Barstar Gene Research Articles

Successful fertility restoration in male-sterile barnase line by optimal expression of barstar gene for hybrid-rice seed production

ABSTRACT The basic requirement for the barnase-barstar-based hybrid-seed production technology is the availability of stable male-sterile barnase and restorer barstar lines. Development of two rice (Oryza sativa) male-sterile barnase lines has been earlier reported from our lab. Complete restoration of male fertility in transgenic male-sterile lines has always been a challenging task for molecular breeders. In the present study, we have reported the successful fertility restoration in male-sterile barnase lines using barstar restorer lines generated through two completely different tapetum-specific promoters. The barstar lines were not able to restore male fertility in one barnase line but did restore it in the other barnase line. The male-sterile barnase line and its corresponding restorer line could be diversified into appropriate combiners for heterosis rice breeding.

Double-seedlings and embryo-free seeds generated by genetic engineering.

Apomixis can fix the heterosis of Hybrid F1, by maintaining its heterozygous genotype, and is an ideal way for the development of hybrid rice. In this paper, we designed an engineering strategy for realizing apomictic reproduction of hybrid rice in the way of induce adventitious embryos. An embryogenesis gene, AtWUS, controlled by the ovule-specific promoter, a ribonuclease gene Barnase driven by the egg cell-specific promoter pDD45, and an inactivation gene ZmAA1 driven by the pollen-specific promoter pG47 were simultaneously integrated into one T-DNA, and co-transformed with the second T-DNA carrying a Barstar gene. Double-seedlings were observed in transgenic line. Whole-genome sequencing and ploidy levels confirmed by flow cytometry showed that one of the double-seedlings was heterozygous diploid and the other seedling was homozygous haploid, which confirmed that embryogenesis in one of the double-seedlings arises from the zygote after fertilization and the other derived from an unfertilized gamete. Meanwhile we obtained embryo-free seeds at frequencies of 2.6% to 3.8% in T1 generation, and 0.75% to 3% in T2 generation. Though we did not obtained adventitious embryos in hybrid rice in this study, the phenomenon of double-seedlings and embryo-free seeds in transgenic line was informative and strongly suggested that endosperm development is an autonomously organized process in rice, independent of egg cell fertilization and embryo-endosperm communication. This provides novel insights into the induction of haploid embryos and lends theoretical support to successful clonal propagation using synthetic apomixis

In‐plant activation of root‐specific expression of a cytotoxic gene disrupts the development of the root‐knot nematode, Meloidogyne javanica

AbstractPlant hosts can be engineered to disrupt the development of sedentary plant‐parasitic nematodes or proper functioning of the feeding sites the nematodes induce. The use of constitutive promoters to express dsRNAs or nematode inhibitor proteins may be unreliable because of possible silencing or yield penalty from continuous expression in a plant host. This ill‐effect can be avoided if a root‐specific, nematode‐responsive promoter (NRP) is used to drive the target nematode‐inhibitory message. This study used the In Plant Activation (INPACT) system to express a barstar‐controlled barnase in galls of Meloidogyne javanica and assessed how the engineered tobacco lines affected the growth and development of the nematodes. Of the 11 combinations of four NRPs and the CaMV 35S promoter assessed, the AtCel1 and TobRB7 combinations activated specific expression of split β‐glucuronidase (GUS) and barnase genes in and around giant cells. The same NRP combination directed expression of the barnase gene in tobacco roots also constitutively expressing the barstar gene (SPBB transgenic lines). On roots of six T1 SPBB lines, there was up to 94% reduction in the number of galls with significantly smaller adult females compared to those on wild‐type plants. Some of the females on lines SPBB4‐1 and SPBB‐4‐2, for example, were not associated with galls. The results indicated the engineered plants disrupted M. javanica development and demonstrate the potential for controlled and localized expression of peptides, such as those that could block specific effectors, to disrupt initiation, formation, establishment, or proper functioning of feeding cells induced by damaging sedentary nematodes.

Comparison of RF3 <i>B. juncea</i> to RF3 <i>B. napus</i>

RF3 Brassica napus L. (InVigorTM Canola) has been in commercial cultivation since 1997. RF3 B. napus was produced by Agrobacterium-mediated transformation to provide glufosniate ammonium herbicide tolerance to the crop. Canola quality (CQ) varieties of Brassica juncea L. (Czern & Coss), a close relative of B. napus, have been available in Canada for more than 20 years. Currently, BASF Agricultural Solutions is developing CQ RF3 B. juncea via conventional breeding with RF3 B. napus to provide similar herbicide tolerance in the B. juncea species. The insertion event RF3 contains the bar gene (origin Streptomyces hygroscopicus) coding for phosphinothricin acetyl transferase (PAT/bar) protein which confers tolerance to glufosinate-ammonium herbicides. RF3 also contains the barstar gene (origin Bacillus amyloliquefaciens), coding for the Barstar protein, which is an inhibitor of the Barnase protein. In the absence of Barnase, there is no impact of Barstar. Many safety studies have been conducted during the development of the canola quality lines of RF3 B. napus and RF3 B. juncea. This report seeks to compare the two species based on the scientific data generated during the safety assessments of RF3 B. napus and RF3 B. juncea and discusses the utility of using studies with one species to support the safety of the other species both containing the same insertion event.

Food/Feed and Environmental Risk Assessment of Genetically Modified Glufosinate-tolerant Oilseed Rape MS8, RF3 and MS8 x RF3 for Import, Processing and Feed Uses under Directive 2001/18/EC (Notification C/BE/96/01)

Food/Feed and Environmental Risk Assessment of Genetically Modified Glufosinate-tolerant Oilseed Rape MS8, RF3 and MS8 x RF3 for Import, Processing and Feed Uses under Directive 2001/18/EC (Notification C/BE/96/01)

Environmental Risk Assessment of Glufosinate-Tolerant Genetically Modified Oilseed Rape MS8, RF3 and MS8 x RF3 for Import, Processing and Feed Uses under Directive 2001/18/EC (Notification C/BE/96/01)

Environmental Risk Assessment of Glufosinate-Tolerant Genetically Modified Oilseed Rape MS8, RF3 and MS8 x RF3 for Import, Processing and Feed Uses under Directive 2001/18/EC (Notification C/BE/96/01)

Identification and characterization of the promoter of a gene expressing mainly in the tapetum tissue of cotton (Gossypium hirsutum L.)

Anther and tapetum-specific genes are important for understanding male gametophyte development, as well as for their use in the development of barnase/barstar-gene based male sterility and restorer system for hybrid seed production. An essential component of the system is the availability of tapetum-specific promoters. In the present study, anther-specific genes were identified in cotton using microarray-based differential expression analysis, some of which show expression specific to the anthers at a stage where tapetum tissue was fully developed. Validation of the identified genes using RT-PCR and in situ hybridization identified one novel gene (AEG—Anther Expressing Gene) encoding a putative lipid binding protein as having a tapetum-specific expression. Further, three paralogs of the gene were identified in the cotton genome out of which the gene AEG1 (Anther Expressing Gene1) was found to express in the tapetum layer. Analysis of transgenic plants developed in cotton using 1.5 Kb promoter region of the of AEG1 gene fused upstream to the reporter gene β-glucuronidase revealed a broad window of expression of the AEG1 promoter in the tapetum tissue from the tetrad stage of anthers till the degeneration of the tapetum cells. Low levels of expression were also observed in the root tissues. Expression was not observed in the stem and leaves. The broad window of expression of AEG1 promoter in the tapetum tissue makes it a suitable candidate for the expression of the barstar gene for effective fertility restoration in the barnase/barstar system.

Analysis of activity driven by upstream regulatory modules (URM) of tapetum specific genes TA29 and A9 at ectopic locations in tobacco transgenics

TA29 and A9 are genes from Nicotiana tabacum and Arabidopsis thaliana respectively, which express in a tapetum specific manner. The upstream regulatory modules (URMs; i.e. the promoter and the 5′UTR) of these genes have been used in development of male sterile and restorer lines expressing the barnase and barstar genes for hybrid seed production. While initial studies show that these URMs drive the expression in a tapetum specific manner, there are no recordings of unintended (leaky) expression driven by these URMs at ectopic locations due to position effect in developed transgenic lines. The information on leaky expression driven by tissue specific URMs is important for their use in developing transgenic plants. The present study records the leaky activity of both these URMs in transgenic tobacco lines using β-glucuronidase as a reporter gene. Leaky activity was observed in about one-fourth of the lines developed with TA29. Most interestingly in these lines, the leaky expression of the reporter gene was observed to be restricted to the meristematic tip region of the roots and at the leaf gap from where leaf trace diverges from stem bundles. Such a restricted and unique pattern of leaky activity of a tissue specific promoter or a URM has never been reported before, including the URM of the A9 gene analyzed in the present study. This observation suggests the presence of cryptic cis-elements within the URM of TA29 gene that can possibly activate it in meristematic tissue when integrated at certain ectopic locations. The URM of the A9 gene was also observed to show leaky activity. However, there was no unique pattern as observed with that of TA29. Further, in the study we also show that while the smaller (290 bp) length of TA29 URM can be used to drive the expression of barnase gene to develop male sterile lines, it adversely affects the regeneration of transgenic tobacco lines due to leaky expression. This adverse effect is significantly reduced when the full length (1.5 kb) URM of the TA29 gene is used.

Safety assessment of transgenic canola RF3 with bar and barstar gene on Sprague-Dawley (SD) rats by 90-day feeding test

Canola is one of the most important plant oilseed crops. To avoid the threat of herbicides, the RF3 line with bar gene and barstar gene was developed, which can act as glufosinate resistance resources and restore fertility in hybrid lines. To assess the food safety of transgenic canola RF3, 2.5%, 5% and 10% GM canola RF3 and its non-GM isogenic line Drakkar were formulated into diet to feed Spragure-Dawley (SD) rats for 90 days. The effects on the general growth and toxicological parameters, as well as gut microbiota of rats, were evaluated. Several significant differences on body weight, feed consumption, relative organ weight, hematology and serum biochemistry were observed among rats in the 90-day feeding test. However, these statistical differences were randomly observed among different groups and were not dose-related, which were not considered to be biologically significant. Furthermore, the results of bacterial 16S rRNA sequencing of fecal samples showed that the diets containing GM canola did not disturb the balance of gut microbiota. In conclusion, the canola RF3 is considered as safe and wholesome as the non-GM canola based on this 90-day feeding test and gut microbiota analysis.

A Set of Genetic Constructs for Binase and Barstar Overproduction

Low molecular weight guanyl-preferring ribonuclease (RNase) secreted by Bacillus pumilus is referred to as binase and regarded as a potential agent to target oncogenic diseases and viral infections. Depending on research purposes, native binase or tagged and mutated forms are required for application. Specific inhibition of binase activity is achieved by binding to RNase inhibitor barstar. Native and mutant forms of binase are routinely obtained by ion-exchange chromatography, while tagged versions of binase, which can be purified faster and less tedious than non-tagged, are currently unavailable. Here, recombinant DNA techniques were employed to improve binase overexpression in bacterial host cells, its purification, and subsequent detection in downstream assays using affinity tag. Here, recombinant DNA techniques were employed to improve binase overexpression in bacterial host cells. Binase-barstar genetic cassette was modified to obtain strong two-gene operon with the possibility of rapid exchange of binase and/or barstar genes with homologous or mutated ones as well as the generation of C-terminally His-tagged binase and/or barstar. The binase-barstar operon was introduced into pET26b vector as well as into pET26b-pET15b hybrid vector that was created in this study. As a result, plasmids allowed the induced production of extracellular non-tagged, N- or C-terminal His-tagged binase and synchronous intracellular production of non-tagged or C-His-tagged barstar. The constructs can be further sub-cloned in gram-positive bacterial hosts to ensure native production conditions.

Effective restoration of male-sterile (barnase) lines requires overlapping and higher levels of barstar expression: A multi-generation field analysis in Brassica juncea

We have earlier reported the deployment of an effective pollination control system for hybrid seed production in Brassica juncea based on use of the barnase-barstar system. The current study analyses the stability and performance of four male-sterile (barnase) and 34 fertility restorer (barstar) lines under containment field conditions. Line (barnase) x tester (barstar) crosses were studied for effective fertility restoration over three successive growing seasons by screening the F1 and representative F2 progeny for three major parameters, namely segregation of Basta resistance and sensitive plants, distribution of male-sterile and fertile plants (vis-a-vis segregation of the barnase and the barstar genes) and pollen viability of the restored events. A total of 29 male sterile/restorer combinations out of 53 obtained in the first growing season were stable for their restoration ability when tested for two subsequent growing seasons. Stable restorers with high pollen viability were obtained at a significantly higher frequency for one of the barnase lines, bn 3.6, out of the four lines tested. Among the barstar lines, constructs carrying two transcription units of the barstar gene provided more effective fertility restoration. Quantitative real-time PCR analysis of restored progeny obtained for all the four barnase lines showed that three barnase lines (bn 3.23, bn 3.4 and bn 3.48) had an early and much higher level of barnase expression than the line bn 3.6 indicating that the level of barnase expression in the line bn 3.6 is optimal for better and stable restoration. It was observed that a high barstar : barnase transcript ratio and an extended window of barstar gene expression are critical parameters for the development of stable male-sterile (barnase) and fertility-restorer (barstar) combinations.

Application (EFSA‐GMO‐RX‐MS8‐RF3) for renewal of the authorisation for continued marketing of existing (1) food and food ingredients produced from genetically modified glufosinate‐tolerant oilseed rape Ms8, Rf3 and Ms8 × Rf3, and (2) feed materials produced from genetically modified glufosinate‐tolerant oilseed rape Ms8, Rf3 and Ms8 × Rf3, under Regulation (EC) No 1829/2003 from Bayer CropScience

Hybrid oilseed rape Ms8 x Rf3 has been produced by conventional crossing between the genetically modified (GM) parental lines Ms8 and Rf3. The hybrid system is achieved using a pollination control system by insertion and expression of barnase and barstar genes into two separate oilseed rape lines; each gene is linked to the bar gene conferring tolerance to glufosinate-containing herbicides. The scope of this application covers the continued marketing of existing food (processed oil) and feeds produced from oilseed rape Ms8, Rf3 and Ms8 x Rf3 which were lawfully placed on the market in the Community before the date of entry into force of Regulation (EC) No 1829/2003. In 2005 the EFSA GMO Panel had issued a scientific opinion on the safety of glufosinate-tolerant oilseed rape Ms8 x Rf3 derived from parental lines Ms8 and Rf3 for import and processing for feed and industrial uses. In delivering the present opinion, the EFSA GMO Panel considered the information provided in the application EFSA-GMO-RX-MS8-RF3 as well as additional information submitted by the applicant. The new data included molecular and bioinformatic analyses and a 42-day feeding study on chickens. The animal study showed that the tested material of oilseed rape Ms8 x Rf3 was nutritionally equivalent to its non-GM counterpart. In addition, bioinformatic studies using updated databases confirmed that no relevant similarities exist between the newly expressed proteins and known allergens or toxic proteins. Therefore, the EFSA GMO Panel reiterates the conclusions of its previous scientific opinion of 2005 that GM oilseed rape Ms8, Rf3 and Ms8 x Rf3 is unlikely to have an adverse effect on human and animal health or, in the context of its proposed uses, on the environment. This also applies to the products which are the subject of the present application.

Transfer and Detection of barstar Gene to Maize Inbred Line 18–599 (White) by Particle Bombardment

Abstract In China, the purity of maize hybrid strain is discomforting to the development of seed industrialization. Finding a new method for reproduction of maize hybrid strain is necessary. In this study, using particle bombardment, barstar gene was transferred into maize inbred line 18–599 (White), which is an antiviral and high quality maize inbred line. By molecular detection of the anther of transgenic maize, two plants transferred with barstar gene were gained in this study, which are two restorer lines. The two plants showed normal male spike, and lively microspores. But the capacity of the two restorer lines should be studied in the future. The aim of this study is to find a new method of reproduction of maize hybrid strain using engineering restorer lines and engineering sterility lines by gene engineering technology.

Retransformation of a male sterile barnase line with the barstar gene as an efficient alternative method to identify male sterile–restorer combinations for heterosis breeding

We report in this study, an improved method for identifying male sterile-restorer combinations using the barnase-barstar system of pollination control for heterosis breeding in crop plants, as an alternative to the conventional line x tester cross method. In this strategy, a transgenic male sterile barnase line was retransformed with appropriate barstar constructs. Double transformants carrying both the barnase and barstar genes were identified and screened for their male fertility status. Using this strategy, 66-90% of fertile retransformants (restored events) were obtained in Brassica juncea using two different barstar constructs. Restored events were analysed for their pollen viability and copy number of the barstar gene. Around 90% of the restored events showed high pollen viability and approximately 30% contained single copy integrations of the barstar gene. These observations were significantly different from those made in our earlier studies using line (barnase) x tester (barstar) crosses, wherein only two viable male sterile-restorer combinations were identified by screening 88 different cross-combinations. The retransformation strategy not only generated several independent restorers for a given male sterile line from a single transformation experiment but also identified potential restorers in the T(0) generation itself leading to significant savings in time, cost and labour. Single copy restored plants with high pollen viability were selfed to segregate male sterile (barnase) and restorer (barstar) lines in the T(1) progeny which could subsequently be diversified into appropriate combiners for heterosis breeding. This strategy will be particularly useful for crop plants where poor transformation frequencies and/or lengthy transformation protocols are a major limitation.

Expression of single-chain antibody-barstar fusion in plants

We successfully cloned and expressed a single-chain antibody (425scFv), that is directed to human epidermal growth factor receptor HER1 (EGFR) in transgenic tobacco plants as a fusion with bacterial barstar gene (425scFv-barstar). Plant-produced recombinant 425scFv-barstar was recovered using barstar–barnase system. Based on barstar–barnase affinity, during purification of the plant-produced 425scFv-barstar, we generated bispecific scFv-antibody heterodimers from individual single-chain fragments initially produced in different host systems with binding activity to both HER1 and HER2/neu tumor antigens. We demonstrated by flow cytometry and indirect immunofluorescent microscopy that both the components of heterodimer retain its specific cell-binding activity.

A new vector for controllable expression of an anti-HER2/neu mini-antibody-barnase fusion protein in HEK 293T cells

Tumor-targeted vectors with controllable expression of therapeutic genes and specific antitumor antibodies are promising tools for the reduction of malignant tumors. Here we describe a new plasmid for the eukaryotic expression of an anti-HER2/neu mini-antibody-barnase fusion protein (4D5 scFv-barnase-His 5) with an NH 2-terminal leader peptide. The 4D5 scFv-barnase-His 5 gene was placed downstream of the tetracycline responsive-element minimal promoter in the vector using the Tet-Off gene-expression system. The Bacillus amyloliquefaciens ribonuclease barnase is toxic for the host cells. To overcome this problem, barstar gene under its own minimal cytomegalovirus promoter was used in designed vector. Barstar inhibits the background level of barnase in the cells in the presence of tetracycline in culture medium. The HEK 293T cells were transfected with the designed vector, and the 4D5 scFv-barnase-His 5 fusion protein was identified by anti-barnase antibodies in cell culture medium and after purification from cell lysates using metal-affinity chromatography. The overexpression of the anti-HER2/neu mini-antibody-barnase fusion protein decreased the intensity of fluorescence of HEK 293T cells co-transfected with the generated plasmid and a plasmid containing the gene of enhanced green fluorescent protein (pEGFP-N1), in comparison with the intensity of fluorescence of HEK 293T cells transfected with pEGFP-N1, in the absence of tetracycline in the medium. The effect of the 4D5 scFv-barnase-His 5 on EGFP fluorescence indicates that the introduced barnase functions as a ribonuclease inside the cells. The anti-HER2/neu mini-antibody could be used to deliver barnase to HER2/neu-positive cells and provide its penetration into the target cells, as HER2/neu is a ligand-internalizing receptor. This expression vector has potential applications to both gene and antibody therapies of cancer.

Double recoverable block of function – a molecular control of transgene flow with enhanced reliability

Despite all the achieved benefits and potential promises from recombinant DNA technology of plants, the potential of transgene spread to wild relatives and to non-transgenic crops is still of a wide-spread concern. We continue to develop recoverable block of function (RBF) technology for gene flow control in transgenic plants. RBF consists of two elements: blocking construct (BC) and recovering construct (RC). Natural expression of the BC (barnase) in embryos and sprouts blocks a physiological function essential for survival or reproduction of the transgenic plant (mRNA synthesis and germination). Artificially induced (heat shock treatment) RC (barstar) recovers the blocked function enabling transgenic plant to reproduce. In natural conditions without artificial induction of RC the transgenic plant can not reproduce itself. However, a single RBF may still fail because of the potential for mutations and gene silencing of the inserted constructs. To minimize the frequency of such an inactivation, we developed a double RBF, in which a single insert comprising two BC, flanking a transgene of interest, was constructed and transferred into tobacco (Nicotiana tabacum (L.)). We used a barstar gene driven by a heat shock or 35S promoter as a RC, and two different promoters were used for barnase genes in the BC. One BC contained a seed germination specific cysteine endopeptidase promoter (BC1) and the other contained the cruciferin promoter (BC2), which is active during fruit development and embryogenesis. Three alternative constructs of double RBF are described, and a segregating two-insert as well as a one-insert cassettes, were compared. One-insert system comprising two BC with different nucleotide sequences but degenerate codons that expressed the same Barnase protein appeared to be the most reliable choice. The biological and molecular data obtained suggest that double RBF is a potent transgene containment technique that can safely be applied in agriculture.

A two gene – two promoter system for enhanced expression of a restorer gene (barstar) and development of improved fertility restorer lines for hybrid seed production in crop plants

We report in this study, strategies for enhancing and extending tissue-specific expression of a restorer gene (barstar) and their use in the development of improved fertility restorer lines for transgenic male sterile (barnase) lines for hybrid seed production. In the first strategy, a chimeric promoter was developed by combining regulatory elements of two overlapping tapetum-specific promoters (A9 and TA29) by placing a 275bp fragment of the A9 promoter (minus the TATA box) upstream to a functional 330bp fragment of the TA29 promoter. Analysis of chimeric promoter activity using the gusA gene showed that it enhanced transgene expression levels in anthers of transgenic Brassica juncea (Indian oilseed mustard) plants by two- to three-fold over those obtained with either promoter alone but retained the temporal expression profile of the TA29 promoter. In the second strategy, the A9 and TA29 promoters were used to independently express two copies of the wild type barstar gene sequence within the same T-DNA borders. Alternatively, one of the promoters was used to express the wild type sequence and the other was used to drive expression of a codon-modified version of the barstar gene designed for enhanced and stable expression in dicot plants. The efficiencies of these constructs for fertility restoration was determined by retransforming a male sterile TA29-barnase line of B. juncea and analyzing the frequency and pollen viability of male fertile (restored) plants thus obtained. A significantly higher frequency of restored plants was obtained with constructs containing two transcription units of the barstar gene (92.9% for constructs with two copies of the wild type gene and 89.8% for constructs using the wild type and codon-modified sequences) as compared to that obtained with the chimeric promoter-barstar construct (77.8%) or the TA29-barstar construct (65.6%). Pollen viability assays on restored plants showed that the desirable extent of restoration is obtained in a higher proportion of male fertile plants generated using the construct containing a combination of the wild type and modified barstar genes. These observations were corroborated by genetic analysis of F1 progeny of crosses between various barstar lines and four different male sterile barnase lines, three of which could not be restored in earlier studies using TA29-barstar constructs. The simultaneous use of the wild type and modified barstar genes with the TA29 and A9 promoters was found to be the most effective method for fertility restoration with efficient restorers being identified for all the male sterile barnase lines tested, including those which could not be restored earlier. The two gene-two promoter strategy could be successfully deployed for enhancing and extending the tissue-specific as well as constitutive expression of other agronomically important transgenes.

A novel strategy for regulated expression of a cytotoxic gene

The tetracycline (Tet) transactivator system is a powerful promoter system to control gene expression. However, expression of a cytotoxic gene in this system has been limited due to the lethal effect caused by low levels of basal expression of the toxic gene. In this report, we describe a novel strategy to express a toxic gene using the Tet system. The barstar gene is placed downstream of a minimal promoter and the barnase gene downstream of the tetracycline responsive element minimal promoter. When barnase is expressed at a basal level, its toxicity in human cell culture is offset by the similar basal level expression of barstar. However, when the barnase expression is induced with the transactivator protein, its overproduction leads to cell death. Therefore, this strategy allows cytotoxicity to be effectively regulated by tetracycline.

Molecular control of transgene escape from genetically modified plants

Potential risks of gene escape from transgenic crops through pollen and seed dispersal are being actively discussed and have slowed down full utilization of gene technology in crop improvement. To ban the transgene flow, barren zones and ‘terminator’ technology were developed as GMO risk management technologies in transgenic crops. Unfortunately, the technologies have not protected reliably the transgene migration to wild relatives. The present study offers a novel molecular technique to eliminate gene flow from transgenic plants to wild relatives by recoverable block of function (RBF). The RBF consists of a blocking sequence linked to the gene of interest and a recovering sequence, all in one transformable construct. The blocking sequence blocks a certain molecular or physiological function of the host plant. Action of the blocking sequence leads to the death of the host plant or to an alteration in its phenotype resulting in inability for sexual reproduction in nature. The recovering construct recovers the blocked function of the host plant. The recovering construct is regulated externally by a specific chemical or physical treatment of the plants and does not act under natural conditions. In nature, hybrids of the transgenic plants with its wild relatives carrying the RBF will die or be unable to reproduce because of the blocking construct action. A working model of RBF is described in this report as one example of the RBF concept. This RBF example is based on barnase (the blocking construct) and barstar (the recovering construct) gene expression in tobacco under sulfhydryl endopeptidase (SH-EP) and a heat shock (HS) promoter, respectively.