Plant regeneration from callus culture of Curcuma aromatica and in vitro detection of somaclonal variation through cytophotometric analysis

Callus cultures initiated from shoot base explants of Curcuma aromatica Salisb. were maintained on Murashige and Skoog (MS) media supplemented with 2 mg dm⁻³ 2,4-dichlorophenoxyacetic acid alone or with 0.5 mg dm⁻³ kinetin. Plantlets were regenerated from 60 and 180-d-old callus on MS media supplemented with 3 mg dm⁻³ benzyladenine and 0.5 mg dm⁻³ α-naphthalene acetic acid. Approximately 8–10 plantlets were produced after 30–40 d of culture per 50 mg of callus inoculated. Out of 113 regenerants analyzed 85 plants were exclusively diploid and 28 were predominantly diploid revealing presence of polyploid nuclei. Frequency of polyploid cells were more in regenerants obtained from 180-d-old callus then from 6-d-old callus which might be attributed to the ageing of callus.     

Development of tetraploidy in V79 spheroids

Summary Chinese hamster V-79-171 cells, when placed in suspension culture, spontaneously form multicell spheroids. As the spheroids enlarge the fraction of polyploid (predominantly tetraploid) cells increases and can approach 100% in very large spheroids. Spheroid size, rather than age, seems to be a major determinant for increased ploidy. When cell separation techniques were used to select enriched populations of diploid and tetraploid cells, the growth rate and plating efficiency of the diploid cells was always marginally higher, and they gradually became predominant in mixed monolayer cultures. Cloned tetraploid cells, however, generally remained quite stable, and no consistent ploidy dependent changes in radiosensitivity were observed relative to normal, diploid cell lines. This research was supported by grants CA 28793 and CA 23511 awarded by the National Cancer Institute, Bethesda, MD.     

Nutritional Requirements for Polyploid Mitoses in Cultured Pea Root Segments

Diploid and polyploid mitoses could be stimulated in excised segments of the mature region of pea roots grown on a sterile culture medium. Diploid mitoses were observed in segments cultured on water alone for 72 hours. Their frequency was increased by the presence of salts, sucrose, vitamins, and any two or all three of the following: an amino mixture, auxins, and kinetin. Polyploid mitoses were observed 72 hours after the beginning of the culture period in segments cultured on salts, sucrose, vitamins, auxins, and kinetin. Polyploid mitoses required the presence of auxins and kinetin in the culture medium. Their frequency was not affected by the presence of a reduced nitrogen source. Light treatments had no effect on the frequency of diploid or polyploid mitoses. Diploid mitoses were first observed about 24 hours after the beginning of the culture and their frequency increased thereafter. Experiments with colchicine showed that diploid cells were entering mitosis for the first time as late as 60 hours after the beginning of the culture. Polyploid mitoses showed a long lag time when compared with diploid mitoses. They began at about 60 hours and their frequency increased thereafter. Experiments with colchicine showed that polyploid cells were entering mitosis for the first time as late as 84 hours after the beginning of the culture. The presence of kinetin in the medium was not required during the first 24 hours in culture for the appearance of polyploid mitoses at 74 hours. However, the presence of kinetin was required after 24 hours. Auxin was required at some time during the first 24 hours of the culture and its continuous presence may be required for the stimulation of polyploid mitoses.     

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.     

In vitro proliferation and differentiation of hemic precursor cells from marrow and blood of naturally chimeric marmosets

Marmosets are unique in that they are “always” blood cell chimeras. When the nucleated cells from the bone marrow and from the peripheral blood of marmosets were incubated in the appropriate culture fluid they were shown capable of extensive proliferation in vitro. Two patterns of cellular proliferation, adherent and nonadherent, occurred in the same culture vessel. Repeated passage of nonadherent cells in RPMI 1640 medium supplemented with calf serum resulted in relatively long-term fluid bulk cultures showing myelocytic differentiation and megakaryocytic maturation. As myelocytic maturation became the predominant feature of cultures mitoses of the precursors diminished. About half of 41 marrow-derived cultures underwent extensive proliferation lasting about two months, as evidenced by an increasing cellularity and the presence of dividing cells. Such active growth occurred in one culture for over 120 days. The natural blood-cell chimerism of marmosets was demonstrated in vitro by cytogenetic analyses of metaphases from four relatively long term marrow cultures. The ratios of male and female cells remained either relatively stable or changed slowly with time in culture. Cells having both diploid and polyploid number of chromosomes were identified male or female, suggesting chimerism in myelocytic and megakaryocytic series. Marmoset lymph node and spleen cells proliferated as lymphoid cultures for various lengths of time up to five weeks but these cells did not differentiate into hemic cell lines. Attempts to culture human and rodent hemic tissue by the procedure used on marmoset tissue were unsuccessful.     

Analysis of the initial stages of plant protoplast development using 33258 Hoechst: reactivation of the cell cycle

A microfluorimetric procedure, employing the fluorescent stain 33258 Hoechst, has been developed for the investigation of the process of DNA synthesis during the initial stages of culture of tobacco ( N. tabacum cv. Xanthi) leaf protoplasts.
In this system, the freshly-isolated protoplasts exhibited a unimodal distribution of nuclear DNA content characteristic of the diploid state. The almost immediate onset of DNA synthesis during culture resulted in a doubling of nuclear DNA levels prior to the first mitoses. Although the majority of the protoplasts subsequently entered into synchronous mitosis and cell division, a proportion of the remainder developed into large polyploid cells. Upon further culture, the polyploid cells became subdivided into clusters of small diploid cells. Measurement of total cell protein and cell volumes during culture indicated that a relationship existed between these parameters and the initiation of mitosis. The significance of these observations is discussed.     

DNA Synthesis, Mitosis and Ploidy of Dividing Cells in Early Crown Gall

DNA synthesis, mitosis and ploidy of dividing cells were studied during 30 h after wounding around wounds inoculated with Agrobacterium tumefaciens, around sterile wounds and in control stems of Vicia faba. DNA synthesis was examined by counting nuclei labelled with ³H-thymidine in slides in which mitoses had been counted and analyzed before autoradiography. The main results were that around inoculated wounds, DNA synthesis and the number of mitoses showed a peak between 12 and 22.5 h. Both types of wounding induced mitoses, many of them polyploid (DNA content higher than 4C), both in the pith and the cortex, whereas in the control stems only diploid mitoses, mostly in the stelar area, were seen. The first polyploid (8C) mitoses around the inoculated wounds took place at 12 h and at 15.5 h 32C mitoses were seen; around the sterile wounds the first 8C divisions occurred at 26 h. The frequency of polyploid mitoses and their degree of ploidy continued to be considerably higher around the crown gall than around the control wounds. When a cell with a higher than 4C content is induced to divide, the 12 chromosomes, as a rule, consist of four, eight, 16 or 32 chromatids, instead of the normal two. The early division of highly polyploid cells around the inoculated wounds is obviously caused by growth factors which are known to be produced by the bacteria. It appears possible that this ability to synthesize excessive amounts of growth factors is subsequently transferred to the host cells through bacterial DNA.     

Enzyme activity during the growth and aging of human cells in vitro

Acid phosphatase, alkaline phosphatase, and lactic dehydrogenase activities have been compared in normal human diploid cell strains and in SV₄₀-transformed heteroploid cell lines derived from them. A higher level of acid phosphatase activity was observed in diploid cultures derived from adult lung than in cultures derived from fetal lung of similar passage levels. The alkaline phosphatase activity of normal diploid fibroblasts was significantly higher than that of SV₄₀-transformed cell lines derived from them. Generally, the lactic dehydrogenase activities of all these cell cultures were similar. Human diploid cells in culture “age,” in the sense that their ability to proliferate decreases with time during serial subcultivation. Evaluation of the activities of these three enzymes during the “aging” process showed that, although alkaline phosphatase and lactic dehydrogenase activities were similar in “young” and “senescent” cells, acid phosphatase showed a small but significant increase in the senescent cells.     

The Effect of Growth Conditions and Origin of Tissue 5Phaseolus vulgaris L.)

Callus cultures isolated from various somatic tissues and anthertissue of Phaseolus vulgaris seedlings on a defined growth mediumcontained few diploid cells. The proportion of diploid cellsdid not alter as cultures lost their ability to form vasculartissue. Meristematic cells of roots initiated after transferto induction medium were diploid. All cultures lost their morphogeneticpotential after five to seven subcultures except anther calluswhich formed vascular tissue over a prolonged period of cultureon maintenance medium. After six subcultures anther callus containedmore polyploid cells than somatic cultures. Callus isolated from bean hypocotyl tissue in the presence ofcoconut milk consisted mainly of diploid cells and retainedits morphogenetic potential for a greater number of subculturesthan callus grown on defined medium. Transfer of callus isolatedon the defined medium to medium containing coconut milk increasedthe proportion of diploid cells and prevented further loss ofinorphogenetic potential. An equivalent concentration of cytokininto that in coconut milk prevented the loss of potential butdid not affect the ploidy of the cultures.     

CELL POPULATION KINETICS IN CALLUS TISSUES OF CULTURED PEA ROOT SEGMENTS

Two callus tissues, one composed of diploid and the other of a mixture of diploid and polyploid cells, were derived by culturing 1-mm pea root segments; the mixed callus tissues were obtained by incorporation of kinetin in the culture medium. The callus tissues were used to determine (a) if cell proliferation was altered with the change in cell constituents of a callus; (b) the rate at which polyploid cells increased after kinetin stimulation; (c) the nature of the mitotic cycle in the diploid and mixed polyploid callus tissues; and (d) if the mitotic cycle changed as the tissue aged. Histological, cytological, radioisotope, and radioautographic analyses were made on callus tissue ranging from 1 to 4 days old. The results indicated that gross morphological changes were associated with the anatomical location of the proliferative cells. They showed that the percentage of polyploid division figures after stimulation by kinetin increased rapidly during the first 6 to 7 days in culture and then continued to increase at a much reduced rate. Cell counts revealed that cell proliferation in the mixed callus tissue was initially delayed when compared with the diploid tissue, but that after the delay was overcome cell number increased in each in similar manner. Analysis of the number of DNA-synthesizing cells showed that their percentage was highest during the first 2 days of culture and then leveled off at a value of about 10%. Mitotic cycle analysis indicated that it could be accurately measured only in the younger diploid callus tissues and that it increased in variability with increased age.     

The effects of growth in vitro on the chromosome complement of Daucus carota (L.) suspension cultures

The chromosome number distributions and modal karyotypes of several suspension culture lines of Daucus carota L. have been analysed at various times after initiation. All lines had stable modal chromosome numbers and karyotypes, with small but significant variation about the modes. Some lines showed a predominance of diploid cells with a karyotype similar to the plant. Polyploid multiples of the modal chromosome number were present in all lines at low frequency. Variation of the 2,4-D concentration in the culture medium produced little alteration of the chromosome number distributions, but omission of 2,4-D produced a significant drop in the frequency of multipolar mitoses in those culture lines in which this treatment induced differentiation. There was no evidence of any direct effect of 2,4-D on general mitotic dynamics. Alteration of the frequency with which cultures were transferred to fresh medium showed that stationary phase was critical in the maintenance of the low frequency of tetraploids present in a predominantly diploid culture line. The results are explicable in terms of a competitive selection for cells with the dominant modal chromosome number in the presence of various mechanisms continuously producing polyploid, aneuploid and structurally altered karyotypes.     

The influence of progressive growth on the specific catalase activity of human diploid cell strains. I. Effect of cellular genotype: Homozygous strains

The specific catalase activity of human diploid cell strains increases with progressive growth of the culture, and falls again following subculture. Although the increase is small, it is readily demonstrable, and is exponential with time. The response of catalase activity to proggressive growth of the culture was studied in three abnormal human cell lines. A diploid cell strain, developed from a patient homozygous for the gene causing acatalasia I, had no detectable catalase activity throughout the life cycle of the culture. Another diploid cell strain, developed from a patient homozygous for the gene causing acatalasia II, had about 5% normal catalase activity, but the proportionate increase in specific activity as the culture grew was the same as for normal cells. Thus the mutation causing acatalasia II does not change the responsiveness of the cell in terms of catalase activity to progressive growth of the culture. The behavior of a heteroploid line was similar to that of the normal diploid strains, but when the growth of the heteroploid cultures reached a plateau, their population densities were four times higher than those of the diploid strains and they had about twice the specific catalase activity.     

MITOSIS IN SUSPENSION CULTURES OF HIGHER PLANT CELLS IN A SYNTHETIC MEDIUM

Torrey , J. G., J. Reinert , and N. Merkel . (Harvard U., Cambridge, Mass.) Mitosis in suspension cultures of higher plant cells in a synthetic medium. Amer. Jour. Bot. 49(4): 420–425. Illus. 1962.—A cytological study was made of plant tissue cultures growing in liquid synthetic medium. Mitoses in cell suspension cultures of root callus tissues of Daucus carota L., Convolvulus arvensis L. and Haplopappus gracilis (Nutt.) Gray were found to occur frequently in the first 2 weeks of culture with the highest frequency at about 7 days. No mitoses were observed after 3 weeks, although fresh weight and the number of free-floating cells in the suspension continued to increase for the entire culture period of 4–6 weeks. Mitoses were most frequent in tissue pieces, but occasional mitoses in single isolated cells in suspension were observed in each type of tissue. Normal mitoses were observed in diploid and polyploid cells of all 3 types of tissues cultures. Little evidence of nuclear or chromosomal aberrations was observed in these cultures.     

Cell-specific endopolyploidy in developing Artemia

Summary Cells in developing Artemia franciscana SFB demonstrated tissue-specific differences in DNA content, as determined by fluorescence intensity of bisbenzimide-stained nuclei and by nuclear area. The general epidermis comprised proliferating diploid (2C) cells. The setal cells had 4C–8C DNA content and did not divide during the first two instars. Salt gland cells were polyploid (>8C) and also did not undergo mitosis. Neural cells in the brain were diploid and were replicating. Cells in the thorax region of the gut had a 4C–8C DNA content and were proliferating. The muscle cells in the cephalic appendages contained 2C non-replicating nuclei. Only diploid epidermal cells were involved in segment morphogenesis. There was no difference in number of chromosomes (n=42) in the epidermal cells and the gut cells, indicating that the tissue-specific endopolyploidy was due to endoreduplication.     

Cytogenetic studies of Haplopappus gracilis in both callus and suspension cell cultures

Summary Investigations have been carried out on karyotype change in both callus and suspension cell cultures of Haplopappus gracilis (2n=4). It has been found that polyploidization arises directly in culture to give up to six times the normal diploid chromosome number in some cultures. In polyploid cultures, both chromosome loss and chromosome rearrangements occur to give rise to aneuploid karyotypes displaying chromosomes which differ in morphology from the diploid set. Whole or partial chromosome loss has been observed in the form of lagging chromosomes and chromosome bridges at anaphase, and micronuclei, ring chromosomes and chromosome fragments at other stages in mitosis. C-banded preparations have confirmed the occurrence of chromosomal rearrangements. Comparative investigations suggest that (i) more polyploidy occurs in callus cultures than in suspension cell cultures, and (ii) the presence of cytokinin (kinetin) in the culture medium may reduce the extent of karyotype change.     

Human hepatocyte polyploidization kinetics in the course of life cycle

The processes of polyploidization in normal human liver parenchyma from 155 individuals aged between 1 day and 92 years were investigated by Feulgen-DNA cytophotometry. It was shown that polyploid hepatocytes appear in individuals from 1 to 5 years old. Up to the age of 50 years the accumulation rate of binucleate and polyploid cells is very slow, but subsequently hepatocyte polyploidization is intensified, and in patients aged 86–92 years the relative number of cells with polyploid nuclei is about 27%. Only a few hepatocytes in the normal human liver reach 16C and 8C×2 ploidy levels for mononucleate and binucleate cells respectively. Using a mathematical modeling method, it was shown that during postnatal liver growth the polyploidization process in human liver is similar to that in the rat, and that polyploid cells are formed mainly from binucleate cells. As in rats, prior to an increase in ploidy level, diploid human hepatocytes can pass several times through the usual mitotic cycles maintaining their initial ploidy level. After birth, only one in ten hepatocytes starting DNA synthesis enters the polyploidization process. At maturity about 60% of 2C-hepatocytes starting DNA synthesis divide by conventional mitosis, the rest dividing by acytokinetic mitosis leading to the formation of binucleate cells. During ageing the probability of hepatocyte polyploidization increases and in this period there are two polyploid or binucleate cells for every diploid dividing by conventional mitosis.     

Physiology of Polyploid Plants: Water Balance in Autotetraploid and Diploid Tomato under Low and High Salinity

Diploid and autotetraploid plants of the cultivated tomato Lycopersicon esculentum cv. Lukullus (Luk) were studied under low and high salinity. Polyploids had a higher water content than diploid plants. Water content in both plant types decreased under salinity, the decrease being smaller in the polyploid plants. Dry weight of whole young plants decreased in both diploid and polyploid plants under salinity, the decrease being smaller in the latter. Transpiration of whole plants, grown in control solution, was lower in polyploid than in diploid plants and decreased more under salinity in the latter. Rate of change of water loss of detached drying leaves was similar in diploid and polyploid plants. Leaves of control diploid plants, however, lost more water per unit leaf area during the phase of stomatal closure apparently due to higher stomatal density. Polyploid plants had fewer but more open stomata per unit leaf area, under both control and saline conditions. Root pressure, determined only under control conditions, seemed to be higher in polyploid plants. No difference in Cl^? concentration per unit leaf dry weight was found between diploid and polyploid plants grown in either control or NaCl solution.     

Induction of polyploid nuclei in Physarum polycephalum by cycloheximide treatment in prophase

Macroplasmodia of the acellular slime mold Physarum polycephalum were treated with pulses of cycloheximide (10 micrograms/ml medium, for 3 h), initiated 10-20 min before metaphase in the synchronous nuclear division cycle. This treatment interfered with normal division of the nuclei, but permitted DNA synthesis in the next S phase. This interpretation is supported by measurements of the DNA content per nucleus in cycloheximide-treated cultures as compared to control cultures, which show that some nuclei after cycloheximide treatment are polyploid. By this method we can produce polyploid strains of Physarum, but the elevated nuclear DNA content is not stable, and after several months the strains have reverted to the normal diploid DNA content.     

The relation of karyotypic change to loss of contact inhibition of division in human diploid cells after SV40 infection

Following in vitro infection of human cell cultures with simian virus 40, karyotypic analyses were performed on the earliest serial culture in which cells were released from contact inhibition of division. In these cultures of diploid fibroblast-like cells, normal karyotypes were found in excess of the statistical expectation for the number of background dividing cells. Thus, loss of contact inhibition of cell division occurs prior to the alteration of chromosome morphology. These events are two of the prime alterations in the series of steps comprising transformation by this virus. The chromosomal changes which were present represent the first cytological alteration detectable. Their distribution in the human karyotype was examined, but was found to have no relation to any specific chromosome or chromosome group.     

Cytogenetics of protoplast cultures of Brachycome dichromosomatica and Crepis capillaris and regeneration of plants

Summary Callus derived protoplasts of Brachycome dichromosomatica (2n=2x=4) and Crepis capillaris (2n=2x=6) have been regenerated into karyologically normal plants, i.e. plants without visible alterations of the diploid chromosome set. However, metaphase analysis of protoplast cultures derived from both callus as well as mesophyll cells showed karyological changes in the overwhelming majority of cells in both species leading to multinucleated, polyploid and aneuploid cells. Furthermore, callus derived protoplasts sometimes exhibited changes at the chromosome level as indicated by translocations. The vast majority of aberrant karyotypes arose from failures during mitosis and cytokinesis, pointing to inadequate microtubules as a possible underlying cause. Karyological events of the kind described herein greatly affect the plating efficiency of isolated protoplasts and the viability of protoplast derived calli. Plant regeneration, although demonstrated in this study for the first time in both species, seems to be limited to rarely occurring, protoplast-derived colonies with a relatively stable genome. Our experiments, performed with chromosomal model species, emphasize the need for controlled, non-mutagenic culture conditions.