In vitro propagation of Farsetia macrantha Blatt. & Hallb.: an endemic and threatened plant of Indian Thar Desert

Plant Cell, Tissue and Organ Culture (PCTOC) - Farsetia macrantha Blatt. & Hallb. is an endemic and critically endangered plant species of Indian Thar desert. Being an endemic and...     

A high-frequency in vitro multiplication,micromorphological studies and ex vitro rooting of Cadaba fruticosa (L.) Druce (Bahuguni): a multipurpose endangered medicinal shrub

Physiology and Molecular Biology of Plants - An efficient and reproducible in vitro propagation protocol has been established for Cadaba fruticosa (L.) Druce. Surface-sterilized nodal stem segments...     

Rapid multiplication of Dalbergia sissoo Roxb.: a timber yielding tree legume through axillary shoot proliferation and ex vitro rooting

An efficient and improved method for in vitro propagation of mature tree of Dalbergia sissoo, an ecologically and commercially important timber yielding species, has been developed through axillary shoot proliferation. Bud breaking occurred from nodal shoot segments derived from rejuvenated shoots produced during early spring from a 20–25-year-old lopped tree, on MS medium containing 8.88 μM benzylaminopurine (BAP). Multiple shoots differentiated (20–21shoots/node) on re-culture of explants on half-strength agar gelled amended MS medium with a combination of 2.22 μM of BAP and 0.002 μM of thidiazuron (TDZ) with 1.0 mM each of Ca(NO₃)₂, K₂SO₄, KCl, and NH₄(SO₄)₂. The maximum shoot multiplication (29–30 shoots/node) was achieved on subculturing in the above mentioned but liquid medium. Furthermore, the problem of shoot tip necrosis and defoliation observed on solid medium were overcome by the use of liquid medium. Ex vitro rooting was achieved on soilrite after basal treatment of microshoots with 984 μM of indole-3-butyric acid (IBA) for 2 min. About 90 % microshoots were rooted on soilrite within 2–3 weeks under the greenhouse conditions. From 20 nodal shoot segments, about 435 hardened plants were acclimatized and transplanted. This is the first report for rapid in vitro propagation of mature trees of D. sissoo on liquid medium followed by ex vitro rooting.     

Rapid in vitro propagation,conservation and analysis of genetic stability of Viola pilosa

A protocol for in vitro propagation was developed for Viola pilosa, a plant of immense medicinal value. To start with in vitro propagation, the sterilized explants (buds) were cultured on MS basal medium supplemented with various concentrations of growth regulators. One of the medium compositions MS basal + 0.5 mg/l BA + 0.5 mg/l TDZ + 0.5 mg/l GA₃ gave best results for in vitro shoot bud establishment. Although the problem of shoot vitrification occurred on this medium but this was overcome by transferring the vitrified shoots on MS medium supplemented with 1 mg/l BA and 0.25 mg/l Kn. The same medium was found to be the best medium for further in vitro shoot multiplication. 100 % root induction from in vitro grown shoots was obtained on half strength MS medium supplemented with 1 mg/l IBA. In vitro formed plantlets were hardened and transferred to soil with 83 % survival. Additionally, conservation of in vitro multiplying shoots was also attempted using two different approaches namely slowing down the growth at low temperature and cryopreservation following vitrification. At low temperature retrieval rate was better at 10 °C than at 4 °C after conservation of in vitro multiplying shoots. In cryopreservation–vitrification studies, the vitrified shoot buds gave maximum retrieval of 41.66 % when they were precooled at 4 °C, while only 16.66 % vitrified shoots were retrieved from those precooled at 10 °C. Genetic stability of the in vitro grown plants was analysed by RAPD and ISSR markers which indicated no somaclonal variation among in vitro grown plants demonstrating the feasibility of using the protocol without any adverse genetical effects.     

In vitro micropropagation of Dracaena sanderiana Sander ex Mast: An important indoor ornamental plant

A protocol has been developed for in vitro plant regeneration from a nodal explant of Dracaena sanderiana Sander ex Mast. Nodal explant showed high callus induction potentiality on MS medium supplemented with 6.78 μM 2,4-dichlorophenoxyacetic acid (2,4-D) followed by 46.5 μM chlorophenoxy acetic acid (CPA). The highest frequency of shoot regeneration (85%) and number of shoots per explant (5.6) were obtained on medium supplemented with 7.84 μM N⁶-benzylaminopurine (BA). Rooting was high on MS solid compared to liquid medium when added with 7.38 μM indole-3-butyric acid (IBA). Fifty percent of the roots were also directly rooted as microcuttings on soil rite, sand and peat mixture (1:1:1). In vitro and ex vitro raised plantlets were used for acclimatization. More than 90% of the plantlets was successfully acclimatized and established in plastic pots. Ex vitro transferred plantlets were normal without any phenotypic aberrations.     

An efficient in vitro regeneration of Ceropegia noorjahaniae: an endemic and critically endangered medicinal herb of the Western Ghats

An efficient protocol was developed for the rapid in vitro multiplication of an endemic and critically endangered medicinal herb, Ceropegia noorjahaniae Ans., via enhanced axillary bud proliferation from nodal explants. The effects of phytohormones [6-benzylaminopurine (BAP), kinetin (Kin) thidiazuron (TDZ), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) or α-naphthalene acetic acid (NAA)] on in vitro regeneration were investigated. The highest number of shoots (18.3 ± 1.3), maximum shoot length (10.1 ± 0.8 cm) and the highest response of shoot induction (95 %) were recorded on MS medium supplemented with 2.0 mg/l BAP. Rooting was best achieved on half-strength MS medium augmented with IBA (1.0 mg/l). Half-strength MS medium supplemented with BAP (4 mg/l) and sucrose (5 %, w/v) produced an average of 5.6 flower buds per microshoots with highest (90 %) flower bud induction response. The plantlets regenerated in vitro with well-developed shoot and roots were successfully established in pots containing sterile sand and coco peat (1:1) and grown in a greenhouse with 85 % survival rate. The regenerated plants did not show any detectable morphological variation. The developed method can be successfully employed for large-scale multiplication and conservation of C. noorjahaniae.     

Micropropagation and in vitro conservation of the rare and threatened plants Ramonda serbica and Ramonda nathaliae

Ramonda serbica and Ramonda nathaliae are rare and endemo relict plant species from Balkan Peninsula. An efficient micro propagation and in vitro conservation method via direct and indirect organogenesis from seed and leaf explants, respectively, was established in this study. The seed of both Ramonda species were collected from different populations in Kosovo, and were germinated in nutrient media JG-B without any phytohormone. The highest number of shoots and multiplication rate was observed on JG-B medium supplemented with BAP and IAA (0.5 mg l⁻¹ each), whereas the highest number of leaves per plantlets was found on WPM and RA medium supplemented with BAP and IAA (0.1 mg l⁻¹ each). During this stage of micro propagation some significant differences were observed in plantlets from different populations. The indirect organogenesis from parts of leaves of natural plants was not successful due to unavailability of established protocol for disinfections of the plant material. On other hand, parts of leaves from micro propagated plantlets, cultured on MS medium supplemented with different ratio of BAP and NAA, resulted in the highest efficiency for shoot regeneration. In vitro conservation of micro propagated plants at the lower temperature (4 °C) had a significantly positive effect for storage of more than 12 months.     

In vitro propagation and withaferin A production in Withania ashwagandha,a rare medicinal plant of India

Withania ashwagandha, belonging to the family Solanaceae, is an important medicinal herb of India with restricted geographic distribution. It is a rich source of withaferin A (WA) and other bioactive withanolides. In the present study a rapid in vitro mass propagation protocol of W. ashwagandha was developed from nodal explants. Nodal explants were cultured on MS medium supplemented with various concentrations and combinations of plant growth regulators (PGRs). The highest number of regenerated shoots per ex-plant (33 ± 2.7) and highest WA (13.4 ± 1.15 mg/g of DW) production was obtained on MS medium supplemented with 5.0 μM 6-benzyladenine (BA) and 1.0 μM Kinetin (Kn). In vitro raised shoots were further rooted on half-strength MS medium containing 2.0 μM Indole-3-butyric acid (IBA) and analyzed for WA production. The rooted plantlets when transferred to poly bags in the greenhouse showed 90 % survival frequency. Levels of WA were higher in the in vitro and ex vitro derived shoot and root tissues as compared to field grown mother plants. In an attempt to further maximize WA production, shoot cultures were further grown in liquid MS medium supplemented with 5.0 μM 6-benzyladenine (BA) and 1.0 μM Kinetin (Kn). Root cultures were grown on half strength MS liquid medium fortified with 2.0 μM of IBA. WA production in the liquid cultures was significantly higher compared to the static composition of the same media. This protocol, first of its kind in this plant, can be successfully employed for conservation, proliferation and large-scale production of WA. The regenerated plants can also be used in traditional medicine as an alternative to naturally collected plants.     

Genome sequence of Ensifer sp. TW10; a Tephrosia wallichii (Biyani) microsymbiont native to the Indian Thar Desert

Ensifer sp. TW10 is a novel N₂-fixing bacterium isolated from a root nodule of the perennial legume Tephrosia wallichii Graham (known locally as Biyani) found in the Great Indian (or Thar) desert, a large arid region in the northwestern part of the Indian subcontinent. Strain TW10 is a Gram-negative, rod shaped, aerobic, motile, non-spore forming, species of root nodule bacteria (RNB) that promiscuously nodulates legumes in Thar Desert alkaline soil. It is fast growing, acid-producing, and tolerates up to 2% NaCl and capable of growth at 40^oC. In this report we describe for the first time the primary features of this Thar Desert soil saprophyte together with genome sequence information and annotation. The 6,802,256 bp genome has a GC content of 62% and is arranged into 57 scaffolds containing 6,470 protein-coding genes, 73 RNA genes and a single rRNA operon. This genome is one of 100 RNB genomes sequenced as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria (GEBA-RNB) project.     

In vitro propagation of the alkaloid producing plant Datura insignis Barb. Rodr.

A method is presented for the in vitro propagation of Datura insignis Barb. Rodr. Nodal explants were cultured on Murashige and Skoog medium supplemented either with BA alone or in combination with 2,4-D or IAA. Shoot multiplication and elongation were obtained in various growth regulator concentrations. Best results were obtained in a medium with 1.0 mgl^-1 of BA. Individual shoots were excised and transfered to growth regulator-free medium for rooting. Additional multiplication was obtained by single-node culture using explants from these in vitro rooted shoots. Rooted plantlets were successfully grown in soil.     

In Vitro Antimicrobial Activity of <Emphasis Type="Italic">Acacia catechu</Emphasis> and Its Phytochemical Analysis

Acacia catechu, commonly known as catechu, cachou and black cutch is an important medicinal plant and an economically important forest tree. The methanolic extract of this plant was found to have antimicrobial activities against six species of pathogenic and non-pathogenic microorganisms: Bacillus subtilis, Staphylococcus aureus, Salmonella typhi, Escherichia coli, Pseudomonas aeruginosa and Candida albicans. The maximum zone of inhibition (20 mm) was found to be exhibited against S. aureus. For this organism the minimum bactericidal concentration (MBC) of the crude extract was 1,000 μg/ml. The extract was found to be equally effective against gram positive and gram negative bacteria. The antimicrobial activity of the extract was found to be decreased during purification. The chemical constituents of organic plant extracts were separated by thin layer chromatography (TLC) and the plant extracts were purified by column chromatography and were further identified by Gas chromatography–mass selection (GC–MS) analysis. The composition of A. catechu extract had shown major components of terpene i.e. camphor (76.40%) and phytol (27.56%) along with other terpenes in minor amounts which are related with their high antibacterial and antifungal properties.     

Establishment of callus and cell suspension culture of Scrophularia striata Boiss.: an in vitro approach for acteoside production

In the present study, a protocol was optimized for establishment of callus and cell suspension culture of Scrophularia striata Boiss. as a strategy to obtain an in vitro acteoside producing cell line for the first time. The effects of growth regulators were analyzed to optimize the biomass growth and acteoside production. The stem explant of S. striata was optimum for callus induction. Modified Murashige and Skoog medium supplemented with 0.5 mg/l naphthalene acetic acid + 2.0 mg/l benzyl adenine was the most favorable medium for callus formation with the highest induction rate (100 %), the best callus growth and the highest acteoside content (1.6 μg/g fresh weight). Incompact and rapid growing suspension cells were established in the liquid medium supplemented with 0.5 mg/l naphthalene acetic acid + 2.0 mg/l benzyl adenine. The optimum time of subculture was found to 17–20 days. Acteoside content in the cell suspension was high during exponential growth phase and decreased subsequently at the stationary phase. The maximum content of acteoside (about 14.25 μg/g cell fresh weight) was observed on the 17th day of the cultivation cycle. This study provided an efficient way to further regulation of phenylethanoid glycoside biosynthesis and production of valuable acteoside, a phenylethanoid glycoside, on scale-up in S. striata cell suspension culture.     

Mother plant age and seasonal influence on in vitro propagation of <Emphasis Type="Italic">Quercus euboica</Emphasis> Pap., an endemic,rare and endangered oak species of Greece

A micropropagation method for Quercus euboica Pap. was developed. Nodal explants from seedlings gave higher multiplication rates than explants from adult plants. Cultures initiated at the beginning of May produced the highest percentage of shoot forming explants and multiplication rate. Woody Plant Medium (WPM) salts, with 100 mg l⁻¹ myoinositol, 1 mg l⁻¹ thiamine, 0.5 mg l⁻¹ pyridoxine, 0.5 mg l⁻¹ nicotinic acid and 3% sucrose was used as basal medium and several cytokinins at various concentrations were evaluated for their effect on shoot multiplication. The highest shoot multiplication rate was obtained with 4.44 μΜ BA. IBA at 9.84 μΜ in the culture medium during the first week of culture, and if followed by culture in hormone-free medium, gave the best rooting results. Darkness at the beginning of the rooting period did not improve rooting. The use of plastic wrap as a cover material of the culture vessels enhanced rooting percentage and root number. Plantlets acclimatized ex vitro in soil from the natural environment of the species survived at a higher percentage (up to 93%) and had more vigorous growth than plantlets grown in a compost–perlite (2:1 v/v) medium (up to 36%).     

In vitro propagation of the medicinal plant Ziziphora tenuior L. and evaluation of its antioxidant activity

Ziziphora tenuior L. (Lamiaceae) is an aromatic herb used for its medicinal values against fungi, bacteria. Micropropagation can be used for large-scale multiplication of essential oil producing plants thus avoiding an overexploitation of natural resources. This work aims to develop a reliable protocol for the in vitro propagation of Z. tenuior, and to compare the antioxidant activity between in vitro propagated and wild plants.The explants were sterilized and cultured on MS medium containing different concentrations of growth regulators naphthalene acetic acid (NAA) or indole-3-butyric acid (IBA) with 0.5 mg/L of kinetin (Kin) callus formation was 70.2% after 45 days of incubation in dark on medium supplemented with 1.5 mg/L of NAA. After one month of callus culture on medium supplemented with 2 mg/L BA the shoot number was 5.12 and for the multiplication stage. The shoot number was 4.21 and length was 6.17 cm on medium supplemented with 1 mg/L Kin + 0.1 mg/L NAA.DPPH• reagent was used to test the antioxidant activity. The aqueous and methanol extracts of in vitro plants which were treated with 1.5 and 1 mg/L of kin plus 0.1 mg/L of NAA showed a strong DPPH• scavenging activity where IC₅₀ was 0.307 and 0.369 mg/ml, respectively, while the IC₅₀ of aqueous and methanol extracts of wild plants was 0.516 and 9.229 mg/ml, respectively. Our results suggested that plant growth regulators and in vitro culture conditions increased the antioxidant activity.     

In vitro propagation from axenic explants of <Emphasis Type="Italic">Lilium oxypetalum</Emphasis> (D. Don) Baker,an endemic bulbous plant of high altitude Himalaya

In vitro propagation protocol for Lilium oxypetalum, a high altitude Himalayan endemic lily, has been developed. Effect of explant types (i.e., callus and in vitro bulblet scales) and sucrose concentration [0–6.0% (w/v)] on in vitro bulblet regeneration of L. oxypetalum was tested in previously optimized Murashige and Skoog basal medium supplemented with 2.0 μM 6-benzyladenine and 0.1 μM α-naphthaleneacetic acid. Callus explants produced significantly (P < 0.01) higher number of bulblets per explant than bulblet scale explants. Of the different concentrations of sucrose tested, 4.5% (w/v) sucrose showed significantly (P < 0.01) higher percentage regeneration (i.e., 70.8 ± 4.2 and 79.2 ± 4.2% regeneration on callus and bulblet scale explants, respectively), and produced higher number of bulblets per explant (i.e., 9.0 ± 0.4 and 5.4 ± 0.5 bulblets on callus and bulblet scale explants, respectively). Regenerated bulblets developed 2–3 leaves when subcultured for 4 weeks and were subsequently transferred ex vitro with a survival rate of 66.7% after 6 weeks. Leaves of the survived plantlets became dry after growing ex vitro for 10 weeks, amongst which 86.4% re-sprouted after remaining dormant for 5–6 weeks and produced 1.5 bulblets per explant. Findings of the present study hold promise for efficiently multiplying the target species in view of its potential economic and conservation significance.     

Synergistic effect of BAP and GA3 on in vitro flowering of Guizotia abyssinica Cass.-A multipurpose oil crop

Apical and axillary buds of Guizotia abyssinica Cass., isolated from seedlings raised in vitro, were cultured. High frequency of shoot regeneration was achieved on MS medium with BAP (1 mgl⁻¹). Effect of BAP, Kn and GA₃ applied successively in culture on shoot regeneration and flower bud formation has been studied. The shoots differentiated in cultures elongated on this medium. These rooted subsequently on half strength MS medium. The shoots flowered in vitro on MS medium with a combination of BAP (0.1mgl⁻¹) + GA₃ (0.1 mgl⁻¹). The plantlets thus formed were successfully hardened with 90 % survival.     

In vitro propagation of Rudraksha (Elaeocarpus sphaericus (Gaertn.) K. Schum): a biotechnological approach for conservation

The species Elaeocarpus sphaericus (Rudraksha) is a religious, medicinally important threatened tree of India. An efficient micropropagation protocol has been developed from nodal explants of this plant species collected from north-east India for large scale production of planting material at favourable sites within the country. Best shoot initiation occurred in MS medium supplemented with 2.2μM BA+2.2μM Kn in combination. Addition of Casein Hydrolysate (CH) (100mg/L) increased the shoot number. Microshoots excised and subcultured in 2.0μM BA further enhanced growth and multiplication. The shoot cultures were maintained in this concentration for 2years with subculturing at 6weeks interval. MS medium containing 5.0μM NAA was most effective for rooting. Successfully acclimatized plants (80%) showed normal growth under suitable habitat conditions.     

Toxoplasma gondii: Effects of Artemisia annua L. on susceptibility to infection in experimental models in vitro and in vivo

Considering that the treatment for toxoplasmosis is based on drugs that show limited efficacy due to their substantial side effects, the purpose of the present study was to evaluate the effects of Artemisia annua on in vitro and in vivo Toxoplasma gondii infection. A. annua infusion was prepared from dried herb and tested in human foreskin fibroblasts (HFF) or mice that were infected with the parasite and compared with sulfadiazine treatment. For in vitro experiments, treatment was done on parasite before HFF infection or on cells previously infected with T. gondii and the inhibitory concentration (IC₅₀) values for each treatment condition were determined. Viability of HFF cells in the presence of different concentrations of A. annua infusion and sulfadiazine was above 72%, even when the highest concentrations from both treatments were tested. Also, the treatment of T. gondii tachyzoites with A. annua infusion before infection in HFF cells showed a dose-response inhibitory curve that reached up to 75% of inhibition, similarly to the results observed when parasites were treated with sulfadiazine. In vivo experiments with a cystogenic T. gondii strain demonstrated an effective control of infection using A. annua infusion. In conclusion, our results indicate that A. annua infusion is useful to control T. gondii infection, due to its low toxicity and its inhibitory action directly against the parasite, resulting in a well tolerated therapeutic tool.     

Primary pharmacological screening of an endemic plant from the Southern Morocco (Tetraena gaetula [Emb. & Maire] Beier & Thulin)

Tetraena gaetula (Emb. & Maire) Beier & Thulin (Zygophyllum gaetulum Emb. & Maire, Zygophyllaceae) is an endemic plant from the southern Morocco. This plant is widely used in Moroccan traditional medicine as an antispasmodic and antidiabetic. Our work aims to evaluate several pharmacological properties of extracts of T. gaetula such pro- or antiproliferative, immunomodulatory, analgesic and antidepressant effects. Initially, we studied intraperitoneally the acute toxicity of aqueous extract of T. gaetula in mice; the lethal dose 50 is 1.2 g/kg of body weight. Our results also showed a stimulating proliferative activity of T. gaetula, particularly at 6 μg/μL of the proteic extract on T lymphocytes. However, this same concentration of proteic extract induced rather cytotoxicity on B cells and macrophages. Our ex vivo results showed a dose-dependent response: (i) stimulation of lymphocyte subpopulations and monocytes in a dose 600 mg/kg, and (ii) immunosuppression at a dose 300 mg/kg. The pharmacological results in vivo showed a pronounced antidepressant effect of the proteic extract at all doses. However, the aqueous extract showed analgesic and anti-depressant effects, mainly at doses 300 and 600 mg/kg.     

High frequency organogenesis in hypocotyl,cotyledon, leaf and petiole explants of broccoli (Brassica oleracea L. var. italica), an important vegetable crop

Broccoli (Brassica oleracea L. var. italica) is an important, nutritionally rich vegetable crop, but severely affected by environmental stresses, pests and diseases which cause massive yield and quality losses. Genetic manipulation is becoming an important method for broccoli improvement. In the present study, a reproducible and highly efficient protocol for obtaining organogenesis from hypocotyl, cotyledon, leaf and petiole explants of broccoli (Brassica oleracea L. var. italica cv. Solan green head) has been developed. Hypocotyl and cotyledon explants were used from 10 to 12 days old aseptically grown seedlings whereas leaf and petiole explants were excised from 18 to 20 days old green house grown seedlings and surface sterilized. These explants were cultured on shoot induction medium containing different concentration and combination of BAP and NAA. High efficiency shoot regeneration has been achieved in hypocotyl (83.33 %), cotyledon (90.11 %), leaf (62.96 %) and petiole (91.10 %) explants on MS medium supplemented with 3.5 mg/l BAP + 0.019 mg/l NAA 2.5 mg/l BAP + 0.5 mg/l NAA, 4.0 mg/l BAP + 0.5 mg/l NAA and 4.5 mg/l BAP + 0.019 mg/l NAA respectively. Petiole explants showed maximum shoot regeneration response as compared to other explants. MS medium supplemented with 0.10 mg/l NAA was found best for root regeneration (100 %) from in vitro developed shoots. The regenerated complete plantlets were transferred to the pots containing cocopeat and successfully acclimatized. This optimized regeneration protocol can be efficiently used for genetic transformation in broccoli. This is the first comparative report on multiple shoot induction using four different types of explants viz. hypocotyl, cotyledon, leaf and petiole.