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Breeding of four-leaf white clover (Trifolium repens L.) through 60Co gamma-ray irradiation

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Abstract

Four-leaf white clover is not found easily in nature due to its low appearance rate (1 in 10,000). Because people believe that it brings good luck and like to either keep it or present it to a loved one, it has commercial and ornamental value. To breed four-leaf clover, we exposed its flowers to γ-rays at the pollination stage. The M1 seeds produced following doses at 25–100Gy showed an approximately 74% germination rate, with seedling survival at 46%. In the M1 generation of plants irradiated within that dose range, we found an increased frequency of four leaflets. One of them, Jeju Lucky-1 (JL-1), had a frequency of about 60%. To see whether that mutation was somaclonal or genetic, we observed its M2 generation and found that such a phenotype reappeared. Although our results demonstrated that the irradiation of fully mature flowers led to a higher frequency of 4-leaflets, we could not clearly explain the genetic mechanism involved. We suggest that JL-1 is valuable as a new variety, without further genetic fixation, because white clover can be propagated vegetatively by stolons.

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... The four-leaf clover is recognized worldwide as a symbol of good fortune. A four-leaf white clover, exhibiting approximately 60% heritable expression, was developed through mutagenesis (Song et al., 2009). Numerous ornamental multifoliolate white clover genotypes were also reported by the Margot Forde Germplasm Centre, New Zealand (Richardson et al., 2008). ...
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... Penelitian Widiastuti et al. (2010) menunjukkan bahwa bentuk dan warna daun pucuk manggis abnormal akibat diiradiasi sinar gamma, hal ini merupakan respon dari terjadinya perubahan proses fisiologi. Song et al. (2009), menjelaskan terjadinya perubahan bentuk dan jumlah helaian daun Trifolium repens L. yang diiradiasi dengan sinar gamma dosis 25-100 Gy. Menurut penelitian ini tanaman mengalami stress akibat iradiasi. ...
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... The c-rays (considered low linear energy transfer radiation) normally induce rearrangements and deletions (Bruggemann et al. 1996;Shikazono et al. 1998Shikazono et al. , 2001. In conventional plant breeding (Chopra 2005), c-irradiation has been used at low doses to induce mutations in mungbean (Sangsiri et al. 2005), miniature tomato (Matsukura et al. 2007), or to produce four-leaf white clover mutants (Song et al. 2009). ...
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The vegetative phenotype of the pea mutant unifoliata (uni) is a simplification of the wild-type compound leaf to a single leaflet. Mutant uni plants are also self-sterile and the flowers resemble known floral meristem and organ identity mutants. In Antirrhinum and Arabidopsis, mutations in the floral meristem identity gene FLORICAULA/LEAFY (FLO/LFY) affect flower development alone, whereas the tobacco FLO/LFY homologue, NFL, is expressed in vegetative tissues, suggesting that NFL specifies determinacy in the progenitor cells for both flowers and leaves. In this paper, we characterised the pea homologue of FLO/LFY. The pea cDNA homologue of FLO/LFY, PEAFLO, mapped to the uni locus in recombinant-inbred mapping populations and markers based on PEAFLO cosegregated with uni in segregating sibling populations. The characterisation of two spontaneous uni mutant alleles, one containing a deletion and the other a point mutation in the PEAFLO coding sequences, predicted that PEAFLO corresponds to UNI and that the mutant vegetative phenotype was conferred by the defective PEAFLO gene. The uni mutant demonstrates that there are shared regulatory processes in the morphogenesis of leaves and flowers and that floral meristem identity genes have an extended role in plant development. Pleiotropic regulatory genes such as UNI support the hypothesis that leaves and flowers derive from a common ancestral sporophyll-like structure. The regulation of indeterminancy during leaf and flower morphogenesis by UNI may reflect a primitive function for the gene in the pre-angiosperm era.
Article
Developmental control of the formation of the serrated margin of leaf blades was investigated. First, the expression was characterized of a marker gene encoding beta-glucuronidase in strain #1-35-38, a transgenic strain of Arabidopsis thaliana (L.) Heynh, derived by the use of a previously described transposon-tagging system. In strain #1-35-38, expression of the marker gene was tissue-specific, being restricted to stipules and the toothed margins of laminae. Using this transgenic marker gene, we examined the development of leaf blade margins in Arabidopsis. We compared the pattern of expression of the marker gene in the leaves of the wild-type plant with that in plants carrying the asymmetric leaves1 (as1) mutation, which causes dramatic changes in leaf-blade morphology in Arabidopsis. The as1 mutant showed normal morphology of early leaf primordia. The mutation affected the development of leaf segmentation in Arabidopsis without any change in the number or morphology of cells in laminae. The as1 mutation affected leaf morphology independently of mutations in other genes known to affect leaf morphogenesis, such as the acaulis1 mutation and the angustifolia mutation. Based upon these results, the development of the morphology of leaf margins in Arabidopsis is discussed.
Article
The polarized processes of cell elongation play a crucial role in morphogenesis of higher plants. We reported previously that the rotundifolia3 (rot3) mutant of Arabidopsis has a defect in the polar elongation of leaf cells. In the present study, we isolated two additional alleles with mutations in the ROT3 gene. The ROT3 gene was cloned by a T-DNA-tagging method and isolation of the gene was confirmed by a molecular analysis of three rot3 mutant alleles obtained from different mutagenesis. The ROT3 gene encodes a cytochrome P-450 (CYP90C1) with domains homologous to regions of steroid hydroxylases of animals and plants. Expression of the ROT3 gene was detected in all major plant organs. Especially, higher expression was detected in the tissues that had high activity of cell division. We confirmed that the ROT3 gene controls polar elongation specifically in leaf cells by an analysis of three rot3 mutants obtained from different mutagenesis experiments. Our results imply that the ROT3 protein is a member of a new class of cytochrome P-450 encoding putative steroid hydroxylases, which is required for the regulated polar elongation of cells in leaves of Arabidopsis.
Article
The CURLY LEAF (CLF) gene in Arabidopsis thaliana (L). Heynh. is required for stable repression of a floral homeotic gene, AGAMOUS in leaves and stems To clarify the function of CLF in organ development, we characterized clf mutants using an anatomical and genetic approach. The clf mutants had normal roots, hypocotyls, and cotyledons, but the foliage leaves and the stems had reduced dimensions. A decrease both in the extent of cell elongation and in the number of cells was evident in the clf mutant leaves, suggesting that the CLF gene might be involved in the division and elongation of cells during leaf morphogenesis. An analysis of the development of clf mutant leaves revealed that the period during which tell division or cell elongation occurred was of normal duration, while the rates of both cell production and cell elongation were lower than in the wild type. Two phases in the elongation of cells were also recognized from this analysis. From analysis of an angustifolia clf double mutant, we found that the two phases of elongation of leaf cells were regulated independently by each gene. Thus, the CLF gene appears to affect cell division at an earlier stage and cell elongation throughout the development of leaf primordia.
Article
We observed induction of additional trichome formation on the adaxial surface of mature leaves of Arabidopsis after massive doses (1-3 kilograys) of gamma-radiation from cobalt-60. A typical increase in trichome number was observed in the seventh leaf when the full expansion of the fifth leaf was irradiated. Under normal growth conditions, trichome numbers on the adaxial surface of seventh leaf of the Arabidopsis ecotypes Columbia (Col) and Landsberg erecta (Ler) were 122.5 +/- 22.7 and 57.5 +/- 14.5, respectively. However, gamma-radiation induced additional trichome formation and the numbers rose to 207.9 +/- 43.7 and 95.0 +/- 27.1 in Col and Ler, respectively. In Col the shape of new trichomes was intact and their formation was spatially maintained at equal distances from other trichomes. In Ler trichome morphology was aberrant and the formation was relatively random. Treatment with antioxidants before gamma-irradiation suppressed the increase in trichome number, and treatment with methyl viologen and light induced small trichomes. These results suggest that gamma-radiation-induced trichome formation is mediated by active oxygen species generated by water radiolysis. gamma-Radiation-induced trichome formation was blocked in the trichome mutants ttg-1, gl1-1, and gl2-1. These results suggest that gamma-radiation-induced trichome formation is mediated by the normal trichome developmental pathway.
Article
In Arabidopsis, the two-dimensional expansion of leaves is regulated via the polarized elongation of cells. The ROTUNDIFOLIA3 (ROT3) protein, a member of the family of cytochromes P450, is involved in this process and regulates leaf length. Transgenic plants that overexpressed a wild-type ROT3 gene had longer leaves than parent plants, without any changes in leaf width. The shapes of floral organs were also altered, but elongation of the stem, roots, and hypocotyls was unaffected. To our knowledge, no similar specific regulation of leaf length has been reported previously. Transgenic plants overexpressing the rot3-2 gene had enlarged leaf blades but leaf petioles of normal length. Morphological alterations in such transgenic plants were associated with changes in shape of leaf cells. The ROT3 gene seems to play an important role in the polar elongation of leafy organs and should be a useful tool for the biodesign of plant organs.
Article
Ionizing radiation produces many chromosome aberrations. A rich variety of aberration types can now be seen with the technique of chromosome painting. Apart from being important in medicine and public health, radiation-produced aberrations act as colorful molecular clues to damage-processing mechanisms and, because juxtaposition of different parts of the genome is involved, to interphase nuclear organization. Recent studies using chromosome painting have helped to identify DNA double-strand-break repair and misrepair pathways, to determine the extent of chromosome territories and motions, and to characterize different aberration patterns left behind by different kinds of radiation.
Article
Heteroblasty in Arabidopsis thaliana was analyzed in a variety of plants with mutations in leaf morphology using a tissue-specific beta-glucuronidase gene marker. Some mutants exhibited their mutant phenotypes specifically in foliage leaves. The phenotypes associated with the foliage-leaf-specific mutations were also found to be induced ectopically in cotyledons in the presence of the lec1 mutation. Moreover, the features of an emfl lec1 double mutant showed that cotyledons can be partially converted into carpelloids. When heteroblastic traits were examined in foliage leaves in the presence of certain mutations or natural deviations by histochemical analysis of the expression of the tissue-specific marker gene, it was found that ectopic expression of the developmental program for the first foliage leaves in lec1 cotyledons seemed to affect the heteroblastic features of the first set of foliage leaves, while foliage leaves beyond the third position appeared normal. Similarly, in wild-type plants, discrepancies in heteroblastic features, relative to standard features, of foliage leaves at early positions seemed to be eliminated in foliage leaves at later positions. These results suggest that heteroblasty in foliage leaves might be affected in part by the heteroblastic stage of the preceding foliage leaves but is finally controlled autonomously at each leaf position.
Article
A cyclic somatic embryogenic system was used to induce mutations in cassava variety PRC 60a in vitro. Globular-stage somatic embryos were selected as suitable experimental materials, and 50 Gy of gamma-rays was determined to be the optimal dose for inducing mutations. During subsequent field trials, more than 50% of the regenerated mutant lines varied morphologically from wild-type plants. Consequently, we used this approach to induce genetic variability for obtaining novel cassava cultivars. Among the different mutant lines obtained, lines S14 and S15 showed large morphological variations. In 10-month-old S14 and S15 mutant lines, storage root yield was reduced 17-fold and 60-fold, respectively, compared to wild-type plants, while the storage roots of S15 mutant plants also exhibited an almost 50% decrease in starch content and a significant reduction (30%) in amylose content. These two features were observed throughout the different developmental stages of the storage roots in S15 plants.
Article
Biodiversity of plant shape is mainly attributable to biodiversity of leaf shape and the shape of floral organs, the modified leaves. However, the exact mechanisms of leaf-shape determination remain unclear due to the complexity of flat-structure organogenesis that includes the simultaneous cell cycling and cell enlargement in primordia. Recent studies in developmental and molecular genetics have revealed several important aspects of leaf-shape control mechanisms. For example, understanding of polar control in leaf-blade expansion has advanced greatly. A curious phenomenon called "compensated cell enlargement" found in leaf organogenesis studies should also provide interesting clues regarding the mechanisms of multicellular organ development. This paper reviews recent research findings with a focus on leaf development in Arabidopsis thaliana.
Variety of four-leaf clover and a method for breeding the same
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Likewise, in pea, the vegetative phenotype of the unifoliata I.-J. Song and H.-G. Kang contributed equally to this work. I.-J. Song Á H.-G. Kang Á T.-W. Bae Á P.-O. Lim Á H.-Y. Lee Subtropical Horticulture Research Institute, Cheju National University, Jeju 690-756, South Korea J.-Y. Kang Á H.-D. Kim Á T. Adachi Á H.-Y. Lee (&) Faculty of Biotechnology, Cheju National University, Jeju 690-756, South Korea e-mail: hyoyeon@cheju.ac.kr S.-Y. Kang Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongeup 580-185, South Korea P.-O. Lim Department of Science Education, Cheju National University, Jeju 690-756, South Korea T. Adachi Institute for Plant Breeding R & D, Ltd., 1-46-901, Hama 5-Chome, Tsurumi-Ku, Osaka 538-0035, Japan References Bae CH, Abe T, Min KS, Kim DC, Jung JS, Lee CH, Lim YP, Lee HY (1998) Mutation induction and selection of salt-tolerant plants by heavy-ion beam irradiation in tobacco proembryo.
The mutant crispa reveals multiple roles for PHANTAS-TICA in pea compound leaf development The structure of the mature plant
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Characterization of the 5-methyltryptophan resistant mutant lines selected by mutagenized seeds in rice
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White clover morphology changes with stress treatments) c-Rays and EMS induced leaf mutants in mungbean (Vigna radiata (L) Wilczek) The growth and asymmetry of neighboring plants of white clover
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The structure of the mature plant
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Characterization of the 5-methyltryptophan resistant mutant lines selected by mutagenized seeds in rice
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