MS Medium Research Articles

CALLUS INDUCTION FROM THE FRONDS OF GREATER DUCKWEED (<i>Spirodela polyrhiza </i>(L.) Schleid) AND ASSESSMENT OF ITS ANTIMICROBIAL EFFICACY

Greater duckweed (Spirodela polyrrhiza (L.) Schleid. are known to propagate excessively in stagnant eutrophic water bodies resulting in the contamination of daughter fronds which can affect their secondary metabolite profile. The present study was aimed in establish organogenic callus from the fronds of greater duckweeds; to optimize a protocol by utilizing different plant growth regulators for future use and to screen the antimicrobially potent metabolites present in callus. The induction of callus was successful using sterile fronds of Spirodela polyrrhiza (L.) Schleid as explants in MS, B5 and SH media fortified with plant growth regulators namely IBA and BAP. SH basal medium fortified with IBA and BA in 2:1 ratio was most effective in callus induction. B5 medium fortified with 2.0 µM IBA + 4.0 µM BAP showed highest callus induction in S. polyrrhiza with lower days to callus induction as reflective in their overall biomass. The callus extract showed efficient antimicrobial effect on Pseudomonas aeroginosa (MTCC accession No. 3451) with ampicillin and chloramphenicol as reference antibiotics. Presence of flavonoids like orientin, genistin and apigetrin were indicated in the LC-MS data which might be responsible for antimicrobial activity in a synergistic manner.

Effects of plant hormones and genotypes on anther culture response of safflower (Carthamus tinctorius L.)

Safflower (Carthamus tinctorius L.) belongs to the composite family with various uses, from the stem to the seeds. In vitro flowering and embryo rescue are common techniques for improving safflower hybrid fertility by overcoming breeding constraints. However, anther culture expedites the production of superior types. The aim of the study was to evaluate the effect of plant hormones and genotypes on anther culture responses and to develop a suitable protocol. In both safflower genotypes, callus, shoot, and root formation were investigated using ten factorial hormone treatments. To promote callus induction and shoot regeneration, variable doses of Thidiazuron (TDZ) at 1 mg/l indole butyric acid (IBA) and variable benzylaminopurine (BAP) at 0.5 mg/l NAA were added to the MS medium, respectively. ½ MS media with varying IBA concentrations at a fixed 0.5 mg/l NAA were used for root regeneration. Results showed that Turkan was superior in all aspects of callus induction, and the highest degree of callus formation (46 %) was observed at 0.5 mg/l TDZ. On the other hand, the local safflower performed better for shoot and root regeneration. The highest shoot regeneration capacity (20 %) was shown at 2.0 mg/l BAP, while shoot rooting was reached (21 %) at 1.0 mg/l IBA of culture media. Despite successful in vitro regeneration, acclimated plantlets did not survive in the glasshouse. In this study, we demonstrated the higher capability for both shoot and root regeneration of calli produced from local safflower anthers. More effort is required to improve shoot and root development for both genotypes.

Efficient and rapid in vitro micropropagation of MD2 and H4 pineapple varieties

Pineapple plays an important role in international trade and supports of more than a million households in Côte d’Ivoire. However, its yield is subject to pedoclimatic problems and erosion of genetic potential of plant material. This study aims to establish an effective in vitro yield protocol for MD2 and H4 pineapple varieties. Therefore, the effects of 6-benzylaminopurine (BAP) combined with different concentrations of 2-isopentenyladenine (2-iP), with variable light intensity and activated charcoal on leaf shoot proliferation and elongation were tested. Analysis of variance revealed that Murashige and Skoog medium (MSB5) supplemented with 2 mg/L BAP combined with 0.25 mg/L 2-iP recorded the highest average number of microshoots (13.50 microshoots) in MD2 variety, while 2 mg/L of BAP combined with 0.5 mg/L of 2-iP recorded the best average number of shoots (15.20 microshoots) in H4 variety. Maximum microshoot elongation (7.81 cm and 7.03 cm for MD2 and H4, respectively) was obtained with 2 mg/L BAP combined with 0.25 mg/L of 2-iP. Light intensity of 1500 lux produced 14.06 and 14.86 microshoots in MD2 and H4, respectively. In contrast, maximum microshoot elongation (4.40 cm and 4.13 cm in MD2 and H4, respectively) was recorded at 2000 lux light intensity. A 2 g/L of activated charcoal inhibited microshoot proliferation. However, it induced an elongation of 7.50 cm in MD2 and 7.07 cm in H4. Microshoots were rooted on MS medium supplemented with 100 mg/L myo-inositol, 30 g/L sucrose, 200 mg/L glutamine, 1 mg/L naphthalene acetic acid (NAA), 2 mg/L indole-3-butyric acid (IBA), 2 g/L activated charcoal, all solidified with 6g/L agar. In conclusion, the combination of 2 mg/L BAP and 2-iP (0.25 - 0.5 mg/L) is recommended for better in vitro shoot proliferation in MD2 and H4 varieties. Light intensities of 1500 - 2000 lux are ideal for better shoot proliferation and elongation. Finally, the addition of 2 g/L activated charcoal to the medium improves shoot elongation and rooting in MD2 and H4 varieties.

Cytotoxic activity of callus extract from Vachellia farnesiana (L) Wight & Arn.

The in vitro cultures of Vachellia farnesiana (L) Wight & Arn. have demonstrated cytotoxic activity through callus extract on the HeLa cell line. Explants excised from in vitro-grown seedlings from seeds of two different locations were inoculated on Murashige and Skoog (MS) culture media containing various concentrations of N-6 benzyladenine (BA) or kinetin with 2,4-dichlorophenoxyacetic acid (2,4-D). Optimal efficiency in friable callus induction (100%) was achieved in leaf explants cultured on MS media containing 2.32µM BA + 13.57µM 2,4-D. Plant tissues (callus and leaf) were extracted and subjected to quantitative phytochemical analysis, revealing the highest total alkaloid and phenolic content in leaf extracts from Queretaro adult specimens (339.5 ± 20.9mg atropine equivalents (AE) per g dry extract (DE) and 158.4 ± 12.5mg gallic acid equivalents (GAE) per g DE, respectively). In contrast, callus cultures exhibited significantly higher total triterpene content (356-381mg ursolic acid equivalents (UAE) per g DE) compared to leaf extracts (208-243mg UAE/g DE). Both leaf and callus extracts displayed cytotoxic activity against the HeLa cell line, with a significantly lower half-maximal inhibitory concentration (IC50) for leaf extracts (28-32µg/mL) compared to callus cultures (43-66µg/mL), suggesting that alkaloids were primarily responsible for the cytotoxic activity. Furthermore, this study provides valuable insights into the controlled production of bioactive compounds with cytotoxic activity, with callus serving as a rich source.

Effect of plant growth regulators in inducing Vitisvinifera Callus and increased production of flavonoids in vitro

This study was aimed to use of plant tissue culture technology to induce callus from grapevine Vitisvinifera and stimulate it to increase the production of flavonoids. The study was carried out in two stages after the sterilization process was carried out: The first included establishing callus farms by cultivating cuttings containing a single node after completing their sterilization and cultivating them on MS medium containing 2,4-D and BA at different concentrations (0, 1, 2, 3,4) mg L -1and BA in concentrations (0, 0.1, 0.2, 0.4) in independent experiments. The second phase included studying the effect of growth regulators on the concentrations of carbohydrates and flavonoids in the callus induced from the first experiment. The results of the study showed the superiority of auxien 2,4-D at a concentration of 2mg L -1as it achieved the highest average fresh and dry weight of callus, reaching (3.32 and 1.03) mg respectively, while BA at a concentration of 0.2 mg L -1 achieved the highest average fresh and dry weight of callus was (1.91 and 0.51) mg respectively. The results also showed that the MS medium prepared with a concentration of 4 mg L -1of 2,4-D was significantly superior in the average concentration of the compounds Hesperdine, Nargnine, Proanthocyanin, and Rutin, reaching (64.94, 62.56, 55.63, and 71.22)% respectively.While the medium prepared with a concentration of 3 mg L -1outperformed 2,4-D achieved the highest concentration of Quercetin compounds reaching 75.36% compared to the neutral treatment, which achieved the lowest concentrations.

Pre-Germinative Treatments and In Vitro Germination of Dianthus caryophyllus and Alstroemeria spp. Seeds

Alstroemeria spp. and carnation (Dianthus caryophyllus L.) have considerable and increasing economic importance in the floriculture market, therefore breeders carry out intense breeding programs to select new superior varieties. However, poor germination of hybrid seeds remains a bottleneck. Based on this assumption, seed pre-treatments and in vitro germination protocols, using different germination substrates, were applied in Alstroemeria spp. and carnation to improve germinability. Seed viability was tested using the 2,3,5-triphenyl tetrazolium chloride (TTC) test, and resulted in 91.10% ± 2.33 and 86.66% ± 3.85 in Alstroemeria and carnation, respectively. In Alstroemeria, pre-treatment with potassium nitrate (KNO3) in combination with modified ½ Murashige and Skoog (MS) medium ensured high germination uniformity combined with high germination percentage, showing significantly higher values than the control. In carnation, a suitable seed sterilization procedure was set up (up to 95.8% sterility); treatments with gibberellic acid (GA3) and KNO3 did not influence germination percentage compared to the control. A high multiplication rate of seedling lines was obtained on hormone-free MS medium.

Seed pretreatment using design of engineering approach for regulating <em>Humulus lupulus</em> seed germination in different media

Humulus lupulus, a plant of commercial benefit, has been widely explored for its therapeutic applications. Plant propagation via seeds is one of the major challenges due to seed dormancy and low germination. The physical dormancy of the seed caused by a hard seed coat makes seeds impervious to water. Different types of methods have been reported to break the seed dormancy. The present study emphasizes the optimized chemical method of seed pretreatment for regulating the germination efficiency in three different media namely, Murashige and Skoog (MS), Hoagland’s media and Leafy 200®. The study explores the interaction between two variables viz., acid treatment time and concentration of sulphuric acid, on germination of Humulus lupulus seeds using the Design of Experiment (DOE) approach. The results showed a higher germination rate in MS media and Leafy200® media (100%) seed germination percentage with respect to Hoagland’s media. The result demonstrates the synergistic role of seed pretreatment and media on the seed germination efficiency. The scanning electron microscopy (SEM) analysis of the seeds pretreated with acid revealed small perforations on the seed coat ranging between 5-15 µm. Furthermore, the elemental analysis of the seed coat surface by Energy Dispersive X-ray (EDX) revealed a substantial decrease in carbon and oxygen (%) and the presence of calcium, magnesium, and sulfur. The results indicate the acid hydrolysis of organic constituents on the seed surface enabling changes in seed porosity. Additionally, factors such as pretreatment and media play a pivotal role in regulating seed germination.

Application of iron oxide nanoparticles improves growth and phytochemical constituents of in vitro cultured Carum copticum L.

Iron oxide nanoparticles (FeONPs) are widely used in various applications, such as biomedicine, environmental remediation, and agriculture. Moreover, FeONPs can affect the production of secondary metabolites in plants, which are compounds that have various biological and pharmacological activities. Therefore, in this study, the ajwain (Carum copticum L.) seeds were grown in MS medium containing different levels of FeONPs (0, 100, 200, and 400 mg l−1). After four weeks, the effects of FeONPs on growth, photosynthesis pigments, total phenolic, and anthocyanin content, activities of some antioxidant enzyme and secondary metabolites of ajwain) were investigated. The results showed that low levels of FeONPs (especially 200 mg l−1) increased fresh weight (231 %), total chlorophyll (49 %), total phenolics (259 %), anthocyanin content (110 %), as well as thymol (8 %) and γ-terpinene (16 %) content. Conversely, 400 mg l−1 FeONPs reduced root length (60 %) and fresh weight (64 %) was associated with a reduction in CAT activity, phenol (55 %), thymol (22 %), and γ-terpinene (18 %) content compared to ajwain treated with 200 mg l−1 FeONPs. This reduction was probably due to the increase in ROS caused by the low catalase enzyme activity and important essential oils compounds such as thymol and γ-terpinene. Thus, our results indicate that FeONPs at certain levels can be used as a new way for in vitro production of valuable essential oils and antioxidant compounds in ajwain.

Induction and Suspension Culture of Panax japonicus Callus Tissue for the Production of Secondary Metabolic Active Substances.

Using Panax japonicus as research material, callus induction and culture were carried out, and high-yielding cell lines were screened to establish a suspension culture system that promotes callus growth and the accumulation of the "total saponins" (total content of triterpenoid glycosides or ginsenosides). Using the root as an explant, the medium for callus induction and proliferation was optimized by adjusting culture conditions (initial inoculation amount, carbon source, shaking speed, hormone concentration, culture time) and a high-yielding cell line with efficient proliferation and high total saponins content was screened out. The conditions of suspension culture were refined to find out the most suitable conditions for the suspension culture of callus, and finally, the suspension culture system was established. We found that the lowest (5%) contamination rate was achieved by disinfecting the fresh roots with 75% alcohol for 60 s, followed by soaking in 10% NaClO for 15 min. The highest induction rate (88.17%) of callus was obtained using the medium MS + 16.11 μmol·L-1 NAA + 13.32 μmol·L-1 6-BA + 30.0 g·L-1 sucrose + 7.5 g·L-1 agar. The callus was loose when the callus subcultured on the proliferation medium (MS + 5.37 μmol·L-1 NAA + 13.32 μmol·L-1 6-BA + 30.0 g·L-1 sucrose + 3.8 g·L-1 gellan gum) for 21 days. The callus growth was cultured in a liquid growth medium (MS + 5.37 μmol·L-1 NAA + 13.32 μmol·L-1 6-BA + 30.0 g·L-1 sucrose) with an initial inoculation amount of 40 g·L-1, a shaking speed of 110 r/min and darkness. Cell growth was fastest with a culture period of 21 days. We replaced the growth medium with the production medium (MS + 5.37 μmol·L-1 NAA + 13.32 μmol·L-1 6-BA + 30.0 g·L-1 glucose) for maximum accumulation of total saponins. [Conclusion] A callus induction and suspension culture system for the root of P. japonicus was established. In this way, we can promote the accumulation of total saponins in callus cells and provide a basis for large-scale cell culture and industrial production of medicinal total saponins.

In Vitro Micropropagation of Eria Lasiopetala (Willd.) Ormerod- A Native Medicinal Orchid in Bangladesh

The present study focuses on the asymbiotic germination of seeds through in vitro culture of Eria lasiopetala, a native medicinal orchid in Bangladesh. Out of the four basal media, MS was found to be the most effective medium, where 93.58±0.374% seed germinations were recorded. Different plant growth regulators (PGRs), e.g. BAP, Kin, NAA and were used for secondary protocorm proliferation, plantlet development, and multiplication of shoot buds through in vitro conditions. The maximum number of secondary protocorms (12.80±0.115) were developed that were derived from primary protocorms on MS medium fortified with 0.5 mg/l BAP and 1.0 mg/l NAA. Data revealed that 0.5 mg/l BAP + 0.25 mg/l NAA showed the best combination for height elongation of plantlets (3.12±0.012 cm) in MS medium. While for multiple shoot buds (MSBs) 1.0 mg/l BAP + 0.5 mg/l NAA showed better (6.8±0.127) performance than other combinations of PGRs. The single shoots were isolated from the multiple shoots and sub-cultured on ½MS medium supplemented with NAA (0.5 - 2.0 mg/l), IAA (0.5 - 2.0 mg/l) and IBA (0.5 - 2.0 mg/l) for root induction. The maximum number of roots (7.53±0.176) and its growth (4.61±0.01 cm) were found in ½MS medium supplemented with 1.0 mg/l IAA. The well rooted plantlets were hardened and successfully transferred after acclimatization in pots containing the mixture of coconut husk, charcoal and brick pieces in the ratio of 2:1:1 and eventually established under natural condition. J. Bio-Sci. 32(1): 45-60, 2024

Clonal propagation of rare orchid species Paphiopedilum spp. (Orchidaceae) to save Indonesian biodiversity

Paphiopedilum spp. is one of the terrestrial orchids that is interesting as an ornamental plant. This orchid flower has a distinctive characteristic, namely its labellum resembles a sac, so it is sought after by ornamental flower collectors because of its uniqueness. This orchid is included in the Appendix I group according to CITES, namely a critical species that is threatened with extinction, so it needs to be conserved both in situ and ex situ. Ex situ conservation can be done by propagating orchids using in vitro culture techniques. The objective of this research is to carry out mass propagation of 3 species of Paphiopedilum spp orchids: P. supardii, P. primulinum, P. glaucophyllum through somatic embryogenesis and to characterize the existence of embryo gene RKD4 homolog in Paphiopedilum orchids. The method is to culture the nodes slices in dark conditions for 2 weeks, and then changed to photoperiod mode 16:8 hours (dark:light) on ½ MS media plus a combination of 2,4-D or NAA with TDZ. The somatic embryo were analyzed anatomically. This method is an improvement over previously reported methods for Paphiopedilum spp. due to its efficiency and reproducibility for conservation. We concluded that ½ MS media with a combination of NAA or 2,4D with TDZ were able to induce somatic embryogenesis. The result show that 3 mg L−1 NAA was the best PGRs for PLBs induction in P. supardii (50 %), 1 mg L−1 2,4-D + 0.5 mg L−1 TDZ was the best PGRs for callus embryogenic induction in P. primulinum (20 %), and 1 mg L−1 NAA was the best PGRs for PLBs induction in P. glaucophyllum (33 %).

An Overview of Pharmaceutical Applications and In vitro Micropropagation Techniques for Rare and Endangered Plant Species

Many rare and endangered plant species possess valuable secondary metabolites with pharmacological applications. These bioactive compounds are often integral to traditional medicine systems, highlighting the cultural significance of these plants. The health benefits of many medicinal species are not fully validated by contemporary scientific research, and some may be facing extinction due to habitat loss, overharvesting or climate change. This situation highlights the urgent need for effective conservation strategies of the species and sustainable cultivation methods. Micropropagation is a valuable technique for producing large numbers of plants from a single explant, significantly aiding in the conservation and commercial cultivation of rare species. Among the various types of explants, shoot tips and nodal segments have been identified as the most effective explants for micropropagation. These explants can be induced to generate multiple shoots in Murashige and Skoog (MS) medium containing Benzyl aminopurine (BAP). Thidiazuron (TDZ), Kinetin (KIN), or 2,4-Dichlorophenoxyacetic acid (2,4-D) are commonly used in MS medium to promote shoot and root development in both direct and indirect organogenesis processes. Rooting of the plantlets was typically achieved using MS medium either supplemented with Indole-3-butyric acid (IBA) or devoid of auxins, depending on the species and the specific requirements for rooting.

DAYA MULTIPLIKASI EKSPLAN KENTANG AR 8 PADA BERBAGAI KONSENTRASI BENZIL AMINO PURIN (BAP) DAN EKSTRAK BAWANG MERAH DALAM MEDIA DASAR MURASHIGE DAN SKOOG (MS) SECARA IN VITRO

The technique of propagating potato seeds through tissue culture has long been used to produce quality and disease-free seeds. This study aims to test the effect of the combination of BAP and Shallot Extract on the growth of potato plant explants on MS media (Murshige & Skooge). Research in experimental form uses a completely randomized design (CRD) in a factorial pattern consisting of two factors. As the first factor, the growth hormone BAP consists of 4 levels, namely: 0 ppm, 2 ppm, 3 ppm and 4 ppm. Meanwhile, the second factor is: shallot extract consists of 4 treatment levels, namely: 0%, 0.2%, 0.4% and 0.6%. Thus, there were 16 treatment combinations which were repeated 6 times so that the total experimental units were 96. The ABA treatment with a concentration of 2 ppm/liter gave the best effect on the shoot formation time and root formation time of AR 8 potato plant explants. However, the treatment without ABA had a good effect on Shoot length and root length of explant potato plant varieties AR 8. Shallot extract treatment with a concentration of 20% had the best effect on shoot formation time, root formation time, shoot length and number of leaves. However, the treatment with a concentration of 40% had a better effect on the parameters of the number of shoots and the number of explant segments of the AR 8 variety of potato plants. The interaction of the ABA treatment with a concentration of 2 ppm and the shallot extract with a concentration of 20% had the best effect on the time of emergence of shoots and roots on the AR 8 Potato Explants. However, the interaction without ABA (0ppm) and shallot extract had a better effect on the parameters of shoot length, number of leaves, and number of explant segments in the AR 8 potato variety.

Sexual and Asexual Propagation of Wild Lime (Zanthoxylum fagara L. Sarg.), a Native Florida Plant with Ornamental and Ecological Value

Abstract A series of four propagation studies were implemented to better understand the seed biology of wild lime (Zanthoxylum fagara L. Sarg. [Rutaceae]) and its adventitious rooting response to auxin treatments. Plant origin (north central vs south Florida ecotypes) did not affect initial seed viability but significantly influenced germination response to seasonal temperatures. Fifty-five days after sowing north central Florida seeds, germination was similar among spring, summer and fall treatments (28.9 to 41.1%), but was reduced by the winter temperature (10.7%). South Florida seeds showed greatest germination under the fall temperature (71.2%), and the least germination under the summer temperature (30.2%). Additional seed treatments including applications of gibberellic acid (GA3) with kinetin nominally improved germination by 1.2 times compared to non-treated seed. Seeds tolerated cryopreservation treatments, including combinations of a plant vitrification solution, liquid nitrogen, phloroglucinol and precooling, suggesting long-term storage capability. As an alternative to seed propagation, cutting propagation was found to be a reliable means of reproducing wild lime with 91.0% rooting success when softwood cuttings were treated with 8,000 mg·kg−1 (0.13 oz·lb−1) indole-butyric acid (IBA), compared to 3,000 mg·kg−1 (0.05 oz·lb−1) IBA (86.3%) or the non-treated control (71.2%). Species used in this study: Wild lime, Zanthoxylum fagara (L. Sarg). Chemicals used in this study: 2,3,5-triphenyl-2H-tetrazolium chloride (TZ); sodium hypochlorite solution (Clorox bleach); gibberellic acid (GA3); kinetin (kinetin), plant vitrification solution 2 (PVS2); liquid nitrogen (LN); phloroglucinol (phloroglucinol); glycerol (glycerol); dimethyl sulfoxide (DMSO); MS media (Murashige and Skoog media), sucrose (sucrose); 15N-5P-15K liquid fertilizer (Peters Excel Cal-Mag Special); indole-3-butyric acid (IBA); 14N-14P-14K slow-release fertilizer (Osmocote).

IN VITRO REGENERATION OF STRIPE EBONY (DIOSPYROS FRUTESCENS BLUME)

The Stripe ebony tree (Diospyros frutescens Blume) is considered a species in danger of extinction due to overexploitation. Therefore, there is an urgent need to regenerate and conserve the species for future generation. In this study micropropagation protocol of Diospyros frutescens Blume was developed using nature zygotic embryos. Zygotic embryos isolated from germinated on MS medium containing 0.4mg L-1 Kinetin + 1.0mg L-1 BA. There were (83.11±0.79%) of explants induced shoots after 30 days of culture. The highest number of shoot on 1/2 MS medium supplemented with 2.5mg L-1 BA + 0.6mg L-1 IBA was 5.84±0.035 shoots per explant after 45 days of culture. The percentage of shoot produced roots the highest 85.52±0.48% after 45 days culture on 1/2 MS medium contain 0.8mg L-1 IBA.

Callus of Mundulea sericea as potential anti-diabetic agents; an in silico approach

Mundulea sericea, a rare plant of the fabaceae family, has high medicinal value but a low regeneration rate in its natural habitat. The present study aims to conduct in vitro propagation and screen the potential bioactive compounds in the callus through in silico analysis. The explants were collected, sterilized, and inoculated in a standardized MS media with growth regulators in vitro to obtain sterile plants. The experiment resulted in the formation of a greenish-brown callus at a rate of 82.41 % on MS medium supplemented with BAP (6.66 µM L−1). The callus was powdered and subjected to GC–MS analysis. A total of 31 compounds were identified in the methanolic extract, along with their corresponding peak values. The compounds underwent ADME analysis for toxicity predictions. Eight compounds were chosen further based on pharmacokinetic and pharmacodynamic properties. The selected ligands were docked with anti-diabetic targets α-amylase and α-glucosidase. As a result, the compound CID 5283663 exhibited a strong binding affinity of −9.3 with the α-amylase receptor. The ligand CID 33010 exhibits a strong interaction of −7.5 with the α-glucosidase receptor. Glibenclamide and acarbose were used as standards. This study is the first attempt to identify compounds in the callus extract that could inhibit α-amylase and α-glucosidase enzymes. The findings from molecular docking analysis suggest these compounds could become potent treatments for diabetics, showcasing the promise of natural sources in drug discovery.

Effects of Nitrogen Content and pH on Micropropagation of Rudraksha (Elaeocarpus ganitrus Roxb.)

This study evaluated the effects of pH and inorganic nitrogen sources (KNO3 and NH4NO3) on the regeneration of Elaeocarpus ganitrus Roxb. The pH and nitrogen concentration in the medium were crucial for the successful micropropagation of E. ganitrus. The concentration of nitrogen in the medium significantly affected the development and growth rate of cultures. As a basal medium, Murashige and Skoog's medium (MS) was employed together with various nitrogen concentrations and plant growth hormones. After 4-5 weeks of inoculation, BAP (0.5 mg/l) and NAA (0.1 mg/l) produced the maximum number of shoots (4.42 ± 2.17) among all the growth hormones tested. To obtain the maximum number of shoots and shoot length, different strengths of KNO3 (500-3500 mg/l), NH4NO3 (500-3500 mg/l), and pH (5-7.5) were adjusted in the medium. The largest number of roots was contained in MS medium with 1.0 mg/l NAA (4.47 ± 1.38) after root induction was carried out on MS with NAA, IBA, and IAA (0.2-2.0 mg/l). When the medium contained 1500 mg/l KNO3 and 2000 mg/l NH4NO3 (3.02 ± 1.60), the highest root initiation was observed. The rooting medium with a pH of 6.0 had the most influence and produced the maximum number of roots (4.8 ± 0.48). The plantlets were allowed to harden and acclimate in a greenhouse. A 76% survival rate was noted in the field.

A protocol for the development and maintenance of Coffea arabica (L.) cell suspension cultures

Coffea spp. are remarkable sources of phytochemicals, but the lack of a well-defined culture medium aimed at the induction of non-embryogenic and friable callus hampers the establishment of plant cell suspension cultures for large-scale production of valuable compounds. In this paper, we describe a one-medium protocol suitable to obtain both callus and cell suspension cultures from leaves of two elite cultivars of C. arabica. The protocol was developed through an iterative process involving the determination of the best concentration of auxin and cytokinin, their optimal ratio, as well as the most effective molecule of either hormone class. Young leaves were found to be a good and easy-to-use explant source for callus induction and proliferation, provided that a cytokinin was present in association with a chlorinated auxin in a full strength, semi-solid MS medium. The best results were obtained by hormone concentration and combination of 1 mg/L of both kinetin and 2,4,5-trichlorophenoxyacetic acid. The same ratio of these growth regulators was conveniently used for the development and stabilization of cell suspension cultures in liquid MS medium. When grown in darkness, stabilized suspension cultures showed a fine and homogeneous texture, with a 10-fold biomass increase within 25 days and a cell viability > 90%. In addition, the phytochemical profile revealed the presence of the most widely studied coffee compounds. The protocol can be applied to obtain adequate amounts of cell biomass for use in physiological studies concerning the production of secondary metabolites.

Tea (Camellia sinensis L. O. Kuntze) callus development based on morphological and phytochemical traits applied by variations in plant growth regulators (PGRs)

A variation of the clonal propagation system for tea has been established through in vitro methods, utilizing shoots and leaves derived from primary explants of mature field-grown plants. The research aimed to assess the diversity of tea calluses based on morphological and phytochemical traits derived from leaf and shoot explants on MS media induced by the application of plant growth regulators, including auxin and cytokinin. The research was conducted at the Seed Technology Unit Tissue Culture Laboratory, Faculty of Agriculture, Jatinangor, from April 2022 to February 2023. The experiment employed MS-modified multiplication media and PGR, with BAP at 9 mg/L, TDZ at 1.0 mg/L, Zeatin at 0.1 mg/L, NAA at 0.01 mg/L, and 1 mg/L. Results indicated that applying PGR, auxin, and cytokinin with MS media influenced morphological and phytochemical traits of tea's callus derived from leaves and shoots. Specifically, the treatment of 1 mg/L NAA + 1 mg/L BAP showed the best performance on callus weight and total phenolic and flavonoid contents compared to other treatments., i.e. 9.038% % and 0.05% %, respectively. The success of clonal propagation in tea through in vitro methods is expected to increase by carefully selecting the appropriate source and type of explant, planting media, PGR, and addressing potential contaminants.

In vitro seed propagation of Dendrobium moschatum as influenced by different plant growth regulators

This study aimed to develop an effective in vitro protocol for germinating and multiplying Dendrobium moschatum seeds by identifying optimal concentrations of plant growth regulators for shoot proliferation and root formation. Five different MS media formulations were utilized, including a control (M?) and those supplemented with varying concentrations of kinetin and NAA (M?, M?, M?) and BAP (M?). Results showed significant variation in seed germination time across media, with the longest duration observed in M? (76 days) and the shortest in M? (55 days). Protocorm formation and plantlet development were quickest in M?, while M? and M? exhibited the longest duration. The initiation of leaves or shoots occurred earliest in M? (103 days) and latest in M? (149 days). Notably, leaf and plantlet growth differed between three- and five-month intervals, with M? demonstrating optimal growth over the longer term. These findings underscore the importance of carefully balanced plant growth regulator concentrations in achieving successful seed germination and subsequent growth in D. moschatum.