Karyotype evolution in a tumour derived plant tissue culture analysed by Giemsa C-banding

Summary A hyperdiploid aneuploid karyotype, consisting of 7 chromosomes, has been found in a tumorous suspension cell culture ofCrepis capillaris (2 n=6). Giemsa C-banding has revealed that these 7 chromosomes show consistent patterns of differential staining in all dividing cells. This stable karyotypic situation has persisted during 18 months of cytological monitoring of the culture. Comparison with the diploid C-banded complement of the root tip indicates that numerous structural rearrangements must have occurred during the formation of the aneuploid complement. A likely pathway for evolution of this karyotype involves initial tetraploidy followed by chromosome loss. Such a mechanism has previously been proposed for a plant tissue culture system (Bayliss andGould 1974) and commonly occurs in animal systems, particularly in animal tumours (Terzi andHawkins 1975). An alternative mechanism, which does not necessarily involve tetraploidy, is also proposed.     

Karyological stability and microevolution of a cell suspension culture ofBrachycome dichromosomatica (Asteraceae)

A long-term suspension culture ofBrachycome dichromosomatica (2n = 4) was induced from a cotyledon-derived callus. Subcultures were obtained every week up to three years. The bulk of the cultures displayed a stable diploid karyotype, while one cell line evolved with 2n = 5 chromosomes in the 86th reinoculation. No further chromosomal change occurred also in that cell line. It is assumed that the fifth chromosome is the expression of a trisomy 2.The chromatin ultrastructure was of the species-specific chromomeric type in the wild-type line, while the trisomic line displayed more condensed chromatin, what probably indicates a rather inactive state of the extra-chromosome.Brachycome dichromosomatica is suggested to represent an ideal species to follow-up karyotype stability and/or variation in cell culture.As a former student W. N. dedicates this paper in gratitude and admiration to Prof. DrElisabeth Tschermak-Woess on the occasion of her 70th birthday. Prof.Woess with her scientific work has stimulated in an unique manner the study of nuclear structures in plants, of endopolyploidy and polytene chromosomes, and has thus established the basis for the rapidly increasing research in these fields.     

Somatic hybridization between potato and Nicotiana plumbaginifolia

Summary Electrofusion was carried out between mesophyll protoplasts from the transformed diploid S. tuberosum clone 413 (2n=2x=24) which contains various genetic markers (hormone autotrophy, opine synthesis, kanamycin resistance, -glucuronidase activity) and mesophyll protoplasts of a diploid wild-type clone of N. plumbaginifolia (2n=2x=20). Hybrid calli were obtained after continuous culture on selection medium containing kanamycin. Parental chromosome numbers, determined at 2 months after fusion, revealed hybrid-specific differences between the individual calli. On the basis of these differences three categories of hybrids were distinguished. Category I hybrids contained between 8 and 24 potato chromosomes and more than 20 N. plumbaginifolia chromosomes; category II hybrids had between 1 and 20 N. plumbaginifolia chromosomes and more than 24 potato chromosomes; category III hybrids contained diploid or subdiploid numbers of chromosomes from both parents. The hybrids were evenly distributed over the three categories. After a 1-year culture of 24 representative hybrid callus lines on selection medium the karyotype of 10 hybrids remained stable, whereas 8 hybrids showed polyploidization of the genome of one parent, together with no or minor changes of the chromosome numbers of the other parent. Six hybrids showed slight changes in the hybrid karyotype. The elimination of chromosomes of a particular parent was not correlated to their metaphase location. The processes of spontaneous biparental chromosome elimination leading to the production of asymmetric hybrids of different categories are discussed.     

Chromosome alterations in tissue culture cells ofAllium fistulosum

Heterochromatin behaviour and structural alterations in chromosomes of cells derived from callus culture ofAllium fistulosum have been studied.The diploid chromosome complement ofAllium fistulosum consists of 16 chromosomes with significant amount of heterochromatin mainly of telomeric nature. In eight collections of callus cells analysed, a high rate of numerical and structural chromosome abnormalities was observed. After 12 months in culture about 20% of metaphase chromosomes possessed distinct signs of mutational events.C-banded preparations revealed that many structural alterations involved regions of heterochromatin. Interchromosomal connections and chromatid fusions occurred at telomeric heterochromatin segments. Also formation of the end-to-end associations and polycentric chromosomes often took place without visible loss of telomeric heterochromatin.     

Ploidy stability of maize anther culture-derived callus lines

Summary Ploidy levels of 26Zea mays L. anther culture-derived callus lines of the F1 hybrids (H99 × Pa91, Pa91 × FR16, and H99 × FR16) were determined at various times after culture initiation using flow cytometry (for 21 lines) or chromosome counting of callus cells or regenerated plants (for the remaining 5 lines). Twenty of the lines remained haploid, whereas 6 were diploid. The results from flow cytometry, after examining the DNA content of 5000 nuclei of each callus line, show that each callus line consisted of homogenous haploid or diploid cells. Thus for diploid callus lines, spontaneous chromosome doubling must have occurred before or in the early stages of androgenesis, before the initiation of callus cultures. These long-term callus cultures (growing for up to 38 mo.) have stably maintained their ploidy levels so it is unlikely that the culture conditions have caused chromosome doubling. The restriction fragment length polymorphism pattern obtained with 52 to 58 markers for each diploid callus line shows that all the diploid lines are homozygous diploid so each originated from a microspore and not from diploid maternal F1 hybrid tissue.     

Molecular cytogenetic analysis of repeated sequences in a long term wheat suspension culture

A rapidly growingTriticum aestivum L. (wheat) derived long term suspension culture (named TaKB1), that is probably not regenerable, was analysed for karyotype rearrangements, stability and changes in repetitive DNA. The cell line has an average chromosome number of 21 and the DNA amount of unreplicated cells of TaKB1 measured by flow cytometry is about 30% lower than an unreplicated (1C) bread wheat genome.In situ hybridization of a repetitive DNA sequence (pSc119.2), which occurs as tandemly repeated blocks (heterochromatin) in wheat, shows that chromosomes from the TakB1 line have fewer and weaker subtelomeric locations of the sequence than wheat, suggesting deletions of distal chromosome segments and a reduction in the sites and copy number of the sequence. Thein situ hybridization pattern and chromosome morphology allowed 27 chromosome types to be identified in the cell line. No two analysed cells contained the same chromosome complement, although some chromosome types were present in every cell. Using Southern hybridization the structure and copy number of a retroelement (Wis-2) and its flanking sequence was shown to be the same in the TaKB1 cell line and wheat. Anin situ analysis of rDNA in the TaKB1 cell line (using the probe pTa71) showed a reduction in number of sites and rRNA genes in each cell from that in wheat. Interphase cells of the cell line showed dispersed signal throughout the nucleolus with no evidence for clusters of condensed and inactive rRNA genes.     

Comparison of triploid and diploid cucumber in long-term liquid cultures

Triploid suspensions generally grew more vigorously in modified MS medium with 2,4-D than those of diploids. The embryogenic potential of 26-month-old auxin-dependent suspension cultures depended on the line. Neither triploid nor diploid BOR (Borszczagowski line) were able to produce somatic embryos. Similarly, 12–20-month-old cytokinin-dependent suspensions from the same triploid line were not capable of regeneration. Only aggregates from 26-month-old auxin-dependent suspension of triploid line 603 differentiated into somatic embryos. In contrast, 18-month-old diploid and triploid liquid cultures of meristematic clumps (LMC) of BOR retained their regeneration potential. The ploidy level of triploid and diploid auxin-dependent suspension cultures was stable during the first 8 months. However, the ploidy level of triploids remained stable over 26 months of culture, whereas 66.7% of diploid cultures underwent chromosome doubling. No ploidy changes were observed among plants regenerated from 18-month-old LMC. Our data suggest that loss of embryogenic potential in suspension culture was independent of ploidy level.     

Chromosomal variation in the scincid genus Mabuya and its arboreal relatives (Reptilia: Squamata)

Karyotypes of four East and Southeast Asian species of the genus Mabuya and representatives of three arboreal genera considered to be closely related to the genus (Apterygodon, Dasia and Lamprolepis) are examined. Mabuya rugifera and M. rudis possess 2n=32 karyotypes, whereas M. longicaudata lacks one small chromosome pair, which makes its diploid number 30. The karyotype of M. macularia quadrifasciata from southern Thailand differs from any known scincid karyotypes, including a previously reported karyotype of the conspecific Indian population, in exhibiting a high diploid number (38) and a gradual change in size from large to small chromosomes. This suggests that M. macularia may consist of more than one biological species. Karyotypes of Apterygodon vittatus, Dasia olivacea and Lamprolepis smaragdinus, while exhibiting a close similarity to most Mabuya karyotypes, suggest the occurrence of slight rearrangements involving either the deletion of two small chromosome pairs (A. vittatus), or the change of centromeric positions in medium and small chromosomes (D. olivacea and L. smaragdinus).     

The genomic instability of yeast cdc6-1/cdc6-1 mutants involves chromosome structure and recombination

When diploid cells of Saccharomyces cerevisiae homozygous for the temperature-sensitive cell division cycle mutation cdc6-1 are grown at a semipermissive temperature they exhibit elevated genomic instability, as indicated by enhanced mitotic gene conversion, mitotic intergenic recombination, chromosomal loss, chromosomal gain, and chromosomal rearrangements. Employing quantitative Southern analysis of chromosomes separated by transverse alternating field gel electrophoresis (TAFE), we have demonstrated that 2N-1 cells monosomic for chromosome VII, owing to the cdc6-1 defect, show slow growth and subsequently yield 2N variants that grow at a normal rate in association with restitution of disomy for chromosome VII. Analysis of TAFE gels also demonstrates that cdc6-1/cdc6-1 diploids give rise to aberrant chromosomes of novel lengths. We propose an explanation for the genomic instability induced by the cdc6-1 mutation, which suggests that hyper-recombination, chromosomal loss, chromosomal gain and chromosomal rearrangements reflect aberrant mitotic division by cdc6-1/cdc6-1 cells containing chromosomes that have not replicated fully.     

Characterization of the spontaneously transformed human endothelial cell line ECV304: 1. Multiple chromosomal rearrangements in ECV304 cells

Using differential G-staining of chromosomes, the karyotype of the endothelial cell line ECV304 obtained from endotheliocytes of the human umbilical vein was studied. The cells have been shown to have a polyploid karyotype with a number of chromosomes ranging from 96 to 112, as well as multiple numerical and structural clonal chromosome abnormalities. The structural rearrangements involve almost all chromosomes of the karyotype. Several paired chromosomal rearrangements have been revealed and include del(9)(p21), as well as two derivates of chromosome 3 with a breakpoint at the p25 locus, i.e., der(3)t(3;12)(3p25;12q11～12;12q21～24.?1) and der(3)t(3;?)(3p25). The role of these rearrangements in the immortalization of endotheliocytes and in angiogenesis is discussed. A comparison of the karyotypes of the cell line ECV304 and of the bladder carcinoma T24 cell line has shown that these karyotypes differ in all of the main cytogenetic characteristics. No identical structural chromosomal rearrangements, nor rearrangements characteristic of bladder carcinoma cells have been revealed. The studied endothelial cell line ECV304 is not identical to the T24 cell line.     

Optimization of growth and jaceosidin production in callus and cell suspension cultures of Saussurea medusa

Calli cultures derived from the leaves of Saussurea medusa were selected on the basis of colour into three callus, A, B and C, which suggested different levels of metabolite accumulation. An improved reversed phase high performance liquid chromatographic method provided selective determination of the jaceosidin content of these samples. The jaceosidin concentration in callus B was higher than that of the callus A and C. By using 12-day old culture and 9-day old inoculum, jaceosidin yield of 72.91 mg l^–1was obtained from cell line B in cell suspension cultures. The influence of some factors affecting jaceosidin formation, i.e. temperature, light, inoculum size, type of media, phytohormones, nitrogen and carbon source etc. were also examined. Light irradiation and combination of 3% (w/v) sucrose with 1% glucose brought about a marked increase of jaceosidin production. The effect of blue light on jaceosidin was markedly superior to other kinds of monochromatic light (red and far-red) or white light. Analysis of growth and jaceosidin content of callus cultures and cell suspension cultures demonstrated that the production of jaceosidin was growth-dependent in both cell solid culture and cell suspension culture.     

Haploid and diploid cell cultures from a haplo-diploid insect

Summary

This is the first report of haploid and diploid cell culture from the haplo-diploid parasitoid wasp, Mormoniella vitripennis. Cells were cultured from haploid and diploid wasps by collecting populations of eggs from virgin females (unfertilized, haploid, parthenogenetic eggs) and mated females (mostly fertilized, diploid eggs). Eggs were surface sterilized in 70% ethanol, followed by 50% Chlorox, and rinsed in phosphate buffered saline; larvae were allowed to hatch in culture. Larval cells were dissociated and cultured at 28°C in the presence of Grace's medium supplemented with fetal bovine serum. Most cells in the HMV (predominantly haploid) and DMV (predominantly diploid) cell cultures grew in suspension in the first week, formed monolayers of fibroblasts and epithelial cells by the second week in culture, and continued to grow in monolayers and vesicle-like structures for up to three months. Chromosome analysis of HMV. cells demonstrated over 70% haploid cells, with five chromosomes (N=5). The remainder were aneuploid. No diploid cells (2N= 10) were found in the HMV cell culture. Chromosome analysis of DMV cultures revealed 62% diploid, with ten chromosomes; 13% were haploid, with five chromosomes; the remainder were aneuploid. These data confirm that haploid and diploid cells can be cultured from a haplo-diploid insect species. The HMV cells which are predominantly haploid, and DMV cells which are predominantly diploid may be valuable models for the study of cellular and gene activity in haploid and diploid genetic milieux.     

In vitro culture ofBellevalia romana (L.) Rchb.

Summary Bellevalia romana (L.) Rchb., a monocotyledonous plant characterized by few (2 n=2 x=8) and very large chromosomes, is a useful subject for studying developmental problemsin vitro. Cytological analysis of callus revealed that the majority of cells were diploid, but the remaining cells had aneuploid nuclei with a wide range of chromosome numbers, tetraploid and haploid nuclei. The frequency of aneuploid and polyploid cells was higher in callus grown in the presence of 2,4-D than in callus grown in NAA plus BAP. These nuclei seemed to increase with the duration of culture. The chromosome number distribution as determined by chromosome counts in calli at different culture times was confirmed by DNA cytophotometry. Chromosome number mosaicism (mixoploidy and aneusomaty) also occurred in all root apices of 9 out of 46 plantlets regenerated from callusvia adventitious shoots.     

Karyotypic changes in callus cultures from haploid and diploid plants of Crepis capillaris (L.) Wallr

Two auxin-heterotrophic callus cultures of Crepis capillaris, one coming from an haploid plant and the other from a diploid one, were studied in regard to karyotypic changes for over a year. The degree of polyploidisation of the originally haploid culture was considerably higher than that of the diploid culture. The frequency of chromosome rearrangements was significantly higher in polyploidised karyotypes than in not polyploidised karyotypes and correspondingly greater in the haploid culture. However, the cytogenetical stability of the cultures cannot be measured only through their degree of polyploidisation: it has been found that new karyotypes also originate through chromosome rearrangements at the same ploidy level as the original explant.     

Karyotaxonomical analysis in the genusAngelica (Umbelliferae)

Morphometric karyotype characters were studied in 25Angelica spp. (Umbelliferae, Apioideae) and in one species of the related genusTommasinia. For three species the chromosome numbers are new. In our study the majority of the species investigated are diploids with 2n = 22, some are tetraploids with 2n = 44 (for these tetraploids also diploid cytotypes are reported in the literature). Among the diploid species,A. miqueliana has a distinct karyotype consisting of submetacentric and acrocentric chromosomes only, the remaining diploids with 2n = 22 as well as tetraploids with 2n = 44 have rather symmetrical karyotypes, consisting of metacentric and submetacentric chromosomes. The very different chromosome number 2n = 28 has been found inA. gmelinii. Its karyotype includes two distinct groups of chromosomes: 8 pairs of rather large metacentrics and submetacentrics and 6 pairs of very short and asymmetrical chromosomes. Chromosome numbers and structures appear to be useful in the taxonomy of some intrageneric taxa inAngelica.     

Fertile wheat (Triticum aestivum L.) regenerants from protoplasts of embryogenic suspension culture

We report regeneration of fertile, green plants from wheat (Triticum aestivum L. cv. Aura) protoplasts isolated from an embryogenic suspension initiated from somatic early-embryogenic callus. The present approach combines the optimization of protoplast culture conditions with screening for responsive genotypes. In addition to the dominant effect of the culture media, the increase in fresh mass and the embryogenic potential of somatic callus cultures varied considerably between the various genotypes tested. Establishment of suspension cultures with the required characters for protoplast isolation was improved by reduction of the ratio between cells and medium and by less frequent (monthly) transfer into fresh medium. A new washing solution was introduced to avoid the aggregation of protoplasts. However, the influence of the culture medium on cell division was variable in the different genotypes. We could identify cultures from cultivar Aura that showed approximately a 9% cell division frequency and morphogenic response. The protoplast-derived microcolonies formed both early and late-embryogenic callus on regeneration medium and green fertile plants were obtained through somatic embryogenesis. The reproducibility of plant regeneration from protoplast culture based on the cultivar Aura was demonstrated by several independent experiments. The maintenance of regeneration potential in Aura suspension cultures required establishment of new cultures within a 9-month period.     

Development and Characterization of a Nonmorphogenetic Cell Line of Wheat (Triticum aestivum)

This study was conducted to compare characteristics of a wheat (Triticum aestivum L.) cell line to those of the maize (Zea mays L.) black Mexican sweet (BMS) cell line and to compare protoplasts isolated from suspension cells of these cell lines. The wheat cell line was established from immature-embryo derived callus of the experimental line ‘ND7532’ and was conditioned for growth in suspension culture. For both cell lines, measurements of packed cell volume (PCV), fresh weight (FW), and dry weight (DW) were taken at 3 day intervals from suspension cultures. Measurements of FW of calluses cultured from suspension cells of both cell lines were taken at 6 day intervals. The morphogenetic potential of the wheat ND7532 cell line was tested in both callus and suspension cultures using media promoting regeneration and/or organogenesis. Growth rates of ND7532 cells in suspension culture were comparable to those of BMS cells. However, relative growth rates of calluses recovered from ND7532 suspension cells were slower than those of calluses recovered from BMS suspension cells. The ND7532 cell line has very limited morphogenetic potential and has been maintained as rapidly growing callus tissue for 11 years. Yields of protoplasts from suspension cells of the two cell lines were comparable, though ND7532 protoplasts were typically smaller. The wheat cell line has is now designated ND7532-NM (nonmorphogenetic) and is available for cellular and molecular biology research.     

CHROMOSOME NUMBERS OF SOME SACCHARUM SPECIES HYBRIDS AND THEIR CELL SUSPENSION CULTURES

Chromosome numbers of five Saccharum species hybrids and their cell suspension cultures were determined. The chromosome number was stable in four parental clones (2n = 122, 114, 114, and 112), but was variable in another (2n = 108-128). The cell suspension cultures have been maintained in a yeast extract-enriched nutrient medium for more than 6 years. These cultures were variable in chromosome number for all clones, with a partial aneuploid series at the haploid and/or polyploid level. Each clone had different chromosomal population modes after 6 years of culture. The loss of chromosomes over a period of time would have an effect on the genetic makeup of a cell population. This has implications in the use and interpretation of data from pathological, cytogenetical, biochemical, and physiological studies using cell cultures and is probably a partial explanation for the loss of totipotency in 6-year-old sugarcane tissue and suspension cultures.     

A study of karyotypes and their alterations in cultured and Agrobacterium transformed roots of Lycopersicon peruvianum Mill

Summary Organ culture, plant regeneration from callus culture, and hairy root disease caused by Agrobacterium rhizogenes were utilized as methods of rapid in vitro propagation in Lycopersicon peruvianum Mill. A detailed and comparative karyotype analysis of the resulting material under such in vitro conditions revealed karyotypic stability under organ culture method, ploidy change in callus derived plants, and minor structural alterations of chromosomes in roots transformed by A. rhizogenes.Abbreviations BAP N6-benzylaminopurine - NAA naphthaleneacetic acid - MS Murashige and Skoog medium - RG regeneration medium - SDS sodium dodecyl sulfate     

The Correlation between the Chromosome Variation in Callus and Genotype of Explants of Arabidopsis thaliana

Twelve callus lines of Arabidopsis thaliana were derived from four types of explants excised from diploid plants of two ecotypes (Columbia and Wilna) and autotetraploid plants of the Wilna ecotype. Cytogenetic analysis of the chromosome variation in particular callus lines was carried out for primary culture and callus during 5 months of culture. Ploidy levels of interphase nuclei were estimated by counting the number and size of chromocentres and nuclei of interphase cells. The first polyploid cells in all callus lines were observed during callogenesis. In primary culture the ploidy level ranged between 2 and 15x (10-75 chromosomes). The frequency of polyploid cells was higher in the 5-month old callus culture, but the ploidy level was the same. In the callus lines derived from autotetraploid plants, cells with reduced chromosome number appeared quite frequently along with diploid and polyploid cells.