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1.
Protoplasts were isolated enzymatically from gametophytes of the marine green algaMonostroma angicava. The protoplasts regenerated in PES medium after gradual reduction of the osmoticum. Three types of developmental process were recognized in the protoplast regeneration: an original type, in which the protoplasts regenerated into leafy gametophytes; an apogamic type, in which they regenerated into sporophytes; a callus type, in which they regenerated into callus-like tissues. The resulting gametophytes and apogamic sporophytes became fertile in successive cultures. 相似文献
2.
Summary The conditions for effective isolation of viable protoplasts from Laminaria japonica with an alginase produced by marine bacterium Alteromonas sp. and a commercially available cellulase were investigated. The highest yields of viable protoplasts (7.910.4x106 cells g–1 FW) were obtained with a hypertonic solution containing 50 % seawater, 25 mM MgCl2, 5 mM HEPES buffer system, and 0.5 M mannitol. Protoplasts were not obtained from thalli of L. japonica when an abalone alginase (abalone acetone powder; AAP: Sigma) was used instead of the bacterial alginase. The isolated protoplasts were cultured in an PESI medium at 5 °C. Complete cell wall formation was observed within 7 days, and dividing cells were first observed in a 9-day-old culture. Some protoplasts regenerated into sheet-shaped thalli and rhizoid structures were also observed on some thalli after 30 to 40 days in culture. This is the first report of protoplast regeneration into plantlets of L. japonica Areschoug (Laminariales, Phaeophyceae).Abbreviations FW
Flesh weight
- AAP
Abalone acetone powder
- HEPES
N-2-hydroxy-ethylpiperazine-N-2-ethanesulfonic acid
- Tris
Tris(hyrdoxymethyl)aminomethane
- PESI
Provasoli's enriched seawater with iodine 相似文献
3.
Aldo Asensi Erwan Ar Gall Dominique Marie Claire Billot Patrick Dion Bernard Kloareg 《Journal of phycology》2001,37(3):411-417
In vitro cultures of Laminaria digitata (Hudson) Lamouroux stipe medullary explants (Laminariales, Phaeophyceae) gave rise to friable morula-like masses of pigmented cells. Upon subculture under continuous white light, they grew out as filaments, which tended to dissociate into spherical isolated cells, leading to the establishment of a suspended cell-filament culture. Depending on culture conditions, this cell-filament suspension underwent various forms of growth, including regeneration of morphologically normal sporophytes, and this developmental pathway was stimulated by blue light. Genotyping with nine polymorphic microsatellite markers indicated that the regenerated sporophytes and the cell-filament suspension shared the same genotype. As assessed by flow cytometric analysis of isolated nuclei, the cell-filament suspension exhibited a 2C nuclear DNA content, whereas the regenerated sporophytes displayed a 4C level. Chromosome counting, however, showed that both the mother suspension and the regenerated sporophytes were diploid, suggesting the involvement of polyteny in the regeneration process. 相似文献
4.
A protocol was developed for the isolation, culture and plant regeneration of protoplasts isolated from suspension cultures of Solanum lycopersicoides Dun. (LA 1990). Protoplasts were isolated by an overnight enzyme digestion, further purified by washing in W5 salts solution, and plated in two modified MS protoplast culture media with and without type VII agarose. The addition of agarose to the two culture media did not enhance plating efficiencies and shoot regeneration percentages and in some cases was even inhibitory. Unlike the experience with some other solanaceous species, the deletion of ammonium from the protoplast culture medium was not found to be beneficial. Protoplasts sustained continuous division in the modified MS media and up to 70% of the protoplast-derived calli readily regenerated shoots on MS salts and vitamins medium containing zeatin and GA. 相似文献
5.
Tomoki Kai Kazumi Nimura Hajime Yasui Hiroyuki Mizuta 《Journal of applied phycology》2006,18(1):95-101
Young sporophytes of Laminaria japonica Areshoug were cultured in six indole-acetic acid (IAA) concentrations (0, 10−8, 10−7, 10−6, 10−5, 10−4 M) to examine the effect of auxin on growth. The effects of auxin on sorus formation were also examined by using discs taken from the adult sporophyte. The auxin contents and IAA oxidase activities in the thallus and sorus parts of the sporophyte were determined with the blade and sporophyll of other Laminariales plants, Undaria pinnatifida (Harvey) Suringar and Alaria crassifolia Kjellman. The young sporophytes of L. japonica showed highest elongation rate in 10−5 M IAA. In contrast, the sorus formation on the discs cultured in 10−5 M IAA was markedly delayed in comparison with other concentrations, indicating that sorus formation was suppressed by IAA. Free and conjugated auxin contents were lower in the reproductive parts than in the vegetative parts. In three Laminariales sporophytes, IAA oxidase activity was about 3–9 times higher in the reproductive parts than in the vegetative parts. Taken together these results suggest that the growth and reproduction of Laminariales sporophytes are regulated by internal auxin levels. Elucidating the regulation mechanism is likely to provide information that is important for the management of plant production and the assessment of the physiological status of plants in the field. 相似文献
6.
Summary Protoplasts were isolated from Agrobacterium rhizogenes A4-transformed cell line of Medicago sativa L. The highest yield of protoplasts (4.2×106 per g fresh weight) was obtained from 12-d-old calluses after being subeultured on fresh medium. The viability of protoplasts
reached over 80%. Protoplasts were induced to undergo sustained divisions when cultured in Durand et al. (DPD) medium supplemented
with 2 mgl−1 (9.05 μM) 2,4-dichlorophenoxyacetic acid, 0,2mgl−1 (0.93 μM) kinetin, 0.3 M mannitol, 2% (w/v) sucrose, and 500 mgl−1 casein hydrolyzate at a plating density of 1.0×105 per ml. An agarose-beads culture method was appropriate for protoplast division of transformed alfalfa. The division frequency
was about 30%. Numerous hairy roots were induced from protocalluses on Murashige and Skoog medium without growth regulators.
Paper electrophoresis revealed that all of the regenerated hairy roots tested synthesized the corresponding opines. This protoplast
culture system would be valuable for further somatic hybridization in forage legumes. 相似文献
7.
Summary We have produced a large number of plants regenerated from protoplasts originally isolated from embryo-derived cell suspensions of wild barley, Hordeum murinum L.. Suspensions initially allowed protoplast isolation and culture 5.5 to 9 months from the date of callus initiation. Colony formation efficiencies ranged from 1.5 to 3.0 % and from 0.1 to 1.4 % for protoplast cultures with and without nurse cells, respectively. Both nurse and non-nurse techniques allowed efficient embryogenesis and plant regeneration. More than 400 shoots/plantlets have been obtained from 6 independent experiments. Over 150 plants have been transferred to the greenhouse. Protoplasts isolated from the youngest suspensions (5.5 months old) gave rise to the largest number of plants. Protoplasts isolated from suspensions as old as 15 months were also regenerable.Abbreviations 2,4-D
2,4-dichlorophenoxyacetic acid
- BAP
6-benzylaminopurine
- NAA
naphthaleneacetic acid
- L1, L2
medium according to Lazzeri et al. 1991
- L3 medium
medium according to Jähne et al. 1991a 相似文献
8.
Cell-suspension cultures were initiated from embryogenic calli of various barley cultivars. Seven fast-growing suspension lines were obtained from four different cultivars (cvs. Dissa, Emir, Golden Promise and Igri). Two of these cell suspensions showed morphogenic capacity. From a cell suspension of cv. Dissa, albino plantlets were regenerated when aggregates were cultured on solid medium. Aggregates of cv. Igri usually stopped differentiation at the globular stage, but occasionally formed scutellum-like structures. Five suspension lines were used for protoplast isolation and culture. Dividing protoplasts were obtained from all lines, but with cv. Igri a few divisions only and no further development were observed. Protoplasts from the various lines differed in the time of first division (2–14 d), division frequency (0.09–70.9%) and efficiency of colony formation (0.09–7.3%). Protoplasts isolated from the morphogenic cell suspension of cv. Dissa developed compact calli which sporadically regenerated albino plantlets.Abbreviations CC, MS, N6, SH, Kao8p
culture media; see Material and methods
- cv
chltivar
- dicamba
3,6-dichloro-o-anisic acid
- 2,4-D
2,4-dichlorophenoxyacetic acid
- picloram
4-amino-3,5,6-trichloropicolinic acid 相似文献
9.
Akira Inoue Chieco Mashino Teina Kodama Takao Ojima 《Marine biotechnology (New York, N.Y.)》2011,13(2):256-263
Functional recombinant abalone alginate lyase (rHdAly) and β-1,4-endoglucanase (rHdEG66) were expressed as secreted proteins
with baculoviral expression systems. The specific activity of each recombinant enzyme, 2,490 and 18.2 U/mg for rHdAly and
rHdEG66, respectively, was comparable to its native form at 30°C. Purified rHdAly and rHdEG66 showed the highest specific
activity both at 35°C and optimum pH 8.7 and 5.9, respectively. These properties were also comparable to those of the native
enzymes. Protoplast isolation was attempted from Laminaria japonica using both rHdAly and rHdEG66. When L. japonica blades were incubated in artificial seawater containing rHdAly and rHdEG66, very low numbers of protoplasts (<1 × 103 protoplasts/g fresh weight) resulted. However, using blades pretreated with proteinase K, the protoplast was increased up
to 5 × 106 protoplasts/g fresh weight. Since the average diameter of isolated protoplasts was 11.6 μm, these cells were mostly derived
from the epidermal layer rather than the cortical layer. Our results suggest that at least three enzymes, alginate lyase,
cellulase, and protease, are essential for effective protoplast isolation from L. japonica. The protoplast isolation method in this study is more useful than earlier methods because it preferentially yielded protoplasts
of the epidermal layer, which are known to be able to be regenerated. 相似文献
10.
Protoplasts were isolated from suspension cultures of various cell lines of Duboisia myoporoides R. Br. There were differences among cell lines with respect to optimal conditions for protoplast isolation including the amount and kind of enzymes and the osmoticum concentration. Protoplasts isolated from one cell line were successfully cultured and induced to form cell colonies in liquid modified B5 medium. Addition of conditioned medium, coconut milk and glucose as an osmoticum to protoplast culture medium as well as maintenance of high protoplast density in culture (> 105/ml) were essential to obtain protocolony formation. Reduction of osmoticum concentration and deletion of coconut milk and conditioned medium from the culture medium were necessary to allow further colony development leading to cellus formation. Intact plants regenerated from calli derived from protoplasts were successfully transferred to pots. 相似文献
11.
Protoplasts isolated from protonemata of Funaria hygrometrica wild type and developmentally abnormal mutants were individually selected and cultured in microdroplets of defined, unconditioned culture medium. Up to 60 % of the microcultured wild type protoplasts regenerated whole plants after 4 weeks. Electrofusion in different combinations of defined protoplast pairs of wild type and mutated forms was performed. The products derived from one-to-one electric field induced fusion were efficiently microcultured and somatic hybrids were regenerated. 相似文献
12.
Mizoguchi M Kitano S Takahashi W Sugawara F Tanaka O 《Bioscience, biotechnology, and biochemistry》2006,70(11):2751-2753
We have developed a simple and efficient method for protoplast isolation from Pleurotus cornucopiae. Protoplasts were isolated from aerial mycelia cultured on potato dextrose agar medium without time-consuming propagation in liquid culture. Protoplast yield was significantly increased by means of a decompressing pretreatment of mycelia in enzyme solution and a subsequent enzyme reaction with vibrational mixing. The isolated protoplasts regenerated mycelia and these mycelia formed fruit-bodies without any morphological abnormalities. 相似文献
13.
Protoplasts isolated from thalli of four Porphyra species regenerated successfully into differentiated plantlets. The efficiency of protoplast isolation and the developmental patterns of the regenerating protoplasts depended on the type of tissues from which they were isolated. However, culture conditions greatly influenced the patterns of development at the cellular and organismal levels. Sorbitol, nitrogen, and agar concentration in the medium controlled rates of cell division, thickening of cell walls, development of rhizoids, and formation of calluses or differentiated blades. Agitation disturbed the attachment of the protoplasts to a substrate. Cells in agitated cultures produced suspensions of single cells and non-polarized small calluses. Calluses which developed from protoplasts survived in storage for over two years. The stored calluses, and cells and protoplasts that were isolated from them, were subcultured successfully. We forsee extensive use of Porphyra cell suspensions for strain selection and vegetative propagation of cultivars. This technology, which makes vegetative cloning of selected Porphyra plants possible, may eliminate the need for cultivation and storage of the conchocelis phase. Protoplasts are also being used as tools for studies in genetic engineering of these commercial species. 相似文献
14.
Rana Inder Singh Kanojiya Aarti Sandhu Sardul Singh 《Indian journal of microbiology》2007,47(4):369-372
Interspecies fusants are formed between Agaricus bisporus and Agaricus bitorquis by protoplast fusion technique. Protoplasts were isolated and regenerated by using Novozyme 234 lytic enzyme. Twenty slow
growing isolates were separated from the protoplast regenerated colonies, which were assumed as homokaryons (putative homokaryons).
These twenty isolates were subjected to growth rate, colony morphology and spawn run studies for screening of true homokaryons.
Antifungal markers were developed for selection of fusants. 相似文献
15.
Mariculture of Laminaria japonica (Laminariales, Phaeophyceae) using protoplast regeneration 总被引:1,自引:0,他引:1
The conditions for culture of viable protoplasts from Laminaria japonica were investigated and the regenerative processes were observed in detail. As a result of culturing at four water temperatures (5, 10, 15, and 18°C), we found that low water temperature was better for survival, division and rhizoidal formation of protoplast‐derived cells. Only epidermis‐derived protoplasts developed into normal sporophytes through a direct developmental process. Protoplast‐derived cells divided after 5 days and 2–10 celled germlings formed the first rhizoids after 15 days. Only initial sporophytes with the first rhizoids grew to normal sporophytes with multilayered blades, stipes and holdfasts. When these young sporophytes were transplanted into the sea, they grew to normal fertile sporophytes. 相似文献
16.
Yu-Ji Lian Xiao-Mei Zhao Guang-Zhe Lin Hak-tae Lim 《Plant Cell, Tissue and Organ Culture》2012,109(3):565-572
Protoplasts were isolated from the young leaves of rapid cycling Brassica rapa and cotyledons and hypocotyls of 10-day-old Brassica juncea seedlings. Protoplasts were fused by 40% polyethylene glycol and cultured in modified K8p medium supplemented with 2.5 mg·l−1 isopentenyladenine (2ip), 0.5 mg·l−1 naphthaleneacetic acid, 1 mg·l−1 2,4-dichlorophenoxyacetic acid (2,4-D), 0.1 mg·l−1 zeatin, 1% dimethyl sulfoxide, and 0.4 M mannitol as osmoticum. After 3 days of initial culture, 3 different culture methods
were employed and evaluated. The highest plating efficiency (1.97%) was obtained with a semi-solid agarose embedding culture
method. Both shoots and somatic embryos formed from protoplast culture-derived calli. The somatic embryos were derived from
asymmetrically divided calli that developed progressively into deep-purple heart shapes as well as the early-torpedo and bipolar
stages to finally form complete plantlets. Thirteen putative somatic hybrids were produced via somatic embryogenesis or organogenesis.
Random amplified polymorphism DNA analysis was performed to identify somatic hybrids. Six regenerated plants had a chromosome
number of 2n = 56 the same as the sum of B. juncea (2n = 36) and B. rapa (2n = 20) chromosomes; 2 plants had a chromosome number of 2n = 54. These regenerated plants exhibited morphology intermediate
to those of their parents. The flowers of somatic hybrids exhibited a range of variation; some were normal, while others were
abnormal. No pollen was produced from regenerated plants. Two plants had crinkled petal-like stamens. 相似文献
17.
Protoplasts were isolated from spongy calli in a well growing state. Protoplasts were induced to undergo sustained divisions and to form colonies in the liquid C81V medium supplemented with 2,4-D and kinetin. When protoplast derived colonies were transferred onto agarsolidified medium, the spongy, white calli developed. After being subcultured on N6 medium plus 6BA and IBA, the light-yellow, granular embryogenic calli emerged on the protoplast regenerated callus surface. A large number of plantlets were obtained on MS medium with NAA and IBA via somatic embryogenesis Cytological observation on the donor calli used for protoplast isolation and plantlets regenerated from protoplasts were carried out. Remarkable variation of nucleus morphology and chromosome numbers were observed in donor calli. However, the cytological abnormalities in plantlets regenerated from protoplasts were comparatively less seen. The reason are discussed. 相似文献
18.
The regeneration of meristematic tissues from sporophytes of Laminaria digitata was studied by protoplast and tissue culture. Sequential treatment of explants in sterile seawater with 1% Betadine for 5 min,
1% commercial bleach for 1–2 min and 2% antibiotic treatment supplemented with 1 μM GeO2 overnight enabled viable explants as high as 55%. Different morphogenetic responses were observed from tissue culture on
media supplemented with plant growth regulators alone or in combination, mainly filamentous calluses up to 50% according to
the media. Dark green compact calluses were observed on two combinations: 4 μM Pi + 2 μM N-(2-chloro-4-pyridyl)-N’-phenylurea
(CPPU) and 0.04 μM Pi + 0.44 μM 6-benzylaminopurine. Thalloid-like structures comparable to adventitious buds were regenerated
on medium supplemented with 4 μM Pi + 0.45 μM zeatin but at low frequency suggesting a strong genotypic effect. Friable calluses
were developed from protoplasts in enriched medium with polyamines and containing 0.40 μM CPPU + 0.45 μM 2,4-dichlorophenoxyacetic
acid. In order to produce protoplasts, a one-step enzymatic protocol was developed and yields reached 22 × 106 protoplasts per gram of fresh weight. 相似文献
19.
Y.-J. Zhang Y.-Q. Qian X.-J. Mu Q.-G. Cai Y.-L. Zhou X.-P. Wei 《Plant cell reports》1998,17(10):819-821
Newly expanded in vitro leaves of Actinidia eriantha were used for protoplast isolation. Protoplasts were cultured in liquid MS medium (lacking NH4NO3) supplemented with 2,4-D (2,4-dichlorophenoxyacetic acid) and 0.4 M glucose. The plating efficiency after 3 weeks of culture was 19.4%, and calli were recovered without addition of fresh medium.
These calli regenerated shoots on transfer to MS medium containing 2.28 μM zeatin and 0.57 μM IAA (indole-3-acetic acid). Regenerated shoots were rooted by immersion in 20 ppm IBA (indole-3-butyric acid) solution before
culturing on half-strength MS medium lacking growth regulators. Somaclonal variation, in terms of chromosome number and nuclei
per cell of protoplast-derived plants, was estimated.
Received: 15 March 1997 / Revision received: 27 January 1998 / Accepted: 7 March 1998 相似文献
20.
Protoplasts were isolated enzymatically from the carrageenophyte red alga Grateloupia turuturu (Halymeniales, Rhodophyta) that occurs along the coast of the French Channel in Normandy. Effects of the main factors on
the protoplast yield were identified to improve the isolation protocol. The optimal enzyme composition for cell wall digestion
and protoplast viability consisted of 2% cellulase Onozuka R-10, 0.5% macerozyme R-10, 2% crude extract from viscera of Haliotis tuberculata, 0.8 M mannitol, 20 mM sodium citrate, 0.3% bovine serum albumin at 25°C, and 4-h incubation period. The protoplasts were
approximately 5–15 μm in diameter, liberated mainly from the surface cell layers. Maximum yield was 1.5 × 107 protoplasts g-1 fresh tissue. The protoplasts underwent initial division after 14 days with a high density level of 1 × 106 cells mL-1 in culture medium and developed into microthalli of a line of two to six cells. 相似文献