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In vitro regeneration and conservation of Wrightia tinctoria Roxb., a medicinally important woody plant
Abstract
Wrightia tinctoria Roxb. (WTR) is a medicinal plant with soft wood that belongs to Apocynaceae family. The therapeutic constituents of this plant are used in treating many human ailments like psoriasis, diabetics and cancer. In view of low percent of natural regeneration, over exploitation due to its medicinal (treatment of human ailments) and commercial importance (toy making and pala indigo dye) and the hurdles faced by the Etikoppaka toy making industry (Vizag, A.P. India), there is dier need for conservation and production of propagules of this valuable soft woody and medicinally important tree. This study is undertaken to optimize and develop large scale regeneration and hardening protocol in Vadlamudi, Guntur Dt., A.P. using nodal explants of fifteen days old (in vitro) seedling. The nodal explants are inoculated on MS media amended with BAP (1.5 mg/L) + NAA (0.1 mg/L), induced 14 shoots per explant in 30 days with a length of 5.2 cm and 85-90% shoot initiation. The shoots when inoculated on Murashige Skoog media reinforce with IBA (3.5mg/L), showed eight roots per one shoot explant with maximum length of 6.9 cm. 99 % shoots initiated roots within 21 days of shoot inoculation. After 28 days of rooting, the plantlets are acclimatized to natural conditions through different stages and the % of survival rates of plantlets in the natural conditions was found to be 55 %.
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A protocol for in vitro production and acclimatization of wrightia tinctoria (rox.) R. Br., is reported. Under the regime used in the study, the most critical factor for rooting was activated charcoal (0.05%) along with 6 μM Naphthalene aceticacid. The plantlets hardened with longer photoperiods had better survival percentages when compared with those acclimatised over shorter photoperiods. Hardened plantlets obtained within 45 days, and replanted with 86.8 % survival. This in vitro method can be used for rapid mass clonal propagation of W. tinctoria for conservation strategies, commercial production irrespective of season and gene transformation studies for the production of phytomedicines.
In this paper, we compared the efficacy of observation based modeling approach using a genetic algorithm with the regular statistical analysis as an alternative methodology in plant research. Preliminary experimental data on in vitro rooting was taken for this study with an aim to understand the effect of charcoal and naphthalene acetic acid (NAA) on successful rooting and also to optimize the two variables for maximum result. Observation-based modelling, as well as traditional approach, could identify NAA as a critical factor in rooting of the plantlets under the experimental conditions employed. Symbolic regression analysis using the software deployed here optimised the treatments studied and was successful in identifying the complex non-linear interaction among the variables, with minimalistic preliminary data. The presence of charcoal in the culture medium has a significant impact on root generation by reducing basal callus mass formation. Such an approach is advantageous for establishing in vitro culture protocols as these models will have significant potential for saving time and expenditure in plant tissue culture laboratories, and it further reduces the need for specialised background.
Wrightia tinctoria (Roxb.) R.Br. extensively used in the Indian system of medicine, is a small deciduous tree of the family Apocynaceae The plant is very useful as stomachic, antidysenteric, carminative, astringent, aphrodisiac and diuretic, used in the treatment of abdominal pain, skin diseases and bilious affections. This plant is reported to have fungicidal, antinociceptive, wound healing, immunomodulatory and antiulcer activity. The major phytoconstituents are triacontanol, tryptanthrin, (â-amyrin, ursolic acid, oleanolic acid, (â-sitosterol, cycloartenone, cycloeucalenol, (â-sitosterol, lupeol, wrightial, 14á-methylzymosterol desmosterol and clerosterol. A number of poly herbal formulations containing W. tinctoria is available in market for psoriasis, diarrhoea and dysentery, dandruff and for rejuvenation of joint function.
The present study was undertaken to investigate the effect of sub-acute administration of W. tinctoria bark extract on some haematological, biochemical, histological and antioxidant enzyme status of rat liver and kidney following 21 and 45 days treatment. The animals were observed for gross physiological and behavioural responses, food and water intake and body weight changes. Free radical scavenging activity and histopathology was done on liver and kidney samples. W. tinctoria showed significant hemopoiesis with increase in body weight signifying anabolic effect. It significantly reduced serum SGOT level and increased glucose levels. W. tinctoria caused increased SOD activity of liver along with catalase of both liver and kidney and decreased liver peroxidase (P< 0.001). These features indicate that W. tinctoria upto 1000 mg/kg daily dose is safe and has potential to be consumed for long time in management of various diseases.
An efficient method for totipotent callus formation and whole plant regeneration has been developed for chicory (Cichorium intybus L.). Totipotent calli of chicory were induced from cotyledon, leaf, hypocotyl and root explants on MS medium supplemented with different concentrations of IAA, IBA, NAA and 2,4-D at 0.5-10 µM in combination with BAP (2 µM). These calli were transferred to shoot regeneration medium containing MS basal medium with different concentrations and combinations of BAP, KIN and IAA. Maximum number of shoots was obtained on MS medium with BAP (4 µM) + IAA (1 µM). The shoots were rooted on MS medium supplemented with IAA, IBA and NAA. Rooted plantlets were successfully established in the field after hardening.
An efficient protocol has been developed for rapid mass propagation of Tylophora indica from leaf derived callus. Optimal callus was developed from leaf explants on Murashige and Skoog (MS) basal medium supplemented with 10 M 2,4,5-T. Adventitious shoots were regenerated (85%) from the surface of the callus on MS medium supplemented with 5 M Kinetin. Individual elongated shoots were rooted on half-strength MS medium containing 0.5 M IBA. Regenerated plantlets with well developed shoots and roots were successfully transferred to soil. The study demonstrated a dedifferentiated callogenic propagation route via adventitious shoot develop-ment in T. indica, which could be useful for large scale multiplication of this endangered medicinal plant. Abbreviations: BA – 6-benzyladenine; 2,4-D – 2,4-dichlorophenoxy acetic acid; IAA – indole-3-acetic acid; IBA – indole-3-butyric acid; Kinetin – 6-furfurylaminopurine; MS – Murashige and Skoog medium; NAA – -naphthalene acetic acid; 2,4,5-T – 2,4,5-trichlorophenoxy acetic acid
Plan: The hydro alcoholic extract of Wrightia tinctoria leaves was evaluated for antipsoriatic activity by mouse tail test.
Methodology: Antipsoriatic activity was performed at a dose 200 mg/kg body weight in mice (25-30 g). Isoretinoic acid (0.5 mg/kg) was used as the standard. Degree of orthokeratosis, drug activity and the relative epidermal thicknesses were calculated and statistically analyzed. The extract was also evaluated for its antioxidant potential by DPPH, nitric oxide and hydrogen peroxide radical scavenging
assays.
Outcome: The extract produced significant (p<0.01) degree of orthokeratosis compared to control and the drug activity was found to be 70.18%, which is more potent than the standard (57.43%).. The extract showed prominent antioxidant activity in all the assays. The present study concludes that the selected plant has antipsoriatic activity and can be used for psoriasis treatment.
Aim of this study was to identify and characterize the bioactive principles from the woody stem of Wrightia tinctoria. For isolation of the compounds, the dried woody stem’s powder of Wrightia tinctoria was subjected to hot extraction with petroleum ether and subjected to chromatography. Three compounds (PEW-1, PEW-2 and PEW-3) were isolated and purified by chloroform. Mass spectrum of PEW-1, PEW-2 and PEW-3 showed a parent molecular ion [M+] peak at mlz 426 which corresponds to the molecular formula C30H50O, 412 corresponds to C29H48O and 400 corresponds to C28H48O. In the 1H-NMR spectrum of PEW-1, H-3 proton appeared as a triplet of a double doublet (tdd) at δ 3.21, H-29 proton gives two multiplates at δ 4.71 and δ 4.56, in lH-NMR spectrum of PEW-2, H-3 proton appeared as a triplet of a double doublet (tdd) at δ3.62 and H-6 olefinic proton showed a multiplet at δ5.14. Two olefenic protons appeared downfield at δ 4.16 (m) and δ 4.14 (m). Six methyl proton appeared at δ 1.27, δ 1.19, δ 1.07, δ 1.00, δ 0.98 and δ 0.91 for methyl group and in the 1H-NMR data of PEW-3, H-3 proton appeared at δ 3.21 as a triplet of a double doublet H-6 olefinic proton showed a multiplet at δ 5.10 and Six methyl proton appeared at δ 1.27, δ 1.14, δ 1.09, δ 1.00, δ 0.98 and δ 0.95 for methyl group. From the physical, chemical and spectral characteristics, PEW-1, PEW-2 and PEW-3were concluded as lupeol, stigmasterol and campetosterol.
Multiple shoots were induced in vitro from nodal shoot segments of a 30-yr-old plus tree having enhanced axillary branching. Initiation of culture was strongly influenced by the nature of explant and the season of harvesting. Nodal segments (91%) from young lateral branches produced an average of 5 shoots per node in 3 weeks on agar solidified MS medium supplemented with 2.0 mg 1-1 benzylaminopurine (BAP). After establishment of cultures and initiation of shoot buds, a cluster of shoots including mother explant, was transferred to medium containing a lower concentration of BAP (1.0 mg 1 -1). A three-fold rate of shoot multiplication during every subculture of 3 weeks was achieved. Nodal segments from in vitro raised shoots were also used to initiate a new culture cycle. The shoots could be multiplied for at least 24 months without loss of vigour. The shoots (71%) were successfully rooted when their lower ends were dipped in pre-autoclaved indole-3-butyric acid solution (100 mg 1-1) for 10 min followed by their implantation on modified MS medium (major salts reduced to 1/4 strength) containing 200 mg 1-1 activated charcoal. Out of 2000 plants, 1600 (80%) were successfully hardened under greenhouse conditions and more than 200 have been planted out in soil where they are growing well.
Objective
To explore the cytotoxic activity of the alcoholic extracts of some medicinal plants used traditionally to treat cancer in Chhattisgarh state, India.
Methods
In-vitro cytotoxicity of alcoholic extracts of five plants i.e. Artocarpus heterophyllus, Alangium salvifolium, Buchanania lanzan, Sesbania grandiflora and Wrightia tinctoria was studied against human breast cancer (MCF-7) and human leukemia (HL-60) tumor cell lines, using the thiazolyl blue test (MTT) assay.
Results
Alcoholic extract of Sesbania grandiflora exhibited a prominent inhibitory effect against MCF-7 (IC50 7.00±0.08 μg/mL) and HL-60 (IC50 18.50±0.60 μg/mL) under in vitro condition.
Conclusions
From the result it can be found that the Sesbania grandiflora extract has potent in vitro cytotoxic activity.
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).
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.
In vitro method has been developed for propagation ofWrightia tinctoria R.Br. using cotyledonary node segments. Murashige and Skoog's (MS) medium supplemented with 5.0 mg dm−3 of 6-benzylaminopurine (BAP) and 0.01 mg dm−3 of naphthaleneacetic acid (NAA) induced up to eight shoots per explant with an average shoot length of 1.4 cm in 21 d. Three
fold multiplication rate was achieved during every subculture of regenerated shoots on the same medium producing an average
of 230 shoots per node within 84 d. Reduction in BAP concentration from 5.0 to 1.0 mg dm−3 during subculture promoted shoot length without affecting the rate of multiplication. The differentiated shoots could be
rooted by a dip treatment into preautoclaved indole-3-butyric acid (IBA-500 mg dm−3 for 5 min) followed by their implantation onto MS medium containing 1/4 salts. Rooting was observed within 8–10 d in approximately
80% of shoots inoculated after IBA treatment. 15 d after rooting, the plantlets were transferred to culture bottles containing
soil-SoilriteTM (1∶1) and liquid nutrient solution comprising 1/4 MS salts. After their partial hardening in these bottles for 10 d they
were transferred to pots containing soil-Soilrite (1∶1) mixture with 60% transplantation success. Methods are being standardized
to improve the rate of survival and large scale field transfer.
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