Slide Processing for the Examination of Male Mammalian Meiotic Chromosomes

A rapid method was devised specifically for the cytological identification of translocations in the male mouse at late prophase to metaphase of meiotic division I, but the method should be useful for less specific objectives requiring examination of mammalian testicular material. For the adult mouse, masses of tubules from a single testis, freed of the testicular tunic, are placed in 3 ml of 0.7% sodium citrate for 15-20 min, and subsequently fixed in 50% acetic acid by the addition of 3 ml of glacial acetic acid to the hypotonic citrate. To facilitate handling of individual tubules by preserving their visible structure, the addition of fixative is at a rate which is grossly adjusted so that 2 ml will have been added at the end of 30 sec and the remaining 1 ml by the end of a minute. A single fixed tubule 1-2 cm long is placed lengthwise on a slide and covered with a drop of lactic-acetic orcein made as follows: Add 2 gm of synthetic orcein (G. T. Gurr) to a mixture of 50.0 ml of glacial acetic acid, 42.5 ml of 85% lactic acid, and 7.5 ml of distilled water. After staining for 10 min, a 22 × 50 mm cover slip is placed over the tubule, and it is allowed to stain for an additional 10 min. The majority of germinal cells will not be in late prophase or metaphase of the first meiotic division, therefore many preparations will be useless; however, slides with division figures are radidly selected as follows: Before squashing, examine under a microscope at a magnification of 150, and upon recognition of a single meiotic division, remove the slide and squash the preparation for subsequent detailed examination. As a consequence of the spermatogenic wave that progresses along the length of a tubule, a given slide will usually have many division figures or none at all, hence the limitation of 1 tubule per slide facilitates efficient discarding. Preliminary work with the Chinese hamster suggests that good preparations might be obtained from testes of various mammals when the volume of hypotonic solution is adjusted so as to compensate for varying testicular sizes by maintaining a 15:1 ratio of fluid to estimated volume of tissue.     

Single-Stage Carbon Replicas of Microspores

Single-stage surface replicas of treated or fresh pollen grains can be made ready for the electron microscope in 1.5 hr. The microspores are discharged into a drop of 50% acetone on a 1 cm square of cleaved mica and air dried. Carbon is evaporated to a film thickness of 35 mμ during rotation of the mica support. The carbon film and microspores are parted from the mica with water and heated in 2-aminoethanol at 145-155 C for 10 min to 3 hr. The replicas are then washed 5 min or longer on water at 90 C and picked up on electron microscope grids. The resulting self-shadowed surface replica can be immediately observed by electron microscopy.     

Chromosomes for molecular hybridization

Summary Specific recombinant DNA sequences (5S rRNA, B1, albumin) were assigned to flow sorted chromosomes of the Chinese hamster cell line CHV79. For this purpose, a rapid protocol was developed using filterbound chromosomal DNA and probing with various nucleic acids, that allows sequence identification in chromosomes. A flow histogram and a flow karyogram of the CHV79 cell line were established by flow analysis in order to calculate the amount of DNA per CHV79 cell and their chromosomes. Subsequently, metaphase chromosomes or chromosomal groups were fractionated by electronic sorting and a defined number of chromosomes was directly bound to nitrocellulose filters for sequence homology analysis by a dot blot hybridization procedure. This procedure not only allows the assigning of specific DNA sequences to particular chromosomes, it is also applicable to studies of changes in karyotypes, for example translocations of given sequences.Some results shown constituted a Diploma Thesis by G.L. submitted to and accepted by the Department of Biology, University of Kaiserslautern     

Vital Fluorescent Staining Technique for Microspores of Brassica Napus

The ontogeny of early microspore-derived embryo development was followed using three stains. The stain 3,3'-diethyloxadicarbocyanine iodide, which previously had been reported to be specific for mitochondria, was observed also to demonstrate the exine of developing microspores of Brassica napus.It provided high contrast when used in combination with Tinapol 5BM, a stain for cellulosic cell walls, and aided identification of microspores with embryogenic potential. Hoechst 33342, a nuclear stain, alone or in combination with either or both of the other stains, could be used to highlight the nuclear developmental stage of the microspores. This paper describes procedures using these materials for the specific staining of exine, cell wall/intine and nucleus, thereby permitting their fate to be followed during the early phases of microspore-derived embryo development.     

Synaptonemal-like complexes in Allium cepa Microspores

The paper describes the synaptonemal-like complexes found in the course of microsporogenesis in Allium cepa, during the young microspore stage. In longitudinal sections these structures are morphologically more or less like the synaptonemal complexes, which appear during the pachytene stage of meiosis, and consist of two regions resembling the lateral elements, separated from one another by a space measuring about 1000 Å, in which a central element is occasionally observed. When observed in transverse or oblique sections, their shape indicates that they are tubular structures with a central axis; they are generally observed as independent units inside the nucleus, sometimes associated with the chromatin masses.By using the uranyl-EDTA-lead staining method, which picks out the RNA the synaptonemal-like complexes, show up at the level of their lateral elements and a similar result can be achieved by using the alcoholic PTA technique, which is believed to be a selective staining method for histones. We suppose that the synaptonemal-like complexes consist, at least partially, of RNP material.     

小麦的小孢子培养

小孢子培养是在花药培养的基础上发展起来的一种高效再生体系,其在植物育种中的作用一直受到关注.但是,由于培养技术的限制,小麦(Triticum aestivum)小孢子培养还未能推广应用.近年来小麦小孢子培养发展很快,展现出广阔的应用前景.     

小麦的小孢子培养

小孢子培养是在花药培养的基础上发展起来的一种高效再生体系, 其在植物育种中的作用一直受到关注。但是, 由于培养技术的限制, 小麦(Triticum aestivum)小孢子培养还未能推广应用。近年来小麦小孢子培养发展很快, 展现出广阔的应用前景。     

Simple Methods for Mammalian Chromosomes

The ordinary Feulgen-squash technic, after acetic-alcohol fixation, provides a simple and reliable method of preparing many mammalian tissues for chromosome counts. Tissues best suited to the technic were the seminiferous tubules. Small pieces of tissue about 3-6 mm. long and 1-2 mm. wide were immersed in a freshly made fixative (composed of 3 parts absolute ethyl alcohol and 1 part glacial acetic acid) immediately after removal by biopsy or from a freshly killed animal. After fixation for 3-12 hours the tissue was stained by the standard Feulgen procedure after hydrolysis for 12 minutes in normal HCl at 60 °C A 1-2 mm. piece was then teased apart on a slide in 45% acetic acid with a pair of mounted needles, and the teased tissue was squashed between the slide and the cover slip. During squashing the pressure was applied by hand and was regulated so as to avoid any excessive scattering of the chromosomes. The preparations were made permanent by dehydrating and mounting in Euparal.     

Incipient Genome Differentiation in Gossypium. III. Comparison of Chromosomes of G. HIRSUTUM and Asiatic Diploids Using Heterozygous Translocations

Hybrids between upland cotton (G. hirsutum, genome constitution 2A_hD_h) and either A-genome or D-genome diploid species exhibit 26 paired and 13 unpaired chromosomes at metaphase I. The A_h and D_h genomes are therefore considered homoeologous with those of the respective diploids. Previous studies, nevertheless, revealed a low level of ("incipient") differentiation between D_h and various diploid D genomes. The diploid A genomes have been regarded as more closely homologous to A_h on the basis of low preferential pairing and autotetraploid segregation ratios in allohexaploids.—The present study addressed the following questions: Are the diploid A genomes differentiated from A_h in meiotic homology? If so, is the differentiation manifested equally by all 13 chromosomes or is it localized in certain chromosomes?—Three diploid A-genome lines representing G. herbaceum and G. arboreum were hybridized by in ovulo culture of embryos (1) with a standard line of G. hirsutum, which differs from G. herbaceum by two and from G. arboreum by three naturally occurring reciprocal translocations involving chromosomes 1–5, and (2) with six lines homozygous for experimental translocations involving chromosomes 6, 7, 10, 11, 12 and 13. Chiasma frequencies in hybrids were compared with those in appropriate G. hirsutum controls. In every comparison overall chiasma frequencies were slightly lower in the hybrids. Therefore A_h appears to be differentiated from the diploid A genomes. No localized differentiation was detected in chromosomes marked by experimental translocations. The differentiation may be localized mainly in chromosomes 4 and 5.     

水稻减数分裂染色体分析方法

减数分裂粗线期染色体研究技术的发展, 很大程度上克服了水稻(Oryza sativa)细胞遗传研究中较小染色体所带来的研究困难。减数分裂染色体的制备与观察已经成为水稻细胞遗传学研究中的常规方法。该文详细描述了水稻中常用的减数分裂染色体制备、荧光原位杂交和免疫荧光染色的实验方法。     

A Modified Technic for Salivary-Gland Chromosomes

This technic was worked out for making permanent mounts of salivary-gland squashes of strains and species of Drosophilidae, which yield material difficult to work with. It includes a prior fixation in a modified Carnoy's fluid, staining in solutions of orcein in 60% or 70% acetic acid, part dehydration in the vapor and liquid of a 95% solution of alcohol, and direct mounting in Euparal by means of the drainage-method.     

水稻游离小孢子培养最新研究进展

水稻小孢子培养技术不但可用来生产单倍体植株和加倍单倍体植株,而且可望成为转基因的理想受体系统。目前转基因技术、分子杂交技术与小孢子培育技术的有机结合已成为加快水稻育种速度的有效途径。本文从水稻小孢子分离纯化、影响愈伤组织或胚状体发生的因素以及植株再生三个方面综述了水稻游离小孢子培养的最新研究进展。最后就水稻小孢子培育技术急需解决的问题进行了讨论,并对小孢子培育技术发展前景进行了展望。