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     

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     

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.     

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.     

Buckwheat (Fagopyrum esculentum Moench) has high capacity to take up phosphorus (P) from a calcium (Ca)-bound Source

Two experiments were carried out in a growth chamber to investigate the phosphorus (P)-uptake efficiency of Fagopyrum esculentum Moench (buckwheat) and Triticum aestivum (spring wheat) from a Ca-bound form. The first experiment was based on a sand-culture system with either rock phosphate (RP) or CaHPO₄ (CaHP) as the P source and nitrate or ammonium nitrate as nitrogen source. A highly calcareous soil was used in the second experiment. Buckwheat was shown to be highly efficient in taking up Ca-bound P compared to spring wheat. When plants were supplied with nitrate, the total P uptake by buckwheat from RP was nearly 10-fold higher than that of spring wheat (20.1 compared with 2.1 mg P pot^–1). Changing nitrogen source from nitrate only to ammonium nitrate increased P uptake by spring wheat substantially, but not buckwheat. High P-uptake efficiency of buckwheat was also demonstrated using the field soil, but to a lesser extent, which may be related to the difference in Zn supply between sand culture and field soil. It is suggested that buckwheat may be included in intercropping or crop rotation systems to activate P sources in calcareous soils. The principal mechanism of P uptake efficiency of buckwheat may be its ability to acidify the rhizosphere; however, further study is needed to unravel the regulation of root excretion of H⁺ and its molecular basis in order to exploit buckwheat's genetic capability to utilise sparingly soluble P from soil.     

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.     

Plant regeneration from cotyledon protoplasts of Brassica carinata

Protoplasts isolated from cotyledons of Brassica carinata, underwent sustained division when cultured at 5.0 × 10⁴ ml^-1 in modified 8p medium (KM8P) with 1.0% (w/v) Seaplaque agarose. Cell colonies produced callus when agarose droplets, in which the protoplasts were embedded, were transferred to K8 medium with 0.6% (w/v) Sigma Type I or Type VII agarose at day 16, giving a plating efficiency of 1.6%. Seventy percent of the protoplast derived-tissues produced shoot buds after subculture to MS medium containing 3.0% (w/v) sucrose, 1.125 mgl^-1 BAP, 0.035 mgl^-1 GA and 0.6% (w/v) Type I agarose, resulting in shoot formation from 1.1% of the protoplasts originally plated. Protoplast-derived colonies transferred to hormone-free MS medium with 1.0% (w/v) sucrose and 0.6% (w/v) Type I agarose produced roots. The latter gave rise to shoots after excision from the parent callus and culture on MS medium with 3.0% sucrose, 0.225 mgl^-1 BAP, and 0.6% (w/v) Type I agarose. Shoots regenerated directly from protoplast-derived calli, or indirectly from roots, developed prolific root systems when placed on hormone-free MS medium with 1.0% (w/v) sucrose and 0.6% (w/v) Type I agarose.Abbreviations BAP 6-benzylaminopurine - CH casein hydrolysate - 2,4-D 2,4-dichlorophenoxyacetic acid - GA gibberellic acid - K kinetin - NAA -naphthaleneacetic acid - MES 2(N-morpholino)ethanesulphonic acid, 2,iP-6(,-dimethylallyamino) purine - IAA indole-3-acetic acid - Z zeatin - ZR zeatin riboside     

Purification,molecular cloning and functional characterization of flavonoid C‐glucosyltransferases from Fagopyrum esculentum M. (buckwheat) cotyledon

C‐Glycosides are characterized by their C–C bonds in which the anomeric carbon of the sugar moieties is directly bound to the carbon atom of aglycon. C‐Glycosides are remarkably stable, as their C–C bonds are resistant to glycosidase or acid hydrolysis. A variety of plant species are known to accumulate C‐glycosylflavonoids; however, the genes encoding for enzymes that catalyze C‐glycosylation of flavonoids have been identified only from Oryza sativa (rice) and Zea mays (maize), and have not been identified from dicot plants. In this study, we identified the C‐glucosyltransferase gene from the dicot plant Fagopyrum esculentum M. (buckwheat). We purified two isozymes from buckwheat seedlings that catalyze C‐glucosylation of 2‐hydroxyflavanones, which are expressed specifically in the cotyledon during seed germination. Following purification we isolated the cDNA corresponding to each isozyme [FeCGTa (UGT708C1) and FeCGTb (UGT708C2)]. When expressed in Escherichia coli, both proteins demonstrated C‐glucosylation activity towards 2‐hydroxyflavanones, dihydrochalcone, trihydroxyacetophenones and other related compounds with chemical structures similar to 2′,4′,6′‐trihydroxyacetophenone. Molecular phylogenetic analysis of plant glycosyltransferases shows that flavonoid C‐glycosyltransferases form a different clade with other functionally analyzed plant glycosyltransferases.     

Plant regeneration from embryogenic cultures of Phragmites australis (Cav.) Trin. Ex Steud.

Embryogenic cultures of the common reed [Phragmites australis (Cav.) Trin. Ex. Steud.] were induced on Murashige and Skoog (1962)-based medium with 2% (w/v) sucrose, B5 vitamins and 4.5 μM 2,4-dichlorphenoxyacetic acid. Four independent culture lines, two initiated from stem nodes and two from roots, were established. These cultures underwent somatic embryogenesis. In one line of stem node origin, the somatic embryos germinated and developed into plants, following transfer of embryogenic cultures to Murashige and Skoog (1962)-based medium lacking growth regulators, with 108 ± 17 plants being recovered per 100 mg fresh weight of culture. In other lines, the somatic embryos developed roots, but not shoots. Shoot regeneration via somatic embryogenesis offers potential as anin vitro system for physiological studies, including assessments of the response of common reed to environmental pollutants. This revised version was published online in June 2006 with corrections to the Cover Date.     

Plant regeneration from seedling explants and cotyledon protoplasts ofGlycine argyrea tind

Summary Plants have been regenerated from nodular, green callus derived from cotyledon, petiole and leaf lamina explants ofG. argyrea, a perennial relative of the soybean (G. max). The degree of response obtained was governed primarily by the genotype used, accession G1626 proving the most responsive. Shoots were also recovered from about 6.0% of cotyledon protoplasts of this genotype. The implications of these results are discussed in relation to genetic manipulations using this species.     

Amino acid sequence of the 26 kDa subunit of legumin-type seed storage protein of common buckwheat (Fagopyrum esculentum Moench): molecular characterization and phylogenetic analysis

The paper describes the amino acid sequence of a 26 kDa basic subunit of 13S globulin of common buckwheat (Fagopyrum esculentum Moench). The protein has 93 and 75% sequence homology with 11S globulin of Coffea arabica and beta subunit of 11S globulin of Cucurbita pepo respectively. The subunit has the "globally conserved" N-terminal sequence consisting of Gly-Ile-Asp-Glu and the cysteine at P7' from the proteolytic processing site. A conserved 7 residue domain of Pro-His-Trp-Asn-Ile-Asn-Ala, characteristic of basic subunits of legumins from non-leguminous angiosperms, is also present in this protein. A distinguishing features of this subunit is the relatively high level of lysine and methionine.     

Salt tolerance conferred by overexpression of Arabidopsis vacuolar Na+/H+ antiporter gene AtNHX1 in common buckwheat (Fagopyrum esculentum)

Agriculture productivity is severely affected by soil salinity. One possible mechanism by which plants could survive salt stress is to compartmentalize sodium ions away from the cytosol. In the present work, transgenic buckwheat plants overexpressing AtNHX1, a vacuolar Na⁺/H⁺ antiporter gene from Arabidopsis thaliana, were regenerated after transformation with Agrobacterium tumefaciens. These plants were able to grow, flower and accumulate more rutin in the presence of 200 mmol/l sodium chloride. Moreover, the content of important nutrients in buckwheat was not affected by the high salinity of the soil. These results demonstrated the potential value of these transgenic plants for agriculture use in saline soil.     

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     

Somatic embryogenesis and plant regeneration from embryonic tissues ofCamellia japonica L.

Somatic embryogenesis and plantlet regeneration were achieved from immature and mature zygoticCamellia japonica embryos cultured on Murashige & Skoog's mineral medium without growth regulators or with various combinations of IBA and BAR The dependence of embryogenesis rates on growth regulator levels was not clear, though high concentrations such as 4 mg 1^-1BAP plus 2 mg 1^-1IBA were definitely inhibitory. BAP at 1 or 2 mg 1^-1 did appear to determine the formation of bud-like embryos. By far the most responsive initial explants were immature embryonic axes collected in September, 94% of which produced somatic embryos as against only 20% for embryonic axes from mature seeds collected in October. Cotyledon explants were also embryogenic. Somatic embryos differentiating directly on the hypocotyl of the embryonic axes or the surface of cotyledons passed through typical stages of embryogenesis. Indirect somatic embryogenesis via callus was also evident. Embryogenic potential was maintained by secondary embryogenesis through the successive generations of embryos.     

Purification and characterization of a flavonol 3-O-beta-heterodisaccharidase from the dried herb of Fagopyrum esculentum Moench

A flavonol-3-O-beta-heterodisaccharide glycosidase (FHG I) was isolated from dried aerial tissues of Fagopyrum esculentum Moench (Fagopyri herba). It has a specific enzyme activity of ca. 3.5 nkat mg(-1) protein in buffered extracts when rutin (quercetin-3-O-rutinoside) was used as substrate and an optimal enzyme activity was seen at around pH 4.8 and 30 degrees C. FHG I was purified about 156-fold to apparent homogeneity by hydrophobic interaction, anion exchange and size exclusion chromatographic steps. The apparent molecular mass of FHG I was 74.5+/-2 kDa as determined by SDS-PAGE and it is a monomeric glycoprotein with a carbohydrate content of 23%. The isoelectric point as determined by isoelectric focusing was 5.7 and the energy of activation was 32 kJ mol(-1). FHG I exhibits a high substrate specificity, preferring flavonol 3-O-glycosides comprising the disaccharide rutinose. The K(m) and V(max) values for the natural substrate rutin were calculated to be 0.561 microM and 745 nkat mg (-1) protein, respectively. Two oligopeptide fragments obtained after enzymatic digestion of FHG I were sequenced and showed similarities to sequences of beta-glucohydrolases from other plant species. Polyclonal antibodies were raised and their specificities determined. Another flavonol 3-O-beta-heterodisaccharide glycosidase (FHG II) could also be detected in buckwheat herb, having a molecular mass of 85.3+/-2 kDa and an isoelectric point between pH 6.0 and 6.5.     

Plant regeneration via somatic embryogenesis in cotton

An efficient in vitro plant regeneration system characterized by rapid and continuous production of somatic embryos using leaf and stem explants of abnormal seedling as an explant have been developed in Gossypium hirsutum L. Embryogenic callus and somatic embryos have been obtained directly from the explants of cotton abnormal seedlings. Plant growth regulators influenced the induction of cotton somatic embryogenesis. The optimal medium for direct somatic embryogenesis was modified MS medium supplemented with 0.1 mg l^-1 ZT and 2 g l^-1 activated carbon. On this medium, an average of 28.0 and 28.1 matured somatic embryos formed from per leaf and stem explants respectively. The highest frequency of somatic embryogenesis was 100%. The somatic embryos were converted into normal plantlets when cultured on modified MS medium supplemented with 0.1 mg l^-1 ZT. Upon transfer to soil, plants grew well and appeared normal. Plants could be regenerated within 60–80 days. The system of cotton somatic embryogenesis and plant regeneration described here will facilitate the application of plant tissue culture and genetic engineering on cotton genetic improvement. This revised version was published online in June 2006 with corrections to the Cover Date.     

In vitro plant regeneration of Arachis correntina (Leguminosae) through somatic embryogenesis and organogenesis

In vitro protocols for plant regeneration of Arachis correntina through both somatic embryogenesis and organogenesis were developed using immature leaves as explants. Morphologically normal somatic embryos were obtained on culture media composed of 20.70 or 41.41 μM picloram (PIC) with the addition of 0.044 μM 6-benzylaminopurine (BA), resulting in a 33 and 24% of conversion into plants, respectively. The source of explants and the developmental stage of the leaves had a marked effect on somatic embryogenesis. The second folded immature leaves from in vitro growing plants were the most responsive producing up to 30% embryogenesis in MS+41.41 μM PIC. Embryos converted into plants after transfer to MS medium devoid of growth regulators and these plants were successfully acclimatised. Adventitious shoots were obtained on culture media supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) or naphthaleneacetic acid (NAA) with or without 0.044 μM BA, achieving plant regeneration in the induction media. The highest percentage of bud formation was obtained on culture medium composed of␣MS+10.74 μM NAA+0.044 μM BA (12.5%). Roots were formed on all culture media tested. Regenerated plants were transferred to pots and grew well under greenhouse conditions.     

Callus induction and high-frequency plant regeneration from hypocotyl and cotyledon explants of Arctium lappa L.

Summary This study describes a protocol for plant regeneration from cultured seedling explants of Arctium lappa. Hypocotyls and cotyledons of A. lappa were induced to form callus by culturing on Murashige and Skoog (MS) medium supplemented with 2.0mg l^−1 2,4-dichlorophenoxyacetic acid and 0.5–2.0 mg l^−1 benzyladenine (BA). Formation of adventitious buds could be induced from calluses or explants directly by culturing on MS medium containing 1.0–2.0 mg l^−1 α-naphthaleneacetic acid (NAA) and 0.5–2.0 mg l^−1 BA. These regenerated shoots were rooted on MS medium with 1.0 mg l^−1 indole-3-butyric acid or indole-3-acetic acid in combination with 1.0 mgl^−1 NAA. The regenerated plants acclimatized in soil were normal morphologically and in growth characters. They flowered and set seeds in the following year after acclimatization.     

Plant regeneration from embryogenic cultures initiated from mature loblolly pine zygotic embryos

Summary Mature zygotic embryos of eight (open-pollinated) families of loblolly pine (Pinus taeda L.) were cultured on eight different basal salt formulations, each supplemented with 36.2 μM 2,4-dichlorophenoxyacetic acid, 17.8 μM 6-benzyladenine, 18.6 μM kinetin, 500 mg l⁻¹ casein hydrolysate, and 500 mg l⁻¹ l-glutamine for 9 wk; embryogenic tissue was formed on cotyledons, hypocotyls, and radieles of mature zygotic embryos. Callus was subcultured on the callus proliferation medium, the same as the induction medium but with one-fifth concentration of auxin and cytokinin for 9 wk. On this medium a white to translucent, glossy, mucilaginous embryogenic callus containing embryogenic suspensor masses (ESMs) was obtained. The highest frequency of explants forming embryogenic tissue, 17%, occurred on a modified Murashige and Skoog salts basal medium containing the concentration of KNO₃, Ca(NO₃)₂·4H₂O, NH₄NO₃, KCl, ZnSO₄·7H₂O, and MnSO₄·H₂O, 720, 1900, 400, 250, 25.8, and 25.35 mg l⁻¹, respectively. Embryogenic suspension cultures were established by culturing embryogenic callus in liquid callus proliferation medium. Liquid cultures containing ESMs were transferred to medium containing abscisic acid, polyethylene glycols, and activated charcoal for stimulating the production of cotyledonary somatic embryos. Mature somatic embryos germinated for 4–12 wk on medium containing indole-butyric acid, gibberellic acid, 6-benzyladenine, activated charcoal, and reduced sucrose concentration (15 g l⁻¹). Two hundred and ninety-one regenerated plantlets were transferred to a perlite:peatmoss:vermiculite (1∶1∶1) mixture, then the plants were transplanted to soil in the earth, and 73 plantlets survived in the field.     

Plant regeneration via direct embryo axis-like shoot and root formation from excised cotyledon explants of ginseng seedlings

Summary Cotyledon explants of Panax ginseng at various developmental stages were cultured on Murashige and Skoog (MS) medium with 0.5 μM indole butyric acid and 8.8 μM N⁶-benzyladenine. Upon culturing of cotyledon explants from mature zygotic embryos, 34% of the explants formed somatic embryos, and 46% formed adventitious shoots. In the cotyledon explants from 1-wk-old seedlings, embryo axis-like shoots and roots developed at a high frequency (79%) near the excised portion of the cotyledon base. The developmental pattern of embryo axis-like organ formation was structurally different from that of somatic embryos and adventitious shoots but similar to that of parts of the embryo axis of zygotic embryos. In the early stages of embryo axis-like organ formation, epicotyl-like shoot primordia were developed directly from the cotyledon base after 2 wk of culture; subsequently roots developed near the base of the epicotyl-like shoots and eventually regenerated into plantlets with both shoots and roots. The frequency of embryo axis-like organ formation declined as the growth of seedlings proceeded. In addition, the frequency of somatic embryo and adventitious bud formation rapidly declined with the age of the cotyledons. Plant regeneration via embryo axis-like organ formation might be a new pattern of morphogenesis in P. ginseng cotyledon culture.