Anthocyanin synthesis in tissue cultures of Callistephus chinensis (China aster)

Callus cultures were derived from stems and leaves of 3 anthocyanin producing and 3 acyanic lines of Callistephus chinensis (Compositae). The tissue cultures of the cyanic lines were shown to produce cyanidin whereas in the calli of the acyanic lines no anthocyanin synthesis occurred Culture conditions were improved in order to enhance both anthocyanin production and growth of the tissue cultures.Abbreviations IAA indoleacetic acid - NAA naphtaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - MS-medium Murashige and Skoog medium     

Marker proteins for embryogenic differentiation patterns in pea callus

Polypeptide pattern alterations during somatic embryogenesis were investigated using callus cultures of two Pisum sativum genotypes. Both genotypes show the formation of two different callus lines from the same explant after six to eight weeks in culture: a nodular yellowish callus line, which forms somatic embryoids in suspension cultures (e⁺) and a white compact callus line with no regenerative capacity (e^–). The cytosol proteins of the two different callus lines were separated in a semi-preparative two-dimensional system and the polypeptide patterns were compared. Two protein bands were found (P1: M_r=45000 D, pI=7.0–7.1; P2: M_r=7000 D, pI=<4.5), which were characteristic of the putatively embryogenic (e⁺) callus line in all tissues investigated (two genotypes × two explant sources). These proteins found in nodular (e⁺) pea cultures are very similar to two proteins found in Daucus carota suspension cultures preceding the formation of somatic embryos.Abbreviations BA 6-benzyl-aminopurine - Bistris 2-(bis(2-hydroxyethyl)imino)-2-(hydroxymethyl)-1.3-propanediol - PAGE polyacrylamide gel electrophoresis - pI isoelectric point - TCA trichloroacetic acid - Tris tris(hydroxymethyl)-aminomethane     

Effect of the genes B and Pl on anthocyanin synthesis in maize endosperm culture

B and Pl are two genes involved in anthocyanin biosynthesis in maize (Zea mays) plant tissues. In this work the effect of B and Pl on pigment accumulation is analyzed in endosperm tissues, either cultured in vitro or scraped off from the seed. The results obtained indicate that the two genes play a different role in callus pigmentation: B exerts a qualitative change in pigment composition, while Pl controls the rate of pigment accumulation in the callus. Anthocyanin synthesis in all strains analyzed appears to be light independent. Two cases of instability in pigment production arisen in the endosperm cultures are described and discussed in relation to epigenetic variation in secondary metabolite content in plant tissue culture.Abbreviations BEAF Benzene/ethyl acetate/formic acid (40:10:5) - 2-4D 2,4-dichlorophenoxyacetic acid - Wi initial weight - Wt total weight     

Plant regeneration from protoplasts of Felicia and Brachycome

Protoplasts were isolated from leaves of axenic shoot cultures of Felicia bergeriana (Kingfisher Daisy) and Brachycome iberidifolia (Swan River Daisy) and from callus cultures of Felicia. Plants were regenerated from all three sources and since both species are of ornamental value (blue flowered) the establishment of plant regeneration provides a basis for their incorporation in somatic hybridisation programmes involving important ornamentals such as Chrysanthemum.Abbreviations BAP 6-benzylaminopurine - IAA indole-3-acetic acid - NAA naphthaleneacetic acid - KIN 6-furfurylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - MS Murashige and Skoog (1962) - FDA fluorescein diacetate - f. wt. fresh weight     

Plant regeneration from callus cultures of Durum and emmer wheat

Callus cultures were initiated from isolated mature embryos of Triticum turgidum L. Thell ssps durum and dicoccum on a basal medium supplemented with 2,4-D, 2,4,5-Cl₃POP or 2,4-D+CM. Shoot bud regeneration was observed on 2,4,5-Cl₃POP medium. In both the cultivars of durum, further development of shoot buds occurred on transfer of tissues to basal medium whereas in dicoccum basal medium supplemented with coconut milk or coconut milk with NAA (0.2 mg/l) was necessary. The regenerated shoot buds were induced to root on basal medium supplemented with NAA. The in vitro obtained plants were transferred to soil and successfully grown to maturity. Chlorophyll variants were observed among the regenerated plants of dicoccum.Abbreviations BA benzyladenine - CM coconut milk - 2,4-D 2,4-dichlorophenoxyacetic acid - 2,iP 6---dimethylallylamine purine - IAA indoleacetic acid - NAA -naphthalene acetic acid - Kn kinetin - 2,4,5-Cl₃POP 2,4,5-trichlorophenoxypropionic acid - MS modified Murashige and Skoog's medium - RH relative humidity - Z zeatin     

Plant regeneration from protoplasts of Dimorphotheca and Rudbeckia

Protoplasts were isolated from leaves, shoots, cotyledons, ray florets and callus cultures of Dimorphotheca aurantiaca (syn. D. sinuata) (Cape Marigold, Star of the Veldt) and Rudbeckia hirta, R. laciniata and R. purpurea; species of ornamental value. For Dimorphotheca, plants were regenerated from protoplasts of all sources apart from the ray floret, whilst for the Rudbeckia species, although protoplast division was induced in most cases, only leaf mesophyll protoplasts of R. hirta c.v. Marmalade gave plants. The establishment of plant regeneration for these ornamental species, from protoplasts, now provides a basis for their somatic hybridisation.Abbreviations BAP 6-benzylaminopurine - IAA indole-3-acetic acid - NAA naphthaleneacetic acid - K kinetin - GA₃ gibberellic acid - MS Murashige and Skoog (1962) - f.wt. fresh weight     

Establishment of new axenic hairy root lines by inoculation with Agrobacterium rhizogenes

Cultured hairy root lines resulting from infection by Agrobacterium rhizogenes are known for approximately thirty plant species. We extend this range by establishing forty original dicotyledonous hairy root lines with A. rhizogenes strain A₄. Hairy roots have been cultured for at least 2–6 years on Murashige & Skoog medium. Some hairy root cultures such as Anagallis arvensis and Antirrhinum majus spontaneously regenerated whole plants.     

Propagation of Indian Rhubarh (Rheum emodi Wall.) using shoot-tip and leaf explant culture

Shoot-tip explants of Rheum emodi Wall. (Polygonaceae) gave rise to multiple shoots when cultured on a Murashige and Skoog (1962) medium (MS) with 2.0 mg/l 6-benzylaminopurine (BAP) and 1.0 mg/l indole-3-butyric acid (IBA). Also, shoot buds developed from leaf explants using MS medium with 2.0 mg/l BAP and 0.25 to 1.0 mg/l indole-3-acetic acid (IAA) or IBA. Roots were induced when the resulting shoots were placed on MS medium with 1.0 mg/l IBA. Both regeneration procedures gave rise to healthy plantlets that were established in soil under glasshouse conditions at 80% frequency after hardening phase of two weeks. Regenerated plants showed a constant chromosome number of 2n=2x=22, same as the parent plant. The use of liquid shake cultures minimized the time and culture medium requirements for propagation. This procedure can be applied for the conservation and utilization of elite clones of R. emodi.Abbreviations BAP 6-benzylaminopurine - Dd H₂O Double glass distilled water - IAA indole-3-acetic acid - IBA indole-3-butyric acid - K Kinetin - MS Murashige and Skoog's (1962) medium - RH Relative humidity CIMAP Publication No. 876     

Effects of gibberellic acid and uniconazole on the activities of some enzymes of anthocyanin biosynthesis in carrot cell cultures

Gibberellic acid (GA₃) inhibition of anthocyanin accumulation by carrot cell-suspension cultures was reversed by supplying dihydroquercitin or naringenin to the culture and not by supplying 4-coumaric acid or malonic acid. This suggested that gibberellic acid was inhibiting chalcone synthase, chalcone isomerase, or acetyl CoA carboxylase. Acetyl-CoA-carboxylase specific activity was the same in GA₃-treated and untreated cultures and was not detected in cultures treated with uniconazole, an inhibitor of gibberellic acid biosynthesis. Chalcone-isomerase specific activity was lower in GA₃-treated cultures than in untreated cultures and was lower in uniconazole-treated cultures than in the GA₃-treated cultures. The total chalcone synthase activity in extracts from GA₃- and from uniconazole-treated cells was not significantly different from that in extracts of untreated tissue. When these extracts were chromatographed on a Mono Q column, three peaks of chalcone synthase activity were found in extracts of nontreated cells, whereas only two of these peaks were detected in extracts of GA₃-treated cells. The extracts from GA₃-treated cells did not contain the peak of chalcone synthase activity that, in untreated cells, preceded the main peak. The correlation between the absence of this peak and the inhibition of anthocyanin accumulation suggests that this form of chalcone synthase is responsible for anthocyanin synthesis and that GA₃ prevents this form from appearing in the cells.     

Plant regeneration from hypocotyl-derived protoplasts of Brassica juncea (L.) Czern and Coss

Protoplasts isolated from etiolated hypocotyls of 6-day-old seedlings of Brassica juncea cv RLM 198 were cultured in a modified V₄₇ medium containing 7% mannitol, 2% sucrose, 1.0 mg/l 2,4-D, 0.1 mg/l NAA and 0.4 mg/l BAP, at a density of 5×10⁴ protoplasts per ml of medium. Cultures were incubated in the dark at 25+1°C. After 7 d of culture, cell colonies were diluted with 8_p medium containing 5% mannitol and a similar hormone combination as described earlier. After 14 d, cell colonies were embedded in 8_p medium containing agarose and 3.5% mannitol. Immediately upon gelling, liquid 8_p medium was added to each Petri dish as an overlayer, and cultures were incubated in the light. After a total of 3 to 4 weeks in culture, microcalli were obtained. A modified MS medium with 2% sucrose, 1.0 mg/l 2,4-D and 0.1 mg/l kinetin solidified with 0.5% agarose was used for growing microcalli into callus lines. On MS medium containing 2% sucrose, 0.1 mg/l IAA, 2.0 mg/l zeatin riboside and 2.0 mg/l BAP, solidified with 0.5% agarose, about 35% of the calli regenerated multiple shoots. The time required from culture of protoplasts to multiple shoot regeneration was about 10 weeks. Regenerated shoots were rooted and plants were re-established in a growth chamber at high frequency.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IAA Indole-3-acetic acid - NAA -naphthaleneacetic acid - BAP 6-benzylaminopurine - IBA Indole-3-butyric acid     

Induction of freezing tolerance in alfalfa cell suspension cultures

The effects of ABA, 2,4-D, kinetin and cold exposure on the cold hardiness of Medicago sativa L. cell suspensions were investigated. Cultures treated with 5×10^–5 M ABA at 2°C for 4 weeks in the absence of kinetin showed a 50% survival after freezing to –12.5°C, whereas cultures grown at 25°C under normal conditions tolerated freezing to only –3°C. The optimum ABA treatment of 5×10^–5 M for 4 weeks was effective only in combination with cold exposure. Of six cell lines tested, all showed different degrees of induced cold hardiness. The results suggest that ABA alone cannot induce freezing tolerance on alfalfa cell suspension cultures and that the deletion of kinetin and combination of low temperature and ABA is critical for the induction of cold hardiness in alfalfa cell suspension cultures.Abbreviations ABA abscisic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - LT₅₀ 50% killing temperature     

Alkaloid production in cell suspension cultures of Thalictrum flavum and T. dipterocarpum

Both cell suspension cultures of Thalictrum flavum and T. dipterocarpum were found to produce berberine (0.3 and 0.4 g/l, respectively) as a main alkaloid. Berberine production in the latter was markedly stimulated by 1-naphthaleneacetic acid in combination with 6-benzylaminopurine, whereas it was rather suppressed by the same auxin in the former. T. flavum cultures accumulated berberine and columbamine in the cells without releasing them into medium. On the other hand, T. dipterocarpum cultures released berberine into medium during the logarithmic growth phase, but thereafter accumulated all the berberine synthesized in the cells.Abbreviations LS Linsmaier and Skoog - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - BA 6-benzylaminopurine - TFG a culture strain of T. flavum ssp. glaucum - TDP a culture strain of T. dipterocarpum     

The effect of dipicolinic acid on maize tissue culture growth is not solely due to inhibition of lysine biosynthesis

Dipicolinic acid, a known inhibitor of an enzyme (dihydrodipicolinic acid reductase) in the maize (Zea mays L.) lysine biosynthetic pathway, inhibits the growth of maize suspension and callus cultures. Inhibited cultures contain somewhat lower free lysine levels, but the inhibition of suspension culture growth was not reversible with simultaneous addition of L-lysine to the culture medium. It is concluded that dipicolinic acid does not act solely as an analog blocking lysine production. Dipicolinic acid thus appears to be unsuitable as a selection for maize tissue culture mutants with lysine overproduction.Abbreviations FW fresh weight - I₅₀ inhibitor concentration at which cell growth is inhibited by 50% - MS Murashige and Skoog (1962) culture medium - ZM Black Mexican Zea mays suspension culture of Chourey and Zurawski (1981)     

Hypoxia enhances prostaglandin synthesis in renal mesangial cell cultures

In view of recent findings which suggest that renal prostaglandins mediate the effect of hypoxia on erythropoietin production, we have studied whether hypoxia is a stimulus for in vitro prostaglandin synthesis. Studies were carried out in rat renal mesangial cell cultures which produce erythropoietin in an oxygen-dependent manner. Production rates of PGE₂ and in specified samples also of 6-keto-PGF_1α, as a measure of PGI₂, and PGF_2α were determined by radioimmunoassay after incubation at either 20% O₂ (normoxic) or 2% O₂ (hypoxic) in gas permeable dishes for 24 hrs. Considerable variation in PGE₂ production was noted among independent cell lines. PGE₂ production appeared to be inversely correlated to the cellular density of the cultures. In addition, PGE₂ production was enhanced in hypoxic cell cultures. The mean increase was 50 to 60%. PGF_2α and 6-keto-PGF_1α increased by about the same rate. These results indicate that hypoxia is a stimulus for in vitro prostaglandin production.     

Anthocyanin production in callus cultures ofDaucus carota as influenced by nutrient stress and osmoticum

Summary Daucus carota callus developed red pigments under the influence of indole-3 acetic acid and kinetin. Maximum yield of anthocyanin at the end of 3 weeks was 5.4% on dry weight basis. The callus subjected to phosphate and nitrate stress produced 7.2% and 8.5% anthocyanin respectively. Feeding of sucrose at 7.5% level resulted in production of 15% anthocyanin. Mannitol as osmoticum had positive influence on anthocyanin production.     

Plant regeneration from leaf explants of Glycine clandestina Wendl

Fully fertile plants with the expected chromosome number 2n=40 were regenerated from excised leaf sections of Glycine clandestina. Shoots formed directly on the explants through organogenesis. Utilizing the same media and procedures fully fertile plants were also regenerated from cotyledon and hypocotyl tissue of the same G. clandestina accession. We were not successful in regenerating plants from root tissue of G. clandestina.Abbreviations 6-BA 6-Benzyladenine - FAA Formalin - NAA Naphthalenacetic acid - IAA Indoleacetic acid - Na₂EDTA Disodium salt Ethylenediamine tetraacetic acid - Fe-NaEDTA Ferric-Sodium salt Ethylenediamine tetraacetic acid     

Nutritional and hormonal requirements of Ginkgo biloba embryo-derived callus and suspension cell culture

Calli were obtained from Ginkgo biloba embryos grown on Murashige and Skoog (MS) medium. The G. biloba cells could grow on either MS or Gamborg B5 mineral salt medium supplemented with sucrose (3% and 2%, respectively) and naphthaleneacetic acid (NAA) and kinetin (K) in concentrations ranging from 0.1 to 2.0 mg·L^–1. Best growth and maintenance of callus cultures were achieved using MS medium supplemented with 2 mg·L^–1 NAA and 1 mg·L^–1 K (N₂K₁MS). Light was required to maintain healthy growth of the callus tissue.In both MS and B5 based media, sucrose was hydrolyzed extracellularly before being taken up by Ginkgo cell suspension cultures. Specific growth rates of 0.13 d^–1 and 0.08 d^–1 were obtained in MS medium supplemented with 1 mg·L^–1 NAA, 0.1 mg·L^–1 K and 30 g·L^–1 sucrose (N₁K_0.1MS) and B5 medium supplemented with the same growth regulator regime and 20 g·L^–1 sucrose (N₁K_0.1B5) respectively. Complete phosphate and ammonium uptake was observed in 11 days when cultured in MS medium and 10 days and 4 days respectively when cultured in B5 medium. During the culture, G. biloba cells consumed only 64% and 29% of the nitrate content of N₁K_0.1MS and N₁K_0.1B5 media respectively. Maximum dry biomass concentrations were 13.4 g·L^–1 and 7.9 g·L^–1, and yields on carbohydrate were 0.39 and 0.45 in N₁K_0.1MS and N₁K_0.1B5 media respectively. The better performance of MS cultures came from the higher sucrose and nitrogen salts concentrations of this medium.Abbreviations B5 Gamborg mineral salt medium - d.w. Dry weight - K Kinetin - MS Murashige and Skoog mineral salt medium - N or NAA Naphthaleneacetic acid - N_iK_jMS i and j are the respective concentrations (mg·L^–1) of NAA and K - n Number of experimental points - r Linear regression correlation coefficient     

Isolation and culture of protoplasts from high anthocyanin-producing callus of sweet potato

Optimum conditions for the isolation and culture of protoplasts from high anthocyanin-producing callus of sweet potato (Ipomoea batatas) were investigated. Growth phase of callus and the ratio of callus-enzyme solution affected the yield of protoplasts. Composition of the medium and protoplast density were examined for protoplast culture.Small colonies were regenerated from the protoplasts. Upon transfer to light a high amount of anthocyanin was accumulated in these colonies.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - MES 2-(N-morpholino)-ethanesulfonic acid     

Somatic embryogenesis and plant regeneration in inflorescence and seed derived callus cultures of Poa pratensis L. (Kentucky bluegrass)

Callus induction and plant regeneration were studied in 15 cultivars of the facultative apomictic species Poa pratensis L. (Kentucky bluegrass).The tissue culture responses of mature seeds and immature inflorescences were compared. Murashige and Skoog's (MS) medium, supplemented with 2 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) was used for callus induction and maintenance. Plants could be regenerated from compact and friable callus on MS medium devoid of 2,4-D. Plants were recovered from 14 cultivars at a high frequency (up to 79% of the callus cultures) when young inflorescences were used as the explant material and from only 3 cultivars, at a low frequency (up to 3%), with seeds. Somatic embryos were observed in callus cultures of many cultivars. Fully developed germinating somatic embryos were occasionally observed. Plant regeneration appeared to take place both via somatic embryogenesis and organogenesis. Plants were generally green but albino shoots developed at a low frequency from friable callus.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog's (1962) medium - IAA indole-3-acetic acid - N₆ medium of Chu et al. (1975)     

Shoot and root organogenesis of Camellia sasanqua

In vitro-derived shoot tips, (10 mm) taken from primary cultures of Camellia sasanqua L., were evaluated for organogenesis when cultured on a half-strength MS medium supplemented with various concentrations of NAA, IBA, BA and GA₃. Maximum shoot proliferation and growth for juvenile and mature tissue was obtained when 0.54 M NAA, 8.8 M BA plus 14.4 to 28.9 M GA₃ was added to the culture media, with a pH between 4.5 and 5.0. In vitro-derived shoots (20 mm) from mature C. sasanqua Day Dream and juvenile C. sasanqua cultures initiated roots in vitro after immersion in 2.5 mM IBA for 30 min. Sixty percent of the mature shoots and 90% of the juvenile shoots initiated roots within 3 weeks of treatment with IBA.Abbreviations MS Murashige and Skoog (1962) - IBA lH-indole-3-butanoic acid - BA N-(phenyl-methyl)-lH-purine-6-amine - GA₃ gibberellic acid - kinetin N-(Z-furanyl-methyl)-lH-purine-6-amine - NAA l-naphthaleneacetic acid - L Linear - Q Quadratic