Callus Initiation Research Articles

Metabolomic profiling of in vitro and in situ grown Nilgiris tea reveals unique signatures for breeding decaffeinated varieties

This study was begun by establishing an in vitro culture in UPASI 9, a Nilgiris tea clone (Camellia sinensis) by optimising various factors. Anatomical studies demonstrated that use of lower carbendazim concentration for sterilisation (0.2%) produced viable and healthy explants for callus initiation. To confirm the genetic consistency of the regenerated plants, gene-specific SSR markers were developed and utilised. GC-MS was employed to analyse volatile metabolites extracted from callus, stem, micro shoots, and leaves of the UPASI 9 tea genotype. The results revealed distinct compositions of metabolites in each sample. More interestingly, caffeine was exclusively detected in leaf samples but absent in all other investigated tissues, despite the presence of Tea Caffeine Synthase (TCS) gene-specific SSRs. Thus, this study provided unique information on the absence of caffeine in in vitro grown Nilgiris tea clone, UPASI 9, as decaffeinated tea has a unique niche in the global market.

The Influence of Sucrose on Biomass and Glycosides Content of Callus Cultured from the Leaves of Stevia rebaudiana Bertoni

Stevia rebaudiana Bertoni contains diterpenoid steviol glycosides that have no adverse impact on blood sugar levels despite being 300 times sweeter than sugar. This study aimed to investigate the rate of callus induction from stevia leaves and the content of glycosides when changing the sucrose percentage in the culture medium.. Murashige and Skoog (MS) culture medium supported by 4.0 mg/l naphthalene acetic acid (NAA) and 1.0 mg/l benzyl adenine (BA) was used, and different concentrations of sucrose (2, 3, 4, 5 and 6%) were tested .The extraction of glycosides from leaf and callus tissues was performed by using methanol. Extracted glycosides were analyzed by high-performance liquid chromatography (HPLC). The results showed significant influences of sucrose on callus initiation. The concentration of 3% sucrose had the highest fresh and dry weight. No callus was induced in the MS medium with a high concentration of sucrose (5% and 6%). However, the highest glycoside content, stevioside and rebaudioside were obtained from callus treated with 4% concentration followed by 3% sucrose the treatment . The highest fresh and dry weight average was obtained with a 3% concentration of sucrose, this treatment also increased the concentration of glycosides in the callus twice as much as in the leaves, and concentrations of 3% and 4% of sucrose gave very similar concentrations of glycosides in callus in terms of being double what was found in the leaves something which may aid in the development of a stevia glycosides based industry.

Nano-Integrated Plant Tissue Culture to Increase the Rate of Callus Induction, Growth, and Curcuminoid Production in Curcuma longa.

Turmeric has attracted a significant amount of interest in recent years due to its strong antimicrobial properties. The tissue culture of turmeric is preferred to obtain disease-free, highest number of plantlets with good uniform chemistry. However, there is a need to increase the speed of the whole process to meet the growing demand for planting materials and to save time and resources. Iron oxide nanoparticles (Fe3O4 NPs) showed positive effects on callus initiation time, proliferation rate, percent root response, shoot length, percent rooting, and number of roots per explant. Highest callus induction, i.e., 80%, was recorded in cultures that were grown in the presence of 15 mg/L of Fe3O4 NPs. Callus initiated earlier in culture tubes that received green synthesized iron nanoparticles in a concentration between 10-15 mg/L. Biofabricated nanoparticles were characterized for their size, physiochemical, and optical properties through UV-Vis spectroscopy, FTIR, XRD, and SEM. Curcuminoids profiling was performed by implementing LC-Ms that revealed increased quantities in plantlets grown in nano-supplemented media when compared to the control.

The Influence of Maturity, Storage, and Embryo Size on Coconut Callus Induction Success

Coconut palms (Cocos nucifera L.) are globally significant palms with both economic and cultural value. Despite the increasing demand for coconut products, production is decreasing globally due to palm senility, pests, and diseases. It has been estimated that over half of the world’s coconut palms need to be replaced immediately. The coconut industry has acknowledged that conventional propagation methods are unlikely to yield sufficient high-quality planting material. Therefore, coconut tissue culture is considered a potential solution to this problem. By using coconut tissue culture, a large number of plantlets can be obtained in a short period of time. In this study, the quality of explants and the development stage (visible shoot/non-visible shoot) of coconut used for micropropagation were examined. To our knowledge, little research has been undertaken on this aspect of coconut micropropagation. Our results indicated that tender coconut fruit exhibited an advantage over mature fruits. In addition, coconut plumule explants subjected to an extended storage of 15 days demonstrated enhanced development compared to those without storage. Notably, smaller embryos utilized as explants displayed superior callus formation compared to their larger counterparts. Finally, embryos possessing shoots exhibited improved callus initiation, albeit accompanied by a more pronounced browning effect. Further investigations are required to obtain more knowledge about the most suitable conditions for plumule explants that lead to optimal callus initiation.

ESR2–HDA6 complex negatively regulates auxin biosynthesis to delay callus initiation in Arabidopsis leaf explants during tissue culture

Plants exhibit an astonishing ability to regulate organ regeneration upon wounding. Excision of leaf explants promotes the biosynthesis of indole-3-acetic acid (IAA), which is polar-transported to excised regions, where cell fate transition leads to root founder cell specification to induce de novo root regeneration. The regeneration capacity of plants has been utilized to develop in vitro tissue culture technologies. Here, we report that IAA accumulation near the wounded site of leaf explants is essential for callus formation on 2,4-dichlorophenoxyacetic acid (2,4-D)-rich callus-inducing medium (CIM). Notably, a high concentration of 2,4-D does not compensate for the action of IAA because of its limited efflux; rather, it lowers IAA biosynthesis via a negative feedback mechanism at an early stage of in vitro tissue culture, delaying callus initiation. The auxin negative feedback loop in CIM-cultured leaf explants is mediated by an auxin-inducible APETALA2 transcription factor, ENHANCER OF SHOOT REGENERATION 2 (ESR2), along with its interacting partner HISTONE DEACETYLASE 6 (HDA6). The ESR2–HDA6 complex binds directly to, and removes the H3ac mark from, the YUCCA1 (YUC1), YUC7, and YUC9 loci, consequently repressing auxin biosynthesis and inhibiting cell fate transition on 2,4-D-rich CIM. These findings indicate that negative feedback regulation of auxin biosynthesis by ESR2 and HDA6 interferes with proper cell fate transition and callus initiation.

Impact of Radiation on Rice Calli and Subsequent In vitro Regeneration

Aromatic rice has high commercial importance with greater economic return. Among the aromatic rice varieties, Kataribhog is a famous local variety that is mostly cultivated in the Dinajpur district northern part of Bangladesh. The yield performance of Kataribhog is lower (<2.50 t/ha) and has shown lodging tendency. Under these circumstances, a research program was taken to improve the existing problem through in vitro culture including nuclear irradiation techniques. Gamma irradiation (Co60) is a powerful tool in crop improvement, and its effects on callus development from kataribhog rice were explored in this research. Three types of media, PT-100G, PT-011G, and PT-100, were utilized to induce callus, which were then subjected to three different doses of radiation (10Gy, 12Gy, and 15Gy). Among the tested media, PT-100G was the most effective, boasting an impressive average of Callus initiation of 79.27% and embryogenic callus induction percentage of 32.93%. In contrast, PT-011G and PT-100 lagged with 52.46% and 44.44% callus initiation; 27.87% and 11.11% embryogenic callus induction, respectively. The radiation dose of 10 Gy was optimal for calli survival, showing an average percentage of 80.00%. 10 Gy is the most effective dose for promoting plant regeneration.

Somatic Embryogenesis and Direct Shoot Bud Formation from in vitro Root Segments of Garlic (Allium sativum L.)

In vitro techniques are unconventionally used for garlic improvement. In garlic tissue culture, numerous roots are produced in vitro. Segments (1 cm) of those in vitro roots were cultivated on MS medium with 5.0 μM 2, 4-D for callus initiation followed by transfer to 0.5 μM 2, 4-D for somatic embryogenesis. Root segments had 51.67% callus and somatic embryo formation. Plantlet regeneration was obtained from 90% calli after being transferred to MS medium supplemented with 5.0 μM kinetin. Within 6 months, 54.8 plants per root segment and 42.2 plants per root tip explant were found. MS media supplemented with 1.0 or 5.0 μM NAA in combination with 10.0 or 50.0 μM BA were used for direct shoot bud induction from root segments. The highest percentage (23.33 %) of direct shoot bud regeneration was found in 5.0 μM NAA with 50.0 μM BA within 2 months. The number of shoots per explant was varied from 1-81 with the highest average of 15.75 shoots per explant. The shoots produced bulblets upon transfer to a medium containing higher amount of sucrose (12%). The largest bulblets weighed 356.57 g in the medium with 5.0 μM NAA and 50.0 μM BA. In both pathways, regeneration occurred from only the apical (distal) end of the root segments. The protocols are promising for continuous regeneration of propagules and genetic transformation study for improvement of garlic. Plant Tissue Cult. & Biotech. 33(2): 135-142, 2023 (December)

Monitoring of the health status of Castanea sativa in the Belasitsa mountain, southwest Bulgaria

In the period 2017-2023, a survey for the assessment of the phytosanitary condition of sweet chestnut (Castanea sativa) was conducted in a permanent sample plot (PSP) Belasitsa, which is part of the European large-scale network for monitoring the health status of forest ecosystems under the International Co-operative Program ‘Forests’. The PSP is set in a natural chestnut stand in Belasitsa mountain at an altitude of 643 m. Data collected from the first years of the monitoring determined a slight deterioration in the health status of the chestnut trees caused by an infection with the fungal pathogen Cryphonectria parasitica. An improvement in trees’ vitality and lack of active necrosis were observed in 2022-2023. On the wounds, initiation of callus along the wounded edges was reported. Currently, the introduction of new and dangerous invasive insect pests Dryocosmus kuriphilus, Corythucha arcuata, etc., has not been detected. Attacks of both pests could further deteriorate the health status of the sweet chestnut in Belasitsa.

Multi-drops and Cross-sections, Efficient Methods for Establishing Cell Suspension Culture of Cuminum cyminum L. and Plant Regeneration

The current study aims of the current study is to establish cell suspension cultures of the medicinal plant, cumin Cuminum cyminum L. by the application of multiple drops and cross-section techniques. These methods were used in cultivating cell suspensions and in vitro plant regeneration. Leaf, stem, hypocotyl and root explants were cultured in Murashige and Skoog (MS) medium containing different concentrations (0.05, 0.5 and 1.0 mg.l-1) of naphthalene acetic acid (NAA) and (0.05, 0.1, 1.0 and 2.0 mg.l-1) benzyl adenine BA for callus production. The results indicated the high response of cumin, as the percentage of callus initiation was 100%.The plant regeneration percentage reached 91.6%. Moreover, a friable callus of hypocotyls was appropriate for initiation of cell suspension culture in MS medium with 0.5mg.l-1 NAA and 1.0 mg.l-1benzyl adenine (BA). The best density for primordial callus formation was 51.0 ×105 cells ml-1 in both multiple-drops and cross-sections embedding methods. Callus that had been produced from multi drops and sectors had the ability for shoot regeneration. This study clarified the efficiency of these techniques in establishing cell suspension culture and shoot regeneration, which could be promising sources for high production of active compounds in cumin plant.

Pengaruh waktu induksi dan ZPT terhadap pertumbuhan kalus kotiledon kacang panjang (Vigna unguiculata subsp. sesquipedalis)

Yard-Long bean plants are an agricultural commodity that has great potential to be developed because they are easy to cultivate, their market share is also quite high but there are still problems in meeting the need for sufficient seeds and they are still susceptible to pests and disease. Therefore, efforts need to be made to increase production and induce variations. One method that can be used is tissue culture for the induction of somaclonal variations and/or the formation of transgenic plants. This study aims to determine the effect of variations in callus initiation time and the effect of Plant Growth Regulators (PGR) administration on the growth of long bean cotyledon explant callus. Seed sterilization was carried out using detergent, 10% sodium hypochlorite, 70% alcohol and sterile distilled water. The results of the research showed that variations in callus initiation time on MS media with the addition of 2,4-D PGR and the use of several variations of PGR in subcultures were able to initiate callus and influence callus size, callus color, and the percentage of browning of Yard-long bean cotyledon explants. Callus initiation time of 4 weeks with a combination of 2 ppm BAP concentration with 0.1 ppm IAA produced the largest callus size and brownish white callus color. The addition of PGR during subculture had a smaller browning percentage than the treatment without PGR.

In Vitro Propagation of Ruta Chalepensis Through and Callus Culture

Plant tissue culture is a field that that enables culturing of various plants and parts of plants usually treated under a nutrient medium and in highly sterile conditions. Out of them callus culture is one of the very interesting arenas of plant biotechnology that encompasses many pivotal benefits. The study focuses on such callus enrichment using different hormones that there by enhance its biological activities. The plant namely Ruta chalepensiswas chosen upon wherein the callus growth was noticed. Ruta chalepensis has multiple medicinal activities like anti-cancer, anti-ulcer, anti-diabetic and many more pharmacological properties that yields in treating and curing of illness. Ruta chalepensis the leaf and internode were taken for the study and to analyse which of those at what concentration of plant growth regulators showed a better callus induction. The MS medium as well as Various hormone concentrations was used for the study like auxin and cytokinin from 0.5mg to 2 mg (2,4 – D, NAA, IAA, IBA). Increased concentration of 2,4-D (1.0 mg/L) alone in the MS medium showed profused callus growth. Both the explants used such as leaf and Internode were also tested in the MS medium which was devoid of hormones/plant growth regulators which was treated as control for comparison. From the data obtained, MS medium supplemented with hormones showed better growth rate and callus induction when compared with that of MS medium without hormones/plant growth regulators. Among the plant growth regulators 2,4-D (1.0 mg/L) showed maximum callus initiation from both leaf and internode explants. Further work was carried out in single and combination of the plant growth regulators for callus proliferation and accumulation. Further analysis is being done to study the growth pattern on combination of hormones and fix the hormone concentrations for the mass propagation of callus from the explants.

Time-Dependent Proteomic Signatures Associated with Embryogenic Callus Induction in Carica papaya L.

Sex segregation increases the cost of Carica papaya production through seed-based propagation. Therefore, in vitro techniques are an attractive option for clonal propagation, especially of hermaphroditic plants. Here, we performed a temporal analysis of the proteome of C. papaya calli aiming to identify the key players involved in embryogenic callus formation. Mature zygotic embryos used as explants were treated with 20 μM 2,4-dichlorophenoxyacetic acid to induce embryogenic callus. Total proteins were extracted from explants at 0 (zygotic embryo) and after 7, 14, and 21 days of induction. A total of 1407 proteins were identified using a bottom-up proteomic approach. The clustering analysis revealed four distinct patterns of protein accumulation throughout callus induction. Proteins related to seed maturation and storage are abundant in the explant before induction, decreasing as callus formation progresses. Carbohydrate and amino acid metabolisms, aerobic respiration, and protein catabolic processes were enriched throughout days of callus induction. Protein kinases associated with auxin responses, such as SKP1-like proteins 1B, accumulated in response to callus induction. Additionally, regulatory proteins, including histone deacetylase (HD2C) and argonaute 1 (AGO1), were more abundant at 7 days, suggesting their role in the acquisition of embryogenic competence. Predicted protein-protein networks revealed the regulatory role of proteins 14-3-3 accumulated during callus induction and the association of proteins involved in oxidative phosphorylation and hormone response. Our findings emphasize the modulation of the proteome during embryogenic callus initiation and identify regulatory proteins that might be involved in the activation of this process.

The HOS1-PIF4/5 module controls callus formation in Arabidopsis leaf explants

ABSTRACT A two-step plant regeneration has been widely exploited to genetic manipulation and genome engineering in plants. Despite technical importance, understanding of molecular mechanism underlying in vitro plant regeneration remains to be fully elucidated. Here, we found that the HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENES 1 (HOS1)-PHYTOCHROME INTERACTING FACTOR 4/5 (PIF4/5) module participates in callus formation. Consistent with the repressive role of HOS1 in PIF transcriptional activation activity, hos1–3 mutant leaf explants exhibited enhanced callus formation, whereas pif4–101 pif5–3 mutant leaf explants showed reduced callus size. The HOS1-PIF4/5 function would be largely dependent on auxin biosynthesis and signaling, which are essential for callus initiation and proliferation. Our findings suggest that the HOS1-PIF4/5 module plays a pivotal role in auxin-dependent callus formation in Arabidopsis.

A Common Molecular Signature Indicates the Pre-Meristematic State of Plant Calli.

In response to different degrees of mechanical injury, certain plant cells re-enter the division cycle to provide cells for tissue replenishment, tissue rejoining, de novo organ formation, and/or wound healing. The intermediate tissue formed by the dividing cells is called a callus. Callus formation can also be induced artificially in vitro by wounding and/or hormone (auxin and cytokinin) treatments. The callus tissue can be maintained in culture, providing starting material for de novo organ or embryo regeneration and thus serving as the basis for many plant biotechnology applications. Due to the biotechnological importance of callus cultures and the scientific interest in the developmental flexibility of somatic plant cells, the initial molecular steps of callus formation have been studied in detail. It was revealed that callus initiation can follow various ways, depending on the organ from which it develops and the inducer, but they converge on a seemingly identical tissue. It is not known, however, if callus is indeed a special tissue with a defined gene expression signature, whether it is a malformed meristem, or a mass of so-called "undifferentiated" cells, as is mostly believed. In this paper, I review the various mechanisms of plant regeneration that may converge on callus initiation. I discuss the role of plant hormones in the detour of callus formation from normal development. Finally, I compare various Arabidopsis gene expression datasets obtained a few days, two weeks, or several years after callus induction and identify 21 genes, including genes of key transcription factors controlling cell division and differentiation in meristematic regions, which were upregulated in all investigated callus samples. I summarize the information available on all 21 genes that point to the pre-meristematic nature of callus tissues underlying their wide regeneration potential.

The nitrate-responsive transcription factor MdNLP7 regulates callus formation by modulating auxin response

Under appropriate culture conditions, plant cells can regenerate new organs or even whole plants. De novo organ regeneration is an excellent biological system, which usually requires additional growth regulators, including auxin and cytokinin. Nitrate is an essential nutrient element for plant vegetative and reproductive development. It has been reported that nitrate is involved in auxin biosynthesis and transport throughout the growth and development of plants. In this study, we demonstrated that the ectopic expression of the MdNLP7 transcription factor in Arabidopsis could regulate the regeneration of root explants. MdNLP7 mainly participated in the regulation of callus formation, starting with pericycle cell division, and mainly affected auxin distribution and accumulation in the regulation process. Moreover, MdNLP7 upregulated the expression of genes related to auxin biosynthesis and transport in the callus formation stage. The results demonstrated that MdNLP7 may play a role in the nitrate-modulated regeneration of root explants. Moreover, the results revealed that nitrate–auxin crosstalk is required for de novo callus initiation and clarified the mechanisms of organogenesis.

Multiplication and Rooting Shoot Tips and Nodes of Petunia Plant (Petunia hybrida)

This study was performed in the cell and plant tissue culture laboratory of the Mosul University Department of Horticulture and Landscape Design, College of Agriculture and Forestry. Shoot tips and nodes were cultured on MS medium supplemented with (0.0, 2.0, 4.0, 6.0, 8.0) mg.l−1 Kin during the multiplication stage of this investigation, and the resulting shoots were grown on 12 MS media for rooting with IBA (0.0, 0.25, 0.5, 0.75, 1.0) mg.l−1. In this experiment, we cultured pieces of leaf clippings from plants grown in the field and exposed them to varying concentrations of 2,4-D (0.0, 0.25, 0.5, 1.0, and 2.0 mg.l−1) in order to initiate and differentiate callus. After eight weeks, MS medium containing 6.0 mg.l−1 Kin resulted in the highest rooting percentage (70%) and highest root number (17.0 root/explant) from cultured shoot tips produced in vitro; in contrast, nodes resulted in 6.0 shoots/explants with a length of 3.8 cm from MS medium containing 6.0 mg.l−1 Kin. Eight weeks after their in vitro production, all of the Plantlets were acclimatized and transferred to the field with 100% survival. The highest callus initiation percentage was obtained from cultured parts of the leaf on MS medium supplemented with 0.5 mg.l−1 2,4-D, while the highest shoots production percentage was 17.857% with 2.0 shoot/explant from callus initiation from MS medium supplemented with 0.25 mg.l-1 2,4-D.

Indirect auxiliary organogenesis of Fraxinus excelsior L. as a tool for ash dieback control

The existence of European ash (Fraxinus excelsior L.) is threatened by fungus-induced ash dieback. It is essential to find effective methods to multiply ash genotypes resistant to ash dieback while preserving the genetic diversity of these tree populations. In this paper the efficient method for production of European ash seedlings using indirect auxiliary organogenesis with multi-factor analysis of its effectiveness is presented. Procedures for a dormancy breaking treatment of seeds and effective disinfection of F. excelsior primary explants, as well as appropriate composition of the culture media taking into account impact of growth regulators and physiological gradient on the micropropagation efficiency were developed. As primary explant for micropropagation of F. excelsior, leaf buds, megagametophytes and zygotic embryos were tested. The best-performing type of primary explant for micropropagation of European ash proved to be zygotic embryos, which were successfully used to regenerate seedlings via indirect auxiliary organogenesis. No statistically significant impact of population origin of F. excelsior explant donor trees was observed on the effectiveness of callus initiation. However, such difference was significant in regard to average productivity of acquired callus cultures (number of seedlings produced) and to average root length of regenerated seedlings. Health condition of explant donor trees and their seeds affects the callus initiation rate from zygotic embryos, but does not affect the productivity of callus lines derived from the seeds and the quality of regenerated seedlings. Indirect auxiliary organogenesis of F. excelsior, developed in our study, not only provides the acquisition of ash seedlings of different genotypes, but also enables rapid selection of desired genotypes already at the callus stage. In this way, the presented method benefits not only profit oriented forestry and wood industry, but also provide the effective and fully controllable tool for reintroduction of various resistant to ash-dieback F. excelsior genotypes without loss of variability and genetic identity of its populations.

Factors Affecting Callus Induction from Anther and Ovary of Okra (Abelmoschus esculentus L.)

Background: Okra [Abelmoschus esculentus (L.) Moench.] is a nutrient-rich vegetable crop widely grown in the tropics and sub-tropics mainly for its edible pods. The haploid technique has been used in plant breeding for the improvements of plants and to develop new varieties in relatively a short time. Hence, we have optimized several factors such as plant growth regulators (PGR), sucrose concentration, cold treatment, type of media and culture conditions for callus induction from the anther and ovary of okra (557 F1 hybrid). Methods: The flower buds of different sizes were collected to determine various stages of development and then subjected to cold pre-treatments. The explants were then cultured on various combinations of PGRs i.e., naphthyloxy acetic (NOA), Indole acetic acid (IAA), 2, 4-Dichlorophenoxy acetic acid (2, 4-D), Benzyl amino purine (BAP), isopentenyl adenine 2iP, Kinetin (KIN) and Thidiazuron (TDZ). Result: The optimum developmental stage of microspore for callus initiation was achieved from flower buds of okra and its size was about 11 mm long. Flower buds that emerged one week after the flowering showed significantly higher percentage of callus induction. The optimum stage for ovary and ovule culture were one or two days prior to anthesis and the flower buds stage was 35±1mm. In conclusion, our study investigated the effect of several factors that affects callus induction in okra and optimized cultured conditions for future haploid development for okra.

Targeting apoptotic anticancer response with natural glucosinolates from cell suspension culture of Lepidium sativum

BackgroundFinding natural products with anticancer activity is an effective strategy to fight this disease. In this respect, Lepidium sativum or garden cress (family Brassicaceae) has been widely used worldwide for its wide therapeutic application, including anticancer and chemoprotective agents. Plant tissue culture techniques hold great promise for natural product enhancement without any climatic boundaries. In this study, glucosinolates and petroleum ether fractions were isolated from in vitro cell cultures and used against different carcinoma cell lines to investigate their anticancer potential. MethodsIn this study, callus cultures from leaf and root explants were initiated, cell suspension cultures were established, and cell growth and viability profiles were characterized. Different amino acids were added as precursors to the cell suspension cultures to enhance glucosinolates accumulation. Gas chromatography–mass spectrometric analysis (GC–MS) of glucosinolates and petroleum ether fractions was performed, and all fractions were tested against different carcinoma cell lines. ResultsThe findings clarified that the maximum callus initiation percentage was obtained in the medium containing 1.0 mg/l 2,4-dichlorophenoxy acetic acid (2,4-D) + 1.0 mg/l kinetin (Kin) (C1). The viable cell number of cell suspension cultures from leaves and roots increased until it reached the maximum values on day 15. Adding tyrosine and methionine to the cell suspension cultures was the most influential and recorded high glucosinolate percentages. 1H-Cyclopenta (b) pyridine-3-carbonitrile-4,5,6,7-tetrahydro-2-methylthio-4-spirocyclohexane was the main glucosinolate compound found in tyrosine-treated leaf suspension (GLT). Fifteen compounds were detected in the petroleum ether fraction in both cell suspensions initiated from the leaf and root (OL and OR). The major compounds were benzene-1,3,5-trimethyl (12.99%) in root cell suspension (OR), and benzene-2-ethyl-1,4-dimethyl (10.66%) in leaf cell suspension (OL). All glucosinolate extracts demonstrated significant anticancer activity against the prostate (PC3), lung (A-549), colorectal (caco2), and liver (HepG2) cell lines. Glucosinolates extracted from leaf cell suspension (GL) were the most active on the hepatocellular carcinoma cell line (HepG2) among all remaining glucosinolate extracts. Treated hepatocellular carcinoma with an IC50 of GL extract (47.5 ug/ml) upregulates pro-apoptotic BAX and downregulates anti-apoptotic BCL2, which disrupts the BAX/BCL2 ratio, leading to activation of caspase 3 inside treated HepG2 cells. ConclusionsThe anticancer action of the GL extract was validated by the cell cycle study of its glucosinolates, which successfully promoted apoptosis and reduced hepatocellular growth by causing S-phase arrest.

Indirect organogenesis of Aceh patchouli leaf explants (Pogostemon cablin Benth) by in vitro

The Aceh patchouli plant (Pogostemon cablin. Benth) is a plantation plant that produces essential oils with great potential, known as patchouli oil. The use of in vitro culture techniques on patchouli plants needs to be developed to meet the needs of patchouli seeds. This study aimed to analyze the effect of plant growth regulators (PGRs) cytokinins 6-Benzyl Amino Purine (BAP) and Thidiazuron (TDZ) and auxin 1-Naphthaleneacetic Acid (NAA) on the organogenesis ability of Aceh patchouli leaf explants var. Lhokseumawe. The treatments analyzed were P1 = MS; P2 = BAP 0.75 mg.l−1, P3 = BAP 1.0 mg.l−1, P4 = TDZ 0.75 mg.l−1, P5 = TDZ 1.0 mg.l−1, P6 = NAA 0.5 mg.l−1, P7 = NAA 1.0 mg.l−1, P8 = BAP 0.75 mg.l−1 + NAA 0.5 mg.l−1, P9 = BAP 0.75 mg.l−1 + NAA 1.0 mg.l−1, P10 = BAP 1.0 mg.l−1 + NAA 0.5 mg.l−1, P11 = BAP 1.0 mg.l−1 + NAA 1.0 mg.l−1, P12 = TDZ 0.75 mg.l−1 + NAA 0.5 mg.l−1, P13 = TDZ 0.75 mg.l−1 + NAA 1.0 mg. l−1, P14 = TDZ 1.0 mg.l−1 + NAA 0.5 mg.l−1, P15 = TDZ 1.0 mg.l−1 + NAA 1.0 mg.l-1. The percentage of callus induction observed, percentage of shoot induction, percentage of browning, percentage of contamination and percentage of live explants at 28 DAI. The results showed that the percentage of explants forming callus was 56.4%, and the percentage of explants forming organ was 54.6%. In these two parameters the treatment of P14 and P15 gave the best results of 6.6% respectively. The percentage of explants experiencing browning was 26.1%, treatment P1 gave the highest response for this parameter, namely 6.6%. The parameter percentage of live explants was 71.3%. The best response was found in treatment P2, P3, P12 and P15, which were 6.6% each. The fastest bud formation occurred at 14 Days After Initiation (DAI) in P4 treatment. The resulting shoots look quite vigor and have formed leaves. In the combination treatment P14, showed the best results, the addition of auxin was able to increase the initiation of embryogenic callus.