL is required for nuclei positioning, cell specification and viability during female gametophyte maturation in maize.

Reversible post-translational modification of numerous proteins by small ubiquitin-related modifiers (SUMOs) represents a major regulatory process in various eukaryotic cellular and developmental processes. To study the role of sumoylation during female gametophyte (FG) development in maize, we identified Zea mays genes encoding SUMO (ZmSUMO1a and ZmSUMO1b) and a diSUMO-like protein called ZmDSUL that contains two head-to-tail SUMO-like domains. Whereas ZmSUMO1a and ZmSUMO1b are almost ubiquitously expressed, ZmDSUL transcripts were detected exclusively in the egg apparatus and zygote. ZmDSUL was selected for detailed studies. ZmDSUL is processed close to the C-terminus, generating a dimeric protein that is similar to animal FAT10 and ISG15, which contain two ubiquitin-like domains. Whereas GFP fused to the ZmDSUL N-terminus was located in the cytoplasm and predominately in the nucleoplasm of some transiently transformed maize suspension cells, C-terminal GFP fusions exclusively accumulated at the nuclear surface. GFP or ZmDSUL-GFP under control of the ZmDSUL promoter first displayed GFP signals in the micropylar-most position of the FG at stage 5/6, when migration of polar nuclei and cellularization occurs. Mature FGs displayed GFP signals exclusively in the egg cell, but the strongest signals were observed shortly after fertilization and disappeared during the first asymmetric zygotic division. RNAi silencing of ZmDSUL showed that it is required for FG viability. Moreover, nuclei segregation and positioning defects occurred at stage FG 5 after mitotic nuclear divisions were completed. In summary, we report a diSUMO-like protein that appears to be essential for nuclei segregation and positioning, the prerequisite for cell specification during FG maturation.

Auxin modulates the transition from the mitotic cycle to the endocycle in Arabidopsis.

Amplification of genomic DNA by endoreduplication often marks the initiation of cell differentiation in animals and plants. The transition from mitotic cycles to endocycles should be developmentally programmed but how this process is regulated remains largely unknown. We show that the plant growth regulator auxin modulates the switch from mitotic cycles to endocycles in Arabidopsis; high levels of TIR1-AUX/IAA-ARF-dependent auxin signalling are required to repress endocycles, thus maintaining cells in mitotic cycles. By contrast, lower levels of TIR1-AUX/IAA-ARF-dependent auxin signalling trigger an exit from mitotic cycles and an entry into endocycles. Our data further demonstrate that this auxin-mediated modulation of the mitotic-to-endocycle switch is tightly coupled with the developmental transition from cell proliferation to cell differentiation in the Arabidopsis root meristem. The transient reduction of auxin signalling by an auxin antagonist PEO-IAA rapidly downregulates the expression of several core cell cycle genes, and we show that overexpressing one of the genes, CYCLIN A2;3 (CYCA2;3), partially suppresses an early initiation of cell differentiation induced by PEO-IAA. Taken together, these results suggest that auxin-mediated mitotic-to-endocycle transition might be part of the developmental programmes that balance cell proliferation and cell differentiation in the Arabidopsis root meristem.

Changes in oil content of transgenic soybeans expressing the yeast SLC1 gene.

The wild type (Wt) and mutant form of yeast (sphingolipid compensation) genes, SLC1 and SLC1-1, have been shown to have lysophosphatidic acid acyltransferase (LPAT) activities (Nageic et al. in J Biol Chem 269:22156-22163, 1993). Expression of these LPAT genes was reported to increase oil content in transgenic Arabidopsis and Brassica napus. It is of interest to determine if the TAG content increase would also be seen in soybeans. Therefore, the wild type SLC1 was expressed in soybean somatic embryos under the control of seed specific phaseolin promoter. Some transgenic somatic embryos and in both T2 and T3 transgenic seeds showed higher oil contents. Compared to controls, the average increase in triglyceride values went up by 1.5% in transgenic somatic embryos. A maximum of 3.2% increase in seed oil content was observed in a T3 line. Expression of the yeast Wt LPAT gene did not alter the fatty acid composition of the seed oil.

The rice transcription factor IDEF1 is essential for the early response to iron deficiency, and induces vegetative expression of late embryogenesis abundant genes.

Higher plants maintain iron homeostasis by regulating the expression of iron (Fe)-related genes in accordance with Fe availability. The transcription factor IDEF1 regulates the response to Fe deficiency in Oryza sativa (rice) by recognizing CATGC sequences within the Fe deficiency-responsive cis-acting element IDE1. To investigate the function of IDEF1 in detail, we analyzed the response to Fe deficiency in transgenic rice plants exhibiting induced or repressed IDEF1 expression. Fe-deficiency treatment in hydroponic culture revealed that IDEF1 knock-down plants are susceptible to early-stage Fe deficiency, in contrast to IDEF1-induced plants. Time-course expression analyses using quantitative reverse-transcriptase PCR revealed that the IDEF1 expression level was positively correlated with the level of induction of the Fe utilization-related genes OsIRO2, OsYSL15, OsIRT1, OsYSL2, OsNAS1, OsNAS2, OsNAS3 and OsDMAS1, just after the onset of Fe starvation. However, this overall transactivation mediated by IDEF1 became less evident in subsequent stages. Microarray and in-silico analyses revealed that genes positively regulated by IDEF1, especially at the early stage, exhibit over-representation of CATGC and IDE1-like elements within the proximal promoter regions. These results indicate the existence of early and subsequent responses to Fe deficiency, with the former requiring IDEF1 more specifically. Proximal regions of IDEF1-regulated gene promoters also showed enrichment of RY elements (CATGCA), which regulate gene expression during seed maturation. The expression of several genes encoding late embryogenesis abundant proteins, including Osem, was induced in Fe-deficient roots and/or leaves in an IDEF1-dependent manner, suggesting a possible function of seed maturation-related genes in Fe-deficient vegetative organs.

Drought tolerance through overexpression of monoubiquitin in transgenic tobacco.

Ubiquitin (Ub) is present in all eukaryotic species examined. It is a multifunctional protein and one of its main known functions is to tag proteins for selective degradation by the 26S proteasome. In this study, Ta-Ub2, a cDNA sequence containing a single Ub repeat and a 3' non-coding region of a polyubiquitin gene, was isolated from wheat (Triticum aestivum) by reverse transcription-polymerase chain reaction (RT-PCR). A PBI sense vector with Ta-Ub2 was constructed and transformed into tobacco plants. Ub expression in wheat leaves, monitored by semi-quantitative RT-PCR, responded to drought stress. In transgenic tobacco, determined by protein gel blot analysis, we found higher amounts of Ub-protein conjugates than in control (tobacco carrying a PBI GUS vector without Ta-Ub2) and wild-type (WT) lines. However, free Ub levels did not significantly differ in the 3 genotypes. Seeds from transgenic, Ub-overexpressing tobacco germinated faster and seedlings grew more vigorously than control and WT samples, both under drought and non-drought conditions. Furthermore, CO(2) assimilation of transgenic plants was significantly higher under drought stress. Our results indicate that Ub may be involved in the response of plants to drought stress and that overexpression of monoubiquitin might be an effective strategy for enhancing drought tolerance.

Specificity of expression of the GUS reporter gene (uidA) driven by the tobacco ASA2 promoter in soybean plants and tissue cultures.

Twelve independent lines were transformed by particle bombardment of soybean embryogenic suspension cultures with the tobacco anthranilate synthase (ASA2) promoter driving the uidA (beta-glucuronidase, GUS) reporter gene. ASA2 appears to be expressed in a tissue culture specific manner in tobacco (Song H-S, Brotherton JE, Gonzales RA, Widholm JM. Tissue culture specific expression of a naturally occurring tobacco feedback-insensitive anthranilate synthase. Plant Physiol 1998;117:533-43). The transgenic lines also contained the hygromycin phosphotransferase (hpt) gene and were selected using hygromycin. All the selected cultures or the embryos that were induced from these cultures expressed GUS measured histochemically. However, no histochemical GUS expression could be found in leaves, stems, roots, pods and root nodules of the plants formed from the embryos and their progeny. Pollen from some of the plants and immature and mature seeds and embryogenic cultures initiated from immature cotyledons did show GUS activity. Quantitative 4-methylumbelliferyl-glucuronide (MUG) assays of the GUS activity in various tissues showed that all with observable histochemical GUS activity contained easily measurable activities and leaves and stems that showed no observable histochemical GUS staining did contain very low but measurable MUG activity above that of the untransformed control but orders of magnitude lower than the constitutive 35S-uidA controls used. Low but clearly above background levels of boiling sensitive GUS activity could be observed in the untransformed control immature seeds and embryogenic cultures using the MUG assay. Thus in soybean the ASA2 promoter drives readily observable GUS expression in tissue cultures, pollen and seeds, with only extremely low levels seen in vegetative tissues of the plants. The ASA2 driven expression seen in mature seed was, however, much lower than that seen with the constitutive 35S promoter; less than 2% in seed coats and less than 0.13% in cotyledons and embryo axes. The predominate tissue culture specific expression pattern of the ASA2 promoter may be useful for genetic transformation of crops.

Red clover (Trifolium pratense).

Genetic modification of plants by the insertion of transgenes can be a powerful experimental approach to answer basic questions about gene product function. This technology can also be used to make improved crop varieties for use in the field. To apply this powerful tool to red clover, an important forage legume, a population of red clover with a high potential for regeneration in tissue culture has been developed. Here we provide a detailed procedure for Agrobacterium-mediated transformation of genotypes derived from this regenerable population. We have successfully used this methodology to express a beta-glucuronidase (GUS) reporter gene and to silence an endogenous polyphenol oxidase gene in red clover.

Expression of ABA 8'-hydroxylases in relation to leaf water relations and seed development in bean.

In plants, the level of abscisic acid (ABA) is determined by synthesis and catabolism. Hydroxylation of ABA at the 8' position is the key step in ABA catabolism. This reaction is catalyzed by ABA 8'-hydroxylase, a cytochrome P450 (CYP). The cDNAs of PvCYP707A1 and PvCYP707A2 were isolated from bean (Phaseolus vulgaris L.) axes treated with (+)-ABA and that of PvCYP707A3 from dehydrated bean leaves. The recombinant PvCYP707A proteins expressed in yeast were biochemically characterized. Yeast strains over-expressing any of the three PvCYP707As were able to convert ABA to phaseic acid (PA). The microsomal fractions from these yeast strains also exhibited ABA 8'-hydroxylase activity. Expression of PvCYP707A3 in primary leaves was strongly increased by water stress, whereas PvCYP707A1 and PvCYP707A2 mRNA levels were rapidly increased by rehydration of water-stressed leaves. Northern blot analysis of PvCYP707As in bean showed a high level of expression in the mature fruits, senescent leaves, roots, seed coats and axes. All three PvCYP707As were expressed at varying intensities throughout seed development. Imbibed seeds also had high PvCYP707A mRNA levels. Thus, expression of PvCYP707As is both environmentally and developmentally regulated. Transgenic Nicotiana sylvestris plants over-expressing PvCYP707As displayed a wilty phenotype, and had reduced ABA levels and increased PA levels. These results demonstrate that expression of PvCYP707As is the major mechanism by which ABA catabolism is regulated in bean.

Regulation of carotenoid and ABA accumulation during the development and germination of Nicotiana plumbaginifolia seeds.

Abscisic acid (ABA) is derived from epoxycarotenoid cleavage and regulates seed development and maturation. A detailed carotenoid analysis was undertaken to study the contribution of epoxycarotenoid synthesis to the regulation of ABA accumulation in Nicotiana plumbaginifolia developing seeds. Maximal accumulation of xanthophylls occurred at mid-development in wild type seeds, when total ABA levels also peaked. In contrast, in ABA-deficient mutants xanthophyll synthesis was delayed, in agreement with the retardation in seed maturation. Seed dormancy was restored in mutants impaired in the conversion of zeaxanthin into violaxanthin by zeaxanthin epoxidase (ZEP), by the introduction of the Arabidopsis AtZEP gene under the control of promoters inducing expression during later stages of seed development compared to wild type NpZEP, and in dry and imbibed seeds. Alterations in the timing and level of ZEP expression did not highly affect the temporal regulation of ABA accumulation in transgenic seeds, despite notable perturbations in xanthophyll accumulation. Therefore, major regulatory control of ABA accumulation might occur downstream of epoxycarotenoid synthesis.

High accumulation of bioactive peptide in transgenic rice seeds by expression of introduced multiple genes.

We have developed a simple binary vector construction system for the simultaneous expression of multiple genes in plants. Up to three independent gene cassettes can be easily integrated into one binary vector using the MultiSite Gateway System. Using this system, we produced transgenic rice plants that accumulated high levels of the hypocholesterolaemic peptide lactostatin (IIAEK) in endosperm. Binary vectors were constructed that could accommodate up to three independent modified glutelin gene cassettes encoding multimer lactostatin in the variable regions. Eight construct permutations were used for rice transformation. We measured the accumulation of lactostatin expressed as a glutelin fusion protein in the mature seeds of 105 independent transgenic rice lines. A general correlation was observed between accumulation level and gene number in the vector constructs, indicating that a higher accumulation of lactostatin was obtained from transgenic rice plants containing the maximum number of gene inserts. These results indicate that this strategy is applicable for the selection of transgenic lines containing large amounts of bioactive peptides in rice seeds.

Expression of the promoter of HyPRP, an embryo-specific gene from Zea mays in maize and tobacco transgenic plants.

zmHyPRP is a gene specifically expressed in maize immature embryos where its transcripts are mainly observed in the scutellum. It has been shown that zmHyPRP expression in the embryo is arrested when ABA levels increase at the beginning of the maturation stage. Here we report the ability of 2 Kb zmHyPRP promoter to reproduce the zmHyPRP gene specific expression pattern in the maize embryo and its repression by ABA at the end of the morphogenetic process. Three different approaches have been used, transient particle bombardment of maize immature excised embryos and stable transformation of maize and tobacco plants with a construct containing 2 Kb of zmHyPRP promoter fused to the GUS gene. This construct has shown to confer specific expression to maize and tobacco embryos but in tobacco expression in the embryo was very low. The same construct was also negatively regulated by ABA in embryos of both species. This suggests that 2 Kb of the zmHyPRP promoter contain all regulatory elements sufficient to confer the developmental expression patterns of the gene characterized to date.

Stable transformation of Theobroma cacao L. and influence of matrix attachment regions on GFP expression.

We describe a protocol for Agrobacterium-mediated genetic transformation of Theobroma cacao L. using cotyledonary explants from primary somatic embryos (SEs) and A. tumefaciens strain AGL1. Transgenic plants carrying the visible marker, gene green fluorescent protein ( EGFP), the selectable marker gene neomycin phosphotransferase II ( NPTII), the class I chitinase gene from cacao ( Chi), and tobacco nuclear matrix attachment regions (MARs) in different combinations were successfully produced via regeneration of secondary SEs. The presence of the Chi gene or MARs did not influence the number of transgenic plants produced compared to the marker genes alone. However, the inclusion of MARs contributed to increased mean GFP expression in the population of transgenics. Additionally, the presence of MARs reduced the occurrence of gene silencing and stabilized high levels of GFP expression in lines of transgenic plants multiplied via reiterative somatic embryogenesis. Ninety-four transgenic plants were acclimated in a greenhouse and grown to maturity. Detailed growth analysis indicated that there were no differences in various growth parameters between transgenic and non-transgenic SE-derived plants. Seeds produced from two genetic crosses with one of the transgenic lines were analyzed for EGFP expression-a near-perfect 1:1 segregation was observed, indicating that this line resulted from the insertion of a single locus of T-DNA.

Control of seed germination in transgenic plants based on the segregation of a two-component genetic system.

We have developed a repressible seed-lethal (SL) system aimed at reducing the probability of transgene introgression into a population of sexually compatible plants. To evaluate the potential of this method, tobacco plants were transformed with an SL construct comprising gene 1 and gene 2 from Agrobacterium tumefaciens whereby gene 1 was controlled by the seed-specific phaseolin promoter modified to contain a binding site for the Escherichia coli TET repressor (R). The expression of this construct allows normal plant and seed development but inhibits seed germination. Plants containing the SL construct were crossed with plants containing the tet R gene to derive plant lines where the expression of the SL construct is repressed. Plant lines that contained both constructs allowed normal seed formation and germination, whereas seeds in which the SL construct was separated from the R gene through segregation did not germinate. The requirements of such a method to efficiently control the flow of novel traits among sexually compatible plants are discussed.