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.     

Analysis of Secondary Metabolite Production in Somatic Embryo of Pasak Bumi (Eurycoma Longifolia Jack.)

Pasak bumi (Eurycoma longifolia Jack.) has been known as a plants that can produce secondary metabolites for medicinal purposes such as: aphrosidiac, antimalaria, dysentri, antitumor, etc. Poor seed germination of pasak bumi will affect the avaibility of plant material for drug extraction. Over exploitation of this plant will also reduce plant population in its natural habitat. In vitro culture, i.e. through somatic embryogenesis, therefore, can be used as one of an alternative method for plant regeneration as well as for in vitro metabolite production. Based on this reason, the research has been done with an objective to analyze the presence of secondary metabolite in somatic embryo of pasak bumi. Seed-derived callus was used as an explant. This callus was maintained to proliferate in MS (Murashige&Skoog, 1962) medium supplemented with 2.25 mg/L 2,4-D and 2.0 mg/L kinetin. A half gram of callus from proliferation medium was transferred into the MS liquid medium containing 1.0 or 2.25 mg/L 2,4-D, and 2.0 mg/L BAP or 2.0 mg/L kinetin. Histochemical examination using Jeffrey's reagen and neutral red showed that alkaloid and terpenoid substances were presence in somatic embryo of pasakbumi. In accordance with histochemical test, GC-MS analyses showed that secondary metabolites was also synthesized by non embryogenic callus and the mixture ofembryogenic callus and somatic embryo, although the concentration in the mixture of embryogenic callus and somatic embryo was lower than those in non-embryogenic callus. Secondary metabolites, including 3-[(cyclohexyl-methyl-amino)-methyl]-3H-benzooxazole-2-one (0.06%) and 2-furancarboxaldehyde, 5-(hydroxymethyl) (43.024%) were found in embryogenic callus and somatic embryo. In addition, the mixture of embryogenic callus and somatic embryo also synthesized fatty acid and lipids (52.751%) which was higher than non-embryogenic callus (24.789%). Based on the result, the mixture of embryogenic callus and somatic embryo could produce secondary metabolites, such as alkaloid, terpenoid subtances, and phenol. The concentration of metabolites in the mixture of embryogenic callus and somatic embryo, however, was lower compare to non-embryogenic callus.     

Somatic Embryogenesis and Synthetic Seed Production—a Biotechnological Approach for True-to-Type Propagation and In Vitro Conservation of an Ornamental Bulbaceous Plant Drimiopsis kirkii Baker.

An efficient plant regeneration protocol through indirect somatic embryogenesis pathway via callus had been developed from the leaf explant of an ornamental bulbaceous plant Drimiopsis kirkii. Optimum friable calli were induced on Murashige and Skoog (MS) basal medium supplemented with 3.0 mg/l of 2,4-dichlorophenoxyacetic acid and 1.0 mg/l of α-naphthalene acetic acid (NAA). On subculturing the callus on MS medium supplemented with 2.5 mg/l of thidiazuron (TDZ), 73.3 % of the cultures responded with 20.4?±?0.3 somatic embryos (SEs) per 500 mg callus at different stages of development after 6 weeks of culture. The highest response of 86.7 % with 28.3?±?0.5 embryos per 500 mg callus was observed on MS medium supplemented with 2.5 mg/l TDZ and 1.0 mg/l NAA. SEs were encapsulated in calcium alginate beads for the production of synthetic seeds (SSs) and their storability was investigated. The highest SS germination (93.3 %) was observed in 1.0 % sodium alginate followed by 86.7 % germination with 2.5 % sodium alginate. The SSs were stored at three different temperatures (4, 15, and 24?ºC) up to 6 months. The SSs kept at 15 °C showed 64.4 % germinability even after 4 months of storage. Both nonencapsulated and encapsulated SE-derived plants were successfully transferred to soil with 93.3 and 88.3 % survival rate accordingly. Randomly amplified polymorphic DNA (RAPD) analysis revealed that there were no somaclonal variations among the plants produced via somatic embryogenesis and they are true-to-type to their parental plant. These results confirmed the most reliable methods, which can be further used for genetic transformation studies as well as for mass propagation of ornamental D. kirkii at a commercial level.     

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.     

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.     

Plantlet Regeneration from Callus Cultures of Selected Genotype of <Emphasis Type="Italic">Aloe vera</Emphasis> L.—An Ancient Plant for Modern Herbal Industries

Aloe vera L., a member of Liliaceae, is a medicinal plant and has a number of curative properties. We describe here the development of tissue culture method for high-frequency plantlet regeneration from inflorescence axis-derived callus cultures of sweet aloe genotype. Competent callus cultures were established on 0.8% agar-gelled Murashige and Skoog’s (MS) basal medium supplemented with 6.0 mg l⁻¹ of 2,4-dichlorophenoxyacetic acid (2,4-D) and 100.0 mg l⁻¹ of activated charcoal and additives (100 mg l⁻¹ of ascorbic acid, 50.0 mg l⁻¹ each of citric acid and polyvinylpyrrolidone, and 25.0 mg l⁻¹ each of l-arginine and adenine sulfate). The callus cultures were cultured on MS medium containing 1.5 mg l⁻¹ of 2,4-D, 0.25 mg l⁻¹ of Kinetin (Kin), and additives with 4% carbohydrate source for multiplication and long-term maintenance of regenerative callus cultures. Callus cultures organized, differentiated, and produced globular embryogenic structures on MS medium with 1.0 mg l⁻¹ of 2,4-D, 0.25 mg l⁻¹ of Kin, and additives (50.0 mg l⁻¹ of ascorbic acid and 25.0 mg l⁻¹ each of citric acid, l-arginine, and adenine sulfate). These globular structures subsequently produced shoot buds and then complete plantlets on MS medium containing 1.0 mg l⁻¹ of 6-benzylaminopurine and additives. A hundred percent regenerated plantlets were hardened in the greenhouse and stored under an agro-net house/nursery. The regeneration system defined could be a useful tool not only for mass-scale propagation of selected genotype of A. vera, but also for genetic improvement of plant species through genetic transformation.     

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.     

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.     

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.     

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.     

Specific Proteins in the Indirect Somatic Embryogenesis of Freesia Refracta

IntroductionHaberlandt[1]proposed the conception of plantso-matic embryo in 1902, which states that plant cellsexhibit totipotency; each cell could divide ceaselesslyand eventually develops into a mature plantlet. Allthese provide a theoretical evidence for somatic embryo-genesis. The related researches reveal thatplantembry-ogenesis occurs as a result of the selective expression ofsets of genes that allowthe synthesis of special proteinsin a temporal and spatial sequential manner under themut…     

A Comparative Study of Anti-Candida Activity and Phenolic Contents of the Calluses from Lythrum salicaria L. in Different Treatments

In the study, anti-Candida activity and phenol contents of Lythrum salicaria L. calli and wild species have been evaluated. The seeds of L. salicaria (Lythraceae), collected from Lahidjan City in the north of Iran, were cultured in Murashige and Skoog medium (MSM) with a supplement, gibberellin, to germinate. Callus inductions were performed from segments of seedling on MSM containing different concentrations of plant growth regulators, 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzylaminopurine (BAP). The activity of calluses extracts, wild plant, gallic acid, and 3,3′,4′-tri-O-methylellagic acid-4-O-β-d-glucopyranoside (TMEG) as the main phenolic compounds against Candida albicans was assessed using cup plate diffusion method. The total phenols contents of calli and wild plant extracts were analyzed using Folin–Ciocalteu reagent. The callus formation in MSM supplemented with various concentrations of 2,4-D and BAP were 0–100 %. Anti-Candida activity of callus extract which obtained from MSM supplemented with 2,4-D and BAP (1 mg?dm^?3) was similar to the wild plant extract. Minimum inhibitory concentration values of gallic acid and TMEG were obtained as 0.312 and 2.5 mg?cm^?3, respectively. Gallic acid equivalent values in all treatments were from 0 to 288 μg GAE mg^?1. Phenolic contents of plant aerial parts (331?±?3.7 μg GAE mg^?1) and the callus, which developed in MSM including 1 mg?dm^?3 of both 2,4-D and BAP, showed the same phenolic value and exhibited anti-Candida extract activity.     

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.     

PLANT REGENERATION FROM IMMATURE COTYLEDONOUS TISSUE CULTURES OF SOYBEAN BY SOMATIC EMBRYOGENESIS

Genetic engineering of soybean is limited because of the difficulty of regeneration of plants via in vitro culture. Here we report that a method of obtaining somatic embryos and regenerated plants of soybean has been developed. It is achieved by culturing immature cotyledons of soybean on modified Murashige-Skoog medium supplemented with high concentration of either napthalene acetic acid (NAA) or 2,4-dichlorophenoxyacetic acid (2,4-D). High induction frequencies of 85% and 94% are reached by using 10mg/l NAA and 5mg/l 2,4-D, respectively. A number of regenerated plantlets of soybean are transplanted to sterilized soil and grown into 15 full plants in pots.     

Ex Situ Conservation of Phyllanthus fraternus Webster and Evaluation of Genetic Fidelity in Regenerates Using DNA-Based Molecular Marker

Germplasm storage of Phyllanthus fraternus by using synseed technology has been optimized. Synseeds were prepared from nodal segments taken from in vitro-grown plantlets. An encapsulation matrix of 3 % sodium alginate and 100 mM calcium chloride with polymerization duration up to 15 min was found most suitable for synseed formation. Maximum plantlet conversion (92.5?±?2.5 %) was obtained on a growth regulator-free ½-strength solid Murashige and Skoog (MS) medium. Multiple shoot proliferation was optimum on a ½ MS medium containing 0.5 mg/l 6-benzylaminopurine (BAP). Shoots were subjected to rooting on MS media containing 1 mg/l α-naphthaleneacetic acid (NAA) and acclimatized successfully. Encapsulated nodal segments can be stored for up to 90 days with a survival frequency of 47.33 %. The clonal fidelity of synseed-derived plantlets was also assessed and compared with that of the mother plant using rapid amplified polymorphic DNA and inter-simple sequence repeat analysis. No changes in molecular profiles were observed among the synseed-derived plantlets and mother plant, which confirms the genetic stability of regenerates. This synseed production protocol could be useful for in vitro multiplication, short-term storage, and exchange of germplasm of this important antiviral and hepatoprotective plant.     

An Efficient Method for Agrobacterium-Mediated Genetic Transformation and Plant Regeneration in Cumin (Cuminum cyminum L.)

Cumin is an annual herbaceous medicinally important plant having diverse applications. An efficient and reproducible method of Agrobacterium-mediated genetic transformation was herein established for the first time. A direct regeneration method without callus induction was optimised using embryos as explant material in Gamborg’s B5 medium supplemented with 0.5-μM 6-benzyladenine and 2.0-μM α-naphthalene acetic acid. About 1,020 embryos (a mean of 255 embryos per batch) were used for the optimisation of transformation conditions. These conditions were an Agrobacterium cell suspension of 0.6 OD₆₀₀, a co-cultivation time of 72 h, 300-μM acetosyringone and wounding of explants using a razor blade. Pre-cultured elongated embryos were treated using optimised conditions. About 720 embryos (a mean of 180 embryos per batch) were used for transformation and 95 % embryos showed transient β-glucuronidase expression after co-cultivation. Putative transformed embryos were cultured on B5 medium for shoot proliferation and 21 regenerated plants were obtained after selection and allowed to root. T₀ plantlets showed β-glucuronidase expression and gene integration was confirmed via PCR amplification of 0.96 and 1.28 kb fragments of the hygromycin-phosphotransferase II and β-glucuronidase genes, respectively. In this study, a transformation efficiency of 1.5 % was demonstrated and a total of 11 transgenic plants were obtained at the hardening stage, however, only four plants acclimatised during hardening. Gene copy number was analysed by Southern blot analysis of hardened plants and single-copy gene integration was observed. This is the first successful attempt of Agrobacterium-mediated genetic transformation of cumin.     

Molecular and Biochemical Characterization in Rauvolfia tetraphylla Plantlets Grown from Synthetic Seeds Following In Vitro Cold Storage

Synseed technology is one of the most important applications of plant biotechnology for in vitro conservation and regeneration of medicinal and aromatic plants. In the present investigation, synseeds of Rauvolfia tetraphylla were produced using in vitro-proliferated shoots upon complexation of 3 % sodium alginate and 100 mM CaCl₂. The encapsulated buds were stored at 4, 8, 12, and 16 °C and high conversion was observed in synseeds stored at 4 °C for 4 weeks. The effect of different medium strength on in vitro conversion response of synseed was evaluated and the maximum conversion (80.6 %) into plantlets was recorded on half-strength woody plant medium supplemented with 7.5 μM 6-benzyladenine and 2.5 μM α-naphthalene acetic acid after 8 weeks of culture. Plantlets with well-developed shoot and roots were hardened and successfully transplanted in field condition. After 4 weeks of transfer to ex vitro conditions, the performance of synseed-derived plantlets was evaluated on the basis of some physiological and biochemical parameters and compared with the in vivo-grown plants. Short-term storage of synthetic seeds at low temperature had no negative impact on physiological and biochemical profile of the plants that survived the storage process. Furthermore, clonal fidelity of synseed-derived plantlets was also assessed and compared with mother plant using rapid amplified polymorphic DNA and inter-simple sequence repeats analysis. No changes in molecular profiles were found among the regenerated plantlets and comparable to mother plant, which confirm the genetic stability among clones. This synseed protocol could be useful for in vitro clonal multiplication, conservation, and short-term storage and exchange of germplasm of this antihypertensive drug-producing plant.     

Antioxidant potential in callus culture of Artemisia amygdalina Decne

This study was conducted to analyse the free radical scavenging potential of callus obtained from nodal segments and leaf explants of Artemisia amygdalina Decne. The explants were inoculated on MS medium augmented with various concentrations of BAP, Kn, NAA and 2,4-D for callus induction. In this study, 12.42?g of callus developed from the leaf explant on MS (NAA 10?+?BAP 7.5?μM) and 8.81?g of callus developed from nodal explant on NAA 2?μM+BAP 2?μM. Callus raised from both explants on all treatments seemed non-regenerative but BAP 2?μM produced 7.33 shoots and BAP 15?μM produced callus and 5 shoots per nodal segment. Callus was analysed for antioxidant activity via DPPH, riboflavin photoxidation and DNA damage assays. Methanol and aqueous extracts show more scavenging in DPPH, deoxyribose assay and in contrast, petroleum ether and ethyl acetate extracts show higher activity in riboflavin photoxidation assay. Tocopherol, ascorbic acid and BHT were used as controls.     

In Vitro Adventitious Shoot Regeneration via Indirect Organogenesis from Petiole Explants of Cassia angustifolia Vahl.—a Potential Medicinal Plant

An effective protocol was developed for in vitro regeneration of the Cassia angustifolia via indirect organogenesis from petiole explants excised from 21-day-old axenic seedlings. Organogenic callus were induced on Murashige and Skoog (MS) medium supplemented with 5.0 μM 2,4-dichlorophenoxy acetic acid and 2.5 μM thidiazuron (TDZ). Adventitious shoot regeneration was achieved on MS medium supplemented with 5.0 μM TDZ as it induced 8.5 ± 0.98 shoots in 85% cultures. The number of shoots and shoot length was significantly enhanced when cultures were subcultured on auxin–cytokinin-containing medium. The highest number of shoots (12.5 ± 1.10) and shoot length (4.3 ± 0.20 cm) was recorded on MS medium supplemented with 5.0 μM TDZ and 1.5 μM indole-3-acetic acid. Regenerated shoots were rooted best on MS medium supplemented with 10.0 μM indole-3-butyric acid followed by their transfer to liquid MS filter paper bridge medium. The plants were successfully hardened off in sterile soilrite followed by their establishment in garden soil with 70% survival rate. The plants showed normal morphological characteristics similar to the field grown plants.     

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.