DNA Methylation and Embryogenic Competence in Leaves and Callus of Napiergrass (Pennisetum purpureum Schum.)

Quantitative and qualitative levels of DNA methylation were evaluated in leaves and callus of Pennisetum purpureum Schum. The level of methylation did not change during leaf differentiation or aging and similar levels of methylation were found in embryogenic and nonembryogenic callus.     

柊叶和象草波式潜流人工湿地的对比研究

为探索柊叶和象草在人工湿地中的应用及其净化机理,该研究以柊叶和象草为人工湿地植物分别构建了波式潜流人工湿地系统,分析了柊叶和象草波式潜流人工湿地对生活污水中COD_(cr)、TN和TP的净化效果,观察了柊叶和象草两种植物在不同季节的生长状况。结果表明:经过15个月的连续运行,在表面水力负荷约0.3 m·d~(-1)的条件下,柊叶和象草波式潜流人工湿地平均去除率是COD_(cr)分别为66.1%和70.1%,TN分别为60.4%和63.7%,TP分别为74.1%和75.1%。两种植物生长良好,根系发达,象草的地上生物量是柊叶的2.1倍,地下生物量相当;冬季象草生长缓慢,柊叶部分叶片的四周干枯,但二者都不会枯亡。这说明两个人工湿地对COD_(cr)、TN和TP都具有较好的去除效果,但无显著性差异,柊叶和象草能明显提高潜流人工湿地的净化效果。     

Derepression of the cell cycle by starvation is involved in the induction of tobacco pollen embryogenesis

Summary Microspectrophotometry following Feulgen staining and autoradiography following (³H)-thymidine labelling were used to study cell-cycle events during pollen development in tobacco (Nicotiana tabacum L.). During normal gametophytic pollen development in the anther and in vitro the generative nucleus passes through the S phase to the G₂ phase soon after microspore mitosis, while the vegetative nucleus remains arrested in G₁ (=G₀). During embryogenie induction by an in vitro starvation treatment of immature pollen ongoing DNA replication in the generative nucleus is completed and followed by DNA replication in the vegetative cell in a large fraction of the pollen grains. Addition of the DNA replication inhibitor hydroxyurea to the starvation medium postpones S phase entry until the pollen is transferred to a rich medium and does not affect embryo formation. These results demonstrate that one of the crucial events of embryogenic induction is the derepression of the G₁ arrest in the cell cycle of the vegetative cell.     

Ultrastructural study of different types of callus from leaf expiants ofAesculus hippocastanum L.

Summary The cell ultrastructure in three types of callus obtained from leaf explants ofAesculus hippocastanum L. has been studied. Remarkable differences have been shown between the cells of the forerunner E₁ callus and those of the callus arising from it, according to the culture conditions.The peculiar characteristics of E₁ are the scarcity of intercellular spaces and the occurrence of autophagic vacuoles in the cells.An embryogenic friable callus (E₂) is formed in time when E₁ is maintained on solid culture medium. The E₂ cells show cytological features typical of a higher metabolic level and contain starch. Diffused middle lamella digestion leads to the detachment of small embryogenic cell aggregates consisting of vacuolated parenchymatous-like cells and small meristematic cells which may be regarded as embryoids initials.Shaking E₁ in the same liquid medium and subsequent culture on solid medium lead to the differentiation of a non-embryogenic callus (NE), whose cells are very large and highly vacuolated, devoid of starch and with organelle-rich cytoplasm. The NE callus shows a high degree of growth, but does not attain embryogenic competence in time.Abbreviations c cell - cr crystal - cw cell wall - d dictyosome - er endoplasmic reticulum - m mitochondrion - mb microbody - n nucleus - p plastid - s starch - v vacuole     

Biochemical and molecular analysis of plants derived from embryogenic tissue cultures of napier grass (Pennisetum purpureum K. Schum)

Summary We have investigated the extent of biochemical and molecular variation in 63 plants of napier grass (Pennisetum purpureum K. Schum.) regenerated from 3- to 24-week-old embryogenic callus cultures. The calli were derived from cultured basal segments of young leaves and immature inflorescences obtained from a single fieldgrown donor plant. The entire population was analyzed for the activity of 14 isozyme systems, but no qualitative variation was found at any of the loci examined. Similarly, no restriction fragment length polymorphisms (RFLPs) were detected in the mitochondrial, plastid and nuclear genomes in a representative sample of regenerated plants. Our results confirm earlier reports of the genetic uniformity of plants derived from somatic embryos and highlight their value both for clonal propagation and for genetic transformation.     

MORPHOLOGY AND ULTRASTRUCTURE OF EMBRYOGENIC CELL SUSPENSION CULTURES OF PANICUM MAXIMUM (GUINEA GRASS) AND PENNISETUM PURPUREUM (NAPIER GRASS)

Morphological and ultrastructural changes during the growth of embryogenic cell suspension cultures of Panicum maximum and Pennisetum purpureum have been studied. The suspensions consist almost entirely of cell aggregates of 50–75 embryogenic cells. The cell aggregates vary in size from 90–400 μm in P. maximum and from 70–340 μm in P. purpureum. Following the period of exponential growth starch grains gradually disappear and vacuolation increases. Ten to 16 days after subculture, P. maximum cells enlarge and separate from each other, and organized embryo-like structures appear. Ultrastructural studies show that the cell aggregates are made up of discrete, individual groups of 2–6 cells each. Each cell group appears to arise from a single cell and breaks away from the ‘mother group’ as cell divisions continue. The embryogenic cells are small (20 μm), isodiametric with many starch grains and contain a large nucleus with a prominent nucleolus. Extensive profiles of endoplasmic reticulum, many small peripheral vacuoles, and several amyloplasts are present. Plasmodesmatal connections exist only between cells within a cell group but not between cells of different cell groups in the large cell aggregates.     

Microspectrophotometric measurements of DNA by automated computerized scanning in cycling cells of theChrysanthemum segetum shoot apex

Summary A new technique of exploitation of the data was proposed after DNA scanning microdensitometry. By using all of the measurements obtained from the seriated sections of a single nucleus, this method made it possible to estimate six characteristic parameters during the different phases of the cell cycle in the various shoot apical cells. The cells whose rate of proliferation was the highest showed the biggest variations of their nuclear and nucleolar volumes during the cell cycle. In the axial zone, where the cells have a slow cell cycle and display the longest duration of the G₁ phase, the volume occupied by dispersed DNA was greater than in the cells of the lateral zone and of the rib meristem, where the cell cycle and the G₁ phase were short. No matter what the cell type, the proportion of the dispersed and condensed DNA varied little when the G₁ and G₂ phases were compared. In the Z phase, characterized by a decondensation of the DNA, the mean DNA amount was 3.4 C. The evolution of the nuclear density during the interphase was also estimated. It is demonstrated that the main feature of the shoot apex zonation was the decondensation of the condensed DNA in the axial zone in both the G₁ and G₂ phases.     

First divisions of somatic embryogenic cells inCichorium hybrid “474”

Summary First divisions of embryogenic cells were studied in leaves of plantlets of aCichorium hybrid (C. intybus L. ×C. endivia L.) cultured in vitro in a liquid agitated medium, at 35 °C in the dark. Stages of reactivation of competent cells were characterized by increase of nucleus and nucleolus diameter, migration of the nucleus in the centre of the cell and thickening of the cell wall. The first division of reactivated embryogenic cells was symmetrical and anticlinal in regard to the xylem vessels orientation. Embryogenic structures consisted in I-type tetrads or in rows of 4–8 cells. Then the divisions occurred in thickness at one end, without polarization or formation of a suspensor-like structure.Abbreviations EC embryogenic cell - ES embryogenic structure     

Involvement of colchicine-sensitive cytoplasmic element in premitotic nuclear positioning ofAdiantum protonemata

Summary When the red-light grown protonema ofAdiantum capillus-veneris was transferred to the dark, the nucleus ceased its migration ca. 5 hours before cell plate formation (Mineyuki andFuruya 1980). To see whether the nucleus was held by some cytoplasmic structure during nuclear positioning, protonemata were treated with various centrifugal forces at different stages of the cell cycle. Nuclei of G₁ phase were easily displaced by centrifugation at 360×g for 15 minutes, but those of G₂ or M phase were not displaced by it, suggesting that the nuclei were held by some cytoplasmic elements in G₂ or M phase. This nuclear anchoring was not detectable in protonemata that were treated with 5mM colchicine. With this treatment, the nucleus did not stop its migration at late G₂ and moved even in prophase. And the retardation of organelle movement which was observed in cytoplasm on the lateral side of the nucleus after the cessation of premitotic nuclear migration (Mineyuki andFuruya 1984) was not observed in the presence of colchicine. Thus the nuclei appear to be held by colchicine-sensitive structure in cytoplasm between the lateral surface of the nucleus and cell wall during the premitotic nuclear positioning. Electron micrographs showing cytoplasmic microtubules were consistent with the idea.Abbreviations PPN Premitotic positioning of the nucleus - L region Cytoplasm between the lateral surface of the nucleus and cell wall (seeMineyuki et al. 1984)     

Nuclear DNA content in differentiated tissues of sunflower (Helianthus annuus L.)

Summary Scanning cytophotometry following Feulgen-staining was used to determine nuclear DNA content in many differentiated tissues of nine cultivars, hybrids or selfed lines ofHelianthus annuus. Apart from such ephemeral tissues as endosperm and anther tapetum, it was found that tissue differentiation in sunflower occurs in the diploid condition, cells being arrested in the DNA presynthetic phase (G₁). In certain cases, however, the nuclear DNA content of differentiated G₁ cells does not exactly match the 2C DNA content found in meristematic cells, but may be either higher or lower. In endosperm and anther tapetum cells, nuclear DNA content may be as high as 24 C and 32 C, respectively. Cytological and autoradiographic analyses after³H-thymidine incorporation reveal that polyploidy in the tapetal cells is due to chromosome endoreduplication. No detectable difference between male-fertile and male-sterile plants exists as far as occurrence and level of cell polyploidy are concerned. The results are discussed in the context of previous investigations on the nuclear condition of differentiatedHelianthus annuus tissue.     

Loss of nuclear DNA in leaves of rye

Summary The nuclear DNA content of rye leaf cells was cytophotometrically determined. At the commencement of differentiation nuclei in rye leaves remain standing at G₁ phase. With further differentiation a remarkable diminution of nuclear DNA content occurs in diploid cells. The largest number of cells showing a loss of nuclear DNA content were found in the top of the leaf. The age of the leaf and the extent of diminution in nuclear DNA content are correlated.Dedicated to Professor Dr. J. Straub in honour of his 70th birthday     

Embryogenic cell suspensions of Triticum aestivum x Leymus angustus F1 hybrids: characterization and plant regeneration

Summary Embryogenic cell suspension cultures were established from Triticum aestivum X Leymus angustus F₁ hybrids, using compact nodular calli derived from inflorescence segments. Calli originating from leaf segments did not give rise to stable cell suspensions. Growth measurements of the cell suspensions revealed that they continued rapid growth up to 10 days after subculturing. Flow cytometric studies of the cell cycle over a 7 day culture period showed that the majority of cells were in G₁ phase while the rest were either in S or G₂. During the 7 days of culture, no significant differences in DNA distribution patterns were observed. The cells from suspension cultures produced somatic embryos when they were transferred to different solid media. The embryos germinated and gave rise to plantlets which were successfully rooted and transferred to soil.     

Endogenous Abscisic Acid and Indole-3-Acetic Acid and Somatic Embryogenesis in Cultured Leaf Explants of Pennisetum purpureum Schum. : Effects in Vivo and in Vitro of Glyphosate, Fluridone, and Paclobutrazol

Effects of application in vivo of glyphosate, fluridone, and paclobutrazol to glasshouse-grown donor plants of Pennisetum purpureum Schum. on endogenous levels of abscisic acid (ABA) and indole-3-acetic acid (IAA) in young leaves and on somatic embryogenesis in cultured leaf explants were studied. Treatment of plants with glyphosate (100 milligrams per liter) resulted in elevated levels of endogenous ABA and IAA in young leaves. In contrast, paclobutrazol (50% active ingredient; 200 milligrams per liter) did not alter the endogenous levels of ABA and IAA. Fluridone (100 milligrams per liter) markedly inhibited synthesis of ABA and leaf explants from fluridone-treated plants lost the capacity for somatic embryogenesis. Explants from glyphosate- or paclobutrazol-treated plants did not show any reduction in embryogenic capacity when compared with untreated control plants. Glyphosate and fluridone were also incorporated into the culture media at various concentrations (0 to 20 milligrams per liter) to study their effects in vitro on somatic embryogenesis in leaf explants from untreated, field-grown plants. Glyphosate was inhibitory to somatic embryogenesis but only at concentrations above 5 milligrams per liter. Fluridone inhibited somatic embryogenesis at all concentrations tested. Inhibition of somatic embryogenesis by fluridone, by either in vivo or in vitro application, could be overcome partially by (±)-ABA added to the culture medium. Exogenous application of (±)-ABA enhanced somatic embryogenesis and reduced the formation of nonembryogenic callus. Application of IAA or gibberellic acid (GA₃; >5 milligrams per liter) was inhibitory to somatic embryogenesis. These results indicate that endogenous ABA is one of the important factors controlling the embryogenic capacity of leaf explants in Napier grass.     

Photosynthetic properties of two different soybean suspension cultures

The photosynthetic properties of two commonly used suspension cultured lines, embryogenic and photoautotrophic (PA, SB-1 line) cells of soybean [Glycine max (L.) Merr.] were characterized. We found that compared to the dark green PA cells, the light green embryogenic cells contained fewer and smaller plastids with less-developed thylakoid membranes. The embryogenic cells also contained much lower contents of both chlorophyll and the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) protein, an undetectable level of Rubisco small subunit protein, and a very low rate of photosynthesis. While the DNA contents of the nuclear genomes were similar in these two types of cultured cells, the embryogenic cells possessed a markedly lower content of plastid DNA. The 18-year-old PA suspension culture, SB-1, continues to evolve with higher Rubisco and plastid DNA contents than leaves, and with small decreases in nuclear DNA content that appears to mimic changes in chromosome numbers. These findings may prove useful in the application of plastid transformation, particularly when non-leaf or non-green tissues must be used as targets for transformation and plant regeneration.     

Cellular responses of leaf explants of Cocos nucifera L. in vitro

Leaf explants of Cocos nucifera L. (coconut palm) were studied in vitro in order to establish whether or not rapid cellular changes contribute to the well known recalcitrance of coconut cells in tissue culture. Segments from the base of immature leaves were cultured on modified Eeuwens' medium at 30°C in darkness. The mitotic index, nuclear DNA amounts, cell and nuclear size were measured both before and during culture (from 0 to 70 days). There was no basipetal gradient of cell division in immature coconut leaves; the mitotic index never exceeded 2% and showed neither a positional nor temporal relationship with leaf development. Moreover the vast majority of cells were in G1 of the cell cycle. This cell cycle pattern was maintained for most of the period in culture although at 70 days there was an increase in the proportion of cells in S- and G2-phases consistent with low rates of callus formation. The nuclear: cell size ratio was constant in cells within the immature leaf irrespective of developmental age. However upon transfer to culture media, cell size but not nuclear size increased. We suggest that this uncoupling of cell and nuclear size disrupts cell co-ordination and is a key contributor to recalcitrant cellular behaviour of this species in vitro.     

Copy numbers of chloroplast and nuclear genomes are proportional in mature mesophyll cells of Triticum and Aegilops species

The possibility of estimating the proportion of chloroplast DNA (ctDNA) and nuclear DNA (nDNA) in nucleic-acid extracts by selective digestion with the methylation-sensitive restriction enzyme PstI, was tested using leaf extracts from Spinacia oleracea and Triticum aestivum. Values of ctDNA as percentage nDNA were estimated to be 14.58%±0.56 (SE) in S. oleracea leaves and 4.97%±0.36 (SE) in T. aestivum leaves. These estimates agree well with those already reported for the same type of leaf material. Selective digestion and quantitative dot-blot hybridisation were used to determine ctDNA as percentage nDNA in expanded leaf tissue from species of Triticum and Aegilops representing three levels of nuclear ploidy and six types of cytoplasm. No significant differences in leaf ctDNA content were detected: in the diploids the leaf ctDNA percentage ranged between 3.8% and 5.1%, and in the polyploids between 3.5% and 4.9%. Consequently, nuclear ploidy and nDNA amount were proportional to ctDNA amount (r₍₁₉₎=0.935, P>0.01) and hence to ctDNA copy number in the mature mesophyll cells of these species. There was a slight increase in ctDNA copy numbers per chloroplast at higher ploidy levels. The balance between numbers of nuclear and chloroplast genomes is discussed in relation to polyploidisation and to the nuclear control of ctDNA replication.Abbreviations ctDNA chloroplast DNA - nDNA nuclear DNA - RuBPCase ribulose-1,5-bisphosphate carboxylase - DAPI 4,6-diamidine-2-phenylindole     

Factors controlling somatic embryogenesis

Histological and ultrastructural, molecular and elemental distribution changes were investigated during the induction of direct somatic embryogenesis using theCamellia japonica leaf culture system. In this culture system, direct somatic embryogenesis is induced in a controlled way in a specific leaf region (leaf blade) within a leaf. Embryogenic and non-embryogenic leaf regions have characteristic energy-dispersive X-ray spectra already before induction. According to these results electron probe X-ray microanalysis (EPMA) can be a tool for early diagnosis of embryogenic competence. Histological studies showed that severe fluctuations in the number of calcium oxalate crystals and in starch accumulation occur after induction but only in induced tissues. Changes in the cell wall composition of competent cells occur shortly after the induction treatment. The induction of morphogenesis is linked to the appearance of callose covering the surface cells of induced leaves and calluses. A 2^nd deposition of material (cutin) is necessary for normal somatic embryogenesis to occur. The involvement of lipid transfer proteins in the appearance of cutin in the embryogenic regions of the explant is suggested.     

The origin and development of somatic embryos following cryopreservation of an embryogenic suspension culture ofPicea sitchensis

Summary The development of somatic embryos in an embryogenic suspension culture ofPicea sitchensis was followed every day for two weeks after thawing from liquid nitrogen (LN₂). Only a few cells, primarily located at the periphery of the embryonic region of the embryos, survived cryopreservation in LN₂. Surviving cells were classified into two groups: embryogenic cells (EC) and non-embryogenic cells (NEC), based on their morphology and embryogenic competence. The dense cytoplasmic EC underwent organized growth and differentiation with first divisions occurring after 24 h, and embryo formation 6–8 days after thawing from LN₂. No evidence of asymmetrical divisions or free-nuclear stages was found during somatic embryo formation. NEC had less dense cytoplasm with numerous small vacuoles. One to five days after thawing the NEC became progressively more vacuolated and elongated. Histological examination revealed no mitotic activity in NEC, and six days after thawing NECs were seen as single cells or unorganized cell aggregates. Two weeks after thawing the appearance of the cryopreserved cultures was comparable to that of the untreated cultures.Abbreviations EC embryogenic cells - ECC embryogenic cell clusters - FDA fluorescein diacetate - GMA glycol methacrylate - LN₂ liquid nitrogen (–196°C) - NEC non-embryogenic cells     

Cell cycle analysis in a multinucleate green alga,Boergesenia forbesii (Siphonocladales,Chlorophyta)*

The cell cycle (nuclear division cycle) of a multinucleate green alga, Boergesenia forbesii (Harvey) Feldmann was studied using microspectrophotometry and BrdU incorporation techniques. Mitosis was observed frequently 1-4 h after the beginning of the light period, on a 16:8 h LD cycle at 25°C. Mitotic nuclei formed discrete patches. Other nuclei remained in the G₁ period. The DNA synthetic phase (S phase) was estimated to last about 12 h from microspectrophotometric study using aphidicolin inhibition just before the S phase and release from it. The G₂ period was estimated to be about 2 h, because a labeled prophase nucleus could be detected when the samples were labeled with BrdU continuously over 3 h. The incorporation pattern of BrdU changed through the S phase nucleus. In early S phase, BrdU staining was detected as many dots in the entire nucleus, while in late S phase, it was detected as several discrete regions along the nuclear membrane. Almost all nuclei in B. forbesii were in the G₁ stage after nuclear division, and the nuclei in several patches of the cell simultaneously initiated DNA synthesis. Once the nuclei entered into S phase, these nuclei continued into G₂ and mitosis. In other words, the cell cycle regulation of entrance into S phase from G₁ is an important factor in the growth and morphogenesis in B. forbesii.     

Promotive and inhibitory effects of diverse arabinogalactan proteins on Daucus carota L. somatic embryogenesis

In the 3-d-old 2-mm root tip of Pisum sativum L. cv. Lincoln the percentage of actively proliferating cells is estimated to be 70%. The remaining cells are non-cycling and arrested with 2C and 4C DNA content in G₀ and in G₂Q, respectively. In this work we studied the kinetic significance of these quiescent cells, using the sorting capabilities of flow cytometry and immunofluorescence techniques to detect the proliferation marker PCNA (proliferating cell nuclear antigen) inside cells within the different cell-cycle compartments. While in animal cells, PCNA is present at a high level only in actively proliferating cells, in 3-d-old pea root tips 95% of the cells are PCNA-positive. After flow cytometry and sorting of pea non-cycling nuclear populations, all G₂Q nuclei appeared strongly PCNA-positive, indicating that these cells had recently left the cell cycle. By contrast, most G₀ nuclei showed a low level of PCNA immunofluorescence intensity, as measured by image analysis, with about 25% of the nuclei being PCNA-negative. This small percentage was found to correspond to root cap cells, as could be observed in the root tip section. These are the only cells in the root apical region which are fully differentiated and which, therefore, lack the competence to enter the cell cycle. In contrast, the more or less PCNA-positive G₀ nuclei could represent a kinetically heterogeneous population of cells competent to proliferate, but which have either recently left the cell cycle or are progressing to the G₀-G₁ transition. Received: 6 November 1996 / Accepted: 14 January 1997