Expression Analysis of Phenylpropanoid Pathway Genes and Metabolomic Analysis of Phenylpropanoid Compounds in Adventitious, Hairy, and Seedling Roots of Tartary Buckwheat

Tartary buckwheat (Fagopyrum tataricum) is an important crop that belongs to the Polygonaceae family, whose roots have received considerable attention due to the presence of compounds with high nutritional and medicinal value. In this study, we aimed to develop an efficient protocol for the culture of adventitious (ARs) and hairy (HRs) roots on a half-strength Schenk and Hildebrandt (SH) medium containing different concentrations of the auxins, α-naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA), and indole-3-acetic acid (IAA). The highest percentage of root induction (91.67%) was achieved with 0.5 mg/L IAA, whereas the greatest number of roots was found in 1 mg/L IAA. In contrast, 0.1 mg/L IBA returned the longest roots. As expected, HRs were obtained from in vitro leaf explants infected with Agrobacterium rhizogenes R1000. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis of 11 phenolic pathway genes revealed that five genes (FtPAL, FtC3H, FtHQT, FtCHS, and FtANS) were highly expressed in HRs, whereas only four (FtC4H, FtFLS2, FtDFR, and FtANR), and three (Ft4CL, FtCHI, and FtF3H) were recognized in the ARs and seedling roots (SRs), respectively. HPLC analysis of phenolic compounds in different root cultures showed that the majority of the phenolic compounds (both individual and total) were significantly accumulated in the HRs. Principal component analysis (PCA) identified differences among the three root types, whereby HRs were separated from ARs and SRs based on the amount of phenolic compounds present. Analysis of the metabolic pathway revealed that among the identified metabolites, the 3, 2, and 1 pathways were associated with flavonoid, flavone and flavonol, and phenylpropanoid biosynthesis, respectively. Hierarchical clustering analysis and the heat map showed that the different root cultures presented unique metabolites.

In vitro Propagation of Banana (Musa paradisiaca L.) Plant Using Shoot Tip Explant

Banana is a fruit crop which has high demand in Ethiopia, but its production is constrained by lack of disease free planting material with conventional propagation methods. For shoot initiation, shoot tip explants were cultured on MS medium supplemented with 0.5, 1.0, 1.5 and 2.0 mg/L BAP. Similarly, MS medium supplemented with BAP at 1.0, 1.5, 2.0 mg/L in combination with IBA at 0.25 and 0.50 mg/L were used for shoot multiplication. Half- strength MS medium augmented with IBA at 1.0, 2.0, 3.0 and 4.0 mg/l were used for root induction. MS medium without PGRs were used as controls. Finally, hardening of the in vitro derived plantlets was carried out in green house both in the primary and secondary acclimatization stages. Results showed that the highest shoot initiation percent (93.40%), highest mean number of shoots per explant (4.67) and lesser day for shoot induction (11.00) were observed in explant cultured on MS + 1.0 mg/L BAP. With shoot multiplication, highest shooting percent (92.60%), maximum number of shoots (7.67) and highest shoot length (5.27 cm) were recorded on MS + 1.5 mg/L BAP + 0.5 mg/L IBA. The highest rooting percent (93.40%), maximum root number per shoot (7.67) and highest root length (11.00 cm) were found on a half strength MS medium + 2.0 mg/L IBA. The survival rate of plantlets were 96.00% in coco peat substrate in primary acclimatization and 97.92% in forest soil, sand and manure substrates mixed at 3:2:1 ratio in secondary acclimatization. Overall, the result showed that the PGRs type, concentrations and combinations used are effective for mass propagation of banana variety studied in this experiment.

High-Frequency Plant Regeneration, Genetic Uniformity, and Flow Cytometric Analysis of Regenerants in Rutachalepensis L.

Ruta chalepensis L., an evergreen shrub in the citrus family, is well-known around the world for its essential oils and variety of bioactivities, indicating its potential medicinal applications. In this study, we investigated the effect of different culture conditions, including plant growth regulators, media types, pH of the medium, and carbon sources, on in vitro regeneration from nodal explants of R. chalepensis. Following 8 weeks of culture, the highest percentage of regeneration (96.3%) and maximum number of shoots (40.3 shoot/explant) with a length of 4.8 cm were obtained with Murashige and Skoog (MS) medium at pH 5.8, supplemented with 3.0% sucrose and 5.0 µM 6-Benzyladenine (BA) in combination with 1.0 µM 1-naphthaleneacetic acid (NAA). For rooting, individually harvested shootlets were transferred on ½ MS (half-strength) supplemented with IAA (indole-3-acetic acid), IBA (indole 3-butyric acid), or NAA, and the best response in terms of root induction (91.6%), number of roots (5.3), and root mean length (4.9 cm) was achieved with 0.5 µM IBA after 6 weeks. An average of 95.2 percent of healthy, in vitro regenerated plantlets survived after being transplanted into potting soil, indicating that they were effectively hardened. DNA assays (PCR-based markers) such as random amplification of polymorphic DNA (RAPD) and directed amplification of minisatellite-region (DAMD) were employed to assess in vitro cultivated R. chalepensis plantlets that produced a monomorphic banding pattern confirming the genetic stability. Additionally, no changes in the flow cytometric profile of ploidy between regenerated plantlets and donor plants were detected. Regeneration of this valuable medicinal plant in vitro will open up new avenues in pharmaceutical biotechnology by providing an unconventional steadfast system for mass multiplication and might be effectively used in genetic manipulation for enhanced bioactive constituents.

Induction and evaluation of secondary metabolite and antioxidant activity in adventitious root of Codonopsis javanica

In this study, the effects of auxin (IBA, NAA), explants, and culture conditions (light/dark) on adventitious root induction of Codonopsis javanica were investigated. The results showed that dark conditions were more suitable for adventitious root induction than light conditions. All three types of explants (internodes, leaves, and nodes) induced adventitious roots, and the appropriate concentration of auxin was 0.5 mg/l IBA. After 4 weeks of incubation under dark conditions, the rooting percentage and number of roots/explant of internode, leaf, and node segments on media supplemented with 0.5 mg/l IBA were 100% and 33.87 roots, 97.78% and 23.48 roots, 100% and 25.20 roots, respectively. These adventitious roots were analysed for the presence of alkaloids, carbohydrates, saponin, fixed oils and fats, phenol, flavonoids, gum, and mucilage. The total polysaccharide content, total phenolic content, and the antioxidant activity (IC50) of C. javanica adventitious root biomass were 16.98%, 1.876 (mg GAE/g DW), and 2.44 (mg/ml), respectively. These results indicate that the adventitious roots of C. javanica contain bioactive compounds, which can be used as a material source for multiplication in large-scale systems.

Micropropagation of Cassava (Manihot esculenta Crantz): Review

Cassava is a vital crop to the food security of millions of people worldwide, particularly in Sub-Saharan Africa. Because the crop produced a reasonable yield on marginal soils, it could help relieve global hunger. As a result, increasing cassava output and its quality attributes are significant. However, the low multiplication rate of this main crop has resulted in the delayed dissemination of improved varieties among farmers. As a result, tissue culture techniques may be a feasible solution for overcoming these challenges. Cassava in vitro propagation had done using either the shoots multiplication technique or somatic embryogenesis. However, the shoot multiplication approach is preferable since it retains clonal fidelity. Plant regeneration via somatic embryogenesis or organogenesis entailed the use of numerous basal media containing various plant growth hormones. Several studies found that each type of cassava clone required a unique protocol to achieve optimal shoot initiation, shoot multiplication, root induction, and elongation. This review describes recent research on cassava micropropagation that makes use of a variety of experimental systems. While each of these systems focuses on a different aspect of technique, they can be significant in understanding the in vitro production of cassava planting material.

A Simple and Efficient Protocol for Hairy Root Culture of Arabidopsis Thaliana

Hairy root culture (HRC) is a valuable biotechnological tool for the production of plant secondary metabolites. Secondary metabolome study of Arabidopsis thaliana can be helpful in understanding the biological roles of various secondary metabolites present in it and HRC constructs the base of such study. The present work deals with the establishment of Agrobacterium rhizogenes strains transformed HRC of A. thaliana with very high transformation frequency resulting in long term hairy root cultures grown in hormone free media. Optimization of culture medium and standardisation of co-cultivation period are the key role players in obtaining high frequency of hairy roots. Four days of preculture in CIM medium and five min of co-cultivation in the bacterial suspension were found to be optimal conditions for root induction. This protocol could become a powerful tool for transcriptomics and proteomics-based studies for different transgenic root lines of A. thaliana.

An Effective Protocol for Carnation (Dianthus caryophyllus L.) cv. 'Geolei' Explants Sterilization for Successful Callusing and Shoot Regeneration

Carnation is a popular floricultural crop grown widely for its attractive cut flowers. Micro-propagation can be used to create large-scale carnation output. For growth and development, plants require some necessary nutrients as well as growth regulators. Due to the importance of carnation, the present work is carried out using leaf and nodal segments to examine the potential of several plant growth regulators for in vitro callus formation and adventitious shoot regeneration. Explants were sterilized properly with bavistin, sodium hypochlorite and mercuric chloride. The minor contaminated cultures were created by consecutively treating the explants with 0.25% bavistin, 0.50% sodium hypochlorite, and 0.1% mercuric chloride for ten, fifteen, and two minutes. MS media with 2.5 mg/l 2,4-dichlorophenoxy acetic acid (2,4-D) in combination with 0.75 mg/l naphthalene acetic acid (NAA) resulted in the maximum callus induction (90.47%) from leaf explants. Maximum shoots (76.47%) were produced in MS media supplemented with 2.0 mg/l Thidiazuron (TDZ) + 0.25 mg/l NAA. NAA at 1.25 mg/l was most efficient for maximum root induction (83.32%). In the present study, an effective protocol of carnation explants sterilization was optimized for successful callusing and shoot regeneration.

ROOT INDUCTION OF PATCHOULI PLANT STEM CUTTINGS USING IBA GROWTH REGULATOR ON SEVERAL PLANT MEDIA

Patchouli (Pogostemon cablin Benth.) plant is one of the essential oil producing plants, that is useful in the chemical industry as a raw material for fragrance products and in the pharmaceutical field. The success rate of patchouli early growth, is largely determined by the cultivation techniques used, including the use of growth regulators, and growing media. This study aims to determine the effect of IBA growth regulators and the composition of plant media on the growth of patchouli plants. This research is carried out in the Greenhouse of the Forestry Study Program of the University of Mataram, using a Factorial Completely Randomized Design (RALF), the first factor is IBA growth regulator with 3 levels, namely 0, 20, 25 ppm and the second factor is composition of plant media with 3 levels, namely forest soil media, soil media:sand:compost (1:1:1), and soil media:sand:compost (2:1:3). The results show that the effect of IBA significantly affect the success percentage of cuttings and the number of roots of patchouli plants. The effect of plant media composition and the interaction between treatment of IBA with plant media composition did not have a significant effect on the growth parameters of patchouli plants.

Clonal propagation of Eucalyptus urophylla under effect of Cyperus rotundus extract and indole-3-acetic acid

Cyperus rotondus tubers possess substances with allelopathic qualities. Some of these substances can act as synergists of plant hormones and be used for root induction in cuttings. This study aimed to shed light on the development and rooting of Eucalyptus urophylla cuttings treated with C. rotondus (nutgrass) tuber extract and IAA (indole-3-acetic acid). The experiments were conducted using a completely randomized design (0; 25; 50; 75 and 100% nutgrass extract and 0; 50; 250; 500; 750 and 1000 mg L-1 of IAA), with four repetitions per treatment and four cuttings per plot. Plantlet height, stem width and root length were measured. The greatest width and height values were recorded for the control (0% nutgrass extract and 0 mg L-1 of IAA), indicating that these substances did not contribute to the development of these variables. However, treatments of nutgrass extract up to 50% were associated with greater root length. C. rotundus extract concentrations of 53% can be used to improve root development in E. urophylla cuttings, and contribute to the production of higher quality plantlets.

Harnessing the Potential of Roots of Traditional Power Plant: Ocimum

Genus Ocimum of Labiatae is well known in all traditional medicinal systems like Ayurveda, Unani, Siddha, and Homeopathy. The pharmaceutical activities of different species of Ocimum attributed to all plant parts. Roots are the most significant vital organ of the plant, as they absorb water and nutrients from soil and transport to aerial parts of the plants. Roots of Ocimum were found helpful with free-radical scavenging activity to improve physical and mental strength as well as to treat diabetes, malaria, and liver problems. Antibacterial activity of Ocimum roots and its main component, rosmarinic acid, is very beneficial to protect against several human pathogens, including bacteria and viruses. Being so important in every way, roots of Ocimum need healthy rhizosphere. Bacteria, fungi, nematodes, types of soil, fungicide, pesticides, salt, radioactive elements, as well as heavy metal contaminations, affect roots and overall growth of Ocimum in positive or negative ways. Each component of rhizosphere (natural, treatment or contamination) affects the roots, which highlights current ecological scenario to discover biosafe and more productive approaches. For such prestigious organ of Ocimum, development of in vitro root cultures and hairy root cultures assists to reduce the efforts and timing of the traditional cultivation process along with elimination of negative factors in rhizosphere. Different strains of Agrobacterium rhizogenes, various media compositions, as well as discrete treatments, like elicitors, on nonidentical species or cultivars of Ocimum boost the root induction, biomass, and accumulation of phytoceuticals differently. Hairy roots and in vitro roots of Ocimum accumulate higher quantity of therapeutic metabolites. These metabolites include several phenolics (like rosmarinic acid, 3-hydroxybenzoic acid, m-coumaric acid, p-coumaric acid, caffeic acid, ferulic acid, vanillic acid, chicoric acid, and lithospermic acid), triterpenes (such as betulinic acid, 3-epimaslinic acid, alphitolic acid, euscaphic acids, oleanolic acid, and ursolic acid) as well as flavonoids (flavones, flavonols, and dihydroflavonols). This review highlights pharmaceutical applications of Ocimum roots, a great deal of rhizosphere components and in vitro culturing techniques to enhance biomass as well as chief phytoceuticals.