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Study of genetic transformation efficiency via organogenesis and embryogenesis in eggplant (Solanum melongena L. cv. Embti): effects of co-culture, temperature and kanamycin and hygromycin-based selection procedures
15-23.Views:359The effects of kanamycin and hygromycin-based selection and co-culture temperature ranging from 22 to 28 °C upon eggplant transformation efficiency were evaluated. Both morphogenic pathways, somatic embryogenesis and organogenesis, were adopted using cotiledonary and hypocotyl explants, respectively. Somatic embryos were recovered in the presence of both antibiotics, although lesser escapes were observed in hygromycin-supplemented medium. Indeed, selection provided by this antibiotic was more efficient compared to kanamycin, nevertheless, shoot regeneration was not observed with hygromycin. Significant difference on the frequency of cotiledonary explants displaying callus (FEC) was observed as embryogenesis was concerned, although a higher number of embryos was observed in hygromycin selective media. The frequency of explants presenting callus (FEC), embryos (FEE) and shoots or buds (FERG) did not differ statistically for the tested co-culture temperatures, although higher regenerant number was observed at 24 °C.
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Aminoglycoside antibiotics affect the in vitro morphogenic response of chrysanthemum and tobacco
93-104.Views:186Broadly the success of genetic transformation of plants requires non-chimeric selection of transformed tissues and its subsequent regeneration. With rare exceptions, most plant transformation protocols still heavily utilize antibiotics for the selection of transgenic cells containing an antibiotic-degrading selectable marker gene. The morphogenic capacity of in vitro chrysanthemum and tobacco stem and leaf explants change with the addition of aminoglycoside antibiotics (AAs). Of 6 antibiotics tested, phytotoxicity occurred at 10-25 and 50-100 pgml-I in chrysanthemum and tobacco explants, respectively, depending on the size of the explant and the timing of application. The presence of light or darkness also had a significant effect. The use of transverse thin cell layers (tTCLs) in conjunction with high initial AA selection levels supported the greatest regeneration of transgenic material (adventitious shoots or callus) and the lowest number of escapes. Flow cytometric analyses demonstrate that regeneration can be predicted in both species, depending on the ploidy level of the callus. Endoreduplication was not observed in chrysanthemum, even at high AA levels, but occurred (8C or more) in tobacco callus, even at low AA concentrations (5-10 pgml-1). The higher the AA level, the greater the DNA degradation and the lower the 2C and 4C values.
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Rhizogenesis in in vitro shoot cultures of passion fruit (Passiflora edulis f. flavicarpa Deg.) is affected by ethylene precursor and by inhibitors
47-54.Views:263The effects of the ethylene precursor ACC and two inhibitors, AgNO3 and AVG, on root formation were tested in in vitro shoots of passion fruit (Passiflora Midis f.flavicalpa Deg.). The organogenic response was assessed on the basis of percentage of shoot-forming. roots, root number and length. The time course of ethylene production was also monitored. ACC inhibited root formation by delaying root emergence and increasine, callus formation at the basis of the shoots. In addition, ACC caused a marked increase in ethylene production, coupled to leaf chlorosis and senescence with lower rooting frequencies, number and length of roots. IAA supplementation increased ethylene production. Both ethylene inhibitors, AgNO3 and AVG, at appropriate concentrations reduced callus formation at the basis of shoots. AVG increased the number of roots per shoot, but drastically reduced length of differentiated roots. Regarding to leaf pigments, ACC promoted a marked reduction on carotenoids and total chlorophyll, whereas AVG and AgNO3 delayed explant senescence and pigments degradation, not differing from IAA supplemented and non-supplemented control treatments. The results confirm previous reports on the beneficial effects of ethylene inhibitors on in vitro rooting and suggest its reliability to be used as an alternative approach to evaluate sensitivity of Passiflora species to ethylene.
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Linking bark anatomy to Eucalyptus Physiological Disorder (EPD) in commercial clones
73-87.Views:380Abiotic stresses trigger the Eucalyptus Physiological Disorder (EPD) which poses a threat to planted and native stands. This research seeks links between eucalyptus bark histological features and EPD, in which the descriptive bark anatomy and histochemistry are approached. Barks from 5-year eucalyptus trees, from commercial clones of E. grandis, E. urophylla and its hybrids, were collected at breast height (DBH), and 50% and 75% of the commercial height, and evaluated. The eucalyptus bark consisted of a periderm (or rhytidome) and a secondary phloem with conspicuous solitary sieve tube elements (STE). The outer bark revealed a secondary phloem with collapsed STE, whereas its inner counterpart displayed non-collapsed STEs. A region crowded with calcium oxalate (CaOx) crystals in axial parenchyma, covering the non-collapsed and partially overlapped collapsed secondary phloem, was observed. Eucalyptus barks exhibited similar anatomical organization at DBH, 50% and 75% of the commercial height, irrespective of expected EPD phenotype or scores. Notwithstanding, there are qualitative differences that are associated with the proportion of non-collapsed phloem and phloem with crystals, which were higher in the tolerant clones and in trees with score 0. The more resistant clones or samples with lower EPD scores exhibited a higher proportion of the regions of living phloem, phloem with CaOx crystals, and non-collapsed phloem. These results support the hypothesis that an increased proportion of STE collapse will occur concurrently with elevated EPD scores and are the basis for an ongoing histometric approach.
