A Schedule Including Cold Treatment to Facilitate Somatic Chromosome Counts in Certain Forage Grasses

Pretreatment at low temperature of root tips of grass grown from tillers in small beakers was found to contract somatic chromosomes and thus facilitate determination of chromosome number. A modification of Randolph's infiltration and card technics has been adapted to working with root tips from large numbers of plants.     

Cytological Methods for Crepis Species

Root tips of Crepis species are fixed in La Cour's “2BE” and dehydrated thru a butyl alcohol series. They are stained in 1% crystal violet for 1 hour, with chromic acid and iodine as pre-and post-staining mordants, respectively, and passed thru dehydrating alcohols containing picric acid and ammonium hydroxide. Differentiation is done in clove oil. The method is rapid; the chromosomes are dark purple; the centromere is not stained; and the cytoplasm is clear. By further controlled destaining the hetero-chromatic segments within the chromosomes may be located.

Pollen mother cells are fixed in acetic alcohol (1:4) and squashed in aceto-carmine. A method is described for making semi-permanent preparations mounted in diaphane.

Pollen grains are mounted in lacto-phenol with acid fuchsin or anilin blue W. S. as the dye.     

Cytological Methods for Crepis Species

Root tips of Crepis species are fixed in La Cour's “2BE” and dehydrated thru a butyl alcohol series. They are stained in 1% crystal violet for 1 hour, with chromic acid and iodine as pre-and post-staining mordants, respectively, and passed thru dehydrating alcohols containing picric acid and ammonium hydroxide. Differentiation is done in clove oil. The method is rapid; the chromosomes are dark purple; the centromere is not stained; and the cytoplasm is clear. By further controlled destaining the hetero-chromatic segments within the chromosomes may be located.

Pollen mother cells are fixed in acetic alcohol (1:4) and squashed in aceto-carmine. A method is described for making semi-permanent preparations mounted in diaphane.

Pollen grains are mounted in lacto-phenol with acid fuchsin or anilin blue W. S. as the dye.     

Chlorazol Black E as A Stain for Root-Tip Chromosomes

A simple method of shining root-tip chromosomes which gives excellent results in photomicrography is described. The schedule is as follows: run paraffin sections of root tips killed in Bouin's fixative down to water; stain for approximately two hours in 1% aqueous chlorazol black E; wash in water; run up to xylene.     

Acettc-Orcein: A New Stain-Fixative for Chromosomes

A new stain-fixative method for chromosomes, namely acetic-orcein, is described, which gives results that are equally good in fresh and permanent preparations. A 45% acetic and 1% orcein content is recommended as a standard solution. For salivary glands of Drosopkila a 2% stain gives the best results, and with the two species D. melanogaster and D. miranda the acetic strength has been raised to 70% with advantage. The addition of chloroform proves necessary for hardening in species of Sciara. Acetic-orcein is equally good for rapid chromosome counts. For root tips the addition of 1 cc. of N HC1 solution to 10 cc. of the standard solution together with gentle heating of the tissues in a drop of the mixture assists in the softening and separation of cells necessary for chromosome study. Orcein can also be used successfully in other combinations such as acetic-propionic or acetic-lactic. The latter is useful for making preparations that do not require ringing. Preparations so made keep from 7 to 14 days.     

Fluorescentin situ hybridization to soybean metaphase chromosomes

Repetitive DNA sequences were detected directly on somatic metaphase chromosome spreads from soybean root tips using fluorescentin situ hybridization. Methods to spread the forty small metaphase chromosomes substantially free of cellular material were developed using protoplasts. The specific DNA probe was a 1.05 kb internal fragment of a soybean gene encoding the 18S ribosomal RNA subunit. Two methods of incorporating biotin residues into the probe were compared and detection was accomplished with fluorescein-labeled avidin. The rDNA probe exhibits distinct yellow fluorescent signals on only two of the forty metaphase chromosomes that have been counterstained with propidium iodide. This result agrees with our previous analyses of soybean pachytene chromosome [27] showing that only chromosome 13 is closely associated with the nucleolus organizer region. Fluorescentin situ hybridization with the rDNA probe was detected on three of the forty-one metaphase chromosomes in plants that are trisomic for chromosome 13.     

Chromosomal instability in lemon grass,Cymbopogon flexuosus (Steudel) Wats

Somatic mitoses in C. flexuosus exhibit a significant degree of chromosomal instability leading to nearly 33% cells with chromosome elimination. A range of chromosome numbers between 20-8 (most common being 2n=20, the somatic number for this species) was encountered from root tip cells. The course of variation suggests a gradual elimination of somatic chromosomes. The larger chromosomes are less stable and are eliminated earlier. The variation in chromosome number in somatic cells within individual plants is possibly controlled by genetic factors, which result from weaker spindle operation and minute chromosomes.CIMAP Publication No. 571 (1984)     

The Smear Technic in Plant Cytology

The following method of making permanent smears of pollen mother cells is in general use and gives excellent results. Determine the stage of meiosis from aceto-carmin mounts. Smear the pollen mother cells on a dry slide. Fix in Navaschin's or a modified Flemming's solution from 1 to 2 hours. Wash in 10 to 20% alcohol from 15 to 30 minutes. Stain in 1% aqueous crystal violet from 1 to 5 minutes. Rinse in water and pass thru 30 to 50% alcohol, about 15 to 20 seconds in each. Transfer to 80% alcohol containing 1% iodine and 1% potassium iodide for 30 seconds. Destain with absolute alcohol, followed by clove oil. xylol, balsam and cover.

Permanent smears for chromosome counts can be quickly made by smearing pollen mother cells on a dry slide, fix and stain with aceto-carmin, dehydrate with mixtures of absolute alcohol and acetic acid, follow with xylol, balsam, and cover.     

芦苇根尖体细胞的染色体数目和形态

本文研究了芦苇(Phragmites communis)幼苗根尖分生组织细胞的染色体数目和形态。其染色体数为2n=96。在有丝分裂中期染色体组中,染色体的大小逐渐地改变,按照染色体的大小和形态,96个染色体能被分成8个同源单倍体染色体组。每一组由11个中部着丝点染色体和1个亚中部着丝点染色体组成。     

芦苇根尖体细胞的染色体数目和形态

本文研究了芦苇(Phragmites communis)幼苗根尖分生组织细胞的染色体数目和形态。其染色体数为2n=96。在有丝分裂中期染色体组中,染色体的大小逐渐地改变,按照染色体的大小和形态,96个染色体能被分成8个同源单倍体染色体组。每一组由11个中部着丝点染色体和1个亚中部着丝点染色体组成。     

An Improved Method for Chromosome Counting in Maize

An improved method for counting chromosomes in maize (Zea mays L.) is presented. Application of cold treatment (5C, 24 hr), heat treatment (42 C, 5 min) and a second cold treatment (5C, 24 hr) to root tips before fixation increased the number of condensed and dispersed countable metaphase chromosome figures. Fixed root tips were prepared by the enzymatic maceration-air drying method and preparations were stained with acetic orcein. Under favorable conditions, one preparation with 50-100 countable chromosome figures could be obtained in diploid maize using this method. Conditions affecting the dispersion of the chromosomes are described. This technique is especially useful for determining the somatic chromosome number in triploid and tetraploid maize lines.     

Notes on Selling'S Green-Light Method for Critical Microscopy (Continued)

Kill root tips in 1 part glacial acetic acid to 3 parts absolute alcohol for 12 or more hours. Remove from killing fluid and place for 5 to 10 minutes in a solution consisting of 1 part 95% alcohol to 1 part concentrated HCl. Transfer to Carnoy's fluid for 5 minutes or longer. Cut a small piece (0.5 mm. or less) off the tip of the root and place on a clean slide in a small drop of iron-aceto-carmin stein. Press directly on the piece of root with a small flat scalpel; the cells will now separate and float free in the stain. Place cover slip over the drop of stain and apply gentle pressure. Heat carefully by passing the slide 3 or 4 times thru the flame of an alcohol lamp. Seal with heated mixture of 1 part Parowax to 1 part gum mastic. Make permanent by the McClintock permanent method.     

SUPERNUMERARY CHROMOSOMES IN SAXIFRAGA VIRGINIENSIS (SAXIFRAGACEAE)

Acetocarmine squashes of root tips have demonstrated that 2n = 20 and 38 in Saxifraga virginiensis. These contrast with the earlier reported count of 2n = 28 for this species. In several populations supernumerary chromosomes were detected. Both intrapopulational and interpopulational variation in supernumerary chromosome number were detected, with the largest number of supernumerary chromosomes observed being six. Because these supernumerary chromosomes are equal in size to many of the smaller A chromosomes during mitotic metaphase, the presence of supernumerary chromosomes in this species could not be ascertained by analysis of mitotic metaphase preparations alone. During mitotic prophase, however, the supernumerary chromosomes of S. virginiensis are highly heterochromatic, appearing more densely coiled and darkly stained than the A chromosomes. This characteristic facilitated the recognition of supernumerary chromosomes in this species. The similarity in size of A and supernumerary chromosomes during mitotic metaphase and the observation of six supernumerary chromosomes in one population suggest that the count of 2n = 28 reported earlier for S. virginiensis may actually be a misinterpretation of 2n = 20 plus 8 supernumerary chromosomes. Furthermore, these findings and the observation of this same supernumerary chromosome phenomenon in other species of Saxifraga raise the possibility that some of the many disparate chromosome counts attributed to aneuploidy in the large genus Saxifraga may also be the result of misinterpretations of supernumerary chromosomes as A chromosomes.     

Alcoholic Hydrochloric Acid-Carmine as a Stain for Chromosomes in Squash Preparations

A regressive bulk carmine staining schedule was adapted from a formula proposed by P. Mayer. The stain is made by boiling gently 4 gm of certified carmine in 15 ml of distilled water to which 1 ml of concentrated HC1 has been added. After cooling, 95 ml of 85% alcohol is added, and the solution filtered. Fixed tissue is soaked in the stain until thoroughly penetrated; squashes are then prepared as usual, but plain 45% acetic acid is used as the temporary mounting medium instead of aceto-carmine. The advantages of this method are: intense, precise staining of chromosomes coupled with a lightly stained cytoplasm; consistency and uniformity of results; simplicity of use.     

Mitotic and meiotic irregularities in somatic hybrids of Lycopersicon esculentum and Solanum tuberosum.

Chromosome numbers were determined in metaphase complements of root-tip meristems of 107 tomato (+) potato somatic hybrids, obtained from five different combinations of parental genotypes. Of these hybrids 79% were aneuploid, lacking one or two chromosomes in most cases. All four hybrids that were studied at mitotic anaphase of root tips showed laggards and bridges, the three aneuploids in a higher frequency than the single euploid. Hybrid K2H2-1C, which showed the highest percentage of aberrant anaphases, possessed 46 chromosomes. Fluorescence in situ hybridization with total genomic DNA showed that this hybrid contained 23 tomato, 22 potato, and 1 recombinant chromosome consisting of a tomato chromosome arm and a potato chromosome arm. The potato parent of K2H2-1C was aneusomatic in its root tips with a high frequency of monosomic and trisomic cells and a relatively high frequency of cells with one fragment or telosome. Meiotic analyses of three tomato (+) potato somatic hybrids revealed laggards, which occurred most frequently in the triploid hybrids, and bridges, which were frequently present in pollen mother cells (PMCs) at anaphase I of hypotetraploid K2H2-1C. We observed putative trivalents in PMCs at diakinesis and metaphase I of eutriploid A7-82A and quadrivalents in part of the PMCs of hypotetraploid K2H2-1C, suggesting that homoeologous recombination between tomato and potato chromosomes occurred in these hybrids. All three hybrids showed a high percentage of first division restitution, giving rise to unreduced gametes. However, shortly after the tetrad stage all microspores completely degenerated, resulting in exclusively sterile pollen.     

Analysis of chromosome number and speculations on the origin of Arundo donax L. (Giant Reed)

Arundo donax (commonly called Giant Reed) is a perennial rhizomatous grass native to Asia, nowadays diffused all over the world. Due to its high biomass production and great adaptability to marginal land, interest in this species is increasing. In fact A. donax could represent an important and promising energy crop for heat and bioethanol second generation production. The propagation of A. donax is strictly agamic by rhizome fragmentation and cane node germination, strongly limiting the possibility of genetic improvement by breeding. The sterility could be caused by the fact that A. donax is a hybrid with uneven ploidy or a triploid species. It is difficult to propose an explanation for its sterility, because the chromosome number of A. donax is still a matter of debate, due to the high number and small size of the chromosomes; in the bibliography different counts ranging from 40 to 110 are reported. With the aim of establishing the chromosome number of A. donax we selected and counted 17 metaphase plates prepared from root tips obtained by hydroponic cultivation of cane nodes; our counts showed that A. donax most probably has 110 chromosomes. Our results suggested us two possible hypotheses, also based on SSR molecular marker results, concerning the evolutionary processes involved in the origins of A. Donax.     

Permanent Mounts Of Chromosomes After 8-Oxyquinoline and Squashing

Fresh young root tips or free-hand cross sections thereof were placed in 0.002 M 8-oxyquinoline (aq.) at 10-14^oC. for 3 hours. After rinsing in water 1-2 minutes, they were soaked in N HC1 at 55^oC. for 25 minutes, rinsed again and squashed under a cover glass on a dry slide. Slide and cover glass were separated by placing in 70% alcohol and allowed to remain therein at least 0.5 hour after separation. Both slide and cover glass were passed through 50% and 30% alcohol to water and stained by the Feulgen procedure (without further hydrolysis) or with crystal violet after mordanting in 1% chromic acid overnight and washing in running water 3-4 hours. Dehydration and mounting in balsam completed the process. The smear on the slide was covered with a clean cover glass and the cover glass, bearing stained material, mounted separately.     

Preliminary Notes on Chromic Fixation in Alcoholic Media

In Note I are pointed out certain chemical difficulties which stand in the way of experiments on chromium fixation in alcoholic media; also a procedure is described by means of which these difficulties may be avoided in the preparation of a chrome-alcohol stock solution.

In Note II it is suggested that the properties of OsO₄ most important in the quick-killing effect conferred by this adjuvant upon aqueous chromic reagents are probably, in addition to its high toxicity, its volatility and its oxidant action. Volatile, toxic oxidants which do not attack alcohol are therefore discussed briefly, with reference especially to their use as quick-killing adjuvants in chrome-alcohol reagents. Iodine is noted as probably the most important inorganic possibility. Reasons are stated for considering the quinones the most promising of numerous possible organic compounds.

Results of fixations of Vicia faba root tips in nine variations of the proportions of acetic acid and iodine in a chrome-alcohol-aceticiodine combination are described. The chrome-alcohol-iodine reagents which proved best proportioned for V. faba root tips preserved some details of the chromosomes better than does Bouin's solution, but did not make certain details of the early prophase as clear as does Benda's modification of the Flemming reagent, according to a comparison with Sharp's figures of Bouin and Benda preparations of root tips of the same species.     

A Modification of the Cresyl Violet Technic for Staining Nerve Cells

Kill root tips in 1 part glacial acetic acid to 3 parba RB Solute alcohol for 12 or more hours. Remove from king fluid a d place for 5 to 10 minutes in a solution consisting of 1 part 95% alcohol to 1 part concentrated HC1. Transfer to Carnoy's fluid for 5 minutes or longer. Cut a small piece (0.5 mm. or less) off the tip of the root Press directly on the piece of root with a small fiat scalpel; the cells will now separate and float free in the stain. Place cover slip over the drop of stain and apply gentle pressure. Heat carefully by paseing the slide 3 or 4 times thru the flame of an alcohol lamp. Seal with heated mixture of 1 part Parowax to 1 part gum mastie. Make permanent by the McClintock permanent method. and place on a clean slide in a small drop of iron-ace-sinin.     

Basic Fuchsin-Crystal Violet; A Rapid Staining Sequence for Juxtaglomerular Granular Cells Embedded in Epoxy Resin

A basic fuchsin-crystal violet staining sequence for demonstration of juxtaglomerular granular cells in epoxy-embedded tissues is rapid and results in slides with excellent contrast and intensity. Procedure: Cut sections 0.3-0.6 μ thick. Hydrate through xylene and alcohol to water. Stain in modified Goodpasture's stain (basic fuchsin, 1; aniline, 1; phenol, 1; 30% alcohol, 100) for 20-30 sec; rinse in tap water; stain in modified Stirling's (crystal violet, 5; alcohol, 10; aniline, 2; water, 88) for 20-30 sec; rinse in tap water and dry on a hotplate; mount in a synthetic resin. Granular cells of the juxtaglomerular apparatus are stained an intense dark blue by the crystal violet. Arterial elastic membranes and collagen are pale blue. Other structures are shades of red.