Meiosis and Pollen Mitosis in Aloe barbadensis Mill. (A. perfoliata var. vera L., A. vera Auth. non Mill.)

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1 Cytologia 40: , 1975 Meiosis and Pollen Mitosis in Aloe barbadensis Mill. (A. perfoliata var. vera L., A. vera Auth. non Mill.) A. B. Sapre Department of Botany, Marathwada University, Aurangabad, , India Received January 22, 1974 Aloe, a genus with about 180 species of xerophytic plants, is indigenous to Africa. Several species of this genus have been introduced into India and they are found to flourish in a variety of climates and soils. Aloe barbadensis Mill., a native of North Africa, is being grown as a horticultural and medicinal plant throughout India. There is some variation in regard to the blooming period from place to place. At this place, the flowering begins sometime in October and continues up to December; in warmer parts it is from March to May. The fruit formation is extremely rare, almost negligible, and, as far as the author is aware, plants are propagated through suckers only. Fruit, whenever formed, is an oblong, trigonous, loculicidally 3-valved capsule, with chaffy flat seeds. The latter do not germinate and are probably without an embryo. Studies on meiosis and pollen-mitosis are presented here from the point of view of failure of fruit formation. Material and methods Flower buds, collected from the Botanical Garden of this Department, were fixed in acetic-alcohol (1:3) for 24 hours and then stored in 70% alcohol at low temperature (5 Ž) until further use. Anthers were squashed in aceto-carmine and the photomicrographs were taken from these temporary preparations. Subsequent ly these slides were made permanent using acetic acid-butyl alcohol grades. Pollen viability tests were carried out using aceto-carmine (Kihara 1958), and well-stained pollens showing sickle-shaped generative cell were taken to be functional, while unstained empty pollens with irregular outline to be sterile. An average of ten microscopic fields (10x ~10x) for five flowers from five different plants was con sidered for calculating percentage of pollen sterility. Results Meiosis Early prophasic studies could not be undertaken because of the extremely sticky nature of the chromosomes. Even in the later stages of meiosis, well-spread chromosomal configurations were rare and the squash preparations resulted in lot of clumping and overlapping, sometimes even making counting difficult. This difficulty was partically overcome through the preparation of a large number of slides for each of the stages, as also by scanning a large number of pollen mother

2 526 A. B. Sapre Cytologia 40 cells (PMCs). Regular meiosis is presented through figures 1 to 14, and as such needs very little description. Figs to 14. Normal meiosis. ~ , leptotene. 2, diplotene, showing chiasmata in seven bivalents. 3, diakinesis, showing seven bivalents with one attached to the nucleolus. 4, metaphase I, polar view. 5-7, metaphase I, equatorial view.

3 1975 Meiosis and Pollen Mitosis in Aloe barbadensis Mill. 527 Figs ~ , telophase I. 11, metaphase II. 12, early anaphase II, note seperation of chromatids. 13 and 14, early and late telophase II.

4 528 A. B. Sapre Cytologia 40 At diplotene, chiasma frequency was calculated from ten PMCs showing well spread chromosomes. Maximum number of chiasmata per PMC was 14 and the chiasma frequency averaged to 1.7. At diakinesis, seven clear bivalents were ob served, with one attached to the nucleolus (Fig. 3). At metaphase I, short chromo somes showed early separation into univalents as compared to long ones. With only a short interphase between meiosis I and II, chromosomes entered metaphase Figs ~ to 23. Meiotic abnormalities. 15, metaphase I, precocious movement of chromosomal arms. 16, 17, 19, chromosomal bridges in metaphase I. 18, telophase I, note laggard. II (Fig. 11), and the separation of sister chromatids with the division of the centro mere (Fig. 12) resulted in four groups of haploid set of chromosomes (Fig. 13 and 14). The division in the PMCs was simultaneous and the cytokinesis, that followed, resulted in either isobilateral or tetrahedral tetrads, the latter pattern of microspores being more frequent. Meiotic abnormalities such as, precocious anaphasic movements of chromo

5 1975 Meiosis and Pollen Mitosis in Aloe barbadensis Mill. 529 Figs , metaphase II, precocious movement of chromosomal arms. ~ , bridges and laggard in Fig. 22. ~ to 25. Pollen mitosis. 24, metaphase, polar view showing a haploid complement. ~ , metaphase idiogram from actual photograph. ~ 1700.

6 530 A. B. Sapre Cytologia 40 Table 1. Karyomorphological studies in Aloe barbadensis Mill. Total chromatin length=10120ƒê T. F.=26.21% (Huziwara 1962) Karyotype formula=2n=14=8l (St.)+4S(Sm.)+2S(St.). somes, formation of bridges and laggards, were very common in both meiosis I and II (Figs ), resulting in a fairly high percentage of pollen sterility. Surprisingly enough, at tetrad stage microspores appeared quite normal. However, during fur ther development towards pollen formation, microspores with unbalanced chromo somal complements did not develop into viable gametophytes but gave rise to collapsed pollen grains. The irregular shape, no affinity for the stain and the empty nature of pollen grains showed clearly that 58.3% pollen grains were non viable. The process of meiosis was asynchronous and anthers from the same flower showed different stages in meiosis. Pollen mitosis The functional microspores enlarged and showed mitotic division of the haploid nucleus into vegetative and generative cells. Clear metaphasic plates during this division (Figs. 24 and 25) showed a genome of seven chromosomes having two distinct sizes, four long and three short and their karyomorphological details are presented in Table 1 and Fig. 26. None of the chromosomes has secondary con striction or a satellite. The size of the chromosomes ranges from 21.44ƒÊ to 7.83ƒÊ. The 'r' value (ratio of long arm/short arm) for all the seven chromosomes lies be tween 1.61 to Karyotype is asymmetrical and bimodal. Discussion Aloe is a native of Africa and is introduced into India. It has been observed in a few species that are growing in our Botanical Garden, as also in the same species growing round about this area, that setting of fruits is extremely rare. Aloe barbadensis shows even difference in its blooming period from place to place, the two main seasons being October-December and March-May. There could be some ecological difference responsible for this varied nature of blooming. African leaf succulents belonging to the tribe Aloinae have mostly red or yellow flowers, adapted to bird pollination (Stebbins 1971). If the pollinator is missing it would naturally result in the failure of fruit-set. Even if the absence of pollinator is taken

7 1975 Meiosis and Pollen Mitosis in Aloe barbadensis Mill. 531 for granted, it has been observed that whole mounts of stigma from open flowers do show a variable amount of pollen loaded on it. Whether the sufficient amount of pollen, and the viable pollen (population effect), is a bottle-neck in fruit formation remains to be investigated. This plant could be designated as a facultative apomict (Swanson 1957), as its only means of reproduction at this place is asexual or vegetative, while under its home conditions it fruits normally and at the same time has the capacity to propa gate vegetatively (Thiselton-Dyer 1898). The vegetative propagation through suckers to maintain itself is possibly a mechanism to exploit ecological conditions, and this is done by suppressing sexual reproduction through failure of meiosis and fertilization together, avoiding segregation and recombination (Darlington 1963). Irregularities during meiosis described and presented through figures give a substantial clue to the high percentage of pollen sterility (58.3%). Pre cocious movements of the chromosomal arms to the poles (Fig. 15 and 20) is pre sumed to be because of neo-centric activity or T-end activity as has been reported in maize (Rhoades 1942, 1952, Rhoades and Vilkomerson 1942) and rye (Praaken and Mi ntzing 1942, Ostergren and Praaken 1946, Rees 1955). Additional chromo somal fibres are developed at certain regions, because the centromere under certain conditions, relinquishes its role to secondary centers of movement, so that neo centric regions move to the poles ahead of the true centromeres. "Neo-centric activity is a response under particular cell conditions of particular regions in parti cular genotypic backgrounds", Lewis and John (1963). In maize, the phenomenon is governed by the presence of an abnormal chromosome 10, which has an extra piece of chromatin attached to the distal end 0of the long arm (Rhoades 1942, Rhoades and Vilkomerson 1942). Frequent occurrence of chromosome bri dges and laggards (Figs , 21-23) is responsible for unbalanced distribution of chromosomes during meiosis I and II, the net result being a high pollen sterility of 58.3per cent. Karyotype is asymmetrical and bimodal as has been described for the entire tribe, Aloinae (Darlington 1963, Stebbins 1971). The origin of bimodal karyotype of the Aloinae is explained (Stebbins 1971) by increasing asymmetry and heterogeneity plus the addition of a chromosome to the complement through fixation of a centric fragment. Marshak (1934) puts a genome of A. vera Fig. 26. Metaphase idiogram, average as an exception to other Aloe species in of seven metaphases. having three long, one intermediate and three short chromosomes and reasons this to be the cause for its wider geographical distribution. However, in my studies

8 532 A. B. Sapre Cytologia 40 scanning of a large number of clear metaphase plates in the first division of micro spore nucleus showed bimodal nature of the karyotype and an average of seven well-spread metaphasic complements revealed four long and three short chromo somes (Fig. 26). Secondary constrictions or satellites have not been recorded in this species, supporting Marshak (1934), though their presence has been noted in other species of Aloe; A. arborescens (Taylor 1925, Heitz 1931), A. saponaria (Taylor 1925) and A. spuria (Sato 1937, 1942). The writer finds that pollen mitotic studies reveal chromosome morphology so well (Fig. 25) as to eliminate the root-tip squashes. It may be concluded that failure of fruit formation is because of the failure of meiosis resulting in a high pollen sterility and also because of self-incompatibility as suggested by Berger (1908) for Aloe species. Germination of apparently normal pollen grains on the stigmatic papillae as a result of selfing was not recorded leading to failure of fertilization. Preliminary attempts to germinate pollen in vitro using different concentrations of sucrose supplemented with boron and calcium have failed. Fruit, whenever formed, may then be parthenocarpic, since the seeds in such fruits are chaffy and do not germinate. Detailed studies involving experimental embryology and breeding are under way to investigate into the causes of failure of fruit formation in Aloe species. Summary Studies on normal meiosis and meiotic abnormalities in Aloe barbadensis have been carried out in detail explaining a high percentage of sterility in the pollen grains. A detailed karyomorphology from pollen mitosis is also presented. The failure of fruit-set is presumed to be because of high pollen sterility (58.3 %) and self-incompatibility, Acknowledgements I am grateful to Prof. K. B. Deshpande, Head, Department of Botany, for facilities and encouragement. I also thank Drs. R. M. Pai and V. N. Naik for stimulating discussions during the present study. References Berger, A Engler's Pflanzenreich IV: 38, III, II, Liliaceae-Asphodeloideae-AIoinae. Darlington, C. D Chromosome Botany and the Origins of Cultivated Plants. George * Heitz Allen and Unvin Ltd. London., E Die Ursache der gesetzmabigen Zahl, Lage, Form, and Gr&Be pflanzlicher Nucleolen. Planta 12: Huziwara, Y Kartyotype analysis in some genera of Compositae VIII. Further studies on the chromosomes of aster. Amer. J. Bet. 49: Kihara, H Fertility and morphological variation in the substitution and restoration back crosses of the hybrids Triticum vulgare ~Aegilops caudataa. X Intern. Genet. Congr., Proc. 1: Lewis, K. R. and John, B Chromosome Marker. J and A Churchill, Ltd. London.

9 1975 Meiosis and Pollen Mitosis in Aloe barbadensis Mill. 533 Marshak, A Chromosomes and compatibility in the Aloinae. Amer. J. Bot. 21: Ostergren, G. and Praaken, R Behaviour on the spindle of the actively mobile chromosome ends of rye. Hereditas 32: Praaken, R. and Muntzing, A A meiotic peculiarity in rye simulating a terminal centromere. Hereditas 28: Rees, H Genotypic control of chromosome behaviour in rye I. Inbred lines. Heredity 9: 93. Rhoades, M. M Preferential segregation in maize. Genetics 27: Preferential segregation in maize. In Heterosis. Iowa State College Press, Ames: and Vilkomerson, H On the anaphase movement of chromosomes. Proc. Nat. Acad. Sci. 28: * Sato, D Karyotype alternation and phylogeny I. Analysis of karyotype in Aloinae with special reference to the SAT-chromosome. Cytologia, Fujii Jubil. Vol.: Karyotype alteration and phylogeny in Liliaceae and allied families. Jap. J. Bot. 12: Stebbins, G. L Chromosomal Evolution in Higher Plants. Edward Arnold Ltd. London. Swanson, C. P Cytology and Cytogenetics. Macmillan and Co., Ltd. Bombay. Taylor, W. R Cytological studies on Gasteria II. A ocmparison of the chromosomes of Gasteria, Aloe and Haworthia. Amer. J. Bot. 12: Thiselton-Dyer, W. T. (Ed.) Flora of Tropical Africa. L. Reeve and Co., Ltd. Kent. * Quoted by Stebbins, G. L Variation and Evolution in Plants. Columbia Univ. Press, N. Y.