Development of a serum-free culture medium for the large scale production of recombinant protein from a Chinese hamster ovary cell line

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     

Evaluation of a simple protein free medium that supports high levels of monoclonal antibody production

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.     

The culture of rat myeloma and rat hybridoma cells in a protein-free medium

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     

A low-cost chemically defined protein free medium for a recombinant CHO cell line producing prothrombin

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.     

Biochemical properties of pigeon milk and its effect on growth

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     

Plantlet regeneration from encapsulated somatic embryos of hybrid Solanum melongena L

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     

Rapid in vitro differentiation of Sesbania bispinosa plants — a leguminous shrub

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.     

Multiplication of the endangered Indian pitcher plant (Nepenthes khasiana) through enhanced axillary branching in vitro

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)     

Micropropagation of Capparis decidua (Forsk.) Edgew. — a tree of arid horticulture

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)     

The use of serum-free medium for the production of functionally active humanised monoclonal antibody from NS0 mouse myeloma cells engineered using glutamine synthetase as a selectable marker

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     

Protoplast culture and plant regeneration ofPinellia ternata

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     

Hydroxylation activity of P450 BM-3 mutant F87V towards aromatic compounds and its application to the synthesis of hydroquinone derivatives from phenolic compounds

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%.     

A reproducible procedure for plant regeneration from seedling hypocotyl protoplasts of Vigna sublobata L.

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     

Plant regeneration in Kentucky bluegrass (Poa pratensis L.) via coleoptile tissue cultures

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     

Plantlet regeneration from immature cotyledon protoplasts of soybean (Glycine max L.)

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     

Adaptation of cholesterol-requiring NS0 mouse myeloma cells to high density growth in a fully defined protein-free and cholesterol-free culture medium

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     

In vitro high frequency plant regeneration of a tree legume Tamarindus indica (L.)

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     

Somatic embryogenesis from leaf- and petiole-derived callus of Vitis rupestris

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     

Repetitive somatic embryogenesis in peanut cotyledon cultures by continual exposure to 2,4-d

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)     

Plant regeneration from mesophyll protoplasts of Williams' Bon Chretien (syn. Bartlett) pear (Pyrus communis L.)

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