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Rapid in vitro propagation system through shoot tip cultures of Vitex trifolia L.-an important multipurpose plant of the Pacific traditional Medicine
Abstract
A rapid and efficient plant propagation system through shoot tip explants was established in Vitex trifolia L., a medicinally important plant belonging to the family Verbenaceae. Multiple shoots were induced directly on Murashige and Skoog (MS) medium consisting of different cytokinins, 6-benzyladenine (BA), kinetin (Kin) and 2-isopentenyl adenine (2-iP), BA at an optimal concentration of 5.0 μM was most effective in inducing multiple shoots where 90 % explants responded with an average shoot number (4.4±0.1) and shoot length (2.0±0.1 cm) after 6 weeks of culture. Inclusion of NAA in the culture medium along with the optimum concentration of BA promoted a higher rate of shoot multiplication and length of the shoot, where 19.2±0.3 well-grown healthy shoots with an average shoot length of 4.4±0.1 cm were obtained on completion of 12 weeks culture period. Ex vitro rooting was achieved best directly in soilrite when basal portion of the shoots were treated with 500 μM indole-3-butyric acid for 15 min which was the most effective in inducing roots, as 95 % of the microshoots produced roots. Plantlets went through a hardening phase in a controlled plant growth chamber, prior to ex-vitro transfer. Micropropagated plants grew well, attained maturity and flowered with 92 % survival rate. The results of this study provide the first report on in vitro plant regeneration of Vitex trifolia L. using shoot tip explants.
... The development of plant regeneration system from shoot tip segment is an effective way to multiply selected variety true to its type showing the same agronomic characteristics (Ahmed and Anis, 2014). In the present experiment, explants cultured on MS basal medium without any PGRs (Plant Growth Regulators) did not induce any morphogenetic response and failed to initiate the shoots. ...
... The capability of BAP in shoot initiation is observed in nodal explants of C. paniculatus (Phulwaria et al., 2013) and other species (Kumar et al., 2010;Singh and Tiwari, 2012;Pathak et al., 2017) as well. The increase in shoot length with BAP than Kin and TDZ may be for, effectiveness of BAP is to stimulate plant tissues to metabolize the natural endogenous hormone to induce the shoot organogenesis (Ahmed and Anis, 2014). It is well known that meta -Topolin plays positive influence than other plant growth regulators in shoot multiplication (Wojtania and Agnieszka, 2010). ...
It is believed and essential that efforts should be made to develop protocols for vulnerable medicinal plants so as to develop new or more safe drugs. In this study, a rapid efficient plant direct propagation through shoot tip explant of Celastrus paniculatus Willd, a medicinal vulnerable plant belongs to the family Celastraceae. Half strength Murashige and Skoog's (MS) medium supplemented with GA 3 showed maximum percentage (82.4 ± 0.50) embryo response through embryo rescue method. Shoot tip explants were transferred from cotyledonary node and inoculated to shoot induction medium supplemented with cytokinin's (BAP, TDZ and Kin) and highest response (87 ± 0.70) with 3.8 shoot number was achieved in BAP 1.0 mg L-1. Shoot multiplication was achieved with combination of BAP (1 mg L-1) with meta-Topolin (1 mg L-1) showed highest response (91.0 ± 1.10) with 10.2 shoots within 10 days after inoculation. The in vitro regenerated shoots were transferred carefully to the half strength and full-strength MS medium supplemented with GA 3 (0.1 to 0.5 mg L-1) for the elongation. The in vitro elongated shoots were treated with different auxins (IAA, IBA and NAA) individually for early rooting and treated shoots were transferred to the half strength MS medium. At the concentration IBA (0.3 mg L-1), 91 % rooting was observed. The regenerated plantlets were acclimatized in pots containing sterilized soil and sand with 3:1 ratio and plantlets were then transferred to the field conditions. Ninty percent of the regenerants survived well. The result of this study revealed the pioneer report on in vitro plant regeneration of Celastrus paniculatus Willd. by using shoot tip explants.
... Vitex trifolia L. (simpleleave chastetree, Labiatae) is a tropical shrub or tree (up to 5 m tall) that grows mainly in the coastal areas and is widely employed by Pacific islanders to cure numerous illnesses [85]. The leaves are used in maceration or decoction, both internally and externally, as a remedy to cure illnesses and especially to abrogate diseases that may involve inflammatory processes [18]. ...
... The leaves are used in maceration or decoction, both internally and externally, as a remedy to cure illnesses and especially to abrogate diseases that may involve inflammatory processes [18]. For example, it can be heated and rubbed on the forehead to treat headaches, and it can be applied topically as an infusion to relieve fever or alleviate pain derived from rheumatism and sprained joints [85,86]. The traditional aqueous decoction of V. trifolia leaves (after lyophilization, 1 mg/mL) has shown the ability to decrease the expression of numerous inflammatory mediators (e.g. ...
Inflammation is a ubiquitous host response in charge of restoring normal tissue structure and function but is a double-edged sword, as the uncontrolled or excessive process can lead to the injury of host cells, chronic inflammation, chronic diseases, and also neoplastic transformation. Throughout history, a wide range of species has been claimed to have anti-inflammatory effects worldwide. Among them, Angelica sinensis, Tropaeolum majus, Castilleja tenuiflora, Biophytum umbraculum, to name just a few, have attracted the scientific and general public attention in the last years. Efforts have been made to assess their relevance through a scientific method. However, inflammation is a complex interdependent process, and phytomedicines are complex mixtures of compounds with multiple mechanisms of biological actions, which restricts systematic explanation. For this purpose, the omics techniques could prove extremely useful. They provide tools for interpreting and integrating results from both the classical medical tradition and modern science. As a result, the concept of network pharmacology applied to phytomedicines emerged. All of this is a step toward personalized therapy.
... However, BA at lower concentrations (1.0 M and 5.0 M) in the MS medium was used in V. negundo for shoot induction and multiplication as reported by several workers (Ahmad et al., 2008;Afroz et al., 2008;Rathore and Shekhawat, 2011), Islam et al. (2009) reported that BA (8.8 M) was found to be most effective for multiple shoot induction from shoot tip explants of V. negundo. Hiregoudar et al. (2006) reported that maximum number of shoots was developed on MS medium supplemeted with BA 5.0 M in V. trifolia (Ahmad et al., 2013a,b;Ahmed and Anis, 2014b). BA proved to be more effective in comparison to Kn and 2iP in all the investigated Vitex species and the order of effectiveness is BA > Kn > 2iP as reported by several workers (Sahoo and Chand, 1998;Sharma et al., 2006;Hiregoudar et al., 2006;Afroz et al., 2008;Ahmad et al., 2008Ahmad et al., , 2013a. ...
... Other combinations investigated include BA + IAA, BA + IBA using various concentrations (Chandramu et al., 2003b;Vadawale et al., 2006;Usha et al., 2007;Ahmad et al., 2008;Rahman and Bhadra, 2011). In V. trifolia the combination of BA and NAA was also very effect as described by Ahmad et al. (2013a,b) and Ahmed and Anis (2014b). It was concluded that the combination of either BA or Kn with IBA was least productive as compared to those with IAA or NAA and the combination of BA and NAA was most effective in all the treatments using nodal explants tried by various workers. ...
... Similar to V. doniana, the effectiveness of BAP in multiple shoot formation has also been reported in other Vitex species, including Vitex negundo (7.4 shoots per explant; Ahmad and Anis, 2011) and V. trifolia (4.4 shoots per explant; Ahmed and Anis, 2014). Furthermore, BAP is widely used for the micropropagation of a number of commercially-important tree species (e.g., Mittal et al., 1989;Barakat and El-Lakany, 1992;Bueno et al., 2003;Anis et al., 2010;Watt, 2014;Vitambas et al., 2020). ...
Propagation of Vitex doniana by seeds has been limited by low yield and a prolonged germination period which, along with over-harvesting, has led to a decline in its natural population. The present study aimed to improve seed germination and to establish in vitro organogenesis protocols to conserve superior genotypes. The effects of seed pre-treatments and environmental conditions on seed germination were investigated through conventional germination methods. Removal of the seed coat followed by germination on filter paper resulted in the highest percentage germination (87 %). The best germination temperature range was 25°C-35°C (80À87 %). A direct organogenesis protocol was established when in vitro nodal explants were cultured for eight weeks on modified Woody Plant Medium (WPM) supplemented with 0.3 mg/L 6-benzyla-minopurine (BAP), resulting in 4 shoots/explant. Shoots were rooted on the modified WPM containing 0.5 mg/L indole-3-acetic acid (IAA), resulting in 80 % rooting and 4 roots/shoot, after four weeks. Successful acclimatisation was achieved, with 93 % survival in the greenhouse. For somatic embryogenesis, pro-embryos that formed from pairing 1 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) with 1 mg/L picloram or each tested auxin with 0.5 mg/L BAP failed to develop into somatic embryos on different maturation media. For time and cost savings and high yield, the present study found that germination be performed using de-coated seeds on filter paper at 25À35°C followed by transfer to soil upon germination. The present study reports the first direct organogenesis protocol for V. doniana, allowing for clonal propagation of high-value genotypes.
... BAP is a cytokine class hormone that stimulates the differentiation of cells in the meristematic tissues and promotes the growth of lateral shoots, apical dominance, and expansion of leaves (Decker et al. 2006;Yuniastuti et al. 2018). The increased shoot induction by BAP may be due to its effectiveness in stimulating plant tissues to metabolize its natural endogenous hormone to induce shoot organogenesis (Ahmed and Anis 2014;Moola and Kumari 2019). Similar to the results of our research, it has been demonstrated by many researchers that BAP is the most effective cytokinin on shoot formation for many plant species and can be used successfully in many species in shoot formation (Ram and Shekhawat 2011;Mayerni et al. 2020). ...
The habitat of the endemic, endangered, and medically important plant, Stachys cretica subsp. kutahyensis belongs to the family Lamiaceae, is threatened by human influence. This study used a shoot tip explant to standardize a simple and proper micropropagation system for relict endemic plant species. Murashige Skoog medium with different concentrations of gibberellic acid and juglone was used for in vitro germination. Seedlings from in vitro germinated plantlets were used as explant sources in tissue culture studies. The highest shoot average per explant (4.60 shoots) and shoot length (2.70 cm) was determined in MS medium with 2.0 mg/L 6-benzyl amino purine with 100% response in all treatments. This study used different rooting mediums: Murashige Skoog and modified Murashige Skoog media. The effects of Murashige Skoog and different concentrations of indole butyric acid, naphthaleneacetic acid, juglone, and modified MS, and different concentrations of juglone were investigated on rooting development. Elongated shoots were successfully rooted in the Mod MS medium with 10–7 M juglone. Rooted plantlets were gradually transferred to the soil by adapted external conditions. The transfer of S. cretica subsp. kutahyensis plants, adapted to the soil and field conditions (Tavşanlı/Nusretler), successfully carried out. This protocol can be used for conservation studies of endemic, endangered, and medicinally important plant species.
... In order to alleviate the damage caused by excess ROS, plants have evolved to develop a wide range of enzymatic defense mechanisms (Li et al., 2011). Similar to the other abiotic stresses, there is an enhancement in the antioxidant enzyme activities during the acclimatization process of in vitro raised plantlets (Dias et al., 2011;Ahmed and Anis, 2014). Evidence demonstrated that the application of T. viride stimulates the antioxidative enzyme activities to minimize the elevated ROS levels produced during stressed conditions (Ahmad et al., 2015;Zhang et al., 2016). ...
Curcuma longa L. is a spice crop with enormous medicinal and cosmetic properties cultivated across the world. It was propagated vegetatively by means of rhizomes, as these were underneath soil prone to soil borne fungal diseases. Treatment of such diseases using chemical fungicides would hinder their nutritional and medicinal value. To overcome such challenges, there are a few alternatives, the major among them was the deployment of disease-free in vitro raised plantlets and the other was biopriming of these plantlets with Trichoderma viride. Besides fungicidal action, T. viride has a prominent role in alleviating several biotic/abiotic stresses and it was more often used during acclimatization. In this study, we studied the role of T. viride on successful acclimatization of in vitro C. longa plantlets by modulating plant antioxidant defense systems. We determined the stress levels (MDA and chlorophyll contents) and monitored the antioxidant enzyme activities (CAT, APX and GR) in both T. viride-treated and untreated micropropagated plantlets at four different time points (0, 15, 30, 45, 60 days) of acclimatization period. Our results depicted clear enhancement in the antioxidant enzyme activities in T.viride treated than untreated plantlets, which signify the role of T. viride in activating defense mechanisms to combat against oxidative stress. Thus this study would give clear understanding of the influence of T. viride on in vitro raised plantlets in sustaining adverse acclimatization conditions.
... The plant regeneration from the nodal segment is one of the most favourable and used explants to multiply the plant, giving similar and identical plants to the mother plant. The nodal segment explants are the favoured explant type, and many researchers have used it to start culturing (Ahmed and Anis, 2014;Nandagopal et al., 2015;Shekhawat et al., 2017). Concerning the establishment nodal segments of A. lanata, data in table 2 showed that growth percentages ranged between 46.67 to 93.33%. ...
... Increase in the activities of antioxidant enzymes during ex vitro acclimatization have been observed in various micropropagated plantlets Ahmed and Anis, 2014). The current study results witnessed increase in all three antioxidative enzymes (CAT, APX and GR) in KIMP over BIMP during acclimatization process (Fig. 3a-c). ...
An improved micropropagation protocol was established from field grown sword suckers of Banana Grand Naine (Musa acuminata AAA) by evaluating the mircopropagules combating reactive oxygen species (ROS) during acclimatization. Optimization of surface sterilization of sprouted apical meristematic buds and pre-culture on Shoot Bud Induction Medium [MS + 4.0 mg l −1 BA under dark conditions for 3 weeks] resulted ideal number of multiple shoot clusters (12.20 ± 0.30). Subculture of these shoot clusters on Shoot Bud Multiplication Medium (SBMM) fortified with 2 mg l −1 BA and 20 mg l −1 AdS induced highest shoot buds (15.00 ± 0.75) in 3 weeks and showed consistent till seven passages. The individual in vitro shoots upon transferring onto MS + 2 mg l −1 KN +1.0 mg l −1 IBA + activated charcoal (3 g l −1) resulted in elongated shoots and in vitro rooting after 3 weeks at a photoperiod of 16 h light and 8 h dark conditions. Evaluation of antioxidant defense status in micropropagated plantlets during acclimatization revealed altered levels of MDA, chlorophyll, catalase, ascorbate peroxidase and glutathione reductase. Our study throws an insight into decisive/differential role of cytokinins (Kinetin) in combating oxidative stress during acclimatization of in vitro regenerated plantlets. The present investigation facilitates the massive propagation (90% survival rate) of healthy and quality in vitro plantlets of banana.
... In tissue cultures, several different types of basal media have been formulated, and the response of different plant species to these varied media is dependent on their nutritional needs [24]. MS medium has been widely used for micropropagation purposes in a number of medicinal plants such as Vitex negundo [25], Albizia lebbeck [26] and Centella asiatica [27], Artemisia sieberi [28], Artemisia pallens [29], Vitex trifolia [30], Ruta graveolens [31], Thymus persicus [32], Cassia alata [33], Bacopa monnieri [34], and Tecoma stans [35,36]. WPM medium induced the highest response in Rauvolfia tetraphylla [37], and B5 medium had the best morphogenic response in Saraca asoca [24]. ...
Ruta chalepensis L., an evergreen shrub in the citrus family, is well-known around the world for its essential oils and variety of bioactivities, indicating its potential medicinal applications. In this study, we investigated the effect of different culture conditions, including plant growth regulators, media types, pH of the medium, and carbon sources, on in vitro regeneration from nodal explants of R. chalepensis. Following 8 weeks of culture, the highest percentage of regeneration (96.3%) and maximum number of shoots (40.3 shoot/explant) with a length of 4.8 cm were obtained with Murashige and Skoog (MS) medium at pH 5.8, supplemented with 3.0% sucrose and 5.0 µM 6-Benzyladenine (BA) in combination with 1.0 µM 1-naphthaleneacetic acid (NAA). For rooting, individually harvested shootlets were transferred on ½ MS (half-strength) supplemented with IAA (indole-3-acetic acid), IBA (indole 3-butyric acid), or NAA, and the best response in terms of root induction (91.6%), number of roots (5.3), and root mean length (4.9 cm) was achieved with 0.5 µM IBA after 6 weeks. An average of 95.2 percent of healthy, in vitro regenerated plantlets survived after being transplanted into potting soil, indicating that they were effectively hardened. DNA assays (PCR-based markers) such as random amplification of polymorphic DNA (RAPD) and directed amplification of minisatellite-region (DAMD) were employed to assess in vitro cultivated R. chalepensis plantlets that produced a monomorphic banding pattern confirming the genetic stability. Additionally, no changes in the flow cytometric profile of ploidy between regenerated plantlets and donor plants were detected. Regeneration of this valuable medicinal plant in vitro will open up new avenues in pharmaceutical biotechnology by providing an unconventional steadfast system for mass multiplication and might be effectively used in genetic manipulation for enhanced bioactive constituents.
... These data due to BAP was involved in the regulation of cell division and adventitious shoot formation. This may be due to its ability to induce the metabolism and production of natural endogenous hormones for morphogenesis (Ahmed & Anis, 2014). These findings were in agreement with those reported by Abd El-Kadder & Hammad (2012) on D. indica L. They showed that the maximum shoot multiplication rate (7.1 shoots per explant) was obtained at 2.0mg L −1 of BAP. ...
IN THIS STUDY, we established of an application protocol for the micropropagation of Dillenia indica with an estimate of betulinic acid from callus, leaf, and regeneration plant. Moreover, the antimicrobial and cytotoxic efficacy of the crude extract and pure compound was compared. The highest number of shoots (3.667 shoots), leaf number per jar (27 leaves) and shoot length (3.667cm) were recorded from the MS medium containing 2.0mg L-1 of benzyl amino purine (BAP). Moreover, different physical elicitors (ultraviolet irradiation, microwave radiation, and light quality) were used on the production and increased the concentration of betulinic acid in callus of D. indica. The data revealed that the highest betulinic acid content (341.775mg/ 100g fresh weight) was recorded in callus microwaved with 200 watts for 10s. Which was three times more than the betulinic acid content recorded in the mother plant. In addition, different crude extracts of D. indica were used to determine their biological activity against six microorganisms. The data showed that the highest diameter of inhibition zones (17.0 mm) was recorded in callus culture extractions incubated under blue light with Candida albicans. The cytotoxic efficacy of betulinic acid and extract of callus, mother plant leaf or in vitro regeneration plants of D. indica were investigated against the Vero cell line by MTT. The results revealed that The IC50 values were 115.7 ± 9.17, 114.98 ± 5.2, 59.45 ± 3.06, and 72.76 ± 6.93μgmL-1 of betulinic acid, callus extract, leaf, and regeneration plant, respectively.
... This unbeneficial effect for addition IAA to the medium in combination with BA was earlier recorded by Ugraiah et al. (2011) on Caralluma bhupenderiana Sarkaria and Ahmed and Anis (2014) on Vitex trifolia L. ...
... After proliferation, the microshoots were excised and allowed for root induction via in vitro and ex vitro methods. Supplementation of auxins in nutrient media promotes rhizogenesis in vitro [38,39] . Rooting percentage (81%) was highest on ½ strength MS medium augmented with 2.46 µM IBA (6.3±0.50 roots with 2.0 cm length) (Fig. 2D) The shoot bases pulse treated with different concentrations of IBA and NAA induced healthy roots from the cut ends of shoots after 4 weeks of incubation in soilrite ® maintained in the greenhouse. ...
The present study describes successful in vitro regeneration systems in Chromolaena odorata (L.) King & H. Rob. by forced axillary bud break from nodal explants. Full strength Murashige and Skoog’s (MS) medium augmented with BAP at 8.88 μM resulted in maximum induction of shoots (4.6±0.25 shoots per explant). The shoots were promoted to proliferate by sub-culturing on MS medium (liquid, agar-gelled and phyta-gelled) fortified with 2.22 μM BAP and 0.571 μM IAA. This combination of MS medium solidified with phytagel yielded 17.6±1.2 shoots per explants per culture vessel after 5 weeks of incubation. Results showed that the quality of shoots on medium gelled with phytagel was better than agar gelled and liquid MS media. Rooting of regenerated shoots was achieved by in vitro and ex vitro methods. About 80% shoots rooted in vitro with 6.3±0.50 roots per shoot on half strength MS medium containing 2.46 μM IBA. Ex vitro method of rooting (12.1±0.10 roots per shoot) was achieved by treating the shoots with 245 μM IBA for 5 min upon 4 weeks of incubation with soilrite® in the greenhouse. Rooted plants were reallocated into paper cups having soilrite®: red soil in equal proportions for hardening. The acclimatized plants were subjected to field trial for 6 weeks and the percentage of survival rate was recorded as 96%. The developed protocol could be utilized for genetic transformation for for development of desired traits in C. odorata.
... Cytokinins are well known for its ability to stimulate plant cell division and promoting axillary bud growth (Silue et al. 2016). The multiple shoot inducing effect of BAP can be attributed to its capability of stimulating the plant tissues to metabolize endogenous hormones and also to induce production of hormones (Ahmed and Anis 2014). Further, BAP is readily metabolized in the plant tissues unlike other synthetic cytokinins (Rai et al. 2010). ...
An efficient in vitro propagation and synthetic seed production protocol was established for the conservation of Decalepis salicifolia (Bedd. ex Hook.f.) Venter, an endemic and critically endangered ethnomedicinal species. Murashige and Skoog (MS) media supplemented with 2.2 μM BAP and 5.7 μM IAA produced shoot tip proliferation with an average shoot length of 5.81 ± 0.03 cm in 3 wk. Axillary bud proliferation was found to be best in 11.1 μM BAP with an average of 3.5 ± 0.06 shoots per explant. Shoot tip and nodal explants were encapsulated in sodium alginate and their regeneration was achieved following storage at 4°C up to 12 wk. Successful rooting was obtained in modified MS medium with low nitrate and high sucrose concentration. Quarter strength MS media containing 2.5 mM each of NH4NO3 and KNO3 along with 8% sucrose produced an average number of 12.4 ± 1.18 roots with an average length of 4.3 ± 0.08 cm. The in vitro derived rooted plants were successfully hardened (92.8%) and established in field with 100.0% survival rate. Inter-simple sequence repeat (ISSR) markers proved the genetic fidelity among the in vitro grown plant showing 99.5% similarity with the mother plant. Micropropagation derived acclimatized plants produced 2-hydroxy-4-methoxybenzaldehyde in amounts similar to seed-derived field-grown plants of the same age. The in vitro propagation protocol developed for D. salicifolia can be utilized to reduce exploitation pressure from their natural habitats and augment ecorestoration, conservation, and cultivation of this critically endangered and industrially valuable plant. The synthetic seeds technique will serve as propagules in ex situ gene banks, as well as supports cost-effective germplasm conservation.
... In this study, Kin and NAA in combination with BAP increased both the number of shoots per explant as well as the shoot length ( Table 2). A similar synergistic effect between BAP and Kin or BAP and NAA in the induction of multiple shoot formation has been demonstrated in several plant species [30,[36][37][38]. ...
Background and objective
Plant tissue culture technology offers a solution for meeting the increasing commercial demand for medicinally important plants, especially cross-pollinating species, whose genetic heterogeneity presents difficulties when using traditional propagation methods. Herein, we describe an effective, rapid, and simple protocol for the micropropagation of anise (Pimpinella anisum).
Materials and methods
We investigated the effect of the type of explant and the type and concentration of plant growth regulator, either individually or in combination, on plant micropropagation.
Results and conclusion
Although multiple shoot formation rate was higher for nodal than for shoot tip explants, there was no significant difference in rooting response between shoots arising from either of them. Maximum shoot response, number of shoots per explant, and shoot length were observed in nodal explants grown on Murashige and Skoog medium supplemented with 5 μmol/l 6-benzylaminopurine, 1 μmol/l kinetin, and 0.5 μmol/l naphthalene acetic acid. The most effective medium for root regeneration contained 3 μmol/l of either indole-3-butyric acid or naphthalene acetic acid. Interestingly, there was no evidence for hyperhydricity, which is commonly found in cultured anise, using our method. Plantlets were successfully hardened and transferred to the greenhouse, with an 85% survival rate. This protocol provides an efficient means for large-scale production of anise, as well a basis for further research aimed at genetic improvement of this plant.
Keywords:
anise, growth regulators, micropropagation, multiple shoot formation, vitrification
... Callogenesis was also observed but there were not significant differences among the different concentrations (data not shown). It seems that Krymsk® 5 responds better to the presence of adenine cytokinins in the medium than phenylureas, which has been observed in other species too (Murai et al., 1997;Ružić and Vujović, 2008;Rafique and Anis, 2014). ...
Krymsk® 5 (VSL-2) is a semi-dwarf cherry rootstock adaptable to a range of climates. The present study aimed to establish the first efficient in vitro propagation protocol for this rootstock. Therefore, six cytokinines, four adenine type (6-benzyladenine, 2-isopentenyladenine, kinetin and meta-topolin) and two phenylureas (thidiazuron and forchlorfenuron) at three (2.4 μΜ, 4.8 μΜ and 9.6 μΜ) concentrations plus three (0.24 μΜ, 0.48 μΜ, 0.96 μΜ) for thidiazuron only were tested during the multiplication stage. 6-Benzyladenine was the most efficient cytokinin, based on the number of shoots produced (3.5 shoots at 9.6 μΜ) and the number of nodes per explant (10 nodes at 9.6 μΜ) whereas the other aromatic adenine tested, i.e. meta-topolin, presented the highest number of nodes per cm and node per shoot. During the rooting stage the synthetic auxins 1-naphthaleneacetic acid (1-NAA) and indolebutyric acid (IBA) were tested at concentrations of 0, 2.5, 5, 10 and 20 μΜ both separately and in all possible combinations. The percentage of successfully rooted explants reached 95% under the combination of 20 μΜ IBA plus 5 μΜ 1-NAA, whereas the highest number of roots recorded was 8.5 roots for the treatment 20 μΜ ΙΒΑ plus 2.5 μΜ 1-NAA. Furthermore, two different carbon sources were compared, the widely used sucrose and the endogenous sugar ratio of mother plant softwood shoot, sampled during late of May. Endogenous sugar ratio proved to be the preferable carbon source, since it increased the number of shoots produced and almost doubled the number of produced nodes per explant.
... indicum (Ahmed & Anis, 2014;Ramadevi, Ugraiah, & Pullaiah, 2012;Tiwari, Tiwari, & Singh, 2001). Each individual adventitious shoots were further separated and subcultured in the same medium (Figure 1e) was successful grown into well developed shoots (Figure 1f). ...
... indicum (Ahmed & Anis, 2014;Ramadevi, Ugraiah, & Pullaiah, 2012;Tiwari, Tiwari, & Singh, 2001). Each individual adventitious shoots were further separated and subcultured in the same medium (Figure 1e) was successful grown into well developed shoots (Figure 1f). ...
This study reports on in vitro regeneration of Abutilon indicum plantlets through callus mediated organogenesis. The leaf explants implanted on Murashige and Skoogs (MS) medium supplemented with 4.52 µM 2, 4-Dicholorophenoxy acetic acid (2,4-D) and 8.88 µM 6 Benzyladenine (BA) showed highest response (70.3%) for callus proliferation, but these callus did not showed any morphogenetic differentiation on the same medium even after 12 weeks. Whereas, subsequent sub-culture of this green proliferated callus on MS medium added with 2.68µM α-Napthalene acetic acid (NAA), 8.88µM BA and 543 µM Adenine sulphate showed the highest frequency (62.2%) of multiple shoot-buds production and also elongation of shoots. Well developed shoots were efficiently rooted in vitro on half strength MS medium supplemented with 7.38 µM Indole-3-butyric acid (IBA). Seventy per cent of in vitro regenerated plantlets were successfully established in garden and were morphologically alike to the donor plants. The genetic homogeneity of these in vitro regenerated plantlets was also affirmed by inter simple sequence repeat (ISSR) analysis using eight ISSR primers. This standardised in vitro organogenesis protocol supplements a good platform for the conservation of A. indicum germplasms and also caters for the needs of the herbal industry.
... Vitex agnus-castus has been historically used for a range of female reproductive conditions in Anglo-American and European practice (van Die et al. 2013). Vitex trifolia Linn leaves are used by the tribes and native medical practitioners for the treatment of various ailments including liver disorders and rheumatic pains in India (Ahmed and Anis 2014). The barks of Vitex polygama are traditionally used as diuretics in several states of Brazil (Meena et al. 2011). ...
Vitex pubescens belonging to the Verbenaceae family, has been historically used as a folk medicine in Indonesia. The dichloromethane extract of V. pubescens stembarks interfered cell proliferation and induced apoptosis in human leukemia HL-60 cells. Our phytochemical investigation resulted in the isolation of several constituents and their chemical structures were determined by spectroscopic analyses. Upon treatment with labdane diterpene lactones, relatively high inhibition of cell proliferation and apoptotic response of the cells were observed. Furthermore, the structure-activity relationship analysis with clerodane diterpenes revealed that a γ-butyrolactone ring contributes their antileukemic potency. Overall, these findings enhanced the ethnopharmacological relevance of V. pubescens for cancer chemotherapy.
... The plant regeneration from shoot tip segments is considered to be one of the most promising ways for multiplying a selected variety true to its type showing the same agronomic characteristics (Ahmed and Anis 2014). Generally, a (Saini and Jaiwal 2002) and Vigna unguiculata (Tang et al. 2012). ...
A rapid, simple and efficient protocol for direct in vitro multiple shoot induction and plantlet recovery was achieved from shoot tip explants of Bambara groundnut [Vigna subterranea (L.) Verdc.]. Shoot tips, were isolated from in vitro-grown seedlings and cultured on Murashige and Skoog basal medium (MS) containing Gamborg’ vitamins (B5) and supplemented with different concentrations of the plant growth regulators Thidiazuron (TDZ), N⁶-benzylaminopurine (BAP), Kinetin (KIN) and Adenine sulfate (ADS). TDZ 0.45 μM was found to be best for shoot multiplication with a mean of 11shoots per explant. Among the carbon sources tested, the best response in terms of mean number of shoots per explant was obtained with sucrose (11). The mean number of shoots per explant induced among the studied landraces of Bambara groundnut varied from 9 to 13. Individual shoots, aseptically excised, produced normal roots within 2 weeks on the basal MS medium supplemented with Indole Butyric Acid (IBA) or Naphthalene acetic acid (NAA). The highest number of roots per shoot and the longest roots were obtained on MS medium with 2 mg/L IBA. Rooted plantlets were successfully hardened under greenhouse conditions and subsequently established in the field conditions, with a recorded survival rate of 70 and 80 %, respectively. The transferred plants in the field were morphologically normal and fertile. This protocol can be efficiently used for mass propagation, germplasm preservation and probably also for gene transfer of Vigna subterranea (L.).
... IBA is more effective than IAA and NAA in root induction in variety of plants, and it is applied economically worldwide (Ranaweeraa et al., 2013). Ex vitro root induction was successfully proved by many researchers in Ceropegia bulbosa (Phulwaria et al., 2013), Momordica dioica (), Caralluma edulis (Patel et al., 2014), Vitex trifolia (Ahmed and Anis, 2014), Acacia nilotica (Rathore et al., 2014Rathore et al., , 2015) etc. It is a cost effective technique and could saveFig. ...
Vitex L. is the largest genus of the Lamiaceae family, and most of its species are used in the traditional medicinal systems of different countries. A systematic review was conducted, according to the PRISMA methodology, to determine the potential of Vitex plants as sources of antimicrobial agents, resulting in 2610 scientific publications from which 141 articles were selected. Data analysis confirmed that Vitex species are used in traditional medicine for symptoms of possible infectious diseases. Conducted studies showed that these medicinal plants exhibited in vitro antimicrobial activity against Bacillus subtilis, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Staphylococcus aureus. Vitex agnus-castus L. and Vitex negundo L. have been the most studied species, not only against bacterial strains but also against fungi such as Aspergillus niger and Candida albicans, viruses such as HIV-1, and parasites such as Plasmodium falciparum. Natural products like agnucastoside, negundol, negundoside, and vitegnoside have been identified in Vitex extracts and their antimicrobial activity against a wide range of microbial strains has been determined. Negundoside showed significant antimicrobial activity against Staphylococcus aureus (MIC 12.5 µg/mL). Our results show that Vitex species are potential sources of new natural antimicrobial agents. However, further experimental studies need to be conducted.
Thepresentstudyaimedtoanalyzetheeffectofsiliconnanoparticles(SiNPs)ontheinvitroproliferationofshoots,biomassproduction,foliarmicro-structuralfeatures,androot-inducingabilityinVitextrifoliaL.Budbreakingwasachievedfromaxillarymeristemson2.5mg/Lof 6 benzylaminopurine(BAP)inMurashigeandSkoog’s(MS)medium.Theshootswereproliferated(18.0§0.39shoots)on0.75mg/L of 1BAPwith0.2mg/L of 11-Naphthaleneaceticacid(NAA).AmongthevariedconcentrationsofAerosil300(hydrophilicsil-ica)used,6.0mg/L of 1SiNPswithoptimalgrowthregulatorsintothemediumhadapositiveimpactonshootproliferation(24.0§0.30shoots)andfreshbiomassproduction.ThelightmicroscopicevaluationshowedthattheleavesdevelopedwithoutSiNPspossessedunderdevelopedcuticle,trichomes,stomata,epidermalcells,andphotosyntheticandvasculartissues.TheSiNPstreatmentresultedinwell-differentiatedtissuesys-temsandfavoredanincreaseinwidthofthelamina,differentiationofpalisadeandspongytissues,thicken-ingofcellwalls,increaseddensityoftrichomes,organizedguardcellsinstomata,andimprovedxylemandphloemtissues.Theshootswereeffectivelyrootedonhalf-strengthMSmediumcontaining3.0mgL1NAA,andtheshootsderivedfromSiNPstreatmenthadthehighestrootingefficiency(8.0rootspershoot).ThefindingshighlightthattheuseofSiNPssignificantlypromotedtissuesystemsandmorphologyofV.trifolia,whichfavoredthesurvivalofplantletsunderinvivoconditions.
Rumex hastatus D. Don, a traditional medicinal plant known for its, antitumorous, antiangiogenic and antibacterial potential is continuously under threat of extinction due to over exploitation and climatic changes. Phytochemical analysis of different plant parts showed that roots had the maximum quantity of two bioactive molecules, luteolin and rutin and thus was selected for establishment of aseptic culture. In vitro technology has been employed for developing an efficient micropropagation protocols for its conservation, large scale production as well elicitation of the valuable bioactive compounds. Murashige and Skoog’s medium supplemented with 5µM TDZ proved best for developing greenish-brown and compact calluses in 98% root cultures within 30 days. Such callus pieces on subculture to same but fresh medium differentiated an average of 10.50 ± 1.29 shoots which were elongated on MS basal medium. Such excised shoots, rooted in MS + 0.1 µM IBA medium with an average of 12.03 ± 2.49 roots per shoot. These plantlets were successfully acclimatized under greenhouse conditions and fields and their genetic fidelity was evaluated employing SRAP and SCoT markers which revealed their high similarity with mother plant proving their true-to-type nature. Interestingly, the in vitro regenerants showed elevated levels of phenols, flavonoids, luteolin and rutin content and higher antioxidant activities over the mother plant as done by phosphomolybdenum and DPPH assays. For elicitation of bioactive compounds, 100 mg/L phenylalanine was best for enhancing optimum luteolin content (407.18 ± 10.58 µg/g d.w.) over control (22.10 ± 0.57 µg/g d.w.) almost, 17.42–fold, whereas for rutin, 25 mg/L chitosan proved most effective enhancing its synthesis up to 1169.07 ± 46.49 µg/g d.w. over control (312.41 ± 12.42 µg/g d.w.), i.e., 2.74–fold. To the best of our knowledge, this is our first report on micropropagation and elicitation of luteolin and rutin in Rumex hastatus through root culture.
Jasminanthes tuyetanhiae is one of the precious herbs and good for human health, discovered in the Da-Bia Mountain (Phu Yen province, Vietnam). The identification of the CTW species (species believed to be J. tuyetanhiae collected in 2019) by molecular indicators showed that this species belongs to the Apocynaceae family. High similarity (98.2%) in the ITS1-5.8SrRNA-ITS2 gene sequence of CTW when compared on Genbank with J. tuyetanhiae and genetic distance difference (1.3%) - in the limit of difference of the same taxon considered on matK sequence shows that CTW is likely to belong to this species. Surface sterilization of ex vitro explants with HgCl2 (0.1%) for 3 min gave the highest sterilization efficiency compared to the remaining treatments with the induction explant rate of 93.33%. BA (0.5 mg l−1) is the most optimal for effective in vitro shoot multiplication in this species. Adding IBA to culture media at the appropriate concentration (0.5 mg l−1) increased rooting ability, shortened rooting time (13 days) and increased root length (6.53 mm). The ventilation culture system improves rooting efficiency, stimulates growth and induces secondary root formation—which is a suitable system for in vitro propagation of the Jasminanthes tuyetanhiae in this study.
Plant tissue culture techniques are used for in vitro culturing of plant parts (plant cells, tissues, organ) with the objective of plant improvement and commercial exploitation. Plant biotechnology is envisaged with the principles of plant tissue culture. Plant genomes respond differently to different culture techniques, conditions, explants, etc. Understanding the innate response conditions to culture techniques opens innumerable avenues for research and standardization. Till date the number of plant species considered recalcitrant is numerous. Plant tissue culture iterates the methods for clonal propagation, creating somaclonal variation and organ culture featuring monoploids, haploids, diploids, dihaploids, somatic embryogenesis, embryo rescue, cybrid developments, harnessing secondary metabolite production and plant transformation. Plant transformation results from targeted gene delivery into plant types for plant improvement and/or recombinant protein production. Transgenic plants are developed through gene delivery into single cell/tissue, regeneration into whole plants that are fertile and adapt to the normal plant propagation process. Delivery methods harness chemical methods, Agrobacterium mediated and particle bombardment, including in-planta transformation techniques. Each of the methods involves specificity with respect to transgene characters and host preferences along with target plant material. Techniques depend on specificity, precision, reliability and scope. Gene transfer techniques can greatly reduce the time taken in crop improvement against conventional breeding procedures and enlarge the array for desired plant improvement.
The present investigation was carried out to develop an efficient protocol for in vitro propagation of Brucea mollis Wall. ex Kurz, an important medicinal plant, confined to only Karbi Anglong district of Assam. The plant is extensively used to cure malaria. Due to several natural factors like inefficient pollination, seed dispersal mechanisms and anthropogenic activities, the plant is facing extinction. Through indirect organogenesis from leaf and internode explants on MS (Murashige and Skoog) media, callus induction was achieved at a maximum (100%). However, the minimum time (7–15 days) was observed for induction of callus on B5 as compared to MS media. The color and texture of the callus varied depending on the type of explants as well as the concentrations of growth regulators used in both the media. Best response for percentage of callus induction (100%) was obtained from the leaf explants on MS media, when the media was supplemented with the four combinations of growth regulators, i.e., (i) BAP (8.88 μM) + NAA (1.61 μM), (ii)BAP (8.88 μM) + NAA (2.68 μM), (iii) BAP (8.88 μM) + 2,4-D (2.27 μM) and (iv) BAP (8.88 μM) + 2,4-D (4.54 μM). While in the case of internode explants, maximum (100%) percentage for callus induction was obtained with BAP (8.88 μM) + NAA (1.61 μM) and BAP (8.88 μM) + NAA (2.68 μM). Multiple shoot regeneration was achieved from the callus of all the three types of explants when transferred to shoot regeneration media (both MS and B5 media) supplemented with BAP, NAA, Kinetin and IBA at different concentrations and combinations. The maximum percentage of shoot regeneration (100%) from the calli of all explant types were obtained with the combination of BAP, NAA and IBA. However, the best response in terms of minimum number of days taken to regenerate shoots and maximum shoot length was obtained using the leaf explants at BAP (8.88 μM) + NAA (2.68 μM) + Kinetin (13.95 μM) on MS media. The regenerated shoots were cultured for rooting on half strength media supplemented with IBA and NAA singly, and the highest rooting percentage was achieved when MS media was supplemented with 14.76 μM IBA. About 60% of the plantlets survived during acclimatization and were successfully transferred to the field. The regeneration protocols standardized for B. mollis could be found most effective for mass propagation of the medicinally important plant as well as its conservation.
Hyperhydricity symptoms are common and significant during the in vitro culture of Dianthus chinensis L. and greatly affect the micropropagation and regeneration of cultured plantlets. However, effective measures for preventing such abnormalities have not been developed for this species. Silver nitrate (AgNO3) has been shown to revert hyperhydric plantlets to a normal state. Nevertheless, the effect of AgNO3 on the prevention of hyperhydricity and the underlying mechanisms remain unclear. In the present study, 98.7% of the Dianthus chinensis L. plantlets cultured in a hyperhydricity induction medium (HIM) developed symptoms of hyperhydricity; however, hyperhydricity symptoms were inhibited to different degrees when D. chinensis L. plantlets were cultured in HIM supplemented with various concentrations of AgNO3. In particular, approximately 97% of the D. chinensis L. plantlets grew normally and did not show any symptoms of hyperhydricity when cultured in HIM supplemented with 30 μmol L⁻¹ AgNO3. Compared with the plantlets cultured in HIM alone, the plantlets cultured in HIM containing AgNO3 displayed dramatic decreases in water content, ethylene content, and reactive oxygen species (ROS) production (particularly regarding H2O2 accumulation in guard cells) and showed increased antioxidant enzyme activity, stoma aperture, and water loss. These changes not only prevented excess water from accumulating in the tissues of plantlets but also improved the antioxidant capacity of plantlets, ultimately resulting in the prevention of hyperhydricity.
Lavenders (Lavandula species) are important aromatic ornamental medicinal plants with wide ranging applications in perfume and pharmaceutical industries. We developed an in vitro propagation protocol for Lavandula coronopifolia. Murashige and Skoog (MS) medium supplemented with 0.5 mg·L⁻¹N⁶ -benzyladenine was the best medium for the proliferation of microshoots, while the highest rooting frequency was obtained using MS medium supplemented with 1.0 mg·L⁻¹ indole-3-butyric acid. Inter-simple sequence repeat analysis revealed that the in vitro-propagated microshoots were highly genetically stable, even after subculture. The highest callus fresh weight (667.9 mg) was obtained by propagating on medium supplemented with a combination of 1.0 mg·L⁻¹ naphthaleneacetic acid and 0.5 mg·L⁻¹ butyric acid. Using the Folin-Ciocalteu method, methanolic extracts of wild L. coronopifolia revealed total phenolic content of 4.9 mg expressed in gallic acid equivalents (GAE) (mg GAE·g⁻¹ dry matter). Radical scavenging activity was estimated at 85% using the free radical 2,2-diphyenyl-picrylhydrazyl assay. Using the brine shrimp assay for cytotoxicity, the methanolic extract was found to be nontoxic. Finally, liquid chromatography tandem mass spectrometry with standard reference compounds was used to quantify the key phenolic compounds in both in vitro and in vivo-grown L. coronopifolia. Six major phenolic compounds were identified: caffeic acid, rosmarinic acid, rutin, quercetin, luteolin, and hesperidin. Levels of these phenolic compounds were highest in wild plant extracts.
Vitex is a large genus consisting of 230 species of trees and shrubs with multiple (ornamental, ethnobotanic and pharmacological) uses. Despite this, micropropagation has been used to effectively propagate and preserve germplasm of only a limited number (six) of Vitex species (V. agnus-castus, V. doniana, V. glabrata, V. negundo, V. rotundifolia, V. trifolia). This review on Vitex provides details of published micropropagation protocols and perspectives on their application to germplasm preservation and in vitro conservation. Such details serve as a practically useful user manual for Vitex researchers. The importance of micropropagation and its application to synthetic seed production, in vitro flowering, production of secondary metabolites, and the use of molecular markers to detect somaclonal variation in vitro, is also highlighted.
Present study reports successful in vitro clonal propagation of a potential medicinal plant, Cassia alata using mature nodal explants. Murashige and Skoog (MS) medium supplemented with different concentrations (0.5, 2.5, 5.0, 7.5, 10.0 and 12.5 μM) of 6-benzyladenine (BA), kinetin and 2-isopentenyl adenine (2-iP) singly as well as in combination with different auxins, α-naphthalene acetic acid (NAA), Indole-3-butyric acid (IBA) or Indole-3-acetic acid (IAA) (0.1, 0.5 and 1.0 μM) were used. MS medium enriched with 7.5 μM BA and 0.5 μM NAA yielded the highest regeneration frequency (92 %) with maximum multiple shoots (12.3 ± 0.6) and shoot length (4.7 ± 0.1 cm) after 12 weeks of culture. Shoots were rooted best on full MS containing 0.5 μM IBA. Ex vitro rooting of in vitro derived microshoots was also achieved in 150 μM IBA treatment for 20 min followed by transfer to thermocol cups containing sterile Soilrite™. About 85 % plantlets survived acclimatization procedure to the field. The genetic fidelity of in vitro regenerated plants was analyzed using random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers. Of the 20 RAPD primers, 18 primers produced clear, reproducible and scorable bands while out of 13 ISSR primers screened, only ten generated well-defined and scorable bands in all the tested plants. A total of 69 and 71 bands were scored with an average of 3.8 and 7.1 bands per primer for RAPD and ISSR primers respectively. All banding profiles from micropropagated plants were monomorphic and similar to of the mother plant, thus confirming the true-to-type nature of the in vitro-raised clones.
Effect of plant growth regulators on axillary shoot multiplication from nodal explants of Ajuga multiflora was studied. Multiple shoots were induced on Murashige and Skoog (MS) medium containing N6-benzyladenine (BA) alone or in combination with indole-3-acetic acid (IAA) or in combination with α-naphthaleneacetic acid (NAA). The maximum shoot induction (79.9%), with a mean number of 9.3 shoots was obtained on MS medium supplemented with 2.0 mg l-1 BA. A combination of BA and auxin was better than BA alone in promoting axillary shoot multiplication from the nodal explants of A. multiflora. The highest mean number of shoots (21.3) was obtained on MS medium supplemented with 2.0 mg l-1 BA and 0.5 mg l-1 NAA.
Direct organogenesis and in vitro flowering was obtained in Vitex trifolia. L an endangered medicinal plant. High frequency and maximum number of multiple shoots were obtained from shoot tip explants on MS medium supplemented with BAP (3.96-15.85 μM) and IAA (5.70-22.83 μM). Regenerates, when transferred to rooting medium with IBA at concentration of 2.46-14.76 μM and IAA at concentration of 2.85-17.13 μM initiated flowering along with rooting. In vitro flowers set viable seeds. Rooted plantlets were hardened and transferred to green house with 100% survivability. This finding has significant role in Pharmaceutical industries and in vitro flowering facilities in vitro pollination and fertilization, further it also facilities in advancing the generation at much faster speed under limited progeny size in the segregating generation of Vitextrifolia. L.
The effect of thidiazuron (TDZ) was investigated on in vitro shoot proliferation from nodal explants of Rauvolfia tetraphylla. Murashige and Skoog (MS) medium containing TDZ (0.5–10 lM) was effective in inducing shoot buds and maintaining high rates of shoot multiplication on hormone free medium. The highest shoot regeneration frequency (90%) and mean number (18.50 ± 1.25) of shoots per explant were achieved from nodal segments cultured on MS medium supplemented with 5 lM TDZ for 4 weeks prior to transfer to MS medium without TDZ for 8 weeks. The regenerated shoots rooted best on MS medium containing 0.5 lM indole-3-butyric acid (IBA). Micropropagated plantlets were hardened to survive ex vitro conditions and were then established into soil. Abbreviations: BA – 6-benzyladenine; IAA – indole-3-acetic acid; IBA – indole-3-butyric acid; Kinetin – 6-furfurylaminopurine; MS – Murashige and Skoog medium; NAA – a-naphthalene acetic acid; TDZ – thid-iazuron
A valuable medicinal plant, Vitex negundo L. has been investigated for its regeneration potential using shoot tip explants. Out of a range of concentrations of cytokinins [6-benzyl adenine (BA), 6-furfurylaminopurine, 2-isopente-nyl adenine] used as supplement to Murashige and Skoog medium (MS), BA at 5.0 lM concentration proved best for multiple shoot induction yielding 3.60 ± 0.50 shoots after 8 weeks of culture. Inclusion of a low concentration of an auxin with optimal cytokinin concentration favoured shoot multiplication and the optimum response was observed on MS medium supplemented with BA (5.0 lM) along with a Naphthalene acetic acid (0.5 lM), where 65.0 ± 1.73 % cultures responded with a mean number of 4.80 ± 0.58 shoots per explants after 8 weeks of culture. Ex vitro rooting of in vitro derived microshoots was achieved upon dipping the cut ends of microshoots in 500 lM indole-3-butyric acid for 10 min followed by transfer to thermocol cups containing sterile soilrite. About 95 % of the plantlets survived the acclimatization procedure and were transferred to greenhouse and finally to field. Screening of the antibacterial activity and estimation of total phenolic content of ethanolic extracts of micropropagated plants were also carried out and compared with that of the mother plant.
This report describes in vitro shoot induction and plant regeneration from mature nodal explants of Vitex trifolia L. on Murashige and Skoog (MS) medium fortified with benzylaminopurine (BAP), kinetin (KN), thidiazuron (TDZ), adenine (ADE),
and 2-isopentenyladenine (2-iP) (0.25 – 10.0 μM). Multiple shoots differentiated directly without callus mediation within
3 weeks when explants were cultured on medium supplemented with cytokinins. The maximum number of shoots (9 shoots per explant)
was developed on a medium supplemented with 5.0 μM BAP. Shoot cultures was established repeatedly subculturing the original
nodal explant on the same medium. Rooting of shoots was achieved on half strength MS medium supplemented with 0.5 μM naphthaleneacetic
acid (NAA). Rooted plantlets transferred to pots containing autoclaved soil and vermiculite mixture (1:1) showed 90 % survival
when transferred to outdoor.
An in vitro propagation technique based on axillary bud proliferation has been developed for matureSapium sebiferum trees. Nodal segments cultured on Murashige and Skoog (MS) medium supplemented with benzyl adenine (1–10 m and -naphthaleneacetic acid (0–0.5 m showed axillary bud proliferation. Shoots proliferated in vitro were multiplied on Murashige and Skoog medium containing 2.5 m benzyl adenine and 0.25 m
-naphthaleneacetic acid. Seasonal changes affected the shoot proliferation potential of the initial explant. Shoots were rooted on a half-strength, growth-regulator-free, agar-gelled, MS medium after a 48-h treatment on half-strength MS liquid medium with 10 m indole-3-butyric acid. Rooted plantlets were potted and acclimatized in a growth chamber and then moved to the greenhouse. Four-month-old plants were transplanted to the field.
Alkannin, a red-purple dye and bioactive compound found in the roots of Arnebia hispidissima has antibiotic and anti-inflammatory properties and is also used in cosmetic and textile industries at a large-scale. In
the present communication, we demonstrate the establishment of callus and cell suspension culture of A. hispidissima with the aim of optimizing the production of alkannin. Highest alkannin content was recorded in cell suspension and callus
culture established on M-9 medium. Production of alkannin was influenced by the different culture medium. Evaluation of alkannin
content of roots of field-grown plants and in vitro grown cell, tissue and organ showed that alkannin production was higher
in all in vitro grown culture systems (cell suspension, callus and roots) than the roots of field-grown plants. The present
investigation may be applicable in designing systems for the large-scale cultivation of A. hispidissima cell suspensions for the production of alkannin.
Keywords
Arnebia hispidissima
–Cell suspension culture–In vitro propagation–Plant growth regulators–Secondary metabolites
An efficient method for cultivation of Vitex negundo L. through axillary shoot (collected from micropropagated plants) proliferation has been successfully developed, which can
be employed at a commercial scale. Axillary shoot induction was most successful using nodal explants for propagation on Murashige
and Skoog (MS) medium supplemented with various concentrations of single cytokinin or in various combination with auxins.
We obtained the maximum percentage (97.6±1.45) response with highest number (16.4±0.60) of shoots per explant on MS medium
supplemented with 6-benzyladenine (BA) and α-naphthalene acetic acid (NAA) at concentrations of 5.0 and 0.5μM, respectively.
Shoot regeneration frequency was optimized by manipulating pH and using various media. MS medium and pH 5.8 was found to be
the optimum for maximum regeneration. Nodal explants from in vitro regenerated microshoots too developed shoots, thus making
the process recurrent. Shootlets with 4–5 nodes were utilized for in vitro rooting, and best response was evaluated on MS
medium supplemented with 1.0μM indole-3-butyric acid (IBA). The well-developed micropropagated plants were acclimatized (97%)
successfully within 2weeks in soilrite and planted ex vitro in normal garden soil, where they grew well without any morphological
and genetic variations. The present regeneration process not only favoured the rapid multiplication but also expressed the
regeneration capability of micropropagated plants.
KeywordsCyclic protocol–Micronodes–Micropropagated plant–Microshoots–
Vitex negundo
An efficient, rapid and large-scale propagation of the woody, aromatic and medicinal shrub, Holarrhena antidysenterica, through in vitro culture of nodal segments with axillary buds, is described. N6-benzyladenine used at 15 μM was the most effective in inducing bud break and growth, and also in initiating multiple shoot proliferation at the rate
of 43 microshoots per nodal explant with axillary buds, after 30 d of eulture. By repeated subculturing of nodal explants
with axillary buds, a high-frequency multiplication rate was established. Efficient rooting was achieved with 35 μM indole-3-butyric acid which was the most effective in inducing roots, as 80% of the microshoots produced roots. Plantlets
went through a bardening phase in a controlled plant growth chamber, prior to ex vitro transfer Micropropagated plants established in garden soil were uniform and identical to donor plants with respect to growth
characteristics and vegetative morphology.
The effect of phytohormones on the breaking of dormancy of axillary buds in Salix pseudolasiogyne and their subsequent proliferation from nodal explants were examined. Nodal explants obtained from a 20–year-old S. pseudolasiogyne tree were cultured either on woody plant basal medium (WPM) or WPM supplemented with benzyladenine (BA, 2.2/4.4μM), zeatin
(1.1/2.2μM), gibberillic acid (GA3, 2.9 and 14.5μM), and GA3+BA (2.9+4.4μM). Although axillary shoots developed in all the media, a higher percentage bud break occurred on BA supplemented
media. To corroborate the results, endogenous levels of cytokinins [Cks, N
6-isopentenyladenine (iP), zeatin riboside (t-ZR), dihydrozeatinriboside (DHZR)] and abscisic acid (ABA) were determined. On BA supplemented media, the levels of zeatin
type (Z-type) of Cks were higher than those of isopentenyladenine type of Ck in the explants, while the ABA level was low.
Axillary shoots did not grow well and became necrotic upon subculture to fresh basal WPM. In order to improve shoot growth,
they were subcultured twice at a 4-week interval on to WPM supplemented with BA (2.2/4.4μM), GA3 (1.4μM), or GA3+BA (1.4+4.4/2.9+4.4μM). Maximal shoot growth (93%) was achieved on WPM supplemented with 2.2μM BA. Comparative analyses
of endogenous Cks revealed that higher Cks (Z-type Cks) were present in actively growing shoots. Rooting was readily achieved
when the shoots were subcultured to WPM without phytohormones. The rooted plants were acclimatized well upon transplantation.
Folk medicinal practitioners, otherwise known as Kavirajes perform a central role in providing primary health-care to the rural inhabitants of Bangladesh. They rely chiefly on medicinal plants for treatment of ailments. However, various ethnomedicinal surveys conducted previously among the Kavirajes have pointed out to major differences in the type of ailments treated and the selection of medicinal plant(s) for treatment of a specific ailment among the Kavirajes. The objective of the present study was to conduct a survey among the two practicing Kavirajes of the adjoining villages of Arpara and Munshefpur in Jessore district, Bangladesh to document any differences among the two Kavirajes as to ailments treated and the medicinal plants used for treatment. Kaviraj 1 (of Arpara village) treated 21 types of ailments, while Kaviraj 2 (of Munshefpur village) treated 10 types of ailments. Only 7 types of ailments were treated in common by both Kavirajes. These ailments included skin diseases, hepatic disorders (jaundice), respiratory tract disorders (coughs, mucus), toothache, gastrointestinal disorders, diabetes, and to stop bleeding from cuts and wounds. Even then the
medicinal plants selected for treatment of these common ailments differed considerably among the two Kavirajes. While Azadirachta indica was used for treatment of skin disorders by Kaviraj 2, Kaviraj 1 used the same plant along with Curcuma longa or with Curcuma longa, Vitex negundo, and Cynodon dactylon. For
treatment of jaundice, both Kavirajes used a combination of the plants, Cajanus cajan and Saccharum
officinarum. However, the dosage differed among the two Kavirajes. While Kaviraj 1 advised drinking only 2 teaspoonful of juice obtained from macerated leaves of Cajanus cajan, Kaviraj 2 advised drinking one glass of juice obtained from macerated leaves of the same plant. The present study highlights several aspects of the
folk medicinal method and the treatment offered by the Kavirajes. First, Kavirajes differ considerably as to the ailments treated, which may be due to experience or may be a result of specialization. Second, Kavirajes vary significantly as to medicinal plants chosen for treatment of any specific ailment, a variation which can extend to dosage even when same plants have been used. The surprising feature of these variations is that they
exist even among Kavirajes of adjoining villages where the availability of medicinal plants is the same. Taken together, the study highlights the importance of conducting surveys among all individual Kavirajes of Bangladesh if one wants to gather in a comprehensive manner, information on medicinal plants used in the folk medicinal system of the country.
A rapid clonal multiplication protocol comprising direct multiple shoot induction from shoot apex of Portulaca grandiflora Hook was developed. Shoot apex from healthy grown plants were used as explants for culturing. Explants were cultured on standard Murashige and Skoog (MS) medium supplemented with different concentrations of benzyl amino purine (BAP) or kinetin (KIN) for primary shoot proliferation. The best shoot proliferation (27.3 per explants with 98% induction) was observed in MS medium containing 2.5mgl-1 BAP. For rooting of microshoots, half strength MS medium supplemented with 0.75mgl-1 Naphthalene acetic acid (NAA) showed best results with 9.2 roots per shoot at an average root length of 6.0 cm with average rooting response of 95%. After acclimatization and transplantation, 100% of the In- vitro derived plants were found healthy in ex vivo conditions.
This study describes in vitro shoot induction and plant regeneration from a mature apical meristem and nodal explants of the endangered medicinal shrub Vitex agnus-castus. Multiple shoots were induced directly from the axis of nodal and apical meristem explants on Murashige and Skoog (MS) medium containing 3% sucrose and different concentrations (1.0, 1.5, 2.0, and 2.5 mg/l) of 6-benzyl aminopurine (BAP) in combination with Kinetin (Kin) and α-naphthalene acetic acid (NAA), both at 0.1 mg/l. BAP and Kinetin were used as supplements to MS basal medium, either individually or in combination with auxins. The optimal concentration of BAP for inducing bud break was found to be 2.0 mg/l when Kinetin was at 0.1 mg/l. Regeneration frequency was highest for both apical meristem and nodal explants (94.5% and 90.3%, respectively) when explants were cultured on MS medium supplemented with BAP (2.0 mg/l) and Kin (0.1 mg/l). A maximum of 7.7±0.4 and 6.7±0.2 shoots were obtained per explant for apical meristem and nodal explants, respectively. Regenerated shoots, transferred to MS medium supplemented with either 1.0 or 1.5 mg/l BAP combined with 0.1 mg/l GA3, showed maximum elongation of 6.7±0.4 and 6.0±1.3 cm in apical meristem and nodal explants, respectively. In vitro regenerated shoots transferred to half-strength MS medium supplemented with 0.1 mg/l IBA induced 90.4% of the shoots to form roots after 30–35 d of culture. Up to 80% of the regenerated shoots were successfully established in soil in the greenhouse.
An in vitro protocol using shoot tip explants from seedling of Adenium obesum (Forssk.) Roem. and Schult. was developed. Explants were cultured on Murashige and Skoog (MS) medium supplemented with various combinations of NAA and BA. The most effective medium for shoot proliferation at a high frequency of 5.20±1.10 shoots per explant consisted of 22.2 μM BA. High rooting and survival were achieved using MS medium supplemented with 0.3% activated charcoal and without any growth regulators. Rooted plants were successfully acclimatized to greenhouse conditions. This study showed that A. obesum could be micropropagated by utilizing multiple shoots derived from seedling shoot tips. A flow cytometric analysis for DNA content revealed no differences among the micropropagated plants and the in vivo natural grown plants. The resulting 2C DNA value (8.35±0.039 pg) of this species was estimated for the first time.
Single medium-based efficient protocols for large-scale multiplication of the rare woody aromatic medicinal plant Rotula aquatica Lour. by means of axillary bud multiplication and indirect organogenesis were established using Murashige and Skoog (MS) medium. There were no significant differences with respect to the induction of shoots per node or callus and roots per shoot on media prepared either with tap water and commercial sugar or those prepared with double distilled water and tissue culture-grade sucrose. The most effective medium for axillary bud proliferation was MS medium fortified with 1.0 mg l(-1 )N(6)-benzylaminopurine (BAP) and 0.5 mg l(-1 )indole-3-butyric acid (IBA), on which shoots were induced at the rate of 15 per node. The excision of node segments from the in vitro-derived shoots and their subsequent culture on medium supplemented with same concentrations of BAP and IBA facilitated enhanced axillary bud proliferation. Callus that developed from the lower cut end of the node explants induced shoots during subculture on half-strength MS medium with 1.0 mg l(-1 )BAP and 0.5 mg l(-1 )kinetin. The shoots developed rooted best on half-strength MS medium supplemented with 0.5 mg l(-1 )naphthaleneacetic acid (NAA). Rooted shoots, following acclimation in the greenhouse, were successfully transferred to field conditions, and 80% of the plantlets survived. When the basal ends of shoots harvested from multiplication medium were dipped in an NAA (0.5 mg l(-1)) solution for 25 days, a mean of 5.6 roots per shoot developed; the transfer to small pots facilitated the survival of 75% of the rooted shoots. Ex vitro rooting by direct transfer of the shoots from the multiplication medium to the greenhouse resulted in a 65% survival. Commercial sugar and tap water and ex vitro rooting make the protocol economically advantageous. About 750 plantlets were procured in a 3-month period starting from a single node explant.
An efficient in vitro multiplication system via multiple shoot bud induction and regeneration has been developed in Chlorophytum arundinaceum using shoot crown explants. Optimum regeneration frequency (87%) and desirable organogenetic response in the form of de novo organized multiple shoot buds without an intervening callus phase was obtained on Murashige and Skoog's (MS) minimal organics medium containing 3% sucrose (w/v) supplemented with 4 x 10(-6) M Kn and 2 x 10(-6) MIBA. Axenic secondary explants with multiple shoot buds on subculturing elicited best response with 1 x 10(-5) M Kinetin (Kn) and 5 x 10(-6) M indole-3-butyric acid (IBA) giving rise to an average of 18.74 shoots per culture with mean shoot length of 7.6 cm +/- 1.73. Varying molar ratios of either Kn/IBA or Kn/NAA revealed statistically significant differences in the regeneration frequencies among the phytohormone treatments. It was observed that the shoot bud differentiation and regeneration was influenced by the molar ratios of cytokinins/auxin rather than their relative concentrations. Healthy regenerated shoots were rooted in half strength MS basal medium containing 3% sucrose (w/v) supplemented with 5 x 10(-6) M IBA. Following simple hardening procedures, rooted plantlets, were transferred to soil-sand (1:1; v/v) with more than 90% success. Genetic fidelity was assessed using random amplified polymorphic DNA (RAPD), karyotype analysis and meiotic behaviour of in vitro and in vivo plants. Five arbitrary decamers displayed same banding profile within all the micropropagated plants and in vivo explant donor. The cytological and molecular analysis complemented and compared well and showed no genomic alterations in the plants regenerated through shoot bud differentiation. High multiplication frequency, molecular, cytological and phenotypic stability ensures the efficacy of the protocol developed for the production and conservation of this important endangered medicinal herb.
The wound healing potency of ethanol leaf extracts of V. trifolia L. and V. altissima L. was evaluated in excision, incision and dead space wound models. Both plants were found to possess significant wound healing activity which was evidenced by a decrease in the period of epithelialization, an increase in the rate of wound contraction, skin breaking strength, granulation tissue dry weight, hydroxyproline content and breaking strength of granulation tissue. Histopathological study of the granulation tissue also showed an increased collagenation when compared with the control group of animals. Of the two extracts, the ethanol leaf extract of V. trifolia showed maximum wound healing activity compared with the leaf extract of V. altissima. However, on comparison with the control group, both leaf extracts were found to possess significant wound healing potency.
The early fourth instar larvae of Culex quinquefasciatus, reared in the laboratory were used for larvicidal assay with leaf extracts of Vitex negundo, Vitex trifolia, Vitex peduncularis and Vitex altissima. The methanol extracts of the four species possessed varying levels of larvicidal nature. The highest larvicidal activity was found with the extract of V. trifolia (LC(50) = 41.41 ppm) followed by V. peduncularis (LC(50) = 76.28 ppm), V. altissima (LC(50) = 128.04 ppm) and V. negundo (LC(50) = 212.57 ppm).
An efficient in vitro method has been established for the production of whole plantlets of Bauhinia tomentosa using cotyledonary node (CN) explants excised from 15 d-old aseptic seedlings. Proliferating shoot cultures were obtained by placing CN explants on Murashige and Skoog (MS) medium supplemented with 6-benzyladenine (BA), kinetin (Kn), and 2-isopentenyl adenine (2-iP), singly or in combination, with either of the auxins, indole-3-acetic acid (IAA) or α-naphthalene acetic acid (NAA). MS medium augmented with 5.0 μM BA was found to be most effective in providing the highest frequency (84%) of shoot regeneration, associated with the maximum number of shoots (9.0 ± 0.6) per explant.The highest efficiency of shoot proliferation was observed using a combination of 5.0 μM BA and 0.5 μM NAA, where the number of shoots increased to 16.2 ± 0.2 per explant after 8 weeks of culture. Improved shoot multiplication and elongation rates were achieved when 20.0 mg l-1 adenine sulphate (AdS) was added to the combined treatment of 5.0 μM BA and 0.5 μM NAA, where an average shoot number of 17.0 ± 0.6 and an average shoot length of 5.8 ± 0.3 cm were obtained.The maximum frequency of root formation (70%) and greatest root lengths (2.3 ± 0.4 cm) were obtained on filter-paper bridges in liquid MS medium augmented with 2.5 μM IBA and 5.0 μM chlorogenic acid (a phenolic compound) after 4 weeks of culture. Regenerated plantlets were successfully acclimatized in a growth room and subsequently transferred to a greenhouse.This is the first report on in vitro plantlet regeneration in B. tomentosa using CN explants.
In vitro propagation technique of Balanites aegyptiaca, a multipurpose woody tree was studied. Nodal segments including axillary bud from mature tree were used as an explant and
their morphogenetic potential was tested on MS media with various concentrations (2.5–15.0μM) of 6-benzyladenine (BA), Kinetin,
and Thidiazuron alone or in combination with different concentrations (0.5–2.5μM) of α-naphthalene acetic acid (NAA). Nodal
segments showed axillary bud proliferation in almost all media tried. MS medium containing 12.5μM BA alone was effective
for inducing multiple shoots (5.0±0.22) with an average shoot length (3.7±0.26cm) in 67% of cultures. A better shoot
differentiation and elongation was achieved in a combined treatment of BA (12.5μM) and NAA (1.0μM). Half strength MS medium
supplemented with Indole-3-butyric acid (IBA) gave the best result for rooting. The maximum frequency of root formation (68%),
number of roots (5.3±0.32) and root length (4.1±0.38cm) was obtained on half strength MS medium containing 1.0μM IBA.
The regenerated plantlets were potted and acclimatized successfully in a growth chamber and then moved to the greenhouse.
An efficient and improved shoot regeneration technique for the micropropagation of Vitex negundo, an aromatic and medicinal shrub through in vitro culture of nodal segments with axillary buds, is described. Thidiazuron
(TDZ) used at 1.0μM was the most effective in inducing bud break and growth, and also in initiating multiple shoot proliferation
at the rate of 25 microshoots per nodal explant with axillary buds, after 4weeks of culture. By repeated subculturing of
nodal explants, a high-frequency multiplication rate was established. Optimum shoot multiplication and elongation was achieved
when TDZ exposed explants were subcultured on Murashige and Skoog (MS) media containing a combination of 1.0μM 6-benzyladenine
(BA) and 0.5μM α-naphthalene acetic acid (NAA). Efficient rooting was achieved directly in soilrite when basal portion of
the shoots were treated with 500μM indole-3-butyric acid for 10min which was the most effective in inducing roots, as 97%
of the microshoots produced roots. Plantlets went through a hardening phase in a controlled plant growth chamber, prior to
ex-vitro transfer. Micropropagated plants grew well, attained maturity and flowered. No phenotypical differences for morphogenesis
were observed among the regenerants.
An efficient and rapid plant regeneration system via direct organogenesis was established for Teucrium stocksianum Boiss. (Lamiaceae), an endangered and valuable medicinal plant. Hypocotyl explants excised from seedlings germinated in vitro
were cultured on Murashige and Skoog (MS) medium supplemented with different concentrations of kinetin and indoleacetic acid
(IAA) to induce shoot formation. Differentiation of multiple shoots was initiated within 3weeks of culture. Optimal regeneration
was achieved on medium containing 3mg/l kinetin and 0.5mg/l IAA. This particular medium composition significantly improved
the production of multiple shoots directly from hypocotyl explants compared to other combinations of plant growth regulators.
Root induction was achieved on half-strength MS medium containing indole-3-butyric acid. Rooted plantlets were successfully
acclimatized, with a survival rate of 75–80%. The protocol developed in this study could be used for long-term in vitro conservation
and mass propagation of this species.
A protocol for the micropropagation of dwarf raspberry (Rubus pubescens) was developed by the establishment of axenic shoot cultures from greenhouse-grown plants, induction of shoot proliferation, and rooting in vitro. Cultures were initiated from shoot tip and nodal explants on 1/2 strength MS (Murashige T. and Skoog F. 1962. Physiol. Plant. 15:473) macro-salts and MS micro-salts and vitamins containing 8.9 M N
6-benzyladenine (BA) and 0.98 M indole-3-butyric acid (IBA). Zeatin was more effective than BA, and induced proliferation of about 1.5–2 times as many shoots as BA in combination with 0.54–1.1 M -naphthaleneacetic acid (NAA) or 0.49–0.98M IBA. With higher zeatin, shoots did not expand and had a high mortality rate. Shoots growing for more than 10weeks on medium that contained 9.1 M zeatin occasionally produced adventitious shoot masses, which appeared to arise from dense calluses growing at the base of the shoots in the medium. Shoots were rooted in vitro in the same medium used for shoot proliferation, but without any growth regulators. Almost all (85–90%)in vitro plantlets survived when transferred to potting medium.
A protocol is described for rapid and large-scale propagation of the woody aromatic and medicinal shrub Vitex negundo by in vitro culture of nodal segments from mature plants. Of the three different cytokinins – N6-benzyladenine (BA), kinetin, and thidiazuron – evaluated as supplements to Murashige and Skoog (MS) medium, BA at an optimal
concentration of 2.0 mg/l was most effective in inducing bud break. Although callus-free multiple-shoot formation was a function
of cytokinin activity alone, faster bud break coupled with an enhanced frequency of shoot development (92%) and internode
elongation were dependent on the synergistic influence of gibberellic acid (GA3) when used at an optimal concentration (0.4 mg/l) along with BA (2.0 mg/l). The frequency of shoot proliferation was markedly
influenced by the explanting season. By repeated subculturing of nodal segments harvested from the in vitro-formed axenic
shoots on MS containing 1.0 mg/l BA and 0.4 mg/l GA3, prolific shoot cultures free from proximal callusing and showing a high-frequency multiplication rate were established.
The percentage shoot multiplication (98–100%) as well as the number of shoots per node (six to eight) were highest during
the first three culture passages, after which there was a gradual decline in shoot development. Rooting was best induced (94%)
in shoots excised from proliferated shoot cultures on half-strength MS medium augmented with an optimal combination of indole-3-acetic
acid and indole-3-butyric acid each at 1.0 mg/l. Vermi-compost was the most suitable planting substrate for hardening inside
a plant growth chamber and its use ensured high-frequency survival (93%) of regenerated plants prior to outdoor transfer.
Micropropagated plants established in garden soil were uniform and identical to the donor plant with respect to growth characteristics
as well as vegetative and floral morphology.
A mass in vitro propagation system for Bacopa monniera (L.) Wettst. (Scrophulariaceae), a medicinally important plant, has been developed. A range of cytokinins have been investigated
for multiple shoot induction with node, internode and leaf explants. Of the four cytokinins (6-benzyladenine, thidiazuron,
kinetin and 2-isopentenyladenine) tested thidiazuron (6.8 μM) and 6-benzyladenine (8.9 μM) proved superior to other treatments. Optimum adventitious shoot buds induction occurred at 6.8 μM thidiazuron where an average of 93 shoot buds were produced in leaf explants after 7 weeks of incubation. However, subculture
of leaf explants on medium containing 2.2 μM benzyladenine yielded a higher number (129.1) of adventitious shoot buds by the end of third subculture. The percentage
shoot multiplication (100%) as well as the number of shoots per explant remained the high during the first 3 subculture cycles,
facilitating their simultaneous harvest for rooting. In vitro derived shoots were elongated on growth regulator-free MS medium and exhibited better rooting response on medium containing
4.9 μM IBA. After a hardening phase of 3 weeks, there was an almost 100% transplantation success in the field.
Nine experiments were conducted to determine effects of various culture medium addenda on inducation of embryogenic calli from immature embryos of a responsive Triticum aestivum L. genotype (PCYT 10). Effects were quantified by counting somatic embryos (embryoids) per callus. Optimal auxin concentrations to induce and maintain somatic embryogenesis were 3.62 M 2,4-dichlorophenoxyacetic acid (2,4-D) or 9.05 M 3,6-dichloro-o-anisic acid (dicamba). In general, dicamba permitted formation of significantly more embryoids than 2,4-D. Kinetin (6-furfurylaminopurine) at 2.56 M or 4.65 M significantly increased percentage scutellar callus when added to 2,4-D or dicamba-containing medium, respectively. Kinetin at 4.65 M significantly increased the numbers of embryoids formed when added to medium containing either synthetic auxin. Significantly fewer embryoids formed when cultures were incubated under diffuse light (16-h photoperiod). Casein hydrolysate (200 mg1-1) or L-arginine (0.23 mM) had no effect on numbers of embryoids formed, whereas L-tryptophan (0.20 mM) enhanced such formation with 2,4-D and decreased such formation with dicamba. Two additional experiments generally demonstrated that response to auxin source in the genotypes ND 7532, PCYT 20, Yaqui 50, and Oasis was similar to that in PCYT 10. The higher molar concentration of dicamba required to induce embryogenic callus coupled with more evident embryoid precocious germination and a more rapid rate of tissue necrosis upon extended incubation without subculture suggests that dicamba is metabolized more rapidly than 2,4-D in T. aestivum callus cultures.
An efficient, rapid and reproducible plant regeneration protocol was successfully developed for Cassia angustifolia using nodal explants excised from 14-day-old aseptic seedlings. Of the two cytokinins, 6-benzyladenine (BA) and thidiazuron
(TDZ) evaluated as supplements to Murashige and Skoog (MS) medium, TDZ at an optimal concentration of 5.0μM was effective
in inducing multiple shoots. The highest rate of shoot multiplication was achieved on MS medium supplemented with 5.0μM TDZ
and 1.0μM indole-3-acetic acid (IAA) at pH 5.8. The regenerated shoots when subcultured on hormone free MS medium considerably
increased the rate of shoot multiplication and shoot length by end of fourth subculture passage. Rooting was achieved on the
isolated shoots using MS medium with 60μM indole -3- butyric acid (IBA) and 1% activated charcoal for 1week and subsequently
transferring the shootlets to half strength MS liquid media without IBA and activated charcoal. The in vitro raised plantlets
with well-developed shoot and roots were successfully established in earthen pots containing garden soil and grown in greenhouse.
Petiolar and distal cotyledonary segments (PCS and DCS) of Albizia chinensis were cultured on Murashige and Skoog's (MS; 1962) medium and induced to form adventitious shoot buds in the presence of either
cytokinins 6-benzylamino purine (BAP), kinetin (KN) or thidiazuron (TDZ). Superiority of BAP in inducing shoot bud and differentiation
was observed. PCS was more morphogenic to shoot bud differentiation than DCS. TDZ was highly effective in inducing shoot buds,
but arrested shoot growth, while KN produced more callus during differentiation of shoots. Rapid and high rate of shoot multiplication
per explant was achieved through subculture in MS medium containing BAP (1.0 mg l−1) and indole-3-acetic acid (IAA) (0.5 mg l−1). BAP at low concentration was required to enhance shoot multiplication and elongation. Successful rooting of regenerated
shoots was carried out in a two-step culture procedure in MS media with indole-3-butyric acid (IBA) (2.0 mg l−1) and subsequent subculture in IBA-free medium.
The use of tissue culture for cloning ornamentals is expensive and presently limited to a certain number of species. However, the introduction of some additional new techniques may possibly reduce the cost and broaden the range of plants that can be propagated economically in vitro. In this report, a survey is given of the methodology followed in our laboratory and its adaptation to commercial practices.Stock plants are grown under controlled conditions prior to in vitro culture in order to obtain healthier explants and uniform response (stage 0). After the establishment of aseptic cultures (stage I), buds are propagated (stage II), and are then prepared to harvest uniform cuttings (stage IIIa). Those cuttings are rooted under in vivo conditions (stage IIIb).
Leaf explants of Dianthus chinensis L. cultured on Murashige and Skoog’s (MS) medium with 6-benzylaminopurine (BAP, -naphthaleneacetic acid (NAA, 0.5 mg/l) or BAP (1 mg/l) produced adventitious shoot buds directly on the surface of the leaf explants without formation of intervening callus. Callus formation occurred after the induction of adventitious buds. Leaf explants cultured on BAP (0.5 mg/l) and 2,4-dichlorophenoxyacetic acid (2,4-D, 1 mg/l) supplemented MS medium also produced adventitious shoot buds along with excessive callus formation. Shoot buds formed on BAP and 2,4-D containing medium need to be subcultured on BAP and NAA supplemented medium for elongation, while the shoot buds induced on BAP and NAA supplemented medium elongated in the primary culture itself. Shoots were rooted on half strength MS medium with IAA (0.05 mg/l). Most rooted shoots grew well and produced viable seeds when grown in field conditions. This protocol is useful for clonal micropropagation and for Agrobacterium-mediated gene transfer in D. chinensis. Adventitious shoot bud formation directly from the leaf explants in D. chinensis is reported here for the first time.
Water and 80% ethanol extracts of 20 Thai medicinal plants used to treat AIDS were tested for their HIV type 1 reverse transcriptase inhibitory activity. The water extracts of Ipomoea carnea subsp. fistulosa (aerial parts), Vitex glabrata (branch), Vitex trifolia (aerial part), Vitex negundo (aerial part), Canna indica (rhizome), and Justicia gendarussa (aerial part) showed HIV-1 RT inhibition ratio (% IR) higher than 90% at a 200 microg/ml concentration. The water extract of Canna indica rhizomes (IC(50) 22.56 microg/ml) was selected for further study, i.e. for its HIV-1 RT inhibition activity and the purification and characterization of the active proteins. Proteins in water extract were fractionated by ammonium sulfate precipitation and separated by sodium dodecyl sulfate acrylamide gel electrophoresis (SDS-PAGE), yielding two proteins, Cip31 (31 kDa) and Cip14 (14 kDa) with IC50 of 17.41 and 19.25 microg/ml and isoelectric point (pI) of 3.5 and 6.35, respectively. Both proteins showed significant HIV-1 RT inhibition.
Six flavonoids, persicogenin (1), artemetin (2), luteolin (3), penduletin (4), vitexicarpin (5) and chrysosplenol-D (6), have been isolated for the first time as new cell cycle inhibitors from Vitex trifolia L., a Chinese folk medicine used to treat cancers, through a bioassay-guided separation procedure. They were identified by spectroscopic methods. The inhibitory effects of 1-6 on the proliferation of mammalian cancer cells have been evaluated by the SRB (sulforhodamine B) method and their effects on cell cycle and apoptosis investigated by flow cytometry with the morphological observation under light microscope and by agarosegel electrophoresis to detect internucleosomal DNA fragmentation. Compounds 1-6 inhibited the proliferation of mouse tsFT210 cancer cells with the IC50s (μg ml-1) > 100 (inhibition rate at 100 μg ml-1, 47.9%) for 1, > 100 (inhibition rate at 100 μg ml-1, 49.6%) for 2, 10.7 for 3, 19.8 for 4, 0.3 for 5, and 3.5 for 6. Flow cytometric investigations for 1-6 demonstrated that 1-5 mainly inhibited cell cycle at the G2/M phase in a dose-dependent manner with a weak induction of apoptosis on the tsFT210 cells, while 6 induced mainly apoptosis of the same tsFT210 cells also in a dose-dependent manner together with a weak inhibition of the cell cycle at the G0/G1 and G2/M phases, demonstrating that 1-6 exert their anti-proliferative effect on tsFT210 cells through inhibiting cell cycle and inducing apoptosis. In contrast to the cell cycle G2/M phase inhibitory main effect on tsFT210 cells, 5 induced mainly apoptosis on human myeloid leukemia K562 cells with a weak inhibition of the cell cycle at the G2/M phase. The present result provides flavonoids 1-6 as new cell cycle inhibitors and 1 and 4 as new anticancer flavonoids, which not only provide the first example of cell cycle G2/M phase inhibitory and apoptosis-inducing constituents of V. trifolia L. but also explain the use of Vitex trifolia L. by Chinese people to treat cancers.
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