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1.
Preparation of protoplasts from Laminaria japonica using native and recombinant abalone alginate lyases 总被引:1,自引:0,他引:1
Laminaria japonica protoplasts were released with high yields using the abalone alginate lyase HdAly in combination with a cellulase and chelating
agents. Addition of EDTA at concentrations higher than 10 mM to Laminaria thalli which had been preincubated with HdAly and Cellulase Onozuka, dramatically improved the yield of protoplasts. EDTA
was far more effective than EGTA, indicating that chelating divalent metal ions such as Mg2+ and Sr2+ in addition to Ca2+ is a key factor for high-yield production of Laminaria protoplasts. Protoplasts had a mean diameter of 27 μm, suggesting that most protoplasts were derived from cortical cells
rather than epidermal layer cells. Recombinant HdAly (rHdAly) was produced from a cDNA clone in the Sf9 insect cell expression
system. rHdAly had substantially the same enzymatic properties and protoplast-producing ability as did native HdAly. The optimal
conditions for high yield production of protoplasts from Laminaria using native and recombinant HdAlys were investigated. 相似文献
2.
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
3.
Vishal Gupta Manoj Kumar Puja Kumari C. R. K. Reddy Bhavanath Jha 《Journal of applied phycology》2011,23(2):209-218
This study reports on the optimization of protoplast yield from two important tropical agarophytes Gracilaria dura and Gracilaria verrucosa using different cell-wall-degrading enzymes obtained from commercial sources. The conditions for achieving the highest protoplast
yield was investigated by optimizing key parameters such as enzyme combinations and their concentrations, duration of enzyme
treatment, enzyme pH, mannitol concentration, and temperature. The significance of each key parameter was also further validated
using the statistical central composite design. The enzyme composition with 4% cellulase Onozuka R-10, 2% macerozyme R-10,
0.5% pectolyase, and 100 U agarase, 0.4 M mannitol in seawater (30‰) adjusted to pH 7.5 produced the highest protoplast yields
of 3.7 ± 0.7 × 106 cells g−1 fresh wt for G. dura and 1.2 ± 0.78 × 106 cells g−1 fresh wt for G. verrucosa when incubated at 25°C for 4–6 h duration. The young growing tips maximally released the protoplasts having a size of 7–15 μm
in G. dura and 15–25 μm in G. verrucosa, mostly from epidermal and upper cortical regions. A few large-size protoplasts of 25–35 μm, presumably from cortical region,
were also observed in G. verrucosa. 相似文献
4.
Ten accessions belonging to the Brassica oleracea subspecies alba and rubra, and to B. oleracea var. sabauda were used in this study. Protoplasts were isolated from leaves and hypocotyls of in vitro grown plants. The influence of selected factors on the yield, viability, and mitotic activity of protoplasts immobilized
in calcium alginate layers was investigated. The efficiency of protoplast isolation from hypocotyls was lower (0.7 ± 0.1 × 106 ml−1) than for protoplasts isolated from leaf mesophyll tissue (2 ± 0.1 × 106 ml−1). High (70–90%) viabilities of immobilized protoplasts were recorded, independent of the explant sources. The highest proportion
of protoplasts undergoing divisions was noted for cv. Reball F1, both from mesophyll (29.8 ± 2.2%) and hypocotyl (17.5 ± 0.3%)
tissues. Developed colonies of callus tissue were subjected to regeneration and as a result plants from six accessions were
obtained. 相似文献
5.
Aoyagi H 《Biotechnology letters》2006,28(20):1687-1694
An index [kv: average isolation rate of viable protoplast (number/ml min)] was established to evaluate the optimal conditions for protoplast isolation from cultured plant cells. The optimal conditions for protoplasts isolation from Nicotiana tabacum BY2 cultured cells could be determined on the basis of the kv [31.7 × 103 (number/ml min)]. The colony-forming efficiency of the protoplasts was about 46%. The optimal conditions for protoplasts isolation from Catharanthus roseus [kv = 38.1 × 103 (number/ml min)] and Wasabia japonica [kv = 14.2 × 103 (number/ml min)] cultured cells could also be determined. Furthermore, a method for rapid regenerating cell wall of protoplast in liquid culture using alginate gel containing locust bean gum was developed. 相似文献
6.
Karin Sonntag Brigitte Ruge-Wehling Peter Wehling 《Plant Cell, Tissue and Organ Culture》2009,96(3):297-305
A method for isolation and shoot regeneration from electrofused protoplasts of L. angustifolius and L. subcarnosus was developed. Viable protoplasts were isolated from leaves of in-vitro grown seedlings at an average yield of 6 × 105 protoplasts g−1 fresh weight. Liquid and agarose solidified B5 media were used for protoplast culture. In the liquid-culture system, all
tested media, VKM, P1 and KM8p, were applicable for inducing cell division (84% of all tested petri dishes at four weeks)
and colony formation. Media containing additional carbohydrates were suitable to produce compact calli with green and brown
pigmentations in different combinations. Analysis of callus with molecular markers allowed to identify six somatic hybrids.
However, none of the parental-protoplast derived cell colonies could develop shoots. This is the first report on protoplast
fusion of L. angustifolius and L. subcarnosus with subsequent shoot regeneration. 相似文献
7.
The ability to measure directly individual protoplast ion fluxes is a valuable addition to patch clamp and other techniques
when using protoplasts to study membrane transporters. Before interpreting observations on protoplasts in terms of behaviour
of intact cells and tissues, some methodological questions should be addressed. These include effects of space and time variations
of transporter activities over the membrane, the osmotic dependence of specific ion transporters and the effect of the regenerating
cell wall. In this study net H+ and Ca2+ fluxes were measured from individual corn (Zea mays L.) coleoptile protoplasts using a non-invasive microelectrode technique for ion flux measurements. For Ca2+, the flux distribution was almost symmetrical, ranging ±30 nmol · m−2 · s−1 around zero. For H+ it was skewed towards efflux ranging from −100 to +10 nmol · m−2 · s−1. The distribution of H+ fluxes through the protoplast surface was a complex mosaic which changed with time, sometimes showing oscillations. These
flux variations with time and position around the surface, apparently driven by endogenous mechanisms, may be relevant to
protoplast pH homeostasis. When the new cell wall was partially regenerated on the next day, the correlation between H+ and Ca2+ fluxes increased, which is consistent with the weak-acid Donnan-Manning model of cell wall ion exchange.
Received: 11 June 1997 / Accepted: 10 July 1997 相似文献
8.
Summary. Amino acids analysis in single wheat embryonic protoplast was performed using capillary electrophoresis equipped with laser-induced
fluorescence (CE-LIF), combination with tissue culture technique. Reagent fluorescein isothiocyanate (FITC) was introduced
into living protoplasts by electroporation for intracellular derivatization. A special osmotic buffer (0.6 mol/L mannitol,
5 mmol/L CaCl2) was used to keep the osmotic balance of embryonic protoplasts during the protoplasts derivatization. After completion of
the derivatization reaction in the protoplasts, a single protoplast was drawn into the capillary tip by electroosmotic flow.
Then a 0.1 M NaOH lysing solution was injected by diffusion. The derivatized amino acids were separated by capillary electrophoresis
and detected by laser-induced fluorescence detection after the protoplast was lysed Nine amino acids were quantitatively and
qualitatively determined and compared in lysate and single protoplast of wheat embryonic cells respectively, with mean concentrations
of amino acids ranging from 2.68×10−5 mol/L to 18.18×10−5 mol/L in single protoplast. 相似文献
9.
A protocol is presented for regenerating plants from leaf protoplasts of Oenothera. The method uses (1) embedding of isolated protoplasts at high cell densities in thin alginate layers, (2) initial culture
in B5 medium containing 3 mg l–1
α-naphthaleneacetic acid (NAA) and 1 mg l-1 6-benzylaminopurine (BAP), (3) reduction of the osmotic pressure of the culture medium at early stages of culture and (4)
plating of microcolonies recovered from the alginate onto solid B5 medium with 3 mg l–1 NAA and 1 mg l–1 BAP. The shortest time required from protoplast isolation to the appearance of shoot initials was 7 weeks. The efficiency
of the procedure for protoplast to cell line formation is high (about 80%).
Received: 17 February 1997 / Revision received: 6 November 1997 / Accepted: 15 November 1997 相似文献
10.
The effects of alginate on the physiological activities of plant cells were studied. Addition of alginate oligomer (AO) to
the suspension culture of Catharanthus roseus L. or Wasabia japonica cells promoted the production of antibiotic enzymes such as 5′-phosphodiesterase or chitinase respectively. Ajmalicine (a
secondary metabolite) production by C. roseus CP3 cells was also promoted when AO was added to the suspension culture. On the basis of these results, we assumed that alginate
is an elicitor-like substance. We therefore compared the effect of AO on C. roseus L. and W. japonica cells with those of chitosan oligomer (CO) and oligo-galacturonic acid (OGA), which are well known as an exogenous elicitor
and endogenous elicitor respectively. The effects of various concentrations of AO, OGA, and CO on the physiological activities,
membrane permeability and protoplast formation of C. roseus L. or W. japonica cells were investigated. AO and OGA showed similar physiological effects, which were quite different from those of CO. Since
alginate appeared to have similar effects to galacturonic acid, we concluded that alginate acts as an endogenous elicitor.
Both alginate and galacturonic acid are uronic acids, and we considered their structural similarity. The effects of esterification
of the carboxylic groups of alginate by propylene oxide were also studied. The greater the degree of esterification, the less
the secretion of 5′-phosphodiesterase. Hence we assumed that carboxylic groups have an important role in the initiation of
the elicitation reaction in plant cells, as shown in the case of galacturonic acid.
Received: 18 January 1999 / Received revision: 2 April 1999 / Accepted: 1 May 1999 相似文献
11.
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
12.
An improved approach for transformation of plant cells by microinjection: molecular and genetic analysis 总被引:4,自引:0,他引:4
Martin Schnorf Gabriele Neuhaus-Url Alessandro Galli Shigeru Iida Ingo Potrykus Gunther Neuhaus 《Transgenic research》1991,1(1):23-30
A new culture method for the injection of tobacco mesophyll protoplasts has been established. The protoplasts are embedded
in a thin layer of alginate and are nourished from the medium in the underlying basislayer. In the alginate layer the protoplasts
regenerate to calli at a frequency of up to 80%. Embedded protoplasts can be selected either with 50 mg l−1 kanamycin or 5 mg l−1 paromomycin. Single resistant cells can be recovered from about 10 000 sensitive cells in one alginate layer. Injection of
theneo gene (coding for neomycin phosphotransferase II) into protoplast derived single cells in the alginate layer results in kanamycin
resistant colonies that can be regenerated to mature plants. These plants express the neomycin phosphotransferase as shown
by enzyme activity assay. The integration of the transgene into the plant genome could be proved by Southern hybridization
to high molecular weight DNA. With this culture method 100 cells can be injected per hour. Transformation frequencies range
from 2 to 20%. In crossing experiments, it was shown that the foreign gene is transmitted to the next generation in a Mendelian
fashion. 相似文献
13.
Barbara Duquenne Tom Eeckhaut Stefaan Werbrouck Johan Van Huylenbroeck 《Plant Cell, Tissue and Organ Culture》2007,91(2):165-173
Vital protoplasts from Spathiphyllum wallisii ‘Alain’ and Anthurium scherzerianum ‘238’ were isolated from both somatic embryos and leaves. The highest yields were obtained when 1.5% cellulase, 0.5% macerase
and 0.5% driselase were used for Spathiphyllum wallisii leaves and 0.5% cellulase, 0.3% macerase and 0.5% driselase for Anthurium scherzerianum embryos. About 1 × 106 protoplasts g−1 and 1 × 105 protoplasts g−1 could be isolated from leaves and embryos, respectively. For protoplast fusion Spathiphyllum wallisii ‘Alain’ and Anthurium scherzerianum ‘238’ were mixed in a 1:1 ratio in a fusion solution containing 1 mM CaCl2·2H2O, 1 mM MES and 0.5 M mannitol. Fusion was performed by protoplast alignment under 500 V cm−1 alternating current for 60 s and subsequent generation of two pulses of 4500 V cm−1 direct current during 50 μs. Development until colony stage was achieved using agarose beads for protoplast culture. 相似文献
14.
Leaves of Solanum virginianum plants were used for protoplast isolation. To support cell wall formation and cell division, protoplasts were cultured in
thin alginate layers floated in liquid medium. When protoplasts were plated at a density of 1.0 × 106/ml in Kao and Michyaluk (KMp8) medium supplemented with 0.5 mg/l zeatin, 1.0 mg/l 2,4-dichlorophenoxyacetic acid, and 1.0 mg/l
α-naphthaleneacetic acid, 42.3% of the dividing cells developed microcalli in 3–4 weeks. Shoot formation via organogenesis
of protoplast-derived calli was achieved for 28% of calli transferred to solidified KMp8 medium supplemented with 2.0 g/l
zeatin and 0.1 mg/l 3-indol acetic acid in about 2 weeks. Further shoot development was observed in Murashige and Skoog (MS)
medium without growth regulators and roots were induced after transfer to MS medium containing 1.0 mg/l 3-indol butyric acid.
Regenerated plants have normal morphology. 相似文献
15.
Ewa Grzebelus Marek Szklarczyk Rafal Baranski 《Plant Cell, Tissue and Organ Culture》2012,109(1):101-109
An easy and effective regeneration system from leaf- and hypocotyl-derived protoplasts was established for carrot. The protoplast
isolation efficiency after preplasmolytic treatment and digestion of source material in enzyme mixture consisted of 1% cellulase
Onozuka R-10 and 0.1% pectolyase Y-23 reached on average 3 × 106 and 106 protoplasts per g of leaf and hypocotyl tissue, respectively. A modified thin alginate layer technique was applied for the
protoplast culture. Direct somatic embryogenesis on a simplified Kao and Michayluk medium in the presence of 2,4-D and zeatin
occurred during cultivation of both leaf- and hypocotyl-derived protoplasts for all accessions used. Morphologically normal
plants were produced at very high efficiency within two months after initiation of the protoplast culture. Ninety three percent
of in vitro derived plants were diploids. Pollen viability and seed set after self-pollination were similar to those of plants
obtained from seeds. 相似文献
16.
Protoplasts isolated from the mycobiont of a cultured lichen Usnea ghattensis were fused with protoplasts of the fungus Aspergillus nidulans in order to increase the growth rate of the cultured lichen mycobiont in vitro. The maximum protoplast yield (102 × 104/g fresh cell mass) was reached in citrate buffer with 50 mmol/L 2-sulfanylethanol (‘2-mercaptoethanol’) containing 0.1 %
Novozym after 1.5 h at pH 5 and ≤25 °C. The increase in the concentration of the above effectors or the addition of others
(e.g., MgSO4) as well as increase in time, shaking frequency, etc. caused the lower yield of protoplasts. The fused protoplasts were regenerated after transfer to malt extract-yeast extract
medium and produced, after a 45-d cultivation, a fresh cell mass of 0.232 g (from starting 0.3 g) along with the lichen substance
usnic acid. 相似文献
17.
RNA isolation is difficult in some plants and algae because phenolics, polysaccharides, or other compounds can bind or co-precipitate
with RNA, and because the success of RNA isolation can be strain-specific and species-specific. To create an improved RNA
isolation protocol for Laminaria japonica Aresch (Laminariaceae, Phaeophyta), four methods for extracting RNA were tested. A cetyltrimethylammonium bromide (CTAB)-based
RNA extraction protocol was developed that clearly showed 28S and 18S ribosomal RNA bands and produced RNA with high yield
(68 μg g−1 fresh weight) and high quality (A
260/280 ratio 1.96 ± 0.05). The isolated RNA was intact, and RT-PCR analysis confirmed that further molecular application is feasible. 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.
M. A. K. Azad S. Yokota F. Ishiguri N. Yoshizawa 《In vitro cellular & developmental biology. Plant》2006,42(6):502-507
Summary A regeneration system from protoplast to plantlet for a medicinal plant species, Phellodendron amurense Rupr., has been developed. Leaves of micropropagated shoots or plantlets were selected as plant materials for protoplast
isolation. The yield and viability of leaf protoplasts were greatly influenced by enzyme combination, treatment time and osmoticum.
The highest viability (86%) with a yield of 7.1×105 protoplasts per gram fresh weight was obtained with a 6-h digestion in 1% Cellulase Onozuka R-10 plus 1% Driselase-20. Sustained
cell division and colony formation from the protoplasts were best supported at a plating density of 4×105−6×105 protoplasts per milliliter using a 0.2% gellan gum-solidified or liquid MS (Murashige and Skoog, 1962) medium containing
0.6M mannitol, 2.0μM 6-benzylaminopurine (BA) with 4.0 μM α-naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA), or 2,4-dichlorophenoxyacetic acid (2,4-D). The protoplast-derived
colonies formed green compact calluses when transferred to a solidified MS medium containing 2.0 μM BA with 4.0μM NAA of IBA. Shoot regeneration from protoplast-derived calluses was induced on MS medium supplemented with 2.0 μM BA and 1.0μM NAA or 2.5μM IBA. Shoot multiplication and elongation occurred on MS medium containing 1.0μM BA. In vitro-grown shoots were rooted on MS medium with either 0.5–4.0μM IBA or NAA. Regenerants were transferred to the Kanuma soil and successfully established under greenhouse conditions. 相似文献
20.
A quantitative study of indole-3-acetic acid (IAA) turnover, and the contribution of tryptophan-dependent and tryptophan-independent
IAA-biosynthesis pathways, was carried out using protoplast preparations and shoot apices obtained from wild-type and transgenic,
IAA-overproducing tobacco (Nicotiana tabacum L.) plants, during a phase of growth when the level of endogenous IAA was stable. Based on the rate of disappearance of [13C6]IAA, the half-life of the IAA pool was calculated to be 1.1 h in wild-type protoplasts and 0.8 h in protoplasts from the
IAA-overproducing line, corresponding to metabolic rates of 59 and 160 pg IAA (μg Chl)−1 h−1, respectively. The rate of conversion of tryptophan to IAA was 15 pg IAA (μg Chl)−1 h−1 in wild-type protoplasts and 101 pg IAA (μg Chl)−1 h−1 in protoplasts from IAA-overproducing plants. In both instances, IAA was metabolised more rapidly than it was synthesised
from tryptophan. As the endogenous IAA pools were in a steady state, these findings indicate that IAA biosynthesis via the
tryptophan-independent pathway was 44 pg IAA (μg Chl)−1 h−1 and 59 pg IAA (μg Chl)−1 h−1, respectively, in the wild-type and transformed protoplast preparations. In a parallel study with apical shoot tissue, the
presumed site of IAA biosynthesis, the rate of tryptophan-dependent IAA biosynthesis exceeded the rate of metabolism of [13C6]IAA despite the steady state of the endogenous IAA pool. The most likely explanation for this anomaly is that, unlike the
protoplast system, injection of substrates into the apical tissues did not result in uniform distribution of label, and that
at least some of the [2H5]tryptophan was metabolised in compartments not normally active in IAA biosynthesis. This demonstrates the importance of using
experimental systems where labelling of the precursor pool can be strictly controlled.
Received: 18 January 2000 / Accepted 24 February 2000 相似文献