Tissue Culture Selection Research Articles

Recent Advances inAgrobacterium-Mediated Maize Genetic Transformation Using Immature Embryos and Seedling Leaf Whorl Explants

The introduction of maize genetic transformation in the 1990s brought forth a powerful tool for crop improvement and a deeper understanding of plant genetics. Despite decades of genetics research, however, and the promise of CRISPR-mediated gene editing, maize transformation currently faces several challenges, such as genotype dependence and limitations in explant availability. Indeed, although the most commonly used method, immature embryo transformation, has been improved through optimization of tissue culture media composition and selection methods, the approach is only applicable to a limited number of public genotypes, including B104 and Hi II. Recently, genotype-flexible methods have been developed using coexpression cassettes of morphogenic transcription factors (MTFs)Baby boom(Bbm) andWushel2(Wus2), which have enabled the successful transformation of many previously recalcitrant maize lines. This MTF-based transformation method has also allowed for the use of alternate explants, such as seedling leaf whorl, whose production is cost-effective and requires only minimum controlled growth space. In this review, we summarize recent advances inAgrobacterium-mediated maize transformation methods that use immature embryos or seedling leaf whorls as starting material.

GWAS determined genetic loci associated with callus induction in oil palm tissue culture.

GWAS identified six loci at 25kb downstream of WAK2, a crucial gene for cell wall and callus formation, enabling development of a SNP marker for enhanced callus induction potential. Efficient callus induction is vital for successful oil palm tissue culture, yet identifying genomic loci and markers for early detection of genotypes with high potential of callus induction remains unclear. In this study, immature male inflorescences from 198 oil palm accessions (dura, tenera and pisifera) were used as explants for tissue culture. Callus induction rates were collected at one-, two- and three-months after inoculation (C1, C2 and C3) as phenotypes. Resequencing generated 11,475,258 high quality single nucleotide polymorphisms (SNPs) as genotypes. GWAS was then performed, and correlation analysis revealed a positive association of C1 with both C2 (R = 0.81) and C3 (R = 0.50), indicating that C1 could be used as the major phenotype for callus induction rate. Therefore, only significant SNPs (P ≤ 0.05) in C1 were identified to develop markers for screening individuals with high potential of callus induction. Among 21 significant SNPs in C1, LD block analysis revealed six SNPs on chromosome 12 (Chr12) potentially linked to callus formation. Subsequently, 13 SNP markers were identified from these loci and electrophoresis results showed that marker C-12 at locus Chr12_12704856 can be used effectively to distinguish the GG allele, which showed the highest probability (69%) of callus induction. Furthermore, a rapid SNP variant detection method without electrophoresis was established via qPCR-based melting curve analysis. Our findings facilitated marker-assisted selection for specific palms with high potential of callus induction using immature male inflorescence as explant, aiding ortet palm selection in oil palm tissue culture.

Quality Enhancement in Forage Crops of Cereals and Legumes

This article focus on quality improvement in forage crops, specifically grasses, cereals, and legumes. As milk and many other by-products obtained from dairy farms, poultry, etc are getting into demand, high-quality forage for livestock production is well recognized, and efforts to improve forage quality can have significant economic and environmental benefits. And an overview of current strategies for quality improvement, including breeding and genetic selection, management practices, and how these practices affect the quality of forage crops such as silica, lignin, and other phenolic components. Use of different breeding methods more over like synthetic cultivars, recurrent selection, etc. the use of biotechnology tools such as RNAi interference, tissue culture, and marker-assisted selection in the particular crop that has been developed for increasing the nutritional value, proteins that present in forage crops, and to decrease the harmful chemicals. Additionally, the paper discusses the challenges associated with quality improvement in forage crops and potential solutions. And about different types of grasses used for different types of cattle as supplements. Limitations that cause obstruction to improving the quality of forage crops. digestibility and how to increase the quality and protein content in milk and cattle. And nutrition that needs to be present in prescribed quantities in forage crops. Overall, this paper highlights the need for continued research and innovation in the field of forage crop quality improvement to support sustainable agriculture and meet the increasing demand for high-quality livestock feed.

Comparison between the growth of Kappahycus alvarezii (Rhodophyta) seed from tissue culture and clone selection cultivated using horizontal net

Kappahycus alvarezii is a species of seaweed belongs to Rhodophyta class which is commonly cultivated by coastal community in tropical areas. This research therefore aimed to clarify the growth of K. alvarezii thallus from culture tissue and cloning selection cultivated using horizontal net. The method was an experimental design, which used a randomized group and Specific Growth Rate (SGR) analysis. Horizontal net was used to avoid pest attack and research failure. The results showed the total growth rate in average within 40 days from the initial 20 g growth to 120,1g and 139,7g of cloning and tissue culture seed, respectively. Meanwhile, the average of SGR of tissue culture and cloning were 6.3% and 6.7%/day, respectively. Statistically, the cloning selection method and tissue culture were not significantly different with good maintenance, especially using the horizontal net. Transparency level had a positive correlation with the growth of cloning selection seeds.

Seasonal variation in growth and carrageenan yield of Kappaphycus alvarezii (Doty) Doty farmed using mass selection in Bungin Permai Coastal Waters, South Konawe District, Southeast (SE) Sulawesi, Indonesia

Two methods, tissue culture, and mass selection are generally used to improve the quality of the seedlings of the red algae K. alvarezii. Mass selection is relatively new to be adapted to the Indonesian seaweed farming. In this study we attempted to cultivate the seaweed for 10 months (February-November 2013) in Bungin Permai coastal waters, South Konawe, Indonesia. Cultivation was performed for 30 days of each cycle, using long-line method. Each rope contained had distance between clumps of 15-, 20-, 25- and 30-cm. The selection was carried out until 9th generation. The results showed that the daily growth rates (DGRs) among treatments were found to vary and tended to have seasonal variation. Minimum DGRs occurred in October while the maximum was in April/May. The DGRs range was from 3.81±1.80%/day to 8.32±1.12%/day while mean annual DGRs of 15-, 20-, 25- and 30- cm PD were 6.46±1.98, 6.62±2.00, 6.74±2.01, and 6.90±2.03%/day, respectively. No significant differences was found among four different planting distance. The mean carrageenan (semi-refined carrageenan/SRC) yields were varied among treatments and tended also to have seasonal variation. Minimum SRC of all treatments was varied and occurred in March/June while the maximum SRC were in May/October/November. The carrageenan yields range were from 31.53±2.04% to 48.91±5.05%. The highest mean annual SRC was recorded for 15-cm planting distance (PD) (42.02 ± 3.38%) followed by 30-cm PD (41.58 ± 2.95%), 20-cm PD (41.56 ± 6.03%) and 25-cm PD (41.45 ± 4.09%). The quality of cultivars produced from the selection met international specifications suggesting that mass selection for longer period could be ‘a new method” to produce better quality of seedlings.

Spontaneous Mutation at Amino Acid 544 of the Ebola Virus Glycoprotein Potentiates Virus Entry and Selection in Tissue Culture.

Ebolaviruses have a surface glycoprotein (GP1,2) that is required for virus attachment and entry into cells. Mutations affecting GP1,2 functions can alter virus growth properties. We generated a recombinant vesicular stomatitis virus encoding Ebola virus Makona variant GP1,2 (rVSV-MAK-GP) and observed emergence of a T544I mutation in the Makona GP1,2 gene during tissue culture passage in certain cell lines. The T544I mutation emerged within two passages when VSV-MAK-GP was grown on Vero E6, Vero, and BS-C-1 cells but not when it was passaged on Huh7 and HepG2 cells. The mutation led to a marked increase in virus growth kinetics and conferred a robust growth advantage over wild-type rVSV-MAK-GP on Vero E6 cells. Analysis of complete viral genomes collected from patients in western Africa indicated that this mutation was not found in Ebola virus clinical samples. However, we observed the emergence of T544I during serial passage of various Ebola Makona isolates on Vero E6 cells. Three independent isolates showed emergence of T544I from undetectable levels in nonpassaged virus or virus passaged once to frequencies of greater than 60% within a single passage, consistent with it being a tissue culture adaptation. Intriguingly, T544I is not found in any Sudan, Bundibugyo, or Tai Forest ebolavirus sequences. Furthermore, T544I did not emerge when we serially passaged recombinant VSV encoding GP1,2 from these ebolaviruses. This report provides experimental evidence that the spontaneous mutation T544I is a tissue culture adaptation in certain cell lines and that it may be unique for the species Zaire ebolavirusIMPORTANCE The Ebola virus (Zaire) species is the most lethal species of all ebolaviruses in terms of mortality rate and number of deaths. Understanding how the Ebola virus surface glycoprotein functions to facilitate entry in cells is an area of intense research. Recently, three groups independently identified a polymorphism in the Ebola glycoprotein (I544) that enhanced virus entry, but they did not agree in their conclusions regarding its impact on pathogenesis. Our findings here address the origins of this polymorphism and provide experimental evidence showing that it is the result of a spontaneous mutation (T544I) specific to tissue culture conditions, suggesting that it has no role in pathogenesis. We further show that this mutation may be unique to the species Zaire ebolavirus, as it does not occur in Sudan, Bundibugyo, and Tai Forest ebolaviruses. Understanding the mechanism behind this mutation can provide insight into functional differences that exist in culture conditions and among ebolavirus glycoproteins.

HIV-1 strains belonging to large phylogenetic clusters show accelerated escape from integrase inhibitors in cell culture compared with viral isolates from singleton/small clusters

Objectives: Viral phylogenetics revealed two patterns of HIV-1 spread among MSM in Quebec. While most HIV-1 strains (n = 2011) were associated with singleton/small clusters (cluster size 1–4), 30 viral lineages formed large networks (cluster size 20–140), contributing to 42% of diagnoses between 2011 and 2015. Herein, tissue culture selections ascertained if large cluster lineages possessed higher replicative fitness than singleton/small cluster isolates, allowing for viral escape from integrase inhibitors. Methods: Primary HIV-1 isolates from large 20+ cluster (n = 11) or singleton/small cluster (n = 6) networks were passagedin vitro in escalating concentrations of dolutegravir, elvitegravir and lamivudine for 24–36 weeks. Sanger and deep sequencing assessed genotypic changes under selective drug pressure. Results: Large cluster HIV-1 isolates selected for resistance to dolutegravir, elvitegravir and lamivudine faster than HIV-1 strains forming small clusters. With dolutegravir, large cluster HIV-1 variants acquired solitary R263K (n = 7), S153Y (n = 1) or H51Y (n = 1) mutations as the dominant quasi-species within 8–12 weeks as compared with small cluster lineages where R263K (n = 1/6), S153Y (1/6) or WT species (4/6) were observed after 24 weeks. Interestingly, dolutegravir-associated mutations compromised viral replicative fitness, precluding escalations in concentrations beyond 5–10 nM. With elvitegravir, large cluster variants more rapidly acquired first mutations (T66I, A92G, N155H or S147G) by week 8 followed by sequential accumulation of multiple mutations leading to viral escape (>10 μM) by week 24. Conclusions: Further studies are needed to understand virological features of large cluster viruses that may favour their transmissibility, replicative competence and potential to escape selective antiretroviral drug pressure.

Relative efficiency of positive selection and tissue culture for generating pathogen-free planting materials of yam (Dioscorea spp.)

Yams are staples in West Africa. They are propagated from tubers in an informal seed system. This encourages a build-up of diseases, and necessitates the rapid development of a formal seed system where certified seeds are functional. Although few reports exist on the use of meristem culture to generate pathogen-free yam, the success rate for the most economically important species in the sub-region, Dioscorea rotundata, for the most prevalent viruses is inadequate. To generate pathogen-free yam planting materials, the relative efficiency of tissue culture and positive selection was compared. Twenty-one asymptomatic yam plants were positively selected from 8187 stands of five landraces. Five of these stands were tested virus-negative by multiplex polymerase chain reaction (PCR) for Yam mosaic virus (YMV), Yam mild mosaic virus (YMMV) and Cucumber mosaic virus (CMV), and by PCR for the genus Badnavirus (BV), giving 0.08% success. Single nodes of the positively selected stands were used to establish in vitro plantlets, which were screened onto bacteriological indexing medium. The same was done for meristem- and node-derived plantlets of the improved variety TDr 95/19158. Incidence of endophytes ranged from 18 to 32% in the nodal plantlets while it was 0% in the meristem-derived plantlets. The effect of meristem culture combined with thermotherapy on the virus infection status was determined using virus-tested, one week old in vitro plantlets of eight improved genotypes. These in vitro plantlets were incubated at 36 ± 0.5°C and 16 h photoperiod for 21 days, after which meristems were excised, regenerated into plantlets and re-tested for viruses. Seventy-three percent of the samples were recovered from YMV but the effect on BV was inconsistent. Positive selection can be used as a palliative in generating quality declared seed but meristem culture combined with thermotherapy is more efficient for generating certified seed tubers of yam.

The M184I/V and K65R nucleoside resistance mutations in HIV-1 prevent the emergence of resistance mutations against dolutegravir.

Recommended treatments for newly diagnosed HIV-positive individuals now focus on the integrase strand transfer inhibitors, raltegravir (RAL), elvitegravir (EVG) and dolutegravir (DTG). In treatment-naive individuals, cases of RAL-based and EVG-based virological failure, although rare, are associated with the occurrence of resistance mutations in integrase and/or reverse transcriptase coding sequences. In such cases, common resistance substitutions in reverse transcriptase that were associated with nucleos(t)ide reverse transcriptase inhibitors included M184I/V and K65R and these occurred together with various mutations in integrase. In some instances, these mutations in reverse transcriptase preceded the emergence of mutations in integrase. In contrast, no resistance substitutions in either integrase or reverse transcriptase have been observed to date in viruses isolated from treatment-naive individuals who experienced treatment failure with DTG-based regimens. The objective of this study was to determine the effects of the M184I/V and K65R substitutions in reverse transcriptase on the ability of HIV-1 to become resistant against RAL, EVG or DTG. We performed tissue culture selection experiments using reverse transcriptase inhibitor-resistant viruses containing resistance substitutions at positions K65R, M184I or M184V in the presence of increasing concentrations of RAL, EVG or DTG and monitored changes in integrase sequences by genotyping. Selections using EVG and RAL led to the emergence of resistance mutations in integrase. In contrast, only the wild-type virus was able to acquire resistance mutations for DTG. Resistance mutations against nucleos(t)ide reverse transcriptase inhibitors antagonized the development of HIV-1 resistance against DTG but not RAL or EVG.

The dolutegravir R263K resistance mutation in HIV-1 integrase is incompatible with the emergence of resistance against raltegravir.

Although the integrase inhibitor dolutegravir (DTG) has demonstrated greater resilience than other antiretroviral drugs at withstanding the emergence of HIV-1 resistance mutations, such substitutions can develop, albeit rarely, in treatment-experienced integrase inhibitor-naïve individuals. The most common substitution in integrase under those circumstances is R263K whereas another substitution that was selected against DTG in tissue culture was G118R. The objective of this study was to determine the effects of these DTG-specific resistance substitutions on the ability of HIV-1 to become resistant against either of two other integrase inhibitors, raltegravir (RAL) and elvitegravir (EVG). We performed tissue culture selection experiments using DTG-resistant viruses containing integrase substitutions at positions R263K, H51Y/R263K, E138K/R263K, G118R and H51Y/G118R in the presence of increasing concentrations of either RAL or EVG. Changes in integrase sequences were monitored by genotyping. The presence of the R263K substitution delayed the emergence of resistance against RAL whereas the simultaneous presence of either the H51Y or E138K secondary substitutions in combination with R263K somewhat mitigated this inhibitory effect. In contrast, resistance against EVG appeared earlier than in wild-type virus in viruses containing the R263K and E138K/R263K DTG-associated resistance substitutions. The DTG-resistant R263K substitution antagonized the development of HIV-1 resistance against RAL while partially facilitating the occurrence of resistance against EVG.

Characterization of the Drug Resistance Profiles of Integrase Strand Transfer Inhibitors in Simian Immunodeficiency Virus SIVmac239.

We previously showed that the simian immunodeficiency virus SIVmac239 is susceptible to human immunodeficiency virus (HIV) integrase (IN) strand transfer inhibitors (INSTIs) and that the same IN drug resistance mutations result in similar phenotypes in both viruses. Now we wished to determine whether tissue culture drug selection studies with SIV would yield the same resistance mutations as in HIV. Tissue culture selection experiments were performed using rhesus macaque peripheral blood mononuclear cells (PBMCs) infected with SIVmac239 viruses in the presence of increasing concentrations of dolutegravir (DTG), elvitegravir (EVG), and raltegravir (RAL). We now show that 22 weeks of selection pressure with DTG yielded a mutation at position R263K in SIV, similar to what has been observed in HIV, and that selections with EVG led to emergence of the E92Q substitution, which is a primary INSTI resistance mutation in HIV associated with EVG treatment failure. To study this at a biochemical level, purified recombinant SIVmac239 wild-type (WT) and E92Q, T97A, G118R, Y143R, Q148R, N155H, R263K, E92Q T97A, E92Q Y143R, R263K H51Y, and G140S Q148R recombinant substitution-containing IN enzymes were produced, and each of the characteristics strand transfer, 3'-processing activity, and INSTI inhibitory constants was assessed in cell-free assays. The results show that the G118R and G140S Q148R substitutions decreased Km' and Vmax'/Km' for strand transfer compared to those of the WT. RAL and EVG showed reduced activity against both viruses and against enzymes containing Q148R, E92Q Y143R, and G140S Q148R. Both viruses and enzymes containing Q148R and G140S Q148R showed moderate levels of resistance against DTG. This study further confirms that the same mutations associated with drug resistance in HIV display similar profiles in SIV. Our goal was to definitively establish whether HIV and simian immunodeficiency virus (SIV) share similar resistance pathways under tissue culture drug selection pressure with integrase strand transfer inhibitors and to test the effect of HIV-1 integrase resistance-associated mutations on SIV integrase catalytic activity and resistance to integrase strand transfer inhibitors. Clinically relevant HIV integrase resistance-associated mutations were selected in SIV in our tissue culture experiments. Not only do we report on the characterization of SIV recombinant integrase enzyme catalytic activities, we also provide the first research anywhere on the effect of mutations within recombinant integrase SIV enzymes on drug resistance.

Dolutegravir resistance mutation R263K cannot coexist in combination with many classical integrase inhibitor resistance substitutions.

The new integrase strand transfer inhibitor (INSTI) dolutegravir (DTG) displays limited cross-resistance with older drugs of this class and selects for the R263K substitution in treatment-experienced patients. We performed tissue culture selections with DTG, using viruses resistant to older INSTIs and infectivity and resistance assays, and showed that the presence of the E92Q or N155H substitution was compatible with the emergence of R263K, whereas the G140S Q148R, E92Q N155H, G140S, Y143R, and Q148R substitutions were not.

Addition of E138K to R263K in HIV integrase increases resistance to dolutegravir, but fails to restore activity of the HIV integrase enzyme and viral replication capacity.

The results of several clinical trials suggest that the integrase inhibitor dolutegravir may be less prone than other drugs to the emergence of HIV drug resistance mutations in treatment-naive patients. We have shown that the R263K mutation commonly emerged during tissue culture selection studies with dolutegravir and conferred low levels of resistance to this drug while simultaneously diminishing both HIV replication capacity and integrase enzymatic activity. E138K has been identified as a secondary mutation for dolutegravir in selection studies and has also been observed as a secondary mutation in the clinic for the integrase inhibitors raltegravir and elvitegravir. We used biochemical cell-free strand-transfer assays and tissue culture assays to characterize the effects of the E138K/R263K combination of mutations on resistance to dolutegravir, integrase enzyme activity and HIV-1 replication capacity. We show here that the addition of the E138K substitution to R263K increased the resistance of HIV-1 to dolutegravir but failed to restore viral replication capacity, integrase strand-transfer activity and integration within cellular DNA. We also show that the addition of E138K to R263K did not increase the resistance to raltegravir or elvitegravir. The addition of the E138K substitution to R263K was also less detrimental to integrase strand-transfer activity and integration than a different secondary mutation at position H51Y that had also been selected in culture. The E138K substitution failed to restore the defect in viral replication capacity that is associated with R263K, confirming previous selection studies that failed to identify compensatory mutation(s) for the latter primary mutation. This study suggests that the R263K resistance pathway may represent an evolutionary dead end for HIV in treatment-naive individuals who are treated with dolutegravir and will need to be confirmed by the long-term use of dolutegravir in the clinic.

Development and phytochemical characterization of high polyphenol red lettuce with anti-diabetic properties.

Polyphenol-rich Rutgers Scarlet Lettuce (RSL) (Lactuca sativa L.) was developed through somaclonal variation and selection in tissue culture. RSL may contain among the highest reported contents of polyphenols and antioxidants in the category of common fruits and vegetables (95.6 mg/g dry weight and 8.7 mg/g fresh weight gallic acid equivalents and 2721 µmol/g dry weight and 223 µmol/g fresh weight Trolox equivalents). Three main compounds accumulate at particularly high levels in RSL: chlorogenic acid, up to 27.6 mg/g dry weight, cyanidin malonyl-glucoside, up to 20.5 mg/g dry weight, and quercetin malonyl-glucoside, up to 35.7 mg/g dry weight. Major polyphenolic constituents of RSL have been associated with health promotion as well as anti-diabetic and/or anti-inflammatory activities. Daily oral administration of RSL (100 or 300 mg/kg) for up to eight days acutely reduced hyperglycemia and improved insulin sensitivity in high fat diet-induced obese hyperglycemic mice compared to vehicle (water) control. Data presented here support possible use of RSL as a functional food for the dietary management of diabetes.

Near Surface Swimming of Salmonella Typhimurium Explains Target-Site Selection and Cooperative Invasion

Targeting of permissive entry sites is crucial for bacterial infection. The targeting mechanisms are incompletely understood. We have analyzed target-site selection by S. Typhimurium. This enteropathogenic bacterium employs adhesins (e.g. fim) and the type III secretion system 1 (TTSS-1) for host cell binding, the triggering of ruffles and invasion. Typically, S. Typhimurium invasion is focused on a subset of cells and multiple bacteria invade via the same ruffle. It has remained unclear how this is achieved. We have studied target-site selection in tissue culture by time lapse microscopy, movement pattern analysis and modeling. Flagellar motility (but not chemotaxis) was required for reaching the host cell surface in vitro. Subsequently, physical forces trapped the pathogen for ∼1.5–3 s in “near surface swimming”. This increased the local pathogen density and facilitated “scanning” of the host surface topology. We observed transient TTSS-1 and fim-independent “stopping” and irreversible TTSS-1-mediated docking, in particular at sites of prominent topology, i.e. the base of rounded-up cells and membrane ruffles. Our data indicate that target site selection and the cooperative infection of membrane ruffles are attributable to near surface swimming. This mechanism might be of general importance for understanding infection by flagellated bacteria.

The role of polymorphisms at position 89 in the HIV-1 protease gene in the development of drug resistance to HIV-1 protease inhibitors

Relatively little is known about the development of resistance to protease inhibitors (PIs) in non-B subtypes. In subtype B viruses, L89 is commonly found at position 89 in the HIV protease (PR) gene, whereas M89 is commonly observed as a polymorphism in other subtypes. We compared the frequencies of substitutions at position 89 in PR in tissue culture selections and in clinical databases of PI-naive and PI-experienced populations. Representative subtype A/CRF01_AE (n = 2 and 3) and subtype C (n = 5) isolates were cultured in MT-2 cells and cord blood mononuclear cells (CBMCs), respectively, under increasing drug pressure with PIs, and drug resistance mutations were identified. The M89 natural polymorphism in non-B subtypes commonly led to the appearance of an M89T mutation in selections with atazanavir in subtypes A/AE and C, and was accompanied by other previously recognized atazanavir mutations. The M89T mutation contributed to phenotypic resistance to atazanavir and cross-resistance to lopinavir and nelfinavir, but not to other PIs. A shift from a L89 natural polymorphism to L89I/M arose in two of five subtype C selections with PIs. M89I/V/T mutations were acquired by 10%-11% of individuals harbouring non-B subtypes who were failing PI-based regimens, but were rarely observed in drug-naive persons and in patients failing non-PI-based regimens. The M/L89 natural polymorphism present in non-B subtypes may lead to the M89T mutational pathway conferring resistance to atazanavir, lopinavir and nelfinavir.

Interactions of different drug resistance mutations in HIV Reverse Transcriptase in defining patterns of drug resistance

Background The M184V mutation in HIV RT confers high-level resistance to both 3TC and FTC and also has a strong negative impact on viral replication capacity. In addition, the presence of M184V is usually preceded by a M1841 mutation that results from a G®A hypermutation. However, viruses that contain M184V usually outcompete M184I due to higher replication ability; therefore M184V becomes predominant. In contrast, the E138K mutation in HIV-1 RT confers resistance to Rilpivirine (RPV) and Etravirine(ETR), two NNRTIs, and also impairs viral replication capacity. Phase III clinical trials (ECHO & THRIVE ) showed that E138K/M184I are the most frequent mutations in patients who received RPV together with co-formulated TDF/ FTC,suggesting that E138K might be a signature mutation for both ETR and RPV. We have assessed the impact of E138K on enzymatic processivity, viral replication capacity, and drug susceptibility in the context of M184I/V. Methods We studied recombinant HIV-1 RT enzymes and viruses containing either the E138K and M184I/V mutations alone and E1 38K-M1 84I/V in combination in regard to enzyme processivity, polymerization rates, replication capacity, and drug susceptibility. Results Tissue culture selections with ETR yielded E138K that conferred modest (3-5-fold) resistance to this drug as well as to efavirenz(EFV) and neviripine (NVP). These susceptibilities were not altered in either viruses or RT enzymes containing E138K together with M184I or M184V. The relative replication capacity of HIV-1 containing either E138K or M184I/V alone was decreased by ~2- fold compared to wild-type (wt). In contrast, the simultaneous presence of E138K and M184I/V was shown to reverse this low fitness, and this result was confirmed in cell-free experiments in which purified RT enzymes containing E138K alone or E138K-M184I/V double mutations displayed higher processivity than eitherwt or M184I/V RT, especially at low dNTP concentrations. No significant differences were observed between M184I and M184V in regard to these characteristics. In addition, RT containing E138K or E138K together with M184I/M184V possessed lower RNase H activity than either wt RT or M184I/V RT. Conclusions

Selection and characterization of a new switchgrass ( Panicum virgatum L.) line with high somatic embryogenic capacity for genetic transformation

Switchgrass ( Panicum virgatum L.) is used horticulturally as an ornamental and agronomically as an animal feedstock and a putative bio-energy crop. Genetic transformation, using somatic embryogenic (SE) callus derived from mature seeds, is one strategy for improving switchgrass traits. A superior switchgrass line, HR8, was developed in this study using recurrent tissue culture selection from cv. Alamo. Eighty two percent of HR8 seeds germinated after harvest comparing to 26.8% for unselected ‘Alamo’. HR8 seeds that germinated produced 84.9% SE callus. HR8 seeds had higher endogenous abscisic acid (ABA) contents and responded differently to exogenous additions of ABA in culture. Endophytes were isolated from switchgrass seeds and callus. HR8 callus had less endophytic contamination than that of ‘Alamo’ callus. HR8 SE calli were genetically transformable using Agrobacterium. Therefore, HR8 is a superior line for generating SE callus and Agrobacterium-mediated transformation.

Medium selection in tissue culture of japonica rice cultivar Nipponbare and preparation of transgenic plants with an elicitor-coding gene from Magnaporthe grisea

AbstractN6 medium, Murashige and Skoog (MS) medium and BNBN medium were adopted in tissue cultures of Nipponbare (japonica) scutellum for performance comparison. Results indicated that, among the three kinds of culture media, the NB medium worked best in callus induction and gene transformation and was most suitable for tissue culture of Nipponbare scutellum. Based on this, the elicitor-encoding gene pemG1 from Magnaporthe grisea was introduced into the genome of Nipponbare via the Agrobacterium-mediated method. Transgenic rice plants were obtained. The integration, transcription and expression of pemG1 in rice were confirmed by polymerase chain reaction (PCR), Northern blot and Western blot, respectively. Genetic analysis showed that the transgenes were segregated normally in the progenies.

Genetic manipulation of the mouse via gene targeting in embryonic stem cells.

Gene targeting applied to totipotent embryonic stem (ES) cells is a very powerful means of creating highly specific mutations of genes in the mouse. The successful application of this technology is however constrained by both the types of mutations that can be generated at a target locus and the ability to reconstruct a germline chimera from the manipulated cells. We have developed two cell lines that can be routinely transmitted through the germline of chimeras after cloning and prolonged selection in tissue culture. We have also established a variety of methods for generating non-selected mutations at the X-linked hprt locus in ES cells. Our observations at this locus have enabled us to generate successfully a subtle mutation at the non-selectable Hox-2.6 locus.