B-chromosomes in some Lachenalia species and hybrids

Varying chromosome numbers within a species and a series of diploid, polyploid and basic numbers have been reported for the genus Lachenalia. In a cytogenetical study conducted on this genus, B-chromosomes were observed in somatic and meiotic material of some species and F1 hybrids. The size of the B-chromosomes in somatic material was found to be similar, or slightly smaller than the smallest chromosome of the normal complement. In pollen mother cells (PMCs), having univalent B-chromosomes, the B-chromosomes divided precociously during the first meiotic division. In a few PMCs of the hybrids where more than one B-chromosome was observed, the B-chromosomes associated to produce bivalents indicating that they are homologous or homoeologous. The B-chromosome bivalents tended to disjoin normally during the first meiotic division and the resulting chromosomes divided normally during the second meiotic division. The discovery of B-chromosomes in the genus helps to account for some of the variation in chromosome number that has been reported for the genus, and specifically for the species L. orchioides (L.) Ait.


Introduction
Lachenalia is a member of the Liliaceae [Hyacinthaceae (sensu stricta)] family. These small bulbous plants are endemic to southern Africa with a concentration of species in the south-western Cape Province. The genus incorporates approximately 90 species. It is currently under revision and according to Duncan (1988), the number of species is more likely to near 1l0. The plants are characterized by tubular or bell-shaped flowers arranged in a spike on a fleshy stem that grows 200 to 250 mm high. The colours of the flowers range from shades of red, green, blue, purple, yellow and white. Breeding work on this little-known, but one of South Africa's most beautiful wild flowers, commenced in 1965 at the Vegetable and Ornamental Plant Research Institute at Roodeplaat near Pretoria.
This paper deals with the presence and behaviour of the Bchromosomes discovered in some of the Lachenalia species and hybrids.

Materials and Methods
The nine FJ hybrids of the six species most frequently used in the breeding programme and their surviving parents which were investigated, are given in Table 1. The numbers allocated to the hybrids, and those in brackets after the specific names are reference numbers used by the Vegetable and Ornamental Plant Research Institute.
For somatic chromosome studies young root tips were pretreated with 0.05% colchicine for 3 h and fixed in 1:3 acetic-alcohol for 24 h. Subsequently, the root tips were hydrolyzed in IN HCI for 5-7 min at 60°C and squashed in propionic acid. For meiotic studies, flower spikes were dissected from the bulbs as soon as the first flowers became visible between the leaves. In this process the plants were  (Table 1). However, in the case of the hybrids, meiotic studies were coducted in PMCs from the same clone that were used for chromosome counts in the root tips, except for the hybrid 7529 namely L. splendida Diels (30) X L. aloides (LJ.) Engl. cv. Pearsonii (22) where two different clones were used (Table 1). For the species it was not possible to do the meiotic studies on the same clone used for chromosome counts as more than one plant had been collected on a locality spot and were allocated the same number. After dissection, the inflorescences were fixed in a 6:3:2 mixture (v/v) of methanol, chloroform and propionic acid (pienaar 1955) and the anthers were squashed in propionic carmine.
As mentioned by Moffet (1936) the chromosomes of Lachenalia fixed and stained poorly at the prophase I stages of meiosis. This investigation was therefore limited to metaphase and anaphase of the first and second meiotic division. At least 100 pollen mother cells (PMCs) of each species and hybrid were studied.

Results and Discussion
Extra chromosomes were observed in some somatic and meiotic material of different Lachenalia species and hybrids ( 14 + 0-2B normal complement in that they are smaller and are heterochromatic. However, these characteristics are not invariable (Jones & Rees 1982). In this investigation of Lachenalia species and hybrids the size of the extra chromosomes was more or less similar to the smallest chromosome of the normal complement. Further, the latter chromosomes have no defmite staining pattern. In some cells they stained slightly lighter or darker and in others similar to the chromosomes of the normal complement. The variation in staining did not correlate with specific material or developmental stages. According to Jones & Rees (1982) the mitotic transmission of B-chromosomes to daughter cells during vegetative growth, is in most species disjunctional such that all cells carry the same number of B-chromosomes. There are exceptions where nondisjunction of the B-chromosomes causes variation in the number of B-chromosomes among cells within the same individual. The extra chromosomes observed in some somatic cells of Lachenalia presumably revealed the latter behaviour. In the species and hybrids investigated most of the cells have the normal chromosome number, but in addition to the normal complement, one or two extra chromosomes were observed in some cells. These unstable distribution patterns of B-chromosomes have been reported in different species by several workers (Price 1963;Shopova 1966;Joshi & Raghuvanshi 1969;Lewis et al. 1971;Sen 1974). This manner of transmission and distribution of the extra chromosome in Lachenalia, accounts for the variation in chromosome number between vegetatively similar plants (fable 1) and is reflected in meiotic material as well. Jones & Rees (1982) stated that while the behaviour and transmission of B-chromosomes during mitosis is, in most cases, orthodox and regular, the distribution of B-chromosomes to gametes during reproduction is unorthodox in the majority of species. They found that the main cause is the nondisjunction of B-chromosomes either during meiosis or, in higher plants particularly, during mitotic divisions in gametophytes subsequent to meiosis. Jones & Rees (1982) are of the opinion that Bchromosomes descended from members of the normal complement, which initially existed as trisomics or trisomic fragments and became modified in structure. This affects their pairing behaviour at meiosis and prevents homologous associations with their ancestral normal chromosomes. These trisomics and trisomic fragment can arise in various ways, e.g. non-disjunction or unequal interchanges. The centric fragment may be lost or evolve into a B-chromosome.
Since the Lachenalia plants with the extra chromosomes did not exhibit phenotypic abnormalities, the variation in basic number in this genus is probably not due to trisomy or partial trisomy, and must be ascribed to B-chromosomes. These extra chromosomes are not particularly small, as mentioned earlier. Mogford (1978) found that the chromosomes of L. aloides have large amounts of centric heterochromatin. Thus, relatively large inert centric fragments could have evolved in some of the species.

B-chromosomes in somatic material of Lachenalia
The somatic chromosome numbers are listed in Table 1 The B-chromosomes therefore account for the variation in chromosome number reported in L. orehioides and probably also in other species of the genus.

B-chromosomes in meiotic material of Lachena/ia
The meiotic chromosome numbers are also listed in Table 1. B-chromosomes were observed in the species L. aloides var. quadricolor (122) and in hybrids 7504, namely L. orchioides (61) X L. aloides var. (41); 7554, namely L. orehioides var. glaueina (143) X L. aloides var. quaddeolor (155) and 7556, namely L. mutabilis Sweet (161) X L. aloides var. quadrieolor (155) ( Table 1). The frequency and behaviour of B-chromosomes observed in the latter species and hybrids are given in Table 2. The total number of pollen mother cells (PMCs) having B-chromosomes ranged from 2.8% in the hybrid 7504, namely L. orehioides(61) XL. aloidesvar. (41) to 6.1 % in the species L. aloides var. quadricolor (122). The meiosis of the hybrids 7556, namely L. mutabilis (161) X L. aloides var. quadricolor (155) and 7529, namely L. splendida (30) X L. aloides cv. Pearsonii (22) are abnormal. Due to the staining and the size of the observed exstra chromosomes it was difficult to determine whether they resulted from the abnormality, or were in fact true B-chromosomes. The statistics of these stages were therefore omitted from the calculation of the total number of PMCs having B-chromosomes for the latter two hybrids; Their B-chromosome values in Table 2 are therefore only approximate, namely 4.1 % and 3.3% respectively.
PMCs having one B-chromosome were most frequently observed. A few PMCs with more than one B-chromosomes were, however, also observed.
The behaviour of B-chromosomes in PMCs having one B-chromosome B-chromosomes were observed as univalents in up to 7.8% of the PMCs at metaphase I (Table 2 and Figure la). The Bchromosomes of hybrids 7504 and 7554 underwent precocious chromatid separation during mataphase I in 1.0% and 0.6% of the PMCs respectively (Table 2 and Figure Ib). Further, up to 11.1 % of the PMCs at anaphase I had a Bchromosome with only one chromatid at each pole as the result of precocious chromatid separation. In up to 2.2% of the PMCs at anaphase I, the B-chromosome laggards were included with the normal chromosomes at a pole. No Bchromosomes laggards were found on the equatorial plate of anaphase I (Table 2). Therefore, the B-chromosome univalents either underwent precocious chromatid seperation or were included intact at one pole, but did not lag during the first meiotic division. In up to 3.4% of the PMCs at metaphase II, B-chromosomes with only one chromatid (resulting from precocious chromatid separation during the first division) were observed (Table 2 and Figure lc). These B-chromosomes were incapable of division at anaphase II and were either included with the normal chromosomes at a pole or remained stranded on the equatorial plate. According to Jones & Rees (1982), B-chromosomes univalents are successfully transmitted to the gametes if they are incorporated undivided within a telophase I nucleus. If not, Table 2 The frequency and behaviour of the 8-chromosomes observed in pollen mother cells (PMCs) of some

Lachena/ia species and F, hybrids. Number of cells (N) and percentage (P)
The B-chromosomes observed as a: they are eliminated. In the case of Lachenalia it appeared that the B-chromosome univalent more often divided during first meiotic division (Table 2). Therefore it could be expected that the latter behaviour of the B-chromosomes in Lachenalia will reduce the transmisssion of B-chromosomes to the next generation especially in plants with only one B-chromosome. However, Jones & Rees (1982) stated that B-chromosomes univalents are not always inevitably eliminated if they do divide at the first division. An exception is described in Festuca pratensis Huds. by Bosemark (1954). He found that in 15 to 30% of the PMCs the B-chromosomes divide at anaphase I. The B-chromosomes with only one chromatid lag at anaphase II, but only a few fail to be incoroporated into telophase II nuclei with the result that the number eliminated was small. It was not possible to make any similar conclusions for the genus Lachenalia from the present results . The exact transmission mechanism of univalent Bchromosomes will have to be determined.
The behaviour of 8-chromosomes in PMCs having more than one 8-chromosome In a few PMCs of the hybrids, more than one B-chromosome was observed and the B-chromosomes appeared as bivalents at metaphase I (Table 2 and Figure Id). In 0.3% of the PMCs at metaphase I of the hybrid 7554, namely L. orchiodes var.
glaucina (143) x L. aloides var. quadricolor (155), three Bchromosomes were observed ( Table 2). The latter associated to produce a bivalent and a univalent. These B-chromosomes are therefore homologous or homoeologous. The disjunction of the B-chromosome bivalents were presumably normal during the first and second meiotic division. In the hybrid 7504 0.5% of the PMCs at metaphase I had precocious 1a 1c 1e Figure 1 The B-chromosomes, marked with an arrow observed (la) as a metaphase I univalent in the hybrid 7556, namely L. mutabilis  Figure Ie). In up to 2.9% of the PMCs at anaphase I, one B-chromosome was observed at each pole as a result-of normal bivalent disjunction (Table 2). However, at metaphase II, only Bchromosomes with one chromatid, (resulting from precocious chromatid separation during the first division) were found. This observation is probably due to the relatively low number of cells studied at metaphase II. In up to 20% of the PMCs at anaphase II, the B-chromosomes divided normally and a Bchromosome was observed at each pole (Table 2). Therefore, it appears that in PMCs having more than one B-chromosome their behaviour during first and second meiotic division are normal and the B-chromosomes are distributed, like the chromosomes of the normal complement, one to each nucleus of the tetrad of microspores. In this way the B-chromosomes could be transmitted from one generation to the next. Jones & Rees (1982) are of the opinion that a higher average number of B-chromosomes in the offspring than in the parental generation indicates a B-chromosome accumulation mechanism. The accumulation mechanisms of Bchromosomes could be operative before, during or after ·meiosis. In this study no accumulation of B-chromosomes occurred prior to or during meiosis. PMCs having more than one B-chromosome were observed in hybrids only (Table 2). Therefore an accumulation mechanism, if present., should be postmeiotic.