Conservation, Regeneration and Genetic Stability of Regenerants from Alginate-Encapsulated Shoot Explants of Gardenia jasminoides Ellis.

The present study demonstrates the potential of the alginate encapsulation of shoot tips and nodal segments of Gardenia jasminoides Ellis, the short-term cold storage of artificial seeds and subsequent successful conversion to desirable, uniform and genetically stable plantlets. Shoot tips and first-node segments below them, derived from shoots of in vitro cultures, responded better than second-to-fourth-node segments on agar-solidified Murashige and Skoog (MS) nutrient medium and thus, they were used as explants for alginate encapsulation. Explant encapsulation in 2.5% sodium alginate in combination with 50 mM of calcium chloride resulted in the production of soft beads, while hardening in 100 mM of calcium chloride formed firm beads of uniform globular shape, suitable for handling. The addition of liquid MS nutrient medium in the sodium alginate solution doubled the subsequent germination response of the beads. The maintenance of alginate beads under light favored their germination response compared to maintenance in darkness. Encapsulated shoot tip explants of gardenia, which were stored at 4 °C for 4, 8 or 12 weeks, showed a gradual decline in their regeneration response (73.3, 68.9, 53.3%, respectively), whereas, non-encapsulated explants (naked), stored under the same time durations of cold conditions, exhibited a sharp decline in regeneration response up to entirely zeroing (48.9, 11.1, 0.0%, respectively). Shoots, derived from 12-week cold-stored encapsulated explants, were easily rooted in solid MS nutrient medium with the addition of 0.5 µM of Indole-3-acetic acid (IAA) and after transplantation of the rooted plantlets individually to pots containing a peat-perlite (3:1, v/v) substrate, they were successfully acclimatized in the greenhouse under the gradual reduction of 75 or 50% shading with survival rates of 95-100%. The genetic stability of the acclimatized plantlets was assessed and compared with the mother plant using inter simple sequence repeat (ISSR) markers. ISSR analysis confirmed that all regenerated plantlets were genetically identical to the mother plant. This procedure of artificial seed production could be useful for the short-term storage of germplasm and the production of genetically identical and stable plants as an alternative method of micropropagation in Gardenia jasminoides.

De novo transcriptome analysis of petal senescence in Gardenia jasminoides Ellis.

BACKGROUND: The petal senescence of ethylene insensitive species has not been investigated thoroughly while little is known about the temporal and tissue specific expression patterns of transcription factors (TFs) in this developmental process. Even less is known on flower senescence of the ornamental pot plant Gardenia jasminoides, a non climacteric flower with significant commercial value. RESULTS: We initiated a de novo transcriptome study to investigate the petal senescence in four developmental stages of cut gardenia flowers considering that the visible symptoms of senescence appear within 4 days of flower opening. De novo assembly of transcriptome sequencing resulted in 102,263 contigs with mean length of 360 nucleotides that generated 57,503 unigenes. These were further clustered into 20,970 clusters and 36,533 singletons. The comparison of the consecutive developmental stages resulted in 180 common, differentially expressed unigenes. A large number of Simple Sequence Repeats were also identified comprising a large number of dinucleotides and trinucleotides. The prevailing families of differentially expressed TFs comprise the AP2/EREBP, WRKY and the bHLH. There are 81 differentially expressed TFs when the symptoms of flower senescence become visible with the most prevailing being the WRKY family with 19 unigenes. No other WRKY TFs had been identified up to now in petal senescence of ethylene insensitive species. A large number of differentially expressed genes were identified at the initiation of visible symptoms of senescence compared to the open flower stage indicating a significant shift in the expression profiles which might be coordinated by up-regulated and/or down-regulated TFs. The expression of 16 genes that belong to the TF families of WRKY, bHLH and the ethylene sensing pathway was validated using qRT--PCR. CONCLUSION: This de novo transcriptome analysis resulted in the identification of TFs with specific temporal expression patterns such as two WRKYs and one bHLH, which might play the role of senescence progression regulators. Further research is required to investigate their role in gardenia flowers in order to develop tools to delay petal senescence.

Desmoid tumor of the abdominal wall: a case report.

INTRODUCTION: Desmoid tumors are rare lesions without any metastatic potential but a strong tendency to invade locally and to recur. These tumors are associated with women of fertile age, especially during and after pregnancy. CASE PRESENTATION: The case of a desmoid tumor of the anterior abdominal wall in a 40-year-old Caucasian man with no relevant family history is presented, describing its appearance on computed tomography and ultrasonography. The patient, who presented with a painless mass in the left anterolateral abdomen, had a history of previous urgent abdominal surgery after a shotgun injury two years earlier. Radical resection of the affected abdominal wall musculature was performed, and the defect was reconstructed with polypropylene mesh. CONCLUSION: The diagnosis of desmoid tumor should be strongly considered even in male patients with an abdominal mass and a history of previous abdominal surgery. The goal of its treatment is complete tumor excision and avoidance of the development of complications such as hernia.

Association of POAG risk factors and the Thr377Met MYOC mutation in an isolated Greek population.

PURPOSE: To characterize the MYOC genotype correlation with phenotypes in an isolated Greek population with a high incidence of glaucoma. METHODS: Five hundred thirty-one villagers were enrolled in the study. Participants underwent a comprehensive ophthalmic examination. All three exons of myocilin were bidirectionally sequenced. Power calculations and measured genotype analysis was conducted using the genetic variance analysis program, SOLAR version 4.2, to account for the relatedness between individuals. RESULTS: The participants, 376 of whom were linked in a single 11-generation pedigree, ranged in age from 10 to 95 years with a mean age of 49. Sixty-five individuals had POAG, and 27 of those carried the Thr377Met MYOC mutation. Both peak intraocular pressure and vertical cup-to dis- ratio were significantly associated with the MYOC Thr377Met variant (P = 9 x 10(-14) and P = 9 x 10(-8), respectively), whereas central corneal thickness showed no significant association (P < 0.7). CONCLUSIONS: This village had a high frequency of glaucoma, with 12% of the participants aged 10 to 95 years having the disease. In this cohort, the Thr377Met MYOC mutation was significantly associated with both high intraocular pressures and high vertical cup-to-disc ratios. No association was found with central corneal thickness.

Ectopic Liver Tissue Attached to the Gallbladder Wall: a case report.

INTRODUCTION: Ectopic liver tissue is a rare entity, reported to occur in several intra-, retro- and extra- peritoneal sites, including the gallbladder. It is usually detected incidentally, during laparoscopy, laparotomy, or autopsy. Several possible mechanisms may explain the development of liver ectopia. Although ectopic liver tissue is usually asymptomatic, it behaves like orthotopic liver, developing the same pathologic conditions. CASE PRESENTATION: We describe the case of a 54-year-old woman who was found to have a nodule attached to the gallbladder wall without any connection with the main liver, during an elective laparoscopic cholecystectomy for gallstone disease. The nodule was removed with the gallbladder and identified histologically as normal ectopic liver tissue. CONCLUSION: It would seem sensible to resect the ectopic tissue if encountered during cholecystectomy for gallstones. Laparoscopic management of ectopic liver can be feasible.

Myocilin variations and familial glaucoma in Taxiarchis, a small Greek village.

PURPOSE: To initiate a prospective study of glaucoma in a Greek village reported over 30 years ago to have several large families with primary open-angle glaucoma (POAG). METHODS: A random group of 126 villagers from Taxiarchis, Greece was examined in the village community center. The detailed evaluation included ophthalmic and general history, measurement of blood pressure, intraocular pressure (IOP), and central corneal thickness (CCT) as well as evaluation of the optic nerve status. RESULTS: The incidence of glaucoma approached 18% in this small isolated village. Myocilin variants were present in almost half of the individuals screened with Arg76Lys and Thr377Met being the most common finding (25% and 17%, respectively). Over half of the individuals with the Thr377Met mutation were diagnosed with glaucoma. Two of these patients were homozygous for the Thr377Met mutation. Three individuals with the Arg76Lys polymorphism had glaucoma; however, two of these individuals also had the Thr377Met mutation. Only two patients with pseudoexfoliation were identified. CONCLUSIONS: The incidence of glaucoma and the Thr377Met MYOC mutation in this population is much higher than that reported for other European populations.

24-Hour control with a latanoprost-timolol fixed combination vs timolol alone.

OBJECTIVE: To evaluate 24-hour intraocular pressure (IOP) control with an evening-dosed latanoprost-timolol maleate fixed combination vs timolol alone in patients with primary open-angle glaucoma. METHODS: After a medicine-free period, qualified patients were randomized to either placebo dosed in the morning with a latanoprost-timolol fixed combination dosed in the evening or timolol alone dosed twice daily for 8 weeks. Patients were then switched to the opposite treatment for 8 weeks. At baseline and at the end of each treatment period, patients underwent IOP measurements. RESULTS: Both treatments reduced the IOP from untreated baseline at each time point and for the 24-hour curve (P<.001). When treatments were compared, the latanoprost-timolol fixed combination decreased the IOP more than timolol alone at each time point and for the 24-hour curve (2.9 mm Hg), and provided a lower absolute IOP at each time point (P<.001) and for the range (fluctuation) in IOP (P = .003) and for the 24-hour curve. Several adverse effects were observed more often with the latanoprost-timolol fixed combination, including ocular stinging (P = .05), conjunctival hyperemia (P = .02), and ocular itching (P = .04). CONCLUSION: The evening-dosed latanoprost-timolol fixed combination may provide better IOP control than timolol alone over 24 hours and may demonstrate a narrower range of IOP fluctuation in patients with primary open-angle glaucoma.

Peroxidases, lignin and anatomy during in vitro and ex vitro rooting of gardenia (Gardenia jasminoides Ellis) microshoots.

In vitro and ex vitro rooting of gardenia (Gardenia jasminoides Ellis) microshoots with or without indolic-3-butyric acid (IBA) was studied in order to improve acclimatization of microplants after root formation and transplantation. Peroxidase (POD) activity and isoforms, lignin content and anatomical observations were evaluated in the course of the three interdependent phases (induction, initiation and expression) of microshoot rooting. Microshoots treated or not treated with IBA achieved high rooting percentages both in vitro and ex vitro. At the end of the 2-week acclimatization period, the percentage of surviving microplants ranged from 80% to 100%, for in vitro and ex vitro rooted microshoots, respectively. Microshoots rooted in vitro and ex vitro showed a relationship between rooting and POD activity but in a different time course. It appeared that root formation occurred after the microshoots had reached and passed a peak of maximum enzyme activity. In all treatments, electrophoretic analysis (native PAGE) of PODs revealed the appearance of one anionic and three cationic POD isoforms (C(1), C(3) and C(4)). An additional cationic POD isoform (C(2)) appeared only in the ex vitro rooting. The lignin content was similar in microshoots rooted both in vitro and ex vitro. The sequential anatomical changes during the rooting process were similar in both in vitro and ex vitro rooting treatments. In the case of in vitro rooting, pith cells had vacuoles entirely filled with a dark substance, while in the case of ex vitro rooting, pith cells contained many amyloplasts. The origin of the adventitious roots, in both rooting conditions, was located in the cambial ring. Roots with organized tissue systems emerged from the microshoot stem 10-14 days after the root induction treatments; on day 10 for rooting in vitro, while a 4-day delay was noted in microshoots rooted ex vitro.

Protein, leucine aminopeptidase, esterase, acid phosphatase and photosynthetic responses of oleander (Nerium oleander L.) during cold acclimation and freezing treatments.

Six-month-old oleander (Nerium oleander L.) pot plants, derived from vegetative propagation by cuttings, were tested for their ability to cold hardening. Damage of the non-acclimated (NA) plants was visible when treated by low freezing temperatures (below -2 degrees C). The responses of total proteins, leucine aminopeptidase (LAP), esterase (EST) and acid phosphatase (ACP) isoforms of NA and cold-acclimated (CA; 4 degrees C for 14 days) plants were compared using polyacrylamide gel electrophoresis. These molecular markers were also compared in NA and CA plants which received for 2h temperatures of 0, -2, -4, -6 and -8 degrees C. A new 38-kDa polypeptide appeared from day 7 to 14 during the acclimation treatment in the bark extracts and on day 14 in the leaf extracts. The above-mentioned polypeptide band (38 kDa) strongly appeared in all freezing treatments (0, -2, -4, -6 and -8 degrees C) in both bark and leaf extracts of the CA plants. Alterations in the number and the intensity of LAP and EST isoforms as well as in the intensity of ACP isoforms were observed in both bark and leaf of the CA oleander plants. A newly expressed EST isoform is proposed as biochemical marker for the cold acclimation treatment. CO2 assimilation rates (A) as well as transpiration rates (E) in NA plants were positive in 0 degrees C and negative in all temperatures below zero in the freezing treatments. In contrast, CO2 assimilation rates (A) and transpiration rates (E) were positive in CA plants in all temperatures of freezing treatment. A significant decrease (P<0.05) in chlorophyll (Chl) a, Chl a+b concentration and Chl a/b ratio were noticed in oleander plants during the acclimation treatment (from day 0 to 14), while Chl b concentration was unchanged at the respective time. On the other hand, no significant (P<0.05) differences were observed in the freezing treatments.

Dissipation of three organochlorine and four pyrethroid pesticides sprayed in a greenhouse environment during hydroponic cultivation of gerbera.

A wide range of pesticides (insecticides, acaricides and fungicides) is used in the hydroponic cultivation of gerbera. The fate and behaviour of some of these commonly used pesticides (organochlorine and pyrethroid insecticides/acaricides) in a greenhouse installation was investigated. The concentrations of endosulfan, dicofol, tetradifon, permethrin, bifenthrin, cypermethrin and deltamethrin in the air of the greenhouse were related to their physicochemical properties and respective application rates. Of these compounds, endosulfan exhibited the highest concentration in the greenhouse air 2 h after application (10 microg m(-3)), due to its highest volatility; afterwards, up to 12 h post-application, a rapid decrease was recorded which finally led to a concentration of 0.41 microg m(-3) at 6 days post-application. Dicofol exhibited the same trend of dissipation in the greenhouse air; its concentration decreased from 0.27 microg m(-3) immediately after the application to 0.12 microg m(-3) at 6 days post-application. Tetradifon, bifenthrin and permethrin were found at concentrations lower than 0.1 microg m(-3) at 2 h post-application, and cypermethrin and deltamethrin could not be detected. The concentrations of all the above pesticides in the drain water of the open hydroponic system were relatively high immediately after application and decreased rapidly during the following 3 days. In the closed hydroponic system, all the pesticides accumulated in the nutrient solution during the first 24 h after application and slowly decreased in concentration over the following 3 days. The spray operators applying these pesticides were exposed to rather high concentrations and the spray deposits were monitored on the chest and forehead of the operator. Endosulfan exhibited the highest concentrations of the seven pesticides. The organochlorine pesticides (endosulfan, dicofol and tetradifon) were usually detected at higher concentrations on the chest than on the forehead of the operator; pyrethroids were detected at higher concentrations on the forehead than on the chest of the operator.

Activity and isoforms of peroxidases, lignin and anatomy, during adventitious rooting in cuttings of Ebenus cretica L.

Adventitious rooting of Ebenus cretica cuttings was studied in order to examine a) the rooting ability of different genotypes in relation to electrophoretic patterns of peroxidases. b) the activity and electrophoretic patterns of soluble and wall ionically bound peroxidases, the lignin content and anatomical changes in the control and IBA treated cuttings of and genotypes in the course of adventitious root formation. In addition, a fraction of soluble cationic peroxidases was separated by gel filtration chromatography from the total soluble peroxidases of a genotype. No rooting occurred in cuttings without IBA-treatment. In both genotypes, electrophoretic patterns of soluble anionic peroxidases revealed two common peroxidase isoforms, while a fast-migrating anionic peroxidase isoform (A3) appeared only in genotypes. Both genotypes showed similar patterns of soluble, as well as wall ionically bound cationic peroxidase isoforms. The number of isoforms was unchanged during the rooting process (induction, initiation and expression phase) but an increase in peroxidase activity (initiation phase) followed by decrease has been found in IBA-treated cuttings. During initiation phase the lignin content was almost similar to that on day 0 in genotype while it was reduced at by about 50% in genotype at the respective time. Microscopic observations revealed anatomical differences between genotypes. According to this study, the and genotypes display differences in anatomy, lignin content, activity of soluble peroxidases and the electrophoretic patterns of soluble anionic peroxidase isoforms. The A3-anionic peroxidase isoform could be used as biochemical marker to distinguish and genotypes of E. cretica and seems to be correlated to lignin synthesis in rooting process.