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  • 1
    ISSN: 1871-4528
    Keywords: epigenetic variation ; leaf ontogeny ; somaclonal variation ; tissue culture
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Summary Potato leaf morphology changes during plant development with the phase shift from vegetative growth to flowering. Image analysis can detect differences in leaf morphology and has been used here to distinguish differences in leaf morphology between potato crops derived from seed tubers and minitubers and between crops derived from different micropropagation protocols. Further, leaf shape parameters can be used to determine the relative maturity of crops. This finding is of economic importance since differences in plant development, for example delayed flowering, are associated with yield parameters. It is hypothesised that image analysis of established microplants can be used as an early evaluation of micropropagation protocols for potato.
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  • 2
    ISSN: 1573-9368
    Keywords: Bacillus thuringiensis ; Borer disease ; Saccharum ; somaclonal variation ; transgenic sugarcane
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A population of 42 transgenic sugarcane ( hybrid, cv. Ja60‐5) clones expressing a truncated cryIA(b) gene from Bacillus thuringiensis was evaluated in field trials under artificial borer (Diatraea saccharalis Fab.) infection. Five clones displaying the highest borer tolerance were selected and analysed with molecular tools (RAPD, AFLP and RAMP) to verify genomic changes. Results of field trials provided evidence both for the expression of the resistance trait and for the occurrence of limited but consistent morphological, physiological and phytopathological variation, as compared with control plants regenerated from dedifferentiated culture without transformation (C1‐control) or with plants that were clonally propagated in the field (C2‐control). The five elite transgenic clones, selected for consistent borer‐resistance and good agronomic traits, were further evaluated in a large scale field trial. It was found that the majority of agronomic and industrial traits were those of the original cv. Ja60‐5, but that a small number of qualitative traits was different. DNA changes were verified in the five selected clones. A total of 51 polymorphic DNA bands (out of the 1237 analysed bands) was identified by extensive AFLP and RAMP analysis, thus showing rare but consistent genomic changes in the transgenic plants, as compared with C1‐ and C2‐control plants. It is proposed that the increased variability verified in transgenic plants by field trials and DNA analysis is essentially correlated with cell growth in the dedifferentiated state during the transformation procedure. The results, which are consistent with those published in the case of other transgenic plant populations, are discussed in the context of selecting approaches to gene transfer that minimize somaclonal variation. This is important especially in cases, such as that of sugarcane, where success of backcrosses to restore the original genotype is made difficult by the complex ploidy state of the plant.
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  • 3
    Electronic Resource
    Electronic Resource
    Springer
    Molecular breeding 5 (1999), S. 11-20 
    ISSN: 1572-9788
    Keywords: plant breeding ; protoplasts ; somatic fusion ; ribosomal RNA genes
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract Ribosomal RNA genes were exploited as markers to identify somatic hybrids between Solanum tuberosum cv. Brodick and wild diploid Solanum species, S. megistacrolobum, S. sanctae-rosae and S. sparsipilum and DNA methylation as a possible regulatory factor in gene expression was investigated. Specific restriction enzyme/probe combinations revealed useful polymorphisms in the conserved coding and variable intergenic spacer regions of the ribosomal RNA genes. Some intermediate ribosomal RNA gene profiles indicate hybridity whereas others were characteristic of S. tuberosum cv. Brodick. This evidence is suggestive of somatic exchange/re-arrangement between the NOR region of S. sanctae-rosae and S. tuberosum cv. Brodick. Ribosomal RNA gene copy number analysis of the somatic hybrids did not reveal hexaploid values suggesting that these products are not symmetric hybrids derived from the parental diploid and tetraploid plants. The results indicate site-specific methylation of ribosomal RNA gene sequences for the parental plants; while some somatic hybrids display a reduction, others show an increase. The significance of the findings for somatic cell genetics and plant breeding studies is discussed.
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  • 4
    ISSN: 1573-5060
    Keywords: rice ; Oryza sativa L. ; photoperiod sensitive genic male sterility (PGMS) ; protoplasts ; flow cytometry ; tetraploid
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract Plants were regenerated from protoplasts isolated from embryonic suspension cultures of N5047S, a photoperiod sensitive genic male sterile (PGMS) Japonica rice line. Flow cytometric analyses of nuclear DNA content identified some tetraploid regenerates whose agronomic traits could be distinguished from diploid regenerates. Pollen and female fertility of diploid protoplast-derived clones grown under different light and temperature conditions was compared. A promising PGMS protoplast clone, ZAU11S, was developed from these clones. Its male sterility was confirmed as a photoperiod × temperature interaction type.
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  • 5
    ISSN: 1573-5028
    Keywords: heartwood constituent ; phytoalexin ; pinosylvin ; Pinus sylvestris ; protoplasts ; stilbene synthase ; transient expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Young pine seedlings respond to environmental stress by induced synthesis of pinosylvin, a stilbene phytoalexin. Heartwood of pine trees is characterized by a high content of pinosylvin. The formation of pinosylvin from cinnamoyl-CoA and three molecules malonyl-CoA catalysed by pinosylvin synthase is typical of the genus Pinus. Its enzyme activity not detectable in unstressed seedlings is substantially increased upon application of stimuli like UV-light or infection with the phytopathogenic fungus Botrytis cinerea. A genomic DNA library was screened with pinosylvin synthase cDNA pSP-54 as a probe. Ten clones were isolated and grouped into five subclasses according to the size of their introns. After subcloning into plasmid T7T3, four different members of the five gene subclasses were characterized by sequencing. Emphasis was put on isolating various promoters and analyzing and comparing their responsiveness. The amino acid sequences deduced from genes PST-1, PST-2, PST-3 and PST-5 shared an overall identity of more than 95%. In gene PST-5, the putative translation start site ATG was replaced by CTG. While promoter regions near the TATAA box were almost identical PST-1, PST-2 and PST-3, further upstream sequences differed substantially. Differences in promoter strength were analysed both in transgenic tobacco plants and by transient expression in tobacco protoplasts. Constructs used contained the bacterial β-glucuronidase under the control of the promoters of pine genes PST-1, PST-2 and PST-3. Upon treatment with UV light or fungal elicitor, the promoter of PST-1 showed highest responsiveness and led to tissue-specific expression in vascular bundles. The data suggest that in pine the gene product of PST-1 is responsible for both the stress response in seedlings and pinosylvin formation in the heartwood.
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  • 6
    ISSN: 1573-5044
    Keywords: adventive somatic embryo ; cell culture ; protoplasts ; regeneration
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Adventive somatic embryos were initiated from the cut edges of juvenile leaf explants of two cultivars of sugarcane (Saccharum spp. hybrid cv. CoL-54 and cv. CP-43/33). This response was achieved using MS medium containing 9 μmol (2 mg l-1) 2,4-D and 500 mg l-1 CH under either continuous or 16-h photoperiod. Regeneration from somatic embryos was achieved under either continuous or 16-h photoperiod on MS basal medium in 5–6 weeks. Using adventive somatic embryos of 20–25 days of age as an explant source, homogeneous cell suspension cultures were initiated in both AA and MS media supplemented with 2 mg l-1 2,4-D and 500 mg l-1 CH. Protoplasts were isolated from homogeneous cell suspension cultures, an average yield being 2.5×107 ml-1 for both the cultivars. The best division efficiency (1.5 and 0.80%) and microcalluses for cv. CoL-54 and cv. CP-43/33, respectively were achieved using modified KPR medium under dark conditions in 6–8 weeks. Microcalluses were proliferated and plant regeneration was achieved from protocalluses.
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  • 7
    Electronic Resource
    Electronic Resource
    Springer
    Plant cell, tissue and organ culture 58 (1999), S. 159-162 
    ISSN: 1573-5044
    Keywords: agronomic traits ; Pennisetum glaucum ; Sclerospora graminicola ; somaclonal variation ; tissue culture
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Plants regenerated from seed-derived callus of a PNMS 6B line of pearl millet (Pennisetum glaucum (L.) R. Br.) were evaluated for their resistance induced by somaclonal variation for downy mildew disease caused by Sclerospora graminicola (Sacc.) Schroter. Among the 201 lines regenerated, only 3 lines consistently proved highly resistant (free from disease incidence) for up to 5 generations; whereas, 17 lines were resistant (disease incidence ranging from 1 to 9%). Resistance was confirmed by testing the plants under both laboratory and field conditions. The plants were evaluated for their agronomic traits.
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  • 8
    Electronic Resource
    Electronic Resource
    Springer
    Plant cell, tissue and organ culture 58 (1999), S. 199-203 
    ISSN: 1573-5044
    Keywords: triploid watermelon ; seedless watermelon ; tetraploid watermelon ; plant breeding ; somaclonal variation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Ploidy of watermelon [Citrullus lanatus (Thunb.) Matsum. and Nakai shoots and plantlets was estimated by painting the lower epidermis of intact in vitro-derived leaves with fluorescein diacetate (FDA) and observing fluorescence of guard cell chloroplasts with a microscope and UV light. Leaves from in vitro shoot-tip cultures of known diploid cultivars and tetraploid breeding lines were used to establish the mean number of chloroplasts per guard cell pair. Leaves from diploid and tetraploid shoot cultures had 9.7 and 17.8 chloroplasts per guard cell pair, respectively. This method then was used to estimate the ploidy of shoots regenerated from cotyledon explants of the diploid cultivar Minilee. Approximately 11% of the 188 regenerated shoots were classified as tetraploid during in vitro culture. Putative tetraploids were transplanted to the field and self-pollinated. About 45% of tetraploids identified in vitro produced fruit and viable seed. Chloroplast counts of R1 progeny were used to confirm their ploidy. All of the putative diploids were confirmed diploid and all putative tetraploids proved to be non-chimeric true breeding tetraploids.
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  • 9
    ISSN: 1573-5060
    Keywords: Erwinia carotovora ; Solanum tuberosum ; somaclonal variation ; RAPD
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract Three somatic hybrid lines between potato (cv. While Lady line no. Ke 79, 2n = 2x = 48) + Solanum brevidens (PI 218228, 2n = 2x = 24) were evaluated using randomly amplified polymorphic DNA (RAPD) markers. The lines originated from the same callus but showed different reactions to Erwinia carotovora ssp. carotovora, the cause of potato soft rot. By the use of 48 oligomer primers producing 99 scorable bands, DNA polymorphism were detected on 7 of 12 S. brevidens chromosomes. Loss of certain DNA segments on chromosome 5, 6, 9 and 11 were observed. Some of the variations could have taken place in early callus stage of development; others may have occurred after initiation of individual shoot regeneration. The possible involvement of missing RAPD products specific to one somatic hybrid that shows decreased resistance to bacterial soft rot is discussed.
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  • 10
    ISSN: 1573-5044
    Keywords: Na+ tolerance ; plant regeneration ; salt stress ; Solanum tuberosum ; somaclonal variation ; RAPDs
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A stable salt-tolerant potato cell line, able to grow on media containing 60–450 mM NaCl (i.e. low to high salinity) was selected. Callus grown on 120 or 150 mM NaCl showed higher fresh weights than the rest of the treatments. Replacing NaCl by KCl or Na2SO4 showed that reductions in fresh weight were mainly due to the presence of Na+ ions. When PEG 6000 was added to the medium instead of salt, the salt tolerant cell lines were unable to overcome the PEG-induced water stress. Whole plants, regenerated from salt tolerant callus, exhibited salt stress tolerance as evidenced by their higher fresh and dry weights when watered with 90 mM NaCl, and they also produced more tubers per plant under salt stress. Salt-tolerant plants differed phenotypically from control plants both in terms of leaf shape, tuber flesh and skin colour, which was reddish. In addition, DNA fingerprinting by RAPDs, with 70 different primers, confirmed that the salt tolerant regenerants also differed genotypically from the control, salt sensitive Kennebec potato plants from which they had been selected.
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  • 11
    ISSN: 1573-5060
    Keywords: Cucumis sativus L. ; rDNA ; regeneration systems ; somaclonal variation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract Somaclonal variation in the Borszczagowski line of Cucumis sativus L. was determined for five regeneration systems: micropropagation (MP), direct leaf callus regeneration (DLR), leaf callus regeneration (LCR), recurrent leaf callus regeneration (RLCR), and direct protoplast regeneration (DPR). The frequency at which new phenotypes appeared in R1 lines and the stability of the rDNA region analysed using of five probes were investigated. MP was not subject to change, while DLR caused only infrequent changes. The highest frequency of change arose through DPR (90% of lines) and RLCR (42.8%), as opposed to 5.9% with LCR. Tetraploids were produced only in the case of LCR (4.7%) and RLCR (28%).
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  • 12
    ISSN: 1573-5060
    Keywords: Tissue culture ; somaclonal variation ; Triticum aestivum L. ; plant breeding
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract Somaclones (R3 and R4 generations) regenerated from five winter wheat (Triticum aestivum L.) genotypes were evaluated for variation in agronomic and morphological characters. Immature embryos were used as initial explant material. Comparisons for plant height, top internode length, spike length, number of seeds per spike and 100 seed weight were made between the somaclones and their parents. Some morphological variations of stem and spike characteristics were registered which demonstrate that plant height and spike length can be changed by using immature embryo culture. The results obtained may be considered a biotechnological contribution to wheat plant improvement.
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  • 13
    Electronic Resource
    Electronic Resource
    Springer
    Plant cell, tissue and organ culture 55 (1998), S. 151-154 
    ISSN: 1573-5044
    Keywords: protoplast ; somaclonal variation ; somatic embryo ; tissue culture ; Umbelliferae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Hypocotyl segments of Bupleurum falcatum L. formed embryogenic calluses when cultured on Murashige and Skoog's (MS) medium supplemented with 9.0 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Suspension cultures were initiated by placing calluses into medium with 0.45 μM 2,4-D. Protoplasts were enzymatically isolated from suspension cultures. They were plated at a density of 5 × 104 protoplasts per ml on MS medium supplemented with 9% mannitol, 9.0 μM 2,4-D, 4.4 μM BA, 4.6 μM kinetin, and 0.6% Seaplaque agarose. After four weeks of culture, microcalluses were formed and subsequently transferred to MS solid medium with 18.1 μM 2,4-D. Upon transfer to MS basal medium, microcalluses gave rise to somatic embryos at a frequency of approximately 10%. They subsequently developed into plantlets. The regenerants were successfully transplanted to potting soil and grown to maturity in a greenhouse. The regenerants had the normal chromosome number of 2n=2x=20 and did not show morphological aberrancy.
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  • 14
    ISSN: 1573-5087
    Keywords: chilling tolerance ; fatty acids ; galactolipids ; phospholipids ; rice ; somaclonal variation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract The relationship between chilling tolerance of six rice cultivars – Facagro 57, Facagro 76, Fujisaka 5, Kirundo 3, Kirundo 9 and IR64 -and the fatty acid composition in total lipids, phospholipids, galactolipids and neutral lipids from leaves was studied. Higher double bond index and proportions of linolenic acid in the phospholipid and galactolipid classes were related to cultivar chilling tolerance, but this was not so for the total lipids nor the neutral lipid class. The somaclonal families derived from Facagro 76, Kirundo 3 and Kirundo 9 that showed enhanced chilling tolerance as compared to their original parental cultivar were analyzed for fatty acid composition in phospholipids and galactolipids from leaves. Altered proportions in fatty acid composition in phospholipids, galactolipids or both were found in the somaclonal families derived from Facagro 76 and Kirundo 9, but not from Kirundo 3. These changes most usually resulted in higher double bond index and higher proportions in linoleic and linolenic acids which were related either to lower ratio of C16 to C18 fatty acids or to higher unsaturation in the C18 fatty acid fraction. Different mechanisms thus seem to be implicated in the altered fatty acid composition of somaclones, which may be related to the chilling tolerance improvement of some somaclonal families.
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  • 15
    ISSN: 1573-5079
    Keywords: C4 photosynthesis ; enzyme kinetics ; protein phosphorylation ; protoplasts
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Kinetic analyses were performed on the nonphosphorylated and in vitro phosphorylated forms of recombinant Sorghum C4 phospho enolpyruvate carboxylase (C4 PEPC). The native enzyme was purified by immunoaffinity chromatography and its integrity demonstrated by Western blot analyses using anti N- and C-terminus antibodies. At suboptimal pH (7.1 to 7.3) and PEP concentration (2.5 mM), phosphorylation, positive metabolite effectors e.g., glucose-6-phosphate, glycine and dihydroxyacetone-phosphate, or an increase in pH strongly activated the enzyme and lowered the inhibitory effect of L-malate. C4 PEPC phosphorylation strengthened the effect of the positive effectors thereby decreasing further the enzyme's sensitivity to this inhibitor. L-malate also decreased the phosphorylation rate of C4 PEPC, a process antagonized by positive metabolite effectors. This was shown both in vitro, in a reconstituted phosphorylation assay containing the catalytic subunit of a cAMP-dependent protein kinase or the Sorghum leaf PEPC-PK and in situ, during induction of C4 PEPC phosphorylation in mesophyll cell protoplasts.
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  • 16
    ISSN: 1573-5060
    Keywords: Malus ; somaclonal variation ; tissue culture ; in vitro
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract To assess somaclonal variation, ‘Gala’ and ‘Royal Gala’ trees obtained via axillary and adventitious bud formation were compared ex vitro to conventionally grafted trees. In general, tissue culture-derived trees were relatively erect in comparison to grafted trees. Their branch angles were narrower than those of grafted trees. All trees that flowered had pink blossoms. There were no obvious differences in flowering time or in floral morphology. Most of the seven-year-old grafted control trees produced more fruits than either axillary or regenerated trees. Although there were differences in the range of fruit color between ‘Royal Gala’ and ‘Gala’ apples in both the control and tissue culture-derived plants (the fruits of ‘Royal Gala’ were darker red and more striped than those of ‘Gala’) and also in the degree of pigmentation from tree-to-tree, none of the variation exceeded that observed among apples harvested from an individual ‘Royal Gala’ or ‘Gala’ control tree for either the plants derived from axillary buds or adventitiously. Since both ‘Gala’ and ‘Royal Gala’ axillary buds showed very little somaclonal variation for the morphological and reproductive traits we studied, it appears that tissue culture may be a useful way to propagate these cultivars.
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  • 17
    ISSN: 1573-5060
    Keywords: apple ; fire blight ; resistance ; somaclonal variation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract Four somaclonal variants regenerated from adventitious buds of the apple variety Greensleeves were preselected on the basis of their reduced fire blight susceptibility. The present study aimed at assessing precisely their level of fire blight resistance through various inoculation techniques (on in vitro leaves and microcuttings, on greenhouse plants and in field conditions). Overall results of these tests indicated that one clone (R 46/3) was clearly less susceptible than the control. This clone was also characterized as a ‘spur’ variant, with a reduced growth which can explain its limited susceptibility to fire blight. A second clone (R 20/63) was slightly less susceptible than the control in greenhouse and field tests, but this low level of resistance was overcome by high concentrations of inoculum. The absence of variation in chromosome number and isozyme patterns confirmed the genetic trueness-to-type of these four somaclones.
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  • 18
    Electronic Resource
    Electronic Resource
    Springer
    Plant growth regulation 25 (1998), S. 135-141 
    ISSN: 1573-5087
    Keywords: ABA synthesis ; Amaranthus ; fluridone ; osmotic stress ; protoplasts ; rose petal
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract All the protoplasts analysed in this study whatever the original plant material were able to accumulate ABA under osmotic stress. The time course of ABA accumulation strongly differed according to the plant material. In both rose petal or Amaranthus leaf protoplasts, the increase in ABA level was significant but transient. Protoplasts prepared from Amaranthus cell suspensions behaved differently, showing a late and durable accumulation of ABA. Similar patterns of changes in ABA accumulation were observed in the original plant material under osmotic stress. A pretreatment of plant material by fluridone induced a strong inhibition of ABA accumulation whatever the origin of protoplasts was. This result suggests that ABA could be synthesised via the carotenoid pathway in the absence of the cell wall.
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  • 19
    ISSN: 1432-203X
    Keywords: Oryza sativa ; rice ; somaclonal variation ; genetic integrity ; RAPD analysis ; DNA markers
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary RAPD analysis was performed among eight rice somaclonal families known to vary for specific characters and four somaclonal families which were phenotypically normal. The parental cultivar,indica rice cv. FR13A, was found to be homogeneous and homozygous at all but one of the 45 RAPD loci. Polymorphisms were found at 28 of the 45 bands among the somaclonal families, including both loss of parental bands, and the appearance of novel non-parental bands. Segregation data revealed both heterozygous and homozygous mutation events, with recessive mutations more prevalent than dominant. All somaclonal families differed significantly from the parental material, indicating that genomic alterations occurred in all families regardless of phenotype. None of the variant families could be regarded as isogenic lines of FR13A at the DNA level. However, some of the DNA level variation may be in highly repeated sequences with no phenotypic effects. The implications for somaclonal breeding and genetic engineering programs are discussed.
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  • 20
    Electronic Resource
    Electronic Resource
    Springer
    Biochemical genetics 35 (1997), S. 189-204 
    ISSN: 1573-4927
    Keywords: somaclonal variation ; tissue culture ; isozymes ; cactus ; genetic diversity
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract Morphological and isozyme variation was observed among plants regenerated from callus cultures of Cereus peruvianus. Different morphological types of shoots (68%) were observed in 4-year-old regenerated plants, while no distinct morphological variants were observed in plants grown from germinated seeds. Isozyme patterns of 633 plants regenerated from calli and of 261 plants grown from germinated seeds showed no variation in isocitrate dehydrogenase isozyme, and the differential sorbitol dehydrogenase, alcohol dehydrogenase, malate dehydrogenase, acid phosphatase, and peroxidase isozyme patterns observed in regenerated plants were attributed to nonallelic variation. Allelic variation was detected at three isoesterase loci. The proportion of polymorphic loci for both populations was 13.6% and the deviation from Hardy–Weinberg equilibrium for the Est-1 and Est-7 loci observed in somaclones was attributed to the manner in which the regenerant population was established. The high values for genetic identity among regenerant and seed-grown plant populations are in accordance with the low levels of interpopulation genetic divergence. In somaclones of C. peruvianus, morphological divergence was achieved within a short time but was not associated with any isozyme changes and also was not accompanied by biochemical genetic divergence.
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  • 21
    ISSN: 1573-5028
    Keywords: movement protein ; protoplasts ; resistance ; tomato spotted wilt virus (TSWV) ; tospovirus ; transgenic plants
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Transgenic Nicotiana tabacum plants expressing RNA sequences of the tomato spotted wilt virus NSM gene, which encodes the putative viral movement protein, were found to be highly resistant to infection with the virus. Expression of untranslatable as well as anti-sense RNA of the NSM gene resulted in resistance levels as high as those in plants expressing translatable RNA sequences. For all three types of transgenic plants resistance levels of up to 100% were reached in the S2 progeny. These results indicate that the resistance mediated by the NSM gene is accomplished by expression of transcripts rather than protein in transgenic plants, similar to previously observed N gene-mediated resistance. Protoplast inoculations revealed that resistant plants expressing NSM are, in contrast to N transgenic resistant plants, not resistant at the cellular level. This suggests the RNA-mediated resistance mechanism against TSWV targets viral mRNAs rather than the viral genome.
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  • 22
    Electronic Resource
    Electronic Resource
    Springer
    Plant molecular biology 35 (1997), S. 231-240 
    ISSN: 1573-5028
    Keywords: insertion mutation ; retrotransposon ; rice ; somaclonal variation ; transposable elements ; transposon tagging
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Retrotransposons were extensively surveyed in rice using two molecular methods. The total copy number of retrotransposons in the rice genome was estimated to be about 1000 and 32 families were isolated, showing that retrotransposons are a major class of transposable elements in rice. Although these retrotransposons appear inactive during normal growth conditions, 5 out of 32 families were active under tissue culture conditions. The most active element, Tos17, was studied in detail. Its activity was show to be regulated mainly at the transcriptional level. The analysis of target sites of transposition indicated that activation of Tos17 is an important cause of tissue culture-induced mutations in rice. Tissue culture-induced activation of Tos17 was used to develop the site-selected mutagenesis system, in which mutants carrying a Tos17 insertion in the gene of interest can be identified among rice plants regenerated from tissue culture by the PCR using one primer for the ends of Tos17 and another for the gene of interest. This system will contribute to understanding the functions of rice genes whose sequences are being determined by the rice genome project.
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  • 23
    Electronic Resource
    Electronic Resource
    Springer
    Plant and soil 192 (1997), S. 23-30 
    ISSN: 1573-5036
    Keywords: aluminium ; callose ; protoplasts ; resistance ; toxicity ; Zea mays
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract The screening of 37 Zea mays L. cultivars in nutrient solution using root elongation (24 h) as a parameter showed large genotypic differences in Al resistance among the genetic material evaluated. Callose concentrations in root tips were closely and positively related to Al-induced inhibition of root elongation. Therefore, Al-induced callose formation in root tips appears to be an excellent indicator of Al injury and can be used as a selection criteria for Al sensitivity. In contrast, aluminium concentrations in root tips were not related to Al-induced inhibition of root elongation, nor to Al-induced callose formation. Callose formation was also induced by short-term A1 treatment in root tip protoplasts, and the response of protoplasts clearly reflected the cultivar-specific response to Al of intact roots. This indicates that in maize, Al sensitivity is expressed on the protoplast level.
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  • 24
    Electronic Resource
    Electronic Resource
    Springer
    Plant cell, tissue and organ culture 51 (1997), S. 43-47 
    ISSN: 1573-5044
    Keywords: Allium cepa ; onion ; organogenesis ; protoplasts ; suspension culture
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Tissue culture techniques involving cell suspension, protoplast fusion and culture in the genus Allium are different as Allium is recalcitrant due to the biological peculiarity of the genus. A procedure is described for the establishment of a regenerable suspension culture and for the isolation and culture of protoplasts of Allium cepa.
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  • 25
    ISSN: 1573-5060
    Keywords: Oryza sativa ; japonica rice cultivar ; mature embryo ; primary calli ; protoplasts ; green plant regeneration
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract A plant regeneration system from rice protoplasts using calli derived from mature embryos was established for the two Brazilian modern rice cultivars IAC-201 and IAC-165. After 30 to 40 days of in vitro culture it was possible to obtain on average 6 million protoplasts per gram of callus. Microscopic selection of embryogenic calli was a key step for protoplast isolation. The production of embryogenic calli increased when L-proline and casein hydrolysate were used in the callus induction medium. The Oc or IR52 nurse cell lines were essential for protoplast division. Different regeneration media were studied and 139 plants were regenerated which set seed. Some of the regenerated plants showed morphological variation such as the presence of awns in spite of the short time of the in vitro culture.
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  • 26
    ISSN: 1573-5087
    Keywords: chloroplasts ; membrane dynamics ; Pisum ; photosymthesis ; protoplasts ; triacontanol
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract The effects of a long chain aliphatic alcohol 1-triacontanol (TRIA) on the photosynthesis and membrane properties of mesophyll protoplasts and chloroplasts isolated from pea leaves were studied. In vitro treatments of isolated protoplasts caused a large enhancement (166% ) of the CO2-fixation rate after 60 min of TRIA (10-6 M) application as compared to the control. An enhanced photosynthetic response was observed in in vitro treated leaf pieces. Application of octacosanol (OCTA) under the same experimental conditions did not result in any stimulating effects. In vivo treatments of pea seedlings also resulted in a significant increase of the net CO2 uptake to 109% and 119% in 10-8 M and 10-6 M TRIA-treated plants respectively. It was demonstrated that the incubation of both protoplasts and chloroplasts with TRIA resulted in a rise of the excimer/monomer (IE/IM ) ratio of pyrene (Py) fluorescence, thus indicating remarkable fluidization and/or disordering of the lipid matrix of their membranes. This effect depended on the incubation time and became evident at very low concentrations of TRIA (0.3 μM). The increase of membrane fluidity was accompanied by TRIA-induced alterations in the dielectric environment in the membrane regions where Py molecules are situated. The results are discussed in terms of specific concentration dependent TRIA-induced alterations of the dynamic properties of protoplast and chloroplast membranes and their possible involvement in the initiation of the integral physiological response to exogenous application of TRIA.
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  • 27
    ISSN: 1573-5044
    Keywords: PEG-mediated transformation ; protoplasts ; transgene inheritance
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A protocol for PEG-mediated transformation of protoplasts is described forA. thaliana ecotype Landsbergerecta and two marker lines derived from it, M4 and M10. The optimal transformation conditions were: 14 µg plasmid DNA and 28 µg carrier DNA per 6 x 105 protoplasts in 15% (w/v) PEG solution. Based on the hygromycin resistance conferred by the transgene, relative transformation frequencies of 2.5–3.2% and absolute transformation frequencies of 1–2 x 10−4, were obtained. Shoot regeneration frequencies of 40–60% were achieved, and fertile transgenic plants of the three tested lines were obtained. Southern blot hybridizations demonstrated multi-copy integration patterns in most cases. Hygromycin resistance segregation patterns of 3:1 and 15:1 were found, as well as unexpected segregation patterns, suggesting that modifications in gene expression took place and that these can progressively occur over successive generations.
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  • 28
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    Euphytica 95 (1997), S. 229-235 
    ISSN: 1573-5060
    Keywords: apomixis ; Hypericum perforatum ; rDNA ; heterogeneity ; RFLP ; progeny ; somaclonal variation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract Three adjacent EcoRI fragments of the rDNA unit from Lycopersicon esculentum, eleven anonymous genomic and two anonymous cDNA clones from Brassica napus and three restriction endonucleases: BamHI, EcoRI and EcoRV were used for RFLP analysis of the genome of Hypericum perforatum L. A polymorphic band identified with EcoRI and two rDNA probes in five somaclones originated from the same genotype was detected in all progenies of two somaclones indicating the inheritance of the molecular changes. rDNA unit heterogeneity represented by two types of RFLP pattern revealed among somaclones and seed-derived control plants using EcoRV and two rDNA probes may indicate an alloploid origin of Hypericum perforatum. The occurrence of the identical RFLP patterns in some R0 somaclones and seed-derived plants and their progenies may be related to the apomictic mode of reproduction which is assumed to be prevalent in Hypericum perforatum. The differences in RFLP patterns of progenies when compared with the maternal plants (1 out of 10 progenies of one control plant and 1 out of 8 progenies of one somaclone) may indicate that some progenies have originated through sexual recombination.
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  • 29
    ISSN: 1573-5060
    Keywords: Sweet potato ; Ipomoea batatas ; plant regeneration ; somatic embryogenesis ; protoplasts ; genetic variation ; somaclonal variation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract The application of new techniques for improvement of sweet potato crops, particularly including the exploitation of somaclonal variation, gene transfer by genetic transformation and somatic hybridization, requires the control of plant regeneration from tissue cultures. Shoots can easily be regenerated from explants of stems, petioles, leaves and roots, while callus cultures do not produce any shoots. The potential of somatic embryogenesis and plant regeneration via embryogenesis was evaluated for 10 cultivars of sweet potato. Protocols for plant regeneration from cultured protoplasts have also been developed. Since mesophyll was resistant to enzyme digestion, fragments of stems and petioles, callus and cell suspensions were used as source of protoplasts of sweet potato. Series of transfers of protoplast-derived calluses, particularly those which had been obtained from in vitro plants, to media containing a high level of zeatin resulted in successful formation of shoots in only two sweet potato cultivars. In addition, the embryogenic potential was irreversibly lost through protoplast culture, since protoplasts isolated from embryogenic cell suspensions developed into non-embryogenic callus. Consequently, an alternative protocol is being successfully developed to improve plant regeneration from cultured protoplasts of sweet potato, involving first root formation from which shoots can then be regenerated. Preliminary evaluation in field conditions in Gabon revealed that plants regenerated from cultured protoplasts exhibited a great genetic variability in their growth and tuber formation in particular.
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  • 30
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    Euphytica 96 (1997), S. 135-142 
    ISSN: 1573-5060
    Keywords: Oryza sativa ; chilling tolerance ; in vitro selection ; rice ; somaclonal variation ; field performance
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract Embryo-derived calli of four rice varieties cultivated at high altitude in Burundi — Facagro 57, Facagro 76, Kirundo 3 and Kirundo 9 — were submitted to different temperature regimes. The percentage of regenerating calli greatly varied depending on variety, length of culture and callus temperature treatment. The reduction of regeneration percentages induced by low temperature was more pronounced in the more sensitive varieties. Regenerated plants (R0) and their progenies in R1, R2 and R3 were cold-screened together with control plants. In all varieties, significantly higher survival rates were obtained in R3 with in vitro plants than with control plants. Such chilling tolerance improvement was not obtained following a massal selection applied during 3 successive generations onto the control plants. In vitro plants regenerated from calli cultivated either at 25 °C, either at 4 °C, were cultivated at different altitudes in Burundi during two successive generations. For most observed traits, the in vitro plants were characterized by lower means, larger variation and higher maximum values than the control plants. The most chilling-tolerant somaclonal families were most usually characterized by extensive differences in fatty acid composition, chilling-induced electrolyte leakage and chlorophyll fluorescence, compared to the varieties they derived from.
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  • 31
    ISSN: 1573-5060
    Keywords: somaclonal variation ; in vitro selection ; herbicides ; wheat ; triticum aestivum
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract The present work was initiated to assess tissue culture techniques as a means of generating and selecting herbicide tolerant genotypes of wheat through exploiting somaclonal variation. Callus was initiated from immature embryos of genetically defined varieties and subcultured onto selective media containing 5 μm, 10 μm and 50 μm concentrations of difenzoquat and atrazine. Plants were regenerated from all the selective media except that media containing the highest herbicide concentration. The progenies of the regenerated plants were tested as whole plants for their response to spray application of the herbicides. For difenzoquat, variation in response from extreme susceptibility to tolerance was observed. However, genetic characterization by progeny testing tolerant lines revealed that the induced variation was not heritable. No plants tolerant to atrazine were obtained. Overall, no clear evidence of heritable mutations was obtained. Alternative strategies to obtain herbicide tolerant genotypes using cell culture techniques are discussed.
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  • 32
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    Plant cell, tissue and organ culture 48 (1997), S. 31-35 
    ISSN: 1573-5044
    Keywords: Cucurbitaceae ; Cucumis melo ; polyploidy ; regeneration ; somaclonal variation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The chromosome number of cells in the shoot primordium aggregates and produced plants of melon [Cucumis melo L. 'Prince' (2n=2x=24)] was examined. Shoot primordium aggregates were induced from shoot-tips cultured in liquid medium and shaken at low speed (2 rpm). They were maintained by subculturing small pieces (5mm〈) every 4 weeks. Shoot primordium aggregates just after induction contained about 97 diploid and 3 tetraploid cells, which was similar to those maintained in shoot primordium cultures for 6 years. This indicates that the ploidy level was maintained stably. On the other hand, plants produced from the shoot primordium aggregates just after induction were either diploid, tetraploid or mixoploid with both diploid and tetraploid cells. These ploidies were again observed among plants produced from shoot primordium cultures that were 2, 3 or 4 years old. A majority of produced plants were diploid while the total frequency of tetraploids and mixoploids was less than 8 of plants produced from all ages. Therefore, the frequency of somaclonal variation with respect to ploidy among plants produced from shoot primordium aggregates is likely to be stable at a low level over the long term.
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  • 33
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    World journal of microbiology and biotechnology 13 (1997), S. 469-473 
    ISSN: 1573-0972
    Keywords: 2-Deoxy-d-glucose ; hydroxylation ; immobilization ; polyoxin ; protoplasts ; steroids
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
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  • 34
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    Biologia plantarum 39 (1997), S. 561-567 
    ISSN: 1573-8264
    Keywords: Beta vulgaris ; plating efficiency ; protoplasts ; putrescine ; spermidine ; spermine
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The influence of the exogenous polyamines: putrescine, spermidine and spermine, on the frequency of protoplast divisions for 2 genotypes of sugar beet (Beta vulgaris L.) was analyzed. Protoplasts were cultured by the agarose disk method on Saunders and Doley medium supplemented with either hormones or polyamines, or hormones combined with polyamines. The latter supplement led to a statistically significant increase in plating efficiency. The improvement in division index was caused mainly by spermine.
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  • 35
    ISSN: 1432-203X
    Keywords: Ty1-copia group retrotransposons ; potato ; protoplasts ; expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The potato (Solanum tuberosum) genome contains a highly heterogeneous population of Ty1-copia group retrotransposons. Here we identify the first such transposable element known to be transcribed in this species. The elements are transcriptionally activated during protoplast isolation. The majority of the activated Ty1-copia sequences are similar to elements which are transcriptionally induced under the same conditions in tobacco (Nicotiana tabacum). We also show that a previously identified potato element M166, which has no known equivalent in tobacco is also transcribed under these conditions. It appears that the control of transcription of this particular Ty1-copia group retrotransposons has been broadly conserved between these two species.
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  • 36
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    Plant cell reports 15 (1996), S. 737-741 
    ISSN: 1432-203X
    Keywords: Zoysia japonica ; embryogenic callus ; protoplasts ; plant regeneration
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Embryogenic callus of Japanese lawngrass (Zoysia japonica Steud.) was induced from sterile mature seeds on LS medium with 5 mg / l of 2,4-D. Embryogenic callus selected visually under microscope was proliferated in liquid N6 medium with amino acids (N6-AA medium). Protoplasts were isolated from suspension cells by the treatment of enzyme mixture containing pectolyase Y-23 and cultured in K8p medium with 2 mg / l of 2,4-D at the density of 106 / ml. Plants were regenerated by transferring the protoplasts derived callus to MS medium and incubating at 28 °C under light for two months. Plantlets were successfully transplanted in the soil.
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  • 37
    ISSN: 1432-203X
    Keywords: Primulaceae ; tissue culture ; regeneration ; somaclonal variation ; organogenesis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary In long-term callus cultures of Cyclamen persicum Mill. two types of tissue could be distinguished. One type featured a brown suberised outer layer and was poorly organogenic. The other type was yellowish in appearance and gave rise to many shoot buds. Both types co-existed on the same callus, the former prevailing. Selection for organogenic tissue resulted in cultures yielding approximately three times more petioles than random subcultures. Callus-derived shoots could be rooted and established in the greenhouse. The method allowed for the production of thousands of plants but the regenerants often showed deviant phenotypes and genotypes.
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  • 38
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    Plant cell reports 16 (1996), S. 88-91 
    ISSN: 1432-203X
    Keywords: Gentiana crassicaulis Duthie ex Burk ; protoplasts ; callus ; plant regeneration
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Fast growing calli induced from hypocotyl segments ofGentiana crassicaulis were used for preparation of protoplasts. High yields of viable protoplasts were produced in an enzyme solution containing 1–2% cellulase, I% pecfinase, and 0.5% Hemicellulase. Protoplasts were cultured in KM8P medium containing 1 mg/l 2,4-D, 0.5 mg/l 6BA, 500 mg/l LH, 0.5 M glucose and 0.1 M mannitol by the solid-liquid dual layer culture method. First division occurred within 4–5 days of culture at a frequency of 17.8%. Sustained divisions led to callus formation. Periodically diluting the cultures with freshly prepared liquid medium containing 1% glucose was critical for colony formation. Protocolonies about 2 mm in size were transferred onto MS medium supplemented with 3 mg/l ZT, 2 mg/l 6BA, 1 mg/l GA3, 1 mg/l NAA and 6% sucrose to obtain embryogenic calli. Plantlets were regenerated via somatic embryogenesis at high frequency on hormone-free MS Medium.
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  • 39
    ISSN: 1573-9368
    Keywords: Transgenic rice ; PCR-RAPD ; arbitrary DNA primers ; genomic changes ; somaclonal variation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The occurrence of genomic modifications in transgenic rice plants recovered from protoplasts and their transmission to the self-pollination progeny has been verfied with the random amplified polymorphic DNA (RAPD) approach. The plant was the Indica-type rice (Oryza sativa L.) cultivar Chinsurah Boro II. The analysed material was: (1) microspore-derived embryogenic rice cells grown in suspension culture, (2) transgenic plants recovered from protoplasts produced from the cultured cells and (3) the self-pollination progeny (two successive generations) of the transgenic plants. DNA purified from samples of these materials was PCR-amplified with different random oligonucleotide primers and the amplification products were analysed by agarose gel electrophoresis. Band polymorphism was scored and used in band-sharing analyses to produce a similarity matrix. Relationships among the analysed genomes were expressed in a dendrogram. The extensive DNA changes evidenced in cultured cells demonstrate the occurrence of somaclonal variation in the material used to produce protoplasts for gene transfer. Quantitatively reduced DNA changes were also found in the resulting transgenic plants and i their self-pollination progenies. While confirming the stability of the foreign gene in transgenic plants, this work gives molecular evidence for the occurrence of stable genomic changes in transgenic plants and points toin vitro cell culture as the causative agent. RAPDs are shown to be a convenient tool to detect and estimate the phenomenon at the molecular level. The methodology is also proposed as a fast tool to select those transgenic individuals that retain the most balanced genomic structure and to control the result of back-crosses planned to restore the original genome.
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  • 40
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    Plant growth regulation 18 (1996), S. 149-154 
    ISSN: 1573-5087
    Keywords: cell culture ; auxins ; cytokinins ; confocal laser scanning microscope ; protoplasts
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract The model system presented here is based on immobilised single cells, derived directly from tobacco mesophyll protoplasts. It allows the adequate steering of cell populations towards expansion, cell cycling or cell resting. Using this approach cells always have the same predictable response to auxins and cytokinins whatever their actual physiological status. This model system opens new ways to study cellular parameters governing these hormone responses, some of which have been explored so far; a) the cytokinin response can equally well be induced by endogenous as by exogenous cytokinins; b) at least two intracellular components, microtubuli and the ER, adapt their architecture to the hormone-induced status of the cell; c) addition of NAA to the cells does not induce a change in the cytoplasmic pH.
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  • 41
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    World journal of microbiology and biotechnology 12 (1996), S. 625-628 
    ISSN: 1573-0972
    Keywords: Mycorrhizae ; protoplasts ; Suillus
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Experiments were performed with the mycorrhizal fungus Suillus granulatus to define the parameters for production and regeneration of protoplasts. Protoplasts were released at frequencies between 1 and 3×107/ml from mycelium 3 to 7 days old. The best osmotic stabilizer for protoplast release was MgSO4 (0.7 m). To optimize protoplast release and regeneration an enzyme (Novozym 234) concentration 1.7 mg/ml was chosen, with a digestion time of 1 to 2 h. Regenerated colonies formed mycorrhizae within 60 days after inoculation in Pinus caribaea var. hondurensis seedlings.
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  • 42
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    Plant cell, tissue and organ culture 46 (1996), S. 179-186 
    ISSN: 1573-5044
    Keywords: cereal ; herbicide-tolerance ; Hordeum vulgare ; somaclonal variation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract In vitro culture of barley calluses has been used to produce plants with increased glyphosate tolerance. Calluses from immature embryos of barleyHordeum vulgare L. (‘Jeff’) were cultured on Murashige and Skoog medium with 10-6, 10-5, 10-4, 5×10-4, 10-3, or 10-2M glyphosate for one, four or thirty months. Plants were regenerated from calluses maintained in glyphosate medium at 10-6, 10-5 or 10-4M for four months, at 10-5 or 5×10-4M for one month and at 10-5M for thirty months. The progeny of each regenerated plant was analyzed for response to glyphosate. Some progenies showed increased tolerance to glyphosate.
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  • 43
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    Plant cell, tissue and organ culture 46 (1996), S. 35-41 
    ISSN: 1573-5044
    Keywords: Elaeis guineensis ; oil palm ; protoplasts
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Procedures are deseribed for the efficient isolation of protoplasts from a variety of oil palm (Elaeis guineensis Jacq.) tissues. Various factors including donor source, composition of enzyme mixture and culture medium affected the yield and viability of the protoplasts Polyembryogenic cultures of oil palm were the most suitable starting material in terms of yield, viability and metabolic competence. Pectolyase Y-23 in association with cellulase and hemicellulase was required for the efficient release of protoplasts from the oil palm tissues. Limited cell division to form microcallus was observed at very low frequency (〈0.01%) when glutathione and catalase were incorporated in the culture medium.
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  • 44
    ISSN: 1573-5060
    Keywords: protoplasts ; potato ; somatic hybrids ; sexual hybrids ; late blight ; Phytophthora infestans
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract Intraspecific tetraploid somatic and sexual hybrid plants have been resynthesised following protoplast fusion and by sexual crosses between two dihaploid potato (Solanum tuberosum) lines each possessing complementary agronomic traits. The dihaploid PDH 40 possesses good tuber shape and yield but has foliage susceptibility to late blight (Phytophthora infestans). On the other hand, the dihaploid PDH 727 possesses resistance to blight in the foliage but has a low yield of small and irregular shaped tubers. Since it was only possible to use a partial selection strategy based on culture media to facilitate recovery of somatic hybrid plants-further morphological and esterase isozyme based characterisations were performed to identify somatic hybrid plants from amongst the non-hybrid plant material. When the blight resistance of both the intraspecific somatic and sexual hybrid plants was assessed there was no significant difference in the mean resistance value and it was intermediate between those of their parents. However, the range of resistance was much wider among the sexual hybrids than among the plants derived from somatic fusion. An assessment of tuber yield between tetraploid sexual and somatic hybrids showed no significant difference and it was higher than that of either parent value. The implication of these results in the context of potato genetics and breeding is discussed.
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  • 45
    ISSN: 1573-5060
    Keywords: Alstroemeria ; genetic fingerprinting ; RAPD ; somaclonal variation ; alstroemeria
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Summary We have used random amplified polymorphic DNA (RAPD) markers to study genetic variation in Alstroemeria. The first objective was to examine the discriminatory power of RAPD markers in different genotypes of Alstroemeria obtained by traditional breeding. All genotypes examined, including commercial Alstroemeria varieties, could be distinguished on the basis of their RAPD profiles. Progeny plants could be distinguished from their parents. A second objective of this study was to investigate whether RAPD markers can be used as a routine tool to detect mutant plants, as an alternative to glasshouse testing. To address this objective, we analysed Alstroemeria plants that carried phenotypically visible mutations that either were induced by irradiation using X-rays or were the result of somaclonal variation. In eight out of a total of 13 mutant Alstroemeria plants obtained after irradiation or tissue culture we detected no polymorphisms when compared to control plants that were considered to be non-mutated. Only in five of the mutant plants analysed we detected one to two polymorphisms. These results suggest that frequent genome rearrangements had not occurred in the mutant plants analysed. These results also demonstrate that the RAPD technique is an inappropriate tool for the rapid screening of Alstroemeria for induced variation. It that the RAPD technique is an inappropriate tool for the rapid screening of Alstroemeria for induced variation. It seems probable that this conclusion would be equally applicable in other plant genera in which induced variation has occurred. However, the RAPD technique is a simple and effective tool for genetic fingerprinting of Alstroemeria varieties, provided their differences are due to sexual propagation.
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  • 46
    ISSN: 1573-5060
    Keywords: Fusarium resistance ; Fusarium spp. ; wheat ; Triticum aestivum ; culture filtrate ; in vitro selection ; somaclonal variation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Summary Calli of resistant, intermediary and susceptible wheat (Triticum aestivum L.) varieties were selected using culture filtrates of Fusarium graminearum and F. culmorum and the regenerants were evaluated for resistance up to R3. Czapek-Dox broth medium was inoculated with mycelia of Fusarium isolates and incubated for 2–6 weeks. Filtrates were added to MS callus growing medium, then 5 weeks-old calli were transferred onto this medium (MST) for 4–5 weeks. MST containing 30% filtrate was found to be suitable for selection. Resistant calli were transferred again to fresh MST for further two selection cycles. The surviving calli produced less fertile regenerated lines (R0) than the non-selected ones. Among 18 R1 lines tested for Fusarium-resistance in the seedling stage by artificial inoculation in the greenhouse, two (11.1%) were significantly more resistant, one (5.6%) was more susceptible than the original cultivar and the rest (83.3%) behaved similarly to the donor plants. Among unselected R3 lines of three varieties, practically the same number of resistant plants were found as among the related selected ones. When the R3 selfed generations obtained through double-layer and culture filtrate selection techniques were tested for Fusarium-resistance, 35.7% of the lines were found to be more resistant than the original cultivars, none was more susceptible and 64.3% had a reaction similar to that of the source materials. Thus, inheritance of the disease reaction was not stable in all cases. Success of in vitro selection for Fusarium-resistance depended also on the genotype, and toxin analysis showed that although being effective, the selective media contained deoxynivalenol only exceptionally. In selecting wheat for Fusarium-resistance in vitro, the culture filtrate technique proved better than the double-layer procedure.
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  • 47
    ISSN: 1573-5087
    Keywords: auxin receptor ; ion channels ; auxin-binding protein ; protoplasts ; electrophysiology
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract Studies of membrane electrical responses of isolated protoplasts to auxin have demonstrated the existence of elementary response chains to auxin at the plasma membrane, presently defined only by their uttermost ends. At one side, as demonstrated by several lines of evidence, the auxin perception unit involves proteins homologous to ZmER-abp1 (abp1), the most abundant auxin-binding protein from maize coleoptiles. At the other side, multiple ion transport proteins appear as targets of the auxin signal; the proton pump ATPase, an anion channel and potassium channels. We investigated early electrical responses to auxin at the plasma membrane of tobacco protoplasts. The work presented here will initially focus on abp1 and its functional role at the membrane. The C-terminus abp1 peptide (Pz151–163) was recently reported to modulate K+ currents at the plasma membrane of intact guard cells from broad bean [23] and induce plasma membrane hyperpolarisation of tobacco mesophyll protoplasts. These results further demonstrate that proteins involved in plasma membrane responses to auxin are related to maize abp1, and provide clues as to the region of the protein possibly involved in the interaction of abp1 with the plasma membrane. Secondly, this report concentrates on one of the targets of auxin, a voltage-dependent and ATP-regulated anion channel that we characterised on protoplasts from tobacco cell suspensions. This anion channel was specifically modulated by auxin, as already observed for the anion channel of guard cells [14]. Further work will be needed to assess if this auxin modulation involves a direct interaction between the hormone and the anion channel protein(s), or follows from the activation of a perception chain including abp1 homologues.
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  • 48
    ISSN: 1432-203X
    Keywords: Cucumis mel ; somaclonal variation ; low-temperature ; selection ; germinability
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Plants were regenerated from adventitious buds and somatic embryos (R0) of melon (Cucumis melo L.), the cultivar Andes. Somaclonal variants of melon with low temperature germinability were selected from the progenies (R1) of R0 plants. Among 5,618 R1 seeds harvested from 23 R0 plants that were regenerated from adventitious buds 4 seeds germinated after 5 days of culture at 15 °C (selection rate; 0.07%). However, among 374 R2 seeds harvested from 2 R1 plants no seed germinated after 7 days of culture at 14 °C. Among 9,181 R1 seeds harvested from 50 R0 plants regenerated from somatic embryos 110 seeds germinated after 5 days of culture at 15 °C (selection rate; 1.20%). Among 3,717 R2 seeds harvested from 17 R1 plants 113 seeds germinated after 7 days of culture at 14 °C (selection rate; 3.04%). R3 seeds were collected from these R2 plants following self-pollination. Forty-five of the 47 lines (R3) originated from 10 R0 plants showed higher germination rates than that of the original cultivar. Selected lines with low-temperature germinability showed greater fruit growth rate than the original cultivar during the middle stage when they were cultivated in a greenhouse under low-temperature conditions. Of fruits harvested from 31 lines, 15 lines showed greater fruit volume than the original cultivar.
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  • 49
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    Plant cell reports 14 (1995), S. 738-742 
    ISSN: 1432-203X
    Keywords: Hyoscyamus muticus ; plant regeneration ; protoplasts ; transformed roots
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Successful plant regeneration was achieved for the first time from hairy root-derived protoplasts of Hyoscyamus muticus. High yields (7 × 106 / g fresh weight) of protoplasts were isolated directly from the transformed roots of Hyoscyamus muticus using an enzyme mixture comprising 1 % macerozyme and 2 % cellulase in an osmoticum consisting of 0.2 M CaCl2 and 0.6 M mannitol. Protoplasts were first cultured in liquid NT/PRO I medium and further on semi-solid NT/PRO II agar medium. The procedure permits highly efficient formation of colonies. The plating efficiency varied from 1–9 %. The small individual colonies regenerated easily into shoots and roots at frequencies of 18 % and 70 %, respectively. The time required for the development of small plantlets from protoplasts was 8–11 weeks. The regenerated plants contained rolB from Ri-T-DNA and exhibited an altered phenotype compared to the control plants.
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  • 50
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    Plant molecular biology 28 (1995), S. 123-136 
    ISSN: 1573-5028
    Keywords: Agrobacterium rhizogenes ; auxin biosynthesis genes ; plant cell division ; protoplasts
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Plant oncogenes aux1 and aux2 carried by the TR-DNA of Agrobacterium rhizogenes strain A4 encode two enzymes involved in the auxin biosynthesis pathway in transformed plant cells. The short divergent promoter region between the two aux-coding sequences contains the main regulatory elements. This region was fused to the uidA reporter gene and introduced into Nicotiana tabacum in order to investigate the regulation and the tissue specificity of these genes. Neither wound nor hormone induction could be detected on transgenic leaf discs. However, phytohormone concentration and auxin/cytokinin balance controlled the expression of the chimaeric genes in transgenic protoplasts. The expression was localised in apical meristems, root tip meristems, lateral root primordia, in cells derived from transgenic protoplasts and in transgenic calli. Histological analysis showed that the expression was located in cells reactivated by in vitro culture. Experiments using cell-cycle inhibitors such as hydroxyurea or aphidicolin on transgenic protoplast cultures highly decreased the β-glucuronidase activity of the chimaeric genes. These results as well as the histological approach suggest a correlation between expression of the aux1 and aux2 genes and cell division.
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  • 51
    ISSN: 1573-5028
    Keywords: Agrobacterium tumefaciens ; extensin ; hydroxyproline-rich cell wall protein ; Nicotiana sylvestris ; protoplasts ; wounding
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A cDNA clone (6PExt 1.2) encoding a novel extensin was isolated from a cDNA library made from 6 h old mesophyll protoplasts of Nicotiana sylvestris. The screening was performed with a heterologous probe from carrot. The encoded polypeptide showed features characteristic of hydroxyproline-rich glycoproteins such as Ser-(Pro)4 repeats and a high content in Tyr and Lys residues. The presence of four Tyr-X-Tyr-Lys motifs suggests the possibility for intramolecular isodityrosine cross-links whereas three Val-Tyr-Lys motifs may participate in intermolecular cross-links. The analysis of genomic DNA gel blots using both the N. sylvestris and the carrot clones as probes showed that the 6PExt 1.2 gene belongs to a complex multigene family encoding extensin and extensin-related polypeptides in N. sylvestris as well as in related Nicotianeae including a laboratory hybrid. This was confirmed by the analysis of RNA gel blots: a set of mRNAs ranging in size from 0.3 kb to 3.5 kb was found by the carrot extensin probe. The 6PExt 1.2 probe found a 1.2 kb mRNA in protoplasts and in wounded tissues as well as a 0.9 kb mRNA which seemed to be stem-specific. The gene encoding 6PExt 1.2 was induced by wounding in protoplasts, in leaf strips and after Agrobacterium tumefaciens infection of stems.
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  • 52
    ISSN: 1432-203X
    Keywords: garlic ; Allium sativum L. ; shoot primordia ; protoplasts ; plant regeneration
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Protoplasts derived from tissue-cultured shoot primordia of garlic (Allium sativum L.) initiated successive cell divisions within 4 days and formed small individual calli (0.2mm in diameter) after 5 weeks of culture on Gamborg's B5 medium supplemented with 0.1% casein hydrolysate, 1mg/1 1-naphthaleneacetic acid and 1mg/1 6-benzylaminopurine. Plating efficiency was roughly 5% at the density of 1x104 protoplasts/ml of medium. Adventitious buds developed from the calli during subsequent subculture on Gamborg's B5 medium supplemented with 40mg/l adenine and 10% coconut milk. When transferred to the same medium without supplements, these buds grew into shoots and rooted. The regenerated garlic plantlets were successfully transferred to the greenhouse and grew into whole plants.
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  • 53
    ISSN: 1573-5176
    Keywords: protoplasts ; flow cytometry ; seaweed ; biotechnology
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The ability to rapidly distinguish viable sub-populations of cells within populations of macroalgal protoplast isolations was demonstrated using flow cytometry. Viable protoplasts from Ulva sp. and Porphyra perforata J. Ag. were distinguished from non-viable protoplasts based on differential fluorescein accumulation. The identities of cortical and epidermal protoplasts from Macrocystis pyrifera (L.) C. Ag. were inferred based on light-scattering and chlorophyll a autofluorescence. Three cell types could be distinguished among protoplasts released from thalli of P. perforata based on chlorophyll a and phycoerythrin autofluorescence. Mixed protoplast populations of Ulva sp. and P. perforata were also discernable based on relative chlorophyll a and phycoerythrin autofluorescence. The ability to screen heterogenous protoplast populations rapidly, combined with the cell sorting capabilities of many flow cytometers, should prove valuable for seaweed biotechnology.
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  • 54
    ISSN: 1573-5044
    Keywords: Hordeum vulgare ; plasmid uptake ; polyethylene glycol (PEG) ; protoplasts ; transient gene expression ; stable transformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Cat gene expression has been investigated following PEG-mediated plasmid uptake into barley protoplasts. The uptake conditions optimised for transient expression were employed for stable transformation. Transformed protoplast-derived calli of the cvs. Dissa and Igri, were selected on medium containing G418 at 40 μg ml−1 or kanamycin sulphate at 250 μg ml−1. Absolute transformation frequencies of 28.9×10−5 and 21.3×10−5 were recorded for Dissa with kanamycin sulphate and G418 selection, respectively. The frequency for Igri was 11.5×10−5 with G418 selection. Antibiotic resistant protoplast-derived colonies expressed NPTII activity; Southern hybridisation confirmed integration of the nptII gene into barley genomic DNA.
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  • 55
    ISSN: 1573-5060
    Keywords: somaclonal variation ; somatic embryogenesis ; tissue culture ; wheat ; Triticum aestivum
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Summary Somatic embryogenesis was initiated from ‘immature embryos’ on Murashige-Skoog (MS) medium plus 2 mg.l-1 2,4-dichlorophenoxyacetic acid, 2% sucrose and 0.6% agarose. Somatic embryos were isolated and regenerated into whole green plants on MS medium devoid of 2,4-D. These regenerants were previously demonstrated to differ in their mitochondrial DNA organization. In order to estimate their characteristics three progenies of short-term culture regenerants and three progenies of long-term culture regenerants were analyzed and compared to the parental line. These somaclones obtained from the wheat variety Chinese Spring were evaluated for variation of 13 agronomic and morphological quantitative characters in comparison to the parental line. Significant variation was observed for plant height, spike length, main tiller diameter, between the somaclones regenerated from long-term culture and their parent. Differences were observed to increase with the duration of culture, leading to a significant modification of the structure of the plants. Several changes occurred during the somatic tissue cultures, but to a lesser extent than has previously been described in the literature.
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  • 56
    ISSN: 1573-5036
    Keywords: Al tolerance ; 1 ; 3-β-glucan ; K leakage ; plasma membrane ; protoplasts
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract Experiments were carried out to identify the primary site for aluminium (Al) toxicity in roots. Al accumulated in large amounts in the younger and outer cells in roots of pea and was retarded when the ionic strength of the Al solution was high. Cell destruction was extensive in the regions with high Al accumulation. The accumulation of Al in, and potassium (K) leakage from, the root tip were in the order pea〉maize〉rice, the same order as their sensitivity to Al. The protoplasts from the root tip portion of pea incubated with Al showed a wrinkled and uneven surface. The protoplasts progressively shrank and eventually collapsed. Viability decreased in this process. In the control protoplasts of maize, β-glucan formation was uniform on the spherical surfaces, whereas it was spotty in the Al-treated protoplasts; the cell wall material of the latter contained partly 1, 3-β-glucan which is known to be synthesised by 1, 3-β-glucan synthase embedded in the plasma membrane. These results suggest that the specific site for Al toxicity is the plasma membrane of younger and outer cells in roots and that Al tolerance depends largely on the integrity of the plasma membrane.
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  • 57
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    Plant cell, tissue and organ culture 41 (1995), S. 41-48 
    ISSN: 1573-5044
    Keywords: callus ; Medicago littoralis ; plant regeneration ; protoplasts
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Plant regeneration from explant and protoplast derived callus has been achieved in Medicago littoralis cv. Harbinger 1886, an annual legume resistant to the fungus Pseudopeziza medicaginis. Callus was induced from different tissue explants and the fastest growth rate was observed for hypocotyls in B5 medium with 2 mg l−1 2,4-dichlorophenoxyacetic acid and 0.5 mg l−1 N6-benzyladenine. Protoplasts were isolated from cotyledons and leaves of sterile plants and from callus; the first two kinds of protoplasts showed a plating efficiency of 5.6% and 5%, respectively, when embedded in agarose. Plant regeneration occurred on media containing % MathType!MTEF!2!1!+-% feaafiart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr% 4rNCHbGeaGqiVu0Je9sqqrpepC0xbbL8F4rqqrFfpeea0xe9Lq-Jc9% vqaqpepm0xbba9pwe9Q8fs0-yqaqpepae9qq-f0-yqaqVeLsFr0-vr% 0-vr0db8meaabaqaciGacaGaaeqabaWaaeaaeaaakeaacaqGobWaaW% baaSqabeaacaqG2aaaaOGaaeOVfiaabs5adaahaaWcbeqaaiaaikda% aaGccaqG+waaaa!3F97!\[{\text{N}}^{\text{6}} {\text{\Delta }}^2 {\text{}}\]isopentenyl-adenine combined with indole-3-acetic acid or 1,2-benzisoxazole-3-acetic acid, and on media with N6-benzyladenine plus α-naphtaleneacetic acid; a cytokinin/auxin ratio higher than 1 induced embryos while a ratio around 1 stimulated shoot formation. Embryo development and rooting of shoots were performed in RL medium without growth regulators.
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  • 58
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    Plant cell, tissue and organ culture 43 (1995), S. 229-235 
    ISSN: 1573-5044
    Keywords: Albinism ; Hordeum vulgare L. ; somaclonal variation ; totipotency
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The use of the synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-d) has played an important role in the production and maintenance of totipotent cereal callus. However, 2,4-d has been implicated in the loss of totipotency from barley callus. To examine the effect of 2,4-d on barley callus, regenerability and karyotype were examined over time as influenced by cultivar differences and 2,4-d levels, during a period in which initially vigorous plant regeneration typically declines dramatically. Higher (20.4–27.1 μM) versus lower (6.8–13.6 μM) concentrations of 2,4-d were positively associated with the number of green plantlets recovered from calli maintained for 10 and 16 weeks before transfer to regeneration media, and with the longevity of regenerability. There was a positive relationship between 2,4-d concentration and normal karyotype. We also investigated the use of phenylacetic acid for the initiation of regenerable barley callus. Very poor callus growth and plant regeneration was supported by phenylacetic acid.
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  • 59
    ISSN: 1573-5087
    Keywords: Gibberellin ; in vitro propagation ; Musa, quantification ; somaclonal variation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract GA3 and GA20 were quantified in leaf extracts from true-to-type and somaclonal variants (dwarf and giant) of Musa AAA cv. ‘Grand nain’ by GC-MS-SIM after purification on reverse- and normal-phase HPLC and detection by ELISA with GA3 antibodies and by a dwarf rice bioassay. GA3 concentration in dwarf plants was 811 ng g−1 dry weight. For normal and giant plants, the endogeneous GA3 levels were respectively 3.6 and 4.6 times higher. The GA20 concentration in the giant plant was 68 ng g−1 of dry weight. This concentration was, respectively, 4.6 and 7.3 times higher than those of normal and dwarf plants. These results suggest that the somaclonal variations affecting banana plant height are associated with modifications in GA metabolism.
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  • 60
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    Journal of industrial microbiology and biotechnology 15 (1995), S. 397-400 
    ISSN: 1476-5535
    Keywords: mycolytic enzymes ; Trichoderma harzianum ; protoplasts ; Rhizopus nigricans ; induction
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The effect of the growth form of the inducing microorganism on specificTrichoderma harzianum mycolytic enzyme production was studied. The pelleted form ofRhizopus nigricans gave a better product concerning protoplast formation ability. The maximum yield of protoplasts from the target fungusCochliobolus lunatus was 1×108 ml−1. Analysis of individual specific enzyme activities inTrichoderma mycolytic enzyme preparations confirms the importance of high chitinase and low protease activity for high protoplast yields. Supplementation of the production medium with chitin increased the chitinase activity in theTrichoderma exoenzyme mixture.
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  • 61
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    Plant cell, tissue and organ culture 41 (1995), S. 9-15 
    ISSN: 1573-5044
    Keywords: birdsfoot trefoil ; cotyledonous protoplasts ; Lotus corniculatus L. cv. Leo ; pretreatment ; regeneration ; somaclonal variation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A method was developed for rapid plant regeneration from protoplasts of birdsfoot trefoil (Lotus corniculatus L. cv. Leo). Green cotyledons from in vitro grown seedlings were preplasmolyzed in CPW salts containing 13% mannitol (CPW 13 M) for 1 h prior to the enzyme treatment. The enzyme formula consisted of 2% (w/v) Onozuka Cellulase R-10, 1% (w/v) Macerase and 0.1% (w/v) Pectolyase Y-23 in CPW 13 M. This method produced high yields of viable protoplasts after purification. The procedure is reproducible and takes approximately 2.5 months from protoplast isolation to plantlet establishment in a greenhouse. More than 100 plantlets were grown in soil. Two somaclonal variants, a chimeric plant for chlorophyll production and an albino cell line, have been obtained by this procedure.
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  • 62
    ISSN: 1573-5044
    Keywords: potato ; protoplasts ; regeneration ; Solanum
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The response to different in vitro methods for use in potato breeding has been evaluated in 11 genotypes of 5 Solanum species, S. etuberosum, S. lycopersicoides, S. maglia, S. rickii, and S. tuberosum. Callus induction and growth, and shoot regeneration were strongly influenced by the genotype, explant source, and medium utilized. Furthermore, considerable differences among the 11 genotypes were found both in plating efficiency and shoot regeneration from protoplast culture. Some interesting correlations were found between different tissue culture responses, suggesting linkage and/or pleiotropic effect of genes. The potential application to potato breeding of the in vitro techniques analyzed is discussed.
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  • 63
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    Plant cell, tissue and organ culture 40 (1995), S. 187-189 
    ISSN: 1573-5044
    Keywords: propagation ; somaclonal variation ; triglycerides
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Plant rgeneration occurred on leaf-and stem-derived callus of Cuphea ericoides Cham. & Schlechtd obtained in Murashige and Skoog medium supplemented with auxins [indole-3-acetic acid (IAA), α-naphthaleneacetic acid (NAA) or 2,4-dichlorophenoxyacetic acid (2,4-d)] plus cytokinins [6-benzyladenine (BA) or kinetin]. These calluses were subcultured and showed vigorous growth. When subcultured on medium containing 2.22 or 4.44 μM BA, the calluses showed profuse regeneration of shoots whereas those subcultured on medium supplemented with 2.69 μM NAA or 0.226 μM 2,4-d produced numerous roots. Isolated shoots rooted on Murashige and Skoog medium lacking growth regulators or containing 0.54 μM NAA or 0.49 μM indole-3-butyric acid (IBA). Plantlets were acclimatized to greenhouse conditions.
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  • 64
    ISSN: 1573-5060
    Keywords: glyphosate ; herbicide tolerance ; non-target effects ; somaclonal variation ; Zea mays ; maize
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Summary The progeny of 104 regenerated maize plants were screened for tolerance to the safe broad-spectrum herbicide glyphosate during seed germination and early growth. Seven somaclones showed varying degrees of resistance to the application of the herbicide at 1.2 mM (0.1 kg a.i. in 400 1 ha-1 of water). Plants capable of a normal growth following treatment with 2.4 mM (0.2 kg ha-1) glyphosate at the three leaf stage were selfed, and their progeny analyzed. A family able to tolerate the exposure to glyphosate at 2.4 mM was isolated and shown to maintain a photosynthetic rate comparable with control after the application of the herbicide. The selfed progeny of the tolerant somaclone was characterized as to the properties of two targets of glyphosate, the shikimate pathway enzymes 5-enol-pyruvyl-shikimate-3-phosphate (EPSP) synthase and 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase. In vitro tests ruled out the possibility that the tolerance was due to altered forms of these enzymes. Families showed significant variability with regard to EPSP and DAHP synthase levels, measured at different stages during seedling growth; however, not even these traits were correlated with in vivo response to glyphosate. The possible role of other physiological processes in determining the increased tolerance to the herbicide is discussed.
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  • 65
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    World journal of microbiology and biotechnology 11 (1995), S. 416-425 
    ISSN: 1573-0972
    Keywords: Genetic transformation ; micropropagation ; somaclonal variation ; synthetic seed
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Many important food and fibre crops have attained close to their maximum yields as a result of conventional breeding approaches and advances in agronomic and horticultural practices. The manipulation of cell and tissue cultures to produce somatic embryos efficiently is one of the keystones of the new technologies that will greatly alter the way crops are planted (as synthetic seed) and genetically altered in the future. Gene transfer into embryogenic plant cells is already challenging conventional plant breeding, and has become an indispensable tool for crop improvement. This review provides a current assessment of the impact of somatic embryogenesis in agriculture.
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  • 66
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    World journal of microbiology and biotechnology 11 (1995), S. 409-415 
    ISSN: 1573-0972
    Keywords: Breeding ; embryo culture ; haploids ; micropropagation ; protoplasts ; synthetic seed ; transformation ; wide hybridization
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Plant tissue culture comprises a set of in vitro techniques, methods and strategies that are part of the group of technologies called plant biotechnology. Tissue culture has been exploited to create genetic variability from which crop plants can be improved, to improve the state of health of the planted material and to increase the number of desirable germplasms available to the plant breeder. Tissue-culture protocols are available for most crop species, although continued optimization is still required for many crops, especially cereals and woody plants. Tissueculture techniques, in combination with molecular techniques, have been successfully used to incorporate specific traits through gene transfer. In vitro techniques for the culture of protoplasts, anthers, microspores, ovules and embryos have been used to create new genetic variation in the breeding lines, often via haploid production. Cell culture has also produced somaclonal and gametoclonal variants with crop-improvement potential. The culture of single cells and meristems can be effectively used to eradicate pathogens from planting material and thereby dramatically improve the yield of established cultivars. Large-scale micropropagation laboratories are providing millions of plants for the commercial ornamental market and the agricultural, clonally-propagated crop market. With selected laboratory material typically taking one or two decades to reach the commercial market through plant breeding, this technology can be expected to have an ever increasing impact on crop improvement as we approach the new millenium.
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  • 67
    ISSN: 1573-5060
    Keywords: protoplasts ; protoclonal variation ; somatic hybridization ; top-fruit trees ; woody ornamentals
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Summary Until recently, temperate fruit trees and woody ornamentals have been regarded as recalcitrant to biotechnological breeding approaches based on protoplasts. This however should no longer be the case, as procedures are now available, not only for the regeneration of complete plants from protoplasts of various tissues of such species, but also for the exploitation of protoplast technology for their genetic manipulation. This paper will examine the recent advances and state of the art in this domain, with particular attention to the use of protoplast technology as a novel tool in the breeding of rosaceous top-fruit tree species and woody ornamentals. Problems and their solutions within the context of regenerating plants from isolated protoplasts of stone (Prunus spp.), pome (Pyrus spp., Malus spp.) and small (Rubus spp.) fruits, and of several shrubby ornamental genotypes (Lonicera spp., Weigela spp., Forsythia spp., Cotoneaster spp.) will be addressed. Interspecific (Prunus spinosa + Prunus cerasifera) and intergeneric (Forsythia spp. + Syringa spp.) somatic hybridization within this group of species, as well as the use of protoplasts for host/pathogen interaction studies (Pyrus/Erwinia amylovora) will also be discussed.
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  • 68
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    Euphytica 85 (1955), S. 295-302 
    ISSN: 1573-5060
    Keywords: tissue culture ; somaclonal variation ; plant breeding
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Summary Somaclonal variation is a tool that can be used by plant breeders. The review examines where this tool can be applied most effectively and the factors that limit or improve its chances of success. The main factors that influence the variation generated from tissue culture are (1) the degree of departure from organised growth, (2) the genotype, (3) growth regulators and (4) tissue source. Despite an increasing understanding of how these factors work it is still not possible to predict the outcome of a somaclonal breeding programme. New varieties have been produced by somaclonal variation, but in a large number of cases improved variants have not been selected because (1) the variation was all negative, (2) positive changes were also altered in negative ways, (3) the changes were not novel, or (4) the changes were not stable after selfing or crossing. Somaclonal variation is cheaper than other methods of genetic manipulation. At the present time, it is also more universally applicable and does not require ‘containment’ procedures. It has been most successful in crops with limited genetic systems and/or narrow genetic bases, where it can provide a rapid source of variability for crop improvement.
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  • 69
    ISSN: 1573-5060
    Keywords: doubled haploids ; micropropagation ; mutant cultivars ; mutation techniques ; somaclonal variation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Summary Conventional mutation techniques have often been used to improve yield, quality, disease and pest resistance in crops, or to increase the attractiveness of flowers and ornamental plants. More than 1700 mutant varieties involving 154 plant species have been officially released. In some economically important crops, e.g. barley, durum wheat and cotton, mutant varieties occupy the majority of cultivated areas in many countries. Mutation techniques have become one of the major tools in the breeding of ornamentals such as alstroemeria, begonia, chrysanthemum, carnation, dahlia and streptocarpus. The use of in vitro techniques such as anther culture, shoot organogenesis, somatic embryogenesis and protoplast fusion can overcome some of the limitations in the application of mutation techniques in both seed and vegetatively propagated crops. In vitro culture in combination with induced mutations can speed up breeding programmes, from the generation of variability, through selection, to multiplication of the desired genotypes. The expression of induced mutations in the pure homozygote obtained through microspore, anther or ovary culture, can enhance the rapid recovery of the desired traits. In some vegetatively propagated species, mutations in combination with in vitro culture technique, may be the only method of improving an existing cultivar. Currently, many molecular studies rely on the induction and identification of mutants in ‘model species’ for construction and subsequent saturation of genetic maps, understanding of developmental genetics and elucidation of biochemical pathways. Once identified and isolated, the genes that encode agronomically-important features can be either introduced directly into crop plants or used as probes to search for similar genes in crop species. It seems most likely that the recent developments based on these technologies will soon provide improved methods for selection of desired mutants.
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  • 70
    ISSN: 1573-5060
    Keywords: barley ; electroporation ; PEG-mediated DNA uptake ; promoter analysis ; protoplasts ; transient expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Summary In some cereal species that are still recalcitrant to stable transformation and regeneration, transient expression in isolated protoplasts is a useful tool for the study of gene expression and regulation. We have successfully applied these techniques to barley protoplasts derived from developing endosperm, aleurone, leaves and roots in order to characterize functionally cis-acting motives in two gene promoters, corresponding to trypsin inhibitor BTI-CMe and to sucrose synthase Ss1. Gene specificity is maintained in transient expression assays with protoplasts isolated from these different barley tissues and the pattern of expression parallels the mRNA levels observed for the corresponding genes in the same tissues.
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  • 71
    ISSN: 1573-5060
    Keywords: aluminium toxicity ; soil acidity ; somaclonal variation ; sorghum ; Sorghum bicolor (L.) Moench ; tissue culture ; salt stress ; drought stress ; variants
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Summary Sorghum bicolor (L.) Moench is generally quite sensitive to salt and acid (high aluminium) soil stresses, but quite tolerant of drought stress. As with any stress phenomenon, intra-specific variability exists within the genus. In vitro cell selection and somaclonal variation offer an alternative to traditional breeding methodology for generating improved breeding lines for hybrid development. A field selection protocol was developed for the three soil stresses and inter-stress evaluations were conducted in an effort to find multiple, stress-tolerant genotypes. The acid soil-drought stress, super-tolerant selections were located by the R7 generation when exposed to a combined aluminium-drought stress field environment and when the regeneration population (number of regenerated lines from one callus source) was maintained at 15,000 plants or higher. A variant frequency of 0.1 to 0.2% for stress tolerance and acceptable agronomic traits among the surviving somaclones, provided an adequate number of phenotypes with desirable agronomic characteristics and a high level of soil stress tolerance. Subsequent research verified that the stress-tolerant regenerants had superior acid soil and drought stress tolerance to that of the donor parents, that their yield capabilities under stress were superior to their parents, and that their stress tolerance attributes were transferred in hybrid combinations. In vitro selection was not effective in increasing the number of field stress survivors. In fact, superior germplasms were developed from non-stressed callus or salt-stressed callus. In vitro selection reduced regeneration frequency and subsequent survival of plants under field stress. In vitro-stressed regenerants should be subjected only to non-stressed environments to maintain population numbers for field selection and thereafter should be subjected to stress environments during later (R5+) generations. The optimal strategy for the exploitation of somaclonal variation may be through short-term cell culture (〈 12 months) with no attempt at in vitro selection.
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  • 72
    ISSN: 1573-5060
    Keywords: Oryza sativa L. cv. Taipei 309 ; rice ; protoplasts ; direct DNA uptake ; kanamycin-resistant transgenic plants ; field trial ; glasshouse trial ; neomycin phosphotransferase II (npt II) gene ; gene expression and inheritance
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Summary The phenotypes of seed progeny (R2 generation) of Oryza sativa L. cv. Taipei 309, which carried the neomycin phosphotransferase II (npt II) gene, were compared with those of non-transformed, protoplast-derived plants of the same generation and non-transformed, seed-derived plants under field and glasshouse conditions. Under both conditions the transgenic plants were generally smaller, took longer to flower and had reduced fertility. Significant differences were observed between individuals within the group of transgenic plants. The npt II gene was present in most of the transgenic plants, but NPT II activity was only detected in a minority of individuals.
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  • 73
    ISSN: 1573-5060
    Keywords: bleomycin ; direct gene transfer ; expression ; irradiation ; petunia ; protoplasts
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Summary The production of transgenic plants by means of direct gene transfer to protoplasts is now a widely-used technique. The biological mechanisms underlying the transformation are still poorly understood, but many investigations have attempted to shed light on some components of this process. Varying the experimental conditions has in some cases led to better transformation rates, but further improvements of the protocols are possible. Such improvements will require a better understanding of how the alien DNA enters the cells, becomes integrated into the chromosomes and is treated as a part of the plant genome. Irradiation with sublethal doses of X-rays or UV-light has been shown to increase the transformation frequency, while certain drugs have been shown to act in a similar manner. The effects of these and other factors are discussed.
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  • 74
    ISSN: 1573-5060
    Keywords: callus culture ; organogenesis ; pea ; Pisum sativum ; somaclonal variation ; somatic embryogenesis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Summary The possibility of producing agronomically-useful somaclones via organogenesis and somatic embryogenesis from callus cultures of pea (Pisum sativum L.) was studied. Organogenic calli were induced from immature leaflets on MSB medium with NAA and BAP. Embryogenic calli were derived either from immature zygotic embryos (using 2,4-D) or from shoot apices (using picloram) of aseptically-germinated seedlings. The seed progenies (T1 to T3-generation) of primary regenerants were grown in field conditions and their phenotypic variation was evaluated and compared with control, non-tissue culture-derived plant material. In addition, electrophoretic analyses of selected isoenzyme systems and total proteins have been done. The results do not show dramatic changes in qualitative and quantitative traits. The evaluation of at least two future generations (T4, T5) is planned.
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  • 75
    Electronic Resource
    Electronic Resource
    Springer
    Euphytica 85 (1955), S. 323-327 
    ISSN: 1573-5060
    Keywords: Brassica napus ; fatty acids ; gas chromatography ; Lunaria annua ; protoplast regeneration ; somaclonal variation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Summary A programme of research was designed to investigate methods for the modification of the fatty acid profiles of high performance lines of oilseed rape (Brassica napus L.) in an attempt to produce lines with enhanced levels of industrially useful fatty acids. The methodology employed to achieve these objectives was based on the exploitation of somaclonal or protoclonal variation, and targeted somatic hybridization using wild cruciferous germplasm as fusion partners. A range of somaclonal lines was produced from shoot regeneration protocols. These lines underwent replicated, randomised glasshouse trials for morphological assessment followed by gas chromatographic analysis to monitor any changes in fatty acid profile. It was found that a small number of lines exhibited potentially useful changes in oleic acid and polyunsaturated fatty acid content. Protoplast regeneration and electrofusion protocols for a range of winter oilseed rape lines were developed, and methods for the isolation and fusion of protoplasts of the wild crucifer Lunaria annua (chosen for its high nervonic acid content) established.
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  • 76
    ISSN: 1573-5060
    Keywords: Linum usitatissimum ; linseed ; mutation breeding ; somaclonal variation ; fatty acids ; genetic engineering
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Summary In the early 1980s the phenomenon of somaclonal variation induced by cell culture was exploited to produce genetic variation in linseed. The linseed variety Andro, derived from the widely grown Canadian variety McGregor, was selected in saline culture and was released for production in Canada. ‘Andro’ possesses traits very different from its parent, such as increased seedling vigour and tolerance to heat stress. Additional stable somaclonal variation in characters such as yield, days to maturity, seed weight and oil content were subsequently induced in ‘McGregor’. However, despite extensive screening of the somaclonal variants, no significant variation in the fatty acid profile was found. Chemical mutagenesis using ethyl methanesulphonate was, however, succesful in modifying the fatty acid profile of McGregor. Initial screening of M2 seed by the thiobarbituric acid colourimetric procedure was followed by gas chromatography to select half-seeds with atypical fatty acid profiles. Two independent, partially dominant genes were identified that were responsible for reducing the linolenic acid (18 : 3) from 50% to 2% while increasing linoleic acid (18 : 2) to 70%. A single, partially dominant gene, inherited independently of the linolenic acid genes, increased palmitic acid (16 : 0) from 7% to 30% and palmitoleic acid (16 : 1) from trace amounts to 4%. Agrobacterium-mediated transformation of linseed has also been successful. Herbicide tolerance genes for glyphosate, sulfonylurea and phosphinothricin have been incorporated into Canadian varieties. Commercially useful levels of tolerance to sulfonylurea herbicides have been achieved with no adverse agronomic affect. It is expected that a transgenic variety containing this resistance will be registered for commercial production in Canada in 1994. Standard breeding techniques, the application of antisense technology and the overexpression of fatty acid synthesis genes are being used to further modify the fatty acid profile of linseed, as well as for the transfer of abiotic stress-related genes identified in bromegrass.
    Type of Medium: Electronic Resource
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