Cell fusion: A possible mechanism for the origin of polyploidy

S. Afr. 1. Bot. 1995,61(2): 60-<55

60

Cell fusion: A possible mechanism for the origin of polyploidy Johan J. Spies' and Susan M.e. van Wyk Department of Botany and Genetics, University of the Orange Free State, P.O. Box 339, Bloemfontein , 9300, Republic of South Africa Received: 23 Seprember 1994; revised 23 November 1994

Different ploidy levels in meiocytes within the same anther are occasionally observed. These result from cell fusion during microsporogenesis. Fu sion usually occurs during early prophase (Ieptonema to zygonema) and may include many cells (in th is study up to 17 cells we re observed). The pOlynucleate fused cell usually forms a single spindle and meiosis appears normaL The end result of meiosis is polyhaploid gametes. Fused cells, and the gametes developing from them, are much larger than the normal (unfused) cells , This phenomenon occurred in one in a hundred to one in a thousand meiocytes and was observed in unrelated taxa, e.g. Poaceae (19 genera representing all subfamilies except 8ambusoideae) and Solanaceae (Lycium). Verskillende ploi'dievlakke word soms in 'n enkele helmknop waargeneem. Oit is die resultaat van selfusie tydens mikrosporogenese. Selfusie vind plaas tydens vroee profase (Ieptoteen tot sigoteen) en baie selle kan betrokke Wees (tot 17 selle is tydens hierdie studie waargeneem). Die veelkernige fusiese l vorm gewoonlik een spoel en meiose ve rtoon normaal. Die eind re sultaat van meiose is polihaplo'(ede gamete. Fusies elle en die gamete wat uit hulle onlwikkel, is baie groter as die normale enversmelte selle. Hierdie verskynsel kem voer by een uit 'n henderd tot een uit 'n duisend meiosiete en is by onverwante taksa waargeneem, bv. Poaceae (by 19 genera wat al die subfamilies met uitsondering van die Bambusoideae verteenwoordig) en Solanaceae (Lycium). Keywords : Cell fusion , meiosis, polyploidy. "To whom co rrespondence shou ld be addressed.

Introduction De Wet (1980) described three different modes of origin of polyploidy. These modes include zygotic chromosome doublin g, meristematic chromosome doubling and gametic chromosome non-reduction. Zygotic chromosome doubling includes the fertilization of tbe occasio nally four.d non-reduced male and fem ale gametes. De Wet (1980) described the occurrence of this phenomenon as rare. Meristematic chromosome doubling in either developing seedlings or in active meristematic tissues was also described as a rare event (Jahr et ai. 1965). Gametic non -red uced chromosomes imply the absence or failure of meios is in either gamete. The frequency of thi s occurren ce was not mentioned by De Wet (1980). Polyploidy is frequently observed in the grass es and Stebbins (1985) postulated that more than 80% of the world's grasses have undergone polyploidy at some time during their phylogenetic development. This figure for polyploidy in the world's grasses is reflected also in the South African grass flora, where cytogenetic studies on various species have revealed th at approximately 81% of the South African grasses studied are polyploid (Moffett & Hurcombe 1949; Pienaar 1959; De Wet 1954, 1960; De Wet & Anderson 1956; Davidse ef al. 1986; Spies & Du Plessis 1986a & b, 1987a & b, 1988; Spies & Jonker 1987 ; Du Plessis & Spies 1988,1992; Spies & Voges 1988; Spies ef al. 1989, 1990, 1991, 1992; Strydom & Spies 1994; Visser & Spies 1994a-<:). In our studies, different ploidy levels within the same anther were occasionally observed. The aim of this paper is to investigate the origin of meiocytes with different ploidy levels within the same anther. Materials and Methods Cytogenetic material was collected and fixed in the field. Cytogenetic voucher specimens are housed in the National Herbarium, Pretoria (PRE) and/or in the Geo Po tts Herbarium, Department of Botany and Genetics, University of the Orange Free State, Bloemfontein (BLFU) .

Young inflorescences were collected and fi xed in Carnoy's fixative (1886). After 24-48 h of fixation, the fixative was replaced by 70% ethanol. Anthers were squashed in 2% acetocarmine (Darlington & La Cour 1976). Slides were made permanent by freezing them with liquid CO, (Bowen 1956), followed by dehydration in ethanol and mounting in Euparal.

Results and Discussion During routine investigations of meiotic material in our laboratory, we occasionally observed different chromosome numbers in different cells of the same anther. The differen ces in chromosome numbers are polyploid in nature. Our first reaction to this phenomenon was to attribute these differences to contamination of the preparations with pollen mother cells of other species. Additional care was given to all instruments used during the preparati on of squashes and, while collecting material in the field, care was taken to collect inflorescenses from single plants only. However, despite the exclusion of all possible forms of contamination, the occurrence of different ploidy levels in the same an ther persisted. The different ploidy levels in a specimen occurred repeatedly in material collected and processed during different seasons (years) by different people. Therefore, we do not consider this phenomenon as a procedural artifact. An example of different ploidy levels in the same anther of a Lolium temulentum specimen (Spies 4722). is presented in Figure IA-C In addition to the normal diploid (n ~ x ~ 7) cells usually observed (Figure 1A), a few octoploid (n ~ 4x ~ 28) cells (Figure lB) and even decaploid (n ~ 5x = 35) cells were observed in the same anther (Figure 1C). A similar situation was observed in variou s species, e. g. Lagurus ovatus (Figure ID), Helictotrichon turg idulum (Figure IE), Lophochioa cristata (Figure IF) and many other genera (Table 1). Careful investigation of this phenomenon revealed the occurrence of interphase ce11s with numerous nuclei (Figure 2). The multinucleate in terphase cells result from the fusion of various cells. Interphase cells in close proximity form cytoplasmic

61

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Figure 1 Cell fusion in the subfamily Pooideae (Poaceae). A-C. Lalium temuienlUm , Spies 4722. A, metapbase I cells with the normal diploid chromosome complement (n = x = 7) and a large polyploid metaphase I cell; B. polyploid metaphase I cell (n = ±4x = ±28) from the same anther as A; C, abnormal metaphase II cell with a very high ploidy level (n = ±7x = ±35). D. Lagurus ovalus, Spies 3894, meiotic cells interspersed with binucleate tapetum cells - the majority of diakinesis cells are diploid (n = x = 7) with a fu sed cell with n = 2x = 14. E. Helictotrichon turgidulum, Spies 2355, diakinesis cells with a fused cell in the lower left corner. F. Lophochloa cristata, Spies 4965, metaphase I in two fused cells where two different spindles fo rmed. Scale bars: 10 J-lm.

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Figure 2 Initial stages of cell fusion. A. Chaetobromus dregeanus, Spies 3366. Cytoplasmic bridges have formed between neigbbouring cells. B. C. dregeanus, Spies 3366. Fused cell with three interphase nuclei. C. C. dregeanus, Spies 3366. Fused cell with nuclei still indicative of cytomixis. D. Avena sativa, Spies 5302. Fused cell with two interphase nuclei. E. Cenchrus ciliaris, Spies 5238. Fused cell with two interphase nuclei. F. Fe stuca scabra, Spies 4452, partially fused cells. Scale bars: 10 J.lIO.

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Figure 3 Meiosis after cell fusion. A. Anapbase I with a 12- 12 segregation in an unfused cell of Lycium afrum specimen, Joubert 371. B. Anapbase I with a 24-24 segregation in a fused cell from the same specimen as in A. C. Metaphase I in a fused cell of L arenicoiwn, Joubert 361, with a single spindle. D. Heiiclolrichon curgiduJum, Spies 2355. Diakinesis showing 14 bivalents. E. H. turgidulum, Spies 4775. Diakinesis with 1411 _ F. Diakinesis with 21111 in the Spies 4775 specimen. G. Lolium lemulentum, Spies 4722, diakinesis with 1411 , H.Andro~ pogan huillensis, Spies 1977, metaphase I. LA. huillensis. Spies 1977. Metaphase I with many more chromosomes. Scale bar: 10 J.llD.

bridges (Figure 2A). The initial stages of the cytoplasmic connections are similar to those described by Heslop-Harrison (1966). The phenomenon of cytoplasmic bridges was already described by Gates in 1908. It is very difficult to compare our study (squashing of cells) with electron microscopic studies of sectioned material (reviewed by Bhandari in 1984). However, we believe that the observations made in our laboratory not only conftrm previous reports, but also extend to include the consequences of the cytoplasmic linking of different cells. In cells linked by cytoplasmic bridges, the nucleus of one cell moves to the other cell by mean s of cytomixis. The cytoplasmic bridges enlarge and eventually the cells become totally fused (Figure 2B- F). These nuclei are usually synchronized in meiotic

development. A single spindle is usually formed for all nuclei (Figures 3, 4A, E-F & 5), although sometimes more than one can be formed (Figure 4B, C). Anaphase I is usually normal and telophase I results in two polybaploid daughter cells with a normal meiosis II. The product is polyhaploid gametes which, although obtained from meiosis, have different numbers of genomes per gamete. The number of genomes per gamete is directly correlated with the number of nuclei present in the interphase cells. The result of cell fusion is an increased ploidy level in the male gamete. This process can double the ploidy level or even increase it many times over. The highest increase we observed was approximately 17-fold. Theoretically a diploid plant can produce up to a 17-p loid gamete. The limitation in the increase in

S , Arc. 1, I)ot., 1995,6 1(2)

63 ploidy levels produced by cell fusi on is the ability of the polyhaploid gametes to achieve pollination. T he polybaploid gametes are larger than the norm aJ haploid gametes and seem fertile (Figure 5H & n, but since all materials in our laboratory were ftxed, it was impossible to determine the viability of the pollen in pollen germination tests. The term cell fusion, incorporating the whole process of the actual fusion and the resulling formation of polybaploid gametes, has been described in the genus Chaetobromus (Poaceae: Danthonieae) (Spies et af. 1990). This phenomenon has since been observed in various specimens. representing unrelated taxa from different locations in South Africa. Cell fusion has been observed in all the genera listed in Table 1. These genera include a variety of grasses and even a member of the Solanaceae, Lycium. No correlation has been observed between habitat and cell fusion. In specimens with cell fusion. fused cells represent one in a hundred to one in a thousand cells. Cell fusion involves the increase of similar genomes and results in autoploidy. A gamete is forme d and fertilization will, therefo re, render the zygote autoploid, segmental alloploid or alloploid. depending on the degree of outbreeding of the taxa. Since cell fusion increases the number of homologous chromosomes in a gamete, autosyndetic pairing in the zygote is relatively certain. A process sim ilar to ce ll fusion may also be present during early macrosporogenesis. The fusion of nuclei in the embryo sac has already been described as restitution nuclei present in several types of embryo sac development, for example the Fritillaria and Plumbagella types (Willemse & Van Went 1984) , In his work on endopolyploidy and polyteny, Nagl (1978) described the occurrence of nuclei of different sizes in the same

Table 1 List of genera in which cell fusion was observed Family: Poaccae Subfamily: Arundinoideac

Tribe: Danthonicac Chaetobromus, MerxmuelJera, Pelltaschistis, Tribolium

Subfamily: Panicoideae Tribe: Paniceae Cenchrus, Urochloa

Tribe: Andropogoncae Andropogon. lIyparrhenia

Subfamily: Chloridoideae

llarpochloa. Microchloa Subfamily: Pooideae Tribe: Hainardieae Parapho/is

Tribe: Poeae Festuca, Latium

Tribe: Bromcae

Brornus Tribe: Aveneae Agros/is, Aira, Avena, Lagurus, ].,fJphochloa

Family: Solanaceae Lycium

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Figure 4 Cell fusion in Chaetobromus dregeanus, Spies 3366. A. Anaphase I with an 18- 18 segregation of c hromosomes. B. Metapbase I in a fused cell with two different spindles - the upper metaphase has c1e.:vly fewer chromosomes than the lower metaphase. C. Metaphase in a fused cell with three spindles. D- F. Metaphase I in cells containing various chromosome numbers. Scale bars: 10 J..Ul1.

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Figure 5 The original plate indicating cell fusion in Chaerobromus dregeanus, Spies 3366. a--e. Metaphase I cells with various chromosome numbers. f. Fused cell containing five nuclei. g. Cytomixis. h. Pollen mother cells. Note the difference in cell size between the 'normal' meiocytes (bottom) and the large 'fused' meiocyte in tbe top right comer. i. Pollen formed from 'fused' cell (top) and 'normal' cell (bottom). Scale bars: 10 Jlm. (Reproduced from Spies et al. 1990, Genome 33: 652).

tissu e of individual specimens belonging to 'various species. The origin of different sizes of nuclei was attributed to endopolyploidy. Failure of either chromosome or chromatid segregation during anaphase, failure of cytokinesis and restitution of nuclei

were described as the main contributors to endopolyploidy. A similar situation was observed in somatic tissue and was described as mixoploidy (Sharma 1956; Reese 1973; Albers & Meve 1991).

S. Afr. 1. Bol., 1995, 61(2)

Cell fusion cannot be regarded as endopolyploidy sensu stricto. because different cells are involved.. It can also not be regarded as restitution of nuclei, since various cells are involved.. Cell fusion is. consequently, a process of putative polyploidization, previously seen but disregarded as possible contamination on the slide. In conclusion it can be stated that ce ll fusion usually occurs during early prophase (leptonema to zygonema) and may include up to 17 cells. The polynucleale celJ forms a single spindle and meiosis appears normaL The final result is gametes with higher ploidy levels than the expected haploid chromosome number. Fused cells and gametes developing from them are much larger than the normal Cunfused) cells. Through fusion of interphase cells, polyhaploid gametes can be fonned. This process may assist in the formation of polyploid specimens.

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