Naphthaleneacetic Acid Research Articles

An efficient in vitro organogenesis protocol for the endangered relic tree species Bretschneidera sinensis and genetic fidelity assessment using DNA markers.

Bretschneidera sinensis is a monotypic species of rare and tertiary relic trees mainly distributed in China. B. sinensis is a potentially valuable horticultural plant, which has significant ornamental and research value, and is a crucial tool for the study of phylogeography. The artificial cultivation of B. sinensis is of great scientific value and practical significance. In this study, we developed a direct organogenesis process of B. sinensis using mature zygotic embryos as initial materials. The highest sterile germination induction (54.5%) from the mature zygotic embryo was obtained in a Murashige and Skoog (MS) medium with 2.0 mg·L-1 6-benzylaminopurine (6-BA) and 0.2 mg·L-1 α-naphthaleneacetic acid (NAA). The highest percentage of shoot regeneration (90.37%) was attained using 1.0 mg·L-1 6-BA and 0.01 mg·L-1 NAA in the MS medium. The Woody Plant Medium (WPM) had the greatest adventitious shoot elongation rate of 93.33%. The most optimized rooting rate was 88.89% in a half-strength MS medium containing 2.0 mg·L-1 indole-3-butyric acid (IBA) and 1.0 mg·L-1 NAA. The genetic fidelity of in vitro regenerated plantlets was assessed using inter-simple sequence repeats and random amplified polymorphic DNA molecular markers, confirming the genetic uniformity and stability of regenerated B. sinensis plantlets. Our research presents an effective in vitro propagation system for B. sinensis, laying the groundwork for its germplasm conservation and large-scale production while maintaining high genetic integrity.

Impact of Micronutrients and Plant Growth Regulators on Brinjal (Solanum melongena L.) Growth, Yield and Quality

The present research work conducted to find out the effect of plant growth regulators and micronutrients on growth, yield, and quality of Brinjal (Solanum melongena L.) under open field condition. The experiment was designed in a Randomized Complete Block Design (RCBD) with three replicates. The experiment was conducted on Horticulture farm at Sher-e-Bangla Agricultural University; Dhaka, Bangladesh from July 2022 to June 2023. The experimental treatments included three levels of PGR i.e. No PGR, 200 ppm gibberellic acid (GA3) and 100 ppm Naphthaleneacetic acid (NAA) and three levels of micronutrients i.e.no micronutrients, 200 ppm Boron and 200 ppm Zn. The treatments had a substantial effect on growth, yield, and quality of Brinjal. GA3 treated plants performed better than NAA and no PGR treated plants and Zn produced higher yields and quality than Boron and no micronutrients treated plants in most of the parameters. Considering treatment combination of PGR and micronutrients, plant height, leaf area (471.33 cm2), SPAD value (52.95), number of flowers plant-1 (65.33), number of fruits plant-1 (58.38), yield plant-1 (3312.8g), lipid (0.35%) were observed highest from the treatment combination of 200 ppm GA3 with 200 ppm Zinc whereas dry weight of whole plant (158.80 g), individual fruit weight (67.33g) and fiber (12.28%) were observed from the treatment combination of 100 ppm NAA with 200 ppm zinc but in case of number of branch (14.00) and Ash (8.44%) were observed from the treatment of 100 ppm NAA application . Maximum parameter showed lowest value under no treatment combination. This study suggests that foliar application of GA3 with micronutrients such as Zinc during flowering stage of eggplant is the most effective strategy for improving the nutritional quality of Brinjal fruits and increasing crop yields, both of which will help to reduce micronutrient malnutrition and hunger worldwide.

Exogenous Cytokinin Induces Callus and Protocorm-Like-Bodies Formation in In Vitro Root Tips of Vanilla planifolia Andrews.

Vanilla is a popular flavouring essence derived from the pods of vanilla orchid plants. Due to the high demand for vanilla flavour, high yielding vanilla plantlets are necessary for establishing vanilla plantations. Clonal micropropagation is a viable technique for the mass production of high yielding vanilla plantlets. This study reports an efficient regeneration protocol by using cytokinin as the sole plant growth regulator to regenerate plantlets from the root tips of a commercial vanilla orchid species, Vanilla planifolia. Most studies to date have reported using seeds and nodes as starting explants for in vitro micropropagation of vanilla orchids. So far, regeneration from roots has not been very successful. Previous studies favoured the use of auxins only or high auxin to cytokinin ratios to induce callus, and sole cytokinins were used for direct shoot regeneration. However, it was sporadically observed in plantlets regeneration of V. planifolia that multiple shoots were regenerated from the tips of intact aerial roots submerged in media. This study therefore investigated the regeneration of excised vanilla root tips through the application of most commonly used auxins (1-naphthaleneacetic acid and 2,4-dichlorophenoxyacetic acid) and cytokinins (6-benzylaminopurine and thidiazuron). High auxin presence is known to promote callusing in in vitro plants. However, in this study, auxin treatment inhibits callusing in root tips. While cytokinin treatments, even at low levels, has promoted high rate of callusing. These callus cells regenerate into protocorm-like-body (PLB) shoots when cytokinin levels are increased to 0.5 mg/mL 6-benzylaminopurine (BAP) under light conditions. The findings of the study have the potential of providing large quantity of high yielding vanilla plantlets through clonal micropropagation.

Regulation of Summer Season Flowering to Enhance the Flowering and Physical Parameters of Mrig Bahar in Guava (Psidium guajava l.) Cv. Lucknow-49 under Sodic Soil

The present investigation entitled “Regulation of summer season flowering to enhance the Flowering and physical parameters of mrig bahar in guava (Psidium guajava L.) cv. Lucknow-49 under sodic soil” was carried out at Production Processing of Usar Waste Land Akma, Department of Fruit Science, Acharya Narendra Deva University of Agriculture and Technology, Narendra Nagar (Kumarganj), Ayodhya (U.P.) during 2021-22. The experiment was conducted in a Randomized Block Design with three replications and ten treatments. The treatments were Urea (15, 20 and 25%), NAA (75, 150 and 225 ppm), 2,4-D (20, 40 and 60 ppm) and control applied on second week of April. The results denoted that all the treatments were emphatic in summer deblossoming and enhanced the physical attribute of fruits throughout both season over the control, however, maximum yield per plant, maximum no. of fruits per plant, maximum fruit set percentage and minimum flower drop percentage was recorded with control in rainy season. The profitable effects of the treatments tried were observed to be more prominent in winter season crop than the rainy season crop. Among the treatments, deblossoming with Naphthalene Acetic Acid (NAA) 225 ppm showed maximum enhancement in Flowering characters viz. Percent flower drop (%), Percent fruit set (%) and fruit physical characters viz. Number of fruits per plant, fruit length (cm), width (cm), weight (g), fruit yield per plant (kg/tree) over the control in winter season as well as in rainy season crop.

Effect of growth promotors on morphogenesis, photosynthetic apparatus, productivity and residual substances content in sweet pepper (Capsicum annuum) fruits

The use of plant growth promoting substances in order to optimize crop performance is one of the leading tasks of modern plant physiology. The effect of 0.005% 1-naphthaleneacetic acid (1-NAA), 0.005% gibberellic acid (GA3), 0.005% 6-benzylaminopurine (6-BAP) foliar treatment on morphogenesis, leaf parameters, photosynthetic pigments content, photosynthetic apparatus activity indices, and residual amounts of the used substances in the sweet pepper Capsicum annuum L. variety Antey fruits was investigated. The field experiment was laid on plots of 32 m2. The treatment of plants was carried out at the budding stage. Morphometric indices were determined at the flowering and fruit formation stages. The mesostructure of the middle tier leaves was studied at the fruit formation stage. The content of the chlorophylls was determined in the fresh leaf tissue by the spectrophotometric method. Indices of photosystem II (PSII) activity were determined by registration of chlorophyll fluorescence induction, using a portable single-beam fluorimeter. The assay of the residual content growth of promotors in the fruits was carried out on a Shimadzu GC gas chromatograph with a mass spectrometric detector – GCMS-QP2020 EI. It was revealed that treatment with GA3 increased plant height. All substances increased the number of leaves on the plant, their fresh and dry weight, the leaf area, and the canopy leaf area index. All growth promoting substances thickened leaf laminas due to the growth of chlorenchyma. GA3 and 6-BAP increased the volume of columnar parenchyma cells, and 1-NAA and GA3 increased the size of leaf spongy parenchyma cells. 6-BAP increased the total chlorophyll (a + b) content in the leaves, and under the GA3 treatment this index tended to decrease. 6-BAP increased the chlorophyll index of plants at the fruit formation stage, and GA3 decreased it at the flowering stage. The net photosynthetic rate increased under the influence of 1-NAA and 6-BAP, and decreased under GA3 treatment. GA3 reduced photorespiration and transpiration in leaves, 6-BAP increased transpiration, and 1-NAA decreased it. All substances increased dark respiration. Treatment with GA3 and 6-BAP reliably increased the maximum quantum efficiency of PSII photochemical reactions. 1-NAA and 6-BAP increased the actual quantum efficiency of PSII photochemistry. The coefficient of chlorophyll fluorescence photochemical quenching significantly increased at flowering under the application of 1-NAA, and tended to increase at the stage of fruit formation under the treatment of 1-NAA and 6-BAP. When GA3 was applied, the rate of chlorophyll fluorescence photochemical quenching was significantly reduced. The index of PSII reaction centers fraction that do not reduce the QB acceptor did not change with the use of 1-NAA and GA3 and tended to decrease after the use of 6-BAP. All growth promotors increased the whole plant dry weight. They reduced the net photosynthetic efficiency at the flowering stage, and at the stage of fruit formation it significantly increased under the treatment with 6-BAP. The 6-BAP caused the redistribution of plastic substances into the fruits due to the decrease in the dry weight of vegetative organs. Treatment with 1-NAA increased the proportion of root dry weight. 1-NAA, GA3 and 6-BAP increased the yield of fruits from one plant by 17%, 22% and 20% respectively due to the increase in the number of fruits per plant. The residual amounts of 1-NAA and 6-BAP in ripe sweet pepper fruits did not exceed the maximum permissible sanitary standards.

Optimization of somatic embryogenesis in Euterpe edulis Martius using auxin analogs and atomic force microscopy

Euterpe edulis Martius is an endangered species of the Atlantic Forest, whose fruits have high antioxidant potential, and propagated exclusively by seeds. The present study assessed the ability of different auxin inducers and picloram analogs to trigger somatic embryogenesis in E. edulis. Immature seeds were harvested, and their zygotic embryos were excised and grown in MS culture medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) or picloram at 150, 300, 450, 600 µM. The activity of picloram analogs triclopyr and clopyralid was evaluated in semisolid MS medium. At maturation and germination, picloram-derived calli and somatic embryos isolated from triclopyr-grown cultures were first transferred to pre-maturation medium and, after 30 days, to basal MS or MS medium supplemented with either 5 µM abscisic acid or 0.53 µM 1-naphthaleneacetic acid plus 12.3 µM 2-isopentenyladenine. Finally, somatic embryos with root protrusions were transferred to MS medium devoid of sucrose for 30 days and then acclimatized ex vitro. Scanning, transmission, and atomic force microscopy revealed that picloram was superior to 2,4-D but less effective than triclopyr (100 µM) in inducing embryogenesis. Maturation and germination of somatic embryos in E. edulis can be maximized by 5 µM abscisic acid, and selecting calli via atomic force microscopy.

Phenolic profiling and bioactivity assessment of in vitro propagated Psidium cattleianum Sabine: A promising study

Psidium cattleianum Sabine (strawberry guava) is an evergreen shrub that is grown as a fruiting hedge and has received significant consideration in the food and pharmaceutical disciplines. This study aims to set a promising protocol for in vitro propagation of P. cattleianum, along with profiling the phenolic content of the original plant (OP), induced callus (IC), and regenerated plantlets (RP) extracts, ultimately, evaluating their anti-inflammatory, antioxidant, and anticancer potential. Seeds were treated with commercial bleaching, HCl, and H2O2 to enhance the germination percentage and minimize the contamination percentage. Culturing sterilized leaf explants onto Murashige and Skoog (MS) medium supplemented with benzyl adenine (BA), 2,4-dichloro phenoxy acetic acid, and kinetin showed the best callus induction, while supplementation of MS media with BA, adenine sulfate, naphthalene acetic acid, and gibberellic acid activated regeneration. Augmentation of MS media with indol-3-butyric acid recorded the maximum rooting percentage. Finally, the obtained rooted shoots were successfully acclimatized in sand and peat moss soil. HPLC-MS/MS profiles of OP, RP, and IC showed a variety of phenolic metabolites. IC extract decreased the viability of MCF-7, HepG2, and K-562 cancer cell lines. Also, OP exhibits strong antioxidant activity. P. cattleianum and its RP are profound sources of phenolic compounds promoted for promising applications in the food and pharmaceutical industries.

Naftalin Asetik Asit, 6-Benzilaminopürin ve İndol-3-Bütirik Asit Kombinasyonlarının Actinidia deliciosa Kallus Gelişimi Üzerine Biyokimyasal Bir Araştırma

Bu çalışmada oksin türevi olan naftalin asetik asit (NAA), sitokinin türevi 6-Benzilaminopürin (BAP) ile indol-3-butirik asit (IBA)’in farklı konsantrasyonları ve kombinasyonları kivi bitkisinin (Actinidia deliciosa) kotiledon eksplantlarına uygulandı. Kallus hücrelerinin fenolik bileşik, antioksidan aktivite, yağ asidi bileşimi ve lipofilik molekül içerikleri incelendi. Kültür ortamında yetiştirilen Actinidia deliciosa sürgünleri 5 santimetreye ulaşınca kotiledonları eksplant kaynağı olarak kullanıldı. Farklı doz ve kombinasyonlarda bitki büyüme düzenleyicileri (BBD) içeren kültür ortamlarında kallus indüksiyonu sağlandı. Kalluslar ile yapılan analiz sonuçlarına göre total fenolik içeriği 0.5 mg L-1 NAA + 1 mg L-1 BAP + 0.5 mg L-1 IBA kombinasyonu içeren F grubunda kontrol grubuna göre azaldığı halde (p

Optimizing callus formation in the endangered medicinal tree Hydnocarpus macrocarpa using phytohormone

Hydnocarpus macrocarpa (Flacourtiaceae) is one of the endemic and endangered medicinal tree, was selected to investigate the influence of plant growth regulators on callus formation. H. macrocarpa is majorly distributed at low and medium elevations of the Southern Western Ghats. The seeds of this plant are used for the treatment of ulcers, biliousness, leprosy and epilepsy. An experiment was carried out with two different methods. In the first method Murashige and Skoog (MS) medium supplemented with cytokinin alone or combined with auxin (2–4-Dichlorophenoxy acetic acid; Naphthalene acetic acid; Indole acetic acid; Indole butyric acid and Gibberellic acid) at different concentrations (1, 2, 3, 4 and 5 µM) were used; in the seond method, MS medium supplemented with coconut endosperm (Solid and liquid) extract (1, 2, 3, 4 and 5 %) with Aloe vera jelly extract at different concentrations (1, 2, 3, 4 and 5 %) in combination and separately were used. Among the different treatments, good callus proliferation was obtained using MS medium supplemented with Benzylaminopurine and in combination with Naphthalene acetic acid and 2–4-Dichlorophenoxyacetic acid (3 µM and 4 µM). But the best callus proliferation, shoot tip proliferation; auxiliary root and shoot formation were observed in MS medium supplemented with Aloe vera jelly extract in combination with coconut endosperm extract. The natural growth regulators supplemented with the media gave a positive response for plant growth. The results showed that the natural growth regulators, such as coconut endosperm and Aloe vera jelly extracts were found to be more efficient than the synthetic growth regulators (auxin and cytokinin). The presence of Indole acetic acid and Indole butyric acid in both Aloe vera jelly extracts and coconut endosperm were confirmed by High performance liquid chromatography and Fourier transform infrared spectroscopy analysis.

Matabolomic Changes Induced by 6-Benzylaminopurine in Polygonatum cyrtonema

Polygonatum cyrtonema Hua (family Asparagaceae) is an endemic plant in China that is valuable for its edible and medicinal uses. Plant growth regulators (PGRs) are natural or synthetic compounds that can regulate plant development and metabolism effectively. To explore potential applications of PGRs for improving the yield and bioactivity of this plant, four PGRs, including gibberellic acid (GA3), 6-benzylaminopurine (6-BA), naphthaleneacetic acid (NAA) and 24-epibrassinolide (EBL), were used in this study and sprayed on the growing seedlings of P. cyrtonema. All of these PGRs did not significantly affect the growth rate of P. cyrtonema, but they had varying effects on the polysaccharide and saponin content in the rhizome. NAA and 6-BA positively affected the polysaccharide content, while most PGR treatments negatively affected the saponin content. Widely targeted metabolomic analysis based on UPLC-MS/MS was conducted and revealed 101 differential metabolites in response to 6-BA, most of which were flavonoids, steroids and lipids. Most of the significantly changed flavonoids decreased under the 6-BA treatment. The study provides insights into the potential use of PGRs for improving the quality of P. cyrtonema, particularly in regulating the content of bioactive compounds.

Plant Growth Regulators and Rooting Media: A Viable Approach for Growth and Performance of Citrus

Plant growth regulators accompanied with appropriate growth medium can boost up the growth and performance of plants to a greater extent. PGRs such as auxin, gibberellin and cytokine play an exaggerative role in growth and development of the citrus. Moreover, the study reflects a clear overview of the impact of PGRs and different growth media to encourage rooting percentage, number of leaves, survival percentage, sprouting time, and root length of citrus cuttings. Citrus is a versatile fruit crop having a huge number of varieties, cultivars, and species; comes in different colours and flavours. Although, there are different methods used in the multiplication of citrus plant which includes budding, grafting, cutting, air layering, etc. However, propagation by cutting technique is the most commercial and farmer’s friendly approach. Different elements such as concentration of PGRs, age of plants and environmental factors are responsible for the successful growth of the citrus cuttings. Naphthalene acetic acid (NAA) and indole-3-butyric acid (IBA) are the two types of auxin, which are most commonly utilized in citrus propagation. Rooting medium plays a crucial role in the supply the minerals and availability of water in citrus. An absolute rooting medium must have more enough nutrients and water holding capacity with better drainage as well as sufficient air spores for excellent gaseous exchange that fruitfully influence the rooting ability and also have a profound effect on growth and development in citrus cutting. The best time for cutting of citrus stems is in between the July and August.

Genome-wide analysis of the KNOX gene family in Moso bamboo: insights into their role in promoting the rapid shoot growth

BackgroundKNOTTED1-like homeobox (KNOX) genes, plant-specific homologous box transcription factors (TFs), play a central role in regulating plant growth, development, organ formation, and response to biotic and abiotic stresses. However, a comprehensive genome-wide identification of the KNOX genes in Moso bamboo (Phyllostachys edulis), the fastest growing plant, has not yet been conducted, and the specific biological functions of this family remain unknown.ResultsThe expression profiles of 24 KNOX genes, divided into two subfamilies, were determined by integrating Moso bamboo genome and its transcriptional data. The KNOX gene promoters were found to contain several light and stress-related cis-acting elements. Synteny analysis revealed stronger similarity with rice KNOX genes than with Arabidopsis KNOX genes. Additionally, several conserved structural domains and motifs were identified in the KNOX proteins. The expansion of the KNOX gene family was primarily regulated by tandem duplications. Furthermore, the KNOX genes were responsive to naphthaleneacetic acid (NAA) and gibberellin (GA) hormones, exhibiting distinct temporal expression patterns in four different organs of Moso bamboo. Short Time-series Expression Miner (STEM) analysis and quantitative real-time PCR (qRT-PCR) assays demonstrated that PeKNOX genes may play a role in promoting rapid shoot growth. Additionally, Gene Ontology (GO) and Protein–Protein Interaction (PPI) network enrichment analyses revealed several functional annotations for PeKNOXs. By regulating downstream target genes, PeKNOXs are involved in the synthesis of AUX /IAA, ultimately affecting cell division and elongation.ConclusionsIn the present study, we identified and characterized a total of 24 KNOX genes in Moso bamboo and investigated their physiological properties and conserved structural domains. To understand their functional roles, we conducted an analysis of gene expression profiles using STEM and RNA-seq data. This analysis successfully revealed regulatory networks of the KNOX genes, involving both upstream and downstream genes. Furthermore, the KNOX genes are involved in the AUX/IAA metabolic pathway, which accelerates shoot growth by influencing downstream target genes. These results provide a theoretical foundation for studying the molecular mechanisms underlying the rapid growth and establish the groundwork for future research into the functions and transcriptional regulatory networks of the KNOX gene family.

EFFICIENT CLONAL PROPAGATION OF GUAVA CULTIVARS BY MINI SOFTWOOD CUTTINGS

Guava (Psidium guajava L.) is commercially propagated by seeds in Punjab and vegetative propagation systems are lacking. Propagation by stem cuttings is handicapped by the non-availability of plant material in bulk for cuttings and season dependency. Hence, juvenile mini softwood (MSW) shoot cuttings (3"-4") of Gola (Round) cultivar having two to three nodes were treated with plant growth regulators (PGRs) including indole butyric acid ‘IBA’ or naphthalene acetic acid ‘NAA’ in combinations with benzyl aminopurine ‘BAP’ and planted in sand, silt and coco peat (40:40:20 v/v) under CRD layout for PGR optimization. Cuttings treated with BAP and IBA (2 mgL-1 + 3 mgL-1) started early sprouting in 8 days and induced a greater number of branches (5-6), number of leaves (11), number of roots (6-7) and long roots (14 cm). Whereas, sprout length was higher up to 14 cm in cuttings treated with BAP and IBA (3 mgL-1 + 1 mgL-1). In the second part of the study, application of the optimized PGR treatment of BAP and IBA (2 mgL-1 + 3 mgL-1) to MSW cuttings of different commercial strains of cultivars Gola (Round) and Surahi (Pyriform) showed a better number of roots (5, 4), root length (13 cm, 11 cm), bud sprouting (100% and 80%) and number of nodes (5 each) in Gola (Sadabahar ‘G1’) and Surahi (Sindhi Bao ‘S2’), respectively. The rooted cuttings were maintained under standard growth room conditions and higher humidity till proper plant growth and the established plantlets were transferred to green house. This study established an efficient clonal propagation system using MSW cuttings thus reducing the plant material and PGRs required for mass multiplication of elite guava cultivars. Keywords: Auxin, cytokinin, softwood cuttings, shoot, rooting.

Integrated Transcriptomic and Metabolomic Analysis of Exogenous NAA Effects on Maize Seedling Root Systems under Potassium Deficiency.

Auxin plays a crucial role in regulating root growth and development, and its distribution pattern under environmental stimuli significantly influences root plasticity. Under K deficiency, the interaction between K+ transporters and auxin can modulate root development. This study compared the differences in root morphology and physiological mechanisms of the low-K-tolerant maize inbred line 90-21-3 and K-sensitive maize inbred line D937 under K-deficiency (K+ = 0.2 mM) with exogenous NAA (1-naphthaleneacetic acid, NAA = 0.01 mM) treatment. Root systems of 90-21-3 exhibited higher K+ absorption efficiency. Conversely, D937 seedling roots demonstrated greater plasticity and higher K+ content. In-depth analysis through transcriptomics and metabolomics revealed that 90-21-3 and D937 seedling roots showed differential responses to exogenous NAA under K-deficiency. In 90-21-3, upregulation of the expression of K+ absorption and transport-related proteins (proton-exporting ATPase and potassium transporter) and the enrichment of antioxidant-related functional genes were observed. In D937, exogenous NAA promoted the responses of genes related to intercellular ethylene and cation transport to K-deficiency. Differential metabolite enrichment analysis primarily revealed significant enrichment in flavonoid biosynthesis, tryptophan metabolism, and hormone signaling pathways. Integrated transcriptomic and metabolomic analyses revealed that phenylpropanoid biosynthesis is a crucial pathway, with core genes (related to peroxidase enzyme) and core metabolites upregulated in 90-21-3. The findings suggest that under K-deficiency, exogenous NAA induces substantial changes in maize roots, with the phenylpropanoid biosynthesis pathway playing a crucial role in the maize root's response to exogenous NAA regulation under K-deficiency.

Ex-situ Conservation of Bulbophyllum leopardinum, A Threatened Medicinal Orchid of Nepal

A successful micropropagation method was developed via the in-vitro seed germination and seedling growth of the epiphytic and/or lithophytic orchid Bulbophyllum leopardinum, a species having horticultural and therapeutic significance. To enhance seed germination, several quantities and combinations of naphthalene acetic acid (NAA), 6-benzyl amino purine (BAP), indole acetic acid (IAA), gibberellic acid (GA3), and coconut water (CW) were added to 0.8% (w/v) agar-solidified MS medium. Half-strength MS medium has been experimented with alone and in combination with BAP, Kinetin (Kn), and GA3 to promote shoot development. In-vitro-developed healthy shoots were chosen to establish roots in a half-strength MS (HMS) medium supplemented with various auxins. The best and earliest seed germination with the greenest protocorms (96.3±0.5% in 7 weeks) was achieved on HMS medium fortified with 15% CW (H15C). Further tests for the shoot as well as root development were continued with an H15C medium. H15C with 1 mg/l BAP and 1.5 mg/l Kinetin was most effective for early in vitro development and differentiation into seedlings with the many long shoots (9.3±0.1 shoots and 2.4±0.1 cm per culture) within 12 weeks of sub-culture. The most suitable rooting hormone was 1 mg/l NAA (4.2±0.26 roots per culture). This medium also produced the longest roots (1.9±0.09 cm per culture). By successfully developing a protocol for the mass propagation of B. leopardinum, this research has enhanced both the cultivation and the commercialization potential of this species.

Induction, Proliferation and Differentiation of Callus in Paris polyphylla Sm. through Leaf Culture

Paris polyphylla Sm. is a vulnerable medicinal plant employed in the treatment of various ailments. This study seeks to establish a protocol for callus induction, proliferation, and differentiation of P. polyphylla. Immature leaf explants were cultured on MS medium with varying concentrations of plant growth regulators (PGRs), including 2,4-dichlorophenoxyacetic acid (2,4-D), kinetin (Kn), 6-benzylaminopurine (BAP), Thidiazuron (TDZ), α-Naphthalene acetic acid (NAA), and Gibberellic acid (GA3), along with 10% coconut water. After 12 weeks of primary culture, the optimal callus induction was observed in MS medium supplemented with 0.25 mg/l 2,4-D + 0.5 mg/l Kn. In the secondary culture at 8 weeks, the best callus proliferation, as determined by callus weight or growth index, occurred in MS medium supplemented with 2.0 mg/l BAP alone, 2.0 mg/l Kn alone, 1.0 mg/l TDZ alone, combinations of 2.0 mg/l Kn + 1.0 mg/l BAP + 2.0 mg/l GA3, and combinations of 0.5 mg/l NAA + 2.0 mg/l BAP + 2.0 mg/l GA3, as well as 10% coconut water. Furthermore, callus differentiation into mini rhizomes with root primordia was successfully achieved in MS media containing 2.5 mg/l Kn and 10% coconut water. This study reports, for the first time, the formation and differentiation of callus from leaf explants in P. polyphylla. Large-scale callus generation from leaf explants has the potential to enhance the production of bioactive secondary metabolites for therapeutic purposes and facilitate the development of plantlets through organogenesis.

In vitro germination and micropropagation of the Balkan endemic Lilium chalcedonicum L., a potential ornamental lily

Lilium chalcedonicum L., a bulbous perennial endemic to the Balkans, is recognized for its considerable ornamental potential. In this study, seeds were gathered from a wild population and subjected to surface disinfection. Subsequently, they were placed in Petri dishes containing a half-strength Murashige and Skoog (MS) nutrient substrate (MS/2), with temperature conditions at 15 °C and 20 °C, followed by cycles of 5 °C and return to 15 °C and 20 °C, respectively. The in vitro-cultivated seedlings were then transplanted for further growth in MS medium enriched with either 30 g L-1 or 60 g L-1 sucrose and 0.1 mg L-1 6-N-benzyladenine (BA). In the following stage, the resulting bulblets were separated and cultivated in MS/2, either without hormones (Hf) or with BA at concentrations of 0.2 mg L-1, 0.5 mg L-1, or 1 mg L-1. Moreover, the combined influence of 1-naphthaleneacetic acid (NAA) at a concentration of 1.0 mg L-1 and 0.2 mg L-1 BA was examined. This stage was succeeded by a further division of the bulblets, and the explants were cultured in Hf, MS/2 or with the inclusion of 0.5 mg L-1 BA or a combination of BA and NAA at ratios of 0.2/1 and 0.5/0.05 mg L-1; zeatin at 0.5 mg L-1 was also used, combined with 0.05 mg L-1 NAA. Notably, all seeds exhibited a 100% germination rate following alternating temperature regimes including periods of low temperature. During the initial cultivation phase, each seedling gave rise to two bulblets measuring 0.6 cm in diameter. Subsequent subcultures on Hf substrates and those containing BA without NAA, resulted in the highest number of bulblets (1.6-2.2 bulbs/plant) of the largest diameter (0.8-0.9 cm). Particularly noteworthy was the substantial increase in the average number of produced bulblets during the subsequent subcultures (multiplication stage), where 16.9 bulbs were formed in MS/2 medium containing 0.5 mg L-1 BA, with a median diameter of 1.7 cm.

Exploring the Production of Secondary Metabolites from a Halophyte Tetragonia tetragonoides through Callus Culture

Considering Korea’s gradual shift toward an aging society, consumer interest in compounds with physiological benefits, including antioxidant and anticancer effects, has surged. This study explored the potential of Tetragonia tetragonoides (Pall.) Kunze, commonly known as New Zealand Spinach (NZS), a halophyte with reported health benefits, including efficacy in treating gastrointestinal diseases, high blood pressure, diabetes, and obesity. This study also introduced a novel callus culture system for NZS, allowing for the rapid in vitro production of secondary metabolites. Optimal callus induction (100%) and biomass production (0.416 g) were achieved by adding 2.0 mg·L−1 6-BA (6-Benzylaminopurine) and 0.5 mg·L−1 2,4-D (2,4-Dichlorophenoxyacetic acid) among five auxin and cytokinin combinations. Two distinct callus types, TGC [TDZ (Thidiazuron)-supplemented Green Callus] and TNYC [TDZ + NAA (Naphthalene acetic acid)-supplemented Yellow Callus], were identified, each with unique characteristics. The calli showed total phenolic and flavonoid contents comparable to those of NZS leaves grown in the greenhouse. An expression analysis of six genes (CHS, CHI, F3H, F3′H, FLS, and DFR) involved in the kaempferol biosynthesis revealed an enhanced flavonoid biosynthesis-related gene expression in TGC, emphasizing its potential for compound production. GC-MS analysis identified distinct compound profiles in TGC and TNYC, with 2,3-butanediol and succinic acid being the predominant compounds among the nine and forty-four components, respectively. These calli offer a stable supply of functional compounds and present an environmentally sustainable solution. The derived callus culture system is anticipated to contribute to the development of healthy functional foods or pharmaceuticals from halophyte NZS.

Establishment of an efficient genetic transformation system for Tanacetum cinerariifolium

The Dalmatian Daisy Tanacetum cinerariifolium is an Asteraceae plant species that produces the natural insecticide “pyrethrum”, which is effective against mosquito disease vectors and household pests. To enhance the content of pyrethrum in flowers, a more detailed understanding of the mechanisms underlying pyrethrum biosynthesis is needed. Even though gene transformation and genome editing techniques are vital for investigating pyrethrin biosynthesis, limited information is available on the transformation of T. cinerariifolium. Furthermore, each seedling possesses a distinct genotype with large variations by self-incompatibility. We herein employed T. cinerariifolium line #14 with weak self-incompatibility to establish a protocol of efficient regeneration from leaf segments and transformation. Leaf segments formed calli on 1/2 Murashige and Skoog’s basal medium (MS) with naphthalene acetic acid 1 mg L−1 and 6-benzylaminopurine (BAP) 2 mg L−1, regenerated shoots from calli on 1/2 MS with BAP 0.5 mg L−1 and GA3 0.2 mg L−1, and elongated shoot stems on 1/2 MS with indole-3-butyric acid 0.5 mg L−1 and BAP 0.5 mg L−1. To establish genetic transformation, Rhizobium radiobacter strain EHA105 with the highest infectivity and the mas1'-2' bidirectional promoter with the highest expression of the nptII resistance gene were used, and the antibiotic G418 was added to medium at a concentration of 10 to 20 mg L−1 to select transformed cells. Using established regeneration techniques, we successfully obtained transformants that highly expressed the transgene gusA. This technique will be useful for creating genetically modified T. cinerariifolium, particularly for elucidating the mechanism of pyrethrin biosynthesis toward the creation of pyrethrin-rich traits.

In Vitro Regeneration of Ragleaf (Crassocephalum crepidioides (Benth.) S.Moore) Using Kinetin

Micropropagation is a valuable means for large-scale production and preservation of invasive alien species such as ragleaf. To determine the concentration of kinetin adequate for in vitro growth of the ragleaf flower, seeds of the plant were propagated in vitro using Murashige and Skoog medium supplemented with basal 0.5 mg L-1 Naphthalene Acetic Acid and incubated for four weeks. The growing shoots were cut into pieces and inoculated into MS media with five kinetin concentrations: 0.0, 1.0, 1.5, 2.0, and 2.5 mg L-1. The tubes were kept in an incubation room for seven weeks. The experiment was arranged in a completely randomized design with ten replications. Shoot length (cm) and the number of leaves, nodes, and roots were measured. The control medium recorded less growth performance across all the variables, indicating the important role of the exogenous kinetin. The plants in 2.50 mg L-1 kinetin were the tallest (7.23±0.87 cm) with the highest number of leaves (28.17±2.57), nodes (10.50±1.02) and shoots (3.83±0.17). The media containing 2.50 mg L-1 kinetin also produced the highest number of roots (9.00), which is necessary for the survival of the seedlings. The results indicate that 2.5 mg L-1 is adequate for cultured C. crepidioides.