Chromosomal Instability in Callus Culture of Pisum sativum

A study on the variation in chromosome number and structure of the cells from 45-days-old embryonal axis explant callus tissue of Pisum sativum L. of four genotypes was undertaken. The chromosome number showed a wide range of variation, however, the majority of cells studied were observed to be diploid (2n= 2x= 14) in nature. Among the numerical aberrations induced, tetraploid cells were the most frequent as compared to aneuploid cells of cells with a higher ploidy level. The frequency of fragments and bridges were highest among other structural changes. The composition and concentration of growth regulators was found to effect chromosomal instability whereas there was no effect of genotype.     

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.     

Chromosome variations in regenerants of Arabidopsis thaliana derived from 2- and 6-week-old callus detected using flow cytometry and FISH analyses

Shoot organogenesis was induced from 2- and 6-week-old callus derived from the leaves of Arabidopsis thaliana ecotype Columbia (2n = 10). Regenerated plants were evaluated for chromosomal variations by means of flow cytometry and fluorescent in situ hybridization (FISH). Flow cytometric measurements revealed the occurrence of diploid, tetraploid, and octoploid plants among the regenerants of 2-week-old calli, whereas only diploid and tetraploid plants were regenerated from the 6-week-old calli. Chromosome counting showed that plants developed from the 2-week-old calli exhibited mixoploidy and a high frequency of aneuploid cells. These plants were infertile and displayed altered morphology. FISH with 5S and 25S rDNA probes allowed to detect some structural chromosomal rearrangements in regenerated plants. Along with cells which exhibited correct localisation of rDNA loci, also cells bearing chromosomal translocations, deletions or duplications were found. The type of structural aberrations varied between diploid and tetraploid regenerants.     

Ploidy of somatic embryos and the ability to regenerate plantlets in melon (Cucumis melo L.)

Summary The number of chromosomes in cells of callus, somatic embryos and regenerated plantlets during somatic embryogenesis were examined in two cultivars of melon (Cucumis melo L.). Somatic embryos were diploid (50.0%/32.1%), tetraploid (38.5%/57.5%) and octoploid (11.5%/10.4%) whereas in callus cells diploidy (41.9%/43.3%), tetraploidy (27.9%/25.8%), octoploidy (11.6%/15.5%) and a low frequency of other types of ploidy and aneuploidy were observed. Mixoploid somatic embryos were not observed. These results suggest that the somatic embryos were selectively differentiated from diploid, tetraploid and octoploid cells, and that endopolyploidization of cultured cells occurred before the start of cell division leading to somatic embryogenesis. The ratio of diploid to tetraploid (1.30/0.55) in somatic embryos was less than that in callus cells (1.50/1.68) while ratios of diploid to octoploid (4.35/3.09) and tetraploid to octoploid (3.35/5.52) in somatic embryos were greater than those in callus cells (3.61/2.80 and 2.40/1.67). Therefore, it appears that the ability of callus cell to differentiate into somatic embryos increases in the following order: octoploid < diploid < tetraploid. Regenerated plantlets were diploid (65.5%/55.1%) and tetraploid (34.5%/44.9%). No octoploid plantlets were observed. The ratio of diploid to tetraploid in regenerated plantlets (1.72/1.23) was greater than that in somatic embryos. Therefore, it appears that the ability of somatic embryos to develop into plantlets increases in the following order: octoploid < tetraploid < diploid.     

Chromosomal changes in three cell strains of the tasmanian rat-kangaroo,Potorous tridactylis,cultured in vitro (Marsupialia)

The chromosomal changes occurring in three strains of cells from Potorous tridactylis, one derived from testis and two of kidney tissue, were followed during the in vitro life of the strains.One kidney cell strain was a slow growing one and died after 23 passages showing aneuploidy with very aberrant metaphases. The strain derived from testis showed aneuploidy after a period of growth retardation, about 50% of the aneuploid cells having 18, 19 or 20 chromosomes. In these cells the chromosomes 1, 2 and 4 were always present in triplicate and the cells always had two X-chromosomes. The second kidney strain showed aneuploidy after a period of growth retardation, cells with 22 and 23 chromosomes being the most frequent ones, but in different proportions. As the number of aneuploid cells gradually decreased, diploid cells appeared in the population. Their number also decreased and a new population of aneuploid cells arose, having 23 chromosomes, missing one chromosome nr. 5 from the tetraploid complement. Then again the cell strain returned to diploidy but as the frequency of these diploid cells decreased, the strain died out.The work was carried out, in part, under the association between Euratom and the University of Leiden, contract No. 052-64-I-BIAN, and it also received support from the Foundation for Basic Medical Research (FUNGO).     

Spontaneous polyploidy,gynogenesis and androgenesis in second generation (F2) koi Cyprinus carpio × goldfish Carassius auratus hybrids

The objective of this study was to characterize the genetics of second generation (F₂) koi Cyprinus carpio × goldfish Carassius auratus hybrids. Spermatozoa produced by a novel, fertile F₁ male were found to be diploid by flow‐cytometric analysis. Backcross (F₁ female × C. carpio male and C. carpio female × F₁ male) juveniles were triploid, confirming that female and male F₁ hybrids both produced diploid gametes. The vast majority of surviving F₂ juveniles was diploid and small proportions were aneuploid (2·1n–2·3n and 3·1n–3·9n), triploid (3n) and tetraploid (4n). Microsatellite genotyping showed that F₂ diploids repeated either the complete maternal or the complete paternal genotype. Fish with the maternal genotype were female and fish with the paternal genotype were male. This demonstrates that F₂ diploids were the result of spontaneous gynogenesis and spontaneous androgenesis. Analysis of microsatellite inheritance and the sex ratio in F₂ crosses showed that spontaneous gynogenesis and androgenesis did not always occur in equal proportions. One cross was found to have an approximate equal number of androgenetic and gynogenetic offspring while in several other crosses spontaneous androgenesis was found to occur more frequently than spontaneous gynogenesis.     

Genetic and chromosomal variation inPetunia hybrida plants regenerated from protoplast and callus cultures

Plants regenerated from callus cultures derived from leaf discs and mesophyll protoplasts ofPetunia hybrida cv. Rose of Heaven exhibit a high frequency of genetic and chromosomal variation. Of twelve leaf disc-derived plants examined, only three had the normal diploid chromosome number (2n=14) while seven were tetraploid and two were aneuploid (16 and 27 chromosomes). Of seventeen plants derived from two protoplasts, none had the diploid chromosome number. Most had 28 chromosomes, one 29, two 27, one 26 and one had variable numbers (14–28) in different root tip cells. In all cases aneuploidy was associated with developmental abnormality. In addition, heritable differences in growth, morphology and flower pigmentation were observed in callus-derived tetraploids and diploids, including one diploid which differed from parent plants in at least four characters. These results are discussed in terms of the importance ofPetunia in genetics research and for studies of somaclonal variation.     

Regenerative callus of <Emphasis Type="Italic">Dianthus</Emphasis> ‘Telstar Scarlet’ showing mixoploidy produce diploid plants

Highly regenerative callus was isolated from the base of adventitious shoots on cotyledon explants of Dianthus hybrida Telstar Scarlet cultured on MS medium supplemented with 1 mg l⁻¹ TDZ and 0.1 mg l⁻¹ NAA. Flow cytometric analysis showed that cotyledon tissue is a mixture of diploid and tetraploid cells. Whereas the regenerative callus consisted of cells showing various ploidy levels of 2C to 16C, their regeneration ability was maintained as long as they were sub-cultured onto fresh media. More than 93% of regenerated shoots from the calluses were diploid. Only a few shoots were revealed as tetraploids and octoploids, suggesting that diploid cells had higher regeneration ability.     

Desynapsis in diploid and tetraploid clones of ryegrass

Desynapsis is described in diploid and tetraploid plants of perennial ryegrass. The plants were derived by repeated cloning of a single mixoploid (2n=14 and 28) detected among colchicine-treated seedlings. The diploid and tetraploid clones varied in degree of desynapsis, chiasma number, and fertility. The variation among the clones was probably environmental. The progeny of the mixoploid parent included dipoids, tetraploids, and an aneuploid. One diploid and the aneuploid were desynaptic and originated perhaps by selfing. Apparently a single recessive gene determined desynapsis. The role of synaptic genes in controlling the chemical structure and function of nucleoprotein macromolecules is discussed.     

ULTRASTRUCTURAL COMPARISION OF POLLEN GRAINS FROM 2n, 3n,AND 4n PLANTS OF EUPHORBIA DULCIS

Pollen development in plants with different ploidy levels of Euphorbia dulcis is similar but some ultrastructural differences do occur. In pollen of diploid plants large aggregations of rough endoplasmic reticulum [RER] are attached to the pollen wall near the young generative cell but such aggregations are not present in other karyotypes. Plastids are detected only in young generative cells of triploid plants. In diploid plants the generative cell becomes spindle-shaped, in triploid and tetraploid plants it remains round during the movement from the pollen wall to the center of the vegetative cell. The intine surrounding the generative cell in 3n plants is thinner than that found in 2n and 4n plants. Pollen grains in tetraploid plants are twice as large as those in diploid plants. Pollen viability is 90% in 2n plants, but only 10% in 4n plants.     

SOMATIC EMBRYOGENESIS AND PLANT REGENERATION IN TISSUE CULTURES OF PANICUM MAXIMUM JACQ.

Callus tissue cultures were initiated from immature embryos, mature embryos and young inflorescences of Guinea grass (Panicum maximum Jacq.) on Murashige and Skoog's (MS) medium supplemented with 2.5–10 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D). Calluses were transferred onto the same nutrient medium with 0.2 mg/l 2,4-D, or without 2,4-D. In callus cultures derived from immature embryos and young inflorescence segments, plantlets were produced via somatic embryogenesis after 3–5 wk. Young plants were successfully transplanted to pots and grown in the greenhouse. Plant development in callus obtained from mature embryos took place through the organization of shoot meristems. Regenerated plants were shown to have the normal tetraploid chromosome number of 2n = 4x = 32.     

In vitro regeneration ability of diploid and autotetraploid plants of Cichorium intybus L.

Polyploidy has played a significant role in the evolutionary history of plants and is a valuable tool for obtaining useful characteristics. Because of the novelty of polyploids, comparison of their in vitro culture responses with diploids would be notable. In this study, leaf explants from diploid, autotetraploid and mixoploid plants of Cichorium intybus L. were cultured in vitro on the similar media and under same conditions. The ploidy level of the obtained calluses and regenerants were determined by flow cytometry analysis. The callogenic response of leaf explants cultured on the callus induction medium did not depend on the ploidy level of their parental plants. According to the flow cytometry analysis, the increased ploidy levels (4x) and (8x) were observed in the callus cultures with diploid and tetraploid origin, respectively. A considerable difference was observed between the ploidy level of mixoploid plants and their calluses, indicating the dominance of diploid cells in the callus tissue. The results showed that polyploidy led to the loss of organogenic potential as the tetraploid origin calluses failed to regenerate, while the diploid origin calluses successfully regenerated to whole plants.     

Arrangement of spindle apparatus in mitoses of different ploidy

In non-hypotonically treated mitoses from tissue cultures of Microtus agrestis, both the constitutive heterochromatin of the sex chromosomes and the spindle apparatus were stained by the Giemsa C-banding technique. By means of counting the heterochromatic chromosomes, we determined the cell ploidy and studied the number of centrioles and the spindle arrangement of diploid, triploid, tetraploid and octoploid mitoses. Diploid and triploid prophases contained 2 centrioles in most cases, tetraploid prophases 4, binucleate cells with 2 diploid nuclei likewise 4 and binucleate cells with 2 tetraploid nuclei 8 centrioles. Nearly 99% of diploid and triploid metaphases were bipolar. Of the tetraploid metaphases only 45% were bipolar, 29.5% tripolar, 7.5% quadripolar and 18% formed as a parallel mitosis. In all examined binucleate cells that had had an asynchronous DNA synthesis, a multipolar mitosis was found.     

Ploidy-dependent genomic stability in the tissue cultures of ornamental Phlox drummondii Hook

Comparisons of the chromosome numbers, 2C nuclear DNA amounts and karyomorphology were made in explant cultures of diploid (2n = 2x = 14) and autotetraploid (2n = 4x = 28) Phlox drummondii. In 6–36 week old calli derived from diploid internodal segment explants, and in cells of root tips regenerated from such callus, marked differences were observed in chromosome number. The chromosome numbers ranged from 2n = 14 to 2n = 100 and DNA amounts from 8.20 to 63.20 pg in the diploid derived callus, while the extent of variation was much reduced in the regenerated roots. In contrast, the autotetraploid cultures were characterised by the maintenance of the same chromosome number and DNA amounts as the mother plant. Modified chromosome structures were not apparent in any of the cultures. The possible reasons for the chromosomal instability at the diploid level and stability at the tetraploid level are discussed.     

Numerical analysis of isozyme variation in Glycine tomentella

Isozyme variation among 114 accessions of the Glycine tomentella Hayata was analysed by single linkage cluster analysis and the unweighted pair group centroid method (UPGMC). The diploid accessions fell into six distinct, well defined groups, which conformed with differences in chromosome number (2n − 38 or 2n − 40) or in geographic origin. The majority of the tetraploid accessions belonged to a large, geographically widespread group, predominantly aneuploid (2n − 78) group. The remaining four tetraploid groups were distinct on the basis of morphology or geographic distribution. The validity of tetraploid isozyme groupings for reflecting subspecific differentiation was supported by the published reports of hybrid fertility. All of the nineteen crosses between isozyme groups have yielded sterile hybrids, whereas five crosses within groups have yielded fertile hybrids. The relationship between diploids and tetraploids was examined either as the similarity between individual accessions, or that between isozyme groups. These analyses indicated that each tetraploid group is closely related to only one or two of the diploid groups or subgroups.     

Chromosomal changes in eight cell strains of the tasmanian rat-kangaroo,Potorous tridactylis (Marsupialia). Male and female heart fibroblasts cultured in vitro

Eight cell strains, derived from the hearts of a male and a female Motorous, were followed during their in vitro cultivation.All three male cell strains started as normal diploid cell strains. One of them, 2Hpo stayed diploid until passage 59, when the cells were frozen and stored at –96°C. After a period of growth retardation, that lasted two months, 1Hpo showed aneuploidy, the cells having 22–24 chromosomes. The cells with 23 chromosomes formed about 30% of the population. These cells predominantly missed the chromosomes 2, 3 and Y₁, from the tetraploid set. In the other cells no consistent pattern was observed. The cell strain 4Hpo did not show aneuploidy after three months of growth retardation. At the last passage (nr. 24) before death, it showed 25% diploid cells and 40% tetraploid cells.Three female strains were initiated on fibrin clot, two on plasm clot. No differences in growth and chromosomal changes, due to the different embedding media, were observed. All the strains started as diploid (2n=12) cell strains. The chromosomal changes that occurred showed many differences. Three cell strains (5Hf, 7Hp, 52Hf) died without showing any pattern in the aneuploid cells. One cell strain (53Hf) showed an aneuploid cell population with a stemline of 14 chromosomes. The cell strain (8Hp) showed different changes in ploidy. After 50 passages, it changed from diploid to aneuploid (19 chromosomes per cell in the stemline). Twenty passages later diploid cells started to dominate the population again (80% at passage 85). Then a new aneuploid population with a stemline of 18 chromosomes (30% triploid cells) arose, and the strain survived as a permanent line.The work was carried out, in part, under the association between Euratom and the University of Leiden, contract Nr. 052-64-I BIAN, and it also received support form the Foundation for Basic Medical Research (FUNGO).     

Ploidy levels of plants regenerated from mixed ploidy maize callus cultures

Summary Haploid and diploid anther-derivedZea mays callus lines were treated with the antimicrotubule herbicide pronamide to produce mixed ploidy callus as determined by flow cytometry. The ploidy levels of the plants regenerated from the callus were determined by counting the leaf epidermal guard cell chloroplast numbers. The proportion of diploid regenerated plants was somewhat lower than the proportion of diploid cells of the callus. The diploid plants regenerated somewhat faster than the haploids. The proportion of tetraploids regenerated from the pronamide treated diploid callus, which originated by spontaneous chromosome doubling, was much lower than the proportion of cells indicating that tetraploid cells survive or regenerate plants at a lower frequency than diploid cells.     

In vitro embryo rescue culture of F<Subscript>1</Subscript> progenies from crosses between diploid and tetraploid grape varieties

The purpose of this study was to understand factors affecting in vitro embryo rescue culture from hybrids between diploid and tetraploid varieties of grape in creation new triploid germplasm resources. The effects of different media, removal ages of immature seeds and reciprocal crosses of parents on the germination and seedling survival of immature seeds from crosses between diploid and tetraploid grape varieties by in vitro embryo rescue culture were investigated. The results indicated that the medium consisting of NN-1969 + IAA 1.75 mg l⁻¹ + GA₃ 0.35 mg l⁻¹ + CH 400 mg l⁻¹ + AC 2.0 g l⁻¹ was better than other media. The optimal removal age of immature seeds for the best development of embryos was 35–45 days after pollination (DAP). The percentage of germination (PG) for immature seeds and the percentage of seedling survival (PSS) for immature seeds for diploid varieties used as female parents were 10.72% and 4.35% higher than when tetraploid varieties were used as female parents respectively. A total of 41 hybrid progenies from eight combinations were obtained, made up of 17 diploid, 9 tetraploid, 14 aneuploid, and 1 triploid progeny as determined by root-tip chromosome identification. The triploid progeny was from Fujiminori (2n = 4x = 76) × Jingxiu (2n = 2x = 38). These results implied that it was feasible to extend the hybridization range of grape and to create new germplasm resources by in vitro embryo rescue based on the conventional hybridization. The NN-1969 medium supplemented with GA₃ and IAA was more propitious to the development of immature seeds sampled at about 45 DAP. It was easier to obtain plants using diploid as female parent, but triploid progeny was only obtained using tetraploid as female parent.     

Etude de l'aneuploïdie dans différents naissains d'Ostreidae (Bivalvia)

Study of aneuploidy in spats of Ostreidae (Bivalvia). Chromosomes of cells from gill tissue of juveniles (5–10 mm) from four different populations of Ostrea edulis and five different populations of Crassostrea gigas were examined in order to study aneuploidy and its significance. Mitotic chromosome counts were made for a sample of spats in each population. Cells with the normal diploid complement (2n=20) and with aneuploid complements (2n different from 20) were scored. The total percentage of aneuploid cells in the studied populations of the two species varies from 9% to 34%. Individual patterns of aneuploidy were examined in three populations of Crassostrea gigas. Some animals showed only normal diploid cells, others having both normal and aneuploid cells in variable proportions. The relationship between aneuploidy and growth rate was studied in experimental spats. Mitotic chromosome counts were made on individuals from two groups of animals: the first group was collected at a precise date at a control size of 8 mm, the second group reaching the same size of 8 mm only one month later. The percentage of animals showing aneuploid cells is much greater in the second group. There is a relationship between occurrence of aneuploid cells and growth rate. The karyotypes of 18 aneuploid sets of Ostrea edulis from different spats were analyzed. Chromosome loss was observed only in the submetacentric chromosome pairs. This loss of chromosomes could have an effect on the percentage of homozygotes observed in electrophoregrams. Thus, the percentage of homozygotes would be higher in juveniles showing the greatest number of aneuploid cells. An excess of homozygosity (=heterozygote deficiency) has been reported at a number of enzyme loci in over two dozen bivalve species (including Ostrea and Crassostrea). We suggest the hypothesis that this excess of homozygosity could be related to the occurrence of aneuploid cells. The relationship between aneuploidy and growth rate may prove to be a genetic factor of importance for oyster culturing.     

Cytogenetic characterization of embryogenic callus and regenerated plants of Pennisetum americanum (L.) K. Schum

Summary Embryogenic calli were derived from cultured segments of immature inflorescences of Pennisetum americanum (pearl millet). The original explants as well as the embryogenic calli and the plants regenerated via somatic embryogenesis were examined cytogenetically. Embryogenic calli were predominantly diploid (2n=14) after one month and six months in culture (92% and 76%, respectively). Tetraploid and aneuploid cells were observed in the original explant (2.5% and 1.2%) as well as in one (4.0% and 4.0%) and six-month-old calli (10.0% and 14.0%). Plants were regenerated from calli that had been in continuous culture for two, four and six months. Of the 101 regenerants, 100 were diploid and 1 was tetraploid. The tetraploid was an albino as were three of the diploid regenerants. Examination of 30 of the regenerants in meiotic diakinesis, anaphase I, anaphase II and quartet stages revealed no cytogenetic differences between control and regenerated plants. Gel electrophoresis for total protein content and alcohol dehydrogenase and malate dehydrogenase activity also did not reveal any differences between the controls and regenerants. The results of this study show that a slight shift toward aneuploidy and polyploidy may occur in embryogenic cultures, but there also is a strong selection in favor of plant regeneration from cytogenetically normal cells.