Abstract

This study was on cytology of visible chromosome formation under the light microscope during cell cycle in rapeseed. There are different shape and size granular appearance in the nucleus at the interphase stages, in which are composed of heterochromatin. The threadlike chromosome formation are closely correlated with that of granules. These granules were disappear by dissociated tight coiling with progressing prophase and at the some time the threadlike chromosome formation by increase coiling in diameted and number. The each threadlike chromosome is composed of two chromatids. The chromosomes are twisted about each other in relational coils, when the chromosomes attain their maximum contraction the nuclear membrane break down. At last in this paper the threadlakes chromosome formation were discussed.

Introduction

There are some paper on the ultrastructure of from chromatin to form chromosome have been obtained by various techniques including X-ray diffraction, chemical analysis, electron microscopy, and autoradigraphy, and have been development some hypothesis. But our fundamental knowledge of the molecular structure of chromosomes is still very incomplete. This is particularly relevant for the more complex chromosomes of higher plants.We expected to study in this aspect. Because of there were a few paper in this one on rapeseed. In the present paper we report the feature on cytology of visible chromosome formation in rapeseed. These studies are important for cell cycle, micronucleus formation and mechanism of chromosomal banding.

Materials and methods

Material offering

There are cultivars Xiang Lon You 2, Xiang You 11,Xiang You 13,Xiang You 14 of B. napus from Hunan Agricultural University Changsha, China.

Chromosome preparation

Seeds were germinated on wet fiter in a petri dish at 25°C Germinating seeds with roots 1.0~1.5 cm long were immersed in 0.002 M 8-hydroxyquinoline solution for 30 min at 25°C, then throughly washed in distilled water. The good root tips were excised and immersed them in 0.075 M KCl solution for 30 min at 25°C, and then macerated in 2.5% of mixture enzymatic solution of cellulase and pectinase for 3~4 h at 25°C.

The macerated roots were rinsed with distilled water 2~ 3 time and then immersed it in the distilled water for 5~10 min. Pour out the distilled water, add 4~5 ml fresh prepared methanol: acetic acid (3:1) to the material. Put on 3~4 root tips on the glass slides, smashed with forceps, and add 2 drops fixation solution on the slide, then bake it on the alcohol burner, The preparations were stained in 1:9 (v/v) Giemsa solution for 20 min, followed by rinsing with distilled water. When the slides became completely dry and mounted in damar balsam.

Main Results

1.Chromatin morphological characteristic analysis during interphase stage of cell cycle in B. napus. The most generally accepted chromatin was chromosomal different movement state. The interphase period is generally subdivided into three phases--the G1 period, the S period (Synthesis),and the G2 period. But it is difficult for recognition according to indices of cytomorphology. The chromatin state appear in interphase nucleus on B. napus were observed. The chromosomes are not visible and the nucleus has different size granular appearance. Their average number in per nucleus were observed that is Xiang Lon You 2 varies from 30±4, Xiang You 11 varies from 31±3, Xiang You 13 varies from 30±4 and Xiang You 14 varies as 29±3 respectively. These granules are composed of heterochromatin and are evenly distributed. They were referred to as similar globular pre-chromosome type. As described in other three varieties of B. napus in 1991. These granules do not undergo despiralization and decondensation at the end of each cell division. Instead, they remain tightly coiled at a time when the rest of the chromosomes are in a relatively uncoiled condition.

Table 1. The number variation on heteromatin granular in B. napus.

2.The morphological variable on chromosome formation with progressing prophase in B. napus. There are a lot of darker staining granules with different shape and size. The darker staining granules joined by chromonematal fibrils. These fibrils between granules are strained to straighten, so that these granules become spindle shape or quite long.

It has been shown the threadlike chromosomes formation are closely correlated with that of darker staining granules. As shown in diagram, one side or two sides of granules may be seen fine thread, these fine thread commence shorter, thinner and lightly stained, then increase longer and thicker, to become visible threadlike chromosomes in the nucleus. These granules were disappear with progressing prophase and at the some time the threadlike chromosome formation. At the end of chromosome can be seen coiling is lax. It shows the chromosome is composed of two chromatids. The chromosomes were stained with Giemsa and showed dark segment and light segment along the entire length of chromosomes.

3.The Variable law on the chromosomal morphology during mitotic prophase in B. napus. Throughout prophase each chromatic develops coils and so becomes shorter, thicker and more readily visible by increased spiralization. The chromatids are seen to be twisted around each other in relational coil. The coils interlock in a manner that the chromatids cannot be separated without unwinding the coil. This kind of twisting is also called plectonemic coiling in mitosis.

At the end of prophase, when the chromosomes attain their maximum contraction, the nuclear membrane breaks down and disappears. There is no longer much relational coiling present, and consequently, the chromatids are no longer twisted about each other but lie side by side. The number of chromosome of cell can be counted as 2n=38 in B. napus .

Discussion

Threadlike chromosomal formation

The cytological morphological characteristics have been shown that there are a process of morphological changes from chromatin to chromosome. This is process from similar globular pre-chromosome type to from darker staining granules with fibrils, and then these fine thread commence shorter, thinner and lightly stained, then increase longer and thicker, to become visible threadlike chromosome in the nucleus. The granules were disappear.

Based on above observation it can be presumed that the movement process from chromatin to form chromosome during extreme early prophase should be included two process: 1) the darkly stained granules in nucleus during interphase stages were dissociated tight coiling with progressing prophase and at the same time form some threadlike chromosomes; 2) the fine thread from granules at fine thread other end formation threadlike chromosomes by increase coiling in number and diameter. This causes an apparent thickening of the chromosomes that is often referred to as contraction. This reason may be as follow:

1) Euchromatin and heterochromatin have same chemical compose. Only the state of existence is different. According to electron micrographs this chromatin fiber has a beaded structure and the components of this structure are spheroid chromatin units called nucleosomes (Olins and olins,1974;Oudet et al.,1975). That is chromatin fibrils are composed of a series of repeating units consisting of tightly packed DNA and associated protein, altercating with more extended DNA and associated protein. Therefore, the nucleosome is the basic unit of these darkly stained granule and chromatin fiber.

Due to these granules are composed by heterochromatin which is regions of tightly folded and highest level of coiling d therefore appear to be darkly stained by Giemsa solution. When threadlike chromosomeformation the granules were dissociated continuously coiling.

2) Maximum condensation heterochromatin (inactive chromatin) can transform into active chromatin or conversion hetero-to euchromatic regions and vice versa under certain condition. These processes are as relation between loop and chromomere in lampbrush chromosome, the loops of lampbrush chromosome appears to be spun out from the chromosome at its thin end and rewound into the chromosome at the other end. But when threadlike chromosome are produced, that is continuos dissociating tight coiling from these granule and then these fiber continually develop coiling according to basic pattern of chromosome which is a shape suitable for transport. This is transmission of genetic information. The granules disappear after threadlike chromosomal formation.

3) The threadlike chromosomal formation takes up a relatively long time at extreme early prophase. The reason of this long period is that there are some very important functions at extreme early prophase taken care of, such as metabolism, synthesis and preparation some material for the next cell division.

According to discuss above. It could therefore be considered as follows: (1) when chromosomes were induced to produce aberration during cell cycle plants, from chromatin to form threadlike chromosome are most easily produced aberration phase. Because of the chromosome are fine thread which is broken easily, or produces some chemical changes in the cell, which in turn cause anomalies when the chromosomes replicate. So in general, dividing cells are much more sensitive to induction factor than non-dividing cells. But due to exist repair function during chromosome replication and may be have some an effect on aberration frequency of chromosomes and chromatic. (2) When induction aberration, the micronuclei were formed by association between heterochromatin granules. (3) These granules may have relation to the number of chromosomes and C-banding. (4) In a lot of hypothesis of chromosome ultrastructure, the solenoid hypothesis (Finch,1975) can explain chromosome ultrastructure more than other one.

Acknowledgement

The authors thank Oil Crops Institute at Hunan Agricultural University provide rapeseed seeds for our research.

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