Internode Explants Research Articles

Effects of different growth hormones on Solanum tuberosum L. callus induction and regeneration

This study was carried out to develop a protocol for callus induction, regeneration, shoot, and root induction in two potato cultivars (Riviera and Almonda) that can be further used to develop genetically modified plants. Callus was induced from leaf and internodal explants on Murashige and Skoog's medium (MS) supplemented with different combinations of hormones. The highest percentage of callus induction was obtained from the internodal explants of Riviera cultivar was 100% in Callus Induction Media I (CIM I) supplemented with 2 mg/L Benzyl Adenine (BA), 0.2 mg/L ?-Naphthalene Acetic Acid (NAA), 2 mg/L 2,4-Dichlorophenoxy acetic acid (2,4-D), 0.2 mg/L Kinetin (Kin) and 0.2 mg/L gibberellic acid (GA3). After one-month calli were shifted to Shoot Induction Media I and II (SIM I and SIM II), the highest percentage for shoot induction was 33.3% with the leaf explants of Riviera cultivar on SIM II supplemented with 2 mg/L BA, 0.2 mg/L NAA, 0.5 mg/L Kin and 0.2 mg/L GA3. The root induction percentage was 100% for both cultivars. Regenerated potato plants had normal leaf shapes with uniform morphology. These findings provide valuable insights for developing tissue culture protocols for potato regeneration, which are essential for genetic engineering and potato breeding programs.

Temperature and LED lightning affect the regeneration of somatic embryo-derived sporophytes on the internode explants of the tree fern Cyathea delgadii Sternb.

The influence of the temperature and different light qualities emitted by light-emitting diodes (LEDs) and fluorescent lamps (Fl) on the micropropagation of the tree fern Cyathea delgadii was evaluated. The most efficient somatic embryo production was obtained on internode explants at 22 °C. The optimal temperature range for obtaining well-developed plants was 24–28 °C. This stimulated the elongation and development of the first leaf and the formation of the next leaf and roots primordia. Temperatures lower than 24 °C and higher than 28 °C inhibited the formation of young sporophyte organs and delayed their development. The RBUV (35% red, 15% blue, and 50% UV) and B (100% blue) lights and Fl light were beneficial for the sporophyte production on internode explants. However, plants obtained under RBUV light were undeveloped. The white LED light stimulated the number of explants capable of gametophyte production and development. The RB light (70% red and 30% blue) enhanced the number of roots of newly-formed plants. Most of the LED lights tested had a good impact on root elongation compared to Fl light and constant darkness. The R light (100% red) benefits leaf development and elongation. Research shows that temperature and LED lightning play a significant role in the process of morphogenesis in C. delgadii, significantly affecting the embryogenic competence of somatic cells and the development of sporophytes.

Optimization of factors affecting Agrobacterium-mediated hairy root induction in Vitex negundo L. (Lamiaceae)

Vitex negundo L. is an aromatic, woody, blooming shrub in the Verbenaceae family which can grow into a small tree. Traditionally, V. negundo root has been used to cure diabetes, colic, boils, leprosy, and rheumatism. Keeping the importance of its roots in mind, an attempt has been taken for development of a protocol for efficient hairy root proliferation system. The Agrobacterium rhizogenes strains (MTCC 532 and MTCC 2364) were used for hairy root induction. For A. rhizogenes infection, both in vitro and in vivo leaves as well as internodes were used as explant. In vitro leaves and internodal explants were obtained by the inoculation of matured nodal segments on the optimum medium [MS + 2.0 mg/L N6-Benzylaminopurine (BAP)] with c.a. 91.6% shoot regeneration and an average of 8.1 shoots per explants. In vitro leaf showed best hairy root induction followed by in vitro internode on ½ MS medium augmented with acetosyringone. Highest transformation efficiency was achieved using MTCC 2364 strain, while no transformation was observed in MTCC 532 strain. Different factors affecting transformation including co-cultivation period, infection time and optical density (O.D.) value were standardized. The highest efficacy, 88.8% hairy root induction was observed in in vitro leaves infected by MTCC 2364 for 60 minutes infection time with an O.D. value of 0.29 maintained over a 44-48 hours of co-cultivation period. The prescribed protocol may be used as a reference for development of industrial scale hairy root production for bioactive compound located in root of V. negundo.

Microtuber Induction and Plant Regeneration of Potato (Solanum tuberosum L.): A Review

Potato (Solanum tuberosum L.) is one of the most economically significant annual vegetable crops occupying the fourth position worldwide among all the food crops. The main drawbacks of the traditional seed production method are the seed tubers' high size, which drives up seed production costs, the ease with which viruses and other diseases can damage them, the slow rate of seed multiplication, and the lengthier time it takes to obtain breeder seed. There are various benefits to the invention and use of microtubers in potato farming. It is not necessary to acclimatize or move to a different medium when growing microtubers because they can be kept for up to a year and then transported straight to the market. By doing this, the field cycle needed to produce a sufficient amount of seed potatoes is greatly reduced, and a high quality of materials is guaranteed. Shoot tip and node or internodal explants, which have a survival probability of about 90%, are frequently used in the process of in vitro plant regeneration. Positive outcomes in stimulating shoot formation have been observed when Murashige and Skoog media enriched with benzyl amino purine (BAP) and kinetin is used. Furthermore, employing the MS medium has been reported to maximize the effectiveness of microtuber induction in potatoes. It's been found that exposing the plants to a 16-hour photoperiod and complete darkness can improve the effectiveness of microtuber induction even further. Ultimately, this review study offers an appropriate method for a successful pathway in the in vitro regeneration of potatoes by carefully examining prior scientific data.

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.

In Vitro Proliferation of Petunia Hybrida Vilm; Worldwide Important Ornamental Plant

This project tries to artificially grow one of the most ornamentally significant Petunia hybrida when it is out of season. Murashige and Skoog Basal Medium (MS Basal Medium) was used for in vitro growth of P. hybrida, supplemented with different Plant Growth Regulators (PGRs). Various physical and chemical factors on in vitro growth of P. hybrida using different explants were analyzed. The type of auxin that proved significant for somatic embryogenesis of P. hybrida Vilm was 2,4-Dichlorophenoxyacetic acid (2.0 mg/L) using the leaf as explant under controlled environmental conditions, i.e., 92% at 23oC with a 5.8 pH and 16 hours photoperiod & 30g/l sucrose in. Friable calli were also developed from leaf, node, and internodal explants on a tissue culture medium (MS basal medium) when supplemented with other PGRs in certain combinations i.e. 2,4-Dichlorophenoxyacetic acid (2.0 mg/L), Benzyl Amino Purine (2.5mg/L), Benzyl Amino Purine + 2,4-Dichlorophenoxyacetic acid (2mg/L +2.5mg/L), Naphthalene Acetic Acid (1.5mg/L), 2,4-Dichlorophenoxyacetic acid + Naphthalene Acetic Acid (2mg/L+1.5mg/L) and Benzyl Amino Purine + Naphthalene Acetic Acid (2.5mg/L+0.5mg/L) at optimal physical factors under strict aseptic environment. These results may enable one to produce the required type of P. hybrida Vilm during off and on seasons on a commercial scale by using plant tissue culture as a tool.

Indirect Organogenesis from in vitro Derived Leaf and Internodes of Coccinia cordifolia (L.) Cogn. - An Important Medicinal Climber

An efficient in vitro regeneration protocol was developed via callus from the explants of leaf and internode of Coccinia cordifolia (L.) Cogn., an important medicinal climber. Among the different combinations of auxins and cytokines used, BAP with NAA were found highly effective for induction of callus from in vitro derived leaf disc and internode explants. Maximum callus formation rates were recorded on MS with 1.0 mg/l BAP and 0.1 mg/l NAA for both leaf disc and internode explants. The best organogenesis via callus was obtained on MS with 2.0 mg/l BAP and 0.5 mg/l NAA. The highest numbers of shoots regenerated per culture were 6.5 ± 0.10 and 5.6 ± 0.55 for leaf disc and internode explants respectively and leaf discs was found to be better for indirect organogenesis. For root induction, half strength of MS medium supplemented with 0.1 mg/l IBA was found to be the most effective. The mean number of roots regenerated per shoot was 6.8 ± 0.10 with an average root length of 2.8 ± 0.20 cm. In vitro derived plantlets were acclimatized successfully. This protocol for in vitro regeneration can be utilized for the improvement of this climber through biotechnological methods. Plant Tissue Cult. & Biotech. 32(2): 127-136, 2022 (December)

Regeneration Efficiency Of Five High Yielding Potato Varieties Of Bangladesh

Experiments were conducted to develop an efficient and reliable protocol for in vitro direct regeneration of potato for future transformation. Five varieties of potato viz. BARI Alu-7 (Diamant), BARI Alu-21 (Provento), BARI Alu-24 (Dura), BARI Alu-25 (Asterix) and BARI Alu-26 (Felsina); 3 levels of Zeatin riboside viz. 3, 4 and 5 mg/l ; 2 types of explants viz. internode and leaf from 19-22 days old micro plants were used in this study. Internode explants of BARI Alu-25 produced the highest number of shoots (40.17) within16 days without callus phase in MS medium supplemented with 5 mg/l ZR + 0.2 mg/l GA3 + 0.01 mg/l IAA followed by BARI Alu-21 (38.13), BARI Alu-7 (37.49), respectively. Leaf explants also produced shoots directly without callus phase in the same medium. In this case, the highest number (7.87) of shoots was found from the variety BARI Alu-25 followed by BARI Alu-21(7.42). ½MS medium supplemented with 0.5 – 1.0mg/l IBA resulted the best performance as to the rooting from ZR treated regenerated shoots having 100% plant survival at ex vitro conditions. This protocol may be used for rapid shoot proliferation and genetic transformation in potato. Bangladesh J. Bot. 51(4): 637-645, 2022 (December)

Enhancement of shoot organogenesis in Polygonum tinctorium by sucrose and gelling agents

Indigo (Polygonum tinctorium), though it is a medicinal and dye crop, can also be grown in temperate areas, especially because it is commonly disseminated in Japan. To date, much research has been considered under investigation, especially for the regeneration of the indigo plant. Here in this study, we investigated the response of sucroses and gelling agents on the shoot organogenesis of the indigo plant. Micropropagation in terms of shoot regeneration and its growth was highly responded to sucrose and gelling agents. While culturing of internode explants on initial shoot regeneration media supplemented with sucrose and gelling agents of phytagar and gelrite significantly upgraded the regeneration efficiency as well as shoot growth. The regeneration capacity of the shoot was augmented with increased levels of sucrose up to 40 g L-1 and then started to decrease, whereas the increasing pattern continued even at the highest concentration (50 g L-1). The highest shoot regeneration (6.0 ± 0.5) was achieved by the treatment of 40 g L-1 giving 7.5 times higher shoot regeneration compared to the control. The increasing pattern for shoot length was more pronounced than that of shoot regeneration. The shoot length ranged from 10.2 mm to 23.5 mm within the sucrose treatments. In this study, the highest shoot length (23.5± 0.21) was observed by the treatment of 50 g L-1 exhibiting 2.3 times higher shoot length compared to the control. Gelling agent gelrite performed better than phytagar for both regeneration and shoot length growth. The shoot regeneration among the phytagar treatments ranged from 3.8 to 6.0 shoots/explant whereas the shoot regeneration ranged from 6.6 to 7.2 among the gelrite treatments. The highest shoots/explant (7.2) and the longest shoot length (22.4 mm) were observed due to the treatment of gelrite 3. The lowest shoot regeneration and shoot length were denoted when phytagar 9 was applied. It is proposed from our study that sucrose and gelling agent especially gelrite 3 could be applied in shoot organogenesis and plant transformation of any plant species, especially for P. tinctorium.

In vitro Callus Induction of Rauvolfia tetraphylla (L.) an Endangered Medicinal Plant and Evaluation of Biological Activities

Rauvolfiatetraphylla (L.) is a critically endangered woody shrub that is one among the most significantApocynaceae family species cultivated for its therapeutic benefits. The goal of this work is to standardizethe micropropagation methodology for Rauvolfiatetraphylla (L.). Explants wereused to make the callus(Leaf, Internode, and Anther). “When leaf explants were grown on Murashige and Skoog (MS) mediasupplemented with Indole-3-Acetic Acid (IAA) (0.5mg/ml), a maximum of 94.2 percent of callus initiationwas observed”.”On MS media with 2,4-dichloro phenoxy acetic acid (2,4-D) + Indole-3-Acetic Acid (IAA)(0.5+0.5mg/ml), internodal explants demonstrated a maximum of callus initiation of77.9%”.”On MS mediawith 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (2,4-D) (0.5 mg/ml) alone,the greatestcallus initiation (89.8%) was found in anther explants”. The qualitative preliminary phytochemical analysiswas carried out from leaf and fruit extracts. Extracts were obtained from various organic solvents by thesequential method. Alkaloids, carbohydrates, fixed oils and fats, phenolic compounds, and tannins wereidentified from various crude extracts such as Hexane, Chloroform, Ethyl acetate, and Methanol. Inantibacterial activity, the maximum inhibition was observed in methanol extract of leaf and fruit againstEscherichia coli (100 g/ml).

A stable method for Agrobacterium-mediated transformation of Mentha piperita and Mentha canadensis using internodal explants

A stable method for Agrobacterium-mediated transformation of Mentha piperita and Mentha canadensis using internodal explants

Effect of silver nitrate and putrescine on in vitro shoot organogenesis of Polygonum multiflorum

Polygonum multiflorum is a flowering plant that belongs to the family Polygonaceae and it is commonly used for medicinal and ornamental purposes. Few studies have been studied about the regeneration of this species. Therefore, we aimed to develop a suitable protocol for regeneration and subsequent growth of shoots by comparing the silver nitrate (AgNO3) (ethylene inhibitor) and the putrescine (polyamine). Internode explants were cultured on shoot regeneration media (Murashige and Skoog (MS) media containing 2 mg L-1 of 6-benzylaminopurine). To analysis, the effect of AgNO3 and putrescine on shoot regeneration and length, different concentrations (mg L-1) of AgNO3 (0, 1, 5, 7, 10, and 20) and putrescine (0, 10, 30, 50, 100, and 200) were added to the MS media. The result showed that at highest concentration (20 mg L-1) of AgNO3 treatment decrease number of shoots (NOS) (1.4 ± 0.2 mm) and shoot length (9.7 ± 1.6 mm) was observed. Putrescine considerably increased the regeneration efficiency, NOS per explant, and shoot length in all the concentrations when compared to AgNO3 treatment. Among the different concentrations, the highest NOS (2.52 ± 0.2 mm) was obtained in cultures supplemented with 30 mg L-1 putrescine, whereas the further increase in putrescine concentration reduced shoot regeneration. The longest shoots (20.5 ± 1.7 mm) were achieved in cultures supplemented with 200 mg L-1 putrescine. The findings of this study indicate that the addition of putrescine to the media could be suitable for P. multiflorum micropropagation and plant transformation.

In vitro production of tropane alkaloids from Brugmansia suaveolens

For thousands of years, secondary metabolites have been utilized as medications, flavors, pesticides, and dyes. For the generation of valuable secondary metabolites, in vitro plant culture techniques have been viewed as beneficial alternatives to whole plants. Brugmansia suaveolens is an ornamental plant including anticholinergic agents which are employed in medicine. Callus induction was performed from leaf and internode explants cultured on Murashige and Skoog’s medium supplemented with different concentrations and combinations of plant growth regulators (PGRs) with 6 treatments. The highest callus induction response was obtained from the leaf-originated explants (73%) on the medium supplemented with 0.4 mg/L KIN and 0.2 mg/L NAA which produced friable callus in 4 weeks. The cell suspension culture of B. suaveolens was established in shake flasks using friable calli. The extraction protocol of tropane alkaloids was optimized, atropine and scopolamine were obtained efficiently. The data could provide technical support for the large-scale production of valuable alkaloids of B. suaveolens in vitro systems with improved strategies.

Biotechnology for propagation and secondary metabolite production in Bacopa monnieri.

Bacopa monnieri (L.) Wettst. or water hyssop commonly known as "Brahmi" is a small, creeping, succulent herb from the Plantaginaceae family. It is popularly employed in Ayurvedic medicine as a nerve tonic to improve memory and cognition. Of late, this plant has been reported extensively for its pharmacologically active phyto-constituents. The main phytochemicals are brahmine, alkaloids, herpestine, and saponins. The saponins include bacoside A, bacoside B, and betulic acid. Investigation into the pharmacological effect of this plant has thrived lately, encouraging its neuroprotective and memory supporting capacity among others. Besides, it possesses many other therapeutic activities like antimicrobial, antioxidant, anti-inflammatory, gastroprotective properties, etc. Because of its multipurpose therapeutic potential, it is overexploited owing to the prioritization of natural remedies over conventional ones, which compels us to conserve them. B. monnieri is confronting the danger of extinction from its natural habitat as it is a major cultivated medico-botanical and seed propagation is restricted due to less seed availability and viability. The ever-increasing demand for the plant can be dealt with mass propagation through plant tissue culture strategy. Micropropagation utilizing axillary meristems as well as de novo organogenesis have been widely investigated in this plant which has also been explored for its conservation and production of different types of secondary metabolites. Diverse in vitro methods such as organogenesis, cell suspension, and callus cultures have been accounted for with the aim of production and/or enhancement of bacosides. Direct shoot-organogenesis was initiated in excised leaf and internodal explants without any exogenous plant growth regulator(s) (PGRs), and the induction rate was improved when exogenous cytokinins and other supplements were used. Moreover, biotechnological toolkits like Agrobacterium-mediated transformation and the use of mutagens have been reported. Besides, the molecular marker-based studies demonstrated the clonal fidelity among the natural and in vitro generated plantlets also elucidating the inherent diversity among the natural populations. Agrobacterium-mediated transformation system was mostly employed to optimize bacoside biosynthesis and heterologous expression of other genes. The present review aims at depicting the recent research outcomes of in vitro studies performed on B. monnieri which include root and shoot organogenesis, callus induction, somatic embryogenesis, production of secondary metabolites by in vitro propagation, acclimatization of the in vitro raised plantlets, genetic transformation, and molecular marker-based studies of clonal fidelity. KEY POINTS: • Critical and up to date records on in vitro propagation of Bacopa monnieri • In vitro propagation and elicitation of secondary metabolites from B. monnieri • Molecular markers and transgenic studies in B. monnieri.

High-efficiency <i>Agrobacterium</i>-mediated transformation of chrysanthemum via vacuum infiltration of internode

Leaf disc transformation is one of the traditional methods that are now widely used in chrysanthemum with highly economical and ornamental value in world flower production, but it depends on plant genotypes and is time consuming and complicated. In addition, the transformation success rate of this method is low, generally ranging from 0.1% to 6.25%. Therefore, a highly efficient transformation system is needed. In this study, we are the first to establish a high-efficient chrysanthemum <i>Agrobacterium</i>-mediated transformation system via vacuum infiltration. Chrysanthemum stem internode explants were used as research material and <i>CmLEC1</i> was used as a reporter gene. After approximately 3 months of culture and selection, the positive transgenic plants were obtained. Additionally, the positive probability was about 42%. The transformation efficiency was up to 37.7%, and if the escapes were removed, it was 16%. Furthermore, stable expression of <i>CmLEC1</i> in transgenic 'Yuhualuoying' was confirmed by qRT-PCR analysis. These results suggest that this genetic transformation system via vacuum infiltration of chrysanthemum stem internode is highly efficient and convenient, and much better than traditional leaf disc transformation, and it will play an important role in chrysanthemum transformation and functional genetics research.

Transcriptomic Changes in Internode Explants of Stinging Nettle during Callogenesis.

Callogenesis, the process during which explants derived from differentiated plant tissues are subjected to a trans-differentiation step characterized by the proliferation of a mass of cells, is fundamental to indirect organogenesis and the establishment of cell suspension cultures. Therefore, understanding how callogenesis takes place is helpful to plant tissue culture, as well as to plant biotechnology and bioprocess engineering. The common herbaceous plant stinging nettle (Urtica dioica L.) is a species producing cellulosic fibres (the bast fibres) and a whole array of phytochemicals for pharmacological, nutraceutical and cosmeceutical use. Thus, it is of interest as a potential multi-purpose plant. In this study, callogenesis in internode explants of a nettle fibre clone (clone 13) was studied using RNA-Seq to understand which gene ontologies predominate at different time points. Callogenesis was induced with the plant growth regulators α-napthaleneacetic acid (NAA) and 6-benzyl aminopurine (BAP) after having determined their optimal concentrations. The process was studied over a period of 34 days, a time point at which a well-visible callus mass developed on the explants. The bioinformatic analysis of the transcriptomic dataset revealed specific gene ontologies characterizing each of the four time points investigated (0, 1, 10 and 34 days). The results show that, while the advanced stage of callogenesis is characterized by the iron deficiency response triggered by the high levels of reactive oxygen species accumulated by the proliferating cell mass, the intermediate and early phases are dominated by ontologies related to the immune response and cell wall loosening, respectively.

Callus induction and chemical characterization of cell suspension cultures of jojoba (Simmondsia chinensis L.)

Jojoba (Simmondsia chinensis L.) oil is also known as liquid wax or fixed oil. It is an important metabolite of jojoba having commercial importance in cosmetics as well as a potential biofuel source. We presented an efficient system for in vitro establishment of cell suspension cultures (CSC) from proliferating friable calluses. For this purpose, cotyledon, internode, and leaf explants were cultured on MS medium + 1, 2, 4, 6, 8 or 10 µM 2, 4-Dichlorophenoxyacetic acid (2, 4-D), α-Naphthalene acetic acid (NAA) alone or in combination with 1 or 2 µM N6-benzylaminopurine (BAP) or Kinetin. Results demonstrated that 100% healthy, friable and variegated calluses were obtained on 8 µM, 10 µM 2, 4-D or 2, 4-D 10 µM + 2 µM BAP and represented as callus lines (CL) CL-1, CL-2 or CL-3, respectively, after 38 days. One-gram callus tissue per CL was then immersed in the respective liquid medium and agitated on an orbital shaker at 60-70 rpm under the growth room conditions (25 ± 2 °C, 16 h light period) for the preparation of CSC. After 15 days, CSC was sieved and large clumps were removed. Growth measurement of CSC was determined by cell counting, packed cell volume (PCV) and cell viability. The highest number of viable cells was obtained at 2.57 OD with CL-3, where PCV was highest (0.35 ml) on CL-1 of 38 days old calluses. 2,3,5-Triphenyltetrazolium chloride was a reliable approach for the determination of cell viability of CSC.

Influence of plant growth regulators and UV light exposure on the formation and phenolic content of stevia rebaudiana bertoni callus: a preliminary study

This study was conducted to evaluate the effect of different combinations and levels of plant growth regulators, 2,4-dichlorophenoxy acetic acid (2,4-D) and 6-benzylaminopurine (BAP), on callus induction of Stevia rebaudiana Bertoni leaf and internode explants, and the total phenolic content of Stevia callus as influenced by the length of exposure to ultraviolet (UV) light radiation. Early callus initiation was recorded in leaf explants inoculated in Murashige and Skoog (MS) medium supplemented with 2,4-D and BAP. After a week of incubation, leaf explants showed callus formation while in internode explants, callus formation was observed 2 weeks after inoculation. The culture medium supplemented with 1.5mg L-1 2,4-D + 2.0mg L-1 BAP (T4) was the best treatment for leaf explants with 95% callus formation and the addition of 1.0mg L-1 2,4-D + 4.0mg L-1 BAP (T3) was the best treatment for internode explant with 90% callus formation. After 4 weeks of incubation, the leaf and internode calli were observed as compact, non-embryogenic, and yellowish green in all treatments except those inoculated in MS medium alone (control). Stevia leaf callus was subjected to UV radiation after 4 weeks at varying time of exposure. The exposed calli turned brown signifying a possible increasing production of secondary metabolites. Furthermore, combination of 1.5mg L-1 2,4-D + 2.0mg L-1 BAP (T4) and 3h of UV light exposure yielded the highest phenolic content of 87.71mg GAE per g callus. The results further revealed that both medium composition and time of exposure affect the production of phenolic content of Stevia leaf callus.

Micropropagation and Secondary Metabolites Content of White-Purple Varieties of Orthosiphon aristatus Blume Miq.

&lt;b&gt;Background and Objective:&lt;/b&gt; The cat whiskers plant (&lt;i&gt;Orthosiphon aristatus&lt;/i&gt; Blume Miq) is a plant that has been widely used as raw material for traditional medicine. The population of white-purple varieties of &lt;i&gt;O. aristatus&lt;/i&gt; is decreasing efforts to maintain the white-purple &lt;i&gt;O. aristatus&lt;/i&gt; need to be done keeping in mind its potential as raw material for traditional medicine. This study aims to determine the composition of a suitable medium in growing plantlet &lt;i&gt;O. aristatus&lt;/i&gt; white-purple varieties and the content of its secondary metabolites. &lt;b&gt;Materials and Methods:&lt;/b&gt; The internode explants were induced on MS medium added by various combinations of zeatin and 2,4-Dichlorophenoxyacetic acid (2,4-D). Root induction was carried out on shoots formed on MS medium with Indole-3-Butyric Acid (IBA). The acclimatization process was carried out using soil media. Determination of secondary metabolite levels was carried out on &lt;i&gt;O. aristatus&lt;/i&gt; (&lt;i&gt;in vitro&lt;/i&gt; culture) and wild-type plants aged ten months using high-performance liquid chromatography (HPLC). &lt;b&gt;Results:&lt;/b&gt; MS+BAP 2ppm+NAA3 ppm media was the optimal medium for growing shoots in leaf explants. Media MS+zeatin 3 ppm+2,4-D 2 ppm produced good shoot growth on internode explants. The best root induction occurred in MS+IBA media of 0.75 ppm. The acclimatization process was successful on shoots originating from the internode, while those from leaf explants had not succeeded in growing and developing. &lt;b&gt;Conclusion:&lt;/b&gt; The levels of rosmarinic acid and sinensetin in the white-purple variety &lt;i&gt;O. aristatus&lt;/i&gt; (&lt;i&gt;in vitro&lt;/i&gt; culture) were 1.08 and 1.62% w/w and higher than those of wild varieties.

Optimization of Modified Murashige and Skoog (MS) Medium Promoting Efficient Callus Induction in Zinnia elegans

Zinnia (Zinnia elegans Jacq.) occupies a pristine place in the garden for its bright colours and long flowering period. Zinnias are propagated by seeds hence genetic variation and a tendency for stock plants to decline in vigor over time is high. Young internodal explants of this species were grown on modified Murashige and Skoog (MS) medium supplemented with various growth hormones to evaluate callus induction. 2,4 dichlorophenoxyacetic acid (2,4-D) at 1.0 mg/L was shown to be the best choice for a high percentage of callus induction (70-73%) and a rapid growth rate of viable and healthy callus. A large-scale and quick callus generation protocol was developed in the lab and might be used for in-vitro micropropagation.