Artificial Chromosome Doubling Research Articles

Artificial Polyploidization Enhances Morphological, Physiological, and Biological Characteristics in Melothria scabra Naudin

Cucamelon (Meltric scabra Naudin, Cucurbitaceae) is a climbing, herbaceous perennial plant with various culinary and medicinal uses. The current study aimed to develop novel autopolyploid genotypes of M. scabra through in vitro polyploidization and assess their morphological and phytochemical characteristics. For polyploid induction, oryzalin was employed as an antimitotic agent, and it was applied at various concentrations (40, 60, and 80 μM) and durations (24 and 48 h). Flow cytometry analysis confirmed the successful induction of polyploids, with polyploidization efficiency ranging from 2.5% to 15%. From a total of 240 treated plants, a total of 20 autotetraploid plants were obtained. The obtained polyploid and control diploid genotypes were cultivated under greenhouse conditions. Further, the plants were transferred to field conditions, and the leaves, flowers, and fruits were harvested to be evaluated for the morphological, biochemical, and biological activity variations among the obtained genotypes. Morphological comparisons between diploid and autotetraploid plants revealed significant differences in flower characteristics, fruit attributes, and leaf morphology. Nutritional evaluation demonstrated enhancement of key parameters in the induced polyploids compared to the diploid plants, including glucose, fructose, carotenoid, polyphenol, and antioxidant contents, highlighting the potential impact of polyploidization on these traits. The results from this study highlight the potential of artificial chromosome doubling as an effective breeding strategy in M. scabra and related plant species.

What Makes Bread and Durum Wheat Different?

Durum wheat (tetraploid) and bread wheat (hexaploid) are two closely related species with potentially different adaptation capacities and only a few distinct technological properties that make durum semolina and wheat flour more suitable for pasta, or bread and bakery products, respectively. Interspecific crosses and new breeding technologies now allow researchers to develop wheat lines with durum or bread quality features in either a tetraploid or hexaploid genetic background; such lines combine any technological properties of wheat with the different adaptation capacity expressed by tetraploid and hexaploid wheat genomes. Here, we discuss what makes bread and durum wheat different, consider their environmental adaptation capacity and the major quality-related genes that explain the different end-uses of semolina and bread flour and that could be targets for future wheat breeding programs.

Triploid forage grass hybrids Festuca apennina\u2009\xd7\u2009F. pratensis display extraordinary heterosis for yield characteristics

Hybrid vigor (or heterosis) is the over-performance of a hybrid over its parents in a specific trait or a set of traits. As such, hybrid breeding serves as a tool to efficiently trigger gains in breeding programs. Moreover, hybrids of genetically distant landraces, varieties or even species may become evolutionary successful. In Swiss alpine swards, we observed frequent prevalence of triploid hybrids of Festuca pratensis × F. apennina with outstanding competitiveness relative to their parental species in the sites of sympatric occurrence. Observations of these highly vigorous hybrids prompted the study on their heterosis across various environmental conditions. Phenotypic observations during 3 years at four locations at different altitudes (from 200 to 1850 m a.s.l.) have shown significant heterosis for dry biomass production at all sites during the first and second year, and at the mid- and high altitude sites also in the third year. At mid-altitude (1000 m a.s.l.), heterosis increased steadily and reached a maximum of + 508% for annual yield (+ 626% for a single cut) in the third year. This is by far the highest value of heterosis ever reported for annual dry matter yield of a forage grass. Further utilization of triploid hybrids in forage grass breeding is hampered by their sterility. Hence there is a need for vegetative propagation. However, artificial chromosome doubling of triploids to create fertile hexaploids, or seeking ways to propagate them vegetatively at an industrial scale might overcome this limitation.

Artificial polyploidy induction for improvement of ornamental and medicinal plants

Green plants provide food, fuel, materials, and medicine for humans. Amongst these, medicinal plants contain unique molecules with therapeutic applications. However, our understanding of these organisms is in its infancy. Landscaping plants also have a great impact on the human soul and emotional perception. These two groups of plants are part of natural wealth and have great economic value. One of the remarkable breeding strategies to improve the valuable properties of plants is artificial polyploidy induction. Medicinal and ornamental plants with duplicated whole sets of chromosomes, although not always, have more distinctive characteristics such as modified phytochemical profile, higher content of desired pharma molecules, plant form, flower color, size and style, fragrance, vase life, and prolonged flowering period. Therefore, artificial chromosome doubling (ACD) of ornamental and medicinal plants could have significant economic consequences. In medicinal plants with an ornamental flower/inflorescence, and/or in ornamental plants bearing essential oils, ACD is a dual beneficial breeding strategy. Working with both in vitro and in vivo chromosome doubling pathways, researchers are able to produce custom-designed plants with higher marketability. Some critical factors must be considered to establish a successful ACD protocol. The plant genotype and explant type are important parameters in this procedure. Type, dosage and duration of application (exposure time) of antimitotic agent (AMA) should also be considered as the main factors. Antimitotic agents can be classified according to their efficacy and toxicity. Although colchicine is the most applied and well-known AMA, there are other alternative mitotic spindle inhibitors with higher specificity for tubulin binding sites in plant materials and possessing less toxicity for animal tubulin. Testing the interaction effects of plant and AMA parameters is necessary for establishing an ACD breeding program and promoting the economic values of medicinal and ornamental plants. This paper reviews significant developments in polyploidization using different antimitotic agents over the last decade in the field of ornamental and medicinal plants. Artificial polyploidy induction is one of the applicable breeding methods in ornamental and medicinal plants. Increasing the efficiency of chromosome doubling and fast-reliable screening of desired phenotypes are prerequisites for production of custom-designed medicinal and ornamental plants through artificial polyploidy induction.

Artificial Chromosome Doubling in Allotetraploid Calendula officinalis.

Calendula officinalis L. is known as an ornamental plant as well as a source of biochemical compounds used in cosmetics and industry. C. officinalis has a complex karyotype. Published chromosome numbers differ between 2n = 4x = 28 or 32. We have estimated genome sizes in nine commercial cultivars and evaluated the ploidy level by karyotyping and fluorescent in situ hybridization (FISH) using 5S and 45S rDNA loci. The detection of chromosome sets of two rather than four homologues would suggest that C. officinalis has an allotetraploid background. In addition, four signals for 45S but only two for 5S were found by using FISH. Artificial chromosome doubling is a common technique in plant breeding, as polyploidization results in several consequences for plant growth and development. Especially the suggested allotetraploid background in C. officinalis is interesting when examining the effect of chromosome doubling on the plant phenotype. Here we describe chromosome doubling of three allotetraploid cultivars of C. officinalis, ‘Nova,’ ‘WUR 1553-7’ and ‘Orange Beauty’. Three antimitotic agents – colchicine, oryzalin and trifluralin - were used in different concentrations to find the combination of the best agent and the best dosage to obtain octaploids. For all three cultivars a few octaploids were obtained. A concentration of 200 and 400 ppm of colchicine was most efficient for chromosome doubling in ‘Nova’ and ‘Orange Beauty,’ respectively. For ‘WUR 1553-7’ the treatment with 20 ppm oryzalin was also effective. Cell numbers and first observations of the phenotype in the chromosome doubled plants show thicker leaves and bigger cells, as commonly observed after ploidy doubling. Due to the low number of chromosome doubled plants obtained more elaborate phenotyping will be performed on following generations cultivated under field conditions.

Fertility recovery of anther-derived haploid plants in Cape gooseberry (Physalis peruviana L.)

The cape gooseberry (Physalis peruviana L.) is one of the most important Colombian exotic fruits. Chromosome doubling of anther-derived plants is a key factor in the application of double haploid technology for the genetic improvement of crops. In the present study, axillary buds from four haploid cape gooseberry genotypes were used to evaluate artificial chromosome doubling induced by colchicine and its effects on ploidy level and pollen fertility. Three concentrations of colchicine (5, 10 and 15 mM) and three exposure times (2, 4 and 6 h) were used to determine the best treatment for the generation of fertileB plants from axillary buds of haploid genotypes. The colchicine increased both the number of chromosomes, from 36 to 129, and the average chloroplasts in stomata guard cell, from 4.5 to 23.8. The optimal chromosome doubling of the haploids was obtained with the 5 mM colchicine solution and 2 h exposure time. This protocol produced chromosome doubling in over 60% of the regenerants of the four haploid genotypes, with a high level of fertility. Morphologically, the fertile mixoploid plants showed variation in the vegetative, flowering and fruit characteristics, as compared to the haploid plants.

Colchicine Alternatives for Chromosome Doubling in Maize Haploids for Doubled‐Haploid Production

In vivo production of doubled‐haploid (DH) lines in maize (Zea mays L.) routinely involves artificial chromosome doubling by colchicine treatment of seedlings. Because colchicine is a hazardous chemical, replacing it by less‐toxic alternatives would be highly desirable. Our objectives were to (i) test the efficacy of various herbicides alone or in combination with other herbicides or phytohormones as chromosome doubling agents, (ii) evaluate the efficacy of application of these chemicals for different durations and methods of delivery to meristems, and (iii) compare colchicine treatment with the most promising alternative treatment under economic aspects. Several antimitotic herbicides with different modes of action and two phytohormones were tested in various combinations and concentrations in four experiments for survival rate (SR) of germinated seedlings, reproduction rate (RR) measured as the proportion of D0 plants with seed set, and overall success rate (OSR) measured as the proportion of D1 ears with seed set obtained from the germinated seedlings. Amiprophos‐methyl (APM) and pronamid, applied with the seedling soaking method, outperformed all other alternative treatments tested and reached almost the same OSR as colchicine. Cost comparison between the best treatment, containing APM and pronamid, and the colchicine control showed that the total production cost per D0 plant with seed set was about 10% higher than for colchicine as a result of slightly lower OSR. In conclusion, APM combined in an optimum dosage with pronamid is a promising alternative to colchicine in view of the lower toxicity and similar rate of chromosome doubling in maize.

Chromosome Doubling of Microspore-Derived Plants from Cabbage (Brassica oleracea var. capitata L.) and Broccoli (Brassica oleracea var. italica L.).

Chromosome doubling of microspore-derived plants is an important factor in the practical application of microspore culture technology because breeding programs require a large number of genetically stable, homozygous doubled haploid plants with a high level of fertility. In the present paper, 29 populations of microspore-derived plantlets from cabbage (Brassica oleracea var. capitata) and broccoli (Brassica oleracea var. italica) were used to study the ploidy level and spontaneous chromosome doubling of these populations, the artificial chromosome doubling induced by colchicine, and the influence of tissue culture duration on the chromosomal ploidy of the microspore-derived regenerants. Spontaneous chromosome doubling occurred randomly and was genotype dependent. In the plant populations derived from microspores, there were haploids, diploids, and even a low frequency of polyploids and mixed-ploidy plantlets. The total spontaneous doubling in the 14 cabbage populations ranged from 0 to 76.9%, compared with 52.2 to 100% in the 15 broccoli populations. To improve the rate of chromosome doubling, an efficient and reliable artificial chromosome doubling protocol (i.e., the immersion of haploid plantlet roots in a colchicine solution) was developed for cabbage and broccoli microspore-derived haploids. The optimal chromosome doubling of the haploids was obtained with a solution of 0.2% colchicine for 9–12 h or 0.4% colchicine for 3–9 h for cabbage and 0.05% colchicine for 6–12 h for broccoli. This protocol produced chromosome doubling in over 50% of the haploid genotypes for most of the populations derived from cabbage and broccoli. Notably, after 1 or more years in tissue culture, the chromosomes of the haploids were doubled, and most of the haploids turned into doubled haploid or mixed-ploidy plants. This is the first report indicating that tissue culture duration can change the chromosomal ploidy of microspore-derived regenerants.

Chromosomal rearrangements directly cause underdominant F1 pollen sterility in Mimulus lewisii-Mimulus cardinalis hybrids.

Chromosomal rearrangements can contribute to the evolution of postzygotic reproductive isolation directly, by disrupting meiosis in F1 hybrids, or indirectly, by suppressing recombination among genic incompatibilities. Because direct effects of rearrangements on fertility imply fitness costs during their spread, understanding the mechanism of F1 hybrid sterility is integral to reconstructing the role(s) of rearrangements in speciation. In hybrids between monkeyflowers Mimulus cardinalis and Mimulus lewisii, rearrangements contain all quantitative trait loci (QTLs) for both premating barriers and pollen sterility, suggesting that they may have facilitated speciation in this model system. We used artificial chromosome doubling and comparative mapping to test whether heterozygous rearrangements directly cause underdominant male sterility in M. lewisii-M. cardinalis hybrids. Consistent with a direct chromosomal basis for hybrid sterility, synthetic tetraploid F1 s showed highly restored fertility (83.4% pollen fertility) relative to diploids F1 s (36.0%). Additional mapping with Mimulus parishii-M. cardinalis and M. parishii-M. lewisii hybrids demonstrated that underdominant male sterility is caused by one M. lewisii specific and one M. cardinalis specific reciprocal translocation, but that inversions had no direct effects on fertility. We discuss the importance of translocations as causes of reproductive isolation, and consider models for how underdominant rearrangements spread and fix despite intrinsic fitness costs.

Early spontaneous diploidization of maternal maize haploids generated by in vivo haploid induction

The production of doubled haploid (DH) lines has become a key technology in maize (Zea mays L.) research and breeding. However, most of the haploid plants are sterile and in many cases artificial chromosome doubling involves the use of costly and toxic chemicals. Here, we report a special kind of doubled haploid named the early doubled haploid (EH) that was generated directly by in vivo haploid induction. We found 83 EH plants induced from the hybrid Zhengdan958, 55 families of its F2:3 population and the parental lines, all of which were confirmed to be homozygous diploids via flow cytometry and 104 SSR markers. The progeny of EH0 (EH1) behaved in the same manner and showed the same potentialities as the parents of Zheng58 and Chang7-2. EH plants were also detected in other genetic backgrounds at a frequency of 1–3.5 % based on the total number of haploid plants. Because the EH lines exhibited completely fertility and were obtained from induction directly in one step, they could be used in DH breeding as a new breeding strategy. According to our observations, it is likely that spontaneous doubling in EH occurred during embryo development when haploid induction. The possible mechanism of EH is also discussed.

Difference in the polysomaty degree during fruit development among plants with different ploidy levels produced by artificial chromosome doubling of a pepper ( Capsicum annuum) cultivar ‘Shishitou No. 562’

Polysomaty status of fruit pericarp was compared between a diploid cultivar of pepper ( Capsicum annuum ‘Shishitou No. 562’) and its artificially induced triploid and tetraploid plants. In pepper fruits of different ploidy levels, fruit size as assessed by fruit length reached a plateau from 3 to 4 weeks after anthesis, and the maximum fruit length of triploid and tetraploid fruits was about 43% and 88% of the diploid counterpart, respectively. However, the maximum fruit diameter of both triploid and tetraploid plants was almost the same as that of diploid plant. Polysomaty status of fruit pericarp increased during fruit development and the maximum DNA content of the pericarp cells in the diploid, triploid and tetraploid fruits showed 64C, 96C and 128C, respectively. These results indicate that the same number of endoreduplication is programmed to occur in pepper fruits irrespective of the difference in the ploidy level of plants.

Synthetic hexaploid wheat and its utilization for wheat genetic improvement in China

Synthetic hexaploid wheat (Triticum turgidumxAegilops tauschii) was created to explore for novel genes from T. turgidum and Ae. tauschii that can be used for common wheat improvement. In the present paper, research advances on the utilization of synthetic hexaploid wheat for wheat genetic improvement in China are reviewed. Over 200 synthetic hexaploid wheat (SHW) accessions from the International Maize and Wheat Improvement Centre (CIMMYT) were introduced into China since 1995. Four cultivars derived from these, Chuanmai 38, Chuanmai 42, Chuanmai 43 and Chuanmai 47, have been released in China. Of these, Chuanmai 42, with large kernels and resistance to stripe rust, had the highest average yield (>6 t/ha) among all cultivars over two years in Sichuan provincial yield trials, outyielding the commercial check cultivar Chuanmai 107 by 22.7%. Meanwhile, by either artificial chromosome doubling via colchicine treatment or spontaneous chromosome doubling via a union of unreduced gametes (2n) from T. turgidum-Ae. tauschii hybrids, new SHW lines were produced in China. Mitotic-like meiosis might be the cytological mechanism of spontaneous chromosome doubling. SHW lines with genes for spontaneous chromosome doubling may be useful for producing new SHW-alien amphidiploids and double haploid in wheat genetic improvement.

Intergeneric hybridization among colchicaceous ornamentals, Gloriosa spp., Littonia modesta and Sandersonia aurantiaca via ovule culture

Colchicaceous plants, Gloriosa spp., Littonia modesta and Sandersonia aurantiaca, are cultivated as ornamentals. However, unfortunately no large variations in horticultural traits are found within each genus. We examined intergeneric hybridization using 6 genotypes of Gloriosa spp., 1 genotype of L. modesta and 2 genotypes of S. aurantiaca to obtain wider variability and to develop novel cultivars in those groups. Following intergeneric cross-pollination, putative hybrid plantlets were obtained via ovule culture in various combinations. Early confirmation of the hybridity of ovule culture-derived plantlets was accomplished by flow cytometry and random amplified polymorphic DNA analyses. Several intergeneric hybrids have so far been produced flowers and subjected to morphological characterization. All the hybrids examined, i.e. L. modesta×S. aurantiaca, L. modesta×S. aurantiaca ‘Phoenix’, L. modesta×G. superba ‘Lutea’, L. modesta×G. ‘Marron Gold’, S. aurantiaca×G. superba ‘Lutea’, S. aurantiaca×G. ‘Marron Gold’ and S. aurantiaca ‘Phoenix’×G. ‘Marron Gold’, had novel morphological characteristics compared with their parents, some of which were horticulturally attractive. The results obtained in our series of studies indicate the validity of intergeneric hybridization in the improvement programs of colchicaceous ornamentals. We are now examining to develop a rapid and efficient micropropagation system and to restore fertility by artificial chromosome doubling of intergeneric hybrids that had been produced in our series of experiments.

Pyramiding of alleles with different rust resistance specificities in Linum usitatissimum L.

Transgenic flax plants expressing flax rust resistance specificities conferred by L2 and L10 alleles of L locus were produced through Agrobacterium tumefaciens-mediated transformation of hypocotyl segments or anther culture derived-calli. Transgenic plants were characterized by PCR amplification and Southern hybridization. Homozygous transgenic lines containing a single locus of effective transgene were isolated by consecutive progeny analyses of transgenic plants. In addition, homozygous lines were directly obtained from transformation of haploid cells followed by spontaneous or artificial chromosome doubling when anther culture derived-calli were used as the explants. All transgenic plants containing L2 transgene expressed L2 flax rust resistance specificity and had unambiguous infection type (IT) “0” (immune) reactions to flax rust race 22, which is virulent to endogenous L6 and K1 genes present in the untransformed Linola™1084. Transgenic plants containing L10 transgene exhibited various levels of resistance to flax rust race 191. Five plants had IT “fleck” (immune) reactions, similar to the reactions with the L10 rust differential line, Bolley Golden Selection. The enhanced resistance to rust race 191 in these transgenic plants was attributed to the expression of L10 rust resistance specificity. Further evaluation of the transgenic plants containing L2 or L10 transgene to flax rust race 258 and race 247 respectively showed that the endogenous rust resistance specificities were not modified. The implication of this study in producing transgenic flax plants with multiple resistance specificities and for crop improvement using molecular breeding strategy is discussed.

INDUCTION OF 2N GAMETES FOR OVERCOMING F1-STERILITY IN LILY AND TULIP

For overcoming F1-sterility in interspecific hybrids, mitotic and meiotic polyploidisation is applied in lily and can result in fertile allopolyploids. The mechanism of viable pollen production of mitotic and meiotic polyploidisation is quite different. Mitotic polyploids are obtained by artificial chromosome doubling and results in normal pairing of homologous chromosome set, which enables the formation of 2x gametes during the meiosis. Meiotic polyploidisation, on the other hand, leads in rare cases of restitutional chromosome division, to the formation of unreduced gametes (2n gametes). In contrast to mitotic doubling, homoeologous recombination can occur in these gametes. Genomic in situ hybridization (GISH) was used to discriminate parental chromosomes in the F1 hybrids and backcross progenies to detect homoeologous recombination. Mitotic polyploidisation showed no homoeologous recombinations between the parental genomes whereas in meiotic polyploids it was detected in a high frequency. The use of 2n gametes is therefore the most promising approach for the introgression of desirable characters in using interspecific hybrids in breeding. In both cases, the frequency of viable gametes appeared to be low and limited to a few F1-hybrids. A new and promising method is developed in which 2n-gametes can be induced by the application of laughing gas (N2O). This method proved to be successful in lily and the occurrence of homoeologous recombination, a characteristic of meiotic polyploidisation, was detected. In tulip where the production of mitotic polyploids requires at least five years, it will speed up breeding with interspecific hybrids enormously

Induction of Fertile Amphidiploids by Artificial Chromosome-doubling in Interspecific Hybrid between Dianthus caryophyllus L. and D. japonicus Thunb

Amphidiploid induction by chromosome-doubling is the most efficient method of utilizing a sterile interspecific hybrid for breeding. Previously, although we successfully obtained an interspecific hybrid between carnation (Dianthus caryophyllus L.) and Dianthus japonicus Thunb., the hybrid was sterile. In the present study, therefore, attempts were mode to induce amphidiploids to restore the fertility by artificial chromosome-doubling, i.e., by applying drops of a colchicine solution on to the shoot tips of greenhouse-grown plants or by treating nodal segments excised from in vitro-grown plants by incubation in a colchicine or amiprophos-methyl (APM) solution. Among 156 hybrid plants regenerated after these treatments, nine were revealed to be tetraploids, in addition to one octoploid and 88 mixoploids. Colchicine dropping treatment at a concentration of 2,000 mg·l−1 for one day led to the highest rate (14%) of tetraploid formation, whereas 10% tetraploid formation was obtained by in vitro shaking culture with 5 and 10 mg·l−1 APM for 24 hrs. The amphidiploids exhibited a larger flower size and later flowering time than the original diploid hybrid, and the fertility of both pollen and seed was restored. These amphidiploids are expected to be used for the breeding of carnation cultivars, which are suitable for growing under the Japanese climatic conditions.

Culture of unpollinated ovules, ovaries, and flower buds in some species of the genus Allium and haploid induction via gynogenesis in onion (Allium cepa L.)

Induction of haploid plants is of great importance for breeding purposes because of the possibility to obtain from haploids homozygous material by artificial chromosome doubling in relatively short times. The present study reports the first evidence of successfull haploid induction in onion. Isolated ovules, ovaries, or whole flower buds of different Allium species were cultured on BDS agar medium. Testa browning in the ovules and an extensive growth of the latter were observed. In cultures of ovaries and flower buds, development of callus and subsequent regeneration of plantlets from the region of the nectaries were observed. In leek, sometimes supernumerary flower organs like ovules were formed in this callus. In onion (Allium cepa L.), plantlets developed from the ovules in all culture methods. Chromosome numbers of these plantlets were counted in root tip squash preparations. They were found to be haploid. Haploid plants were significantly smaller than diploid ones. They were transferred to soil and developed until bulb formation. Because of their importance for breeding, haploid plants obtained by gynogenesis are further stored in vitro.

Induced autotetraploidy in Matricaria chamomilla L.

Matricaria chamomilla L. is a diploid species (2n=18) the flowers of which are important source of an oil commonly known as ‘Blue oil’. Improvement through artificial chromosome doubling gave favourable response resulting in larger and longer lasting capitula. Tetraploid showed typical autoploid meiotic behaviour and normal fertility.