Tetraploidization events by chromosome doubling of nucellar cells are frequent in apomictic citrus and are dependent on genotype and environment

Background and Aims

Polyploidy is a major component of plant evolution. The citrus gene pool is essentially diploid but tetraploid plants are frequently encountered in seedlings of diploid apomictic genotypes. The main objectives of the present study were to establish the origin of these tetraploid plants and to ascertain the importance of genotypic and environmental factors on tetraploid formation.

Methods

Tetraploid seedlings from 30 diploid apomictic genotypes were selected by flow cytometry and genotyped with 24 single sequence repeat (SSR) markers to analyse their genetic origin. Embryo rescue was used to grow all embryos contained in polyembryonic seeds of ‘Tardivo di Ciaculli’ mandarin, followed by characterization of the plantlets obtained by flow cytometry and SSR markers to accurately establish the rate of tetraploidization events and their potential tissue location. Inter-annual variations in tetraploid seedling rates were analysed for seven genotypes. Variation in tetraploid plantlet rates was analysed between different seedlings of the same genotype (‘Carrizo’ citrange; Citrus sinensis × Poncirus trifoliata) from seeds collected in different tropical, subtropical and Mediterranean countries.

Key Results

Tetraploid plants were obtained for all the studied diploid genotypes, except for four mandarins. All tetraploid plants were identical to their diploid maternal line for SSR markers and were not cytochimeric. Significant genotypic and environmental effects were observed, as well as negative correlation between mean temperature during the flowering period and tetraploidy seedling rates. The higher frequencies (20 %) of tetraploids were observed for citranges cultivated in the Mediterranean area.

Conclusions

Tetraploidization by chromosome doubling of nucellar cells are frequent events in apomictic citrus, and are affected by both genotypic and environmental factors. Colder conditions in marginal climatic areas appear to favour the expression of tetraploidization. Tetraploid genotypes arising from chromosome doubling of apomictic citrus are extensively being used as parents in breeding programmes to develop seedless triploid cultivars and have potential direct use as new rootstocks.     

Morphological, physiological, cytological and phytochemical studies in diploid and colchicine-induced tetraploid plants of Echinacea purpurea (L.)

Echinacea purpurea (L.) is one of the important medicinal plant species. To obtain the tetraploid plants of Echinacea purpurea with improved medicinal qualities, the root tips of two true leaves seedlings were imbibed in 0.25 % (w/v) colchicine solution for 24, 48, 72, 96 and 168 h. The ploidy level of plants was determined by chromosome counting of root tip cells, and confirmed by flow cytometric analysis. Tetraploid induction occurred in seedlings treated for 24, 48 and 72 h at colchicine solution. The morphological, physiological, cytological, and phytochemical characteristics of diploid and colchicine-induced tetraploid plants were compared. Results indicated that tetraploid plants had considerable larger stomata, pollen grain, seed and flower. Moreover, chloroplast number in guard cells, amount of chlorophyll (a, b, and a + b), carotenoids as well as width and thickness of leaves were increased in tetraploids. However, stomata frequency, leaf index, plant height, and quantum efficiency of photosystem II in tetraploid were lower than diploid plants. High-performance liquid chromatography analysis showed that leaves of the tetraploid plants had more cichoric acid (45 %) and chlorogenic acid (71 %) than diploid plants. It was concluded that morphological and physiological characteristics can be used as useful parameters for preliminary screening of putative tetraploids in this species.     

山金柑实生后代四倍体发掘及形态和代谢评价

以多胚系山金柑（Fortunella hindsii Swingle）为材料,采用“观根辨叶看油胞”形态初选法,从1289株实生后代筛选出疑似四倍体（双二倍体）8株,流式细胞仪检测和SSR分子鉴定表明它们均为同源四倍体,初选准确率100%,群体自然发生率0.62%。对其形态和初生代谢物进行检测,结果显示：山金柑四倍体株高、茎粗、节间数、节间长、气孔密度均显著低于二倍体,而叶片厚度、气孔大小显著高于二倍体;GC-MS分析鉴定到24种初生代谢物,四倍体叶片奎宁酸含量显著高于二倍体,肌醇、4-氨基丁酸和1-棕榈酸单甘油酯含量显著低于二倍体。     

Genetic diversity in sexual diploid and apomictic tetraploid populations of Paspalum notatum situated in sympatry or allopatry

Paspalum notatum is a subtropical grass widely distributed in the temperate areas of America. Diploids are sexual while polyploids give rise to clonal seeds through aposporous apomixis. RAPD markers were used to analyze the genetic structure of three natural populations: i) diploids reproducing sexually (R2X); ii) sympatric apomictic tetraploids collected in the vicinity of the diploids (R4X); iii) allopatric apomictic tetraploids growing in isolation (C4X). The apomictic reproduction rate was evaluated by the use of molecular markers in progeny tests, while chromosome-counting allowed the verification of ploidy levels. Data revealed that the R4X group presented a variation considerably higher than that observed for C4X. Jaccards coefficients were used to produce a cluster diagram using the UPGMA method. All but one tetraploid genotypes grouped together and were associated to diploid genotype A21. The possibility of occasional generation of novel tetraploid clones from the interaction between tetraploid and diploid individuals is discussed.     

Phytogeographical studies ofCalamagrostis sachalinensis (Gramineae)

Chromosome numbers were determined on 223 collections ofCalamagrostis sachalinensis from 18 localities in Japan. The plants were found to be tetraploid (2n=28), hexaploid (2n=42) or octoploid (2n=56). A few collections were found to include one or two B-chromosomes. The tetraploid collections were made from central Honshu and Mt. Apoi in Hokkaido, while the hexaploids and the octoploids were detected in many localities. Pollen examination of these collections showed that the tetraploids with but one exception have good pollen and the hexaploids and the octoploids have no pollen or have bad pollen with stainability less than 10%. With the help of pollen examination of a number of herbarium specimens, the distribution of the tetraploids and that of the assemblage of the hexaploids and octoploids were delineated. Morphological studies indicated that the tetraploid, hexaploid and octoploid plants can not be separated in gross and spikelet morphology and that the tetraploids in central Honshu and those in Mt. Apoi are significantly different in leaf features. It was concluded thatC. sachalinensis represents an apo-amphimictic complex, which includes the following four races: 1) tetraploid, amphimictic, having thin leaf blades 5–10 mm broad and growing on the subalpine conifer forest belt and the conifer forest-alpine ecotone in the mountains of central Honshu; 2) tetraploid, amphimictic, having hard leaf blades 2–6 mm broad and growing on the stony, arid and exposed alpine belt on Mt. Apoi in Hokkaido; 3) hexaploid, mainly apomictic, the most variable ecologically, widely distributed; 4) octoploid, mainly apomictic, frequent in the upper montane to alpine belts, probably widely distributed.     

Evaluation of isozyme systems in Citrus to facilitate identification of fusion products

Summary Ten isozymes were analyzed in nucellar calli of nine Citrus species and cultivars and roots of the corresponding apomictic seedlings. The zymograms obtained can be divided into three groups: a) isozyme patterns similar in both calli and roots, b) isozyme patterns similar in calli but variable in roots, and c) isozyme patterns variable in both calli and roots. Analysis of these ten isozyme systems may facilitate identification of fusion products in Citrus.Contribution from the Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel. No. 354-E, 1982 series     

IDENTICAL-TWIN HYBRIDS OF CITRUS X PONCIRUS FROM STRICTLY SEXUAL SEED PARENTS

Tree characters of 13 sets of twin and triplet hybrids from single seeds of crosses of Citrus × Poncirus and characters of three other Citrus twin sets are described. Evidence from tree size and habit, leaf size, shape, and thickness, and time of leafing and leaf fall indicates that these twins and triplets are probably all genetically identical within sets. Recent cytological evidence is cited which indicates that supernumerary sexual embryos can occur in Citrus by a process of budding from the primary embryo. The twin and triplet hybrids are clearly of separate origin from multiple asexual seedlings, also normally genetically identical, which arise fram cells of the nucellus in many Citrus varieties.     

When sexual meets apomict: genome size,ploidy level and reproductive mode variation of Sorbus aria s.l. and S. austriaca (Rosaceae) in Bosnia and Herzegovina

Background and Aims Allopolyploidy and intraspecific heteroploid crosses are associated, in certain groups, with changes in the mating system. The genus Sorbus represents an appropriate model to study the relationships between ploidy and reproductive mode variations. Diploid S. aria and tetraploid apomictic S. austriaca were screened for ploidy and mating system variations within pure and sympatric populations in order to gain insights into their putative causalities.Methods Flow cytometry was used to assess genome size and ploidy level among 380 S. aria s.l. and S. austriaca individuals from Bosnia and Herzegovina, with 303 single-seed flow cytometric seed screenings being performed to identify their mating system. Pollen viability and seed set were also determined.Key Results Flow cytometry confirmed the presence of di-, tri- and tetraploid cytotype mixtures in mixed-ploidy populations of S. aria and S. austriaca. No ploidy variation was detected in single-species populations. Diploid S. aria mother plants always produced sexually originated seeds, whereas tetraploid S. austriaca as well as triploid S. aria were obligate apomicts. Tetraploid S. aria preserved sexuality in a low portion of plants. A tendency towards a balanced 2m : 1p parental genome contribution to the endosperm was shared by diploids and tetraploids, regardless of their sexual or asexual origin. In contrast, most triploids apparently tolerated endosperm imbalance.Conclusions Coexistence of apomictic tetraploids and sexual diploids drives the production of novel polyploid cytotypes with predominantly apomictic reproductive modes. The data suggest that processes governing cytotype diversity and mating system variation in Sorbus from Bosnia and Herzegovina are probably parallel to those in other diversity hotspots of this genus. The results represent a solid contribution to knowledge of the reproduction of Sorbus and will inform future investigations of the molecular and genetic mechanisms involved in triggering and regulating cytotype diversity and alteration of reproductive modes.     

Genome accumulation in eastern gamagrass,Tripsacum dactyloides (L.) L. (Poaceae)

Intermatings between sexual diploids (2n=36) and facultative apomictic tetraploids (2n=4n=72) of Tripsacum dactyloides var. dactyloides, (L.), L., have been generated. Subsequent exchange and addition of genetic material between the two cytotypes, via a backcrossing program, provides one method for the manipulation of genetic elements involved in apomictic reproduction (diplospory) of this species.Identification and development of fertile triploids, and their innate tendency to exhibit a high frequency of unreduced egg cells which remain receptive to fertilization by a sperm nucleus, allows for the unique development of B _III (2n=3x+n) tetraploids by genome accumulation. The resultant B _III progeny provide new breeding stock for investigating the inheritance and generation of such materials also allows for the occurrence of genetic recombination between sexual diploids and apomictic tetraploids. Potential use of fertile triploids, their tendency for genome accumulation and their use in exploiting apomictic tetraploid germplasm resources are also discussed.All programs and services of the U.S. Department of Agriculture are offered on a nondiscriminatory basis without regard to race, color, national origin, religion, sex, age, marital status, or handicap.     

In vitro induction of tetraploids in Arachis paraguariensis

In this study, an efficient procedure was established for successful induction of tetraploid Arachis paraguariensis by treating diploid explants with colchicine. Quartered-seed, callus and shoot-tips were treated with colchicine at concentrations of 0.05, 0.1, 0.2 and 0.5?% (w/v) for 4, 8, 16, 20 and 24?h before they were transferred unto modified Murashige and Skoog medium for either callus induction or shoot regeneration. Results showed that quartered-seed displayed the highest frequency of in vitro plantlet regeneration and tetraploid induction, as well as the lowest mortality rate. Flow cytometric analysis also confirmed that the induced tetraploids from quartered-seed were true-to-type. The 0.5?% colchicine treatment for 4 to 8?h gave the best results with 39 and 43?% of the explants yielding tetraploid plants, respectively. Two?months following transfer to ex vitro environment, morphological and growth characteristics of the induced tetraploids were measured. Overall, increasing the ploidy level from 2× to 4× resulted in fewer stomata but more trichomes per unit leaf area. Tetraploid plants obtained in this study should expand the genetic base of Arachis, and can also be used in overcoming the existing hybridization barriers that may be due to ploidy differences within the genus Arachis.     

Apomixis and ploidy barrier suppress pollen-mediated gene flow in field grown transgenic turf and forage grass (Paspalum notatum Flüggé)

Bahiagrass (Paspalum notatum Flüggé) is the predominant forage grass in the southeastern US. The commercially important bahiagrass cultivar ‘Argentine’ is preferred for genetic transformation over sexual diploid cytotypes, since it produces uniform seed progeny through apomixis. Pseudogamous apomictic seed production in Argentine bahiagrass may contribute to transgene confinement. It is characterized by embryo development which is independent of fertilization of the egg cell, but requires fertilization with compatible pollen to produce the endosperm. Pollen-mediated gene transfer from transgenic, glufosinate-resistant apomictic bahiagrass as pollen donor at close proximity (0.5–3.5 m) with non-transgenic sexual or apomictic bahiagrass cultivars as pollen receptors was evaluated under field conditions. Hybridization frequency was evaluated by glufosinate herbicide resistance in >23,300 seedlings derived from open-pollinated (OP) pollen receptor plants. Average gene transfer between transgenic apomictic, tetraploid and sexual diploid bahiagrass was 0.03%. Herbicide-resistant hybrids confirmed by immuno-chromatographic detection of the PAT protein displayed a single copy bar gene identical to the pollen parent. Hybrids resulting from diploid pollen receptors were confirmed as triploids or aneu-triploids with significantly reduced vigor and seed set as compared to the parents. Transmission of transgenes to sexual bahiagrass is severely restricted by the ploidy difference between tetraploid apomicts and diploid sexual bahiagrass. Average gene transfer between transgenic apomictic tetraploid and non-transgenic, apomictic tetraploid bahiagrass was 0.17%, confirming a very low frequency of amphimixis in apomictic bahiagrass cultivars. While not providing complete transgene containment, gene transfer between transgenic apomictic and non-transgenic bahiagrass occurs at a much lower frequency than reported for other cross-pollinating or facultative apomictic grasses.     

In vitro induction of tetraploids in an interspecific hybrid of Calanthe (Calanthe discolor × Calanthe sieboldii) through colchicine and oryzalin treatments

Tetraploids were successfully produced from diploid seeds obtained through interspecific crossing between Calanthe discolor and Calanthe sieboldii by treating with colchicine or oryzalin. Colchicine was tested at concentrations of 0.05 and 0.1 % for 0, 3, or 7 days and oryzalin was tested at a concentration of 0.003 % for 1, 2, 4, and 7 days, and the ploidy of the seedlings was determined by flow cytometry. Tetraploids (4×) were obtained from the interspecific hybrid seeds treated with all colchicine and oryzalin concentrations. The most efficient condition for inducing tetraploids seemed to be treated with 0.003 % oryzalin for 1 or 2 days. Cytological and morphological evidence confirmed the results of flow cytometric analysis. The stomatal density and sizes of the tetraploid plants were significantly higher and larger than those of the diploid plants. Differences in leaf shape were found between the tetraploid and diploid plants under the same growing conditions: the leaves of the diploids were elongated and those of the tetraploids were round.     

LEAF ISOZYMES AS GENETIC MARKERS IN CITRUS

The genetic control of isozymes from Citrus and its near relatives was determined for three gene/enzyme systems: glutamate oxaloacetate transaminase, phosphoglucose isomerase and phosphoglucose mutase. These enzymes are controlled by four genes having 19 codominant alleles, 12 of which occur in Citrus subg Citrus. Formal genetic studies were carried out with F, biotypes and F₁ populations of known origin. When biotypes were grouped into traditionally recognized species to examine genetic affinities within and between species, a remarkable pattern of uniformity of genotype combinations was found within a species, and every species had an unique combination. Because many economically important cultivars produce asexual(nucellar) as well as sexual(zygotic) embryos, a central problem of the breeder is to distinguish these when plants are young, long before fruiting. Isozyme markers can be used with varying degrees of efficiency, depending on the genotypes of the particular parents, to distinguish nucellar seedlings from those of zygotic origin.     

Genetic analysis of apomixis in Citrus and Poncirus by molecular markers

Propagation of citrus rootstocks depends upon the production of clonal plants from nucellar seedlings. This makes apomixis one of the host important traits in breeding programs for citrus rootstocks. The genetic control of apomixis was studied in a 50-tree progeny derived from the cross C. volkameriana×P. trifoliata using 69 molecular markers and bulked segregant analysis. The proportion of nucellar seedlings was estimated by isoenzymatic analysis of 25 seedlings per tree for 2 consecutive years. The type of embryony (polyembryonic versus monoembryonic seeds) was also determined for fruit-yielding trees. Separate genetic maps for each parental species were developed. The integration and comparison of these maps could be accomplished using common multiallelic segregant loci. Differences in gene synteny between the two species-specific genetic maps were shown. Important distortions in the segregation of markers at several genomic regions, some of them also involving differences in the C-methylation pattern, have been observed, especially for the pollen parent. Analysis of quantitative trait loci (QTLs) revealed the presence of six genomic positions (two in P. trifoliata and four in C. volkameriana) contributing individually up to 24% of the total variation for apomixis. Within the same species, QTLs with positive and negative allele effects were present, even in the same linkage group. One of the markers associated to apomixis (Apo2) is also associated to embryony type. Therefore, the genetic control of apomictic reproduction found in citrus (nucellar embryony) is quite complex compared to what has been reported for gametophytic apomixis. Molecular markers linked to QTLs governing apomixis will be useful to assist selection of future apomictic rootstocks for citrus varieties. Received: 30 November 1998 / Accepted: 28 December 1998     

Induction of tetraploidy using high temperature exposure during the first zygote division in Populus adenopoda Maxim

Triploid breeding has proven to be a valuable approach in Populus improvement. An ideal way to achieve triploid Populus is to cross diploids with tetraploids. In this study, we attempted to induce tetraploids of Populus adenopoda by inhibiting the first zygote division with high temperature exposure and increase the efficiency of tetraploid production. The relationship between zygote division stages and developmental changes of the cotton-like fiber in ovaries were analyzed, helping to guide the treatment towards the most accurate cytological stage. Among the 3,582 offspring, 32 teraploids were confirmed by both flow cytometric analysis and chromosome number counting. The highest efficacy of teraploid induction was 14.12 %. Significant differences between diploids and tetraploids were observed between the mean sizes and densities of leaf stoma. The tetraploid plants showed modified morphological characteristics, such as thicker leaves, modified leaf morphology, and stunted growth. Our finding showed that exposure during the first zygote division is an ideal method for teraploid induction in Populus.     

Why only tetraploid Solidago gigantea (Asteraceae) became invasive: a common garden comparison of ploidy levels

Many studies have compared the growth of plants from native and invasive populations, but few have considered the role of ploidy. In its native range in North America, Solidago gigantea Aiton (Asteraceae) occurs as a diploid, tetraploid and hexaploid, with considerable habitat differentiation and geographic separation amongst these ploidy levels. In the introduced range in Europe, however, only tetraploid populations are known. We investigated the growth performance and life history characteristics of plants from 12 European and 24 North American (12 diploid, 12 tetraploid) populations in a common garden experiment involving two nutrient and two calcium treatments. Twelve plants per population were grown in pots for two seasons. We measured 24 traits related to leaf nutrients, plant size, biomass production and phenology as well as sexual and vegetative reproduction. Native diploid plants had a higher specific leaf area and higher leaf nutrient concentrations than native tetraploids, but tetraploids produced many more shoots and rhizomes. Diploids grown with additional calcium produced less biomass, whereas tetraploids were not affected. European plants were less likely to flower and produced smaller capitulescences than North American tetraploids, but biomass production and shoot and rhizome number did not differ. We conclude that a knowledge of ploidy level is essential in comparative studies of invasive and native populations. While clonal growth is important for the invasion success of tetraploid S. gigantea, its potential was not acquired by adaptation after introduction but by evolutionary processes in the native range.     

In vitro regeneration and cytological characterization of shoots from leaf explants of three accessions of Solanum commersonii

In vitro culture of explants were used to apply genetic or cell engineering techniques to the sexually incompatible potato relative Solanum commersonii (2n=2x=24) Three accessions of S. commersonii were tested for regeneration from leaf explants using six different protocols. A two step-regeneration procedure gave the best results. Genetic variability for regeneration ability was found between accessions, and between clones within accessions. The accession PI 472834 regenerated at highest frequency. Clones with high regeneration ability were selected. Approximately 60% of regenerated plants were diploids and 40% were tetraploids. A very low frequency of chimeras was found. Leaf shape and chloroplast counts in guard cells were shown to be quick and reliable methods for estimating ploidy levels. Use of the diploid and tetraploid regenerants obtained for potato breeding is discussed.Abbreviations BAP 6-benzylaminopurine - EBN Endosperm Balance Number - GA₃ gibberellic acid - IAA indole-3-acetic acid - MS Murashige and Skoog - NAA 1-naphthalene-acetic acid - ZEA zeatin     

Factors affecting the frequency of tetraploid cells in a predominantly diploid suspension culture ofDaucus carota

Summary Tetraploid clones were isolated from a predominantly diploid culture line ofDaucus carota by plating alone or after colchicine treatment. Although individually these tetraploid clones had similar growth rates to the diploid culture, they were progressively eliminated from mixtures with the diploid line. As the diploid culture line always contained a low frequency of tetraploids, the selective elimination of tetraploids must have been balanced by their continuous production. The frequency of endoreduplication detected was too low to maintain the equilibrium frequency of tetraploids and it is proposed that polyploidisation also occurred by endomitosis. The mean frequency of tetraploid metaphases within the diploid culture line was reduced by shortening the interval between subcultures from 14 to 7 days. This 7-day regime also eliminated linear growth and stationary phase periods, but did not alter the maximum growth rate or mitotic index of the culture. It is argued that the change in mean tetraploid metaphase frequency is indicative of a change in the number of tetraploid cells within the culture, brought about by an alteration in the equilibrium between production and elimination of tetraploid cells.     

Biogeographic variation in genetic variability,apomixis expression and ploidy of St. John's wort (Hypericum perforatum) across its native and introduced range

Background and Aims

St. John''s wort (Hypericum perforatum) is becoming an important model plant system for investigations into ecology, reproductive biology and pharmacology. This study investigates biogeographic variation for population genetic structure and reproduction in its ancestral (European) and introduced (North America) ranges.

Methods

Over 2000 individuals from 43 localities were analysed for ploidy, microsatellite variation (19 loci) and reproduction (flow cytometric seed screen). Most individuals were tetraploid (93 %), while lower frequencies of hexaploid (6 %), diploid (<1 %) and triploid (<1 %) individuals were also identified.

Key Results

A flow cytometric analysis of 24 single seeds per individual, and five individuals per population demonstrated opposite patterns between ploidy types, with tetraploids producing more apomictic (73 %) than sexual (24 %) seed, while hexaploids produced more sexual (73 %) than apomictic (23 %) seed. As hexaploids are derived from tetraploids, these data imply that gene dosage, in addition to the effects of hybridization, influences the switch from apomictic to sexual reproduction. No significant differences in seed production were found between Europe and North America. An analysis of population structure based upon microsatellite profiling demonstrated three major genetic clusters in Europe, whose distribution was reflective of Pleistocene glaciation (e.g. refugia) and post-glacial recolonization of Europe.

Conclusions

The presence of pure and mixed populations representing all three genetic clusters in North America demonstrates that H. perforatum was introduced multiple times onto the continent, followed by gene flow between the different gene pools. Taken together, the data presented here suggest that plasticity in reproduction has no influence on the invasive potential of H. perforatum.     

A cytotaxonomic study of theCalamagrostis purpurea —langsdorffii—canadensis complex in the lowlands of hokkaido

A chromosome survey of 278 individuals from 52 localities confirmed, that the principal components of theCalamagrostis purpurea—langsdorffi—canadensis complex in the lowlands of Hokkaido are tetraploid (2n=28) and octoploid (2n=56). A few hexploids (2n=42), were also found, but they may be either a triploid of the tetraploid plants or a hybrid between the tetraploid and octoploid plants. These hexaploids clearly differ in origin from the North American plants with 2n=42 which are generally placed underC. canadensis. The tetraploids and the octoploids are significantly different in pollen size but resemble each other in other morphological features and ecological requirements. These two races are also not disparate in distribution within Hokkaido. The tetraploids in Hokkaido are closely related to the tetraploids known in Alaska, and these tetraploid plants may be continuously distributed from Hokkaido to Alaska. The octoploids in Hokkaido have shown good pollen. They seem to have had a somewhat different course of evolution fromC. purpurea s. str. which principally reproduces in an apomictic manner.