Auxin-cytokinin synergism in vitro for producing genetically stable plants of Ruta graveolens using shoot tip meristems

An efficient micropropagation protocol was developed for Ruta graveolens Linn. using shoot tip meristems derived from a 4-month-old field grown plant. In vitro shoot regeneration and proliferation was accomplished on Murashige and Skoogs (MS) semi-solid medium in addition to different doses of cytokinins viz.6- benzyl adenine (BA), Kinetin (Kn) or 2-isopetynyl adenine (2iP), singly or in combination with auxins viz. indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) or α-naphthalene acetic acid (NAA). Highest regeneration frequency (27.6%) was obtained on (MS) medium composed of BA (10 µM) with maximum number (9.4) of shoots and 4.3 cm shoot length after 4 weeks of incubation. Among various combinations tried best regeneration frequency (71%) of multiple shoot formation with highest number (12.6) of shoots per shoot tip explants were achieved in MS medium augmented with a combination BA (10.0 µM) and NAA (2.5 µM) after 4 weeks of incubation. The optimum frequency (97%) of rhizogenesis was achieved on half-strength MS medium having 0.5 µM IBA after 4 weeks of incubation. Tissue culture raised plantlets with 5–7 fully opened leaves with healthy root system were successfully acclimatized off in Soilrite? with 80% survival rate followed by transportation to normal soil under natural light. Genetic stability among in vitro raised progeny was evaluated by ISSR and RAPD markers. The entire banding pattern revealed from in vitro regenerated plants was monomorphic to the donor. The present protocol provides an alternative option for commercial propagation and fruitful setting up of genetically uniform progeny for sustainable utilization and germplasm preservation.     

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

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

Rapid plant regeneration,validation of genetic integrity by ISSR markers and conservation of Reseda pentagyna an endemic plant growing in Saudi Arabia

Reseda pentagyna is the only endemic species among the seven species of the genera Reseda found in Saudi Arabia. Probably no information is available on regeneration by conventional method of regeneration through seeds or cuttings. Therefore, alternative method of tissue culture was attempted to regenerate and multiply the plant. High shoot regeneration (14.44 shoots/explant) was obtained after four weeks, when shoot cuttings cultured on MS containing BA at 1.0 µM. Other cytokinins e.g., Kn, 2iP and TDZ found to be less effective in bud induction and shoot multiplication. Individual shoots were rooted on MS medium supplemented with various auxins at 0.5–5.0 µM concentrations. The IBA (1.5 µM) supplemented MS media induced maximum (83.3%) rooting. The plantlets were acclimatized and hardened under greenhouse conditions in plastic pots containing soil and farm yard manure with 95.0% success. The protocol developed would help to multiply the plant as well as conserve them in natural habitat. This can also be utilized to obtain active constituents for pharmaceutics and genetic manipulations.     

Indirect organogenesis from various explants of Hildegardia populifolia (Roxb.) Schott & Endl. – A threatened tree species from Eastern Ghats of Tamil Nadu,India

Hildegardia species are an important resource for fiber industry. This investigation was conducted to develop a plant regeneration protocol for Hildegardia populifolia (Roxb.) Schott & Endl. via indirect organogenesis Callus was obtained from leaf, internode and petiole explants, among these explants internode explant gave best result on MS medium supplemented with different concentrations of 2,4-Dichlorophenoxy acetic acid (2,4-D). The highest percentage (100%) of regeneration was obtained with benzyladenine (BA) (2.0 mg/l) + indole-3-acetic acid (IAA) (0.1 mg/l) + glutamine (25 mg/l) + thidiazuron (TDZ) (0.5 mg/l) from internode explants. Shootlets were highly rooted on MS medium supplemented with 3.0 mg/l indole-3-butyric acid (IBA). In vitro rooted seedlings were successfully acclimatized. This in vitro regeneration system will facilitate further development of reliable procedures for this genus.     

In vitro cultures of Silybum marianum and silymarin accumulation

In this study, a protocol for initiation of callus and shoot cultures from leaves and shoot tips explants of different silybium genotypes collected from different locations in Egypt was established. Callus cultures were initiated from leaves explants and exposed to different concentrations of the precursor (coniferyl alcohol). Shoot cultures were initiated from shoot tips explants. Moreover, the produced plants of the different Silybium shoots as well as intact plants were subjected to protein screening using SDS–PAGE analysis.Results obtained revealed that the optimum medium for growth and maintenance of friable callus was MS medium supplemented with 0.25 mg L⁻¹ 2,4-Dichlorophenoxy acetic acid (2,4-D) + 0.25 mg L⁻¹ Kinetin (Kin). The best medium for proliferation of high number of shoots was MS-medium with 0.25 mg L⁻¹ each of Benzyl Adinine (BA) and Naphthalene Acetic Acid (NAA). Coniferyl alcohol in concentration of 30 μM caused an increase in accumulation of silymarin contents in most callus cultures. SDS–PAGE of different Silybium shoots revealed that the protein profiles of 100% of in vitro produced plantlets similar to their control.     

Rapid in vitro micropropagation of Agapanthus praecox

In vitro micropropagation and acclimatization for the ornamental Agapanthus praecox, are reported. The influence of different growth regulators on shoot multiplication from shoot-tip explants of A. praecox was investigated. Prolific shoot multiplication (47.3 ± 1.96 shoots per explant) was achieved on Murashige and Skoog (MS) medium supplemented with 22.2 μM benzyladenine (BA), 2.9 μM indole-3-acetic acid (IAA), and 4.5 μM thidiazuron (TDZ). Shoots were rooted on half-strength MS basal medium supplemented with 5.7 μM IAA and 2.5 μM 2-isopentenyladenine (2iP) with 11.3 ± 0.78 roots per shoot. The in vitro-raised plants were established successfully in a 1:1 (v/v) vermiculite:sand mixture when maintained in a greenhouse with 100% survival. The elongated shoots (more than 5 cm in length) were treated for rooting and acclimatization in a moistened (5.7 μM IAA and 2.5 μM 2iP) vermiculite:sand (1:1 v/v) mixture, first in the misthouse and then in the greenhouse. Rooting and acclimatization was achieved simultaneously (100%) in the misthouse which was followed by greenhouse cultivation. This system can be used for rapid mass clonal propagation of A. praecox, for conservation strategies, commercial production, gene transformation studies and to produce phytomedicines.     

Ex vivo implications of phytohormones on various in vitro responses in Leptadenia reticulata (Retz.) Wight. & Arn.—An endangered plant

Leptadenia reticulata (Retz.) Wight. & Arn. is an important medicinal plant, belongs to the family Asclepiadaceae. This plant is known for its medicinal uses since 4500 BC. Presently this is an endangered species (Arya et al., 2003). Six shoots (2–4 cm long) per node differentiated on MS medium + 5.0 mg/l of BAP + additives. Incorporation of additives in the culture medium promoted growth of cultures. The shoots differentiated per explant were repeatedly transferred on to fresh MS + 1.0 mg/l of BAP + 0.1 mg/l of NAA and additives. The regenerated shoots were subcultured for further multiplication on MS + 1.0 mg/l BAP + 0.5 mg/l Kin + 2-iP (0.5 mg/l) and 0.1 mg/l of NAA + additives regularly after an interval of 3 weeks. Addition of ammonium sulphate in the medium resulted in increase in shoot number and promoted elongation also growth of cultures was sustained even if subculturing was delayed (26 ± 2 days). Success was also achieved in defining protocol for in vitro regeneration of shoots from petiole derived callus. Shoots regenerated in vitro by both processes were rooted in vitro on 1/4 strength of MS medium + 3.0 mg/l of IBA after 15–20 days. Cent percent of the shoots rooted ex vitro, if the in vitro regenerated shoots were treated with 200 mg/l of IBA. The in vitro–ex vitro rooted plantlets were hardened under different regimes of temperature and humidity in a greenhouse. The hardened plantlets were transferred to soil in polybags. More than 95% plants survived in field conditions. Total dry biomass harvested per year was 2800 kg/acre.     

Optimization of multiple shoot induction and plant regeneration in Indian barley (Hordeum vulgare) cultivars using mature embryos

Barley is the fourth most important crop in the world. Development of a regeneration system using immature embryos is both time consuming and laborious. The present study was initiated with a view to develop a regeneration system in six genotypes of Indian barley (Hordeum vulgare) cultivars as a prerequisite to transformation. The mature embryos were excised from seeds and cultured on MS medium supplemented with high and low concentrations of cytokinins and auxins respectively. The MS medium containing 3 mg/L N⁶-benzylaminopurine (BA) and 0.5 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) was found to be the most effective for multiple shoot formation in HOR7231 cultivar that could produce 12 shoots per explant. The other cultivars HOR4409 and HOR3844 produced a minimum number of adventitious shoots (1.33 and 1.67 respectively) on MS medium supplemented with 1 mg/L BA and 0.3 mg/L 2,4-D. The elongated shoots were separated and successfully rooted on MS medium containing 1 mg/L indole-3-acetic acid (IAA). The response of different barley cultivars was found to be varying with respect to multiple shoot production. This is the first report of multiple shoot induction and plantlet regeneration in Indian cultivar of barley which would be useful for genetic transformation.     

Identification of new potent inhibitor of aldose reductase from Ocimum basilicum

Recent efforts to develop cure for chronic diabetic complications have led to the discovery of potent inhibitors against aldose reductase (AKR1B1, EC 1.1.1.21) whose role in diabetes is well-evident. In the present work, two new natural products were isolated from the ariel part of Ocimum basilicum; 7-(3-hydroxypropyl)-3-methyl-8-β-O-d-glucoside-2H-chromen-2-one (1) and E-4-(6′-hydroxyhex-3′-en-1-yl)phenyl propionate (2) and confirmed their structures with different spectroscopic techniques including NMR spectroscopy etc. The isolated compounds (1, 2) were evaluated for in vitro inhibitory activity against aldose reductase (AKR1B1) and aldehyde reductase (AKR1A1). The natural product (1) showed better inhibitory activity for AKR1B1 with IC₅₀ value of 2.095 ± 0.77 µM compare to standard sorbinil (IC₅₀ = 3.14 ± 0.02 µM). Moreover, the compound (1) also showed multifolds higher activity (IC₅₀ = 0.783 ± 0.07 µM) against AKR1A1 as compared to standard valproic acid (IC₅₀ = 57.4 ± 0.89 µM). However, the natural product (2) showed slightly lower activity for AKR1B1 (IC₅₀ = 4.324 ± 1.25 µM). Moreover, the molecular docking studies of the potent inhibitors were also performed to identify the putative binding modes within the active site of aldose/aldehyde reductases.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of northern red oak (<Emphasis Type="Italic">Quercus rubra</Emphasis> L.)

In vitro propagation of northern red oak (Quercus rubra) shoots was successful from cotyledonary node explants excised from 8-wk-old in vitro grown seedlings. Initially, four shoots per explant were obtained on Murashige and Skoog (MS) medium supplemented with 4.4 μM 6-benzylaminopurine (BA), 0.45 μM thidiazuron (TDZ), and 500 mg l⁻¹ casein hydrolysate (CH) with a regeneration frequency of 64.7% after 3 wk. Subculturing explants (after harvesting shoots) to fresh treatment medium significantly increased shoot bud regeneration (16.6 buds per explant), but the buds failed to develop into shoots. A higher percentage (73.3%) of the explants regenerated four shoots per explant on woody plant medium (WPM) supplemented with 4.4 μM BA, 0.29 μM gibberellic acid (GA₃), and 500 mg l⁻¹ CH after 3 wk. Explants subcultured to fresh treatment medium after harvesting shoots significantly increased shoot regeneration (16 shoots per explant). Shoot elongation was achieved (4 cm) when shoots were excised and cultured on WPM supplemented with 0.44 μM BA and 0.29 μM GA₃. In vitro regenerated shoots were rooted on WPM supplemented with 4.9 μM indole-3-butyric acid. A higher percentage regeneration response and shoot numbers per explant were recorded on WPM supplemented with BA and GA₃, than on MS medium containing BA and TDZ. Lower concentrations of BA and GA₃ were required for shoot elongation and prevention of shoot tip necrosis. Each cotyledonary node yielded approximately 20 shoots within 12 wk. Rooted plantlets were successfully acclimatized.     

Synthesis of alpha amylase inhibitors based on privileged indole scaffold

Twenty five derivatives of indole carbohydrazide (1–25) had been synthesized. These compounds were characterized using ¹H NMR and EI-MS, and further evaluated for their α-amylase inhibitory potential. The analogs (1–25) showed varying degree of α-amylase inhibitory potential.ranging between 9.28 and 599.0 µM when compared with standard acarbose having IC₅₀ value 8.78 ± 0.16 µM. Six analogs, 25 (IC₅₀ = 9.28 ± 0.153 µM), 22 (IC₅₀ = 9.79 ± 0.43 µM), 4 (IC₅₀ = 11.08 ± 0.357 µM), 1 (IC₅₀ = 12.65 ± 0.169 µM), 8 (IC₅₀ = 21.37 ± 0.07 µM) and 14 (IC₅₀ = 43.21 ± 0.14 µM) showed potent α-amylase inhibition as compared to the standard acarbose (IC₅₀ = 8.78 ± 0.16 µM). All other analogs displayed good to moderate inhibitory potential. Structure-activity relationship was established through the interaction of the active compounds with enzyme active site with the help of docking studies.     

In vitro induction of tetraploid <Emphasis Type="Italic">Ziziphus jujuba</Emphasis> Mill. var. <Emphasis Type="Italic">spinosa</Emphasis> plants from leaf explants

The genetic manipulation of Capsicum has been unsuccessful, and a large bottleneck to transferring the desired genes is due to the difficulty in regenerating whole plants through tissue culture because of its highly recalcitrant and high genotype specificity. This study aimed to investigate and establish rapid shoot regeneration from the proximal ends of the leaves of Capsicum frutescens KT-OC and BOX-RUB varieties. A maximum of 8–10 shoot buds were obtained from the margins of the proximal portion of a cotyledonary leaf explant of C. frutescens variety KT-OC on medium I containing 44.44 µM 6-benzylaminopurine (BA), 5.71 µM indole-3-acetic acid (IAA), 10 µM silver nitrate (AgNO₃) and 1.98 mg L^?1 2-(N-morpholine) ethane sulphonic acid within 4 weeks of incubation, of which 60% of explants responded in terms of shoot buds. Petiole explants (40%) cultured on the same medium produced 2–4 shoots per explant from the distal portion. The cut portions of the cotyledonary leaf proximal portions responded well to shoot bud formation in the presence of 22.20 µM BA and 14.68 µM phenyl acetic acid (PAA), wherein 100% of explants responded in terms of shoot bud formation, with an average of 10?±?1.7 and 8?±?1.9 shoot buds per explant in KT-OC and BOX-RUB varieties, respectively. The differentiated shoots grew well and proliferated in the presence of 14.68 µM PAA?+?22.20 µM BA and 10 µM AgNO₃. Shoot elongation was obtained in presence of 1.44 µM gibberellic acid (GA₃) and 10 µM AgNO₃. These shoots were rooted on plant growth regulator-free half-strength MS medium and upon hardening; field survival rate was 70%. This reproducible regeneration method for C. frutescens, especially the Indian high pungent variety, from proximal portion of cotyledonary leaf and petiole explants, can be used for biotechnological improvement.     

New 1,3,4-oxadiazole/oxime hybrids: Design,synthesis, anti-inflammatory,COX inhibitory activities and ulcerogenic liability

A series of new 1,3,4-oxadiazole/oxime hybrids were synthesized and designed as potent COX inhibitors. The prepared compounds were evaluated for their anti-inflammatory, antioxidant and ulcerogenic activities. The results indicated that the prepared compounds exhibited remarkable anti-inflammatory activity with (69.60–109.60% of indomethacin activity) after 4 h. In vitro COX inhibitory assay showed that compounds 6d and 7h are potent COX inhibitors with IC₅₀ of (1.10–0.94) and (2.30–5.00) µM on both COX-1 and COX-2 respectively. Compound 7h was found to inhibit both COXs non-competitively with K_i values of 73 µM and 89 µM. Most of the tested compounds showed ulcer-free stomachs compared to indomethacin.     

Effects of electromagnetic fields exposure on rapid micropropagation of beach plum (Prunus maritima)

In this research, electromagnetic fields of strength gradient 48–115 kA/m were applied, and it was found that the increase in the field strength stimulated the regeneration and growth in the explants. The effect of a 10-min treatment of explants with a strength of 97 kA/m on the regeneration and growth was highest, and the number of the sprouts induced (17.2 ± 1.74) was 2.3 times greater than that in the control group (7.40 ± 0.51). The fresh weight, dry weight and height of the sprouts from the explants treated with a strength of 97 kA/m, in culture medium 2 (MS + 2.0 mg/L ZT + 0.2 mg/L IBA + 30 mg/L Vc), were higher than those in culture medium 1 (MS + 2.0 mg/L ZT + 0.2 mg/L NAA + 30 mg/L Vc) and the control group, and the mean number of the sprouts in culture medium 2 increased 5.2 times, but 3.1 times in culture medium 1. Root length of plantlets from explants treated with a strength of 97 kA/m and optimized rooting medium 1/2MS + 0.1 mg/L NAA + 0.1 mg/L IBA + 30 mg/L bovine serum albumin (BSA) was significantly larger, compared with the control. The average root length was 5.39 ± 0.68 cm. It was concluded that the optimization of culture medium and treatment of explants with a certain magnetic field strength could increase the number of regeneration sprouts and growth.     

Carbohydrazones as new class of carbonic anhydrase inhibitors: Synthesis,kinetics, and ligand docking studies

Discovery and development of carbonic anhydrase inhibitors is crucial for their clinical use as antiepileptic, diurectic and antiglaucoma agents. Keeping this in mind, we have synthesized carbohydrazones 1–27 and evaluated them for their in vitro carbonic anhydrase inhibitory potential. Out of twenty-seven compounds, compounds 1 (IC₅₀ = 1.33 ± 0.01 µM), 2 (IC₅₀ = 1.85 ± 0.24 µM), 3 (IC₅₀ = 1.37 ± 0.06 µM), and 9 (IC₅₀ = 1.46 ± 0.12 µM) have showed carbonic anhydrase inhibition better than the standard drug zonisamide (IC₅₀ = 1.86 ± 0.03 µM). Moreover, compounds 4 (IC₅₀ = 2.32 ± 0.04 µM), 5 (IC₅₀ = 3.96 ± 0.35 µM), 7 (IC₅₀ = 2.33 ± 0.02 µM), and 8 (IC₅₀ = 2.67 ± 0.01 µM) showed good inhibitory activity. Cheminformatic analysis has shown that compounds 1 and 2 possess lead-like properties. In addition, kinetic and molecular docking studies were also performed to investigate the binding interaction between carbohydrazones and carbonic anhydrase enzyme. This study has identified a novel and potent class of carbonic anhydrase inhibitors with the potential to be investigated further.     

Effects of nutrient media,different cytokinin types and their concentrations on in vitro multiplication of G × N15 (hybrid of almond × peach) vegetative rootstock

The objective of this research was to assess the effects of different media i.e. Murashige and Skoog (MS) and Quoirin and Lepoivre (QL), cytokinin type i.e. 6-Benzyladenin (BA) and 6-Benzylaminopurine (BAP) and cytokinin concentration on in vitro proliferation of the G × N15 rootstock. To evaluate the effects of different media and cytokinin type, two separate experiments were conducted as factorial based on completely randomized design, and single nodes were used as explants. The results showed that MS nutrient medium was found to be superior to QL nutrient medium. Regarding the interaction between media and growth regulators, the best interaction was found in MS medium supplemented with 1 mg l⁻¹ BAP resulting in 8.5 new micro shoots/explant while 7.75 shoots were observed in MS medium containing 1.25 mg l⁻¹ BA. The longest length of new micro-shoots (2.10 cm) was obtained in hormone-free MS medium. Findings of this study showed that there is a significant correlation between the hormone level and plantlet height and formed callus weight so that an increase in BAP and BA levels in both of MS and QL media resulted significantly in height decrease and callus weight increase. The results also suggest that the best and the worst plantlets in terms of quality were observed in hormone-free QL medium and MS medium supplemented with 1.25 mg l⁻¹, respectively. These results reflect the fact that the presence of high amounts of NH₄NO₃ and cytokinin especially BAP in culture medium triggered inhibitory effect on shoot growth.     

Somatic embryogenesis and plantlet regeneration from mature zygotic embryos of Manchurian ash (Fraxinus mandshurica Rupr.)

Using mature cotyledonary explants of Fraxinus mandshurica, an efficient plant regeneration system was developed via somatic embryogenesis. More than 67 % of mature cotyledons of zygotic embryos yielded 23–159 somatic embryos (SEs) per explant when incubated on medium consisting of half-strength Murashige and Skoog (MS) salts and vitamins (MS1/2) supplemented with 8.88 μM 6-benzyladenine (BA), 26.84 μM naphthaleneacetic acid (NAA), 75 g L^?1 sucrose, and 400 mg L^?1 casein hydrolysate (CH). Approximately, 82 % of induced SEs were observed on browning cotyledonary explants. Histological studies of cotyledon explants at various stages of somatic embryogenesis revealed that the SEs originated from single epidermal cells and developed to the globular, heart, torpedo, and cotyledonary stage embryos. Secondary somatic embryos (SSEs) formed on the surface of radicle tips of the SEs. Addition of low concentrations of NAA and 200–400 mg L^?1 CH to MS1/2 medium increased SSE induction. Cotyledonary SSEs were cultured on MS1/2 medium with 10 mM abscisic acid in the presence of light to promote maturation, and >92 % of mature SSEs were able to germinate with normal shoots. After 8 weeks in culture in the presence of light on medium with one-third of the MS macroelements as well as 0.06 μM NAA, >94 % of the germinated SSEs converted into plantlets. Plantlets acclimatized successfully to ex vitro conditions and developed normal phenotypes under field conditions.