Showing papers on "Murashige and Skoog medium published in 1973"

Induction of pollen plants from anthers of triticum aestivum l. cultured in vitro

Abstract: Anthers of Triticum aestivum L. were cultured in vitro on the MS medium supplemented with auxins, with or without kinetin. Then the pollen grains were successfully, induced to develop into intact plants through a stage of callus formation. Experiments indicated that pollen grains at the mid-uninucleate stage were most favourable for induction to form calli, and that addition of lactalbumin hydrolysate and appropriate increase of the sucrose coneentration had some promoting effects on pollen callus formation. The pollen callus with relatively great differentiating capacity was generally compact in texture and possessed the histological characteristics of the primary meristem. It was noted that the results obtained with different materials (different varieties or hybrids of different crosses) varied considerably. The progeny of the plant induced from F 1, hybrid pollen showed no segregation of characters, suggesting that application of the anther culture method to plant breeding is very promising.

Ultrastructural aspects of shoot initiation in tobacco callus cultures

Abstract: An ultrastructural investigation of shoot initiation in tobacco (Nicotiana tabacum L. var. W. 38) callus cultures was made. Zones of preferential division were observed in the basal portion of the tissue by eight days in culture and these led, sequentially, to meristemoids, primordia, and shoots. During the initial stages of meristemoid formation, protein inclusions and large accumulations of plastid starch were present in the cells, while vacuoles were filled with membranous and cytoplasmic protrusions. At later stages of meristemoid development, these features were not observed in the cells, which were also smaller in size and possessed numerous small, peripheral vacuoles. It appears that the membranous and cytoplasmic protrusions are involved in vacuolar reduction during meristemoid formation. It would also appear that the storage materials supply the energy and other reserves needed for the organogenetic process. By contrast, tissue cultured under nonshoot-forming conditions and nonmeristemoid regions of shoot-forming tissue remained parenchymatous over the same time period. ZONES OF preferential cell division occur in the parenchymatous tissue of callus cultures of tobacco by day 8 (Thorpe and Murashige, 1970). Meristemoids arise from these areas, although not all areas of preferential cell division give rise to these structures. Meristemoids (Torrey, 1966) are the meristem-like aggregations of small, nonpolar cells which appear to be nonvacuolate at the light microscope level and from which primordia and ultimately leafy vegetative shoots are formed (Murashige, 1964; Thorpe and Murashige, 1970). The mechanism of meristemoid formation in tissue cultured under organ-forming conditions is not yet known, although it has been suggested that these could arise from single cells (Torrey, 1966). Associated with meristemoid development is a rapid increase and decrease in the amount of stored starch in tobacco callus (Thorpe and Murashige, 1968, 1970). This starch may serve as a source of energy for shoot formation which, judging by the respiratory activity of the tissue (Thorpe and Meier, 1972; Ross and Thorpe, 1973), has a high energy requirement. In this study, ultrastructural changes during meristemoid formation and shoot initiation were investigated. MATERIALS AND METHODS-Tobacco (Nicotiana tabacum L. var. 'Wisconsin 38') callus was isolated from stem pith segments and maintained on three-quarter strength Murashige-Skoog (MS) 1 Received for publication 27 November 1972. Supported by NRC of Canada grant no. A-6467 to T.A.T. medium (Murashige and Skoog, 1962). For shoot production, the tissue was grown on the modified MS medium reported earlier (Thorpe and Murashige, 1970) except that the concentrations of Ltyrosine, adenine sulphate, and NaH2PO4 H20 were reduced by half. The medium contained indole-3-acetic acid and kinetin in final concentrations of 10-5M. Cultures were maintained in darkness in 125-ml Erlenmeyer culture vessels containing 50 ml of medium. Sections of tobacco callus, each measuring ca. 3 x 3 x 2 mm, were used as inoculum. Thin sections, cut by hand, parallel to the surface in contact with the medium were irrigated with fixative (2/2 % glutaraldehyde) and scanned under a binocular microscope. Areas of preferential division, with prominent nuclei, and areas of shoot initiation were excised and prepared for electron microscopy. Each piece of tissue was ca. 1 mm3. Tissue was sampled from 8-, 10-, 12-, and 14-day-old shoot-forming tissue and from 8-, and 14-day-old nonshoot-forming tissue. Freehand monitor sections were also stained with iodinepotassium iodide (Jensen, 1962). Heavy blueblack staining for starch occurred in the regions visually determined as areas of preferential cell division or meristemoids (Thorpe and Murashige, 1968). The tissue samples were prefixed in 21?% glutaraldehyde in 0.1 M phosphate buffer, pH 6.8 -+ 0.5 for 4 hr. Following buffer washes, the material was fixed in 1 % unbuffered Os04 for 50 min. The tissue was then dehydrated in an ethanolic series after a further buffer wash and embedded in ERL-4206 (Spurr, 1969). Staining was

Shoot differentiation in callus cultures of Datura innoxia

Abstract: Datura innoxia Mill. callus cultures formed shoots in 2–4 weeks on media containing; a) gibberellic acid, b) indoleacetic acid, c) low concentrations of naphthylacetic acid, d) low concentrations of 2,4-dichlorophenoxyacetic acid, e) benzylaminopurine, f) no growth substance. Benzylaminopurine promoted shoot differentiation. Gibberellic acid inhibited shoot formation weakly, but inhibited proper leaf blade formation. Root differentiation was rare. The callus cultures of Datura innoxia grew rapidly (100-fold in 4 weeks) on a slightly modified Murashige and Skoog medium (0.5 mg/l thiamin · HCl, pH 5.5, no glycine) in light at 30°C. Callus grew well on any single one of the growth substances NAA (10−5M), 2,4-D (10−6M) or BAP (3 × 10−6M). Growth was less and more erratic on GA or IAA. The callus cultures did not grow significantly better when BAP was combined with one of the auxins or with GA.

5′-Phosphodiesterase Formation by Cultured Plant Cells

Abstract: 5′-Phosphodiesterase (5′-PDase) which degrades RNA to nucleoside-5′-monophosphates was investigated in various kinds of plant calli, and the calli of Vinca rosea and Phytolacca americana were found to have the high activity. The liquid culture conditions of the cells of V. rosea were examined. Three mg of kinetin and 0.5 mg of 2,4-dichlorophenoxyacetic acid per liter in the Murashige and Skoog medium were optimal for the growth and the 5′-PDase formation. Under the optimal conditions, time courses of the cell growth and the enzyme formation were measured.The 5′-PDase of the cultured cells of V. rosea in suspension showed the maximal activity at pH 8.0 and 60°C. A comparison of 5′-PDase of the cultured cells and of the mother plant of V. rosea was carried out and it was found that the cultured cells had more than 30 times as much 5′-PDase activity as the mother plant on dry cell weight basis.

Nutritional requirements for ovule formation in excised pistils of nigella

Abstract: The nutritional requirements for ovule formation in Nigella sativa L. were investigated by growing excised pistils on defined media. Pistils grown on a medium containing the minerals of Murashige and Skoog produced significantly more ovules than on a medium containing the minerals of Bilderback. When the nitrogen, sulfate, and phosphate of the Bilderback medium were adjusted to levels comparable to those of the Murashige and Skoog medium, a similar number of ovules was formed. The effect of different forms and concentrations of nitrogen on ovule formation and pistil growth was investigated. High concentrations of nitrate (40 mM) favored pistil growth and ovule formation, but comparable levels of ammonium were toxic. When ammonium at concentrations above 10 mM was added to nitrate media, ovule formation was inhibited. A medium containing low concentrations of ammonium (10 mM) and nitrate (5 mM) supported more ovule formation and pistil growth in young pistils than a low-nitrate (5 mM) medium without ammonium. However, ovule formation on a medium containing 10 mM ammonium and 5 mm nitrate was significantly less than on a medium containing only 15 mM nitrate. Low concentrations of organic nitrogen in the form of a-alanine (1 mM) and ,y-aminobutyric acid (5 mM) supported ovule formation and pistil growth similar to a high

Investigations on the induction and morphogenesis of wheat(triticum vulgare)pollen plants

Abstract: The development of haploid plants has been successfully induced from wheat (Triticum vulgare)pollens by means of the anther cultured in vitro.On the MS medium supplemented with 2-20mg/l 2,4-D and various organic addenda,out of 21094 inoculated anthers taken for culture from 27 hybrids or varieties,103 produced calli.All the calli developed from dehisced anther sacs.Microscopical examinations indicated that these calli were formed by repeated divisions of the pollen grains.5 days after,the pollen calli were transferred into the medium containing either 0.2-2mg/l NAA and 0.2-2mg/l kinetin or 0.5mg/l IAA and 15% cocoanut milk,differentiation of shoots began to occur.It was also found that if the anthers were inoculated on the MS medium containing either 20% cocoanut milk or 2mg/l IAA and 2mg/l kinetin,shoots would develop directly from the anther sacs.The chromosome counts in root tips of 6 plantlets examined gave the number 21,confir- ming that they are haploids.The haploid plants produced spikes but did not set seeds.