Showing papers on "Kinetin published in 2010"

The role of cytokinins in shoot organogenesis in apple

Abstract: Effective regeneration in vitro is a necessary precondition for the implementation of different biotechnological approaches in plant breeding. Numerous studies have reported on regeneration from apple somatic tissues, and organogenesis has been proved to be influenced by several factors including mother shoots (genotype, size, type, and age of explant), in vitro conditions (dark period, light intensity, and quality), and others (wounding, orientation of leaf explants). However, one of the most important factors before and during the regeneration process is the type and concentration of cytokinin applied. Thidiazuron and benzyladenine are the most frequently used cytokinins in the regeneration systems, but their efficiency depends on genotype and other factors. Other cytokinins (e.g., zeatin and kinetin) have also been tested in several experiments and they were found in general to be less active. The organogenic ability of explants can also be increased by a properly selected cytokinin pre-treatment. Cytokinins applied in the pre-treatments can influence the leaf structure, which in turn can alter the regeneration capacity of the leaf explant. Interactions between factors of pre-treatments (hormones, light, and culture conditions) and factors of the regeneration phase should be considered. This review brings into focus the role of different cytokinins during in vitro shoot development, discussing their effects on the histology of leaves developed in vitro, and how this affects the subsequent regeneration process.

In vitro propagation of the endangered plant Centaurea ultreiae: assessment of genetic stability by cytological studies, flow cytometry and RAPD analysis

Abstract: The effect of different cytokinins on multiple shoot regeneration from shoots of Centaurea ultreiae was studied. The culture system consisted of solid basal half-strength Murashige and Skoog medium supplemented with one of four cytokinins [6-benzyladenine (BA), zeatin, kinetin, or N6-(2-isopentyl) adenine (2-iP)] at each of five different concentrations. The highest multiplication rate (5.52 shoots per explant) was obtained in the medium supplemented with 4.44 μM BA. Shoots were successfully rooted (91% success) by dipping the basal end into a solution containing 10 M 1-naphthaleneacetic acid for 30 s. Genetic stability of the regenerated plants was assessed by random amplified polymorphic DNA (RAPD) analysis and flow cytometry. In the initial randomly selected plant material (control) and 20 of its regenerants, 2,688 bands were generated by RAPD with 12 different primers, and the same banding profiles were exhibited. Molecular and cytological analyses did not reveal genomic alterations in any of the regenerated plants obtained on medium containing 4.44 μM BA. The success of acclimatization to environmental conditions—100% of plants were successfully acclimatized—suggests that the micropropagation system described is a reliable method for propagation of C. ultreiae.

Effect of foliar applied kinetin and indole acetic acid on maize plants grown under saline conditions

Abstract: This study examined the effects of kinetin (KIN) and indoleacetic acid (IAA) sprayed on the leaves of salinity- stressed maize (Zea mays L. cv., DK 647 F1) plants grown in field conditions. Salt stress was created by adding 100 mM NaCl to the irrigation water through a drip irrigation system during the irrigation schedule. Kinetin (KIN), indole acetic acid (IAA), and their combinations were sprayed foliarly. Salt stress (S) reduced the total dry matter, grain yield, chlorophyll content, and relative water content (RWC), but increased electrolyte leakage and proline accumulation in the maize plants. Foliar applications of both KIN and IAA treatments overcame to variable extents the adverse effects of NaCl stress on the earlier mentioned physiological parameters. However, the combination of KIN plus IAA did not improve salinity tolerance in maize plants. Salt stress increased Na + concentration, and reduced those of Ca 2+ and K + in the leaves of maize plants. Foliar application of both KIN and IAA significantly reduced Na + concentration and increased those of Ca 2+ and K + . Foliar application of KIN and IAA, especially at 2 mM, counteracted some of the salt induced adverse effects by enhancing essential inorganic nutrients as well as by maintaining membrane permeability.

Plant regeneration through the direct induction of shoot buds from petiole explants of Jatropha curcas: a biofuel plant

Abstract: An efficient and reproducible method for the regeneration of Jatropha curcas plants has been developed. The method employed direct induction of shoot buds from petiole explants, without the formation of an intervening callus using a Murashige and Skoog (MS) medium supplemented with different concentrations of thidiazuron (TDZ). The best induction of shoot buds ( 58.35 % ) and the number of shoot buds per explant (10.10) were observed when in vitro petiole explants were placed horizontally on MS medium supplemented with 2.27 μM TDZ after 6 weeks. The induced shoot buds were transferred to MS medium containing 10 μM kinetin (Kn), 4.5 μM 6-benzyl aminopurine (BAP) and 5.5 μM α-naphthaleneacetic acid (NAA) for shoot proliferation. The proliferated shoots could be elongated on MS medium supplemented with different concentrations and combinations of BAP, indole-3-acetic acid (IAA), NAA and indole-3-butyric acid (IBA). MS medium supplemented with 2.25 μM BAP and 8.5 μM IAA was found to be the best combination for shoot elongation and 3.01-3.91 cm elongation was achieved after 6 weeks. However, significant differences in plant regeneration and shoot elongation were observed among the genotypes studied. The orientation (horizontal or vertical) and source (in vitro or in vivo) of explants also significantly influenced plant regeneration. The elongated shoots could be rooted on half-strength MS medium supplemented with 2% sucrose, different concentrations and combinations of IBA, IAA and NAA, and 0.25 mg L -1 activated charcoal. Half-strength MS medium supplemented with 2 % sucrose, 15 μM IBA, 5.7 μM IAA, 5.5 μM NAA and 0.25 mg L -1 activated charcoal was found to be the best for promoting rooting. The rooted plants could be established in soil with more than 90% survival.

A Comparative Study of the Antibacterial, Antifungal and Antioxidant Activity and Total Content of Phenolic Compounds of Cell Cultures and Wild Plants of Three Endemic Species of Ephedra

Abstract: Investigations were carried out to determine antimicrobial and antioxidant properties and total phenol content of three wild species of Ephedra compared with their respective callus cultures. Callus induction was performed in a standard Murashige and Skoog (MS) medium with the following hormonal ranges (mg/L) for every species NAA:1.5, Kin:1 for Ephedra strobiliacea, NAA:2, Kin:1 for Ephedra procera and NAA:2, Kin:0.5 for Ephedra pachyclada. These ranges of PGPR (Plant Growth Promote Regulators) were chosen based on callus induction rates, RGR (Relative Growth Rate) and their fresh weights. An antimicrobial test against five gram negative and two gram positive bacteria and two fungi was performed using the disc diffusion method. All methanolic extracts showed antimicrobial activity, but the antimicrobial activity of the callus cultures was lower than those of the wild plants. E. strobilacea showed the highest antimicrobial activity, and all methanolic extracts of the wild plants and callus cultures unexpectedly showed the highest antimicrobial activity against Pseudomonas aeruginosa. A FRAP (Ferric Reducing Antioxidant Power) test was conducted to evaluate extracts for antioxidant activity. E. strobilacea with 1.61 +/- 0.08 mmol eq quercetin/g extract and 0.278 +/- 0.02 mmol eq quercetin/g extract for the wild plant and callus, respectively, showed the highest results.The total phenol content of extracts was measured by a Folin Ciocalteau test. All the chosen species displayed phenol contents but E. strobilacea had the highest amount (504.9 +/- 41.51 micromol eq catechin/g extracts and 114.61 +/- 15.13 micromol eq catechin/g extracts for the wild plants and callus, respectively).

Elimination of virus and rapid propagation of disease-free sugarcane (Saccharum spp. cultivar NCo376) using apical meristem culture

Abstract: The use of apical meristem culture for simultaneous virus elimination and shoot proliferation in sugarcane was assessed. Virus-free plants were propagated from Sugarcane mosaic virus and Sugarcane yellow leaf virus-infected material of the South African commercial cultivar, NCo376. A combination of thermotherapy by hot water treatment of stem sections (nodes) and subsequent germination of vegetative buds at 40°C and optimal meristem size were key factors for the production of virus-free plants. Only meristems of 2 mm in length or of a smaller size (but >0.5 mm) resulted in virus-free sugarcane. Shoot induction and proliferation via direct organogenesis were achieved on Murashige and Skoog nutrient medium supplemented with 0.1 mg l−1 6-benzyladenine and 0.015 mg l−1 6-furfurylaminopurine (KIN). The established protocol provides for the rapid proliferation of virus-free shoots from infected sugarcane plants and approximately 1,300 shoots were propagated from a single 2 mm meristem in 11 weeks. Plants remained virus-free when tested 12 months later.

In vitro production of stevia rebaudiana bertoni

Abstract: Experiments were conducted for the standardization of in vitro culture technique for themass propagation of Stevia rebaudiana, a medicinally important, zero-calorie value, sweet tastedand an antidiabetic herb. Shoot tip, nodal segment and leaf were used as explants and they werecultured on Murashige and Skoog (MS) medium supplemented with different concentrations ofBA, Kn and IAA both in individual and in combined form for shoot inductions and the best resultswere obtained from MS medium supplemented with BA+ IAA at the concentrations of 1.0 mg/land 0.5 mg/l respectively. Among the explants used, shoot tip stood first in inducing shootdevelopment. Best root formation of in vitro developed shoots could be achieved on half-strengthNitsch (N6) medium supplemented with IAA at concentration 1.0 mg/l. The in vitro developedplantlets were transferred to pot and they were grown in greenhouse for hardening and finally theywere planted in the open filed. Around 82% of plants were successfully established in natural fieldcondition.

Effects of sucrose and pH levels on in vitro shoot regeneration from leaf explants of Bacopa monnieri and accumulation of bacoside A in regenerated shoots

Abstract: Brahmi (Bacopa monnieri) is an important medicinal plant mainly used for the treatment of neurological disorders and depression. Recent investigations revealed that bacoside A is major chemical component shown to be responsible for memory facilitating action of brahmi. The current investigation was carried out to assess the potential for increasing biomass and the concentration of bacoside A in the in vitro regenerated shoots by varying sucrose and pH levels of shoot regeneration medium. The leaf explants were cultured on the Murashige and Skoog (MS) medium supplemented with 2 mg l−1 kinetin (KN) and with varying concentrations of sucrose (0, 1, 2, 3, 4, 5 and 6% at pH 5.8) and pH (4.5, 5.0, 5.5, 6.0 and 6.5 with 2% sucrose) with the objective of verifying the effects of sucrose and pH level on shoot regeneration and to verify the accumulation of bacoside A in the regenerated shoots. The shoot biomass increased (150.50 ± 2.84 shoots per explant, fresh wt 6.31 ± 0.12 g and dry wt 250 ± 5.00 mg) on the medium supplemented with 2% sucrose and pH which was set at 4.5. The results of HPLC analysis indicate that increase in sucrose concentration (0, 1, 2, 3, 4, 5 and 6% at pH 5.8) lead to decrease in the bacoside A content (39.51, 22.43, 13.05, 12.17, 10.73, 9.56 and 8.93 mg g−1 dry wt, respectively) in regenerated shoots. These findings provide evidence that stressful condition of inadequate supply of carbon elevated synthesis of bacoside A in brahmi shoots. However, 2% sucrose is found suitable for biomass accumulation. Therefore, medium supplemented with 2% sucrose and pH set at 4.5 was found suitable for both biomass (6.31 ± 0.12 g fresh wt and 250 ± 5.00 mg dry wt) and bacoside A accumulation (13.09 mg g−1 dry wt).

Growth, secondary metabolite production and antioxidant enzyme response of Morinda citrifolia adventitious root as affected by auxin and cytokinin

Abstract: Morinda citrifolia adventitious roots were cultured in shake flasks using Murashige and Skoog medium with different types and concentrations of auxin and cytokinin. Root (fresh weight and dry weight) accumulation was enhanced at 5 mg l−1 indole butyric acid (IBA) and at 7 and 9 mg l−1 naphthalene acetic acid (NAA). On the other hand, 9 mg l−1 NAA decreased the anthraquinone, phenolic and flavonoid contents more severely than 9 mg l−1 IBA. When adventitious roots were treated with kinetin (0.1, 0.3 and 0.5 mg l−1) and thidiazuron (TDZ; 0.1, 0.3 and 0.5 mg l−1) in combination with 5 mg l−1 IBA, fresh weight and dry weight decreased but secondary metabolite content increased. The secondary metabolite content (including 1,1-diphenyl-2-picrylhydrazyl activity) increased more in TDZ-treated than in kinetin-treated roots. Antioxidative enzymes such as catalase (CAT) and guaiacol peroxidase (G-POD), which play important roles in plant defense, also increased. A strong decrease in ascorbate peroxidase activity resulted in a high accumulation of hydrogen peroxide. This indicates that adventitious roots can grow under stress conditions with induced CAT and G-POD activities and higher accumulations of secondary metabolites. These results suggest that 5 mg l−1 IBA supplementation is useful for growth and secondary metabolite production in adventitious roots of M. citrifolia.

Callus induction and plant regeneration in Dorem ammoniacum D., an endangered medicinal plant

Abstract: Dorema ammoniacum D. Don. (Apiaceae), a native medicinal plant in Iran, is classified as a vulnerable species. Root, hypocotyl, and cotyledon segments were cultured on Murashige and Skoog (MS) (1962) medium supplemented with either 2,4-dichlorophenyoxyacetic acid (2,4-D) or naphathalene acetic acid (NAA), at 0–2 mg l−1, alone or in combination with either benzyladenine (BA) or kinetin (KN), at 0–2 mg l−1 for callus induction. The best response (100%) was observed from root segments on MS medium containing 1 mg l−1 NAA and 2 mg l−1 BA. The calli derived from various explants were subcultured on MS medium supplemented with BA (1–4 mg l−1) alone or in combination with NAA or indole-3-butyric acid (IBA), at 0.2 or 0.5 mg l−1 for shoot induction. Calli derived from hypocotyl segments showed significantly higher frequency of plantlet regeneration and number of plantlets than the calli derived from root and cotyledon segments. Therefore, MS medium supplemented with 2 mg l−1 BA and 0.2 mg l−1 IBA produced the highest frequency of shoot regeneration (87.3%) in hypocotyl-derived callus. The optimal medium for rooting contained 2.5 mg l−1 IBA on which 87.03% of the regenerated shoots developed roots with an average number of 5.2 roots per shoots within 30 days. These plantlets were hardened and transferred to the soil. The described method can be successfully employed for the large-scale multiplication and conservation of germplasm this plant.

High frequency plant regeneration from leaf derived callus of high Δ9-tetrahydrocannabinol yielding Cannabis sativa L.

Abstract: An efficient in vitro propagation protocol for rapidly producing Cannabis sativa plantlets from young leaf tissue was developed. Using gas chromatography-flame ionization detection (GC-FID), high THC yielding elite female clone of a drug-type CANNABIS variety (MX) was screened and its vegetatively propagated clones were used for micropropagation. Calli were induced from leaf explant on Murashige and Skoog medium supplemented with different concentrations (0.5, 1.0, 1.5, and 2.0 µM) of indole- 3-acetic acid (IAA), indole- 3- butyric acid (IBA), naphthalene acetic acid (NAA), and 2,4-dichlorophenoxy-acetic acid (2,4-D) in combination with 1.0 µM of thidiazuron (TDZ) for the production of callus. The optimum callus growth and maintenance was in 0.5 µM NAA plus 1.0 µM TDZ. The two-month-old calli were subcultured to MS media containing different concentrations of cytokinins (BAP, KN, TDZ). The rate of shoot induction and proliferation was highest in 0.5 µM TDZ. Of the various auxins (IAA, IBA, and NAA) tested, regenerated shoots rooted best on half strength MS medium (1/2 - MS) supplemented with 2.5 µM IBA. The rooted plantlets were successfully established in soil and grown to maturity with no gross variations in morphology and cannabinoids content at a survival rate of 95 % in the indoor growroom.

Shoot regeneration from cotyledonary leaf explants of Jatropha curcas: a biodiesel plant.

Abstract: A simple, high frequency, and reproducible method for plant regeneration through direct organogenesis from cotyledonary leaf explants of Jatropha curcas was developed using Murashige and Skoog (MS) medium supplemented with different concentrations of thidiazuron (TDZ) or 6-benzyl aminopurine (BAP). Medium containing TDZ has greater influence on regeneration as compared to BAP. The induced shoot buds were transferred to MS medium containing 10 μM kinetin (Kn), 4.5 μM BAP, and 5.5 μM α-naphthaleneacetic acid (NAA) for shoot proliferation. The proliferated shoots could be elongated on MS medium supplemented with different concentrations and combinations of BAP, indole-3-acetic acid (IAA), NAA, and indole-3-butyric acid (IBA). MS medium with 2.25 μM BAP and 8.5 μM IAA was found to be the best combination for shoot elongation. However, significant differences in plant regeneration and shoot elongation were observed among the genotypes studied. Rooting was achieved when the basal cut end of elongated shoots were dipped in half strength MS liquid medium containing different concentrations and combinations of IBA, IAA, and NAA for 4 days, followed by transfer to growth regulators free half strength MS medium supplemented 0.25 mg l−1 activated charcoal. Elongated shoot treated with 15 μM IBA, 5.7 μM IAA, and 11 μM NAA resulted in highest percent rooting. The rooted plants could be established in soil with more than 90% survival rate. The method developed may be useful in improvement of J. curcas through genetic modification.

In vitro screening of rice (oryza sativa l.) callus for drought tolerance

Abstract: While drought resistance is become of increasing importance in rice (Oryza sativa L.), selection under actual field conditions is tedious due to low heritability and time required. Selection in tissue culture is thought to be one way to improve selection efficiency, but this requires standardized protocols. Rice cultivars PAU 201 and PR 116 showed significant callus induction, but the capacity for callus induction and regeneration decreased under polyethylene glycol (PEG) (6000) stress in both cultivars. Calli were induced on semisolid Murahige and Skoog (MS) medium supplemented with 2.5 mg l-1 2, 4-dichlorophenoxy acetic acid (2,4-D) + 0.5 mg l-1 kinetin (kin) + 560 mg l-1 proline + 30 g l-1 sucrose + 8 g l agar-1. Embryogenic calli showed shoot regeneration on MS medium supplemented with 2.0 mg l-1 benzyl aminopurine (BAP) + 0.5 mg l-1 kinetin + 0.5 mg l-1 napthalene acetic acid (NAA) + 30 g l-1 sucrose + 8 g l-1 agar. Increased levels of PEG (6000) (0, 0.5, 1.0, 1.5, 2.0 %) were used to create water stress. There was reduction in callus induction ability and plant regeneration efficiency with increasing levels of PEG (6000) stress. These results indicated that PEG (6000) can be used as water stress creating agent under in vitro conditions and rice variety PR 116 was relatively tolerant to drought stress as compared to PAU 201. This study will serve as a base line for in vitro screening of drought tolerant transgenic rice.

Cytokinin Biosynthesis and Metabolism

Abstract: Since the discovery of cytokinins in the 1950s, it has been clearly established that they play an important role in various processes in the growth and development of plants, including the promotion of cell division, the counteraction of senescence, the regulation of apical dominance and the transmission of nutritional signals. Kinetin (Fig. 1a) was the first substance to be identified as a cytokinin, and although it was isolated from autoclaved herring sperm DNA (34) it is not naturally produced and has not been found in living plants. The naturally occurring cytokinin trans-zeatin (tZ, Fig. 1b) was first isolated from immature maize endosperm in the early 1960s (26). In the following 40 years, several cytokinin species have been identified from various plant species (35, 43). These studies demonstrated that natural plant cytokinins are adenines which have substituted at the N6 terminal either an isoprene-derived side chain (isoprenoid cytokinins), or an aromatic derivative side chain (aromatic cytokinins).

Effects of plant growth regulators and l-glutamic acid on shoot organogenesis in the halophyte Leymus chinensis (Trin.)

Abstract: The halophyte Leymus chinensis (Trin.) is a perennial rhizome grass (tribe Gramineae) that is widely distributed in China, Mongolia and Siberia, where it is produced as a forage product. In this report, we establish a highly reproducible plant regeneration system through somatic embryogenesis. Two explants, mature seeds and leaf base segments were used; these parts displayed different responses to combinations of growth factors that affect embryogenic callus induction, callus type optimization and plant regeneration. The highest callus induction frequency was obtained on Murashige and Skoog (MS) medium supplemented with 2.0 mg l−1 2,4-dichlorophenoxyacetic acid (2,4-D) in the presence of 5.0 mg l−1 l-glutamic acid. The inclusion of 5.0 mg l−1 l-glutamic acid was found to significantly promote primary callus induction, embryogenic callus formation and callus status improvement. Subculturing on maintenance medium for 1–2 months before plant regeneration was found to be essential for the optimization of callus type and the maturation of embryogenic callus. Callus relative water content and growth rate were simultaneously investigated during callus maintenance, and found to possibly be related to callus type. Shoots were differentiated from the embryogenic callus on the optimal medium with MS salts containing 0.2–0.5 mg l−1 α-naphthalene acetic acid (NAA), 2.0 mg l−1 kinetin (Kn) and 2.0 g l−1 casamino acids in 71.0 and 69.2% of wild-type (WT) and Jisheng No.1 (JS) plants, respectively. Plant regeneration was variable depending on NAA levels, and the addition of casamino acids stimulated the maturation of embryogenic callus and plant regeneration. Transferring callus with shoots onto half-strength MS medium resulted in rooting within 1 week. The growth of regenerated plants was also surveyed in the field. This is the first report of plant regeneration through somatic embryogenesis from mature seeds and leaf base segments of L. chinensis.

Effect of Plant Growth Regulators on Callus, Cell Suspension and Cell Line Selection for Flavonoid Production from Pegaga (centella asiatica L. urban)

Abstract: Problem statement: Considering pegaga medicinal properties and over-exploitation, the requirement for a tissue culture technique as an alternative production system was crucial. Approach: Investigation of cell suspension culture response to different plant growth regulators (PRGs) for flavonoid production from elite cell line was carried out. Callus cultures were initiated from the leaf explants of Centella asiatica on Murashige and Skoog (MS) medium containing B5 vitamins and 30 g L−1 sucrose supplemented with different concentrations (0.5-2.5 mg L−1) of 2,4-D, NAA, Dicamba, Picloram and IBA supplied singly and in combination with different concentrations (0.5-1.5 mg L−1) of kinetin, BAP and TDZ. Results: Callus induction was observed for all the PGRs tested. The highest callus induction frequency (86.67%) was observed in MS medium containing 2.0 mg L−1 2,4-D while the combination of 2.0 mg L−1 2,4-D and 1 mg L−1 kinetin in MS medium gave the highest biomass yield (0.27 g dry weight culture−1). This combination was also found to be best for callus proliferation for all the accessions investigated. Among the four accessions tested, UPM03 was found to have the highest biomass yield (0.041 g DW culture−1) and hydrolysed flavonoid content (10.75 mg g−1 DW) after the 12th day of culture. The flavonoids present in the four accessions were quercetin, kaempherol, luteolin and rutin based on high performance liquid chromatography (HPLC) analysis. These results indicated that C. asiatica accession UPM03 was the potential elite cell line in mass production of flavonoid, especially luteolin. Coclusions/Recommendations: In the establishment of cell suspension culture, 2 mg L−1 2,4-D and 1 mg L−1 kinetin were the best PGRs in supporting the cell growth and flavonoid production. This is the first report on the use of PRGs on the establishment of cell suspension cultures in flavonoid production of C. asiatica.

Activity of antioxidant enzyme during in vitro organogenesis in Crocus sativus

Abstract: The effect of various hormonal combinations on regeneration of shoots and roots from meristem-derived callus of Crocus sativus L. and activities of antioxidant enzymes have been studied. The most efficient regeneration occurred with 1.0 mg dm−3 1-naphthaleneacetic acid (NAA) + 1.0 mg dm−3 thidiazuron and 1.0 mg dm−3 NAA + 2.0 mg dm−3 kinetin. For sprouting, regenerated shoot were subcultured on Murashige and Skoog medium containing 1.0 mg dm−3 NAA + 1.0 mg dm−3 benzylaminopurine (BAP). Protein content and superoxide dismutase activity decreased in regenerated shoots and roots and increased in sprouting shoots, while catalase (CAT), peroxidase (POX) and polyphenol oxidase (PPO) activities increased during organogenesis and decreased in sprouting shoots. High CAT and PPO activities were detected in regenerated roots, whereas high POX activity was observed in regenerated shoot.

Adventitious root suspension cultures of Hypericum perforatum : effect of nitrogen source on production of biomass and secondary metabolites

Abstract: The present study investigated the effect of nitrogen source (NH4+; NO3−) at different concentrations on the accumulation of biomass and secondary metabolites in adventitious root cultures of Hypericum perforatum L. Cultures were initiated in shake flasks by using half-strength Murashige and Skoog (MS) medium with B5 vitamins, 1.0 mg l−1 indole-3-butyric acid, 0.1 mg l−1 kinetin, 3% (w/v) sucrose, and different ratios of ammonium and nitrate (0:30, 5:25, 10:20, 15:15, 20:10, 25:5, and 30:0 mM, using NH4Cl and KNO3). The cultures were maintained in darkness. The medium supplemented with 5:25 (mM) NH4+/NO3− resulted in the optimum accumulation of biomass and total phenols and flavonoids. The antioxidant potential of a methanolic extract, measured as the 1, 1-diphenyl-2-picrylhydrazyl and 2, 2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) radical scavenging activities, of H. perforatum adventitious roots showed that antioxidant activity was high from root extracts that were grown on higher concentrations of NO3− nitrogen (15, 20, and 25 mM). Further, assessment of hydrogen peroxide (H2O2) and malondialdehyde content of the root extracts revealed that cultures supplemented with higher levels of NO3− nitrogen (15–30 mM) were under oxidative stress, which boosted the levels of secondary metabolites in the adventitious roots. These results suggest that optimal adventitious root biomass could be achieved with the supplementation of cultures with 5:25 ratios of MS nitrogen sources.

Direct plant regeneration from encapsulated nodal segments of Vitex negundo

Abstract: An efficient protocol for encapsulation of nodal segments of Vitex negundo L. has been developed for the production of non-embryogenic synthetic seeds. The encapsulations of nodal segments were significantly affected by the concentrations of sodium alginate and calcium chloride. A 3 % Na2-alginate with 100 mM CaCl2 has been found to be optimum concentration for the production of uniform synthetic seed. For germination, the synseeds were cultured on Murashige and Skoog (MS) basal medium supplemented with kinetin (KIN) and α-naphthalene acetic acid (NAA) either singly or in various combinations. MS medium containing 2.5 μM KIN in combination with 1.0 μM NAA was found to be the optimum for maximum (92.6 ± 3.71 %) plantlet conversion frequency. Well developed regenerated plantlets were hardened, acclimatized and established in field, where they grew well without any detectable variation.

Effect of the different auxins and cytokinins in callus induction, shoot, root regeneration in sugarcane

Abstract: Studies on micropropogation of callus culture was undertaken. In which expalnts were inoculated in MS medium fortified with various concentration of 2,4-D, auxin, cytokinin, sucrose at different pH level .The best callus induction was observed at 3.0mg/L, 2,4-D with 10% coconut milk (CM). Best regeneration of shoot was achieved when they were cultured on MS medium supplemented with BAP 1.0mg/L and IBA 0.5mg/L. Among the different media tested with 3mg/L NAA and 5% sucrose supplemented media proved best production of roots.

Adventitious shoot induction from cultured internodal explants of Malaxis acuminata D. Don, a valuable terrestrial medicinal orchid

Abstract: Malaxis acuminata is a terrestrial orchid that grows in shady areas of semi-evergreen to shrubby forests. It is highly valued for its medicinal properties as dried pseudo-bulbs are important ingredients of several Ayurvedic preparations. In this study, adventitious shoot buds were induced from internodal explants of M. acuminata grown on Murashige and Skoog (MS) medium supplemented with different concentrations of 6-benzyladenine (BA), kinetin (Kn), and thidiazuron (TDZ). Of the three cytokinins used, TDZ at 3 mg l−1 induced the highest frequency (82%) of organogenic explants. However, all responding explants produced only a single adventitious shoot irrespective of the type and concentration of the cytokinin. Adding 0.5 mg l−1 α naphthaleneacetic acid (NAA) to the medium enhanced adventitious shoot formation. In the presence of 3 mg l−1 TDZ and 0.5 mg l−1 NAA, frequency of organogenesis was 96% with a mean number of 6.1 shoots per explant. Prolonged culture or subculture on the same medium did not promote further shoot production. However, transfer of these cultures to MS medium supplemented with 3 mg l−1 TDZ and 0.5 mg l−1 NAA and various concentrations of different polyamines (PAs), including spermine, spermidine, and putrescine, significantly increased mean shoot number per explant. The highest frequency of shoot induction (100%) and mean shoot number per explant (14.6) was observed on MS medium with 3 mg l−1 TDZ, 0.5 mg l−1 NAA, and 0.4 mM spermidine. Regenerated shoots were excised and subcultured on an elongation medium consisting of MS medium with 3 mg l−1 BA. Moreover, the highest frequency of rooting (96%) and mean number of roots per shoot (3.3) was observed on MS medium with 4 mg l−1 indole-3-butyric acid (IBA) and 1.5 mg l−1 activated charcoal (AC). Almost 90% of rooted shoots were successfully acclimatized and established ex vitro.

In vitro plant regeneration system for Cassia siamea Lam., a leguminous tree of economic importance

Abstract: A method for rapid in vitro propagation of Cassia siamea Lam. using cotyledonary node explants, excised from 14-day old aseptic seedlings, has been established. Murashige and Skoog (MS) medium supplemented with different concentrations of 6-benzyladenine (BA), kinetin (Kn) and thidiazuron (TDZ) singly or in combination with auxins was used for regeneration studies. Among the single treatment of three cytokinins BA at 1.0 µM was found to be optimum for direct shoot regeneration as it induced an average of 8.20 ± 0.66 shoots per explant. The regeneration frequency further enhanced with the application of auxin along with optimal BA concentration. The highest frequency for shoot regeneration (90%), the maximum number of shoots per explant (12.20 ± 0.73) and the maximum shoot length (6.40 ± 0.07) cm were obtained on the medium consisted of MS + 1.0 µM BA + 0.5 µM NAA. Successful in vitro rooting was induced from cut end of the microshoots when placed on half-strength MS + IBA (2.5 µM). The regenerated shoots with well developed root system were successfully acclimatized and established in pots containing sterilized garden soil and garden manure (1:1) and grown under greenhouse conditions with 85% survival rate.

In vitro propagation for the conservation of a rare medicinal plant Justicia gendarussa Burm. f. by nodal explants and shoot regeneration from callus

Abstract: Justicia gendarussa is a valuable medicinal plant and various parts of this plant are pharmaceutically used for the treatment of different diseases. In vitro regeneration of shoot buds was obtained from culture of nodal cuttings as well as shoot regeneration from callus. The nodal cuttings differed in shoot proliferation in terms of percentage of explants that responded and average shoot length with various concentrations (4.4, 8.9, 13.3, 17.7, 22.2 μM) of 6-benzyladenine (BA), kinetin (Kn) and thidiazuron. In all treatments, one shoot was invariably present. Optimum 87% of cultures responded with an average shoot length of 4.4 cm on Murashige and Skoog (MS) medium supplemented with 17.7 μM BA. Callus was induced from the mature leaf segments on MS medium supplemented with Kn (4.7, 13.9, 23.2 μM) alone or in combination with 2, 4-dichlorophenoxyacetic acid (2, 4-D; 2.3 μM, 4.5 μM). Optimum callus induction (78%) was obtained on MS medium supplemented with 14 μM Kn and 4.5 μM 2, 4-D. When the callus was subcultured on MS medium fortified with BA (8.9, 17.7, 26.6 μM) or Kn (9.3, 18.6, 27.9 μM) alone or in combination with α naphthalene acetic acid (NAA; 2.7, 5.4 μM), shoot regeneration was obtained. The highest response (92%) was observed on MS medium containing 17.7 μM BA and 5.4 μM NAA. On this medium, an average number of 12.2 shoots were obtained per responding callus. The shoots obtained from callus and nodal cuttings were rooted with a frequency of 73% on MS medium augmented with 9.8 μM indole-3-butyric acid. The rooted shoots were successfully transplanted to soil and sand mixture (1:1) with 90% survival rate. The protocol standardized for shoot proliferation and regeneration in J. gendarussa from nodal cuttings and leaf-derived callus is suitable for micropropagation and conservation of this essential medicinal plant.