Establishment Of Callus Cultures Research Articles

Establishment of callus cultures and quantification of Caffeic acid using HPTLC from Desmodium gangeticum (L.)

Desmodium gangeticum (L.) belonging to family Fabaceae is an economically important medicinal plant which isutilised in Dashmoolarishta. Various bioactive compounds have been isolated from whole plant and roots, and one of them is an important phenolic compound – caffeic acid (CA). This phenolic acid and its derivatives have antioxidant, anti-inflammatory, anticarcinogenic and hepatocarcinoma, a highly aggressive and causing considerable mortality across the world. In the present study, leaf explants were placed on MS medium fortified with different concentration of cytokinin (BA/Kn) and auxin (IAA/NAA) for establishing callus cultures. MS medium fortified with BA (20 µM) and IAA (2 µM) was optimised for the same. Methanolic extracts of in vivo leaf sample (DG1) and in vitro sample (leaf derived callus) (DG2) were assessed for CA quantification using HPTLC. Thus, the chemical fingerprint that was obtained, confirmed that DG 2 of D. gangeticum exhibited the potency to synthesise more amount of CA (316 ± 7.5 µg/g DW) in comparison to DG1 which was 194 ± 2.3 µg/g DW.

Establishment of callus-cultures of the Argentinean mistletoe, Ligaria cuneifolia (R. et P.) Tiegh (Loranthaceae) and screening of their polyphenolic content

Ligaria cuneifolia (R. et P.) Tiegh (Loranthaceae), known as liga, muerdago criollo, or Argentinean mistletoe, is a hemiparasitic plant with a broad distribution in central and northern Argentina. Pharmacological studies showed that L. cuneifolia extracts have hypolipemic, antioxidant, antibacterial, and immunomodulatory effects. We have established callus cultures from embryo and haustoria fragments. The highest frequency of callus formation from embryos (85%) was obtained on White medium with 4% (w/v) sucrose and 2.5 µM 1-naphtalene acetic acid and 9.2 µM kinetin as plant growth regulators (PGRs). From haustoria, the best result (35%) was obtained on Gamborg medium with 3% (w/v) sucrose and 0.45 µM 2,4-dichlorephenoxyacetic acid and 0.47 µM zeatin as PGRs. Thin layer chromatography showed that callus methanolic extract (2.5% w/v) had a lower content of flavonoids and proanthocyanins as compared to the wild plant (5% w/v for leaves, stems, and flowers), but a higher content of hydroxycinnamic acids. High performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) showed the presence of quercetin glycosides and phenolic acids in the methanolic extracts both from the parent plant and the callus obtained from embryo. Callus cultures were established from embryo and haustorium explants of Ligaria cuneifolia. Leaves, stems, and meristems were recalcitrant to in vitro culture. Callus tissues contained quercetin glycosides and phenolic acids.

Influence of thidiazuron on callus induction and crocin production in corm and style explants of Crocus sativus L.

Crocus sativus L., mostly famous as saffron, has gained more attention due to its crocin (crocetin ester) pigment responsible for its extensive pharmaceutical properties. In this study, we established two different callus cultures from corm and style explants of saffron to find out the best explant as a suitable source for crocin production. Comparative analyses of total phenolic, flavonoid, carotenoid and anthocyanin contents were also performed in the two callus cultures. For callus induction, different combinations of MS medium with name thidiazuron (TDZ), benzylaminopurine (BA), 1-naphthaleneacetic acid (NAA), and 2,4-dichlorophenoxyacetic acid (2,4-D) alone or in combination were tested. Of the used media, all the combinations containing TDZ and NAA gave 100% callus induction. HPLC-DAD and HPLC–ESI-MS analysis were used for identification of crocin esters in established callus cultures. The highest amount of 0.35 mg g−1 DW crocin was detected in style originated calli grown on the medium containing 3 mg L−1 NAA + 1 mg L−1 TDZ while the corm calli showed the most abundant total carotenoid (0.73 mg g−1 DW), phenolic (15.04 mg gallic acid equivalent g−1 DW) and flavonoid (0.76 mg rutin equivalent g−1 DW) contents. In general, style-derived calli showed longer time survival with a fine texture and good quality compared to corm-derived calli.

Total phenolics, resveratrol content and antioxidant activity of seeds and calluses of pinto peanut (Arachis pintoi Krapov. & W.C. Greg.)

Arachis pintoi is a peanut species native to Brazil, which is cultivated in many countries for animal forage, soil cover, landscaping, and recovery of degraded areas. Tissue culture studies for this species have been focused in plant production, whereas works on in vitro secondary metabolites production are scarce. The goal of the present work was to establish callus cultures from different seed explants of A. pintoi, aiming at evaluating the potential for metabolites production and antioxidant activity. Embryonic axes, leaflets, and cotyledons were cultured on solidified MS medium supplemented with picloram (PIC), 2,4-dichlorophenoxyacetic acid (2,4-D), thidiazuron (TDZ) or different combinations of 6-benzyladenine (BA) and α-naphthaleneacetic acid (NAA), under light or dark conditions. Friable calluses with a high biomass (4.3 ± 0.3 g FW per callus) were obtained from embryonic leaflets cultured on medium supplemented with 17.6 µM BA plus 5.4 µM NAA, in the dark. Cotyledons and embryonic axes cultured in the presence of 4.4 µM BA combined with 10.8 µM NAA formed heterogeneous calluses with a compact base and a large friable surface. Trans-resveratrol and other stilbenes that were not found in seeds were detected in callus extracts, especially those originated from cotyledons, although these materials showed lower total phenolic contents (TPC) when compared with seeds with and without testa, as well as cotyledons. Extracts from seeds with testa and from calluses derived from cotyledons and embryonic axes showed the highest EC50 in DPPH assays. No correlation between TPC, trans-resveratrol and antioxidant activity was observed.

Establishment of Callus Induction and Cell Suspension Cultures of Dendrathema Indicum var. Aromaticum a Scented Chrysanthemum

Dendranthema indicum var. aromaticum is an important aroma plant in genus Dendrathema, and the establishment of callus cultures and cell suspension cultures is the basement of further protoplast fusion studies, which make it possible to breed new fragrant chrysanthemum. In this study, the effects of different plant growth regulating substances in different concentrations on callus induction were investigated with stem segments, leaves, petioles as explants. The results showed that the optimal explants were lower stem segments according to the percentage of callus formation, callus hardness, growth potential and shoot differentiation. The optimal induction mediums were MS supplemented with 1.0 mg．l-12.4 D and 0.2 mg．l-1 6-BA. The cell suspension culture system was established by using the subculture calli. The results showed that the suitable inoculum size was 2g and the suitable cell suspension culture medium was MS supplemented with 0.2 mg．l-1 6-BA and 0.5 mg．l-1 2,4-D. The time course of cell growth showed that the greatest cell fresh weight appeared on day 14 and the highest cell viability on day 3.

Stepharine production in morphogenic cell cultures of Stephania glabra (ROXB.) Miers

Young leaves of Stephania glabra (ROXB.) Miers. (Menispermaceae) were used to establish callus cultures with the goal of studying their ability to produce the valuable alkaloid stepharine. The establishment of callus cultures from this plant is extremely difficult because primary calli are not viable and must be transferred to a liquid culture medium for subsequent subculturing. All obtained cultures possessed high morphogenic or rhizogenic potential. The methanolic extracts of two examined cell lines were analyzed by HPLC with high-resolution mass spectrometry to determine the alkaloid composition. Eleven alkaloids were detected in both cultures, including stepharine, magnoflorine, menisperine, roemerine, palmatine, corydalmine, N-methylcorydalmine, columbamine, tetrahydropalmatine, jatrorrhizine and tetrandrine. The predominant alkaloid was stepharine, the structure of which was identified through 1H and 13C NMR, UV, HRMS and tandem MS. Quantitative HPLC-UV analysis showed that stepharine was produced at high levels (0.9 % dry cell weight) in the morphogenic callus culture S2-L cultivated in liquid media. To our knowledge, this is the highest level of stepharine production identified in plant cell cultures. The overall production parameters of the culture have remained stable over a long-term cultivation period for 3 years.

Mucilage Synthesis in Callus Cultures of Plantago ovata Forsk

The seed husk of Plantago ovata known as psyllium (Isabgol) yields medicinally important mucilage. The amount of mucilage produced is about 25 % (by weight) of the total seed yield. In the present study, an attempt was made to increase the amount of mucilage through callus cultures of P. ovata. The first step involved establishment of callus cultures in P. ovata. Leaf explants from 10 to 20 day old seedlings were cultured on MS basal medium supplemented with various concentrations of different plant growth regulators. The highest rate of callus induction (89 %) was obtained on MS medium containing 0.5 mg l−1 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.0 mg l−1 Kinetin. The mucilage content was estimated from the callus obtained in different media. The best mucilage production was obtained in the MS medium supplemented with 0.25 mg l−1 2,4-D and 0.25 mg l−1 thidiazuron. Significant differences with regard to the total mucilage content were recorded. Overall, the callus produced nearly five times more mucilage than the seeds. The present technology provides an alternative route to production of large quantities of mucilage without plants.

Isolation, quantification and anti‑microbial screening of flavonoids from callus of Vitex negundo Linn

Context: Natural habitats for medicinal plant are disappearing fast and together with environmental instabilities, it is increasingly difficult to acquire plant‑derived compounds. This has prompted scientists to consider the possibilities of using cell cultures as an alternative supply for the production of plant natural products. Aim: The present investigation deals with the establishment of callus cultures from leaf explants of Vitex negundo, to extract flavonoids from the raised callus and to assess their anti‑microbial potential. Materials and Methods: Callus culture of Vitex negundo has been established on Murashige and Skoog’s medium supplemented with 2, 4‑D using leaf explants. Growth index (GI) was calculated for the tissues obtained after 2, 4, 6 and 8 weeks, respectively. Calli of 2, 4, 6 and 8 weeks old were used for extraction of total flavonoids (free and bound) using standard method. Flavonoid extract of 6 weeks old callus with maximum GI and maximum flavonoid content was then tested against Staphylococcus aureus, Agrobacterium tumefaciens (bacteria), Candida albicans (yeast) for their anti‑microbial potential. Minimum inhibitory concentration, minimum bactericidal/fungicidal concentrations and total activity were also evaluated. Results: GI was found maximum in 6 weeks old tissue (0.76). Total flavonoids from 6 week old tissue showed maximum zone of inhibition against A. tumefaciens. Kaempferol was identified from total flavonoids of 6 week old callus through, thin layer chromatography (TLC), preparative TLC, Melting point and Infra red spectral studies. Content of kaempferol in 6‑week‑old calli was also estimated using standard Spectrophotometric method, which came out to be 54.25 μg/mg d.wt (dry weight) of calli. Conclusion: The protocol of callus induction of V. negundo opens new vistas that could facilitate phytochemical production, extraction of pharmaceuticals and their screening against different human pathogens from the callus without harvesting the plant itself. Key words: Anti‑microbial potential, callus, flavonoids, Vitex negundo

Influence of different sterilization methods on callus initiation and production of pigmented callus in Arnebia densiflora Ledeb.

We analyzed the effects of sodium hypochlorite and Plant Preservative Mixture (PPM) on surface sterilization. We also examined the effects that the addition of an antibiotic-antimycotic solution to the culture medium had on callus induction. Explants were initially sterilized with different concentrations of sodium hypochlorite and cultured on MS media containing kinetin (0.29 \muM) and naphthalene acetic acid (NAA, 10 \muM). No calluses were produced, either because of contamination of the explants, or loss of explants as a result of the high levels of sodium hypochlorite. The application of PPM and antibiotics at different concentrations reduced contamination and led to callus induction from shoot apexes and young root explants. The best callus responses were obtained using PPM at 1%-2%, whereas callus induction on shoot apexes diminished at higher concentrations (4% PPM). This is the first report of successful sterilization and reduced contamination of explants from naturally field grown A. densiflora by PPM. Moreover, established callus cultures produced pigmented calluses, which were analyzed spectrophotometrically.

Production of anti-HIV-1 calanolides in a callus culture of Calophyllum brasiliense (Cambes)

Calophyllum brasiliense (Cambes) produces calanolide secondary metabolites that are active against human immunodeficiency virus type 1 reverse transcriptase. In this study, it was demonstrated that plant tissue culture is a useful technique for producing these metabolites. Different concentrations and combinations of plant growth regulators were tested in leaf and seed explants to establish callus cultures capable of producing calanolides. Highest callus induction (100%) was achieved when seed explants were incubated in a medium consisting of 8.88 μM 6-benzyladenine and 20 μM picloram. Highest callus induction (80.67%) was observed when leaf explants were incubated on a medium consisting of 0.46 μM kinetin and 5.37 μM α-naphthaleneacetic acid. High-performance liquid chromatography quantitative analysis revealed higher calanolide B and calanolide C production in calluses from seed explants than those developed from leaves (309.25 vs. 8.70 mg kg−1 for calanolide B; 117.70 vs. 0.0 mg kg−1 for calanolide C).

Biosynthesis and accumulation of a medicinal compound, Picroside-I, in cultures of Picrorhiza kurroa Royle ex Benth

Establishment of callus cultures and plant regeneration from different explants coupled with estimation of Picrosides in morphogenetically different developmental stages showed that Picroside-I accumulates in shoot cultures of Picrorhiza kurroa with no detection of Picroside-II. The Picroside-I content was 1.9, 1.5, and 0.04 mg/g in leaf discs, stem and root segments, respectively. The Picroside-I content declined to almost non- detectable levels in callus cultures derived from leaf discs, stem segments with no change in Picroside-I content in root segments or calli derived thereof. The biosynthesis and accumulation of Picroside-I started in callus cultures differentiating into shoot primordia and reached to the concentrations comparable to original explants of leaf discs and stem segments in fully developed shoots with contents of 2.0 and 1.5 mg/g, respectively. The shoots formed from root-derived callus cultures were relatively slow in growth as well as the amount of Picroside-I content was comparatively low (1.0 mg/g) compared to shoots derived from callus cultures of leaf and stem segments, respectively. The current study concludes that the biosynthesis and accumulation of Picroside-I is developmentally regulated in different morphogenetic stages of P. kurroa tissue cultures.

Chrysophanol glycosides from callus cultures of monocotyledonous Kniphofia spp. (Asphodelaceae).

We established callus cultures of the monocotyledonous plants Kniphofia foliosa and K. tuckii (Asphodelaceae), which produce the anthraquinone derivatives chrysophanol and its glycosides. The minor product chrysophanol 8-O-beta-gentiobioside was fully characterized by spectroscopic analysis and synthesis.

Endogenous hormone concentrations and embryogenic callus development in wheat

Immature zygotic embryos of two wheat (Triticum aestivum L.) genotypes, known for their different ability to generate embryogenic callus, were used as initial explants to establish callus cultures. Embryogenic and non-embryogenic calluses were obtained from the competent genotype (`Combi'), while only non-embryogenic callus was produced by the incompetent one (`Devon'). The morphogenetic competence of each callus type was evaluated by transferring some segments to regeneration conditions. The endogenous hormone concentrations (free indole-3-acetic acid [IAA], abscisic acid [ABA], gibberellins 1, 3 and 20 [GAs], zeatin/zeatin riboside [Z/ZR] and N6[Δ2-isopentenyl] adenine/ N6[Δ2-isopentenyl] adenosine; [iP/iPA]) of the initial explants were determined by means of radio-immunoassay and showed that the only difference was the higher concentration of ABA found in the embryos of the most competent genotype; whose embryos showed a reduced rate of precocious germination. When analysing the endogenous hormone concentrations in the various callus types generated in each genotype, it was found that only differences in the free IAA concentrations were associated with variations in the morphogenic properties of the calluses. Higher concentrations of endogenous free IAA were typical of embryogenic callus cultures. It was also observed that a loss in the embryogenic competence of the calluses, due to a prolonged time of culture, occurred concomitantly with a reduction in free IAA concentrations, practically to the concentrations found in the non-embryogenic calluses.

Hormonal status of maize initial explants and of the embryogenic and non-embryogenic callus cultures derived from them as related to morphogenesis in vitro

Endogenous hormone levels (indole-3-acetic acid [IAA], abscisic acid [ABA], gibberellins 1, 3 and 20 [GAs], zeatin/zeatin riboside [Z/ZR] and N 6[Δ 2-isopentenyl] adenine/ N 6[Δ 2-isopentenyl] adenosine; [iP/iPA]) were analysed in immature maize zygotic embryos of two maize ( Zea mays L.) genotypes, known for their distinct ability to generate embryogenic (E) callus. No differences were found among genotypes in the hormone contents of the embryos. These embryos were also used as initial explants to establish callus cultures. E and non-embryogenic (NE) calli were obtained from the competent genotype (A188), while only NE callus was produced by the incompetent one (B73). The morphogenetic competence of each callus type was evaluated by transferring some segments to regeneration conditions. When analysing the endogenous hormone levels in the various callus types generated in each genotype, it was found that only differences in the IAA levels accounted for variations in the morphogenic properties of the calli. Higher levels of endogenous IAA were typical of embryogenic callus cultures. It was also observed, that a loss in the embryogenic competence of the calli, due to a prolonged time of culture, occurred concomitantly with a reduction in the IAA levels, practically to the levels found in the non-embryogenic calli.

Differential expression of a cell wall-localized peroxidase isoenzyme capable of oxidizing 4-hydroxystilbenes during the cell culture of grapevine (Vitis vinifera cv. Airen and Monastrell)

Differential expression and localization of peroxidase isoenzymes capable of oxidizing 4-hydroxystilbenes was studied during establishment of callus cultures from Vitis vinifera ‘Airen’ (anthocyanin-non accumulating) and ‘Monastrell’ (anthocyanin-accumulating) berries. Callus formation from mesocarp tissues was accompanied by differential expression of several peroxidase isoenzyemes located in cell walls, among which only peroxidase isoenzyme A1 was capable of oxidizing 4-hydroxystilbene to any great extent. Likewise, grape cell cultures were capable of accumulating the grape stilbene phytoalexin, resveratrol. However, e-viniferin, the most powerful phytoalexin in grapevines and previously considered as the product of peroxidase-mediated oxidative coupling of two resveratrol moieties, was only detectable in trace amounts. Since grapevine suspension cell cultures were unable to produce H2O2 as revealed by the luminol test, H2O2 production by the cultured cells appears to be one of the main factors which limits resveratrol oxidation in the cell walls of grapevine cells cultured in suspension.

Callus production, suspension culture and in vitro alkaloid yields of Ephedra

The effects of a range of plant growth regulators on callus production in various Ephedra species were examined. Species examined were E. andina, E. distachya, E. equisitina, E. fragilis var, camplyopoda, E. gerardiana, E. intermedia, E. major ssp procera, E. minima and E. saxatilis. All species produced callus on modified MS medium supplemented with 0.25 μM kinetin and 5.0 μM 2,4-dichlorophenoxyacetic acid or 1-naphthaleneacetic acid. Neither indole-3-acetic acid nor 3-indolebutyric acid induced significant callus formation but the latter maintained growth of established callus cultures in several species. Suspension cultures of several species were established in MS medium supplemented with 0.25 μM kinetin and 5.0 μM 2,4-dichlorophenoxyacetic acid or 1-naphthaleneacetic acid. Sustained fresh weight doubling times of 70±7h were recorded for cell suspension cultures of E. andina grown in a semi-continous air-lift bubble bioreactor and a minimum doubling time of 56 h was recorded for E. andina in batch culture. It also proved possible to immobilise E. andina batch cultures in sodium alginate beads.

Characterization and mapping of a gene controlling shoot regeneration in tomato

SummaryThe superior regeneration capacity of Lycopersicon peruvianum was introduced into the cultivated tomato Lycopersicon esculentum by backcrossing hybrid material with the tomato genotype VF11. In segregating material derived from these backcrosses, the ability to regenerate shoots on root explants cultured on a zeatin‐containing medium, was highly correlated with the ability to regenerate shoots on established callus cultures. The efficient shoot‐regenerating root explant system permitted us to study the genetics of this trait and to locate the genes involved, using a set of morphological markers defining all 12 tomato chromosomes. Depending on the tomato genotype, mono, ‐di‐ or trigenic ratios were observed. It is concluded that a dominant L. peruvianum allele at a locus (Rg‐1) near the middle of chromosome 3 determines efficient shoot regeneration on root explants in tomato in combination with dominant alleles at one or two other loci of either L. peruvianum or L. esculentum origin. The map location of the Rg‐1 locus was refined further using a number of chromosome‐3‐specific RFLPs. The addition of new classical and RFLP linkage data to existing literature data and subsequent processing resulted in a revised and integrated map of tomato chromosome 3. From a morphological and physiological analysis of genotypes differing in Rg phenotype, it is concluded that the genetic component associated with regeneration determines the maintenance of morphogenetic competence and not the sensitivity to hormones.

Plant regeneration capacity of callus derived from leaf, stem, and root segments of Populus alba L. x P. grandidentata Michx.

Expiants for establishing callus cultures originated from in vitro cultured hybrid poplar (Populus alba L. X P. grandidentata Michx.). Plant regeneration was achieved from established callus cultures derived from stem internodes (SI), leaf discs (LD), and root segments (RS). Shoot regeneration from callus occurred within 4 weeks of culture on most of the media tested. Frequency of shoot formation was greatly increased by subculturing the selected organogenic calli on regeneration media. The highest rate of multiple shoot formation (an average number of 7/SI, 11/LD, and 8/RS) was obtained by using 0.05 μM IBA in combination with 22.5 μM 2iP, 22.5 μM zeatin, and 12.5 μM 2iP, respectively. Regenerated shoots were easily rooted in polyterra(™) peat plugs in transparent plastic boxes. The rooted plantlets were subsequently transferred to pots containing an artificial potting mix.

Chromosomal instability in cell- and tissue cultures of tomato haploids and diploids

The effect of the tissue culture system, the genotype and the ploidy level of the plant material used as explant source on the stability of the ploidy level of plants regenerated fromcell and tissue cultures of tomato was investigated. In addition the use of the chloroplast number in guard cells as a measure for ploidy level was evaluated. Haploids of tomato were very instable, which instability was observed already in somatic root tip and leaf cells. The number of regenerated plants that retained the original ploidy level differed significantly between the tested haploids. The plants that were regenerated from leaf explants of diploids were predominantly diploid in contrast to the plants regenerated from established callus cultures and protoplast where the majority was tetraploid.

Stable ploidy levels in long-term callus cultures of loblolly pine

Ploidy levels were calculated for callus cultures of loblolly pine (Pinus taeda L.), based on nuclear DNA content measured by Feulgen cytophotometry. The nuclear DNA content of initial stem explants showed a predominant 2C condition with less 3C and 4C, in ratios approximating those expected from diploid cells as they replicate DNA in the mitotic cell cycle. Cells with higher ploidy were produced during callus initiation, as indicated by a sharp reduction in the 2C population and a concomitant increase in higher DNA levels up to 8C. A gradual decrease in the higher ploidy levels occurred in subsequent subculture intervals, so that by 18 weeks the diploid nuclear DNA distribution was again observed, with complete elimination of DNA levels greater than 4C. Established callus cultures derived from stem or embryo explants and cultured on three different nutrient media for 48-76 weeks also showed the diploid nuclear DNA distribution with no indication of polyploid cells.