Showing papers on "Kinetin published in 2021"

CALLUS FORMATION, PHENOLICS CONTENT AND RELATED ANTIOXIDANT ACTIVITIES IN TISSUE CULTURE OF A MEDICINAL PLANT COLOCYNTH (Citrullus colocynthis)

Abstract: Callus cultures from stems, leaves and roots of colocynth were initiated on MS media supplemented with various combinations of 2,4 dichlorophenoxyacetic acid (2,4-D) with kinetin (KIN) and benzyladenine (BA) with α-naphthaleneacetic acid (NAA). The highest percentage of callus formation frequency (98.9%) was obtained from stem explants grown on MS media supplemented with (1.0 mg/L) 2,4-D + (1.0 mg/L) KIN. The total phenolics and flavonoid content of the colocynth callus cultures were measured. The results showed that the MS medium supplemented with 6.0 mg/L 2,4-D + 2.0 mg/L KIN (MD3) gave the highest content of total phenolics (19.2 mg/100g d.w.) in leaf-derived calli. The highest content of flavonoids (47.3 mg/100g d.w.) was obtained in stem derived calli grown on the same medium (MD3). Antioxidant activities of extracts were determined using different assays, including DPPH radical scavenging activity, hydrogen peroxide (H2O2) scavenging activity and ferric reducing power. Leaf-derived calli cultured on MS medium + 2.0 mg/L 2,4-D + 1.0 mg/L KIN (MD1) showed the highest DPPH radical scavenging activity (85.3%). The highest percentage of H2O2 scavenging activity (61.4%) was detected in leaf explant-derived calli growing on MD1. The leaf-derived calli growing on (MD3) gave the highest ferric reducing power (22.3 µg/g d.w.), compared to the activities of stems, leaves and roots of in vitro grown seedlings (3.28, 12.9 and 2.85 µg/g d.w.), which were used as controls. On the basis of the current findings, we conclude that MS media supplemented with different combinations of 2,4-D and KIN yields higher phenolics, flavonoids contents and antioxidant activities than MS media supplemented with BA and NAA.

Short-term cold storage of encapsulated somatic embryos and retrieval of plantlets in grey orchid (Vanda tessellata (Roxb.) Hook. ex G.Don)

Abstract: Vanda tessellata (Roxb.) Hook. ex G.Don (grey orchid) is a horticultural and medicinally potent, and conservation prioritized orchid species. The present study investigates asymbiotic seed germination, callus induction, somatic embryogenesis, encapsulation of somatic embryos (SEs), and retrieval of plantlets of V. tessellata from the artificial seeds after short-term cold storage at − 4 °C for 12 months. Seeds cultured on full strength Murashige and Skoog’s (MS) medium containing 2.0 mg L−1 6-benzylaminopurine (BAP) had the highest frequency of asymbiotic seed germination (94%) as compared with half- and one-fourth strength MS media supplemented with various concentrations and combinations of 6-furfurylaminopurine (kinetin) and BAP. The protocorm like bodies (PLBs) gained dedifferentiation and proliferated into embryogenic calli on MS medium containing 2.0 mg L−1 BAP and 0.5 mg L−1 indole-3 acetic acid (IAA). Somatic embryos (SEs) were differentiated from the callus when cultured on MS medium in combination with 1.0 mg L−1 BAP and 0.5 mg L−1 IAA. The morpho-anatomical elucidations authenticated somatic embryogenesis and various developmental stages of SEs from embryogenic calli. Short-term cold storage of SEs was carried out by encapsulating with 2% sodium alginate and 100 mM calcium chloride and stored under dark at − 4 °C for 12 months to conserve this valuable germplasm. The stored artificial seeds were germinated (91%) on MS medium incorporated with additives and 0.5 mg L−1 each of BAP, kinetin, and IAA. Complete retrieval of plantlets was achieved in 8 week with formation of shoots (4.2 cm average length) and roots (2.5 cm). The well developed plantlets were acclimatized in the greenhouse with 90% survival rate. Vanda tessellata (grey orchid) is a horticultural and medicinally potent, and conservation prioritized orchid species. A reproducible protocol has been successfully established for asymbiotic seed germination, callus induction, somatic embryogenesis, encapsulation of somatic embryos, and retrieval of plantlets of V. tessellata from the artificial seeds after short-term cold storage at − 4 °C for 12 months.

The development of callus and cell suspension cultures of Sabah Snake Grass (Clinacanthus nutans) for the production of flavonoids and phenolics

Abstract: Clinacanthus nutans (Burm.f.) Lindau is a valuable medicinal plant that has gained interest as a side treatment for cancer in Southeast Asia. Phenolic and flavonoid compounds identified in this plant have been linked to anti-cancer properties. However, quantification of such metabolites in plants varies depending on cultivation methods and conditions resulting in inconsistent yield. This study aims to establish cell suspension culture of C. nutans, evaluate the accumulation of phenolics, flavonoids and to assess the antioxidant activity of extracts from in vitro cultures. Callus was induced from leaf explants of C. nutans and proliferated on Murashige & Skoog (MS) medium supplemented with different combinations of plant growth regulators (2,4-dichlorophenoxyacetic acid, 6-benzylaminopurine and kinetin). Assessment of growth kinetics for the suspension culture was performed. The total phenolic, flavonoid contents and antioxidant activity of the extracts were evaluated followed by high-performance liquid chromatography (HPLC) to detect selected flavonoids. MS medium supplemented with 0.25 mg/L 2, 4-D and 0.25 mg/L BAP was optimal for callus induction, proliferation and suspension cultures. The highest total phenolic content was obtained from suspension cells (55.35 mg GAE/g DW) whereas leaf showed the highest flavonoid content (25.13 mg QE/g DW). Leaf extract demonstrated the strongest antioxidant activity with the lowest IC50 value (117.42 μg/mL). HPLC analysis revealed the presence of catechin, luteolin, quercetin and kaempferol in the suspension cells and the leaf. The present study indicated that cell suspension cultures are able to accumulate higher phenolic compounds and possess all four selected flavonoids similar to the outdoor grown plant.

Cell Suspension Culture and In Vitro Screening for Drought Tolerance in Soybean Using Poly-Ethylene Glycol.

Abstract: Soybean (Glycine max (L) Merrill) is used in India mostly as a substantial fund of protein and oil, which makes the crop significantly important. Somaclonal variation has been researched as a base of additional variability for drought in soybean. In the present experiment calli/cell clumps/embryoids rose from immature and mature embryonic axis and cotyledons explants were exposed to different concentrations of polyethylene glycol (PEG6000). A discontinuous method proved to be superior as it permitted the calli/embryoids/cell clumps to regain their regeneration competence. A total of 64 (12.21%) plantlets of genotype JS335 and 78 (13.13%) of genotype JS93-05 were regenerated after four consequent subcultures on the selection medium with an effective lethal concentration of 20% PEG6000, and proliferated calli/embryoids/cell clumps were further subcultured on Murashige and Skoog regeneration medium supplemented with 0.5 mgL−1 each of α-napthalene acetic acid (NAA), 6-benzyladenine (BA) and Kinetin (Kn), 20.0 gL−1 sucrose and 7.5 gL−1 agar. Putative drought-tolerant plantlets were acquired from genotype JS93-05 (38) in more numbers compared to genotype JS335 (26). Random decamer primers confirmed the presence of variability between mother plants and regenerated plants from both the genotypes. Since these plantlets recovered from tolerant calli/embryoids/cell clumps selected from the medium supplemented with PEG6000, the possibility exists that these plants may prove to be tolerant against drought stress.

Salicylic acid in combination with kinetin or calcium ameliorates heavy metal stress in Phaseolus vulgaris plant

Abstract: It has been well documented that phytohormones plays a pivotal role in combating ill effects of various abiotic stresses in crop plants. Out of various phytohormones, worldwide researcher has recognized salicylic acid as potent stress alleviator through modulation in plant metabolic processes. Therefore, present study was designed with an aim to dissect out the role of salicylic acid (through seed) mediated amelioration of heavy metal stress in the presence and absence of kinetin or calcium in Phaseolus vulgaris plants. Seeds were soaked in salicylic acid (0.1 mM) either alone or in combination with kinetin (30 ppm) or calcium chloride (40 mM) before sowing and then seedlings were exposed to toxic concentration of nickel (2.5 mM) and lead (0.5 mM). Salicylic acid alone and in combination with kinetin or calcium improved growth traits, photosynthetic pigment, carbohydrate contents, and nitrogenous constituents along with activities of carbonic anhydrase, nitrate reductase, catalase, peroxidase, and superoxide dismutase of Phaseolus vulgaris plants. However, nickel and/or lead showed oxidative damage through increased electrolyte leakage, malondialdehyde and reduced uptake of mineral ions. Moreover, plants raised from seeds soaked in salicylic acid in combination with kinetin or calcium showed enhanced activities of antioxidant enzymes and proline accumulation under nickel and/or lead stress in Phaseolus vulgaris plants. This study revealed the efficiency of pre-sowing seed soaked in salicylic acid either alone or in alternate combination with kinetin and calcium on neutralizing the toxic effect generated from nickel and/or lead in Phaseolus vulgaris plants and could be employed as sustainable agricultural technique in removal of nickel and lead stresses from plants.

Development of a Cell Suspension Culture System for Promoting Alkaloid and Vinca Alkaloid Biosynthesis Using Endophytic Fungi Isolated from Local Catharanthus roseus

Abstract: Cell and tissue cultures of Catharanthus roseus have been studied extensively as an alternative strategy to improve the production of valuable secondary metabolites. The purpose of this study was to produce C. roseus callus and suspension cell biomass of good quality and quantity to improve the total alkaloids and bis-indole alkaloids. The young stem derived-callus of C. roseus variety Quang Ninh (QN) was grown on MS medium supplemented with 1.5 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) plus 1.5 mg/L kinetin, and the growth rate increased by 67-fold after 20 days. The optimal conditions for maintaining the cell suspension culture were 150 mg/50 mL cell inoculum, a medium pH of 5.5 and a culture temperature of 25 °C. The low alkaloid content in the culture was compensated for by using endophytic fungi isolated from local C. roseus. Cell extracts of endophytic fungi—identified as Fusarium solani RN1 and Chaetomium funicola RN3—were found to significantly promote alkaloid accumulation. This elicitation also stimulated the accumulation of a tested bis-indole alkaloid, vinblastine. The findings are important for investigating the effects of fungal elicitors on the biosynthesis of vinblastine and vincristine, as well as other terpenoid indole alkaloids (TIAs), in C. roseus QN cell suspension cultures.

Exploring potential of copper and silver nano particles to establish efficient callogenesis and regeneration system for wheat (Triticum aestivum L.).

Abstract: In vitro recalcitrance of wheat to regeneration is the major bottleneck for its improvement through callus-based genetic transformation. Nanotechnology is one of the most dynamic areas of research, which can transform agriculture and biotechnology to ensure food security on sustainable basis. Present study was designed to investigate effects of CuSO4, AgNO3 and their nanoparticles on tissue culture responses of mature embryo culture of wheat genotypes (AS-2002 and Wafaq-2001). Initially, MS-based callus induction and regeneration medium were optimized for both genotypes using various concentrations of auxin (2,4-D, IAA) and cytokinins (BAP, kinetin). The genotypes differed for embryogenic callus induction and regeneration potential. Genotype AS-2002 yielded maximum embryogenic calli in response to 3.0 mg/l 2,4-D, whereas Wafaq-2001 offered the highest embryogenic calli against 3.5 mg/l 2,4-D supplemented in the induction medium. Genotype AS-2002 showed maximum regeneration (59.33%) in response to regeneration protocol comprising 0.5 mg/l IAA, 0.3 mg/l BAP and 1.0 mg/l Kin, while Wafaq-2001 performed best in response to 0.5 mg/l IAA, 0.3 mg/l BAP and 1.5 mg/l Kin with 55.33% regeneration efficiency. The same optimized basal induction and regeneration medium for both genotypes were further used to study effects of CuSO4, AgNO3 and their nano-particles employing independent experiments. The optimized induction medium fortified with various concentrations of CuSO4 or CuNPs confirmed significant effects on frequency of embryogenic callus. Addition of either 0.020 mg/l or 0.025 mg/l CuSO4, or 0.015 mg/l CNPs showed comparable results for embryogenic callus induction and were statistically at par with embryogenic callus induction of 74.00%, 75.67% and 76.83%, respectively. Significantly higher regeneration was achieved from MS-based regeneration medium supplemented with 0.015 mg/l or 0.020 mg/l CuNPs than standard 0.025 mg/l CuSO4. In another study, the basal induction and regeneration medium were fortified with AgNO3 or AgNPs ranging from 1 to 7 mg/l along with basal regeneration media devoid of AgNO3 or AgNPs (control). The maximum embryogenic calli were witnessed from medium fortified with 3.0 mg/l or 4.0 mg/l AgNPs compared with control and rest of the treatments. The standardized regeneration medium fortified with 5.0 mg/l AgNO3 or 3.0 mg/l AgNPs showed pronounced effect on regeneration of wheat genotypes and offered maximum regeneration compared with control. The individual and combined effect of Cu and Ag nanoparticles along with control (basal regeneration media of each genotype) was also tested. Surprisingly, co-application of metallic NPs showed a significant increase in embryogenic callus formation of genotypes. Induction medium supplemented with 0.015 mg/l CuNPs + 4.0 mg/l AgNPs or 0.020 mg/l CuNPs + 2.0 mg/l AgNPs showed splendid results compared to control and other combination of Cu and Ag nanoparticles. The maximum regeneration was achieved by co-application of 0.015 mg/l CuNP and 4.0 mg/l AgNPs with 21% increment of regeneration over control. It is revealed that CuNPs and AgNPs are potential candidate to augment somatic embryogenesis and regeneration of mature embryo explants of wheat.

Modulatory and Toxicological Perspectives on the Effects of the Small Molecule Kinetin.

Abstract: Plant hormones are small regulatory molecules that exert pharmacological actions in mammalian cells such as anti-oxidative and pro-metabolic effects. Kinetin belongs to the group of plant hormones cytokinin and has been associated with modulatory functions in mammalian cells. The mammalian adenosine receptor (A2a-R) is known to modulate multiple physiological responses in animal cells. Here, we describe that kinetin binds to the adenosine receptor (A2a-R) through the Asn253 residue in an adenosine dependent manner. To harness the beneficial effects of kinetin for future human use, we assess its acute toxicity by analyzing different biochemical and histological markers in rats. Kinetin at a dose below 1 mg/kg had no adverse effects on the serum level of glucose or on the activity of serum alanine transaminase (ALT) or aspartate aminotransferase (AST) enzymes in the kinetin treated rats. Whereas, creatinine levels increased after a kinetin treatment at a dose of 0.5 mg/kg. Furthermore, 5 mg/kg treated kinetin rats showed normal renal corpuscles, but a mild degeneration was observed in the renal glomeruli and renal tubules, as well as few degenerated hepatocytes were also observed in the liver. Kinetin doses below 5 mg/kg did not show any localized toxicity in the liver and kidney tissues. In addition to unraveling the binding interaction between kinetin and A2a-R, our findings suggest safe dose limits for the future use of kinetin as a therapeutic and modulatory agent against various pathophysiological conditions.

meta-Topolin-induced enhanced biomass production via direct and indirect regeneration, synthetic seed production, and genetic fidelity assessment of Bacopa monnieri (L.) Pennell, a memory-booster plant

Abstract: The present study reports enhanced in vitro mass propagation, synthetic seed production and its regeneration, acclimatization, and genetic fidelity assessment of in vitro regenerants of Bacopa monnieri (L.) Pennell; a medicinal plant renowned for its memory-enhancing property. For the first time, meta-Topolin (mT) as well as N6-benzyladenine (BA) and kinetin at their variable concentrations (0.5, 1. 1.5, 2 and 2.5 mg/l) were supplemented individually in Murashige and Skoog (Physiol Plant 15:473–497, 1962) (MS) basal medium to induce multiple shoots from shoot tip explants, initially collected from two-month-old vegetatively propagated plants. The best result in terms of multiplication (~ 7 shoots/explant) and biomass accumulation (~ 451.3 mg fresh weight, ~ 104.3 mg dry weight) was obtained in MS medium supplemented with 1 mg/l mT. For rooting of in vitro shoots, supplementation of 1.5 mg/l IAA and 0.5 mg/l IBA in MS media resulted in induction of the maximum number (~ 20) and highest length (~ 5.5 cm) of roots, respectively. Most efficient indirect regeneration and biomass production from green and hard basal calli (induced spontaneously in BA-supplemented media) was recorded in a simple MS medium. Synthetic seeds were produced from in vitro shoot tips using 2.5% sodium alginate and 75 mM calcium chloride solutions and were successfully regenerated in half-strength MS medium. Six-week-old in vitro regenerants were transferred to different substrates for acclimatization, among which cocopeat was found to be the best exhibiting the highest survival and growth in external conditions. Finally, molecular marker-(ISSR) and phenology-based genetic fidelity assessment of the in vitro regenerants exhibited that the in vitro regenerants were true-to-type in nature within themselves as well as with the mother plant.

Evaluation of antioxidant potential and reducing power of callus induced from leaves of asystasia gangetica (l.) t.anderson

Abstract: Objective: To evaluate the bioactive molecules and antioxidant potential of callus induced from leaves of Asystasia gangetica. Methods: In this report, the leaves of A.gangetica (AG) were incubated with Murashige and Skoog (MS) medium supplemented with combinations of auxins and cytokinins for callus induction. The qualitative estimation of bioactive molecules like flavonoids, phenolics, tannins and their antioxidant potential were investigated. The ability of radical scavenging activity and reducing power of methanolic, ethanolic and aqueous extract using DPPH, FRAP and Phopshomlybdate assay were carried out. Results: Callus was induced on MS medium supplemented with various concentration and combination of auxins and cytokinins.Maximum percentage of callusing was seen on media supplemented with 2,4-Dichlorophenoxyaceticacid 5mg/L or combination of Kinetin 2mg/L and 2mg/L Napthaleneaceticacid.The total phenolic content, flavonoids and tannins in callus were estimated in various solvents. Further, the callus showed the FRAP values of 17.67 ± 0.0, 17.30 ± 1.830 and 23.81 ± 0.945 µg AAE / mg extract for methanolic, ethanolic and aqueous extract respectively. Methanolic extract showed highest DPPH scavenging activity and reducing abilty. Conclusion: A.gangetica callus had substantial amount of bioactive molecules exhibiting potent antioxidant activity and reducing ability. Development of appropriate strategies for enhancing the bioactive molecules in callus could have far-reaching implications for isolation of novel antioxidant molecules for human health.

Optimization of efficient direct organogenesis protocol for Punica granatum L. cv. Kandhari Kabuli from mature leaf explants

Abstract: Punica granatum L. is an important horticultural fruit crop with high medicinal and economic value. Its rising commercial demand necessitates the production of high-quality planting material. Here, we describe an efficient protocol for direct organogenesis in Punica granatum L. cv. Kandhari Kabuli from mature leaf explant. The optimized sterilization procedure for explant includes sequential treatment with 70% ethanol (0.75 min), 0.2% Bavistin (15 min), and 0.5% sodium hypochlorite (2 min), which resulted in 83% axenic cultures. The accumulation of phenolics was effectively controlled by subculturing of leaf explants three to four times at a regular interval of 24 h. The organogenic capability of leaf segments was investigated on full-strength Murashige and Skoog (MS) medium supplemented with different plant growth regulators (PGRs), including the cytokinins 6-benzylaminopurine (BAP) and thidiazuron (TDZ) alone or in combination with α-naphthaleneacetic acid (NAA). BAP promoted the greatest morphogenic response as compared to that from TDZ. However, the greatest frequency of shoot induction (43%) was achieved on MS medium supplemented with 10 μM BAP and 2.5 μM NAA under dark incubation for 2 wk. Furthermore, micro-shoot proliferation and elongation were achieved on multiplication medium consisting of MS medium supplemented with 9.0 μM BAP, 2.5 μM Kinetin (KN), and 0.5 μM gibberellic acid (GA3) up to the third subculturing. However, further subculturing resulted in vitrification. A hormone-free medium containing 300 mg L−1 activated charcoal (AC) was found to be effective to reduce vitrification and promote shoot multiplication. In vitro rooting was carried out on the ½ MS basal medium containing 500 mg L−1 AC using shoots from different subculture passages. Successive subculturing tends to have a positive effect on in vitro rooting and increased rooting up to 70.62%. Well-rooted plantlets were acclimatized successfully in the small plastic pots containing sterilized sand and later shifted to the soil. This optimized protocol can be routinely used for rapid large-scale propagation of pomegranate and is a prerequisite for trait improvement via genetic engineering.

Effect of Culture Media and Plant Growth Regulators on Shoot Proliferation and Rooting of Internode Explants from Moroccan Native Almond (Prunus dulcis Mill.) Genotypes

Abstract: In this study, several methods have been used to facilitate shoot formation from nodal explants of local almond ecotypes known as “Beldi” grown in Eastern Morocco. Nodal segments of divers old local genotypes were cultured on various concentrations of auxin (indole-3-butyric acid (IBA)) and cytokinins (6-benzyl-aminopurine (BAP), thidiazuron (TDZ), and kinetin (KIN)) added to two different media (Murashige and Skoog (MS) and Heller medium). The results showed that TDZ was more effective than the other tested hormones for in vitro proliferation of the “Beldi” ecotype. TDZ at the concentration of 1 mg/L significantly improved the nodal shoot proliferation rate, with the highest percentage (63.6% ± 0.63) and number of regenerated shoots (13 ± 0.54) recorded for S1 genotype inoculated on MS medium, while the most significant rooting rate (60.41% ± 0.81) of proliferated shoots and number of roots per shoot (7.3 ± 1.36) were achieved for S2 genotype on 1 mg/L of IBA incorporated to a half-strength MS medium. With 80% of plantlets survival, the rooted shoots were successfully adapted to the in vivo conditions and were grown vigorously in the greenhouse without any morphological abnormalities.

Variables Affecting Shoot Growth and Plantlet Recovery in Tissue Cultures of Drug-Type Cannabis sativa L.

Abstract: Tissue culture approaches are widely used in crop plants for the purposes of micropropagation, regeneration of plants through organogenesis, obtaining pathogen-free plantlets from meristem culture, and developing genetically modified plants. In this research, we evaluated variables that can influence the success of shoot growth and plantlet production in tissue cultures of drug-type Cannabis sativa L. (marijuana). Various sterilization methods were tested to ensure shoot development from nodal explants by limiting the frequency of contaminating endophytes, which otherwise caused the death of explants. Seven commercially grown tetrahydrocannabinol (THC)-containing cannabis genotypes (strains) showed significant differences in response to shoot growth from meristems and nodal explants on Murashige and Skoog (MS) medium containing thidiazuron (1 μM) and naphthaleneacetic acid (0.5 μM) plus 1% activated charcoal. The effect of Driver and Kuniyuki Walnut (DKW) or MS basal salts in media on shoot length and leaf numbers from nodal explants was compared and showed genotype dependency with regard to the growth response. To obtain rooted plantlets, shoots from meristems and nodal explants of genotype Moby Dick were evaluated for rooting, following the addition of sodium metasilicate, silver nitrate, indole-3-butyric acid (IBA), kinetin, or 2,4-D. Sodium metasilicate improved the visual appearance of the foliage and improved the rate of rooting. Silver nitrate also promoted rooting. Following acclimatization, plantlet survival in hydroponic culture, peat plugs, and rockwool substrate was 57, 76, and 83%, respectively. The development of plantlets from meristems is described for the first time in C. sativa and has potential for obtaining pathogen-free plants. The callogenesis response of leaf explants of 11 genotypes on MS medium without activated charcoal was 35% to 100%, depending on the genotype; organogenesis was not observed. The success in recovery of plantlets from meristems and nodal explants is influenced by cannabis genotype, degree of endophytic contamination of the explants, and frequency of rooting. The procedures described here have potential applications for research and commercial utility to obtain plantlets in stage 1 tissue cultures of C. sativa.

Arnica montana Cell Culture Establishment, and Assessment of Its Cytotoxic, Antibacterial, α-Amylase Inhibitor, and Antioxidant In Vitro Bioactivities.

Abstract: Arnica montana cell suspension culture could be a sustainable source of a vegetal material producer of secondary metabolites (SMs) possessing biological effects. Different plant growth regulator concentrations (0-5 mg/L) were tested in foliar explants to induce a callus that was used to establish a cell suspension culture. Growth kinetics was carried out for 30 days. A methanolic extract obtained from biomass harvested at 30 days of growth kinetics was fractionated, and three fractions were tested for bioactivities. We induced a callus with 1 mg/L of picloram and 0.5 mg/L of kinetin in foliar explants, which allowed for the establishment of a cell suspension culture, and the latter had the highest total SMs contents at day 30. Three fractions showed differences in total SMs contents, with the highest values per gram as follows: 270 mg gallic acid equivalent for total phenolic content, 200 mg quercetin equivalent for total flavonoid content, 83 mg verbascoside equivalent for total phenolic acid content, and 396 mg parthenolide equivalent for total sesquiterpene lactone content. The best bioactivities were 2-6 µg/mL for the 50% inhibition of 2,2-diphenyl-1-picrylhydrazyl radical, 30% cellular viability of lymphoma cells at 40 µg/mL, 17% inhibition against Escherichia coli and Staphylococcus aureus at 8 µg/disk, and α-amylase inhibition at 12% with 10 µg/mL. The total SMs contents were correlated with bioactivities.

Improved protocol for micropropagation of genetically uniform plants of commercially important cardamom (Elettaria cardamomum Maton)

Abstract: Cardamom (Elettaria cardamomum Maton) is a highly valuable commercial spice crop, originating from the Western Ghats of southern India. It has importance both as a spice and for its medicinal properties. An efficient and reliable micropropagation protocol using shoot tips as explants is presented which can be utilized for development of elite planting material. Growth patterns of cardamom shoots were analyzed on 45 media treatments and best shoot proliferation was recorded on Murashige and Skoog (MS) medium supplemented with 4.4 μM 6-benzylaminopurine (BAP) and 2.32 μM kinetin (Kn) with 5.83 shoots regenerated per explant. The longest shoots (6 cm) were observed on 0.44 μM BAP and 2.32 μM Kn containing medium. Regenerated shoots rooted well on full strength MS basal medium, generating 3.50 roots per explant with an average root length of 4.33 cm within 4 wk. Successful hardening with a field survival percentage of 80% was achieved after acclimatization of well-developed plantlets. Genetic stability of the micropropagated plants was confirmed using 13 ISSR markers. This study reports an improved micropropagation protocol for cardamom with an enhanced rate of shoot multiplication leading to the production of healthy shoots which can be easily rooted and successfully transferred to the field.

6-Benzylamino purine outperforms Kinetin and Thidiazuron in ameliorating flower longevity in Calendula officinalis L. by orchestrating physiological and biochemical responses

Abstract: In view of extending the relatively brief postharvest life of flowers by a range of technologies, the present study elucidates the implication of 6-benzylamino purine (BAP), kinetin (KN) and thidiazuron (TDZ) on postharvest performance and flower longevity of isolated flowers of Calendula officinalis . BAP and KN belong to adenine group cytokinins while as TDZ is a diphenyl urea compound having cytokinin like activity. The harvested flowers were supplemented with BAP, KN and TDZ at various concentrations viz., 25, 50, 75 and 100 µM at one day before anthesis (cup shaped) stage. The control was designated by a distinct set of flowers held in distilled water (DW). Our findings revealed substantial enhancement in flower longevity by application of various growth regulators as compared to the control. Vase solutions containing BAP and KN at 50 µM and TDZ at 75 µM (individually) were most effective in improving the longevity of cut Calendula flowers. Improvement in flower longevity was primarily associated with high membrane stability index (MSI), upregulated activities of various antioxidant enzymes viz., catalase (CAT), superoxide dismutase (SOD) and ascorbate peroxidase (APX), besides an attenuated lipoxygenase (LOX) activity in the petals. As compared to control, the treated flowers exhibited higher values of soluble proteins, total phenols and total sugars, besides lower α-amino acid content in the petal tissues. However, BAP outplayed TDZ and KN in improving the flower longevity of Calendula officinalis by maintaining higher physiological and biochemical stability in petals.

Production of Virus-Free Garlic Plants through Somatic Embryogenesis

Abstract: The present study was conducted to establish a protocol for the regeneration of virus-free garlic plants through somatic embryogenesis of two Croatian garlic ecotypes. Basal parts of cloves from mother plants were cultured on a full Murashige and Skoog (MS) or modified MS medium (¼ of KNO3 and NH4NO3 and 2xMgSO4) containing 0.1 mg L−1 2,4-dichlorophenoxyacetic acid (2,4-D) or 1 mg L−1 2,4-D + 0.5 mg L−1 kinetin (Kin) and representing four different treatments. Plants were regenerated in MS medium containing 0.1 mg L−1 2,4-D and rooted in a medium containing 0.05 mg L−1 1-naphthaleneacetic acid (NAA) + 0.005 mg L−1 6-(γ,γ-dimethylallylamino)purine (2iP). The presence of viruses (i.e., sanitary status) of the mother plants and regenerants was checked by enzyme-linked immunosorbent assay (ELISA) and reverse transcription-polymerase chain reaction (RT-PCR). The mother plants were infected with onion yellow dwarf virus (OYDV) and leek yellow stripe virus (LYSV). In addition, the presence of garlic common latent virus (GCLV) was confirmed in four mother plants. Embryogenic callus developed in all four treatments with success ranging from 55% to 81% depending on treatment and ecotype. Plant conversion was significantly higher in somatic embryos developed in media containing 0.1 mg L−1 2,4-D than those developed in media containing 1 mg L−1 2,4-D + 0.5 mg L−1 Kin. Virus elimination success ranged from 13.3% up to 62.5% depending on garlic ecotype and treatment. The overall rate of virus elimination by somatic embryogenesis for both treatments and ecotypes were 20.7%, 22.9%, and 30.5% for OYDV, GCLV, and LYSV, respectively. Based on these results, somatic embryogenesis has been shown to be equally or more successful in eliminating garlic viruses compared to other in vitro methods.

Effect of Growth Regulators on In Vitro Micropropagation of Potato ( Solanum tuberosum L.) Gudiene and Belete Varieties from Ethiopia.

Abstract: Aim Potato (Solanum tuberosum L) is one of the important crops in Ethiopia which has a crucial role in nutritional security, poverty alleviation, and income generation The aim of the present investigation is to develop an efficient in vitro propagation protocol for Belete and Gudiene potato varieties by using lateral bud as explants Materials and methods Shoot initiation was achieved by inoculating buds on full-strength MS Murashige and Skoog medium (MS) fortified with variable concentrations of BAP and NAA Basal MS was used as control throughout the experiment Results Results of our study showed that best shoot initiation was obtained on MS medium supplemented with 15 mg/l BAP + 30 mg/l NAA for Gudiene variety, whereas 10 mg/l BAP and 20 mg/l NAA produced more shoots in Belete variety The initiated shoots increased two- to three-fold upon subculture on the MS medium fortified with varying concentrations of BAP and Kinetin The highest numbers of multiple shoots were obtained in the MS medium containing 25 mg/l Kinetin The combined effect of BAP and Kinetin did not produce any additional positive effect for shoot multiplication Rooting percentage and number of roots/shoot were found best on the MS medium fortified with 10 mg/l IBA + 05 IAA Conclusions The variety Gudiene was found best for shoot initiation and root formation, while Belete variety proved its superiority for multiple shoot formation A total number of 8266% of plantlets were acclimatized under field conditions This work indicates the practical applicability of plant tissue culture using lateral bud as explants is effective for micropropagation of potato in vitro

Somatic Embryogenesis and Plant Regeneration in Viola canescens Wall. Ex. Roxb.: An Endangered Himalayan Herb

Abstract: Viola canescens Wall. ex. Roxb. is an important but threatened medicinal herb found at 1500-2400 m above mean sea level in the Himalayas. Overexploitation and habitat preference have put the plant under serious threat. Thus, the present study was undertaken to develop an efficient protocol for in vitro propagation via somatic embryogenesis. The results revealed that plant can be regenerated successfully through somatic embryogenesis using leaf derived calli. Regular subculturing of calli on Murashige and Skoog (MS) medium with 2,4-dichlorophenoxyacetic acid (2,4-D)/indole-3-butyric acid (IBA)/kinetin (Kn) and varying combinations of 2,4-D+Kn induced somatic embryogenesis. The maximum average number of somatic embryos (SE) (19.15 ± 2.66) was induced on the medium with 0.15 + 0.05 mg L-1 of 2,4-D and Kn, respectively, and this medium was used as a control. To enhance somatic embryo induction, the control MS medium was supplemented with l-glutamine (200-400 mg L-1) and casein hydrolysate (1-4%). The maximum average number of SE (27.66 ± 2.67) and average mature SE (13.16 ± 3.48) were recorded on the medium having 2 % l-glutamine and 50 mg L-1 casein hydrolysate. The induced SE were asynchronous, so, to foster their maturation, the culture medium (free from growth regulators) was supplemented with abscisic acid (ABA) and silver nitrate (AgNO3). The maximum average number (35.96 ± 3.68) of mature SE was noticed on MS medium supplemented with 1.5 mg L-1 ABA. Mature embryos had two well-developed cotyledons and an elongated hypocotyl root axis. The development of SE into plantlets was significant for embryos matured on the medium with AgNO3 and ABA, with 86.67% and 83.33% conversion on the medium with 0.20 mg L-1 6-benzylaminopurine (BAP). The plantlets thus produced acclimatized in a growth chamber before being transferred to the field, which showed 89.89% survival. The plants were morphologically similar to the mother plant with successful flowering.

A regeneration system using cotyledons and cotyledonary node explants of Toona ciliata

Abstract: We used the cotyledons and cotyledonary nodes of Toona ciliata (Chinese mahogany) as explants to examine callus and adventitious shoot induction when exposed to different ratios of hormones. We also investigated the effects of seedling age, inoculation method, and genotype on the efficient regeneration of T. ciliata. The results showed that different genotypes exhibited significantly different callus induction efficiency. The cotyledons and cotyledonary nodes of 20-day seedlings inoculated onto MS medium with 0.5 mg/L 6-benzylaminopurine (6-BA), 0.5 mg/L kinetin (KT) and 0.05 mg/L 1-naphthylacetic acid (NAA) achieved a greater regeneration rate than did other concentrations of cytokinin and auxin. The numbers of shoots per cotyledon and cotyledonary node explant were 7.33 and 6.67. The optimal inoculation method for cotyledons was that the distal end of the explants was placed in contact with the medium. The optimal adventitious shoot differentiation medium for cotyledon explants was MS medium containing 0.3 mg/L 6-BA and 0.2 mg/L NAA, producing a 3.4 cm height of shoot on average. This study established an efficient regeneration system for T. ciliata with cotyledons and cotyledonary nodes as explants.

Alterations in oleanolic acid and sterol content in marigold (Calendula officinalis) hairy root cultures in response to stimulation by selected phytohormones

Abstract: Hairy root cultures are an efficient tool for the biotechnological production of plant metabolites and a convenient experimental model for analyzing the effect of various compounds on plant metabolism. In contrast to many other types of in vitro plant cultures, hairy roots do not require an external supply of phytohormones to the medium. Consequently, plant growth regulators such as auxins and cytokinins are rarely used as elicitors in hairy root in vitro cultures; however, they can strongly influence plant defense responses. The aim of this study was to investigate the influence of two auxins: natural indole-3-acetic acid (IAA) and synthetic 1-naphthaleneacetic acid (NAA), as well as two cytokinins: natural kinetin and synthetic 6-benzylaminopurine (BAP) at a concentration of 0.75 mg/L on the metabolism of sterols and triterpenoids in Calendula officinalis hairy roots. Auxins prevented the accumulation of triterpenoid saponins (oleanolic acid glycosides), while cytokinin BAP increased their accumulation by 17% and their release into the culture medium by a factor of 10. Other cytokinins and kinetins increased the sterol levels by 17%, the level of stigmasterol by 15%, and the level of isofucosterol by 7 times.