Leaf-derived Callus Cultures Research Articles

Influence of elicitors on the enhancement of camptothecin accumulation and antioxidant potential in callus cultures of Chonemorpha fragrans

• Effect of elicitors on production of biomass and synthesis of secondary metabolites in Chonemorpha fragrans were explored • Antioxidant activity was found to be enhanced in elicitor treated callus • Higher content of secondary metabolites was noticed in chitosan and salicylic acid elicited callus extract. • Camptothecin content was maximum in yeast extract and glycine elicited callus extract Chonemorpha fragrans (Moon) Alston is a valuable medicinal plant with various applications in pharmaceuticals for various diseases. The current study was designed to examine the role of elicitors on biomass, camptothecin production and the antioxidant capacity of callus culture. The different elicitors such as chitosan, salicylic acid, yeast extract, and glycine were employed in this study. The phytoconstituents were characterized and quantified by GC-MS and LC-MS analysis. Antioxidant capacity was assessed by DPPH, ABTS, reducing power, and phosphmolybdenum assay. Maximum biomass was achieved in 100 mg/L Gl elicited callus on Gamborg's medium augmented with 0.5 mg/L of 2, 4-D, and BAP. The higher antioxidant and secondary metabolites were higher in the Ct elicited callus. GC-MS analysis revealed the presence of phytoconstituents like β – sitosterol, stigmasterol, and squalene. Higher Camptothecin (CPT) content was noticed in Ye supplemented callus. Therefore, our results are demonstrated that the elicitor supplements have the potential to enhance biomass and phytoconstituents in leaf-derived callus cultures of C. fragrans.

Synergistic effects of salicylic acid and light stress on bioactive metabolites in basil callus cultures

Thai basil is a well-known medicinal plant that is rich in bioactive antioxidant compounds having a variety of health benefits, including cancer, diabetes, and cardiovascular disease prevention. Plant cell and tissue culture technologies can be systematically developed as a valuable, low-cost biomass supply for the production of high-value phytochemicals. The aim of this study is to create an effective protocol for producing “Thai basil” leaf derived callus cultures with a high yield of biomass and antioxidants as an alternative to growing plants using salicylic acid and various light regimes as elicitors. Salicylic acid (SA) and light significantly affects many metabolic processes coupled with biosynthesis of secondary metabolites in plants. Callus was exploited with various SA concentrations under 3 different light regimes. Indeed, the results of our research disclose that continuous light with moderate SA concentration (10 μM) significantly increased total phenolic content (18.7 ± 1.13 mg/g DW), flavonoid content (7.2 ± 0.21 mg/g DW), eugenol (0.56 ± 0.015 mg/g DW), rosmarinic acid (54.35 ± 1.85 mg/g DW), chicoric acid (64.46 ± 6.71 mg/g DW) cyanidin (0.42 ± 0.021 mg/g DW) and peonidin (0.32 ± 0.002 mg/g DW) production followed by photoperiod and dark. SA treatment (25 μM) under photoperiod showed maximum biomass accumulation (FW: 226.6 ± 3.91 g/L and DW: 16.44 ± 0.59 g/L) and caffeic acid production (0.54 ± 0.025 mg/g DW). Moreover in vitro antioxidant potential (DPPH and ABTS) and cellular antioxidant capacity was found higher in continuous light treated callus along with 10 μM SA treatment. Callus extracts also showed biocompatibility with human erythrocytes that indicates their nontoxic nature and moderate potential against alpha-amylase inhibition which will help in regulating glucose level. Compared to commercial leaves, callus extracts showed higher production of chicoric acid and rosmarinic acid associated with higher antioxidant capacity. In addition, this biological system also has a large capacity for continuous biomass production, thus demonstrating its high potential for possible nutraceutical applications.

Elicitor-mediated enhancement of biomass, polyphenols, mangiferin production and antioxidant activities in callus cultures of Salacia chinensis L.

The present investigation aimed to improve callus biomass, polyphenolic content, biosynthesis of mangiferin and biological potential following application of different elicitor treatments for medicinally important Salacia chinensis L. The leaf-derived callus cultures were established on Murashige and Skoog's (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D: 2.0mg/l) and 6-benzylaminopurine (BAP: 1.5mg/l). These cultures were treated with different elicitors viz. jasmonic acid (JA), methyl jasmonate (MeJA) and yeast extracts (YE). The highest calli biomass (five-fold increase within 4weeks) was achieved when callus was treated with JA (75µM). The callus obtained on MS medium supplemented with 2,4-D (2.0mg/l), BAP (1.5mg/l) and treated with JA (75µM) displayed augmented values for total phenolics, flavonoids and mangiferin contents. Besides, same treatment elicits the calli for antioxidant properties as evaluated by 2,2-diphenyl-2-picrylhydrazyl (DPPH), ferric-reducing antioxidant power (FRAP) and metal chelating assays. This is the first report on the elicitation study in genus Salacia and, therefore, the discoveries suggested that, S. chinensis calli might be a perfect source for large-scale production of industrially important secondary metabolites. Concurrently data provide accumulated information demonstrating its prominent antioxidant effect revealing its potential without disturbing natural resources.

Callus Culture of Thai Basil Is an Effective Biological System for the Production of Antioxidants.

Thai basil is a renowned medicinal plant and a rich source of bioactive antioxidant compounds with several health benefits, with actions to prevent of cancer, diabetes and cardiovascular disease. Plant cell and tissue culture technologies can be routinely established as an important, sustainable and low-cost biomass source to produce high-value phytochemicals. The current study aimed at developing an effective protocol to produce Thai basil leaf-derived callus cultures with sustainable and high production of biomass and antioxidants as an alternative of leaves production. MS basal medium with various concentrations of plant growth regulators (PGRs) compatible with nutraceutical applications (i.e., gibberellic acid (GA3) and 6-benzylaminopurine (BAP) either alone or in combination with naphthalene acetic acid (NAA)) were evaluated. Among all tested PGRs, the combination BAP:NAA (5 mg/L:1 mg/L) yields the maximum biomass accumulation (fresh weight (FW): 190 g/L and dry weight (DW): 13.05 g/L) as well as enhanced phenolic (346.08 mg/L) production. HPLC quantification analysis indicated high productions of chicoric acid (35.77 mg/g DW) and rosmarinic acid (7.35 mg/g DW) under optimized callus culture conditions. Antioxidant potential was assessed using both in vitro cell free and in vivo cellular antioxidant assays. Maximum in vitro antioxidant activity DPPH (93.2% of radical scavenging activity) and ABTS (1322 µM Trolox equivalent antioxidant capacity) was also observed for the extracts from callus cultures grown in optimal conditions. In vivo cellular antioxidant activity assay confirmed the effective protection against oxidative stress of the corresponding extract by the maximum inhibition of ROS and RNS production. Compared to commercial leaves, callus extracts showed higher production of chicoric acid and rosmarinic acid associated with higher antioxidant capacity. In addition, this biological system also has a large capacity for continuous biomass production, thus demonstrating its high potential for possible nutraceutical applications.

Thidiazuron-induced efficient biosynthesis of phenolic compounds in callus culture of Ipomoea turbinata Lagasca and Segura

Ipomoea turbinata Lagasca and Segura (Purple Moonflower) belongs to the largest flowering genus Ipomoea in the Convolvulaceae family. Ipomoea turbinata has not been previously explored for its in vitro potential. This is the first study focused on thidiazuron-induced callus culture for efficient biosynthesis of commercially important phenolic compounds in this plant species. Among the two plant growth regulators tested on leaf, stem, and root explants, 5 mg L−1 thidiazuron (TDZ) induced the highest biomass accumulation (61.4 g L−1 fresh weight, 6.3 g L−1 dry weight) in leaf-derived callus cultures after 5 wk of culture. The highest total phenolic and flavonoid contents recorded were 9.04 mg g−1 and 1.16 mg g−1, respectively, in optimized callus cultures. High-performance liquid chromatography analysis indicated high levels of pharmacologically important anticancer compounds such as chlorogenic acid (13.48 mg g−1), arctigenin (11.67 mg g−1), quercetin (6.19 mg g−1), and kaempferol (5.48 mg g−1), along with other phenolic acids. Furthermore, the antioxidant activity was also evaluated, and leaf-derived callus culture displayed a maximum of 62.6% antioxidant potential. The induction of improved biomass accumulation in callus culture and the production of multipotent bioactive metabolites shows the potential of the multifunctional thidiazuron hormone as an efficient elicitation tool in callus culture of I. turbinata.

Exogenous melatonin trigger biomass accumulation and production of stress enzymes during callogenesis in medicinally important Prunella vulgaris L. (Selfheal).

The objective of the current study was to monitor the variations caused by the application of exogenous melatonin on growth kinetics and production of stress enzymes in Prunella vulgaris. Leaf and petiole explants were used for callogenesis. These explants were inoculated on Murashige and Skoog media containing various concentrations of melatonin alone or in combination with 2.0mg/l naphthalene acetic acid. Herein, a maximum of 3.18-g/100ml fresh biomass accumulation was observed on day 35 during log phase of growth kinetics at 1.0mg/l melatonin concentration from leaf explants. While 0.5 and 1.0mg/l melatonin enhanced the biomass accumulation from petiole explants. Moreover, the synergistic combination of melatonin and naphthalene acetic acid also promoted growth from leaf and petiole explants. Leaf derived callus cultures treated with 1.0mg/l melatonin induced the production of total proteincontent (90.47μgBSAE/mgFW) and protease activity (4.77U/gFW). While the calli obtained from petiole explants have shown highest content of total protein (160.8μg BSAE/mgFW) and protease activity (5.35U/gFW) on media containing 0.5mg/l melatonin. Similarly, 0.5mg/l melatonin enhanced superoxide dismutase (3.011nM/min/mgFW) and peroxidase (1.73nM/min/mgFW) enzymes from leaf derived callus cultures. The combination of 1.0 and 1.5mg/l naphthalene acetic acid enhanced content of total protein and protease activity in leaf and petiole derived cultures. These results suggested that the application of melatonin play a positive role in biomass accumulation and production of stress enzymes in P. vulgaris.

Differential accumulation of silymarin induced by exposure of Silybum marianum L. callus cultures to several spectres of monochromatic lights

Silybum marianum L. (Milk thistle) is one of the most extensively studied medicinal herbs with well-known hepatoprotective activity. Light is considered as a key abiotic elicitor influencing several physiological processes in plants, including the biosynthesis of secondary metabolites. In this study, we investigated the influence of light quality on morphological and biochemical aspects in in vitro grown leaf-derived callus cultures of S. marianum. Combination of 6-benzylaminopurine (BAP 2.5 mg/L) and α-naphthalene acetic acid (NAA 1.0 mg/L) resulted in optimum callogenic response (97%) when placed under cool-white light with 16 h light and 8 h dark. Red light significantly increased the total phenolic content (TPC), total flavonoid content (TFC), antioxidant and superoxide dismutase (SOD) activities while highest peroxidase (POD) activity was recorded for the dark grown cultures, followed by green light grown cultures. HPLC analysis revealed enhanced total silymarin content under red light (18.67 mg/g DW), which was almost double than control (9.17 mg/g DW). Individually, the level of silychristin, isosilychristin, silydianin, silybin A and silybin B were found greatest under red light, whereas green spectrum resulted in highest accumulation of isosilybin A and isosilybin B. Conversely, the amount of taxifolin was found maximum under continuous white light (0.480 mg/g DW) which was almost 8-fold greater than control (0.063 mg/g DW). A positive correlation was found between the TPC, TFC and antioxidant activities. This study will assist in comprehending the influence of light quality on production of valuable secondary metabolites in in vitro cultures of S. marianum L.

Plant Regeneration from Leaf-derived Callus Cultures of Primrose (Primula vulgaris)

Efficient micropropagation of Primula species is important both for fundamental scientific studies and commercial applications. Primula vulgaris (Huds), along with other Primulaceae species, exhibits floral heteromorphy with two distinct forms of hermaphroditic flower. Studies to identify genes that control heteromorphic flower development require propagation of floral mutants, and efficient regeneration is a key requirement for plant transformation. Several species, including P. vulgaris cultivars and P. ×polyantha hybrids, are important horticultural crops in Europe, United States, and Japan and semidouble/double Primula varieties offer a high-end product. Vegetative propagation of sterile double forms, and as a means to increase numbers of inbred parent plants for F1 seed production is, however, slow. Micropropagation offers the most efficient way of increasing these varieties quickly and efficiently. To date, most Primula micropropagation protocols require explant material derived from in vitro grown seedlings or use floral parts as donor material with seasonal limitations. Therefore, an effective and efficient protocol was developed for in vitro regeneration of P. vulgaris via indirect organogenesis from adult leaf–derived explants. Exposure of leaf explants of P. vulgaris to media containing synthetic cytokinin, thidiazuron (TDZ), and auxin [1-naphthylacetic acid (NAA) or 2,4-dichlorophenoxyacetic acid (2,4-D)] resulted in undifferentiated cell proliferation and followed by differentiated growth as shoot organogenesis. Silver nitrate improved in vitro callus growth and increased shoot regeneration further, with up to 72% of explants producing shoots. Regenerated plants developed normally and produced normal fertile flowers within 7 months. The system was also successfully applied for the micropropagation of sterile double-flowered P. vulgaris ‘Sue Jervis’. The protocol reported here enables propagation of P. vulgaris without seasonal limitation or destruction of valuable parent donor material. The protocol, with further development, has the potential to underpin development of a transformation system for Primula, which would be of value in studies on flower development and disease resistance in laboratory grown plants.

ED-XRF spectrometry-based comparative inorganic profile of leaf-derived in vitro calli and in vivo leaf samples of Phyllanthus amarus Schum. & Thonn.—a hepatoprotective herb

The Energy Dispersive X-ray Fluorescence (ED-XRF) set-up incorporating a molybdenum secondary exciter was used for quantitative determination of major and minor elements in leaves of in vivo grown medicinal herb Phyllanthus amarus vis-á-vis its leaf-derived in vitro callus culture. The elements such as K, Ca, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Se, Rb, Sr and Pb were identified, quantified and compared between both the sources. Experimental results revealed that, compared to the naturally grown herb, in vitro leaf-derived callus cultures were more efficient in accumulating inorganic elements, especially trace elements, which are essential for growth and development and more importantly for prevention and cure of diseases. This investigation on a medicinal plant species is the first of its kind to have used the ED-XRF technique to demonstrate a comparative account of the elemental profile of in vitro callus cultures with their in vivo donor in order to explore the possibility of exploiting the former as a viable alternative and a renewable source of phytochemicals.

Production of pyrroloquinazoline alkaloid from leaf and petiole-derived callus cultures of Adhatoda zeylanica

Callus cultures of Adhatoda zeylanica Medicus were established from leaf and petiole explants. Accumulation of a bioactive pyrroloquinazoline alkaloid, vasicine, in callus cultures was detected and confirmed by thin layer chromatography, electron-ionization mass spectra, 13C NMR and high-pressure liquid chromatography analysis. The mass of vasicine obtained from leaf-derived callus cultures was found as 188 and this is comparable to that of the authentic sample. The retention time for leaf-derived extract was 10.065 and for the petiole-derived extract was 9.78 (authentic sample had 9.6 retention time) on high-performance liquid chromatography. The mass and NMR spectra were compared with the spectra obtained from the authentic sample of vasicine. Different growth regulators greatly influenced the growth of callus cultures. The accumulation of vasicine was more in leaf-derived callus grown on Murashige and Skoog (MS) medium with 2.3 μM kinetin, and 4.5 μM 2,4-dichlorophenoxyacetic acid. This is the first report on in vitro production of a pharmacologically important compound vasicine and its characterization by mass spectrometry and 13C NMR studies from callus cultures of Adhatoda zeylanica.

Plant regeneration from callus and suspension cultures of Valeriana edulis ssp. procera via simultaneous organogenesis and somatic embryogenesis

Plants of the genus Valeriana produce irioid esters of biotechnological interest, known as valepotriates. The regeneration of Valeriana edulis ssp. procera via indirect organogenesis and somatic embryogenesis from leaf-derived callus and suspension cultures, is presented. For induction of callus, leaves were cultured on semisolid MS medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin. Embryogenic and organogenic callus development was observed after 16 weeks. Calli were transferred to semisolid and liquid Murashige and Skoog (MS) medium supplemented with kinetin and 1-naphthaleneacetic acid (NAA). Appearance of shoot and somatic embryo occurred 4 weeks later. Shoots were rooted and somatic embryos were germinated and were transferred to the greenhouse. A better organogenic and embryogenic response was observed from suspension cultures. Histological observations of morphogenic callus revealed that both somatic embryos and shoots arose from the same type of callus.

Comparative salt responses of callus cultures of Vigna radiata L. Wilczek to various osmotic and ionic stresses

Summary Leaf derived callus cultures of Vigna radiata (L.) Wilczek cv. K-851 were grown on modified PC-L2 medium salinized with various concentrations of NaCl, KCl, Na2SO4 or their mixture and mannitol to study the effect of osmotic and different types of salt stresses on callus growth and ion accumulation. The callus responded differently with stress and salt types. Callus growth was inhibited more severely with Na2SO4, followed by KCl, salt mixture, NaCl and mannitol. A correlation between growth inhibition and the concentration of inorganic solutes in callus tissue was noted. Accumulation of Na+ in callus was accompanied by loss of K+ at all levels of Na2SO4, NaCl and salt mixture. Severe inhibition of cell growth with Na2SO4 was due to more uptake of Na+ by cells under Na2SO4 medium than any other salt and their mixture. SO4−2 ion content in Na2SO4 grown callus was 60–70 times higher than Na+. Similarly, K+ and Cl− contents of KCl callus were greater than Na+ and Cl− of NaCl grown callus. A comparison between salt mixture and its constituent different salts shows that some single salts (Na2SO4 and KCl) were more toxic than a salt mixture. An increase in the osmotic stress (by mannitol) inhibited callus growth less severely than any of the salts. This may be due to higher K+ content of the cells under mannitol stress than iso-osmotic concentration of any of the salts.

Variation among somaclonal progenies from three species of Stylosanthes

Plants were regenerated from leaf-derived callus cultures of the diploid species Stylosanthes guianensis (four genotypes), and the tetraploid species S. scabra (four genotypes), and S. hamata (one genotype). No morphological variation was observed in the 97 regenerated plants (SC1) of S. scabra, but 23 of the 104 SC1 plants of S. hamata were sterile dwarfs. In a field experiment with 42 SC2 families of S. scabra, four were low yielding and five segregated for late flowering. Dwarfs were again found in SC2 families of S. hamata, and both high and low yielding families were observed. Glasshouse inoculations with various pathogenic races of Colletotrichum gloeosporioides were performed to assess the reactions of 276 S. guianensis, 56 S. scabra and 62 S. hamata SC2 families. A major gene mutation from dominant to recessive appeared to confer increased susceptibility in S. hamata SC2 families. Eight of twelve tetraploid S. guianensis cv. Graham families exhibited partial resistance to Type B race 3 anthracnose. Two S. scabra cv. Fitzroy families segregated for reaction to Type A race 1 anthracnose. Selfed progenies (SC3 generation) of two of the three partially resistant SC2 individuals maintained their partial resistance, indicating stable inheritance. Segregation data suggested that changes in minor genes caused this variation.

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.

Differential solasodine accumulation in photoautotrophic and heterotrophic tissue cultures of Solanum laciniatum

Foliage from a Solanum laciniatum plant contained 7.64 mg solasodine per g dry weight. In contrast, leaf-derived callus cultures incubated under light yielded only 0.09 mg/g solasodine. A similar low level was recovered from shoots regenerated from this callus and cultured under heterotrophic conditions. However, shoots cultured photoheterotrophically or photoautotrophically yielded solasodine concentrations approaching those of field grown plants. Solasodine biosynthesis in S. laciniatum is therefore promoted by actively photosynthesising chloroplasts, and cell cultures yield only low solasodine levels as a consequence of their heterotrophic mode of nutrition.

Comparison of Proanthocyanidins and Related Compounds in Leaves and Leaf-Derived Cell Cultures of Ginkgo bioloba L., Pseudotsuga menziesii Franco, and Ribes sanguineum Pursh

Proanthocyanidins, flavan-3-ols, and their flavanoid precursors in leaves and leaf-derived callus and cell suspension cultures have been isolated and analyzed by high performance liquid chromatography with C(18) columns, paper chromatography, and by chemical and spectrophotometric methods. Cultures of Ginkgo biloba and Pseudotsuga menziesii (Douglas-fir) produced much greater amounts of proanthocyanidins than leaves per milligram dry weight. In cultures, however, the prodelphinidin component relative to that of procyanidins decreased; this was most pronounced in Pseudotsuga. In contrast, callus cultures of Ribes sanguineum accumulated proanthocyanidins in amounts about equal to those in intact leaves per milligram dry weight and the prodelphinidin content remained high. Although Ginkgo and Ribes leaves contained major amounts of flavan-3-ols and dimers with the 2,3-cis-stereochemistry, their cultures tended to synthesize 2,3-trans-isomers instead. Glycosides of flavanone and 3-hydroxyflavanone precursors accumulated in medium to high amounts on a dry weight basis in leaves and cultures of Ribes and Pseudotsuga, and the 3'-glycosidic linkage predominated when the latter species was cultured with 2,4-dichlorophenoxyacetic acid rather than naphthaleneacetic acid.

The effect of phosphate, nitrogen and sucrose on the production of phenolics and solasodine in callus cultures of solanum laciniatum

In leaf derived callus cultures of Solanum laciniatum Ait. both phenolics and solasodine concentrations increased when medium phosphate or nitrogen concs. were reduced to one-eighth or when sucrose concentration was increased from 3 to 4-8 %. Under these conditions growth was reduced and final FW:DW fell. Growth was inhibited by sucrose depletion and nitrogen supple -mentation. On additional nitrogen the concentrations of phenolics and protein significantly increased, FW:DW was reduced and solasodine concentration was unaffected. In seedling derived cultures phosphate depletion resulted in a significant increase in phenolics concentration, an inhibition of growth and a rise in solasodine concentration.