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1.
Fabiana R. X. Batista Carlos A. Pereira Ronaldo Z. Mendon?a Angela M. Moraes 《Cytotechnology》2005,49(1):1-9
Animal cells can be cultured both in basal media supplemented with fetal bovine serum (FBS) and in serum-free media. In this
work, the supplementation of Grace’s medium with a set of nutrients to reduce FBS requirements in Spodoptera frugiperda (Sf9) cell culture was evaluated, aiming the production of Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) at a cost lower than those for the production using Sf900 II medium. In Grace’s medium supplemented
with glucose, Pluronic F68 (PF68) and yeast extract (YE), the effects of FBS and milk whey ultrafiltrate (MWU) on cell concentration
and viability during midexponential and stationary growth phase were evaluated. In spite of the fact that FBS presented higher
statistical effects than MWU on all dependent variables in the first cell passage studies, after cell adaptation, AgMNPV polyhedra
production was comparable to that in Sf900 II. Batch cultivation in Grace’s medium with 2.7 g l−1 glucose, 8 g l−1 YE and 0.1% (w/v) PF68 supplemented with 1% (w/v) MWU and 3% (v/v) FBS increased viable cell concentration to about 5-fold
(4.7×106 cells ml−1) when compared to Grace’s containing 10% (v/v) FBS (9.5×105 cells ml−1). AgMNPV polyhedra (PIBs) production was around 3-fold higher in the MWU supplemented medium (1.6×107 PIBs ml−1) than in Grace’s medium with 10% FBS (0.6×107 PIBs ml−1). This study therefore shows a promising achievement to significantly reduce FBS concentration in Sf9 insect cell media,
keeping high productivity in terms of cell concentration and final virus production at a cost almost 50% lower than that observed
for Sf900 II medium.
C.A. Pereira is recipient of a CNPq fellowship. 相似文献
2.
Summary New cell lines, designated as ML-DmDl≈10, were established from dissociated imaginal discs ofDrosophila melanogaster. The culture medium was prepared by mixing in a 1:1 ratio Cross and Sang’s M3(BF) medium, supplemented with 10% heat inactivated
fetal bovine serum (FBS), with the supernatant of a primary embryonic cell culture made in the M3(BF) medium and supplementing
this mixture with insulin. One cell line was established in the medium containing larval hemolymph instead of the primary
culture supernatan, and another was established in fresh M3(BF) medium supplemented with insulin and FBS. In these mediums,
imaginal disc cells first formed aggregates and cellular vesicles within a few weeks followed by the proliferation of thin-layered
cells around them after about 1 mo. Ten cell lines have so far been established from two kinds of imaginal discs and disc
mixtures. The ploidy of these cell lines was predominantly diploid. Population doubling time was about 50 to 70 h at 3 to
10 mo. after initiation of the culture. When the cell aggregates formed in vitro were implanted in metamorphosing larvae,
they differentiated at high frequency into adult cuticular strutures in the early phase of the primary culture. This differentiation
of aggregates was also observed, though at low frequency, in a culture maintained by dilution-transfer for 6 to 15 mo. in
vitro. 相似文献
3.
Pankaj K. Sikka Kenneth E. McMartin 《In vitro cellular & developmental biology. Animal》1996,32(5):285-291
Summary Anin vitro model to establish primary and subcultures of rat kidney proximal tubule (RPT) cells is described. After excising the kidneys
and separating the cortex, the cortical tissue is digested with the enzyme DNAse-collagenase (Type I) resulting in a high
yield of viable RPT Cells. The isolated RPT cells are then seeded onto rat tail collagen-coated surfaces and grown to confluency
in a serum-free, hormonally defined medium. The cell yield can be increased by transfering the conditioned medium on Day 1
to more rat tail collagen-coated surfaces. RPT cell attachment and morphology was better on rat tail collagen-coated surfaces
than on bovine collagen Type I coated surfaces. The culture medium was a 1∶1 mixture of Ham’s F-12 and Dulbecco’s modified
Eagle’s medium supplemented with bovine serum albumin, insulin, transferrin, selenium, hydrocortisone, triiodothyronine, epidermal
growth factor, and glutamine. The RPT cells became confluent in 7–10 d, at which point they could be subcultured by trypsinizing
and growth in the same medium. In some studies, 10 ng/ml cholera toxin was added to the culture medium. We could passage the
RPT cells up to 14 times in the presence of cholera toxin. The cells were investigated for activity of several markers. The
cells were histochemically positive for alkaline phosphatase and γ-glutamyl transpeptidase activity and synthesized the intermediate
filament pankeratin. The RPT cells displayed apically directed sodium-dependent active glucose transport in culture. Hence,
the RPT cells retain structural and functional characteristics of transporting renal epithelia in culture. This rat cell culture
model will be a valuable tool for substrate uptake and nephrotoxicity studies. 相似文献
4.
Stephen M. Ferkovich Herbert Oberlander Charles Dillard Eddie Leach 《In vitro cellular & developmental biology. Animal》1994,30(4):279-282
Summary Embryos of the parasitoidMicropolitis croceipes develop from pregerm band stage to first larval instar in cell culture medium conditioned by a cell line (IPLB-LdFB) derived
from fat body from an atypical hostLymantria dispar. However, the percentage of eggs that develop normally to the first larval instar stage is significantly less than for those
maintained in IPL-52B medium conditioned with host fat body tissue. Therefore, we examined the capacity of five insect cell
lines to promote growth and development of pregerm band eggs in five media, IPL-52B, TC-199, TC-100, Grace’s, and ExCell 400.
The developmental response ofM. croceipes was dependent both on the cell line and the cell culture medium used. TC-100, TC-199, and Grace’s media promoted development
to the germ band stage without the need for conditioning with host tissue. IPL-52B supported development to the germ hand
stage when a defined lipid concentrate was added. In IPL-52B medium, the IPLB-LdFB cell line promoted a significantly higher
number of eggs developing to germ band relative to the other cell lines; however, none of the cell line-conditioned IPL-52B
medium significantly stimulated egg hatch relative to the control medium. None of the cell line-conditioned Grace’s media
had a significant effect on eggs attaining germ band stage compared with the Grace’s control medium. However, Grace’s medium
conditioned with the IAL-TND1 and IPLB-LdFB cell lines promoted development beyond germ band, resulting in a significantly
higher percentage of hatching eggs than the Grace’s control medium. Although the BCIRL-HZ-AMI cell line, which is derived
from the parasitoid’s typical host, did not induce hatch in either IPL-52B medium or Grace’s medium, it promoted hatch in
TC-199 and Excell 400 media. Fat body taken from the same species that the cell lines were derived from was a better predictor
of a cell line’s embryotrophic activity in Grace’s medium rather than in IPL-52B medium. Thus, the composition of the medium
and the species and tissue type of the cell line source must be evaluated interactively to determine optimal conditions for
promoting development ofM. croceipes in vitro. 相似文献
5.
Selection of a standard culture medium for primary culture of <Emphasis Type="Italic">Limulus polyphemus</Emphasis> Amebocytes 总被引:1,自引:0,他引:1
Hurton LV Berkson JM Smith SA 《In vitro cellular & developmental biology. Animal》2005,41(10):325-329
Summary This study provides information relevant to future research aimed at producing Limulus Amebocyte Lysate (LAL) in vitro, which would potentially reduce the need to harvest and bleed horseshoe crabs as in the current
methods of LAL production. To address the need for primary culture of horseshoe crab amebocytes, this study tested the effects
of a variety of standard insect cell culture media on amebocyte morphology and viability after 7 d of maintenance. Amerbocyte
morphology was least altered from in vivo form in Grace’s Modified Insect Medium, with no observed degranulation of cells,
as compared to the other media tested. There were significant differences in amebocyte viability among the six insect cell
culture media tested. Grace’s Modified Insect Medium sustained viability of 77.2±5.1% (mean ± standard deviation) of amebocytes,
followed distantly by Grace’s Insect Medium with 35.1±8.7% amebocyte viability. Results indicate that Grace’s Modified Insect
Medium with horseshoe crab serum supplementation was the best candidate of the six media tested for future medium optimization
for Limulus amebocyte requirements. 相似文献
6.
The impacts of cryoprotectants (CP) and cell status during the growth cycle on Panax ginseng cell viability during cryopreservation were investigated. The ginseng cells used had a 5–7 times proliferation rate (compared
with inoculum) in 2–3 weeks and were subcultured at 2- and 4-week intervals in liquid and on solid media, respectively. After
testing various CP solutions of glycerol, dimethylsulphoxide, ethylene glycol and sucrose, a combination of 10% (v/v) glycerol
and 4% (w/v) sucrose was selected for its least cytotoxicity and highest cell viability after thawing. With this CP solution,
cells throughout the growth cycle exhibited a ‘U’-shaped fluctuation of post-thaw cell viability. The highest viability (86.5%)
occurred during the lag phase from cells already maintained in suspension culture and then in the late exponential phase (61.7%);
the lowest level of 15.4% was in the mid-exponential phase. Callus freshly transferred to liquid medium showed a less obvious
fluctuation pattern. The recovered cells were brown-to-reddish at first and gradually returned to a light yellow colour after
several subcultures.
Received: 1 October 1998 / Revision received: 6 January 2000 / Accepted: 11 January 2000 相似文献
7.
Tzu-Ping Lin Yun Kit Hom James Richards Satyabrata Nandi 《In vitro cellular & developmental biology. Animal》1991,27(3):191-196
Summary Free radical damage has the potential to significantly affect the behavior of cells in culture. In this study the effects
of antioxidants (superoxide dismutase, catalase, and vitamin E) and lowered oxygen tension (1% oxygen) on primary culture
of rat mammary epithelial cells were examined. Rat mammary epithelial cells were dissociated in collagenase with or without
the addition of antioxidants and low oxygen tension, then cultured for 10 d in rat-tail collagen gel matrix and fed with Dulbecco’s
modified Eagle’sF12 medium supplemented with various hormones and growth factors. Growth potential of the mammary cells was
enhanced when antioxidants and low oxygen tension were used, alone or in combination, during the cell dissociation period.
Using antioxidants and low oxygen tension during the culture period failed to improve growth potential regardless whether
cells were dissociated in standard conditions or with antioxidants and low oxygen tension. The use of antioxidants and low
oxygen tension during the cell dissociation period also reduced the degree of keratinization of the cells after 10 d of culture.
Using antioxidants and low oxygen tension during the cell culture period did not further reduce keratinization if antioxidants
and low oxygen tension were used during the dissociation period, but were effective in reducing keratinization if cells were
dissociated in standard condition. In this system, antioxidants and low oxygen tension reduced lipid peroxidation during the
cell dissociation period. An iron chelator, desferal, can also reduce lipid peroxidation and enhance growth when used during
cell dissociation, suggesting the enhanced growth potential by the addition of antioxidants and low oxygen to be due to the
reduction of lipid peroxidation.
This study is supported by grants CA05388 and GM11903 to Y. K. H. from the Public Health Service, U.S. Department of Health
and Human Services, Washington, DC. 相似文献
8.
Establishment and characterization of two embryonic cell lines of <Emphasis Type="Italic">Bombyx mori</Emphasis> 总被引:1,自引:0,他引:1
Pan MH Xiao SQ Chen M Hong XJ Lu C 《In vitro cellular & developmental biology. Animal》2007,43(2):101-104
Two cell lines, i.e., BmE-SWU1 and BmE-SWU2, were established from silkworm embryonic tissues of the reversion phase through
primary culture in Grace’s medium supplemented with 20% fetal bovine serum. The BmE-SWU1 cell line mainly included diploid
spindle cells and round cells, which were large and had severe heteroploidy karyotypes. The population doubling time of the
30th passage of the cell line was 58.7 hr. BmE-SWU2 cells were oblong or round, and small. The population doubling time for
the 30th passage of the cell line was 46.6 hr. Of BmE-SWU2 cells 89.9% were diploid (2n = 56). Both strains were attached to epithelial-like cell lines and were susceptible to Bombyx mori nucleopolyhedroviruse (BmNPV). Inter simple sequence repeat (ISSR) fingerprinting of silkworm embryonic cell line was obtained. 相似文献
9.
Cytokine effect on ex vivo expansion of haemopoietic stem cells from different human sources 总被引:3,自引:0,他引:3
Sandro Eridani Umberto Mazza Paolo Massaro Maria Luisa La Targia Anna Teresa Maiolo Andrea Mosca 《Biotherapy》1998,11(4):291-296
Human pluripotential stem cells (PSC) are currently the target for transplantation attempts and genetic manipulation. We have
therefore investigated the frequency and the expansion potential of PSC’s in different types of blood samples. CD 34+ cells were thus obtained from human bone marrow (BM), as well as from peripheral blood (PB) and cord blood (CB) samples.
After immuno-magnetic separation the highest yields of CD 34+ cells were from BM (1.08–2.25%) and CB (0.42–1.32%) while PB samples gave much lower values. Suspension cultures of PSC’s
from the three sources were then set up, in the presence of combinations of haemopoietic growth factors. A remarkable amplification
of the nucleated cell pool was observed reaching a maximum between 10 and 15 days of culture; earliest and maximum expansion
(up to 220-fold) was achieved when Erythropoietin (Epo) was added to the culture medium, but this resulted in reduction of
colony-forming cells and differentiation into erythroid progenitors. Clonogenic tests for BFU-E’s derived colonies showed
a peak value at 5 days of liquid culture. Further studies are advisable to establish the best cytokine combination for a valuableex vivo expansion, coupled with preservation of stem cell properties. 相似文献
10.
Summary Isolation and maintenance of porcine embryonic stem (pES) cells have been hindered by the inability to inhibit differentiation
of the porcine inner cell mass (pICM) in vitro. Culture conditions currently in use have been developed from mouse ES cell culture and are not effective for maintaining
the pICM. Optimizing culture conditions for the pICM is essential. We have developed a grading system to detect changes in
the differentiation status of in vitro cultured pICM. Porcine ICMs (Day 7) were isolated by immunosurgery and cultured for 4 d in either Dulbecco’s modified Eagle’s
medium (DMEM)-based medium (D medium) or DMEM/Ham’s F-10 (1:1)-based medium (D/H medium) with or without human Leukemia Inhibitory
Factor (hLIF, 1000 iu/ml). Colonies were photographed daily for morphological analysis. pICMs were categorized into one of
two types based on their morphological profile: type A, nonepithelial or type B, epithelial-like. Eight investigators evaluated
pICM differentiation using standardized differentiation profiles. Each pICM series was graded on a scale of 1 (fully undifferentiated)
to 5 (fully differentiated) for each time point. Differentiation was verified by alkaline phosphatase activity, cytokeratin
staining, and scanning electron microscopy. Neither hLIF nor culture medium delayed differentiation of pICMs (P=0.08 and P=0.25, respectively). The grading system employed was an effective tool for detecting treatment effects on differentiation
of the developing pICM. These results demonstrate that hLIF cannot significantly inhibit differentiation of the pICM, and
is unlikely to assist in porcine ES cell isolation. Future experiments utilizing homologous cytokines may prove more beneficial. 相似文献
11.
Bernd Fischer Manfred Lambertz Christa Hegele-Hartung 《In vitro cellular & developmental biology. Animal》1992,28(5):337-347
Rabbit morulae and blastocysts were cultured in conventional culture media [Ham’s F10 or BSM II supplemented with bovine serum
albumin (BSA) or serum] or in Ham’s medium supplemented with synchronous or asynchronous uterine flushings, mostly for 2 days,
and afterwards investigated by light and electron microscopy and by autoradiography. Ultrastructure and cell proliferation
differed considerably between cultured embryos and noncultured controls. Cultured embryos displayed more dead cells. They
were developmentally retarded (predominance of smooth endoplasmic reticulum rather than the age-specific rough endoplasmic
reticulum, mitochondria still round to ovoid shaped) and showed nonspecific signs of cells damage (swollen mitochondria and
Golgi complex vesicles, increased number of lysosomes). All these features were also present in embryos grown in uterine flushing-supplemented
media, but were less pronounced. Cell damage and impaired cell proliferation had affected trophoblast cells more than embryoblast
cells. Endoderm could be differentiated only if culture had been started with blastocysts—not with morulae—and seems to require
uterine secretions. No significant ultrastructural differences were observed between embryos cultured in synchronous or in
asynchronous uterine flushings. Present results indicate that cultured preimplantation rabbit embryos deviate clearly from
those grown in vivo and maintain, for some time, a better cellular structure—and probably function —in the presence of uterine
flushings than in conventional culture media. Specific abnormal morphologic features related to a particular medium could
not be identified. 相似文献
12.
Eun Jung Kim No Soo Kim Gyun Min Lee 《In vitro cellular & developmental biology. Animal》1999,35(4):178-182
Summary To develop serum-free (SF) medium for dihydrofolate reductase-deficient Chinese hamster ovary cells (DG44), a statistical
optimization approach based on a Plackett-Burman design was adopted. DG44 cells which were normally maintained in 10% serum
medium were gradually weaned to 0.5% serum medium to increase the probability of successful growth in SF medium. A basal medium
was prepared by supplementing Dulbecco’s modified Eagle’s medium and Ham’s nutrient mixture F12 with hypoxanthine (10 mg/l)
and thymidine (10 mg/l). Twenty-eight different supplements were selected as variables on the basis of their growth-promoting
abilities. From statistical analysis, leucine, tryptophan, lysine, proline, histidine, hydrocortisone, ethanolamine, and phosphatidylcholine
were identified as important components showing positive effects on cell growth. A new SF medium (SF-DG44) was formulated
by supplementing the basal medium with these components. When the weaned cells were inoculated at 1.0 × 105 cells/ml, a maximum viable cell concentration of 6.4×105 cells/ml was achieved in SF-DG44 medium. In contrast, when the unweaned cells were used, a concentration of only 4.1×105 cells/ml was reached under the same culture conditions, indicating that weaning of cells improves cell growth in SF medium.
In summary, we found that development of a novel SF medium for DG44 cells was facilitated using a Plackett-Burman design technique
and weaning of cells. 相似文献
13.
A cell line designated as ADG was established from an abalone digestive gland using ERDF medium supplemented with 8% fetal
bovine serum (FBS), 8% abalone hemolymph, and high concentrations of NaCl, KCl, MgCl2, MgSO4, and CaCl2. ADG cells proliferated better in protein-free medium than in FBS-supplemented medium. Among 9 kinds of media examined, ERDF
medium was shown to be optimal for cell growth. ADG cells secreted 13 different kinds of glycosidases in protein-free medium:
α-L-fucosidase, β-L-fucosidase, α-D-galactosidase, β-D-galactosidase, N-acetyl-α-D-galactosaminidase, N-acetyl-β-D-galactosaminidase,
α-D-glucosidase, β-D-glucosidase, N-acetyl-α-D-glucosaminidase, N-acetyl-β-D-glucosaminidase, α-D-mannosidase, β-D-mannosidase,
β-D-xylosidase, and 1-3 xylanase. When ADG cells were cultured in Grace’s insect cell medium, the activity of some secreted
glycosidases increased 25-fold to 65-fold per cell as compared with control cells cultured in ERDF medium. ADG - abalone digestive
gland; ERDF - enriched RDF; FBS - fetal bovine serum; L-15 - Leibovitz’s L-15 media; DME - Dulbecco’s modified Eagle medium;
F-12 - nutrient mixture (Ham); LDF - L-15; DME: F-12 = 10 : 7 : 3; MEM - minimum essential medium; RPMI - RPMI medium 1640;
199 - media 199; GIC - Grace’s insect cell medium; pNP -p -nitrophenol.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
14.
M. S. Noël-Hudson I. Dusser I. Collober M. P. Muriel F. Bonté A. Meybeck J. Font J. Wepierre 《In vitro cellular & developmental biology. Animal》1995,31(7):508-515
Summary Cell suspensions of human keratinocytes seeded onto cell culture inserts may undergo terminal differentiation in the absence
of fibroblasts. Among the parameters that control these morphogenic events, exposure to air and the composition of the culture
medium were investigated. In the latter case, three media were considered DMEM:Ham’s F12, MCDB 153, and keratinocyte SFM medium
at equivalent calcium (1.5 mM) and fetal calf serum (5%) concentrations.
Immunochemical methods and transmission electron microscopy show that cells cultured in DMEM:Ham’s F12 medium, and then raised
at the air-liquid interface, form a basal layer plus suprabasal cell layers corresponding to thestratum spinosum, stratum granulosum, andstratum corneum. The suprabasal keratinocyte layers show morphologies that resemble intact skin in which cells are connected by desmosomes
and contain intermediate filaments and keratohyalin-filaggrin granules. When the cultures are kept submerged, the keratinocytes
show occasional keratohyalin granules and are connected by fewer desmosomes. Additionally, no properstratum corneum is formed. In keratinocyte SFM medium and MCDB 153, cultures raised at the air-liquid interface are not able to form an epithelium
of normal architecture and do not express terminal differentiation markers. Differentiation is initiated, however, since desmosomes
and bundles of keratin filaments appear; on the other hand, filaggrin is not expressed even after 28 d in culture. Membrane-bound
transglutaminase is expressed throughout the entire suprabasal compartment in MCDB153 and DMEM:Ham’s F12 media but never appears
in keratinocyte SFM medium.
These studies show the relative independence of epidermal differentiation program to the composition (including the calcium
concentration) of the media contacting the dermis and filling the extracellular space. Conversely, differentiation appears
to depend on elements of basal medium and/or components synthesized by keratinocytes under the influence of the culture medium. 相似文献
15.
A. C. Bonfils S. C. Gleddie J. A. Webb W. A. Keller 《In vitro cellular & developmental biology. Plant》1992,28(3):137-142
Summary Rapidly growing cell suspension cultures of shepherd’s purse (Capsella bursa-pastoris L. Medic.) were established from leaf-derived calli. These suspensions remained unorganized in the presence of 2,4-D, but
underwent extensive root organogenesis in a growth regulator-free liquid medium. Attempts to induce direct embryogenesis in
liquid cultures were unsuccessful, but numerous embryos were obtained from cells plated onto growth-regulator-free solid medium.
These embryos were frequently abnormal, and secondary embryogenesis was problematic for plant recovery but fertile plants
were recovered. Viable protoplasts could readily be isolated from these cell suspensions. After 1 wk of culture, protoplast
viability was 62%, and 7% of the cells had divided. Embryogenesis was observed from protoplast-derived microcolonies, plated
on growth-regulator-free medium. Although these somatic embryos were difficult to root, plants were recovered. New cell suspensions
were more recently established, which were only 4 to 6 mo. old when plant regeneration was attempted. Numerous shoots were
obtained when these cells were plated onto growth-regulator-free solid media. However, these shoots differed from the embryos
previously obtained in that they readily rooted and rapidly developed into plantlets. This system may allow the use of shepherd’s
purse as a gene source for introgression of agronomically interesting traits intoBrassica crop species through protoplast manipulation and somatic hybridization. 相似文献
16.
Kathryn Kamo Janet Chen Roger Lawson 《In vitro cellular & developmental biology. Plant》1990,26(4):425-430
Summary Inflorencence stalks from greenhouse-grownGladiolus plants of the cultivars ‘Blue Isle’ and ‘Hunting Song’ cultured on a Murashige and Skoog basal salts medium supplemented
with 53.6 μM 1-napthaleneacetic acid formed a compact, not friable type of callus that regenerated plantlets. Cormel slices and intact
plantlets of three cultivars (‘Peter Pears’, ‘Rosa Supreme’, ‘Jenny Lee’) propagated through tissue culture formed a friable
type of callus when cultured on Murashige and Skoog basal salts medium supplemented with 2,4-dichlorophenoxyacetic acid. This
friable callus readily formed a cell suspension when the callus was placed in a liquid medium. Plants were regenerated from
two-month-old suspension cell cultures of the commercial cultivar ‘Peter Pears’ after the suspension cells had been cultured
on solid medium. 相似文献
17.
Summary A melanoma cell clone was isolated from cultured B16 mouse melanoma cells. This clone,conv, which was characterized by rounded and spindle-shaped cell morphology, was not highly melanotic under the usual culture
condition but had high tyrosinase (dopa oxidase) activity.
When the cells were seeded to form colonies on a plastic culture dish in Eagle’s minimum essential medium supplemented with
10% bovine calf serum, two kinds of cell types always appeared. One was cytochemically dopa-positive and spindle-shaped (S
type cell) with the same phenotypes as those of the parental cells. The other was dopa-negative and fibroblastlike (F type
cell) containing no melanosomes. It was observed that the conversion from S type to F type occurred with a high frequency.
The conversion from F type to S type also occurred but with a low frequency. 相似文献
18.
To develop an in vitro culture system for bovine oocytes and early embryos, we examined the effects of co-culture of in vitro matured and in vitro fertilized embryos with trophoblastic vesicles and cumulus cells. We also studied the effects of culture medium components and oxygen gas pressure by modifying TCM-199 medium and using a gas-tight chamber. We found that co-culture with trophoblastic vesicles or cumulus cells promoted early embryos to develop beyond the eight-cell block; 17 to 19% of the initial oocytes developed to the morula stage. The effects of removing glucose and other energy sources from the medium, adding EDTA to the medium, reducing the concentration of serum, and reducing the oxygen gas pressure on the development of embryos were also examined. These modifications during the initial phase of co-culture greatly increased the rate of embryo development to the morula (36 to 38% of oocytes developed to morulae) and blastocyst stages. 相似文献
19.
Summary Anthers and ovaries of six grapevine cultivars (three Vitis vinifera L., two V × Labruscana L. H. Bailey, and one complex hybrid) were extracted from flower buds over 2 yr and cultured on three media reported to promote
somatic embryogenesis in Vitis tissues. The highest percent embryogenesis from the hybrid ‘Chancellor’ and V. vinifera ‘Chardonnay’, ‘Merlot’, and ‘Pinot Noir’ occurred on medium C [Nitsch and Nitsch, 1969, basal medium with 3.0% (w/v) sucrose,
0.01% (w/v) inositol. 0.3% (w/v) Phytagel, 2.5 μM 2.4-dichlorophenoxyacetic acid, 2.5μM β-naphthoxyacetic acid, 5.0μM N-(2-chloro-4-pyridyl)-N′-phenylurea, and 0.05% (w/v) glutamine]. Regardless of the media, the labrusca cultivars ‘Concord’ and ‘Niagara’ produced
soft non-embryogenic callus that was sometimes mixed with well-developed somatic embryos. Nine vinifera genotypes were further
tested for several different years on medium C. Embryogenic cultures suitable for transformation were obtained from all genotypes
in more than 1 yr. The average percent embryogenesis from ovaries was 7-fold higher than from anthers. There was significant
annual variation in percent embryogenesis, demonstrating the need for media comparisons to be replicated for more than one
season. Suspension cultures suitable for use in genetic transformation were initiated from ‘Chardonnay’, ‘Merlot,’ and ‘Pinot
Noir’ pro-embryogenic masses. ‘Chardonnay’ suspension cultures plated and grown under conditions developed for recovery of
plants after biolistic transformation yielded approximately 500 non-transformed embryos per plate after 4 mo. of culture,
with 68.6% of the embryos converting to plants. This is the first reported protocol for embryogenesis from ‘Concord,’ ‘Cabernet
Franc,’ and ‘Pinot Noir’ grapevines. 相似文献
20.
Zita Demeter Gyula Surányi V. Attila Molnár Gábor Sramkó Dániel Beyer Zoltán Kónya Gábor Vasas Márta M-Hamvas Csaba Máthé 《Plant Cell, Tissue and Organ Culture》2010,100(3):349-353
Crocus heuffelianus belongs to the C. vernus (Iridaceae) species aggregate. In the Carpathian Basin and particularly in Hungary it is considered an endangered species. Therefore
our aim was to establish a tissue culture system with potential of germplasm preservation of this taxon. For in vitro culture
experiments, shoot primordia from corms were the most suitable. We induced an embryogenic callus line from those explants
on basal Murashige-Skoog (MS) medium supplemented with Gamborg’s vitamins, 2% (w/v) sucrose, 10 mg l−1 (53.7 μM) α-naphthaleneacetic acid (NAA) and 1 mg l−1 (4.44 μM) 6-benzyladenine (BA). Globular stage embryos developed on this medium and several culture conditions were used
in an attempt to obtain mature embryos and plant regeneration. Firstly a decrease of auxin/cytokinin concentration and ratio,
then secondly a decrease in the strength of culture medium and the concentration of carbon source was used, which was effective
in embryogenesis and the production of plants. Regeneration medium used in the second step was fourfold diluted MS medium
and Gamborg’s vitamins supplemented with 1% (w/v) sucrose, 0.05 mg l−1 (0.26 μM) NAA and 0.5 mg l−1 (2.22 μM) BA, with a 14/10 h photoperiod. Under these conditions we could detect all the stages of somatic embryo development
characteristic for Iridaceae. This is the first report demonstrating the production of stable tissue culture of C. heuffelianus with potential use in germplasm preservation via plant regeneration. This study could also contribute to a better understanding
of somatic embryogenesis in the Crocus genus. 相似文献