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Breeding glittering carnations by an efficient mutagenesis syst
Abstract
We have developed a systematic and directed method to create novel glittering mutants in carnation (Dianthus caryophyllus) by combining the advantages of ion-beam breeding and genomic information. The method is a series of steps that include: (i) establishing a basic strategy to select appropriate genotypes for specific breeding aims using genomic information, (ii) identifying factors that induce anthocyanic vacuolar inclusions (AVIs), (iii) using ion-beam irradiation consecutively to modify pigment glycosylation and/or acylation, (iv) tuning shading of color, and (v) selecting stable mutants with markers. During the course of this work, we identified a factor that causes AVIs and analyzed the content of anthocyanins and related compounds in the flowers of these mutants. Applying the method, we have created two highly novel carnations with the most glittering color ever reported.
... Ion beam technology was developed in Japan and is characterized by high mutagenesis efficiency, even at low doses, with minimal adverse effects on growth because it can deliver higher amounts of energy more locally than X-rays and γ-rays [6], which have been used for conventional mutagenesis [7,8]. There are many examples of mutants created by ion beam irradiation in plants, such as torenia [9,10], verbena [11,12], gentian [13], cyclamen [14], petunia [15], tricyrtis hirta [16], carnation [17,18], and Colocasia [19]. And the most common case of mutant production by ion beam irradiation is in chrysanthemum [20][21][22][23][24][25][26][27][28][29], which are in the same family as gerbera, but it has not been reported in gerbera. ...
... Ion beam technology was developed in Japan and is characterized by high mutagenesis efficiency, even at low doses, with minimal adverse effects on growth because it can deliver higher amounts of energy more locally than Xrays and γ-rays [6], which have been used for conventional mutagenesis [7,8]. There are many examples of mutants created by ion beam irradiation in plants, such as torenia [9,10], verbena [11,12], gentian [13], cyclamen [14], petunia [15], tricyrtis hirta [16], carnation [17,18], and Colocasia [19]. And the most common case of mutant production by ion beam irradiation is in chrysanthemum [20][21][22][23][24][25][26][27][28][29], which are in the same family as gerbera, but it has not been reported in gerbera. ...
Gerbera in vitro shoots were irradiated using three types of ion beams with different line energy transfers (LETs) to investigate the effective LET and absorbed doses for mutagenesis. Furthermore, genomic mutation analyses were conducted on the obtained mutants. Survival rate analysis showed a lower lethal dose 50% (LD50) with ion beams with higher LETs. Trait/morphological mutations exhibited changes in the color and shape of petals and male sterility. Irradiation conditions with the highest growth change and trait/morphological mutation rates in each ion were C irradiation at 10 Gy, Ar irradiation at 5 Gy, and Fe irradiation at 5 Gy, with a range of absorbed dose of around LD50 to about 10 Gy lower. The highest trait/morphological mutation rate was 14.1% with Ar irradiation at 5 Gy, which was one of the criteria for ion beam irradiation of gerbera in vitro shoots. Furthermore, the genomic mutation in the flower color, petal shape, and male sterile mutants were confirmed by genotype analysis using Genotyping by Random Amplicon Sequencing-Direct technology. This is the first study to report the efficient production of gerbera mutants that could be analyzed. Our findings may lead to more efficient gerbera mutant production and analysis technology.
... Orange flowers arise due to the coexistence of Pg-based anthocyanin and Ch2'G (Gonnet and Hieu, 1992;Morimoto et al., 2019). There are a handful of commercial carnations that display colors distinct from general carnations, and the wellknown dusky-purple-flowered carnation 'Nazareno' has a deacyl anthocyanin, pelargonidin 3,5-O-diglucoside (Pg3,5dG), as the major flower pigment (Okamura et al., 2012). There are other carnations with distinct flower colors that have been generated by ion-beam breeding and these varieties accumulate either pelargonidin 3-O-glucoside (Pg3G), cyanidin 3-O-glucoside (Cy3G) or cyanidin 3,5-O-diglucoside (Cy3,5dG) Okamura et al., 2013). ...
... According to the chromatographic analysis, cyaniccolored carnations basically had one major anthocyanin type in the petals and accumulated either Pg-or Cybased anthocyanins dominantly as reported in previous studies (Ootani and Miura, 1961;Okamura et al., 2012Okamura et al., , 2013. This study also indicated that no carnation cultivars except for a variegated carnation 'Pink Montezuma' (No. 60) had both Pg-and Cy-based anthocyanins as the major flower pigment in a petal. ...
Current carnation cultivars have a wide range of flower colors, which is one of the important traits for the flower market. Since large numbers of commercial carnation cultivars bearing various flower colors and flower color patterns have been developed over the last few decades, a comprehensive understanding of the diversity of flower color characteristics has become difficult to achieve. In this study, 56 standard carnation cultivars and 54 spray carnation cultivars were collected and evaluated in terms of flower color and flower color pattern, two visual traits, and flower pigments. Visual flower color analysis indicated that the flower color pattern of the 110 cultivars could be categorized into five typical types and two minor types, and the five typical types were further classified into 16 sub-types. Additionally, flower colors of these carnations could be categorized into 15 hues. High-performance liquid chromatography (HPLC) analysis indicated that carnation flower color is basically determined by the combination of pelargonidin and cyanidin-based anthocyanins, chalcononaringenin 2'-O-glucoside and chlorophyll, giving cyanic, yellow and green color, respectively. Microscopic observation of the petal epidermis indicated that formation of anthocyanin aggregates in the vacuoles and cell shape affect color perceptions, either metallic or velvety, and this is involved in flower color diversification. A fundamental investigation of flower color characteristics will support further developments in flower-color breeding.
... Pigment inclusions were investigated in several flower species and referred to as anthocyanic vacuolar inclusions or AVIs (Markham et al. 2000). However, information on the relationship between AVIs and peculiar colors is limited (Morita et al. 2005, Zhang et al. 2006, and there are no reports on the hereditary pattern of peculiar coloration and AVIs other than our preliminary report (Okamura et al. 2012). Furthermore, there are very few kinds of peculiar color phenotypes in carnation. ...
... Stability of the unique flower color was confirmed after vegetative propagation and then we designated the individual as 'Line A' (Fig. 2a). We further tried to diversify the metallic flower color by the ion-beam breeding technique (Okamura et al. 2012). Petal segments of 'Line A' were irradiated with 320 MeV carbon ions and cultured in vitro to obtain regenerated plants. ...
In general, carnations (Dianthus caryophyllus) have each of four kinds of anthocyanins acylated by malic acid. A few carnation cultivars are known to display a peculiar dusky color supposedly caused by anthocyanic vacuolar inclusions (AVIs). The hereditary pattern suggests that the peculiar color is controlled by a single recessive factor tightly linked with existence of AVIs containing non-acylated anthocyanins. To diversify the peculiar color carnation, we produced a bluish purple line displaying a highly novel metallic appearance by crossbreeding. By subjecting the line to ion-beam irradiation, we generated metallic reddish purple, metallic crimson and metallic red lines. The major anthocyanin of the metallic bluish purple and reddish purple lines was pelargonidin 3,5-diglucoside, whereas that of the metallic crimson and red lines was pelargonidin 3-glucoside. All four metallic lines did not have transcripts for anthocyanin malyltransferase. Metallic crimson and red lines did not express the acyl-glucose-dependent anthocyanin 5-O-glucosyltransferase gene. In contrast to the dusky color types, metallic lines have highly condensed AVIs and water-clear vacuolar sap in the petal adaxial epidermal cells. Differences in the number of AVIs on the abaxial side were observed within mutants containing the same anthocyanin, thereby affecting their shade and hue. We demonstrated that (1) a factor generating the AVIs is inactivated anthocyanin malyltransferase gene, (2) AVIs in water-clear vacuolar sap in the adaxial epidermal cells generate the novel metallic appearance, and (3) ion beam breeding is a useful tool for increasing metallic colors by changing anthocyanin structure and the level of AVIs.
... The Dianthus is commonly known as "carnation," which refers to Dianthus caryophyllus and several intra/interspecific hybrids. Dianthus flowers vary widely in colors and shapes [24][25][26][27]. Molecular understanding of physiological mechanism underlying environmental stimulus effects on flower morphology will facilitate to develop new varieties. ...
Touch stimulus responses are common in plants. Some flowering plants sense the arrival of their pollinators and secrete nectar or release pollen sacs, facilitating successful pollination. Molecular mechanisms for mechanical stimulus responses in plants are well characterized in Arabidopsis leaves, but not in non-model plants or other organs such as flowers. Here, we performed RNA-seq analysis of touched flower buds of Dianthus hybrida, a major ornamental plant. Upon touch treatment, 931 and 132 genes were upregulated and downregulated, respectively. GO enrichment analysis revealed that genes encoding serine/threonine protein kinases were significantly abundant among the upregulated genes, which is consistent with previous studies that demonstrated the pivotal role of protein phosphorylation in the touch stimulus response of Arabidopsis leaves. In comparison with the gene expression profile of touched Arabidopsis leaves, the same families but different homologs of the representative touch-induced genes encoding protein kinases were upregulated, showing that phosphorelay signaling was the common mechanism for touch stimulus response in flowers and leaves, but the players of the phosphorelay signaling were different. These results will contribute to further studies on the mechanical stimulus responses of ornamental flowers and the utilization of this mechanism for breeding programs.
... Induction of mutation using ion beams has been attempted with many ornamental plant species produced and sold as cut flowers, potted plants, and bedding plants, including chrysanthemum (Asami et al. 2011, Furutani et al. 2008, Hisamura et al. 2016, Matsumura et al. 2010, Nagatomi et al. 1997, Okada et al. 2010, Okamura et al. 2015, Sakamoto et al. 2016, Shirao et al. 2007, Suzuki et al. 2005, Tamaki et al. 2017, Tamari et al. 2017, Wakita et al. 2008, carnation (Okamura et al. 2003(Okamura et al. , 2012(Okamura et al. , 2013, rose (Hara et al. 2003, lily (Lilium spp.; Chiba et al. 2007, Kondo et al. 2008, Limonium spp. , Ogawa et al. 2014, Gypsophila spp. ...
Ornamental plants that have a rich variety of flower colors and shapes are highly prized in the commercial flower market, and therefore, mutant cultivars that produce different types of flowers while retaining their growth habits are in demand. Furthermore, mutation breeding is well suited for ornamental plants because many species can be easily vegetatively propagated, facilitating the production of spontaneous and induced mutants. The use of ion beams in mutation breeding has rapidly expanded since the 1990s in Japan, with the prospect that more ion beam-specific mutants will be generated. There are currently four irradiation facilities in Japan that provide ion beam irradiation for plant materials. The development of mutant cultivars using ion beams has been attempted on many ornamental plants thus far, and some species have been used to investigate the process of mutagenesis. In addition, progress is being made in clarifying the genetic mechanism for expressing important traits, which will probably result in the development of more efficient mutation breeding methods for ornamental plants. This review not only provides examples of successful mutation breeding results using ion beams, but it also describes research on mutagenesis and compares results of ion beam and gamma ray breeding using ornamental plants.
... Carnation flowers contain the following four kinds of major anthocyanins, all of which have glucose moieties acylated by malic acid: pelargonidin 3-malylglucoside (Pg3MG) (Figure 4A), cyanidin 3-malylglucoside (Cy3MG) ( Figure 4B), pelargonidin 3,5-cyclicmalyldiglucoside (Pg3,5cMdG) ( Figure 4C), and cyanidin 3,5-cyclicmalyldiglucoside (Cy3,5cMdG) ( Figure 4D) (Bloor 1998;Nakayama et al. 2000;. Each anthocyanin is responsible for a speci c ower color (Figure 4) as described in another review described by Okamura et al. (2012) in this issue. In our work, mutants containing a non-acylated anthocyanin corresponding to each of the carnation ower anthocyanins, pelargonidin 3-glucoside (Pg3G) (Figure 4E), cyanidin 3-glucoside (Cy3G) ( Figure 4F), pelargonidin 3,5-diglucoside (Pg3,5dG) ( Figure 4G), and cyanidin 3,5-diglucoside (Cy3,5dG) ( Figure 4H), were obtained by loss of the acylation enzyme (Abe et al. 2008) and glucosyltranferase (Matsuba et al. 2010) activities by ion-beam irradiation (Okamura et al. 2003). ...
We analyzed flower color mutants of cyclamen (Cyclamen spp.) and carnation (Dianthus caryophyllus) obtained by ion-beam irradiation with an idea that a comprehensive analysis of anthocyanin and its biosynthetically related compounds, such as flavonols and cinnamic acid derivatives, is necessary in order to understand flower color expression mechanism. In this review, we discuss mechanisms for flower color mutation and deduce the following ideas: anthocyanin and its biosynthetically related compounds are cooperatively and compensatively regulated; multiple factors are often concerned in the expression of the same color phenotypes; and changes in chemical structure of a pigment induces new properties that generate novel phenotypes.
Carnation stands in the top ten of the most cultivated and economically important flowers in the world. Since its first selective cross in the 1700s, hundreds of new varieties with unusual characteristics have emerged through conventional and unconventional methods, giving rise to single-flowered, multiple-flowered, and/or miniature-flowered. Most varieties generated have been focused on searching for new colors, giving way to purple or metallic varieties, in a wide range of colors. The search for new varieties that meet market demands continues. Thanks to the increasing development of biotechnological and molecular tools for plant breeding, the generation of new carnation varieties has been increasing. However, there is still a need for varieties that can tolerate climatic conditions, better quality flowers with longer shelf-life, plants resistant to diseases and pests, and plants that will keep carnations of the greatest economic relevance and consumer interest. This chapter presents different breeding methods and cultivation methods used in carnations, focusing on new challenges and areas of breeding opportunity.
Ornamental crops are primarily celebrated for their aesthetic features and allure. The development of new or improved varieties is important for enhancing their aesthetic appeal and substantially increasing their value. Several constraints impede the application of traditional breeding methods in the development of new ornamental varieties. Transgenesis presents a significant opportunity for crafting novel ornamental crop varieties, effectively overcoming the limitations posed by conventional breeding techniques. Transgenesis also provides access to gene pool across the species and organisms to allow the gene transfer from microbes, insects or unrelated plants to ornamental crops to obtain the desirable phenotype. Transgenic technology has been extensively applied to acquire stress-resistant ornamental varieties capable of withstanding adverse climate conditions. Moreover, it has effectively showcased its utility in introducing novel traits such as flower colour, anatomy, fragrance, and plant architecture. Although transgenic technology has been in use for ornamental crops for quite some time, its commercial success has been limited, despite a few notable examples like blue-coloured roses and carnations. Additionally, there is a scarcity of documented instances showcasing its application in modifying other commercially valuable ornamental traits. The development of genomic resources in ornamental plants is anticipated to expedite the integration of transgenesis in this field. This chapter delves into the significance of ornamental plants in the market, the constraints of traditional breeding methods, and successful instances of employing transgenic technology in the development of ornamental crop varieties.
The chapter covers mutation work (mutagens, working dose, mutants) carried out throughout the world on approximately 120 ornamental crops.
Marker-assisted selection (MAS) is a selective method that is not affected by environmental factors. The success of MAS-based breeding programs depends on the selection and validation of the markers used. In addition to the MAS technique, the evaluation of biochemical behaviors, functional evaluation, and gene expression of the lines under stress conditions will be effective for better identifying salinity-tolerant lines. In this regard, to identify mutant lines (M9) rice of saline tolerant, valuation of some effective biochemical traits (seedling phase) as a split factorial experiment based on a randomized complete blocks design with three replications in the greenhouse of Genetics and Agricultural Biotechnology Institute of Tabarestan (GABIT), Sari Agricultural Sciences and Natural Resources University (SANRU), was done in 2019. The main factor included sampling time (3, 6, and 9 days after the stress), and sub-factors included salinity stress in three levels of NaCl (0, 4 dS/m, and 8 dS/m (Adding salt to the water and paddy soil and then saturated salt extraction)) and genotypes (14 mutants (M9), two susceptible controls of Sepidrood, IR29, and two controls tolerant of Dailamani, Nonabokra). The total grain weight per plant and STI index were evaluated as factorial experimental. To validate the gene (s) associated with salinity stress and evaluate allelic diversity of these markers across mutant lines rice, the leaf samples of 14 mutant lines (M9) rice and four sensitive and tolerant controls were collected. Then Band pattern of 18 SSR markers was studied. Also, after validation and identification of some genes affecting salinity stress, molecular evaluations were performed using cgSSR microsatellite to identify new salinity lines in mutant populations (60 lines). In order to evaluate the expression of 5 salinity tolerant genes, four genotypes of IR29 (sensitive control), Nonabokra (tolerant control), G7 and G8 (mutant lines) were used. Leaf samples at 8 dS / m level were harvested in four time courses of 0 (without stress), 3, 6, and 9 days after salinity stress. Analysis of variance showed that the main and the interaction effects were significant for all traits at 1% probability level. At the time of sampling (9 days after stress) and salinity of 8dS/m, the proline concentration in tolerant genotypes increased, and malondialdehyde content decreased. At nine days after stress and salinity 8 dS/m, cluster analysis divided the genotypes into five groups and four genotypes along with the tolerant G17 were in the fifth group. Four genotypes at salinity 8dS/m had a higher total grain weight per plant than Dailamani. 11 primers were selected based on band pattern analysis in susceptible/tolerant cultivars at the molecular analysis. The molecular analysis results showed that OsMAPK4, OsCML11 and OsCPK17 had the highest polymorphic information content (PIC). OsMAPK4 and OsCML11 had the highest marker index (MI) at a rate of 0.23. Cluster and biplot analysis of molecular data of mutant lines studied (14 lines) were divided respectively into 3 and 4 groups. Cluster analysis of molecular data using three candidated markers divided the 60 mutant lines (related to mutant populations) studied in this research into three groups. Expression pattern of OsCPK17 gene at the beginning of stress, IR29 sensitive control showed a significant increase in expression (73) and maintained this increase until the end of stress with less intensity. The expression pattern of OsRacB (T) at nine days after stress application showed an increase in expression (42) sensitive IR29 control and a significant decrease in expression in G8 mutant (-149). Also, the expression pattern of OsMAPK4 gene Nonabokra tolerant control at nine days after stress showed an increase in high expression (45) compared to the reference gene. In total, four SSR primers including OsPEX11-1, OsRacB (T), OsCPK17 and OsMAPK4 were proposed as informative markers for screening salt susceptible/tolerant lines. In the evaluation of 14 mutant lines (M9) studied, eight lines (G1 genotypes from Sang-Tarom mutant, G2 from Rashti mutant, G4 and G8 from Tarom Hashemi mutant and G9 from Chalousi mutant, G12 and G13 from mutant Nemat and G14 from mutant khazar) and in the evaluation of mutant populations M9 (60 lines), 13 lines (code lines 1, 2, 3, 4 from Sang -Tarom mutant, code lines 11, 13, 14, 16, 17, 18 from Tarom Hashemi mutant and codes line (38, 39 and 40 of the Khazar mutant) were identified as new lines tolerant to salinity stress that can be suggested that these lines be used as promising saline-tolerant lines in farms facing salinity stress (soil and water) to increase productivity and increase yield.
The ornamental plant industry is a dynamic and diverse sector worldwide. Plant breeders develop a great number of new cultivars each year to increase production and supply market demands of the ornamental plants. Mutation breeding is a highly effective method for creating genetic variability in ornamental plants with desirable characters expected within a given species’ genetic scope. A mutation creation is called induced mutagenesis of which results are varied according to mutagens and the type of the technique and it is a random process. The target-selected mutagenesis including the random mutagenesis and selection of mutants at a selected locus belongs to this category. By the present, mutations were induced by treatments with physical and chemical mutagens and sometimes their combination. However, biotechnological approaches such as transposable elements, disrupting the gene through the insertion of a DNA fragment and using molecular techniques to create a mutation at a defined site in a DNA molecule have been used to obtain mutations since the development of recombinant DNA technology. There are about 720 ornamental mutant cultivars developed by mutation breeding studies that have been accelerated with tissue culture techniques since the 1970s. In vitro mutagenesis provides for the isolation of chimeric tissues and the propagation of irradiated tissues in mainly vegetatively propagated plants. In addition, in vitro techniques can be supported to the breeding program before, after, and during the mutagen treatments, allowing the scientist to perform the studies timeless and independently of environmental conditions. In vitro techniques have been commonly used in mutation breeding since in vitro technologies were extensively developed. This chapter presents a brief history of mutation breeding in ornamentals and in vitro mutagenesis strategies in ornamental plants.
Marker-assisted selection (MAS), a selective method which is not influenced by environmental factors. The success of MAS-based breeding programs depends on
the selection and validation of the markers used. In this study, to validate the gene(s) associated with salinity stress and evaluation of allelic diversity of these markers in mutant rice lines, Band pattern of 18 SSR markers on a leaf sample of 14 mutant lines (M9) of rice, along with 2 susceptible controls (IR29 and Sepidrood) and 2 tolerant controls (Nonabokra and Dylmani) in 1398 in Genetics & Agricultural Biotechnology Institute of Tabarestan (GABIT). 11 primers were selected based on band pattern analysis in susceptible / tolerant cultivars. The molecular analysis results showed that OsMAPK4, OsCML11 and OsCPK17 had highest polymorphic information content (PIC). OsMAPK4 and OsCML11 had highest marker index (MI) at a rate of 0.23. The lowest PIC (0.05) and MI (0. 11) was accounted for OsCAX (D). Cluster analysis of molecular data, divided rice
genotypes into three distinct groups. However, analysis of Biplot classified the genotypes into four different groups. In this study, 3 genes OsCML11, OsMAPK4 and OsCPK17 were identified on chromosomes 1, 6 and 7 respectively, as the most efficient primers in
identifying the genetic diversity between the rice genotypes, considering that these primers have a very high linkage with salinity resistance genes, can be predicted that 3 lines G1 (M9-P1-7-2-1), G8 (M9-P3- 21-1-1) and G9 (M9-P6 -7-1-1) have high tolerance to
salinity stress.
Keywords: MAS, mutants, Rice, salinity stress.
To identify mutant lines (M9) rice of saline tolerant, and evaluation of some effective biochemical traits (seedling phase) an experiment as a split factorial based on a randomized complete block design with three replications in the greenhouse of Genetics and Agricultural Biotechnology Institute of Tabarestan (GABIT), was done in 2019. The main factor included sampling time (3, 6 and 9 days after stress) and sub-factors included salinity stress in three levels of NaCl (0, 4 dS/m and 8 dS/m (Adding salt to the water and paddy soil and then saturated salt extraction)) and genotypes (14 mutants (M9), 2 susceptible controls of Sepidrood, IR29 and 2 controls tolerant of Dailamani, Nonabokra). Also, identification of some genes effective in salinity stress was performed using three cgSSR microsatellite primers and then, the total grain weight per plant and STI index were evaluated as factorial. Analysis of variance showed that the main and the interaction effects were significant for all traits at 1% probability level. At the time of sampling (9 days after stress) and salinity of 8dS/m, proline concentration in tolerant genotypes increased whereas malondialdehyde content decreased. In nine days after stress and salinity 8 dS/m, cluster analysis divided the genotypes into five groups
and four genotypes along with the tolerant G17 were in the fifth group. Molecular analysis of the band pattern of OsPEX11-1, OsNac5 and OsRacB (T) primers showed 6, 2 and 7 genotypes, respectively, in the same order as the tolerant genotypes. Four genotypes at salinity 8dS/m had higher total grain weight per plant than Dailamani. Finally, considering the effective biochemical properties and cluster analysis results, and with emphasis on molecular and quantitative evaluation it can be suggested that G1 genotypes from Sang-Tarom mutant, G2 from Rashti mutant, G4 from Hashemi mutant and G9 from Chalousi mutant in the first priority and G8 genotypes from Hashemi mutant, G12 and G13 from mutant Nemat in the second priority can be used as promising saline-tolerant lines in farms facing salinity stress (soil and water).
Keywords: Biochemical, Salinity stress, Cluster analysis, Mutant and Molecular.
Mutation breeding is advantageous and essential for carnation to extend the variation of genetic information and create novel traits. We have demonstrated that tissue culture combined with ion beam irradiation can create a wider spectrum in flower phenotype than that with gamma irradiation, which has been the most commonly used mutagen worldwide. We showed that chronic gamma irradiation also has the potential to generate a wide spectrum of flower color mutants comparable to ion beam irradiation. Furthermore, we found that non-acylated type anthocyanin is the key determinant in creating metallic coloration in carnation. We succeeded in diversifying the metallic coloration by ion beam irradiation. Through these studies, eight new varieties of carnation have been developed so far and sold worldwide for 8–15 years. Despite the relatively narrow genetic background of carnation, our research suggested that novel mutant varieties can be created by ion beam breeding system and also by finding key determinants that control flower phenotypes.
Pigments responsible for the appearance of colors in higher plants are classified as chlorophylls, carotenoids, flavonoids and betalains. Pigments not only endow nature with attractive colors but also contribute towards improving health and lower the risk of diseases. The antioxidant properties of pigments have been suggested as being the main mechanism by which they impart their beneficial effects. Anthocyanin compounds have high free radical scavenging capacity and play an essential role in the prevention of cardiovascular disease, obesity, cancer, diabetes and other diseases. Similarly, carotenoids have health-promoting effects: immune enhancement and reduction of the risk of developing degenerative diseases such as cancer, cardiovascular diseases, cataract and macular degeneration. In spite of the health benefits the natural pigments accrue, the use of synthetic pigments comprising of heavy metals is rampant in the food and beverage industry. In this era of going back to nature it is imperative to use the natural pigments in pharmaceutical and food industry. Pigment profiling involves the isolation, separation and characterization of pigments. Trends in pigment analysis reflect not only advances in analytical methodology and instrumentation, but also on current knowledge of their functions or actions. The biology and health benefits of plant pigments have lead researchers to discover modify and utilize techniques for the extraction, separation and quantification of these compounds from natural sources. Techniques like HPLC-MS, HPLC-PAD and HPLC-NMR are most effective techniques for characterization of pigments. In recent years, research on plant pigments attracted more and more attention, especially their isolation and purification in some of the pigment rich fruit and vegetable crops. In contrast, there have been few studies on flower crops which are otherwise well known for the presence of a variety of pigments with myriad colours.
Mutagenesis is an effective approach to expand flower color variation in ornamental plants. Ion beams generated by accelerators are useful mutagens in plant mutation breeding, although the mutability often differs, probably depending on the genetic background of the parental plants. This chapter summarizes the analyses of flower color mutants of cyclamen and carnation obtained by ion beams. Accumulated knowledge regarding the relationship among flower colors, pigments, and associated genes will help our understanding of their genetic background and the choice of suitable starting materials. Our research demonstrated that mutants with altered anthocyanin compositions corresponded to mutation in genes in the biosynthetic pathway and that further understanding of biosynthetic genes associated with the expression of flower colors is important to create novel flower varieties.
To examine flux regulation in the flavonoid pathway of tobacco flowers, we suppressed two genes for dihydroflavonol 4-reductase (NtDFR 1 and 2) by RNA interference (Ri)-mediated post transcriptional gene silencing in pink-flowered tobacco. Two phenotypes were observed, pale pink (DFR-Ri_PP)- and white (DFR-Ri_W)-flowered lines. The relative mRNA levels of NtDFR genes in DFR-Ri_PP and DFR-Ri_W lines were reduced by 79%–95% relative to non-transformed (NT) plants. DFR-Ri_W lines had five-fold higher levels of small interference RNAs compared to DFR-Ri_PP lines. Expression of eight structural genes in the flavonoid pathway was significantly increased in DFR-Ri_W lines but not in DFR-Ri_PP lines based on quantitative RT-PCR. Anthocyanin contents correlated with flower color, with a reduction of 72%–97% in DFR-Ri_PP and DFR-Ri_W lines. Decreases in anthocyanin in flower were proportional with reductions of proanthocyanidin content in seeds. Two pale pink lines, DFR-Ri_PP 17 and 20, with anthocyanin decreases and the lowest level of DFR gene silencing, had higher (dihydro) flavonol production than a white flowered line, DFR-Ri_W 67. This finding suggests that suppression of DFR can increase the total levels of flavonoids due to (dihydro) flavonol biosynthesis. Our observations that higher suppression of DFR had a greater influence on the expression of flavonoid biosynthetic genes demonstrates the key role of DFR in the pathway and allows selection among DFR-Ri lines for plants with specific gene expression profiles to fine-tune flux through the pathway.
Ion beams have been used as a mutagen to improve the efficiency of plant mutation breeding. Mutation breeding is sometimes perceived as a random process. In this review, we describe our recent progress in developing a more efficient mutagenesis technique using ion beam irradiation combined with sucrose pretreatment or subsequent re-irradiation. To shorten the time required for breeding new cultivars of cyclamen, we identified anthocyanin biosynthesis genes and examined the effectiveness of PCR screening of irradiated deletion-mutant candidates at early growth stages. We believe this research is a step toward more efficient and controlled mutation breeding using ion beams.
Global environmental dissociative changes are now in steady state. Its negative impacts were gradually imposed on a wide range of crops and thus crop improvement was hindered as well. Given this challenge, existing and new, appropriate technologies need to be integrated for global crop improvement. Among the different present approaches, mutagenesis and mutation breeding and the isolation of improved or novel phenotypes in conjunction with conventional breeding programmes
can result in mutant varieties endowed with new and desirable variation of agrometrical traits. Induced mutations and its related technologies play very well in this ground and this overall strategy helps to trace the crop genetic diversity along with its biodiversity maintenance. Such induced mutagenesis, a crucial step in crop improvement programme, is now successful in application due to the advancement and incorporation of large-scale selection techniques, micropropagation
and other in vitro culture methods, molecular biology tools and techniques in modern crop breeding performance. Time to time, different mutation techniques and their application processes are changing significantly; in this perspective, insertional mutagenesis and retrotransposons are taking more supports for mutational tagging and new mutation generation. For details investigation on plant structure and function, mutagenic agents and their precise role are much essential as it can produce mutants with some phenotypic changes. Functional genomics
studies make the ultimatum platform on this field of study where few crop plants were used for mutational experimentation on some prime agronomic traits till now. This is a prerequisite step and is applying on diverse crop for further improvement. High throughput DNA technologies for mutation screening such as TILLING
(Targeting Induced Limited Lesions IN Genomes), high-resolution melt analysis (HRM)
, ECOTILLING
etc. are the key techniques and resources in molecular mutation breeding. Molecular mutation breeding will significantly increase both the efficiency and efficacy of mutation techniques in crop breeding. Such modern and classical technologies are using for the development of mutation induction with the objective of using a set of globally important crops to validate identified relevant novel techniques and build these into modular pipelines to serve as technology packages for induced crop mutations. Thus, mutation assisted plant breeding will play a crucial role in the generation of ‘designer crop varieties’ to address the uncertainties of global climate variability and change, and the challenges of global plant-product insecurity.
The efficiency of mutation generation by ion beams, the characteristics of the mutants induced by the combined method of ion-beam irradiation with in vitro cell and tissue culture are investigated. Ion beam breeding can induce a wide variety of flower-color and-shape mutants, and that the combined method of ion beam irradiation with tissue culture is useful to obtain commercial varieties in a short time. The potential of ion beam breeding and its application to ornamentals is discussed. Transposable elements are considerably related to mutations because of their potential ability to transpose. Increasing knowledge on the relationship between mutated genes and transposable elements would improve mutation breeding.
Two cDNAs with homology to glutathione S-transferase (GST) were isolated from the carnation (Dianthus caryophyllus); these cDNAs are termed here DcGSTF1 and DcGSTF2. Phylogenetic analysis suggested that both DcGSTF1 and DcGSTF2 belonged to the Phi class of GSTs. DcGSTF2 showed high levels of transcription at late stages of petal development when anthocyanin biosynthesis is most active. Sequencing of DcGSTF2 indicated that it consisted of three exons and two introns. A truncated DcGSTF2 gene, resulting from the insertion of a CACTA-type transposable element, was found in the genome of a mutable flower line bearing deep pink sectors on pale pink petals. A full length DcGSTF2 gene driven by a continuous expression promoter was introduced into the epidermal cells of carnations with pale pink petals. The transformed cells were deep pink. These results suggest that the DcGSTF2 gene is responsible for flower color intensity in carnations.
The efficiency of ion-beam irradiation combined with tissue culture in obtaining floral mutants was investigated and compared with those of gamma rays and X-rays in carnation. Leaf segments of carnation plants in vitro were irradiated with the 220 MeV carbon ions, and cultured till the shoot regenerated. The carbon ion had the highest effect in reducing the regeneration frequency, and the RBE value with respect to gamma-rays was four. The higher mutation frequency and the wider mutation spectrum were obtained in plants irradiated with the carbon ions than low LET radiations. Three new carnation varieties developed by ion-beam irradiation were applied for the registration of the Japanese Ministry of Agriculture, Forestry and Fisheries. The results indicate that ion beam irradiation could induce wide variety of flower-color and -shape mutants, and that the combined method of ion-beam irradiation with tissue culture is useful to obtain the commercial varieties in a short time.
Glucosylation of anthocyanin in carnations (Dianthus caryophyllus) and delphiniums (Delphinium grandiflorum) involves novel sugar donors, aromatic acyl-glucoses, in a reaction catalyzed by the enzymes acyl-glucose-dependent anthocyanin 5(7)-O-glucosyltransferase (AA5GT and AA7GT). The AA5GT enzyme was purified from carnation petals, and cDNAs encoding carnation Dc AA5GT and the delphinium homolog Dg AA7GT were isolated. Recombinant Dc AA5GT and Dg AA7GT proteins showed AA5GT and AA7GT activities in vitro. Although expression of Dc AA5GT in developing carnation petals was highest at early stages, AA5GT activity and anthocyanin accumulation continued to increase during later stages. Neither Dc AA5GT expression nor AA5GT activity was observed in the petals of mutant carnations; these petals accumulated anthocyanin lacking the glucosyl moiety at the 5 position. Transient expression of Dc AA5GT in petal cells of mutant carnations is expected to result in the transfer of a glucose moiety to the 5 position of anthocyanin. The amino acid sequences of Dc AA5GT and Dg AA7GT showed high similarity to glycoside hydrolase family 1 proteins, which typically act as β-glycosidases. A phylogenetic analysis of the amino acid sequences suggested that other plant species are likely to have similar acyl-glucose-dependent glucosyltransferases.
IntroductionThe Anthocyanin Biosynthetic PathwayGlycosylation of AnthocyanidinsAcylation of Anthocyanin GlycosidesTransport of Anthocyanins from Cytosol to VacuolesConcluding RemarksReferences
We examined the effect of pretreatment on the frequency of flower-color mutants induced by ion beams. We found that petunia seedlings treated with 3% sucrose from 8 days after sowing accumulated significant amount of pigments within 4 days compared to non-treated control seedlings. The petunia seedlings treated with sucrose were exposed to 320-MeV carbon ions. The sucrose treatment did not affect the survival rate and seed fertility of the M1 plants. In the M2 lines obtained by self-pollination of individual M1 plants, chlorophyll mutants were obtained in both treated and non-treated groups with a similar frequency. Flower-color mutants that included magenta, purple, light pink and white were obtained from the original violet color. The frequency of flower-color mutants was significantly higher in the sucrose-treated group than in the non-treated group. These results suggest that sucrose pretreatment specifically increases the frequency of flower-color mutation following ion beam irradiation.
Carnations have anthocyanins acylated with malate. Although anthocyanin acyltransferases have been reported in several plant species, anthocyanin malyltransferase (AMalT) activity in carnation has not been identified. Here, an acyl donor substance of AMalT, 1-O-β-d-malylglucose, was extracted and partially purified from the petals of carnation. This was synthesized chemically to analyze AMalT activity in a crude extract from carnation. Changes in the AMalT activity showed close correlation to the accumulation of pelargonidin 3-malylglucoside (Pel 3-malGlc) during the development of red petals of carnation, but neither AMalT activity nor Pel 3-malGlc accumulation was detectable in roots, stems and leaves.
Recently, heavy ions or ion beams have been used to generate new mutants or varieties, especially in higher plants. It has been found that ion beams show high relative biological effectiveness (RBE) of growth inhibition, lethality, and so on, but the characteristics of ion beams on mutation have not been clearly elucidated. To understand the effect of ion beams on mutation induction, mutation rates were investigated using visible known Arabidopsis mutant phenotypes, indicating that mutation frequencies induced by carbon ions were 20-fold higher than by electrons. In chrysanthemum and carnation, flower-color and flower-form mutants, which are hardly produced by gamma rays or X rays, were induced by ion beams. Novel mutants and their responsible genes, such as UV-B resistant, serrated petals and sepals, anthocyaninless, etc. were induced by ion beams. These results indicated that the characteristics of ion beams for mutation induction are high mutation frequency and broad mutation spectrum and therefore, efficient induction of novel mutants. On the other hand, PCR and sequencing analyses showed that half of all mutants induced by ion beams possessed large DNA alterations, while the rest had point-like mutations. Both mutations induced by ion beams had a common feature that deletion of several bases were predominantly induced. It is plausible that ion beams induce a limited amount of large and irreparable DNA damage, resulting in production of a null mutation that shows a new mutant phenotype.
Flower colour is determined primarily by the production of pigments, usually anthocyanins or carotenoids, but the shade and intensity of the colour are often changed by other factors such as vacuolar compounds, pH and metal ions. Pigmentation can also be affected by the shape of epidermal cells, especially those facing prospective pollinators. A conical shape is believed to increase the proportion of incident light that enters the epidermal cells, enhancing light absorption by the floral pigments, and thus the intensity of their colour. We have identified a gene (mixta) that affects the intensity of pigmentation of epidermal cells in Antirrhinum majus petals. The cells of the corolla lobes fail to differentiate into their normal conical form in mixta mutants. We have cloned the mixta gene by transposon tagging; its sequence reveals that it encodes a Myb-related protein that probably participates in the transcriptional control of epidermal cell shape.
Plant compounds that are perceived by humans to have color are generally referred to as 'pigments'. Their varied structures and colors have long fascinated chemists and biologists, who have examined their chemical and physical properties, their mode of synthesis, and their physiological and ecological roles. Plant pigments also have a long history of use by humans. The major classes of plant pigments, with the exception of the chlorophylls, are reviewed here. Anthocyanins, a class of flavonoids derived ultimately from phenylalanine, are water-soluble, synthesized in the cytosol, and localized in vacuoles. They provide a wide range of colors ranging from orange/red to violet/blue. In addition to various modifications to their structures, their specific color also depends on co-pigments, metal ions and pH. They are widely distributed in the plant kingdom. The lipid-soluble, yellow-to-red carotenoids, a subclass of terpenoids, are also distributed ubiquitously in plants. They are synthesized in chloroplasts and are essential to the integrity of the photosynthetic apparatus. Betalains, also conferring yellow-to-red colors, are nitrogen-containing water-soluble compounds derived from tyrosine that are found only in a limited number of plant lineages. In contrast to anthocyanins and carotenoids, the biosynthetic pathway of betalains is only partially understood. All three classes of pigments act as visible signals to attract insects, birds and animals for pollination and seed dispersal. They also protect plants from damage caused by UV and visible light.
Pigment Biosynthesis I
- Y Ozeki
- Y Matsuba
- Y Abe
- Umemoto Nsasaki
Ozeki Y, Matsuba Y, Abe Y, Umemoto NSasaki N (2011) Pigment
Biosynthesis I. Anthocyanins In: Ashihara H, Crozier A,
Komamine A (Eds) Plant Metabolism and Biotechnology. John
Wiley & Sons, pp 313-333
Carnation serine carboxypeptidase-like acyltransferase is important for anthocyanin malyltransferase activity and formation of anthocyanic vacuolar inclusions
- N Umemoto
- Y Abe
- E A Cano
- M Okamura
- N Sasaki
- S Yoshida
- Y Ozeki
Umemoto N, Abe Y, Cano EA, Okamura M, Sasaki N, Yoshida
S, Ozeki Y (2009) Carnation serine carboxypeptidase-like
acyltransferase is important for anthocyanin malyltransferase
activity and formation of anthocyanic vacuolar inclusions. 5th
International Workshop on Anthocyanins, 2009, in Japan p 115.