Somatic Chromosome Counts from Leaf Meristems in the Tribe Triticeae

The Feulgen procedure was applied to chromosome preparations of leaf meristems from growing plants of wheat, barley, and wheat-wheatgrass hybrids. Leaf primordia from the base of secondary tillers were pretreated in cold water (overnight, 2 C), fixed in glacial acetic acid (20 min, 2 C), hydrolyzed in 1 N HCl (14 min), stained in leuco-basic fuchsin (about 15 min) and squashed in 1% acetocarmine. The chromosome spreads from leaf meristems were generally superior to those of the root meristems of the same plant. Mitotic index in leaf meristems was higher than in root meristems in some species. The method appears useful for counting the chromosome number of growing plants, detecting chimeras, and verifying root tip chromosome counts.     

Genetic and biochemical analysis of Hypericum perforatum L. plants regenerated after cryopreservation

Shoot-tips from in vitro cultured Hypericum perforatum L. genotypes were subjected to assessments of developmental competence, genetic stability, and biosynthetic ability to identify critical points during cryopreservation. Survival rate, chromosome number stability, alteration in VNTR sequences and hypericin content were evaluated, in plants after pre-culture, and two subsequent cryogenic steps (cryoprotection and cooling) and those recovered from cryopreserved meristems. Pre-culture and cryoprotection treatments, did not reveal any significant differences, in these studied characteristics. Genetic stability was assessed by chromosome counts and analysis of variability in the VNTR sequences. No changes in chromosome number were detected in comparison with the untreated control but minor alterations were revealed in non-coding sequences. The content of hypericin after the recovery of cryopreserved meristems remained comparable with the unfrozen control. The controlled rate freezing technique used for cryopreservation was relevant for restoration of genetic and biochemical stability in Hypericum perforatum L. shoot-tips.     

Involvement of knob heterochromatin in mitotic abnormalities in germinating aged seeds of maize.

Seeds of three inbred lines of maize, with contrasting numbers of heterochromatic knobs and stored under two different ageing treatments, were analyzed for the occurrence of abnormalities at mitotic anaphase in root meristems 3, 7, 21,42, and 56 days after germination, and in root meristems of their freshly harvested selfed progeny. The largest category of detectable aberrations involved breakage of knobbed chromosome arms. We have obtained evidence that knob heterochromatin plays a central role in the origin of primary chromosome bridges. The initial event responsible for the occurrence of breakages and lagging chromosomes was characterized by the nondisjunction of newly replicated sister chromatids, which was observed to occur preferentially at the knob level. Such configurations, and all the other types of abnormalities (as for example, lagging chromosomes, typical chromosome bridges, fragments, and micronuclei), were observed at decreasing frequencies throughout root growth. Nevertheless, we have detected the occurrence of breakage-fusion-bridge cycles that were initiated by broken chromosomes. The relationship between late-replicating DNA in maize knob heterochromatin and the vulnerability of such regions to breakage is discussed. Our observations suggest a similarity between the mechanisms involved or associated with the origin of the described abnormalities and those reported to occur in cultured maize cells.     

Refined examination of plant metaphase chromosome structure at different levels made feasible by new isolation methods

Two methods for isolation of plant metaphase chromosomes are described. The first, micromanipulation, allows the isolation of a number of individual chromosomes, which may be used as templates for the generation of chromosome specific DNA libraries and for physical sequence mapping by the polymerase chain reaction (PCR). The second provides, from synchronized meristems, pure chromosome suspensions suitable for flow cytometric analysis and chromosome sorting. Restriction endonuclease banding, immunostaining of chromosomal antigens, as well as fluorescence in situ hybridization at high signal to noise ratio were successfully performed on the isolated chromosomes. Chromosomes obtained by both protocols were suitable for scanning electron microscopy, the methods should also prove useful for refined analyses of the karyotypes of other plant species.by D. Schweizer     

Chromosome number stability and mitotic activity of cryopreserved Hypericum perforatum L. meristems

Meristems of in vitro-grown Hypericum perforatum L. plants were precultured for 3, 10, or 14 days in the presence of 0.5 M mannitol, or 0.076 µM or 0.76 µM abscisic acid, in RM basal liquid culture medium supplemented with 0.5 mg/l 6-benzylaminopurine and subsequently subjected to cryopreservation by the slow freezing method. The survival rate - determined as the percentage of meristems capable of differentiating plantlets - varied between 10% and 48%. Chromosome number stability of the cryopreserved meristems was determined by chromosome counting. The mitotic index of the control did not significantly differ from that of the treated samples.     

Somatic chromosome number doubling of selected potato genotypes using callus culture or the colchicine treatment of shoot nodes in vitro

Experiments were carried out to double the somatic cell chromosome numbers of a monoploid and dihaploid of Solanum tuberosum and a genotype of S. circaeifolium subsp. quimense. Colchicine was used in vitro on shoot nodes from which the axillary meristems had been removed. Plants with doubled chromosome numbers were obtained from shoots grown from the tertiary, sub-axillary meristems of all three genotypes. The callus culture of stem and leaf explants was found to produce more shoots with doubled chromosome numbers than the colchicine treatment in the case of the dihaploid and quimense genotypes but no shoots were obtained from callus culture of the monoploid. Fifty-two % of the shoots from the dihaploid and 63% from the quimense clone were ploidy doubled in the case of the best callus culture system. Using a sub-lethal dose of colchicine, the dihaploid yielded 37% ploidy-doubled shoots whereas all the shoots produced from the monoploid were doubled and the quimense clone produced 27% doubled plants. Callus culture was highly dependent upon the type of growth medium and other, unknown, factors. The colchicine treatment, although yielding fewer products, was more reliable for achieving ploidy doubling in selected clones if the number of plants produced is not important.     

The Nuffield O-level Biology Project,Examinations and Teachers in Training

A procedure is described for providing preparations of dividing cells from root apical meristems, requiring only inexpensive equipment and minimal experimental skill. 8-Hydroxyquinoline is used instead of the more common colchicine, and Toluidene-blue is used as a chromosome stain. The method has been successfully tested in schools and also yields permanent preparations of adequate quality for university use.     

Survival and growth of eastern white pine shoot apical meristemsin vitro

Shoot apical meristems of seedling and mature eastern white pine trees were excised and grownin vitro. Placing the meristems on filters instead of directly on agarose-solidified nutrient medium enhanced survival of both juvenile and mature meristems. Applying forcing treatments to mature branches improved survival and growth of dissected meristems compared with meristems from non-forced branches in experiments conducted over two years. No consistent differences were observed among 2-, 4-, and 6-week forcing treatments. Including 5.37 nM (0.001 mg l^-1) l-naphthaleneacetic acid in the culture medium did not affect meristem survival or growth. Some meristems from seedlings grew rapidly, produced primary leaves, underwent internode elongation, and in three cases, produced adventitious roots. Meristems from mature trees did not grow as rapidly as seedling meristems. The leaves produced by mature meristems appeared to be scale leaves and a few of these had brachyblast primordia in the axils. The shoots derived from mature meristems did not produce adventitious roots.     

The plant nucleolar cycle under hypoxia

Summary Hypoxia disturbs the nucleolar cycle inAllium cepa L. meristems by diminishing the disorganizing stage and increasing the nucleologenesis time.Though nucleolar remnants persist in the enlarged metaphase recorded under hypoxia, prenucleolar bodies appear at the same time than in control meristems if related to the timing of nucleolar envelope breakage. Such appearance is apparently independent of the chromosome condensation cycle, since it took place in anaphase and not in midtelophase as in controls.Finally, the involvement of NORs in segregation is questioned since prenucleolar bodies are also segregated under hypoxia, both when integrated in the reforming nuclei or when dispersed in cytoplasm.     

Genetic and morphological characterization of the barley <Emphasis Type="Italic">uniculm2</Emphasis> (<Emphasis Type="Italic">cul2</Emphasis>) mutant

Axillary meristem growth and development help define plant architecture in barley (Hordeum vulgare L). Plants carrying the recessive uniculm2 (cul2) mutation initiate vegetative axillary meristem development but fail to develop tillers. In addition, inflorescence axillary meristems develop into spikelets, but the spikelets at the distal end of the inflorescence have an altered phyllotaxy and are sometimes absent. Double mutant combinations of cul2 and nine other recessive mutations that exhibit low to high tiller number phenotypes resulted in a uniculm vegetative phenotype. One exception was the occasional multiple shoots produced in combination with granum-a; a high tillering mutant that occasionally produces two shoot apical meristems. These results show that the CUL2 gene product plays a role in the development of axillary meristems into tillers but does not regulate the development of vegetative apical meristems. Moreover, novel double-mutant inflorescence phenotypes were observed with cul2 in combination with the other mutants. These data show that the wild-type CUL2 gene product is involved in controlling proper inflorescence development and that it functions in combination with some of the other genes that affect branching. Our genetic analysis indicates that there are genetically separate but not distinct regulatory controls on vegetative and inflorescence axillary development. Finally, to facilitate future positionally cloning of cul2, we positioned cul2 on chromosome 6(6H) of the barley RFLP map.     

An Improved Method for Preparing the Chromosomes of Pines and other Gymnosperms

A simple technic is described to produce well spread gymnosperm chromosomes. Root tip meristems are digested with a pectinase:cellulase mixture to produce a cell suspension which then is squashed to yield flat, well spread chromosome complements that can be stained or used for in situ hybridization.     

Fluorescent labeling of plant chromosomes in suspension by FISH

By optimizing the concentration and time of treatment with hydroxyurea (HU), a DNA synthesis inhibitor, and trifluralin, a microtubule inhibitor, a highly effective (over 60%) cell cycle synchronization method for rye and barley meristem cells was developed. Chromosome suspensions containing highly purified and morphologically intact rye and barley chromosomes were prepared from the meristems of their root tips by homogenization. Digoxigenin-labeled 5S rDNA was used as a probe in FISH for the rye chromosomes in the suspension, and biotin-labeled 17S rDNA and centromeric DNA were used in FISH for the rye and barley chromosome suspensions, respectively. Bright signals were detected at the specific regions of interest on the chromosomes. The results indicate that the method developed in this study is useful for selection and sorting of chromosomes that are not distinguishable by other means, using specific fluorescent labeling by FISH of the chromosomes in suspension.     

Clastogenic effects produced by black pepper in mitotic cells of Vicia faba

Black pepper, as is well known, is an important spice widely used in the cooking and processing of meat and fish. The aim of the present investigation was to evaluate the clastogenic potential of black pepper. This was accomplished by treating root meristems of Vicia faba with aqueous extracts of black pepper. Examination of the treated roots showed the presence of chromatid breaks, chromosome breaks, gaps and exchanges. Statistically significant differences from controls were observed. Experiments to evaluate its clastogenic potential in mouse systems are in progress and the results will be published elsewhere.     

Growth kinetics of the Golgi apparatus during the cell cycle in onion root meristems

In onion root meristems, the number of dictyosomes per cell shows a kinetics of growth strongly related to the cell cycle. During the interphase of steady-state proliferative cells, the volume density and numerical density of the Golgi apparatus decrease to reach minimum values in late-interphase cells, characterized by their greatest length. This pattern is also found in the total volume occupied by Golgi apparatus. Once in mitosis, the above-mentioned parameters begin to increase reaching maximum mean values in telophase. After the experimental uncoupling of chromosome and growth cycles by presynchronization with hydroxyurea, we found a similar behaviour pattern in the Golgi apparatus: decreasing values during interphase and a triggering of Golgi-apparatus growth in prophase independently of the bigger cell sizes reached in mitosis as an effect of pretreatment with hydroxyurea. These results indicate a cyclic kinetics of this subcellular component in higher-plant meristems, coupled with early mitotic events.     

Natural variation in nuclear characters of meristems in Vicia faba

The natural variation in chromosome size in root-tip and shoot apex meristems of Vicia faba has been studied. Chromosomes in main root-tips of one week old plants are 2–3 times larger than those in small lateral root-tips of 3 week old plants. While the nuclear DNA content remains constant, the nuclear RNA and nuclear histone contents show a positive linear correlation with chromosome volume. The DNA: histone ratio varies and is lowest in cells with large chromosomes. Nuclear volume and chromosome volume are not linearly related and changes in nuclear density therefore seem likely. A positive linear relationship between chromosome volume and mitotic index in colchicined squashes is demonstrated. The results strongly suggest that variation in chromosome size indicates a corresponding change in the rate of cell metabolism and may well reflect change in genetic activity.     

利用氟乐灵进行同源四倍体萝卜种质创制研究

本研究应用除草剂氟乐灵处理两叶一心幼苗生长点,进行同源四倍体萝卜种质诱导,对变异植株进行形态、细胞学鉴定和花粉母细胞染色体数目鉴定。结果表明,应用 0.2 mmol/L 和 1.0 mmol/L 氟乐灵处理,6个萝卜品种都获得同源四倍体植株,10 mmol/L 处理仅在 Nau-zhqh 得到同源四倍体;其中 0.2 mmol/L处理 Nau-dy 和 1.0 mmol/L 处理 Nau-xbch 获得四倍体最高诱导率(40%);四倍体种质与二倍体种质相比,形态性状、气孔大小、保卫细胞内叶绿体数目、花器官大小、花粉粒大小及花粉萌发率都存在显著差异,将形态、气孔鉴定和染色体计数结合可以准确确定变异株的倍性。研究表明利用氟乐灵诱导是进行萝卜同源四倍体种质创新的有效途径之一。本研究应用除草剂氟乐灵处理两叶一心幼苗生长点,进行同源四倍体萝卜种质诱导,对变异植株进行形态、细胞学鉴定和花粉母细胞染色体数目鉴定。结果表明,应用 0.2 mmol/L 和 1.0 mmol/L 氟乐灵处理,6个萝卜品种都获得同源四倍体植株,10 mmol/L 处理仅在 Nau-zhqh 得到同源四倍体;其中 0.2 mmol/L处理 Nau-dy 和 1.0 mmol/L 处理 Nau-xbch 获得四倍体最高诱导率(40%);四倍体种质与二倍体种质相比,形态性状、气孔大小、保卫细胞内叶绿体数目、花器官大小、花粉粒大小及花粉萌发率都存在显著差异,将形态、气孔鉴定和染色体计数结合可以准确确定变异株的倍性。研究表明利用氟乐灵诱导是进行萝卜同源四倍体种质创新的有效途径之一。     

The effects of colchicine-induced autotetraploidy on selected characteristics of nuruozak (<Emphasis Type="Italic">Salvia leriifolia</Emphasis>)

Nuruozak (Salvia leriifolia Benth), is a perennial herbaceous plant that is endemic to Iran and has recently been introduced as a medicinal plant. Artificial polyploidy is an efficient method to increase the production of secondary metabolites and can result in a whole spectrum of genetic, molecular and physiological modifications. In order to produce an autotetraploid population of nuruozak, various concentrations of colchicine (0.00, 0.05, 0.10, 0.20 or 0.50% w/v) were applied to the seeds and shoot apical meristems of young seedlings at the fourth leaf-stage. Microscopic studies, flow cytometry analysis and chromosome counting were conducted to select tetraploid nuruozak plants. Furthermore, the effects of ploidy level on the essential oil content and composition and biomass production of nuruozak plants, as well as selected structural and physiological characteristics were studied. Based on the number of the obtained tetraploids, treatment of shoot apical meristems was more efficient than seed treatment. Structural and phytochemical characteristics, chlorophyll content and photosynthetic rate were affected by the increase in ploidy level. In addition to the higher potential in biomass production, tetraploid plants produced eight new compounds which were absent in diploids.     

Combined Cycloheximide and 8-Hydroxyquinoline Pretreatment for Study of Plant Chromosomes

The actions of cycloheximide and 8-hydmxyquinoline on dividing cells of root meristems of Zea mays L. have been studied during the development of a new cytological technique for sugar cane (Saccharum) root tips. The determination of mitotic phase indices revealed that combined treatment with cycloheximide (70 ppm) plus 8-hydroxyquinoline (250 ppm) was superior to treatments with either chemical separately. After the combined treatment, the preparations contained nearly ten times more cells in prophase and metaphase that were suitable for chromosome counting than those given a single pretreatment with 8-hydmxyquinoline. This new pretreatment has been developed especially for chromosome studies in tropical grasses with a large number of small chromosomes. However, both chemicals are active in a wide range of plant species.     

Combined cycloheximide and 8-hydroxyquinoline pretreatment for study of plant chromosomes.

The actions of cycloheximide and 8-hydroxyquinoline on dividing cells of root meristems of Zea mays L. have been studied during the development of a new cytological technique for sugar cane (Saccharum) root tips. The determination of mitotic phase indices revealed that combined treatment with cycloheximide (70 ppm) plus 8-hydroxyquinoline (250 ppm) was superior to treatments with either chemical separately. After the combined treatment, the preparations contained nearly ten times more cells in prophase and metaphase that were suitable for chromosome counting than those given a single pretreatment with 8-hydroxyquinoline. This new pretreatment has been developed especially for chromosome studies in tropical grasses with a large number of small chromosomes. However, both chemicals are active in a wide range of plant species.