Tetraploid Plants Research Articles

Cell line screening of Catharanthus roseus for high yield production of ajmalicine

Cell line screening of Catharanthus roseus for high yield production of ajmalicine was conducted through subculturing of callus that induced from tetraploid plant leaves. The cell line was characterized as green colour with photosynthetic capacity. The content of ajmalicine reached 2.578 and 0.895 mg/g DW in callus and in cell suspension culture, respectively. The cell suspension culture treated with methyl jasmonate (MeJA) harvested a drastic increase of ajmalicine yield (2.2 mg/g DW in cell and 2 mg/L in culture medium). Therefore, the cell line had potential commercial value. Key words: Catharanthus roseus, ajmalicine production, callus culture, cell suspension culture, methyl jasmonate.

Induction of Anthurium andraeanum “Arizona” tetraploid by colchicine in vitro

Tetraploids plants of Anthurium andraeanum “Arizona” were successfully induced after treating diploid tissue masses with colchicine. Masses originating from diploid aerial roots were treated with colchicine at three different concentrations (i.e., 0.1, 0.2, 0.3%) for about 3, 5 and 7 h, and then were transferred into Murashige and Skoog medium containing 3 mg/l BAP + 0.2 mg/l 2,4-D. After 60 days, the survival rate and numbers of regenerative shoots were scored. The high concentration and longer duration sharply reduced survival rate. In contrast, the regeneration of plantlets was not noticeably affected by colchicine. Tetraploid plants were obtained in all treatments, but the percentage of induced tetraploids ranged from 0.2 to 7.6%. The best induction was obtained with a 5-h, treatment with 0.3% colchicine. The stomatal size of tetraploid plants was larger than in diploid plants; however, the stomatal density was lower than in diploid plants. Tetraploid plants possessed stronger petioles, thicker and deeper green leaves, and thicker and longer lived spathes in comparison with diploid plants. Abnormal spathes, such as double spathes or those lacking pedicels, were observed in tetraploid plants. Tetraploid plantlets could be regenerated via aerial roots; this technique could be applied to tetraploid plant propagation.

Ribosomal FISH mapping reveals hybridity in phytoestrogen producing Curcuma species from Thailand

Species in the genus Curcuma (Zingiberaceae) that are cultivated widely in Thailand for their phytoestrogen-producing rhizomes are called wan-chak-motluk. Five cultivars belonging to Curcuma comosa (cultivars with 2n = 42 and 63) and Curcuma elata (2n = 63) were examined using the molecular cytogenetic method of fluorescence in situ hybridisation (FISH) in order to identify genetic relationships among these cultivars based on chromosomal maps of the 18S–25S ribosomal loci. The results revealed hybrid features in this Curcuma species group and a significant similarity among wan-chak-motluk cultivars. The main features included: (1) the presence of the single largest ribosomal site, assigned the Cc1 marker site, in the somatic 2n complement of all cultivars, and (2) the odd numbers of ribosomal sites in the complements, most often in sets of three. We therefore propose that the cultivar with 2n = 42 (C. comosa) is a homoploid hybrid species comprised of two different ancestral genomes and has a diploid status with the basic chromosome number x = 21. The cultivars with 2n = 63 (C. comosa and C. elata) are most probably triploids arising within the 2n = 42 diploid species/cultivars via a meiotic modification, rather than from hybridisation between diploid and tetraploid plants. The knowledge about genetic and genomic relationships among wan-chak-motluk cultivars will be important in the research projects that aim to explore and promote new plant materials for cultivation.

DNA Methylation in Genomes of Several Annual Herbaceous and Woody Perennial Plants of Varying Ploidy as Detected by MSAP

Polyploidization is known to accompany altered DNA methylation in higher plants, which plays an important role in gene expression regulation and maintaining genome stability. While the characteristics of DNA methylation in different polyploid plants are still to be elucidated; here, status of genomic DNA methylation in a series of diploid, triploid, and tetraploid annual herbaceous plants (watermelon and Salvia) and woody perennials (pear, Poplar, and loquat) were explored by methylation-specific amplified polymorphism analysis. The results indicated that levels of DNA methylation in triploid watermelon and Salvia were lower than their diploid parents. In triploid Poplar and pear, higher levels of DNA methylation were detected, and no significant difference was observed between triploid and tetraploid in all tested materials. Further data analysis suggested that about half of the total detected sites underwent changes of DNA methylation patterns in triploid watermelons and Salvia, as well as an obvious trend towards demethylation. However, the changes of DNA methylation patterns in three triploid woody perennials were only 17.54–33.40%. This implied that the characteristics of DNA methylation are significantly different during the polyploidization of different plant species. Furthermore, the results suggested that the level of DNA methylation was nonlinearly related to the ploidy level, and triploid plants displayed more interesting DNA methylation status. The characteristics and possible functions of DNA methylation in different ploidy series are further discussed.

Comparative assessment of genetic and epigenetic variation among regenerants of potato (Solanum tuberosum) derived from long-term nodal tissue-culture and cell selection

Three long-term nodal tissued cultured Russet Burbank potato clones and nine thaxtomin A-treated regenerant lines, derived from the nodal lines, were assessed for genetic and epigenetic (in the form of DNA methylation) differences by AFLP and MSAP. The treated regenerant lines were originally selected for superior resistance to common scab disease and acceptable tuber yield in pot and field trials. The long-term, tissue culture clone lines exhibited genetic (8.75-15.63% polymorphisms) and epigenetic (12.56-26.13% polymorphisms) differences between them and may represent a stress response induced by normal plant growth disruption. The thaxtomin A-treated regenerant lines exhibited much higher significant (p < 0.05) genetic (2-29.38%) and epigenetic (45.22-51.76%) polymorphisms than the nodal cultured parent clones. Methylation-sensitive mutations accumulated within the regenerant lines are significantly correlated (p < 0.05) to disease resistance. However, linking phenotypic differences that could be of benefit to potato growers, to single gene sequence polymorphisms in a tetraploid plant such as the potato would be extremely difficult since it is assumed many desirable traits are under polygenic control.

The Effects of Induction of Autotetraploidy on the Essential Oil Content and Compositions of Dragonhead (Dracocephalum moldavica L.)

Induction of autotetraploidy in dragonhead (Dracocephalum moldavica L.) had a significant effect on its essential oil content and compositions. The seedlings of dragonhead were treated with colchicine by applying dropping method in the stage of emergence of two true leaves. Ploidy level in the treated plants was estimated by flow cytometric analysis. The essential oils of both tetraploid plants and diploid parents were extracted by hydro distillation technique and were analyzed by capillary GC and GC-MS. The essential oil content of herb was increased 27.5 % in tetraploid plants. 30 components were identified in the oils of both diploid and tetraploid plants. The major components of the oil of diploid plants were geranyl acetate (40.4 %), geraniol (16. 3%), Z-citral (14.1 %), E-citral (9.9 %), nerol (5.8 %) and neryl acetate (5.4 %), While the main components of the oil of tetraploid plants were geranyl acetate (36.3 %), geraniol (19.5 %), Z-citral (15.4 %), E-citral (9.1 %), nerol (5.5 %) and neryl acetate (4.7%).

Meiose analyses of sugar beet triploids

During the seed crop growing there can be also problems with uncontrolled pollination if in the previous years there was seed shedding during the harvest, which can cause growth and flowering of diploid, triploid and tetraploid plants and that can present serious problem for the next sugar beet seed crop. Meiosis of triploid hybrids is irregular and can result in development of gametes with different chromosome number. Sugar beet triploids in metaphase I have 9 univalents and 9 bivalents and therefore can be source of aneuploid gametes. After cytological analysis that was performed on 2124 meiocytes, in 14.64% different types of irregularities were detected, such as dislocated chromosomes and chromosome bridges.

Generation of autotetraploid plant of ginger (Zingiber officinale Rosc.) and its quality evaluation.

Background:Zingiber officinale Rosc. is not only an important medical plant in China, but also one of the most commonly used plant spices around the world. Early researches in Z. officinale Rosc. were focused on rapid propagation, germplasm preservation, and somatic embryogenesis, only a few reports focused on the generation of tetraploid ginger plants with colchicines treatment in vitro.Materials and Methods:The adventitious buds were submerged into different concentrations of colchicine water solution for different time to induce polyploid plants, and the induced buds were identified by root-tip chromosome determination and stomatal apparatus observation. Eighteen selected tetraploid lines were transferred to the field, and the leaf characteristics, rhizome yield, contents of volatile oil and gingerol were respectively evaluated to provide evidence of high-yield and good qualities of tetraploid ginger.Results:The induction rate reached as high as 33.3% of treated buds. More than 48 lines of autotetraploid plants were obtained. All tetraploid plants showed typical polyploidy characteristics. All of the 18 selected tetraploid lines possessed higher rhizome yield and overall productivity of volatile oil and gingerol than those of the control.Conclusion:Five elite lines have been selected for further selection and breeding new varieties for commercial production in agricultural production.

Induction and flow cytometry identification of tetraploids from seed-derived explants through colchicine treatments in Catharanthus roseus (L.) G. Don.

The tetraploid plants of Catharanthus roseus (L.) G. Don was obtained by colchicine induction from seeds explants, and the ploidy of the plants was identified by flow cytometry. The optimal treatment is 0.2% colchicine solution treated for 24 hours, and the induction rate reaches up to 30%. Comparing with morphological characteristics and growth habits between tetraploids and the control, we found that tetraploids of C. roseus had larger stoma and more branches and leaves. HPLC analysis showed tetraploidization could increase the contents of terpenoid indole alkaloids in C. roseus. Thus, tetraploidization could be used to produce higher alkaloids lines for commercial use. QRT-PCR results showed that the expression of enzymes involved in terpenoid indole alkaloids biosynthesis pathway had increased in the tetraploid plants. To our knowledge, this was the first paper to explore the secondary metabolism in autotetraploid C. roseus induced by colchicine.

Development of Autotetraploid Perennial Rye (Secale montanum Guss.) and Selection for Seed Set

Different concentrations of colchicine solution (0. 1% and 0.2%) and temperatures (24 and 27 o C) were applied to wild diploid rye and autotetraploid forms were produced. Five hundred seedlings were placed into Petri dis hes for each treatment (total 2000). Cytological research was ca rried out on root tips of plants in C 0 and C 1 generations. Live seedling rate and tetraploid plant rate were change d by 6.4 ‐ 37.4 % and 8.75 ‐ 37.04%, respectively based on treatments. Plants forming the C1 generation were determined as 68.12% tetraploid, 14.71% diploid, 16.89% aneuploid (13.35% hypoploid, 3.57% hyperploid) and 0.27% triploid. Chromosome numbers of aneuploid plants ranged between 2n=19 - 29 and it was determined that plants having a chromosome number of 2n=27 were the most common. Other aneuploid plants (2n=19 - 26) were rare. Prior to chromosome counting in the C 1 generation, the seed lots were divided into five seed size groups (9-11 g, 11-13 g, 13-19 g, 15-17g, 17-19 g). Chromosome numbers were determined by a modification of the conventional root tip squash technique. Chromosome numbers varied with seed size. The rate of tetraploids, diploids and aneuploi ds were varied between 17.86-87.25%, 2.04-42.86%, 10.71- 39.29 %, respectively. As the size of seed was increased, th e rate of diploid and aneuploids decreased. Seed se t rate, spike length and the number of spikelets in plants belonging to different generations (C1, C 2, C 3 and C 4) were investigated. Seed set rate was increased from 30.51% to 61.45% with the selection of plants passing over general average in respect to seed set in each generation, however, it was determined that spike length and number of spikelets decreased. The differences were generally highly significant.

Influence of ploidy level on morphology, growth and drought susceptibility in Spathiphyllum wallisii

In this study, we analysed morphological, anatomical and physiological effects of polyploidisation in Spathiphyllumwallisii in order to evaluate possible interesting advantages of polyploids for ornamental breeding. Stomatal density was negatively correlated with increased ploidy level. Stomatal size increased in polyploids. Tetraploid Spathiphyllum plants had more ovate and thicker leaves. The inflorescence of tetraploids had a more ovate and thicker spathum, a more cylindrical spadix and a thicker but shorter flower stalk. Biomass production of the tetraploids was reduced, as expressed by lower total dry weights, and tetraploids produced fewer shoots and leaves compared with their diploid progenitors. Furthermore, tetraploid Spathiphyllum plants were more resistant to drought stress compared with diploid plants. After 15 days of drought stress, diploids showed symptoms of wilting, while the tetraploids showed almost no symptoms. Further, measurements of stomatal resistance, leaf water potential, relative water content and proline content indicated that the tetraploid genotypes were more resistant to drought stress compared with the diploids.

Highly efficient plant regeneration and in vitro polyploid induction using hypocotyl explants from diploid mulberry (Morus multicaulis Poir.)

Efficient plant regeneration is essential for successful transformation and in vitro polyploidy induction in mulberry. A high frequency (80%) of plant regeneration from hypocotyls occurred under in vitro conditions in mulberry (Morus multicaulis Poir.). We identified three key factors for enhancing successful regeneration based on earlier work: (1) hypocotyl position, (2) the combination and concentration of growth regulators, and (3) the addition of AgNO3. The highest frequency of shoot regeneration was achieved using hypocotyl segments, which are proximal to apical meristems, and the optimal culture conditions were Murashige and Skoog’s (MS) (Murashige and Skoog, 1962) basal medium supplemented with 3.0 mg l−1 6-benzylamino purine, 0.3 mg l−1 indole-3-acetic acid, 0.1% polyvinypyrrolidone, and 1.0 mg/l silver nitrate (AgNO3) under subdued light at 25 ± 2°C. Treating the shoots with 0.2% colchicine (dipping for 72 h) resulted in a 14% tetraploid frequency, whereas a 20% tetraploid frequency resulted from using a 0.25% colchicine (dripping for 5 d) treatment, as determined by chromosome number counts. The diploid plant chromosome number was 28 (2n = 2x = 28) and that of tetraploid plants was 56 (2n = 4x = 56). Regenerated shoots rooted easily in 8–10 d using half-strength basal MS medium with 0.5 mg l−1 indole-3-butyric acid and were successfully established in the soil.

Breeding barriers at a diploid–hexaploid contact zone in Aster amellus

Polyploidization is an important mechanism of sympatric speciation, but few studies have addressed breeding barriers between polyploids and their diploid progenitors in the field, and the available data have been mainly obtained from diploid-tetraploid contact zones. In contrast to diploid-tetraploid complexes, hybridization between diploid and hexaploid individuals may lead to viable fertile tetraploid offspring, and thus the interactions between these ploidy levels can be more complex. We investigated the breeding barriers operating between diploid and hexaploid individuals of Aster amellus at a contact zone in Central Europe to understand the absence of hybrids (i.e., tetraploids) and mixed populations. Phenological segregation, assortative mating mediated by pollinators and crossing ability were assessed under natural and controlled conditions in diploid and hexaploid populations growing in close proximity. The results revealed low levels of reproductive isolation (RI) due to flowering phenology (RI = 11–45%) and pollinator behavior (RI = 17%), so that pollen transfer between diploids and hexaploids is possible. In contrast, almost complete reproductive isolation was observed due to a series of post-pollination barriers that significantly reduced the production of offspring from inter-cytotype crosses (RI = 99.9%), even though some tetraploids were detected in seeds and seedlings. We conclude that the absence of tetraploids at the contact zone is probably due to a combination of several factors, including spatial segregation, strong post-pollination barriers (such as gametic isolation, low viability of tetraploid seeds and/or inability of tetraploid plants to reach the flowering stage), and to a lesser extent, temporal and behavioral segregation. Future studies should explore the fitness of tetraploids and the effect of different traits on the reproductive success and fitness of each cytotype. This will enable a fuller understanding of the dynamics and mechanisms acting in contact zones.

Genome Size and Chromosome Analyses in Prairie Cordgrass

The next generation of bioenergy crops will be grown on marginal lands. Prairie cordgrass (Spartina pectinata Link) is well suited to marginal land because of its tolerance to stresses such as salinity and water fluctuations. Developing prairie cordgrass as an energy crop requires genomic information such as genomic size and ploidy level. The 2C nuclear genome size of 11 prairie cordgrass populations, originating from Illinois, North Dakota, and South Dakota, were determined by flow cytometry using somatic G1 nuclei, and the ploidy variation confirmed by counting the chromosome number. One population from each of North Dakota and South Dakota, as well as the Red River, had genome sizes of 3.1 pg of DNA per G1 somatic nucleus whereas, the majority of the eight populations from Illinois had a genome size of 1.6 pg. One plant from one of the Illinois populations had an estimated genome size of 2.3 pg. These genome sizes of 3.1 and 1.6 pg, along with their respective chromosome numbers of 80 and 40 indicate octoploid and tetraploid plants. The chromosome number of 60 observed in the plant with 2.3 pg indicated that this plant was hexaploid. This study provides the first estimates of genome size for prairie cordgrass and is also the first to report observed hexaploidy in this species. This genomic information provides critical knowledge for designing efficient breeding schemes for plant improvement.

Induction of tetraploidy in Juncus effusus by colchicine

Tetraploidy was induced in vitro in mat rush (Juncus effusus L.) cultivar Nonglin-4 by exposure to colchicine (0, 50, 100 and 500 mg dm−3) for 6, 12 and 24 h. Flow cytometric analysis was used to confirm the ploidy level. Anatomical and ultrastructural analyses at cellular and subcellular levels in tetraploid and diploid control plants revealed differences between diploid and tetraploid plants. The leaf epidermis had larger stomata but lower stomatal density in tetraploid plants. In addition, mesophyll cells in tetraploid plants appeared more compact and showed less intercellular spaces along with increased size of vascular bundles. However, a significant reduction of chlorophyll content was observed in tetraploid plants that might be the result of structural modification in the lamellar membranes of chloroplasts.

Meiotic behavior in Panicum maximum Jacq. (Poaceae: Panicoideae: Paniceae): hybrids and their genitors

Panicum maximum Jacq. is one of the most cultivated grasses in the world. The intraspecific hybridization breeding program underway at Embrapa Beef Cattle aims at combining several agronomic characteristics in a single plant with high seed production. Four intraspecific hybrids originated from the cross between sexual tetraploid female plants with apomictic tetraploid male plants, including the genitors, were cytologically evaluated in the meiotic process throughout conventional methodology. Hybrids and genitors showed high meiotic stability. The percentage of abnormal cells among them ranged from 6.7 to 14.2%. Abnormalities recorded were irregular chromosome segregation, chromosome stickiness, and absence of the first cytokinesis, but the latter did not compromise pollen viability. Multivalent chromosome configurations at diakinesis, ranging from one to three quadrivalents, suggest that genetic recombination and introgression of some genes can be expected in the hybrids. Based on meiotic stability, the hybrids should be selected for high seed production, and the genitors could remain in the program participating in other intraspecific crosses.

Effect of ploidy increase on transgene expression: example from Citrus diploid cybrid and allotetraploid somatic hybrid expressing the EGFP gene

Polyploidization is an important speciation mechanism for all eukaryotes, and it has profound impacts on biodiversity dynamics and ecosystem functioning. Green fluorescent protein (GFP) has been used as an effective marker to visually screen somatic hybrids at an early stage in protoplast fusion. We have previously reported that the intensity of GFP fluorescence of regenerated embryoids was also an early indicator of ploidy level. However, little is known concerning the effects of ploidy increase on the GFP expression in citrus somatic hybrids at the plant level. Herein, allotetraploid and diploid cybrid plants with enhanced GFP (EGFP) expression were regenerated from the fusion of embryogenic callus protoplasts from 'Murcott' tangor (Citrus reticulata Blanco × Citrus sinensis (L.) Osbeck) and mesophyll protoplasts from transgenic 'Valencia' orange (C. sinensis (L.) Osbeck) expressing the EGFP gene, via electrofusion. Subsequent simple sequence repeat (SSR), chloroplast simple sequence repeat and cleaved amplified polymorphic sequence analysis revealed that the two regenerated tetraploid plants were true allotetraploid somatic hybrids possessing nuclear genomic DNA of both parents and cytoplasmic DNA from the callus parent, while the five regenerated diploid plants were cybrids containing nuclear DNA of the leaf parent and with complex segregation of cytoplasmic DNA. Furthermore, EGFP expression was compared in cells and protoplasts from mature leaves of these diploid cybrids and allotetraploid somatic hybrids. Results showed that the intensity of GFP fluorescence per cell or protoplast in diploid was generally brighter than in allotetraploid. Moreover, same hybridization signal was detected on allotetraploid and diploid plants by Southern blot analysis. By real-time RT-PCR and Western blot analysis, GFP expression level of the diploid cybrid was revealed significantly higher than that of the allotetraploid somatic hybrid. These results suggest that ploidy level conversion can affect transgene expression and citrus diploid cybrid and allotetraploid somatic hybrid represents another example of gene regulation coupled to ploidy.

Copper Uptake Is Differentially Modulated by Phenylalanine Ammonia-lyase Inhibition in Diploid and Tetraploid Chamomile

The effect of phenylalanine ammonia-lyase (PAL) inhibition by 2-aminoindane-2-phosphonic acid (AIP) in copper-exposed diploid and tetraploid chamomile (Matricaria chamomilla ) roots has been studied in a short-term experiment (24 h). Cu evoked stronger induction of PAL activity and accumulation of soluble phenols, flavonols (quercetin and kaempferol), and lignin in diploid plants, whereas AlCl3-reactive flavonoids and phenolic acids did not differ with respect to ploidy. Amounts of hydrogen peroxide and superoxide also preferentially increased in diploid. Surprisingly, PAL activity was restored in both +AIP cultivars, being inversely correlated with the accumulation of free phenylalanine. Notwithstanding this, total soluble phenols and flavonols were more depleted in Cu+AIP diploid roots. Soluble proteins decreased in response to Cu, and AIP had no effect. Among free amino acids, proline increased more visibly in Cu+AIP diploid, suggesting that this could be a protective mechanism in conditions with depleted content of phenols. Decrease in potassium content was ploidy-independent, calcium increased in all Cu variants, and Fe increased in Cu-exposed tetraploid. Shoot Cu content did not differ in Cu-exposed cultivars, but diploid roots contained more Cu. AIP decreased root Cu but increased shoot Cu amounts in diploid, whereas tetraploid plants did not exhibit similar responses. These data indicate that inhibition of root phenolic metabolism by AIP was effective enough, allowing Cu to accumulate in diploid shoots. The present findings are discussed in the context of available data about AIP effects and with respect to the role of phenols in metal uptake.

STUDY ON THE EMBRYOGENESIS OF CROSSES OF TETRAPLOID WITH DIPLOID IN CUCUMBER

To investigate the reasons behind the seeding difficulty, pollination and embryonic developments of cucumber from crosses of autotetraploid with diploid (4xx4 x , 2xx2x, 4xx2x, 2x×4x) were studied. Results showed: 1) pollen germination rate on the stigma of self-pollinated tetraploid (4x×4x) were the lowest, and in 2x×2x, 2x×4x, the percentage of pollen germination on stigma were higher; 2) tetraploid plant have a higher abnormal embryo sacs rate (from 17.0s to 24.90%) than that of diploid (from 4.8 to 6.5%); 3) the fertilization process of self-pollinated diploid, self-pollinated tetraploid, crosses of tetraploid with diploid (4xx2x) were from 24 to 36 h, from 48 to 60 h, from 36 to 48 h respectively. No fertilization was observed in crosses of diploid with tetraploid (2X×4X). The fertilization rates of self-pollinated tetraploid were lower (19.17 to 25.39%) than that of 4x×2x (30.25 to 48.08%); 4). The embryo developments of self-pollinated diploid and tetraploid were normal, whereas in 4xx2x, the embryo development was abnormal and developed very slowly, which were the key reason of sterility. Therefore, no mature triploid seeds have been obtained; and 5) In 4xx2x, the obvious degeneration of the endosperm was the important factor of the slow embryo development.

A new cytotype of Jacobaea vulgaris (Asteraceae): frequency, morphology and origin

Jacobaea vulgaris subsp. vulgaris constitutes an intricate polyploid complex distributed in Europe. Four chromosome numbers have been reported in this species: 2n=20, 40, 80 and 32. Chromosome number 2n=20 reported from Bulgaria is due to misidentification with J. aquatica. We discovered new hexaploid (2n=6x=60) cytotype occuring within four sympatric populations of otherwise tetraploid and octoploid plants in the Czech Republic, Slovakia and Ukraine. The frequency of hexaploid individuals within 76 studied populations is very low (only 10 of 693 analysed plants), and hexaploids probably represent hybrids between tetraploid and octoploid plants. Multivariate morphometric analysis reveals partial separation of tetraploid and octoploid plants, whereas hexaploid individuals are similar to octoploids. Octoploids and hexaploids exhibit slightly longer ray florets, involucral bracts and tubular florets and more hairy outer achenes. Hexaploid plants display larger pollen grains and lower pollen fertility.