Ex vitro rescue,physiochemical evaluation,secondary metabolite production and assessment of genetic stability using DNA based molecular markers in regenerated plants of <Emphasis Type="Italic">Decalepis salicifolia</Emphasis> (Bedd. ex Hook.f.) Venter

To ensure replenishment, a refine protocol for micropropagation of Decalepis salicifolia (Bedd. ex Hook.f.) Venter a critically endangered and endemic medicinal plant was developed using mature nodal explants. A high frequency shoot regeneration system was obtained on Murashige and Skoog (MS) medium comprised of 6- benzyladenine (BA) (5.0 µM)?+?α-naphthalene acetic acid (NAA) (0.5 µM)?+?adenine sulphate (ADS) (30.0 µM) corresponds to a highest mean number of 9.97?±?0.01 shoots/explants with maximum shoot length of 6.46?±?0.1 cm. Successful rooting in microshoots was achieved on half strength MS medium supplemented with indole-3-butyric acid (IBA) (2.5 µM). A maximum of 6.10?±?0.07 roots/microshoot with average root length of 2.30?±?0.06 cm was obtained. As much as 90% plantlets survived when Soilrite^? was used as planting substrate and finally established in soil without any casualty and morphological variation. Acclimatized plantlets were screened for pigment content, net photosynthetic rate (PN), stomatal conductance (Gs) and transpiration rate (E) during subsequent days of acclimatization as well as the changes in antioxidant was also evaluated. A steady rise was observed in the activity of superoxide dismutase (SOD) for initial 21 days and then after a decrease was found showing improved acclimatization efficiency of the plant. Similarly, the activities of catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR) enzyme shows reliable increase as the days of acclimatization advanced which play their precautionary role against oxidative damage to the plant. The genetic fidelity of the in vitro raised plantlets with that of mother plant was further confirmed using random amplified polymorphic DNA (RAPD) and inters simple sequence repeats (ISSR) analysis. Additionally, the effect of acclimatization on the biosynthesis of 2-hydroxy-4-methoxybenzaldehyde (2H4MB) in the root system was also evaluated in relation to their biomass production. Maximum fresh weight (4.9 g/plant), dry weight (0.65 g/plant) of roots and 2H4MB content (6.8 µg ml^?1 of root extract) was obtained after 10 weeks of acclimatization. Accelerated multiplication rate with the stability of genetic virtue, physiological and biochemical parameter assure the efficacy of the protocol developed for the propagation of this critically endangered medicinal plant.     

Change in total phenolic content and antibacterial activity in regenerants of Vitex negundo L.

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-isopentenyl adenine] used as supplement to Murashige and Skoog medium (MS), BA at 5.0 μM 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 μM) along with α Naphthalene acetic acid (0.5 μM), 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 μM 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.     

Photosynthetic apparatus performance in function of the cytokinins used during the in vitro multiplication of <Emphasis Type="Italic">Aechmea blanchetiana</Emphasis> (Bromeliaceae)

During the in vitro multiplication phase, the employment of cytokinins may be necessary to induce side shoots of many plant species. However, the mechanism by which cytokinins influence the physiology of plants in vitro is not well understood. Therefore, the objective of this study was to assess the influence of two cytokinins in function of concentration on the o photosynthetic apparatus performance and the stomatal functionality of Aechmea blanchetiana during in vitro multiplication. Plants previously established in vitro were transferred to MS culture media supplemented with 6-benzylaminopurine (BAP) or 6-furfurylaminopurine (kinetin—KIN) at concentration of 0, 5, 10, 15 or 20 µM. After 60 days of exposure to the plant growth regulators, the multiplication rate, photosynthetic apparatus performance and stomatal functionality were assessed. The use of KIN did not induce the formation of microshoots. On the other hand, the shoot number increased with rising BAP concentration. There was a reduction of the maximum fluorescence (F_m) and maximum quantum yield (φP₀) as a function of concentration of cytokinins. The most pronounced decrease was observed in the microshoots grown with KIN. The increase in concentration of cytokinins induced greater absorption flux (ABS/RC), trapping flux (TR₀/RC) and dissipation flux (DI₀/RC) of energy per reaction center. The stomatal functionality declined with rising cytokinin concentration. The use of KIN is not recommended for in vitro multiplication of this species. The use of BAP at low concentrations assures a multiplication rate with lower degree of disorders in the photosynthetic apparatus of the formed microshoots.     

High-frequency clonal propagation of <Emphasis Type="Italic">Curcuma angustifolia</Emphasis> ensuring genetic fidelity of micropropagated plants

A high-frequency clonal propagation protocol was developed for Curcuma angustifolia Roxb., a high valued traditional medicinal plant. Axillary bud explants of C. angustifolia were explanted on Murashige and Skoog (MS) medium fortified with 4.4–22.2 µM 6-benzyladenine (BA), 2.9–5.7 µM indole-3-acetic acid (IAA), 2.3–23.2 µM kinetin (Kin), 2.7–5.4 µM naphthalene acetic acid (NAA) and 67.8-271.5 µM adenine sulphate (Ads) in different combinations. The maximum number of shoots per explants (14.1?±?0.55) and roots per shoot (7.6?±?0.47) was achieved on media containing 13.3 µM BA, 5.7 µM IAA and 135.7 µM Ads. Stability in phytomedicinal yield potential of micropropagated plants was assessed through GC–MS and HPTLC. Gas chromatogram of essential oil of conventional and micropropagated plants of C. angustifolia had similar essential oil profile. HPTLC analysis of rhizome extracts of in vitro and field grown plants revealed no significant differences in the fingerprint pattern and in curcumin content. Genetic integrity of in vitro and field grown derived plants were evaluated with inter-simple sequence repeat (ISSR) primers and flow cytometry using Glycine max as an internal standard. A total of 1260 well resolved bands were generated by 12 ISSR primers showing monomorphic banding patterns across all plants analyzed. The mean 2C DNA content of conventionally and micropropagated plant was estimated to be 2.26 pg and 2.31 pg, respectively. As no somaclonal variations were detected in tissue culture plantlets, the present micropropagation protocol could be applied for in vitro conservation and large-scale production of C. angustifolia.     

Micropropagation of mature <Emphasis Type="Italic">Quercus ilex</Emphasis> L. trees by axillary budding

This paper reports the successful micropropagation of mature Quercus ilex trees known as reluctant to in vitro propagation. Crown branch segments collected from 30 to 100 year-old trees were forced in order to promote the production of sprouting shoots that were used as a source of explants for initiating the cultures. Sterilization was critical and required low-level disinfestation protocols. Six out of the eight mature genotypes attempted were successfully inoculated and then maintained in culture with varying responses. Shoot proliferation of holm oak was influenced by BA concentration, with improved multiplication and shoot appearance when the BA concentration was sequentially reduced over the culture period. Micropropagation by axillary budding was achieved by culturing shoots on a sequence of cytokinin-enriched Lloyd and McCown (WPM) media alternating 2 week-long subcultures on 0.44 µM benzyadenine (BA) first, followed by 0.22 µM BA, then 0.044 µM BA plus 0.46 µM zeatin. Sucrose concentration and agar brand affected shoot proliferation, and the best results were obtained on WPM medium supplemented with 8 g L^?1 Sigma agar (A-1296; Sigma-Aldrich) and 30 g L^?1 sucrose. Addition of 20 µM silver thiosulphate had a significant positive effect on the appearance and development of shoots with a higher number of shoots being healthy and showing reduced shoot tip necrosis and early senescence of leaves. The 18.8% of the microshoots obtained for one clone could be rooted within 15 days on a half-strength Murashige and Skoog medium containing 14.8 µM or 24.6 µM indole-3-butyric acid and 0.54 µM α-naphthalene acetic acid.     

A liquid culture system for shoot proliferation and analysis of pharmaceutically active constituents of Catharanthus roseus (L.) G. Don

An efficient and cost effective micropropagation protocol using liquid medium was developed for Catharanthus roseus, a commercially important medicinal plant. Comparative analysis of shoot growth and proliferation in liquid Murashige and Skoog (MS) medium supplemented with different concentrations of cytokinins [6-Benzyladenine (BA), Kinetin (KN) and Thidiazuron (TDZ)] was conducted. Better response in terms of shoot proliferation, shoot diameter, number of leaves/shoot, number of branches/shoot, fresh weight and dry weight was observed in a liquid medium vis-à-vis solid medium. A sample of 20 ml of liquid medium supplemented with 5 ??M of BA was optimized for propagation of C. roseus by a liquid culture system. Among various concentrations of auxins tried, 1-Naphthaleneacetic acid (NAA) 5 ??M was found to be the best for root induction. Quantification of pharmaceutically important constituents (vincristine and vinblastine) and total alkaloid content of microshoots grown in solid and liquid medium as well as in vitro raised plants and mother plant was also conducted, hitherto unreported in this high-value medicinal plant. This work further lays the foundations for the shifting of plant production from small to commercial scale.     

Rapid in vitro propagation system through shoot tip cultures of Vitex trifolia L.–an important multipurpose plant of the Pacific traditional Medicine

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.     

Direct plant regeneration from different explants through micropropagation and determination of secondary metabolites in the critically endangered endemic Rhaponticoides mykalea

Direct plant regeneration from different explants, micropropagation and determination of secondary metabolites were studied in the critically endangered endemic Rhaponticoides mykalea (Hub.-Mor.) M.V. Agab & Greuter. Seed germination was achieved by damaging the seed coat and cultivating the embryos on Woody Plant Medium (WPM), of which 40% germinated. The epicotyls and cotyledonary petioles of seedlings were used as initial explants and direct shoot regeneration was obtained on WPM containing 2.22 μM 6-benzyladenine (BA). WPM medium supplemented with 2.22 μM BA and 4.92 μM indole-3-butyric acid (IBA) significantly improved the production of multiple shoots, resulting in an average of 5.6 shoots per explants. The highest rooting of shoots (35.6%) was observed with WPM medium containing 19.68 μM IBA with 990 μM putrescine. Plantlets with well-developed roots were transferred to soil and acclimatised within a plant growth chamber. Acclimatised plants showed 100% survival rate and remained healthy. As a part of our study, the content of secondary metabolites in three tissue culture regenerated lines were determined by HPLC analysis. Chlorogenic acid, Quercetin and scutellarin were confirmed secondary metabolites of R. mykalea.     

Tissue culture and metabolome investigation of a wild endangered medicinal plant using high definition mass spectrometry

An increasing effort is dedicated to investigate the potential of native plants used in traditional medicine as a source of bioactive compounds for numerous industries. The bioprospection of the metabolome of medicinal and/or endangered plants has two important merits: confirming or revealing the biotechnological potential of that species, and assisting in its conservation. In addition, biotechnological techniques, such as tissue culture, are key strategies in conservation and multiplication of medicinal plants. This is the first in vitro development and non-targeted metabolome study by UPLC–QTOF–MS^E of extracts from C. menthoides, an endangered medicinal plant. In vitro development investigation with a wide range of plant growth regulators resulted in maximum survival rate (81%) and the highest growth rate (1.74 cm?±?0.36) for plantlets cultured on Murashige and Skoog medium, supplemented with 1 µM gibberellic acid. Maximum rooting occurred on medium supplemented with 4.4 µM 6-benzyladenine, which also resulted in more leaves per plantlet (10.16?±?1.7). We developed a protocol that can be used for the clonal propagation and ex situ conservation of this species. In terms of metabolome analysis, a total of 107 metabolites from several classes were detected and identified in its hydrophilic extract (HE), including organic acids and derivatives, glucosinolates, terpenes, phenolic compounds as well as other polar metabolites. The metabolites in HE with the greatest signal intensity included the isoquinoline alkaloid magnoflorine; the coumaric acid rosmarinic acid; the steroid-cardanolide convallatoxin; two anthraquinones including the poorly investigated ventinone A. Several molecules identified here carry potential pharmacological benefits such as anti-inflammatory and anticancer applications.     

Putrescine and polyamine inhibitors in culture medium alter in vitro rooting response of <Emphasis Type="Italic">Decalepis hamiltonii</Emphasis> Wight &amp; Arn.

The influence of polyamine putrescine (PUT), and polyamine inhibitors were tested for in vitro rooting response from micro shoots that initially established on Murashige and Skoog (MS) medium comprising 2.7 µM α-Naphthaleneacetic acid (NAA) and 8.9 µM 6-Benzylaminopurine (BA) by using nodal explants of Decalepis hamiltonii. Incorporation of putrescine alone in rooting medium devoid of auxins supported the best response for in vitro rooting qualitatively and quantitatively. Incorporation of putrescine at 50 µM able to induce 8.62?±?1.93 roots with a maximum root length of 9.10?±?1.65 cm wherein, the root fresh weight was also found to be high compared to all other treatments (5.248?±?1.71 g). Addition of putrescine inhibitor cyclohexylamine (CHA) in medium curtailed rooting response from microshoots. Among the three polyamine inhibitors, CHA in presence of 9.8 µM Indole-3-butyric acid (IBA) outperformed α-DL-difluromethylarginine (DFMA) and α-DL-difluoromethylornithine (DFMO) combination with 9.8?µM IBA. The least response for root number (1.55?±?0.72), root length (1.96?±?0.45 cm), and root weight (1.94?±?0.35 g) was found for IBA?+?PUT?+?DFMA and the best response was noted for IBA?+?PUT?+?CHA (2.6?±?1.1, 2.92?±?0.73 cm, 3.03?±?0.75 g) respectively. Endogenous content of putrescine, spermidine and spermine supported the rooting response from in vitro shoots. These results have clearly demonstrated that putrescine plays a crucial role in rooting of D. hamiltonii. Plantlets were transferred to micro-pots for a short acclimatization stage in greenhouse where they survived at 90?%. This highly reproducible procedure can be adopted for large scale swallow root propagation. Overall, supplementing putrescine in the rooting medium enhances the quantity and quality of roots in D. hamiltonii, thus confirming its role.     

Rapid plant regeneration and analysis of genetic fidelity in micropropagated plants of Vitex trifolia: an important medicinal plant

An efficient in vitro regeneration protocol of a valuable medicinal plant, Vitex trifolia has been successfully established using nodal segments as explants. Three different cytokinins (BA, Kn, 2iP) and auxins (NAA, IAA, IBA) in different concentrations and combinations, evaluated as supplements to Murashige and Skoog’s medium showed to have a marked influence on the regeneration output. Among all the single cytokinin treatments MS medium supplemented with 5.0 μM BA produced the maximum number of shoots yielding 8.20 ± 0.37 shoots per explant with 4.8 ± 0.43 cm shoot length after 8 weeks of culture. Combined with low auxin concentrations, all the three cytokinins at their optimal concentrations synergistically enhanced the regeneration credentials. However, MS medium enriched with 5.0 μM BA and 0.5 μM NAA yielded the best possible regeneration in the species with a regeneration percentage of 97.33 ± 2.67 % and amounting to 16.80 ± 0.58 shoots per explant with 6.20 ± 0.25 cm mean shoot length at the end of 8 weeks in culture. Ex vitro rooting of in vitro derived microshoots was achieved by 20 min 500 μM IBA treatment followed by transfer to thermocol cups containing sterile soilrite. A 95 % plantlets survived acclimatization procedure to the field. Genetic conformity of the regenerated plants was established through RAPD. All the bands visualized on agarose gels were monomorphic with that of the donor plant indicating the clonal nature of the regenerants.     

Thidiazuron: a potent cytokinin for woody plant tissue culture

Thidizuron (TDZ) is among the most active cytokinin-like substances for woody plant tissue culture. It facilitates efficient micropropagation of many recalcitrant woody species. Low concentrations (<1 µM) can induce greater axillary proliferation than many other cytokinins; however, TDZ may inhibit shoot elongation. In some cases it is necessary to transfer shoots to an elongation medium containing a lower level of TDZ and/or a less active cytokinin. At concentrations higher than 1 µM, TDZ can stimulate the formation of callus, adventitious shoots or somatic embryos. Subsequent rooting of microshoots may be unaffected or slightly inhibited by prior exposure to TDZ. The main undesirable side effect of TDZ is that cultures of some species occasionally form fasciated shoots. The high cytokinin activity and positive response of woody species to TDZ have established it as among the most active cytokinins forin vitro manipulation of many woody species.     

Establishment of an in vitro propagation protocol for <Emphasis Type="Italic">Thymus lotocephalus</Emphasis>, a rare aromatic species of the Algarve (Portugal)

The aim of this work was to develop an in vitro propagation protocol for the endangered species Thymus lotocephalus using seedlings as explants. Several macronutrient concentrations of Murashige and Skoog medium (MS), cytokinin types and concentrations, and cytokinin/auxin combinations were tested to assess the shoots’ proliferation capacity. Although the best proliferation results were obtained with 6-benzyladenine, high percentages of hyperhidric shoots were observed. Because high proliferation of healthy shoots was observed in MS medium that was free of plant growth regulators, this medium was chosen for proliferation studies. The best rooting results were achieved in ¼MS medium without auxins (92.00 ± 6.11%, 6.54 ± 0.52 and 1.60 ± 0.10 cm regarding rooting frequency, number of roots per shoot and longest roots, respectively) or supplemented with 0.5 mg l^?1 indole-3-acetic acid (98.00 ± 2.11%, 11.14 ± 0.75 and 2.40 ± 0.24 cm, respectively). Plantlets were successfully acclimatised to ex vitro conditions with a survival rate of 93.33%. With the development of this micropropagation protocol, an important contribution has been made to the conservation of the endangered species T. lotocephalus.     

Seasonal and micro-environmental factors controlling clonal propagation of mature trees of marwar teak [<Emphasis Type="Italic">Tecomella undulata</Emphasis> (Sm.) Seem]

A simple method has been developed for clonal propagation of mature trees of Tecomella undulata (Sm.) Seem, a medicinally important deciduous timber tree of hot arid regions, via multiple shoot proliferation from axillary buds after examining the role of season influences and physico–chemical conditions on micropropagation. Spring season (March–April) was the best period for contamination free establishment of explants and maximum sprouting of healthy axillary buds. Shoots proliferated directly from the explant nodes cultured on Murashige and Skoog’s medium containing cytokinins, BAP supporting better growth compared to kinetin during shoot induction as well as multiplication phase. Cytokinin concentration influenced the bud induction frequency and optimal response of 2.6 buds per explant was achieved in 86.66% explants on media supplemented with 10 µM BAP. Stunted shoot buds with excessive callus were observed when cytokinin concentration was increased beyond optimal levels. Ascorbic acid (50 mg/l), arginine and citric acid (25 mg/l each) were added to proliferation and multiplication media for reducing callus proliferation and better shoot growth. Among the media (B5, MS, NN, WPM and SH) tested, SH was best for shoot multiplication. Shoot cultures were multiplied by regular subculture of axillary shoots on SH medium containing 5.0 µM each of BAP and kinetin. Shoots produced roots when cultured on ½× SH medium + 10 μM IBA. Regenerated plantlets were successfully transferred to field after hardening and acclimatization. Genetic homogeneity of tissue culture raised plants was confirmed by generation of monomorphic DNA fragments with Start codon targeted and intersimple sequence repeat (ISSR) markers.     

Effect of cytokinins on shoots proliferation and rosmarinic and salvianolic acid B production in shoot culture of <Emphasis Type="Italic">Dracocephalum forrestii</Emphasis> W. W. Smith

The current study estimates the effect of different cytokinins on shoot proliferation and biosynthesis of caffeic acid derivatives in Dracocephalum forrestii in vitro culture. The shoots were grown on Murashige and Skoog (MS) agar medium with 1 µM indole-3-acetic acid (IAA) and different content of 6-benzyloaminopurine (BAP), zeatin, kinetin (1, 2, 4, 8, 18 µM) or thidiazuron (TDZ) (0.1, 0.2, 0.5, 1, 2 µM). The highest multiplication rate (about seven shoots and/or buds per explant) was obtained after 4 weeks of culture on MS medium with 1 µM IAA and 8 or 16 µM BAP. Optimal biomass of plant material was also received on the same media. The identity of the compounds present in the hydromethanolic extracts from D. forrestii shoots grown on cytokinin-supplemented media was confirmed using UPLC–PDA–ESI–MS method. The analysis revealed the presence of nine metabolites recognized as caffeic acid derivatives. The content of the predominant phenolic acids in the extracts, i.e. rosmarinic acid (RA) and salvianolic acid B (SAB), was determined with UHPLC. The highest yield of RA was found in shoots cultivated in the medium containing 1 µM IAA and 2 µM BAP (18.7 mg/g DW). The highest level of SAB (5.3–5.9 mg/g DW) was identified in multiple shoots grown in the presence of 1 µM IAA and 0.5–1 µM TDZ or 2 µM BAP.     

Somatic embryogenesis and plant regeneration of blackberry using the thin cell layer technique

Limonium ‘Misty Blue’ is an interspecific hybrid of Limonium latifolium and L. bellidifolium and has a huge demand in floriculture business as both fresh and dry flowers with stunning purple-blue blooms. The propagation only through vegetative means restrict the popularization of this plant to the flower growers. We therefore optimized an efficient micropropagation protocol for direct organogenesis from root explants, as leaf is not conducible to respond in culture. 61.43% of root explants directly formed shoot buds on their surface after 4-weeks of culture in media containing ½ MS, 43.82 mM sucrose 2.22 µM BA and 1.07 µM NAA. The shoot buds failed to differentiate into healthy shoots unless the previous medium was replaced by full strength MS, and 87.64 mM sucrose along with 0.44 µM BA and 1.07 µM NAA. Encapsulations of juvenile shoots were carried out by 3% sodium alginate and 100 mM CaCl₂ which were again successfully stored at 4?°C for 30 days along with 56.79% of plant recovery in MS?+?0.44 µM BA?+?4.5 µM IBA?+?87.64 mM sucrose containing medium. 150 synthetic seed derived full grown plants were successfully acclimatized in green house, where a total of 101 plants survived after secondary hardening. The ISSR analysis revealed genetic homogeneity of synthetic seed derived hardened plants.     

Efficient in vitro propagation of <Emphasis Type="Italic">Artemisia nilagirica</Emphasis> var. <Emphasis Type="Italic">nilagirica</Emphasis> (Indian wormwood) and assessment of genetic fidelity of micropropagated plants

A reliable protocol has been established for in vitro propagation of Artemisia nilagirica var. nilagirica (Indian wormwood), a valuable medicinal plant from India. A highly proliferating organogenic callus was obtained on Murashige and Skoog (MS) medium supplemented with 2.5 µM IAA when nodal explants were cultured on MS medium supplemented with various growth regulators. Further, highest regeneration frequency (83.3 %) of adventitious shoots was observed, when the callus was sub-cultured on MS medium supplemented with 6-benzylaminopurine (BAP; 2.5 µM) along with 7.5 µM 2-isopentenyl adenine (2-iP). An optimal of 10.16 ± 2.24 shoots were regenerated on medium supplemented with 2.5 µM BAP + 7.5 µM 2-iP. Quarter strength MS medium supplemented with 10 µM IBA was effective for rooting of the shoots. Ex-vitro plants were normal and were established successfully. Cytological and molecular marker studies showed that regenerated plants showed genetic stability in micro-propagated plants.     

In vitro conservation of <Emphasis Type="Italic">Alectra chitrakutensis</Emphasis>: a critically endangered root parasitic plant of high medicinal importance

The present study provides an effective in vitro propagation method for critically endangered parasitic plant Alectra chitrakutensis (M.A. Rau) R. Prasad & R.D. Dixit (Scrophulariaceae). The plant comprises tremendous medicinal properties to treat many diseases such as leprosy, leucoderma, tuberculosis and paralysis. The protocol has been developed using rhizome explant on the modified Murashige and Skoog medium containing various phytohormones to raise complete plantlets without any host interaction. 0.5 mg/l kinetin and 1.0 mg/l NAA was found to be the most excellent medium combination to obtain plantlets with an average shoot length of 11 ± 1.04 cm; and 100% response for rooting. The same medium combination also supported flowering in vitro. Further, comparative molecular and chemical characterization of in vitro and in vivo grown plants were carried out by performing RAPD and HPTLC analysis, respectively, to make sure the genetic as well as chemical stability among the plants. RAPD analysis with MAP (01-20) and OPJ primers (01-20) exhibited a monomorphic band pattern which revealed the genetic similarities among the tested plants. Similarly, presence of azafrin, the most important pharmaceutically active compound in in vitro generated plants indicates the efficacy of protocol to fulfill the objective of in vitro propagation of this critically endangered plant species. Moreover, the present study is the first report towards in vitro conservation of parasitic plant A. chitrakutensis without host from the rhizome explant.     

Auxin and nutritional stress coupled somatic embryogenesis in <Emphasis Type="Italic">Oldenlandia umbellata</Emphasis> L.

Somatic embryos were induced from internodal segment derived callus of Oldenlandia umbellata L., in MS medium supplemented with different concentrations of 2,4-Dichlorophenoxy acetic acid (2,4-D). Initially calli were developed from internodes of microshoots inoculated in 2.5 µM NAA supplemented medium. Then calli were transferred to 2,4-D added medium for somatic embryogenesis. Nutritional stress coupled with higher concentration of 2,4-D triggered somatic embryogenesis. Nutritional stress was induced by culturing callus in a fixed amount of medium for a period up to 20 weeks without any external supply of nutrients. Addition of 2.5 µM 2,4-D gave 100% embryogenesis within 16 weeks of incubation. Callus mass bearing somatic embryos were transferred to germination medium facilitated production of in vitro plantlets. MS medium supplemented with 2.5 µM benzyl adenine and 0.5 µM α-naphthalene acetic acid produced 15.33 plants per culture within 4 weeks of culture. Somatic embryo germinated plants were then hardened and transferred to green house.     

Organogenesis and efficient in vitro plantlet regeneration from nodal segments of <Emphasis Type="Italic">Allamanda cathartica</Emphasis> L. using TDZ and ultrasound assisted extraction of quercetin

The present investigation was carried out to evaluate the instigative effect of thidiazuron (TDZ) on multiple shoot induction from nodal segments of Allamanda cathartica and estimated the flavonoid yield among the regenerants. High rate of shoot bud induction was achieved on Murashige and Skoog (MS) medium augmented with 0.3 µM TDZ from nodal segments exposed for 30 days. However, for shoot proliferation and elongation, TDZ exposed cultures were further cultured on MS medium devoid of TDZ and/or supplemented with different concentration of 6-benzyladenine (BA) and Kinetin (Kn). BA at 2.5 µM gave the maximum mean number of shoots (44.00?±?1.30) and shoot length (7.50?±?0.21 cm) per explant after 12 weeks of incubation in the secondary medium. The response of explant was influenced by the collection time. The highest rooting in the microshoots (5 cm) was achieved on 1/2 MS liquid medium supplemented with 0.5 µM Indole-3 butyric acid (IBA) which produced 4.50?±?0.16 mean roots/shoot with 4.05?±?0.17 cm mean root length. The leaves of 30 day old acclimatized plantlets were used for phytochemical screening. Ultrasonication mediated extraction and quantification of bioactive flavonoid namely quercetin through colorimetry and mass spectrometry analysis from the leaves of regenerants. Extraction was processed in methanol using 2 g leaf sample through sonication. Total yield of flavonoids and quercetin content was found to be maximum in 2.5 µM BA treated plants with respect to control and other treated samples. The concentration of total flavonoids was estimated to be 172.90 mg QE/g which yielded 51.39 mg/g quercetin. The study ensures a rapid cultivation of plantlets, thus enhancing the biomass production which may be utilized in the isolation and quantification of other biological potential compound for the use in treatment of various ailments.