Chromosomal Analysis of Cultured Cells of Barley (Hordeum vulgare L.): Chromosome Number Variation

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1 _??_ 1990 by Cytologia, Tokyo Cytologia 55: , 1990 Chromosomal Analysis of Cultured Cells of Barley (Hordeum vulgare L.): Chromosome Number Variation B. D. Mohanty1 Department of Botany, University of Kalyani, Kalyani , India Accepted February 23, 1990 It is well known that plant cells and tissues show genomic instability in an in vitro system (Sunderland 1977). Barley, being a basic diploid, expresses marked phenotypic changes at the primary trisomic stage, and cannot tolerate deficiencies at the diploid level (Tsuchiya 1960, Singh and Tsuchiya 1977). Mixing of the polyploid and aneuploid cells disturbs the physi ological and genetical balance of the callus, leading to loss of the capacity to regenerate plants (Torrey 1967, Singh 1986). Thus, plant regeneration appears to be linked with the chromo some behaviour of the source callus culture (Jha and Roy 1982). Barley tissue culture has proved to be an ideal system, as the barley plants generated by tissue culture exhibited a variability which could be traced to that of the source culture (Orton 1980). Predominant occurrence of aneuploid karyotypes is reported in ovule cultures of barley (Orton 1980). Persistence of high frequency diploid karyotypes in cells of the embryo callus has also been reported (Ruiz and Vazquez 1981). In another report, an increase of tetraploid cells with progressive subcultures from stem node cultures of barley has been men tioned (Ruiz and Vazquez 1982). Singh (1986) observed a majority of diploid karyotypes in morphogenetic callus cells, while non-morphogenetic, immature, embryo calli of barley were dominated by aneuploid karyotypes. However, the spectrum of chromosomal status during successive subcultures was not examined. Even callus cultures derived from different explants of the same species are often described to be karyologically different (Novak and Vyskot 1975). The present study attempts to enumerate systematically the variation of chromosome number in callus cultures derived from three different explants, viz., embryo, mesocotyl and leaf from barley, and further to observe the quantitative karyotyic changes during a course of 6 successive subcultures. Materials and methods Mature embryo, mesocotyl, and leaf-base explants of barley (Hordeum vulgare L. cv. Karan 92) were cultured on MS (Murashige and Skoog 1962) basal medium supplemented with 2, 4-D (1, 2 and 4mg1-1). The detailed procedures of isolation of explants, culture etc. have been described elsewhere (Mohanty 1987, Mohanty and Ghosh 1987). The cul tures were kept in growth chambers at 20 }2 Ž, and illuminated with Philips (India) fluo rescent lamps (25,umol m-2s-1) for 8hr daily. Each callus was subcultured, at 30-day intervals, to the same freshly prepared medium, and maintained upto 6 months. Net age of the callus was counted from the date of 1st subculture. For cytological study, 5 calli from each subculture were collected from 3-5 culture tubes, and were separately pretreated with a-bromonaphthalene for 3hr, washed with distilled 1 Present address: Fruits and Vegetables Technology Central Food Technological Research Institute, Mysore , India.

2 400 B. D. Mohanty Cytologia 55 water, fixed in aceto-ethanol (1:3) for 8hr, and stained according to the orcein-hcl procedure. Data were analysed from 5-15 preparations per callus. For control preparation, root tips from germinating barley seedlings were processed as described for callus pieces. All mitotic metaphase cells of root tip showed 2n=14 chromosome counts; photographs etc, were taken from temporary slide preparations. Each experiment was repeated three times, and the results were reported as mean value+standard deviation. Duncan's New Multiple Range test was applied to tset the significance of reported differences. Figs. 1A-F. Chromosome number variation in callus culture of Hordeum vulgare L. A, callus cell showing normal diploid (2n=14) chromosomes. B, callus cell showing tetraploid chromo somes 2n=28. C, a hyper diploid callus cell showing 2n=19 chromosomes. D, hypodiploid callus cell showing 2n=5 chromosomes. E and F, callus cells showing high polyploid cells (2n_??_ 100).

3 1990 Chromosomal Analysis of Cultured Cells of Barley 401 Results Barley callus cultures, obtained from embryo, mesocotyl and leaf explants, revealed, in general. a wide variation in chromosome number (Fig. IA-F); however, a majority of the cell populations in each culture type were observed to be diploid in nature (Tables 1-3). The frequency and behaviour of each class of ploidy during subsequent subcultures were specific to each culture type. In embryo callus culture, the diploid cell frequency was observed to vary from 70-79%. But there was no sequential increase or decrease in diploid cell frequency with increase in the culture age. However, the frequencies of occurrence of tetraploid cells were 3%, 4% and 6% after 30, 60 and 90 days of culture respectively; with further increase Table 1. Variation in ploidy level in embryo callus tissues of Hordeum vulgare Any two means in the same column not followed by a common superscript differ significantly (P_??_0.05). * Values in the parentheses indicate the percentage (%) of the corresponding mean values. Table 2. Variation of ploidy level in mesocotyl callus tissues of Hordeum vulgare Any two means in the same column not followed by a common superscript differ significantly (P_??_0.05). * Values in the parentheses indicate the percentage of corresponding mean values. in culture age, the frequency of tetraploid cells increased significantly (Table 1). After 90 days, the frequency with which aneuploid and high polyploid cells occurred started decreasing, or else, such cells were completely eliminated from the culture. With increase in culture age, the range of variation in chromosome number also narrowed down, showing complete elimination of hypoploid cells (Table 1). In callus cultures derived from mesocotyl explants, the frequency of diploid mitosis showed a significant increase from 120 days onwards. There was an instant, significant increase of tetraploid cells in the interval from 120 to 150 days of culture, but there was no significant increase thereafter. Aneuploid and higher ploidy cells showed a decreasing tendency with increased age of the culture (Table 2).

4 402 B. D. Mohanty Cytologia 55 Table 3. Variation in ploidy level in leaf callus tissues of Hordeum vulgare Any two means in the same column not followed by a common superscript differ significantly (P_??_0.05). * Values presented in the parentheses indicate the percentage of the corresponding mean values. Figs. 2A-F. Interphase abnormalities in callus cells of Hordeum vulgare L. A, binucleate callus cells. B, multinucleate callus cells. C, callus cells showing micronuclei formation. D and E, amitotic nuclear division in callus cells. F, callus cell showing chromsomes condensed to dot like structures.

5 1990 Chromosomal Analysis of Cultured Cells of Barley 403 In leaf callus cultures, the diploid cell frequency ranged between 76-81% during subse quent subcultures (Table 3). There was no correlation between increase or decrease of the diploid cells and the culture age. Tetraploid cells showed a similar trend as the diploid cells. Aneuploid frequency was initially high, but gradually started decreasing with the culture age. However, there always existed a low, and almost constant, frequency of high polyploid cells in this type of cultures (Table 3). Besides the variation in chromosome number, all the culture types also showed mitotic and interphase abnormalities, like anaphase bridges, chromosomal fragments, clumping of chromosomes, asynchronous divisions, laggards, binucleate to multinucleate cells, one or several micronuclei, and extrusion of chromatin matter from the cells (Fig. 2A-F). Several types of nuclear morphology, suggesting amitotic nuclear division, were also observed (Fig. 2D, E). In some cases, one or more nuclei in a cell were found in divisional phase, while the rest were in the interphase state. Occasionally, chromosomes were condensed even to dot like structures (Fig. 2F). Discussion Present findings tend to differ from the observation of Orton (1980) regarding the predom inance of aneuploid karyotypes in an ovary culture of barley. However, his method of count ing chromosome number has been considered to be erroneous (Ruiz and Vazquez 1982). Examination of cultures from different explant sources, and their subsequent analysis at regular intervals, have revealed the predominance of the diploid cell population in barley callus cul tures. Occurrence of high frequency diploid mitosis, in an upto two-year-old, immature, embryo callus of barley has also been reported (Ruiz and Vazquez 1981). The same authors in another report have described the decrease of diploid cells with simultaneous increase of tetraploids in the stem node callus of barley (Ruiz and Vazquez 1982). Present experiments have revealed, however, that in no case was there any gradual decrease of diploid mitosis with increase in age of the calli. It has been suggested that generation of tetraploid cells in culture might be due to the division of medullary parenchyma (Ruiz and Vazquez 1982), which bears tetraploid compo nents in some plant stems (D LAmato 1952, Tabata et al. 1968, Brossard 1976). In the present fi ndings, the increase of tetraploid cells at a specific age of the culture supports this view. Only in embryo callus, did the tetraploid cells increase significantly from 120 days onwards. However, there there was no simultaneous decrease of diploid mitosis. Thus, a change of cell population from diploid to tetraploid, with increasing age of the culture, is not probable. Karyotypic instability in an in vitro system has been attributed to an interaction of explant growth potentiality with the environmental culture conditions (Orton 1980). The more important question here is the type of tissue of a particular explant which contributes to callus formation. This again depends upon the concentration of growth hormone used and age of the culture. Thus, with the change of the explant source, the concentration of growth hor mone required for callus formation is also changed, so much so that the type of cells in a par ticular explant, which ultimately contributes to the callus formation, also becomes uncertain. Present findings indicate the operation of a selection pressure in the establishment of dominant diploid karyotypes in barley callus cultures. However, the frequency of diploid cells in each callus type varied with the explant source and age of the culture. Various reasons-such as spindle abnormalities, chromosome lagging at anaphase, asyn chronous division, fragmentation and loss of chromosome content through micronuclei forma tion-have been mentioned for numerical variation in chromosome number (Bayliss 1973, Dutta Gupta and Ghosh 1883, Dutta et al. 1983, Mohanty et al. 1986).

6 404 B. D. Mohanty Cytologia 55 The aneuploid and highly polyploid cells were eliminated with progressive age of the callus. However, at a given period, cells of different ploidy levels give scope for each type of culture to function as a unique store house of genotypes. Plant regeneration from these cultures would help us to know the role of ploidy level on quantity and quality of the regen erants produced. Summary A study on the variation in chromosome number of the cells from a nodular morphogenic callus (Mohanty and Ghosh 1987), derived from embryo or mesocotyl, or leaf, was undertaken. This study also covered the extent of variation in relation to repeated subculturing for 6 months. The chromosome number showed wide variations (2n=5 to 100); the diploid cells (2n=2x= 14), however, were dominant in all the cultures obtained from each type of explant. In an embryo callus, the tetraploid (2n=4x=28) cells increased significantly after 90 days of cultur ing, but no significant rhythmic increase of tetraploid cells with age could be observed in mes ocotyl and leaf calli. The frequency of aneuploid and high polyploid cells decreased with increase in age of the culture. References Bayliss, M. W Origin of chromosome number variation in cultured plant cells. Nature 246: Brossard, D The influence of kinetin on formation and ploidy levels of bud arising from Nicotiana tobacum pith tissue grown in vitro. Z. Pflanzenphysiologie 78: D'Amato, F Polyploidy in the differentiation and function of tissues and cells in plants. A critical ex amination of the literature. Caryologia 4: Dutta, A. K., Biswas, A. K. and Ghosh, P. D Chromosomal variation in callus tissues of two species of Nigella. The Nucleus 26: Dutta Gupta, S. and Ghosh, P. D Chromosome analysis in callus culture of Triticum aestivum. The Nucleus 26: Jha, T. B. and Roy, S. C Chromosomal behaviour in cultures of Vicia faba. Cytologia 47: 465. Mohanty, B. D Cytomorphogenetic studies in callus cultures of Hordeum vulgare L. and Sorghum bicolor (L.) Moench. Ph. D. Thesis, Kalyani University, India, pp and Ghosh, P. D Somatic embryogenesis and plant regeneration from leaf callus of Hordeum vulgare L. Ann. Bot. 61: , Paul, N. K. and Ghosh, P. D Chromsomal behaviour in callus culture of Zea mays L. Cytologia 51: Murashige, T. and Skoog, F A revised medium for rapid growth and bioassay with tobacco tissue culture. Physiol. Plant. 15: Novak, F. J. and Vyskot, B Karyology of callus cultures derived from Nicotiana tabacum L. haploids and polyploidy of regenerants. Z. Pflanzenzucht. 75: Orton, T. J Chromosomal variability in tissue cultures and regenerated plants of Hordeum. Theor. Appl. Genet. 56: Ruiz, M. L. and Vazquez, A. M Cell population evoution in tissue cultures from embryo barley (Hor deum vulgare L.) after caffeine treatment. Protoplasma 107: and Chromosome number evolution in stem derived calluses of Hordeum vulgare L. cultured in vitro. Protoplasma 111: Singh, R. J Chromosomal variation in immature embryo derived calluses of barley (Hordeum vulgare L.). Theor. Appl. Genet. 72: and Tsuchiya, T Morphology, fertility and transmission in seven monotelotrisomics of barley. Z. Pflanzenzicht. 78: Sunderland, N Nuclear cytology. in Plant Tissue Culture. (H. E. Street, ed.), Blackwell Scientific Pub lishers, Oxford, ppp Tabata, M., Yamamoto, H. and Hiraoka, N Chromosome constitution and nicotine formation of matured plants derived from cultured pith of tobacco. Jap. J. Genet. 43: Torrey, J. G Morphogenesis in relation to chromosomal constitution in long-term plant tissue culture. Physiol. Plant. 20: Tsuchiya, T Cytogenetic studies of trisomics in barley. Jap. J. Bot. 17: