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Breeding of four-leaf white clover (Trifolium repens L.) through 60Co gamma-ray irradiation
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.
... 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). ...
Background
The genus Trifolium is characterized by typical trifoliolate leaves. Alterations in leaf formats from trifoliolate to multifoliolate, i.e., individual plants bearing trifoliolate, quadrifoliolate, pentafoliolate or more leaflets, were previously reported among many species of the genus. The study is an attempt to develop pure pentafoliolate plants of T. alexandrinum and to understand its genetic control.
Methods
The experimental material consisted of two populations of T. alexandrinum with multifoliolate leaf expression, i.e.,interspecific hybrid progenies of T. alexandrinum with T. apertum , and T. alexandrinum genotype Penta-1. Penetrance of the multifoliolate trait was observed among multifoliolate and trifoliolate plant progenies. In vitro culture and regeneration of plantlets from the axillary buds from different plant sources was also attempted.
Results
The inheritance among a large number of plant progenies together with in vitro micro-propagation results did not establish a definite pattern. The multifoliolate leaf formation was of chimeric nature, i.e., more than one leaf format appearing on individual branches. Reversal to normal trifoliolate from multifoliolate was also quite common. Penetrance and expression of multifoliolate leaf formation was higher among the plants raised from multifoliolate plants. Multifoliolate and pure pentafoliolate plants were observed in the progenies of pure trifoliolate plants and vice-versa. There was an apparent increase in the pentafoliolate leaf formation frequency over the years due to targeted selection. A few progenies of the complete pentafoliolate plants in the first year were true breeding in the second year. Frequency of plantlets with multifoliolate leaf formation was also higher in in vitro axillary bud multiplication when the explant bud was excised from the multifoliolate leaf node.
Conclusion
Number of leaflets being a discrete variable, occurrence of multifoliolate leaves on individual branches, reversal of leaf formats on branches and developing true breeding pentafoliolates were the factors leading to a hypothesis beyond normal Mendelian inheritance. Transposable elements (TEs) involved in leaf development in combination with epigenetics were probably responsible for alterations in the expression of leaflet number. Putative TE’s movement owing to chromosomal rearrangements possibly resulted in homozygous pentafoliolate trait with evolutionary significance. The hypothesis provides a new insight into understanding the genetic control of this trait in T. alexandrinum and may also be useful in other Trifolium species where such observations are reported.
... 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. ...
p>ABSTRACT
Handeuleum (Graptophyllum pictum L.Griff) is a medicinal plant widely used as a traditional medicine due to its benefecial content. Therefore, it should be developed as one of the leading Indonesian medicinal plants. The purpose of this research was to study the effect of gamma ray irradiation on morphological alteration and growth of Handeuleum accession from Bogor. The doses of gamma rays were 0, 15, 30, 45, 60, 75, 90, and 105 Gy. The results of the research showed that gamma-ray irradiation induced morphological changes and influences the growth of Handeuleum. Irradiation dose at rate of 105 Gy produced new leaf morphology in Handeuleum, namely cordate. Doses of 60, 75, 90, and 105 Gy produce stunted plants with stiff and yellowish-green leaves. The dose of 45 Gy produced relative green color index which was higher than the control plants.
Keywords: handeuleum, gamma irradiations</p
... 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). ...
The poor stability and off-flavors of soybean oil and protein products can be reduced by eliminating lipoxygenases from soybean seed. Mature seeds of OX948, a lipoxygenase triple null mutant line, do not contain lipoxygenase proteins. The objective of this study was to determine the molecular basis of the seed lipoxygenase null traits in OX948. Comparisons of the sequences for lipoxygenase 1 (Lx1) and lipoxygenase 2 (Lx2) genes in the mutant (OX948) with those in a line with normal lipoxygenase levels (RG10) showed that the mutations in these genes affected a highly conserved group of six histidines necessary for enzymatic activity. The OX948 mutation in Lx1 is a 74 bp deletion in exon 8, which introduces a stop codon that prematurely terminates translation. A single T-A substitution in Lx2 changes histidine H532 (one of the iron-binding ligands essential for L-2 activity) to glutamine. The mutation in the lipoxygenase 3 (Lx3) gene in OX948 is in the promoter region and represents two single base substitutions in a cis-acting AAATAC paired box. All three mutations would result in the loss of lipoxygenase activity in mature seed. The seed lipoxygenase gene mutation-based molecular markers could be used to accelerate and simplify breeding efforts for soybean cultivars with improved flavor.
Cymbidium goeringii is one of the most important and popular species in the orchid family in north-east Asia. In the present study, we prepared multiplex PCR system, and used it for the genotyping of eight simple sequence repeats (SSR) markers (CG409, CG415, CG709, CG722, CG787, CG1023, CG1210, and CG1281) in subject with 40 samples of cultivated varieties. All the analyzed samples showed different combined genotypes. The average combined power of discrimination was very high value of 7.14 x 10(-10), and observed heterozygosity (Ho = 0.466) was similar with two wild populations of C. goeringii, which may indicate no or little occurrence of genetic change after collection from wild habitats. The present study also developed a two-dimensional barcode to express information of genotype results of eight SSR markers (SSR DNA ID). The discrimination power of DNA ID between two individuals will be statistically more than 99.999999%. The SSR DNA ID and two-dimensional barcode may be very usefully applied for the discrimination and maintence of cultivars of C. goeringii.
Gamma rays and EMS induced chlorophyll mutations in mungbean Abstract The chlorophyll mutations were investigated in a population of established cultivars of mungbean namely, T44, PDM 11 and PDM 54, induced through gamma rays (10, 20, 30 and 40 kR), EMS (0.01, 0.02, 0.03 and 0.04 M) and combination of 10, 20, 30 and 40 kR gamma rays with 0.02 M EMS. The frequency and spectrum for different types of chlorophyll mutations viz. albina, xantha, chlorina and viridis in M 2 generation were observed. The frequency and spectrum of chlorophyll mutations were marked difference between three cultivars under present investigation and observed that, 30kR gamma rays and 0.02 M EMS in T44, 20 kR gamma rays and 0.03 M EMS in PDM 11 and 20 kR gamma rays and 0.02 M EMS in PDM 54 induce maximum chlorophyll mutations. Combined treatments produced higher mutation frequencies than the individual doses of mutagens. Highest chlorophyll mutations frequencies were observed in combination of 20 kR gamma rays + 0.02 M EMS in all the three cultivars. Genotypic differences were observed with regard to frequencies and spectrum. Chlorina and viridis appeared to be more frequently, relative to other chlorophyll mutations. Inheritance of albina, xantha, chlorina and viridis was monogencally controlled and was recessive in nature. Independent response towards the mutagenic treatments for chlorophyll mutations was observed among all the three cultivars.
2 Abstract: In the present research, the effects of gamma radiation on mitotic index have been investigated. Seeds of Hordeum vulgare L. were treated with different doses of gamma ray (50, 100, 150, 200, 250, 300 Gy). MI (mitotic index) at embryonic root tip of barley seedling has been decreased with increasing doses of gamma radiation. The inhibitory effects of gamma rays on the mitotic index indicated that gamma rays have mutagenic effects on embryonic roots of barley.
Seeds of mungbean varieties KPS 2, VC 6468-11-1B, their F1 and F2 were treated with gamma rays (Cs-137 source) at the dose of 500 Gy. The M1 seeds were sown in the field with the controls (non-irradiated seeds) and bulk-harvested. The M2 seeds were sown to observe their characters and number of mutants in each population. Among over 430,000 plants observed, irradiated F1 population gave the highest frequency of mutants at 0.168%, followed by F2, VC 6468-11-1B, and KPS 2 at 0.165%, 0.152%, and 0.142%, respectively. Mutant characters were grouped as chlorophyll, leaf, flower, and pod mutants. Chlorophyll mutations included albino, coppery leaf, light-green leaf, variegated leaf, waxy leaf, white streak leaf, and xantha leaf. Leaf mutations were lanceolate leaf, narrow-rugose leaf, multiple leaflet, round-cuneate leaf, unifoliate leaf, and wrinkled leaf. The flower mutant was cock's comb raceme while the pod mutant was a lobed one. All mutants were purified for genetic study and possible uses of the traits.
Due to the non-availability of sources of resistance in Sri Lanka to Phytopbthora blight in sesame (Sesamum indicum) caused by Phytopbthora nicotianae var. parasitica, a mutation breeding programme was initiated. Seeds of three genotypes were subjected to six doses of gamma rays from 100 Gy to 750 Gy from a 60Co source. Seeds of M2 bulks, sampled by variety and treatment from the first five capsules formed on M1 plants, were grown in a field with a history of repeated incidence of the disease. The best 21 lines, having the highest survival and seed production, identified in the screening of M3 and M4 progeny rows of selected M2 single plants were then tested in a replicated field trial with the recommended cultivar ‘MI 3’ as a control. The plant survival in selected lines averaged 43.3 percent as against 7.2 percent in the recommended variety. Eight selections recorded significantly higher seed yield than ‘MI 3’ at P < 0.01 and another three at P < 0.05. Gamma ray treatments of 450 Gy and 600 Gy produced more lines tolerant to the disease than the other doses used.
The plasticity of white clover (Trifolium repens L.) results in changes in plant habit in response to different environmental stresses. The objective of this research was to characterize those morphological changes associated with plasticity in white clover clones derived from ‘Osceola’, ‘Grassland Huia’, and SRVR germplasm. Clones were exposed to treatments in the following two-way factorial design: no clipping or clipping on 7-d intervals and barrier to stolon rooting or no barrier. In a three-season greenhouse test, 90 clones were measured for leaf dry weight (DW), stolon DW, stolon length, root DW, and apex number. Generated parameters were herbage DW (leaf DW + stolon DW), biomass DW (root DW + herbage DW), leaf-to-stem ratio (leaf DW ÷ stolon DW), and herbage-to-root ratio (herbage DW ÷ root DW). Plant means were significantly reduced for all traits and ratios with repeated clipping. The rooting barrier significantly reduced apex number, root DW, and the leaf-to-stem ratio while increasing the stolon DW and the herbage-to-root ratio. The interaction of root barrier and repeated clipping was significant for apex number, root DW, and the herbage-to-root ratio. Near zero correlations for the same measurements made on the same clone grown with different stress treatments suggest genetic differences in the magnitude of plasticity. Path analysis further described changes in relationships among traits for plants exposed to different stresses. The magnitude of plasticity appeared to be a clone-specific phenomenon that may frustrate verifying gain with selection for progenies of selections evaluated in different environments.
We report two new recessive mutations in Arabidopsis, mgoun1 and mgoun2 which cause a reduction in the number of leaves and floral organs, larger meristems and fasciation of the inflorescence stem. Although meristem structure is affected in the mutants, we provide evidence that its overall organisation is normal, as shown by the expression patterns of two meristem markers. Microscopical analyses suggest that both mutations affect organ primordia production. mgo1 strongly inhibits leaf production in a weak allele of shoot meristemless, stm-2. In addition, mgo1 and 2 severely reduce the ability of the fasciata1 and 2 mutants to initiate organs, although meristem formation per se was not inhibited. The strong allele, stm-5, is epistatic to mgo1, showing that the presence of meristematic cells is essential for MGO1 function. These results suggest a role for the MGO genes in primordia initiation although a more general role in meristem function can not be excluded. We describe a form of fasciation which is radically different from that described for clavata, which is thought to have an increased size of the meristem centre. Instead of one enlarged central meristem mgo1 and 2 show a continuous fragmentation of the shoot apex into multiple meristems, which leads to the formation of many extra branches. The phenotype of mgo1 clv3 and mgo2 clv3 double mutants suggest that the MGO and CLV genes are involved in different events. In conclusion, our results reveal two new components of the regulatory network controlling meristem function and primordia formation. A model for MGO genes is discussed.
We previously showed that the ANGUSTIFOLIA (AN) gene regulates the width of leaves of Arabidopsis thaliana, by controlling the polar elongation of leaf cells. In the present study, we found that the abnormal arrangement of cortical microtubules (MTs) in an leaf cells appeared to account entirely for the abnormal shape of the cells. It suggested that the AN gene might regulate the polarity of cell growth by controlling the arrangement of cortical MTs. We cloned the AN gene using a map-based strategy and identified it as the first member of the CtBP family to be found in plants. Wild-type AN cDNA reversed the narrow-leaved phenotype and the abnormal arrangement of cortical MTs of the an-1 mutation. In the animal kingdom, CtBPs self-associate and act as co-repressors of transcription. The AN protein can also self-associate in the yeast two-hybrid system. Furthermore, microarray analysis suggested that the AN gene might regulate the expression of certain genes, e.g. the gene involved in formation of cell walls, MERI5. A discussion of the molecular mechanisms involved in the leaf shape regulation is presented based on our observations.
An early genetic study showed that most radiation-induced mutations are not transmitted to progeny. In recent molecular studies in plants, mainly M2 plants or their progeny, which contain only transmissible mutations, have been analyzed, but the early results imply that these studies are insufficient as comprehensive descriptions of radiation-induced mutations. To study radiation-induced mutations caused by low-LET gamma-rays and high-LET carbon ions at the molecular level, we used the pollen-irradiation method and the plant Arabidopsis thaliana to study various mutations, including nontransmissible mutations. This analysis revealed that most mutants induced with irradiation with gamma-rays (150-600 Gy) or carbon ions (40-150 Gy) carried extremely large deletions of up to >6 Mbp, the majority of which were not transmitted to progeny. Mutations containing 1- or 4-bp deletions, which were transmitted normally, were also found. Comparison of the deleted regions in the mutants showing various manners of transmission suggests that the nontransmissibility of the large deletions may be due to the deletion of a particular region that contains a gene or genes required for gamete development or viability.
Pinnate compound leaves have laminae called leaflets distributed at intervals along an axis, the rachis, whereas simple leaves have a single lamina. In simple- and compound-leaved species, the PHANTASTICA (PHAN) gene is required for lamina formation. Antirrhinum majus mutants lacking a functional gene develop abaxialized, bladeless adult leaves. Transgenic downregulation of PHAN in the compound tomato (Solanum lycopersicum) leaf results in an abaxialized rachis without leaflets. The extent of PHAN gene expression was found to be correlated with leaf morphology in diverse compound-leaved species; pinnate leaves had a complete adaxial domain of PHAN gene expression, and peltate leaves had a diminished domain. These previous studies predict the form of a compound-leaved phan mutant to be either peltate or an abaxialized rachis. Here, we characterize crispa, a phan mutant in pea (Pisum sativum), and find that the compound leaf remains pinnate, with individual leaflets abaxialized, rather than the whole leaf. The mutant develops ectopic stipules on the petiole-rachis axis, which are associated with ectopic class 1 KNOTTED1-like homeobox (KNOX) gene expression, showing that the interaction between CRISPA and the KNOX gene PISUM SATIVUM KNOTTED2 specifies stipule boundaries. KNOX and CRISPA gene expression patterns indicate that the mechanism of pea leaf initiation is more like Arabidopsis thaliana than tomato.
This experiment was carried out to increase the cultivation area of rape (Brassica napus) and develop this plant as a biodiesel so dry seeds of 3 cultivars, 'Naehan', 'Hanla', and 'Tammi' were irradiated with proton ion and gamma-ray at range of 0 to 2,000 Gy and then radiosensitivity of M, generation were investigated. The germination rate were more than 95.3% regardless of radiation source, dosage and cultivar and emergence rate at 6 days after sowing were decreased by increasing dosage but those of 12 days as more than 91.7% were similar all together. Survival rate in 1,000 Gy and below at 7 weeks after sowing were similar but remarkably reduced 38.1 similar to 84.1% in 1,500 Gy and 2,000 Gy. The length and area of cotyledon, and hypocotyl length in all 3 cultivars were badly reduced with high significancy by increasing dosage of proton ion and gamma-ray irradiation and sensitive responses were showed in order of 'Naehan' 'Hanla', and 'Tammi'. On the other hand, there was hormesis that the length and area of cotyledon were more increased at 100 similar to 200 Gy in the proton ion than control in 'Tammi'. Chlorophyll content, number of true leaves and leaf area at I month after sowing were decreased by increasing radiation dosage regardless of radiation source so had high significancy. As the cotyledons at I month after sowing almost showed chlorosis in more than 800 Gy and chlorosis was especially serious in "Tammi'. The number of true leaves was about 2.3 in control and the low level of dosage but cotyledon didn't grow in the high level dosage of 1,500 Gy and over. And the dosage in which leaf area was reduced to 50% compared to that of control were 800 Gy in 'Nachan' and 1,500 Gy in 'Tammi' and 'Hanla' by proton ion and 400 Gy in 'Tammi', 1,500 Gy both 'Naehan' and 'Hanla' by gamma-ray. The fresh weight of top land than root was more sensitive so dosage in which fresh weight reduced to 50% compared to that of control were 600 Gy in 'Naehan' and 1,000 Gy both 'Tammi' and 'Hanla'. As the results, growth of seedling decreased over 50% in more than 1,000 Gy with high sensitivity to radiation and so it was appeared that proper dosage for mutation breeding in rape was 1,000 Gy and below.
Three Basmati rice varieties (Basmati 370, Basmati Pak, and Super Basmati) were used to examine varietal differences in radiosensitivity to gamma radiation. Dry seeds of rice varieties were exposed to 150, 200, 250 and 300 Gy of gamma rays. Sensitivity to dose was determined by various measurements on the M1 generation and on the basis of frequency of various types of chlorophyll mutations obtained in the M2 generation. With the increase in radiation dose a decrease in germination, seedling height, root length and emergence under field conditions was observed in M1 generation. In contrast, the gamma rays doses had some stimulatory effects on total spikelets at the maturity stage. These effects were observed in all the gamma radiation treatments in case of Basmati 370 where total spikelets increased above the non-irradiated control. Plant height and seed fertility decreased with increase in gamma radiation dose in an approximately linear fashion. The LD50 values for seed fertility were 238, 232 and 223 Gy for Basmati 370, Basmati Pak and Super Basmati, respectively. The effectiveness of the dose in inducing genetical changes was estimated by counting the number of chlorophyll mutations in the M2 generation. The frequency of chlorophyll mutations increased with the radiation dosage upto 250 Gy which sharply decreased thereafter. Gamma ray dose of 200 and 250 Gy produced the highest mutation frequency for Basmati 370 followed by Basmati Pak and Super Basmati. The albina type of mutation was most frequent in all the three varieties.
The plant leaf provides an ideal system to study the mechanisms of organ formation and morphogenesis. The key factors that control leaf morphogenesis include the timing, location and extent of meristematic activity during cell division and differentiation. We identified an Arabidopsis mutant in which the regulation of meristematic activities in leaves was aberrant. The recessive mutant allele blade-on-petiole1-1 (bop1-1) produced ectopic, lobed blades along the adaxial side of petioles of the cotyledon and rosette leaves. The ectopic organ, which has some of the characteristics of rosette leaf blades with formation of trichomes in a dorsoventrally dependent manner, was generated by prolonged and clustered cell division in the mutant petioles. Ectopic, lobed blades were also formed on the proximal part of cauline leaves that lacked a petiole. Thus, BOP1 regulates the meristematic activity of leaf cells in a proximodistally dependent manner. Manifestation of the phenotypes in the mutant leaves was dependent on the leaf position. Thus, BOP1 controls leaf morphogenesis through control of the ectopic meristematic activity but within the context of the leaf proximodistality, dorsoventrality and heteroblasty. BOP1 appears to regulate meristematic activity in organs other than leaves, since the mutation also causes some ectopic outgrowths on stem surfaces and at the base of floral organs. Three class I knox genes, i.e., KNAT1, KNAT2 and KNAT6, were expressed aberrantly in the leaves of the bop1-1 mutant. Furthermore, the bop1-1 mutation showed some synergistic effect in double mutants with as1-1 or as2-2 mutation that is known to be defective in the regulation of meristematic activity and class I knox gene expression in leaves. The bop1-1 mutation also showed a synergistic effect with the stm-1 mutation, a strong mutant allele of a class I knox gene, STM. We, thus, suggest that BOP1 promotes or maintains a developmentally determinate state in leaf cells through the regulation of class I knox genes.
One direct way to identify a gene and its function is a forward genetic approach based on mutation analyses. To accumulate genetic resources for breeding and functional genomics in the tomato, we generated 6,347 lines of an M2 population with 300 Gy of gamma-ray irradiation in the inbred miniature dwarf variety 'Micro-Tom.' In total, 6,301 M2 lines were screened based on morphological alteration and brix-aberration, and 237 lines were selected as mutant candidates at the first screening. Subsequent screening of the self-fertilized M3 and M4 progeny yielded 24 lines of morphological mutants and 11 lines of aberrant brix mutants. Segregation data suggested that most of the mutant lines had single recessive mutations, with the exception of two lines. The chlorophyll mutation ratio in germinated M 2 seedlings was 0.37% and the actual mutant frequency was 0.5%. The selected mutant lines exhibited a wide range of mutations, including whole plant properties with a severe phenotype, which allowed for more efficient screening of knockout mutants. Three characterized mutants, pale leaf, pink, and short root are also described.
We observed induction of additional trichome formation on the adaxial surface of mature leaves of Arabidopsis after massive doses (1–3 kilograys) of γ-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, γ-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 γ-irradiation suppressed the increase in trichome number, and treatment with methyl viologen and light induced small trichomes. These results suggest that γ-radiation-induced trichome formation is mediated by active oxygen species generated by water radiolysis. γ-Radiation-induced trichome formation was blocked in the trichome mutants ttg-1, gl1-1, andgl2-1. These results suggest that γ-radiation-induced trichome formation is mediated by the normal trichome developmental pathway.
In order to increase the efficiency of induction of mutations by chemical muta-gens, the fertilized egg cells at 6 hours after flowering were treated with MNU. The frequency of mutations and the efficiency in the treatment of fertilized egg cell greatly exceeded those in the treatment of dry seeds. The possible concentration of MNU applied on the fertilized egg cell was estimated less than 1.0 mM.
A rice plant resistant to 5-methyltryptophan (5MT) was selected from mutagenized M3 seeds (Oryza sativa L. var. Sasanishiki) originating from panicles treated with ethylene imine (0.2%) 2 h after flowering. When germinated on 5MT-containing medium, the seeds (M4) from selfed plants segregated with a 3 resistant:1 sensitive ratio, indicating that the plant was heterozygous for a resistance gene and that the resistance was dominant. The resistance was also expressed in callus derived from seeds. Analysis of the free amino acids in seeds, seedlings, and calli showed that homozygous resistant plants (TR1) contained higher levels of total free amino acids than sensitive plants. In particular the levels of tryptophan, phenylalanine, and histidine were, respectively, 8.5, 5.4, and 4.9 times higher than those in the sensitive plants.
The effects of spring fainfall, critical levels of summer moisture stress, and sheep Ovis aries stocking rates on white clover (Trifolium repens cv. Huia) have been evaluated in a 30-yr experiment (1964-93) Late summer (January-March) moisture stress was critical in determining white clover presence in the following spring (September). These climate-based relationships reinforce the importance of stolon growth and survival as a regenerative strategy for white clover. However, over the 30 yr, the species showed decreasing resilience post drought, which suggests a long-term failure of seed-based regeneration. Interactions between seasonal moisture stress, sheep stocking rate, interspecific plant competition, and seed pool dynamics, are proposed to explain the nature of long-term decline in white clover presence in well-fertilised, sown pastures in the Northern Tablelands of New South Wales. -from Authors
There has been little study on the growth and morphology of individual plants constituting the population of white clover in mixed swards under grazing. Such information is required if the mechanisms governing plant productivity and persistence are to be understood.
Intact white clover plants were sampled from intensively sheep-grazed pastures under set stocking, rotational grazing, and a combination of both systems, by taking turves (250 × 250 mm), and washing out the plants, every month for a year. Characters measured for every stolon of each plant were: presence of a growing point; numbers of leaves, roots and axillary buds; stolon length. Total plant leaf and stolon dry weight were also recorded. Plants were classified according to degree of branching, and the contribution of each branching order to the population determined.
There were strong seasonal variations in plant size (leaf and stolon dry weight, stolon length, and numbers of stolons and leaves per plant) which showed a significant decrease in spring with recovery over the following summer. This was paralleled by a rapid increase in the proportion of less branched plants (1st and 2nd branching order) in the population from 60 to 80% in spring, as higher-order plants broke up into smaller- and lower-ordered plants at this time. Numbers of roots per plant increased over winter to peak in early spring then declined in the following summer-autumn. While system of grazing management had no significant effect on branching structure of plants, it had a large effect on plant dry weight; rotationally grazed plants were 2·5 times larger than set stocked plants (0·182 cf. 0·073 g respectively).
Other general features of plant morphology were that each successive order of branch stolons was shorter and length before branching was less than that of their preceding parent stolon. The highest branching order observed was 6th order. There was no relationship between branching and numbers of roots; in branched plants only 55% of stolons were rooted regardless of plant order, but rooted stolons accounted for 85% of total stolon length and carried 62, 48 and 90% of the leaves, growing points and axillary buds per plant, respectively.
Comparison with other studies suggests that the processes outlined in this report may be common to white clover growth under grazing over a wide range of favourable environments.
The discoveries during the late 1920s that the genetic material is amenable to changes excited geneticists who saw new opportunities for both basic and practical applications. During the 1950s, induced mutagenesis was widely pursued in the US, Europe, Japan and China. In India, Swaminathan and his team at the Indian Agricultural Research Institute, New Delhi initiated a major programme on mutagenesis in crop plants. These studies were broadly aimed at understanding the process of mutation, testing the efficacy of various mutagens, identifying optimum dose and the best method of treatment for different crop species; isolation of mutants of basic and applied value; elucidating the biological effects of radiation-treated media, seeds and vegetative propagules on the organisms consuming them.
Transplants of white clover (Trifolium repens L.) were grown isolated from each other and in pairs placed at different distances apart. The paired plants developed asymmetrically and at the interface between paired clones both the density of nodes and of stolons appeared to reach ceiling values that were of the same order as those achieved in isolated clones. It is argued that the growth of plants of T. repens is controlled by the local conditions experienced by the plant parts and not by integrated growth of the whole. Transplants of three different genotypes of T. repens, which differed in growth form, were grown as neighbouring pairs and the calculated asymmetry of the plants was used to compare their mutual aggressivenes. The more compact (phalangeal) genotypes induced greater asymmetry in their neighbours than the more diffuse forms.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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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