DNA replication in maize leaf protoplasts

Maize leaf protoplasts were investigated for their metabolic competence and capacity to synthesize DNA. When protoplasts were incubated at elevated temperatures, they exhibited a heat shock response with specific proteins being preferentially synthesized. This indicated that the protoplasts were fully metabolically functional and capable of responding to environmental stimuli. Significant DNA synthesis was observed in these protoplasts after incorporation of ³H-thymidine into chromatin by trichloroacetic acid precipitation and by incorporation of 5-bromo-2-deoxyuridine (BrdU), an analog of thymidine, detected by immunofluorescence. The immunocytochemical method revealed that about 50% of nuclei in the maize leaf protoplasts were labelled after 3 days of culture and that most of these nuclei were labelled as intensely as normal mitotic cells. Aphidicolin, an inhibitor of DNA polymerase-, decreased the percentage of labelled nuclei, demonstrating that the labelling was substantially due to replicative DNA synthesis. However, chromosome condensation was not observed. It is proposed that these protoplasts are capable of DNA synthesis, but incapable of nuclear division. Effects of media additives on the number of nuclei entering S phase in these protoplasts were also assessed by the immunocytochemical method. Inclusion of 80mM Ca²⁺ in the enzyme solution increased protoplast yield and also appeared beneficial to DNA synthesis. The antioxidant, n-propyl gallate, which was used to stabilize the protoplasts, delayed the onset of DNA synthesis. Arginine and spermidine produced a slight increase in DNA synthesis.Abbreviations BrdU 5-bromo-2-deoxyuridine - DMSO dimethyl sulfoxide - n-PG n-propyl gallate - PBS phosphate-buffered saline Dedicated to Dr. Friedrich Constabel on the occasion of his 60th birthday     

Ultrastructural research on cell wall regeneration by cultured pollen protoplasts of Lilium longiflorum

Summary Protoplasts from pollen grains of Lilium longiflorum regenerate amorphous cellulosic cell walls in culture, during which some precursors of cellulose are polymerized, thus producing progressively harder cellulosic cell walls as the period of culture continues. It is presumed that the components of the cell wall regenerated during 1 week in culture differ from those of the intine of the pollen grain wall. The regenerated cell wall is formed by means of large smooth vesicles; in addition, numerous coated vesicles and pits aid in wall regeneration. The pollen tube that germinates from the 8-day-old cultured protoplast has numerous Golgi bodies and many vesicles which build the pollen tube wall. The tube wall has two layers just like a normal pollen tube wall.     

Plant regeneration from protoplasts isolated from embryogenic suspension cultured cells of Cinnamomum camphora L.

An efficient and reproducible protocol is described for the regeneration of Cinnamomum camphora protoplasts isolated from cultured embryogenic suspension cells. Maximum protoplast yield (13.1±2.1×10⁶/g FW) and viability (91.8±3.8%) were achieved using a mixture of 3% (w/v) cellulase Onozuka R10 and 3% (w/v) macerozyme Onozuka R10 in 12.7% (w/v) mannitol solution containing 0.12% (w/v) MES, 0.36% (w/v) CaCl₂·2H₂O, and 0.011% (w/v) NaH₂PO₄·2H₂O. First divisions occurred 7–10 days following culture initiation. The highest division frequency (24.6±2.9%) and plating efficiency (6.88±0.8%) were obtained in liquid medium (MS) supplemented with 30 g l^–1 sucrose, 0.7M glucose, 0.1 mg l^–1 NAA, 1.0 mg l^–1 BA, and 1.0 mg l^–1 GA₃. After somatic embryo induction and then shoot induction, the protoplast-derived embryos produced plantlets at an efficiency of 17.5%. Somatic embryos developed into well-rooted plants on MS medium supplemented with 1.0 mg l^–1 3-indole butyric acid (IBA). Regenerated plants that transferred to soil have normal morphology.     

Comparison between maize root cells and their respective regenerating protoplasts: wall polysaccharides

The cell-wall polysaccharides from different parts of maize roots have been analysed. The arabinose, galactose and mannose contents are influenced by cell differentiation, whereas xylose, rhamnose and uronic-acid contents are not. In cap cells, the pectin content is low but rhamnose and fucose are present in larger quantities. The cell-wall polysaccharides from cells of the elongation zone and their respective regenerating protoplasts were also analysed. The walls of the protoplasts contained higher xylose and mannose levels and a much lower level of cellulose than the cells from which they were derived.     

Cell wall regeneration and galactomannan biosynthesis in protoplasts from carob

Protoplast isolation from endosperms of developing carob (Ceratonia siliqua L.) seeds is reported for the first time. These protoplasts regenerated cell walls within 12 h. In order to assess their potential for galactomannan biosynthesis, the incorporation of radioactivity in the regenerated cell wall polysaccharides (CWP) and extracellular polysaccharides (ECP), after feeding these protoplasts with D-[U-¹⁴C]glucose or D-[U-¹⁴C]mannose was studied. The pattern of the radioactive label distribution in the neutral sugars of the trifluoroacetic acid (TFA) hydrolysate of CWP was different from that of the ECP. In the TFA hydrolysis products of the CWP, immediately after protoplast isolation, the greatest level of radioactivity (approximately 90%) was detected in glucose, galactose and mannose. After 2 days protoplast culture, the label in mannose increased. In contrast, immediately after protoplast isolation, approximately 90% of radioactivity of the ECP was detected in galactose and mannose. However, during culture, the radioactivity incorporation in mannose dropped to one third, while that in galactose and arabinose increased significantly. Hydrolysis of the CWP and ECP with -galactosidase and endo--mannanase confirmed that, at least part of mannose and galactose belonged to galactomannan molecules. These results were compared with those obtained upon feeding developing endosperm tissue with D-[U-¹⁴C]mannose. From our results we concluded that protoplasts from endosperm tissues of developing carob seeds, retained the ability of their original explant to synthesize galactomannan, making protoplasts candidates for the study of galactomannan biosynthesis.     

Effect of N deficiency on leaf growth and cell wall peroxidase activity in contrasting maize genotypes

Two maize genotypes differing in leaf elongation rate (high-LER and low-LER) were used for the investigation of the effects of nitrogen deficiency on leaf growth and development and activity of enzyme cell wall peroxidase in the leaf growth zone. Plants were grown in a growth cabinet in perlite as a substrate and watered with complete N-NO₃ solution (+N) and N-NO₃ deficient solution (–N). Comparison between the investigated genotypes showed that final leaf length in both N treatments was related with LER, but not with the duration of leaf elongation. Faster leaf elongation rate in high-LER compared with low-LER genotype, was associated with longer growth zone, a bigger number of cells in it, and higher cell flux rate, although cell elongation rate was similar in both genotypes. These lines of evidence indirectly indicated that leaves of the faster growing genotype were characterized by higher meristematic activity. Nitrogen deficiency reduced the flux of cells and cell elongation rate, length of cell division zone and the number of cells in whole zone, significantly for both genotypes, although duration of cell elongation was increased and final epidermal cell length was unchanged. These results showed that N deficiency reduced both cell division and cell elongation, which in turn resulted in decreased leaf length and prolonged time for leaf development. Nitrogen deficiency significantly increased both bulk and segmental cell wall peroxidase activity in the growth zone of both investigated genotypes, thus showing an interaction between leaf growth cessation and enzyme activity.     

Plant regeneration from cultured protoplasts of Moricandia nitens

A protoplast culture procedure was developed for the wild species Moricandia nitens that is a C₃-C₄ intermediate with a close relationship to Brassica species. Protoplasts derived from leaf mesophyll, grown on B5 medium, were cultured at a density of 1 × 10⁵ ml^-1 in modified KM8p medium supplemented with 0.5 mg l^-1 BA, 1.0 mg l^-1 2,4-D and 0.1 mg l^-1 α-NAA. After 3 w of culture in the dark, microcalli were plated on an amplification medium in dim light. Calli larger than 3 mm in diameter were transferred to the regeneration medium in light. Twenty-three percent of the calli could regenerate plants from the media using a range of growth regulator combinations. Two or more shoots often grew from one callus. The procedure allowed hundreds of plantlets to develop from protoplast culture within a period of 10 w. This practical system provides the opportunity to transfer the C₃–C₄ intermediate character to Brassica crops via somatic hybridization. This revised version was published online in June 2006 with corrections to the Cover Date.     

Transformation of maize (Zea mays L.) protoplasts and regeneration of haploid transgenic plants

Transgenic haploid maize (Zea mays L.) plants were obtained from protoplasts isolated from microspore-derived cell suspension cultures. Protoplasts were electroporated in the presence of plasmid DNA containing the gus A and npt II genes encoding ß-glucuronidase (GUS) and neomycin phosphotransferase II (NPT II), respectively. Transformed calli were selected and continuously maintained on kanamycin containing medium. Stable transformation was confirmed by enzyme assays and DNA. analysis. Stably transformed tissue was transferred to regeneration medium and several plants were obtained. Most plants showed NPT II activity, and some also showed GUS activity. Chromosome examinations performed on representative plants showed that they were haploid. As expected, these plants were infertile.     

The effect of gamma radiation and N-ethyl-N-nitrosourea on cultured maize callus growth and plant regeneration

Regenerable maize calli of two inbred lines were exposed to 0 to 100 Gy of gamma rays or treated with 0 to 30 mM of N-ethyl-N-nitrosourea (ENU) to determine their effect on growth and plant regeneration capability. Both growth and plant regeneration capacity decreased with increasing levels of either gamma radiation or ENU; however, plant regeneration capacity was more sensitive to either agent than growth. The 50% inhibition dose (I₅₀) for callus growth (fresh-weight gain) was approximately 100 Gy of gamma radiation and 30 mM ENU. The I₅₀ for plant regeneration capacity of treated callus was approximately 25 Gy of gamma radiation and 2.5 mM ENU. The decrease in plant regeneration capacity correlated with a change in tissue composition of the treated callus from a hard, yellow and opaque tissue to a soft, grayish-yellow and translucent tissue. This change was quantified by measuring the reduction of MnO₄ ^- to MnO₂ (PR assay) by the callus. These results suggest that the effect of gamma radiation or ENU on plant regeneration capacity must be taken into consideration if these potentially mutagenic agents are to be used on maize callus cultures, for the purpose of producing useful mutations at a whole plant level. The data also suggest that the PR assay may be useful for predicting the actual plant regeneration capacity of maize callus.Abbreviations g f.w. gram fresh weight - ENU N-ethyl-N-nitrosourea - PR assay permanganate reduction assay - I₅₀ 50% inhibition dose     

Ultrastructural characterization and three-dimensional reconstruction of isolated maize (Zea mays L.) egg cell protoplasts

Summary Isolated egg cell protoplasts ofZea mays L., inbred line A 188, have been studied at the transmission electron microscope level. Their preparation for electron microscopy has been performed by embedding in ultra-low gelling agarose as a preliminary step. Five isolated egg cell protoplasts were serially ultrathin sectioned and studied in detail. One of these protoplasts was reconstructed in three dimensions to provide additional information on its structure. After enzymatic digestion and microdissection, isolated egg cells are true, highly vacuolized protoplasts. The structure of their organelles agrees with in situ observations, indicating an ultrastructural intactness after isolation: the mitochondria are polymorphic, form reticulate networks, and have well developed cristae; the plastids contain starch grains; and the spherical nucleus is euchromatic. As in situ, the organelles of the isolated egg cell protoplasts are aggregated near the nucleus. The complete picture provided by this work should serve as a comparative base for studies on in vitro fertilization products.     

Modeling the water use efficiency of soybean and maize plants under environmental stresses: application of a synthetic model of photosynthesis-transpiration based on stomatal behavior

Understanding the variability of plant WUE and its control mechanism can promote the comprehension to the coupling relationship of water and carbon cycle in terrestrial ecosystem, which is the foundation for developing water-carbon coupling cycle model. In this paper, we made clear the differences of net assimilation rate, transpiration rate, and WUE between the two species by comparing the experiment data of soybean (Glycine max Merr.) and maize (Zea mays L.) plants under water and soil nutrient stresses. WUE of maize was about two and a half times more than that of soybean in the same weather conditions. Enhancement of water stresses led to the marked decrease of Am and Em of two species, but water stresses of some degree could improve WUE, and this effect was more obvious for soybean. WUE of the two species changed with psiL in a second-order curve relation, and the WUE at high fertilization was higher than that at low fertilization, this effect was especially obvious for maize. Moreover, according to the synthetic model of photosynthesis-transpiration based on stomatal behavior (SMPTSB) presented by Yu et al. (2001), the WUE model and its applicability were discussed with the data measured in this experiment. The WUE estimated by means of the model accorded well with the measured values. However, this model underestimated the WUE for maize slightly, thus further improvement on the original model was made in this study. Finally, by discussing some physiological factors controlling Am and WUE, we made clear the physiological explanation for differences of the relative contributions of stomata- and mesophyll processes to control of Am and WUE, and the applicability of WUE model between the two species. Because the requirement to stomatal conductance by unit change of net assimilation rate is different, the responses of opening-closing activity of stomata to environmental stresses are different between the two species. To obtain the same level of net assimilation rate, soybean has to open its stomata more widely to keep small stomatal resistance, as compared with maize.     

Adventitious shoot regeneration from leaf explants of tissue cultured and greenhouse-grown raspberry

Adventitious shoot regeneration was observed using leaf-petiole explants from shoot-proliferating cultures of Comet red raspberry (Rubus idaeus L.). A maximum regeneration rate of 70% (3.7 shoots/explant) was obtained using 4.5–9.1 M (1–2 mg l^–1) N-phenyl-N-1,2,3-thiadiazol-5-ylurea (thidiazuron or TDZ) with 2.5–4.9 M (0.5–1 mg l^–1) 1H-indole-3-butanoic acid (IBA) or 2.3 M (0.5 mg l^–1) TDZ with 4.9 M (1 mg l^–1) IBA in modified Murashige-Skoog medium. TDZ was more effective than N-(phenylmethyl)-1H-purin-6-amine (BA) at promoting regeneration in combinations tested with IBA (maximum 50% regeneration rate; 1.8 shoots/explant). Variation in the agar concentration or incubation temperature, orientation or scoring of the leaf-petiole explants and use of separate leaf or petiole explants had no effect on shoot regeneration. Incubation in the dark for 1, 2 or 3 weeks prior to growth in the light did not influence the percent regeneration rate but depressed the number of adventitious shoots. Explant source, from micropropagated shoots or greenhouse-grown plants, had an effect on shoot regeneration that was genotype dependent. Only 8 of 22 (36%) raspberry cultivars were capable of regeneration from leaf explants derived from greenhouse-grown plants.     

Composition of the cell wall formed by protoplasts isolated from cell suspension cultures of Vinca rosea

The biochemistry of cell-wall regeneration in protoplasts obtained from Vinca rosea L. (Catharanthus roseus (L.) G. Don) cells grown in suspension culture by isolating the regenerated wall and the extracellular polysaccharides of protoplasts cultured for various periods, and investigating their composition. Gas-liquid chromatography and tracer studies with D-[U-¹⁴C]glucose showed that the sugar composition of the extracellular polysaccharides was similar to that of the original cell culture, consisting mainly of polyuronide and 3,6-linked arabinogalactan. the regenerated cell wall was composed of non-cellulosic glucans having 1,3- and 1,4-linkages, while its content in pectic and hemicellulosic components was very low.     

Construction and characterization of normalized cDNA library of maize inbred MO17 from multiple tissues and developmental stages

A comprehensive complementary DNA (cDNA) library is a valuable resource for functional genomics. In this study, we set up a normalized cDNA library of Mo17 (MONL) by saturation hybridization with genomic DNA, which contained expressed genes of eight tissues and organs from inbred Mo17 of maize (Zea mays L.). In this library, the insert sizes range from 0.4 kb to 4 kb and the average size is 1.18 kb. 10 830 clones were spotted on nylon membrane to make a cDNA microarray. Randomly picked 300 clones from the cDNA library were sequenced. The cDNA microarray was hybridized with pooled tissue mRNA probes or housekeeping gene cDNA probes. The results showed the normalized cDNA library comprehensively includes tissue-specific genes in which 71% are unique ESTs (expressed sequence tags) based on the 300 sequences analyzed. Using the BLAST program to compare the sequences against online nucleotide databases, 88% sequences were found in ZmDB or NCBI, and 12% sequences were not found in existing nucleotide databases. More than 73% sequences are of unknown function. The library could be extensively used in developing DNA markers, sequencing ESTs, mining new genes, identifying positional cloning and candidate genes, and developing microarrays in maize genomics research.__________From Molekulyarnaya Biologiya, Vol. 39, No. 2, 2005, pp. 198–206.Original English Text Copyright © 2005 by Z. Zhang, F. Zhang, Tang, Pi, Zheng.This article was submitted by the authors in English.     

Influence of 2,4-D,TIBA and 3,5-D on the growth response of cultured maize embryos

Induction of embryogenic calli from immature zygotic embryos of maize requires the presence of 2,4-D or similar auxin-like growth regulators in the culture medium. Pulse-chase experiments with 2,4-D, using various concentrations of 2,4-D in the induction medium were tested in relation to induction of callus in the embryogenic inbred line A188 and the non-embryogenic inbred A632. Interactions of 2,4-D, 3,5-D and the auxin transport inhibitor TIBA were also studied. Pulse-chase experiments showed that exposure to 2,4-D influenced the culture response from 0.5 h onwards. After a pulse of 0.5 h, shoot and root elongation of the embryo was stimulated. A pulse of 16 h or longer induced outgrowths and callus formation at the basal side of the scutellum. Pulses of 7 days and longer resulted in the induction of friable embryogenic Type II callus in A188. Embryos were cultured at 2,4-D concentrations ranging from 0.002 to 2000 mg l⁻¹ and optimal concentration for the induction of embryogenic callus in A188 was 2 mg l⁻¹. At lower concentrations there was a transition between callus formation and germination; at increasing concentrations, callus induction was reduced and finally growth responses became blocked. When TIBA was added to medium without 2,4-D, root elongation decreased in a dose-dependent way suggesting the need of polar transport of endogenous auxins for root elongation. When added to medium with 2,4-D, TIBA caused suppression of callus formation, again pointing to the necessity of polar transport of 2,4-D. In combination with 2,4-D, cultures with 3,5-D resembled cultures at lower 2,4-D concentrations, pointing to a competitive interaction between 3,5-D and 2,4-D. This revised version was published online in June 2006 with corrections to the Cover Date.     

Efficient plant regeneration from cell suspension protoplasts of the woody medicinal plant Solanum dulcamara L. (bittersweet,woody nightshade)

Large populations of viable protoplasts were released from suspension cultured cells of the woody medicinal plant Solanum dulcamara (bittersweet, woody nightshade) when the cells were harvested 3 to 7 months after culture initiation and 4 to 5 days after transfer to fresh medium. A Bio-Gel p6 purified enzyme mixture enhanced the protoplast plating efficiency 6 fold compared to the unpurified mixture, without affecting protoplast yield. Agarose-solidified medium markedly improved protoplast division and colony formation, and enabled protoplasts to be plated at lower densities than in liquid medium. All protoplast-derived tissues produced shoots on MS based medium with 1.0 mgl^-1 zeatin. Shoots rooted readily on medium lacking phytohormones. Cytological examination revealed high chromosome stability of suspension cultured cells, of plants derived from such cells, and of protoplast-derived plants. The implication of these results is discussed in relation to the genetic manipulation of this pharmaceutically important plant.     

O-Acetylation of plant cell wall polysaccharides: identification and partial characterization of a rhamnogalacturonan O-acetyl-transferase from potato suspension-cultured cells

 A microsomal preparation from suspension-cultured potato stem cells (Solanum tuberosum L. cv. AZY) was incubated with [¹⁴C]acetyl-CoA resulting in a precipitable radiolabeled product. Analysis of the product revealed that it consisted mostly of acetylated proteins and cell wall polysaccharides, including xyloglucan, homogalacturonan and rhamnogalacturonan I. Thus, acetyl-CoA is a donor-substrate for the O-acetylation of wall polysaccharides. A rhamnogalacturonan acetylesterase was used to develop an assay to measure and characterize rhamnogalacturonan O-acetyl transferase activity in the microsomal preparation. Using this assay, it was shown that the transferase activity was highest during the linear growth phase of the cells, had a pH-optimum at pH 7.0, a temperature optimum at 30 °C, an apparent K _m of 35 μM and an apparent V _max of 0.9 pkat per mg protein. Further analysis of the radiolabeled acetylated product revealed that it had a molecular mass >500 kDa. Received: 3 July 1999; Accepted: 27 September 1999     

A coupled model of photosynthesis-transpiration based on the stomatal behavior for maize (Zea mays L.) grown in the field

The study presents a theoretical basis of a stomatal behavior-based coupled model for estimating photosynthesis, A, and transpiration, E. Outputs of the model were tested against data observed in a maize (Zea mays L.) field. The model was developed by introducing the internal conductance, g _ic, to CO₂ assimilation, and the general equation of stomatal conductance, g _sw, to H₂O diffusion, into models of CO₂ and H₂O diffusion through the stomata of plant leaves. The coupled model is easier for practical use since the model only includes environmental variables, such as ambient CO₂ concentration, leaf temperature, humidity and photosynthetic photon flux received at the leaves within the canopy. Moreover, concept of g _ic, and factors controlling A and E were discussed, and applicability of the model was examined with the data collected in the maize field.