Dry Callus Research Articles

Phenolic and flavonoid amounts and antioxidant capacity of Lavandula officinalis (lavender) callus grown in different growth regulator combinations

In this study, it was aimed to determine the total antioxidant capacity and phenolic and flavonoid amounts of Lavandula officinalis (lavender), which is an aromatic plant, callus extracts. Combinations of Naphthalene Acetic Acid (NAA), Benzylaminopurine (BAP), Kinetin (KIN) and 2,4-Dichlorophenoxy Acetic Acid (2,4-D) plant growth regulators at different concentrations were used in the growth medium. The specified analyzes were carried out for the extracts obtained from the callus of plants grown in different media. The callus were extracted with water and ethanol solvents. Total phenolic amount analyzes were accomplished using the Folin-Ciocalteu method. The spectrophotometric AlCl3 method was used for the total flavonoid amount analysis and the antioxidant capacity of lavender callus extracts was measured considering the radical cation capture ability of 2,2′-azino-bis -3- ethylbenzthiazoline -6- sulphonicacid (ABTS). The highest antioxidant capacity (9.24 ± 0.14 mmol TEAC/g dry callus weight) was obtained from the callus of plants grown in medium containing 0.5 mg/L BAP + 0.5 mg/L 2,4-D combination; the highest amount of phenolic substance (35.74± 0.48 mg GAE/g dry callus weight) was obtained from the callus of plants grown in medium containing the combination of 0.5 mg/L BAP + 0.5 mg/L NAA; the highest amount of flavonoid substance (32.42 ± 0.46 mg QE/g dry callus weight) was obtained from the callus of plants grown in medium containing the combination of 0.5 mg/L BAP + 1 mg/L 2,4-D. The results are compared for the combination of plant growth regulators and the effects of the different growth medium ingredients were specified.

Soybean Callus—A Potential Source of Tocopherols

In vitro cultures have been used as an effective means to achieve a high level of secondary metabolites in various plants, including soy. In this study, the contents of α-, γ-, and δ- tocopherol were quantified in soybean callus, and their amounts were compared to those of soybeans cultivated using the conventional tillage system with three weed controls (respectively without herbicide and with two variants of herbicide). Soybean callus was produced using Murashige and Skoog 1962 (MS) medium supplemented with 0.1 mg/L 6-Benzylaminopurine (BAP) and 0. 1 mg/L Thidiazuron (TDZ). The highest amount of fresh callus was obtained from soybeans from the conventional tillage system with second weed control (S-metolachlor 960 g/L, imazamox 40 g/L, and propaquizafop 100 g/L) respectively 13,652.4 ± 1177.62 mg. The analyzed tocopherols were in much higher content in soy dry callus than the soybean seeds (5.63 µg/g compared with the 0.35 α-toco in soybean, 47.57 µg/g compared with 18.71 µg/g γ-toco or, 5.56 µg/g compared with 1.74 µg/g β-toco). The highest content of the three analyzed tocopherols was γ -tocopherol, both in callus and soybeans. Furthermore, the data showed that herbicides used in soybean culture significantly influenced both the in vitro callus production and the tocopherol callus content (p ˂ 0.05). Altogether, soybean callus can be an important source of tocopherols, and herbicides significantly influence in vitro callus production and the tocopherol callus content.

Callus inducement of Toona sinensis: Potential agents against SARS-Corona virus replication

Bioprospecting of anti-SARS-corona virus phytochemical agents is an important issue today as an alternative to chemical drugs whose effectiveness has not been fully successful with no light effect. Quercetin, a component of Toona sinensis young leaves extract, was reported to have anti-viral activity against SARS-corona virus replication in vitro. Currently, the low efficiency of secondary metabolite production is an obstacle. Production of secondary metabolites from callus induction in vitro is considered more efficient and in a short time for commercial applications. The composition of hormones in callus media affects the secondary metabolites formed. The study was conducted to determine the effect of synthetic hormones auxin (BAP) and cytokinin (NAA) on callus percentage, fresh and dry callus cell weight and the average time of callus formation. The call percentage (100%) was obtained in different combinations of BAP and NAA in MS medium. The highest fresh weight of callus (165.50 gL−1) and dry cell weight (28.47 gL−1) were observed in MS medium (1.5 mgL−1 BAP and 1 mgL−1 NAA) within 7 days initial formation time of callus. The results showed that all the indices measured were positively correlated with callus induction in T. sinensis.

ANALYSIS OF SECONDARY METABOLITES OF CALLUS OF RAMBUTAN Nephelium lappaceum L

Rambutan plant (Nephelium lappaceum L.) is a member of the Sapindaceae family. The rambutan plant is one of the natural ingredients that can be developed as traditional medicine. Rambutan peel has the potential for good antioxidant and anticancer activity. Rambutan fruit does not grow every time it needs efforts to produce the active substance in rambutan, using plant tissue culture techniques. The use of the correct variety of mediums and hormones at the right concentration is the key to thriving tissue culture. Explants derived from rambutan leaves were planted precisely on solid media Murashige and Skoog (MS) and WoddyPlant Medium (WPM) containing Indole-3-Butyric Acid (IBA) and Kinetin. After seven days, the callus was subcultured, then after 35 days, the subculture callus was collected and dried. Dry callus and rambutan leaves (Wild type) were macerated with n-hexane, ethyl acetate, and ethanol. The concentrated extract was then applied to a GF 254 silica gel plate with the mobile phase Toluene-Acetone (7: 3) and n-hexane-EthylAsetate (3: 7). The results showed that the concentration of IBA 2 ppm and kinetin three ppm was the best combination because it produced callus. TLC results of rambutan leave with plant tissue culture containing flavonoids and triterpenoids. This study provides new information regarding the induction of rambutan callus and can become the basis for producing active metabolites in rambutan with cell suspension culture development.

Effect of light quality and tissue origin on phenolic compound accumulation and antioxidant activity in Camellia japonica calli

In Camellia japonica callus culture, the effects of light quality on the accumulation of phenolic compounds were examined. To this end, the calli were cultured under three monochrome [white (W), red (R), and blue (B)] and two mixed [red/blue (RB) and red/green/blue (RGB)] light sources. After 4 weeks of culture, fresh and dry callus weights were determined, and phenolic content was analyzed using high-performance liquid chromatography. We also examined the effects of RGB light on calli derived from four origins (leaf: CL; root: CR; petal: CP; and ovary: CO). Total phenolic and flavonoid content and DPPH radical scavenging activity were highest in calli exposed to RB and RGB. Catechin was detected only in calli exposed to mixed light (RB and RGB). Interestingly, red pigmentation development in CL and CR calli after 2 weeks of culture and organogenic calli in CR and CP samples was observed. Furthermore, expression of the phenylalanine ammonia lyase and chalcone synthase genes was found to be higher in the CR line than in other lines, and total phenolic and flavonoid content was also higher in the CR and CP lines. The findings of the present study revealed that the accumulation of phenolics and flavonoids in callus tissue varies depending on light quality, which stimulates cell division, and may thus affect the proliferation and organogenesis of C. japonica callus. We also established that the effects of light on red pigment expression and the accumulation of bioactive compounds are dependent on the tissue origin of callus material.

Labellar secretion and secretory trichomes of Rhetinantha cerifera (Barb. Rodr.) M.A.Blanco (Orchidaceae, Maxillariinae): micromorphology and composition

The structure and composition of the labellar secretory trichomes of Rhetinantha cerifera (Barb. Rodr.) M.A.Blanco were analyzed by scanning electron and light microscopy, as well as histochemistry and chemistry. Histological analyses revealed that the chlorophyllated and dry callus at the basal region of labellum is non-secretory. The white sticky secretion is produced by unicellular secretory trichomes (not papillae), occupying the V-shaped ridge at the apical and median regions of the labellum, and the central portion of the labellar adaxial base, behind the callus. Chemical analyses of dichloromethane extracts of the secretion detected several long carbon chain constituents common in plant waxes (n-alkanes, carboxylic acids, alcohols, esters) and phytosteroids, predominantly cycloartenol derivatives. Histochemical tests showed that the secretion contains terpenoids (oleoresin), free fatty acids, phenolic compounds (including flavonoids), and polysaccharides (mainly mucilage); the results were negative for alkaloids. The secretory unicellular trichomes can concomitantly activate different metabolic pathways, and the exudate should be characterized as “heterogeneous mixtures,” consisting of lipophilic and hydrophilic compounds. Therefore, the labellar secretion is chemically more complex than plant waxes composition. The specific epithet cerifera is thus misleading, and previously reported interpretations regarding the secretion are equivocated. Based on the present results and those from the literature, it is suggested that R. cerifera and R. notylioglossa (Rchb.f.) M.A.Blanco are taxonomical entities that should be merged into a single species, as has been suggested in Flora Brasil 2020 under construction.

Callus induction and somatic embryogenesis in Stevia rebaudiana Bertoni as a medicinal plant

Stevia rebaudiana (Bert.) from Asteraceae family is a useful medicinal plant that prevents and cures diabetes, blood pressure, weight gain and tooth decay. Due to self-incompatibility in stevia, somatic embryo investigation for artificial seed production is valuable in this plant. In order to evaluate the callus induction characteristics in stevia, a factorial experiment was laid out based on a completely randomized design with three replications. The factors included ten hormone combinations and control, two kinds of media (MS and B5) and two types of explants (leaf and internode). Callus induction characters including the percentage of callus formation, days to callus induction, fresh and dry callus weight were recorded. Analysis of variance showed significant differences (p<0.01) among hormone combinations, media and explant types as well as their interactions. The best treatment for callus induction with minimum time to callus formation was 1 mg/l NAA+1 mg/l BAP. The highest fresh and dry callus weight were obtained on B5 medium supplemented by 1 mg/l 2,4-D+1 mg/l BAP (in leaf explant) and 0.25 mg/l 2,4-D+ 0.1 mg/l BAP (in internode explant). These results can be used in suspension culture. To induce somatic embryogenesis in suspension culture, six hormone treatments were investigated. The highest somatic embryogenesis percentage was obtained in MS medium supplemented by 2 mg/l 2,4-D+ 0.5 mg/l NAA+0.5 mg/l BAP.

Changes in biochemical constituents, enzyme activities and protein profiles during root-shoot differentiation in callus culture of Dioscorea alata

The changes in biochemical constituents and polypeptide (PP) bands were studied during root-shoot differentiation in Dioscorea alata (greater yam). Among different explants used - leaf, node, internode and tuber, node explants were found to be the best for induction and growth of callus. Depending on weights of fresh and dry callus, high growth and best callusing were observed on MS medium supplemented with 2.5 µM NAA. This high growth value callus was subsequently supplemented with various concentrations and combinations of growth regulators to identify the suitable regeneration media. Regeneration of roots occurred in 12-16 days and was the best on MS medium having 2.0 µM NAA and 0.5 µM IBA. Shoots regenerated in 16 days in MS medium supplemented with 2.0 μM BAP and 0.5 µM NAA. The biochemical constituents such as total soluble sugars, reducing sugars, total soluble proteins and total phenols decreased whereas free amino acids increased during root and shoot differentiation of D. alata. Activities of enzymes, viz. α-amylase, acid invertase, acid phosphatase, acid protease and peroxidase decreased during callus differentiation. While four PP bands (25.56, 24.35, 19.13 and 18.2 kDa) appeared during root differentiation, only three PP bands (53.7, 25.12 and 19.13 kDa) were noted during shoot differentiation. One common PP band (19.13 kDa) appeared during both root and shoot differentiation. There was disappearance of four common bands (89.13, 69.8, 36.3 and 27.43 kDa) during differentiation of root and shoot. To conclude, changes in biochemical constituents and expression of root/shoot specific PP may be used as markers to characterize differentiation pathway and to augment the selection of regenerating potential callus for rapid in vitro propagation.

In vitro Accumulation of Polyphenols in Tea Callus Derived from Anther.

Background:Tea is an economic important crop with high medicinal value due to rich polyphenols content. In the present research we studied the accumulation of polyphenols of in vitro regenerated callus from anthers.Objective:Callus induction of tea anthers and in vitro accumulation of phenolic compounds from the anther-derived callus.Materials and Methods:Standardization of callus induction for tea anthers. In vitro generated callus was screened for in vivo accumulation of catechins and its isomers were screened by FC reagent staining technique. The methanol extract of dry and green callus obtained were estimated qualitatively by Fourier transform infrared spectroscopy (FTIR)–alternative total reflection (ATR) and quantitatively by HPLC method.Results:Anthers inoculated on half strength MS media fortified with 2,4-dichloro acetic acid (2 mg/L), Kn (1 mg/L), and BAP (1 mg/L) induced callus under photoperiod of 9:15 h light. The in vivo histochemical studies revealed the accumulation of polyphenols in the callus. The in vitro generated fresh and dry callus were used for extraction and screened for accumulated polyphenols [galic acid, (+)-catechin (C), (−)-epicatechin, (−)-epigallocatechin, (−)-epigallocatechin gallate, (−)-gallocatechins, (−)-epicatechin gallate] were estimated qualitatively by FTIR–ATR method and quantitatively by HPLC method.Conclusion:The FC staining technique used here helps in localization of polyphenol compounds accumulation in the tissues by instant microscopic studies. The study have scope in large-scale isolation of various medicinally important flavonol by using anther culture.Abbreviations used: HPLC: high pressure liquid chromatography; FTIR: Fourier transform infrared spectroscopy; 2,4-D: 2,4-dichloro acetic acid; BAP: N6-benzyl amino purine; kn: kinetin

Gamma radiation induced variation in growth characteristics and production of bioactive compounds during callogenesis in Stevia rebaudiana (Bert.)

BackgroundStevia rebaudiana Bertoni is an important anti-diabetic medicinal herb containing non-caloric sweet compounds. In this study, the effect of gamma irradiation on growth kinetics and accumulation of various bioactive compounds were investigated during callogenesis. ResultsCallus was developed from leaf pieces inoculated on Murashige and Skoog (MS) medium containing combination of 6-benzyladenine (BA; 1.0mgl−1), α-naphthalene acetic acid (NAA), indole butyric acid (IBA) and gibberellic acid (GA3; 0.3mgl−1). After 30-days, vigorous calli were transferred to fresh medium and exposed to various gamma irradiations (5.0, 10, 15 and 20Gy). It has been observed that the increasing doses of gamma rays inhibited callus proliferation (88.61–79.16%) as compared to control (95.83%). Similarly, 10, 15 and 20Gy doses induced friable, granular and spongy callus as compared to control (compact). Furthermore, 5.0, 10 and 20Gy doses significantly reduced the fresh callus biomass (FCB), however, 15Gy dose enhanced FCB (1660mg) and dry callus biomass (DCB; 159.36mg) than control (1520; 145.92mg). The chromatographic data revealed that 15Gy dose slightly enhanced stevioside content (0.251mg/g-DCB) than control (0.232mg/g-DW), while other doses showed a negative effect on stevioside content. Higher antioxidant activity (88.73%) was observed in 20Gy treated callus cultures. However, higher total phenolic content (TPC; 43.90mg/g DCB) and total flavonoids content (TFC; 6.87mg/g DCB) were observed in 15Gy treated callus cultures. ConclusionsThe application of gamma irradiation did not show major variation in biomass and bioactive compounds production in callus cultures of S. rebaudiana.

Effect of Salt Stress on Some Growth Indicators and Cellular Components of Wheat (<i>Triticum aestivum</i> L.) Callus

The experiment was conducted to study the effect of sodium chloride (NaCl) at the concentrations of 0.0, 0.5, 1.0 and 1.5% on the callus cells. The Iraq wheat variety was grown in vitro for the purpose of knowing the effect of salt stress on some indicators and cellular components of callus by using a randomized complete design, at the laboratories of tissue culture propagation date palm unit in the College of Agriculture / University of Kufa during the period 2014-2015. Fresh and dry weight, the rate of absolute growth, percentage of dry matter of callus, content of the callus cells of proline, total soluble carbohydrates, sodium and potassium ions, effectiveness of the enzymes catalase and peroxidase study shock salt proteins in callus were determined as indicators of the effect of NaCl on the callus culture cells in the above salty medium and the results can be summarized, to control treatment achieved the highest average of (fresh callus weight, dry callus weight, the absolute growth rate of potassium ion), which was (0.0805 g, 6.13 mg, 3.147 mg / day and 1.125%) respectively, while the conc. 1.5% NaCl gave highest average in callus content of (proline, total soluble carbohydrates, sodium ion, the effectiveness of Catalase and Peroxidase enzyme and Salt shock proteins), which (0.134 mg / g, 2.9527 mg / g, 0.1050%, 0.1510 Micromol / protein / min, 0.1127 Micromol / protein / min and the emergence of small molecular weights), respectively.

Response of four medical plants of Euphorbia species in callus initiation in vitro

Callus cultures were initiated for four Euphorbia species. Nodule explants cultured on Murashige and Skoog (MS) medium, supplemented with different concentrations 0,0.5,1,1.5,2 mg/l of the auxin 2,4-Dichlorophenoxy acetic acid 2,4-D. Half of the cultures were incubated for16 hrs/day photoperiod, while the other half was incubated under complete darkness. The incubation temperature was 25±1ºC. Observations on number of nodule explants initiated callus were recorded at 2,4,6,8 weeks of culture. For callus maintenance, 50mg of callus produced were re-cultured on MS medium supplemented with different concentrations of 2,4-D 0,0.4,0.8,1.2,1.6 mg/l. Callus fresh and dry weights were recorded after 4 weeks. Results showed that nodule explants of Epeplus and Ehirta incubated under light conditions achieved the highest response to callus initiation 75-100% compared with the other species under experimental conditions. E.Helioscopia incubated under light conditions achieved the lowest response for callus initiation 25-75%. Results also showed significant differences between Euphorbia species in fresh and dry callus weights, E. Ehirta produced the highest fresh and dry weight of callus reaching 1.410 and 0.046 mg respectively. The amount of fresh and dry weight of callus produced under dark conditions was significantly higher than that produced under light conditions.

Purification and Analysis of Abscisic Acid-Specifically-Inducible Proteins from Rice Callus

Two ABA-specifically-inducible proteins from rice callus were isolated and purified by precipitation with 65–100% saturated (NH 4) 2SO 4, followed by the DEAE-sepharose, TSK-gel, and two-dimension electrophoresis. Iso-electric points (pI) of the proteins with the same molecular mass (24.5 kD) were 6.1 and 6.9, respectively. The Western blot analysis indicated that the proteins expressed in different tissues were obviously different. The A1 (pI 6.1) protein was only detected in calli treated with ABA and seed embryos (SE). However, the A2 (pI 6.9) protein was found not only in the calli treated with ABA and SE, but also in the white dry callus. Thus it suggested that the two proteins might play some important roles in the processes of seed embryo (or somatic embryo) formation.