Skoog Nutrient Medium Research Articles

Haploid plant regeneration from unpollinated ovule cultures of niger (Guizotia abyssinica (L. f.) Cass.)

Haploid plants were regenerated in vitro from unpollinated ovules of niger (Guizotia abyssinica (L. f.) (Cass.) on Murashige and Skoog nutrient medium (MS) supplemented with 10 μM naphthaleneacetic acid or 10 μM NAA + 1.5 μM kinetin and 30 g/l sucrose. Gamborg (B5) medium was the best for plant regeneration (in comparison with MS, Nitsch and Nitsch (NN), and Chu (N6) media) from cultured ovules, and 6.66 and 7.33 ovules of JNC-6 and Ootacamund cultivars were involved in direct plant regeneration on this medium. Matured ovules (ovules collected one day before anthesis or on the day of anthesis) only responded to cultural regimes and involved in direct plantlet development. Cytological preparation of root tips and chloroplast counts in the guard cells of leaf stomata of regenerated plants confirmed their haploid nature.

Protein pattern changes in tomato under in vitro salt stress

The investigation of salt-induced changes in the proteome would highlight important genes because of a high resolution of protein separation by two-dimensional gel electrophoresis (2-DE) and protein identification by mass spectrometry and database search. Tomato (Lycopersicon esculentum Mill.) is a model plant for studying the mechanisms of plant salt tolerance. Seeds of tomato cv. Shirazy were germinated on water-agar medium. After germination, seedlings were transferred to Murashige and Skoog nutrient medium supplemented with 0, 40, 80, 120, and 160 mM NaCl. After 24 days, leaf and root samples were collected for protein extraction and shoot dry weight measurement. Alterations induced in leaf and root proteins under salt stress treatments were studied by one-dimensional SDS-PAGE. Leaf proteins were also analyzed by 2-DE. With increasing salt concentration in the medium, shoot dry weight decreased. SDS-PAGE showed induction of at least five proteins with mol wts of 30, 62, and 75 kD in roots and 38 and 46 kD in leaves. On the 2-DE gel, more than 400 protein spots were reproducibly detected. At least 18 spots showed significant changes under salt stress. Three of them corresponded to new proteins, while six proteins were up-regulated and five proteins were down-regulated by salt stress. In addition, salinity inhibited the synthesis of four leaf proteins. Ten spots were analyzed by matrix-assistant laser desorption/ionization-time of flight (MALDI-TOF), which led to the identification of some proteins, which could play a physiological role under salt stress. The expression of new proteins(enoyl-CoA hydratase, EGF receptor-like protein, salt tolerance protein, phosphoglycerate mutase-like protein, and M2D3.3 protein) under salt stress indicates that tomato leaf cells respond to salt stress by changes in different physiological processes. All identified proteins are somehow related to various salt stress responses, such as cell proliferation.

Galanthamine production by Leucojum aestivum in vitro systems

The callus induction from young fruits of Leucojum aestivum was performed on Murashige–Skoog nutrient medium supplemented with 4 mg/L 2,4 dichlorphenoxyacetic and 2 mg/L 6-benzylamynopurine. Further, by planting the obtained calluses on the same nutrient medium supplemented with 1.15 mg/L α-naphtylacetic acid and 2.0 mg/L 6-benzylamynopurine shoot cultures were established. The growth and galanthamine accumulation of obtained L. aestivum in vitro systems were studied. It was established that the amount of accumulated galanthamine strongly depended on the level of the differentiation. The maximum yield of biomass (17.8 g/L) and the maximum amount of accumulated galanthamine (2.5 mg/L) were achieved after day 35 of submerged cultivation of L. aestivum 80 shoot culture, performed under illumination. Data concerning the time courses of the utilization of the main nutrient components of the medium during cultivation of L. aestivum shoot culture are presented as well.

A novel in vitro hydroponic culture system for potato (Solanum tuberosum L.) microtuber production

An innovative in vitro hydroponic culture system used in potato (Solanum tuberosum L.) microtuber production is described in this paper. In vitro potato plantlets, 6–8 cm in height, derived from meristems of potato tubers cultured on 1/2 Murashige and Skoog (MS) nutrient medium after 30 days culture were cut into 1.5 cm stem node segments and used as explants. These stem nodes were cultured in a novel system called in vitro hydroponic culture system containing 1/2 MS medium supplemented with 0.5 μM naphthaleneacetic acid (NAA), 0.3 μM gibberellic acid (GA3), 3.7 μM adenine sulfate, 10% coconut water, 0.5 g/l activated charcoal, 80 g/l sucrose with or without 8 g l−1 agar. Liquid medium was distributed to the carrier substrates in each storey of the system with the aid of capillary robes. In the present paper, the effects of porous material used as substrate carrier and the number of storeys involved in the culture system on microtuber formation and their morphological characteristics are reported. Cotton layer substrate is more stable for organogenesis of potato microtubers. Microtubers, 3.19 mm in diameter and 49.82 mg in weight, could be harvested from a one-storey in vitro hydroponic culture system containing filter paper as substrate. However, microtubers cropped from three-storey in vitro hydroponic culture system with cotton layer were bigger and weightier than those from three-storey system containing filter paper. The above results of the in vitro hydroponic system examined in this study might open up a new approach in producing potato and other hygrophilous microtuber.

Effect of Rol Transgenes, IAA, and Kinetin on Starch Content and the Size of Starch Granules in Tubers of In Vitro Potato Plants

Stem cuttings were produced from Solanum tuberosum L., cv. Desiree, plants and their transgenic forms harboring rolB and rolC genes from Agrobacterium rhizogenes. Plants were cultured on hormone-free Murashige and Skoog nutrient medium (MS) and on MS supplemented with IAA or kinetin. In microtubers developed on these cuttings, we estimated the content of starch and the number and size of starch granules. Expression of rol genes changed these indices: in tubers of rolC transformants, a greater number of small granules were produced, whereas in tubers of rolB transformants, a fewer number of large granules were developed as compared with wild-type plants. Expression of rol genes did not affect starch content during the first three weeks of cutting culturing but increased it by 15–30% in five-week-old tubers. IAA addition to MS medium increased starch content and the size of starch granules in control plants and rolB tubers by 10–30%, whereas kinetin did not exert any significant influence. The effects of rol transgenes on the initiation and termination of starch granule development are discussed.

Photoperiodic and Hormonal Control of Tuberization in Potato Plants Transformed with the PHYB Gene from Arabidopsis

We studied photoperiodic and hormonal regulation of tuberization in wild-type potato (Solanum tuberosum L., cv, Desiree) plants and derivative transgenic plants harboring the PHYB gene from Arabidopsis, which encodes the phytochrome B apoprotein, under the control of the cauliflower mosaic virus 35S promoter. Plants were cultured on hormone-free Murashige and Skoog nutrient medium containing 5% sucrose or on the same medium supplemented with 1 mg/l kinetin under conditions of long day (LD, 16 h), short day (SD, 10 h), or SD with interrupted long night. We estimated cytokinins (zeatin and zeatin riboside) in underground and aboveground plant organs by the ELISA technique and GA activity in a bioassay with dwarf pea seedlings. Under LD conditions, transgenic plants produced substantially less tubers than wild-type plants. Kinetin addition to the culturing medium resulted in stimulation of tuberization under LD conditions, especially pronounced in the PHYB plants. The content of cytokinins and the activity of GA were much higher under LD conditions, especially in leaves. The total level of both phytohormones was higher in transformed as compared to wild-type plants. A relation of phytochrome-dependent tuberization to the hormonal status of underground and above-ground plant organs and possible reasons for kinetin stimulatory effect on this process are discussed.

The Effect of Tobacco Plant Transformation with a Gene for Acyl-Lipid Δ9-Desaturase from Synechococcus vulcanus on Plant Chilling Tolerance

Tobacco plants with the introduced desC gene for acyl-lipid Δ9-desaturase from the thermophilic cyanobacterium Synechococcus vulcanus were cultivated on agar-solidified Murashige and Skoog nutrient medium supplemented with ferulic acid and antibiotics at 22°C and a 16-h photoperiod. Control plants were transformed with an empty pGA482 vector. The analysis of fatty acids (FAs) showed that, in transgenic plants, the level of 16:0 and 18:0 FAs decreased substantially, whereas the levels of di- and trienoic FAs increased. Transformed plants were more cold-tolerant. The tolerance to chilling was evaluated from electrolyte leakage from tissues damaged by cold treatments and from the accumulation of a product of lipid peroxidation, malondialdehyde. It was concluded that acyl-lipid Δ9-desaturase was actively expressed in transgenic tobacco plants and converted stearic acid into oleic acid, thus producing a substrate for further synthesis of di- and trienoic FAs. An increased proportion of polyunsaturated FAs in membrane lipids resulted in improved tobacco plant tolerance to chilling.

In vitro culture of Pandanus amaryllifolius and enhancement of 2‐acetyl‐1‐pyrroline, the major flavouring compound of aromatic rice, by precursor feeding of L‐proline

AbstractShoots, plantlets and semi‐differentiated callus (SDC) cultures of Pandanus amaryllifolius capable of producing high levels of basmati rice flavour were established in vitro using Murashige and Skoog nutrient medium. A total of 10% of the initial explants responded to produce shoot cultures in the presence of benzylamino purine (BAP) (0.5 mg L−1) and glutamine (100 mg L−1). Leaf explants and basal portions of shoots produced SDC whereas elongated in vitro shoots could be continuously multiplied, using BAP (1.5 mg L−1) and kinetin (Kn) (1.0 mg L−1), and rooted in half‐strength medium for ex vitro cultivation leading to a process of micropropagation. Steam‐distillation extraction (SDE) followed by gas chromatography‐mass spectrometry (GC‐MS) analysis of various cultured organs and spent liquid medium used for SDC revealed the presence of 2‐acetyl‐1‐pyrroline (2‐AP) to various extents. This 2‐AP compound has been identified as the major flavouring compound of scented basmati and other scented rice varieties. 2‐AP was found to be highest, on a fresh weight basis, in SDC (19.7 mg kg−1) on the 40th day, whereas in vitro roots, shoots and field leaves (of one‐year‐old plant) had lower levels of 15, 6.8 and 14 mg kg−1, respectively. Further enhancement of 2‐AP in SDC using precursor was possible by feeding into medium 1 mmol L−1 of L‐proline where a highest level of 21.67 ppm of 2‐AP accumulated on the seventh day whereas a higher level of 2 mmol L−1 of L‐proline suppressed 2‐AP levels. The present report is the first on the tissue culture studies of P. amaryllifolius where continuous production of plantlets as well as synthesis of high levels of 2‐AP has been documented. Copyright © 2005 Society of Chemical Industry

The effect of seed cryopreservation on the development of protocorms by the hybrid orchid Bratonia

The aim of this work was to study the influence of storage in liquid nitrogen on the viability of seeds of the hybrid orchid Bratonia and further development of its protocorms in vitro. Seeds were frozen in ampoules by direct immersion in liquid nitrogen and stored in the cryobank for a month. The germination rates of cryopreserved and control (nonfrozen) seeds did not differ and remained as high as 100%. The protocorms derived were cultured on the agar-solidified Murashige and Skoog nutrient medium (MS), half-strength MS and Knop media and also in Morel liquid medium. During the first 45 days of culturing, protocorms derived from cryopreserved seeds grew faster than control protocorms on the MS and half-strength MS media but, at longer culturing (496 days), the size of control protocorms was significantly larger. After 639 days of culturing, there was no difference in the amount of perished, budding, and newly formed protocorms obtained from cryopreserved and control seeds, except half-strength MS medium where the number of budding protocorms in the case of cryopreserved seeds was a little greater than in the control treatment. After seed cryopreservation, the frequency of budding and newly formed protocorms was greater on the agarized MS and in liquid Morel media. Cryopreservation had little effect on the subsequent growth of protocorms in vitro. The preferable nutrient media for culturing the protocorms have been suggested.

The effect of boron on calcium uptake and growth in micropropagated potato plantlets

Boron (B) requirements differ widely among plant species and the concentration range between toxicity and deficiency is less for B than for any other nutrient. Excess B can adversely affect calcium (Ca) uptake and plant growth. Potato cvs Bintje and Norland plantlets were micropropagated on Murashige & Skoog (MS) nutrient medium, supplemented with 3 (MS control level) or 9 mM Ca, and a range of H3BO3 levels (0.025 to 0.300 mM B). Medium B levels of 0.100 and 0.300 mM decreased Ca content in leaves and shoots of cv. Norland, but not Bintje. Medium B level of 0.025 mM, which is 25% of the control MS level, enhanced Ca uptake in cv. Norland and did not compromise normal plantlet growth in either cultivar. This lower H3BO3 level (0.025 mM B), and a B-free gelling agent such as Gelrite, could be used for micropropagation of potato and possibly other species susceptible to Ca defifiency disorders.

In vitro propagation of Petasites hybridus (Asteraceae) from leaf and petiole explants and from inflorescence buds.

In vitro shoot regeneration from sterile leaf and petiole explants and from in-situ-collected inflorescence buds of Petasites hybridus was achieved by a simple two-step protocol. Murashige and Skoog (MS) nutrient medium was supplemented with 17.6 μM benzyladenine (BA)+0.54 μM naphthaleneacetic acid (NAA) to induce shoots. After 5 weeks of culture, 40% of the petiole and 27% of the leaf explants produced shoots compared to 76% of the inflorescence buds. Single shoots were excised and subcultured on MS medium supplemented with various cytokinins (N6-(Δ2-isopentenyl)adenine, BA, kinetin and thidiazuron). A concentration of 8.8 μM kinetin+0.54 μM NAA performed best in terms of shoot multiplication rate, average shoot length and spontaneous root induction.

IN VITRO PROPAGATION OF AN EGYPTIAN LANDRACE OF GREEN PEPPER AND BREEDING FOR TOLERANCE TO HEAT AND DROUGHT STRESSES

There has been a renewed interest in landraces and local cultivars of crop species as source of genetic variation for tolerance to biotic and abiotic stresses. In our study we used a pepper (Capsicum annuum L.) landrace collected from different farms at Assiut. Shoot-buds on the basal 5 to 7 stem nodes from plants at the end of the growing season were used to propagate desirable individuals in vitro. The greatest number of shoots were produced in cultures on Murashige and Skoog (MS) nutrient medium containing 3 μM benzyladenine (BA) alone or plus 0.9 μM Indole-3-acetic acid (IAA) and on the medium with 5 μM BA alone or plus 1.5 μM IAA. Useful variation was observed under heat and drought stress conditions. Population raised from bulked seeds of tested progenies showed significant increase in percentage of fruit set, fruit weight, number of fruits, and fruit yield compared to population of the original landrace collection. This in vitro method would be useful to conserve field collections from such germplasm. Copies of desired variants can be grown under favorable conditions for producing explants of anthers to use in gamete sorting and developing double-haploid lines. Chemical names used: N-(phenylmethyl1H-[benzyladenine, BA], 1H-indole-3-acetic acid (IAA), α-naphthaleneacetic acid (NAA).

Rapid clonal multiplication through in vitro axillary shoot proliferation of Aegle marmelos (L.) Corr., a medicinal tree.

Rapid clonal multiplication of Aegle marmelos (L.) Corr. (Rutaceae), a medicinal tree, was achieved by enhanced axillary bud proliferation in young single-node segments of a 25-year-old tree cultured in Murashige and Skoog (MS) nutrient medium. Bud break was dependent on cytokinin supply, but the synergistic combination of 2.5 mg l-1 6-benzylaminopurine (BAP) and 1.0 mg l-1 indole-3-acetic acid (IAA) induced the formation of 12.1 shoots of up to 5.2 cm length in 48% of the explants after 7 weeks of culture. Explants of in-vitro-grown shoots - node, whole leaf, shoot tip and internode - were subcultured in the presence of 0.05-2.5 mg l-1 BAP to produce 11.3, 18.4, 5.3 and 3.2 shoots and shoot buds at a 100%, 70%, 95% and 40% rate respectively, in 7 weeks. Different shoot nodes and leaves were equally regenerative and adventitious organogenesis in the latter was confined to cut petiolar ends. Nodal explants responded most favourably at low BAP (0.05-0.1 mg l-1) and produced uniform (3.8-5.3 cm) shoots facilitating their simultaneous harvest for rooting. Repeated subculturing through five cycles of nodes and leaves of shoot cultures enabled continuous production of healthy callus-free shoots without any sign of decline. Shoot cuttings (3.0-5.2 cm) were best rooted in half-strength MS medium with 0.5 mg l-1 IAA (70%) or 10.0 mg l-1 indole-3-butyric acid (90%). Eighty-eight percent of the rooted plants were established in polybags after hardening.

Cryopreservation of Chrysanthemum cinerariaefolium Vis. cells and its impact on their pyrethrin biosynthesis ability.

An efficient protocol for cryogenic storage of high-pyrethrin-producing cell lines of Chrysanthemum cinerariaefolium is described. Optimal survival (92%) was obtained with cells precultured in 1/2 Murashige and Skoog nutrient medium containing 180 g ⋅ l-1 sucrose for 30 days, then incubated in the same medium in the presence of 5% DMSO for 1 h in an ice bath, cooled slowly to -20°C and immersed for 30 min in liquid nitrogen. After cryopreservation, the cells conserved the same growth pattern, but displayed different biochemical properties. The subculture derived from the thawed cells was characterized by a lower chlorophyll content and a higher pyrethrin biosynthesis ability.

Somatic embryogenesis and plant regeneration from immature embryos of five families of Quercus acutissima.

Immature embryos of sawtooth oak (Quercus acutissima Carruth.) were obtained from five seed families and cultured on modified Murashige and Skoog nutrient medium containing 1 g/l L-glutamine and 5 mM proline and supplemented with 1.0 mg/l indole-3-butyric acid and 1.0 mg/l 6-benzylaminopurine. The frequency of somatic embryogenesis from immature embryos was a function of the collection date and seed family. The highest frequency of explants forming somatic embryos was obtained with seeds of family Chungnam 11, i.e. 7 weeks post-fertilization (90.9%) and 9 weeks post-fertilization (91.2%). No response was shown by families Chungnam 14, 15 and Jeonbook 29 (0%), at 10 weeks post fertilization. During germination, the highest frequency of epicotyl formation was obtained with Chungnam 11 (44.0%) or Chungnam 15 (43.5%), and the highest rate of radicle formation was shown by Chungnam 11 (26.1%). The most responsive seed family with respect to the formation of both epicotyl (43.5%) and radicle (26.1%) was Chungnam 11. Twenty plantlets were transplanted to a perlite:peatmoss:vermiculite (1:1:1) soil mixture, and 8 plants survived in the field.

Embryogenic Callus Production, Plant Regeneration and Transient Gene Expression Following Particle Bombardment in the Pasture Grass, Cenchrus ciliaris (Gramineae)

Callus initiated from surface sterilised, mature seeds of buffel grass (Cenchrus ciliaris L.) gave rise to an embryogenic form when cultured on Murashige and Skoog's nutrient medium supplemented with 3% sucrose, 5% coconut water and 4 mg L-1 2,4-D. Multiple green shoots regenerated on 20% to 50% of embryogenic calli after transfer to hormone-free medium and incubation in the light. Variations in cytokinin concentration and light intensity during regeneration did not significantly increase the regeneration frequency or the number of shoots produced. Regenerated plants developed normally when transplanted to soil. A high frequency of transient expression of the β-glucuronidase gene resulted following transfer into embryogenic callus by particle bombardment. This is a promising system for production of transformed buffel grass plants, if the frequency of shoot production can be increased.

Study of the In Vitro Rooting of Kazanlak Oil-Bearing Rose (Rosa damascenaMill.)

ABSTRACT The effect of several factors on in vitro rooting of the Kazanlak oil-bearing rose has been studied. Using the Murashige Skoog nutrient medium, the concentrations of naphthalene acetic acid (0.05 and 0.1 mg/L), the presence of phloroglucinol and variation in the quantity of mineral salts used were evaluated. Also, the conditions of microplant cultivation such as temperature, light and photoperiod, as well as the changes during growing in a two-phase-nutrient medium (solid, including the tested variants, plus liquid) were studied. Optimum results were obtained using a nutrient medium with 25% of the recommended mineral salts normally encountered in the medium plus 0.1 mg/L naphthalene acetic acid and 126 mg/L phloroglycinol in the presence of light. Analogous results were obtained for both the single and two-phase-nutrient media.

Cryopreservation of immature spring wheat zygotic embryos using an abscisic acid pretreatment

Spring wheat (Triticum aestivum L.) zygotic embryos were successfully cryopreserved, without the addition of exogenous cryoprotectants, using only an abscisic acid (ABA) pretreatment. Optimum survival was obtained when embryos were cultured in vitro for 10 days on semisolid Murashige and Skoog (MS) nutrient medium supplemented with 0.5 mg/L (±) ABA prior to cryopreservation. The embryos resumed growth within three days when returned to MS medium devoid of ABA but containing 2mg/L 2,4-dichlorophenoxyacetic acid. The embryogenic calli produced from these embryos exhibited normal plant regeneration on auxin-free media. Changes in dw/fw ratio, as well as the esterified fatty acid and sucrose concentrations correlated positively with the development of tolerance to cryopreservation.

Regeneration of Sideritis angustifolia (Labiatae) Plants from Single Cell Cultures

Mechanically isolated cells from hypocotyl-derived calli of Sideritis angustifolia Lag. were inoculated on agar-solidified Murashige and Skoog (MS) nutrient medium containing CH and different concentrations and combinations of NAA and BA. Isolated cells underwent continued divisions giving rise to calli after 30 days of culture. Best results were obtained when medium was supplemented with 0.5 μM NAA and 4.4 or 8.8 μM BA or with 5 μM NAA and 4.4μM BA. Calli obtained in the presence of 0.5 μM NAA and 8.8 μM BA regenerated shoot-buds when transferred to MS medium without CH and supplemented or not with these growth regulators. Once shoots were excised, the calli maintained their morphogenic competence along several subcultures in hormone-free MS medium. Regenerated shoots were easily rooted on MS medium without growth regulators.

In Vitro Propagation of Sideritis angustifolia Lag.

Summary Hypocotyl, root and cotyledon explants from 30 day old seedlings of Sideritis angustifolia Lag. were cultured on Murashige and Skoog nutrient medium. Organogenesis of roots and shoots was induced with treatments containing BA or KIN in combination with NAA, IAA or 2,4-D, however, those combinations including NAA and BA were optimal. Most regenerated shoots developed roots when transferred to medium without growth regulators. Rooted plantlets were successfully transplanted into soil.