The effect of light and 2,4-D on anthocyanin production in Oxalis reclinata callus

In vitro cultures of O. reclinata accumulate red anthocyanin pigments. Two callus lines were established from O. reclinata, one red and the other non-pigmented. The red callus accumulated cyanidin-3-glucoside as a major pigment. Light irradiation induced anthocyanin synthesis in white callus, resulting in a heterogenous red callus line being formed. The incubation of red and white callus cultures in the dark or at low-light resulted in the repression of red pigment accumulation. The application of 2,4-D (1.0 mg l^-1) inhibited pigment production in the white callus and decreased anthocyanin accumulation in the red callus. The polypeptide composition of the red and white callus lines from O. reclinata were compared using two-dimensional electrophoresis. The red callus had a larger subset of neutral and acidic polypeptides.     

In vitro anti-microbial activity of extracts from the callus cultures of some Nigella species

Crude methanol extracts from callus cultures of Nigella arvensis, N. damascena, N. hispanica, N. integrifolia, and N. sativa were investigated for their anti-microbial activity. Growth inhibition was determined in Gram-positive and Gram-negative bacterial strains as well as in yeast by using a broth-microdilution method. The results showed that the extracts of all calli tested exhibited significant anti-microbial activity, especially against Bacillus cereus, Staphylococcus aureus and Staphylococcus epidermidis. Compared with other Nigella species, a callus culture of N. hispanica was the most effective against the microorganisms used in this study.     

The production of strawberry plants from callus cultures

Shoots were regenerated from callus of the commercially important strawberry varieties Bogota, Brighton, Cambridge Favourite, Hapil, Ostara, Rapella, Red Gauntlet and JILA33 which is a promising selection from a current breeding programme.The callus was initiated from explants of petiole or lamina of leaves of micropropagated shoots in vitro or of lamina or peduncle from greenhouse plants. There was more shoot regeneration with callus from lamina than from petiole although with the variety Hapil, regeneration occurred only with callus from peduncle.With seven of the varieties, shoot regeneration occurred on culture media with BAP and 2,4-D whilst with the remaining variety, Cambridge Favourite, it occurred only with medium which contained 1AA- alanine conjugate in place of 2,4-D.Regenerated shoots rooted readily and the plants produced are being studied for somaclonal variation.     

Effect of sucrose on polyploidization in early callus cultures of Solanum tuberosum

Leaf segments of a monohaploid, dihaploid and tetraploid genotype of the potato (Solanum tuberosum; x = 12) were cultured on callus-inducing medium with 10, 20, 30 or 40 gl^–1 sucrose. After 5 and 7 days of culture, metaphases contained the somatic or polyploidized number of mono- or diplochromosomes. The percentages of polyploidized metaphases were inversely correlated with the number of chromosome sets of the genotypes. In monohaploid leaf segments the percentages of polyploidized metaphases and of metaphases with diplochromosomes increased when the sucrose concentration was raised from 10 or 20 to 30 gl^–1 and remained constant or decreased from 30 to 40 gl^–1. Higher concentrations of sucrose but not higher osmolalities of the medium due to mannitol induced endoreduplication in more cells. The frequency of polyploidized metaphases and metaphases with diplochromosomes in dihaploid and tetraploid leaf segments remained constant through increases in sucrose concentrations.     

Molecular characterization of Mutator systems in maize embryogenic callus cultures indicates Mu element activity in vitro

Summary Active Mutator lines of maize (Zea mays L.) are characterized by their ability to generate new mutants at a high rate and by somatic instability at Mutator-induced mutant alleles. Mutagenically active lines with fewer than ten Mu elements per diploid genome have not been observed. Alteration of Mutator activity has been shown to correlate with the state of modification of Hinfl restiction sites that lie within inverted terminal repeats of Mu elements. To determine whether active Mutator systems can be established and maintained in culture, copy number and modification state of Mu elements were investigated in embryogenic callus lines derived from F₁S of crosses of active Mutator stock with the inbred lines A188 and H99. All callus lines studied maintain high Mu-element copy numbers, and more than half show a continued lack of modification at the Mu element Hinfl sites; thus, parameters associated with mutagenic activity in planta are present in some, but not all, callus lines. Mutator activity was then tested directly by restriction fragment analysis of subclonal populations from A188/Mu ² and H99/Mu ² embryonic cultures. Novel Mu-homologous restriction fragments occurred in 38% of the subpopulations which contained unmodified Mu elements, but not in control cultures containing modified, genetically inactive Mu elements. We conclude that Mu elements from active Mutator parents can remain transpositionally active in embryogenic cell culture. Active Mutator cell lines may be useful for the production of mutations in vitro.     

In vitro organogenesis from leaf explants of Annona squamosa Linn

Multiple shoot formation was induced from excised leaf explants of Annona squamosa Linn. (custard apple) seedlings on a Murashige and Skoog basal medium containing benzylaminopurine and kinetin. Various auxins in combination with the above medium produced callusing of the explants. In an investigation of environmental factors affecting shoot induction it was seen that the maximum number of shoots were obtained using the leaf base with petiole at a temperature of 27°C and a light intensity of 1000 lux. Roots were initiated erratically when individual shoots were treated with an auxin and then transferred to an auxin free medium. The process of the development of adventitious buds in leaf culture was analysed histologically.NCL Communication No. 3104.     

Ploidy levels of plants regenerated from mixed ploidy maize callus cultures

Summary Haploid and diploid anther-derivedZea mays callus lines were treated with the antimicrotubule herbicide pronamide to produce mixed ploidy callus as determined by flow cytometry. The ploidy levels of the plants regenerated from the callus were determined by counting the leaf epidermal guard cell chloroplast numbers. The proportion of diploid regenerated plants was somewhat lower than the proportion of diploid cells of the callus. The diploid plants regenerated somewhat faster than the haploids. The proportion of tetraploids regenerated from the pronamide treated diploid callus, which originated by spontaneous chromosome doubling, was much lower than the proportion of cells indicating that tetraploid cells survive or regenerate plants at a lower frequency than diploid cells.     

Optimisation of the second phase of a two phase growth system for anthocyanin accumulation in callus cultures of Rudbeckia hirta

A two-phase growth system composed of modified Schenk-Hildebrandt medium and enriched Miller's medium was used to grow callus of R. hirta L. The second phase of the system has been optimised and the callus synthesised a 12-compound set of anthocyanins, comprising 4.97% of tissue mass (tubular flowers of the plant in nature only contain one compound comprising 0.28%). The beneficial effect of high content of cysteine on anthocyanin accumulation was noted. The predominant element of the anthocyanin set obtained in vitro is cyanidin-3-O-(6-O-malonyl-β-D-glucopyranoside), a relatively stable compound. This revised version was published online in June 2006 with corrections to the Cover Date.     

In vitro isoflavonoid production in callus from different organs of Pueraria lobata (Wild.) Ohwi

Pueraria lobata (Wild.) Ohwi is a medicinal plant producing large amounts of isoflavonoid glycosides. Here, the ability of in vitro callus cultures to synthesize isoflavonoids was tested. Callus cultures have been initiated from different explants of in vitro germinated plants using modified MS medium. Roots, leaves and stem segments were the best sources of callus tissue. The isoflavonoid profile and content was determined by means of chromatographic methods. Callus from all organs contained isoflavonoid aglycones: genistein and daidzein and daidzein glycosides: daidzin, puerarin and 3'-methoxypuerarin. The differences between each kind of explant were observed in both the total amount of isoflavonoids and in the proportion of individual compounds. The highest content was in root callus, followed by leaf- and stem callus.     

Plant regeneration from callus cultures of several soybean genotypes via embryogenesis and organogenesis

Using callus derived from immature embryos, regeneration of viable plants was obtained in soybean (Glycine max (L.) Merr.). Depending on the composition of the medium, regeneration occurred via embryogenesis or via organogenesis. Embryogenesis resulted when embryos were plated on Murashige and Skoog (MS) medium containing 43 M -naphthaleneacetic acid. In work with the cultivar Williams 82, the addition of 5.0 M thiamine HCl increased embryogenesis from 33% to 58% of the embryos plated. Addition of 30 M nicotinic acid to the MS medium enhanced embryogenesis further to 76%. Organogenesis was obtained when medium containing 13.3 M 6-benzylaminopurine, 0.2 M and -naphthaleneacetic acid and four times the normal concentration of MS minor salts was used. Histological studies of these cultures confirmed the organogenic and embryogenic nature of the cultures, by demonstrating the formation of shoot buds and somatic embryos, respectively. Similar responses were obtained in all 54 genotypes tested in this manner. The cultures retained the ability to regenerate complete plants for at least 12 months and 12–15 subcultures. Seeds have been obtained from several regenerated plants and when grown in the field these produced normal-appearing fertile plants.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetio acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MS Murashige and Shoog (1962) medium - NAA -naphthaleneacetic acid - picloram 4-amino-3,5,6-trichloropicolinic acid     

Flavonoid production in Scutellaria baicalensis callus cultures

St-20 and St-7 lines were isolated from the stem callus of Scutellaria baicalensis Georgi on indole-3-acetic acid and 2,4-dichlorophenoxyacetic acid media, respectively. The flavonoid content of St-20 line was superior to that of St-7 line. The growth and flavonoid (baicalin, baicalein, wogonin and wogonin-7-0-glucuronide) content in St-20 line were best on Linsmaier-Skoog's basal medium containing 10^-7 M–10^-5 M kinetin. St-20 line showed the same flavonoid content and pattern as the root of parent plant after the culture period of 70 days.     

In vitro plant regeneration of spinach from mature seed-derived callus

Summary A system for the regeneration of spinach (Spinacia oleracea L.) from mature dry seed explants has been established. The response of two commercial spinach cultivars, ‘Grandstand’ and ‘Baker’, was examined. Callus proliferation was most prominent on MS medium supplemented with 9.3 μM of 6-furfurylaminopurine (kinetin) and 3.39 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Adventitious shoot formation was observed within 8 wk after callus was transferred onto regeneration medium. Shoot regeneration was best from callus induced on 9.3 μM kinetin and 4.56 μM 2,4-D. The regeneration medium contained 9.3 μM kinetin, 0.045 μM 2,4-D, and 2.89 μM gibberellic acid (GA₃). Shoots were rooted on hormone-free medium, and plants grown in a greenhouse showed normal phenotype. This system is beneficial in rapid propagation of spinach plants, particularly when only a limited number of seeds are available.     

Origin of plantlets and callus obtained from chile pepper anther cultures

Summary Androgenesis occurred from chile pepper (Capsicum annuum L.) anthers incubated in a continuous warm environment (29° C) with continuous light. Forty plantes and embryoids were retrieved from anther cultures and anllyzed for isozyme markers. Of these, 35 exhibited a single allele for markers suggesting microspore origin, while 5 were heterozygous indicating somatic tissue origin. Chromosome numbers were confirmed for 21 plantlets, of which 16 were haploid and 5 were diploid. However, two plants exhibited a single allele for an isozyme marker but possessed the diploid chromosome number, suggesting spontaneous doubling. Anther cultures also produced callus. Nearly 92% of the slow-growing calli sampled were heterozygous for the isozyme marker, suggesting somatic tissue origin. More than 46% of the fast-growing calli exhibited only one allele for the marker, indicating microspore origin. Callus did not regenerate plantlets. The occurrence of both heterozygous and homozygous diploid plantlets from pepper anther cultures has important implications for applied breeding programs.     

Effects of incubation temperature on microspore callus production and plant regeneration in wheat anther cultures

Anthers of wheat cultivars Orofen and Pitic 62 were incubated for 8 days at 15, 20, 25, 30, 35 and 40°C before transfer to 25°C. Compared with anthers cultured at 25°C constantly, anthers treated at 30°C produced 40% more microspore callus and green plants in both cultivars whereas those treated at 35°C produced 2–3 fold more green plants. Treatment at 40°C was deleterious. Possible modes of action of high temperature on callus production and albinism were discussed.     

Differential callus type formation by auxins and cytokinin in in vitro cultures of pepper (Capsicum annuum L.)

ABSTRACT

Two types of callus were produced by pepper explants cultured in various media containing auxins, the cytokinin 6-benzylaminopurine (BAP) and the auxin transport inhibitor 2,3,5-triiodobenzoic acid (TIBA). Callus produced on media containing auxins alone was friable, grey-green or green-orange in colour and more compact, whereas when BAP was added to culture media with a low concentration of auxin or when the medium contained TIBA alone, the callus produced was white and very hard. This type of callus was also produced in cultures of older tissues and of young tissues cultured on hormonefree medium. Results are discussed in relation to the role of cytokinins in wounding, phenylpropanoid metabolism and lignin biosynthesis.     

Attempts to produce solasodine in callus and suspension cultures of Solanum mauritianum Scop.

Callus cultures of Solanum mauritianum Scop. were initiated from green berry explants on a hormone-free Murashige and Skoog (1962) medium excluding glycine, and containing 0.1 g L^–1 myo-inositol and 3% sucrose. Such cultures contained 10.08±0.59 g g^–1 DW of solasodine, which is equivalent to that in the leaves of mature S. mauritianum plants, but far less than that extracted from the green berries (185 g g^–1 DW). In vitro solasodine productivity could be increased by reducing the strength of the medium by half, substituting 3% glucose for 3% sucrose as carbon source, or by the addition of certain combinations of BA and NAA. Phosphate limitation and alterations in the carbon: nitrogen ratio were not able to increase solasodine productivity. Suspension cultures of S. mauritianum were initiated and maintained in a Murashige and Skoog (1962) medium with the RT vitamins of Khanna and Staba (1968), 0.1 g L^–1 myo-inositol, 3% sucrose and 1 mg L^–1 2,4-D. No solasodine was detectable in these cultures, or slight modifications thereof.Abbreviations BA benzyladenine - NAA naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog's (1962) medium     

Organogenesis and somatic embryogenesis from callus cultures in Muscari armeniacum Leichtl. ex Bak.

Summary Establishment of fast-growing, highly regenerable callus cultures was examined in Muscari armeniacum Leichtl. ex Bak. in order to develop an efficient genetic transformation system. High-frequency callus formation was obtained from leaf explants of cv. Blue Pearl on media containing 2,4-dichlorophenoxyacetic acid (2,4-D), α-naphthaleneacetic acid (NAA) or 4-amino-3,5,6-trichloropicolinic acid (picloram, PIC). Fast-growing, yellowish nodular callus lines and white friable callus lines containing a few somatic embryos were established on initiation medium supplemented with 4.5 μM 2,4-D and with 54 μM NAA, respectively. The yellowish nodular calluses vigorously produced shoot buds after transfer to media containing 0.44–44 μM 6-benzyladenine (BA), whereas the white friable calluses produced numerous somatic embryos upon transfer to plant growth regulator-free (PGR-F) medium. Histological observation of shoot buds and somatic embryos indicated that the former consisted of an apparent shoot meristem and several leaf primordia, and the latter had two distinct meristematic regions, corresponding to shoot and root meristems. Both shoot buds and somatic embryos developed into complete plantlets on PGR-F medium. Regenerated plants showed no observable morphological alterations. High proliferation and regeneration ability of these calluses, were maintained for over 2 yr.     

The role of calcium channels in anthocyanin production in callus cultures of Daucus carota

The involvement of Ca²⁺ ATPases in anthocyanin accumulation in callus cultures of Daucus carota was investigated under the influence of calcium and calcium channel modulators. Ionophore (I) treatment enhanced callus growth and anthocyanin accumulation. Increasing the amount of calcium applied to cultures enhanced the anthocyanin level. Ionophore treatment influenced the enhancement of Ca²⁺ATPase and endogenous titres of PAs. Addition of the calcium channel blocker verapamil or the calmodulin antagonist chlorpromazine to the A23187 (ionophore) treated cells caused a reduction in anthocyanin levels. Channel blockers reduced Ca²⁺ATPase activity, which was restored by ionophore treatment, showing the importance of calcium in anthocyanin production. Higher ethylene levels were also found in treatment with ionophore or 2X calcium. Thus the influence of ionophore in anthocyanin production and its inhibition by calcium channel modulators suggests that calcium plays an important role in the production of anthocyanin by carrot callus cultures.     

Growth Performance of Cuttings Raised from in vitro and in vivo Propagated Stock Plants of Rosa damascena Mill.

Comparative studies on rooting and growth performance of cuttings raised from in vitro and in vivo grown plants of Rosa damascena are described. Cuttings were treated with different auxins and upon transfer to soil their growth performance was recorded. Overall, the auxin treated cuttings of in vitro raised plants responded better than the cuttings of in vivo raised plants. Optimal response for percentage of rooting, root number, root length and bottom breaks was observed at 100 mg dm^–3 IBA. The cuttings derived from in vitro raised plants showed a significantly better response for percent rooting, root number, root length and bottom buds in control treatments.     

Plant regeneration from leaf-derived callus cultures of the tropical pasture legume Stylosanthes scabra Vog.

Callus cultures of 5 genotypes of S. scabra Vog. were optimally established from leaf tissue on Murashige and Skoog (MS) basal medium supplemented with 0.5–2.0 mg l^-1 2, 4-Dichlorophenoxy acetic acid (2, 4-D) and 1.0–2.0 mg l^-1 6-benzylaminopurine (BAP). On media containing 2, 4-D only, calli were soft, and rhizogenesis occurred on calli of 4 genotypes. Calli formed on media containing BAP only, but not with kinetin only. In the presence of 2, 4-D, BAP inhibited rhizogenesis and promoted better callus growth than kinetin. High frequency shoot induction was achieved for 3 genotypes on MS +2.0 mg l^-1 BAP. Roots formed on shoots when sub-cultured on half-strenght MS without growth regulators. The form of cytokinin used in the callus induction media appeared to affect subsequent shoot organogenesis. Genotypic differences were observed for shoot organogenesis. There was some morphological variation evident among regenerants.