Restorer Gene Research Articles

Comparative genomic analysis of the compound Brassica napus Rf locus.

BackgroundThe plant trait of cytoplasmically-inherited male sterility (CMS) and its suppression by nuclear restorer-of-fertility (Rf) genes can be viewed as a genetic arms race between the mitochondrial and nuclear genomes. Most nuclear Rf genes have been shown to encode P-type pentatricopeptide repeat proteins (PPRs). Phylogenetic analysis of P-class PPRs from sequenced plants genomes has shown that Rf-proteins cluster in a distinct clade of P-class PPRs, RFL-PPRs, that display hallmarks of positive evolutionary selection. Genes encoding RFL-PPRs (RFLs) within a given plant genome tend to be closely related both in sequence and position, but a detailed understanding of how such species-specific expansion occurs is lacking. In the canola, (oilseed rape) species Brassica napus, previous work has indicated the nuclear restorer genes for the two native forms of CMS, Rfn (for nap CMS) and Rfp (pol CMS), represent alternate haplotypes, or alleles, of a single nuclear locus.ResultsFine genetic mapping indicates that Rfn does indeed localize to the same genomic region as Rfp. We find this region is enriched in RFL genes, three of which, based on their position and expression, represent potential candidates for Rfn; one of these genes, designated PPR4, is a preferred candidate in that it is not expressed in the nap CMS line. Comparison of the corresponding regions of the genomes of B. rapa, B. oleracea, Arabidopsis thaliana and A. lyrata provides insight into the expansion of this group of RFL genes in different lines of evolutionary descent.ConclusionsUnlike other nuclear restorer loci containing multiple RFL genes, the RFL genes in the Rf region of B. napus are not present in tandem arrays but rather are dispersed in genomic location. The genes do not share similar flanking non-coding regions and do not contain introns, indicating that they have duplicated primarily through a retrotransposition-mediated process. In contrast, segmental duplication has been responsible for the distribution of the 10 sequences we annotated as RFL genes in the corresponding region of the A. lyrata genome. Our observations define the Brassica Rf locus and indicate that different mechanisms may be responsible for the proliferation of RFL genes even among closely related genomes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3117-0) contains supplementary material, which is available to authorized users.

Discovery, utilization and molecular mechanisms of CMS-WA in rice

The discovery and exploitation of the Wild Abortive type cytoplasmic male sterility (CMS-WA) is a key issue in hybrid rice breeding for utilization of heterosis, which is the best successful case for exploitation of wild plant resources in agriculture. In this review, we traced the history of discovery and research of CMS-WA, and introduced the latest progresses on study of the molecular basis of CMS-WA and its restoration as well as the evolution of the related genes. The Chinese scientist Yuan LongPing pioneered the study of male sterility in rice since 1964. In 1970s, Yuan’s group discovered a male sterile wild rice ( Oryza rufipogon Griff.) plant in Hainan Island of China, and designated this material as “Wild Abortive (WA)”. Genetic study showed that this male sterility is conferred by the cytoplasm, thus is called CMS-WA. Chinese breeders made great efforts to breed CMS lines by introgression of the CMS-WA cytoplasm into a number of rice cultivars via backcrossing. Accordingly, related maintainer lines and restorer lines were bred to successfully develop the “three-line” system for hybrid rice production. The commercial hybrid rice was released in 1976 in China, and has occupied about 50% of the total rice planting area since the late 1980s, which increased grain yield by 20%–30%. In recent years, scientists have made great progress on the molecular studies of CMS-WA and its restoration, and the evolution of the genes for this system. As representative in this field, LIU YaoGuang’s group isolated the CMS-WA gene WA352 from the mitochondrial genome, which is a new gene consisting of multiple mitochondrial genomic segments of unknown function. WA352 is expressed constitutively into three transcripts, but the WA352 protein accumulates specifically in the anther tapetal cells at the microspore mother cell stage, and interacts directly with a nucleus-encoded protein COX11, which is the assembly factor for cytochrome c oxidase and also has a role in scavenge of reactive oxygen species (ROS) and inhibition of programmed cell death (PCD). When WA352 arrests COX11 by the interaction, the metabolism of ROS is affected, leading to ROS burst and cytochrome c release to the cytosol and triggering premature PCD in the tapetum. This abnormal tapetal degeneration eventually causes pollen abortion. Two loci, Rf3 and Rf4 , were mapped on chromosome 1 and 10, respectively, as the restorer genes for CMS-WA. Rf4 was firstly cloned recently by Liu’s group. Rf4 encodes a pentatricopeptide repeat (PPR) protein (PPR9-782-M) possessing a mitochondrial transit signal and 18 PPR motifs, with high similarity to PPR3-791-M encoded by Rf1a of the CMS-BT system. The Rf4 protein decreases the mRNA level of WA352 , thereby restoring WA352 -mediated male sterility probably in a post-transcriptional mechanism. However, Rf3 does not affect the WA352 transcript abundance but impairs the production of WA352 protein. Thus, the studies indicate that plant CMS/restoration systems comprises multiple layers of cytoplasmic-nuclear gene interactions in the molecular level. Recently, LIU’s group further studied the evolutionary trajectory of WA352 by identification and characterization of a number of mitochondrial genomic recombinant structures related to WA352 in the wild rice. These structures originated and evolved through complex evolutionary routes by multiple rearrangements in the mitochondrial genome of O. rufipogon. The study revealed that functional CMS genes originated from non-CMS protogenes by sequence variation-based functionalization as well as substoichiometric shifting (i.e., copy number variation). Finally, this review discussed the key issues and future directions of the hybrid rice breeding programs.

Development and validation of candidate gene-specific markers for the major fertility restorer genes, Rf4 and Rf3 in rice

Two major nuclear genes, Rf3 and Rf4, are known to be associated with fertility restoration of wild-abortive cytoplasmic male sterility (WA-CMS) in rice. In the present study, through a comparative sequence analysis of the reported putative candidate genes, viz. PPR9-782-(M,I) and PPR762 (for Rf4) and SF21 (for Rf3), among restorer and maintainer lines of rice, we identified significant polymorphism between the two lines and developed a set of PCR-based codominant markers, which could distinguish maintainers from restorers. Among the five markers developed targeting the polymorphisms in PPR9-782-(M,I), the marker RMS-PPR9-1 was observed to show clear polymorphism between the restorer (n = 120) and maintainer lines (n = 44) analyzed. Another codominant marker, named RMS-PPR762 targeting PPR762, displayed a lower efficiency in identification of restorers and maintainers, indicating that PPR9-782-(M,I) is indeed the candidate gene for Rf4. With respect to Rf3, a codominant marker, named RMS-SF21-5 developed targeting SF21, displayed significantly lower efficiency in identification of restorers and non-restorers as compared to the Rf4-specific markers. Validation of these markers in a F2 mapping population segregating for fertility restoration indicated that Rf4 has a major influence on fertility restoration and Rf3 is a minor gene. Further, the functional marker RMS-PPR9-1 was observed to be very useful in identification of impurities in a seed lot of the popular hybrid, DRRH3. Interestingly, when RMS-PPR9-1 and RMS-SF21-5 were considered in conjunction with analysis, near-complete, marker–trait co-segregation was observed, indicating that deployment of the candidate gene-specific markers both Rf4 and Rf3, together, can be helpful in accurate identification of fertility restorer lines and can facilitate targeted transfer of the two restorer genes into elite varieties through marker-assisted breeding.

Fine mapping of a fertility restoring gene for a new CMS hybrid rice system

The Fujian Abortion cytoplasmic male sterility (CMS-FA) system, a new type of sporophytic CMS system in indica rice (Oryza sativa L.), was developed using the cytoplasm and the corresponding fertility-restoring gene from a wild rice (O. rufipogon L.), which originated from Fujian Province, China. Previous studies in combination with several years of production practice demonstrated that CMS-FA hybrid rice was superior to CMS-WA hybrid rice, a prevailing hybrid rice worldwide, and that the male fertility restoration was controlled by a pair of dominant alleles. We tentatively designated the fertility restoration gene as Rf(fa). The analysis of the polymorphism between the fertile and sterile pool DNAs from a mapping segregation population (BC1F1) indicated that Rf(fa) was located on rice chromosome 10. We further delimited the Rf(fa) locus to a 121.1-kb region flanked by RM6100 and MM2023, which were approximately 0.26 cM and 0.18 cM away from Rf(fa), respectively, by simple sequence repeat molecular marker linkage genetic analysis. These results would facilitate the map-based cloning of Rf(fa), the elucidation of a novel molecular mechanism underlying cytoplasm–nucleus interaction in the CMS-FA system, and the production application of this hybrid rice.

Development of Gene-Pyramid Lines of the Elite Restorer Line, RPHR-1005 Possessing Durable Bacterial Blight and Blast Resistance.

RPHR-1005, the stable restorer line of the popular medium slender (MS) grain type rice hybrid, DRRH-3 was improved in this study for resistance against bacterial blight (BB) and blast diseases through marker-assisted backcross breeding (MABB). In this study, four major resistance genes (i.e., Xa21 and Xa33 for BB resistance and Pi2 and Pi54 for blast resistance) have been transferred to RPHR-1005 using RPBio Patho-1 (possessing Xa21 + Pi2), RPBio Patho-2 (possessing Xa21 + Pi54) and FBR1-15EM (possessing Xa33) as the donors. Foreground selection was carried out using PCR-based molecular markers specific for the target resistance genes and the major fertility restorer genes, Rf3 and Rf4, while background selection was carried out using a set of parental polymorphic rice SSR markers and backcrossing was continued uptoBC2 generation. At BC2F2, plants possessing the gene combination- Xa21 + Pi2, Xa21 + Pi54 and Xa33 in homozygous condition and with >92% recovery of the recurrent parent genome (RPG) were identified and intercrossed to combine all the four resistance genes. Twenty-two homozygous, pyramid lines of RPHR-1005 comprising of three single-gene containing lines, six 2-gene containing lines, eight 3-gene containing lines, and five 4-gene containing lines were identified among the double intercross lines at F3 generation (DICF3). They were then evaluated for their resistance against BB and blast, fertility restoration ability and for key agro-morphological traits. While single gene containing lines were resistant to either BB or blast, the 2-gene, 3-gene, and 4-gene pyramid lines showed good level of resistance against both and/or either of the two diseases. Most of the 2-gene, 3-gene, and 4-gene containing pyramid lines showed yield levels and other key agro-morphological and grain quality traits comparable to the original recurrent parent and showed complete fertility restoration ability, with a few showing higher yield as compared to RPHR-1005. Further, the experimental hybrids derived by crossing the gene-pyramid lines of RPHR-1005 with APMS6A (the female parent of DRRH-3), showed heterosis levels equivalent to or higher than DRRH-3. The results of present study exemplify the utility of MABB for targeted improvement of multiple traits in hybrid rice.

A Gene Encoding Pentatricopeptide Repeat Protein Partially Restores Fertility in RT98-Type Cytoplasmic Male-Sterile Rice.

Cytoplasmic male sterility (CMS) lines in rice, which have the cytoplasm of a wild species and the nuclear genome of cultivated rice, are of value for the study of genetic interactions between the mitochondrial and nuclear genomes. The RT98-type CMS line RT98A and the fertility restorer line RT98C carry the cytoplasm of the wild species Oryza rufipogon and the nuclear genome of the Taichung 65 cultivar (Oryza sativa L.). Based on a classical crossing experiment, fertility is reported to be restored gametophytically by the presence of a tentative single gene, designated Rf98, which is derived from the cytoplasm donor. Fine mapping of Rf98 revealed that at least two genes, which are closely positioned, are required for complete fertility restoration in RT98A. Here, we identified seven pentatricopeptide repeat (PPR) genes that are located within a 170 kb region as candidates for Rf98 Complementation tests revealed that the introduction of one of these PPR genes, PPR762, resulted in the partial recovery of fertility with a seed setting rate up to 9.3%. We conclude that PPR762 is an essential fertility restorer gene for RT98-type CMS. The low rate of seed setting suggested that some other genes near the Rf98 locus are also necessary for the full recovery of seed setting.

Water-Use-Efficient Cytoplasmic Male Sterility Analogs in Sunflower

ABSTRACTCytoplasmic male sterility (cms) and associated restorer genes have been the major promoters for the development of commercial hybrids in sunflower (Helianthus annuus L.) around the world. Attempts have been made in the past to find new cms sources to broaden the genetic base and reduce the vulnerability of the cultivated crop to environmental stresses and diseases. However, these diverse cms sources could not be used for hybrid development because of non-availability of effective restorers. Further, it is important to study the agronomic value of the new cms sources, which could help breeders improve the hybrid performance by transferring only the nuclear genotype into an elite cytoplasmic background. To study the influence of different cms sources on quantitative characters, nine alloplasmic male-sterile lines (cms analogs) of the inbred line NC-41B were developed. The objective was to investigate the extent of diversity among these cms analogs in relation to their agronomic performance and to identify water-use-efficient cms sources that could be exploited for development of hybrids suitable for growing under moisture-stress conditions. These cms analogs were evaluated for divergence on the basis of 21 characters comprising morpho-physiological, yield, and quality aspects, under water-stress conditions. Water-stress was induced by withdrawing irrigation after anthesis. The analysis of variance revealed significant differences among the cms analogs for all the traits. On the basis of Mahalanobis D2 statistic, nine alloplasmic lines, along with a common maintainer, were grouped into four clusters. A critical examination of clusters indicated the presence of a high level of diversity in the material. CMS ARG-3A (Helianthus argophyllus) was found to be the most diverse cms source that performed well under water-stress conditions, which could be used as a new cms source to develop high-yielding, water-use-efficient sunflower hybrids with diverse cytoplasmic backgrounds.

Development of Cytoplasmic Male Sterile IR24 and IR64 Using CW-CMS/Rf17 System.

BackgroundA wild-abortive-type (WA) cytoplasmic male sterility (CMS) has been almost exclusively used for breeding three-line hybrid rice. Many indica cultivars are known to carry restorer genes for WA-CMS lines and cannot be used as maintainer lines. Especially elite indica cultivars IR24 and IR64 are known to be restorer lines for WA-CMS lines, and are used as male parents for hybrid seed production. If we develop CMS IR24 and CMS IR64, the combination of F1 pairs in hybrid rice breeding programs will be greatly broadened.FindingsFor production of CMS lines and restorer lines of IR24 and IR64, we employed Chinese wild rice (CW)-type CMS/Restorer of fertility 17 (Rf17) system, in which fertility is restored by a single nuclear gene, Rf17. Successive backcrossing and marker-assisted selection of Rf17 succeeded to produce completely male sterile CMS lines and fully restored restorer lines of IR24 and IR64. CW-cytoplasm did not affect agronomic characteristics.ConclusionsSince IR64 is one of the most popular mega-varieties and used for breeding of many modern varieties, the CW-CMS line of IR64 will be useful for hybrid rice breeding.Electronic supplementary materialThe online version of this article (doi:10.1186/s12284-016-0097-2) contains supplementary material, which is available to authorized users.

598. TARGTCNS for the Treatment of Central Nervous System Disorders

Diseases and disorders of the central nervous system (CNS) represent a large field of unmet need that significantly impacts the lifespan and quality of life of many patients. The ability to deliver therapeutic proteins from the systemic circulation to the CNS is hampered by the blood-brain barrier (BBB). A number of strategies have been developed to allow therapies to cross the BBB, but to date with limited success. We have developed a novel localized approach to deliver protein and peptide therapeutics to the CNS based on our proprietary Transduced Autologous Restorative Gene Therapy system (TARGT™) platform. TARGT is based on autologous dermal micro-organs (MO) harvested and transduced ex-vivo to produce a therapeutic protein and then re-implanted in the patient. In this study we assessed whether TARGT can survive and deliver sustained localized protein post implantation into the CNS. In rats, MOs, 2×1mm each, were harvested from Lewis rat skin, characterized in-vitro, and then implanted in the Lewis rats’ cisterna magna. The procedure was well-tolerated and without observed behavioral change in all implanted rats. Histopathology of MOs excised several weeks post implantation, demonstrated viable and integrated MOs without signs of implant rejection. In the second phase, rat MOs were processed ex-vivo into TARGT secreting erythropoietin (TARGTEPO) by transduction with Helper Dependent Adenoviral vector encoding human erythropoietin (HDAd-HuEPO). Rat TARGTs secreting human EPO were then implanted into Lewis rats’ cisterna magna. Post implantation, cerebrospinal fluid (CSF) and serum were collected and EPO levels were measured by HuEPO ELISA. Results obtained suggest that implantation was well tolerated with no observed signs of rejection. Measurable human EPO levels were detected in rat CSF for the duration of the experiment. Detectable but low levels of Hu-EPO were also measured in the rat serum, as expected, since CSF drains into the peripheral blood. In pigs we have tested the viability and monoclonal antibody secretion from TARGTs into the CNS. Humira, an established antibody drug, was selected as a proof of concept. Pig MOs were harvested and transduced with the HDAd vector carrying the Humira sequence. Two TARGTHumira were implanted in the pig subdural space at the parietal convexity region and the pig was followed for one week post implantation. Results obtained post implantation suggests no observed pig's behavioral change. Humira was measured in CSF samples taken from the implantation area and the lumbar space. One week post implantation, TARGTs were excised out of the pig brain when they are viable and no signs of inflammation or damage to the brain tissue were observed. These results demonstrate that TARGT is a promising novel therapeutic platform with the potential for treatment of CNS disorders. We are now actively studying TARGTCNS treatment of lysosomal storage diseases and brain malignancies.

Microsatellite-Aided Screening for Fertility Restoration Genes (Rf) Facilitates Hybrid Improvement

DNA markers enabled to determine the chromosomal locations of the two Rf genes (Rf3 and Rf4) in the wild-abortive cytoplasmic male sterility (WA-CMS) system. Four simple sequence repeats (SSRs) RM171, RM258, RM315 and RM443 were used to detect the allelic status with respect to the fertility restoration genes (Rf3 and Rf4) in 300 rice cultivars or breeding lines. The results revealed that out of 300 lines, 90 lines screened had Rf3, 65 lines had Rf4, and 45 lines had Rf3 and Rf4 alleles. Furthermore, 45 lines selected using SSR markers were mated with a CMS line (IR58025A) to analyze their restoring ability. Offspring of all the test lines except HHZ8-SAL9DT1-Y1, HHZ5-SAL9-Y3-1 and IDSA77 exhibited higher pollen and spikelet fertility (> 80%), thus confirming they bear the Rf alleles. The hybrid offspring of ARH12-6-1-1-B-3-1, IR32307-10-3-2-1 and Sahel 329 had the highest pollen fertility (97.39%, 98.30% and 97.10%, respectively) and spikelet fertility (95.10%, 97.07% and 96.10%, respectively).

Особливості формування елементів структури продуктивності кукурудзи залежно від наявності в їх материнському компоненті генетичних маркерів

Особливості формування елементів структури продуктивності кукурудзи залежно від наявності в їх материнському компоненті генетичних маркерів

Gene Mapping of Fertility Restorer Gene(s) in Rice using STS and SSR Markers

Gene Mapping of Fertility Restorer Gene(s) in Rice using STS and SSR Markers

The restorer gene for soybean M‐type cytoplasmic male sterility, Rf‐m, is located in a PPR gene‐rich region on chromosome 16

AbstractOver the past decade, M‐type cytoplasmic male sterility (CMS) line W931A and a variety of restorer lines have been exploited for the release of hybrid seeds in soybean (Glycine max). However, the identities of restorer genes in the nuclei of soybean restorer lines are still unclear. In this study, we analysed the inheritance pattern of restorer locus Rf‐m from restorer line WR016 and constructed a high‐resolution map of this locus. Results showed that Rf‐m in WR016 is a monogenic dominant gene located within a 162.4‐kb region on chromosome 16, which is flanked on each side by new developed simple sequence repeat (SSR) markers GmSSR1602 and GmSSR1610 at a distance of 0.11 and 0.25 cM, respectively. Nineteen open reading frames (ORFs) were predicted in this region. Of these, seven genes arranged in tandem on chromosome 16 encode pentatricopeptide repeat (PPR) proteins, which is similar to other reported restorer loci in plants. These results lay a solid foundation for map‐based cloning of the Rf‐m gene and will be helpful for marker‐assisted selection of elite CMS restorer lines.

In Silico Identification of Candidate Genes for Fertility Restoration in Cytoplasmic Male Sterile Perennial Ryegrass (Lolium perenne L.).

Perennial ryegrass (Lolium perenne L.) is widely used for forage production in both permanent and temporary grassland systems. To increase yields in perennial ryegrass, recent breeding efforts have been focused on strategies to more efficiently exploit heterosis by hybrid breeding. Cytoplasmic male sterility (CMS) is a widely applied mechanism to control pollination for commercial hybrid seed production and although CMS systems have been identified in perennial ryegrass, they are yet to be fully characterized. Here, we present a bioinformatics pipeline for efficient identification of candidate restorer of fertility (Rf) genes for CMS. From a high-quality draft of the perennial ryegrass genome, 373 pentatricopeptide repeat (PPR) genes were identified and classified, further identifying 25 restorer of fertility-like PPR (RFL) genes through a combination of DNA sequence clustering and comparison to known Rf genes. This extensive gene family was targeted as the majority of Rf genes in higher plants are RFL genes. These RFL genes were further investigated by phylogenetic analyses, identifying three groups of perennial ryegrass RFLs. These three groups likely represent genomic regions of active RFL generation and identify the probable location of perennial ryegrass PPR-Rf genes. This pipeline allows for the identification of candidate PPR-Rf genes from genomic sequence data and can be used in any plant species. Functional markers for PPR-Rf genes will facilitate map-based cloning of Rf genes and enable the use of CMS as an efficient tool to control pollination for hybrid crop production.

Marker-assisted introgression of the major bacterial blight resistance gene, Xa21 and blast resistance gene, Pi54 into RPHR-1005, the restorer line of the popular rice hybrid, DRRH3

The elite Indian rice hybrid, DRRH3 is highly susceptible to two major diseases, bacterial blight (BB) and blast, which limit its productivity significantly. In the present study, we have introgressed two major genes, viz., Xa21 and Pi54 conferring resistance against BB and blast, respectively into RPHR-1005, the male parent of DRRH3 through marker-assisted backcross breeding (MABB) and analyzed the backcross derived plants for their resistance against BB and blast. RPBio Patho-2 was used as a donor for both the resistance genes. Gene-specific markers were used for the foreground selection of Xa21 and Pi54 at each stage of backcrossing and markers specific for the major fertility restorer genes, Rf3 and Rf4 were used only at BC1F1 generation for foreground selection. Background selection was done using 62 polymorphic SSR markers and marker-assisted backcrossing was continued till BC3 generation. At BC3F4, through intensive phenotype-based selections 15 promising lines (ABLs) possessing high level of resistance against BB and blast, high yield, fine-grain type, complete fertility restoration along with better panicle exsertion and taller plant type as compared to RPHR-1005 were identified and test crossed with APMS 6 A, the female parent of DRRH3. The newly derived hybrids (i.e. improved versions of DRRH3) were observed to possess high level of resistance against BB and blast along with medium-slender grain type and yield level better than or equivalent to that of DRRH3. Our study exemplifies the utility of MABB for targeted improvement of multiple traits in hybrid rice.

The effect of disease on the evolution of females and the genetic basis of sex in populations with cytoplasmic male sterility.

The evolution of separate males and females is an important evolutionary transition that has occurred multiple times in flowering plants. While empirical studies have stressed the potential importance of natural enemies and organismal interactions in the evolution of separate sexes, there has been no treatment of natural enemies in the theoretical literature. We investigated the effects of disease on the evolution of females in gynodioecious populations composed of females and hermaphrodites, where sex is determined by the interaction of cytoplasmic male sterility (CMS) and nuclear restorer genes. When females are significantly more resistant than hermaphrodites, disease drives an increase in the frequency of females and sex determination becomes nuclear, creating the pre-conditions for the evolution of separate males and females. However, when females are only moderately more resistant, disease drives changes in the frequency of CMS and restorer alleles, but has little effect on the frequency of females. We discuss our results in the context of the evolution of mating systems and cyto-nuclear epistasis.

Achievements in NS rapeseed hybrids breeding

The increased production of oilseed rape (Brassica napus L.) is evident on a global scale, but also in Serbia in the last decade. Rapeseed is used primarily for vegetable oil and processing industry, but also as a source of protein for animal feed and green manure. Following the cultivation of varieties, breeding and cultivation of hybrid rapeseed started in the 1990's, to take advantage of heterosis in F1 generation, while protecting the breeder's rights during seed commercialization. The breeding of hybrid oilseed rape requires high quality starting material (lines with good combining abilities) for introduction of male sterility. Ogura sterility system is primarily used at the Institute of Field and Vegetable Crops, Novi Sad, Serbia. To use this system, separate lines are modified with genes for cytoplasmic male sterility (cms female line - mother line) and restoration of fertility (Rf male lines - father line). In order to maintain the sterility of the mother line it is necessary to produce a maintainer line of cytoplasmic male sterility. Creation of these lines and hybrids at the Institute of Field and Vegetable Crops was successfully monitored with intense use of cytogenetic laboratory methods. The structure and vitality of pollen, including different phases during meiosis were checked so that cms stability was confirmed during the introduction of these genes into different lines. Rapeseed breeding program in Serbia resulted in numerous varieties through collaboration of researchers engaged in breeding and genetics of this plant species. So far, in addition to 12 varieties of winter rapeseed and two varieties of spring rapeseed, a new hybrid of winter rapeseed NS Ras was registered in Serbia. NS Ras is an early-maturing hybrid characterized by high seed yield and oil content. Average yield of NS Ras for two seasons and three sites was 4256 kg ha-1 of seed and 1704 kg ha-1 of oil. Three promising winter rapeseed hybrids are in the process of registration in the Republic of Serbia and one in the EU. While addressing many challenges in breeding, especially resistance to biotic and abiotic stress, in addition to classical breeding, it is necessary to apply the cytogenetic and molecular analysis to increase the efficiency of producing competitive rapeseed genotypes.

Influence of Soil Moisture and Air Temperature on the Stability of Cytoplasmic Male Sterility (CMS) in Maize (<i>Zea mays</i> L.)

Cytoplasmic male sterility (CMS) is a maternally inherited trait that suppresses the production of viable pollen. CMS is a useful biological tool for confinement strategies to facilitate coexistence of genetically modified (GM) and non-GM crops in case where it is required. The trait is reversible and can be restored to fertility in the presence of nuclear restorer genes (Rf genes) and by environmental impacts. The aim of this study was to investigate the influence of the level of irrigation on the stability of CMS maize hybrids under defined greenhouse conditions. Additionally the combination of irrigation and air temperature was studied. Three CMS maize hybrids were grown with different levels of irrigation and in different temperature regimes. Tassel characteristics, pollen production and fertility were assessed. The CMS stability was high in hot air temperatures and decreased in lower temperatures. The level of irrigation had no major effect on the level of sterility. The extent of these phenomena was depending on the genotype of CMS maize and should be known before using CMS for coexistence purposes.

Development of a platform for breeding by design of CMS restorer lines based on an SSSL library in rice (Oryza sativa L.).

Exploitation of the heterosis of hybrid rice has shown great success in the improvement of rice yields. However, few genotypes exhibit strong restoration ability as effective restorers of cytoplasmic male sterility (CMS) in the development of hybrid rice. In this study, we developed a platform for the breeding by design of CMS restorer lines based on a library of chromosomal single segment substitution lines (SSSLs) in the Huajingxian74 (HJX74) genetic background. The target genes for breeding by design, Rf34 and Rf44, which are associated with a strong restoration ability, and gs3, gw8, Wxg1 and Alk, which are associated with good grain quality, were selected from the HJX74 SSSL library. Through pyramiding of the target genes, a restorer line, H121R, was developed. The H121R line was then improved regarding blast resistance by pyramiding of the qBLAST11 gene. Hence, a new restorer line with blast resistance, H131R, was developed. The platform involving the Rf34 and Rf44 restorer genes would be used for the continuous improvement of restorer lines through breeding by design in rice.

Preclinical and preliminary clinical evaluation of genetically transduced dermal tissue implants for the sustained secretion of erythropoietin and interferon α

Protein drugs are currently delivered by bolus injection and although treatment frequently is successful, these methods also have major drawbacks, which call for the development of alternative technologies allowing prolonged delivery of these drugs. We developed a new ex vivo gene therapy platform called Transduced Autologous Restorative Gene Therapy (TARGT) for sustained long term production and secretion of autologous therapeutic proteins. A biopsy of dermal tissue taken from the patient is transduced ex vivo with a viral vector encoding the required gene under a constitutive promoter. Following measurement of protein secretion ex vivo, the transduced dermal tissue is implanted back into the patient, where it secretes the therapeutic protein into the circulation for several months or longer. A major hurdle to this approach is potential immunogenicity of the transduced tissue following implantation. In this paper we describe the preclinical and early clinical development of this technology, which allowed overcoming these hurdles. To that end, we have used the helper dependent (HD) adenoviral vector with newly designed expression cassette containing genetic elements to optimize transgene expression. Moreover, we have developed procedures for TARGT tissue implantation, with measures to improve engraftment and reduce inflammation and rejection. Implantation of human TARGT to immune-deficient SCID mice indicated long-term production of active proteins in the blood. Preliminary results of a clinical trial from two anemic end-stage renal disease patients, implanted with TARGTs expressing the human erythropoietin (EPO) gene, demonstrated prolonged secretion with physiologic blood level of the hormone and Hemoglobin maintenance in the desired range, for a period of at least five months without exogenous EPO administration. We believe that the TARGT technology has the potential to become a platform for the sustained delivery of therapeutic proteins in various clinical indications.