Deoxyribonucleotide synthesis and DNA polymerase activity in plant cells (Vicia faba and Glycine max).

Enzymes of deoxyribonucleotide and DNA biosynthesis, which are little known in plants, were studied in root tips of germinating broad beans (Vicia faba) and in fast-growing cultures of soybean cells (Glycine max). The plant cells contain a ribonucleoside 5'-diphosphate reductase which is detected in vitro only during a limited period of growth, viz. 30--32 h after inhibition of Vicia seeds, and between the second and third day after inoculation of soybean cultures. In both species ribonucleotide reductase activity precedes maximum DNA synthesis. The reductases could be precipitated with ammonium sulfate but were not purified further due to the extremely low enzyme content of the plant extracts. Therefore the reductive pathway of deoxyribotide formation was also established in Vicia root tips by efficient labeling of the plant DNA with a ribonucleoside, [5-3H]cytidine, which reaches a maximum at the same time as the reductase activity measured in vitro. Cycloheximide inhibits this process, indicating the need for de novo enzyme induction. In contrast, DNA polymerase is present in the tissue throughout the entire development and rises only 2-fold in activity during the S phase. The soluble polymerases were partially characterized in both legume species and were found very similar to the DNA polymerase of pea seedlings. Ribonucleotide reductase is more likely a limiting component of DNA formation during the plant cell cycle than DNA polymerase.     

Studies on the Micronuclei Induced by Irradiation in Vicia faba

In this experiment ,the remits of observing under the light microscope and electron microscope, and statistical analysis showed that micronucleus had a certain shape and structure. There was a linear correlation between the micronudeated cell frequency and dosage or the chromosomal aberration frequency in the root tip cells. The micronudeated cell frequency changed according to the different periods of time after irradiation. At the same time, micronuclei that might be synchronous, or not, with the main nucleus for the cell cycle were seen. That means it is possible for some micronuclei induced by irradiation to carry on division. Therefore, the advantages of micronucleus test lies not only in simply and effectively evaluating radiation damage to cells but also in exploring the mechanism of radiation genetics in plant.     

Studies on the Mitotic Prophase Chromosomal Fibers in Vicia faba

The observations have been made on the structures of mitotic prophase nuclei and chromosomes in Vicia faba root meristematic cells. Two methods, i.e., the cell squashing and the nucleus isolation methods, were applied in present study to prepare the specimen of chromosomes and nuclei. Chromosomal fibers 0.3—0.5 μm in diameter were observed in the squashed preparations stained with Giemsa, and in the isolated nucleus preparations treated with 0.05% EDTA followed by Giemsa staining. Using Feulgen reaction, it has been demonstrated that these fibers are nuclear origin containing DNA. The results suggest that this order of chromosomal fiber may be one structural level in the chromosomes in Vicia faba. This conclusion is in support of the view which holds that there exists an intermediate level of structure between the 250–300Å chromatin fiber and the chromosome.     

Legumin Synthesis in Developing Cotyledons of Vicia faba L

The synthesis of legumin in developing cotyledons of Vicia faba L. has been examined as a potential system for approaching the problem of differential gene expression. The pattern of legumin synthesis was determined during the growth of the cotyledon by microcomplement fixation which provided a sensitive and specific assay for legumin in the presence of vicilin. Legumin was detected even in young cotyledons. However, when the cotyledons were about 10 millimeters long, and cell division was essentially complete, there was a sharp increase in the rate of legumin accumulation.     

Studies on the movement and distribution of ethylene in Vicia faba L.

In Vicia faba ethylene does not appear to move between different parts of the plant in physiologically significant amounts. The resistance to longitudinal movement is such that lateral emanation effectively isolates different parts of the plant from each other. When emanation is prevented, ethylene can be channelled to any part of the plant. Exposure of one section of a plant to ¹⁴C-labelled ethylene (up to 200 l/l) increased the internal concentration in other parts with ethylene that did not originate from the feeding chamber. A basipetal gradient of endogenous ethylene concentration was found in the lacuna of intact plants, the source of ethylene being the stem tissue. The permeability of stem tissue to ethylene decreases with age. The concentration of ethylene in tissues surrounding the lacuna is always higher than that in the lacuna and it is argued that compartmentation of ethylene occurs within these tissues.     

蚕豆特矮秆突变体的遗传研究

用在国内首次发现的蚕豆特矮秆突变体的优良后代“鄂农 82矮”及高秆品种“启豆1号”为亲本, 研究了株高及其与株高密切有关的两个性状──节间长和节数的遗传。结果表明:株高、节间长、节数3个性状受核基因控制,不存在任何形式的母性遗传。供试双亲株高性状存在一对基因的差异,我们用Df-df表示这一基因座的两个等位基因,高秆亲本基因型为Df Df,矮秆亲本的基因型为dfdf,高秆对矮秆为显性。数量遗传分析表明,3个性状的遗传均符合加性显性模型,株高、节长的加性效应和显性效应都很重要,显性度接近于1。节数仅受加性效应控制,显性效应不显著。 Abstract:DwarF Enong 82,which derived from the unique dwarf mutant discovered in China,was crossed with the commercial broad bean cultivar Qidou 1.From the crossing six basic generations were generated for studing the genetical behaviors of plant height and two related characters i.e. node length and number of nodes.The results showed that the three characters were governed by nuclear genes.The difference in plant height between two parents was related to a loci,which was designated Df-df.Generation mean analyses revealed that the additive-dominance model was adequate to interpreting the inheritance of the three characters.The estimates of number of genes controlling these characterswere the same astheresults with Mendlian methods.     

Apical Dominance in Vicia faba

Apical dominance phenomena have been studied in seedlings ofVicia faba particularly in relation to the movement about theplant of uracil-2-¹⁴C applied to the cotyledons. Decapitationjust below the second node releases the growth of the lowermostlateral bud and inhibition is completely reimposed by applicationof indole-3-acetic acid (IAA) to the cut surface. Uracil-2-¹⁴Capplied in solution to the cotyledons is distributed in thestems of all experimental seedlings with no consistent differencesdue to decapitation or IAA application. On the other hand, decapitationresults in a rapid increase in uracil-2-¹⁴C content in the lateralbuds which far exceeds their promoted growth. This uptake iscompletely suppressed by IAA application. A ring of tri-iodobenzoicacid (TIBA)-lanolin paste around the stem above the bud suppressesIAA action both in bud growth and on uracil-2-¹⁴C uptake, andalso on the movement of IAA-1-¹⁴C down the stem. TIBA-application to the base of the bud does not prevent IAAaction on bud growth, but also does not prevent the movementof IAA-1-¹⁴C (or a water soluble product of its metabolism)into the bud. Direct application of kinetin to the lateral bud of intact plantscauses a short-lived release of growth. Gibberellic acid producesa smaller and scarcely significant increase which is additiveto the kinetin effect. Neither has any effect on uracil-2-¹⁴Cmovement into the bud. The implications of these findings are discussed in relationto various existing theories of the mode of auxin action inapical dominance and it is concluded that their strongest supportis for a mechanism involving the suppression of phloem differentiationin the vascular supply to the bud.     

Studies on pollination and fertilization in the field bean (Vicia faba L.)

The percentage of cross-fertilization in the field bean was found to be around 45%, and to be higher on the lower nodes of the plants than on the upper. No differences in the relative pollen tube growth rates of self and foreign pollen were observed. The different lines and genotypes investigated responded differently to open pollination, selfing with manual tripping, and selfing without tripping. Fertilizer application and date of sowing affected the number of flowers produced and the number of seeds set, but did not affect the ratio of seeds to flowers, i.e. the index of self fertility.     

First DNA Synthesis around Sterile and Crown Gall-Inoculated Wounds in Vicia faba

DNA synthesis was measured during a 30 hour period in stem segments containing a wound — either sterile or inoculated with Agrobacterium tumefaciens— and in control stems of Vicia faba. An identical series of experiments was conducted both on upper, still growing internodes and on lower, mature ones. The incorporation of ³H-thymidine was determined from extracted DNA with a liquid scintillation spectrometer. The results from the two internodes were fairly similar. DNA synthesis was not significantly different in the two types of wounds. It rose above control level at about 10 hours after wounding and reached its peak at about 19 hours. Roughly, this pattern of DNA synthesis seems to correspond to the minimum conditioning and transformation time as found in other plants. However, the decisive factor need not be DNA synthesis, but could be some other process coinciding with it.     

Studies of Chromosome Banding with Giemsa in Vicia faba and Allium cepa

Giemsa dye is a complex mixture containing methylene blue, its oxidation products-azure Ⅰ, Ⅱ, Ⅲ, and their eosinate. The results of our experiments have demonstrated that staining with methylene blue alone can give a faint trace of banding as well as azure Ⅰ, Ⅱ. No bands are obtained with eosin. Nevertheless, good chromosome bandings can be often produced by staining with methylene blue-eosinate or azure Ⅱ-eosinate. These data indicate that eosinate has an important effect for the formation of C-banding on plant chromosomes. In our experiments, the treatments of chromosomes with trypsin or papain have also resulted in good C-banding pattern when slides are stained with Giemsa. We found that the slides untreated with proteinase showed homogeneous intense chromosome staining and, on the contrary, the slides treated with proteinase led to palestaining chromosomes and presenting bandings. It has shown that proteinase, especially trypsin, not only can remove a large amount of chromosomal protein but also can remove DNA and results in C-bandings. Treated properly with trypsin and followed by the Feulgen staining, chromosomes can also produce the C-bandings, but chromosomes treated overtime with trypsin are stained more palely in Feulgen reaction or lead to colourlessness. The above results have further proved that trypsin technique removes large amounts of chromosome DNA and removes less from the C-band regions than from the non-band regions. In this paper we mainly discussed the effects of protein on mechanism of plant chromosome banding. We consider that the production of plant C-banding is probably due to the differential accessibility of nucleoprotein between euehromatin and heteroehromatin regions. It brings about selective removal of nucleoprotein from the chromosome arms. We have compared the effect of trypsin with papain and pepsin on producing bands. Good bands are produced by Giemsa staining chromosomes with trypsin, but no bands are obtained by staining chromosomes treated with pepsin. So the results have expressed that histones are possibly playing more important role in C-bandings.     

Sister chromatid exchanges in Vicia faba

Evolutionary loss of the Y chromosome has occurred in Climacia areolaris (Hagen) of the neuropteran family Sisyridae. The diploid set comprises 6 pairs of autosomes, plus 2 X chromosomes in the female and 1 X in the male. The Y is retained in Sisyra vicaria (Walker) of the same family: its chromosome number is 14 in both sexes including 2X chromosomes in the female and 1X plus Y in the male. Two alternative pathways for the segregation of the sex chromosomes-distance segregation and sex bivalent formation-co-exist in the latter species in a ratio of approximately 1 to 6; the possible phylogenetic significance of this feature is discussed.     

Agrobacterium-mediated transformation of Vicia faba

Among the major grain legume crops, Vicia faba belongs to those where the production of transgenic plants has not yet convincingly been reported. We have produced stably transformed lines of faba bean with an Agrobacterium tumefaciens-mediated gene transfer system. Stem segments from aseptically germinated seedlings were inoculated with A. tumefaciens strains EHA101 or EHA105, carrying binary vectors conferring (1) uidA, (2) a mutant lysC gene, coding for a bacterial aspartate kinase insensitive to feedback control by threonine, and (3) the coding sequence for a methionine-rich sunflower 2S-albumin, each in combination with nptII as selectable marker. Kanamycin-resistant calluses were obtained on callus initiation medium at a frequency of 10–30%. Shoot regeneration was achieved on thidiazuron containing medium in a second culture step. A subsequent transfer of shoots to BA-containing medium was necessary for stem elongation and leaf development. Shoots were rooted or grafted onto young seedlings in vitro and mature plants were recovered. Molecular analysis confirmed the integration of the transgenes into the plant genome. Inheritance and expression of the foreign genes was demonstrated by Southern blot, PCR, western analysis and enzyme activity assays. Although at present the system is time-consuming and of relatively low efficiency, it represents a feasible approach for the production of genetically engineered faba beans.     

Nitrogen Metabolism of Vicia faba

Vicia faba plants were grown for four and six weeks without externally supplied nitrogen. Some nitrogen was transported to the plant axis from the cotyledons throughout this period, but the amount available was insufficient to support maximum shoot growth. During this period the protein content of the shoot declined whilst the free amino acids, especially aspartic acid, glutamic acid, histamine and the combined pool for threonine, serine, asparagine and glutamine and ammonia, increased in amount. In contrast to the shoot the protein content of the root increased as did their free amino acid content, but the increase in the latter was less than in the shoot and only the combined value for threonine, serine, asparagines and glutamine increased significantly. During tbe last two weeks growth, some soluble non-amino acid compound appeared to donate nitrogen to the pool of free amino acids in the root and shoot.     

Sister chromatid exchanges in Vicia faba

The relationship between chromosomal aberrations and sister chromatid exchanges (SCE's) after treatment of Vicia faba root tips with thiotepa, caffeine and 8-ethoxycaffeine (EOC) was studied by using a modified fluorescent plus Giemsa (EPG) technique. At concentrations which had little effect on the frequency of chromosomal aberrations, thiotepa strongly increased the frequency of SCE's, provided that the chromosomes were allowed to replicate between treatment and fixation. Frequently, the size of the exchanged material was smaller than the diameter of the chromatid. Post-treatments with caffeine of roots previously exposed to thiotepa strongly increased the frequency of aberrations, but had little effect on the frequency of SCE's. In contrast to thiotepa, EOC caused only a slight increase in the frequency of SCE's even at concentrations which produced a high frequency of chromosomal aberrations. Thus, there was not a close correlation between SCE's and chromosomal aberrations. Single-strand exchanges between the DNA double helices in sister chromatids were not detected.     

Biosynthetic studies on chlorinated indoles in Vicia faba

We identified L-4-chlorotriptophan (L-4-Cl-Trp) in two week old plants of Vicia faba. Deuterium labeled indole was incorporated to 4-chloroindole in these plants. Deuterium labeled L- and D-4-Cl-Trp were both synthesized and, when supplied to the plants only L-4-Cl-Trp was incorporated to 4-Cl-indoleacetic acid, although the incorporation ratio was very low.     

蚕豆染色体的复制带显带技术

本文采用作者首创的双周期BrdU二次标记法研究了蚕豆根尖细胞染色体的复制带,得到了分布在整条染色体上的清晰稳定的多条带纹.这是复制带首次在植物染色体上取得的具有实用意义的带型.为进一步制定植物染色体的标准带型和研究植物染色体的复制方式提供了一条途径.     

Studies on induced changes in growth patterns and polarity in roots of Vicia faba L.

Summary Roots of Vicia faba were treated with colchicine (0.025%), or IAA (4.7×10^-6 M), or both, for 3 hours and fixed at various intervals over the following 11 days. The axis of spindle orientation and the distribution of mitotic figures, lateral root primordia and xylem vessel elements was examined in the apical 10 mm of median longitudinal sections of these roots.No effect of IAA was found on the orientation of the spindle. However, evidence was obtained indicating that the systems controlling the polarity of cell division and cell expansion differ in some way.The number of lateral root primordia formed was greater in roots treated with IAA or colchicine than in control roots. These primordia were always initiated adjacent to a xylem vessel. Thus, no primordium was closer to the apex than the most apical xylem vessel, suggesting that an endogenous factor involved in primordia initiation is transported in the xylem. The primordia which develop after colchicine treatment grow out as lateral roots; this is in contrast with those which form after IAA treatment and which do not undergo elongation. These results, which it must be emphasized apply only to the apical 1 cm of treated roots, indicate that lateral root primordia become sensitive to IAA at a certain stage in their development. Exogenous IAA acts as an inhibitor.The new meristem, which forms in the primary root apex after colchicine treatment, contains both diploid and polyploid cells, i.e. it was formed from cells that were unaffected and from cells that were affected by colchicine. Following colchicine treatment the size of the meristem shrinks and this can be prevented by treatment with IAA. This and other evidence presented here, suggests that IAA is a factor involved in the control of the size of the apical meristem in normal roots.     

Structural variations in Vicia faba mitochondrial genome

Summary A comparative analysis of the presence of minicircular DNA CCCIB in 16 different lines and cultivars of fertile Vicia faba L. plants was conducted. It was found that copy number of CCCIB ranged from several copies per mitochondrial genome to — probably — zero, depending on cultivar or line. Fertility of plants in these cases was not altered. We chose 10 cultivars and lines among 16 analysed. Mitochondria of five cultivars and lines contained about two CCCIB molecules per one CCCIA. The sixth cultivar contained CCCIB at copy number several times lower. In the last four cultivars CCCIB could not be identified. Copy number analysis of CCC2 in ten chosen cultivars and lines revealed that in eight cases the quantitiy of CCC2 was equal to CCCIA. However, two other cultivars contained about two times lower quantity of CCC2. Parallel to that we observed an increase in quantity of one sequence homologous to CCC2, which in the first eight cultivars and lines could be found only in minor quantities. Comparative restriction analysis revealed notable rearrangement events in mitochondrial DNAs of ten cultivars and lines being investigated. We did not find any correlations between patterns of restriction fragments and copy number of CCCIB. In some cases, rearrangements in Vicia faba mitochondrial genomes caused a duplication of sequences homologous to the Zea mays coxII gene.