Leaf Callus Research Articles

A novel micropropagation of Lycium ruthenicum and epigenetic fidelity assessment of three types of micropropagated plants in vitro and ex vitro.

Lycium ruthenicum is an excellent eco-economic shrub. Numerous researches have been conducted for the function of its fruits but scarcely focused on the somaclonal variation and DNA methylation. An efficient micropropagation protocol from leaves and stems of L. ruthenicum was developed in this study, in which not only the leaf explants but also the stem explants of L. ruthenicum were dedifferentiated and produced adventitious buds/multiple shoots on one type of medium. Notably, the efficient indirect organogenesis of stem explants was independent of exogenous auxin, which is contrary to the common conclusion that induction and proliferation of calli is dependent on exogenous auxin. We proposed that sucrose supply might be the crucial regulator of stem callus induction and proliferation of L. ruthenicum. Furthermore, results of methylation-sensitive amplified polymorphism (MSAP) showed that DNA methylation somaclonal variation (MSV) of CNG decreased but that of CG increased after acclimatization. Three types of micropropagated plants (from leaf calli, stem calli and axillary buds) were epigenetically diverged more from each other after acclimatization and the ex vitro micropropagated plants should be selected to determine the fidelity. In summary, plants micropropagated from axillary buds and leaves of L. ruthenicum was more fidelity and might be suitable for preservation and propagation of elite germplasm. Also, leaf explants should be used in transformation. Meanwhile, plants from stem calli showed the highest MSV and might be used in somaclonal variation breeding. Moreover, one MSV hotspot was found based on biological replicates. The study not only provided foundations for molecular breeding, somaclonal variation breeding, preservation and propagation of elite germplasm, but also offered clues for further revealing novel mechanisms of both stem-explant dedifferentiation and MSV of L. ruthenicum.

MiR858b Inhibits Proanthocyanidin Accumulation by the Repression of DkMYB19 and DkMYB20 in Persimmon.

Persimmon proanthocyanidin (PA) biosynthesis is controlled by structural genes and regulated by transcription factors (TFs). MicroRNAs are a key factor involved in regulating gene expression at the posttranscriptional level whose functions in persimmon PA biosynthesis are poorly understood. Here, we identified a microRNA, miR858b, that putatively targets two R2R3-MYB TFs, DkMYB19 and DkMYB20. DkMYB19, DkMYB20, and miR858b showed divergent expression patterns during fruit development, and the interaction between miR858b and DkMYB19 or DkMYB20 was experimentally validated by 5′ RNA ligase-mediated RACE, LUC enzyme activity analysis, and GFP signal detection. The DkMYB19 localized to the nucleus as well as the cytoplasm and DkMYB20 localized to the nucleus. The overexpression of miR858b led to the downregulation of DkMYB19 and DkMYB20, which reduced the content of PA, whereas a reduction in miR858b activity upregulated DkMYB19 and DkMYB20, resulting in a high content of PA in leaves transiently expressing a small tandem target mimic construct for blocking miR858 (STTM858b) in vivo. The transient transformation of miR858b in fruit discs in vitro also reduced the content of PA, while the content of PA increased under the transient transformation of fruit discs with STTM858b, DkMYB19, or DkMYB20. A similar phenomenon was observed upon the overexpression of miR858b in wild-type (WT) Arabidopsis and DkMYB19 or DkMYB20 in persimmon leaf calli. These findings suggested that miR858b repressed the expression of DkMYB19 and DkMYB20, which contributed to the PA accumulation in persimmon.

BRIDELIA SCANDENS WILD. A NEW SOURCE FOR PODOPHYLLOTOXIN PRODUCTION IN VITRO BY FEEDING CONIFERYL ALCOHOL

Objective: In the present study, a new method for the production of anticancerous compound podophyllotoxin (PTOX) was developed for Bridelia scandens Wild. by feeding coniferyl alcohol.
 Methods: The production of anticancerous compound PTOX through leaf explant derived calli of B. scandens. Murashige and Skoog (MS) medium fortified with 0.5 mg/l 6-Benzylaminopurine (BAP) and 0.5 mg/l 2,4-D (2,4-Dichlorophenoxyacetic acid) induced luxuriant mass of callus growth. Suspension culture was initiated by sterile MS media fortified with 0.1–1.0 mg/l BAP and 0.1–1.0 mg/l 2,4-D. and growth product was analyzed by the high-pressure liquid chromatography method.
 Results: Phytochemical analysis of the B. scandens leaf and leaf calli showed the presence of PTOX at the concentrations of 0.69 and 1.81, respectively. The callus cell suspension was established with the same callogenic media also it is augmented with 10–70 mg/l of coniferyl alcohol to elicit the biosynthesis of PTOX. Successive cultures of the calli suspension yielded stable production of PTOX of 3.91 mg/g dry cell weight at 50 mg/l coniferyl alcohol in the media. The biosynthesis of PTOX was ideal when plant cells were cultivated in the dark with an agitation speed of 100 rpm.
 Conclusion: The growth and production of PTOX were found to be better with glucose than with sucrose as the medium carbon source. The harvesting of the secondary metabolite from the in vitro grown leaf calli of B. scandens is a better way to stop the exploitation of medicinal plants.

In vitro regeneration technique in Rauwolfia serpentina and quantification of reserpine

The reliable technique for the indirect regeneration of R. serpentina was standardised in the present investigation. The secondary metabolites extracted from callus using methanol and analysed using Thin Layer Chromatography (TLC) and UV-Vis Spectrophotometer. The in vitro induction of callus influenced by several factors such as media composition and explants quality. From the various media combinations of 2, 4-D and BAP the frequency of highest callus induction (94.67%) was observed on MS + 2.0 mgL-1 2.4-D + 1.0 mgL-1 BAP when leaf disc used as explant. Throughout the organogenic callus induction, different calli were observed having variation in colour and texture. The shoot regeneration frequency was observed highest (98.33%) in 4.5 mgL-1 BAP + 0.5 mgL-1 NAA. Maximum shoots (15.33 per callus) were observed in leaf callus. The rooting was induced (95.33%) in in vitro regenerated shoots in Murashige and Skoog (MS) medium which contain 1.0 mgL-1 NAA + 0.1 mgL-1 BAP + 1 gL-1 activated charcoal (AC). The in vitro regenerated plantlets through callus culture were hardened in the greenhouse with 75 per cent establishment. The quantity and quality of reserpine was measured in callus and root of R. serpentina. The TLC reveals the accumulation of reserpine at callus as well as roots of cultivated plant. Spectrophotometric estimation showed that concentration of reserpine observed in callus extract was higher (6.8 µgml-1) than the root extract (6.4 µgml-1).

Evaluation of α-amylase expression and analysis of phytochemical in the leaf callus tissue of Rauvolfia serpentina (Linn.) Benth. Ex Kurz exposed to Cyclodextrin

Evaluation of α-amylase expression and analysis of phytochemical in the leaf callus tissue of Rauvolfia serpentina (Linn.) Benth. Ex Kurz exposed to Cyclodextrin

Evaluation of α-amylase expression and analysis of phytochemical in the leaf callus tissue of Rauvolfia serpentina (Linn.) Benth. Ex Kurz exposed to Cyclodextrin

Rauvolfia serpentina (Linn.) Benth. Ex Kurz belongs to the family Apocynaceae. The objective of the present study was to establish an effective protocol for the regeneration of leaf explants from R. serpentina and to study the α-amylase expression and phytochemical profiling by gas chromatography and mass spectrometry (GC-MS). Further antibacterial activity was studied using silver, gold, and copper nanoparticles. The leaf explants were cultured on Murashige and Skoog (MS) medium containing Benzyl amino purine (BAP) (2.0 mg/L-1) and Naphthalene acetic acid (NAA) (1.0 mg/L-1) induced the formation of callus and expressed α-amylase with underexposed to Cyclodextrin. Explants growing on MS medium fortified with 2,4-Dichlorophenoxy acetic acid (2,4-D) (1.0 mg/L-1), NAA (1.0 mg/L-1) with and without BAP (2.0 mg/L-1) and showed a maximum concentration of protein on the 75th day. Synthesis of a-amylase enzyme was expressed 40 days old culture and were confirmatic by western, further Silver, gold, and copper nanoparticles were synthesized using the ethyl acetate extract of callus tissue and subjected to thin-layer chromatography (TLC), which resolved 5 bands. These five bands were characterized by Fourier Transform Infrared (FTIR) Spectroscopy and screened for antimicrobial and antioxidant activities. TLC band 4 alone showed inhibitory activity against both Gram-negative and positive bacteria and potent antioxidant activity. For the first time, α-amylase was found in the callus extract by SDS-PAGE and confirmed by Western blot. The fourth band of TLC from the ethyl acetate extract as well as silver and gold nanoparticles synthesized using this extract revealed pronounced antimicrobial and antioxidant activities. GC-MS analysis revealed 26 compounds, which included mainly the phytosterols and fatty acid esters.

Study on the Establishment of a Rapid Propagation System for Succulent Plant Haworthia heidelbergensis

This study took the inflorescence and leaves of the succulent plant Haworthia heidelbergensis as explants, and explored the effects of different mediums with different hormone ratios on the rapid propagation of Haworthia heidelbergensis. The results showed that the optimal medium component for inflorescence callus induction was MS (Murashige and Skoog)+2.0 mg/L 6-BA+1.0 mg/L 2,4-D+0.2 mg/L NAA, the callus induction rate was 90%; the optimal medium component for leaf callus induction was MS+0.5 mg/L 6-BA+2.0 mg/L KT+0.3 mg/L NAA; the optimal medium for callus differentiation was MS+1.0 mg/L 6-BA+2.0 mg/L KT+0.3 mg/L NAA, the clump bud differentiation rate was 50%; the optimal medium for clump bud proliferation was MS+0.5 mg/L 6-BA+1.0 mg/L KT+0.05 mg/L NAA, the proliferation rate was 600%; the best medium for rooting was 1/2 MS+ 0.2 mg/L NAA+0.3 g/L AC. In conclusion, this study selected the explants and culture mediums, established an aseptic propagation system and provided a reference for the in vitro culture and rapid propagation of Haworthia heidelbergensis.

Induction of growth and antioxidant defense mechanisms in Matricaria chamomilla L. callus by vibration

Effect of sinusoidal vibration on the activity of some antioxidative enzymes, pigments, membrane stability, and total phenolic was investigated in callus tissues of Matricaria chamomilla L. Two calli sources were initiated from leaf and root explants, and different frequencies of sinusoidal vibration (0, 5, 10, 15, 50, and 100 Hz) for 3 min were applied to stimulate callus tissues of M. chamomilla. According to the obtained findings in two calli of Matricaria chamomilla, enhancement of growth by vibration was observed at 15 and 50 Hz. Improvement of growth by vibration can be explained by induction in protein, proline total phenol contents, superoxide dismutase, peroxidase, catalase and ascorbate peroxidase activities, reduction in malondialdehyde, and hydrogen peroxide levels. Carotenoid significantly augmented at 15 and 50 Hz frequencies. The electrophoresis profile showed different isoform patterns, and the intensity of bands was promoted by vibration. Moreover, a new superoxide dismutase band appeared at 15 and 50 Hz frequencies in leaf calli. These results may provide insight into antioxidant defense mechanisms of callus tissue to vibration.

MICROPROPAGATION OF AN ENDANGERED AND ENDEMIC MEDICINAL PLANT CAYRATIA PEDATA VAR. GLABRA

Objective: The objective of the present study was to develop standardization protocol for the successful in vitro mass propagation of Cayratia pedata var. glabra through leaf and stem explants, since it is a rare, endangered, and endemic medicinal plant using biotechnological involvements and to conserve this endangered species.
 Methods: The application of biotechnological principles for the establishment of micropropagation under in vitro conditions has been studied by following the methods. The explants, namely, leaf and stem harvested from in vivo plants were thoroughly washed and properly sterilized with sterilients. The explants were transferred to Murashige and Skoog (MS) medium supplemented with growth regulators 6-benzyladenine (BAP) and naphthalene acetic acid (NAA) in the concentration range of 0.5–3.0 mg/l which were tested for callus induction and morphogenesis. The elongated shoots were transferred to MS medium supplemented with NAA at different concentrations for root induction.
 Results: The explants collected from the field (shola) were treated in different steriliants with various concentrations at different time for sterilization. Among the various combinations tried, the Teepol treatment for 10 min followed by bavistin 20 min, antibiotics, namely, ampicillin and rifampicin for 20 min, 70% alcohol for 30 s, and 0.12 % HgCl2 for 3 min was found to be effective. The explants were cultured in MS medium supplemented with various concentrations of BAP and NAA. The results noted that an increase in the concentration of BAP concomitantly reduced the frequency of callus formation. The maximum callusing frequency and more number of shoot formation was observed in the lower concentration of BAP (0.5 mg/l) in combination with NAA (0.2 mg/l). The callus obtained from all the above combinations was sub-cultured on MS medium with same combinations of BAP and NAA. The maximum frequency of root formation in leaf callus was 85% and 75% in stem callus and both were achieved on MS medium with NAA (1 mg/l) after 2 weeks.
 Conclusions: The current investigation provides a competent in vitro propagation method for C. pedata var. glabra which could be commercialized for developing identical plants with high-quality mass multiplication rate and for better conservation of the germplasm. Both the methods described here are well suited for the mass multiplication of this critically endangered and endemic climber species.

Callus induction and indirect regeneration of Thunbergia coccinea Wall.

In vitro regeneration of Thunbergia coccinea Wall. ex D. Don via organogenesis was obtained from leaf callus tissue of T. coccinea, a perennial climbing medicinal vine plant with attractive red scarlet blooming in particular season. Highest weighted callus was obtained from leaves of T. coccinea on Murashige and Skoog basal medium supplemented with 5.0 mg/L NAA. Embryogenic callus were observed on MS basal media containing NAA rather than 2,4-D. Somatic embryogenesis was demonstrated by field emission scanning electron microscopic studies of some embryogenic callus. Highest frequency of somatic embryos was obtained in 3.0 mg/L NAA. Indirect shoot bud organogenesis and rhizogenesis were also observed on some embryogenic callus. Plantlets were regenerated from both somatic embryos and shoot buds. Highest number of shoots was noted in 0.1 mg/L NAA and 2.0 mg/L BAP combination while transferring in regeneration media. Regenerated plantlets were hardened successfully on pot. The survival rate of regenerated plants after hardening was 50%.

Biomass and terpenoids profile of callus extract of Piper betle L. var. Nigra with abiotic elicitor cobalt (II) chloride

Black betel ( Piper betle L. var. Nigra) is traditional medicinal plant potentially produce secondary metabolites. Its secondary metabolite content can be promoted using callus culture with abiotic elicitor. This study was aimed to determine the effect of cobalt (II) chloride (CoCl 2 ) concentration towards biomass and terpenoids profile of leaf callus of Piper betle L. var. Nigra. Leaf explants of Piper betle L. var. Nigra were cultured in solid Murashige and Skoog (MS) medium supplemented with growth regulators 2,4-D (0.5 mg/L) and BAP (2.0 mg/L) to induce callus formation. Elicitation was performed after callus reached five weeks of age by performing subculture on MS medium added with CoCl 2 (0.5 mg/L; 1.0 mg/L; 2.5 mg/L). Calli of Piper betle L. var. Nigra were harvested after one, two, or three weeks before they were extracted using methanol and analyzed of its terpenoids content using Gas Chromatography-Mass Spectrometry (GC-MS) method. Results showed that calli had compact and crumb texture with green, white, brown, and white color. Callus added 1.0 mg/L CoCl 2 at two weeks age produce terpenoids at 5.95%. Callus with 1.0 mg/L CoCl 2 at three weeks of age produced higher fresh and dry weight at 916.4±162.3 mg and 93.3±11.7 mg respectively. Callus supplemented with 1.0 mg/L CoCl 2 at two weeks of age had higher terpenoids percentage compared to control. Two of the highest terpenoid types found in the callus of black betel leaf explant were 1,2-epoxy-1-vinylcyclododecene and hexadecanoic acid.

Comparative Antioxidant Activity of Ethanolic extracts of Whole Plant and Leaf Callus of Mollugo oppsitifolia L. A Potent Traditional Medicinal Herb

Natural products from dietary components such as Indian species and medicinal plants are known to possess antioxidant activity. Antioxidants are inhibitors of oxidation are compounds which prevent the oxidation and in general prolong the life of the oxidizable matter. Majority of the diseases/disorders are mainly linked to oxidative stress due to free radicals. The free radicals (oxidants) are species with very short half-life, high reactivity and damaging activity towards macromolecules like proteins, DNA and lipids. In general, the reactive oxygen species circulating and react with the electron of other molecules in the body and these also affect various enzyme systems and cause damage which may further contribute to conditions such as cancer, ischemia, ageing, adult respiratory distress syndromes, rheumatoid arthritis etc. Dietary plants contain variable amounts of antioxidants. It has been proved that plant antioxidants may contribute to the beneficial health effects of dietary plants. The present study was to evaluate antioxidant activity of ethanolic extract of iv vivo whole plant and in vitro leaf callus of Mollugo oppositifolia L. is an important traditional medicinal herb belonging to the family Molluginaceae using 2,2-diphenyl-1-Picrylhydrazyl (DPPH) radical scavenging assays. The results obtained showed that the ethanolic extracts of whole plant in vivo and in vitro leaf callus showed significant DPPH activity with IC50 value of 52.82± 0.0017 μg/mL and 58.66±0.004μg/mL respectively, while IC50 of vitamin C as standard was 84.84±11.54μg/mL. Present study revealed that an antioxidant activity was higher leaf callus extract compare to whole plant extract of Mollugo oppositifolia L.

Получение и фитохимический скрининг каллусных и суспензионных культур клеток вздутоплодника сибирского Phlojodicarpus sibiricus (Steph. ex Spreng.) К.-Pol.

Callus and suspension cell cultures have been obtained from aseptic seedlings of Siberian bloat fruit (Phlojodicarpus sibiricus), a producer of coumarins. P. sibiricus callus cultures were characterized by white-yellow coloration, a combination of loose and dense cell aggregates, and satisfactory growth. The dry biomass growth index for cultures of leaf, hypocotyle or root origin was 7-9, 10-12 and 11-13, respectively. Suspension cell cultures were initiated from calluses of leaf and hypocotyle origin; these cultures were also white-yellow and consisted mainly of cell aggregates of the meristem-like and parenchyma-like types with different aggregation degrees depending on the origin. Cell viability during the growth cycle was at the level of 70-80%. In contrast to the original callus cultures, the suspension culture of leaf callus origin had the highest growth characteristics (growth index of about 10). Preliminary phytochemical screening by UPLC ESI MS showed the presence of khellactone-group coumarins in the biomass of P. sibiricus primary calluses (1st-3rd growth cycle) and suspension cultures derived from them. Sibirian bloat fruit, Phlojodicarpus sibiricus, callusogenesis, suspension cell culture, coumarins The works on obtaining and growing P. sibiricus cells, as well as studying their growth characteristics were financed by the Russian Scientific Foundation grant no. 74-00097. The study of phytochemical characteristics of P. sibiricus cell cultures was supported by the state assignment of the Ministry of Education and Science of Russia (FSRG-2020-0019).

PHYTOCHEMICAL AND ANTIOXIDANT ACTIVITY OF BALIOSPERMUM MONTANUM (WILLD.) MUELL. ARG.

Objective: The present study was carried out to evaluate the phytochemical constituents and antioxidant potential of the leaf, stem, root, and stem-derived callus extracts of Baliospermum montanum.
 Methods: An in vitro regeneration protocol was developed for the induction of callus from stem segments cultured on Murashige and Skoog (MS) medium supplemented with different concentrations and combination of 6-benzylaminopurine (BAP), 6-furfurylaminopurine (KIN), 1-naphthaleneacetic acid, and 2, 4-dichlorophenoxyacetic acid. The total phenol, flavonoid, and tannin content were determined according to standard methods. The antioxidant activity of methanolic extract was evaluated using 2, 2-diphenyl-l-picrylhydrazyl (DPPH) and reducing power assays.
 Results: The maximum callus induction was observed on MS medium supplemented with a combination of KIN (0.5 mg/L) + BAP (3.0 mg/L). Methanolic extract of root and aqueous extract of leaf exhibited higher content of phenols. Whereas total flavonoids and tannin were maximum in methanolic extract of leaf compared to other extracts. The methanolic extract of B. montanum leaf had greater antioxidant activity than stem, root, and callus by DPPH and reducing power assays.
 Conclusion: Our study suggests that the methanolic extracts of B. montanum showed potent free radical scavenging activity. Further studies are necessary for isolation and characterization of phytochemical compounds.

Increasing anthraquinone compounds on callus leaf Morinda citrifolia (L.) by elicitation method using chitosan shell of shrimps (Penaeus monodon)

Elicitation is one method to increase the content of secondary metabolite compounds in plant tissues. Anthraquinone is one of the secondary metabolite compounds that have potential of being a medicinal agent and produced by plants. The purpose of this research is to increase anthraquinone content in Morinda citrifolia (L.) leaf callus using elicitation method with chitosan from shrimp shell extract (Penaeus monodon) as elicitors. M.citrifolia (L.) callus was obtained from young leaf explants grown on Murashige and Skoog (MS) medium with 2.4-D addition of 1.75 mg / L and Kinetin 1.5 mg / L aseptically. The second subculture of callus is elicited by elicitor of chitosan each of 0.5 ml. The concentration of elicitor used was 0, 0.5,1,1.5,2 and 2.5 mg / ml. Harvesting of elicitation result done on day 0, 2, 4 and day 8 after elicitation. The results showed that callus containing anthraquinone could be induced in MS medium with addition of 2,4-D 1.75 and Kinetin 1.5 mg / L. and addition of chitosan at some concentrations may increase anthraquinone. The highest anthraquinone content was produced at concentration 2 mg / ml and 4 days harvest time

In-vitro Callus Induction of Durian (Durio zibethinus Murr.) Leaves Using Kinetin and 2,4-D (Dichlorophenoxyacetic acid)

Durian (Durio zibethinus Murr.) is a tropical fruit grown in Southeast Asia and it has high nutritional and economic values and highly appreciated by consumers in Indonesia. Unfortunately, there are several obstacles in planting durian in Indonesia, specifically in Aceh. One of the abstacles is the scarcity of superior durian plants. Generally, plant conservations done conventionally through vegetative propagation. However, this technique has disadvantage such as harming the initial plants. Therefore, plant culture tissue has been used as a modern technique to develop durian plants. The objective of this research was to evaluate the effect of kinetin and 2,4 D on development of durian leaf explants through in-vitro bioassay. The research was conducted at Tissue Culture Laboratory, Faculty of Agriculture, Malikussaleh University from January to April 2019 using Completely Randomized Design (CRD) Factorial with 2 factors observed. The first factor was Kinetin (K): 0.0, 0.1 and 0.5 ppm. The second factor was auxin 2,4 D: 0.0, 0.5 and 1.0 ppm. The results showed that the in-vitro application of kinetin affected the induction of leaf callus. The application of 0.5 ppm of kinetin gave the best result compared to others. The in-vitro application of 2,4 D 0.5 ppm also possessed the best result compared to others. There was no interaction beween in-vitro application of kinetin and 2,4 D on the growth of durian leaf callus at all parameters observed.

Enhanced production of podophyllotoxin, kaempferol, and quercetin from callus culture of Dysosma pleiantha (Hance) Woodson: An endangered medicinal plant.

Dysosma pleiantha (Hance) Woodson is one of the endangered traditional Chinese medicinal herbs, highly valued for its medicinal properties by Taiwan's mountain tribes. The present study aims to develop an efficient protocol for callus biomass by optimizing suitable culture medium, carbon source culture condition, and enhanced production of pharmaceutically important podophyllotoxin, kaempferol, and quercetin from callus culture of D. pleiantha under the influence of different additives. Best callus induction was achieved in Gamborg's medium (B5) with 1 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) along with 0.2 mg/L kinetin under dark condition. Tender leaves of D. pleiantha showed the maximum of 86% callus induction among the different explants tested. Highest leaf callus proliferation was noted in B5 medium with 1 mg/L 2,4-D incubated under complete darkness. In addition, it was found that B5 medium with 1 mg/L 2,4-D along with 2 g/L peptone produced more leaf callus biomass and enhanced production of podophyllotoxin (16.3-fold), kaempferol (12.39-fold), and quercetin (5.03-fold) compared to control. Therefore, D. pleiantha callogenesis can provide an alternative source for enhanced production of secondary compounds regardless of the exploitation of its natural plant population.

Thymoquinone Effects on Cell Viability, Apoptosis and VEGF-A Gene Expression Level in AGS(CRL-1739) Cell Line.

Cancer is one of the most fatal diseases across the world and it was reported that 90% of cancer fatality depends on its angiogenesis potential. Black seed or Nigella sativa L. is a medicinal plant native to southwest Asia. N. sativa has been used for medicinal purposes for centuries and predominantly has bioactive components like Thymoquinone, which is used as a candidate for anti-cancer and anti-angiogenesis drugs. Callus was induced from leaf tissue, after that alcoholic extracts were prepared from three-month-old calluses. Thymoquinone content was measured by HPLC methods. AGS cell line was cultured and treated with standard Thymoquinone and extracts from callus. Then, cell proliferation, expression of angiogenic factor (VEGF-A gene), and apoptosis test were done by MTT assay, real-time PCR and Annexin-v kit, respectively. HPLC found the maximum amount of Thymoquinone in the extract of leaf calluses, which were grown in the dark. MTT assay revealed that particular doses of extracts reduced cell proliferation. Real-time and Fluorescence- Activated Cell Sorting (FACS) results demonstrated that standard Thymoquinone and callus extracts down-regulated the VEGF-A gene expression, and all three induced apoptosis in the AGS cell line. It has been shown that TQ has pro-apoptotic and anti-metastatic effects on stomach cancer cell line, and these properties can introduce it as an anti-cancer drug in the near future.

Identification of lupeol produced by Vernonanthura patens (Kunth) H. Rob. leaf callus culture

The lupeol detection in callus of Vernonanthura patens (Kunth) H. Rob. leaves is discussed. Leaf segments previously treated with sodium hypochlorite, ethanol, and distilled water were placed in MS basal medium (Murashige and Skoog) for 7 days. Next, callus induction were done in two complemented MS medium for 6 weeks. Then, callus propagation were performed in MS medium supplemented with 1.0 mg/L of benzylaminopurine (BAP) and 0.5 mg/L of 2,4-dichlorophenoxyacetic acid (2,4-D) for 50 days. Fresh callus were extracted every 10 days in an ultrasonic bath using ethyl acetate (1.0 g/10 mL). The identification was carried out by Gas Chromatography-Mass Spectrometry (GC-MS) using selected ion monitoring (SIM) acquisition mode with characteristic ions of lupeol. The results obtained indicate the occurrence of lupeol in callus extract after twenty days of proliferation. These findings could be use in subsequent scale-up studies for biomass production containing this active compound in order to replace conventional methods.

Anti-inflammatory activity of ethanol extract of leaf and leaf callus of basil (Ocimum basilicum L.) on RAW 264.7 macrophage cells

Basil (Ocimum basilicum L.) has been used not only as culinary herb for flavor, but traditional medicines such as antiseptic, antispasmodic, digestive regulatory, anti-oxidant and antimicrobial properties. However, the anti-inflammatory activity of plant tissue cultures developed from Ocimum basilicum L. remains unknown. This study aimed to investigate the effect of callus induction and the ethanol extract of in vivo leaf and in vitro (leaf, callus light and dark condition) of Ocimum basilicum L. to examine its anti-inflammatory activity on LPS-stimulated RAW 264.7 macrophage cells in vitro. Callus induction from leaf explants of Ocimum basilicum L. was conducted by incubating leaf explants on MS medium supplemented with various concentrations of KIN in combination with 2,4-D. The constituents of leaf and leaf callus ethanol extracts of Ocimum basilicum L. were quantified using GC–MS analysis. Additionally, cell viability was determined using an MTT assay and anti-inflammatory effects were investigated by measuring NO production. The results showed that the leaf callus was induced on MS medium supplemented with various combination of KIN and 2,4-D over a short time period. Analyses confirmed that in vivo leaf contained many of the constituents than in vitro leaf and callus. Moreover, the ethanol extracts of leaf and leaf callus of Ocimum basilicum L. exhibited non-cytotoxicity and reduced NO production in LPS-stimulated RAW 264.7 macrophage cells. Thus, these results suggest that Ocimum basilicum L. may have potential benefits in preventing pathological inflammation.