μM Α-naphthaleneacetic Acid Research Articles

A system for the transformation and regeneration of the recretohalophyte Limonium bicolor

Limonium bicolor, a typical recretohalophyte, has a specialized salt-secreting structure in the epidermis called the salt gland and plays a significant role in improving saline land. Understanding the molecular mechanisms of salt secretion and salt gland development requires an efficient L. bicolor transformation system, which is described in this report. Leaf explants were incubated with Agrobacterium tumefaciens strain EHA105 harboring the plasmid pTCK303 containing the β-glucuronidase gene (GUS) as the transgene reporter and the hygromycin B resistance gene as a selectable marker. Up to 96.9% of leaves were induced to regenerate shoots on an Murashige and Skoog (MS) medium supplemented with 4.4 μM 6-benzyladenine and 1.1 μM α-naphthaleneacetic acid; roots were induced on the MS medium containing 2.5 μM indole-3-butyric acid. This tissue culture system was suitable for Agrobacterium-mediated transformation of L. bicolor. Pre-cultivated explants (2 d old) were incubated with Agrobacterium (0.6–0.7 at OD600) in a shaking culture for 20 min; the explants and bacterium were co-cultivated for 4 d in the dark before the explants were transferred to a selection medium containing 8 mg/L hygromycin B and 600 mg/L piperacillin sodium (added to prevent continued Agrobacterium growth). Histochemical assays and PCR to detect the GUS gene showed that transformation frequency was 4.43%. Quantitative PCR and Northern blotting further verified the integration and presence of the GUS gene in L. bicolor. This is the first report of an Agrobacterium-based transformation system for L. bicolor. The system will facilitate a research on the identity and function of genes involved in salt gland development and salt secretion.

In vitroregeneration ofCinnamomum tamalaNees from cotyledonary segments

Cinnamomum tamala Nees., a valuable spice-yielding plant of Himalaya, is vulnerable in its natural habitat due to its over-collection for spice and medicinal purpose. In the present study, a successful attempt was made for in vitro shoot bud induction from cotyledons and plant regeneration. The cultured cotyledons of 10–11 wk after flowering induced meristematic response within 4–5 wk of culture where ∼43% explants responded positively on MS medium for tified with 3% sucrose, polyvinyl pyrollidone (100mgL−1) and 6 μM benzyl adenine wherein ∼5–6 shoot buds developed cotyledon−1. Themicro-shoots/shoot buds, developed on initiationmedium, were maintained on MS medium fortified with 3% sucrose and 3 μM kinetin where shoot buds converted into plantlets and supported culture proliferation. Shoots (4 cm long) with fully open leaves were rooted by maintaining on MS medium enriched with 3 μM α-naphthalene acetic acid where 5–6 roots developed per micro-shoot. The rooted plants were transferred to potting mix and maintained in agro-shade ca75% shading where ∼65% transplants survived after two months of transfer.

In vitro regeneration through direct shoot organogenesis in Honey Orange (<i>Citrus tangerina</i>)

Rapid propagation of Honey orange (Citrus tangerina) was achieved by induction of shoots from epicotyl and cotyledonary node explants and rooting of cotyledonary node derived shoots. Significant explant differences were observed in the induction of direct shoots. Cotyledonary node explant is the most efficient in regeneration frequency followed by epicotyl explant. Cotyledonary node explants cultured on Murashige and Skoog (MS) medium supplemented with 8.88 µM N6-benzyladenine (BA) and 0.54 µM α-naphthaleneacetic acid (NAA) developed more than five shoots per explant. The isolated shoots transferred onto the MS medium supplemented with 5.4 µM NAA rooted 100% within 30 days.

Somatic embryogenesis in Manchurian ash: performance of various explant stock trees, optimisation of the basal medium, and simplified plant growth regulator combinations

SummaryManchurian ash (Fraxinus mandshurica Rupr.) is an ecologically and economically important hardwood tree species distributed in Northeast China. Somatic embryogenesis has been achieved in this species. However, to improve the efficiency of somatic embryogenesis in this species, the effects of explant stock tree, basal medium type, and plant growth regulator combination were investigated. Somatic embryos (SEs) of Manchurian ash were induced on cotyledons from young zygotic embryos, and the highest rate of induction was obtained from zygotic cotyledons cultured on 0.5× Murashige and Skoog (MS) medium. When cultured on 0.5× MS medium containing 8.1 µM αnaphthaleneacetic acid (NAA) plus 2.2 µM 6-benzyladenine (BA), the cotyledon explants derived from different stock trees had varying potentials to produce somatic embryogenic tissue. Somatic embryos were formed on only 33 of the 46 stock trees (numbered 1 – 46) tested, and the mean rates of induction ranged from 3 – 67%. Significant differences were observed in the combination of plant growth regulators required for maximum induction of SEs on cotyledon explants obtained from four additional stock trees (numbered 59 – 62), for which the optimal combination was 5.4 – 10.7 µM NAA plus 2.2 µM BA. These tree-specific results imply that breeding source needs to be considered before the potential application of somatic embryogenesis for rapid and large-scale propagation of superior lines of Manchurian ash.

Rapid regeneration and analysis of genetic fidelity and scopoletin contents of micropropagated plants of Spilanthes oleracea L.

SummarySpilanthes oleracea L. is an important medicinal plant used in traditional medicine for curing toothache, stammering, and stomatitis. Axillary shoots were induced from nodal explants on semi-solid and liquid 1.0 × Murashige and Skoog (MS) media supplemented with 1.0, 2.0, 5.0, or 1.0 µM 6-benzylaminopurine (BAP), kinetin (Kn), 2-isopentenyl adenine (2-iP), or thidiazuron (TDZ). The highest number of axillary shoots (mean = 74.3 shoots per explant) were regenerated on liquid 1.0 × MS medium supplemented with 2.0 µM Kn. Liquid medium was found to be more suitable for shoot induction compared to semi-solid medium (mean = 45.5 shoots per explant). Shoots were rooted on semi-solid 0.5 × MS medium supplemented with 10 µM α-naphthaleneacetic acid (NAA). The resulting plantlets were acclimatised in a growth chamber. Random amplified polymorphic DNA (RAPD) marker analysis revealed that the regenerated plants did not exhibit any genetic polymorphism. Scopoletin (6-methoxy-7-hydroxycoumarin) concentrations were determined in regenerated plants as well as in in vivo plants using high performance liquid chromatography (HPLC). Analysis revealed that in vitro-regenerated plants had higher amounts (mean = 3.42 µg g–1 DW) of scopoletin compared to in vivo plants (range 0.01 – 3.04 µg g–1 DW). Liquid 1.0 × MS medium could therefore be used for large-scale propagation of this plant, and the regenerated plants could serve as raw material for the extraction of scopoletin.

Shoot organogenesis and somatic embryogenesis from leaf explants of Valeriana jatamansi Jones

The in vitro leaves of Valeriana jatamansi Jones were used as explants to induce adventitious shoots and somatic embryos from embryogenic callus. Both morphogenic processes could be induced on Murashige and Skoog (MS) basal medium supplemented with different concentrations and combinations of plant growth regulators. Embryogenic callus, which was induced exclusively in the presence of auxins, namely 1.0–7.5μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 5.0μM α-naphthaleneacetic acid (NAA), differentiated into adventitious shoots and somatic embryos, respectively when left on the same medium. Callus, when placed on MS medium containing 5.0μM indole-3-butyric acid (IBA), 5.0μM NAA or 1.0–5.0μM 2,4-D, induced adventitious roots. Cytokinins that were added singly to callus induction medium, including 6-benzyladenine, kinetin and thidiazuron, could not induce callus at 5.0μM except when combined with 0.25–1.0μM NAA.

Direct and indirect shoot and bulblet regeneration from cultured leaf explants of Lilium pumilum, an endangered species

Alternative methods of in vitro cloning that involve both adventitious (direct) and callus intermediate (indirect) pathways were investigated for the endangered species Lilium pumilum. Plantlet regeneration was obtained from leaf explants, cultured on Murashige and Skoog (MS) basal medium supplemented with various combinations of auxins and cytokinins at different concentrations. About 30% of the explants directly formed adventitious shoots on MS medium containing 8.88 μM 6-benzyladenine (BA) and 2.69 μM α-naphthaleneacetic acid (NAA). For production of regenerable callus, callus formation followed by shoot induction was best when explants were initially cultured on MS medium supplemented with 9.05 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Regenerable calli were yellow or purple and readily regenerated shoots when subcultured onto MS medium containing 2.22 μM BA and 1.61 μM NAA. About 78% of the calli were able to produce adventitious shoots. Shoots were rooted on half-strength MS medium supplemented with 1.34 μM NAA and were successfully acclimatized to greenhouse conditions. This report describes an efficient method for the in vitro multiplication of whole plants from leaf explants of the endangered species L. pumilum.

Encapsulation of nodal segments of Cassia angustifolia Vahl. for short-term storage and germplasm exchange

The present study described the encapsulation of nodal segments of Cassia angustifolia Vahl. excised from 1-month-old in vitro raised cultures for short-term conservation and propagation. Various concentrations and combinations of gelling matrix (sodium alginate) and complexing agents (calcium chloride) were tested to prepare uniform beads. The ideal beads were obtained through a combination of 3 % sodium alginate and 100 mM calcium chloride. The maximum conversion response (94 %) of encapsulated beads was obtained in Murashige and Skoog’s medium (MS medium) supplemented with 2.5 μM benzyladenine (BA) and 0.4 μM α-naphthalene acetic acid (NAA) after 6 weeks of culture. The encapsulated and non-encapsulated nodal segments were also stored at 4 °C for different time periods (0, 1, 2, 4, 6 and 8 weeks). The regenerated microshoots were best rooted in optimized rooting medium that comprised half-strength MS + 1.0 μM indole-3-butyric acid (IBA) + 5.0 μM phloroglucinol (PG) for the production of complete plantlets. The regenerated plantlets were successfully hardened and acclimatized in natural conditions with 70 % survival rate.

Rapid regeneration and ploidy stability of 'cv IR36' indica rice (Oryza Sativa. L) confers efficient protocol for in vitro callus organogenesis and Agrobacterium tumefaciens mediated transformation.

BackgroundCereal crops are the major targets for transformation mediated crop improvement and IR36 is an early maturing, high yielding, insect and disease resistant rice variety however, it is abiotic stress sensitive. Hence, development of an efficient and reproducible micropropagation system via somatic embryogenesis and Agrobacterium tumefaciens mediated transformation is prerequisite to develop abiotic stress tolerant IR36. Further, Genetic stability of analysis of plantlets through RAPD and ISSR and Ploidy level through Flow cytometry (FCM) measurement of 2C DNA content is necessary for future application of transformed IR36.ResultsIn this study, Mature seeds inoculated on (Murashige and Skoog) MS medium with 11.31 μM 2, 4-dichlorophenoxyacetic acid (2, 4-D) and 0.3 μM Kinetin (Kn) had highest callus induction frequency (98%). The highest regeneration frequency (80%) was observed in MS + 13.28 μM Benzyladenine (BA) with 8.06 μM α-naphthalene acetic acid (NAA). Randomly Amplified Polymorphic DNA (RAPD), Inter Simple Sequence Repeat (ISSR) and Flow Cytometry (FCM) analysis showed no significant variation in the 2C DNA (0.81 pg/2C) content and Ploidy level between wild type IR36 and in vitro maintained rice lines. Of the various OD bacterial culture, an optimum OD of 0.4 and inoculation duration of 10 min resulted in efficient Agrobacterium-mediated transformation. β-glucuronidase activity was maximum in callus (99.05%).ConclusionsThese results described here confirm the reliability of this protocol for micropropagation and delivery of desirable gene using A. tumefaciens into indica rice.Electronic supplementary materialThe online version of this article (doi:10.1186/1999-3110-54-47) contains supplementary material, which is available to authorized users.

Ιn vitro plant regeneration from leaf explants of the cherry rootstocks CAB-6P, Gisela 6, and MxM 14 using sodium nitroprusside

Conventional multiplication of cherry (Prunus cerasus L.) rootstocks utilizes division, cuttings, and propagation through seed, which are relatively slow and labor intensive and result in genetic variability. Tissue culture, on the other hand, ensures rapid, large-scale, and low-cost production of genetically identical, physiologically uniform, and pathogen-free plants. In the cherry rootstocks CAB-6P, Gisela 6, and MxM 14, sodium nitroprusside (SNP) promoted callus induction, in vitro shoot proliferation, and rooting from leaf explants in a medium containing 17.6 μM benzyladenine and 2.68 μM α-naphthaleneacetic acid. CAB-6P explants treated with 10 μM SNP gave the maximum shoot number (5), whereas 30 μM SNP gave the longest shoots and the greatest shoot induction rate (26.67%). Best rooting was obtained with 50 μM SNP. In Gisela 6 rootstock, the shoot number (10) and shoot length (20.5 mm) were maximal in the control group without plant growth regulators. The shoot induction rate was enhanced (40%) with 40 μM SNP. SNP at 40 μM resulted in root formation, while 30 μM produced the largest callus size, and 10 μM SNP resulted in the maximum callus fresh weight. MxM 14 leaves treated with 30 μM SNP gave the maximum shoot number (3), root number (7.56), and shoot induction rate (40%), whereas 40 μM SNP gave the longest shoots (12 mm) and roots (20 mm). Best results for callus size, callus fresh weight, and callus induction rate (100%) in the CAB-6P and MxM 14 rootstocks were observed with 30 and 40 μM SNP, respectively. Rooted explants with shoots were gradually acclimatized to the external environment with a high survival percentage (85%). An efficient protocol of indirect organogenesis was established for the three cherry rootstocks using SNP.

In vitro plantlet regeneration and Agrobacterium tumefaciens-mediated genetic transformation of Indian Kino tree (Pterocarpus marsupium Roxb.)

The objective of the present study was to develop a protocol for in vitro plantlet regeneration and Agrobacterium tumefaciens-mediated genetic transformation using immature cotyledon explants of Indian Kino tree (Pterocarpus marsupium Roxb.). Immature cotyledon explants excised from 9-day-old axenic seedlings produced optimal callus on Murashige and Skoog (MS) medium supplemented with 1.07 μM α-naphthalene acetic acid (NAA), after 2 weeks of culture. When the above said callus was incubated on MS + 8.90 μM 6-benzylaminopurine (BAP) + 1.07 μM NAA, a regeneration frequency of 60.41 % with shoot number and length 12.2 ± 0.85 and 1.4 ± 0.13, respectively, was observed. For further shoot multiplication and elongation, these cultures were transferred onto MS + 4.40 μM BAP. Elongated shoots dipped in 19.60 μM indole-3-butyric acid (IBA) for 24 h and then cultured on ½MS + 2.85 μM IBA, 75 % shoots developed roots and 95 % of plantlets survived in field condition. Organogenic callus was co-cultivated with the A. tumefaciens strain LBA4404 harboring the binary plasmid pCAMBIA1301with s-glucuronidase (uidA) and hygromycin phosphotransferase (hpt) genes and grown on MS + 8.90 μM BAP + 1.07 μM NAA (RM) + 200 μM acetosyringone for 2 days and then transferred to MS + 8.90 μM BAP + 1.07 μM NAA + 20 mg/l hygromycin + 250 mg/l cefotaxime (SIM) and 4.40 μM BAP + 15 mg/l hygromycin + 200 mg/l cefotaxime (SEM). The putatively transformed shoots were subsequently rooted on ½MS + 2.85 μM IBA + 20 mg/l hygromycin (SRM), after pulse treatment for 24 h with 19.60 μM IBA. Successful gene transfer into putatively transformed plantlets was confirmed by histochemical GUS assay, PCR and RT-PCR analysis. Southern blot analysis of regenerated plantlets confirmed the integration of hpt gene in transgenic plantlets. In the present study, a rate of 20.92 % transformation frequency was achieved and the genetic transformation protocol presented here may pave way for genetic manipulation of this multipurpose legume tree.

Plant regeneration from cell suspension-derived protoplasts of Populus × beijingensis

A protocol for plant regeneration from cell suspension-derived protoplasts of Populus × beijingensis is described. Protoplasts were isolated from cell suspension cultures 6 d after subculture and further cultured in liquid NH4NO3-free Murashige and Skoog (MS) medium supplemented with 0.6 M glucose, 9.05 μM 2,4-dichlorophenoxyacetic acid, and 0.89 μM 6-benzyladenine at a density of 2 × 105 protoplasts per milliliter. The initial plating efficiency and final plating efficiency recorded after 10 and 30 d reached 33.7 and 1.07%, respectively. The proliferated calli transferred to regeneration medium supplemented with 2.22 μM 6-benzyladenine and 0.54 μM α-naphthaleneacetic acid gave the highest rate of shoot formation (44.4%). All protoplast-derived shoots were able to form roots on half-strength MS medium supplemented with 2.46 μM indole-3-butyric acid.

Purple-leaved Ficus lyrata plants produced by overexpressing a grapevine VvMybA1 gene

This study established an efficient method of regenerating plants of Ficus lyrata and producing purple-leaved F. lyrata plants through genetic transformation using a VvMybA1 gene of grapevine. Ficus lyrata, a species with unique violin- or guitar-shaped leaves, was regenerated from leaf-derived calli cultured on Murashige and Skoog (MS) basal medium supplemented with 4.5 μM N-phenyl-N'-1, 2, 3-thiadiazol-5-yl urea (TDZ) and 0.5 μM α-naphthalene acetic acid (NAA). Leaf discs were inoculated with Agrobacterium tumefaciens strain EHA 105 harboring a binary vector DEAT that contains the VvMybA1 gene and neomycin phosphotransferase (npt II) gene and subsequently cultured on the established regeneration medium supplemented with 100 mg l(-1) kanamycin. Results showed that 87.5 % of the leaf discs produced kanamycin-resistant callus, and 68.8 % of them produced adventitious shoots. Transgenic plants with three leaf colors including green, green-purple, and purple were produced. Regular and quantitative real-time PCR analyses confirmed the integration of transgenes into the host genome. Semi-quantitative RT-PCR analysis indicated that the VvMybA1 gene was responsible for the purple-colored phenotype. Purple-leaved plants with strong color stability grew vigorously in a greenhouse. This study illustrated the feasibility of using a genetically engineered VvMybA1 gene for drastic modification of leaf color of an important woody ornamental plant.

Somatic embryogenesis of Neoglaziovia variegata (Arruda) Mez, an important source of fiber from native Brazilian bromeliads

Neoglaziovia variegata (Arruda) Mez, a Bromeliad endemic to the Brazilian Caatinga and source of fiber for many different products, is considered an endangered species. Leaf and stem segments were cultivated on Murashige and Skoog (MS) medium with combinations of 2,4- dichlorophenoxyacetic acid (2,4-D) (0, 9, 18 and 27 µM) and glutamine (0, 50, 100, 150, 200 mg L-1) for the induction of embryogenic calli. Leaf segments did not present a favorable response for the combinations analyzed. The best response for embryogenic calli frequency with the best distribution of somatic embryos on the surface and better conversion rate was obtained using the stems explants in the medium containing 18 µM 2,4-D and 100 mg L-1 glutamine. The embryogenic calli obtained from the different induction media were transferred to MS medium supplemented with 30 g.L-1 sucrose, 2.4 g.L-1 Phytagel®, 0.53 µM α-naphthalene acetic acid (NAA) and 0.88 µM of 6-benzylaminopurine (BAP). The regenerated plants presented normal growth; however, many embryos did not fully develop.

Development of an in vitro protocol for a difficult-to-propagate endemic Australian dryland sedge species Mesomelaena pseudostygia (Cyperaceae)

In vitro propagation for Mesomelaena pseudostygia a difficult-to-propagate dryland sedge species (Cyperaceae) endemic to Western Australia is described. Multiple avenues to in vitro propagation were investigated: shoot culture, organogenesis and somatic embryogenesis, with zygotic embryos as initiation material. The highest multiplication rate for shoots was 3.4 ± 1.0 after 6 wk on basal medium (1/2 strength Murashige and Skoog) with 2.5 μM kinetin and 0.5 μM 6-benzylaminopurine. Shoots achieved peak rooting (83%) following a pulse treatment on basal medium containing 10 μM indolebutyric acid and 2 μM α-naphthaleneacetic acid for 7 wk, followed by transfer to medium (without growth regulators) for a further 7 wk. Alternatively, in vitro grown shoots were pulse treated on basal medium with both 100 μM indolebutyric acid and 20 μM α-naphthaleneacetic acid for 1 wk then placed in Rockwool plugs (under propagation house conditions) for another 7 wk resulting in 63% root induction. Rooted plantlets were also successfully transferred to potting mixture either in Rockwool plugs or bare rooted and maintained in propagation house conditions with ≥95% survival after 7 wk. These results indicate that micropropagation of M. pseudostygia is feasible for small to medium scale restoration purposes. The highest frequency of callus induction was from cultured zygotic embryos on basal medium with 5 μM α-naphthaleneacetic acid, whereas 2,4-dichlorophenoxacetic acid (2 or 5 μM) produced the largest callus sizes. A low frequency of shoot regeneration occurred in zygotic callus tissues in basal medium treatments containing cytokinin (kinetin or thidiazuron at 1 μM). A small proportion (<20%) of zygotic embryo callus explants from 2,4-dichlorophenoxyacetic acid treatments were found to be embryogenic, firstly developing embryo-like structures after 2 wk on basal medium (minus plant growth hormones), that continued to develop with approximately one in twenty germinating after a further 4 wk on basal medium to form small plantlets. Further optimisation is needed to improve somatic embryogenesis efficiency for mass propagation.

In vitro propagation, genetic and secondary metabolite analysis of Aconitum violaceum Jacq.: a threatened medicinal herb

Aconitum violaceum Jacq. is an important medicinal species used for various health ailments including renal pain, rheumatism and high fever. In the present report, a reproducible in vitro regeneration system for Aconitum violaceum Jacq. has developed from the nodal segment of the plant. Induction of shoot buds was achieved on basal Murashige and Skoog (MS) medium. The shoots were elongated on MS medium supplemented with 0.5 μM 6-benzylaminopurine (BAP) and 0.1 μM α-napthaleneacetic acid (NAA) and subsequently transferred to rooting medium. In vitro grown microshoots of A. violaceum were encapsulated in the alginate beads. The success rate of their re-growth was found to be approximately 85.43 %. Of the encapsulated microshoots, 39.86 % exhibited formation of multiple shoots following re-growth on plant growth regulator free MS medium. Healthy root formation was observed in all microshoots following 2 weeks of transfer on half-strength MS medium containing 0.1 μM indole-3-acetic acid (IAA) and 1.0 μM α-naphthalene acetic acid (NAA). These plants were subsequently transferred to pots containing a mixture of soil, sand and compost (1:1:1 v/v), and same were then shifted in the greenhouse with 87 % survival rate. The molecular analysis was carried out using 35 random amplified polymorphic DNAs (RAPD) primers and 25 inter simple sequence repeats (ISSR) primers. Cluster analysis of the RAPD and ISSR profile revealed an average similarity coefficient of 0.966 and 0.974, respectively, confirming genetic stability of tissue culture-raised (TR) plants and synthetic seed-derived plants (SR). The phytochemical analysis of tissue culture-raised and synthetic seeds-derived plants showed higher aconitine content than control plant. The propagation protocol developed in this study provides a basis for germplasm conservation and harnessing the medicinally active compounds of A. violaceum.

In vitro propagation and conservation of Diaphanoptera khorasanica (Caryophyllaceae), a threatened endemic and potential ornamental species in northeast of Iran

Tissue culture methods provide tools to supplement traditional methods for collection, propagation and preservation of endangered plant species. In this study, in vitro propagation of Diaphanoptera khorasanica Rech.f., a rare and threatened plant species with limited distribution range and population was investigated. This species has a potential as an ornamental plant. Single node explants were provided from both adult and seedling sources. Several disinfection treatments were tried to permit selection of a suitable method. Different growth regulators were used for establishment, proliferation and rooting stages. Explants showed the highest establishment percentage after 5 min treatment with 1% sodium hypochlorite (NaOCl), cultured in MS medium containing 2.2 μM 6-benzylaminopurine (BAP) and 2.4 μM indole-3-butyric acid (IBA). The highest proliferation of explants from both adult and seedling source explants was obtained from media supplemented by BA treatment in contrast to TDZ. No significant differences were found between different concentrations of BAP and TDZ. Proliferated shoots in TDZ were longer and had more internode length and less vitrification, in comparison with those in BAP. In vitro rooting of proliferated shoots just induced in liquid half-strength MS medium and rooting was not observed in solid medium. The shoots that originated from adult plants gave rise to the highest rooting rate with 4.8μM α-naphthalene acetic acid (NAA) and 2.4 μM, but NAA rooted plantlets showed higher survival percentage in acclimatization step. This study was aimed towards developing an efficient protocol for in vitro propagation of D. khorasanica and conservation of this vulnerable species.

Lipid peroxidation, H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt; content, and antioxidants during acclimatization of Abrus precatorius to ex vitro conditions

An efficient, rapid, and reproducible plant regeneration protocol was successfully developed for Abrus precatorius L. using mature nodal explants excised from a 5-year-old field grown plant. The highest shoot regeneration frequency (87 %) with maximum number of multiple shoots (15.0) and shoot length (4.8 cm) were recorded on Murashige and Skoog (MS) medium amended with 2.5 μM thidiazuron, 120 mg dm−3 polyvinylpyrrolidone, and 0.5 μM α-naphthalene acetic acid. The best treatment for maximum root (4.0) induction was half strength MS medium supplemented with 1.5 μM indole-3-butyric acid. The in vitro plantlets with well-developed shoots and roots were successfully transferred into plastic cups with Soilrite and acclimatized in a culture room under photon flux density (PFD) of 150 μmol m−2 s−1, thereafter transferred to a greenhouse with PFD of 300 μmol m−2 s−1, and finally to a field with 70 % survival rate. During the acclimatization period (0–49 d), leaf chlorophyll and carotenoid content increased whereas malondialdehyde and H2O2 content decreased probably due to increasing activities of antioxidant enzymes (catalase, superoxide dismutase, glutathione reductase, and ascorbate peroxidase). Our work suggests that micropropagated plants developed an antioxidant enzymatic protective system to avoid oxidative stress during establishment under ex vitro environment.

Extracts of dedifferentiated cultures of Spilanthes acmella Murr. possess antioxidant and anthelmintic properties and hold promise as an alternative source of herbal medicine

This study reports the antioxidant and anthelmintic activities of Spilanthes cell cultures. Murashige and Skoog medium supplemented with 5.0 μM N6-benzylaminopurine, 1.0 μM 2,4-dichlorophenoxyacetic acid and 1.0 μM α-naphthaleneacetic acid was found to be the best medium to induce dedifferentiation from the leaf cells and for further maintenance of the callus cultures. These cell cultures and in vivo leaves were subjected to solvent extraction by hexane, ethyl acetate, methanol and water, and their extraction yield and total phenolic contents were evaluated. The antioxidant activity of extracts was determined by using 2,2-diphenyl-1-picrylhydrazyl free radical scavenging assay. The methanol extracts of in vitro callus and in vivo leaf showed an apparent antioxidant activity and their IC50 values were 1342.9 and 1085.1 μg/ml, respectively. The anthelmintic efficacy of the extracts was also tested using live trematode (fluke) parasites of cattle as the model test material. The aqueous extract of dedifferentiated callus showed a strong anthelmintic activity, which was higher than the activity of the water extract of the field-grown plant. The results of this study verify the potential of Spilanthes as a reservoir of bioactive agents and also substantiate the value of callus cultures as a new source of anthelmintic and antioxidant compounds.

In vitro organogenesis from leaf and transverse thin cell layer derived callus cultures of Talinum triangulare (Jacq.) Willd.

Talinum triangulare is an important medicinal herb used traditionally in the treatment of various diseases. The present study was intended to develop a rapid and efficient protocol for indirect organogenesis from leaf discs and transverse thin cell layer (tTCL) of internodal explants of T. triangulare. Best callusing response (100 %) was observed with tTCL explants on Murashige and Skoog (MS) medium supplemented with 4.44 μM 6-benzyl amino purine and 5.37 μM α-naphthalene acetic acid (NAA). High frequency shoot regeneration (96.67 %) was obtained from tTCL derived calli on MS medium supplemented with a combination of 0.45 μM thidiazuron and 0.27 μM NAA, by producing 9.20 ± 0.35 shoots with a shoot length of 2.74 ± 0.03 cm. In vitro rooting of the microshoots was recorded on half-strength MS medium containing 2.46 μM indole-3-butyric acid by eliciting 15.20 ± 0.27 roots with a length of 4.25 ± 0.11 cm. The rooted shoots were acclimatized on garden soil, sand and coco pith (1:1:3 v/v) planting substrate. The plantlets were successfully established under field conditions with 100 % survival rate. The hardened plants exhibited homogeneity and no observable morphological variations were detected among the regenerants and the mother plants of T. triangulare.