In vitro Regeneration of Japanese Burdock (Arctium lappa L.) from Cotyledon and Leaf Derived Explants

Considering the vegetable and medicinal values, a micropropagation protocol has been established for Japanese Burdock (Arctium lappa L.) by culturing the explants of cotyledon and leaf obtained from in vitro grown seedlings. Direct shoot regeneration was achieved from cotyledon and leaf explants on MS fortified with 4.0 μM BAP and 2.0 μM IBA or NAA after 5 weeks of culture. In addition, both the explants also formed callus from their cut margins within 6 weeks of cultivation on medium complemented with 6.0 μM BAP and 4.0 μM IBA or NAA. Adventitious shoots were also redeveloped through indirect organogenesis from the cotyledon and leaf-derived callus within 10 weeks of culture on MS containing 4.0 μM BAP and 2.0 μM IBA or NAA. The highest rate of shoot reproduction was attained at the third subculture, and more than 12.6 shoots were formed per callus clump. Within 4 weeks of transfer to the rooting medium on MS containing 6.0 μM IBA, the cultured micro-shoots produced highest 5.3 roots per cultured shoot. Rooted plantlets were successfully established on a soil-composed-sand mixture under natural condition with 93.3% survival rate Plant Tissue Cult. & Biotech. 31(2): 123-134, 2021 (December)

Development of an Efficient In vitro Regeneration Protocol for Chrysanthemum (Chrysanthemum morifolium Ramat)

An efficient and rapid in vitro regeneration protocol was developed for chrysanthemum (Chrysanthemum morifolium Ramat) using two local varieties of Bangladesh namely, BARI Chrysanthemum-2 (BARI Chry-2) and local yellow (Y). MS medium supplemented with nine different concentrations and combinations of BAP and IAA was employed to optimize regeneration protocol using young in vitro derived leaf explants. Direct organogenesis was observed from the leaf explants on MS medium supplemented with 0.5 mg/l BAP and 2.0 mg/l IAA (T6) for both the varieties. This treatment (T6) induced shoot buds directly on the adaxial surface of the leaf providing the highest regeneration percentage (90% for BARI Chry-2 and 94.73% for Y), the highest number of shoot/explant (7.6 for BARI Chry-2 and 8.6 for Y) and maximum length of the shoot after six weeks (3 cm for BARI Chry-2 and 2.9 cm for Y) of culture. Explants with initially regenerated shoots were subculture on hormone free MS medium for shoot elongation after 4 weeks of their inoculation. During elongation of shoots, 90-95% of the regenerated shoots produced roots spontaneously in hormone free MS medium within 7-8 weeks of their inoculation. Rooted plantlets were transplanted to the field following hardening where 100% plantlets were survived and produced flower without any variation. Plant Tissue Cult. & Biotech. 31(2): 161-171, 2021 (December)

Efficient Plant Regeneration via Indirect Organogenesis in Carnation (Dianthus Caryophyllus Semperflorens flore pleno) Cultivars

ABSTRACT Callus induction and plant regeneration are important steps of in vitro plant breeding of ornamental plants. In this study, the effects of different combinations of plant growth regulators (PGRs), promoters, and minerals on callus induction and plant regeneration in different carnation cultivars were studied in a completely randomized design with three replications. For callus induction, 16 different combinations of 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzylaminopurine (BA), 1-naphthaleneacetic acid (NAA), and casein hydrolysate (CH) were studied using in vitro leaf explants. The Murashige and Skoog (MS) medium supplemented with 0.2 mg·dm-3 of 2,4-D and 200 mg·dm-3 of CH showed the highest frequency of callus induction. Among the cultivars, ‘Noblesse’ showed the highest rate of callus induction (91.67%). Regarding regeneration, BA, NAA, silver nitrate (AgNO3), and adenine hemisulfate (As) were used in ten different combinations. The ‘Cameron’, ‘Tabasco’, and ‘Noblesse’ cultivars with 95.24% regeneration percentage showed the highest rate of plant regeneration. Generally, in most cultivars, the highest regeneration rate and shoot number per explant were found in the MS medium supplemented with 3 mg·dm-3 of BA, 0.6 mg·dm-3 of NAA, 5 mg·dm-3 of AgNO3, and 40 mg·dm-3 of As. According to the results, the highest regeneration frequency was obtained when 40 mg·dm-3 of As was added to the medium. Finally, the flow cytometry analysis indicated that there were no significant differences between in vitro regenerated and control plants in terms of DNA ratios.

In Vitro Amphidiploid Induction of A Distant Hybrid Populus Simonii × P. Euphratica Cv. ‘Xiaohuyang-2’ and its Effect on Plant Morphology and Anatomy

In plants, highly gametic sterility of distant hybrids usually restricts their utilization in breeding programs. Amphidiploid induction produced by somatic chromosome doubling of distant hybrids can effectively restore their gametic fertility. In this study, nodal-segment and leaf explants of a distant hybrid Populus simonii × P. euphratica cv. ‘Xiaohuyang-2’ were used to induce chromosome doubling with colchicine in vitro. Although chromosome doubling of the nodal-segment explants only produced mixoploids, the treatments of leaf explants on adventitious bud regeneration medium successfully produced 4 amphidiploids, which might be attributed to the direct organogenesis of the adventitious buds on the leaf explants. The highest amphidiploid induction frequency was 16.7%. Both the explant survival rate and polyploidization frequency were significantly affected by colchicine concentration and exposure time. The amphidiploid plants were significantly differed from the diploid and mixoploid plants on morphological and anatomical characteristics. They had larger, thicker, and greener leaves than the diploids and mixoploids. The changes of stomatal features also accompanied with increase of ploidy level. The induced amphidiploid plants of the distant hybrid ‘Xiaohuyang-2’ are expected to play important roles in breeding programs of Populus in future, which can be used as a bridge parent with ability of unreduced gamete formation to cross with fast-growth germplasms to produce triploids pyramiding desirable traits of fast growth, easy cutting propagation, and salt and drought tolerances.

Spontaneous Regeneration of Plantlets Derived from Hairy Root Cultures of Lopezia racemosa and the Cytotoxic Activity of Their Organic Extracts

A histological analysis was performed with the aim of elucidating the spontaneous regeneration process of the hairy root lines LRT 2.3 and LRT 6.4, derived from Lopezia racemosa leaf explants and genetically transformed with the Agrobacterium rhizogenes strain ATCC15834/pTDT. The analysis showed both lines regenerate via indirect somatic embryogenesis; LRT 6.4 also regenerated by direct organogenesis. The morphogenic characteristics of the regenerated plantlets from both lines showed the typical characteristics, described previously, including a higher number of axillary shoot formation, short internodes, and plagiotropic roots compared with wild-type seedlings. The regeneration process occurred without the addition of plant growth regulators and was linked to the sucrose concentration in the culture medium. Reducing the sucrose concentration from 3% to 2%, 1%, and 0.5% increased the regeneration rate in LRT 6.4; the effect was less pronounced in LRT 2.3. The cytotoxic activity of different organic extracts obtained from roots and shoots were evaluated in the cancer cell lines HeLa (cervical carcinoma), HCT-15 (colon adenocarcinoma), and OVCAR (ovary carcinoma). The hexane and dichloromethane extracts from roots of both lines showed cytotoxic activity against the HeLa cell line. Only the dichloromethane extract from the roots of PLRT 2.3 showed cytotoxic activity against the OVCAR cell line. None of the methanol extracts showed cytotoxic activity, nor the shoot extracts from any solvent.

Transcriptomic and physiological analysis identifies a gene network module highly associated with brassinosteroid regulation in hybrid sweetgum tissues differing in the capability of somatic embryogenesis

Somatic embryogenesis is a preferred method for large-scale production of forest trees due to its high propagation efficiency. In this study, hybrid sweetgum leaves with phase changes from mature to embryogenic state were selected as experimental material to study somatic embryo initiation. Embryogenicity ranged from high to low, i.e. from 45%, 25%, and 12.5% to 0, with the samples of embryogenic callus (EC), whiten leaf edge (WLI), whiten leaf (WLII), and green leaf (GL) respectively. High correlations existed between embryogenicity and endogenous brassinosteroids (BRs) (r = 0.95, p < 0.05). Similarly, concentrations of endogenous BRs of the sample set correlated positively (r = 0.93, 0.99, 0.87, 0.99, 0.96 respectively, P < 0.05) to expression of somatic embryo (SE)-related genes, i.e. BBM, LEC2, ABI3, PLT2, and WOX2. Hierarchical cluster and weighted gene coexpression network analysis identified modules of coexpressed genes and network in 4820 differentially expressed genes (DEGs) from All-BR-Regulated Genes (ABRG). Moreover, exogenously-supplemented epiBR, together with 2,4-D and 6-BA, increased embryogenicity of GL-sourced callus, and expression of SE- and auxin-related genes, while brassinazole (BRZ), a BR biosynthesis inhibitor, reduced embryogenicity. Evidences obtained in this study revealed that BRs involved in phase change of leaf explants and may function in regulating gene expression and enhancing auxin effects. This study successfully established protocols for inducing somatic embryogenesis from leaf explants in hybrid sweetgum, which could facilitate the propagation process greatly, and provide theoretical basis for manipulating SE competence of explants in ornamental woody plants.

A fast and effective protocol for obtaining genetically diverse stevia (Stevia rebaudiana Bertoni) regenerants through indirect organogenesis

Plant regeneration through indirect organogenesis allows obtaining genetic variability that can be used in the creation of new cultivars. The study presents a fast and effective protocol of one-step preparation of stevia (Stevia rebaudiana Bertoni) regenerants. To obtain callus tissue and shoot regeneration, leaves and nodal segments were used as primary explants, which were placed on MS (Murashige and Skoog) medium supplemented with plant growth regulators (PGRs): NAA (1-naphthaleneacetic acid – 2.0 mg·dm–3, BA (6-benzylaminopurine – 4.0 mg·dm–3), 2,4‑D (2,4-dichlorophenoxyacetic – 2.0 mg·dm–3). Callus tissue was formed on both types of explants, however, only those derived from nodal segments were proliferating. An average of 3.92 shoots per explant were obtained from leaf explants on the applied medium after 6 weeks of culture. The analysis of the morphogenetic capacity of the obtained regenerants was carried out on MS medium supplemented with PGRs – kinetin (0.25 mg·dm–3), BA (0.5 mg·dm–3). The evaluation of the mean number of shoots, mean shoot length (cm), and the mean number of nodes per shoot indicated phenotypic variability of regenerants. The use of RAPD (randomly amplified polymorphic DNA) markers confirmed the differences also at the DNA level. The proposed one-step indirect organogenesis regeneration protocol induced somaclonal variation of Stevia rebaudiana Bertoni and the obtained regenerants, after selection, could be used in the breeding of this species.

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.

Development of An Efficient Protocol for Rapid Propagation of Curculigo orchioides Using Leaf Explant Culture

Curculigo orchioides is one of the most common medicinal plants used by diverse cultures and tribal groups. The roots of the plant are used medicinally in Asian countries. Curculigo orchioides have the ability to regenerate through seeds and tubers, but the regeneration rate is low. Plant tissue culture method was believed to have potential for rapid multiplication of this medicinal plant. An efficient protocol for rapid propagation of Curculigo orchioides, of the family Amaryllidaceae, was developed using leaf explants culture. The leaf explants (1 cm x 1 cm squares) cultured on Murashige and Skoog (MS) basal medium were supplemented with various concentrations and combinations of auxins and cytokinins with temperature 25 ± 2°C, relative humidity 85-90% and photoperiod of 12 hours light (2000-3000 lux). Callus induction was obtained within 4 weeks, 2,4-D at 3 mg/l formed profuse callus and the degree was found to be the highest (+++) among all the treatments. The best response to shoot induction, with maximum shoot number 5.33 (mean number of shoots per explant) was obtained using 1.0 mg/l 6-benzyl aminopurine (BAP) in combination with 1.0 mg/l Kinetin. In vitro shoots were induced for rooting on 0.5 mg/l of NAA supplemented medium. In order for seedlings propagated in vitro to adapt to natural conditions, plants were growned on a substrate coir: husk ash: sand (with the ratio of 0.5: 0.5: 1) in a greenhouse (humidity: 70%, temperature: 28-300C) gave 88.33% survival rate after 8 weeks of culture. With the results received, this is an effective approach to propagating Curculigo orchioides.

Lippia graveolens (Lamiales: Verbenaceae) and Oryganum vulgare (Lamiales: Lamiaceae) obtained by means of the in vitro propagation technique

Objective: The objective of this study was to establish the method of propagation of Oryganum vulgare and Lippia graveolens employing a plant tissue culture technique that decreased the phenolization percentages and increased the multiplication coefficients. Design/ methodology/ approach: The in vitro germination percentage was evaluated in both MS and MS medium + activated carbon. Microcuttings (small shoots) of both species were established in base medium added with different antioxidant agents to decrease the phenolization of explants; the treatments were arranged in a completely randomized block design. For the propagation phase, a completely randomized factorial design was used, where the auxin/cytokinin phytoregulators, type of explants (axillary buds and leaves), and the species (Lippia graveolens and Oryganum vulgare) were considered as factors. Results: maximum germination (63.3% ±12.5) was obtained on day 15 ​​in both culture media for L. graveolens and O. vulgare. The use of antioxidant agents mainly activated carbon, increased the in vitro establishment and activation of vegetative buds in both species by up to 90%. There were significant differences in the variables evaluated regarding the treatments, the explant, and the species in the multiplication phase. The combination 1.0/ 0.5 mg L-1 BA/AIB induces callus formation for both species. When used as leaf explants, callus formation was potentiated. Study Limitations / Implications: The results presented are advances from a long-term experiment. Findings/conclusions: The germination of L. graveolens seeds can be achieved in MS medium after 15 days. Microcuttings of both L. graveolens and O. vulgare were successfully established in MS basal medium enriched with 1 g L-1 charcoal that showed low oxidation percentages and induced up to 90% the production of shoots in the explants. The mixture of 1.0/0.5 mg L-1 BA/AIB induces callus formation for both species; when this medium is in contact with leaves as an explant, its formation is potentiated, achieving diameters up to 15 mm. In order to achieve the induction of shoots and roots, buds should be established in MS medium enriched with 0.5 mg L-1 IBA for both species; this mixture encreased the multiplication coefficients