Agrobacterium-mediated transformation and plant regeneration of buckwheat (Fagopyrum esculentum Moench.)

Genetic transformation of buckwheat (Fagopyrum esculentum Moench.) and regeneration of transgenic plants were obtained by using Agrobacterium tumefaciens strains as vectors. Buckwheat cotyledons were excised from imbibed seeds, co-cultivated with A. tumefaciens and subjected to previously reported protocols for callus and shoot regeneration. The transformation with oncogenic strains was confirmed by opine and DNA analyses of tumour tissue extracts. Plants were regenerated on cotyledon fragments incubated with strain A281, harboring pGA472, which carries the neomycin phosphotransferase II gene for kanamycin resistance. The transformation of resistant shoot clones was confirmed by NPTII enzyme assay and DNA hybridization. A large number of transformed shoots were rooted and fertile plantlets were raised in the greenhouse. Transgenic plants comprised pin and thrum clones, which were allowed to cross-pollinate. In about 180 R₂ seeds tested for kanamycin resistance, the ratio of resistant to sensitive seedlings was roughly 3:1.Abbreviations BAP 6-benzylaminopurine - 2,4-D dichloro-phenoxyacetic acid - 2iP 6-(, ,-dimethylallyl-amino)-purine - IBA indole-3-butyric acid - IAA indole-3-acetic acid - Km kanamycin - NPTII neomycin phosphotransferase II     

Anther culture and androgenetic plant regeneration in buckwheat (Fagopyrum esculentum Moench)

Anther culture for haploid induction of buckwheat was studied over a period of five years. Approximately 24,000 anthers were isolated and cultured on different culture media. The regeneration capacity was generally very low. Data are presented for experiments that included 7278 anthers on which 99 calluses were formed and 20 buds regenerations were noted. Regeneration occurred most readily on gellan-gum solidified media, with 90 g l^-1 maltose, 2.5 mg l^-1 BA, 0.5 mg l^-1 IAA, and preferably in darkness. Haploid cells, as established by chromosome counts, were observed in eight regenerants. Several abnormalities of pollen development in vitro were detected. Starch presence in pollen as a possible sign of androgenic capacity was studied. Microspores in uninucleate and early binucleate stages contained only proplastids, while in adult pollen grains a number of amyloplasts were present.Abbreviations BA benzyladenine - IAA indole-3-acetic acid - 2,4 D-2,4-dichloro-phenoxyacetic acid - IBA indole-3-butyric acid - 2iP 6- c,c-dimethylallylaminol-purine - NAA -naphthalene acetic acid     

Plant regeneration from cotyledon tissues of common buckwheat (Fagopyrum esculentum Moench)

Summary Plants were regenerated from cotyledon tissue of greenhouse grown seedlings of common buckwheat (Fagopyrum esculentum Moench.). Maximum callus regeneration was induced on Murashige and Skoog (MS) medium containing 2,4-D (2.0 mg l⁻¹) and kinetin (KIN) (0.2 mg l⁻¹) and either 3 or 6% sucrose. Friable callus was transferred to MS media containing KIN and benzylaminopurine (BAP) at varied concentrations for embryogenic callus induction. The optimum medium for embryogenic callus induction was found to be MS medium supplemented with 0.2 mg l⁻¹ KIN, 2.0 mg l⁻¹ BAP and 3% (w/v) sucrose. Variation of sucrose from 3 to 6% did not show any significant effect on callus induction or embryogenesis. Regeneration of embryonic callus varied from 13 to 32%. Whole plants were obtained at high frequencies when the embryogenic calluses with somatic embryos and organized shoot primordia were transferred to half-strength MS media with 3% sucrose. Regenerated plants after acclimation were transferred to greenhouse conditions, and both vegetative and floral characteristics were observed for variation. This regeneration system may be valuable for genetic transformation and cell selection in common buckwheat.     

Pb hyperaccumulation and tolerance in common buckwheat (Fagopyrum esculentum Moench)

Common buckwheat grown in Pb-contaminated soil was found to accumulate a large amount of Pb in its leaves (8,000 mg/kg DW), stem (2,000 mg/kg DW), and roots (3,300 mg/kg DW), without significant damage. This indicates that buckwheat is a newly recognized Pb hyperaccumulator, which is defined as a plant containing over 1,000 mg/kg of Pb in its shoots on a dry-weight basis. Moreover, it was shown that application of the biodegradable chelator methylglycinediacetic acid trisodium salt at concentrations of up to 20 mmol/kg resulted in a more than five times higher concentration of Pb in the shoot without notable growth inhibitation at up to 10 mmol/kg. These results indicate that buckwheat is a potential phytoremediator of Pb-contaminated soils.     

外源蔗糖对盐胁迫荞麦幼苗根系生长的缓解效应

实验结果表明,盐胁迫下,添加外源蔗糖后,荞麦根系伸长生长和根系活力增加,超氧化物歧化酶（SOD）和过氧化物酶（POD）活性下降,过氧化氢酶（CAT）活性升高,丙二醛（MDA）含量下降,总黄酮含量和苯丙氨酸解氨酶（PAL）活性极显著升高。     

An angiotensin-I converting enzyme inhibitor from buckwheat (Fagopyrum esculentum Moench) flour

A compound that inhibited angiotensin-I converting enzyme (ACE) activity was isolated from buckwheat powder. This compound is thought to be the hydroxy derivative of nicotianamine and its chemical structure is 2'-hydroxynicotianamine. This compound showed a very high inhibitory activity toward ACE, and the IC(50) was 0.08 microM. Only this hydroxy analog was found in buckwheat powder, at about 30 mg/100g, and no nicotianamine was detected. However, nicotianamine was detected in the buckwheat plant body. 2'-hydroxynicotianamine was also found in other polygonaceous plants.     

Form of aluminium for uptake and translocation in buckwheat (Fagopyrum esculentum Moench)

 The forms of Al for uptake by the roots and translocation from the root to the shoot were investigated in a buckwheat (Fagopyrum esculentum Moench, cv. Jianxi) that accumulates Al in its leaves. The Al concentration in the xylem sap was 15-fold higher in the plants exposed to AlCl₃ than in those exposed to an Al-oxalate (1:3) complex, suggesting that the roots take up Al in the ionic form. The Al concentration in the xylem sap was 4-fold higher than that in the external solution after a 1-h exposure to AlCl₃ solution and 10-fold higher after a 2-h exposure. The Al concentration in the xylem sap increased with increasing Al concentration in the external solution. The Al uptake was not affected by a respiratory inhibitor, hydroxylamine, but significantly inhibited by the addition of La. These results suggest that Al uptake by the root is a passive process, and La³⁺ competes for the binding sites for Al³⁺ on the plasma membrane. The form of Al in the xylem sap was identified by ²⁷Al-nuclear magnetic resonance analysis. The chemical shift of ²⁷Al in the xylem sap was around 10.9 ppm, which is consistent with that of the Al-citrate complex. Furthermore, the dominant organic acid in the xylem sap was citric acid, indicating that Al was translocated in the form of Al-citrate complex. Because Al is present as Al-oxalate (1:3) in the root, the present data show that ligand exchange from oxalate to citrate occurs before Al is released to xylem. Received: 10 December 1999 / Accepted: 3 February 2000     

Characterization of heat-induced aggregates of globulin from common buckwheat (Fagopyrum esculentum Moench)

Some physicochemical properties and the microstructure of heat-induced aggregates of globulin from common buckwheat (Fagopyrum esculentum Moench) (BWG) formed at 100 °C in 0.01 M phosphate buffer containing 1.0 M NaCl, pH 7.4 were studied. Differential scanning calorimetric (DSC) analysis shows a re-distribution of native and extensively denatured proteins in the heat-induced aggregates of BWG, particularly in the ISA fraction. Sodium dodecyl sulfate polyacrylamide gel electrophoretic (SDS-PAGE) analysis suggests the occurrence of both dissociation and association of molecules and the involvement of intermolecular disulfide linkages during thermal aggregation. Transmission electron microscopy (TEM) reveals that native BWG appeared as uniform compact globules with diameters ranging between 11.7 and 12.5 nm. TEM examination of the buffer-soluble aggregates, fractionated by sucrose density gradient ultracentrifugation, demonstrates the formation of strand-like small aggregates and large compact globular soluble macroaggregates.     

甜荞FaesSTK基因在长花柱长雄蕊突变体lpls中的表达分析

采用RACE技术,从甜荞（Fagopyrum esculentum Moench）中克隆获得3种花型的STK同源基因FaesSTK,并对其序列特征进行分析。结果显示,甜荞3种花型植株STK同源基因序列一致,全长为967 bp,包含长689 bp的完整开放阅读框,编码一个由225个氨基酸残基组成的D类MADS-box转录因子。蛋白序列比对及系统发育分析结果表明,FaesSTK蛋白属于MADS-box转录因子中的STK进化系。包含1个由57个氨基酸残基组成的高度保守的MADS结构域;1个由82个氨基酸残基组成的次级保守区域的K结构域,在C端的转录激活区还含有另外2个高度保守的基序（AGⅠ和AGⅡ）。实时荧光定量检测结果显示,FaesSTK基因主要在甜荞lpls突变体的雄蕊、雌蕊和不同发育时期的幼果中表达,在根和花被片中仅能检测到微弱的转录信号,在叶和茎中不表达,其中在雌蕊和果实中的表达量极显著高于其他组织。推测该基因在花发育过程中可能主要参与调控甜荞lpls突变体雌蕊和果实的发育。     

荞麦与大豆叶片乙醇酸氧化酶的纯化和性质比较研究

分别从荞麦与大豆叶片中部分纯化了乙醇酸氧化酶 (GO ,EC1 .1 .3 .1 ) ,并研究其部分性质。结果显示荞麦与大豆叶片中GO的催化特性有明显差异 :大豆叶片中GO对乙醇酸Km值为 0 .3 1mmol/L ,对乙醛酸Km值为 1 .98mmol/L。外源草酸对GO氧化乙醇酸活性影响很小 ,但对其氧化乙醛酸活性抑制明显 ,5mmol/L草酸可抑制 44%。而荞麦叶片中GO性质有所不同 :GO对乙醇酸Km为 0 .46mmol/L ,对乙醛酸Km为 0 .85mmol/L。草酸对荞麦GO氧化乙醇酸活性影响也很小 ,对其氧化乙醛酸活性的抑制作用明显小于大豆 ,5mmol/L草酸只抑制 2 4%。上述研究结果表明 ,荞麦GO对乙醛酸的亲和力明显强于大豆 ,并且草酸对其GO氧化乙醛酸活性影响较小。因此相对于大豆而言 ,GO可能在荞麦叶片草酸合成中起重要作用。     

Identification and characterization of a rhamnosyltransferase involved in rutin biosynthesis in Fagopyrum esculentum (common buckwheat)

Rutin, a 3-rutinosyl quercetin, is a representative flavonoid distributed in many plant species, and is highlighted for its therapeutic potential. In this study, we purified uridine diphosphate-rhamnose: quercetin 3-O-glucoside 6″-O-rhamnosyltransferase and isolated the corresponding cDNA (FeF3G6″RhaT) from seedlings of common buckwheat (Fagopyrum esculentum). The recombinant FeF3G6″RhaT enzyme expressed in Escherichia coli exhibited 6″-O-rhamnosylation activity against flavonol 3-O-glucoside and flavonol 3-O-galactoside as substrates, but showed only faint activity against flavonoid 7-O-glucosides. Tobacco cells expressing FeF3G6″RhaT converted the administered quercetin into rutin, suggesting that FeF3G6″RhaT can function as a rhamnosyltransferase in planta. Quantitative PCR analysis on several organs of common buckwheat revealed that accumulation of FeF3G6″RhaT began during the early developmental stages of rutin-accumulating organs, such as flowers, leaves, and cotyledons. These results suggest that FeF3G6″RhaT is involved in rutin biosynthesis in common buckwheat.     

二倍体普通荞麦异常花朵及其结构的研究

在二倍体普通荞麦BW1 9 1的自交后代群体中有一些植株上的很多花朵形态及其结构发生变异。对这些异常花朵的形态进行了观察 ,统计了各变异花朵的被片数目 (x1)、花柱数目 (x2 )、胚珠数目 (x3 )和雄蕊数目 (x4)等参数。结果发现 ,该群体植株的花朵类型多达 2 5种以上。同一植株上花朵的被片数目 (x1)、花柱数目 (x2 )、胚珠数目 (x3 )和雄蕊数目 (x4)等参数变异广泛。大多数花朵为 5被片 3花柱 1胚珠 8雄蕊 ,但是被片数变幅为 3～ 8,雄蕊数变幅为 3～ 1 1 ,花柱数变幅为 2～ 8,胚珠数变幅为 1～ 3。当花柱数分别为 2、3、4、5、6、7或 8时 ,胚珠数分别是 1 (正常 )、1 (正常 )、1 (正常 ) +0 (未发育胚珠 )、1 (正常 ) +1 (小型胚珠 )、2 (正常 )、2 (正常) +0 (未发育胚珠 )或 2 (正常 ) +1 (小型胚珠 )。偏相关分析表明 ,被片数与花柱数存在显著相关 (r12 .3 4 =0 2 3 0 2 ) ,被片数与雄蕊数存在极显著相关 (r14 .2 3 =0 .472 7 )以及花柱数与胚珠数之间存在极显著相关(r2 3 .14 =0 .7787 ) ,这暗示被片、花柱、胚珠、雄蕊在遗传或发育上是相关的     

Aluminium localization in root tips of the aluminium-accumulating plant species buckwheat (Fagopyrum esculentum Moench)

Aluminium (Al) uptake and transport in the root tip of buckwheat is not yet completely understood. For localization of Al in root tips, fluorescent dyes and laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) were compared. The staining of Al with morin is an appropriate means to study qualitatively the radial distribution along the root tip axis of Al which is complexed by oxalate and citrate in buckwheat roots. The results compare well with the distribution of total Al determined by LA-ICP-MS which could be reliably calibrated to compare with Al contents by conventional total Al determination using graphite furnace atomic absorption spectrometry. The Al localization in root cross-sections along the root tip showed that in buckwheat Al is highly mobile in the radial direction. The root apex predominantly accumulated Al in the cortex. The subapical root section showed a homogenous Al distribution across the whole section. In the following root section Al was located particularly in the pericycle and the xylem parenchyma cells. With further increasing distance from the root apex Al could be detected only in individual xylem vessels. The results support the view that the 10 mm apical root tip is the main site of Al uptake into the symplast of the cortex, while the subapical 10-20 mm zone is the main site of xylem loading through the pericycle and xylem parenchyma cells. Progress in the better molecular understanding of Al transport in buckwheat will depend on the consideration of the tissue specificity of Al transport and complexation.     

Identification and characterization of granule bound starch synthase (GBSS-I) from common buckwheat (Fagopyrum esculentum Moench)

Starch grains present in the endosperm of grains of common buckwheat (Fagopyrum esculentum Moench) show a monomodal distribution with size ranging from 4 to 10 μm. SDS-PAGE analysis of starch granule bound proteins revealed the presence of a single band corresponding to molecular mass of 59.7 kDa. The protein is localized within the central core of the starch grains. Antisera raised against the 59.7 kDa protein cross reacted with the 61 kDa GBSS-I from endosperm starches of maize and the 60 kDa GBSS-I from endosperm starches of rice and wheat, thereby indicating serological homology between the 59.7 kDa buckwheat starch granule bound protein and GBSS-I of wheat, maize and rice. 2D-PAGE of starch granule bound proteins of common buckwheat resolved the fraction into 7 spots with pI ranging from 5.2 to 5.6. N-terminal amino acid sequence for 25 residues of two immunoreactive proteins separated by 2D PAGE showed 94 % homology with N-terminal amino acid sequence of GBSS-I from Hordeum vulgare, Triticum spp. and Phaseolus vulgaris. Even though analysis of the sequence alignment revealed a clear diversification into monocotyledonous and dicotyledonous groups, the protein from buckwheat showed similarities with GBSS-I from both dicots as well as monocots. As is the case with dicots, the sequence of GBSS-I from buckwheat has valine as the 11th residue. GBSS-I from majority of monocots has methionine at this position. The sequence also showed similarities with monocots with valine at P’5 from the N-terminus. GBSS-I from majority of dicots has isoleucine at this position. The significance of these substitution remains to be ascertained.     

Somatic embryogenesis and bud formation from immature embryos of buckwheat (Fagopyrum esculentum Moench.)

Immature embryos of Fagopyrum esculentum cv. Pennquad were isolated from field-grown plants and cultured on media containing a high benzylaminopurine to indole-3-acetic acid ratio. Part of the embryos were grown in the presence of 2,4-dichlorophenoxyacetic acid and kinetin for the first 5 days, and then transferred to benzylaminopurine + indole-3-acetic acid medium. From callus tissues developed on hypocotyls and cotyledons, 3 types of tissue were selected in later subcultures: (a) callus tissue strains that produced buds, (b) embryogenic tissue, and (c) unorganized callus tissue, lacking any organogenic capacity. Pretreatment with 2,4-dichlorophenoxyacetic acid increased the number of explants which gave rise to bud forming and embryogenic tissue, but was not essential for morphogenesis. Somatic embryogenesis was confirmed by histological observation. Plantlets could be easily obtained by inducing adventitious roots on shoots, but spontaneous root development in somatic embryos was infrequent.Abbreviations BAP benzylaminopurine - IAA indole-3-acetic acid - 2,4-D dichlorophenoxyacetic acid - IBA indole-3-butyric acid     

Inflorescence structure and control of flowering time and duration by light in buckwheat (Fagopyrum esculentum Moench)

Morphogenesis of the reproductive structure of buckwheat and the impact of light conditions on flowering time and duration have been investigated using the variety 'La Harpe'. Inflorescences were initiated acropetally, in leaf axils, by the shoot apical meristem until its arrest of functioning which was accompanied by the abortion of the last inflorescence produced. The buckwheat inflorescence is a compound raceme that produces laterally flowered cymose clusters, the number of which was affected by the position of the inflorescence along the main stem. Similarly, the number of flowers in a lateral cluster was dependent on the inflorescence's position on the stem. The development of each inflorescence was stopped as its meristem stopped functioning and, in a situation reminiscent of the shoot apical meristem, the latest initiated cyme aborted. The development of each cyme was also terminated with the abortion of a few young flowers. The variety 'La Harpe' is a facultative short-day plant: the number of nodes generated before the initiation of the first inflorescence and the number of days from sowing to macroscopic appearance of this inflorescence were reduced in 8 h days as compared with 16 h days. The number of inflorescences, and thus flowering duration, was also strongly reduced by short days. It was unaffected by light irradiance in 8 h days while, in 16 h days, it was prolonged when light intensity was increased, suggesting the interaction of two different mechanisms for its regulation. Buckwheat is a distylous species, but inflorescence structure and flowering behaviour were not affected by floral morph.     

Homostyly as an efficient tool for development of homozygous lines of buckwheat (Fagopyrum esculentum Moench)

A genetic scheme of pollination in buckwheat Fagopyrum esculentum Moench and the specificity of work with its recessive mutations identified on the basis of heterostyly are considered. The possibility of obtaining a genetically determined donor of self-fertility on the basis of S1 allele controlling the development of the homostyle flower type in buckwheat is examined. The possibility of using homostyly as an efficient tool for the development of self-fertile homozygous lines and making their genetic analysis is also discussed.     

Effects of methyl jasmonate on accumulation of flavonoids in seedlings of common buckwheat (Fagopyrum esculentum Moench)

The jasmonates, which include jasmonic acid and its methyl ester (MJ), play a central role in regulating the biosynthesis of many secondary metabolites, including flavonoids, and also are signaling molecules in environmental stresses. Synthesis of anthocyanins pigments is a final part of flavonoids pathway route. Accumulation of the pigments in young seedlings is stimulated by various environmental stresses, such as high-intensity light, wounding, pathogen attack, drought, sugar and nutrient deficiency. The anthocyanins take part in defense system against excess of light and UV-B light, and therefore it is probably main reason why young plant tissues accumulate enlarged levels of the pigments. The effects of exogenously applied MJ on level of anthocyanins, glycosides of apigenin, luteolin, quercetin and proanthocyanidins in seedlings of common buckwheat (Fagopyrum esculentum Moench) were studied. MJ decreased contents of all the found cyanidin glycosides and its aglycone in hypocotyls of buckwheat seedlings. However contents of particular anthocyanins in cotyledons of buckwheat seedlings treated with the plant hormone were not significantly different from the control. Applied doses of MJ did not affect levels of quercetin, apigenin and luteolin glycosides in the analyzed parts of buckwheat seedlings: cotyledons and hypocotyls. On the other hand, treatment of buckwheat seedlings with MJ clearly stimulated of proanthocyanidins biosynthesis in hypocotyls. We suggest that methyl jasmonate induces in hypocotyls of buckwheat seedlings the leucocyanidin reductase or anthocyanidin reductase, possible enzymes in proanthocyanidins synthesis, and/or inhibits anthocyanidin synthase, which transforms leucocyanidin into cyanidin. According to our knowledge this is the first report regarding the effect of methyl jasmonate on enhancing the accumulation of proanthocyanidins in cultivated plants.