Showing papers on "Kinetin published in 2018"

Modification of cadmium toxicity in pea seedlings by kinetin

Abstract: The effect of foliar application of kinetin on the growth and some physiological processes of pea plants growing in soil supplemented with 25 or 50μM Cd were studied. Cadmium treatment inhibited the growth rate, chlorophyll (Chl) content, net photosynthesis (PN), content of soluble sugars and free amino acids of either shoots or roots. The application of kinetin (kin) enhanced the growth rate, Chl content, PN, soluble sugars and free amino acids content of shoots and roots; dark respiration rate (RD), contents of soluble protein and proline were increased by cadmium treatment. The addition of kinetin to Cd-stressed plant reduced RD, soluble protein and proline content. Considerable variations in the content of Na+, K+, Ca2+ and Mg2+ were induced by Cd-treatments. Foliar application of kinetin exhibited a favorable effect on the accumulation of some ions and antagonized or ameliorated the inhibitory effect of Cd stress on some others.

Efficient Callus Induction and Regeneration in Selected Indica Rice

Abstract: An efficient callus induction and in vitro regeneration were developed using plant growth regulators, carbon sources, and basal media for three selected Malaysian wetland rice varieties (MR220, MR220-CL2, and MR232) and one upland variety (Bario). Effect of plant growth regulator (PGR) was carried out using four different concentrations (1–4 mg/L) of 2,4-D (2, 4-dichlorophenoxyacetic acid), and NAA (1-naphthalene acetic acid) (2.5, 5.0, 7.5, and 10 mg/L) with optimized 2,4-D. Effects of carbon sources (maltose and sorbitol), and basal media (MS, N6, and LS) were also studied with optimized PGR to maximize the induction of regenerable calli. This study found that all four varieties exhibited high frequency of callus induction on MS (Murashige and Skoog) medium that was supplemented with 3 mg/L 2,4-D and 30 g/L maltose. Callus induction frequencies in the cases of MR220, MR220-CL2, MR232, and Bario were found to be 76%, 94%, 85%, and 42% respectively. Morphological analysis through scanning electron microscopy (SEM) and histological analysis revealed the embryogenicity of the induced callus. In the regeneration study, it was observed that combination of 2 mg/L BAP (6-benzylaminopurine), 2 mg/L Kin (Kinetin) and 0.5 mg/L NAA supplemented MS medium has the potential to promote regeneration of selected indica rice varieties with higher regeneration percentage, i.e., 82% (MR220-CL2), 68% (both in MR220 and MR232), and 40% (Bario). The optimized conditions for callus formation and regeneration can be useful for biotechnological practices for the genetic improvement of Malaysian indica rice.

Imperative roles of halotolerant plant growth-promoting rhizobacteria and kinetin in improving salt tolerance and growth of black gram (Phaseolus mungo)

Abstract: The salinity stress causes a major threat for plant growth, yield, and biomass production. The present study was designed to assess the effect of exogenously applied kinetin and halotolerant plant growth-promoting rhizobacteria (H-PGPR) on alleviation of salt stress in black gram (Phaseolus mungo). A total of 15 rhizobacterial isolates obtained from a salt-affected area were analyzed for their capability to improve growth of P. mungo plants growing in greenhouse conditions. Out of the tested rhizobacteria, the two bacterial isolates which exhibited maximum growth potential were screened and their growth-promoting attributes were evaluated. The role of screened H-PGPR and/or kinetin (8 and 10 μM) was evaluated in P. mungo plants irrigated with three levels of brackish water (S1 = 3, S2 = 5, and S3 = 7 dSm−1) under field condition. Salt stress reduced transpiration rate, stomatal conductance, salt tolerance index, growth, leaf area, photosynthetic pigments, leaf relative water content (LRWC), biomass production, and seed yield in subjected plants. Conversely, the salinized plants treated with kinetin and/or H-PGPR exhibited improved levels of chlorophyll contents, LRWC, root growth, shoot growth, biomass production, and seed yield. The H-PGPR and/or kinetin supplementation also reduced electrolyte leakage in salt-stressed plants. Overall, the present findings will be of great value to recognize the mechanism of salt stress alleviation in P. mungo plants under the influence of H-PGPR and/or kinetin.

Effect of TIBA, fluridone and salicylic acid on somatic embryogenesis and endogenous hormone and sugar contents in the tree fern Cyathea delgadii Sternb.

Abstract: Somatic embryogenesis (SE) in the tree fern Cyathea delgadii was first described in 2015 and since then has been used to exploration of this phenomenon in cryptogamic plants. To deepen the knowledge about the hormonal control of SE, stipe explants were cultured on media supplemented with hormone biosynthesis and transport inhibitors (HBTIs). In the presence of 30 µM 2,3,5-triiodobenzoic acid (TIBA), or 40 µM fluridone or 125 µM salicylic acid (SA), somatic embryo production was totally inhibited. The quantitative analysis of the changes in endogenous hormone and sugar contents was conducted every 2 days within 10-day-long initial culture. The results showed that the concentrations of endogenous indole-3-acetic acid (IAA), abscisic acid (ABA), cytokinins (CKs) and soluble sugars were strongly modified either by TIBA and fluridone. Under their influence, the contents of cytokinins such as c-Z, c-ZR, t-Z, t-ZR, KinR were reduced to barely detectable levels. Treatment with SA results in the changes in endogenous IAA and sugar contents. It also modifies the IAA/CKs ratio; however, excluding the first 2 days of culture, the concentrations of ABA and cytokinins were kept on the control level. All HBTIs significantly increased the kinetin (Kin) content. Our work sheds new light on the relationships between the biosynthetic inhibitors and phytohormones and sugars in the process of early SE. It can be helpful to study the role of hormones in acquisition of embryogenic competence.

Cadmium toxicity and its amelioration by kinetin in tomato seedlings vis-à-vis ascorbate-glutathione cycle.

Abstract: The supplementation of plant hormones may enhance the tolerance capacity of plants against certain environmental stresses by increasing their physiological functioning and detoxification capacity. To answer the question that whether a phytohormone 'kinetin' (KN, 6-furfuylaminopurine), one of the artificial cytokinins could ameliorate the cadmium induced toxicity in tomato seedlings, the effect of KN was assessed in differentially cadmium (Cd1: 3mgkg-1 sand and Cd2: 9mgkg-1 sand) intoxicated tomato seedlings by estimating the changes in reactive oxygen species (ROS, viz. superoxide radical and H2O2 generation) and probable alteration in photosystem II photochemistry, ascorbate-glutathione cycle enzymes and their metabolites. Accumulation of Cd in tomato seedlings increased the production of ROS by negatively impacting PS II photochemistry (decrease in Fv/Fm (ϕP0), Ψ0, ϕE0 and PIABS and increase in energy fluxes per reaction centre: ABS/RC, ET0/RC, TR0/RC and DI0/RC) manifested by lowered fresh mass despite the accelerated activity of AsA-GSH cycle enzymes (viz. ascorbate peroxidase, APX; glutathione reductase, GR; dehydroascorbate reductase, DHAR and monodehydroascorbate reductase; MDHAR). Simultaneous application of kinetin (10μM) alleviated the negative effects on the fresh mass and lowered the ROS level by positively affecting PS II photochemistry and further rise in AsA-GSH cycle enzymes and their metabolites.

Enhanced production of phenolic acids in cell suspension culture of Salvia leriifolia Benth. using growth regulators and sucrose

Abstract: Salvia leriifolia Benth. (Lamiaceae) is an endangered medicinal plant with hypoglycemic, anti-inflammatory and analgesic properties. Many of the beneficial effects of Salvia spp. are attributed to the phenolic compounds. In the present study, an efficient procedure has been developed for establishment of cell suspension culture of S. leriifolia as a strategy to obtain an in vitro phenolic acids producing cell line for the first time. The effect of growth regulators and various concentrations of sucrose have been analyzed, to optimize biomass growth and phenolic acids production. The callus used for this purpose was obtained from leaves of 15-day-old in vitro seedlings, on Murashige and Skoog (MS) basal medium supplemented with different hormone balances including benzylaminopurine (BAP) and indole butyric acid (IBA); 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin (KIN); naphthaleneacetic acid (NAA) and BAP. Modified MS medium supplemented with 5 mg/L BAP and 5 mg/L NAA was the optimal condition for callus formation with the highest induction rate (100%), the best callus growth and the highest phenolic acids content. No callus induction was observed in combinations of IBA and BAP. Cell suspension cultures were established by transferring 0.5 g of callus to 30 mL liquid MS medium supplemented with 5 mg/L BAP and 5 mg/L NAA. Dynamics of phenolic acids production has been investigated during the growth cycle of the suspension cultures. The maximum content of caffeic acid and salvianolic acid B were observed on the 15th day of the cultivation cycle while the highest amount of rosmarinic acid was observed on the first day. In response to various sucrose concentrations, cell cultures with 40 g/L sucrose not only produced the highest dry biomass but also the highest induction of caffeic acid and salvianolic acid B. The highest amount of rosmarinic acid was observed in media containing 50 g/L sucrose. These prepared cell suspension cultures provided a useful system for further enhanced production of phenolic acids at a large scale.

Kinetin and Gibberellic acid (GA3) act synergistically to produce high value polyunsaturated fatty acids in Nannochloropsis oceanica CASA CC201

Abstract: Two functionally different plant growth regulators, Kinetin and GA3, were evaluated individually or in combination for its effect on growth, lipid yield, PUFAs and EPA accumulation in Nannochloropsis oceanica CASA CC201 . It was observed that the treatment with 0.215 ppm Kinetin resulted in high cell number of 521 × 10 6 cells per mL than the control (398 × 10 6 cells per mL), but GA3 had an adverse effect on cell number. Kinetin increases the specific growth rate to 0.24/day and doubling time to 2.86 days than control (4.38 days). Treatment with GA3 at a concentration of 50 ppm gives the highest cellular lipid accumulation of 61.5% DCW than the control (35.5% DCW). The combination of Kinetin and GA3 exhibited a synergistic effect on total lipid yield of 246.25 mg/L compared with control (121.5 mg/L). The addition of Kinetin increases the percentage of EPA 4 times as compared to the control.

Image Processing and Artificial Neural Network-Based Models to Measure and Predict Physical Properties of Embryogenic Callus and Number of Somatic Embryos in Ajowan ( Trachyspermum ammi (L.) Sprague)

Abstract: Trachyspermum ammi (L.) Sprague (Ajowan) is an endangered medicinal plant with useful pharmaceutical properties. Ex situ conservation of this medicinal plant needs the development of an in vitro regeneration protocol using somatic embryogenesis. In the present study, a high-precision image-processing approach was successfully applied to measure physical properties of embryogenic callus. Explant age and the concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), kinetin (Kin), and sucrose were used as inputs, and an artificial intelligence technique was applied to predict physical properties of embryogenic callus, and the number of somatic embryos produced. Artificial neural network (ANN) models were tested to find the best combinations of input variables that affected output variables. The lower values of root mean square error, and mean absolute error, and the highest values of determination coefficient, were achieved when all four input variables were applied to predict the number of somatic embryos, the area of the callus, the perimeter of the callus, the Feret diameter of the callus, the roundness of the callus, and the true density of the callus in ANN models. The highest measured and predicted number of somatic embryos were achieved from the interaction of 15-d-old explants × 1.5 mg L−1 2,4-D × 0.5 mg L−1 Kin × 2.5% (w/v) sucrose. Based on sensitivity analysis, the 2,4-D concentration was the most important component in the culture medium that affected the number of somatic embryos and physical properties of the embryogenic callus tissue.

Kinetin and Indole Acetic Acid Promote Antioxidant Defense System and Reduce Oxidative Stress in Maize ( Zea mays L.) Plants Grown at Boron Toxicity

Abstract: A study was conducted to assess the influence of boron (B) toxicity on functioning of antioxidant machinery to counteract oxidative stress in maize (Zea mays L.) plants as well as the mitigating effect of kinetin (KIN) and indole acetic acid (IAA) on these phenomena. Plants of maize cv. DK 647 F1 were exposed to 0.05 and 2 mM boron in nutrient solution 8 days after germination, and the plants were grown for a further 7 days in these conditions. After 15 days growth, deionized water (control), 1.0 or 2.0 mM of KIN, or IAA were applied to the leaves of maize plants once each 7 days. After 21 days of these treatments, the plants were harvested to evaluate growth, water relations, and oxidative and antioxidative systems. Boron toxicity significantly reduced dry matter, efficiency of photosystem II (Fv/Fm), and leaf relative water content in the maize plants when compared to those in non-stressed plants, but in contrast, it enhanced electrolyte leakage (EL), hydrogen peroxide (H2O2), free proline, malondialdehyde (MDA) and the activities of peroxidase, superoxide dismutase, and catalase in the maize plants. However, KIN or IAA applied as a foliar spray to maize plants grown at excess B caused a significant improvement in growth attributes, plant water status and the activities of various antioxidant enzymes as well as proline content, but they lowered EL, and H2O2 and MDA contents. Boron toxicity increased leaf B and reduced leaf K+, Ca2+, and P contents when compared to those in the control plants. Foliar applied KIN or IAA to the plant leaves lowered tissue B levels, but in contrast, it resulted in significant increases in Ca2+, K+ and P levels. The results of the study indicated that the spray of KIN and IAA, particularly at 2 mM, can mitigate to a significant extent the adverse effects of B toxicity on maize plants, which was found be associated with reduced content of B, H2O2, MDA as well as EL, and increased activities of key antioxidant enzymes in maize plants.

Kinetin Regulates UV-B-Induced Damage to Growth, Photosystem II Photochemistry, and Nitrogen Metabolism in Tomato Seedlings

Abstract: Cytokinins are a class of plant growth regulators that regulate several developmental processes in plants, and recently their role in counteracting the deleterious effects of abiotic stresses has been noted. The impacts of kinetin (10 µM, KN; an artificial cytokinin) on growth, photosystem II photochemistry, and nitrogen metabolism in tomato seedlings exposed to two levels (UV-B1, ambient+ 1.2 kJ m−2 day−1, and UV-B2, ambient+ 2.4 kJ m−2 day−1) of enhanced UV-B radiation were analyzed under open field condition. The growth, pigment contents, carbonic anhydrase activity, photosynthetic O2 yield, and values of chlorophyll a fluorescence parameters: F v/F 0, F v/F m or φP0, ψ 0, φE 0, and PIABS declined, whereas the values of energy flux parameters (ABS/RC, TR0/RC, ET0/RC, and DI0/RC) of PS II, efficiency of water splitting complex (F 0/F v), and respiratory rate of O2 uptake increased under UV-B stress. Likewise, UV-B exposure at both doses significantly inhibited the activity of enzymes involved in nitrogen metabolism: nitrate reductase, nitrite reductase, glutamine synthetase, and glutamate synthase. In contrast, an enhancing effect on glutamate dehydrogenase activity was observed under UV-B stress. Exogenous KN resulted in a significant attenuation in UV-B-induced negative effects on growth, pigments, photosynthesis, and nitrogen metabolism. The study concludes that exogenous KN improved the growth performance of tomato seedlings by attenuating the damaging effects of UV-B radiation on photochemistry of PS II and nitrogen metabolism, and the alleviating effect against the low dose (UV-B1) of UV-B was more pronounced.

Plant regeneration via direct somatic embryogenesis from leaf explants of Tolumnia Louise Elmore ‘Elsa’

Abstract: Tolumnia genus (equitant Oncidium) is a group of small orchids with vivid flower color. Thousands of hybrids have been registered on Royal Horticulture Society and showed great potential for ornamental plant market. The aim of this study is to establish an efficient method for in vitro propagation. Leaf explants taken from in vitro-grown plants were used to induce direct somatic embryogenesis on a modified 1/2 MS medium supplemented with five kinds of cytokinins, 2iP, BA, kinetin, TDZ and zeatin at 0.3, 1 and 3 mg l−1 in darkness. TDZ at 3 mg l−1 gave the highest percentage of explants with somatic globular embryos after 90 days of culture. It was found that 2,4-D and light regime highly retarded direct somatic embryogenesis and showed 95–100% of explant browning. Histological observations revealed that the leaf cells divided into meristematic cells firstly, followed by somatic proembryos, and then somatic globular embryos. Eventually, somatic embryos developed a bipolar structure with the shoot apical meristem and the root meristem. Scanning electron microscopy observations showed that the direct somatic embryogenesis from leaf explants was asynchronously. The somatic embryos were found on the leaf tip, the adaxial surface and also the mesophyll through a cleft, and it reflected the heterogeneity of the explant. The 90-day-old globular embryos were detached from the parent explants and transferred onto a hormone-free 1/2 MS medium in light condition for about 1 month to obtain 1-cm-height plantlets. After another 3 months for growth, the plantlets were potted with Sphagnum moss and were acclimatized in a shaded greenhouse. After 1 month of culture, the survival rate was 100%. In this report, a protocol for efficient regenerating a Tolumnia orchid, Louise Elmore ‘Elsa’, was established via direct somatic embryogenesis and might reveal an alternative approach for mass propagation of Tolumnia genus in orchid industry.

Comparative analysis of the effects of chitosan and common plant growth regulators on in vitro propagation of Ipomoea purpurea (L.) Roth from nodal explants.

Abstract: This study aimed to evaluate the effects of partially N-acetylated chitosans with a degree of acetylation (DA) of 10% on in vitro propagation of an ornamental plant, Ipomoea purpurea, by emphasizing the importance of the degree of polymerization (DP) on in vitro plant development. The effects of either a chitosan oligomer mixture with a DP between 2 and 15 (5.0, 10.0, and 20.0 mg L−1) or chitosan polymer with a DP of 70 were compared with commonly used cytokinins [6-benzylaminopurine (BAP) and kinetin (KIN) at 0.5, 1.0, 2.0, and 4.0 mg L−1] and auxins [indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) at 0.25, 0.5, 1.0, and 2.0 mg L−1]. The nodal explants used in this study were taken from donor plants obtained by germination of the seeds. The results indicated that all chitosan treatments had positive effects on the shoot induction, but only the oligomer mixture at 5 mg L−1 gave the best results for mean shoot number, shoot length, and leaf number, compared to the other treatments and control. Also, all chitosan treatments increased mean number of roots and triggered adventitious root induction. However, root elongation was decreased in the presence of chitosan in the medium. The root elongation-inhibitory effects of chitosan become clearer in the presence of oligomer mixture. In general, chitosan had similar effects with tested cytokinins rather than auxins. The results of this study suggested that the application of chitosan oligomers—rather than polymers—can be an eco-friendly and effective alternative to synthetic cytokinins in horticulture.

Regeneration of plantlets through somatic embryogenesis from root derived calli of Hibiscus sabdariffa L. (Roselle) and assessment of genetic stability by flow cytometry and ISSR analysis.

Abstract: Induction of somatic embryogenesis and complete plantlet regeneration from callus culture of Hibiscus sabdariffa L. var. HS4288 has been made. Leaf and root explants were cultured on Murashige and Skoog (MS) and Driver-Kuniyuki Walnut (DKW) basal media supplemented with different concentrations of synthetic auxins and cytokinins. Root explants on DKW medium supplemented with 2.26μM 2, 4-Dichlorophenoxyacetic acid (2, 4-D) and 4.65μM kinetin (KIN) induced highest percentage (70%) of embryogenic calli. Average number of globular embryos per root derived callus produced within 6 weeks of culture initiation on MS media with different plant growth regulators (PGRs) ranged from 2.27±0.12 to 8.80±0.17 and that of cotyledonary embryos ranged from 0.00 to 2.53±0.20. On DKW medium comparatively more globular embryos (2.70±0.15 to 14.53±0.23) and cotyledonary embryos (0.00 to 8.90±0.17) were produced than that of MS medium. Regeneration of complete plantlets was highest (76.67%) when embryogenic calli with mature somatic embryos were grown on DKW medium containing 2.32μM KIN and 2.22μM 6-Benzyladenine (BA). Plants were primarily hardened in humidity, temperature and light controlled chamber and finally in a greenhouse showed 70% survival ability. Different stages of somatic embryogenesis process in the root derived embryogenic calli were elaborated in detail by morphological, histological and SEM study. The data were statistically analyzed by Duncan Multiple range test (p ≤ 0.05) and Principal component analysis (PCA). Flow cytometry and Inter-simple sequence repeats (ISSR) marker analysis confirmed that there was no genetic variation within the regenerated plants.

A combined pathway of organogenesis and somatic embryogenesis for an efficient large-scale propagation in date palm (Phoenix dactylifera L.) cv. Mejhoul.

Abstract: An efficient regeneration system via a combined pathway of organogenesis and somatic embryogenesis was developed for date palm (Phoenix dactylifera L.) cv. Mejhoul. Adventitious buds were obtained from shoot-tip explants with a frequency of 53.3% after 9 months of culture: 6 months on half-strength Murashige and Skoog (MS/2) medium containing 14.2 µM indole-3-acetic acid (IAA), 13.4 µM 1-naphthaleneacetic acid (NAA) and 0.5 µM 6-(dimethylallylamino) purine (2iP), and 3 months on MS/2 medium supplemented with 1.1 µM IAA, 1.1 µM NAA, 0.5 µM 2iP, 2.2 µM 6-benzyladenine (BA) and 0.4 µM kinetin. Adventitious bud segments were used as explants to induce somatic embryogenesis, and the effects of different concentrations (22.5, 45, 90, 225 or 450 µM) of 3,6-dichloro-o-anisic acid (dicamba) and 4-amino-3,5,6-trichloropicolinic acid (picloram) were evaluated. The optimal medium for somatic embryogenesis induction was MS medium supplemented with 45 µM picloram and 5 µM 2iP, in which the somatic embryogenesis rate was 70%. For somatic embryo maturation, the effects of sorbitol, mannitol, polyethylene glycol (PEG) and abscisic acid (ABA) were tested. The highest maturation rate (88.6 mature somatic embryos per 100 mg fresh weight callus) was observed on liquid MS medium supplemented with 20 g L−1 PEG. Subsequent somatic embryo germination was achieved with up to 52.0% in MS medium containing 2.5 µM NAA and 2.5 µM BA. The regenerated plantlets were transferred to the glasshouse where 76.0% of them survived.

Synergistic dynamics of light, photoperiod and chemical stimulants influences biomass and lipid productivity in Chlorella singularis (UUIND5) for biodiesel production

Abstract: Microalgae have emerged as a potential alternative for the production of many useful compounds like protein, carbohydrate and lipid. Lipid-rich microalgae are important and rich source for alternative energy production. In order to commercially utilize microalgae for energy production, the lipid productivity should be enhanced. Keeping in view the above-mentioned potentials of microalgae, in the present study, we have attempted to display the role of chemical stimulants and light in the growth and lipid production of the microalgae Chlorella singularis (UUIND5). During the present investigations, effect of varying photoperiods and different types of lights and chemical stimulants, viz. CaCl2 and kinetin on growth rate and lipid production, was studied. The maximum growth rate recorded was 166 ± 0.3 mg/L/d, when 0.80 g/l CaCl2 and 0.5 mg/l kinetin were added to Bold’s basal medium. C. singularis was then cultivated in this medium for 14 days under sunlight +LED (10-h sunlight + 14-h LED light) at photoperiod 24-h light/0-h dark. The maximum lipid yield 30.2% of dry wt. was obtained under sunlight +LED. Further, the gas chromatography analysis also showed the presence of fatty acid methyl esters (FAME). FAMEs profile was analyzed according to ASTM D6751 specification. Thus, it was concluded that sunlight +LED at 24-h light/0-h dark (100 μmol photons m−2 s−1) photoperiod with CaCl2 and kinetin is an effective strategy to boost lipid productivity in C. singularis (UUIND5).

Silver nitrate-induced in vitro shoot multiplication and precocious flowering in Catharanthus roseus (L.) G. Don, a rich source of terpenoid indole alkaloids

Abstract: Catharanthus roseus (L.) G. Don is an economically and medicinally important plant since its leaves and flowers contain terpenoid indole alkaloids. The present study, for the first time, encompasses the influence of silver nitrate (AgNO3), in consort with cytokinins like N 6-benzyladenine (BA) and 6-furfurylaminopurine (kinetin), to regenerate multiple shoots from nodal segments explants and to induce high-frequency precocious flowering of C. roseus under in vitro condition. Synergistic effect of equal concentrations of BA and kinetin was enhanced following the amalgamation of AgNO3. As high as 98% explants responded to multiple shoot initiation and proliferation in Murashige and Skoog medium supplemented with 3 µM BA, 3 µM kinetin and 0.1 µM AgNO3. As many as 7 shoots were developed per explant following 12 days of inoculation. Continuous culture in the same medium for 21 days induced precocious flowering from 75% shoots, wherein a maximum of ~ 6 (5.67 ± 0.88) flowers was observed per in vitro shoot. On the other hand, in the combinations of BA and kinetin excluding AgNO3, a maximum of 6.67% explants responded and initiated merely 3.33 shoots per explant. Nevertheless, no induction of flower was observed in the media devoid of AgNO3. Our results on the induction and proliferation of multiple shoots with simultaneous flowering would help the global pharmaceutical industry to produce in vitro shoots and flowers in bulk, as an alternative source of alkaloids.

Effect of cytokinins on in vitro multiplication, volatiles composition and rosmarinic acid content of Thymus leucotrichus Hal. shoots

Abstract: An efficient in vitro multiplication protocol was designed to Thymus leucotrichus, a subshrub and perennial herb growing naturally in the Northwest of Turkey. Of all basal media studied, Murashige and Skoog medium was found to be superior to the others, providing higher shoot formation and the maximum shoot length. Varying concentrations of cytokinins, i.e., 6-benzyladenine, thidiazuron, 2-isopentenyladenine and kinetin were supplemented in the nutrient media to observe their effects on shoot development and biomass. Rosmarinic acid content and volatile compositions of both naturally growing plants and in vitro multiplied plantlets were also evaluated. 6-benzyladenine (1.0 mg/L) and kinetin (0.5 mg/L) were found to be optimum for shoot number and shoot elongation, respectively. Thidiazuron (1.0 mg/L) was superior for biomass production. Rosmarinic acid content of in vitro multiplied plants was found to be higher than that of wild plants, reaching a maximum with 0.5 mg/L 2-isopentenyladenine, which yielded 10.15 mg/g dry weight. The highest thymol content was obtained with 1.0 mg/L kinetin (55.82%), while thidiazuron (0.1 mg/L) increased carvacrol production (12.53%). Overall, Murashige and Skoog medium supplemented with 1.0 mg/L kinetin was determined to be the most favorable medium studied.

Phycomolecule-coated silver nanoparticles and seaweed extracts induced high-frequency somatic embryogenesis and plant regeneration from Gloriosa superba L.

Abstract: An efficient protocol for somatic embryogenesis and plant regeneration from rhizome explants of Gloriosa superba L. was developed using various plant growth regulators (PGRs), Ulva lactuca extracts (ULE), and phycomolecule-coated U. lactuca silver nanoparticles (ULAgNPs). Callus was initiated from rhizome explants on the Murashige and Skoog (MS) medium supplemented with various concentrations of 2,4-dichlorophenoxyacetic acid (1.0–5.0 mg L−1), α-naphthaleneacetic acid (NAA) (0.5 mg L−1), ULE (10–50%), and ULAgNPs (0.1–0.5 mg L−1). Callus was cultured on MS medium fortified with various concentrations and combinations of 6-benzylaminopurine (BAP) (0.5–2.5 mg L−1), kinetin (KIN) (0.5–2.5 mg L−1), ULAgNPs (0.1–0.5 mg L−1), and 20% ULE along with 0.5 mg L−1 NAA for the development of somatic embryos (SEs). The maximum percentage of embryo maturation (100%) was observed on the medium containing 0.5 mg L−1 ULAgNPs, 2.0 mg L−1 BAP, 0.5 mg L−1 abscisic acid (ABA), 0.5 mg L−1 silver nitrate (AgNO3), and 20% ULE. The highest percentage of embryo germination (86.1%) was noticed on the MS medium containing 0.3 mg L−1 ULAgNPs, 5.0 mg L−1 gibberellic acid (GA3), 2.0 mg L−1 BAP, 0.5 mg L−1 adenine sulfate (AdS), and 20% ULE. Well-rooted plantlets were successfully acclimatized in the greenhouse with 70% survival rate. Results suggest that U. lactuca extract-derived silver nanoparticles could be used as biostimulants for the enhancement of somatic embryogenesis and plant regeneration rate in G. superba.

Optimization of factors affecting in vitro regeneration, flowering, ex vitro rooting and foliar micromorphological studies of Oldenlandia corymbosa L.: a multipotent herb

Abstract: The conditions were optimized for efficient in vitro regeneration of shoots and roots of Oldenlandia corymbosa L. using nodal shoot explants. Murashige and Skoog’s (MS) medium augmented with additives and 2.0 mg L−1 BAP was recorded optimum for shoot bud induction from the nodal meristems. The shoots were proliferated by subsequent subcultures on half strength MS medium fortified with 1.0 mg L−1 BAP and 0.5 mg L−1 kinetin + additives. This media combination yielded maximum number of shoots (223 ± 4.12 shoots/culture bottle) with 13.4 cm average length. Flower buds were induced (4.2 ± 0.28 flowers) from the in vitro multiplied shoots on MS medium contained 1.0 mg L−1 BAP and 0.5 mg L−1 of kinetin and IAA under 50 µmol m−2 s−1 SFPD light intensity for 12 h/day photoperiod. In vitro regenerated shoots were rooted on half strength MS medium conjunct with IAA, IBA and NAA singly at different concentrations. The best rooting response was observed on half strength MS medium containing IBA at 2.0 mg L−1 with activated charcoal. Roots were also induced from the cut ends of the shoots using ex vitro rooting techniques in O. corymbosa by pulse treating the shoots with 300 mg L−1 IBA for 4 min. Better roots were achieved in this method than the in vitro roots in terms of numbers (14.7 ± 0.21) and firmness. The foliar micromorphological studies could help to understand the structural adaptations of micropropagated O. corymbosa plantlets towards field environments. The in vitro induced anomalies in stomatal apparatus were repaired during hardening of plantlets in the greenhouse and after field transfer. Decrease in stomatal density (from 70.78 ± 0.55 to 55.6 ± 0.10), and increase in veins, trichomes and crystals/raphides densities revealed the developments of structural changes in the leaves to withstand in the harsh field conditions. The acclimatized plantlets with well developed root systems were successfully shifted to the natural soils with 98% survival rate.

Effect of cytokinins on shoots proliferation and rosmarinic and salvianolic acid B production in shoot culture of Dracocephalum forrestii W. W. Smith

Abstract: 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.

Salicylic acid and kinetin mediated stimulation of salt tolerance in cucumber ( Cucumis sativus L.) genotypes varying in salinity tolerance

Abstract: Greenhouse studies were undertaken to evaluate the genetic performance of two cucumber genotypes (Long Green and Summer Green) at four salinity levels (0, 25, 50, and 100 mM NaCl). Seeds were pretreated with 50 mg salicylic acid (SA) L−1 and 25 mg kinetin (Kin) L−1. Under hydroponic conditions, seed pretreatment with Kin significantly increased shoot and root dry biomass and reduced the salt injury index in both genotypes. SA reduced the salt injury index of Long Green cucumbers. In a pot experiment, Kin treatment reduced Na+ and increased K+ concentration, photosynthesis, and chlorophyll content in both genotypes, compared to SA under saline soil conditions. Kin treatment improved fruit yield in both genotypes, while SA had a statistically significant effect on Long Green fruit yield. The application of SA and Kin enhanced salinity tolerance in both genotypes by the activation of antioxidants, especially superoxide dismutase, peroxidase, and catalase, which offset oxidative injury. Summer Green exhibited better salt tolerance and improved osmoregulation that resulted in higher fruit yield than Long Green. It was concluded that cucumber genotypes differed in salt tolerance, and seed pre-treatment with Kin minimized salt stress injury, even in sensitive genotype which could sustain crop production under saline conditions.

Improvement of seed dehiscence and germination in ginseng by stratification, gibberellin, and/or kinetin treatments

Abstract: Korean ginseng (Panax ginseng C.A. Meyer) is a largely sought after resource due to its substantial health benefits. However, large-scale culture of ginseng seeds is limited because of its long maturation time and triple-dormancy. To develop new strategies to improve the dehiscence, germination, and breaking dormancy, we investigated the effects of stratification, gibberellin (GA3), and/or kinetin treatments on the dehiscence, development and germination of P. ginseng. Indehiscent seeds for the dehiscence test and dehiscent seeds for the germination test were immersed in a solution containing 288.7 μM GA3 and/or 232.3 μM kinetin for 24 h, respectively, at 25 °C. After dehiscence, a cold stratification experiment was conducted at 2 °C for 15, 30, 45, 60, and 90 days in the dark. Exogenous GA3 and kinetin treatments significantly improved the dehiscence rate. In addition, both plant growth regulators (PGRs) appeared to enhance the zygotic embryo development of ginseng seeds. GA3 on its own had a slight effect on breaking dormancy and germination, but the effect was not sufficient to serve as a complete substitute for the cold stratification requirement due to the deep physiological dormancy characteristic of P. ginseng. Kinetin treatment had a significant effect on germination and breaking dormancy, although a short period of cold stratification was also required. The combination of GA3 and kinetin treatments was more effective at enhancing both seed dehiscence and germination than either PGR treatment alone.

Establishment of an efficient in vitro propagation system for Iris sanguinea.

Abstract: Iris sanguinea is a perennial flowering plant that is typically cultivated through seeds or bulbs. However, due to limitations in conventional propagation, an alternate regeneration system using seeds was developed. The protocol included optimization of sterilization, stratification and scarification methods as iris seeds exhibit physiological dormancy. In addition to chlorine-based disinfection, alkaline or heat treatment was used to break seed dormancy and reduce contamination. When seeds were soaked in water at 80 °C overnight, and sterilized with 75% EtOH for 30 s and 4% NaOCl solution for 20 minutes, contamination was reduced to 10% and a 73.3% germination was achieved. The germinated seedlings with 2-3 leaves and radicle were used as explants to induce adventitious buds. The optimal MS medium with 0.5 mg L−1 6-benzylaminopurine, 0.2 mg L−1 NAA, and 1.0 mg L−1 kinetin resulted in 93.3% shoot induction and a proliferation coefficient of 5.30. Medium with 0.5 mg L−1 NAA achieved 96.4% rooting of the adventitious shoots. The survival rate was more than 90% after 30 days growth in the cultivated matrix. In conclusion, a successful regeneration system for propagation of I. sanguinea was developed using seeds, which could be utilized for large-scale propagation of irises of ecological and horticultural importance.

Organogenesis and efficient in vitro plantlet regeneration from nodal segments of Allamanda cathartica L. using TDZ and ultrasound assisted extraction of quercetin

Abstract: 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.

Production of doubled haploids in sugar beet ( Beta vulgaris ): an efficient method by a multivariate experiment

Abstract: The present paper describes a detailed study of a highly efficient protocol to multiply the number of haploids in sugar beet production and subsequent chromosome doubling. The protocol involves an experiment investigating factorial interactions between cold pretreatment, seven genotypes of sugar beet, and kinetin to improve haploid embryo induction. In addition, the effects of color of ovules and flower bud position on haploid embryo induction were investigated. After subjecting the data to analysis of variance or Student’s t test (P < .05), the effect sizes of the independent variables were also estimated. Cold pretreatment was effective in stimulating the ovules. The haploid embryo induction rate for 1-week cold pretreated ovules (9.01%) was higher than that of freshly cultured ones (6.15%). In comparison with hormone-free medium (5.16%), the gynogenesis rate for the media supplemented with 0.05 or 0.5 mg L−l kinetin increased to 7.58 and 10.05%, respectively. The genotype responses were significantly different. Interactions of kinetin × cold pretreatment, genotype × hormonal treatment, genotype × cold pretreatment, and the three-way interaction were statistically significant. Moreover, the main effects of flower bud position, ovule color, and comma-form ovule on gynogenic response were significant. After investigating the effect of 5 g L−l colchicine for 3, 5, or 7 min on one genotype’s (SG2) specimens, all the haploid plantlets from the other genotypes were treated for 5 min as the best treatment. The paper discusses interactions of the factors, which may be interesting for others aiming to breed doubled haploid sugar beet or possibly other related plant species.

In vitro Induction and Generation of Tetraploid Plants of Sophora tonkinensis Gapnep.

Abstract: Background: Sophora tonkinensis Gapnep. is an important medical plant in China. Early researches of S. tonkinensis were focused on rapid propagation and quality analysis of in vitro tissue culture plantlet, and still no research focuses on the plant breeding of and there were no excellent varieties for artificial cultivation of S. tonkinensis. Objective: To set up a method to generate and select the best varieties of S. tonkinensis by polyploid breeding after induction by colchicine treatment. Materials and Methods: The adventitious buds were submerged in different concentrations of aqueous colchicine solution for different lengths of time to induce polyploidy in the plants, and the induced buds were identified by root-tip chromosome determination and leaf characteristics comparison. The contents of matrine and oxymatrine of radix ex rhizoma in 13 selected tetraploid lines were collected after 90 days in vitro rooting culture and were evaluated to provide evidence of good qualities of tetraploid S. tonkinensis. Results: The results showed that the highest percentage of tetraploid induction was 23.33% and occurred in the 0.2% (w/v) colchicine treatment for 30 h. Fifty lines of tetraploid plants were obtained and 12 of the 13 selected tetraploid lines exhibited higher productivity of total contents of matrine and oxymatrine when compared to controls. Conclusion: The data demonstrate that polyploidy induction can be beneficial for improving the medicinal value of S. tonkinensis. Abbreviations used: MS medium: Murashige and Skoog medium; BAP: 6-benzylaminopurine; NAA: A-naphthaleneacetic acid; IAA: Indole-3-acetic acid; KT: Kinetin; IBA: Indole-3-butyric acid; ABT: Rooting power.

Antimitotic and hormone effects on green double haploid plant production through anther culture of Mediterranean japonica rice

Abstract: Rice double haploid (DH) plants are produced mainly through anther culture. In order to improve the anther culture protocol, microspores of two japonica rice genotypes (NRVC980385 and H28) were subjected to three growth regulator combinations and four colchicine treatments on induction medium. In addition, a post anther culture procedure using colchicine or oryzalin was tested to induce double haploid plantlets from haploid plantlets. A cold pre-treatment of microspores for 9 days at 10 °C increased callus induction 50-fold in the NRCV980385 genotype. For both genotypes, 2 mg L−1 2,4-D and 1 mg L−1 kinetin on colchicine-free induction medium gave the best culture responses. The culturability of both genotypes changed on colchicine-supplemented induction media. A high genotype dependency was recorded for callus induction, callus regenerating green plantlets and regeneration of green double haploid plantlets. Colchicine at 300 mg L−1 for 48 h enhanced callus induction 100-fold in H28. Colchicine-supplemented media clearly improved green double haploid plantlet regeneration. We showed that the post-anther culture treatment of haploid plantlets at 500 mg L−1 of colchicine permitted fertile double haploid plantlets to be generated. Finally, an enhanced medium-throughput flow cytometry protocol for rice was tested to analyse all the plantlets from anther and post anther culture.

Kinetin improves motility, viability and antioxidative parameters of ram semen during storage at refrigerator temperature.

Abstract: The present experiment was conducted to evaluate the effect of kinetin on ram semen quality during cold storage. Ejaculates were collected using an artificial vagina from five Qezel rams. Ejaculates which met the criteria (volume of 0.75–2 ml; minimum spermatozoa concentration of 2.5 × 109 spermatozoa/ml and forward progressive motility of 80%), were pooled, diluted with extender without kinetin (control) or enriched with 25 (K 25), 50 (K 50), 100 (K 100) and 200 (K 200) μM kinetin at a final concentration of 500 × 106 spermatozoa per mL. Spermatozoa motion characteristics were evaluated by computer-assisted sperm analysis. In addition, percent of viability (spermatozoa showing no color was considered to be alive) and spermatozoa with intact plasma membrane (spermatozoa with curled/swollen tail was considered healthy) were determined. Moreover, amounts of malondialdehyde (MDA), total antioxidant activity (AOA), nitric oxide (NO) and superoxide dismutase (SOD) activity were determined in the seminal plasma and spermatozoa at 0, 24, 48 and 72 h of storage. Higher percent of total and forward progressive motility was observed in K 25, K 50 and K 100 groups compared to control group at 72 h of storage (P < 0.001). Moreover, K 25 (78.61 ± 1.11%), K 50 (82.46 ± 1.08%) and K 100 (82.96 ± 1.49%) groups showed higher percentage of viability compared to control (72.38 ± 1.49%) at 72 h of storage (P < 0.05). Semen enrichment with kinetin resulted in the higher percent of intact plasma membrane of spermatozoa at 48 and 72 h (P < 0.001). Amounts of MDA were lower and amounts of AOA were higher in K 50 and K 100 groups compared to control at 48 and 72 h (P < 0.05). There were no significant differences in NO levels and SOD activities of seminal plasma and spermatozoa among groups during the experiment. The present experiment revealed that kinetin at proper concentration could enhances spermatozoa kinematics, viability, spermatozoa plasma membrane functionality and amounts of AOA and reduces the level of lipid peroxidation during chilled storage of ram semen.

A highly efficient method for somatic embryogenesis of Kelussia odorotissima Mozaff., an endangered medicinal plant

Abstract: Kelussia odoratissima Mozaff. (or Kelus) is a medicinal plant native to the Zagros Mountains in Iran. This plant is widely used as a food flavoring and for its health-promoting properties. It has been considered an endangered species by the United Nations Development Programme. In this study, a somatic embryogenesis (SE) method was developed for mass propagation of Kelus. The green globular embryogenic callus was induced on cotyledonary leaves using the Murashige and Skoog (MS) medium supplemented with 1 mg/l 2,4-dichlorophenoxyaceticacid (2,4-D) and 0.25 mg/l Kinetin. Different treatments were assayed for proliferation of the embryogenic callus. The calli remained embryogenic in an MS medium containing 2,4-D (1 mg/l). The light treatments and carbon source showed significant effects (P ≤ 0.05) on the proliferation and development of somatic embryos. These treatments improved the conversion rate of the cotyledonary-stage embryos by 100%. The average numbers of embryos in the globular, heart, torpedo, and cotyledonary stages decreased by the addition of 3 g/l case in hydrolisate. The genetic stability among tissue culture-derived plants and the mother plant were assessed using the amplification fragment length polymorphism. No polymorphic band was observed among all the plants, exhibiting the genetic stability during in vitro multiplication. This research provides a promising approach for true-to-type plant multiplication of K. odoratissima through SE.