Somatic polyploidy and its consequences for flower coloration and flower morphology in azalea

Flower colour variegation is not only a phenomenon of importance to horticulture, the phenotype involved is also often used as a scientific model system for the study of complex gene regulation processes. In the course of such studies on azalea, we observed a correlation between flower colour patterns, flower morphology and somatic polyploidy. Using high-resolution flow cytometry of nuclear DNA, the ploidy level was determined in flowers of different azalea sport families. Sports exhibiting variegated flowers with broad (>7 mm), differently coloured, petal edges (picotee type) proved to be tetraploid in the petal edge but diploid in the rest of the flower tissue. Neither flower colour pattern nor ploidy differences are periclinal chimeric in origin, but seem to be correlated with the topographic location of the cells within the flower tissue, i.e. the margin of the petals. The possible role of gene dosage effects and cell size involved in the remarkable correlation between somatic polyploidy, (flavonoid) gene expression and flower morphology is discussed.     

Cytotaxonomy and breeding system of the genusBiscutella (Cruciferae)

Chromosome counts were determined for 46 populations ofBiscutella representing 28 taxa. The genus was found to contain diploid taxa with 2n = 12, 16 and 18, tetraploid taxa with 2n = 36 and hexaploid taxa having 2n = 54.B. laevigata L. s. l. consists of diploid and tetraploid populations which are poorly differentiated morphologically. TetraploidB. laevigata s. l. and hexaploidB. variegata Boiss. & Reuter (s. l.) are characterized by chromosomal instability. The variation in chromosome numbers and the occurrence of polyploidy is discussed in relation to the taxonomy of the genus. An investigation of the breeding system showed that most of the annual species were self-compatible and partly inbreeding and most of the perennial species self-incompatible and, therefore, outbreeding, while one annual species,B. cichoriifolia Loisel., showed both systems.     

Ploidy, geographical distribution and morphological differentiation of Parasenecio auriculata (Senecioneae; Asteraceae) in Japan

Parasenecio auriculata is a woodland perennial herb widely distributed in Northeastern Asia, constituted by a poorly understood polyploidy with a diploid (2n=2x=60) and a tetraploid (2n=4x=120). In this study, for a better understanding of the polyploidal evolution, cytogeography and morphological variation were analyzed in Japanese P. auriculata, including two varieties; var. bulbifera endemic to central Hokkaido and var. kamtschatica widely distributed in northern Honshu and Hokkaido. The occurrence of two polyploidal levels was reconfirmed. While var. bulbifera is predominantly tetraploid, var. kamtschatica is comprised of diploid and tetraploid. Morphological variation among 22 quantitative characteristics is continuous and not distinctive among cytotypes or varieties, but plant size tended to be larger in the order, diploid of var. kamtschatica, var. bulbifera, and tetraploid of var. kamtschatica. The cytotype distribution showed a conspicuous geographical pattern. Besides var. bulbifera endemic to the central Hokkaido, the diploid of var. kamtschatica is mainly found in Southern Hokkaido, and the tetraploid has a disjunct distribution in eastern and northern Hokkaido and northern Honshu. Such a geographical pattern is possibly attributable to the differentiation of climatic preference among cytotypes and varieties, and may have been established in association with the climatic cline along the Japanese archipelago.     

Chromosome doubling of 2x Solanum species by oryzalin: method development and comparison with spontaneous chromosome doubling in vitro

A potato breeding scheme implies the possibility of ploidy level manipulation either by reducing the chromosome number of cultivars from 48 to 24 to be able to cross them with diploid related species or by doubling diploid material to reach the generally optimal tetraploid level. In vitro spontaneous chromosome doubling is widely used but can lead to somaclonal variation. Since oryzalin has proven to be efficient as a chromosome doubling agent on potato cell suspension cultures, we tried this herbicide on various Solanum species and interspecific diploid hybrids. A 24 h dip in a 28.8 M aqueous oryzalin solution applied on apical buds was the most efficient treatment in terms of tetraploid plant production (mean = 4.1 tetraploid plants for 10 treated buds over 4 genotypes). However 50–100% of the regenerated tetraploid plants acclimatized after in vitro treatment proved to be chimaeric. Consequently, a selection procedure in the progeny was necessary to obtain real and stable doubled clones and final yields were low. This technique is easy to apply and could be a good alternative to chromosome doubling by spontaneous in vitro regeneration in the case of refractory genotypes especially where somaclonal variation is problematic. Percentage of tetraploids among the regenerated plants varied from 6 to 29% with the oryzalin doubling technique while it varied from 20 to 78% by in vitro spontaneous doubling for five diploid genotypes. An observation of the progeny indicated that chimaeras were more frequent using oryzalin (50–100% of the initially supposed tetraploid plants) than when chromosomes doubled spontaneously (4–67% of the initially supposed tetraploid plants).     

Origin of chloroplast DNA diversity in the Andean potatoes

Summary Wide chloroplast DNA (ctDNA) diversity has been reported in the Andean cultivated tetraploid potato, Solanum tuberosum ssp. andigena. Andean diploid potatoes were analyzed in this study to elucidate the origin of the diverse ctDNA variation of the cultivated tetraploids. The ctDNA types of 58 cultivated diploid potatoes (S. stenotomum, S. goniocalyx and S. phureja), 35 accessions of S. sparsipilum, a diploid weed species, and 40 accessions of the wild or weed species, S. chacoense, were determined based on ctDNA restriction fragment patterns of BamHI, HindIII and PvuII. Several different ctDNA types were found in the cultivated potatoes as well as in weed and wild potato species; thus, intraspecific ctDNA variation may be common in both wild and cultivated potato species and perhaps in the higher plant kingdom as a whole. The ctDNA variation range of cultivated diploid potatoes was similar to that of the tetraploid potatoes, suggesting that the ctDNA diversity of the tetraploid potato could have been introduced from cultivated diploid potatoes. This provided further evidence that the Andean cultivated tetraploid potato, ssp. andigena, could have arisen many times from the cultivated diploid populations. The diverse but conserved ctDNA variation noted in the Andean potatoes may have occurred in the early stage of species differentiation of South American tuber-bearing Solanums.     

Hybridization and speciation in the genusDryopteris (Pteridophyta:Dryopteridaceae) on Pico Island in the Azores

The cytology of wild hybrids ofDryopteris from Pico, Azores has been examined. This has confirmed the parentage of the hybrids, and provided evidence to support the view that the tetraploid speciesD. crispifolia has evolved in the Azores from two diploid species,D. aemula andD. azorica, by hybridization and polyploidy.     

Cytogenetic characterization of embryogenic callus and regenerated plants of Pennisetum americanum (L.) K. Schum

Summary Embryogenic calli were derived from cultured segments of immature inflorescences of Pennisetum americanum (pearl millet). The original explants as well as the embryogenic calli and the plants regenerated via somatic embryogenesis were examined cytogenetically. Embryogenic calli were predominantly diploid (2n=14) after one month and six months in culture (92% and 76%, respectively). Tetraploid and aneuploid cells were observed in the original explant (2.5% and 1.2%) as well as in one (4.0% and 4.0%) and six-month-old calli (10.0% and 14.0%). Plants were regenerated from calli that had been in continuous culture for two, four and six months. Of the 101 regenerants, 100 were diploid and 1 was tetraploid. The tetraploid was an albino as were three of the diploid regenerants. Examination of 30 of the regenerants in meiotic diakinesis, anaphase I, anaphase II and quartet stages revealed no cytogenetic differences between control and regenerated plants. Gel electrophoresis for total protein content and alcohol dehydrogenase and malate dehydrogenase activity also did not reveal any differences between the controls and regenerants. The results of this study show that a slight shift toward aneuploidy and polyploidy may occur in embryogenic cultures, but there also is a strong selection in favor of plant regeneration from cytogenetically normal cells.     

Current status in production and utilization of dihaploids from somatic hybrids between eggplant (Solanum melongena L.) and its wild relatives

The major constrains for practical exploitation of the somatic hybrids between eggplant and its wild relatives have been their sterility and tetraploidy which prevented their incorporation into breeding programs. Here we demonstrate that anther culture was successfully utilized to bring back the ploidy level to the diploid status in tetraploid interspecific hybrids between eggplant and the allied species S. integrifolium and S. aethiopicum gr. gilo. Both the relative species are resistant to Fusarium oxysporum f. sp. melongenae and to some strains of bacterial wilt (Ralstonia solanacearum) which are very destructive diseases of eggplant. Dihaploid androgenetic plants were obtained from the somatic hybrids, from the “double somatic hybrid” obtained by sexual cross of the two somatic hybrids [(eggplant + S. aethiopicum) × (eggplant + S. integrifolium)], and from tetraploid backcrossed plants between the somatic hybrid with S. aethiopicum and eggplant. Phenotypical, molecular, biological and biochemical characterization, and also artificial inoculation with Fusarium oxysporum are consistent with a recombination between the genomes of the species involved in the hybridizations. Dihaploids resistant to Fusarium were successfully backcrossed with eggplant. Besides their utility as potential valuable breeding materials, the introgressed lines obtained may be utilized in genetic and molecular studies about the resistance to Fusarium from S. integrifolium and S. aethiopicum gr. gilo.     

Ploidy of somatic embryos and the ability to regenerate plantlets in melon (Cucumis melo L.)

Summary The number of chromosomes in cells of callus, somatic embryos and regenerated plantlets during somatic embryogenesis were examined in two cultivars of melon (Cucumis melo L.). Somatic embryos were diploid (50.0%/32.1%), tetraploid (38.5%/57.5%) and octoploid (11.5%/10.4%) whereas in callus cells diploidy (41.9%/43.3%), tetraploidy (27.9%/25.8%), octoploidy (11.6%/15.5%) and a low frequency of other types of ploidy and aneuploidy were observed. Mixoploid somatic embryos were not observed. These results suggest that the somatic embryos were selectively differentiated from diploid, tetraploid and octoploid cells, and that endopolyploidization of cultured cells occurred before the start of cell division leading to somatic embryogenesis. The ratio of diploid to tetraploid (1.30/0.55) in somatic embryos was less than that in callus cells (1.50/1.68) while ratios of diploid to octoploid (4.35/3.09) and tetraploid to octoploid (3.35/5.52) in somatic embryos were greater than those in callus cells (3.61/2.80 and 2.40/1.67). Therefore, it appears that the ability of callus cell to differentiate into somatic embryos increases in the following order: octoploid < diploid < tetraploid. Regenerated plantlets were diploid (65.5%/55.1%) and tetraploid (34.5%/44.9%). No octoploid plantlets were observed. The ratio of diploid to tetraploid in regenerated plantlets (1.72/1.23) was greater than that in somatic embryos. Therefore, it appears that the ability of somatic embryos to develop into plantlets increases in the following order: octoploid < tetraploid < diploid.     

Evolutionary patterns in the antR-Cor gene in the dwarf dogwood complex (Cornus, Cornaceae)

The evolutionary pattern of the myc-like anthocyanin regulatory gene antR-Cor was examined in the dwarf dogwood species complex (Cornus Subgenus Arctocrania) that contains two diploid species (C. canadensis and C. suecica), their putative hybrids with intermediate phenotypes, and a tetraploid derivative (C. unalaschkensis). Full-length sequences of this gene (∼4 kb) were sequenced and characterized for 47 dwarf dogwood samples representing all taxa categories from 43 sites in the Pacific Northwest. Analysis of nucleotide diversity indicated departures from neutral evolution, due most likely to local population structure. Neighbor-joining and haplotype network analyses show that sequences from the tetraploid and diploid intermediates are much more strongly diverged from C. suecica than from C. canadensis, and that the intermediate phenotypes may represent an ancestral group to C. canadensis rather than interspecific hybrids. Seven amino acid mutations that are potentially linked to myc-like anthocyanin regulatory gene function correlate with petal colors differences that characterize the divergence between two diploid species and the tetraploid species in this complex. The evidence provides a working hypothesis for testing the role of the gene in speciation and its link to the petal coloration. Sequencing and analysis of additional nuclear genes will be necessary to resolve questions about the evolution of the dwarf dogwood complex.     

<Emphasis Type="Italic">In vitro</Emphasis> induction and identification of autotetraploids of <Emphasis Type="Italic">Dioscorea zingiberensis</Emphasis>

Dioscorea zingiberensis is an important medicinal plant and a source of diosgenin in China. We report research on the induction, characteristics, and chemical assays of polyploid plants of D. zingiberensis. Immersing calli in 0.3% colchicine solution for 16 h prior to culture induced a high number of autotetraploid plants. The induction rate reached as high as 36.7% of treated calli. More than 50 lines of autotetraploid plants were obtained. All tetraploid plants showed typical polyploidy characteristics. Twenty selected tetraploid lines were transferred to the field for determination of morphological characteristics and for chemical assays. Six elite lines have been selected for further selection and breeding into new varieties for commercial production.     

Reducing the tetraploid non-nodulating alfalfa (Medicago sativa) MnNC-1008(NN) germ plasm to the diploid level

MnNC-1008(NN) (referred to as MN-1008) is a tetraploid alfalfa mutant with two recessive genes (nn ₁ and nn ₂ )conditioning the non-nodulating trait. The tetraploid level (2n=4x=32) of this Medicago sativa germ plasm was reduced to the diploid (2n=2x=16) level using the 4x-2x genetic cross originally described as a workable method for the induction of haploidy in alfalfa by T. E. Bingham. In our experiments more than 7000 emasculated flowers of a single non-nodulating MN-1008 mutant alfalfa plant with purple petals were cross-pollinated with pollen from a single, diploid, yellow-flowered alfalfa plant. Mature seeds from these crosses were collected and germinated, after which the plants were subjected to morphological and cytogenetic analyses as well as to DNA fingerprinting. Out of 26 viable progeny, 6 were hybrid plants, 19 proved to be self-mated derivatives of MN-1008, while one descendant turned out to be a diploid (2n=2x=16), purple flowered, non-nodulating plant denoted as M. sativa DN-1008. This diploid, non-nodulating alfalfa plant can serve as starting material to facilitate the comprehensive morphological, physiological and genetic analysis (gene mapping and cloning) of nodulation in order to learn more about the biology of the symbiotic root nodule development. To produce diploid, nodulating hybrid F₁ plants, DN-1008 was crossed with a diploid, yellow-flowered M. sativa ssp. quasifalcata plant. An F₂ population segregating the nn ₁ and nn ₂ genes in a diploid manner, in which the genetic analysis is more simple than in a tetraploid population, can be established by self-mating of the F₁ plants.     

Ribosomal RNA genes in biotypes ofScilla peruviana (Liliaceae)

Scilla peruviana biotypes have different chromosome numbers due to changes in the nucleolar chromosomes and polyploidy. We have examined two diploid (2n = 15 and 2n = 16) and two tetraploid biotypes (2n = 28 and 2n = 32). From the results of rRNA/DNA filter hybridizations it appears that rDNA percentages of the diploid biotypes are, approximately, 2.2-fold higher than those of the tetraploid biotypes. To examine the rRNA gene structure we have utilizedSouthern blot hybridization after DNA digestions with three restriction enzymes: Eco RI, Hind III and Bam HI. From the band analysis of both single and double digestions it has been possible to reveal the presence, in the diploid biotypes, of three gene types, heterogeneous both for length and for nucleotide sequences in the external spacer. The three rRNA genes are 12 600, 12 700, and 12 800 base pairs long and they have a different position of the Hind III sites in the external spacer. On the other hand, a single gene type of 12 600 base pairs, identical to the first type of the diploid biotypes, surprisingly exists in the tetraploid biotypes. Considerations on the rRNA gene regulation and evolution are made.     

Selection of somatic hybrids after fusion of protoplasts from Datura innoxia Mill. and Atropa belladonna L.

After fusion of protoplasts from a diploid (2n=24) and a tetraploid (4n=48) chlorophyll-deficient mutant of Datura innoxia Mill. with diploid (2n=72) green wild-type protoplasts of Atropa belladonna L. thirteen somatic hybrids could be selected, most of which had already started to produce leaves and shoots. Hybrid calli were recognizable by the production of hairs, typical for Datura innoxia, and the green colour, derived from Atropa belladonna. Further proof for the hybrid nature was furnished by cytological investigations. The metaphase chromosomes of both species are easily distinguishable in their size: chromosomes of Datura innoxia are about twice as large as those of Atropa belladonna. The chromosome numbers of the hybrids varied from ca. 84 to ca. 175.Abbreviations BAP 6-benzylaminopurine - NAA -naphthalene acetic acid     

濒危植物丰都车前的繁育系统与传粉生物学研究

丰都车前为三峡库区特有珍稀濒危植物,目前仅在重庆市江津支坪镇涂家村附近大中坝上有一小种群分布。以野生种群和移栽种群为对象,通过野外观察和人工授粉实验对丰都车前传粉生物学及繁育系统进行研究。结果表明:(1)丰都车前单花花期约4 d,花序花期约8 d,种群花期约3个月;单花开花进程可分为6个时期:花蕾期、雌蕊伸长期、雄蕊初露期、花瓣展开期、花瓣反折期、枯萎凋谢期。(2)花瓣展开前,柱头先伸出花冠,达最高可受性,之后花瓣展开雄蕊才露出,而此时大部分柱头已枯萎失活丧失可受性,只有少部分具可受性。表现出雌雄异熟的特征,一定程度避免了自交的发生。(3)花粉活力始于雄蕊初露期,花瓣展开、花药开裂时花粉活力最高,至枯萎凋谢前均具有活力。花粉活力最高期避开了柱头可受性最佳期,仅有少部分重叠。(4)繁育系统检测显示,花粉/胚珠比为15373.29,杂交指数为3。结合套袋实验表明,丰都车前可能同时具有风媒和虫媒传粉机制,传粉昆虫为中华蜜蜂、食蚜蝇,但数量偏少,不存在无融合生殖,异交为主,部分自交亲和,有时需要传粉者。(5)开花时期避开了其分布地洪水淹没期,能够顺利完成开花授粉及座果。综上所述,丰都车前的花部特征、开花物候和繁育系统为适应分布地夏季洪水水淹胁迫提供了一定的生殖保障,促进了该种群的续存。     

Chemically Induced Polyploidization in Spathiphyllum wallisii Regel through Somatic Embryogenesis

Polyploid Spathiphyllum wallisii Regel 'Speedy' plants were obtained from somatic embryos induced on anther filaments exposed to mitosis inhibitors. Primary embryos yielded less polyploid plantlets than secondary embryos. Colchicine could be efficiently replaced by oryzalin or trifluralin (10 μM), resulting in an average yield of 5% polyploids. The mitosis inhibitors were directly added to the induction medium. Morphological differences between diploid and tetraploid S. wallisii hybrids were observed. Throughout our experiments flow cytometry was applied as an unambiguous screening tool. Ploidy breeding within the genus Spathiphyllum holds promises towards the development of tetraploid hybrids with an altered morphology, or triploids with a reduced fertility.     

Genetic variation in sympatric populations of diploid and polyploid brine shrimp (Artemia parthenogenetica)

We examined genetic variation in sympatric diploid and polyploid brine shrimp Artemia parthenogenetica from each of three populations (China, Italy and Spain). Italian and Spanish tetraploids are closely related (I=0.964). Diploids and tetraploids within each of the two European populations are also closely related (mean I=0.905). Most alleles found in diploids also exist in sympatric polyploids. In contrast, the asexual Artemia (2N, 4N and 5N) in our study share few alleles with their close sexual relative, A. tunisiana (mean I=0.002). These results, as well as the work of other authors, strongly suggest that at least the tetraploid Artemia in our study have an autopolyploid origin.Clonal diversity of polyploid Artemia can be very high at least in some population. Both diploids and polyploids had low clonal diversities in the populations dominated by polyploids and high clonal diversities in the population dominated by diploids.The most common genotypes of sympatric diploid and polyploid Artemia frequently differed. Some alleles occurred only in diploids, while others were restricted to polyploids. These results suggest that polyploidy in Artemia has led to genetic divergence from diploid progenitors, and that ploidy-level variation must also be considered in developing an understanding of spatial and temporal allozyme polymorphism in asexual populations.     

DasFestuca vivipara-Problem in den Alpen

The differences betweenFestuca vivipara (tetraploid) andF. ovina subsp.supina (diploid), two often confused taxa, are demonstrated in regard to morphology, leaf anatomy, ecology and distribution. New maps illustrate the distribution ofF. vivipara in the Alps and the Northern Hemisphere. The development of different polyploidy levels withinF. vivipara as a consequence of occasional sexual processes is discussed. (English Summary on p. 39.)

     

Molecular,cytological and morpho-agronomical characterization of hexaploid somatic hybrids in Medicago

Somatic hybrid plants produced by protoplast fusion between tetraploid Medicago sativa (2n= 4x=32) and the diploid species Medicago coerulea (2n= 2x=16) have been RFLP fingerprinted to establish their nuclear composition. Although all of the chromosomes were present, molecular analysis revealed an incomplete incorporation of the alleles of the diploid parent in the fusion products. In the polycross progeny the alleles of both parents segregated in a Mendelian mode. Cytological observations indicated that in the somatic hybrid population minor abnormalities are present; these are restricted mainly to the formation of univalents and lagging chromosomes. Meiosis appeared to be more stable than has been previously reported in the hexaploids of alfalfa. The somatic hybrids grown in the field had a rather vigorous aspect, particularly with respect to the vegetative organs. Forage yield was comparable to that of thmore productive parent. The results are discussed with a view to utilizing the somatic hybrids as starting material for breeding alfalfa at the hexaploid level.This paper was supported by the National Research Council of Italy, Special Project RAISA, Sub-project No.2 paper No. 1911     

Ploidy doubling by in vitro culture of excised protocorms or protocorm-like bodies in <Emphasis Type="Italic">Phalaenopsis</Emphasis> species

Endopolyploidy was observed in the protocorms of diploid Phalaenopsis aphrodite subsp. formosana with ploidy doubling achieved by in vitro regeneration of excised protocorms, or protocorm-like bodies (PLBs). Thirty-four per cent of the PLBs regenerated from the first cycle of sectioned protocorms were found to be polyploids with ploidy doubled once or twice as determined by flow-cytometry. The frequency of ploidy doubling increased as the sectioning cycles increased and was highest in diploid followed by the triploid and tetraploid. Regeneration of the endopolyploid cells in the tissue of the protocorms or PLBs is proposed as the source of the development of ploidy doubled plantlets. The frequency of ploidy doubling was similar in seven other Phalaenopsis species, although the rate of increase within cycles was genotype specific. In two species, a comparison of five parameters between 5-month-old diploid and tetraploid potted plants showed only the stomata density differed significantly. The flowers of the tetraploid plant were larger and heavier than those of the diploids. This ploidy doubling method is a simple and effective means to produce large number of polyploid Phalaenopsis species plants as well as their hybrids. The method will be beneficial to orchid breeding programs especially for the interspecific hybridization between varieties having different chromosome sizes and ploidy levels.