Tetraploid Mutant Research Articles

Characterisation of a Spontaneous Mutant of Lemna gibba G3 (Lemnaceae).

A spontaneous mutant of the duckweed Lemna gibba clone no. 7796 (known as strain G3, WT) was discovered. In this mutant clone, L. gibba clone no. 9602 (mt), the morphological parameters (frond length, frond width, root length, root diameter) indicated an enlarged size. A change in the frond shape was indicated by the decreased frond length/width ratio, which could have taxonomic consequences. Several different cell types in both the frond and the root were also enlarged. Flow cytometric measurements disclosed the genome size of the WT as 557 Mbp/1C and that of the mt strain as 1153 Mbp/1C. This represents the results of polyploidisation of a diploid clone to a tetraploid one. The mutant clone flowered under the influence of long day-treatment in half-strength Hutner's medium in striking contrast to the diploid WT. Low concentration of salicylic acid (<1 µM) induced flowering in the tetraploid mutant but not in the diploid plants. The transcript levels of nuclear-encoded genes of the photosynthetic apparatus (CAB, RBCS) showed higher abundance in light and less dramatic decline in darkness in the mt than in WT, while this was not the case with plastid-encoded genes (RBCL, PSAA, PSBA, PSBC).

Structural Maintenance of Chromosomes 5/6 Complex Is Necessary for Tetraploid Genome Stability in Arabidopsis thaliana.

Polyploidization is a common phenomenon in the evolution of flowering plants. However, only a few genes controlling polyploid genome stability, fitness, and reproductive success are known. Here, we studied the effects of loss-of-function mutations in NSE2 and NSE4A subunits of the Structural Maintenance of Chromosomes 5/6 (SMC5/6) complex in autotetraploid Arabidopsis thaliana plants. The diploid nse2 and nse4a plants show partially reduced fertility and produce about 10% triploid offspring with two paternal and one maternal genome copies. In contrast, the autotetraploid nse2 and nse4a plants were almost sterile and produced hexaploid and aneuploid progeny with the extra genome copies or chromosomes coming from both parents. In addition, tetraploid mutants had more severe meiotic defects, possibly due to the presence of four homologous chromosomes instead of two. Overall, our study suggests that the SMC5/6 complex is an important player in the maintenance of tetraploid genome stability and that autotetraploid Arabidopsis plants have a generally higher frequency of but also higher tolerance for aneuploidy compared to diploids.

Apple varieties of natural breeding and their role in assortment improvement

The results of studies and use of apple varieties of natural breeding in the long-term breeding are given. At the Russian Research Institute of Fruit Crop Breeding (RRIFCB), a large apple gene pool has been collected, which is presented by varieties and forms of diff erent genetic and geographical origin including a collection of natural breeding varieties. The purpose of these studies is to identify apple varieties of natural breeding that are the best in the main economic and biological parameters for use in breeding to create new varieties adapted to local conditions. It has been found that the varieties of natural breeding, as a rule, are characterized by valuable economic and biological traits, which provide them with high environmental adaptability. The use of varieties of natural breeding has been analyzed. We have revealed the varieties that are more often used as parent forms in breeding: ‘Antonovka Obyknovennaya’, ‘Borovinka’, ‘Papirovka’ and some others. The sources of the most valuable economically useful traits have been identifi ed, such as high winter hardiness, productivity, resistance to diseases, long storage life and improved biochemical composition of fruit. Tetraploid forms of ‘Papirovka’ have been studied for using as donors of diploid gametes. Tetraploid mutant of ‘Antonovka Obyknovennaya’, i.e. ‘Antonovka Ploskaya’ has been studied. The description of apple varieties of natural breeding, which can yet be met in old orchards of Central Russia, is given. 20 apple varieties obtained with using varieties of natural breeding have been created at RRIFCB and included in the State register of breeding achievements. Varieties ‘Avgusta’, ‘Bolotovskoye’, ‘Vavilovskoye’, ‘Imrus’, ‘Osipovskoye’, ‘Svezhest’ and ‘Yablochny Spas’ already occupy large areas in the commercial orchards.

Successful induction and the systematic characterization of tetraploids in cerasus humilis for subsequent breeding

The Chinese dwarf cherry (Cerasus humilis; 2n = 16) has been increasingly prized for its colorful fruits, high economic values, nutraceutical benefits, and wide adaptability for diverse soil and environmental conditions. Polyploid induction is an efficient way to develop new selections and cultivars of horticultural crops. In this study, successful induction of tetraploids (2n = 32) in C. humilis (cultivar: ‘Nongda 7’) was achieved and the tetraploid plants were subsequently characterized. The highest mutagenesis rate reached in our study was 30.0 % with 80 mg/L colchicine added to the solid Murashige and Skoog (MS) media for both the embryo and stem tip. In liquid media, the highest mutagenesis rate achieved was 16.7 % with the addition of 60 mg/L colchicine. Systematic examination of the induced tetraploid mutants, significant morphological differences were observed in leaves, stems, and roots. Tetraploid mutants exhibited larger leaves, shorter internodes, and thicker roots than their diploid counterparts. Moreover, average stomatal size of the tetraploid mutants was significantly larger than that of the diploids. At the same time, the stomatal density of tetraploid plants decreased significantly in comparison to the that of diploids. The tetraploid mutants have been and will be used in our breeding efforts of new cultivars with higher yields, better fruit quality and wider adaptability.

Generation of stable nulliplex autopolyploid lines of Arabidopsis thaliana using CRISPR/Cas9 genome editing.

RNA-guided endonuclease-mediated targeted mutagenesis using the clustered regularly interspersed short palindromic repeats (CRISPR)/Cas9 system has been successful at targeting specific loci for modification in plants. While polyploidy is an evolutionary mechanism enabling plant adaptation, the analysis of gene function in polyploid plants has been limited due to challenges associated with generating polyploid knockout mutants for all gene copies in polyploid plant lines. This study investigated whether CRISPR/Cas9 mediated targeted mutagenesis can generate nulliplex tetraploid mutant lines in Arabidopsis thaliana, while also comparing the relative efficiency of targeted mutagenesis in tetraploid (4x) versus diploid (2x) backgrounds. Using CRISPR/Cas9 genome editing to generate knockout alleles of the TTG1 gene, we demonstrate that homozygous nulliplex mutants can be directly generated in tetraploid Arabidopsis thaliana plants. CRISPR/Cas9 genome editing now provides a route to more efficient generation of polyploid mutants for improving understanding of genome dosage effects in plants.

Polyploidy Induction of Moth Orchid (Phalaenopsis amabilis (L.) Blume) by Colchicine Treatment on Pollinated Flowers

The effectiveness of colchicine treatment at inducing polyploidy in Phalaenopsis amabilis (2n=2x=38) was investigated. Colchicine was applied on P. amabilis pollinated flowers and the development of the pods (fruit) was examined. Colchicine concentration in the experiment were 0, 50, 500, 1000, and 2000 mg L-1, with three and five days duration. The result showed that higher colchicine concentration decreased pod length and pod number. Visual screening of the seedlings obtained from colchicines treatments were classified into normal seedlings (NS), i.e. those having similar morphology to control, and putative polyploidy mutant seedlings (PMS). All colchicine-treated PMS were significantly different compared to control in the basal organ of the protocorm (BOP) length and width, leaf length, and root length and diameter. The PMS chromosome number analysis revealed that 50 mg L-1 for three or five days colchicine application and 500 mg L-1 for five days colchicine application produced 60 and 100% tetraploid mutant seedlings, respectively.

Tetraploid Induction and Identification of &amp;lt;i&amp;gt;Gossypium arboreum&amp;lt;/i&amp;gt;

Gossypium arboreum (2n = 26, A2) is a diploid species with limited production in acreage com-pared with G. hirsutum and G. barbadense. However, its unique traits such as insect and disease resistance contribute an important germplasm to cotton breeding. So polyploid manipulation for G. arboreum is an effective approach of germplasm development. This research focused on tetraploid induction of G. arboreum by colchicine. Morphology and cytology identifications for obtained mutants were also conducted. The seedling growth and development of all mutants was more stunted than controls. According to preliminary morphological characteristics, mutant rates in different treatment were statistically estimated and the highest mutant rate was 42.31% under the treatment of 0.1% colchicine for 24 hours. The chromosome number of most mutants was 2n = 4x = 52, while the chromosome number of diploid controls was 2n = 2x = 26 by cytology observation of root tip cells. By microscope observation of low leaf epidermis, there were significant differences for stoma area between tetraploids and diploids. The meiosis behavior of the induced tetraploid was much more complex than that of the diploid. At diakinesis, some univalent, trivalent and polyvalent were also observed besides bivalent and quadrivalent. There were different kinds of polyad in tetraspore period of mutants. The dissociate chromosomes existed during metaphase I and II, the unbalance separation of chromosomes existed during anaphase I and II. As a result, tetraploid mutants of G. arboreum were identified and their desirable traits would be further evaluated to incorporate into next breeding program.

A modified TILLING approach to detect induced mutations in tetraploid and hexaploid wheat

BackgroundWheat (Triticum ssp.) is an important food source for humans in many regions around the world. However, the ability to understand and modify gene function for crop improvement is hindered by the lack of available genomic resources. TILLING is a powerful reverse genetics approach that combines chemical mutagenesis with a high-throughput screen for mutations. Wheat is specially well-suited for TILLING due to the high mutation densities tolerated by polyploids, which allow for very efficient screens. Despite this, few TILLING populations are currently available. In addition, current TILLING screening protocols require high-throughput genotyping platforms, limiting their use.ResultsWe developed mutant populations of pasta and common wheat and organized them for TILLING. To simplify and decrease costs, we developed a non-denaturing polyacrylamide gel set-up that uses ethidium bromide to detect fragments generated by crude celery juice extract digestion of heteroduplexes. This detection method had similar sensitivity as traditional LI-COR screens, suggesting that it represents a valid alternative. We developed genome-specific primers to circumvent the presence of multiple homoeologous copies of our target genes. Each mutant library was characterized by TILLING multiple genes, revealing high mutation densities in both the hexaploid (~1/38 kb) and tetraploid (~1/51 kb) populations for 50% GC targets. These mutation frequencies predict that screening 1,536 lines for an effective target region of 1.3 kb with 50% GC content will result in ~52 hexaploid and ~39 tetraploid mutant alleles. This implies a high probability of obtaining knock-out alleles (P = 0.91 for hexaploid, P = 0.84 for tetraploid), in addition to multiple missense mutations. In total, we identified over 275 novel alleles in eleven targeted gene/genome combinations in hexaploid and tetraploid wheat and have validated the presence of a subset of them in our seed stock.ConclusionWe have generated reverse genetics TILLING resources for pasta and bread wheat and achieved a high mutation density in both populations. We also developed a modified screening method that will lower barriers to adopt this promising technology. We hope that the use of this reverse genetics resource will enable more researchers to pursue wheat functional genomics and provide novel allelic diversity for wheat improvement.

Development of a Range of Polyploid Lines in Petunia hybrida and the Relationship of Ploidy with the Single-/Double-flower Trait

A set of Petunia hybrida plants encompassing a range of ploidy levels was developed through colchicine-mediated induction of chromosome doubling. The resulting double-flower tetraploid plants were cross-hybridized with inbred single-flower diploid lines to generate F1 populations with segregation for ploidy level and flower type. The initial in vivo application of colchicine to seedling apical tips produced mixoploid plants of petunia at a high rate of efficiency. Thus, 95% of the shoot tips treated with colchicine for 48 h resulted in polyploid mutant plants, and no difference in this efficiency was observed using concentrations of colchicine between 0.2 and 2.0 mg·mL−1. Of the polyploid plants, 10% were found to be tetraploid and 85% were mixoploid (chimeric). Compared with their diploid counterparts, polyploid plants underwent reduced elongation growth during the first 2 weeks and had thicker stems and shorter internodes resulting in dwarfing of the whole plant. In extreme cases, very slow growth rates produced stunted plantlets. Polyploid plants also had larger, thicker leaves and, in some cases, the leaves that developed after 1 month of growth appeared seriously malformed. Octoploid plants were also obtained and these tended to have more extreme phenotypes. Pure tetraploid plants of double-flower petunia were isolated by the in vitro culture of explants from the initial chimeric tetraploid mutants. These were crossed with three inbred single-flower diploid lines (S1, S2, and S3) thereby generating F1 populations that showed segregation for flower type and ploidy level and included the generation of triploid plants. In the tetraploid plants, flower diameter and the number of flower petals were not changed significantly (P &gt; 0.05) compared with the original diploid double-flower plants, but observation of the pollen grains revealed segregation for size consistent with the increased ploidy level. Analysis of the F1 progeny plants also indicated that chromosome number is not necessary but sufficient to cause the production of semidouble-flowered plants. Flower color and flower diameter were also analyzed in the F1 progeny and complex patterns of inheritance were inferred. In addition to single and double flowers, semidouble-flowered plants were also suggested to be generated by the hybridization of 2n or 3n pollen from the double-flower tetraploid plants with the single-flower diploid lines.

Ginger (Zingiber officinale) autotetraploids with improved processing quality produced by an in vitro colchicine treatment

Ginger autotetraploids were produced by immersing shoot tips in a 0.5% w/v colchicine, 2% v/v dimethyl sulfoxide solution for 2 h. Stomatal measurements were used as an early indicator of ploidy differences in culture with mean stomata length of tetraploids (49.2 μm) being significantly larger than the diploid (38.8 µm). Of the 500 shoot tips treated, 2% were characterised as stable autotetraploid lines following field evaluation over several seasons. Results were confirmed with flow cytometry and, of the 7 lines evaluated for distinctness and uniformity, 6 were solid tetraploid mutants and 1 was a periclinal chimera. Significant differences were noted between individual tetraploid lines in terms of shoot length, leaf length, leaf width, size of rhizome sections (knob weight) and fibre content. The solid autotetraploid lines had significantly wider, greener leaves than the diploids, they had significantly fewer but thicker shoots and, although ‘Queensland’ (the diploid parent from which the tetraploids were derived) had a greater total rhizome mass at harvest, its knob size was significantly smaller. From the autotetraploid lines, one line was selected for commercial release as ‘Buderim Gold’. It compared the most favourably with ‘Queensland’ in terms of the aroma/flavour profile and fibre content at early harvest, and had consistently good rhizome yield. More importantly it produced large rhizome sections, resulting in a higher recovery of premium grade confectionery ginger and a more attractive fresh market product.

Analysis of karyotypic stability of homoeologous-pairing (ph) mutants in allopolyploid wheats

Karyotypic analysis of wheat lines with different genotypes for the homoeologous-pairing loci Ph1 and Ph2 was carried out by means of a genomic in situ hybridization method that allowed unequivocal identification of the A, B and D genomes. Chromosomal rearrangements mainly affecting the A and D genomes were found in all plants of allohexaploid wheat (AABBDD) lacking Ph1 activity. The frequency of intergenomic exchanges per plant in ph1b mutant and nulli-5B lines was 4.31 and 3.40, respectively. In addition, an unbalanced genomic constitution was found in a few plants, some even showing a euploid chromosomal number. By contrast, rearranged karyotypes were detected neither in the ph1 mutant line (ph1c) of allotetraploid wheat (AABB) nor in the allohexaploid wheat lines lacking Ph2 activity, namely ph2b mutant and nulli-3D lines. These results were compared with the chromosomal pairing behaviour displayed by mutant lines ph1c, ph1b and ph2b at first meiotic metaphase. Despite the finding of standard, nonrearranged karyotypes in the phlc tetraploid mutant, the frequency of A-B homoeologous metaphase I association was similar to that observed in the ph1b hexaploid mutant. The results presented clearly demonstrate that inactivity of the Ph1 locus induces karyotypic instability in wheat. Intergenomic exchanges have probably been accumulating since the original ph1 mutant and aneuploid lines were obtained, which should be taken into account when it is planned to use these lines for basic research on Ph1 function or in applied wheat breeding programmes.

Tetraploid `Planters Jumbo' Melon Lines C883-m6-4x and 67-m6-100-4x

Lines C883-m6-4x and 67-m6100-4X are spontaneous tetraploid mutants from the melon cultivar Planters Jumbo (Cucumis melo L.). ‘Planters Jumbo’ is the result of a series of crosses and “selected generations involving several cultivars and plant introductions.These tetraploid selections were approved for release in 1991 by the Agricultural Research Service, U.S. Dept. of Agriculture, in response to seed industry requests. These 4x lines can be used as a tool in tetraploid melon research and for 3x melon production feasibility studies.

Chromosomal instability in mutagen-sensitive mutants isolated from mouse lymphoma L5178Y cells I. Five different genes participate in the formation of baseline sister-chromatid exchanges and spontaneous chromosomal aberrations

In a search for cell mutants that show an increase or a decrease in the frequency of baseline sister-chromatid exchanges (SCEs) or spontaneous chromosomal aberrations (CAs), large numbers of mutagen-sensitive clones previously isolated from mouse lymphoma L5178Y cells were analyzed. In addition to two SCE mutants (ES 4 and AC 12) previously reported, three other mutants were identified as an SCE mutant. An ehtyl methanesulfonate-sensitive mutant ES 2 and an alkylating agent-sensitive mutant MS 1 exhibited, respectively, 1.4-fold and 1.8-fold higher baseline SCE frequencies than did the parental L5178Y. In contrast, M10, which is sensitive to X-ray and 4-nitroquinoline 1-oxide, showed a reduced frequency of baseline SCEs (0.65-fold). These 5 mutants including ES 4 and AC 12 had 3–9-fold increases in spontaneous CA frequencies. Measurement of baseline SCE formation in inter-mutant hybrids revealed that M10 mutation is dominant, MS 1 and ES 4 mutations are semidominant, and ES 2 and AC 12 mutations are recessive. Because SCE frequencies in hybrids formed between pairs of 4 mutants (ES 2, MS 1, ES 4 and AC 12) were significantly lower than those in the tetraploid mutant cells, these 4 mutants probably belong to different complementation groups. Since M10 behaved dominantly with respect to SCE phenotype, it was not possible to determine by complementation test whether it belongs to a different group from the other mutants. However, the finding that M10 is complemented by other mutants for EMS sensitivity indicates that the M10 mutation is different from the other mutations. From these results, it is concluded that at least 4 different genes participate in the formation of high levels of baseline SCEs. The defects in ES 2, MS 1, ES 4, and AC 12 produce common lesions responsible for the formation of both SCEs and CAs. In contrast, the defect in M10 is associated with a high increase in spontaneous CA frequency, but conversely associated with a decrease in baseline SCE frequency. This suggests that M10 is defective in the process involved in the formation of baseline SCEs.

Amanitin binding to RNA polymerase II in α-amanitin-resistant rat myoblast mutants

A series of independent α-amanitin-resistant (Ama R ) mutants have been isolated from diploid and tetraploid strains of L6 rat myoblast cells. With one exception, these mutants contain both α-amanitin-sensitive wild-type and α-amanitin-resistant mutant RNA polymerase II activities. The relative resistance of the mutant enzymes in extracts of the different Ama R isolates, determined by measuring the inhibition constant, K 1 , for α-amanitin, parallels their relative resistance to growth inhibition by the toxin. The binding affinities of the different mutant forms of RNA polymerase II for γ-amanitin, determined by measuring the equilibrium dissociation constant, K d , for γ-[ 3 H]amanitin§ bound to the enzyme, are also reduced relative to the wild-type enzyme, and the K d values are proportional to the K 1 values. Thus the spectrum of Ama R phenotypes in these mutant strains likely reflects the presence of mutations at different sites in the structural gene coding for the α-amanitin binding subunit of RNA polymerase II. No difference has been found between mutant and wild-type forms of the enzyme with respect to their turnover numbers, K m values for UTP, or thermal stabilities. Diploid mutants contain equal amounts of wild-type and mutant forms of RNA polymerase II. This indicates that they possess one wild-type and one mutant allele for the α-amanitin binding subunit of the enzyme, and that both alleles are expressed in a codominant fashion. Similarly, the tetraploid mutants possess three copies of the wild-type and one of the mutant allele. One tetraploid mutant has been isolated which is only slightly resistant to growth inhibition by α-amanitin and does not contain a detectably altered form of RNA polymerase II. This strain may be defective in α-amanitin transport.