Thidiazuron-Induced Changes in Biomass Parameters,Total Phenolic Content,and Antioxidant Activity in Callus Cultures of Artemisia absinthium L.

Callus culture of Artemisia absinthium L. was established for enhanced production of phenolics and higher antioxidant activity. Callus was induced from seed-derived leaf explants, incubated on to MS media supplemented with thidiazuron (TDZ; 0.5–5.0 mg/l) either alone or in combination with α-naphthalene acetic acid (NAA; 1.0 mg/l). These callus cultures were investigated for their growth kinetics, total phenolic content, and antioxidant activity on weekly basis for a period of 49 days. Maximum dry biomass accumulation of 8.73 g/l was observed on day 42 in response to 1.0 mg/l TDZ and 1.0 mg/l NAA. Furthermore, maximum level of total phenolic content of 8.53 mg GAE/g DW and highest 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity of 72.6 % were observed in calli formed in response to 1.0 mg/l TDZ on day 42. The results showed a positive correlation of total phenolic content and DPPH radical scavenging activity in most of the callus cultures of A. absinthium L.     

In Vitro Propagation of Dioscorea alata var. purpurae

Dioscorea alata var. purpurae (Indian purple yam) is an important source of diosgenin, a triterpenoid that is used as a raw material in the synthesis of corticosteroid hormones. These drugs are used for the treatment of pharmacological conditions such as arthritis. This paper reports in vitro propagation of explants of various parts of Dioscorea—tuber, leaves, and nodes. Murashige and Skoog media supplemented with hormones and additives was used to get maximum callus initiation and shoot/root induction. All the cultures were maintained at 25?±?2 °C under cool-white fluorescent tubes with 16-h photoperiod. Callus initiation was observed from 8th to 11th day of inoculation, and subsequent root/shoot was initiated in nodal callus after 21 days. Hormones such as kinetin, indole-3-acetic acid, indole-3-butyric acid, α-naphthalene acetic acid, and thiadizuron did not show significant enhancement. Also, there was no need for supplementing additives (silver nitrate, glutamine, l-asparagine monohydrate, polyethylene glycol). Combination of 6-benzylaminopurine (0.2 ppm) and 2,4-dichlorophenoxyacetic acid (2 ppm) hormones gave the best results, and all parts of the plants gave similar callus induction.     

Effect of Various Growth Hormone Concentration and Combination on Callus Induction,Nature of Callus and Callogenic Response of Nerium odorum

Nerium odorum, Linn. (Apocynaceae) is an important evergreen shrub. It is heat, salinity and drought tolerant. Plants with milky sap have medicinal value, mainly cardenolides, flavonoids and terpenes. It is used for wastewater purification and for restoration of riparian woodlands. In view of these facts, the study was conducted for micropropagation of N. odorum. Murashige and Skoog (MS) media supplemented with different concentrations (0.5–10.0 mg/l) of 1-naphthaleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzylaminopurine (BAP) and kinetin (Kin) were used singly and in combinations. Among all the growth hormones, 2,4-D was the best for callus induction (75 % in stem and 79 % in leaf) and in combination 2,4-D and BAP (78 % in stem and 81 % in leaf). The day of callus induction started from the 19th to the 37th day. This variation is due to the differences in culture conditions and the age of explants. The fresh and dry weight and moisture content showed good growth of callus, which is used in further studies of alkaloid production. Micropropagation of this plant allows the production of clones at a fast rate and in continuous manner. This work can lead to the development of an efficient protocol for callus induction and other issues.     

Acceleration of Adventitious Shoots by Interaction Between Exogenous Hormone and Adenine Sulphate in Althaea Officinalis L.

In the current study attempts were made to investigate the effects of three different phases of callus induction followed by adventitious regeneration from leaf segments (central and lateral vein). Callus induction was observed in Murashige and Skoog’s (MS) medium supplemented with 15.0 μM 2,4-dichloro phenoxy acetic acid (2,4-D). Adventitious shoot buds formation was achieved on MS medium supplemented with 7.5 μM 2,4-D and 20.0 μM AdS in liquid medium as it induced 19.2?±?0.58 buds in central vein explants. Addition of different growth regulators (cytokinins—6-benzyladenine, kinetin and 2-isopentenyl adenine alone or in combination with auxins—indole-3-acetic acid, indole-3-butyric acid and α-naphthalene acetic acid, improved the shoot regeneration efficiency, in which 5.0 μM 6-benzyl adenine along with 0.25 μM α-naphthalene acetic acid was shown to be the most effective medium for maximum shoot regeneration (81.3 %) with 24.6 number of shoots and 4.4?±?0.08 cm shoot length per explant. Leaf culture of central veins led to better shoot formation capacity in comparison to lateral vein. Rooting was readily achieved on the differentiated shoots on 1/2 MS medium augmented with 20.0 μM indole-3-butyric acid. The plants were successfully hardened off in sterile soilrite followed by their establishment in garden soil with 80 % survival rate.     

Shoot organogenesis from root-derived callus of Rhinacanthus nasutus (L.) Kurz. and assessment of clonal fidelity of micropropagted plants using RAPD analysis

An efficient regeneration system was established for an ethnomedicinal shrub Rhinacanthus nasutus from root-derived callus organogenesis. The root segments were cultured on MS medium supplemented with various concentrations of Kn (1.0–4.0 μM) alone or in combination with IBA (0.2–0.6 μM) or 2, 4-D (0.5–1.5 μM). The optimum frequency (94 %) of callus induction was recorded on MS medium supplemented with 3.0 μM Kn and 0.4 μM IBA. For shoot regeneration from callus, MS medium supplemented with different concentrations (1.0–7.0 μM) of BA or TDZ alone or in combination with NAA (0.2–1.0 μm) was employed. The highest frequency of shoot regeneration (91 %) and mean number of shoots (28.3) were observed on MS medium supplemented with 5.0 μM BA and 0.7 μM NAA. The shoots were excised and cultured on MS medium with 4.0 μM IBA produced 3.4 roots per shoot in 88 % cultures. Of the 65 plants transferred to soil 54 survived (83 %). The plants were transferred to field after successful hardening. RAPD analysis of the regenerated plants showed high similarity with the mother plant.     

Production of a new mucilage compound in Lepidium sativum callus by optimizing in vitro growth conditions

The mucilage in Lepidium sativum L. is considered a biologically active compound with diverse medicinal properties. Different explants (hypocotyls and leaf) were transferred to Murashige and Skoog (MS) medium supplemented with twelve different plant growth regulator combinations under two different incubations (light and dark). The best mucilage production from callus (36.76% g g^?1 dry weight) was obtained in the MS medium supplemented with 1 mg L^?1 of 2, 4-D and 2 mg L^?1 of BAP under the light condition. The mucilage produced by callus culture was nearly three times more than the mucilage yield of the seeds. The glucose, arabinose + mannose and galactose were 43.4 (mg g^?1 DW), 195.3 (mg g^?1 DW) and 86.2 (mg g^?1 DW) in the mucilage originated from seed, callus leaf and callus hypocotyl, respectively. The present study proposes an efficient method for producing large scales of mucilage with a favorable sugar aimed at food or pharmaceutical industries.     

Optimization of Adventitious Root Culture for Production of Biomass and Secondary Metabolites in Prunella vulgaris L.

Adventitious root cultures of Prunella vulgaris L. were established in shaking flask system for the production of biomass and secondary metabolites. Adventitious root cultures were induced from callus cultures obtained from leaf explants on solid Murashige and Skoog (MS) medium containing combination of 6-benzyladenine (BA; 1.0 mg l^?1) and naphthalene acetic acid (NAA; 1.5 mg l^?1). Thereafter, 0.49 g inoculum was transferred to liquid MS medium supplemented with different concentrations of NAA (0.5–2.0 mg l^?1). Growth kinetics of adventitious roots was recorded with an interval of 7 days for 49 days period. Highest biomass accumulation (2.13 g/l) was observed in liquid medium containing 1.0 mg l^?1 NAA after 21 days of inoculation. However, other concentrations of NAA also showed similar accumulation pattern but the biomass gradually decreases after 49 days of inoculation. Adventitious roots were collected and dried for investigation of total phenolics (TP), total flavonoids (TF), and antioxidant activities. Higher TPC (0.995 GAE mg/g-DRB) and TFC (6.615 RE mg/g-DRB) were observed in 0.5 mg l^?1 NAA treated cultures. In contrast, higher antioxidant activity (83.53 %) was observed 1.5 mg l^?1 NAA treated cultures. These results are helpful in up scaling of root cultures into bioreactor for secondary metabolites production.     

Effects of polysaccharide elicitors from endophytic Fusarium oxysporium Dzf17 on growth and diosgenin production in cell suspension culture of Dioscorea zingiberensis

Three polysaccharides, namely exopolysaccharide (EPS), water-extracted mycelial polysaccharide (WPS) and sodium hydroxide-extracted mycelial polysaccharide (SPS), were prepared from the endophytic fungus Fusarium oxysporium Dzf17 isolated from the rhizomes of Dioscorea zingiberensis. The effects of the time of addition and polysaccharide concentration on the growth and diosgenin accumulation in cell suspension culture of D. zingiberensis were studied. Among them, WPS was found to be the most effective polysaccharide. When WPS was added to the medium at 20 mg/L on the 25th day of culture, the cell dry weight was increased 1.34-fold, diosgenin content 2.85-fold, and diosgenin yield 3.83-fold in comparison to those of control. EPS and SPS showed moderate and relatively weak enhancement effects on cell growth and diosgenin accumulation, respectively. The dynamics of cell growth and diosgenin accumulation when WPS was added to the medium at 20 mg/L on the 25th day of culture were investigated, and results showed that dry weight of cells reached a maximum value on day 30 but the maximum diosgenin content was achieved on day 31.     

Plant Regeneration Through Callus Organogenesis and True-to-Type Conformity of Plants by RAPD Analysis in Desmodium gangeticum (Linn.) DC.

An efficient plant regeneration protocol was established for an endangered ethnomedicinal plant Desmodium gangeticum (Linn.) DC. Morphogenic calli were produced from 96 % of the cultures comprising the immature leaf explants on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid (4.0 mg?l^?1) in combination with 6-benzylaminopurine (BA; 0.8 mg?l^?1). For callus regeneration, various concentrations of BA (1.0–5.0 mg?l^?1) or thidiazuron (TDZ; 1.0–5.0 mg?l^?1) alone or in combination with indole-3-acetic acid (IAA; 0.2–1.0 mg?l^?1) were used. Highest response of shoot regeneration was observed on MS medium fortified with TDZ (4.0 mg?l^?1) and IAA (0.5 mg?l^?1) combination. Here, 100 % cultures responded with an average number of 22.3 shoots per gram calli. Inclusion of indole-3-butyric acid in half MS medium favored rooting of recovered shoots. Out of 45 rooted plants transferred to soil, 40 survived. Total DNA was extracted from the leaves of the acclimatized plants of D. gangeticum. Analysis of random amplified polymorphic DNA using 13 arbitrary decanucleotide primers showed the genetic homogeneity in all the ten plants regenerated from callus with parental plant, suggesting that shoot regeneration from callus could be used for the true-to-type multiplication of this plant.     

Callus induction and plant regeneration from different explants of Actinidia deliciosa

In this study, an efficient procedure was developed for callus induction and regeneration of kiwifruit (Actinidia deliciosa) using different organs of shoots developed under in vitro conditions. Effects of explants source and media (M₁, 1.0 mg l⁻¹ BA + 2.0 mg l⁻¹ 2,4-D–M₂, 1.0 mg l⁻¹ NAA + 2.0 mg l⁻¹ 2,4-D) on initiation of callus were examined in order to obtain callus for organogenesis. The best callus for plant regeneration was obtained from leaf explants on Murashige and Skoog’s medium (MS) supplemented with M₂. Formation of callus from leaf of kiwifruit (A. deliciosa) was cultured in MS medium containing different concentration of N⁶-benzylaminopurin (BA; 0.0, 1.0, 2.0, 4.0, 6.0, 8.0 mg l⁻¹) for callus proliferation and plant regeneration. Although the first shoot formation was appeared in medium containing 6.0 and 8.0 mg l⁻¹ BA, the best shoots formation was obtained in medium with 4.0 mg l⁻¹ BA.     

Shoot Multiplication and Callus Induction of Labisia pumila var. alata as Influenced by Different Plant Growth Regulators Treatments and Its Polyphenolic Activities Compared with the Wild Plant

This study aims to investigate whether the in vitro-cultured L. pumila var. alata has higher antioxidant activity than its wild plant. An 8-week-old L. pumila var. alata nodal segment and leaf explants were cultured onto Murashige and Skoog (MS) medium supplemented with various cytokinins (zeatin, kinetin, and 6-benzylaminopurine (BAP)) for shoot multiplication and auxins (2,4-dichlorophenoxyacetic acid (2,4-D) and picloram) for callus induction, respectively. The results showed that 2 mg/L zeatin produced the optimal results for shoot and leaf development, and 0.5 mg/L 2,4-D produced the highest callus induction results (60%). After this, 0.5 mg/L 2,4-D was combined with 0.25 mg/L cytokinins and supplemented to the MS medium. The optimal results for callus induction (100%) with yellowish to greenish and compact texture were obtained using 0.5 mg/L 2,4-D combined with 0.25 mg/L zeatin. Leaves obtained from in vitro plantlets and wild plants as well as callus were extracted and analyzed for their antioxidant activities (DPPH and FRAP methods) and polyphenolic properties (total flavonoid and total phenolic content). When compared with leaf extracts of in vitro plantlets and wild plants of L. pumila var. alata, the callus extract displayed significantly higher antioxidant activities and total phenolic and flavonoid content. Hence, callus culture potentially can be adapted for antioxidant and polyphenolic production to satisfy pharmaceutical and nutraceutical needs while conserving wild L. pumila var. alata.     

Improved Protocol for Somatic Embryogenesis and Calcium Alginate Encapsulation in Anethum graveolens L.: A Medicinal Herb

An improved procedure has been developed for efficient somatic embryogenesis in Anethum graveolens. Green friable embryogenic callus was obtained from hypocotyl segments on medium augmented with 2,4-dichlorophenoxyacetic acid (2,4-D). The highest embryogenic callus induction frequency of 87 % was obtained on Murashige and Skoog (MS) medium containing 1.13 μM 2,4-D. At lower concentration of 2,4-D (0.34 μM) callus turned dark in color and slow growing. Embryogenic cultures (76 %) responded with a mean number of 43 globular and 18 heart stage embryos. Somatic embryo maturation and subsequent conversion into plantlets took place on MS lacking growth regulators. Maximum number of somatic embryos developed on MS medium was 128.3 (per flask) and a plantlet conversion of 82 % was observed. Calcium alginate beads were produced by encapsulating somatic embryos. Highest percent germination (83 %) was observed on 0.8 % agar solidified MS medium with the plantlets acquiring an average length of 2.1 cm. Encapsulated somatic embryos could be stored at 4 °C up to 60 days with a conversion frequency of 49.3 %. Highest protein and proline content has been observed in embryogenic callus with small globular embryos. During morphological differentiation of the somatic embryos, changes in the antioxidant enzymatic system were observed. Superoxide dismutase (SOD) activity increased during initial stages and decreased catalase (CAT), peroxidase (POD), and ascorbate peroxidase (APX) activities were detected.     

Regeneration and Agrobacterium-mediated transformation of the apomictic species Eulaliopsis binata

Protocols for regeneration and Agrobacterium-mediated transformation of the apomictic species Eulaliopsis binata were developed. Initially, seeds of four genotypes of E. binata were incubated on a callus induction Murashige and Skoog (MS) basal medium supplemented with three concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D). It was found that 36.2 % of explants developed highly friable callus on medium containing 3.0 mg l^?1 2,4-D. Based on frequency of callus induction, the genotype Neixiang was selected for regeneration and transformation. Callus incubated on MS basal medium supplemented with 0.2 mg l^?1 α-naphthalene acetic acid and 6.0 mg l^?1 6-furfuryl-aminopurine developed shoots. Subsequently, Agrobacterium tumefaciens strain EHA105—harboring a plasmid pCAMBIA1381 carrying a hygromycin phosphotransferase (hpt) resistance gene and a synthetic green fluorescent protein (GFP) gene, both driven by the cauliflower mosaic virus 35S promoter—was used for transformation system. Putative transgenic callus was obtained following two cycles of hygromycin selection. Expression of the transgene(s) in putative transgenic callus was analyzed using the GFP detection. Molecular identification of putative transformed shoots was performed by polymerase chain reaction and Southern blot analysis to confirm presence and integration of the hpt gene.     

Effect of Adenine Sulphate Interaction on Growth and Development of Shoot Regeneration and Inhibition of Shoot Tip Necrosis Under In Vitro Condition in Adult Syzygium cumini L.—a Multipurpose Tree

An efficient method for cloning Syzygium cumini (above 40 years old) through mature nodal segments has been successfully developed and that could be exploited for large-scale production of this valuable multipurpose tree. Nodal segments from mature tree were taken as explants and cultured on MS basal medium with different cytokinins (BA, Kin, AdS). The application of BA proved to be the best responsive cytokinin for the induction of shoot buds and shoots, but the proliferated shoots exhibited slower and stunted growth accompanied with abscission of leaves and shoot tip necrosis (STN). The problem of leaf abscission and STN was considerably reduced by the application of an adjuvant, adenine sulphate (AdS) in the optimal medium which led to the production of a maximum of 14 shoots. Further improvement in shoot bud regeneration and improved growth pattern of the regenerating tissue was obtained on the media comprised of MS?+?BA (10 μM)?+?GA₃ (2.5 μM). A total number of 15 shoots with mean shoot length of 5.9 cm was obtained. The healthy elongated shoots were then rooted on MS basal augmented with NAA (5 μM). The plantlets obtained were healthy and were successfully acclimatized and transferred under field condition with 70 % survival rate.     

Scarlet Flax Linum grandiflorum (L.) In Vitro Cultures as a New Source of Antioxidant and Anti-Inflammatory Lignans

In vitro cultures of scarlet flax (Linum grandiflorum L.), an important ornamental flax, have been established as a new possible valuable resource of lignans and neolignans for antioxidant and anti-inflammatory applications. The callogenic potential at different concentrations of α-naphthalene acetic acid (NAA) and thidiazuron (TDZ), alone or in combinations, was evaluated using both L. grandiflorum hypocotyl and cotyledon explants. A higher callus induction frequency was observed on NAA than TDZ, especially for hypocotyl explants, with a maximum frequency (i.e., 95.2%) on 1.0 mg/L of NAA. The presence of NAA (1.0 mg/L) in conjunction with TDZ tended to increase the frequency of callogenesis relative to TDZ alone, but never reached the values observed with NAA alone, thereby indicating the lack of synergy between these two plant growth regulators (PGRs). Similarly, in terms of biomass, NAA was more effective than TDZ, with a maximum accumulation of biomass registered for medium supplemented with 1.0 mg/L of NAA using hypocotyls as initial explants (DW: 13.1 g). However, for biomass, a synergy between the two PGRs was observed, particularly for cotyledon-derived explants and for the lowest concentrations of TDZ. The influence of these two PGRs on callogenesis and biomass is discussed. The HPLC analysis confirmed the presence of lignans (secoisolariciresinol (SECO) and lariciresinol (LARI) and neolignan (dehydrodiconiferyl alcohol [DCA]) naturally accumulated in their glycoside forms. Furthermore, the antioxidant activities performed for both hypocotyl- and cotyledon-derived cultures were also found maximal (DPPH: 89.5%, FRAP 866: µM TEAC, ABTS: 456 µM TEAC) in hypocotyl-derived callus cultures as compared with callus obtained from cotyledon explants. Moreover, the anti-inflammatory activities revealed high inhibition (COX-1: 47.4% and COX-2: 51.1%) for extract of hypocotyl-derived callus cultures at 2.5 mg/L TDZ. The anti-inflammatory action against COX-1 and COX-2 was supported by the IC₅₀ values. This report provides a viable approach for enhanced biomass accumulation and efficient production of (neo)lignans in L. grandiflorum callus cultures.     

Direct and Indirect Organogenesis of Alpinia galanga and the Phytochemical Analysis

Alpinia galanga is a rhizomatous herb rich in essential oils and various other significant phytoconstituents. Rapid direct regeneration was obtained from the rhizome explants (15.66 ± 0.57 shoots) on MS media supplemented with zeatin at a concentration of 2 mg/l. The callus cultures of A. galanga were initiated from the rhizome explants on MS media supplemented with 2 mg/l each of BAP, 2,4-D, and NAA. The callus was analyzed for the presence of a vital phytoconstituent--acetoxychavicol acetate (ACA) associated with various biological properties. ACA was detected in the young friable callus as well as the stationary phase callus. Moreover, the induction of morphogenetic response in callus resulted in higher accumulation of ACA. The phytohormone withdrawal from the propagation media and the subsequent transfer of callus to BAP (2 mg/l) containing MS media has resulted in multiple shoot induction. The regenerated (indirect) plants have shown 1.6-fold higher ACA content (1.253%) when compared to the control plant (0.783%). Micropropagation of such conventionally propagated plants is very essential to meet the commercial demand as well as to ensure easy storage and transportation of disease free stocks.     

Piper nigrum: Micropropagation, Antioxidative enzyme activities, and Chromatographic Fingerprint Analysis for Quality Control

A reliable in vitro regeneration system for the economical and medicinally important Piper nigrum L. has been established. Callus and shoot regeneration was encouraged from leaf portions on Murashige and Skoog (MS) medium augmented with varied concentrations of plant growth regulators. A higher callus production (90 %) was observed in explants incubated on MS medium incorporated with 1.0 mg?L^?1 6-benzyladenine (BA) along with 0.5 mg?L^?1 gibberellic acid after 4 weeks of culture. Moreover, a callogenic response of 85 % was also recorded for 1.0 mg?L^?1 BA in combination with 0.25 mg?L^?1 α-naphthalene acetic acid (NAA) and 0.25 mg?L^?1 2,4-dichlorophenoxyacetic acid or 0.5 mg?L^?1 indole butyric acid (IBA) along with 0.25 mg?L^?1 NAA and indole acetic acid. Subsequent sub-culturing of callus after 4 weeks of culture onto MS medium supplemented with 1.5 mg?L^?1 thiodiazoran or 1.5 mg?L^?1 IBA induced 100 % shoot response. Rooted plantlets were achieved on medium containing varied concentrations of auxins. The antioxidative enzyme activities [superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX)] revealed that significantly higher SOD was observed in regenerated plantlets than in other tissues. However, POD, CAT, and APX were higher in callus than in other tissues. A high-performance liquid chromatography (HPLC) fingerprint analysis protocol was established for quality control in different in vitro-regenerated tissues of P. nigrum L. During analysis, most of the common peaks represent the active principle “piperine.” The chemical contents, especially piperine, showed variation from callus culture to whole plantlet regeneration. Based on the deviation in chromatographic peaks, the in vitro-regenerated plantlets exhibit a nearly similar piperine profile to acclimated plantlets. The in vitro regeneration system and HPLC fingerprint analysis established here brought a novel approach to the quality control of in vitro plantlets, producing metabolites of interest with substantial applications for the conservation of germplasm.     

Regeneration-Based Quantification of Coumarins (Scopoletin and Scoparone) in <Emphasis Type="Italic">Abutilon indicum</Emphasis> In Vitro Cultures

Abutilon indicum exploited for its immense value has been propagated successfully through multiple shoot induction and somatic embryogenesis. Direct regeneration (8.20?±?0.83 shoots) was achieved from nodal explants using 0.5 mg/l kinetin (Kn) in MS media. The basal callus from nodal explants turned embryogenic on subsequent introduction of 0.2 mg/l TDZ into the Kn-supplemented media, giving rise to somatic embryos. The embryogenic potential of calli expressed in terms of embryo-forming capacity (EFC) increased from 8.15 EFC to 20.95 EFC after plasmolysis. The phytochemical analysis (HPLC) for the presence of scopoletin and scoparone has revealed a unique accumulation pattern, with higher levels of scopoletin during the earlier stages and scoparone in the later stages of development. The embryogenic calli contained the highest amount of coumarins (99.20?±?0.97 and 61.03?±?0.47 μg/gFW, respectively) followed by regenerated plant (9.43?±?0.20 and 36.36?±?1.19 μg/gFW, respectively), obtained via somatic embryogenesis. Rapid multiplication of A. indicum equipped with two potent coumarins is important in order to meet the commercial demand for combat against dreadful diseases, thereby providing a new platform for plant-based drugs and their manufacture on a commercial scale.     

Linnaea borealis L. var. borealis—In Vitro Cultures and Phytochemical Screening as a Dual Strategy for Its Ex Situ Conservation and a Source of Bioactive Compounds of the Rare Species

Linnaea borealis L. (Twinflower)—a dwarf shrub in the Linnaeeae tribe of Caprifoliaceae family—is distributed across the Northern Hemisphere. By means of this study, a reliable protocol for efficient micropropagation of uniform L. borealis L. var. borealis plantlets has been provided for the first time; callus culture was also established. Different initial explants, types of cultures, media systems, and plant growth regulators in Murashige and Skoog (MS) media were tested. Agitated shoot cultures in the liquid media turned out to be the best system for the production of sustainable plant biomass. After stabilization of the callus lines, the highest growth index (c.a. 526%) was gained for callus maintained on MS enriched with picloram. TLC and UHPLC-HESI-HRMS analysis confirmed the presence of phenolic acids and flavonoids, and for the first time, the presence of iridoids and triterpenoid saponins in this species. Multiplication of L. borealis shoot culture provides renewable raw material, allowing for the assessment of the phytochemical profile, and, in the future, for the quantitative analyses and the studies of the biological activity of extracts, fractions, or isolated compounds. This is the first report on in vitro cultures of traditionally used L. borealis rare taxon and its biosynthetic potential.     

The Study of Ascorbate Peroxidase,Catalase and Peroxidase During In Vitro Regeneration of Argyrolobium roseum

Here, we demonstrate the micropropagation protocol of Argyrolobium roseum (Camb.), an endangered herb exhibiting anti-diabetic and immune-suppressant properties, and antioxidant enzymes pattern is evaluated. Maximum callogenic response (60 %) was observed from leaf explant at 1.0 mg L^?1 1-nephthalene acetic acid (NAA) and 0.5 mg L^?1 6-benzyl aminopurine (BA) in Murashige and Skoog (MS) medium using hypocotyl and root explants (48 % each). Addition of AgNO₃ and PVP in the culture medium led to an increase in callogenic response up to 86 % from leaf explant and 72 % from hypocotyl and root explants. The best shooting response was observed in the presence of NAA, while maximum shoot length and number of shoots were achieved based on BA-supplemented MS medium. The regenerated shoots were rooted and successfully acclimatized under greenhouse conditions. Catalase and peroxidase enzymes showed ascending pattern during in vitro plant development from seed while ascorbate peroxidase showed descending pattern. Totally reverse response of these enzymes was observed during callus induction from three different explants. During shoot induction, catalase and peroxidase increased at high rate while there was a mild reduction in ascorbate peroxidase activity. Catalase and peroxidase continuously increased; on the other hand, ascorbate peroxidase activity decreased during root development and acclimatization states. The protocol described here can be employed for the mass propagation and genetic transformation of this rare herb. This study also highlights the importance and role of ascorbate peroxidase, catalase, and peroxidase in the establishment of A. roseum in vitro culture through callogenesis and organogenesis.