Highest Multiplication Rate Research Articles

Recent advances in production of healthy planting material for disease management in Potato

Unlike most crops where ’true‘ seed is used for cultivation, potato is usually propagated through tubers. This vegetative mode of propagation is beset with many problems; the most important of which is a progressive and significant decline in the yield potential of seed tubers over the years. Use of high yielding varieties and quality planting materials are two important prerequisites for healthy seed potato production. Low rate of tuber multiplication, high seed (tuber) rate, and progressive accumulation of degenerative viruses, perishability and bulkiness are inherent problems in potato seed production. This may results in non-availability of adequate quantities of quality seeds at affordable price and seed cost alone reflects 40–50% to the total costs of cultivation in potato. To circumvent some of these problems, several modifications such as tuber indexing for virus freedom, seed multiplication stages and seed certification standards have been developed and integrated with conventional potato seed production programmes. The advent of tissue culture, in which virus-free plants can be produced through meristem culture, maintained indefinitely under controlled conditions and multiplied in artificial media under sterile conditions in the laboratory throughout the year irrespective of growing season, has revolutionized seed production in potato world over. Recent developments in automation of minitubers production have further enhanced adaptability of these techniques in potato seed production. In addition to quality assurance through meristem culture, aeroponic technique of minitubers production ensures high multiplication rate at initial stages of quality seed potato production.

In vitro arrow cane (Gynerium sagitatum Aubl.) multiplication in double phase medium

Arrow cane (Gynerium sagitatum Aubl.) is a Poaceae species used as fiber source to make traditional and valuable handmade craftsmanship by indigenous communities in Northern Colombia. Since no commercial crops are established fiber needs are taken from natural plant populations affecting ecosystem. A micropropagation protocol to clonally multiply large quantities of arrow cane plant material for planting commercial crops has been developed; however, micropropagated plants are costly compared to naturally extracted plant material. To reduce micropropagated plants costs, in the present research a double phase medium formulation along with continuous shoot culture with no periodic transfers to fresh medium was compared to semisolid medium system with subculture every four weeks with respect to multiplication rate and costs of micropropagated plants. The results showed that continuous culture of explants with double phase medium and no periodic transfers resulted in higher multiplication rates and larger shoots compared to shoots cultured using the conventionalsemisolid medium system and transfer to fresh medium every four weeks. Plants from both, semisolid and double phase culture system, fully adapted and recovered when transferred to ex vitro conditions. The cost analysis showed that double phase cultured shoots are ≥20% less expensive.

SEQUENCING OF MATK GENE FOR TAXONOMIC IDENTIFICATION AND IN VITRO PROPAGATION OF A LOCAL YAM (DIOSCOREA SP.) CULTIVAR GROWN AT MUONG KHUONG, LAO CAI

Khoai mo, local name of Yam species belonging to the Dioscoraceae family, is widely grown at Muong Khuong, Lao Cai province because of their important food and pharmaticeutical valuables. This work aimed to sequence the MatK gene obtained from leaves of a local yam grown in Muong Khuong, Lao Cai. The sequencing result showed a partial sequence of the MatK gene which was submitted to GenBank with accession MF494702 provided. The length of this partial sequence is 916 nucleotides. Phylogeny analysis indicated that local yam cultivar grown at Muong Khuong, Lao Cai belonged to Dioscorea alata species. Nodal segments collected from this local yam were used as initial material for in vitro vegetative multiplication. In the shoot formation step, BAP or BAP and NAA complex were used in shoot formation experience. In vitro shooting effect was higher on MS medium supplemented with BAP at 0.3 mg/l of contrentration in compared to other studied BAP concentrations. The presence of NAA at 0.5 mg/l in medium containing BAP promoted a high rate of shoot multiplication. Highest value of shoot production was obtained on MS medium fortified with BAP (1.5 mg/l) and NAA (0.5 mg/l) from nodal segments. In next step, shoots were successfully rooted in vitro. A high number of roots per shoot and highest length of roots were obtained on MS medium containing 0.2 or 0.3 mg/l of NAA. High acclimatization frequencies were obtained when transferred rooted explants to rich soil, yellow clay soil and soil+sand mixture (1:1). Best survival rate (100%) was achieved on rich soil substrate during ex vitro acclimatization process.

In vitro propagation of Rosa ‘Konstancin’ (R. rugosa × R. beggeriana), a plant with high nutritional and pro-health value

Abstract The aim of the study was to develop an efficient micropropagation system for Rosa ‘Konstancin’, an interspecific hybrid between R. rugosa and R. beggeriana, whose fruits have high pro-health value. Shoot cultures were initiated from shoot buds collected in May and August from 15-year-old field-grown Rosa ‘Konstancin’ shrubs. The effect and interaction of different concentrations of phytohormones, sucrose and iron sources on in vitro initiation, multiplication and rooting of shoots were studied. The time of collecting explants from donor plants significantly affected the initiation of shoot culture of Rosa ‘Konstancin’. Considerably higher frequency of bud break (100%) was obtained in explants isolated in August as compared to those collected at the end of May (30%). All buds developed into single shoots after 2-4 weeks of growing on the basal Murashige and Skoog medium containing 2.2 µM BAP, 0.3 µM GA3 and 88 mM of sucrose. The highest multiplication rate (4.8 shoots/explant) in a 5-week period was obtained on MS medium containing 50% of nitrogen salts, 3.1 µM BAP, 0.9 µM GA3 and 58 mM sucrose. High rooting frequency (100%) and quality of rooted plantlets was obtained on a medium containing 0.5 µM IBA, 138 µM Fe-EDDHA and 88 mM sucrose. Fe-EDDHA had a beneficial effect on the growth and photosynthetic activity of Rosa ‘Konstancin’ plantlets, which were successfully acclimatized ex vitro, with a more than 90% survival rate.

Development of Nolana carnosa micropropagation protocol

Nolana carnosa (Lindl.) Miers ex Dunal, (Fam.: Solanaceae), is a bushy plant native from Chile's north coast. It grows in the Atacama Desert in salty soils under extremely dry conditions. This understudied species is an interesting new ornamental option because of its morphological characteristics like abundant white to deep blue flowering, evergreen foliage and compact growth. With the aim to develop an efficient in vitro culture protocol for this species, during autumn 2015 shoots were collected in Bahia Inglesa, Atacama Region, Chile. Materials were surface sterilized and then initiated to in vitro culture in the lab. Multiplication and rooting trials were done during 2016 once the materials were finally free from fungi and bacteria by surface sterilization and subculture. The multiplication trial used five MS based medium where only BAP content varied (treatments were: 0.0, 0.5, 1.0, 1.5 and 2.0 mg L-1 of BAP). Under the flow cabin 50 plants composed by 3-4 nodes were established. Growth measurements were taken after a 5-weeks cycle. Number of sprouts, multiplication rate, plant's height, fresh and dry weight were measured and water content (%) was calculated. A second trial to enhance rooting was carried out, using 5 MS based media with different contents of IBA in which 50 plants were established. The trial was measured once a week for three weeks counting the number of rooted plants per media. The highest multiplication rate (3.26) and plant size (56.03 mm) were obtained in hormone free media. No statistical difference was observed in plant's weight. Plants grown in 1.5 and 2.0 mg L-1 BAP showed higher dry matter production. Similar results were obtained in rooting experiments where hormone free media enhanced root production in plants (84 and 87%). Finally, both hormone free media used in trials showed the best results.

Effect of cytokinins on shoots proliferation and rosmarinic and salvianolic acid B production in shoot culture of Dracocephalum forrestii W. W. Smith

The current study estimates the effect of different cytokinins on shoot proliferation and biosynthesis of caffeic acid derivatives in Dracocephalum forrestii in vitro culture. The shoots were grown on Murashige and Skoog (MS) agar medium with 1 µM indole-3-acetic acid (IAA) and different content of 6-benzyloaminopurine (BAP), zeatin, kinetin (1, 2, 4, 8, 18 µM) or thidiazuron (TDZ) (0.1, 0.2, 0.5, 1, 2 µM). The highest multiplication rate (about seven shoots and/or buds per explant) was obtained after 4 weeks of culture on MS medium with 1 µM IAA and 8 or 16 µM BAP. Optimal biomass of plant material was also received on the same media. The identity of the compounds present in the hydromethanolic extracts from D. forrestii shoots grown on cytokinin-supplemented media was confirmed using UPLC–PDA–ESI–MS method. The analysis revealed the presence of nine metabolites recognized as caffeic acid derivatives. The content of the predominant phenolic acids in the extracts, i.e. rosmarinic acid (RA) and salvianolic acid B (SAB), was determined with UHPLC. The highest yield of RA was found in shoots cultivated in the medium containing 1 µM IAA and 2 µM BAP (18.7 mg/g DW). The highest level of SAB (5.3–5.9 mg/g DW) was identified in multiple shoots grown in the presence of 1 µM IAA and 0.5–1 µM TDZ or 2 µM BAP.

Large-scale propagation of Myrobolan (Prunus cerasifera) in RITA® bioreactors and ISSR-based assessment of genetic conformity

An efficient protocol for the micropropagation of Prunus cerasifera using temporary immersion bioreactors was described. One-node cuttings excised from field-grown plants were successfully disinfected using 12% sodium hypochlorite for 10 min. Immersion duration, frequency and medium composition in Bioreactors type RITA® were evaluated. A high multiplication rate was obtained using RITA® bioreactors containing Murashige and Skoog’s medium (MS) supplemented with 1 mg L−1 N6-benzyladenine (BAP) and 1 mg L−1 indole-3-butyric acid (IBA) with 20 min/12 h as immersion time. RITA®-derived shoots were found to be more vigorous than those regenerated using the standard procedure. Higher levels of photosynthetic pigments were observed in shoots cultured in bioreactors, during the proliferation and rooting stages, which proved a certain degree of photoautotrophy of RITA®-derived vitroplants. A high proportion of shoots was successfully rooted in an MS liquid medium supplemented with 1 g L−1 activated charcoal in RITA® bioreactors. During plant acclimatization, survival rates exceeding 80% were recorded. The genetic fidelity of micropropagated plants was investigated using inter simple sequence repeat (ISSR) markers. Results proved the generation of homogenous amplification profiles and thus supported the clonal fidelity of regenerated vitroplants.

Optimising Gelling Agents, Light Source and After-care to Commercialise Teak Tissue Culture

Mass propagation of Teak (Tectona grandis Linn. f.) a commercial species is a priority to increase multiplication rate and meet the growing demand for planting material. The present study has assessed the in vitro performance of Teak Tissue Cultures with different gelling agents, different light conditions, and aftercare of ex vitro rooted shoots to enhance their survival rate. Multiple shoot formation was induced from excised seedling nodal explants on MS supplemented with BA and Kn and about 5 - 10 shoots were obtained from each explant. Significant variation (p > 0.05) was observed in the number of shoots produced by the different clones. Three gelling agents (agar, phytagel and gellan gum), and two light sources (tube light and LED) were tested for enhancing shoot multiplication. No significant difference in in vitro growth was observed between clones with different solidifying agents. Teak, however, did not respond favourably to LED lights. Rooting-acclimatization phase was achieved in the nursery with 80 - 95 per cent success. The rooted plants were sprayed with DAP and Humaur to assess the growth performance following transplanting. Significant variations in rooting indicate the existence of physiological variations among the clones. Application of fertilizers promoted an initial boost followed by a steady increase during the rest of the study period. Clones with high multiplication rates under in vitro conditions could be selected for commercialization of teak multiplication. Under ex vitro conditions, a spray of fertilizers during the initial establishment phase would result in increased vigour of transplantable plants. This would ensure better survival on out planting.Plant Tissue Cult. & Biotech. 28(1): 13-24, 2018 (June)

Revision Anterior Cruciate Ligament Reconstruction Outcomes in Younger Patients: Medial Meniscal Pathology and High Rates of Return to Sport Are Associated With Third ACL Injuries

Background: There are limited data evaluating the outcomes of revision anterior cruciate ligament (ACL) reconstruction surgery in younger patients despite recent reports that the rates of graft rupture are higher in young cohorts. Purpose: To explore the outcomes of revision ACL reconstruction surgery in younger patients with the specific aims of determining the rates of third ACL injury and whether knee pathology at the time of revision surgery and return to sport were associated with further injury. Study Design: Case-control study; Level of evidence, 3. Methods: The study cohort consisted of 151 consecutive patients who were aged 25 years or younger at the time of their first revision ACL reconstruction. The number of subsequent ACL injuries (graft rerupture or contralateral injury to the native ACL) was determined at a mean follow-up time of 4.5 years (range, 2-9 years). Surgical details were recorded, along with a range of sport participation outcomes. Contingency tables were used to assess the associations between subsequent ACL injury and return to sport, knee pathology, and the drilling of new femoral or tibial tunnels at revision surgery. Results: The follow-up rate was 85% (128/151). Graft reruptures occurred in 20 patients (16%) at a mean time of 2 years after revision surgery. Contralateral ACL injuries occurred in 15 patients (12%) at a mean time of 3.9 years. The total number of patients who had a third ACL injury was 35 (27%). There was a significant association between having medial meniscal pathology and sustaining a graft rerupture (P = .03), but there was no association between graft rerupture and using the same tunnels from the primary procedure at revision surgery. After revision reconstruction, 68% of patients (95% CI, 55%-71%) returned to their preinjury level of sport, compared with 83% (95% CI, 69%-84%) after primary reconstruction in the same patients. Those who had a third ACL injury had a significantly higher rate of return to preinjury sport (83%) after the revision procedure than did the group that did not have further ACL injuries (62%, P = .02). Conclusion: Younger patients are at significant risk of having multiple ACL injuries. The high rate of third ACL injuries presents a significant issue for future knee health in these young athletes. Medial meniscal pathology and returning to high-risk sport are factors that are significantly associated with the high multiple ACL injury rate in the young.

LED lighting affects plant growth, morphogenesis and phytochemical contents of Myrtus communis L. in vitro

The influence of light quality and cytokinin content in media on growth, development, photosynthetic pigments and secondary metabolite content of Myrtus communis L. was evaluated in an in vitro culture. Various treatments with light emitting diodes (LEDs): 100% blue (B), a mix of 70% red and 30% blue (RB) and 100% red were applied and compared with a traditional fluorescent lamp as control. Axillary shoots were incubated on Murashige and Skoog medium with 30 g dm−3 sucrose, 0.5% BioAgar, 0.5 μM 1-naphthaleneacetic acid and different concentrations of 6-benzyladenine (BA): 1, 2.5 and 5 µM. Cultures were maintained for 6 weeks in 23/21 ± 1 °C (day/night), 80% relative humidity and 16/8 h photoperiod; photosynthetic photon flux density (PPFD) was 35 µmol m−2 s−1 in all treatments. Light spectra and BA content in media affected biometrical and phytochemical M. communis properties. Red LEDs and 5 µM BA resulted in the highest multiplication rate. The highest shoots were obtained under red LEDs, but with the lowest concentration of cytokinin in media. Fresh weight was greatest on LEDs containing blue light in the spectrum (B and RB); moreover, 5 µM BA increased dry weight. Photosynthetic pigment levels were lower under LED light compared to control lamps. Phenolic acids and flavonoids were identified in M. communis leaf extracts. Myricetin was the major constituent with highest concentration under red LEDs and highest BA level.

Patient and cycle characteristics predicting high pregnancy rates with single-embryo transfer: an analysis of the Society for Assisted Reproductive Technology outcomes between 2004 and 2013

To analyze factors associated with high live birth rate and low multiple birth rate in fresh and frozen-thawed assisted reproductive technology (ART) cycles. Retrospective cohort analysis. Not applicable. The study population included 181,523 women undergoing in vitro fertilization with autologous fresh first cycles, 27,033 with fresh first oocyte donor cycles, 37,658 with fresh second cycles, and 35,446 with frozen-thawed second cycles. None. Live birth rate and multiple birth rate after single-embryo transfer (SET) and double embryo transfer (DET) were measured, in addition to cycle characteristics. In patients with favorable prognostic factors, including younger maternal age, transfer of a blastocyst, and additional embryos cryopreserved, the gain in the live birth rate from SET to DET was approximately 10%-15%; however, the multiple birth rate increased from approximately 2% to greater than 49% in both autologous and donor fresh and frozen-thawed transfer cycles. This study reports a 10%-15% reduction in live birth rate and a 47% decrement in multiple birth rate with SET compared with DET in the setting of favorable patient prognostic factors. Our findings present an opportunity to increase the rate of SET across the United States and thereby reduce the multiple birth rate and its associated poor perinatal outcomes with assisted reproductive technology pregnancies.

Establishment of an efficient in vitro regeneration and Agrobacterium rhizogenes-mediated genetic transformation protocol for safed musli (Chlorophytum borivilianum Santapau & R.R.Fern.)

Safed musli (Chlorophytum borivilianum Santapau & R.R.Fern.) is a medicinal herb valued for its tuberous roots that contain several bioactive compounds. Conventionally, safed musli tubers are harvested for secondary metabolites 9 mo after planting. Alternatively, in vitro culture can be used for mass production and to shorten the time required to produce secondary metabolites. This study was designed to develop an efficient hairy root induction protocol for safed musli. Shoot bud explants of various lengths were tested on Murashige and Skoog medium with 3 mg L−1 6-benzylaminopurine. Shoot buds >4 cm resulted in 100% multiplication, but 2- to 4-cm shoot buds produced a high multiplication rate (92%), the highest number of multiple shoots (15.55 ± 0.37), and the longest shoots (6.83 ± 0.08 cm) after 30 d of culture. In vitro shoots were infected with one of five strains of Agrobacterium rhizogenes. Only strains MAFF106590, MAFF106591, and MAFF720002 induced the hairy root phenotype indicative of genetic transformation. MAFF720002 strain had a positive transformation response in all in vitro raised safed musli stem segments of different lengths. Stems 2.5 cm in length had the highest transformation percentage (76 to 81%), greatest number of hairy roots per explant (5.02 ± 0.06), and the longest hairy roots (23 ± 0.49 cm). PCR analysis detected the rolB gene in the transformed samples. The A. rhizogenes strain played a significant role in producing and promoting the growth of hairy roots in safed musli. This approach can potentially be utilized for the replacement of conventional field methods for secondary metabolite production from safed musli.

Effects of plant growth regulators on multiple shoots regeneration of in vitroChrysanthemum morifoliumRamat

This study aimed to compare effects of different plant growth regulators on multiple shoot production of Chrysanthemum morifolium Ramat. Nodal segments and shoots of chrysanthemum were cultured on (a) Murashige and Skoog (MS, 1962), (b) MS supplemented with benzylaminopurine (BAP), (c) MS supplemented with BAP and indole acetic acid (IAA), (d) MS supplemented with IAA and BAP, (e) MS supplemented with kinetin (Kin) and indolebutyric acid (IBA) in different concentrations. The shoot cultured on MS supplemented with 0.5 mg L-1 BAP showed the highest multiple shoots (4.91 shoots segment-1). Micropropagation using shoots is the most desirable and safe technique, as it does not only minimize genetic variation but also helps in the formation of healthy shoots. It also provides high rate of multiplication. The seedlings induced from shoots cultured on modified ½MS supplemented with activated charcoal showed higher growth rate than the shoots cultured on modified ½MS without activated charcoal.

Biochemical and morphometric analysis of Rosa tomentosa and Rosa rubiginosa during application of liquid culture systems for in vitro shoot production

ABSTRACTTwo liquid culture systems, rotary shaker (RS) and temporary immersion system (TIS) for the propagation of Rosa rubiginosa and Rosa tomentosa were investigated and compared with solid medium culture. Shoot tips were grown on basic Murashige and Skoog medium with 20 mg dm−3 Fe EDDHA, 1 µM BA, 1.5 µM GA3 and 3% sucrose for six weeks. Of the different culture systems, liquid cultures resulted in better growth and development of roses. Both RS and TIS cultures improved biomass growth, multiplication rate, leaf blade area and plant height; however, RS reduced the shoot dry mass content. There were no changes in the content of chlorophyll a, b, a + b in plants developed in the tested systems. Use of a solid medium favoured the accumulation of phenolic compounds, soluble sugars, and carotenoids, but it was accompanied by browning and necrosis of shoots. Plants propagated in TIS were characterised by the high content of phenolic compounds and soluble sugars, but they had the highest multiplication rate. Use of TIS resulted in eight times the number of shoots for R. tomentosa and twice as many for R. rubiginosa compared to solid medium.

In vitro propagation of Leucocroton havanensis Borhidi (Euphorbiaceae): A rare serpentine-endemic species of Cuba

Leucocroton havanensis Borhidi is an endemic plant species of Cuba able to hyperaccumulate nickel. In order to sustain the conservation of this species, an efficient protocol for its micropropagation, via axillary bud proliferation, is described. We placed apical segments from aseptic seedlings on basal medium supplemented with indole-3-acetic acid (IAA) and 6-benzylaminopurine (BAP) or thidiazuron (TDZ), individually or in combination. On a medium containing 0.5 mg L−1 IAA and 1.0 mg L−1 BAP explants (65.5%) developed axillary buds. Nevertheless, combinations of 0.5 mg L−1 IAA with 0.1 mg L−1 TDZ was the most effective treatment producing the highest number of buds per explant (30.3); while the control treatment, without growth regulators, produced no buds at all. Transfer of buds to medium supplemented with indole-3-butyric acid, indicated that 0.25 mg L−1 is the amount of hormone required to generate roots on young buds (100%). In order to assess DNA variations in micropropagated plants, an Amplified Fragment Length Polymorphism analysis was performed and no genetic variation was detected. This study demonstrates that a high multiplication rate can be obtained by means of the reported protocol, and that plantlets can be readily hardened (96% survival) in a greenhouse by transplanting them on serpentine soil.

Refinement of protocol for rapid clonal regeneration of economical bamboo, Bambusa balcooa in the agroclimatic conditions of Bihar, India

Bambusa balcooa is a commercially important bamboo. The present study was undertaken for refinement of protocol for the rapid and mass production of this species of bamboo in agroclimatic condition of Bihar. Micro-clonal propagation techniques have been employed for the study. This technique is the only method for the large scale production of B. balcooa. Explants were collected from Bamboo setum from TNB College campus. Combined effect of 6-benzylaminopurine (BAP) and Kinetin (Kn) (BAP 2 mg/l + Kn 0.5 mg/l) resulted in 85% bud breakage during initiation of cultures. One remarkable feature that was observed is that microshoots in medium without supplementation of naphthalene acetic acid (NAA) had necrotic shoots and they had less multiplication rate in liquid media. High multiplication rate with comparatively longer shoots were observed in liquid media. NAA (2.5 mg/l) along with half strength of MS showed 100% rooting but it was less than 50% when Murashige and Skoog (MS) media was supplemented with indole 3-butyric acid (IBA). Remarkably, in half strength of MS, clumps dried at first within 7 days in rooting media, however, new green healthy shoots proliferated later (after 21 days). The data obtained from this study has set a refined protocol for the clonal regeneration of B. balcooa for the state of Bihar. The current study will help to produce this species of bamboo on large scale, and thereby help to boost rural economy of Bihar. Key words: In vitro regeneration, clonal propagation, bamboo, Bambusa balcooa, micropropagation.

English

This work aimed to develop a protocol for the in vitro establishment, multiplication, rooting and ex vitro acclimatization of Mimosa pudica L., a species used in folk medicine and with pharmacological activity. Aseptic cultures were established from seeds inoculated in MS medium, without growth regulators, followed by an in vitro stabilization phase in culture medium supplemented with 2.22 μM BAP. The cultures were transferred to MS medium supplemented with different cytokinins, combined or not with an auxin, aiming its large-scale propagation. The culture medium supplemented with 5 µM BAP plus 0.5 µM NAA provided the highest multiplication rate and top quality plantlets. The combination of 0.6 µM TDZ plus 0.05 µM NAA resulted in higher multiplication rates than in response to combination of BAP plus NAA, although the subsequent maintenance of the cultures in a medium without growth regulators has resulted in low regenerative response. In vitro rooting of micro-cuttings was high even in the absence of auxins. Over 90% of plantlets transferred to the greenhouse survived after the acclimatization phase. Acclimatized plants presented normal vegetative and reproductive development. The procedures established in the present study allow a massive production of M. pudica plants for further pharmacological studies. Key words: Biodiversity conservation, ex vitro acclimatization, in vitro rooting, micropropagation, Mimosa pudica.

<i>In Vitro</i> Propagation and Recovery of Eight Apple and Two Pear Cultivars Held in a Germplasm Bank

Eight apple (Malus x domestica) and two pear (Pyrus communis) traditional cultivars of commercial interest were selected from the germplasm bank CIAM (Centro de Investigaciones Agrarias de Mabegondo, NW of Spain) one of the most important collections of The Iberian Peninsula. In order to define the establishment protocol, apical buds 1.0 - 1.5 millimetre length of the selected cultivars were established in solid mineral medium Murashige and Skoog (MS) with modified vitamins (thiamine 10X) and supplemented with 1 mg·L-1 6-benzyladenine (BA), 0.2 mg·L-1 gibberellic acid (GA3) and 0.3 mg·L-1 of indole-3-butyric acid (IBA). The highest shoot initiation rates were achieved with apple cultivar “Principe Grande” (55%) and “Barburina” pear cultivar (50%); the lowest rate was obtained with apple cultivar “Camoesa” (11%). Initiation rates for shoots culture varied between 22% and 37% for the other cultivars. The cytokinins BA, zeatin, 2-isopentyladenine (2iP) and thidiazuron (TDZ) were tested at concentrations of 0, 0.25, 0.5 and 1 mg·L-1 in the absence or presence of IBA (0.1 mg·L-1) for the multiplication step. For all cultivars, the highest multiplication rates were obtained with medium supplemented with TDZ and also in media containing BA. The in vitro rooting success of shoots in medium with 0.1 mg·L-1 IBA without cytokinins, and the survival rate of plantlets acclimatized under greenhouse conditions depended on the genotype of the different cultivars. In vitro propagation of these ten traditional apple and pear cultivars in NW Spain has not previously been reported.

Analysis of the prevalence of drug-resistant hepatitis B virus in patients with antiviral therapy failure in a Chinese tertiary referral liver centre (2010-2014).

To study the prevalence of drug-resistant HBV in patients with therapy failure in a Chinese tertiary referral liver centre. 1223 HBV-infected patients who underwent genotypic resistance testing between 2010-2014 were studied. 3TC genotypic resistance was the most common (46.5%), followed by LdT resistant (46.2%), ETV intermediate (37.9%), ADV resistant (11.4%), TDF intermediate (11.4%) and ETV resistant (1.7%). The 3TC resistance rate increased from 39.8% in 2010 to 56.6% in 2013, before decreasing to 49.5% in 2014, evidence of a lagging effect of l-nucleoside consumption. M204I, N236T and L180M+M204V+V173L/S202G were the most common substitutions for l-nucleoside (3TC and LdT), ADV and ETV genotypic resistant phenotypes, respectively. 3TC-exposed patients showed a high multiple genetic resistance rate (3TC-resistant+LdT-resistant+ETV intermediate; 58.8%). Resistance rates to 3TC, LdT and ETV in HCC patients were significantly higher than in cirrhosis and CHB patients. Resistance rates to different drugs showed no statistical difference between genotype B and C patients, whilst some amino acid substitution showed genotype bias, e.g. N236T incidence in genotype B was significantly higher than in genotype C (43.2% vs. 5.9%; P<0.0001), and genotype C isolates had a significantly higher A181V/T incidence than genotype B (54.9% vs. 19.3%; P<0.0001). 3TC genotypic resistance was most common in this centre, whilst ETV had the lowest resistance rate. HBV genotypes had no impact on antiviral drug resistance, except for some drug resistance substitutions bias. Optional initial therapy and subsequent rescue treatment should be based on knowledge of nucleos(t)ide analogue resistance.

High-performance micropropagation of dendroenergetic poplar hybrids in photomixotrophic Temporary Immersion Bioreactors (TIBs)

By the first time an efficient methodology for micropropagation of poplar hybrids by temporary immersion in liquid medium was developed under photomixotrophic conditions. Influence of factors, such as plant regulator/balance in culture medium, number of explants or inoculums per bottle, immersion frequencies in Temporary Immersion Bioreactors (TIBs), reduced sucrose as priming treatment and photoperiod were studied in TIBs with 0.4MPa CO2 at a light intensity of 100μMm−2s−1. Protocol was standardized in three poplar clones recommended for their dendroenergetic traits. The highest multiplication rates were obtained by inoculating 20 and 24 nodal segments in TIBs with 200ml of medium MS+0.22μM TDZ. During winter (8h photoperiod), the highest multiplication rates in TIBs were obtained with 8h immersion frequencies, while in summer (14h photoperiod) immersions every 6h showed the best multiplication rates. The main percent of ex vitro rooted plants was recovered when plantlets were micropropagated in liquid medium supplemented with 1.5% sucrose. A drying pretreatment for 20min followed by 10min inoculation with a mycorrhiza solution (Scleroderma spp.-Pisolithus tinctorius) allowed us to obtain 97.3% of poplars adapted to ex vitro environmental conditions. DNA patterns amplified with the ISSR primer (CTC)5 showed high genomic similarity without polymorphic bands between samples of the same hybrid, which indicated the non-occurrence of genomic changes in the studied populations. Overall, TIBs technology constitute an advance to the industrial development of clonal propagation of poplars for biomass production, in comparison with the classical micropropagation.