Rapid in vitro multiplication and restoration of Celastrus paniculatus Willd. sub sp. paniculatus (Celastraceae), a medicinal woody climber

Nodes, shoot tips, internodes and leaf bases (approximately 1.0 cm) excised from young vines of the flowering woody climber, Celastrus paniculatus WilId. sub. sp. paniculatus (Celastraceae) were cultured in Murashige and Skoog (MS) medium containing agar (0.6%), sucrose (3%) and varied concentrations of 6-benzyl aminopurine (BAP) and kinetin. All the explant types were regenerative and maximum number (3.6) and frequency (94%) of axillary shoot formation of (5.08 cm long) was recorded in the nodes cultured in BAP (1 mg L(-1)) after 6 weeks. Combinations of BAP (1 mg L(-1)) and indole-3-acetic acid/l-naphthalene acetic acid (0.01-1 mg L(-1); IAA/NAA) tested with nodes induced formation of less number (3 and 2.2) of shoots at same frequency (94%). All the explant types viz. node, shoot tip, internode and leaf base of in vitro derived shoots responded earlier and better in lower concentrations of BAP (0.5-2 mg L(-1)) with formation of 8, 3.1, 6.4 and 1.8 shoots respectively during the same period. In spite of the advanced and increased caulogenic responses, differences in cytokinin requirements between different explants observed during culture initiation still persisted with the nodes, shoot tips, internodes and petiole segments responding best at 0.5, 1 and 2 mg L(-1) BAP, respectively. The repeated reculture up to 10 cycles of the nodes from the shoot cultures each at 6-week intervals enabled multiplication and stocking of shoots without decline. Rooting of 3-7 cm shoot cuttings was induced in half-strength MS liquid medium containing IAA (1 mg L(-1)) with formation of 7.25 roots of 2.41 cm length within 6 weeks. Rooted plants were established at 84-96% rate in community pots without hardening, the least value (84%) being obtained with NAA- induced thick and calloid rooted plants. Four month old community potted plants were reintroduced into native forest habitats at 95% efficiency and 8 months after restoration, the plants were uniform in morphological, growth, cytological and peroxidase and esterase isozyme characteristics.     

Cryopreservation and genetic stability assessment of regenerants of the critically endangered medicinal plant Dioscorea deltoidea Wall. ex Griseb. for cryobanking of germplasm

In Vitro Cellular & Developmental Biology - Plant - Dioscorea deltoidea Wall. ex Griseb. is a critically endangered, commercially important crop of high medicinal value. Several accessions of...     

Micropropagation and assessment of genetic fidelity of Henckelia incana: an endemic and medicinal Gesneriad of South India

Physiology and Molecular Biology of Plants - Henckelia incana is an endemic medicinal plant used for the treatment of fever and skin allergy. In the present study shoot regeneration was evaluated...     

Micropropagation of an endangered plant species: Coronopus navasii (Brassicaceae)

Plantlets of Coronopus navasii, an endangered species from SE Spain, were successfully regenerated from shoot and root segments excised from young seedlings. Initiation of multiple buds and development of leaves were obtained in MS modified medium plus l mg/l BAP and 0.1 mg/l NAA. Rooting was achieved by transfer of the isolated shoots to fresh MS medium without plant growth regulators. Plant survival of 47% was obtained six weeks after removal from in vitro culture conditions.Abbreviations BAP 6-benzylaminopurine - NAA naphthaleneacetic acid - MS medium see Murashige and Skoog 1962     

Micropropagation and in vitro flowering of endemic and endangered plant Ceropegia attenuata Hook

Factors affecting in vitro propagation were evaluated for Ceropegia attenuata Hook., an endemic and endangered plant having ornamental potential but a limited reproductive capacity. Rapid shoot multiplication from nodal explants was established using varying concentrations of cytokinins and auxins either alone or in combinations. The highest frequency of shoot induction was achieved when nodal explants were inoculated on Murashige and Skoog (MS) medium supplemented with 13.31 μM 6-benzylaminopurine with a mean of 12.9?±?0.5 shoots per explant. High concentrations of TDZ (6.81–11.35 μM) and KN (6.78–11.61 μM) resulted in stunted and vitrified shoots. Factors implicated in the promotion of floral transition of the C. attenuata have been identified which are 4-amino-3, 5, 6-trichloropicolinic acid (picloram), 6-benzylaminopurine, sucrose and photoperiod. The highest frequency of flowering (100%) was obtained when axillary shoot explants were transferred to MS medium supplemented with picloram (4.14 μM) within 4 weeks of culture. Transfer of in vitro regenerated shoots to half strength MS medium with 2.46 μM indole-3-butyric acid (IBA) showed maximum root induction. The in vitro grown plantlets were successfully acclimatized in the glasshouse with 85% of survival and showed normal development. The developed protocol provided a simple, cost-effective approach for the conservation of endangered plant C. attenuata for replenishing its declining populations.     

Micropropagation of the medicinal plant, Scilla natalensis Planch.

Primary bulb explants of Scilla natalensis were cultured in vitro on modified MS medium. Some of these explants initiated shoots, which provided a sterile source of secondary leaf and bulb explants. The secondary explants responded similarly to various combinations of plant growth regulators. Shoots were initiated spontaneously on medium containing no plant growth regulators. The number of shoots initiated was increased by the addition of kinetin or thidiazuron (TDZ) alone, but was reduced by the addition of indole-3-acetic acid (IAA) or naphthalene acetic acid (NAA) alone. Optimal shoot initiation occurred on medium containing 1 to 2 mg l^–1 kinetin and 1 to 2 mg l^–1 IAA. These shoots were rooted on medium containing 1 mg l^–1 IAA. The plantlets were successfully acclimatised in the misthouse/shadehouse.     

Micropropagation of a medicinal plant, Plantago major L.

An efficient micropropagation protocol was developed for an important medicinal plant, Plantago major L. For this purpose, it is recommended to culture shoot-tips on modified MS medium [412.5 mg dm-3 NH4NO3 and 340 mg dm-3 KH2PO4] supplemented with 50 g dm-3 glucose and 0.5 μM 6-benzylaminopurine. Maximum rooting frequency was obtained at 1 μM naphthaleneacetic acid. This revised version was published online in August 2006 with corrections to the Cover Date.     

In vitro shoot bud differentiation and plantlet regeneration in Celastrus paniculatus Willd

Multiple shoot buds could be induced directly from internode explants of Celastrus paniculatus inoculated on Murashige and Skoog’s (MS) medium containing different growth regulators. The best response was obtained when 4.44 μM 6-benzylaminopurine (BAP) was incorporated in the medium. Incorporation of indole-3-acetic acid (IAA) and α-naphthalene acetic acid (NAA) did not improve response, rather promoted callusing. Adventitious shoot buds could be multiplied and elongated on MS medium containing 2.22 μM BAP. Rooting of shoots (80 %) was obtained when their bases were dipped in pre-autoclaved indole-3-butyric acid (IBA) solution (2.45 mM) for 10 min followed by their implantation on medium containing 1/4 MS salts, 1.0 % sucrose and 0.6 % agar. Out of 500 plantlets subjected to hardening, 410 were successfully hardened under greenhouse conditions. Twenty plants were established in field while remaining of them were transferred to nursery conditions without any mortality.     

Micropropagation protocol for Antigonon leptopus an important ornamental and medicinal plant

The effect of some factors on in vitro consecutive micropropagation behavior of Antigonon leptopus was examined including those of culture establishment, shootlets multiplication, rooting and acclimatization stages. The highest percent of aseptic cultures and survival of explants (100%) were obtained as a result of using Clorox 10% for 3?min followed by MC 0.1% for 2?min while, using each of them individually (Clorox 20% or MC 0.1%) for 5?min caused the highest percent of shoot formation. During the multiplication stage, the highest percent of shoot formation was reached to 100% with repeating culture of explants (two times) on MS medium supplemented with 2ip at 1.0 and IBA at 0.2?mg/l. The highest numbers of shootlets/explant were obtained when 2.0?mg/l of BAP or 0.5?mg/l BA?+?0.2?mg/l of IBA were added to MS culture medium. Culturing the explants on MS medium supplemented with 2ip at 0.5 or 1.0?mg/l each combined with 0.2?mg/l of IBA showed the longest shootlets. Reducing the strength of culture media to ½ or ¾ had promotion effect on rooting formation of shootlets. The best results of plant acclimatization (survival percent, plant height and root length) were obtained by using sand or peat moss soil. The amplified DNA fragments using B7, B9 and C19 primers for mother and micropropagated plants showed that the produced pattern by primer B7 had a maximum number of 10 bands of DNA fragments with molecular size ranging between 1025.57 and 176.36?bp, micropropagated plants showed 95.2% similarity in relation to mother plant.     

Cytotoxic constituents from Celastrus paniculatus induce apoptosis and autophagy in breast cancer cells

Celastrus paniculatus is a traditional medicinal plant with diverse pharmacological activities. To identify its bioactive constituents, three new β-dihydroagarofuranoid sesquiterpenes were isolated from the whole plant, of which the major constituent is (1α,2α,8β,9β)-1,8-bis(acetyloxy)-2,9-bis(benzoyloxy)-14-hydroxy-β-dihydroagarofuran. It was assessed for its antiproliferative activity, and it suppressed the viability of MCF-7 breast cancer cells with an IC₅₀ of 17 ± 1 μM. This growth inhibition was, in part, attributable to apoptosis. Moreover, this drug treatment led to LC3B-II accumulation, indicative of autophagy. Western blot analysis established its ability to target a broad range of signaling effectors related to survival and cell cycle progression, including Akt, NF-κB, p53, and MAP kinases. In addition, flow cytometry analysis indicates increased reactive oxygen species production in response to this compound. Taken together, these findings suggest a pleiotropic mode of mechanism that underlies the antiproliferative activity of this compound in MCF-7 breast cancer cells.     

Antioxidant property of Celastrus paniculatus willd.: a possible mechanism in enhancing cognition.

In the present study aqueous, methanolic, chloroform and petroleum ether extracts of seeds of Celastrus paniculatus were investigated for their effect on cognitive functions in rats. Male Wistar rats weighing 200-250 g each were used to study effect on learning and memory through use of the shuttle-box, step-through, step-down and elevated plus maze paradigms. Only the aqueous seed extract (200 mg/kg body wt. for 14 days) showed an improvement in learning and memory in both the shuttle-box and step-through paradigms. Therefore, further experiments were conducted using the aqueous extract at 100, 200 and 300 mg/kg body wt. doses in different paradigms of cognition. All three doses of the aqueous extract increased the number of avoidances in the shuttle-box and step-through latency the in step-through apparatus, but no significant difference was observed between the doses tested. In the step-down apparatus, the 200- and 300-mg/kg body wt. doses of aqueous extract showed a significant increase in step-down latency, whereas no significant difference was observed in the elevated-plus-maze paradigm between drug-treated and vehicle-treated groups. Since the behavioral impairments are associated with oxidative stress, we investigated the effect of the aqueous extract on oxidative stress parameters. Among the three doses tested, only 200 and 300 mg/kg body wt. stimulated a significant decrease in the brain levels of malondialdehyde, with simultaneous significant increases in levels of glutathione and catalase. The present findings indicate that the aqueous extract of Celastrus paniculatus seed has cognitive-enhancing properties and an antioxidant effect might be involved.     

Micropropagation of Cunila galioides,a popular medicinal plant of south Brazil

Axillary buds of field plants of Cunila galioides Benth. were used to evaluate the effect of growth regulators and culture media on the in vitro shoot proliferation and growing. The highest multiplication rate was obtained using Murashige and Skoog (MS) medium supplemented with 8.8 M of benzyladenine. Repeated subcultures of shoot tips and single nodes at 4-week intervals for eight months on the above medium enabled mass multiplication of shoots without any evidence of decline. The best conditions for rooting were MS medium plus 0.5 to 2.5 M of indolebutyric acid. The rooted plants were successfully transferred to soil, exhibiting a normal development.     

Low-temperature storage of Rauvolfia serpentina Benth. ex Kurz.: An endangered,endemic medicinal plant

On a standard shoot culture medium, nodal cultures of Sarpagandha (Rauvolfia serpentina) could be maintained for nine months at 25° C by replacing cotton plugs with polypropylene caps as enclosures for culture tubes. Low temperature incubation of in vitro cultures appeared highly promising because cultures exhibited normal health even after 15 months of storage at 15° C; while 10°C and 5°C were found deleterious to growth of the cultures of R. serpentina.     

Micropropagation of Allium wallichii kunth, a threatened medicinal plant of Nepal

Summary Bulbs and aerial parts of the Nepalese plant Allium wallichii are widely used for medicinal purposes and as a spice. Due to overharvesting the natural populations of the species have been increasingly reduced and the domestication of the species should be considered. For the purpose of the production of plantlets suitable for field culture, a micropropagation procedure based on multiple shoot culture has been established. Multiplication factors of 4.6 on average were possible on MS medium supplemented with 20 μM zeatin. After rooting on MS medium with 10 μM indolebutyric acid, plantlets were acclimatized to greenhouse conditions and transferred to the field with good success. Part of the PhD thesis of P. R. Malla.     

Micropropagation and genetic stability of a Dendrobium hybrid (Orchidaceae)

Summary Dendrobium hybrids have great economic importance in a number of countries. Asymbiotic seed germination and the conventional vegetative method have been commonly used by growers to propagate these plants. To overcome somaclonal variation, which is commonly exhibited by Dendrobium (Nobile group) when micropropagated from protocorm-like bodies, a protocol for propagating Dendrobium Second Love in vitro using axillary buds in the presence of thidiazuron was developed. Random amplified polymorphic DNA analysis was also carried out to check for possible genetic alterations in plants originating from six consecutive subcultures. The results revealed that the established protocol was efficient for the in vitro cloning of this orchid hybrid and the plants obtained from the six subcultures did not exhibit any type of polymorphism.     

Efficient micropropagation and assessment of genetic fidelity of Boerhaavia diffusa L- High trade medicinal plant

Physiology and Molecular Biology of Plants - Boerhaavia diffusa L is a medicinal herb with immense pharmaceutical significance. The plant is used by many herbalist, Ayurvedic and pharmaceutical...     

Regeneration of Plantlets via Callus Cultures in Celastrus paniculatus Willd-A Rare Endangered Medicinal Plant

Plantlet regeneration in Celastrus paniculatus Willd (Celastraceae), an endangered medicinal plant has been achieved from cotyledonary leaf-derived callus. The cotyledonary leaves from mature embryo callused on MS medium supplemented with NAA (5 × 10^-6 M) and Kinetin (5 × 10^-6 M) with a doubling time of 16.2 days. Well developed multiple shoots of 5.6 cm could be obtained via organogenesis within 28 days on MS + BA (2 × 10^-5 M). Altered shoot morphology occurred with the change in the concentration of auxins and cytokinins. Rooting (85%) of isolated shoots was achieved on McCown medium (WPM) containing IBA (5 × 10^-6 M). Rooted plantlets were transferred in small pots containing sand, garden soil and vermiculite mix. The procedure can serve as a reliable and reproducible protocol for propagation and ex situ conservation ofthis species.     

Micropropagation,antioxidant properties and phytochemical assessment of Swertia corymbosa (Griseb.) Wight ex C. B. Clarke: a medicinal plant

Swertia corymbosa (Griseb.) Wight ex C. B. Clarke, a valuable medicinal plant, has been investigated for its regeneration potential using nodal explants. Out of a range of concentrations of cytokinins [6-benzyl adenine (BA), 6-furfurylaminopurine (Kn), 2-isopentenyl adenine (2iP), thidiazuron (TDZ), and zeatin (Z)] used as supplements with MS, BA at 4.40 μM concentration proved best for multiple shoot induction yielding 26.50 ± 0.26 shoots after 12 weeks of culture. Addition of low concentration of NAA (1.3 μM) in MS medium supplemented with the cytokinin BA (4.40 μM) favoured shoot multiplication. A mean number of 35.78 ± 0.81 shoots were produced per explant. Additive effect of BA (4.40 μM) in combination with Kn (4.64 μM) produced highest number of shoots (83.20 ± 4.29). Addition of GA₃ (1.4 μM) to the above medium not only favored shoot elongation but also enhanced the number of shoots (113.98 ± 3.80). The microshoots were rooted successfully on half-strength MS medium supplemented with 9.8 μM of IBA. The plantlets were successfully transferred to hardening medium containing vermiculite with 87 % survival rate. Screening of the antibacterial, antioxidant activity and estimation of total phenolic and flavonoid content of methanolic extracts of micropropagated plants were also carried out and compared with that of the wild-grown plants. In all the tests, methanolic extract from wild-grown plants showed higher antioxidant, antimicrobial activity, total phenolic and flavonoid content than in vitro propagated plants. The content of secondary metabolites in wild-grown plants and in vitro propagated plants was determined by HPLC coupled with ESI-MS and the presence of loganic acid, swertiamarin, sweroside, gentiopicroside, isovitexin, amoroswertin, amarogentin, gentiacaulein, decussatin, and swertianin in the samples were confirmed. Gentiopicroside (40.726 mg/g) and swertianin (29.598 mg/g) were found to be the major compounds which may be responsible for the antimicrobial and antioxidant activities. The results of the present study confirmed the therapeutic potency of S. corymbosa used in the traditional medicine; in addition, the protocol for in vitro production developed in the present study could be applied for mass multiplication and for the conservation of germplasm.