DNA synthesis, cell division and specific cytodifferentiation in cultured pea root cortical explants

Pea root segments cut 10–11 mm behind the tip of germinating seedlings were prepared by removal of the central cylinders with a tissue punch. These cortical explants were cultured aseptically on nutrient medium containing auxin with and without added cytokinin. In the absence of kinetin, the cortical cells enlarged and separated but failed to show DNA synthesis, mitosis, cell division or subsequent cytodifferentiation. In the presence of 1 ppm kinetin, cortical nuclei showed ³H-thymidine incorporation beginning between 24 and 32 hr; mitoses began about 48 hr, reaching a maximum of 6% at 60 hr. From an initial number of 8000 cells per segment, the cell count increased to 37,000 by day 7 and 140,000 by day 21. At the outset all mitoses were tetraploid; with time the proportion of tetraploid mitotic cells decreased and an octaploid population increased. A frequency of less than 10% diploid mitoses was observed after day 5. Only 25% of the cortical cells showed initial labeling. Beginning on day 7 tracheary elements differentiated from cortical derivatives. By day 14 about 25% and by day 21 about 35% of the total cell population had formed tracheary elements. As a system for analysis in biochemical and cytological terms, pea cortical explants represent an excellent system for the study of cytodifferentiation.     

Growth regulation, plastid differentiation and the development of a photosynthetic system in cultured carrot root explants as influenced by exogenous sucrose and various phytohormones

Chromoplasts, which exist in the cells of freshly isolated carrot root explants, seemed to be transformed in thylakoid containing plastids, and chlorophyll formation was initiated if the explants were cultured in a liquid medium containing inositol and IAA as a hormonal supplement. This process was intensified when kinetin was also added, but no dependence on a sucrose supply could be found.A sucrose supply of 2% in conjunction with the combination of all three hormones, however, was needed to achieve maximal thylakoid formation including stacking in individual chloroplasts and for the very extensive chloroplast multiplication in explants growing with high cell division activity. It should be noted that the number of plastids per cell is strongly increased by the sucrose supplement which leads also to starch accumulation. However, no transformation into chloroplasts occurred without the hormonal stimulus.     

Growth regulation,plastid differentiation and the development of a photosynthetic system in cultured carrot root explants as influenced by exogenous sucrose and various phytohormones

Chromoplasts, which exist in the cells of freshly isolated carrot root explants, seemed to be transformed in thylakoid containing plastids, and chlorophyll formation was initiated if the explants were cultured in a liquid medium containing inositol and IAA as a hormonal supplement. This process was intensified when kinetin was also added, but no dependence on a sucrose supply could be found. A sucrose supply of 2% in conjunction with the combination of all three hormones, however, was needed to achieve maximal thylakoid formation including stacking in individual chloroplasts and for the very extensive chloroplast multiplication in explants growing with high cell division activity. It should be noted that the number of plastids per cell is strongly increased by the sucrose supplement which leads also to starch accumulation. However, no transformation into chloroplasts occurred without the hormonal stimulus.     

Biochemical and histological changes associated with <Emphasis Type="Italic">in vitro</Emphasis> responses in sunflower cotyledonary explants

In vitro shoot regeneration from sunflower cotyledonary explants can be obtained in the presence of kinetin and indole-3-acetic acid. In contrast, callus proliferation is obtained in the presence of 2,4-dichlorophenoxyacetic acid on culture medium. The purpose of this study was to investigate changes in protein profiles during callus and shoot development from cotyledonary explants and to correlate them with ontogenic stages during in vitro culture. Cotyledons cultured in the presence of 2,4-dichlorophenoxyacetic acid produced friable callus as a result of early division of parenchymatic cells associated with the vascular bundles of the explant. The callogenic ability was independent of the cotyledonary region used as starting explant. Direct shoot organogenesis was observed from the same type of cells growing in culture media supplemented with kinetin and indole-3-acetic acid. In this case, the regeneration potential varied among regions from which the explants were obtained. Protein profiles revealed differences associated with shoots or callus developmental programs. A 27-kDa polypeptide was uniquely detected in the explants undergoing shoot organogenesis. The amount of this polypeptide during the first 4 d of culture increased and was followed by the appearance of meristematic centers in histologically analyzed samples. This polypeptide could be used as a specific marker for in vitro shoot development in this species.     

Hormonal control of deoxyribonucleic Acid and protein syntheses in pea root cortical explants

The hormonal control of DNA and protein syntheses in cortical explants taken at 10 to 11 mm from the tip of 3-day-old seedling roots (Pisum sativum cv. Little Marvel) was examined. On the auxin medium, S2M, the cortical cells began to enlarge at day 4 in culture, with no DNA synthesis or cell division throughout the 7-day culture period. With the addition of kinetin to this medium, S2M + K, the DNA content of the explants increased about three times by day 3, with further increases thereafter. This DNA increase was followed by cell division activity and subsequent tracheary element differentiation initiated at day 5. At least two divisions per parent cortical cell were required prior to this cytodifferentiation. The absolute hormonal requirements for the DNA synthesis and cell division responses were substantiated by the lack of either response in explants cultured on basal (S2M medium minus auxins) or basal + K medium for 7 days. On the auxin medium, there was no protein accumulation in the cortical explants over the 7-day period. On S2M + K medium, protein accumulation began after day 2 with a steady rate of increase until day 4, and some fluctuation thereafter. The pattern of increasing uptake of ¹⁴C-leucine was similar for days 0 to 4 in explants on either medium. After day 4 on S2M, the uptake continued to increase coincident with cell enlargement initiation, whereas on S2M + K there was a decline. Incorporation of ¹⁴C-leucine into trichloroacetic acid-precipitates of the total buffered homogenate from explants on both media exhibited a similar pattern, i.e. an increase during days 0 to 3 and then a decline to a level about three times higher than day 0. Incorporation into the homogenate soluble fraction also showed a similar pattern in explants cultured with or without kinetin. From the differences in net protein accumulation and the incorporation data, speculation on a cytokinin effect on protein synthesis and degradation rates is presented.     

Shoot regeneration and somatic embryogenesis from different explants of Brahmi [Bacopa monniera (L.) Wettst.]

The morphogenetic potential of node, internode and leaf explants of Brahmi [Bacopa monniera (L.) Wettst.] was investigated to develop reliable protocols for shoot regeneration and somatic embryogenesis. The explants were excised from shoots raised from axillary buds of nodal explants cultured on Murashige and Skoog (MS) basal medium. Presence of 6-benzylaminopurine (BA) or kinetin influenced the degree of callus formation, from which a large number of shoot buds regenerated. Leaf explants gave the largest number of shoot buds followed by node and internode explants. BA was superior to kinetin; BA at 1.5 – 2.0 mg/l appeared to be optimum for inducing the maximum number of shoot buds. MS + 0.1 mg/l BA + 0.2 mg/l indole-3-acetic acid was the most suitable for shoot elongation. Elongated shoots were rooted on full- or half-strength MS medium with or without 0.5 – 1.0 mg/l indole-3-butyric acid or 0.5 – 1.0 mg/l α-naphthaleneacetic acid. The rooted plants were successfully established in soil. Calli derived from nodal explants cultured on MS medium containing 0.5 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), when subcultured on MS medium containing 0.1 or 0.5 mg/l BA or 0.2 mg/l 2,4-D + 0.1 or 0.5 mg/l kinetin, developed somatic embryos. The somatic embryos germinated either on the same media or on MS basal medium, and the resulting plantlets were successfully transplanted to soil. Received: 25 September 1996 / Revision received: 23 October 1997 / Accepted: 12 November 1997     

Cell proliferation in ectodermal explants from Xenopus embryos

Summary Dissociated prospective ectoderm cells from Xenopus laevis embryos divide autonomously up to the 17th division cycle of the embryo. To examine the requirements for the further proliferation of these cells, the continuation of cell division in compact ectodermal explants beyond the 17th division cycle has been studied. Such explants develop into aggregates of epidermal cells, as can be shown immunohistochemically with an anti-serum against Xenopus epidermal cytokeratin. Cell division in these explants is comparable to the in vivo proliferation rate at least during the first 24 h of cultivation, that is, well beyond the 17th division cycle. Thus, epidermal cells are provided with all the factors necessary for continued proliferation, but these can be effective only when the cells form tight aggregates. The long-term changes in cell number are complex. Mitotic figures are present until the explants disintegrate after 3–4 days. However, the total cell number per explant does not increase during later development. The production of cells by mitotic divisions is likely to be countered by the loss of cells due to cell death, which is indicated by the presence of pyknotic nuclei.     

In vitro regeneration of plantlets in Brassica alboglabra

Different vegetative parts of Brassica alboglabra seedlings and mature plants were used as explants in culture.A high frequency (60–100%) of shoot regeneration was obtained from hypocotyl explants, nodal stem segments, internodal segments and shoot apices cultured on Murashige-Skoog basal medium. Addition of 6-benzylaminopurine and kinetin increased the average number of shoots per explant. When detached and transferred to basal medium, the shoots readily developed roots. Regenerated plantlets could be successfully transplanted in soil.     

Regulation of Brassica rapa chloroplast proliferation in vivo and in cultured leaf disks

Summary. To understand the regulatory mechanisms of chloroplast proliferation, chloroplast replication was studied in cultured leaf disks cut from plants of 25 species. In leaf disks from Brassica rapa var. perviridis, the number of chloroplasts per cell increased remarkably in culture. We examined chloroplast replication in this plant in vivo and in culture media with and without benzyladenine, a cytokinin. In whole plants, leaf cells undergo two phases from leaf emergence to full expansion: an early proliferative stage, in which mitosis occurs, and a differentiational stage after mitosis has diminished. During the proliferative stage, chloroplast replication keeps pace with cell division. In the differentiational phase, cell division ceases but chloroplast replication continues for two or three more cycles, with the number of chloroplasts per cell reaching about 60. In the leaf disks, the number of chloroplasts per cell increased from about 18 to 300 without benzyladenine, and to over 600 with benzyladenine, indicating that this cytokinin enhances chloroplast replication in cultured tissue. We also studied changes in ploidy and cell volume between in vivo cells and cells grown in culture with and without benzyladenine. Ploidy and cell volume increased in a manner very similar to that of the number of chloroplasts, suggesting a relationship between these phenomena.Correspondence and reprints: Department of Biological Sciences, Graduate School of Science, University of Tokyo, Hongo, Tokyo 113-0033, Japan.     

Tiba inhibition of <Emphasis Type="Italic">in vitro</Emphasis> organogenesis in excised tobacco leaf explants

Summary Triiodobenzoic acid (TIBA), an anti-auxin, was found to inhibit both shoot and root formation in cultured excised leaf explants of tobacco (Nicotiana tabacum L.). The shoot formation (SF) medium used required only exogenous cytokinin (N⁶-benzyladenine) and the root formation (RF) medium required both auxin (indole-3-butyric acid) and cytokinin (kinetin). By transferring the explants from SF or RF media to SF or RF media with TIBA (4.0×10⁻⁵ M), respectively or vice versa, at different times in culture, it was found that TIBA inhibition was at the time of meristemoid formation and after determination of organogenesis. This indicates that TIBA interfered with endogenous auxin involvement in organized cell division.     

Rooting and the Metabolism of Nicotine in Tobacco Callus Cultures

The usefulness of exogenous nicotine as a factor in the induction of morphogenesis in a tobacco tissue culture medium has been demonstrated. Nicotiana rustica callus cell cultures were grown on a modified Murashige and Skoog medium with 2 mg/l indoleacetic acid (IAA) and 0.2 mg/l kinetin (MMS). Root morphogenesis was induced in roller tube callus cell cultures and solid callus cell cultures grown on MMS without kinetin supplemented with 10–100 mg/l nicotine. Optimal nicotine concentration for root induction was 50 mg/l. Other tests using varying combinations of IAA, kinetin and nicotine produced no obvious morphogenesis, although some changes in the amount of callus growth and endogenous protein concentration did correlate with nicotine concentration relative to the presence of IAA and/or kinetin. In liquid MMS medium, ¹⁴C-nicotine was primarily incorporated into the protein fraction of cultured cells while primarily incorporated into the cell wall and/or cell membrane fraction of cells cultured on MMS without kinetin in the medium. In MMS without IAA and MMS without both IAA and kinetin, there was incorporation, but to a lesser extent in both the protein and the cell wall and/or cell membrane fractions.     

NaCl胁迫对宁夏枸杞叶和幼根显微及超微结构的影响

以宁夏枸杞为材料,采用超薄切片技术制备样品,应用光学显微镜和透射电镜分析了不同浓度NaCl胁迫条件下宁夏枸杞叶和幼根显微及超微结构的变化。结果表明：随着NaCl胁迫的加重,(1)叶片上表皮细胞增厚,栅栏组织细胞出现缩短现象,排列疏松且紊乱;幼根的初生结构无明显变化。(2)叶片栅栏组织中叶绿体不再紧靠在细胞膜上,叶绿体双层膜破坏,基粒片层松散排列,杂乱无章,出现膨胀和空泡现象,淀粉粒和嗜锇颗粒增多,叶肉细胞中线粒体发生轻微变化;幼根中皮层薄壁细胞线粒体形状发生改变,结构破坏,内膜和外膜模糊甚至破裂,大多数嵴模糊,出现空泡现象;细胞核解体,基质外溢。研究表明, 不同浓度的NaCl胁迫对宁夏枸杞叶片和幼根细胞的显微及超微结构影响不同,NaCl浓度大于200 mmol/L时,宁夏枸杞叶片和幼根细胞的显微及超微结构发生了明显变化,且叶肉细胞中线粒体的变化没有叶绿体的变化显著,推测叶肉细胞中线粒体的耐盐性比叶绿体强。     

Effects of noradrenaline exposure on rat brown adipocytes in cultures. An ultrastructural study

Adipocytes in intact brown adipose tissue show multivacuolar lipid deposit and mitochondria of 'typical' morphology. Cultured brown adipocytes retain the multivacuolar lipid deposit, while 'typical' mitochondria degenerate and 'atypical' organelles appear instead of the former. Since evidence exists that catecholamines deeply influence brown adipose tissue morphology and function in vivo, we undertook the present ultrastructural investigation to assess whether exposure of cultured brown fat cell to noradrenaline could prevent (or induce regression of) the in vitro morphological modifications of brown adipocytes. Brown adipocytes cultured for 8 h in the presence of noradrenaline (5 X 10(-5) M) had a larger mitochondrial area (i.e. a larger percentage of cytoplasm occupied by non-degenerating mitochondria) in comparison with control cells, as assessed by morphometry; this was due to larger number of mitochondria in noradrenaline-treated cells. Moreover, a number of cells with mitochondria strictly resembling those of the intact tissue were visible in noradrenaline-treated cultured after 8 hr, while 'typical' mitochondria were no longer observed in parallel control cultures. After 5 days of culture without hormone addition, exposure to noradrenaline (5 X 10(-5) M) did not induce quantitative modifications of 'atypical' mitochondria or changes of their ultrastructure up to 12 hr. However, reduction in size of the lipid deposit and activation of both rough endoplasmic reticulum and Golgi apparatus were evident in noradrenaline-treated adipocytes in comparison with non-treated cells.     

Somatic embryogenesis in perennial statice Limonium bellidifolium,Plumbaginaceae

Cotyledon explants from perennial statice Limonium bellidifolium (Statice caspia Willd.) were cultured on Murashige and Skoog's basal medium (MS) supplemented with various levels of 2,4-D, kinetin and sucrose. Embryogenic calluses developed over a period of 10 days with the highest response at 4.5 M (1 mg l^–1) 2,4-D, 0.5 M (0.1 mg l^–1) kinetin and 117 mM sucrose. Following induction, the calluses were transferred to MS media supplemented with 88 or 117 mM sucrose and 0 or 0.5 M kinetin. Somatic embryos at the globular, heart-shaped, torpedo, and cotyledonary stages developed. Fully germinated plantlets developed with the best response in medium supplemented with 117 mM sucrose and 0.5 M kinetin. Direct somatic embryogenesis without a callus phase was observed with some of the cotyledon explants. Induction, maturation and germination of somatic embryos on the optimized media were equally effective using cotyledon, hypocotyl and root explants. Serial sections of L. bellidifolium cotyledon explants cultured for two weeks indicated that pro-embryogenic masses originated from parenchyma cells below the epidermis. Further histological observations of embryogenic calluses confirmed the initiation and development of globular and heart-shaped embryos and repetitive somatic embryogenesis. Ultrastructural observations indicated that the embryogenic cells were less vacuolate with abundant organelles compared to the cells of the explant. This is the first report of somatic embryogenesis in the Plumbaginaceae.     

Control by cytokinins of the cellular behavior in the plate meristem of zucchini cotyledons

The temporal and spatial effects of exogenous cytokinins on both cell expansion and division activity in the plate meristem of cultured zucchini cotyledons were studied. N ⁶-benzylaminopurine (1–100 μM) and N-(2-chloro-4pyridyl)-N′-phenylurea (4PU-30) (0.1–100 μM) greatly stimulated the cell growth and division. They provoked multiple cell cycles, formation of larger clusters of daughter cells and an increase of the final number of cells. Both cytokinins led to earlier achievement of final cotyledon size and shortened the cell doubling time. By contrast to the purine cytokinin, phenylurea cytokinin 4PU-30 enlarged the cotyledon predominantly in length. Zeatin and kinetin were less effective, particularly in stimulating cell expansion. In low concentrations, all cytokinins were more effective in stimulating division activity rather than expansion. The cells in the cotyledon margins displayed a higher division activity, especially when treated with exogenous cytokinins. The final cotyledon and cluster areas were not of the strict proportional dependence upon the number of their cells. These results provide a novel example where stimulated cell division fails to evoke a respective increase in the final organ size.     

Ultrastructural Changes of Rice Pollen Callus Cell Grown on Differentiation Medium

A comparative observation on the rice pollen callus cultured on the medium supplemented with 2 mg/L 2,4-D and omitted 2,4-D but contained 0.5 mg/L kinetin and 0.2 mg/L NAA respectively was made by electron microscope. The callus cells when transferred to the medium containing kinetin show some changes at ultrastructural level. The numbers of mitochondria, plast and ribosome show an increase in some epidermal cells which kept an ability of active division and may be differentiated into a primordial bud. The storage materials such as lipid bodies and starch grains show sharp decrease or disappearance and the degree of vacuolation decrease in the parenchyma cells of internal callus. Besides vessel differentiation, some sieve elements appeared in the callus. The types of callus cell became various and some electron-dense cell occurred. Although above-mentioned ultrastructural changes appeared in callus cells grown under differentiation condition were true but we do not think those are specific mark for differentiating cell. More work is needed with cytochemistry and molecular biological methods to study callus cell differentiation.     

Investigations on the cell cycle of haploid and diploid tissue cultures of Datura innoxia Mill. and its synchronization

Using a ¹⁴C/³H double-labelling technique, the influence of kinetic on the length of the cell cycle of meristematic cells in haploid and diploid callus cultures of Datura innoxia was determined. The total length of the cell cycle of haploid cells as compared to that of diploid cells was reduced by 2.3 h (-kinetin) or 1.4 h (+kinetin). Furthermore, the addition of kinetin to the nutrient solution also reduces cell cycle duration at both ploidy levels. For synchronization of the cell cycle, a fluorodesoxyuridine/thymidine system was successfully employed. Apparently, the reduction of total cell cycle duration of cycling cells due to treatment with kinetin occurred at the expense of the G₁phase. Nevertheless, kinetin seems to exert an influence on the transition of cells from the G₂ into the M phase as well.Abbreviations FUdR fluorodeoxyuridine - HU hydroxyurea - IAA nidole acetic acid     

Coumarin effects on Glycine max hypocotyl explants

Coumarin induced root formation and stimulated fresh weight production in hypocotyl explants of Glycine max L. cultured in vitro. All stimulatory effects caused by coumarin were induced within a relatively narrow range of concentrations between 1–500 μM, yielding optimum dose response curves. When coumarin was combined with kinetin fresh weight increased considerably, at optimum concentrations to a level almost as high as that obtained with NAA (10 μM) and kinetin (10 μM). Root formation was almost completely inhibited when kinetin was added in combination with coumarin. NAA + coumarin had small stimulatory effects on fresh weight. but were inhibitory in root formation. The frequency of rooting per explant, texture and pigmentation were also affected by different treatments.     

Culture of intramural cardiac ganglia of the newborn guinea-pig

The ultrastructure of cultured intrinsic neurones and SIF (small intensely fluorescent) cells dissociated from the atria and interatrial septum of newborn guinea-pig heart has been studied for the first time and compared with these cells in situ. Mononucleate and binucleate neuronal somata and their processes were observed in the culture preparation; their ultrastructure was similar to that of neurones in intracardiac ganglia observed in situ. The number of neurites associated with neuronal cell bodies increased after the first week in culture. A subpopulation of intracardiac neurones showed abnormalities in culture, comparable to the changes previously described in neurones of the monkey heart after unilateral vagotomy in situ. Small granule-containing cells were observed in culture, corresponding to those described in the heart in situ. One type of large process in the culture preparation containing densely packed mitochondria has not been seen in situ, suggesting that changes in cell ultrastructure due to the conditions of culture cannot be discounted. However, the ultrastructure of the cultured cells was, for the most part, consistent with that of the same cell type in situ, indicating that the culture preparation may be a useful model for investigation of the roles and interactions of intramural neurones in the heart, which are inaccessible for such studies in situ.     

马占相思子叶离体培养中细胞早期变化的研究

用光学和电子显微镜观察马占相思子叶在离体培养中的细胞早期变化,结果表明,未培养的子叶薄壁细胞内充满大量贮藏物质,在脱分化过程中,贮藏物质逐渐减少,细胞核体积增大,液泡蛋白体出现,质体转化为原质体,线粒体,内质网等细胞器数量增加,马脱分化启动后的细胞分裂方式进行了讨论。