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1.
Development of a serum-free culture medium for the large scale production of recombinant protein from a Chinese hamster ovary cell line 总被引:4,自引:0,他引:4
A serum-free medium, WCM5, has been developed for the large scale propagation of CHO (Chinese hamster ovary) cells which express recombinant protein using dihydrofolate reductase as a selectable marker. WCM5 was prepared by supplementing Iscoves medium without lecithin, albumin or transferrin with a number of components which were shown to benefit growth. WCM5 medium contained 5 mg l–1 human recombinant insulin (Nucellin) but was otherwise protein-free. CHO 3D11* cells which had been engineered to express a humanised antibody, CAMPATH*-1H, were routinely grown using serum-containing medium. From a seeding density of 105 cells ml–1, cells grown in static culture with serum reached a maximal cell density of 6.5×105 cells ml–1 after 6 days in culture and produced a maximal antibody concentration of 69 mg l–1 after 11 days in culture. CHO 3D11* cells grown with serum were washed in serum-free medium then cultured in WCM5 medium. Following a period of adaptation the cell growth and product yield was superior to that achieved with serum-containing medium. CHO cells producing CAMPATH-1H grown in an 8000 l stirred bioreactor seeded with 2×105 cells ml–1 reached a maximal viable cell density of 2.16×106 cells ml–1 after 108 h in culture and a maximal antibody concentration of 131.1 mg l–1 after 122 h in culture.Abbreviations CHO
Chinese hamster ovary
- dhfr
dihydrofolate reductase
- dhfr
dihydrofolate reductase deficient
- MTX
methotrexate
- H
hypoxanthine
- T
thymidine
- T/V
trypsin versene
- F12
Hams F12 medium
- NEAA
non essential amino acids 相似文献
2.
A simple protein free medium was formulated and tested in suspension culture using three hybridoma cell lines. The medium, referred to as CDSS (Chemically Defined Serum Substitutes), consisted of the basal medium DMEM:Ham F12, 1:1, with HEPES (D12H), plus pluronic F68, trace elements, ferric citrate, ascorbic acid, and ethanolamine. No protein or lipid components were added. All three cell lines were weaned off serum using CDSS and a commercially available protein free medium PFHM-II. Data shown here indicated that normally cells took 1–7 weeks to wean off serum and an additional 2–7 weeks to adapt to suspension culture. After adaptation the cells were able to grow well in suspension culture using both protein free media and in the main performed better than serum containing controls. The stability of the three hybridoma cells for antibody production following freeze/thaw procedures and long term subculturing was also tested. All three lines were frozen using our protein free CDSS medium (containing 0.75% bovine serum albumin and 10% dimethyl sulfoxide) in liquid nitrogen for up to one year. Cells thawed from these stocks recovered well and were able to maintain good growth and antibody production characteristics. One line was shown to grow using our protein free CDSS medium in suspension culture for 12 weeks without loss of antibody productivity. 相似文献
3.
Keen MJ 《Cytotechnology》1995,17(3):193-202
Y0 is a rat x rat hybridoma cell line, which does not secrete immunoglobulin, produced using a fusion partner derived from the Y3 (Y3,Ag.1.2.3) rat myoloma cell line. Y0 and Y3 have both been widely used as fusion partners in the production of rat x rat hybridomas. Y0 has also been used in recombinant gene technology. Y0 cells grown in shake flask culture, using RPMI 1640 medium with 4mM l-glutamine and 5% foetal bovine serum, reached a maximal cell density of 1.5×106 cells ml–1 with 86% viability. Y0 cells which has been adapted to grow in ABC protein-free medium reached a maximal density, in shake flask culture, of 8.75×105 cells ml–1 with 79% viability. An improved protein-free medium, designated W38 medium, was developed. In shake flask culture, W38 medium supported Y0 cell growth to a density of 2.02×106 cells ml–1 with 96% viability. Two Y3 hybridomas, YID 13.9.4 cells and SAM 618 cells were adapted to growth in W38 medium. For both hybridomas, cell growth and product yield in shake flask culture using W38 medium was superior to that obtained with serum-containing RPMI 1640 medium.Abbreviations F12
Hams F12 medium
- DMEM
Dulbeccos medium
- RPMI
RPMI 1640 medium
- FBS
foetal bovine serum 相似文献
4.
A low-cost chemically defined protein free medium for a recombinant CHO cell line producing prothrombin 总被引:4,自引:0,他引:4
A chemically defined protein free medium, DF6S, was developed for the cultivation of a recombinant Chinese hamster ovary cell line (CHO2DS) producing human prothrombin in suspension batch culture. DF6S was formulated by optimizing DME/F12 with amino acids and supplementing the optimized DME/F12 with aurintricarboxylic acid, ethanolamine, ferric sulfate, Pluronic F68, putrescine and sodium pyruvate. From a seeding density of 2.3 × 105 cells ml–1, CHO2DS cells grown in suspension in DF6S medium reached a maximal cell density of 1.92 × 106 cells ml–1 with an accumulated prothrombin concentration of 16.7 mg l–1 after 6 days in culture. 相似文献
5.
S. Shetty L. Bharathi K. B. Shenoy S. N. Hegde 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》1992,162(7):632-636
Summary The avian juvenile food pigeon milk was studied for its chemical composition and effect on growth in vivo and in vitro. Pigeon milk on a wet weight basis consisted of 9–13% protein, 9–11% fat, 0.9–1.5% carbohydrate, 0.8–1.1% ash, 0.10–0.12% non-protein nitrogen, energy content 5.6–6.8 kcal·g-1. Except for proteins there was little or no decrease in pigeon milk constitutents during the first week of secretion. Pigeon milk proteins consisted of trichloroacetic acid (precipitable), trichloroacetic acid (soluble), and free amino acid components in the ranges 8.4–12.1%, 0.5–0.7% and 1.4–2.5%, respectively; whereas the level of trichloroacetic acid (precipitable) and trichloroacetic acid (soluble) components decreased by about 30%, that of the free amino acids increased by 9% in the first week. About 0.6–1.0% of pigeon milk sugar was found in the trichloroacetic acid (soluble) fraction and increased by 67% in the first week. The remainder was found in the trichloroacetic acid (precipitable) fraction and did not change during this period. Major lipids of pigeon milk were the neutral lipids (7.8–8.4%); the minor lipids were glycolipids (0.9–1.6%), phospholipids (0.5–1.4%) and cholesterol (0.5–0.6%). Squabs fed pigeon milk increased their body weight by 22-fold in the first 3 weeks after hatching, and crude extracts of pigeon milk stimulated the growth of cultured hamster ovary cells. These results reflect the ability of pigeon milk to stimulate growth both in vivo and in vitro.Abbreviations AOAC
association of official analytical chemists
- BRIT
board of radiation and isotope technology
- CHO
chinese hamster ovary
- DNA
deoxyribonucleic acid
- EDTA
ethylenediaminetetra-acetic acid
- FCS
foetal calf serum
- GF
growth factor
- GS
goat serum
- MEM
minimum essential medium
- NPN
nonprotein nitrogen
- PBS
phosphate-buffered saline
- PM
pigeon milk
- TCA(P)
trichloroacetic acid precipitable fraction
- TCA(S)
trichloroacetic acid soluble fraction 相似文献
6.
High frequency somatic embryogenesis was induced from leaf expiants of F1 hybrid Solanum melongena L. on Murashige and Skoog's medium supplemented with 8.0 mg/1 NAA and 0.1 mg/1 Kn. The somatic embryos were encapsulated in various concentrations (2–6%) of sodium alginate and complexed with calcium chloride (25–100mM): 3% sodium alginate and 75 mM calcium chloride were found to be optimal for encapsulation. The encapsulated somatic embryos were transferred to various conversion media in vitro and in vivo. The frequency of plantlet regeneration varied from 27.0–49.7% in vitro and 2.0–4.5% in vivo.Abbreviations IAA
indole-3-acetic acid
- IBA
indole-3-butyric acid
- Kn
Kinetin
- MS
Murashige and Skoog (1962)
- NAA
naphthalene acetic acid 相似文献
7.
Multiple shoots differentiated from hypocotyl explants of Sesbania bispinosa (Jacq.) W.F. Wight, a leguminous woody shrub, when cultured on Gamborg's basal medium alone or in combination with 6-benzyl aminopurine (10–7–10–4 M). For cotyledonary explants 6-benzyl aminopurine (10–6–10–4 M) was necessary. The shoots rooted when cultured on Gamborg's basal medium containing indole-3-butyric acid (10-5 M). Plantlets thus formed were transferred to soil where they have flowered and also set fruits. 相似文献
8.
Axenic seedling-derived two- to three-node stem segments of Nepenthes khasiana Hook.f. were successfully cultured on Woody Plant Medium containing 2.2 M benzyladenine to produce a 0.5–1.5 cm axillary shoot from each node in 7–8 weeks. The rapid growth along with the axillary branching of this shoot enabled amassing of 6–12 shoots during subculture. Excised shoots transferred to basal medium or rooted in medium containing 2.7 M naphthaleneacetic acid produced typical pitchers at leaf tips. Rooted plants were established in pots at 90–95% survival rate.Abbreviations AA
ascorbic acid
- AC
activated charcoal
- CA
citric acid
- BA
6-benzyladenine
- IAA
indole-3-acetic acid
- NAA
-naphthaleneacetic acid
- KC
Knudson-C (1946) basal medium
- MS
Murashige & Skoog (1962) basal medium
- WPM
Woody Plant basal medium (Lloyd & McCown 1980) 相似文献
9.
A method for micropropagation of mature trees of Capparis decidua was developed. Multiple shoots were obtained from nodal explants on Murashige and Skoog's (1962) medium+0.1mgl–1 NAA+5.0mgl–1BAP+additives (50mgl–1 ascorbic acid and25 mgl–1 each of adenine sulphate, L-arginine and citric acid) at 28 ± 2°C, 12 h/dphotoperiod and 35–40 mol m-2s–1 photon flux density. The shoots were multiplied by (i) subculture of nodal shoot segments onto MS +0.1 mgl-–1 IAA+1.0mgl–1 BAPH+additives, and (ii) repeated transfer of original explant onto MS+ 0.1mgl–1 IAA+mg l–1 BAP+additives, at intervals of 3 weeks. Sixty to 70% of the shoots rooted when pulse treated with 100 mg l–1 IBA in half strength MS liquid medium for 4h, and then transferred onto hormone-free half-strength agar-gelled MS basal saltmedium. Incubation in dark at 33 ± 2°C for 6d favoured root induction. In vitro hardened plants were transferred to pots.Abbreviations IAA
Indole-3-aceticacid
- IBA
Indole-3-butyric acid
- NAA
-naphthaleneacetic acid
- BAP
6-benzylaminopurine
- Kn
6-furfurylaminopurine
- 2-ip
Isopentenyl adenine
- B5
Gamborg et al. (1968) medium
- MS
Murashige and Skoog's (1962) medium
- WP
Woody plant medium (Lloyd and McCown 1981) 相似文献
10.
A protein-free growth medium (W38 medium) had previously been developed for the NS0 mouse myeloma cell line which is cholesterol-auxotrophic. This paper describes the development of a protein-free growth medium for NS0 cells expressing humanised monoclonal antibody using GS (glutamine synthetase) as a selectable marker. Several GS-engineered NS0 cell lines expressing humanised monoclonal antibody grew in a modification of W38 medium which maintained GS-selection, supplemented with cholesterol, phosphatidylcholine and -cyclodextrin. Further studies showed that additional glutamic acid, asparagine, ribonucleosides and choline chloride improved cell growth. Amino acid analysis identified a number of amino acids that were being depleted from the culture medium. NS0 cell lines 9D4 and 2H5 expressing CAMPATH-1H* were adapted to enable them to grow serum-free in the absence of cholesterol and -cyclodextrin. Cholesterol-independent 9D4 (9D4.CF) cells grown in shake flask culture using an enriched protein-free medium (WNSD medium), supplemented with human recombinant insulin (Nucellin), reached a maximum cell density to 1.86×106 cells ml–1 producing 76.6 mg l–1 of antibody. CAMPATH-1H antibody produced using serum-free medium was found to be functionally activein vitro in the Antibody Dependant Cellular Cytotoxicity (ADCC) assay.Abbreviations C
cholesterol
- CD
cyclodextrin
- dhfr
dihydrofolate reductase
- F68
Pluronic F68
- GS
glutamine synthetase
- MSX
methionine sulphoximine
- P
phosphatidylcholine
- PC-FBS
phosphatidylcholine, cholesterol and foetal bovine serum
- RPMI
RPMI 1640 medium
- ADCC
Antibody-dependant cellular cytotoxicity 相似文献
11.
A study was undertaken to develop a protoplast regeneration system for pinellia. A yield of 19 29 x 105 protoplasts/g F. W. could be obtained from cell suspension cultures incubated in a digestion enzyme solution with 2% cellulase Onzuka R-10, 10% pectinase (Sigma), 0.01% pectolyase Y23. K8P and modified MS media were used to culture protoplasts in: a) liquid, b) liquid-solid double layer, or c) agarose embedded protoplast culture. The former two were conducive to colony formation from protoplast-derived cells. The frequency of cell division was about 8% after 3 days in culture. Gradually adding fresh medium of lower osmotic pressure into the medium for protoplast culture favored cell division. Calli (1–2 mm in diameter) formed after 30–40 days in culture. The calli transferred onto medium supplemented with KT (0.5 mg 1–1) and NAA (0.2 mg 1)–1) could regenerate plants after 40–50 days. Of 47 plantlets transplanted into plots, 29 flowered and were fertile.Abbreviations 2,4-D
2,4-dichlorophenoxyacetic acid
- NAA
-naphthaleneacetic acid
- KT
kinetin
- CH
casein hydrolysate 相似文献
12.
Sulistyaningdyah WT Ogawa J Li QS Maeda C Yano Y Schmid RD Shimizu S 《Applied microbiology and biotechnology》2005,67(4):556-562
Cytochrome P450 BM-3 from Bacillus megaterium is a fatty acid hydroxylase exhibiting selectivity for long-chain substrates (12–20 carbons). Replacement of Phe87 in P450 BM-3 by Val (F87V) greatly increased its activity towards a variety of aromatic and phenolic compounds. The apparent initial reaction rates of F87V as to benzothiophene, indan, 2,6-dichlorophenol, and 2-(benzyloxy)phenol were 227, 204, 129, and 385 nmol min–1 nmol–1 P450, which are 220-, 66-, 99-, and 963-fold those of the wild type, respectively. These results indicate that Phe87 plays a critical role in the control of the substrate specificity of P450 BM-3. Furthermore, F87V catalyzed regioselective hydroxylation at the para position of various phenolic compounds. In particular, F87V showed high activity as to the hydroxylation of 2-(benzyloxy)phenol to 2-(benzyloxy)hydroquinone. With F87V as the catalyst, 0.71 mg ml–1 2-(benzyloxy)hydroquinone was produced from 1.0 mg ml–1 2-(benzyloxy)phenol in 4 h, with a molar yield of 66%. 相似文献
13.
Viable protoplasts of Vigna sublobata L. were isolated enzymatically from hypocotyls of axenic seedlings. Protoplast yields were dependent upon seedling age, with maximum yields (2.25 ± 0.35 × 106 g fwt–1) from seedlings aged 6 d. Protoplasts regenerated cell walls and underwent sustained divisions when cultured in either agarose-solidified or liquid K8P medium. The plating density affected the division frequency and plating efficiency; the division frequency (68 ±0 6.0%) was maximum at 4.0 × 104 ml–1 while plating efficiency was maximum (1.3 ± 0.1%) at 5.0 × 104 ml–1. Dividing protoplasts developed into microcalli, which produced glossy green compact nodular calli on transfer to 8.0 gl–1 w/v agar-solidified medium containing MS salts, B5 organic components, 30 g l–1 sucrose, NAA (0.2–0.5 mg l–1), zeatin riboside (0.5–2.0 mg l–1) and GA3 (0.5–1.0 mg l–1). These calli, after sub-culture on the same medium, produced shoot buds which underwent elongation following transfer of tissues to 6.0 g l–1 agar-solidified B5 medium containing 30g l–1 sucrose, IBA (0.01 mg l–1) and BAP (1.0 mg l–1). Elongated shoots developed roots after transfer to 8.0g l–1 agar-solidified, hormone-free MS medium with 30 g l–1 sucrose.Abbreviations IAA
indole-3-acetic acid
- IBA
indole-3-butyric acid
- BAP
6-benzyladenine or benzylaminopurine
- B5
medium after Gamborg et al (1968)
- 2,4-D
2,4-dichlorophenoxyacetic acid
- GA3
gibberellic acid
- 2,i-P
6-(--dimethylallylamino) purine
- MS
medium after Murashige and Skoog (1962)
- NAA
1-naphthaleneacetic acid 相似文献
14.
Plant regeneration in Kentucky bluegrass (Poa pratensis L.) via coleoptile tissue cultures 总被引:1,自引:0,他引:1
Summary Plant regeneration in Kentucky bluegrass (Poa pratensis L. cv. Touchdown) via culture of seedling tissues was investigated. When coleoptile, leaf, and stem sections of dark-germinated seedlings were cultured on Murashige and Skoog (MS) medium, different types of callus were produced, depending on the expiant source and growth regulator combinations. Only compact-friable callus (type 3) and moderately compact, friable callus (type 2) produced shoots upon subculture. The nonstructured watery callus (type 4) produced roots without shoots. Shoot differentiation from callus tissues was highest when the culture medium contained 0.2 mgL–1 picloram + 0.01 mgL–1 -naphthaleneacetic acid (NAA). Calli grown from coleoptiles had higher shoot regeneration frequency (32%) than that obtained from either stem sections (12%) or young leaf tissues (2%) of the same seedlings. Some organogenic callus lines produced exclusively green plants, while others produced albino shoots or a mixture of green and albino shoots. The green plants were multiplied in a medium containing 0.1 mgL–1 BAP plus either 0.2 mgL–1 picloram or 0.1 mgL–1 indole-3-acetic acid (IAA). Over 90% of the cultures in the shoot proliferation medium produced roots in 4 weeks. The rooted plants were successfully established in soil medium and grown in the greenhouse.Abbreviations BAP
6-benzylaminopurine
- 2,4-D
2,4-dichlorophenoxyacetic acid
- IAA
indole-3-acetic acid
- MS
Murashige and Skoog (1962) medium
- NAA
-naphthaleneacetic acid
- picloram
4-amino-3,5,6-trichloropicolinic acid
- TDZ
thidiazuron 相似文献
15.
Protoplasts were isolated from immature cotyledons of Glycine max L. Merr. cv. Clark 63 and cultured in liquid or in agarose-gelled modified KP8 medium. Plating efficiencies of 45–50% were obtained in liquid medium and 55–60% in 1.2% (w/v) agarose beads. Upon regular dilution with K8 medium rapidly growing green microcalli (1–2 mm in size) were obtained in 5–6 weeks, which upon transfer to MSB medium with 0.5 mg 1–1 each of 2,4-D, BA, Kn and 500 mg 1–1 CH produced compact green calli in 4–6 weeks. After 3–4 regular subcultures of 14 days each on MSB medium containing 0.5 mg 1–1 each of BA, Kn, ZT, 0.1 mg 1–1 NAA and 500 mg 1–1 CH, about 21% of the compact calli formed multiple shoots. Addition of glutamine, asparagine and GA3 enhanced shoot regeneration up to 30%. Shoots of 0.5–1.0 cm length were transferred to 1/2 MS medium with 0.01 mg 1–1 TH and 0.5 mg 1–1 GA3 for elongation. In 2 to 3 weeks, approximately 60% of the shoots were 2–3 cm in length. These shoots were rooted on 1/2 MS with 1% sucrose and 0.2 mg 1–1 IBA or 0.5 mg 1–1 NAA. So far, twenty six plants have been transferred to the greenhouse, where they all have set seed.Abbreviations BA
6-benzyladenine
- CH
casein hydrolysate
- 2,4-D
2,4-dichlorophenoxyacetic acid
- FDA
fluorescein diacetate
- GA3
gibberellic acid
- IBA
indole-3-butyric acid
- Kn
kinetin
- MES
2[N-morpholino] ethane sulfonic acid
- NAA
naphthaleneacetic acid
- TH
thidiazuron
- ZT
zeatin 相似文献
16.
NS0 has been used as a fusion partner for the production of hybridomas and has more recently been engineered to produce recombinant protein. A protein-free culture medium, designated W38 medium, has previously been developed which supported high density growth of rat myeloma and hybridoma cell lines. NS0 cells failed to grow in W38 medium and in a number of protein-free culture media which support the growth of other myeloma cell lines. NS0 cells are derived from the NS-1 cell line, which is known to require exogencus cholesterol. It was found that NS0 cells grew in W38 medium supplemented with phosphatidylcholine, cholesterol, and albumin and that NS0 were auxotrophic for cholesterol. Protein-free growth of NS0 cells was achieved by using -cyclodextrin to replace albumin as a lipid carrier. The maximal cell density reached in this protein-free medium was in excess of 1.5×106 cell ml–1. The lipid supplements in the medium precipitated after a few days storage at +4°C. In order to overcome this problem a protocol was developed which allowed NS0 cells to be adapted to cholesterol-independent growth in W38 medium. NS0.CF (cholesterol-independent NS0 cells) were cultured continuously in W38 medium for several months. In shake flask culture a cell density of 2.4×106 cells ml–1 was achieved in W38 medium compared with 1.41×106 cells ml–1 in RPMI 1640 medium containing 10% foetal bovine serum. NS0.CF cells readily grew in a 1 litre stirred bioreactor using W38 medium supplemented with Pluronic F68 reaching a density of 3.24×106 cells ml–1. NS0.CF were cloned protein-free by limiting dilution in W38 medium, giving colonies in wells that were seeded at an average density of 0.32 cells per 200 l. This study has demonstrated for the first time the growth of a cholesterol-requiring mouse myeloma cell line in a completely defined protein-free medium and its subsequent adaptation to cholesterol-independence.Abbreviations BSA
bovine serum albumin
- C
cholesterol
- CD
cyclodextrin
- F68
Pluronic F68
- GS
glutamine synthetase
- P
phosphatidylcholine
- PC-FBS
phosphatidylcholine, cholesterol and foetal bovine serum
- RPMI
RPMI 1640 medium
- MSX
methionine sulphoximine 相似文献
17.
Optimal culture conditions for high frequency plant regeneration from excised cotyledons of Tamarindus indica were established. Maximum shoot bud differentiation (100%) occurred when the adaxial surface of the entire cotyledon (excised from 12-d old seedlings) was in contact with MS medium containing 5×10–6M BAP. On MS alone only roots were formed. Shoot or root formation was confined to nodal tissue at the top of the notch present on the adaxial surface at the proximal end of the cotyledon. Thirty-four to 95 shoots were regenerated in a 4 month period from individual cotyledons. Shoots were rooted on MS with 5.7×10–6M IAA. IAA (5.7×10–7M) alone induced complete plant formation. Regenerated plants were established in the soil with 70% success.Abbreviations BAP
6-benzylaminopurine
- KIN
kine-tin
- 2-iP
6-Y-Y-dimethylallyl aminopurine
- AdS
adenine sulphate
- IAA
indole-3-acetic acid
- IBA
indole-3-butyric acid
- NAA
1-naphthalene acetic acid
- MS
Murashige and Skoog (1962) medium 相似文献
18.
Lucia Martinelli Paola Bragagna Valentino Poletti Attilio Scienza 《Plant cell reports》1993,12(4):207-210
Somatic embryogenesis from leaf- and petiole-derived calli of Vitis rupestris was obtained with an efficiency of 3.2% and 4.2% of plated explants, respectively on two combinations of 6-benzyladenine and 2,4-dichlorophenoxyacetic acid (1/0.1 and 1/1 mgl–1) added to MS medium. Embryogenic callus, embryo subcultures and somatic embryogenesis from somatic embryos were obtained either in the presence of 1 mgl–1 indole-3-acetic acid or 0.1 mgl–1 indole-3-butyric acid added to MS or NN media. Within a 4-month culture, embryo germination occurred at a frequency of 13% of explanted embryos when chilling at 4°C was provided for two weeks and a combination of 6-benzyladenine (1 mgl–1) with indole-3-butyric acid (0.1 mgl–1) was added to NN medium supplemented with casein hydrolysate (250 mgl–1). A higher frequency (51%) was obtained in a longer culture time (9 months) when only indole-3-butyric acid was present in the medium and in absence of chilling.Abbreviations BA
6-benzyladenine
- 2,4-D
2,4-dichlorophenoxyacetic acid
- GA3
gibberellic acid
- IAA
indole-3-acetic acid
- IBA
indole-3-butyric acid
- MS
Murashige and Skoog (1962)
- NN
Nitsch and Nitsch (1969)
- NOA
2-naphthoxyacetic acid 相似文献
19.
Repetitive somatic embryogenesis in peanut cotyledon cultures by continual exposure to 2,4-d 总被引:1,自引:0,他引:1
Somatic embryos from immature cotyledons in peanut (Arachis hypogaea) were initiated on media supplemented with 2,4-dichlorophenoxyacetic acid (2,4-d). Over 90% primary embryogenesis and 41–46% repetitive embryogenesis were obtained 12 weeks after initiation by maintaining embryogenic cultures on medium containing 20 mg 1-1 2,4-d. Maintenance of cultures on medium with 30 or 40 mg I-1 2,4-d resulted in lower primary and secondary embryogenesis, and proliferation of nonembryogenic callus. Transfer of embryogenic cultures to a secondary medium with 10 or 20 mg I-1 2,4-d significantly enhanced secondary embryogenesis compared to basal medium without the growth regulator. The use of Murashige & Skoog versus Finer's media had no significant effect on embryogenesis (85–95%), repetitive embryogenesis (11–37%) or mean embryo number. Secondary embryogenesis was also maintained for over one year by repeated subculture of isolated somatic embryos on medium with 20 mg I-1 2,4-d.Abbreviations B5
Gamborg et al. medium (Gamborg et al. 1968)
- 2,4-d
2,4-dichlorophenoxyacetic acid
- FN
Finer & Nagasawa medium (Finer & Nagasawa 1968)
- MS
Murashige & Skoog medium (Murashige & Skoog 1962) 相似文献
20.
Leaf protoplasts of axenic shoot cultures of Pyrus communis L. cv. Williams' Bon Chretien (syn. Bartlett) underwent cell wall regeneration and division to give multicellular colonies in a modified Murashige and Skoog medium which lacked ammonium ions, but supplemented with 1-naphthaleneacetic acid (NAA), 4-indole-3yl-acetic acid, 6-benzylaminopurine (BAP) and casein hydrolysate. Protoplast-derived colonies gave callus on Murashige and Skoog salts medium with NAA and BAP and exhibited shoot regeneration on half-strength Murashige and Skoog medium supplemented with 0.2 mg 1–1 4-indole-3yl-butyric acid, 2.0 mg 1–1 BAP, 0.2 mg 1–1 gibberellic acid, 50 mg 1–1 casein hydrolysate and 10 mg 1–1 Ca-pantothenate. Following rooting, protoplast-derived plants of pear were transferred to the glasshouse where they completed acclimatization.Abbreviations BAP
6-benzylaminopurine
- FPE
final plating efficiency
- GA3
gibberellic acid
- IAA
4-indole-3yl-acetic acid
- IBA
4-indole-3yl-but yric acid
- IPE
initial plating efficiency
- NAA
1-naphthaleneacetic acid
- f.wt.
fresh weight
- MES
2-N-morpholinoethane sulfonic acid
- MS
Murashige and Skoog (1962)
- %PE
% plating efficiency
- PVP-10
polyvinylpyrrolidone (Av. MW 10,000)
- FDA
fluorescein diacetate 相似文献