_??_1991 by Cytologia, Tokyo Cytologia 56: 31-41, 1991 Karyomorphology of Amaryllis Hybrids Tasneem F. Khaleel, Susan Haven and Tim Gilg Department of Biological Sciences, Eastern Montana College Billings, Montana 59101, U. S. A. Accepted July 20, 1990 The genus Amaryllis (Amaryllidaceae) has attracted horticulturists and taxonomists alike because of the extensive hybridization of its species and the nomenclatural disputes (Traub 1983, Goldblatt 1984). In addition to the amazing diversity of hybrids , a large number of cultivated wild species are in circulation. The present day Amaryllis is the result of selections that have continued for over 200 years using whatever species were available. The quest for size and colour of flower led to indiscriminate use of hybrids and wild species in breeding, con sequently some species hybrids display low fertility and often will not set seed. According to Bell (1973) the genus Amaryllis illustrates a contemporary horticultural problem. Since hybrids are easier to grow than the species, the latter are frequently lost. Considering the importance of elemental species, an attempt should be made to conserve the valuable germ plasm for future hybridization. If parental species in the breeding programs are known, new varieties could be developed by systematic and organized hybridization programs. Where parental species are not well documented, pedigrees might possibly be traced or determined through cytological analysis. Cytological and embryological studies on the genus Amaryllis, are of immense value in assessing and evaluating the breeding potential of wild species and hybrids. They provide valuable information about the constitution of germ plasm, genetic compatibility of taxa involved in hybridization, reasons for low fertility and lack of or low seed set. A review of literature indicates that 25 of the 76 species have been studied (Flory and Coulthard 1981); although hundreds of hybrids are in circulation, only few have been studied (Guha 1979, Khaleel and Siemsen 1989). The basic chromosome number in the genus is x=11. While most species are diploid, the widely available hybrids are nearly uniformaly tetraploid. Other polyploids with 2n=33, 66, and 77 are also on record (Guha 1979). The present study involv ing nine hybrids Apple Blossom, Bold Leader, Cocktail, Desert Dawn, Dutch Belle, Intokazi, Miracle, Tangerine and Zanzibar is an attempt to furnish more comprehensive and exact information on the cytology and embryology of these hybrids. Since all of them show pollen sterility, degeneration of female gametophytes and low percentage of seed set, the data may be useful in evaluating the breeding potential of these hybrids and tracing their pedigrees. All hybrids more or less conform to the Division 4 (Reginae-type hybrids distinguished by open faced flowers with short tepal tubes, markedly imbricate or less markedly imbricate segments involving A. reginae, A. coreiensis and similar species), or Division 5 (Leopaldii type hybrids showing wide open flowers held horizontally indicative of A. leopaldii and A. paridina) of Traub's (1958) classification of cultivated Amaryllis (Table 1). Materials and methods The material used in the present study includes 192 bulbs of nine horticultural varieties of Amaryllis grown in the greenhouse of Eastern Montana College, Billings, Montana for the past five-seven years. Bulbs of Apple Blossom, Desert Dawn, Dutch Belle, Intokazi and Zanzibar were obtained from the Brown Bulb Ranch, Washington, and Bold Leader, Cocktail, 32 Tasneem F. Khaleel, Susan Haven and Tim Gilg Cytologia 56 Miracle and Tangerine from Park Seed Company, N. Carolina. Excised root tips from each bulb were pretreated with 0.25% colchicine for three and a half to four hours at room tem perature and fixed in Carnoy's fluid (3 ethanol: 1 glacial acetic acid V: V) for 24 hours, hy drolysed in IN HCl at 60•Ž for 8-9 minutes, stained with Schiff's reagent and squashed in 2% acetocarmine. Root tips of all varieties were treated similarly for karyological studies. Slides were made permanent by removing the coverslip by the dry ice method and processing through an n-butanol series, and mounting in euparal. Young anthers from each hybrid were fixed in modified Carnoy's fluid (6 ethanol: 1 glacial acetic acid: 3 chloroform, V:V:V) for 24 hours. They were squashed in 2% acetocarmine for meiotic studies. Permanent slides were made following the same steps as for the root tips. Camera lucida drawings of chromosomes were made from five mitotic metaphase plates of each variety. Chromosomes were karyotyped according to the increasing length of the short arm. Arm ratio was obtained by dividing the long arm by short arm and centromeric index was calculated by dividing short arm by the total length and x 100. The arm ratio and the centromeric index were both considered in determining the position of the centromere. Ave rage chromosome length was calculated by dividing the total chromatin length by the number of chromosomes in the karyotype. Photomicrographs were taken with Zeiss microscope. One thousand pollen grains from each hybrid were stained in 1% acetocarmine for 8-12 hours and scanned to determine fertility. Those appearing dense and darkly stained were considered fertile. Customary methods of dehydration, infiltration, microtoming and staining were followed for embryological studes of each hybrid. Since no significant differences were found among the hybrids in the present study or the ones studied previously (Khaleel and Siemsen 1989), results have not been described under the observations. However, pertinent reference has been made in the text. Voucher specimens of each variety have been placed in the Eastern Montana College herbarium. Observations Morphology: Table 1 indicates some of the morphological features of the nine hybrids. The measurements given are means obtained from 8-10 mature flowering bulbs of approximate ly the same age for each variety. Two distinct behavior patterns were observed over a period of six years. Bold Leader, Desert Dawn, Dutch Belle, Intokazi, and Miracle flowered every year regardless of fruit set while Apple Blossom, Cocktail, Tangerine and Zanzibar flowered following a season of rest after fruiting. The classification used follows Traub's (1958) di visions of cultivated Amaryllis. Cytology: All nine hybrids are tetraploid with 2n=44 (Figs. 1-12). Tables 2 and 3 show the karyotype and genome analysis of each hybrid. The chromosomes can be clustered into three groups according to arm ratio, and centromeric index, which ranges from 1:3 to 1:6 (centromeric index: 1-25: acrocentric) in the fist group, 1:1.5 to 1:2.9 (centromeric index: 25.1-40: submetacentric) in the second, and 1:1 to 1:1.49 (centromeric index:40.1 50:metacentric) in the third. Chromosome length ranges from 4.3 to 17.7ƒÊm, with a bimodal distribution favoring long and short lengths (Figs. 1-12). Four length classes were recognized: small (4.3-6.6ƒÊm), medium (6.7-8.3ƒÊm), large (8.4-10ƒÊm), and very large (10 .1-17.7ƒÊm). All hybrids show various combinations of the three types and four sizes of chromosomes (Talbes 2 and 3). Secondary constrictions are terminal or near terminal leading to very small, inconspicuous satellites. Meiosis is normal and is characterized by the presence of bivalents , trivalents and quad rivalents. 1991 Karyomorphology of Amaryllis Hybrids 33 Appleblossom: Karyotype (Figs. 1, 4 and Table 2) consists of 22 pairs of chromosomes. Four pairs (2, 4-6) of chromosomes have subterminal constrictions (one small and three medium), 10 pairs (1,3 and 15-22) have submedian constrictions (2 small, 1 medium and 7 large) and eight pairs (7-14) show median or near median constrictions (all eight small). The Figs. 1-3. Photomicrographs of somatic metaphase plates of Amaryllis hybrids. All figures •~1700. 1, Apple Blossom. 2, Bold Leader. 3, Cocktail. 34 Tasneem F. Khaleel, Susan Haven and Tim Gilg Cytologia 56 toatl chromatin length is 307.6ƒÊm with chromosome lengths from 4.33ƒÊm to 10ƒÊm. The average chromosome length is 6 .7ƒÊm. Four haploid genomes can be recognized (Table 3), each consisting of two acrocentric, five submetacentric and four metacentric chromosomes respectively. The karyotype formula for this hybrid, therefore is 4 (2A5SM4M)=44. Figs. 4-12. Karyotypes of Amaryllis hybrids . All figures •~1750 . 4, Apple Blossom . 5, Bold Leader . 6, Desert Dawn. 7, Dutch Belle. 8, Intokazi . 9, Cocktail. 10 , Miracle. 11, Tangerine. 12, Zanzibar. Bold Leader: Karyotype (Figs. 2, 5 and Table 2) consists of 22 pairs of chromosomes -5) of chromosomes have subterminal . Fourpairs(2 constrictions (2 medium, 1 large and 1 very large) , 9 pairs (1, 6-10, 20-22) have submedian constrictions (1 small , 2 medium, 3 large and 3 very large) and nine pairs (11-19) have median or near median constrictions (8 small and Table 1. Classification, morphology, and behavior of Amaryllis hybrids Table 2. Karyotype analysis of Amaryllis hybrids All hybrids 2n=44; All Measurements are in micrometers. S=Small (4.3ƒÊ-6.6ƒÊ) A=Acrocentric M=Medium (6.7ƒÊ-8.3ƒÊ) SM=Submetacentric L=Large (8.4ƒÊ-10ƒÊ) M=Metacentric VL=Very large (10.1ƒÊ-18ƒÊ) 36 Tasneem F. Khaleel, Susan Haven and Tim Gilg Cytologia 56 Table 3. Genome analysis of * Since genomes I and II are the same , composition is only given once. 1 medium). The total chromatin length is 350.7ƒÊm with lengths from 5.3ƒÊm to 13 .7ƒÊm. The average chromosome length is 7.9ƒÊm. Four haploid genomes are recognized (Table 3) . Two of these consist of two acrocentric, five submetacentric and four metacentric chromo somes; the other two are composed of two acrocentric, four submetacentric and five metacen tric chromosomes respectively. The karyotype formula is 2(2A5SM4M)+2(2A4SM5M)= 44. Desert Dawn: Karyotype (Fig. 6 and Table 2) is composed of 2n=44 in 22 pairs . Ten pairs (1-4, 10, 13, 15, 19, 21 and 22) show subterminal constrictions (2 large and eight very large), 10 pairs (5, 6, 8, 9, 11, 12, 16-18, and 20) show submedian constrictions (1 small , 4 medium, 1 large and 4 very large) and 2 pairs (7 and 14) show median or near median con strictions (1 small and I medium).