Tetraploid Plants Research Articles

Cytogenetic characterization of induced autotetraploids in grass pea (Lathyrus sativusL.)

Abstract Induction of polyploidy was successfully achieved in grass pea (Lathyrus sativus L.) variety BioR-231 using 0.10% (eight hours three days), 0.20% (six hours two days and eight hours two days), 0.30% (eight hours one day) and 0.40% (six hours one day) colchicine solution by apical shoot tip treatment of young seedlings. Compared to diploid variety the tetraploid plants were morphologically distinguished by luxuriant vegetative growth showing broader and thicker leafl ets and stipules, fl attened stems, increased number of branches, enhanced length of tendril as well as peduncle, delayed fl owering and reduced pollen fertility. Number of sto-mata decreased but its size increased signifi cantly in tetraploids. Presence of 7II and 7–7 separation at meiosis-I were the usual feature in diploids (2n=2x=14) while occurrences of 28 chromosomes in different combinations of multivalents, bivalents and univalents at metaphase-I and unequal separation of chromosomes toward the spindle poles, bridge formation,...

Tissue culture and generation of autotetraploid plants of Sophora flavescens Aiton.

Background:Sophora flavescens Aiton is an important medicinal plant in China. Early in vitro researches of S. flavescens were focused on callus induction and cell suspension culture, only a few were concerned with in vitro multiplication.Objective:To establish and optimize the rapid propagation technology of S. flavescens and to generate and characterize polyploid plants of S. flavescens.Materials and Methods:The different concentrations of 6-benzylaminopurine (BAP), indole-3-acetic acid (IAA) and kinetin (KT) were used to establish and screen the optimal rapid propagation technology of S. flavescens by orthogonal test; 0.2% colchicine solution was used to induce polyploid plants and the induced buds were identified by root-tip chromosome determination and stomatal apparatus observation.Results:A large number of buds could be induced directly from epicotyl and hypocotyl explants on the Murashige and Skoog medium (MS; 1962) supplemented with 1.4–1.6 mg/l 6-benzylaminopurine (BAP) and 0.3 mg/l indole-3-acetic acid (IAA). More than 50 lines of autotetraploid plants were obtained. The chromosome number of the autotetraploid plantlet was 2n = 4× = 36. All tetraploid plants showed typical polyploid characteristics.Conclusion:Obtained autotetraploid lines will be of important genetic and breeding value and can be used for further selection and plant breeding.

Chromosome duplication in Brachiaria (A. Rich.) Stapf allows intraspecific crosses

Brachiaria decumbens cv. Basilisk is the single most important forage grass used for pastures in the tropics. Breeding to produce improved cultivars has been impossible until now due to the lack of compatible sexual ecotypes. This paper reports the success of somatic chromosome duplication of sexually reproducing diploid plants of B. decumbens and of a diploid hybrid between B. decumbens and B. brizantha, which should allow intraspecific crosses with natural apomictic tetraploid accessions of either species. Polyploidization was induced in explants cultured in vitro on a medium supplemented with colchicine at 0.01% for 48 hours, transferred to the same medium without colchicine until shoot regeneration occurred. Five sexual tetraploid plants (3.9% of plants recovered) were obtained. Crosses with apomictic cultivars recovered 14 seeds. The novel sexual tetraploids generated were unique and represented a major breakthrough in breeding B. decumbens to obtain superior hybrids.

Cytogenetical investigations in colchicine-induced tetraploids of Cyamopsis tetragonoloba L.

Successful induction of autotetraploidy has been achieved in five accessions of Cyamopsis tetragonoloba L. The diploid seedlings of these accessions were treated with different concentrations of aqueous colchicine using the cotton-swab method for 10–18 hours within 2–3 days. The highest percentage of success was recorded when the seedlings were treated with 0.2% colchicine for 10 h within two days. The synthesized plants showed remarkable enhancement in several morphological and floral characters making them more robust and better plants from the food and feed aspect. Cytologically, quadrivalent frequency ranging from 3.18 to 5.45 and univalent frequency ranging from 0.08 to 1.10 were characteristic of the colchicine-induced tetraploids. Among all the associations, bivalent chromosome associations were observed more frequently (2.95 to 6.04). The anaphase I and II disjunction of bivalents/chromosomes was leading more or less regularly and equally to the formation of at least few seeds from some of the synthesized plants. Significant enhancement in morphological traits as revealed in colchicine-induced tetraploid plants having a better food and feed value and normal meiotic behaviour of synthesized autotetraploids leading to a good seed set may ultimately result in the genetic improvement of Cyamopsis tetragonoloba.

In vitro induction of tetraploids in crape myrtle (Lagerstroemia indica L.)

A protocol for in vitro induction of crape myrtle tetraploids using nodes from in vitro-grown shoots (2n = 48) was established. Nodal buds were excised from in vitro-grown shoots, maintained on proliferation medium containing Murashige and Skoog medium supplemented with 4.44 μM 6-benzyladenine , 0.54 μM α-naphthaleneacetic acid, and treated with a range of concentrations of colchicine under three different conditions. Nodal bud explants treated in liquid proliferation medium supplemented with either 15 or 20 mM colchicine for 24 h turned necrotic and died; whereas, those cultured on solid proliferation medium supplemented with either 125 or 250 μM colchicine for 30 days survived, but no tetraploid plants were obtained. However, when explants were cultured in liquid proliferation medium containing 250, 500 or 750 μM colchicine for 10 days, tetraploid plants (2n = 96) were obtained. Incubation of explants in medium containing 750 μM colchicine promoted the highest frequency of survival (40%) of explants and of recovered tetraploids (60%). Morphological and anatomical characteristics of leaves, including leaf index, stomata size and number, stomata index (length/width), and number of chloroplasts in guard cells correlated with ploidy of crape myrtle plants. The number of chloroplasts in guard cells of stomata was a stable and reliable marker in discriminating plants of different ploidy levels. Chromosome counts and flow cytometry confirmed these findings.

Induced Polyploidy in Diploid Ornamental Ginger (Hedychium muluense R. M. Smith) Using Colchicine and Oryzalin

The ploidy level of H. muluense, a diploid (2n = 2x = 34) and dwarf ornamental ginger species, has been determined and is reported for the first time. Oryzalin and colchicine were successfully used to induce polyploidy in Hedychium muluense in vitro. Embryogenic cell lines were treated with oryzalin (30, 60, or 120 μM) and colchicine (2.5, 5, or 10 mm) for 24, 48, or 72 h. The control contained no antimitotic agent. Flow cytometry, chloroplast count, and stomatal frequency were more effective and reliable than stomatal length as methods for assessing ploidy. Overall, oryzalin was more effective than colchicine in inducing polyploidy. The highest induction frequency (15%) of tetraploidy was achieved when embryogenic callus was exposed to 60 μM oryzalin for 72 h. For colchicine, exposure of embryogenic callus to the 2.5 mm colchicine for 24 h was the most effective in creating tetraploid (13%) plants.

Production of tetraploid plants of non apomictic citrus genotypes

Ploidy manipulation in Citrus is a major issue of current breeding programs aiming to develop triploid seedless mandarins to address consumer demands for seedless fruits. The most effective method to obtain triploid hybrids is to pollinate tetraploid non apomictic cultivars with pollen of diploid varieties. Such non apomictic tetraploid lines are not found in the citrus germplasm and need to be created. In this work we describe a new methodology based on in vitro shoot-tip grafting combined with treatment of the micro-grafted shoot-tip with colchicine and oryzalin to achieve chromosome doubling and a dechimerization procedure assisted by flow cytometry. Stable tetraploid plants of Clemenules, Fina and Marisol clementines and Moncada mandarin have been obtained directly from shoot tip grafting combined with colchicine and oryzalin treatments or after dechimerization of mixoploids plants (2x-4x). These stable tetraploid plants have been used in 4x x 2x hybridizations, to recover over 3,250 triploid hybrids in 3 years.

Cytological and RAPD data revealed genetic relationships among nine selected populations of the wild bramble species, Rubus parvifolius and R. coreanus (Rosaceae)

Rubusparvifolius L. and R. coreanus Miq. are the two morphologically distinct, endemic wild brambles in East Asia that may be useful in raspberry breeding and cultivar development. In this study, genetic relationships among a total of nine populations of these two species, collected from Sichuan Province in southwestern China, were investigated by cytological and RAPD analyzing. With the exception of ploidy levels, R. parvifolius and R. coreanus had similar cytological features. Six R. parvifolius populations included diploids, triploid, tetraploid, and mixed diploid-tetraploid cytotype plants; among them, the population that produced the fewest seeds was the triploid, while the three R. coreanus populations were all diploid. But obvious genetic differentiation between R. parvifolius and R. coreanus was revealed by RAPD markers, the Euclidean’s GS coefficient generated by UPGMA cluster analysis between R. parvifolius and R. coreanus was only 0.442, about half of that observed within species. Both genetic relationships among these populations and the origin of polyploidy in R. parvifolius populations are also discussed.

Induction of tetraploid poplar and black locust plants using colchicine: chloroplast number as an early marker for selecting polyploids in vitro

Tetraploid plants were produced by inducing chromosome doubling using colchicine in in vitro shoot tips of poplar and black locust clones. Many of the plants treated with colchicine showed modified morphological characteristics like stunted growth, thicker leaves and modified leaf morphology. The counting of chloroplast number in the epidermal guard cells of stomata was used for the rapid screening of tetraploids. The differences in mean chloroplast numbers between diploid and tetraploid plants were highly significant. For all plants tested, the tetraploid genotype had almost double the number of chloroplasts per guard cell compared to the diploid origin. Some plants were further analysed by flow cytometry to verify their ploidy status that was determined by chloroplast numbers. The results of this study demonstrated for the first time that chloroplast counting in poplar and black locust could be an effective and reliable method for pre-screening large numbers of plants for their ploidy level. The protocol might be applicable in a wide scope of breeding programs.

Risk assessment of transgenic apomictic tetraploid bahiagrass, cytogenetics, breeding behavior and performance of intra-specific hybrids

Pollen-mediated gene transfer from stress tolerant or herbicide-resistant transgenic plants may cause environmental or agronomic problems. Apomictic seed production found in some bahiagrass cultivars may serve as a natural transgene containment system. Under greenhouse conditions, the average gene transfer frequency from an herbicide-resistant apomictic tetraploid to a population of sexual diploid bahiagrass genotypes or apomictic tetraploid bahiagrass was 0.16% when the transgenic pollen donor was placed at 0.5-1.5 m distance from the non-transgenic pollen receptors. The herbicide-resistant hybrids were characterized for transgene integration, expression and ploidy, by Southern blot analysis, immuno-chromatography and flow cytometry, respectively. Hybrids resulting from open pollination of non-transgenic diploid female plants with transgenic tetraploid male plants were triploids or near-triploids, with 2n = 26-34. These hybrids displayed a wide range of phenotypic variability, including some non-persistent or non-flowering dwarf-type hybrids with good vigor, or hybrids with vegetative growth similar to non-transgenic plants, but with significantly reduced seed set. Non-flowering aneu-triploids with good vigor/field performance will provide the highest level of transgene containment. Embryo sac analysis of pollinated spikelets confirmed a high proportion of aborted ovules. An apospory-linked RFLP marker was detected in 13 of the 15 near-triploid hybrids. All flowering aneuploid hybrids displayed significantly reduced seed set, and none of the sexual near-triploid hybrids produced any seeds. All tetraploid gene transfer events carried the apospory-linked RFLP marker, suggesting that despite the presence of the aposporus locus, a low degree of sexuality co-exists in apomictic tetraploid cultivars. Thus, tetraploid apomictic bahiagrass does not provide complete transgene containment, although intra-specific gene transfer is drastically reduced compared to sexually reproducing perennial grasses.

Tetraploidization of diploid Dioscorea results in activation of the antioxidant defense system and increased heat tolerance

Polyploidy is reported to show increased tolerance to environmental stress. In this work, tetraploid plants of Dioscorea zingiberensis were obtained by colchicine treatment of shoots propagated in vitro. The highest tetraploid induction rate was achieved by treatment with 0.15% colchicine for 24 h. Diploid and tetraploid plants were exposed to normal (28 °C) and high temperature (42 °C) for 5 d during which physiological indices were measured. Compared with diploid plants, relative electrolyte leakage and contents of malondialdehyde, superoxide anions and hydrogen peroxide were lower in tetraploids, while activities of antioxidant enzymes, such as superoxide dismutase, peroxidase, catalase, ascorbate peroxidase and glutathione reductase, were stimulated and antioxidants (ascorbic acid and glutathione) were maintained at high concentrations. These results indicate that tetraploid plants possess a stronger antioxidant defense system and increased heat tolerance.

Induction of tetraploid ruzigrass (Brachiaria ruziziensis) plants by colchicine treatment of in vitro multiple‐shoot clumps and seedlings

AbstractTetraploid plants of ruzigrass (Brachiaria ruziziensis) have been obtained by colchicine treatment of in vitro multiple‐shoot clumps and germinated seedlings. Multiple‐shoot clumps were induced and maintained on MS basal medium with 0.5 mg L−1 2,4‐dichlorophenoxyacetic acid (2,4‐D) and 2.0 mg L−1 6‐benzylaminopurine (BAP) according to a previous report. The colchicine treatment consisted of culturing multiple‐shoot clumps on MS basal medium containing 0.0125–0.1% colchicine for 12 and 48 h. Surviving multiple‐shoot clumps were regenerated on MS basal medium containing 2.0 mg L−1 BAP. The ploidy level of plants after colchicine treatment was determined by flow cytometry. Eight tetraploid plants were obtained from multiple‐shoot clumps treated with 0.1% colchicine for 12 h. This treatment was identified as the optimum treatment and resulted in the highest frequency (28.6%) of tetraploid plants among the treatments tested on multiple‐shoot clumps. Five tetraploid plants (31.3%) were obtained when seedlings were treated with 0.1% colchicine for 3 h. Although higher concentrations of colchicine and longer durations of colchicine treatment reduced the survival rate of the explants, such treatments increased the frequency of tetraploid plants. A total of 23 tetraploid plants were obtained from 900 different colchicine‐treated multiple‐shoot clumps, and a total of five tetraploid plants were obtained from 300 colchicine‐treated seedlings. Cytological analysis affirmed the results of flow cytometry. Significant differences in leaf blade stomata were observed between diploid and tetraploid ruzigrass.

Induced polyploidy with gigas expression for root traits inCapsicum annuum(L.)

With 1 figure and 1 table Polyploidy was induced in hot pepper (Capsicum annuum cv. ‘GVC-111’) using colchicine and tetraploid plants with robust root size system were obtained. Chromosome doubling was achieved by different concentrations of colchicine (0.05%, 0.1%, 0.2% and 0.4% aqueous solution) to either presoaked seeds or to the shoot tip of young seedlings for 24, 48 and 72 h, respectively. A total of 313 plants with significantly larger average stomata length (48.6%), lower frequency of stomata per square mm (41.7%) and a higher number of chloroplast in guard cells (47.3%) were selected as putative polyploids for further analysis by flow cytometry. Out of 313 putative polyploids, 31 were tetraploid, 270 were mixoploid and 12 were, in fact, diploid. Six tetraploid plants were selected for robust root system observations, longer primary root length and higher density of lateral roots. This study demonstrates successful utilization of colchiploidy to create novel mutations for root size systems.

MORPHOLOGICAL AND ANATOMICAL CHARACTERISATION OF CHEMICALLY INDUCED POLYPLOIDS IN SPATHIPHYLLUM WALLISII

ISHS XXIII International Eucarpia Symposium, Section Ornamentals: Colourful Breeding and Genetics MORPHOLOGICAL AND ANATOMICAL CHARACTERISATION OF CHEMICALLY INDUCED POLYPLOIDS IN SPATHIPHYLLUM WALLISII

Genetic diversity and differentiation in natural and reintroduced populations ofBencomia exstipulataand comparisons withB. caudata(Rosaceae) in the Canary Islands: an analysis using microsatellites

Variation at five polymorphic microsatellite loci was used to investigate genetic diversity and differentiation of two tetraploid Canarian endemics, Bencomia exstipulata and B. caudata. Data were analysed and are discussed in terms of tetrasomic (autotetraploid) and disomic (allotetraploid) inheritance. In both cases, genetic diversity values were similar to those described in other tetraploid plant species. High genetic differentiation between the only two described natural populations of B. exstipulata was detected (FST = 0.411). Bayesian cluster analysis revealed a geographical structure with distinct genetic groups from each island. High genetic differentiation and low genetic diversity of the B. exstipulata population from Tenerife suggest a recent population bottleneck, perhaps caused by the most recent major volcanic eruption, for this natural locality. This may be heightened by possible inbreeding depression and the monoecy of these species. Polymorphic microsatellite loci were also tested across all species in the Bencomia alliance. These reliably amplified the target sequence, suggesting a high degree of conservation of the sequences flanking the microsatellites. © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 160, 429–441.

Dosage and parent-of-origin effects shaping aneuploid swarms in A. thaliana

Changes in chromosome number have a critical role in the evolution and formation of plant species. Triploids, which carry three complete sets of chromosomes, in particular produce offspring with different chromosome numbers, including diploid and tetraploid progeny, as well as a swarm of aneuploid progeny, which carry incomplete chromosome sets. In this study, we investigated the mechanisms shaping these swarms at the population level through a detailed characterization of the progeny of triploid Arabidopsis thaliana. We report that triploid meiosis predominately produced aneuploid gametes, most of which were viable. We performed reciprocal crosses between triploid and either diploid or tetraploid plants and karyotyped all surviving individuals. This allowed us to dissect the parent-of-origin (cross-direction) effects and also the effect of the dosage of the crossing partner on the inheritance of each chromosome type. Overall, our data indicate that the chromosomal composition of the swarms produced by the triploid A. thaliana were strongly influenced by selection acting against specific gamete combinations, but not necessarily associated with aneuploidy. Finally, each of the five chromosome types responded differently to this selection, suggesting the presence of dosage-sensitive factor(s) critical for viability and encoded on different chromosomes.

Karyotype and cytogeography of the genusHeracleum(Apiaceae) in the Hengduan Mountains

Abstract In the present study, the karyotypes of 34 populations belonging to 11 species and one variety ofHeracleumfrom the Hengduan Mountains in China were examined. Chromosome numbers and the karyotypes of three species (H. souliei,H. kingdoni, andH. wenchuanense) are reported for the first time, as are the karyotypes ofH. moellendorffiiandH. henryi(tetraploid). Populations ofH. candicans,H. franchetii, andH. kingdoniin the Hengduan Mountains were found to consist of a mixture of diploid and tetraploid plants. Except for four species ofHeracleum, namelyH. candicans,H. franchetii,H. henryi, andH. kingdoni, which have both diploid and tetraploid karyotypes, all other species ofHeracleumare were found to be diploid. All karyotypes were found to belong to the 2A type of Stebbins, with the exception ofH. candicansvar.obtusifolium, which belongs to 2B, andH. hemsleyanumandH. franchetii(Mt. Dujuan, Daocheng, Sichuan, China), which belong to 1A. There was only a slight difference in the karyotype asymmetry index, which suggests a close kinship for species ofHeracleumand that the entire phylogenetic development ofHeracleumis relatively primitive. Species that exhibited advanced morphological features were also more advanced in karyotype structure, with the order of karyotype evolution being 1A→2A→2B. This phenomenon indicates that the species distributed in the Hengduan Mountains have not diverged completely and that the Hengduan Mountains are a relatively young and active area for the evolution ofHeracleum. Polyploidization inHeracleummay be an important evolutionary mechanisms for some species, generating diversity. The biological attributes, distribution range, and the geological history of the genus have all played a part in accelerating the evolution through polyploidization or aneuploidization. It is known that as the distribution latitude ofHeracleumdecreases from north to south, the chromosome number, ploidy level, and asymmetry structure appear to increase. In the Hengduan Mountains, these tendencies are also evident. Finally, based on all the available cytogeographic data, we speculate that the more advanced tetraplont or aneuploid species ofHeracleumin India may be derived from early diplont species that were distributed in the Caucasus region and Hengduan Mountains. The dispersal ofHeracleumwas from Eurasia to India, because this correlates with the emergence of the Himalayan Mountains through tectonic movement. Thus, the Hengduan Mountains are not only a center of diversity forHeracleum, but also a center of active speciation in modern times.

Mode of Reproduction of Colchicine‐Induced Paspalum plicatulum Tetraploids

Paspalum plicatulum Michx. is a wild forage grass species. The common races are tetraploid and apomictic, while sexual diploid representatives have been reported sporadically. Objectives of this study were to induce sexual 4x individuals from sexual diploids, determine the capacity for hybridization with other apomictic 4x species closely related with P. plicatulum, and thus create a tetraploid sexual material cross compatible with apomictic 4x species of the Plicatula group of Paspalum. Two induced tetraploid plants were recovered from germinating 2x seeds treated for 24 h with colchicine. Bivalent and quadrivalent chromosomes were the most common association at meiosis in these autotetraploids. Embryological analysis and progeny tests using molecular markers revealed that both induced tetraploids reproduced sexually. Single‐seed screening by flow cytometry confirmed full sexual reproduction. These plants retained the high self‐incompatibility system of the diploids, but set seed after reciprocal crosses and when crossed with pollen of apomictic 4x P. guenoarum Arechav. of the Plicatula group. Both sexual 4x plants constitute the foundational material for plant improvement through gene exchange and selection in apomictic 4x P. plicatulum and possibly in several apomictic species of the Plicatula group.

Ploidy doubling by in vitro culture of excised protocorms or protocorm-like bodies in Phalaenopsis species

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

Cybrid/hybrid plants regenerated from somatic fusions between male sterile Satsuma mandarin and seedy tangelos

Somatic hybridization provides an alternative for transferring mitochondria-encoded cytoplasmic male sterility (CMS). Herein, symmetric protoplast electrofusion was conducted between embryogenic callus protoplasts of Citrus unshiu Marc. cv. Guoqing No. 1 (G1), a CMS cultivar, and mesophyll-derived protoplasts of seedy ‘Page’ tangelo [ C. reticulata Blanco × ( C. reticulata Blanco × C. paradisi Macf.)] or ‘Nova’ tangelo [ C. reticulata Blanco × ( C. reticulata Blanco × C. paradisi Macf.)], to transfer CMS trait. Flow cytometry analysis showed that 14 plants recovered from G1 + ‘Page’ tangelo that displayed typical morphological character of ‘Page’ were diploid, and 6 plants regenerated from G1 + ‘Nova’ tangelo were tetraploid. Genetic compositions of regenerated plants from the two fusions were determined by SSR, CAPS and chloroplast-SSR analysis. Cybrid nature of diploids from G1 + ‘Page’ tangelo with nuclear DNA from ‘Page’, mitochondrial DNA (mtDNA) from the G1 and chloroplast DNA (cpDNA) derived from either parent was confirmed. Tetraploid plants from G1 + ‘Nova’ tangelo were identified as somatic hybrids with random cpDNA inheritance. The regenerated cybrid and hybrid plants hold great potential for Citrus seedless breeding at diploid or triploid levels.