POLYPLOIDY IN THE VICIA SATIVA AGGREGATE

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1 New Phytol. (1982) 91, POLYPLOIDY IN THE VICIA SATIVA AGGREGATE BY G. LADIZINSKY AND Y. SHEFER The Hebrew University, Faculty of Agriculture, Rehovot, Israel {Accepted 31 December 1981) SUMMARY Polyploidy is rare in the genus Vicia and is absent in the Vicia sativa aggregate which is composedof types with 2w = 10, 2n = 12and2w = 14. Spontaneous amphidiploids were selected among 2n = 10 x 2n = 14 and 2M = 12 x 2n = 14 hybrid derivatives. Chmosome association at meiosis was mainly as bivalents in these amphidiploids; occasionally they were hetemorphic. The spontaneous amphidiploids were inferior to their diploid parental lines in gwth rate, vigour and seed set over three generations. The behaviour of the spontaneous amphidiploids was compared with artificially induced autotetraploids. Meiosis in the autotetraploids was characterized by a lower number of associations per chmosome and small number of multivalents. The gwth rate of the autotetraploids was retarded and seed set was lower in comparison with the diploid lines. The diploid and the autotetraploid lines could be easily separated fm one another by the shape of their pollen grains; elliptical in the diploid plants and tetrahedral in the autotetraploids. No such modification of the pollen grains was observed in amphidiploids. The potential for polyploidy in Vicia sativa and possible reasons for its absence in that aggregate are discussed. INTRODUCTION The rapid evolution of the Vicia sativa aggregate can be inferred fm the distribution range of the aggregate, its adaptive radiation and chmosomal variation. Three chmosome numbers, 2w = 10, 2w = 12 and 2«= 14 have been recorded. Furthermore, in each diploid type karyotypic variation was observed (Mettin and Hanelt, 1964, 1973; Hollings and Stace, 1974; Ladizinsky, 1968). In wild populations of V. sativa in Israel the 2«= 10 types occupy mainly secondary and man-made habitats, the 2«= 12 are confined mainly to natural vegetation among dwarf shrubs, or in the maquis and the 2«= 14 is found mainly in dry habitats in steppes bordering the mediterranean vegetation zone and in xeric niches within that zone. The various 2n types and karyotypes are css compatible, the F^ hybrids are vegetatively normal but only partially fertile, due to meiotic irregularities (Ladizinsky and Temkin, 1968). However, chmosomally stable derivatives have been selected fm the F^ (Ladizinsky, 1982). Polyploidy as a natural phenomenon is absent in the V. sativa aggregate and is ''are in the entire genus. This paper describes some features of spontaneous ^ttiphidiploids, resulting fm interkaryotypic hybridization, and a comparison With artificially induced autotetraploids. MATERIALS AND METHODS ^he spontaneous amphidiploids 2n = 24 and 2n = 26 were derived fm ^" = 10 X 2M = 14 and 2n = 12x2n = 14 artificial hybrids respectively. Autotetra- Ploids were obtained by treating young seedling shoots with aqueous 0*02% '^ X/82/O7O S03.00/ The New Phytologist

542 G. LADIZINSKY AND Y. SHEFER colchicine solution for 8 h. Flowering autotetraploid sectors were identified by the lower pportion of stainable pollen grains and the shape of these grains.

2 542 G. LADIZINSKY AND Y. SHEFER colchicine solution for 8 h. Flowering autotetraploid sectors were identified by the lower pportion of stainable pollen grains and the shape of these grains. Chmosome association at meiosis and fertility were stained according to Ladizinsky and Temkin (1968). In order to compare chmosome association in plants with different chmosome numbers, the results were expressed as number of associations per chmosome calculated as follows: no. of chisamata per cell chmosome number RESULTS Spontaneous amphidiploids were found by counting chmosome numbers in F2 populations of hybrids between V. sativa plants having 2w = 10, 2n = 12 and 2«= 14 chmosome number. The development, cytology and fertility of these polyploids are as follows. Amphidiploid 2n = 24 This plant was originated fm 2n=10x2n= 14 css. The Fj hybrid developed normally but was semisterile due to meiotic irregularities (Table 1). Two hybrid combinations of this css were studied. They differed fm each other in the karyotypes of the 2?2 = 10 parent which were designated A and B by Ladizinsky (1968). Chmosome counts were made in 58 F2 plants of the 2n = loa x2n 14 hybrid; among them two were partial polyploids, 2«= 19 and 2«= 22. In 16 Fg seedlings of the 2n = lob x2n = 14 hybrid, two were complete polyploids with 2n = 24. Since both seedlings came fm the same pod it is reasonable to assume chmosome doubling took place at mitosis in the F^ hybrid. The polyploid plants were gwn in the greenhouse. They developed normally and were similar to the F^ hybrid. No difference in leaf shape and size was noted between the polyploid plants and the 2«= lob parent. Stomata and pollen grains of the polyploids were only slightly longer than those of the diploids (Table 2). Chmosome pairing in the 2n = 24 amphidiploids was considerably impved by comparison with the F^ hybrid. Most of the chmosomes formed homomorphic bivalents but, occasionally, hetemorphic bivalents were observed (Table 1). About two thirds of the bivalents were d shaped and the number of associations per chmosome was much lower as compared with the diploid parental lines. Thirty seeds collected fm the 2n = 24 plants were planted in the greenhouse and transferred later to the field together with the diploid lines. Polyploid plants in the field developed more slowly in comparison to their diploid lines and, at maturity, they were inferior in total green matter and pduced less seeds. Chmosome pairing at MI examined in nine plants was similar to that observed in the first generation. The slow gwth and reduced seed set was even more extreme in the third generation. Amphidiploid 2n = 26 The origin of this polyploid was fm 2w = 12 x 2n = 14 hybrid. This hybrid developed nornmally but was semisterile due to irregular chmosome pairing ^^ meiosis (Table 1). Among the 23 Fg seedlings originating fm this hybrid one was polyploid, 2w = 26. This polyploid plant developed normally and morphologic^"^ was similar to the Fj hybrid. The stomatal size of the polyploid was within tne

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4 544 G. LADIZINSKY AND Y. SHEFER range of the 2w = 12 parental line but the pollen grains were slightly bigger (Table 2). Chmosome association at MI was considerably impved compared with the Fj hybrid; but the number of associations per chmosome was much lower than that of the diploid parental lines. Also, the fertility was much impved and relatively many seeds were collected fm the 2n = 26 polyploid. A second generation of this amphidiploid was gwn in the field. Chmosome counts made on seedlings were all 2n = 26. The development of the second generation plants were slower compared with the 2w = 12 and 2n 14 parental Table 2. Stomata and pollen grain size mm) in 2x and 4x Vicia sativa Stomata Pollen grains Line no. 2n No. cells Mean size No. cells Mean size (4x) 232(4x) Amphidiploid (136x161) Amphidiploid (169x 161) xl x x xl-6 26 x x x x2-l X X X X x X X X 3-7 lines. Although chmosome pairing was similar to that observed in the first generation, seed set was reduced. In a third generation plant development was more retarded and seed set even lower. Induced autotetraploids Induction of polyploidy by colchicine treatment was attempted in the three parental lines involved in the formation of the spontaneous amphidiploids. In the 2«= 14 and 2n 10 lines polyploid plants were obtained but only in the latter did they reach maturity and pduce seeds. The development of the autotetraploid plants was retarded compared with the diploid plants, and they flowered later. The leaves and the leaflets of the autotetraploids were slightly bigger than those of the diploid plants but this could not be taken as a unique criterion for separating 2x and 4x plants. A significant difference between the diploid and the tetraploid plants was the shape of the pollen grains. It was elliptical oblong in the 2x plants and rhomboid tetrahedral in the 4x plants (Fig. 1). Similar differences in pollen shape were observed between 2n = 10 V. sativa (No. 232) and 4x plants derived fm them in material pvided by Pfessor Yamamoto. DISCUSSION Induction of polyploidy in plants occasionally includes enlargement of vegetative organs and increased size of stomata and pollen grains. Changes in these traits can only be used as one factor for separating 2x fm 4x plants in V. sativa. Modification of pollen grain shape, on the other hand, is a clear cut indication oi autopolyploidy in V. sativa. Although we have noticed this in only two 2w = ^^ lines, similar behaviour has been observed by Pfessor Yamamoto (pers. com-/

$Polyploidy in the Vicia sativa aggregate 545 Fig. 1. Pollen grains of Vicia sativa: (a) diploid 2n = 10, (b) autotetraploid 2n = 20. following doubling of chmosome number in In \1 and In = \A lines.$

5 Polyploidy in the Vicia sativa aggregate 545 Fig. 1. Pollen grains of Vicia sativa: (a) diploid 2n = 10, (b) autotetraploid 2n = 20. following doubling of chmosome number in In \1 and In = \A lines. Such a change in pollen shape following autopolyploidy is apparently very rare. In fact, we came acss only one report of variation in pollen shape following autopolyploidy (Joshi and Raghuvanshi, 1966) but in that case the aberrant shape was related to spindle irregularities in meiosis. In V. sativa, on the other hand, all the pollen grains of the 4x plants were uniform and differed markedly fm those of the 2x plants. Since the pollen shape of the spontaneous amphidiploids was similar to that of the diploid lines, it can be concluded that the modified pollen shape in the autotetraploids was not due to the effect of higher chmosome number but apparently to a quadruple combination of a specific gene or genes in the 2n = 10 line. Similar genes apparently exist also in the 2n = 12 and 2n = 14 V. sativa. Further, it can be assumed that these factors in the various diploid lines are not compensating for each other in a simple manner and, therefore, pollen shape is not affected in the amphidiploids. Another typical feature of autotetraploids was reduced chmosome association m Ml, resulting mainly in d bivalents and univalents, which were unrelated to trivalent configuration. Apparently, the low rate of chiasmata per chmosome was the main reason for the low multivalent association and the generally diploid like ^I of the autotetraploid. Similar behaviour was observed in diakinesis in several pollen mother cells. Earlier stages of meiosis are unsuitable for cytological examination and it cannot be concluded whether the low rate of chiasmata in MI IS due to early terminalization at diplotene, or reduction of cssing over. Chmosome association in the MI of the spontaneous amphidiploids was considerably impved compared with the F, interkaryotypic hybrids but was ^uch lower than that of the diploid parental lines. These values however, were ^'^ry close to those observed in the autopolyploids. It seems that polyploidy per ^e aifects chmosome association in V. sativa; similar behaviour was reported '"ix)lyploid Tulipa (Upcott, 1939).

6 G. LADIZINSKY AND Y. SHEFER Polyploidy has played a major le in the evolution of many gups of plants but affected others little. The establishment of polyploidy in annuals which are predominantly selfers, stems fm interspecific sterile diploid hybrids in which chmosome number was spontaneously doubled. Doubling of chmosome number alone is inadequate for ensuring complete restoration of fertility and it must be accompanied by various means affecting homologous chmosome pairing and regular separation to the poles as essential requirements for viable gamete pduction. Finally, the success of the new polyploid is conditioned by the availability of a habitat where it is selectively advantageous. Polyploidy has been almost insignificant in the evolution of the genus Vicia, and the tribe Vicieae as a whole. Among 87 species of Vicia polyploidy was recorded only in 10 (Kupicha, 1977). These are perennial species whose polyploid origin is not yet known. Rousi (1966) suggested that lack of amphidiploidy in Vicia was due to inviability of the interspecific F^ hybrids. As a natural phenomenon, polyploidy is absent in the V. sativa aggregate but this is not due to hybrid inviability. On the other hand, some features of V. sativa aggregate apparently can permit the formation of polyploids. The occurrence of three chmosome numbers and several karyotypes are clear indication of chmosomal divergence. Further, the association of types with specific chmosome numbers to particular ecological envinments (Ladizinsky, 1968) indicates the evolutionary potential if this variation was assembled in one plant thugh polyploid. Stands of plants having different chmosome numbers are common in natural populations and these plants are css compatible; the F^ hybrids are vegetatively normal and spontaneous doubling of chmosome number in these hybrids has been found in this experimental study and elsewhere (Yamamoto, 1966). The amphidiploids are vegetatively normal and chmosome pairing is impved compared with the diploid hybrids. However, the predominantly bivalent pairing in the amphidiploids is at least in part a consequence either of reduction in chiasmata formation, or early terminalization. Homeologous pairing can be inferred fm the occurrence of hetemorphic bivalents and is apparently the main reason for the segregation observed in vigour and seed set among their pgeny. Thus lack of natural polyploidy in V. sativa might be due to the absence of genes pmoting homologous bivalent pairing at the polyploidy level. Another hypothesis is that even when such a meiotically stable amphidiploid is obtained it is no more successful in the habitats presently occupied by the various karyotypes of the V. sativa aggregate and is inferior to them in other habitats. It could be that, instead of evolving polyploids which are adapted to a range of habitats, the evolutionary success of the V. sativa aggregate has been achieved though the formation of several chmosomally divergent types each adapted to somewhat different habitat. The maintenance of the evolutionary dynamics of this system is ensured by occasional interkaryotypic hybridization which pduce new recombinants that can be tested in new habitats. ACKNOWLEDGEMENT This study was supported by the U.S.-Israel Binationial Science Foundation. REFERENCES HoLLiNGS, E. & STACE, C. A. (1974). Katype variation and evolution in the Vicia sativa aggregate. Phytologist, 73,

7 Polyploidy in the Vicia sativa aggregate 547 JosHi, S. & RAGHUVANSHI, S. S. (1966). Polyploidy and pollen variability in Pempinella monoecia. Biologia Plantarum, 8, KuPiCHA, F. K. (1977). The delimitation of the tribe Vicieae (Leguminosae) and the relationships of Cicer L. Journal of the Linnean Society (Botany), 74, 131^162. LADIZINSKY, G. (1968). Chmosomal polymorphism in wild populations of Vicia sativa. L. Caryologia, 31, LADIZINSKY, G. (1981). Consequences of hybridization in Vicia sativa aggregate. Heredity, 47, LADIZINSKY, G. & TEMKIN, R. (1968). The cytogenetic structure of Vicia sativa aggregate. Theoretical and Applied Genetics, 53, METTIN, D. & HANELT, P. (1964). Cytosystematische Untersuchungen in der Artengruppe um Vicia sativa L. Kulturpflanze, 7, METTIN, D. & HANNELT, P. (1973). Uber Speziationsvorgange in der Gattung Vicia. Kultupflanze, 21, Rousi, A. (1961). Cutotoxonomic studies on Vicia cracca L. and V. tenuifolia Roth. I. Chmosome number and karyotype evolution. Hereditas, 47, UPCOTT, M. (1939). The genetical structure of Tulipa III. Meiosis in polyploids. Journal of Genetics, 37, YAMAMOTO, K. (1966). Studies on the hybrids among the Vicia sativa L. and its related species. Memoirs of the Faculty of Agriculture, Kagawa, University, 21,

Artificial Triploids in Luffa echinato Roxb. P. K. Agarwal,1 R. P. Roy and D. P. Mishra Department of Botany, University of Patna, Patna-5, India

Cytologia 44: 739-743, 1979 Received April 10, 1975 Artificial Triploids in Luffa echinato Roxb. P. K. Agarwal,1 R. P. Roy and D. P. Mishra Department of Botany, University of Patna, Patna-5, India Luffa