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     

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.     

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.     

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.     

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.     

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     

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     

Somatic embryogenesis from immature zygotic embryos ofGinkgo biloba

Immature zygotic embryos ofG. biloba were taken, at various developmental stages, from ovules harvested in November 1993. Zygotic embryos showing the beginning of the cotyledonary development cultured on modified Murashige & Tücker (1969) media proliferated intensely. In fact, 98.5% of the immature zygotic embryos produced embryogenic and undifferentiated tissues (calluses), in proportions varying depending on the hormonal composition of the induction media. After two weeks of culture, direct embryogenesis was observed on the hypertrophic cotyledons when benzyladenine 10 M was used as the sole plant growth regulator in the induction media. The addition of different concentrations of NAA (5–10–20 M) and of BA (5 M) to the induction media led to an indirect embryogenesis after two months, when the calluses were transferred to the development media without auxin. The highest frequency of embryogenic tissues (90–95%) and the highest number of somatic embryos per explant (9.6) were obtained with benzyladenine (10 M) as the sole exogenous growth factor. Some embryos isolated mechanically or in situ on the callus developed as far as the later cotyledonary stage.Abbreviations AUX Auxin - BA Benzyladenine - CYT Cytokinin - IZE Immature zygotic embryo - MT Murashige & Tücker (1969) medium - NAA Naphtaleneacetic acid     

Somatic embryogenesis from immature embryos of redbud (Cercis canadensis)

Somatic embryos developed directly from 96 and 110 day post-anthesis Cercis canadensis L. (redbud) zygotic embryos from one of two trees sampled that were explanted onto modified Schenk and Hildebrandt medium amended with either 1, 2, 3 or 5 mg/1 2,4-D in combination with either 7.6 or 12. 6 mM ammonium ion. Although somatic embryogenesis was expressed on most media, the number of explants that produced somatic embryos and the mean number of embryos formed per explant were greatest on media that contained either 2 or 3 mg/1 2,4-D; 12.6 mM ammonium ion inhibited embryogenesis from 96 day post-anthesis explants. Zygotic embryos explanted 117 days after anthesis produced only callus and roots. Somatic embryos that were bottle-shaped or had distinct cotyledons organized roots on germination media, but only one embryo formed a shoot. No additional development occurred. Histological examination of somatic embryos showed that shoot apical meristems were poorly developed.Abbreviations 2,4-D 2, 4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - BAP 6-benzylaminopurine - FPA Formalin-propionic acid-ethanol (50%)     

Somatic embryogenesis and plant regeneration from immature zygotic embryos of papaya (Carica papaya L.)

Summary Immature zygotic embryos from open-pollinated and selfed Carica papaya L. fruits, 90 to 114 days post-anthesis, produced 2 to 20 somatic embryos on apical domes, cotyledonary nodes, and radicle meristems after culture for three weeks on half-strength Murashige and Skoog (MS) medium supplemented with 0.1 to 25 mg l^–1 2,4-dichlorophenoxyacetic acid (2,4-D), 400 mg l^–1 glutamine, and 6% sucrose. After six weeks of culture, about 40 to 50% of the zygotic embryos had become embryogenic, and each embryogenic embryo yielded hundreds of somatic embryos within five months of culture on media supplemented with 2,4-D. Somatic embryos matured on half-strength MS medium, germinated on MS medium containing 5 mg l^–1 kinetin, and grew large enough for greenhouse culture on MS medium. Shoots were rooted in vermiculite and grown in the greenhouse.Journal Series no. 3449 of the Hawaii Institute of Tropical Agriculture and Human Resources     

Somatic embryogenesis from immature zygotic embryos of oil palm

Immature zygotic embryos at different developmental stages were used for callus induction and regeneration studies. Immature embryos excised from fruits 77, 91, 100, 114, 128, 140 and 193 days after pollination and mature embryos were cultured on modified Y3 medium containing 500 mgl^–1 cysteine, 0.5% (w/v) PVP-40, 500 M 2,4-d and 0.3% (w/v) charcoal. Compact embryogenic tissue began differentiating directly from embryo explants after 2 weeks of culture. The percentage of embryos forming compact embryogenic tissue ranged from 28.6% for 91-day-old embryos to 0% for 140-day-old and older embryos. Friable embryogenic tissue was observed in callus cultures derived from 100-day-old embryos. Although both compact and friable embryogenic tissues were successfully isolated, normal embryo and plantlet development was observed only from friable embryogenic tissue.Abbreviations ABA abscisic acid - 2,4-d 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - PVP polyvinylpyrollidone     

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 from immature embryos in meadowfoam (Limnanthes alba)

Developing embryos from immature seeds were excised and cultured. Optimal proliferation of differentiated secondary embryos occurred on Murashige-Skoog media containing 7% sucrose, 0.1 M 2,4-D, and 0.1–1.0 M zeatin. Higher levels of auxins inhibited embryo proliferation. Secondary embryos were subcultured to produce more embryos. The results indicate the feasibility of clonal propagation of meadowfoam.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BA 6-Benzyladenine     

Somatic embryogenesis and plant development from immature zygotic embryos of seedless grapes (Vitis vinifera L.)

Summary Somatic embryo formation occurred from immature zygotic embryos within ovules of stenospermocarpic seedless grapes (Vitis vinifera L.), when cultured for two months on liquid Emershad/Ramming medium. Somatic embryos continued to proliferate after excision and transfer to Emershad/Ramming medium supplemented with 1 M benzylaminopurine and 0.65% TC agar. Plant development from somatic embryos was influenced by genotype, medium, phase (liquid, agar), stage (torpedo, mature) and their interactions. Optimal plant development occurred on Woody Plant Medium supplemented with 1.5% sucrose + 1 M benzylaminopurine + 0.3% activated charcoal and 0.65% TC agar.Abbreviations ABA abscisic acid - BAP benzylaminopurine - ER Emershad/Ramming - GLM general linear model - MGLH multivariate general linear hypothesis - MS Murashige/Skoog - NaClO sodium hypochlorite - TC tissue culture - WP woody plant     

New Amides from Buckwheat Seeds (Fagopyrum esculentum Moench)

Two new amino acid amides which yield in acid hydrolysis isomeric hydroxybenzylamines and amino acids have been isolated from the achenes of Fagopyrum esculentum Moench. One of them called BN-II is composed of salicylamine and allo-4-hydroxy-l-glutamic acid, and the other, BN-III, p-hydroxybenzylamine and l-glutamic acid. These coupled compounds link one another to form an amide respectively. Finally the structures of BN-II and BN-III were determined to be N⁵-(2′-hydroxybenzyl)-allo-4-hydroxy-l-glutamine and N⁵-(4′-hydroxybenzyl)-l-glutamine respectively from their chemical and spectrometry properties.     

Somatic embryogenesis from immature zygotic embryos of Rosa bourboniana desp

Summary A protocol for the inducton of somatic embryogenesis from immature zygotic embryos of Rosa bourboniana, a scented rose species, was established. Somatic embryos were induced after 8wk of inoculation of zygotic embryos on MS medium supplemented with different concentrations of 2,4-dichlorophenoxy acetic acid (5–15 μM). In addition to 2,4-dichlorophenoxy acetic acid concentrations, somatic embryogenesis was also influenced by the month of collection of the explant and the stage of maturity of the hip. Maximum embryogenic response (16.6%) was observed using 2,4-dichlorophenoxy acetic acid (15 μM), from green hips in the month of September. The use of l-proline (800 mg l⁻¹) was found to be optimum for secondary embryogenesis. On periodic subculturing, the cultures formed somatic embryos sustainably over a period of 18 mo. For somatic embryo germination, 6-benzylaminopurine (5 μM) was found to be most suitable. Rooted plants were transferred successfully to soil and appear morphologically normal under greenhouse conditions. Transfer of plants for hardening was most suitable during the active growth period between June and September. IHBT Publication No: 0447     

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.     

Somatic embryogenesis and plant regeneration from immature embryos of western larch

Somatic embryogenesis was initiated from immature embryos of western larch (Larix occidentalis Nutt.) on media containing 2,4-dichlorophenoxyacetic acid and N6- benzyladenine. The effects of explant type and ammonium nitrate and glutamine concentrations on initiation were tested. Although 21–93% of explants rendered cultures in various experiments, only 3% yielded sustainable embryogenic lines. Excised embryos at the early cotyledonary stage were optimal for initiation. Maturation of somatic embryos was promoted by abscisic acid. Response to abscisic acid concentrations and duration of exposure to abscisic acid varied with genotype. Maximal results were obtained with 0.025 M abscisic acid for 1 to 2 weeks followed by individual culture on medium without growth regulators. Mature somatic embryos developed into shoots with roots. Plantlets have been established in peat.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BA N⁶-benzyladenine - IBA indole-3-butyric acid - ABA abscisic acid     

Efficient flower induction from cultured buckwheat (Fagopyrum esculentum L.) node segments in vitro

Flower induction from shoot segments of buckwheat seedlings was examinedin vitro. Cytokinin, (especially kinetin at 0.1M), short day conditions and a high concentration ofsolidifying agent improved the flower induction from node segments invitro, in up to about 50% of node segments. The use of anaeration membrane on bottle caps and a high content of sucrose in the mediumimproved flower induction in vitro considerably. In theimproved conditions, flowers were induced from 100% cultures and 10bloomed flowers per explant were induced in vitro in 8weeks. Both long and short types of stigmas, and normal set of flowers wereobserved under the microscope. When pollen produced invitro was cultured on an artificial medium, 70% of the pollengrains germinated, indicating normal viability of in vitropollen, and indicating the potential for artificial pollination invitro. All the varieties examined flowered at a similar percentage,suggesting that the process was independent of variety and that flowers couldbeproduced in vitro. Flower induction from buckwheat plantsin vitro and possible cross breeding invitro are also discussed.