Induce Callus Formation Research Articles

In vivo tetraploid induction of mono-embryonic citrus genotypes by colchicine treatment

Polyploid holds great significance in citrus breeding, to produce dwarf tetraploid rootstocks with wide adaptability and triploid scion varieties bearing seedless fruits. In citrus, tetraploids have been generated through somatic hybridization and autonomous chromosome doubling of nucellar embryos. However, most tetraploids were recovered from poly-embryonic (apomictic) citrus, while tetraploids of mono-embryonic (non-apomictic) citrus were seldomly reported. Herein, we developed an in vivo tetraploid induction method by colchicine treatment, which is especially high-efficient in mono-embryonic citrus. The epicotyl of citrus seedling was decapitated and incubated in dark for different time durations (0–15 d) to induce callus formation on the epicotyl wound. The callus on wound was then immersed in different concentrations (0.0125 %-0.100 %) of colchicine for 2 h, enabling regeneration of tetraploid shoots. We achieved high tetraploid induction rate (20.69 %) but low mixoploid induction rate (1.72 %), when 0.025 % colchicine was applied on wound cultured in dark for 9 d By using this method, 147 tetraploids were recovered from mono-embryonic mandarin, tangerine, sweet orange, pummelo and citron. Our method exhibited high tetraploid induction efficiency (14.28 %-20.69 %), low rate of mixoploid (1.72 %-5.30 %), short recovery period (45 d), and no need of tissue culture. The majority (75 %-100 %) of the recovered tetraploids maintained stable ploidy during subsequent growth, and the mono-embryonic nature of the tetraploids was identified by InDel marker analysis. The mono-embryonic tetraploids obtained herein enriched polyploid germplasm pool for citrus breeding.

Plantlet Regeneration from Callus Cultures of Kappaphycus alvarezii for Cultivation in Coastal Waters at Myeik Archipelago, Myanmar.

<b>Background and Objective:</b> The cultivation of <i>Kappaphycus alvarezii</i>, the most economically valuable rhodophyte seaweed species, began in Myanmar in 2004, primarily on islands in the Myeik Archipelago. Since <i>K. alvarezii</i> is not native to Myanmar, it was initially imported from the Philippines and Indonesia. This study aimed to develop a tissue culture procedure for the generation of <i>K. alvarezii</i> plantlets to ensure a continuous supply of seaweed for commercial farming in the coastal waters of Myanmar. <b>Materials and Methods:</b> Specimens of <i>K. alvarezii</i>, two-month-old, were procured in the Myeik Archipelago, Myanmar. After being cleared epiphytes and clinging materials, the specimens were placed in glass aquarium tanks with circulating seawater. Axenic explant culture was established using 1% povidone-iodine for 1 min and a 1% antibiotic mixture for 24 hrs. Plant growth regulators, 6-benzyl aminopurine (BAP) and indole 3 acetic acid (IAA) were supplemented in solid Provasoli's enriched seawater (PES) media to induce callus formation and somatic embryogenesis. <b>Results:</b> The optimal culture conditions were incubation at 22-25°C under cool-white fluorescent-light (15-20 μmol photons/m<sup>2</sup>/sec) with a 12:12 hrs light and dark cycle. Water quality during the culturing process was maintained at a pH of 8 and salinity of 30 PSU. The tissue-cultured <i>K. alvarezii</i> plantlets exhibited an average daily growth rate of 9.70±0.25% over the growth period. <b>Conclusion:</b> Therefore, plantlet regeneration from <i>K. alvarezii</i> callus cultures can be utilized as seedlings for revenue-generating cultivation along the Myanmar coastline.

Antioxidant and Anticancer Activity of Tannins Isolated from Callus Cultures of Achyranthes aspera L.

In the current study, the impact of oxidative stress induced by free radicals generated during metabolic processes was investigated. It was found to be linked to a variety of diseases due to their detrimental effects on nucleic acids and proteins. The primary focus remained on investigating the free radical-scavenging properties and potential anticancer activities of tannins found in callus cultures of A. aspera. It was observed that the induction of callus formation was notably successful when using leaf and root explants, resulting in a callus index as high as 160, as compared to stem explants in the presence of auxins. The extraction yield of callus tannins was the most abundant in chloroform extracts, although the overall antioxidant activity was comparable among chloroform, methanol, and petroleum ether callus extracts. Among these extracts, the chloroform extract from stem callus cultures grown on the MSDN medium showed the highest total antioxidant activity. Notably, tannins extracted from A. aspera leaf callus extracts displayed significant anticancer effects against the Jurket cell line. These effects were evaluated through measures such as cell viability and colony formation. The anticancer activity was notably higher in callus culture extracts as compared to the control. In summary, the results showed that in vitro biomass production can be a valuable approach for augmenting the production of bioactive compounds, such as tannins, from selected medicinal plants including A. aspera. Tannins extracted from A. aspera hold promise for their potential role in the development of new medicines.

Induction and multiplication of callus of AS2K clones Coffea arabica L. through 2,4-D and BAP combination

Abstract Arabica coffee (Coffea arabica L.) has dominated international trade with more than 70% of global coffee production because of it’s taste was more preferred than the other types of coffee. The AS2K clone of C. arabica is a superior clone with high productivity, resistance to pests and diseases, and has a good taste. To maintain these characters, the appropriate propagation is by vegetatively. The aim of this research was to induce callus formation from C. arabica leaves, and to multiplicate the callus on twelve (12) combinations of 2,4-D and BAP as an effort to develop a method for propagating Arabica coffee. Callus induction was conducted on Murashige and Skoog (MS) medium containing 2,4-D 2 mg/L combined with BAP 1 mg/l, and then the multiplication of these callus on MS medium with 12 combinations of 2,4-D (2, 3, or 4 mg/L) and BAP (0, 0.25, or 1 mg/L). The research design used a completely randomized design with 3 replications, and each replication contained 5 callus pieces. The results showed that the combination of 2 ppm 2,4-D and 1 ppm BAP was able to induce callus formation from leaf explants by 49% at week 3 culture, yellowish white in color and friable in texture. Combination of 2,4-D 3 mg/L and BAP 0.5 mg/L provide the best result for callus multiplication.

An Efficient Agrobacterium-Mediated Genetic Transformation System for Gene Editing in Strawberry (Fragaria × ananassa).

The octoploid-cultivated strawberry variety Benihope (Fragaria × ananassa Duch cv. Benihope) is an important commercial plant. It is highly susceptible to different diseases, which ultimately leads to a reduction in yield. Gene-editing methods, such as CRISPR/Cas9, demonstrate potential for improving disease resistance in the strawberry cv. Benihope. Establishing a plant regeneration system suitable for CRISPR/Cas9 gene editing is crucial for obtaining transgenic plants on a large scale. This research established a callus induction and plant regeneration system for Agrobacterium-mediated CRISPR/Cas9 gene editing in strawberry cv. Benihope by evaluating multiple types of explants and various plant growth regulators throughout the entire tissue culture process. The results showed that the efficiency of callus induction is strongly influenced by the type of explant and is highly sensitive to the combination of plant growth regulators. Among the different plant growth regulators employed, thidiazuron (TDZ), in combination with 2,4-dichlorophenoxyacetic acid (2,4-D), effectively induced callus formation and plant regeneration from explants derived from nutrient tissues such as runner tips and crowns. In addition, the regeneration experiment demonstrated that the addition of polyvinylpyrrolidone (PVPP) to the shoot regeneration medium could inhibit tissue browning. The gene-edited plants in which some or all of the Fvb7-1, Fvb7-2, Fvb7-3, and Fvb7-4 genes in the MLO (Mildew resistance Locus O) gene family were knocked out by CRISPR/Cas9 system were obtained by applying the plant regeneration system developed in this study.

In Vitro Germination of Dumbaya Seeds (Momordica cochinchinensis (Lour.) Spreng: A Unique Medicinal Plant of Gorontalo

"Dumbaya (Momordica cochinchinensis (Lour.) Spreng) is one of the plant species believed by the Gorontalo community to be a traditional remedy for various ailments such as inflammation, liver disorders, spleen issues, hemorrhoids, bruising, and infected wounds. Currently, the dumbaya plant is difficult to find and is even considered nearly extinct by the Gorontalo community. This is due to the challenging conventional cultivation of dumbaya plants from seeds. Dumbaya seeds have a hard, stone-like texture, making germination in the wild difficult and requiring special treatment. Regeneration of dumbaya can be achieved through tissue culture techniques, replacing the conventional cultivation system.Through tissue culture, dumbaya seeds are grown in specialized planting media and controlled environments. Based on observations, the propagation of dumbaya plants using in vitro culture techniques has proven successful and effective as a solution for propagating dumbaya seedlings. Dumbaya seeds begin to germinate two weeks after planting (2 WAP). The grown dumbaya seedlings can then be used as explants for further dumbaya propagation by inducing callus formation."

Optimization of Plant Growth Regulators for In Vitro Mass Propagation of a Disease-Free 'Shine Muscat' Grapevine Cultivar.

This study addresses the propagation challenges faced by 'Shine Muscat', a newly introduced premium grapevine cultivar in South Korea, where multiple viral infections pose considerable economic loss. The primary objective was to establish a robust in vitro propagation method for producing disease-free grapes and to identify effective plant growth regulators to facilitate large-scale mass cultivation. After experimentation, 2.0 µM 6-benzyladenine (BA) exhibited superior shoot formation in the Murashige and Skoog medium compared with kinetin and thidiazuron. Conversely, α-naphthaleneacetic acid (NAA) hindered shoot growth and induced callus formation, while indole-3-butyric acid (IBA) and indole-3-acetic acid (IAA) demonstrated favorable root formation, with IBA showing better results overall. Furthermore, inter simple sequence repeat analysis confirmed the genetic stability of in vitro-cultivated seedlings using 2.0 μM BA and 1.0 μM IBA, validating the suitability of the developed propagation method for generating disease-free 'Shine Muscat' grapes. These findings offer promising prospects for commercial grape cultivation, ensuring a consistent supply of healthy grapes in the market.

Callus Induction and Bioactive Compounds Production from Various Cultivars of Medicago sativa L. (alfalfa)

Alfalfa (Medicago sativa L.) belongs to fabacaea family widely grown in Turkey. It is rich in bioactive compounds such as phenolic compounds, flavonoid, essential amino acids (threonine, leucine, lysine, and valine) and tannins, vitamins (A, B1, B2, B6, B12, C and E) or β-carotene. In this study, it was aimed to investigate the impact of secondary metabolite content of explants on callus biomass. For this purpose, cotyledon explants were obtained under sterile conditions, and transferred to standard MS medium containing 1 mg/L 2,4-D (Dichlorophenoxy Acetic Acid) and 0.0125 mg/L kinetin to induce callus formation. The phenolic, flavonoid and tannin contents of the explants were also determined. Leaves and cotyledons explants of 74 M. sativa L. cultivars have been used for callus biomass. The 74 tested alfalfa cultivars varied in their callus growth and callus biomass formation. Van-22, Konya-Ereğli, Alsancak, Gözlü-1 and Iside cultivars were observed with higher callus biomass: Conversely, Van Gevaş, Bitlis Hizan and Van-Çaldıran responded with lower callus biomass in tissue culture. A high-callus biomass cultivar of alfalfa has been shown to have higher total phenolic, flavonoid and tannin content activity than the lower-callus biomass cultivar in terms of leaf explants under tissue culture conditions. Total phenolic content activity was significantly increased in cotyledon explants with higher callus biomass as compared to lower callus biomass. The accumulation of leaf tannin and flavonoid was strongly linked to callus biomass. Cotyledon phenolic and flavonoid content exhibited an increasing trend in response to the increasing biomass of callus.

In Vitro Induction of Interspecific Hybrid and Polyploidy Derived from Oryza officinalis Wall.

Oryza officinalis Wall is a potential genetic resource for rice breeding; however, its distant genome limits its crossing ability with cultivated rice. The interspecific hybridization of O. officinalis and cultivated rice, establishment of its tissue culture, and induction of polyploidy are ways to improve O. officinalis's poor crossability. We developed an interspecific hybrid and studied its reproductive pollen development process in this work, and the results showed that abortive pollens (81.94%) and embryo sac abnormalities (91.04%) were the key causes of its high sterility. In order to induce callus formation in interspecific hybrid explants, two different culture media, namely Chu's N-6 medium (N6) and 1/2 Murashig and Skoog medium (1/2 MS), were employed. Additionally, two plant growth regulators (PGRs), namely 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzylaminopurine (BA), along with L-proline (Pro) and acid hydrolyzed casein, were utilized in the experiment. The optimal N6 medium, supplemented with 2.0 mg·L-1 2,4-D, produced the highest induction rate (80.56 ± 5.44)%. For callus differentiation and proliferation, the MS medium supplemented with 2.0 mg·L-1 BA + 0.2 mg·L-1 NAA produced the highest differentiation rate (75.00 ± 4.97)% and seedling emergence ratio (28.97 ± 4.67)%. The optimal combination for seedling rooting was the 1/2 MS medium supplemented with 2.0 mg L-1 NAA and 0.2 mg L-1 BA, which produced an average of 13.95 roots per plant. For polyploidy induction in the interspecific hybrid, the concentration of colchicine treatment at 400 mg·L-1 for three days is an ideal protocol. We devised tissue culture and interspecific hybrid polyploidy induction to overcome O. officinalis' poor crossability and introduce its favorable features into cultivated rice.

Identification of a pluripotency-inducing small compound, PLU, that induces callus formation via Heat Shock Protein 90-mediated activation of auxin signaling.

Plants retain the ability to generate a pluripotent tissue called callus by dedifferentiating somatic cells. A pluripotent callus can also be artificially induced by culturing explants with hormone mixtures of auxin and cytokinin, and an entire body can then be regenerated from the callus. Here we identified a pluripotency-inducing small compound, PLU, that induces the formation of callus with tissue regeneration potency without the external application of either auxin or cytokinin. The PLU-induced callus expressed several marker genes related to pluripotency acquisition via lateral root initiation processes. PLU-induced callus formation required activation of the auxin signaling pathway though the amount of active auxin was reduced by PLU treatment. RNA-seq analysis and subsequent experiments revealed that Heat Shock Protein 90 (HSP90) mediates a significant part of the PLU-initiated early events. We also showed that HSP90-dependent induction of TRANSPORT INHIBITOR RESPONSE 1, an auxin receptor gene, is required for the callus formation by PLU. Collectively, this study provides a new tool for manipulating and investigating the induction of plant pluripotency from a different angle from the conventional method with the external application of hormone mixtures.

EFFECT OF THIDIAZURON AND 2,4-DICHLORO PHENOXY ACETIC ACID ON CALLUS DEVELOPMENT IN TEA CLONE UPASI 9 TDZ AND 2,4-D ON TEA CALLUS INITIATION

A commonly cultivated tea clone, UPASI 9, is currently propagated through cuttings, an endeavor that has been proved to be environmentally hazardous (due to the use of large quantities of excavated soil) and unprofitable. As an alternative, this study highlights tissue culture as a viable option for the robust and easy regeneration strategy for the tea clone UPASI 9. A study was initiated to establish an in vitro culture protocol for Nilgiris tea (Camellia sinensis) by standardizing explant sources, disinfestation methods, and the constitution of culture media. Segments were dissected from greenwood stem (current year growth) internodes of field-growntea plants. Disinfestation was achieved by successive treatments of fungicide (bavistin), 0.1% mercuric chloride, and 7.5% CaCl2 . MS medium supplemented with sucrose (30 g/l), citric acid (100 mg/l), and ascorbic acid (100 mg/l) was used with combinations of the auxins (2,4- dichlorophenoxyacetic acid; 2,4-D) and cytokinin (Thidiazuron; TDZ). It was found that 1.1 mg of 2,4-D and 1.1 mg of TDZ induced the most callus proliferation. Though equal proportions of TDZ (1.1, 2.2, 3.3 mg) and 2,4-D (1.1, 2.2, 3.3 mg) were effective in inducing callus formation, it was noticed that 1.1 mg each of TDZ and 2,4-D made a major impact by reducing the time required for the callus initiation and proliferation

Каллусные культуры Spergularia marina (L.) Griseb.: получение и фитохимический анализ

In recent decades, interest in halophyte plants has increased due to their high content of biologically active substances with powerful antioxidant, antimicrobial, anti-inflammatory and antitumor properties and promising for the prevention of various diseases. Several species of halophytes grow on the territory of the Kaliningrad region, among which the rare species Spergularia marina (L.) Griseb. is of particular interest, the biological activity and content of secondary metabolites of which have not been studied sufficiently. The purpose of this study was to obtain callus cultures, to study the content of some groups of phenolic compounds and the antioxidant activity of extracts. The study carried out the selection of growth regulators and concentrations that induce callus formation. 19 nutrient media were selected for the induction of S. marina callus cultures. The conducted phytochemical analysis showed a significant content of phenolic compounds and hydroxycinnamic acids, as well as a high level of antioxidant activity of extracts of callus cultures. Out of 19 callus cultures, cultures obtained on Murashige and Skoog nutrient media containing the following combinations of growth regulators are promising for obtaining target secondary metabolites: 0.25 mg/l TDZ and 1 mg/l 2,4-D; 0.1 mg/l TDZ and 1.5 mg/l 2,4-D; 0.25 mg/l TDZ and 0.25 mg/l IBA; 0.5 mg/l TDZ and 0.25 mg/l IBA; 0.25 mg/l KinN and 0.5 mg/l 2,4-D.

The effect of 2,4-D, BA and Thidiazuron on somatic embryo induction of liberica coffee of Tungkal Composite from Jambi

The Tungkal Composite of liberica coffee rejuvenation program at Jambi Province is urgently needed due to the latest status of the plants are old and showing diseased attacked. To support the rejuvenation, the provision of seedlings could be done by tissue culture approach through somatic embryogenesis. This research aimed to study the effect of 2,4-D, BA and TDZ on callus formation and embryo induction from liberica coffee leaf explants. Explants used were leaf sections with or without veins. Explants were grown on Murashige Skoog (MS) basal medium with 2,4-D + BA and 2,4-D + TDZ. A completely randomized design was employed in the trial, and each experimental unit was replicated three times. Results showed that callus could be formed on both types of explants, but leaf explants with veins could produce callus faster. The use of BA in medium could induce callus formation faster than TDZ. The higher the concentration of 2,4-D, BA and TDZ, the slower callus initiation. The addition of TDZ in the medium resulted in mostly callus, while callus from medium with BA showed a friable structure. Globular somatic embryos were formed in all treatments with varying amounts. TDZ can induce more globular somatic embryo than BA.

Effect of Growth Regulators on Callus Formation and Alkaloids Production on Vinca Rosea Plant (Catharanthus roseus L.)

Vinca alkaloids are a subset of drugs obtained from the periwinkle plant they are naturally extracted from the pink periwinkle plant (Catharanthus roseus), and they have been used to treat diabetes, high blood pressure and have been used as disinfectant. The vinca alkaloids are also important for being Cancer resistant there are four major vinca alkaloids in clinical use: Vinblastine (VBL), vinorelbine (VRL), vincristine (VCR) and vindesine (VDS). Vinca alkaloids are the second most used class of cancer drugs and will stay among the original cancer therapies. Catharanthus roseus is still source used for the powerful antitumor drugs. Callus culture had been done on MS-medium containing Different Concentrations from BA – 2,4-D – NAA – IAA. Where the different effects of growth regulators were studied when different parts of the plant have been cultured from leaves and stems this is to induce callus formation and encourage growth and the concentrations that were prepared: BA (0,1 – 0,3 – 1 – 2 mg/l) - NAA (0,1 – 1 mg/l) - IAA (0,1 mg/l) - 2,4-D (0,75 – 1 mg/l). And after culture the explants we transferred it to the growth room with a temperature ranging from 27 to 29 ° C relative humidity around 80% And in complete darkness. The results from HPLC analysis for the dry weight of callus were as follows ajmalicine (0.18 mg/g DW), catharanthine (0.07 mg/g DW), serpentine (0.37mg/g DW), vindoline (0.11 mg/g DW).

Submergence promotes auxin-induced callus formation through ethylene-mediated post-transcriptional control of auxin receptors

Plant cells in damaged tissue can be reprogrammed to acquire pluripotency and induce callus formation. However, in the aboveground organs of many species, somatic cells that are distal to the wound site become less sensitive to auxin-induced callus formation, suggesting the existence of repressive regulatory mechanisms that are largely unknown. Here we reveal that submergence-induced ethylene signals promote callus formation by releasing post-transcriptional silencing of auxin receptor transcripts in non-wounded regions. We determined that short-term submergence of intact seedlings induces auxin-mediated cell dedifferentiation across the entirety of Arabidopsis thaliana explants. The constitutive triple response 1-1 (ctr1-1) mutation induced callus formation in explants without submergence, suggesting that ethylene facilitates cell dedifferentiation. We show that ETHYLENE-INSENSITIVE 2 (EIN2) post-transcriptionally regulates the abundance of transcripts for auxin receptor genes by facilitating microRNA393 degradation. Submergence-induced calli in non-wounded regions were suitable for shoot regeneration, similar to those near the wound site. We also observed submergence-promoted callus formation in Chinese cabbage (Brassica rapa), indicating that this may be a conserved mechanism in other species. Our study identifies previously unknown regulatory mechanisms by which ethylene promotes cell dedifferentiation and provides a new approach for boosting callus induction efficiency in shoot explants.

Agrobacterium tumefaciens mediated genetic transformation of Tripterygium wilfordii and its application to enhance the accumulation of triptolide

Triptolide, an important representative active compound of Tripterygium wilfordii, is hopeful to be developed into modern drug. However, the content of triptolide in natural plants is very low, which seriously restricts its development in the pharmaceutical industry. As a perennial woody vine, research on the genetic transformation of T. wilfordii is challenging and slow, which limits the process of improving triptolide content through biotechnology. In this study, the concentrations and combinations of growth regulators for T. wilfordii callus induction and regeneration were firstly optimized. The 2,4-dichlorophenoxyacetic acid (1 mg/L) was efficient to induce callus formation from leaf explants, while 2.0 mg/L 6-benzylaminopurine with 0.1 mg/L α-naphthaleneacetic acid had the best effect on regeneration from callus. Subsequently, the effect of different concentrations of screening agent (kanamycin or hygromycin B) on callus induction rate was determined. Then, Agrobacterium tumefaciens mediated genetic transformation was established and validated by two different plant expression vectors, pCambia1301 and pJCV53, corresponding to β-glucuronidase histochemical assay and monomeric red fluorescent protein fluorescence detection, respectively. It took 3–4 months from A. tumefaciens infection to complete transgenic plant regeneration. Based on this transformation system of T. wilfordii, the overexpression of miltiradiene synthase gene (TwMS) was performed. Real-time quantitative PCR analysis indicated that the expression levels of TwMS were highly up regulated in the transgenic plants compared with control line. The detection of high-performance liquid chromatography showed that overexpression of TwMS resulted in a 1.20–1.51-fold increase of triptolide in the transgenic lines. These results indicated that this efficient A. tumefaciens mediated genetic transformation provides a useful tool for future T. wilfordii genetic engineering studies to enhance the accumulation of triptolide in this economically important medicinal plant.

Auxin-Induced WUSCHEL-RELATED HOMEOBOX13 Mediates Asymmetric Activity of Callus Formation upon Cutting.

Plants have the regenerative ability to reconnect cut organs, which is physiologically important to survive severe tissue damage. The ability to reconnect organs is utilized as grafting to combine two different individuals. Callus formation at the graft junction facilitates organ attachment and vascular reconnection. While it is well documented that local wounding signals provoke callus formation, how callus formation is differentially regulated at each cut end remains elusive. Here, we report that callus formation activity is asymmetrical between the top and bottom cut ends and is regulated by differential auxin accumulation. Gene expression analyses revealed that cellular auxin response is preferentially upregulated in the top part of the graft. Disruption of polar auxin transport inhibited callus formation from the top, while external application of auxin was sufficient to induce callus formation from the bottom, suggesting that asymmetric auxin accumulation is responsible for active callus formation from the top end. We further found that the expression of a key regulator of callus formation, WUSCHEL-RELATED HOMEOBOX 13 (WOX13), is induced by auxin. The ectopic callus formation from the bottom end, which is triggered by locally supplemented auxin, requires WOX13 function, demonstrating that WOX13 plays a pivotal role in auxin-dependent callus formation. The asymmetric WOX13 expression is observed both in grafted petioles and incised inflorescence stems, underscoring the generality of our findings. We propose that efficient organ reconnection is achieved by a combination of local wounding stimuli and disrupted long-distance signaling.

Effect of Growth Regulators and Nacl Stress on Callus Formation and Alkaloids Production on Vinca Rosea Plant (Catharanthus Roseus L)

Vinca alkaloids are a subset of drugs obtained from the periwinkle plant they are naturally extracted from the pink periwinkle plant (Catharanthus roseus), and they have been used to treat diabetes, high blood pressure and have been used as disinfectant. The vinca alkaloids are also important for being Cancer resistant there are four major vinca alkaloids in Clinical Use: Vinblastine (VBL), vinorelbine (VRL), vincristine (VCR) and vindesine (VDS). Vinca alkaloids are the second most used class of cancer drugs and will stay among the original cancer therapies. Catharanthus roseus is still source used for the powerful antitumor drugs. Callus culture had been done on MS-medium containing Different Concentrations from BA-2, 4-D-NAA-IAA. Where the different effects of growth regulators were studied when different parts of the plant have been cultured from leaves and stems this is to induce callus formation and encourage growth and the concentrations that were prepared: BA (0,1-0, 3-1-2 mg/l)-NAA (0,1-1mg/l)-IAA (0,1 mg/l)-2, 4-D (0,75-1mg/l). And after culture the explants we transferred it to the growth room with a temperature ranging from 27 to 29°C relative humidity around 80% And in complete darkness then we applied the resulting callus tissue under the influence of different concentrations of NaCl as (50-75-100-150mM/L). The results from HPLC analysis for the dry weight of callus were as follows ajmalicine (0.18 mg/g DW), catharanthine (0.07 mg/g DW), serpentine (0.37mg/g DW), vindoline (0.11 mg/g DW).

In vitro culture of Zingeria biebersteiniana (Claus) P. Smirn. as a method for conservation and restoration of genetic diversity

Aim. The aim of the work was to determine the conditions for development of in vitro culture, induction of callus formation, and long-term tissue culture of Zingeria biebersteiniana (Claus) P. Smirn. Methods. In vitro clonal propagation, tissue culture techniques. Results. The seed germination rate was found to increase significantly after long-term cold stratification. The protocol for seed sterilization was developed, which yielded 57.3% of aseptic plants. Collections of in vitro and pot cultured plants were created. Experiments on the adaptation of in vitro propagated plants to pot culture conditions revealed a high level of their survival. The optimal medium for in vitro clonal propagation was MS, supplemented with 0.1 mg/L NAA; while the most effective media for induction of callus formation and for long-term tissue culture was B5 supplemented with 2 mg/L 2,4-D and 0.1 mg/L BAP. Conclusions. The protocols and conditions for seed germination, in vitro clonal propagation, induction of callus formation, as well as long-term tissue culture of Z. biebersteiniana have been developed. The developed techniques of in vitro culture can be used for conservation and restoration of genetic diversity of the species, as well as to obtain sufficient plant material for further studies.

Anti-Oxidant and Anti-Aging Activities of Callus Culture from Three Rice Varieties

The aims of this study were to induce calli from the seeds of three rice varieties (Hommali 105, Munpu, and Niawdum) and investigate their anti-aging potential. First, rice seeds were cultured on a Murashige and Skoog medium (MS medium) supplemented with 2 mg/L of 2,4-Dichlorophenoxyacetic acid (2,4-D), 1 mg/L of 1-Naphthalene acetic acid (NAA), and 1 mg/L of 6-Benzylaminopurine (BAP). After three weeks, the calli were extracted with ethanol. Then, their phenolic contents were determined by spectrophotometer and the amino acids were identified by ultra-performance liquid chromatography (UPLC). Their cytotoxicity, anti-oxidant (potassium ferricyanide reducing power assay (PFRAP), DPPH radical scavenging assay (DPPH), lipid peroxidation inhibition (LPO), and superoxide dismutase activity (SOD)), and anti-aging (keratinocyte proliferation, anti-collagenase, anti-inflammation, and anti-tyrosinase) activities were also investigated. Munpu callus (385%) was obtained with a higher yield than Hommali (322%) and Niawdum (297%) calli. The results revealed that the phenolic and amino acid contents were enhanced in the calli. Moreover, the calli were rich in glutamic acid, alanine, and gamma aminobutyric acid (GABA). The callus extracts showed no cytotoxic effects at a concentration of equal to or lower than 0.25 mg/mL. The highest anti-oxidant activities (PFRAP (0.81 mg AAE/mL), DPPH (68.22%), LPO (52.21%), and SOD (67.16%)) was found in Munpu callus extract. This extract also had the highest keratinocyte proliferation (43.32%), anti-collagenase (53.83%), anti-inflammation (85.40%), and anti-tyrosinase (64.77%) activities. The experimental results suggest that the amounts of bioactive compounds and anti-aging activities of rice seeds can be enhanced by the induction of callus formation.