© 2015 The Japan Mendel Society Cytologia 80(4): 415–418 Spontaneous Inversion Heterozygote in Haworthia limifolia Ramesh Ahirwar*, Pratibha Shrivastava and Rakesh Chandra Verma School of Studies in Botany, Vikram University, Ujjain (M.P.) 456010, India Received December 28, 2014; accepted June 25, 2015 Summary A population of cultivated Haworthia limifolia displayed various types of chromosomal configurations at anaphase/telophase-I and -II due to inversion heterozygosity. It was characterized by the presence of bridge and fragment because of various numbers and positions of crossovers in the inversion loop. The heterozygous condition led to reduced pollen fertility. Key words Haworthia limifolia, Inversion heterozygote, Chromosomal configuration, Pollen fertility. Haworthia is a genus of the tribe Aloineae in the family Liliaceae (Brandham 1974). There are about 60 species native to Southern Africa (Bayer and Manning 2012). Haworthia limifolia is one of the most important ornament plant species. It is a xerophytic, succulent, perennial herb with fleshy leaves arising in a rosette from a short stem. The chromosome number is (2n=28) and 16 chromosomes are long and 12 small. There have been very few studies on spontaneous mutants in Aloineae (Brandham 1975, 1977a, b). In the present investigation an inversion heterozygote was found from a cultivated population which was confirmed on the basis of the presence of bridge and fragment at anaphase-I/-II. Materials and methods Twenty clones of Haworthia limifolia were collected from Malwa Nursery, Ujjain and planted in pots. For meiotic studies, young flower buds of appropriate size were collected and fixed in Carnoy’s fluid. Anthers were separated, teased in a drop of 2% iron acetocarmine on a clean slide and squashed under a cover glass. PMCs were analyzed for suitable stages of meiosis. Slides with well spread cells and clear chromosomes were selected for scoring and photomicrographs are taken from temporary slides under oil immersion and magnified to ×1000. Results Meiosis in normal plants Haworthia limifolia has chromosome number n=14. Among these, there are four long and three short chromosome pairs. Meiosis in the normal plants is regular with 14 bivalents at diaki- nesis. Chromosomes are equally distributed (14 : 14) at each pole at anaphase/telophase-I and -II (Figs. 1, 2). Meiosis in inversion heterozygote The inversion heterozygote showed bridge/fragment configuration at anaphase/telophase-I/-II of meiosis, and PMCs analyzed in the plant were found to have various configurations of chro- * Corresponding author, e-mail: [email protected] DOI: 10.1508/cytologia.80.415 416 R. Ahirwar et al. Cytologia 80(4) Figs. 1–12. Meiosis in control and inversion heterozygote in Haworthia limifolia. 1–2. Meiosis in control. 1. Telophase-I. 2. Telophase-II. 3–12. Meiosis in inversion heterozygote. 3. Bridge at telophase I. 4. Fragment at telophase-I. 5. Bridge+fragment at telophase-I. 6. Two bridges+two fragments at anaphase-I. 7. Micronucleus at telophase-I. 8. Bridge at telo- phase-II. 9. Fragment at telophase-II. 10. Bridge+fragment at telophase-II. 11. Bridge+two fragments at telophase-II. 12. Micronucleus at telophase-II (Scale bar=4 µm). mosomes confirming the presence of inversion heterozygosity (Table 1). At anaphase/telophase-I, bridge, fragment, bridge+fragment and micronucleus were observed in 61.93% of PMCs (Table 1, Figs. 2–6). At anaphase/telophase-II, fragment, bridge, bridge+fragment, micronucleus, unipolar, and tripolar configurations were observed in 76.64% of PMCs (Table 1 and Figs. 7–12). Pollen fer- tility was studied in anthers taken from different buds. It was remarkably low in the abnormal plant (43%). 2015 Spontaneous Inversion Heterozygote in Haworthia limifolia 417 Table 1. Various types of chromosomal configurations at anaphase/telophase-I/-II in meiosis of inversion heterozygote in Haworthia limifolia. S. No. Abnormalities Number of cells (%) A. Anaphase/Telophase-I 1. Normal cells 134 38.0 2. Laggard 63 17.8 3. Bridge 51 14.4 4. Bridge+Fragment 56 15.9 5. 2Bridges+2Fragments 12 3.4 6. Micronucleus 36 10.2 Total number of abnormal cells 218 61.9 B. Anaphase/Telophase-II 7. Normal cells 117 24.78 8. Laggard 108 22.88 9. Bridge 124 26.27 10. Bridge+Fragment 50 10.59 11. Bridge+2Fragments 21 4.44 12. Micronucleus 52 11.01 Total number of abnormal cells 355 75.21 C. Pollen grains 13. Fertile 166 43.00 14. Sterile 220 56.99 Total 386 Discussion For the development of a new species, the first basic requirement is availability of genetic variability at the gene/chromosome level, which may either be spontaneous or induced. Sev- eral workers have found spontaneous inversion heterozygotes in various plant species from natural populations, such as in Aloe barbenensis Mill (Ahirwar and Verma 2013, Kaul 1965, Vig 1968), in Haworthia reinwardtii (Brandham 1974), Haworthia glauca (Riley and Majumdar 2011) and in Paeonia decomposita (Wang et al. 2008). The origin of dicentric bridges through heterozygosity was cytologically investigated by McClintock (1931) in Zea mays showing all the possible cytological manifestations of inversion bridges at anaphase-I and anaphase-II. According to Rothfels and Mason (1975) bridges may originate from chiasma formation in heterozygous inversions. When cell division occurs, a broken chromosome with two centromeres is pulled to the opposite poles of the cell, forming a long chro- mosome bridge called chromatid bridge (Zhang et al. 1997). Bridges and fragments are the results of spontaneous breakage and fusion of the chromosomes. The inversion crossing over hypothesis is a highly improbable phenomenon. In the present investigation an inversion heterozygote was found in the cultivated population of Haworthia limifolia, and that was confirmed on the basis of chromosomal configurations at meiosis division. The pachytene stage is not suitable for cytological analysis because of the dense appearance of this stage (Ekberg 1974). Various types of configura- tions were found in Haworthia limifolia. Their modes of origin are discussed below according to Ekberg (1974). The formation of these bridge/loop fragments greatly depends upon the position of crossover in the pachytene loop. A single crossover inside the inversion loop results in a bridge and a frag- ment at anaphase-I, which gives a single fragment without bridge at anaphase-II. A four-strand crossover inside the inversion loop yields two bridges and two fragments at anaphase-I and simply two fragments at anaphase-II. A single crossover inside the inversion loop and a single crossover outside the inversion loop give a loop and fragment at anaphase-I and a bridge and a fragment at 418 R. Ahirwar et al. Cytologia 80(4) anaphase-II. Two crossovers inside the inversion loop and a single crossover outside the inver- sion loop gives two loops and two fragments at anaphase-I and two bridges and two fragments at anaphase-II. It is important to note that the most common configuration of the plant heterozygous for para- centric inversion is bridge and fragment at anaphase-I, the result of a crossover inside the loop. The acentric fragment will be formed either a micronucleus or an extra microspore in tetrad stage (Sjodin 1971). The acentric fragment will form a micronucleus. As expected, in the present case too, one or two micronuclei were observed. Further, micronuclei will form abnormal tetrads as observed in the present case. Regarding the ultimate effects on pollen fertility, the pollens were highly sterile. The most obvious genetic effect of inversions is formation of unbalanced gametes that often causes microspore sterility (Wang et al. 2008). As is expected, the loss of chromosomal fragment as a result of paracentric inversion leads to deficient gamete and high sterility (Ahirwar and Verma 2013). References Ahirwar, R. and Verma, R. C. 2013. Spontaneous Inversion Heterozygote in Aloe barbadensis Mill. Cytologia 78: 1–4. Bayer, M. B. and Manning, J. C. 2012. The Haworthia Nomenclature: A List of Accepted Species with Some Guidelines for Intra-Specific Names. South Africa National Biodiversity Institute, Claremont. Brandham, P. E. 1974. Interchange and inversion polymorphism among populations of Haworthia reinwardtii var. chalumnensis. Chromosoma 47: 85–108. Brandham, P. E. 1975. Stabilised breakage of a duplicated chromosome segment in Aloe. Chromosoma 51: 269–278. Brandham, P. E. 1977a. 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