Thin Cell Layer Explants Research Articles

Cellular changes in early development of regenerating thin cell layer-explants of rapeseed analyzed by light and electron microscopy

Cellular changes in early development of regenerating thin cell layer-explants of rapeseed analyzed by light and electron microscopy

Immunological Evidence of Thaumatin-Like Proteins during Tobacco Floral Differentiation

Tobacco proteins that share homology with thaumatin, a sweet protein of Thaumatococcus daniellii Benth., are produced in various physiological situations such as pathogenesis-related stress or water deficit stress. Using purified polyclonal anti-thaumatin antibodies, we have detected other thaumatin-like proteins in tobacco (Nicotiana tabacum var Samsun) that have been related with floral differentiation. Thaumatin-like proteins with apparent molecular masses of 42.6, 31.6, and 26.3 kilodaltons were found in immature and mature flower organs in vivo, and others of 46.7, 41.7, and 27.5 kilodaltons were exclusively detected in thin cell layer explants forming flowers. In situ immunolocalization revealed their synthesis in newly differentiated floral meristems, in tracheids, and in parenchyma cells.

Growth Stimulation by Catecholamines in Plant Tissue/Organ Cultures

Addition of catecholamines at micromolar concentrations caused a dramatic stimulation of growth of tobacco (Nicotiana tabacum) thin cell layers (TCLs) and Acmella oppositifolia "hairy" root cultures. A threefold increase in the rate of ethylene evolution was observed in the catecholamine-treated explants. Aminooxyacetic acid and silver thiosulfate, inhibitors of ethylene biosynthesis and action, respectively, reduced the growth-promoting effect of dopamine. However, these compounds alone could also inhibit the growth of the TCL explants. When ethylene in the culture vessel was depleted by trapping with mercuric perchlorate, dopamine-stimulated growth was still obtained, suggesting that ethylene does not mediate the dopamine effect. Dopamine potentiated the growth of TCLs grown in Murashige and Skoog medium supplemented with indoleacetic acid (IAA) and kinetin. When IAA was replaced by 2,4-dichlorophenoxyacetic acid, dopamine addition showed no growth-promoting effect. Instead, 2,4-dichlorophenoxyacetic acid stimulated the growth of TCL explants to the same extent as that obtained with IAA plus dopamine. Because synthetic auxins do not appear to be substrates for IAA oxidizing enzymes, we hypothesized that catecholamines exert their effect by preventing IAA oxidation. Consistent with this explanation, dopamine (25 micromolar) inhibited IAA oxidase activity by 60 to 100% in crude enzyme extracts from tobacco roots and etiolated corn coleoptiles, but had no effect on peroxidase activity in the same extracts. Furthermore, addition of dopamine to TCL cultures resulted in a fourfold reduction in the oxidative degradation of [1-(14)C]IAA fed to the explants. Because the growth enhancement by catecholamines is observed in both IAA-requiring and IAA-independent cultures, we suggest that these aromatic amines may have a role in the regulation of IAA levels in vivo.

The early development in regeneration of rapeseed from thin cell layer explants analyzed by light and electron microscopy

The early development in regeneration of rapeseed from thin cell layer explants analyzed by light and electron microscopy

Chitinase, beta-1,3-glucanase, osmotin, and extensin are expressed in tobacco explants during flower formation.

Sequence analysis of five gene families that were isolated from tobacco thin cell layer explants initiating floral development [Meeks-Wagner et al. (1989). Plant Cell 1, 25-35] showed that two encode the pathogenesis-related proteins basic chitinase and basic beta-1,3-glucanase, while a third encodes the cell wall protein extensin, which also accumulates during pathogen attack. Another sequence family encodes the water stress-induced protein osmotin [Singh et al. (1989). Plant Physiol. 90, 1096-1101]. We found that osmotin was also induced by viral infection and wounding and, hence, could be considered a pathogenesis-related protein. These genes, which were highly expressed in explants during de novo flower formation but not in explants forming vegetative shoots [Meeks-Wagner et al. (1989). Plant Cell 1, 25-35], were also regulated developmentally in day-neutral and photoresponsive tobacco plants with high expression levels in the roots and moderate- to low-level expression in other plant organs including flowers. An unidentified gene family, FB7-4, had its highest level of expression in the basal internodes. Our findings indicate that these genes, some of which are conventionally considered to encode pathogen-related proteins, also have a complex association with normal developmental processes, including the floral response, in healthy plants.

Control of morphogenetic pathways in thin cell layers of tobacco by pH

Thin cell layer explants of tobacco were floated in vitro on the surface of liquid culture media. The initial exogenous concentrations of indolyl-3-butyric acid, and kinetin, the initial medium pH, and the explant density were varied. Various patterns of de novo and direct differentiation without any intermediate callus (flower, vegetative bud, root) as well as the absence of morphogenesis and callus formation without any subsequent organogenesis were separately controlled on 100% of the explants. On the same exogenous combination of glucose, indolyl-3-butyric acid, and kinetin, changes in initial medium pH changed the pattern of morphogenesis. For a given initial exogenous indolyl-3-butyric acid concentration, vegetative buds were obtained at either pH 6.1 or 7.8, whereas a mixture of flowers and vegetative buds was obtained at pH 6.8. Furthermore, changes in explant density changed the morphogenetic response. It is suggested that the effects of the initial medium pH and explant density on morphogenesis may be related partially to modifications of the physicochemical properties of the cell wall and (or) plasmalemma.

Tobacco genes expressed during in vitro floral initiation and their expression during normal plant development.

Since the transition from vegetative to floral development in plants is likely to be influenced by gene expression in several plant organs, we have used an in vitro system, the tobacco "thin cell layer" system as a model for investigating gene expression associated with the initiation of flowering in higher plants. cDNA cloning has been used to identify mRNAs abundant during thin cell layer floral initiation. These genes are expressed in thin cell layer explants initiating floral meristems but not in thin cell layer explants initiating vegetative shoot meristems or possessing roots. Two of these genes are expressed transcriptionally in incipient floral apices during normal plant development. Transcripts of these genes, plus a third gene, occur at low levels in several plant organs and at high levels in the roots, with the maximum levels of root expression reached just prior to the formation of floral meristems.

Cytokinin Habituation in Juvenile and Flowering Tobacco

Abstract The possible link between cytokinin and flowering was examine in tobacco. The degree of cytokinin autotrophy and the competence for cytokinin habitution were measured in callus derived from pith tissue of Nicotiana tabacum cvs. H425 an W38. Explants were taken from internodes at all positions up the stem in juvenile and mature plants. To test whether the competence of cells to form flowers was linked with crtokinin habituation, thin cell layer explants from comparable internodes were tested for their ability to form floral buds. Callus derived from the upper parts of plants showed cytokinin autotrophy whether or not the plants were flowering. Flower buds were formed only on thin cell layer explants from the upper part of plants which were already flowering. Cytokinin habituation and competence to flower are therefore not directly linked although cytokinin habituation could be a prerequisite for meristematic activity and for flowering. Pith from internodes in the lower half of mature pants formed callus which was cytokinin-dependent, although these same internodes in juvenile plants were cytokinin-autotrophic. The ability to form cytokinin-autotrophic callus was therefore greatest in the meristematic regions and was lost as the pith cells aged. Competence to habituate after 35 °C treatment was also shown by pith callus from a few internodes in the middle of the plant below those already forming cytokinin-autotropic callus.

Agrobacterium-mediated transformation of thin cell layer explants from Brassica napus L.

Agrobacterium-mediated transformation of thin cell layer explants (Klimaszewska and Keller 1985) yielded large numbers of transgenic plants of a major Canadian rapeseed cultivar Brassica napus ssp. oleifera cv Westar. The morphology and fertility of these plants were indistinguishable from controls. The Ti plasmid vector, pGV3850 (Zambryski et al. 1983) was used as a cis vector and as a helper plasmid for the binary vector pBin19 (Bevan 1984). Selectable marker genes that conferred resistance to high levels of kanamycin (Km) on Nicotiana tabacum were less efficient in the selection of transgenic B. napus. At low levels of Km (15 μg/ml) large numbers of transgenic plants (50%) were identified among the regenerants by nopaline synthase activity and several of these were confirmed by Southern blot analyses. Only a small number were resistant to higher levels of Km (80 μg/ml). Preliminary analyses indicated that resistance to Km was transmitted to the selfed progeny. Chimeric chloramphenicol acetyl transferase genes were ineffective biochemical markers in transgenic B. napus.

Direct Organogenesis from Petiole and Thin Cell Layer Explants in Sugar Beet CulturedIn Vitro

Direct Organogenesis from Petiole and Thin Cell Layer Explants in Sugar Beet Cultured<i>In Vitro</i>

High frequency plant regeneration from thin cell layer explants of Brassica napus

Explants composed of the epidermis and 4–9 layers of subepidermal cells were excised from internodes of Brassica napus L. ssp. oleifera cv. Westar and cultured on modified Murashige and Skoog (MS) medium. The three or four terminal internodes excised from plants at an early stage (before any flower buds had opened) were shown to be the best explant source. Both cytokinin and auxin were required for induction of shoot organogenesis. Of six auxins tested, only naphthaleneacetic acid (NAA) was effective in shoot bud initiation. All four cytokinins tested (when associated with 0.5 mgl-1 NAA) promoted organogenesis, but at differing frequencies. The highest shoot induction frequency was obtained at 10–15 mgl-1 benzyladenine (BA). The organogenic response was strongly affected by the nitrogen content of the medium. The best response was observed when NO3 - was the sole nitrogen source (supplied as KNO3) in the range 30–90 mM. Sucrose and glucose were equally supportive in shoot regeneration with the optimal levels at 0.12 M and 0.15 M, respectively. Shoots were rooted on medium free of growth regulators and mature plants were grown in the greenhouse. Plants were also recovered from leafy structures which differed morphologically and histologically from shoot buds.