Direct somatic embryogenesis and plant regeneration from mature sugarbeet (Beta vulgaris L.) zygotic cotyledons

An in vitro protocol has been developed for direct somatic embryogenesis of zygotic cotyledons from mature sugarbeet (Beta vulgaris L.) embryos. Explants were sequentially cultured on modified Murashige and Skoog (MS) medium supplemented with different combinations of 2,4-D, NAA, BAP and TIBA. Somatic embryogenesis was induced within 4 weeks of culture on embryogenesis induction medium which contained MS medium supplemented with BAP and TIBA. Proliferation of somatic embryos was observed on embryo proliferation medium, which contained MS medium supplemented with BAP and NAA within 4 weeks of culture. Plants were regenerated on hormone free half; strength MS medium containing a low sucrose concentration. With some sugarbeet lines, high frequencies of plant regeneration in excess of 90percnt; were observed. The incorporation of TIBA in the media was essential for successful regeneration.     

Antiauxin enhanced microshoot initiation and plant regeneration from epicotyl-originated thin-layer explants of sugarbeet (Beta vulgaris L.)

An in vitro method was developed for microshoot initiation from thin-layer explants prepared from the elongated epicotyls of sugarbeet (Beta vulgaris L.). Intact epicotyls of 14-day-old seedlings were excised from the hypocotyls above the cotyledons and allowed to elongate on De Greef and Jacobs (1979) medium supplemented with 0.2 mg/l 6-benzyladenine, 0.2 mg/l gibberellic acid and 0.1 mg/l indole-3-acetic acid in darkness. After a 21-day-incubation, the elongated epicotyls were halved to obtain apical and basal segments prior to removing the leaves and lateral buds. Subsequently, 5–8 mm long, 2–3 mm wide and 0.8–1.0 mm thick tangential sections were prepared longitudinally from the exterior parts of the halved epicotyls. These thin-layer explants were incubated on microshoot initiating media containing various growth regulators. The combination of 1.0 mg/l 6-benzyladenine and the antiauxin 2,3,5-triiodobenzoic acid (1.0 mg/l) resulted in maximum microshoot development (6.3±0.2 microshoots/thin-layer explant). The final efficiency of our tissue culture system was significantly increased by the NaCl (100 mg/l) initiated in vitro rooting of microshoot originated plantlets.Abbreviations AC activated charcoal - asdp apical segment derived plantlet - asTLE apical segment derived thin-layer explant - BA-6 benzyladenine - bsdp basal segment derived plantlet - bsTLE basal segment derived thin-layer explant - EEM1-4 epicotyl elongation media - GA₃ gibberellic acid - GM germinating medium - IAA indole-3-acetic acid - IBA indole-3-butyric acid - KN kinetin - MES morpholino-ethanesulfonic acid - MSI1-6 microshoot initiating media - NAA -naphthalene acetic acid - PG_oB De Greef and Jacobs (1979) medium - RM1-3 rooting media - SDM shoot developing medium - SE standard error - TIBA 2,3,5 triiodobenzoic acid - TLE thin-layer explant - ZEA zeatin     

Glutamine synthetase oligomers and isoforms in sugarbeet (Beta vulgaris L.)

The -amino-N compounds that accumulate in the thickening storage root of sugarbeet (Beta vulgaris L.) were synthesized in the leaves (NO ₃ ^– nutrition) and also in the lateral roots (NH ₄ ⁺ nutrition). Ammonium stimulated glutamine synthetase (GS, EC 6.3.1.2) activity, especially in the lateral roots. With non-denaturing polyacrylamide-gel isoelectric focussing, simultaneously active charge-isomers of GS were separated in both leaves and roots. The leaf isoforms were active in an octameric and also in a tetrameric form. In the root only octameric isoforms were found. The tetramer was more active than the octamer in the leaf blade and vice versa in the leaf stem. Only the tetramer needed -mercaptoethanol for activity stabilization in vitro. A reactivation, however, of an inactive tetramer by the addition of thiol/thioredoxin was not possible. The same isoforms of GS were separated in different organs of sugarbeet but with different patterns of relative activity. The activity pattern depended also on the N-source of the plant. With increasing age of the plant the number of active GS isoforms declined in both leaves and roots although the in-vitro activity remained unchanged (NO ₃ ^– -fed plants) or even increased (NH ₄ ⁺ -fed plants).Abbreviations GS glutamine synthetase (E.C. 6.3.1.2.) - IEF isoelectric focussing - PAGE polyacrylamide gel electrophoresis This work was supported by a grant from Bundesministerium für Forschung und Technologie and by Kleinwanzlebener Saatzucht AG, Einbeck.     

In planta 2,3,5 truodobenzoic acid treatment promotes high frequency and routine in vitro regeneration of sugarbeet (Beta vulgaris L.) plants

Summary The effect ofin planta treatments with auxin inhibitors such as 2,3,5 triiodobenzoic acid (TIBA) on regeneration of plantsin vitro is not known. Here, we show the beneficial effect of preconditioning sugarbeet plants in the greenhouse with TIBA (3 mg/1) for efficientin vitro plant regeneration via a callus phase from cultured leaf explants. Without this treatment, no shoot developed on the control leaf-calluses. Several hundred plants were routinely regenerated using this protocol. More importantly, the number of shoots per explantcallus increased drastically over the subsequent subculture period. The most favorable media for callus induction contained a combination of an auxin and a cytokinin (0.1 mg/1 2,4-dichlorophenoxyacetic acid and 1 mg/1 N-6 benzylaminopurine) or a cytokinin alone (2.2 mg/1 thidiazuron). However, only the callus derived from leaves of TIBA-treated genotypes and induced on thidiazuron-medium produced numerous shoots. Histological studies showed the formation of meristematic zones only in the organogenic callus developed on thidiazuron-coutaining medium. The analysis of peroxidase activity showed that the activity was higher for the TIBA-treated plants than for the untreated control plants.     

In vitro induction of haploid plants from unpollinated ovules and ovaries of the sugarbeet (Beta vulgaris L.)

Summary Haploid plantlets from male fertile and male sterile sugarbeet plants could be induced at frequencies up to 2.2% using ovule culture. Ovary culture on media without charcoal resulted in a similar induction frequency. Plant development was inhibited by callus development originating from the mother tissue. When the callus parts were removed and the ovule transferred to a new medium without 2,4 D, callus formation could be inhibited by adding 0.5% charcoal to the medium. Up to 6.1% haploids were induced. Chromosome counts in leaf tips, chloroplast counts and isozyme patterns revealed that all plants were haploid and originated from the haploid cells of the embryo sac. Root tips showed spontaneous polyploidisation.     

Production of transgenetic sugarbeet (Beta vulgaris L.) plants resistant to phosphinothricin

A method of Agrobacterium-mediated genetic transformation of sugarbeet (Beta vulgaris L.) with vacuum infiltration has been developed. Aseptic 3-weeks old etiolated seedlings of two diploid O-type sugarbeet lines (KS3 and KS7) have been used for genetic transformation. Transgenic sugarbeet plants carrying the reporter beta-glucuronidase gene have been selected for their resistance to glufosinate ammonium herbicide. Integration of transgenes into sugarbeet genome was confirmed with GUS assay and PCR using primers for bar and gusA genes.     

Production of transgenic sugarbeet plants (Beta vulgaris L.) using Agrobacterium rhizogenes

Normal phenotype sugarbeet plants transformed with Agrobacterium rhizogenes were produced using direct regeneration from explants without hairy root phase. Kanamycin resistant plants and Ri-roots carrying the genes of neomycin phosphotransferase II and b-glucuronidase have been obtained. Integration of transgenes into sugarbeet genome was confirmed with GUS-assay and PCR using primers for the introduced genes.     

Sucrose-dependent H+ transport in plasma-membrane vesicles isolated from sugarbeet leaves (Beta vulgaris L.)

The mechanism of sucrose transport across the plasma membrane (PM) was investigated in membrane vesicles isolated from sugarbeet (Beta vulgaris L.) leaves. In the presence of a membrane potential () generated as a K⁺-diffusion potential, negative inside, sucrose induced a rapid and transient alkalization of the medium. Alkalization was inhibited by carbonyl cyanide m-chlorophenylhydrazone, was specific for the sucrose sugar and was dependent on the sucrose concentration with a K_m of approx. 1 mM. Sucrose-induced alkalization and sucrose transport were inhibited by the sulfhydryl-reactive reagent, p-chloromercuribenzene sulfonic acid, and by the histidine-reactive reagent, diethyl pyrocarbonate. Parallel analysis of sucrose uptake and alkalization indicated that the stoichiometry of sucrose uptake to proton consumed was 11. These results provide clear evidence that the saturable mechanism of sucrose transport across the PM in plants is a coupled H⁺-sucrose symport.Abbreviations and Symbols CCCP carbonyl cyanide m-chlorophenylhydrazone - DEPC diethyl pyrocarbonate - PCMBS p-chloromercuribenzene sulfonic acid - pH pH gradient - membrane potential difference - PM plasma membrane The financial support for a portion of thus study was provided by the Deutsche Forschungsgemeinschaft. We thank Kimberly A. Mitchell for her excellent technical assistance and dedicate this report to the memory of Mr. William A. Dungey.     

The effect of antibiotics on the inhibition of callus induction and plant regeneration from cotyledons of sugarbeet (Beta vulgaris L.)

Callus induction and plantlet regeneration from cotyledonary expiants of sugarbeet was observed utilizing two media formulations, MS and a modified MS termed RVIM both supplemented with 1.0 g/ml BAP as the sole growth regulator. Callus induction was genotype dependent The USDA line 8787 produced the highest response for callus induction followed by Betaseed 4587 and the USDA line C600. This order was conserved on both media formulations. Shoot induction was consistently higher averaging 32% from the RVIM formulation over the 3 genotypes compared to 25% from MS. The antibiotics geneticin, gentamycin, hygromycin, kanamycin and phleomycin were screened with the modified RV system utilizing Betaseed 4587. Callus growth was inhibited by levels of 50 g/ml geneticin, 150 g/ml gentamycin, 10 g/ml hygromycin, 150 g/ml kanamycin and 20 g/ml phleomycin. The results indicate that the concentrations of antibiotics used to inhibit callus induction will be sufficient for use as selectable markers in transformation experiments with Beta vulgaris.Abbreviations B₅ basal medium (Gamborg et al, 1968) - BAP N⁶-Benzylaminopurine - IBA Indole-3-butanoic acid - MS basal medium (Murashige and Skoog 1968) - RVIM modified MS basal medium (Freytag et al, 1988) - MES (2[N-Morpholino] ethanesulfonic acid     

Production of transgenic sugarbeet (Beta vulgaris L.) plants of O-type using Agrobacterium tumefaciens

A method of Agrobacterium-mediated genetic transformation of sugarbeet (Beta vulgaris L.) by vacuum infiltration has been developed. Transgenic sugarbeet plants of Ukraine breeding were selected for their resistance either to the antibiotic kanamycin or to the herbicide glufosinate ammonium. Integration of transgenes was confirmed by PCR and GUS-assay.     

Hormone-free medium will support callus production and subsequent shoot regeneration from whole plant leaf explants in some sugarbeet (Beta vulgaris L.) populations

Sugarbeet plants representing 14 of 16 germplasm sources (4 to 5 plants per source) produced callus from leaf disks on a hormone-free Murashige and Skoog based medium. Overall, 49.2% of explants from partially expanded leaves of whole plants initiated callus (53 of 74 plants tested), in an average time of 96.7 days. The time to callus was considerably longer than the 4–6 weeks observed when 1 mg/L N⁶-benzyladenine has been used in the medium. Shoots were regenerated on the hormone-free medium without subculture from callus of eight individual genotypes, representing 3 of the 14 populations that produced callus. Shoots produced by Gartons White Knight and L53 appeared to be of somatic embryo origin. Rhizogenic calli were also produced by the same three populations that regenerated shoots. Significant differences among populations were found for frequency of root formation from leaf disks and time to callus. Variation among plants within a population was significant for four of the five traits examined. The results indicate the ease of hormone autonomization in sugarbeet, and should be of value in designing regeneration media for a wider range of beet germplasm.Abbreviations BA N⁶-benzyladenine - MS Murashige and Skoog basal medium Cooperative investigations of the Agricultural Research Service, U.S. Department of Agriculture and the Michigan Agricultural Experiment Station, East Lansing, Michigan 48824. Journal article No. 12603.     

Beet necrotic yellow vein virus coat protein-mediated protection in sugarbeet (Beta vulgaris L.) protoplasts

Transformed Beta vulgaris L. suspension cultures were obtained after cocultivation of sugarbeet cells with Agrobacterium tumefaciens harbouring a binary vector containing the coat protein gene of beet necrotic yellow vein virus inserted between the kanamycin resistance gene and a ß-glucuronidase reporter gene. Protoplasts were isolated both from untransformed cells, and from transformed cells expressing the viral coat protein, and both were then infected with beet necrotic yellow vein virus. Comparison of the levels of infectivity shows that the expression of the coat protein gene in sugarbeet protoplasts mediates high levels of protection against infection by beet necrotic yellow vein virus.Abbreviations TMV Tobacco Mosaic Virus - CP Coat Protein - BNYVV Beet Necrotic Yellow Vein Virus - ß-Glu ß-glucuronidase - MS Murashige and Skoog (1962) - PEG Polyethylene glycol - npt neomycin phosphotransferase - nos nopaline synthase - FITC fluoresceine isothiocyanate - IAA indole acetic acid - BAP benzyl amino purine - MES 2-[N-Morpholino]ethane sulfonic acid - IgG Immunoglobulin G - nt nucleotide     

Plant regeneration from protoplasts of sugar beet (Beta vulgaris)

Mesophyll protoplasts from two of five sugar beet lines tested were regenerated into plants. Mesophyll protoplasts of all lines showed high plating efficiencies up to 4.0% developed hard compact callus, and two of the lines also developed white, soft and friable callus consisting of starch grain-containing cells. Whereas the compact callus never regenerated into plants, the white friable ones frequently developed globular structures, which became green in the light and formed adventitious shoots after cytokinin (BAP or thidiazuron) treatment. Genetic analysis by PCR-fingerprinting and flow cytometry showed uniformity and unchanged ploidy levels in 15 independently regenerated plantlets in line NF. but altered ploidy level (from diploid to triploid) in a regenerated plantlet of clone VRB.     

Growth habit and sugar accumulation in sugarbeet (Beta vulgaris L.) transformed with a cytokinin biosynthesis gene

Expression of a bacterial cytokinin biosynthesis gene fused to a patatin gene promoter was studied in sugarbeet (Beta vulgaris L.). Two independent transformants, Pat-ipt 1 and 2, exhibited a number of distinguishable morphological alterations commonly induced by cytokinins, i.e. less root growth, reduced leaf surface area, and increased axillary shoot development. Concentrations of the cytokinins zeatin and zeatin riboside were increased by twofold in taproots and 7- to 18-fold in leaves. Leaf sucrose and glucose concentrations were not significantly different from those in control plants except in Pat-ipt 2 where glucose levels were elevated ninefold. Since normal taproot development was severely inhibited, sucrose concentrations in the taproots were significantly reduced.     

Plant regeneration from cultured immature embryos of Sorghum bicolor (L.) Moench

Summary Immature embryos of 20 sorghum genotypes were cultured on MS 5 medium containing MS mineral salts supplemented with 2,4-D, zeatin, glycine, niacinamide, Ca-pantothenate, L-asparagine, and vitamins. For regeneration, calli were transferred onto the same medium with the exception that IAA was substituted for 2,4-D. In general, immature embryos obtained 9–12 days after pollination resulted in the best redifferentiation. Ability of calli to regenerate varied among genotypes; cultivars C401-1 and C625 had the highest redifferentiation frequencies. Ability to redifferentiate was heritable and acted as a dominant trait. At least two gene pairs were involved. Regenerated R₀ plants were planted in a greenhouse and their selfed (R₁ and R₂) progenies were planted in the field and examined for morphological and cytological variations. The majority of the phenotypic variations noted in R₀ were not transmitted to later generations. However, variants for plant height, degree of fertility, and midrib color persisted in R₁ and R₂ generations. A variation in tallness was attributable to one dominant mutant gene. Short stature and male sterility variants appeared to be consequences of recessive mutant genes controlling those traits. Minor variations in peroxidase banding patterns were found among R₀ plants.This study was supported by a research grant from Kansas Sorghum Commission and by a Research Fellowship to the senior author from the Ministry of Agriculture, Animal Husbandry, and Fisheries, China. Contribution 86-456-J from the Kansas Agricultural Experiment Station     

甜菜胞质型谷氨酰胺合成酶基因组DNA的克隆

以甜菜叶片为材料,用CTAB法提取基因组DNA.以分段PCR法扩增得到了完整的甜菜胞质型谷氨酰胺合成酶(GS1)基因组DNA.采用RT-PCR法扩增此GS1基因(GS1)的cDNA序列应用于对照.获得了长度为9 606bp的完整的GS1 DNA序列和长度为1 068 bp的GSI cDNA序列.分析GS1基因组DNA序列表明,它包含13个外显子,被12个内含子分隔开.其外显子区与已公布的GS1 mRNA序列的相似性达99.5%.RT-PCR法获得的cDNA序列与已知的GS1 mRNA序列相似性达99.6%.而2次实验中GS1基因组DNA外显子区与GS1 cDNA序列的相似性达99.9%.GenBank登录号为EU370974.     

Restriction fragment map of sugar beet (Beta vulgaris L.) chloroplast DNA

Summary A restriction endonuclease fragment map of sugar beet chloroplast DNA (ctDNA) has been constructed with the enzymes SmaI, PstI and PvuII. The ctDNA was found to be contained in a circular molecule of 148.5 kbp. In common with many other higher plant ctDNAs, sugar beet ctDNA consists of two inverted repeat sequences of about 20.5 kbp separated by two single-copy regions of different sizes (about 23.2 and 84.3 kbp). Southern hybridization analyses indicated that the genes for rRNAs (23S+16S) and the large subunit of ribulose 1,5-bisphosphate carboxylase were located in the inverted repeats and the large single-copy regions, respectively.     

DNA methylation of retrotransposons,DNA transposons and genes in sugar beet (Beta vulgaris L.)

The methylation of cytosines shapes the epigenetic landscape of plant genomes, coordinates transgenerational epigenetic inheritance, represses the activity of transposable elements (TEs), affects gene expression and, hence, can influence the phenotype. Sugar beet (Beta vulgaris ssp. vulgaris), an important crop that accounts for 30% of worldwide sugar needs, has a relatively small genome size (758 Mbp) consisting of approximately 485 Mbp repetitive DNA (64%), in particular satellite DNA, retrotransposons and DNA transposons. Genome‐wide cytosine methylation in the sugar beet genome was studied in leaves and leaf‐derived callus with a focus on repetitive sequences, including retrotransposons and DNA transposons, the major groups of repetitive DNA sequences, and compared with gene methylation. Genes showed a specific methylation pattern for CG, CHG (H = A, C, and T) and CHH sites, whereas the TE pattern differed, depending on the TE class (class 1, retrotransposons and class 2, DNA transposons). Along genes and TEs, CG and CHG methylation was higher than that of adjacent genomic regions. In contrast to the relatively low CHH methylation in retrotransposons and genes, the level of CHH methylation in DNA transposons was strongly increased, pointing to a functional role of asymmetric methylation in DNA transposon silencing. Comparison of genome‐wide DNA methylation between sugar beet leaves and callus revealed a differential methylation upon tissue culture. Potential epialleles were hypomethylated (lower methylation) at CG and CHG sites in retrotransposons and genes and hypermethylated (higher methylation) at CHH sites in DNA transposons of callus when compared with leaves.