Somatic embryogenesis in Rosa chinensis cv. ‘Old Blush’

Plant Cell, Tissue and Organ Culture (PCTOC) - Rose is one of the most widely used ornamental flowers in the world. However, the low induction rates for most rose somatic embryos means that it is...     

Effect of liquid culture on the growth and development of miniature rose (Rosa chinensis Jacq. ‘Minima’)

The growth of miniature rose (Rosa chinensis Jacq. Minima) shoots cultured on liquid medium was greater relative to those cultured on two-phase (solid + liquid) medium or solid medium alone. Shoot multiplication ratio (number of multiple shoots per explant per subculture) on liquid medium was higher with 17.8–26.6 M 6-benzyladenine at compared to that at 0–8.9 M. Shoots grown on 30 ml or more of liquid medium had a higher multiplication ratio than those grown on 10 or 20 ml. The growth and multiplication ratio increased when the culture period was extended from 3 to 6 weeks, although plantlets began to exhibit some chlorosis by the 6^th week. These conditions were maintained over four subcultures for cultivars Baby Katie, Lavender Jewel, Red Sunblaze and Royal Sunblaze, with no significant change in multiplication ratio over time.Abbreviations BA 6-benzyladenine - NAA 1-naphthaleneacetic acid     

Cryopreservation by encapsulation-dehydration of ‘Christmas bush’ (<Emphasis Type="Italic">Ceratopetalum gummiferum</Emphasis>) shoot tips

Summary Christmas bush (Ceratopetalum gummiferum Sm) is a shrubby tree species of the east coast of New South Wales in Australia. It is much prized as a cut flower crop because of its bright, pinky red floral calyces. New varieties are being developed, the storage of which is an important issue. In this study, it was shown that shoot tips sampled from in vitro plantlets withstood cryopreservation using the encapsulation-dehydration technique. The protocol leading to optimal regrowth was the following: excised shoot tips were pretreated for 1 d in the dark on hormone-free Murashige and Skoog (MS) medium with 0.3 M sucrose, then encapsulated in 3% calcium alginate and precultured in liquid MS medium with 0.5 M sucrose for 3 d. Precultured beads were dehydrated for 6 h in the air current of the laminar flow cabinet to 24.3% moisture content (fresh weight basis) before rapid immersion in liquid nitrogen. Under these conditions, regrowth of shoot tips after cryopreservation reached 61.4%. Regrowth of cryopreserved shoot tips was not affected by the period of cold acclimation of in vitro mother plants.     

Cryopreservation of apple (Malus×domestica Borkh.) shoot tips following encapsulation-dehydration or encapsulation-vitrification

Axillary shoot tips of apple cv. Golden Delicious isolated from shoot cultures were successfully cryopreserved using the encapsulation-dehydration technique. After encapsulation in alginate gel, embedded shoot tips were dehydrated by exposure to a sterile air flow before being frozen in liquid nitrogen and subsequent slow thawing. A preculture on modified MS medium containing 0.75 M sucrose followed by 6 h of dehydration (21% residual water) led to the highest shoot regrowth of frozen, coated shoot tips (83.7%). Among the sugars tested, sucrose and sorbitol presented the best cryoprotective effect. Four other scion apple varieties and rootstocks were also successfully cryopreserved. Axillary shoot tips of five apple (Malus×domestica Borkh.) scion and rootstock cultivars were cryopreserved using the encapsulation-vitrification technique. Using a one-step freezing method, we successfully cryopreserved axillary shoot tips without the requirement of a cold hardening pretreatment of the shoot cultures. Cryopreserved shoot tips treated with aqueous cryoprotective mixture IV containing 180% (w/v) sucrose and 120% (v/v) ethylene glycol showed the highest shoot regrowth rates, which varied from 64% to 77%, depending on the cultivar. Received: 29 July 1999 / Revision received: 24 September 1999 / Accepted: 26 November 1999     

‘Ins’ and ‘outs’ of triple therapy

Chronic oral anticoagulant treatment is obligatory in patients (class I) with mechanical heart valves and in patients with atrial fibrillation with CHADS2 score >1. When these patients undergo percutaneous coronary intervention with placement of a stent, there is also an indication for treatment with aspirin and clopidogrel. Unfortunately, triple therapy is known to increase the bleeding risk. For this group of patients, the bottom line is to find the ideal therapy in patients with indications for both chronic anticoagulation therapy and percutaneous intervention to prevent thromboembolic complications such as stent thrombosis without increasing the risk of bleeding. (Neth Heart J 2010;18:444-50.)     

Fruit load modulates flowering-related gene expression in buds of alternate-bearing ‘Moncada’ mandarin

Background and Aims

Gene determination of flowering is the result of complex interactions involving both promoters and inhibitors. In this study, the expression of flowering-related genes at the meristem level in alternate-bearing citrus trees is analysed, together with the interplay between buds and leaves in the determination of flowering.

Methods

First defruiting experiments were performed to manipulate blossoming intensity in ‘Moncada’ mandarin, Citrus clementina. Further defoliation was performed to elucidate the role leaves play in the flowering process. In both cases, the activity of flowering-related genes was investigated at the flower induction (November) and differentiation (February) stages.

Key Results

Study of the expression pattern of flowering-genes in buds from on (fully loaded) and off (without fruits) trees revealed that homologues of FLOWERING LOCUS T (CiFT), TWIN SISTER OF FT (TSF), APETALA1 (CsAP1) and LEAFY (CsLFY) were negatively affected by fruit load. CiFT and TSF activities showed a marked increase in buds from off trees through the study period (ten-fold in November). By contrast, expression of the homologues of the flowering inhibitors of TERMINAL FLOWER 1 (CsTFL), TERMINAL FLOWER 2 (TFL2) and FLOWERING LOCUS C (FLC) was generally lower in off trees. Regarding floral identity genes, the increase in CsAP1 expression in off trees was much greater in buds than in leaves, and significant variations in CsLFY expression (approx. 20 %) were found only in February. Defoliation experiments further revealed that the absence of leaves completely abolished blossoming and severely affected the expression of most of the flowering-related genes, particularly decreasing the activity of floral promoters and of CsAP1 at the induction stage.

Conclusions

These results suggest that the presence of fruit affects flowering by greatly altering gene-expression not only at the leaf but also at the meristem level. Although leaves are required for flowering to occur, their absence strongly affects the activity of floral promoters and identity genes.     

Cryopreservation of small leaf squares-bearing adventitious buds of <Emphasis Type="Italic">Lilium</Emphasis> Oriental hybrid ‘Siberia’ by vitrification

We report a new cryopreservation method for Lilium Oriental hybrid ‘Siberia’. Adventitious buds were induced from leaf segments cultured for 12 days on adventitious bud induction medium composed of half-strength Murashige and Skoog medium (MS) supplemented with 1 mg L^?1 α-naphthalene acetic acid and 0.5 mg L^?1 thidiazuron. Small leaf squares (SLSs, 3?×?4 mm), each bearing at least one adventitious bud, were cut from leaf segments, precultured on medium with 0.5 M sucrose for 1 day, and then treated for 20 min with a loading solution containing 0.4 M sucrose and 2 M glycerol, followed by exposure to plant vitrification solution 2 for 7 h at 0 °C. Dehydrated SLSs were directly immersed in liquid nitrogen for 1 h. Cryopreserved SLSs were re-warmed in MS medium containing 1.2 M sucrose for 20 min at room temperature, followed by post-thaw culture for recovery. With this procedure, 85% survival and 72% shoot regrowth were achieved following cryopreservation. The use of SLSs bearing adventitious buds for cryopreservation reported in the present study eliminates the time-consuming and labour-intensive step of shoot tip excision, and has great potential to facilitate cryopreservation in other plant species.     

Structural and Narrative Reconstruction of Rural Residents’ Representations of ‘Nature’, ‘Wildlife’, and ‘Landscape’

The objective of this paper is the structural and narrative reconstruction of representations of ‘nature’, ‘wildlife’ and ‘landscape’, held by rural residents of the Dadia Forest Reserve. Data collection involved in-depth interviews. Employing a social representations’ approach, we recovered representational elements that are expected in the case of rural belief systems, such as negative dispositions towards wolves and foxes, as well as elements of an urban adherence, such as nature’s independence. Representational elements refer to visual aspects of the countryside, which seem compatible with the figurative nucleus of the rural idyll. Concerning ‘wildlife’, residents focused on vultures, which comprise the main tourist attraction of the reserve. Scientific knowledge adds to the complexity of the narrative schema, which corresponds to the representation of ‘wildlife’. Interviewees perceived the rural landscape as an interface between the natural and the human-conditioned environment. Our study shows that interviewees make no reference to environmental conservation or quality of life issues, as it could be expected according to relatively wide definitions of the term ‘environmentalism’. Environmental messages reinforced by ecotourism development seem to be recalled primarily in terms of their compatibility with the perceived economic benefit of local people. Despite ecotourism development, representational elements that diverge from a tourist version of ‘nature’, ‘wildlife’ and ‘landscape’ were not pronounced within rural belief-systems. Further interventions within the study area are needed, in order to address a variety of topics under the environmental conservation discourse and raise the environmental awareness of rural residents.     

Organogenesis from ‘Passe Crassane’ and ‘Old Home’ pear (Pyrus communis L.) protoplasts and isoenzymatic trueness-to-type of the regenerated plants

Summary Large numbers of highly viable mesophyll protoplasts were isolated from shoot cultures of the scion cv Passe Crassane and the rootstock genotype Old Home of common pear (Pyrus communis L.). Protoplasts were cultured for both genotypes either as liquid layers or as liquid-over-agar cultures, in ammonium-free MS medium with 0.5 M mannitol, 50 mg/l casein enzymatic hydrolysate (CEH), 2.0 mg/l NAA and 1.0 mg/l BAP, plus either 0.5 mg/l IAA (for Old Home) or 2.0 mg/l IAA (for Passe Crassane). Protoplast microcalli, obtained by day 60 (Passe Crassane) or day 80 (Old Home), were transferred for further growth to ammonium-free MS medium with 2.0 mg/l NAA and 1.0 mg/l BAP. Shoot bud regeneration from the protoplastderived callus was first attempted between 100 (Passe Crassane) and 120 (Old Home) days after protoplast isolation. For Passe Crassane, shoot buds were regenerated (day 130) on a half-strength MS medium with 0.1 mg/l IBA, 0.5 mg/l BAP, 50 mg/l CEH and 20 mg/l Ca-panthotenate. For Old Home, shoot but regeneration only occurred 30 days later and on the same medium as above, which was additionally supplemented with double the concentration of the group B vitamins found in the original MS formulation and 0.05 mg/l GA₃. Following micropropagation and in vitro rooting of shoots, the plants were transferred to soil following standard procedures. Trueness-to-type of the regenerated plants was assessed by analysing their leaf isozyme banding profiles (for EST, AP, PRX, SOD, ENP, LAP, PGI, AAT, ADH, MDH and PGM) and comparing them to those corresponding to the original shoots that provided the protoplasts. No differences between the mother shoots and the protoclones were observed for any one of the 11 isozyme systems studied.     

Importance of the iron chelate formula for micropropagation of Rosa hybrida L. ‘Moneyway’

In vitro propagation of the rose rootstock Moneyway was investigated on the following media: Murashige and Skoog (MS), Quoirin and Lepoivre (QL) and Woody Plant (WP). Growth, which was measured as length of shoots after a 6-week period, was faster on MS and QL than on WP. In spite of the better growth, chlorosis of newly formed leaves occurred from the third week on and was correlated with a lower chlorophyll content of shoots. Replacement of FeEDTA by FeEDDHA in QL and MS resulted in the development of green shoots for more than 3 months. The occurrence of chlorosis was not pH directed since the pH of QL with FeEDTA or FeEDDHA had not changed after 6 weeks of growth. Addition of the light absorbing dye fast yellow 9 to QL with FeEDTA also resulted in green shoots with a higher chlorophyll content. It is suggested that FeEDDHA is a more photostable chelate than FeEDTA, resulting in a higher availability of iron for the rose shoots. The impact of the iron chelate formula on the micropropagation of plant species that are susceptible to iron deficiency is discussed.Abbreviations BA 6-benzyladenine, fast yellow 9-4-amino-1,1-azobenzene-3,4-disulfonic acid - FeEDTA ferric ethylenediamine tetraacetate - FeEDDHA ferric ethylenediamine di(o-hydroxyphenylacetate) - IAA indole-3-acetic acid - IBA indole-3-butyric acid - LSD least significant difference - NAA -naphthaleneacetic acid - P probability     

Modulation of tau protein aggregation using ‘Trojan’ sequences

BackgroundThe abnormal assembly of tau into neurofibrillary tangles has been associated with over 30 debilitating disorders known as tauopathies. Tauopathies affect millions of people worldwide, yet no clinically approved solution for tau aggregation is currently available.MethodsWe employed a structure-based design approach to make a series of short peptide-based perturbants (Trojans), that can interact with the core hydrophobic fragment of tau protein. Through a combination of various biophysical methods, serum stability, toxicity, and blood-brain barrier translocation assays, we have assessed the efficacy of these designed peptides to intervene the aggregation of tau protein fragment.ResultsOur observations suggest that Trojan peptides could modulate the aggregation of the Ac-VQIVYK-NH₂ peptide by either accelerating or arresting its self-assembly and reduce the neurotoxicity of the fibrils formed. The designed perturbant peptides showed three essential pre-requisites such as negligible cytotoxicity, high proteolytic stability in serum, and an ability to cross human blood-brain barrier (BBB). Furthermore, the Trojans could disassemble the pre-formed fibrillar assemblies.ConclusionsThese designed Trojan peptides can serve as a potential therapeutic option for tauopathies, modulating post as well as pre-aggregation leading to the diseases condition.General significanceTauopathies are a group of over 20 progressive neurodegenerative disorders that affect millions of people worldwide. The available therapies of tau-linked neurodegenerative syndromes are limited and mostly symptomatic and therefore there is an urgent need for a cost-effective treatment option. We are presenting a series of structure-based, de novo designed, short peptides that can potentially modulate tau protein aggregation.     

Somatic embryogenesis and shoot regeneration from excised adventitious roots of the rootstock Rosa hybrida L. ‘Moneyway’

Plants were regenerated from excised adventitious roots of the rose rootstock Moneyway via a three step procedure: callus induction, induction of somatic embryos and shoot development. Callus was induced on excised roots after incubation for 4 weeks in the dark on SH-medium (Schenk and Hildebrandt) containing 50 M 2,4-dichlorophenoxyacetic acid. For embryo induction, calluses were transferred to hormone-free SH-medium and incubated for 8 weeks. The use of Gelrite instead of agar during callus induction stimulated somatic embryogenesis (up to 16% of the explants formed organized structures), whereas the presence of 6-benzylaminopurine in this phase inhibited subsequent regeneration. Yellow solid calluses with embryo-like cotyledons or primordia and friable calluses with embryos were selected, and upon incubation in the light shoots developed. Shoot development was faster and more frequent on solid callus than on friable callus (64% and 21% of the calluses finally formed one or more shoots, respectively). Eleven out of thirteen regenerants developed similarly to control shoots. Finally this regeneration method is compared with other systems for somatic embryogenesis and opportunities for the production of transgenic rose rootstocks and rose cultivare are discussed.Abbreviations BAP 6-benzylaminopurine - BM basal medium - BM+ enriched basal medium - 2,4-D 2,4-dichlorophenoxyacetic acid - DAPI-4,6 diamidino-2-phenylindole - FeEDDHA ferric ethylenediamine di(ohydroxyphenylacetate) - FeEDTA ferric ethylenediamine tetraacetate - IBA indole-3-butyric acid     

Mouse Sperm Cryopreservation and Recovery using the I·Cryo Kit

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies. Many of these valuable animals exist only as live animals, with no backup plan in case of emergency. Cryopreservation of embryos can provide this backup, but is costly, can be a lengthy procedure, and generally requires a large number of animals for success. Since the discovery that mouse sperm can be successfully cryopreserved with a basic cryoprotective agent (CPA) consisting of 18% raffinose and 3% skim milk, sperm cryopreservation has become an acceptable and cost-effective procedure for archiving, distributing and recovery of these valuable strains.Here we demonstrate a newly developed I•Cryo kit for mouse sperm cryopreservation. Sperm from five commonly-used strains of inbred mice were frozen using this kit and then recovered. Higher protection ratios of sperm motility (> 60%) and rapid progressive motility (> 45%) compared to the control (basic CPA) were seen for sperm frozen with this kit in 5 inbred mouse strains. Two cell stage embryo development after IVF with the recovered sperm was improved consistently in all 5 mouse strains examined. Over a 1.5 year period, 49 GM mouse lines were archived by sperm cryopreservation with the I•Cryo kit and later recovered by IVF.     

The effects of exogenous auxin and rol genes on root formation in Rosa hybrida L. ‘Moneyway’

The effect of indole-3-butyric acid (IBA) on the formation of non-transformed and rol gene transformed roots on stem slices of in vitro cultured shoots of Rosa hybrida L. Moneyway was examined. Formation of adventitious roots on this rootstock was dependent on the IBA dose; it was not affected by the presence of other root primordia on the same explant. Application of 0.32 to 1 M IBA during 5 days, followed by transfer to medium without hormones resulted in maximum root formation (90%) after three weeks. The formation of such untransformed roots was completely inhibited by transfer to medium with 5 mg 1^–1 kanamycin two days after excision. Ri roots were formed upon inoculation with A. rhizogenes LBA9402 harbouring two plasmids: pRi1855, comprising the rol genes and the binary plasmid p 35Sgusintron with the nptII gene for kanamycin resistance and the CaMV 35Sgusintron gene. The formation of these Ri roots on kanamycin-containing medium was independent of the presence of IBA. Stem slices inoculated with a disarmed A. tumefaciens GV3101, harbouring only the nptII gene, formed callus and subsequently roots in the presence of kanamycin exclusively on medium with high IBA concentrations (10 or 100 M). Root formation at 100 M IBA was considerably improved by transformation with the rolB gene under the influence of the strong CaMV 35S promoter. In addition, low IBA (0.1 and 1 M) stimulated the formation of roots only on stem slices transformed with A. tumefaciens harbouring the rolA+rolB+rolC genes; the rooting response at 10 M IBA was much improved. It was concluded that the 35SrolB gene and especially a combination of rolA, B and C genes promote the rooting response.Abbreviations BAP 6-benzylaminopurine - FeEDDHA ferric ethylenediamine di(o-hydroxyphenylacetate) - IBA indole-3-butyric acid - LSD least significant difference - RIM root induction medium - SE standard error - X-gluc 5-bromo-4-chloro-3-indolyl glucuronide     

Direct somatic embryogenesis from leaf and petiole explants of Spathiphyllum ‘Supreme’ and analysis of regenerants using flow cytometry

This study established a method of regenerating Spathiphyllum ??Supreme?? through direct somatic embryogenesis. Somatic embryos occurred in leaf and petiole explants cultured in the dark on a Murashige and Skoog basal medium supplemented with 2.27, 4.54, or 9.08???M N-phenyl-N??-1,2,3-thiadiazol-5-ylurea (TDZ) in combination with 1.08???M ??-naphthalene acetic acid or 2.26???M 2,4-dichlorophenoxyacetic acid (2,4-D). Explants with somatic embryos were transferred to fresh medium containing the same concentrations of growth regulators under lighted conditions for embryo conversion. The highest frequencies of leaf explants with somatic embryos and embryo conversion were both 84.4?%, which were induced by 9.08???M TDZ with 2.26???M 2,4-D. The frequencies for somatic embryo induction and embryo conversion were both 100?% when petiole explants were induced by 4.54???M TDZ with 2.26???M 2,4-D. The number of plantlets produced per leaf explant and petiole explant were as high as 67.4 and 74.4, respectively. Plantlets after transplanting to a soilless substrate grew vigorously in a shaded greenhouse. Liners were stable without phenotypic variation. Flow cytometry analysis of randomly selected plants showed that they all had a single identical peak. The mean nuclear DNA index for ??Supreme?? was 1.568, and the nuclear DNA content was 14.222?pg 2C^?1. The estimated genome size for ??Supreme?? was 6,954.5?Mbp 1C^?1 with a CV at 4.008?%. The results suggest that the regenerated plants have a stable ploidy level and this established regeneration method can be used for highly effective propagation of uniform Spathiphyllum ??Supreme??.     

Development of genetic maps of the citrus varieties ‘Murcott’ tangor and ‘Pêra’ sweet orange by using fluorescent AFLP markers

The progeny of 87 BC(1) hybrids of 'Murcott' tangor and 'Pera' sweet orange, genotyped with fluorescent amplified fragment length polymorphism (fAFLP) markers, was used for the construction of genetic maps for both citrus varieties. Mapping strategies, considering the progeny as a result of backcrossing and cross-pollination, were exploited in Mapmaker 2.0 (LOD score >or= 3.0 and or= 3.0 and theta 相似文献