The aim of this study was to develop a new micropropagation system for Cassia angustifolia Vahl.,... more The aim of this study was to develop a new micropropagation system for Cassia angustifolia Vahl., an important medicinal legume using root explant as starting material. Root explants taken from 30-day-old aseptic seedlings were cultured on Murashige and Skoog (MS) medium supplemented with different plant growth regulators: 6-benzyladenine (BA), kinetin (Kn), and thidiazuron (TDZ). Organogenic nodular calli obtained on MS + TDZ (1.0 μM) were transferred to shoot regeneration medium supplemented with different cytokinins (BA, Kn or TDZ) either alone or in combination with auxin:indole-3-acetic acid or α-naphthalene acetic acid. Maximum shoot regeneration frequency (90%) was obtained on MS + BA (2.5 μM) + NAA (0.6 μM) wherein a maximum of 42.76 ± 1.47 shoot buds per explant were induced with a maximum conversion rate of 35.63 ± 0.75 shoots per explant and average shoot length of 5.43 ± 0.20 cm. Elongated microshoots were successfully rooted under ex vitro conditions by pulse treatment in 200 μM of indole-3-butyric acid for half an hour. Microshoots were rooted, acclimatized and hardened off simultaneously in sterilized soilrite inside the growth room and then established in pots containing sterilized soil and manure (1:1) and grown under greenhouse condition with 90% survival rate. The histological sections at different developmental stages of shoot buds revealed the organization of nodular meristematic zone leading to the orientation and differentiation of shoot buds in large number and thereafter conversion into healthy shoots.
A method for rapid in vitro propagation of Cassia siamea Lam. using cotyledonary node explants, e... more A method for rapid in vitro propagation of Cassia siamea Lam. using cotyledonary node explants, excised from 14-day old aseptic seedlings, has been established. Murashige and Skoog (MS) medium supplemented with different concentrations of 6-benzyladenine (BA), kinetin (Kn) and thidiazuron (TDZ) singly or in combination with auxins was used for regeneration studies. Among the single treatment of three cytokinins BA at 1.0 µM was found to be optimum for direct shoot regeneration as it induced an average of 8.20 ± 0.66 shoots per explant. The regeneration frequency further enhanced with the application of auxin along with optimal BA concentration. The highest frequency for shoot regeneration (90%), the maximum number of shoots per explant (12.20 ± 0.73) and the maximum shoot length (6.40 ± 0.07) cm were obtained on the medium consisted of MS + 1.0 µM BA + 0.5 µM NAA. Successful in vitro rooting was induced from cut end of the microshoots when placed on half-strength MS + IBA (2.5 µM). The regenerated shoots with well developed root system were successfully acclimatized and established in pots containing sterilized garden soil and garden manure (1:1) and grown under greenhouse conditions with 85% survival rate.
Cassia sophera Linn. is an important medicinal plant belonging to family Fabaceae. It is extensiv... more Cassia sophera Linn. is an important medicinal plant belonging to family Fabaceae. It is extensively used in Homeopathy and is well known for its medicinal properties. The present study describes a simple, efficient and reproducible regeneration system for in vitro propagation of C. sophera through cotyledonary node (CN) explant excised from 21 d old axenic seedlings. Explants were cultured on Murashige and Skoog (MS) medium supplemented with different concentrations of thidiazuron (TDZ). Multiple shoots were produced on all the concentrations of TDZ; however 2.5 μM concentration proved to be optimal for the production of maximum number of shoots. To avoid adverse effects of prolonged exposure to TDZ in long term establishment, the cultures were transferred to TDZ free MS medium fortified with various concentrations of 6- benzyl aminopurine (BA) for multiplication, proliferation and elongation of induced shoots. Emergence of new shoot buds and multiplication continued up to second subculture passage and maximum number (14.9 ± 1.4) of shoots obtained on MS + BA (1.0 μM). Best rooting response was observed on half strength MS containing Indole-3-butyric acid (IBA) (1.0 μM). Regenerated plantlets were successfully acclimatized and hardened off inside the culture room and then transferred to green house with 100 % survival rate.
a b s t r a c t Progress in biotechnological research over the last two decades has provided grea... more a b s t r a c t Progress in biotechnological research over the last two decades has provided greater scope for the improvement of crops, forest trees and other important plant species. Plant propagation using synthetic seeds has opened new vistas in the field of agriculture. Synseed technology is a highly promising tool for the management of transgenic and seedless plant species, polyploid plants with elite traits and plant lines that are difficult to propagate through conventional propagation methods. Delivery of synseeds also alleviates issues like undertaking several passages for scaling up in vitro cultures as well as acclimatization to ex vitro conditions. Optimization of synchronized propagule development followed by automation of the whole process (sorting, harvesting, encapsulation and conversion) can enhance the pace of synseed production. Cryopreservation of encapsulated germplasm has now been increasingly used as an ex vitro conservation tool with the possible minimization of adverse effects of cryoprotectants and post-preservation damages. Through synseed technology, germplasm exchange between countries could be accelerated as a result of reduced plant quarantine requirements because of the aseptic condition of the plant material.
This paper reports an efficient regeneration protocol through parallel organogenic and embryogeni... more This paper reports an efficient regeneration protocol through parallel organogenic and embryogenic pathways from green root segments (GRSs) of Tylophora indica (Burm.f) Merrill. GRSs explants from one year old in vitro cultures were cultured on Murashige and Skoog (MS) medium containing various cytokinins. Five mmol/L of 6-benzyladenine (BA) was most responsive for organogenesis in 1.5 cm long GRSs. Repeated subculture on medium containing both BA (5 mmol/L) and 1-naphthleneacetic acid (NAA) (0.1 mmol/L) promoted multiplication and proliferation of direct shoot buds (46.80 AE 0.96) and callus mediated somatic embryogenesis (18.07 AE 0.33). Germinated embryos isolated from callus were transferred onto maturation medium consisting of half-strength MS medium either devoid of plant growth regulators (PGRs) or with various concentrations of gibberellic acid (GA). Microshoots were excised during subculture and transferred onto root induction medium, thus ensuring a continuous supply of germplasm. Morphogenic variations were noticed in types of roots induced on various auxins. Regenerated plantlets and emblings hardened best on vermiculite with a survival rate of 90% and 70% respectively. However, the emblings were healthier in comparison to the regenerated plants. Histological analysis showed the origin and development of organogenesis.
The aqueous and alcoholic extract of fresh Berberis aristata DC roots, as well as aqueous extract... more The aqueous and alcoholic extract of fresh Berberis aristata DC roots, as well as aqueous extract of dried roots were compared for their antibacterial and antifungal activities by the disc diffusion method. All three extracts showed wide antibacterial activity against Gram-positive bacteria. Among the Gramnegative bacteria tested, the antibacterial activity was limited to Escherichia coli, Salmonella typhimurium, Shigella dysenteriae type 1 and Vibrio cholerae; with the best activity against V. cholerae. MICs of the alcoholic extracts against Gram-positive bacteria ranged between 3.8 ×10 -3 to 6.1 ×10 -3 mg/ml and for Gram-negative bacteria from 6.1 ×10 -3 to 7.6 10 -3 mg/ml. The MICs for Candida species ranged between 0.02 to 3.8 ×10 -3 mg/ml and for Aspergillus species, it was 3 × 10 -3 mg/ml. All three extracts also had antifungal activity against the fungal species tested, except Candida krusei. The extracts of B. aristata also demonstrated anti-inflammatory, analgesic, and antipyretic activities. Chemical analysis revealed the presence of alkaloids, amino acids, tannins, terpenes, resins, phenols and reducing sugars as major compounds. FTIR-spectral analysis of all the extracts revealed the presence of berberine, as a major constituent, along with other chemical constituents.
Pterocarpus marsupium, Clitoria ternatea, and Sanseveiria cylindrica are some of the important an... more Pterocarpus marsupium, Clitoria ternatea, and Sanseveiria cylindrica are some of the important and endangered medicinal plant species of India. Despite of medicinal properties, antibacterial potential of the plants have not yet been explored. The present study was designed to optimize the in vitro technique for micropropagation and to screen the extracts from leaves and in vitro raised calli for antibacterial properties. Excised leaf-explants from the parent plants were surface sterilized and cultivated on Murashige & Skoog's (MS) medium containing N 6 -benzyladenine (BA) in concentrations of 1, 2, 5, and 10 µM. Optimal growth of calli was noticed at a concentration of 5 µM, therefore the extracts from calli grown at this concentration were further studied for antibacterial activity. Both alcoholic and aqueous extracts from leaves of respective plants, and their in vitro raised calli were tested for antibacterial activity by agar well diffusion method against a range of Gram-positive and Gram-negative bacteria. Aqueous extracts showed antibacterial activity against limited number of bacterial species; notably the extracts of C. ternatea which showed antibacterial activity against Streptococcus pyogenes, Bacillus subtilis and Bacillus cereus. Alcoholic extracts of all three plants showed antibacterial activity against a wider range of bacteria. Among the Gram-positive bacteria, extracts from C. ternatea showed strong antibacterial activity against Bacillus spp., whereas the extracts of S. cylindrica showed good antibacterial potential for Staphylococcus aureus, S. epidermidis and S. pyogenes. The extracts from all three plants showed antibacterial activity against Gram-negative bacteria, including, Salmonella spp. and Shigella dysenteriae; organisms causing enteric fever and dysentery. In most of the cases, the extracts from respective calli showed comparable, and in some cases better, result in comparison to the extracts from parent leaves. To the best of our knowledge this is the first preliminary report on antibacterial potential, especially through calli extracts, of these plants; and in vitro cultivation of the explants may be used to obtain phytotherapeutic compounds.
in Vitro Cellular & Developmental Biology-plant, 2007
An efficient protocol was developed for micropropagation of an economically important timber-yiel... more An efficient protocol was developed for micropropagation of an economically important timber-yielding multipurpose tree, Pterocarpus marsupium Roxb. Multiple shoots were induced from cotyledonary nodes (CNs) derived from 18-d-old axenic seedlings on Murashige and Skoog (MS) medium supplemented with thidiazuron (TDZ) (0.1–10 μM). The highest shoot regeneration frequency (90%) and maximum number (15.2 ± 0.20) of shoots per explant was recorded on MS medium amended with 0.4 μM TDZ. Continuous presence of TDZ inhibited shoot elongation. In the primary medium, TDZ-initiated cultures were transferred to the secondary medium supplemented with another cytokinin, 6-benzyladenine (BA), for shoot growth and elongation. Maximum (90%) shoot elongation with an average shoot length of 5.4 ± 0.06 cm was observed at 5 μM BA. To further enhance the number of shoots per explant, mother tissue was repeatedly subcultured on fresh shoot induction medium after each harvest of newly formed shoots. Thus, by adopting this strategy, an average of 44 shoots per explant could be obtained. About 65% of in vitro regenerated shoots produced a maximum number (4.4 ± 0.2) of roots per shoot by a two-step culture procedure employing pulse treatment and subsequent transfer of treated shoots to a low concentration of 0.2 μM indole-3-butyric acid along with phloroglucinol (3.96 μM). The in vitro-raised plantlets were successfully acclimatized first under culture room conditions, then to greenhouse with 70% survival rate.
... well known biotechnological tool for the rapid propagation of medicinal plants for the purpos... more ... well known biotechnological tool for the rapid propagation of medicinal plants for the purpose of commercialization (Kitto, 1997), conservation (Anis et al ... photoperiod with a photosynthetic photon flux density (PPFD) of 50 µM m-2 s-1 irradiance provided by cool white fluorescent ...
Pterocarpus marsupium (Bijasal) is a valuable multipurpose forest tree. The regeneration rate in ... more Pterocarpus marsupium (Bijasal) is a valuable multipurpose forest tree. The regeneration rate in natural habitat is low and the tree is overexploited. An in vitro propagation protocol has been developed from nodal explants obtained from in vitro raised 18-day-old axenic seedlings. The highest shoot regeneration frequency (85%), maximum number of multiple shoots (8.6) as well as length (4.8 cm) were induced from nodal explants on Murashige and Skoog (MS) medium amended with 4.0 μM 6-benzyladenine (BA), 0.5 μM indole-3-acetic acid (IAA) and 20 μM adenine sulphate (AdS). The percentage of shoot multiplication as well as the number of shoots per node remained the same during the first two subculture, afterwards a decline was recorded. Rooting was best induced in microshoots excised from proliferated shoot cultures on semisolid hormone-free half-strength MS medium, after a pulse (dip) treatment for 7 days in half-strength MS liquid medium containing 100 μM indole-3-butyric acid (IBA) and 15.84 μM phloroglucinol (PG). The in vitro-raised plantlets were potted and acclimatized under culture room conditions for 4 weeks before their transfer to a greenhouse, where the established plants showed 75% survival.
An efficient protocol has been developed for high-frequency shoot regeneration and plant establis... more An efficient protocol has been developed for high-frequency shoot regeneration and plant establishment of Clitoria ternatea – a potential medicinal legume. Adventitious shoots were regenerated from young excised root segments of aseptic seedlings on Murashige and Skoog (MS) medium supplemented with various concentrations of 6-benzyladenine (BA), kinetin, α-naphthalene acetic acid (NAA) or 2,4-dichlorophenoxy acetic acid (2,4-D) either singly or in various combinations. The highest frequency (100%) of shoot regeneration and maximum number (16.4 ± 0.24) of shoots per explant was obtained on MS medium supplemented with 20 μm BA and 2.0 μm NAA. Organogenic calli were produced on a medium containing 2,4-D (10 or 20 μm) and BA (5.0 μm). The calli were differentiated into multiple shoots on MS medium supplemented with 2.5–10 μm BA and 2.0 μm NAA. The microshoots were rooted on half-strength MS medium supplemented with 5.0 μm indole-3-butyric acid and transplanted successfully in field conditions. After 12 months of transfer to ex vitro conditions, the performance of micropropagated plants were evaluated on the basis of some physiological and biochemical parameters and compared with the in vivo–grown plants of the same age. The sodium dodecyl sulphate polyacrylamide gel electrophoresis protein profile was same between regenerated and naturally growing shoots. Total soluble protein in aerial part as well as in seeds of in vitro–regenerated and in vivo–grown plants was almost the same. The mitotic study showed normal chromosomal movement and numbers (2x = 16).
Long-term culture establishment and efficient in vitro regeneration protocol for Sansevieria cyli... more Long-term culture establishment and efficient in vitro regeneration protocol for Sansevieria cylindrica Bojer ex Hook was developed using leaf derived callus and nodule culture. Profuse callus induction on leaf discs was achieved on Murashige and Skoog (MS) medium supplemented with 10 µM indole-3-butyric acid (IBA), while a high frequency of nodulation was induced on 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) containing media. Shoot regeneration ability from cultured tissues occurred at varying degrees on all media. Through callus culture a maximum of 17.6 ± 0.14 shoots per culture was formed on medium containing 5µM 6-benzyladenine (BA) and 2 µM α-naphthaleneacetic acid (NAA). Among nodule cultures, the 2,4-D generated nodules were more proliferative and regenerative as compared to 2,4,5-T induced nodules and a maximum of 25 ± 0.16 shoots per culture was produced on a medium containing 5 µM BA plus 1 µM NAA. The regenerated shoots were successfully rooted on a semi-solid half strength MS medium containing 5 µM IBA with an average root number 3.5 ± 0.18 and root length 6.5 ± 0.14 cm. The regenerative ability of callus tissues was steady upto one year, while the nodules retained the totipotency to regenerate on optimal medium even after 3 years of subculturing. The histological sections of nodules confirm the typical anatomy exhibiting the vascular elements in bundles with well demarcated cortex and epidermal covering.
Leaves, stem, and flowers of Saraca asoca, an endangered medicinal plant in India, and young expl... more Leaves, stem, and flowers of Saraca asoca, an endangered medicinal plant in India, and young explants cultivated on Murashige & Skoog's medium containing 6-benzylaminopurine were analyzed for antibacterial potential. Alcoholic and aqueous extracts from parent explants and their in vitro raised calli were tested by an agar well diffusion method. Minimal inhibitory concentrations (MICs) of the extracts were determined by broth microdilution method. Aqueous extracts showed antibacterial activity against limited bacterial species, whereas alcoholic extracts were active against a wider range of bacteria. Although the alcoholic extracts of all the explants and calli showed antibacterial activity, the extracts derived from flowers and their calli showed better results. Extracts derived from calli showed comparable results to the extracts from explants. Overall, the MICs of the extracts ranged from 0.039 to 1.25 mg/mL. MICs against gram-positive bacteria ranged from 0.078 to 1.25 mg/mL, whereas they ranged between 0.039 and 0.625 mg/mL against gram-negative bacteria. A MIC distribution plot showed that gram-negative bacteria were more susceptible to the extracts than gram-positive bacteria. It is concluded that extracts of S. asoca contain antibacterial agent, and as the calli gave good results, in vitro cultivation of the explants may be used to obtain antibacterial compounds. This is the first report on antibacterial activity of S. asoca, especially through in vitro raised calli.
Somatic embryogenesis (SE) has been achieved from hypocotyl-derived callus culture in Pterocarpus... more Somatic embryogenesis (SE) has been achieved from hypocotyl-derived callus culture in Pterocarpus marsupium. Ninety percent of hypocotyl explants (excised from 12-day-old in vitro germinated axenic seedlings) produced callus on Murashige and Skoog medium supplemented with 5 μM 2,4-dichlorophenoxyacetic acid and 1 μM a 6-benzyladenine (BA). Induction of SE occurred after transfer of callus clumps (200 ± 20 mg fresh mass) to MS medium supplemented with BA at 2.0 μM, where a maximum of 23.0 ± 0.88 globular stage embryos per callus clump were observed after 4 weeks of culture. Subculturing of these embryos on MS medium supplemented with 0.5 μM BA, 0.1 μM α-naphthalene acetic acid and 10 μM abscisic acid significantly enhanced the maturation of somatic embryos to early cotyledonary stage, where 21.4 ± 0.32 embryos per callus clump were recorded after 4 weeks of culture. Of 30-well developed somatic embryos, 16.6 ± 0.33 germinated and subsequently converted into plantlets on half-strength MS medium supplemented with 1.0 μM BA. The morphologically normal plantlets with well-developed roots were first transferred to 1/4-liquid MS medium for 48 h and then to pots containing autoclaved soilrite and acclimatized in a culture room. Thereafter, they were transferred to a greenhouse, where 60% of them survived.
The aim of this study was to develop a new micropropagation system for Cassia angustifolia Vahl.,... more The aim of this study was to develop a new micropropagation system for Cassia angustifolia Vahl., an important medicinal legume using root explant as starting material. Root explants taken from 30-day-old aseptic seedlings were cultured on Murashige and Skoog (MS) medium supplemented with different plant growth regulators: 6-benzyladenine (BA), kinetin (Kn), and thidiazuron (TDZ). Organogenic nodular calli obtained on MS + TDZ (1.0 μM) were transferred to shoot regeneration medium supplemented with different cytokinins (BA, Kn or TDZ) either alone or in combination with auxin:indole-3-acetic acid or α-naphthalene acetic acid. Maximum shoot regeneration frequency (90%) was obtained on MS + BA (2.5 μM) + NAA (0.6 μM) wherein a maximum of 42.76 ± 1.47 shoot buds per explant were induced with a maximum conversion rate of 35.63 ± 0.75 shoots per explant and average shoot length of 5.43 ± 0.20 cm. Elongated microshoots were successfully rooted under ex vitro conditions by pulse treatment in 200 μM of indole-3-butyric acid for half an hour. Microshoots were rooted, acclimatized and hardened off simultaneously in sterilized soilrite inside the growth room and then established in pots containing sterilized soil and manure (1:1) and grown under greenhouse condition with 90% survival rate. The histological sections at different developmental stages of shoot buds revealed the organization of nodular meristematic zone leading to the orientation and differentiation of shoot buds in large number and thereafter conversion into healthy shoots.
A method for rapid in vitro propagation of Cassia siamea Lam. using cotyledonary node explants, e... more A method for rapid in vitro propagation of Cassia siamea Lam. using cotyledonary node explants, excised from 14-day old aseptic seedlings, has been established. Murashige and Skoog (MS) medium supplemented with different concentrations of 6-benzyladenine (BA), kinetin (Kn) and thidiazuron (TDZ) singly or in combination with auxins was used for regeneration studies. Among the single treatment of three cytokinins BA at 1.0 µM was found to be optimum for direct shoot regeneration as it induced an average of 8.20 ± 0.66 shoots per explant. The regeneration frequency further enhanced with the application of auxin along with optimal BA concentration. The highest frequency for shoot regeneration (90%), the maximum number of shoots per explant (12.20 ± 0.73) and the maximum shoot length (6.40 ± 0.07) cm were obtained on the medium consisted of MS + 1.0 µM BA + 0.5 µM NAA. Successful in vitro rooting was induced from cut end of the microshoots when placed on half-strength MS + IBA (2.5 µM). The regenerated shoots with well developed root system were successfully acclimatized and established in pots containing sterilized garden soil and garden manure (1:1) and grown under greenhouse conditions with 85% survival rate.
Cassia sophera Linn. is an important medicinal plant belonging to family Fabaceae. It is extensiv... more Cassia sophera Linn. is an important medicinal plant belonging to family Fabaceae. It is extensively used in Homeopathy and is well known for its medicinal properties. The present study describes a simple, efficient and reproducible regeneration system for in vitro propagation of C. sophera through cotyledonary node (CN) explant excised from 21 d old axenic seedlings. Explants were cultured on Murashige and Skoog (MS) medium supplemented with different concentrations of thidiazuron (TDZ). Multiple shoots were produced on all the concentrations of TDZ; however 2.5 μM concentration proved to be optimal for the production of maximum number of shoots. To avoid adverse effects of prolonged exposure to TDZ in long term establishment, the cultures were transferred to TDZ free MS medium fortified with various concentrations of 6- benzyl aminopurine (BA) for multiplication, proliferation and elongation of induced shoots. Emergence of new shoot buds and multiplication continued up to second subculture passage and maximum number (14.9 ± 1.4) of shoots obtained on MS + BA (1.0 μM). Best rooting response was observed on half strength MS containing Indole-3-butyric acid (IBA) (1.0 μM). Regenerated plantlets were successfully acclimatized and hardened off inside the culture room and then transferred to green house with 100 % survival rate.
a b s t r a c t Progress in biotechnological research over the last two decades has provided grea... more a b s t r a c t Progress in biotechnological research over the last two decades has provided greater scope for the improvement of crops, forest trees and other important plant species. Plant propagation using synthetic seeds has opened new vistas in the field of agriculture. Synseed technology is a highly promising tool for the management of transgenic and seedless plant species, polyploid plants with elite traits and plant lines that are difficult to propagate through conventional propagation methods. Delivery of synseeds also alleviates issues like undertaking several passages for scaling up in vitro cultures as well as acclimatization to ex vitro conditions. Optimization of synchronized propagule development followed by automation of the whole process (sorting, harvesting, encapsulation and conversion) can enhance the pace of synseed production. Cryopreservation of encapsulated germplasm has now been increasingly used as an ex vitro conservation tool with the possible minimization of adverse effects of cryoprotectants and post-preservation damages. Through synseed technology, germplasm exchange between countries could be accelerated as a result of reduced plant quarantine requirements because of the aseptic condition of the plant material.
This paper reports an efficient regeneration protocol through parallel organogenic and embryogeni... more This paper reports an efficient regeneration protocol through parallel organogenic and embryogenic pathways from green root segments (GRSs) of Tylophora indica (Burm.f) Merrill. GRSs explants from one year old in vitro cultures were cultured on Murashige and Skoog (MS) medium containing various cytokinins. Five mmol/L of 6-benzyladenine (BA) was most responsive for organogenesis in 1.5 cm long GRSs. Repeated subculture on medium containing both BA (5 mmol/L) and 1-naphthleneacetic acid (NAA) (0.1 mmol/L) promoted multiplication and proliferation of direct shoot buds (46.80 AE 0.96) and callus mediated somatic embryogenesis (18.07 AE 0.33). Germinated embryos isolated from callus were transferred onto maturation medium consisting of half-strength MS medium either devoid of plant growth regulators (PGRs) or with various concentrations of gibberellic acid (GA). Microshoots were excised during subculture and transferred onto root induction medium, thus ensuring a continuous supply of germplasm. Morphogenic variations were noticed in types of roots induced on various auxins. Regenerated plantlets and emblings hardened best on vermiculite with a survival rate of 90% and 70% respectively. However, the emblings were healthier in comparison to the regenerated plants. Histological analysis showed the origin and development of organogenesis.
The aqueous and alcoholic extract of fresh Berberis aristata DC roots, as well as aqueous extract... more The aqueous and alcoholic extract of fresh Berberis aristata DC roots, as well as aqueous extract of dried roots were compared for their antibacterial and antifungal activities by the disc diffusion method. All three extracts showed wide antibacterial activity against Gram-positive bacteria. Among the Gramnegative bacteria tested, the antibacterial activity was limited to Escherichia coli, Salmonella typhimurium, Shigella dysenteriae type 1 and Vibrio cholerae; with the best activity against V. cholerae. MICs of the alcoholic extracts against Gram-positive bacteria ranged between 3.8 ×10 -3 to 6.1 ×10 -3 mg/ml and for Gram-negative bacteria from 6.1 ×10 -3 to 7.6 10 -3 mg/ml. The MICs for Candida species ranged between 0.02 to 3.8 ×10 -3 mg/ml and for Aspergillus species, it was 3 × 10 -3 mg/ml. All three extracts also had antifungal activity against the fungal species tested, except Candida krusei. The extracts of B. aristata also demonstrated anti-inflammatory, analgesic, and antipyretic activities. Chemical analysis revealed the presence of alkaloids, amino acids, tannins, terpenes, resins, phenols and reducing sugars as major compounds. FTIR-spectral analysis of all the extracts revealed the presence of berberine, as a major constituent, along with other chemical constituents.
Pterocarpus marsupium, Clitoria ternatea, and Sanseveiria cylindrica are some of the important an... more Pterocarpus marsupium, Clitoria ternatea, and Sanseveiria cylindrica are some of the important and endangered medicinal plant species of India. Despite of medicinal properties, antibacterial potential of the plants have not yet been explored. The present study was designed to optimize the in vitro technique for micropropagation and to screen the extracts from leaves and in vitro raised calli for antibacterial properties. Excised leaf-explants from the parent plants were surface sterilized and cultivated on Murashige & Skoog's (MS) medium containing N 6 -benzyladenine (BA) in concentrations of 1, 2, 5, and 10 µM. Optimal growth of calli was noticed at a concentration of 5 µM, therefore the extracts from calli grown at this concentration were further studied for antibacterial activity. Both alcoholic and aqueous extracts from leaves of respective plants, and their in vitro raised calli were tested for antibacterial activity by agar well diffusion method against a range of Gram-positive and Gram-negative bacteria. Aqueous extracts showed antibacterial activity against limited number of bacterial species; notably the extracts of C. ternatea which showed antibacterial activity against Streptococcus pyogenes, Bacillus subtilis and Bacillus cereus. Alcoholic extracts of all three plants showed antibacterial activity against a wider range of bacteria. Among the Gram-positive bacteria, extracts from C. ternatea showed strong antibacterial activity against Bacillus spp., whereas the extracts of S. cylindrica showed good antibacterial potential for Staphylococcus aureus, S. epidermidis and S. pyogenes. The extracts from all three plants showed antibacterial activity against Gram-negative bacteria, including, Salmonella spp. and Shigella dysenteriae; organisms causing enteric fever and dysentery. In most of the cases, the extracts from respective calli showed comparable, and in some cases better, result in comparison to the extracts from parent leaves. To the best of our knowledge this is the first preliminary report on antibacterial potential, especially through calli extracts, of these plants; and in vitro cultivation of the explants may be used to obtain phytotherapeutic compounds.
in Vitro Cellular & Developmental Biology-plant, 2007
An efficient protocol was developed for micropropagation of an economically important timber-yiel... more An efficient protocol was developed for micropropagation of an economically important timber-yielding multipurpose tree, Pterocarpus marsupium Roxb. Multiple shoots were induced from cotyledonary nodes (CNs) derived from 18-d-old axenic seedlings on Murashige and Skoog (MS) medium supplemented with thidiazuron (TDZ) (0.1–10 μM). The highest shoot regeneration frequency (90%) and maximum number (15.2 ± 0.20) of shoots per explant was recorded on MS medium amended with 0.4 μM TDZ. Continuous presence of TDZ inhibited shoot elongation. In the primary medium, TDZ-initiated cultures were transferred to the secondary medium supplemented with another cytokinin, 6-benzyladenine (BA), for shoot growth and elongation. Maximum (90%) shoot elongation with an average shoot length of 5.4 ± 0.06 cm was observed at 5 μM BA. To further enhance the number of shoots per explant, mother tissue was repeatedly subcultured on fresh shoot induction medium after each harvest of newly formed shoots. Thus, by adopting this strategy, an average of 44 shoots per explant could be obtained. About 65% of in vitro regenerated shoots produced a maximum number (4.4 ± 0.2) of roots per shoot by a two-step culture procedure employing pulse treatment and subsequent transfer of treated shoots to a low concentration of 0.2 μM indole-3-butyric acid along with phloroglucinol (3.96 μM). The in vitro-raised plantlets were successfully acclimatized first under culture room conditions, then to greenhouse with 70% survival rate.
... well known biotechnological tool for the rapid propagation of medicinal plants for the purpos... more ... well known biotechnological tool for the rapid propagation of medicinal plants for the purpose of commercialization (Kitto, 1997), conservation (Anis et al ... photoperiod with a photosynthetic photon flux density (PPFD) of 50 µM m-2 s-1 irradiance provided by cool white fluorescent ...
Pterocarpus marsupium (Bijasal) is a valuable multipurpose forest tree. The regeneration rate in ... more Pterocarpus marsupium (Bijasal) is a valuable multipurpose forest tree. The regeneration rate in natural habitat is low and the tree is overexploited. An in vitro propagation protocol has been developed from nodal explants obtained from in vitro raised 18-day-old axenic seedlings. The highest shoot regeneration frequency (85%), maximum number of multiple shoots (8.6) as well as length (4.8 cm) were induced from nodal explants on Murashige and Skoog (MS) medium amended with 4.0 μM 6-benzyladenine (BA), 0.5 μM indole-3-acetic acid (IAA) and 20 μM adenine sulphate (AdS). The percentage of shoot multiplication as well as the number of shoots per node remained the same during the first two subculture, afterwards a decline was recorded. Rooting was best induced in microshoots excised from proliferated shoot cultures on semisolid hormone-free half-strength MS medium, after a pulse (dip) treatment for 7 days in half-strength MS liquid medium containing 100 μM indole-3-butyric acid (IBA) and 15.84 μM phloroglucinol (PG). The in vitro-raised plantlets were potted and acclimatized under culture room conditions for 4 weeks before their transfer to a greenhouse, where the established plants showed 75% survival.
An efficient protocol has been developed for high-frequency shoot regeneration and plant establis... more An efficient protocol has been developed for high-frequency shoot regeneration and plant establishment of Clitoria ternatea – a potential medicinal legume. Adventitious shoots were regenerated from young excised root segments of aseptic seedlings on Murashige and Skoog (MS) medium supplemented with various concentrations of 6-benzyladenine (BA), kinetin, α-naphthalene acetic acid (NAA) or 2,4-dichlorophenoxy acetic acid (2,4-D) either singly or in various combinations. The highest frequency (100%) of shoot regeneration and maximum number (16.4 ± 0.24) of shoots per explant was obtained on MS medium supplemented with 20 μm BA and 2.0 μm NAA. Organogenic calli were produced on a medium containing 2,4-D (10 or 20 μm) and BA (5.0 μm). The calli were differentiated into multiple shoots on MS medium supplemented with 2.5–10 μm BA and 2.0 μm NAA. The microshoots were rooted on half-strength MS medium supplemented with 5.0 μm indole-3-butyric acid and transplanted successfully in field conditions. After 12 months of transfer to ex vitro conditions, the performance of micropropagated plants were evaluated on the basis of some physiological and biochemical parameters and compared with the in vivo–grown plants of the same age. The sodium dodecyl sulphate polyacrylamide gel electrophoresis protein profile was same between regenerated and naturally growing shoots. Total soluble protein in aerial part as well as in seeds of in vitro–regenerated and in vivo–grown plants was almost the same. The mitotic study showed normal chromosomal movement and numbers (2x = 16).
Long-term culture establishment and efficient in vitro regeneration protocol for Sansevieria cyli... more Long-term culture establishment and efficient in vitro regeneration protocol for Sansevieria cylindrica Bojer ex Hook was developed using leaf derived callus and nodule culture. Profuse callus induction on leaf discs was achieved on Murashige and Skoog (MS) medium supplemented with 10 µM indole-3-butyric acid (IBA), while a high frequency of nodulation was induced on 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) containing media. Shoot regeneration ability from cultured tissues occurred at varying degrees on all media. Through callus culture a maximum of 17.6 ± 0.14 shoots per culture was formed on medium containing 5µM 6-benzyladenine (BA) and 2 µM α-naphthaleneacetic acid (NAA). Among nodule cultures, the 2,4-D generated nodules were more proliferative and regenerative as compared to 2,4,5-T induced nodules and a maximum of 25 ± 0.16 shoots per culture was produced on a medium containing 5 µM BA plus 1 µM NAA. The regenerated shoots were successfully rooted on a semi-solid half strength MS medium containing 5 µM IBA with an average root number 3.5 ± 0.18 and root length 6.5 ± 0.14 cm. The regenerative ability of callus tissues was steady upto one year, while the nodules retained the totipotency to regenerate on optimal medium even after 3 years of subculturing. The histological sections of nodules confirm the typical anatomy exhibiting the vascular elements in bundles with well demarcated cortex and epidermal covering.
Leaves, stem, and flowers of Saraca asoca, an endangered medicinal plant in India, and young expl... more Leaves, stem, and flowers of Saraca asoca, an endangered medicinal plant in India, and young explants cultivated on Murashige & Skoog's medium containing 6-benzylaminopurine were analyzed for antibacterial potential. Alcoholic and aqueous extracts from parent explants and their in vitro raised calli were tested by an agar well diffusion method. Minimal inhibitory concentrations (MICs) of the extracts were determined by broth microdilution method. Aqueous extracts showed antibacterial activity against limited bacterial species, whereas alcoholic extracts were active against a wider range of bacteria. Although the alcoholic extracts of all the explants and calli showed antibacterial activity, the extracts derived from flowers and their calli showed better results. Extracts derived from calli showed comparable results to the extracts from explants. Overall, the MICs of the extracts ranged from 0.039 to 1.25 mg/mL. MICs against gram-positive bacteria ranged from 0.078 to 1.25 mg/mL, whereas they ranged between 0.039 and 0.625 mg/mL against gram-negative bacteria. A MIC distribution plot showed that gram-negative bacteria were more susceptible to the extracts than gram-positive bacteria. It is concluded that extracts of S. asoca contain antibacterial agent, and as the calli gave good results, in vitro cultivation of the explants may be used to obtain antibacterial compounds. This is the first report on antibacterial activity of S. asoca, especially through in vitro raised calli.
Somatic embryogenesis (SE) has been achieved from hypocotyl-derived callus culture in Pterocarpus... more Somatic embryogenesis (SE) has been achieved from hypocotyl-derived callus culture in Pterocarpus marsupium. Ninety percent of hypocotyl explants (excised from 12-day-old in vitro germinated axenic seedlings) produced callus on Murashige and Skoog medium supplemented with 5 μM 2,4-dichlorophenoxyacetic acid and 1 μM a 6-benzyladenine (BA). Induction of SE occurred after transfer of callus clumps (200 ± 20 mg fresh mass) to MS medium supplemented with BA at 2.0 μM, where a maximum of 23.0 ± 0.88 globular stage embryos per callus clump were observed after 4 weeks of culture. Subculturing of these embryos on MS medium supplemented with 0.5 μM BA, 0.1 μM α-naphthalene acetic acid and 10 μM abscisic acid significantly enhanced the maturation of somatic embryos to early cotyledonary stage, where 21.4 ± 0.32 embryos per callus clump were recorded after 4 weeks of culture. Of 30-well developed somatic embryos, 16.6 ± 0.33 germinated and subsequently converted into plantlets on half-strength MS medium supplemented with 1.0 μM BA. The morphologically normal plantlets with well-developed roots were first transferred to 1/4-liquid MS medium for 48 h and then to pots containing autoclaved soilrite and acclimatized in a culture room. Thereafter, they were transferred to a greenhouse, where 60% of them survived.
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