Diploid Cytotypes Research Articles

Problems Involved in the Proper Identification of Ferns

The paper illustrates how the meiotic Counts of six species of wild ferns namely, Hicriopteris glauca Copel. showing n=56, Pteris vittata L. sensu Holttum (1954) showing n=58 (Figs. 4 & 4a), Pteris biaurita L. sensu lato showing n=3n=87 (Figs. 2, 2a, 3 & 3a), Blechnum orientale L. sensu lato showing n=2n=34, Cyclosorus dentatus Forsk. sensu lato showing n=36 (Figs. 1 & 1a) and Polystichum aculeatum (L.) Roth sensu lato showing n=82, prove useful in their identifications and in finding out their modes of reproduction i.e. sexual or apogamous. These observations also confirm in general the Previous findings by few other workers on the materials from Darjeeling, Ceylon and West Africa. So far as the investigator is aware, this is the first report on the signs of apogamy on the diploid level in B. orientale L., on the triploid level in P. biaurita L. and on the availability of a new diploid cytotype provisionally placed under C. dentatus Forsk. among ferns collected from Shillong and some adjacent areas on the Khasi hills.

Ecological niche modeling of diploid flowering rush (Butomus umbellatus L.) in the United States

Flowering rush (Butomus umbellatus L.) is an invasive perennial monocot found along the United States (U.S.).–Canadian border which can grow into large monotypic mats that can cause water use issues. Currently, there are two known cytotypes, diploid and triploid, within the invaded range; however, basic ecological research is lacking on the diploid cytotype. Ecological niche modelling (ENM) was done on three known populations of the diploid cytotype, alongside a global site model, to determine site-specific abiotic influences and potential suitability within the U.S. The ENM was constructed using climatic and soil variables from public sources with resultant models compared to currently known populations of flowering rush throughout the U.S. Diploid flowering rush populations and the global site models were highly reliant on precipitation in the driest month (27–39% model contribution) and one site was highly reliant on precipitation seasonality (38% model contribution). Diploid flowering rush populations in this study displayed different responses towards abiotic factors; however, seasonal signaling of precipitation patterns are highly important. Furthermore, diploid flowering rush is predicted to invade numerous areas with less than suitable habitat which should warrant further monitoring to prevent further spread.

High endoreduplication after drought-related conditions in haploid but not diploid mosses.

Endoreduplication, the duplication of the nuclear genome without mitosis, is a common process in plants, especially in angiosperms and mosses. Accumulating evidence supports the relationship between endoreduplication and plastic responses to stress factors. Here, we investigated the level of endoreduplication in Ceratodon (Bryophyta), which includes the model organism Ceratodon purpureus. We used flow cytometry to estimate the DNA content of 294 samples from 67 localities and found three well-defined cytotypes, two haploids and one diploid, the haploids corresponding to C. purpureus and Ceratodon amazonum, and the diploid to Ceratodon conicus, recombination occurring between the former two. The endoreduplication index (EI) was significantly different for each cytotype, being higher in the two haploids. In addition, the EI of the haploids was higher during the hot and dry periods typical of the Mediterranean summer than during spring, whereas the EI of the diploid cytotype did not differ between seasons. Endopolyploidy may be essential in haploid mosses to buffer periods of drought and to respond rapidly to desiccation events. Our results also suggest that the EI is closely related to the basic ploidy level, but less so to the nuclear DNA content as previously suggested.

Does the evolution of micromorphology accompany chromosomal changes on dysploid and polyploid levels in the Barnardia japonica complex (Hyacinthaceae)?

BackgroundChromosome number and genome size changes via dysploidy and polyploidy accompany plant diversification and speciation. Such changes often impact also morphological characters. An excellent system to address the questions of how extensive and structured chromosomal changes within one species complex affect the phenotype is the monocot species complex of Barnardia japonica. This taxon contains two well established and distinct diploid cytotypes differing in base chromosome numbers (AA: x = 8, BB: x = 9) and their allopolyploid derivatives on several ploidy levels (from 3x to 6x). This extensive and structured genomic variation, however, is not mirrored by gross morphological differentiation.ResultsThe current study aims to analyze the correlations between the changes of chromosome numbers and genome sizes with palynological and leaf micromorphological characters in diploids and selected allopolyploids of the B. japonica complex. The chromosome numbers varied from 2n = 16 and 18 (2n = 25 with the presence of supernumerary B chromosomes), and from 2n = 26 to 51 in polyploids on four different ploidy levels (3x, 4x, 5x, and 6x). Despite additive chromosome numbers compared to diploid parental cytotypes, all polyploid cytotypes have experienced genome downsizing. Analyses of leaf micromorphological characters did not reveal any diagnostic traits that could be specifically assigned to individual cytotypes. The variation of pollen grain sizes correlated positively with ploidy levels.ConclusionsThis study clearly demonstrates that karyotype and genome size differentiation does not have to be correlated with morphological differentiation of cytotypes.

Antimicrobial and Hepatoprotective Activity of Different Cytotypes of Taraxacum officinale from the Indian Himalayas

ABSTRACT Three cytotypes (2×), (3×), and (4×) of Taraxacum officinale (Dandelion) from the Lahaul-Spiti region of Himachal Pradesh, India, were examined for their phytochemical composition and antimicrobial and hepatoprotective properties. The diploid cytotype had the highest concentration of cichoric acid (1.20% w/w), followed by the triploid and tetraploid cytotypes (0.21 and 0.14% w/w, respectively). The cichoric acid content decreased with increasing ploidy level. The ethanol extract of leaves from diploid cytotype exhibited the highest antibacterial activities against Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Klebsiella pneumonia, and Pseudomonas aeruginosa. As shown by the reduction in the activity of marker enzymes (AST, ALT, and ALP) and the restoration of total protein, bilirubin levels, and liver weight, the hepatoprotective activity was the greatest in the diploid cytotype. Although polyploidy is a key factor in plant evolution, it does not always have the same impact on secondary metabolites.

A sexual highly diploidized autotetraploid plant induced from the diploid cytotype of Paspalum chaseanum Parodi

AbstractPaspalum is one of the most common native grass genera of the New World grasslands, and it includes many wild natural forage species and rare domesticated ones. Polyploidy is widespread among the genus. Many species are multiploid, which basically involve two components: a sexual self‐incompatible diploid cytotype and a conspecific apomictic tetraploid, pseudogamous, and self‐compatible cytotype, though other apomictic polyploid levels may occur in the same species. Tetraploid individuals, reproducing exclusively sexually, have never been identified in a natural apomictic tetraploid Paspalum species. Doubling the chromosome number of a 2x cytotype is used to obtain a sexual autotetraploid to carry out sexual × apomictic crosses in breeding programs for plant improvement. After numerous attempts we generated only one autotetraploid plant of P. chaseanum (I4C) which reproduced sexually and was highly self‐incompatible. An unexpected preferential chromosome pairing resulted in the cytological diploidization of I4C. Despite each chromosome having three copies in I4C, meiotic chromosome configurations showed just 9.9% chromosomes forming multivalents (trivalents and quadrivalents), 82.1% bivalents, plus 7.9% univalents. The possible causes of this preferential pairing were analyzed. Although highly self‐incompatible, it was fertile in crosses following intra‐ and interspecific pollinations, but with variable seed set, from over 45% when crossed with an apomictic natural tetraploid strain of P. chaseanum, to 0.9% with a strain of P. wrightii. Thus, a novel autotetraploid P. chaseanum was synthesized with very interesting characteristics: sexual reproduction, high self‐incompatibility, variable cross‐compatibility, and high cytological diploidization. All valuable attributes for breeding programs in a forage grass.

Uniparentality: advantages for range expansion in diploid and diploid-autopolyploid species

AbstractUniparental reproduction, the capacity of an individual to produce offspring autonomously, is expected to facilitate range expansion of populations. Paspalum spp. reproduce uniparentally by sexual (self-fertility) and asexual (apomixis) reproduction and biparentally by sexual (self-sterility) reproduction. We evaluated the relationship between contrasting reproductive strategies (uni- and biparentality) and their impact on the colonizing ability and geographical range sizes of populations. We determined the cytotype composition of 16 populations of P. indecorum, P. cromyorhizon, P. pumilum and P. maculosum and assessed the sexual (self-fertile and self-sterile) and apomictic proportions by cyto-embryological analyses, fertility rates and seed flow cytometry. Data obtained regarding reproductive modes were compared to the distribution range of each cytotype and species. Sexual diploids with moderate degrees of self-fertility and mixed pollination syndromes showed wider distribution ranges than self-sterile diploids. In sexual diploids, increased rates of self-fertility relate to larger distribution areas. In agamic complexes, self-fertility reduces the differences in range sizes between biparental diploids and uniparental tetraploids. In such complexes, the range size of diploid cytotypes explains the range size and dispersal of apomictic tetraploids. Thus, uniparental reproduction via self-fertility and apomixis describes patterns of geographical parthenogenesis in South American species.

Intra-specific Variability in Anti-Diabetic Activity and UPLC Quantification of Oleanolic Acid from Two Morphotypes and Three Cytotypes of Achyranthes aspera

Achyranthes aspera is an economically important medicinal plant, exhibiting a wide range of cytomorphological diversity. Taking into account, its pharmacological activities, as well as variability, found within the species, the present attempt was made to study the detailed meiotic course in 13 plant samples collected from different areas of North India. The anti-diabetic activity of three different cytotypes (diploid, tetraploid, and hexaploid) and two different morphotypes (obovate-leaf morphotype and ovate-leaf morphotype) was investigated in alloxan-induced Swiss albino male rats. Additionally, phytochemical analysis of aerial parts of these cytotypes and morphotypes was performed using the UPLC technique. The meiotic analysis depicts the presence of three cytotypes with 2n=14, 28, and 42. The results indicated that the diploid cytotype of A. aspera has maximum activity against diabetes in alloxan-induced diabetic rats. Out of the two morphotypes, the obovate-leaf morphotype showed a potent anti-diabetic effect as compared to the ovate-leaf morphotype. UPLC results showed that the maximum percentage of active component oleanolic acid was present in diploid cytotype followed by tetraploid and hexaploid cytotypes. A comparative study of the two morphotypes within the tetraploid cytotype revealed that the obovate-leaf morphotype possessed a higher percentage of oleanolic acid as compared to the ovate-leaf morphotype.

Distinct Genetic Structure Reflects Ploidy Level Differentiation in Newly Discovered, Extremely Small Populations of Xanthocyparis vietnamensis from Southwestern China

Population genetic assessment is crucial for the conservation and management of threatened species. Xanthocyparis vietnamensis is an endangered species that is currently restricted to karst mountains in southwestern China and Vietnam. This rare conifer was first recorded in 2002 from northern Vietnam and then in 2013 from Guangxi, China, yet nothing is known about its genetic diversity nor ploidy level variation, although previous cytological study suggest that Vietnamese populations are tetraploids. There have been about 45 individuals found to date in Guangxi, China. Here, we genotyped 33 X. vietnamensis individuals using 20 newly developed, polymorphic microsatellite loci, to assess the genetic variability of its extremely small populations. The genetic diversity of X. vietnamensis (H E = 0.511) was lower than that of two other heliophile species, Calocedrus macrolepis and Fokienia hodginsii, which have similar distribution ranges. This is consistent with the signature of a genetic bottleneck detected in X. vietnamensis. Although the population genetic differentiation coefficient across loci is moderate (F ST = 0.125), STRUCTURE analysis revealed two distinct genetic clusters, namely the northern and southern population groups; DAPC analysis grouped the southern populations together in one cluster separate from the northern populations; AMOVA analysis detected a significant genetic differentiation between the two population groups (F RT = 0.089, p < 0.05), and BARRIER analysis detected a genetic barrier between them. Moreover, we detected differentiation in ploidy level between northern and southern populations, sampled individuals from the former and the later are all diploid and tetraploid cytotypes with mean genome sizes of 26.08 and 48.02 pg/2C, respectively. We deduced that heterogeneous geomorphology and historical events (e.g., human deforestation, Quaternary climate oscillations) may have contributed to population fragmentation and small population size in X. vietnamensis. Considering both genetic and ploidy level differentiation, we propose that two different management units (northern and southern) should be considered and a combination of in situ and ex situ conservation measures should be employed to preserve populations of this endangered species in southwestern China in the light of our findings.

Integrated analysis of mRNA-seq and miRNA-seq reveals the advantage of polyploid Solidago canadensis in sexual reproduction

BackgroundThe invasion of Solidago canadensis probably related to polyploidy, which may promotes its potential of sexual reproductive. S. canadensis as an invasive species which rapidly widespread through yield huge numbers of seed, but the mechanism remains unknown. To better understand the advantages of sexual reproduction in hexaploid S. canadensis, transcriptome and small RNA sequencing of diploid and hexaploid cytotypes in flower bud and fruit development stages were performed in this study.ResultsThe transcriptome analysis showed that in the flower bud stage, 29 DEGs were MADS-box related genes with 14 up-regulated and 15 down-regulated in hexaploid S. canadensis; 12 SPL genes were detected differentially expressed with 5 up-regulated and 7 down-regulated. In the fruit development stage, 26 MADS-box related genes with 20 up-regulated and 6 down-regulated in hexaploid S. canadensis; 5 SPL genes were all up-regulated; 28 seed storage protein related genes with 18 were up-regulated and 10 down-regulated. The weighted gene co-expression network analysis (WGCNA) identified 19 modules which consisted of co-expressed DEGs with functions such as sexual reproduction, secondary metabolism and transcription factors. Furthermore, we discovered 326 miRNAs with 67 known miRNAs and 259 novel miRNAs. Some of miRNAs, such as miR156, miR156a and miR156f, which target the sexual reproduction related genes.ConclusionOur study provides a global view of the advantages of sexual reproduction in hexaploid S. canadensis based on the molecular mechanisms, which may promote hexaploid S. canadensis owing higher yield and fruit quality in the process of sexual reproduction and higher germination rate of seeds, and finally conductive to diffusion, faster propagation process and enhanced invasiveness.

Breeding Polyploid Varieties of Acacia: Reproductive and Early Growth Characteristics of the Allotetraploid Hybrid (Acacia mangium × A. auriculiformis) in Comparison with Diploid Progenitors

Diploid clones of the hybrid acacia (Acacia mangium × A. auriculiformis) are widely planted in Vietnam because of their high productivity, adaptability, and commercial wood yields. Polyploid breeding offers possibilities for further enhancing hybrid vigor and generating new high value genotypes. In a field trial, we compared three diploid hybrid clones with their respective colchicine-induced tetraploid lines. Flowering and seed production of each cytotype were observed and open pollinated seed collected for determination of outcrossing rate and ploidy, inbreeding depression and marker inheritance in the progeny. Comparisons are also made with published characteristics of autotetraploids derived from A. mangium. Compared with their corresponding diploid cytotypes, the allotetraploids flowered slightly later but more intensely; produced the same number of seeds per pod but larger seeds; and showed a greatly reduced level of outcrossing (an average of 14% compared with 87%). Inbreeding depression for height growth was less for progeny from the allotetraploid lines (17%) than for those from the original diploids (33%). 96% of seeds from the allotetraploid clones were also tetraploid, but we observed triploids at low frequency at both the seed and field progeny stages. The segregation of the molecular markers in outcrossed allotetraploid progenies demonstrated both disomic and tetrasomic inheritance, indicating that the hybrid behaves as a segmental allotetraploid. Results suggest that an open pollinated breeding strategy is a practical option for improving polyploid acacia hybrids.

Solidago canadensis, a Flower or Weed?

Figure 1. Flowers or weeds? S. canadensis is one of the worst exotic weeds in China (A). Yet, farmers grow it as a cut flower (B). Because diploid cytotypes grow faster and flower earlier than other polyploids, all cultivated S. canadensis cytotypes for cut flowers are diploids. We found that diploids are sterile under hot-summer temperature south of 24°C-isotherm range and cultivation of S. canadensis will not lead to naturalization. However, the risk for naturalization increases in regions north of 20ºC-isotherm range. Photo credit: Sheng Qiang. Figure 2. Population of Solidago canadensis in native and introduced range in North America and China, respectively. S. canadensis plants are significantly taller and denser in the introduced range in China (Jiangsu) (A) than in their native range of North America (Pennsylvania, USA) (B). S. canadensis plants often outcompete native plants in invasive habitats and essentially become monoculture-type communities. Photo credit: Sheng Qiang. Figure 3. Dramatic difference in height of Solidago canadensis between native and invasive populations. S. canadensis plants can often be over 4m in height in China (Nanjing) (A), but are usually below 2m in North America (Ottawa, Canada) (B). Photo credit: Sheng Qiang. Figure 4. Field sampling site diagram. At each site, sampling plots were separated by at least 10 m to minimize the probability of sampling multiple ramets from individual genets. Photo credit: Guoqi Chen. Figure 5. Pollen germination and pollen tube growth on a stigma. (A) Pollen germination; (B) pollen tube growth; (C) penetration of pollen tubes into an ovary. Photo credit: Beibei Yao. These photographs illustrate the article “Solidago canadensis, a flower or weed?” by Sheng Qiang, Jiliang Cheng, Guoqi Chen, Beibei Yao published in Ecological Monographs. https://doi.org/10.1002/ecm.1445.

Genesis, Evolution, and Genetic Diversity of the Hexaploid, Narrow Endemic Centaurea tentudaica

Within the genus Centaurea L., polyploidy is very common, and it is believed that, as to all angiosperms, it was key in the history of its diversification and evolution. Centaurea tentudaica is a hexaploid from subsect. Chamaecyanus of unknown origin. In this study, we examined the possible autopolyploid or allopolyploid origin using allozymes and sequences of three molecular markers: nuclear-ribosomic region ETS, and low-copy genes AGT1 and PgiC. We also included three species geographically and morphologically close to C. tentudaica: C. amblensis, C. galianoi, and C. ornata. Neighbor-Net and Bayesian analyses show a close relationship between C. amblensis and C. tentudaica and no relationship to any of the other species, which suggest that C. tentudaica is an autopolyploid of C. amblensis. Allozyme banding pattern also supports the autopolyploidy hypothesis and shows high levels of genetic diversity in the polyploid, which could suggest multiple origins by recurrent crosses of tetraploid and diploid cytotypes of C. amblensis. Environmental niche modeling was used to analyze the distribution of the possible parental species during the present, Last Glacial Maximum (LGM), Last Interglacial Period (LIG), and Penultimate Glacial Maximum (PGM) environmental conditions. Supporting the molecular suggestions that C. tentudaica originated from C. amblensis, environmental niche modeling confirms that past distribution of C. amblensis overlapped with the distribution of C. tentudaica.

Taxonomic evaluations on the anatomical characters of leaf and achene in Turkish Tripleurospermum with its relative Matricaria (Asteraceae)

Tripleurospermum is a typically Mediterranean genus with ca. 40 species. It is widespread and diverse in Turkey with ca. 32 taxa, of which 12 are provided anatomical traits of both leaf and achene/cypsela, previously. In addition, the leaf anatomy of endemic T. baytopianum as well as the achene anatomy of endemic T. rosellum var. album with diploid cytotype are presented from Turkey. In this study, the anatomy of both leaf and achene of 15 taxa as well as achene anatomy of T. baytopianum and T. rosellum var. album with tetraploid cytotype were described in detail from Turkey. The leaf and achene anatomical characteristics of Turkish Tripleurospermum and its closely related genus Matricaria using multivariate analyses (cluster analysis, principal components analysis) were evaluated in taxonomic context. Additional data of further species of Tripleurospermum as well as Matricaria (four taxa) were taken from the literature for multivariate analyses. All studied taxa have the basic leaf structure with lower and upper epidermis including anomocytic stomata, parenchymatic mesophyll, and vascular bundle. The pericarp is composed of several layers of parenchymatous and sclerenchymatous cells with one vascular bundle in each rib. The thickness of palisade sclerenchyma in the pericarp, the vascular bundle length in the midrib of the leaf, and presence or absence of a slime envelope have high taxonomic value at interspecific/intergeneric levels.

Cytotypes of bracken (Pteridium aquilinum) in Europe: widespread diploids and scattered triploids of likely multiple origin

Polyploidization is an important speciation and evolution mechanism in ferns. Initially, new cytotypes face challenges in maintaining themselves within the majority cytotype populations. Unlike in most even-ploidy cytotypes, fern triploids are often apomictic or infertile, due to genetic imbalance. An interesting opportunity to study these phenomena has emerged with the discovery of triploid bracken (Pteridium aquilinum), a triploid fern that is fertile but not apomictic. Originally found in one Welsh population, the distribution of this cytotype in Europe is unknown as is its origin and how it maintains itself among the presumed diploid majority. We sampled 135 populations of P. aquilinum, focusing on Central Europe. Ploidy level of all samples was analyzed by flow cytometry. We compared the two cytotypes via micromorphological characters (spore and stomata size), fertility characteristics (spore abortion and proportion of populations with sporangia-bearing fronds). Additionally, genetic difference between ploidy levels was tested as well. The diploid cytotype of P. aquilinum is dominant in continental Europe with 121 entirely diploid populations found, but we also found 9 mixed and 5 entirely triploid populations. Fertile diploid and triploid plants were found only in 17.7% and 21.4% of populations, respectively. The cytotypes are distinguishable using both tested micromorphological characters, but stomata are more reliable due to overall reduced fertility. Unlike the Welsh specimen, our tested triploid has most spores aborted, ca 97.4%, compared to mean 6.0% of spores aborted in diploids. The triploid cytotype is rare and likely originated multiple times from the diploids and relies on clonal and possibly limited sexual reproduction to maintain itself. However, diploids and triploids are often genetically different within a population, indicating that the triploid may migrate between populations. Due to its vegetative growth and presumed continuous formation, the triploid cytotype is likely to remain established in Central Europe, although in small numbers.

Morphological and cytogenetic analysis of different cytotypes of Boerhaavia diffusa L. and their evaluation for biological activity

Boerhavia diffusa is an herbaceous plant of the family Nyctaginaceae. It is mentioned to have anti-aging, disease prevention, and life-enhancing activities that hold enormous influence on disease burden and affordability/availability of healthcare in the world. In the present study, various populations of this plant species were characterized using morphological markers, cytogenetic and phytochemical analyses. Besides, biological activity was also studied for anti-diabetic and anti-bacterial effects using rats and bacterial systems respectively. For the three cytotypes i.e. diploid, tetraploid and octaploid, twelve morphological traits were studied. HPLC system was used for the identification of bioactive compounds present in extracts. The in vivo anti-hyperglycemic effects of the extract in alloxan- induced diabetes were determined using Swiss albino rats. Rats with fasting plasma glucose vary ≥ 300–315 mg/dl were treated to be diabetic and utilized for the examination. Anti-bacterial activities of three cytotypes of B. diffusa were screened by using the Disc diffusion method against two strains of Gram-positive bacteria and three strains of Gram-negative bacteria. The present study reports the existence of diploid, tetraploid and octaploid cytotypes which was new report from India. The diploid cytotype of B. diffusa showed maximum activity against diabetes in alloxan-induced diabetic rats and more anti-bacterial activity in Gram-positive and Gram-negative bacteria as compared to tetraploid and octaploid cytotypes. HPLC results showed that the concentration of phytoconstituents showed a negative correlation with the ploidy level being maximum in plants with lower ploidy levels. The present paper represents the first report of the pharmacognostic study based on different plant parts and cytotypes in B. diffusa. Results of the present study will be useful for exploiting appropriate cytotypes for various purposes such as formulating new anti-diabetic and anti-bacterial drugs from B. diffusa.

Disparity between morphology and genetics inUrtica dioica(Urticaceae)

AbstractPolyploidization is generally considered a major evolutionary force that can alter the genetic diversity, morphology, physiology and ecology of plants. One striking example is the polyploid Urtica dioica complex, in which diploid taxa are often found in remote and partly relictual geographical ranges, in contrast to tetraploid individuals, which have an unknown evolutionary history and occur in a variety of synanthropic habitats. We used a set of 279 plants, evenly representing the geographical and morphological variation of U. dioica s.l. in Europe and Southwest Asia, and employed multivariate and geometric morphometrics and Hyb-Seq sequencing to estimate the extent of differentiation of diploid taxa and the ubiquitous tetraploid cytotype. Diploid subspecies form more-or-less separate clusters in morphological analyses, but our molecular evaluation did not reveal any structure. Moreover, tetraploids coalesced with diploids in both morphological and molecular analyses. This disparity between morphological and molecular data might be driven by (1) local adaptation of the diploid cytotype that is mirrored in specific phenotypes, (2) only recent genetic diversification of the group and (3) homoploid and heteroploid hybridization events.

Genetic transfer from several apomictic tetraploid Paspalum species to an elite group of sexual plants

AbstractBasic findings from classical genetic studies are available for exploiting apomixis in the breeding of several forage grass genera. Most Paspalum species are multiploid with a sexual diploid cytotype and conspecific apomictic polyploid (mainly tetraploid) cytotypes. Experimental tetraploidized diploids reproduce sexually and, when crossed with natural apomictic tetraploids, yield hybrid populations that segregate for reproductive mode. Genetic studies indicated that apomixis is inherited as a monogenic dominant factor. We recombined 50 selected sexual hybrids obtained from crosses between a tetraploidized sexual genotype of P. plicatulum and 9 natural apomictic tetraploid accessions of 6 species of the Plicatula group. A synthetic sexual tetraploid population (SSTP) of 600 individuals from mixed seed of the 50 intercrossed hybrids was space‐planted in the field. Based on evaluations of plant vigor, seed set, ergot tolerance, regrowth after flowering, and cold tolerance, 31 plants were selected. Crosses between most selected plants and two testers, which belonged to P. guenoarum, were performed, and the generated progeny was planted into the field following a randomized block design with 3 replications. The progeny test was evaluated for seed fertility, biomass yield, and cold tolerance. This procedure allowed selection of 10 elite plants from the SSTP. These plants should contain genes recombined from six apomictic species, without the genetic determinants for apomixis. They may be polycrossed to generate an improved sexual population, or crossed with other apomictic genotypes to obtain improved apomictic hybrids.

UPLC-DAD Assisted Phytochemical Quantitation Reveals a Sex, Ploidy and Ecogeography Specificity in the Expression Levels of Selected Secondary Metabolites in Medicinal Tinospora cordifolia: Implications for Elites' Identification Program.

Medicinal phytochemistry involving UPLC-DAD in an exhaustive analysis involving quantification of eight commercially important phytochemicals viz. syringin, cordifolioside A, magnoflorine, tinocordiside, palmatine, 20β-hydroxyecdysone, L-tetrahydropalmatine and berberine has been done in 143 accessions from eight states and the union territories of Delhi and Jammu & Kashmir of India representing three different ploidy levels viz. diploid (2x), triploid (3x) and synthetic tetraploid (4x). The study was done to assess the effect of sex, ploidy level and ecogeography on the expression level of secondary metabolites in stems of dioecious, medicinally important shrub Tinospora cordifolia. Two different UPLC-DAD methods were used for the quantification of eight selected phytochemicals from the alcoholic stem extracts of T. cordifolia accessions. The Waters Acquity UPLC system hyphenated to the QTOF micromass system, equipped with PDA and ESI-Q-TOF detectors was utilized for the quantitative analysis, Mass Lynx v 4.0 software was used for data analysis. Significant quantitative changes were observed in the analysed secondary metabolites among different accessions of T. cordifolia. The triploid (3x) cytotypes revealed higher amounts of seven out of eight analysed secondary metabolites than diploids and only 20β-hydroxyecdysone was observed to be present in significantly higher amount in diploid cytotypes. Further, at the tetraploid level, novel induced colchiploid (synthetic 4x) genotypes revealed increase in the yield of all of the analysed eight phytochemicals than their respective diploid counterparts. The quantity of active principles in tetraploid cytotypes were also higher than the average triploid levels at multiple locations in five out of eight tested phytochemicals, indicating the influence of ploidy on expression levels of secondary metabolites in T. cordifolia. Additionally, at each of the three ploidy levels (2x, 3x and synthetic 4x), a significant sex specificity could be observed in the expression levels of active principles, with female sex outperforming the male in the content of some phytochemicals, while others getting overexpressed in the male sex. The manifestation of diverse ecogeographies on secondary metabolism was observed in the form of identification of high yielding accessions from the states of Madhya Pradesh, Delhi and Himachal Pradesh and the Union territory of Jammu & Kashmir. Two triploid female accessions that contained approximately two- to eight fold higher amounts of five out of the eight analysed phytochemicals have been identified as superior elites from the wild from the states of Delhi and Madhya Pradesh. The paper shows the first observations of ploidy specificity along with subtle sex and ecogeography influence on the expression levels of secondary metabolome in T. cordifolia.

Ploidy-Dependent Effects of Light Stress on the Mode of Reproduction in the Ranunculus auricomus Complex (Ranunculaceae).

Polyploidy in angiosperms is an influential factor to trigger apomixis, the reproduction of asexual seeds. Apomixis is usually facultative, which means that both sexual and apomictic seeds can be formed by the same plant. Environmental abiotic stress, e.g. light stress, can change the frequency of apomixis. Previous work suggested effects of stress treatments on meiosis and megasporogenesis. We hypothesized that polyploidy would alter the stress response and hence reproductive phenotypes of different cytotypes. The main aims of this research were to explore with prolonged photoperiods, whether polyploidy alters proportions of sexual ovule and sexual seed formation under light stress conditions. We used three facultative apomictic, pseudogamous cytotypes of the Ranunculus auricomus complex (diploid, tetraploid, and hexaploid). Stress treatments were applied by extended light periods (16.5 h) and control (10 h) in climate growth chambers. Proportions of apomeiotic vs. meiotic development in the ovule were evaluated with clearing methods, and mode of seed formation was examined by single seed flow cytometric seed screening (ssFCSS). We further studied pollen stainability to understand effects of pollen quality on seed formation. Results revealed that under extended photoperiod, all cytotypes produced significantly more sexual ovules than in the control, with strongest effects on diploids. The stress treatment affected neither the frequency of seed set nor the proportion of sexual seeds nor pollen quality. Successful seed formation appears to be dependent on balanced maternal: paternal genome contributions. Diploid cytotypes had mostly sexual seed formation, while polyploid cytotypes formed predominantly apomictic seeds. Pollen quality was in hexaploids better than in diploids and tetraploids. These findings confirm our hypothesis that megasporogenesis is triggered by light stress treatments. Comparisons of cytotypes support the hypothesis that ovule development in polyploid plants is less sensitive to prolonged photoperiods and responds to a lesser extent with sexual ovule formation. Polyploids may better buffer environmental stress, which releases the potential for aposporous ovule development from somatic cells, and may facilitate the establishment of apomictic seed formation.