Clone C2 Research Articles

Quality of Tectona grandis for sawn wood production

Forestry companies have invested in genetic improvement to increase wood production in a shorter amount of time. Thus, studies are needed to compare the properties of clonal and seminal wood materials. The objective of this study was to analyze physical and mechanical properties of Tectona grandis from clonal (C1 and C2) and seminal (S) origin and evaluate the yield and quality of sawn wood subjected to outdoor and oven drying. Genetic material was collected from six, 15-year-old trees. Clone C2 presented the lowest amount of bark, and 51 % heartwood up to half the commercial height, while the heartwood of C1 and S went up to 25 % of the height. The three materials did not differ statistically for maximum angular deviation, pith eccentricity, basic density, Janka hardness, anisotropy, commercial income of sawn wood and the presence of knots. After the drying processes, the bowing and crooking indexes were less than 5 mm.m-1, however, the seminal material showed a higher cracking incidence after outdoor and oven drying. In conclusion, the wood properties of the three materials are similar. In addition, the oven drying process is recommended.

Abstract P5-02-28: p27Kip1 V109G single-nucleotide polymorphism (SNP): pinpointing the hormone-receptor positive breast cancer subpopulation that requires CDK4/6 inhibitors in addition to endocrine therapy

Abstract Background: CDK4/6 inhibitors benefit a limited percentage of hormone receptor-positive breast cancer (HRPBC) patients in the adjuvant setting: according to the MonarchE study, from all patients treated with the endocrine plus CDK4/6 inhibitor combination, 84% were adequately treated with endocrine therapy alone, ~5% experienced benefit from the combination, and 11% were not rescued from relapse by abemaciclib. Given the side effects and the cost, biomarkers to guide treatment decisions in this setting are appealing. We found that the p27Kip1 V109G SNP was enriched in HRPBC patients experiencing relapse despite endocrine treatment. p27Kip1 binds to cyclins and CDKs, restraining cells from cycling by inhibiting the formation of CDK/cyclin complexes and their kinase activity, resulting in less phosphorylation of Rb. A functionally impaired p27Kip1 could render tumor cells insensitive to endocrine therapy, while being rescued by CDK4/6 inhibitors. Thus, this SNP could narrow down the patient population that requires adjuvant CDK4/6 inhibitors. Methods: Isogenic HRPBC cell lines, wild-type or polymorphic homozygous for the p27Kip1 V109G SNP were generated with CRISPR-Cas9. Cell cycle and cell viability were assessed with BRDU incorporation and colony assays. Immunoprecipitation coupled with western blot (WB) was used to measure the formation of CDK/Cyclin complexes; Rb phosphorylation was assessed by WB. An in vitro kinase assay was set up to measure the CDK4 activity of p27Kip1/CDK/Cyclin complexes. Patients (n=115) with metastatic, HRPBC receiving endocrine monotherapy or in combination with CDK4/6 inhibitors were genotyped for the p27Kip1 V109G SNP, and PFS by genotype and therapy compared with the Kaplan-Meier method. All statistical tests were two-sided. Results: three isogenic polymorphic clones were generated from the wild-type T47-D hormone-positive cell line. The three clones were resistant to hormonal deprivation compared to wild-type cells. The relative plating efficiency (RPE) in the colony assays of the polymorphic clones exposed to hormonal deprivation compared to that of deprived T47-D cells was 550% (clone C1), 165% (clone E1) and 100% (Clone F5); P< 0.005. The three clones were also resistant to fulvestrant (Fulv) (300%, 170% and 180%, respectively); P< 0.005. Cell cycle (positive BRDU cells) decreased ~3 fold in wild type cells (18% to 6.5%) when exposed to hormonal deprivation or Fulv, but remained unaltered in the polymorphic clones. However, when palbociclib was added to hormonal deprivation or Fulv, the effects in RPE increased and were similar in polymorphic clones and parental cells (>5% RPE compared to vehicle, both in polymorphic and wild-type cells). The p27Kip1 V109G SNP was found in homozygosity in ~15% of metastatic HRPBC patients. When patients received endocrine monotherapy in the first-line setting, polymorphic patients experience rapid failure (N=51) compared to wild-type/heterozygous patients (4.3 vs. 21.1 months; P < 0.0001). However, when patients received hormonal plus CDK4/6 inhibitors, the differences disappeared (18.3 vs. 24.3 months; P=0.85). Mechanistically, we observed that the formation of CDK2/CyclinA, CDK2/CyclinE and CDK4/Cyclin D1 complexes was >200% higher in polymorphic than in wild-type cells (P< 0.05). Regarding CDK4 kinase activity of p27Kip1/CDK/Cyclin complexes, as opposed to wild-type p27Kip1, p27Kip1 V109G was unable to suppress the kinase activity of CDK4 in presence of Fulv or hormonal deprivation. However, palbociclib was able to fully suppress CDK4 kinase activity regardless of the p27Kip1 genotype. Conclusion: Germline p27Kip1 genotyping can constitute a tool for treatment selection: whereas wild-type patients are adequately treated with endocrine monotherapy, polymorphic patients are inherently resistant, but are rescued with CDK4/6 inhibitors. Thus, hormonal+CDK4/6 inhibitor combos could be reserved for the polymorphic patients. Citation Format: Miguel Quintela-Fandino, Silvana Mouron, Maria J. Bueno, Manuel Muñoz, Raul Torres, Sandra Rodriguez, Rodrigo Sánchez-Bayona, Luis Manso, Jorge Silva, Marcos Malumbres. p27Kip1 V109G single-nucleotide polymorphism (SNP): pinpointing the hormone-receptor positive breast cancer subpopulation that requires CDK4/6 inhibitors in addition to endocrine therapy. [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-02-28.

Respostas morfofisiológicas de plantas matrizes de cafeeiro conilon em jardim clonal superadensado

Abstract The objective of this work was to evaluate the morphophysiological responses and cutting production of clones of Conilon coffee (Coffea canephora) cultivars in a super-dense clonal garden in the state of Espírito Santo, Brazil. The super-dense clonal garden was built in 2019 using 39 clones: 9, 9, 9, and 12 of cultivars Centenária ES8132, Diamante ES8112, ES8122 (Jequitibá), and Marilândia ES8143, respectively. The experiment was carried out in a randomized complete block design, with three replicates. Cutting production and the following morphophysiological traits were evaluated at 9 and 18 months after planting: chlorophyll index, normalized difference vegetation index, plant height, canopy height, canopy diameter, number of shoots, number of viable cuttings, number of leaves, fresh leaf mass, and plant fresh and dry matter mass. The super-dense clonal garden caused different morphophysiological responses among the studied clones. In general, clones C2, C5, C6, C8, D1, D8, D9, J8, M2, M9, M10, and M12 showed a higher mean cutting production, whereas C4, J1, J4, M4, and M5 were the most sensitive to the super-dense regime. Under these conditions, it is recommended to increase the proportion of matrix plants of the latter clones.

Energy potential of wood from clones of Eucalyptus and Corymbia in different spacings

This study aimed to determine the influence of spacing and different genetic materials of Eucalyptus and Corymbia on wood production for energy purposes. At 24 months, two Eucalyptus clones (C1 and C2) and two Corymbia clones (C3 and C4) implanted in three different spacings (3.0 × 3.0, 3.0 × 1.5, and 3.0 × 1.0 m) were evaluated, totaling 12 treatments. In each treatment, 4 trees were selected, and 5 disks were removed from each tree, which were used to determine the basic density, the higher calorific value and to quantify the elemental carbon in the wood. Based on these characteristics, it was possible to estimate the dry mass, the carbon mass, and the amount of energy per hectare. It was observed that there was an effect of spacing, clone and interaction for the basic density of wood, being observed in clone C3 (C. citriodora × C. torelliana) the highest values of this property. As for the higher calorific value and the carbon content, no significant effect was found. It was also verified that the production of dry mass, carbon mass, and the amount of energy available per hectare increased with the densification of the spacings for all clones studied. The highest values of the amount of energy per hectare, in the different spacings, were attributed to clones C2 and C3 (3.0 × 3.0 m), clone C3 (3.0 × 1.5 m), and clones C1 and C3 (3.0 × 1.0 m). The C3 clone (Corymbia hybrid) can be a good alternative for energy use, due to its wood quality and productivity similar, or even superior, to Eucalyptus clones already used for this purpose.

Bioprospecting by Phage Display of Mimetic Peptides of Chlamydia trachomatis for Use in Laboratory Diagnosis.

BackgroundChlamydia trachomatis infection is a major public health problem and the most common sexually transmitted infection in the world. Although highly prevalent, 70% to 80% of cases are asymptomatic and undiagnosed.PurposeTo overcome some limitations in terms of rapid diagnosis, phage display technology was used to bioprospect peptide mimetics of C. trachomatis immunoreactive and immunogenic antigens to be selected for the production of synthetic peptides.MethodsInitially, IgG from 22 individuals with C. trachomatis and 30 negative controls was coupled to G protein magnetic beads. The phage display technique consisted of biopanning, genetic sequencing, bioinformatics analysis and phage ELISA.ResultsClones G1, H5, C6 and H7 were selected for testing with individual samples positive and negative for C. trachomatis. Reactions were statistically significant (p < 0.05), with a sensitivity of 90.91, a specificity of 54.55, and AUC values >0.8. One-dimensional analysis with C. trachomatis components indicated that the G1 clone aligned with cell wall-associated hydrolase domain-containing protein, the H5 clone aligned with glycerol-3-phosphate acyltransferase PlsX protein, the C6 clone aligned with a transposase and inactivated derivatives, and the H7 clone aligned with GTP-binding protein. Molecular modeling and three-dimensional analysis indicated the best fit of the four clones with a protein known as chlamydial protease/proteasome-like activity factor (CPAF), an important virulence factor of the bacterium.ConclusionThe peptides produced by phage display are related to the metabolic pathways of C. trachomatis, indicating that they can be used to understand the pathogenesis of the infection. Because of their high sensitivity and AUC values, the peptides present considerable potential for use in platforms for screening C. trachomatis infections.

Targeted Profiling of Epitranscriptomic Reader, Writer, and Eraser Proteins Accompanied with Radioresistance in Breast Cancer Cells.

Epitranscriptomic reader, writer, and eraser (RWE) proteins recognize, install, and remove modified nucleosides in RNA, which are known to play crucial roles in RNA processing, splicing, and stability. Here, we established a liquid chromatography-parallel-reaction monitoring (LC-PRM) method for high-throughput profiling of a total of 152 epitranscriptomic RWE proteins. We also applied the LC-PRM method, in conjunction with stable isotope labeling by amino acids in cell culture (SILAC), to quantify these proteins in two pairs of matched parental/radioresistant breast cancer cells (i.e., MDA-MB-231 and MCF-7 cells and their corresponding radioresistant C5 and C6 clones), with the goal of assessing the roles of these proteins in radioresistance. We found that eight epitranscriptomic RWE proteins were commonly altered by over 1.5-fold in the two pairs of breast cancer cells. Among them, TRMT1 (an m2,2G writer) may play a role in promoting breast cancer radioresistance due to its clinical relevance and its correlation with DNA repair gene sets. To our knowledge, this is the first report of a targeted proteomic method for comprehensive quantifications of epitranscriptomic RWE proteins. We envision that the LC-PRM method is applicable for studying the roles of these proteins in the metastatic transformation of cancer and therapeutic resistance of other types of cancer in the future.

Targeted Proteomic Analysis Revealed Kinome Reprogramming during Acquisition of Radioresistance in Breast Cancer Cells.

Radiotherapy constitutes a major therapeutic modality for early management of breast cancer. Despite the high efficacy in treating breast cancer (BC), radiation resistance and tumor recurrence are major hurdles in breast cancer radiotherapy. Herein, stable isotope labeling by amino acids in cell culture (SILAC) was employed, along with the parallel-reaction monitoring (PRM)-based targeted quantitative proteomic method, to examine the differences in kinase protein expression in MCF-7 and MDA-MB-231 breast cancer cells and their corresponding radioresistant C6 and C5 clones. We quantified the relative protein expression levels of 300 and 281 kinases in C5/MDA-MB-231 and C6/MCF-7 pairs of breast cancer cells, respectively. We also showed that TAF9, which was one of the differentially expressed kinases, enhances radiation resistance in breast cancer cells. Moreover, a correlation analysis of gene expression suggested TAF9's role in upregulating the expression of genes involved with radioresistance. Overall, our study uncovered a large number of differentially expressed kinases accompanied with the acquisition of radioresistance and revealed a role of TAF9 in promoting radioresistance in breast cancer.

Anti-tumor effect of single-chain antibody to Reg3a in colorectal cancer

BackgroundRegenerating protein 3a (Reg3a) is a trophic factor that functions as a stimulus in cell proliferation and neogenesis. Previous studies showed that Reg3a is ectopically upregulated in a majority of colorectal cancers (CRC) and detectable in the serum. MethodsSingle-chain variable fragment targeting Reg3a (scFv-Reg3a) was screened from a phage library. The bioactivity of recombinant Reg3a (rReg3a) and scFv-Reg3a were tested in LoVo and RKO cell lines using MTT, flow cytometry, wound healing and transwell analyses. Whether scFv-Reg3a inhibits tumor growth and enhances 5-fluorouracil (5-FU)-caused cell death were further examined in LoVo cell-transplanted nude BALB/c mice. ResultsA scFv-Reg3a from clone C2 was obtained and its binding affinity (KD) to rReg3a was determined to be 4.44 × 10-10. In cultured LoVo and RKO cells, rReg3a promoted but scFv-Reg3a inhibited cell proliferation, survival, migration and invasion. In LoVo cell-xenografted nude mice, administration of rReg3a accelerated tumor growth while scFv-Reg3a suppressed cell proliferation and reinforced 5-FU-induced cell death. ConclusionThe newly developed scFv-Reg3a is an anti-cancer agent which is potent to suppress CRC cell proliferation and survival. The use of scFv-Reg3a could enhance the effectiveness of 5-FU-based chemotherapy in the cancerous treatment.

Severity of rust (Olivea tectonae) in different clones of teak in northern Mato Grosso

The fungus Olivea tectonae , has been increasing attention from different countries of course have teak or commercial plantations with the species, as it causes severe defoliation of plants, contributing to the reduction of production. The objective was to monitor different teak clones, with five months in Sinop / MT, with respect to the fungus O. tectonae , causal agent of rust. The study of disease progress was made between the months of May 2009 to July 2010, evaluating the severity visually in three regions of the plant: upper, middle and lower. We analyzed ten different clones C1, C2, C3, C4, C5, C6, C7, C8, C9, C10 and, after completion of data collection built up the area under the disease progress curve (AUDPC). The results showed that clone C7 was most likely at the top (AAPCD 35,45) and clone C1 was the most resistant (AACPD 22,37). The C9, C8 and C10 clones are statistically equal, more likely in the middle region (AAPCD 6.332,02; 5.873,34; 5.759,75, respectively) and C5 more resistant clone in this region (AACPD 1.872,70). In the lower region the clone C5 was the toughest (AACPD 5.691,06) and the C9 clone was the most likely (AACPD 15.746,73). In the analysis of the entire plant, the clone C5 was tougher (AACPD 27.603.889,00) and C9 clones C8 and C10 were statistically equal, proving to be the most susceptible to rust (AACPD 78.872.378,00; 68.335.115,00 e 65.686.893,00 respectively).

Enhanced Plantlet Regeneration in Two Cacao (Theobroma cacao) Clones from Immature Inflorescence Explants

Theobroma cacao L. (cacao) is a major tropical crop, grown for its oil-rich seed, which is used in the manufacture of chocolate, its derivatives, and cosmetics. Cacao is cultivated mainly by smallholders and represents a significant export commodity for some developing countries such as Côte d’Ivoire. It is conventionally propagated by seeds, grafting, and cuttings. Somatic embryogenesis offers an alternative method for propagation where large-scale production of planting materials is possible. In the current study, the effect of different concentrations of 2, 4-dichlorophenoxyacetic acid (2, 4-D) and kinetin on induction of somatic embryogenesis and plantlet regeneration in two cocoa clones (coded as C1 and C14) were evaluated. Flowers were collected early in the morning, sterilized, explants excised and cultured on Driver, and Kuniyuki Walnut (DKW) media supplemented with different concentrations of 2, 4-D (9, 10, and 20 µM) and kinetin (0.5, 1, 2.5, 5, 10, and 25 µM) in separate experiments. The frequently used media in somatic embryogenesis of cacao [DKW supplemented with 0.022 µM thidiazuron (TDZ) and 9 µM 2, 4-D] was used as a control. The results of the study showed that explants cultured on media supplemented with 10 µM 2, 4-D and 5 µM kinetin produced the highest (28.0 ± 1.1) mean number of embryos/explant in C1 and this was a 9-fold increase in the number of embryos compared with the control. Explants cultured on media supplemented with 10 µM 2, 4-D and 2.5 µM kinetin produced the highest (7.0 ± 4.0) mean number of embryos/explant in C14 whereas the explants cultured on media supplemented with 20 µM 2, 4-D and 2.5 µM kinetin gave the highest (22.0 ± 1.7) mean number of embryos in clone C1 and C14. The regenerated embryos were germinated and successfully weaned in the green house with a survival rate of 70% being recorded. The paper describes an improved protocol compared with previous work in terms of embryo recovery and survival rate of the elite clones of cocoa through somatic embryogenesis. The results of the current study confirm that somatic embryogenesis of cacao clones is genotype dependent.

Gene expression levels of 4-galactosyltransferase 5 correlate with the tumorigenic potentials of B16-F10 mouse melanoma cells

Our previous studies showed that mouse β4-galactosyltransferase 5 (β4GalT5) is a lactosylceramide (Lac-Cer) synthase, and that its gene expression increases by 2- to 3-fold upon malignant transformation of cells. In the present study, we examined whether or not the tumorigenic and metastatic potentials of B16-F10 mouse melanoma cells can be suppressed by reducing the expression of the β4GalT5 gene. We isolated a stable clone named E5 whose β4GalT5 gene expression level was reduced to 35% that of a control clone C1 by transfection of its antisense cDNA. Thin-layer chromatography analysis of glycosphingolipids showed that the amounts of Lac-Cer and ganglioside GM3 are significantly less in clone E5 than in clone C1. Clone C1 and E5 cells were each transplanted subcutaneously or injected intravenously into C57BL/6 mice, and the sizes of tumors and numbers of colonies formed in the lungs were determined. The average tumor size and average number of colonies formed with clone E5 were decreased to 44 and 49%, respectively, of those formed with clone C1. Furthermore, the numbers and sizes of colonies formed in the soft agarose gels, and the volumes of tumors formed in athymic mice with fibroblasts from wild type, heterozygous and homozygous β4GalT5-knockout mouse embryos upon transformation with the polyoma virus oncogene correlated with the β4GalT5 gene dosage. These results strongly indicate that the amounts of Lac-Cer synthesized by β4GalT5 correlate with the tumorigenic potentials of malignantly transformed cells.

Formation of hydrogen-producing granules and microbial community analysis in a UASB reactor

The effect of substrate concentration on continuous H2 production from a sucrose-rich synthetic wastewater was investigated in this work. The maximum H2 production rate of 2.89 m3 H2/m3/d achieved at 7000 mg COD/L while the peak ethanol concentration of 2840 mg/L was observed at 5000 mg COD/L. The H2-producing granules with ethanol-type fermentation were successfully cultivated in an upflow anaerobic sludge blanket (UASB) reactor. Denaturing gradient gel electrophoresis analysis demonstrates that the substrate concentration had a considerable effect on the microbial community. After the mature H2-producing granules were formed, the microbial community structure in the UASB reactor tends to stabilize. The dominant bacterium in the mature H2-producing granules were composed of Janthinobacterium sp. WPCB148, Clostridium sp. HPB-4, Variovorax paradoxus strain SJ100, Variovorax sp. CN3b and Uncultured bacterium clone C2.

Transcriptional Activation by an URE4-like Sequence in the EhPgp1 Gene Core Promoter

EhPgp1 is one of the multidrug resistance genes expressed in drug resistant trophozoites from Entamoeba histolytica. Previous studies in our laboratory have demonstrated that two C/EBP sites participate in the transcriptional activation of this gene. However there is other relevant region that also governs the regulation of EhPgp1 expression in clone C2. In this report we provide evidence that transcription of the EhPgp1 gene is at least partly regulated by the cis-acting R9 repeated sequences and EhEBP1 protein. Structural analysis of the region from -234 to -197 bp shows the presence of two repeated sequences of 9 bp [R9(1) and R9(2)] located at -226 to -203 bp. Deletions and mutations analysis of the R9 motifs significantly reduced promoter activity in trophozoites from clone C2. EMSA experiments revealed specific binding of nuclear proteins from E. histolytica to the R9 sequence. While competition assays showed that the presence of more than one R9 sequence is necessary for a strong DNA-protein interaction. Moreover, Western blot experiments with partially-purified proteins interacting with the R9 motif and antibodies against EhEBP1, recognized a 28 kDa protein. Interestingly, this antibody in supershift assays prevented the DNA-protein interactions formation, of the R9 sequences and nuclear proteins from amoeba, indicating that one of the proteins that interact with the R9 element is an EhEBP1-like one. In conclusion, we demonstrate that R9 motifs are recognized by an EhEBP1 protein and activate the EhPgp1 gene expression.

Resistance to a bacterial parasite in the crustacean Daphnia magna shows Mendelian segregation with dominance

The influence of host and parasite genetic background on infection outcome is a topic of great interest because of its pertinence to theoretical issues in evolutionary biology. In the present study, we use a classical genetics approach to examine the mode of inheritance of infection outcome in the crustacean Daphnia magna when exposed to the bacterial parasite Pasteuria ramosa. In contrast to previous studies in this system, we use a clone of P. ramosa, not field isolates, which allows for a more definitive interpretation of results. We test parental, F1, F2, backcross and selfed parental clones (total 284 genotypes) for susceptibility against a clone of P. ramosa using two different methods, infection trials and the recently developed attachment test. We find that D. magna clones reliably exhibit either complete resistance or complete susceptibility to P. ramosa clone C1 and that resistance is dominant, and inherited in a pattern consistent with Mendelian segregation of a single-locus with two alleles. The finding of a single host locus controlling susceptibility to P. ramosa suggests that the previously observed genotype-genotype interactions in this system have a simple genetic basis. This has important implications for the outcome of host-parasite co-evolution. Our results add to the growing body of evidence that resistance to parasites in invertebrates is mostly coded by one or few loci with dominance.

OVERVIEW OF THE INVESTIGATIONS OF TRANSGENIC PLUMS IN ROMANIA

Transgenic plums of Prunus domestica L. transformed with the Plum pox virus coat protein gene (PPV-CP) were the subjects of three experiments undertaken in Romania. In the first experiment PPV-CP transgenic clones C2, C3, C4, C5, C6 and PT3 were evaluated for Sharka resistance under high natural infection pressure. Transgenic clone C5, subsequently named HoneySweet, showed high resistance to PPV. None of the C5 trees became naturally infected by aphids for more than ten years. The resistance of C5 was based on post-transcriptional gene silencing (PTGS). In the second experiment we assessed the effect of two heterologous viruses (Prunus necrotic ringspot virus and Prune dwarf virus) on the efficacy and stability of PTGS - mediated resistance to PPV displayed by the C5 plum. The engineered resistance to PPV in C5 transgenic plums was stable and was not suppressed by the presence of the assayed heterologous viruses. Some PPV-CP transgenic plum clones that are susceptible to PPV including C2, C3, C4 and PT3 display a constitutive transcription of PPV-CP sequence. In the third experiment we used these plants to assess the environmental safety issues related to potential hazards concerning the emergence of PPV variants. The serological and molecular variability of PPV detected in transgenic and in conventional plums revealed that the transgenic plums do not affect the diversity of indigenous PPV populations. We suggest that the safety and efficacy data developed in Romania and other European countries and in the United States of America over the last 15 years justifies an expansion of the field tests in Romania and in other countries that are experiencing the damages to plum production caused by PPV.

Overview of the Investigations of Transgenic Plums in Romania

Transgenic plums of Prunus domestica L. transformed with the Plum pox virus coat protein gene (PPV-CP) were the subjects of three experiments undertaken in Romania. In the first experiment PPV-CP transgenic clones C2, C3, C4, C5, C6 and PT3 were evaluated for Sharka resistance under high natural infection pressure. Transgenic clone C5, subsequently named „HoneySweet‟, showed high resistance to PPV. None of the C5 trees became naturally infected by aphids for more than ten years. The resistance of C5 was based on post-transcriptional gene silencing (PTGS). In the second experiment we assessed the effect of two heterologous viruses (Prunus necrotic ringspot virus and Prune dwarf virus) on the efficacy and stability of PTGS - mediated resistance to PPV displayed by the C5 plum. The engineered resistance to PPV in C5 transgenic plums was stable and was not suppressed by the presence of the assayed heterologous viruses. Some PPV-CP transgenic plum clones that are susceptible to PPV including C2, C3, C4 and PT3 display a constitutive transcription of PPV-CP sequence. In the third experiment we used these plants to assess the environmental safety issues related to potential hazards concerning the emergence of PPV variants. The serological and molecular variability of PPV detected in transgenic and in conventional plums revealed that the transgenic plums do not affect the diversity of indigenous PPV populations. We suggest that the safety and efficacy data developed in Romania and other European countries and in the United States of America over the last 15 years justifies an expansion of the field tests in Romania and in other countries that are experiencing the damages to plum production caused by PPV.

Construction of a Metagenomic Library from Compost and Screening of Cellulase- and Xylanase-positive Clones

Metagenomic library was constructed from compost made with pig manure and mushroom cultural waste using fosmid vector. Composting was carried out using moving roller system with intermittent mixing and forced aeration, and samples were taken from 7th- and 14th-day spots, which represent the mid and late stages of the process, respectively. DNAs of about 40 kb were obtained by preparative electrophoresis on 0.4% low-melting agarose gel to fractionate DNA fragments and to remove humic substances. Total 12,380 fosmid clones were obtained. Restriction analysis of randomly selected clones showed that most clones had different inserts, and average size of inserts was about 35 kb. Two cellulase-positive and five xylanase-positive clones were selected from metagenomic library. Cellulase of clone C1 showed maximal activity at 50°C and pH 6.0, and retained its original activity after 30 min of heat treatment at 60°C. Optimum temperature for xylanases of clones X1, X2, X3, and X4 was 50°C, and that of clone X5 was 55°C. Thermostabilities of xylanases were in the order of X4>X5>X1, X2, and X3. Optimum pH of xylanases of X1, X2, and X3 was 6.0, that of X4 was 5.5, and that of X5 was 5.5∼8.0. Xylanasepositive clones could be divided into three groups, X1/X2/X3, X4, and X5, based on influences of temperature and pH on enzyme activity. Sequence analysis of positive subclone of clone C1 showed cellulase, Cel6H, had the highest similarity of 64% to that of Cellulomonas fimi (P07984), suggesting cellulase and xylanase from metagenomic library are novel enzymes.

Growth, chlorophyll fluorescence and biochemical markers in clonal ramets of shisham (Dalbergia sissoo Roxb.) at nursery stage

About 19 clones of shisham (Dalbergia sissoo Roxb.) were raised by using healthy coppice shoot cuttings. After 2 year of establishment and growth, they were examined to identify suitable clone of shisham at nursery stage. Clones were studied for growth (height and basal diameter), chlorophyll fluorescence (Fv/Fm) and biochemical contents (chlorophyll a, b, total chlorophyll, free amino acids, total soluble proteins, total soluble sugars, starch and reducing sugar) in the leaves. Clone C2 and C3 (Gonda, Uttar Pradesh, India) had the highest growth and the chlorophyll fluorescence rate among examined clones. Between these two clones, C2 gave the highest response on height, basal diameter and Fv/Fm. Except free amino acids, all other biochemical contents were greater in the clones belonging to Gonda, Uttar Pradesh, India. Growth parameters, i.e. height and basal diameter increment at nursery stage had a positive correlation with all the studied parameters, except free amino acid and total soluble protein. These findings have demonstrated that there are remarkable clonal variations in growth, physiological and biochemical responses at nursery stage. Thus, physiological and biochemical markers can enhance selection efficiency, in addition to growth characteristics such as height and basal diameter as these have practical value in the tree improvement. The study suggests that clones of Gonda, Uttar Pradesh, India had the best performance and could be further tested in plantation programs.

Efficient genetic transformation of Impatiens hawkerii Bull. (Balsamiaceae) using agrobacterium rhizogenes

Transformation of Impatiens hawkerii Bull. mediated by Agrobacterium rhizogenes strain A4M70GUS was studied. Hairy roots developed 10 days after inoculation were excised from the shoot explants and transferred onto Murashige and Skoog's (MS) basal medium lacking plant growth regulators. More than 20 hairy root clones were established and eight of them were further analyzed. Each clone differed significantly from the others in growth capacity and lateral branching. Clone C2 showed the highest biomass (20.6 g L-1) as well as the highest number of lateral roots (37 ? 2.2). The transgenic nature of the established hairy root clones was confirmed by GUS assay and PCR analysis. In conclusion, hairy roots were developed for the first time in I. hawkerii Bull., and transgenic hairy root clones showed a distinct morphological nature and growth patterns.

Field release of transgenic plums in Romania.

Transgenic clones C2, C3, C4, C5, C6, PT3 and PT5 of Prunus domestica L. transformed with the Plum pox virus coat protein gene (PPV-CP) were evaluated for Shar ka resistance under high infection pressure in field natural conditions in Romania. Tr ansgenic clone C5, recently named HoneySweet, showed high resistance to PPV. None of the C5 trees became s naturally infected by aphids for more than ten ye ars. Known to develop the post-transcriptional gene sile ncing (PTGS) mechanism, we assessed the effect of heterologous viruses on the efficacy and stability of PTGS displayed by the C5 plum against PPV. In this way, C5 trees were graft-inoculated with different combi nations of Prunus necrotic ringspot virus (PNRSV), Prune dwarf virus (PDV) and PPV-D strain. The potential for suppress ion of the silencing mechanism mediated by these ilarviruses was evaluated in orchard and nurs ery. Both trials showed that, the engineered resist ance to PPV in C5 transgenic plums was stable and was not suppr essed by the presence of the assayed heterologous v iruses over a three-year experimental period.