Efficient somatic embryogenesis in sugar beet (<Emphasis Type="Italic">Beta vulgaris</Emphasis> L.) breeding lines

Efficient regeneration via somatic embryogenesis (SE) would be a valuable system for the micropropagation and genetic transformation of sugar beet. This study evaluated the effects of basic culture media (MS and PGo), plant growth regulators, sugars and the starting plant material on somatic embryogenesis in nine sugar beet breeding lines. Somatic embryos were induced from seedlings of several genotypes via an intervening callus phase on PGo medium containing N⁶-benzylaminopurine (BAP). Calli were mainly induced from cotyledons. Maltose was more effective for the induction of somatic embryogenesis than was sucrose. There were significant differences between genotypes. HB 526 and SDM 3, which produced embryogenic calli at frequencies of 25–50%, performed better than SDM 2, 8, 9 and 11. The embryogenic calli and embryos produced by this method were multiplied by repeated subculture. Histological analysis of embryogenic callus cultures indicated that somatic embryos were derived from single- or a small number of cells. 2,4-dichlorophenoxyacetic acid (2,4-D) was ineffective for the induction of somatic embryogenesis from seedlings but induced direct somatic embryogenesis from immature zygotic embryos (IEs). Somatic embryos were mainly initiated from hypocotyls derived from the cultured IEs in line HB 526. Rapid and efficient regeneration of plants via somatic embryogenesis may provide a system for studying the molecular mechanism of SE and a route for the genetic transformation of sugar beet.     

Efficient transformation of beet (<Emphasis Type="Italic">Beta vulgaris</Emphasis>) and production of plants with improved salt-tolerance

Small bud tips of 1–3 mm in length were taken from multiple shoot clumps that derived from immature inflorescence cultures of beet as recipient for the Agrobacterium-mediated transformation and transgenic plants were obtained from eight genotypes. The optimal genetic transformation protocol was established as followed: the buds were immersed in Agrobacterium suspension of OD₆₀₀ =0.3–0.5 for 5–10 min, with vacuum infiltration (0.3–0.5 × 10⁵ Pa) or supplemented with 0.01% Silwet L-77, co-cultured for 2–4 days and followed by 10-day culture on medium containing 100 mg l⁻¹ cefotaxime, then the buds were selected on medium containing 10 mg l⁻¹ hygromycin B for three consecutive generations. The percentage of hygromycin-resistant buds after three selections varied from 13.3 to 30.6% with genotypes. The results of PCR and further Southern blotting of genomic DNA of hygromycin-resistant buds or plants showed that the exogenous hpt and AtNHX1 gene had been integrated into the genomes of some transformed buds or plants. The transgenic buds or plants with AtNHX1 gene encoding Na⁺/H⁺ antiport on the vacuole membrane of Arabidopsis showed improved salt-tolerance than the controls. AtNHX1gene inherited in some transgenic lines as Mendelian segregation. This result revealed that it was feasible to improve salt-tolerance of beets by the introduction of AtNHX1 gene into cultured buds.     

Multiplexed,linkage group-specific SNP marker sets for rapid genetic mapping and fingerprinting of sugar beet (<Emphasis Type="Italic">Beta vulgaris</Emphasis> L.)

A set of single nucleotide polymorphism (SNP) markers has been developed for each of the nine linkage groups of sugar beet. Each set can monitor the polymorphic state at five to six linked marker loci. In each set, the loci selected for marker development are first amplified in a multiplexed reaction. These amplification products are the basis for sequence-specific elongation of primers adjacent to SNP positions. The extension step revealing SNP loci is based on fluorescently labelled nucleotides. In each set, primers developed to reveal SNP alleles differ in length to allow clear peak resolution in capillary electrophoresis. The nine linkage group (LG) –specific sets provide information on the polymorphism at a total of 52 SNP marker loci. Using the SNP-based tool, groups of concerned loci have been anchored to three different linkage maps of sugar beet. In a second experiment, sugar beet breeding lines have been fingerprinted. The use of the nine sets of LG-specific markers in sugar beet genetics and breeding is discussed. The information necessary to specify the 52 marker loci, as well as their map location, and all details concerning SNP assays, including allele type and nature of mutation, are reported.     

High frequency <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation and plant regeneration via direct shoot formation from leaf explants in <Emphasis Type="Italic">Beta vulgaris</Emphasis> and <Emphasis Type="Italic">Beta maritima</Emphasis>

We have developed a new procedure for Agrobacterium-mediated transformation of plants in the genus Beta using shoot-base as the material for Agrobacterium infection. The frequency of regeneration from shoot bases was analyzed in seven accessions of sugarbeet (Beta vulgaris) and two accessions of B. maritima to select materials suitable for obtaining transformed plants. The frequency of transformation of the chosen accessions using Agrobacterium strain LBA4404 and selection on 150-mg/l kanamycin was found to be higher than that in previously published methods. Genomic DNA analysis and -glucuronidase reporter assays showed that the transgene was inherited and expressed in subsequent generations. In our method, shoot bases are prepared by a simple procedure, and transformation does not involve the callus phase, thus minimizing the occurrence of somaclonal variations.     

A BAC library of <Emphasis Type="Italic">Beta vulgaris</Emphasis> L. for the targeted isolation of centromeric DNA and molecular cytogenetics of <Emphasis Type="Italic">Beta</Emphasis> species

We constructed a sugar beet (Beta vulgaris) bacterial artificial chromosome (BAC) library of the monosomic addition line PAT2. This chromosomal mutant carries a single additional chromosome fragment (minichromosome) derived from the wild beet Beta patellaris. Restriction analysis of the mutant line by pulsed-field gel electrophoresis was used to determine HindIII as a suitable enzyme for partial digestion of genomic DNA to generate large-insert fragments which were cloned into the vector pCC1. The library consists of 36,096 clones with an average insert size of 120 kb, and 2.2% of the clones contain mitochondrial or chloroplast DNA. Based on a haploid genome size of 758 Mbp, the library represents 5.7 genome equivalents providing the probability of 99.67% that any sequence of the PAT2 genome can be found in the library. Hybridization to high-density filters was used to isolate 89 BACs containing arrays of the centromere-associated satellite repeats pTS5 and pTS4.1. Using the identified BAC clones in fluorescent in situ hybridization experiments with PAT2 and Beta patellaris chromosome spreads their wild beet origin and centromeric localization was demonstrated. Multi-colour FISH with differently labelled satellite repeats pTS5 and pTS4.1 was used to investigate the large-scale organization of the centromere of the PAT2 minichromosome in detail. FISH studies showed that the centromeric satellite pTS5 is flanked on both sides by pTS4.1 arrays and the arms of the minichromosome are terminated by the Arabidopsis-type telomeric sequences. FISH with a BAC, selected from high-density filters after hybridization with an RFLP marker of the genetic linkage group I, demonstrated that it is feasible to correlate genetic linkage groups with chromosomes. Therefore, the PAT2 BAC library provides a useful tool for the characterization of Beta centromeres and a valuable resource for sugar beet genome analysis.     

Leaf area prediction model for sugar beet (<Emphasis Type="Italic">Beta vulgaris</Emphasis> L.) cultivars

In two successive years (2003 and 2004), a set of 16 commercial sugar beet cultivars was established in Randomized Complete Block experiments at two sites in central Greece. Cultivar combination was different between years, but not between sites. Leaf sampling took place once during the growing season and leaf area, LA [cm²], leaf midvein length, L [cm] and maximum leaf width, W [cm] were determined using an image analysis system. Leaf parameters were mainly affected by cultivars. Leaf dimensions and their squares (L², W²) did not provide an accurate model for LA predictions. Using L×W as an independent variable, a quadratic model (y = 0.003 x² − 1.3027 x + 296.84, r ² = 0.970, p<0.001, n = 32) provided the most accurate estimation of LA. With compromises in accuracy, the linear relationship between L×W and LA (y = 0.5083 x + 31.928, r ² = 0.948, p<0.001, n = 32) could be used as a prediction model thanks to its simplicity.     

Crystals in sugar beet (<Emphasis Type="Italic">Beta vulgaris</Emphasis> L.) leaves

Crystal-containing cells (C-cells) are widely spread in plant tissues; however, the origin of the crystals and their functions remain a subject of discussion. In sugar beet leaves, the membrane vesicles seen in an electron microscope appear in the cytoplasm and penetrate the vacuole by pinocytosis with the participation of tonoplast. In a light microscope, the vesicles fluoresce like crystals in C-cells. These crystal vesicles also fill the C-cells. The content of crystal vesicles is electron-transparent at all stages of leaf development. It is suggested that both individual crystal vesicles in the cytoplasm and in vacuoles and their agglomerations in C cells, vascular bundles, and epidermal cells are lytic compartments. Later, true crystals seem to be formed.     

Regeneration of different cultivars of common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.) via indirect organogenesis

A protocol for in vitro regeneration via indirect organogenesis for Phaseolus vulgaris cv. Negro Jamapa was established. The explants used were apical meristems and cotyledonary nodes dissected from the embryonic axes of germinating seeds. Several auxin/cytokinin combinations were tested for callus induction. The best callus production was obtained with medium containing 1.5 μM 2,4-dichlorophenoxyacetic acid. After 2 weeks of growth calli were transferred to shooting medium containing 22.2 μM 6-benzylaminopurine. Shoots regenerated with a frequency of approximately 0.5 shoots per callus, and upon transfer to rooting medium these shoots produced roots with 100% efficiency. Histological analyses of the regeneration process confirmed the indirect organogenesis pattern. Greenhouse grown regenerated plants showed normal development and were fertile. The protocol was reproducible for other nine P. vulgaris cultivars tested, suggesting a genotype independent procedure.     

Using a competent tissue for efficient transformation of sugarbeet (<Emphasis Type="Italic">Beta vulgaris</Emphasis> L.)

Summary Despite intensive efforts, a reproducible and reliable method for transformation of sugarbeet plants is still lacking. Having examined several explants, we found that cells around the main vein of leaves of plantlets reared from tissue-cultured apical meristems are sufficiently competent for transformation and subsequent regeneration. A transformation protocol was designed by evaluating alterations in several parameters such as plant genotype, Agrobacterium strain, antibiotics, darkness and duration of co-culture period. An average transformation rate of 6.2% transformed shoots per explant was achieved as judged by Southern blotting. Consistent inactivation of reporter genes was correlated to multiple copies of transgenes present in some transformants. The necessary steps for rooting and planting of transformed shoots were also established.     

Multi-trait association mapping in sugar beet (<Emphasis Type="Italic">Beta vulgaris</Emphasis> L.)

Association mapping promises to overcome the limitations of linkage mapping methods. The main objective of this study was to examine the applicability of multivariate association mapping with an empirical data set of sugar beet. A total of 111 diploid sugar beet inbreds was selected from the seed parent heterotic pool to represent a broad diversity with respect to sugar content (SC). The inbreds were genotyped with 26 simple sequence repeat markers chosen according to their map positions in proximity to previously identified quantitative trait loci for SC. For SC and beet yield (BY), the genotypic variances were highly significant (P < 0.01). Based on the global test of the bivariate mixed-model approach, four markers were significantly associated with SC, BY, or both at a false discovery rate of 0.025. All four markers were significantly (P < 0.05) associated with BY but only two with SC. The identification of markers associated with SC, BY, or both indicated that association mapping can be successfully applied in a sugar beet breeding context for detection of marker-phenotype associations. Furthermore, based on our results multivariate association mapping can be recommended as a promising tool to discriminate with a high mapping resolution between pleiotropy and linkage as reasons for co-localization of marker-phenotype associations for different traits.     

Molecular genetic investigation of sugar beet (<Emphasis Type="Italic">Beta vulgaris</Emphasis> L.)

Molecular genetic studies of sugar beet (Beta vulgaris L.) are reviewed as a basis for the development of genomics of this species. The methods used to study structural and functional genomics are considered. The results and their application to increase the efficiency of sugar beet breeding are discussed.     

Morphometric and biochemical characterization of red beet (<Emphasis Type="Italic">Beta vulgaris</Emphasis> L.) hairy roots obtained after single and double transformations

It is known that T-DNA of Agrobacterium rhizogenes affects processes of plant development and activates the synthesis of secondary metabolites in transformed plant cells. In the present investigation, we provide evidence that different strains of A. rhizogenes significantly affect morphometric, morphological and functional characteristics of hairy roots of red beet (Beta vulgaris L.). Infection with four strains of A. rhizogenes (A4, A 2/83, A 20/83 and LMG-150) resulted in ten clones of hairy roots, which were named accordingly as A4(1), A4(2), A4(3), A 2/83(1), A 2/83(2), A 2/83(3), A 20/83(1), A 20/83(2), A 20/83(3) and LMG-150. Their growth characteristics, pigment content, levels of endogenous auxin and T-DNA copy number showed significant differences probably due to the physiological status of the host cell rather than the T-DNA copy number. Although A 2/83 showed highest hairy root induction capacity, the best hairy root clone was obtained with strain LMG-150 that produced highest biomass and pigments. In this root clone, the enzyme peroxidase was found involved in altering the endogenous auxin pool. When root clone LMG-150 was re-transformed to insert additional individual rol genes, two double transformed clones were obtained, one for rolABC and the other for rolC gene where the former produced higher biomass and betalaine than the latter. Despite the established fact that rol genes of T-DNA influence endogenous phytohormones, no direct correlation among the single transformants and the double transformants was found. This is the first report, in our knowledge, where a hairy root clone has been used to obtain double transformants.     

The Novel Use of a Combination of Sonication and Vacuum Infiltration in <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated Transformation of Kidney Bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.) with <Emphasis Type="Italic">lea</Emphasis> Gene

An efficient gene transfer system without tissue culture steps was developed for kidney bean by using sonication and vacuum infiltration assisted, Agrobacterium-mediated transformation. Transgenic kidney bean with a group 3 lea (late embryogenesis abundant) protein gene from Brassica napus was produced through this approach. Among 18 combinations of transformation methods, Agrobacterium-mediated transformation combined with 5 min sonication and 5 min vacuum infiltration turned to be optimal, resulting in the highest transformation efficiency. Transgenic kidney bean plants demonstrated enhanced growth ability under salt and water deficit stress conditions. The increased tolerance was also reflected by delayed development of damage symptoms caused by drought stress. Transgenic lines with high level of lea gene expression showed higher stress tolerance than lines with lower expression level. Stress tolerance of transgenic kidney bean correlated much better with lea gene expression levels than with gene integration results. There is no prior report on the production of transgenic kidney bean using both ultrasonic and vacuum infiltration assisted, Agrobacterium-mediated transformation.     

Efficient procedures for callus induction and adventitious shoot organogenesis in sugar beet (<Emphasis Type="Italic">Beta vulgaris</Emphasis> L.) breeding lines

Summary Improved in vitro tissue culture systems are needed to facilitate the application of transgene technology to the improvement of sugar beet germplasms. Several commercially important sugar beet breeding lines (SDM, 3, 5, 8, 9, 10, 11, HB 526, and CMS 22003) and commercial varieties (Roberta and Gala) were tested for their regeneration capacity through adventitious shoot organogenesis from cotyledons, hypocotyls, root/hypocotyl/shoot transition zone tissues, and leaf lamina and petiole via an intervening callus phase. Callus induction and adventitious shoot regeneration was dependent on genotype and combinations of plant growth regulators. With cotyledon or hypocotyl explants, SDM 3 and 10 showed a better response on adventitious shoot regeneration in medium containing benzyladenine (BA) and 2,3,5-triiodobenzoic acid or 1-naphthaleneacetic acid (NAA) than SDM 11, 5, and 9. Shoot regeneration was obtained from hypocytyl-root or hypocotyl-shoot transition zone tissue in SDM 9, 10, and HB 526 grown on PGo medium supplemented with BA to induce callus, and the regeneration frequency was 25%. Adventitious shoots were also regenerated from leaf explants of SDM 3 and 9 cultured on medium containing NAA for callus induction and BA and NAA to induce shoot regeneration, and in SDM 10 and CSM 22003 cultured on medium containing BA for callus induction and to induce shoot regeneration.     

Insect feeding-induced differential expression of <Emphasis Type="Italic">Beta vulgaris</Emphasis> root genes and their regulation by defense-associated signals

Root responses to insect pests are an area of plant defense research that lacks much information. We have identified more than 150 sugar beet root ESTs enriched for genes responding to sugar beet root maggot feeding from both moderately resistant, F1016, and susceptible, F1010, genotypes using suppressive subtractive hybridization. The largest number of identified F1016 genes grouped into the defense/stress response (28%) and secondary metabolism (10%) categories with a polyphenol oxidase gene, from F1016, identified most often from the subtractive libraries. The differential expression of the root ESTs was confirmed with RT-PCR. The ESTs were further characterized using macroarray-generated expression profiles from F1016 sugar beet roots following mechanical wounding and treatment of roots with the signaling molecules methyl jasmonate, salicylic acid and ethylene. Of the examined root ESTs, 20, 17 and 11% were regulated by methyl jasmonate, salicylic acid and ethylene, respectively, suggesting these signaling pathways are involved in sugar beet root defense responses to insects. Identification of these sugar beet root ESTs provides knowledge in the field of plant root defense and will lead to the development of novel control strategies for control of the sugar beet root maggot.Electronic Supplementary Material Supplementary material is available in the online version of this article at and is accessible for authorized users     

Highly efficient <Emphasis Type="Italic">in vitro</Emphasis> adventitious shoot regeneration of peppermint (<Emphasis Type="Italic">Mentha</Emphasis> x <Emphasis Type="Italic">piperita</Emphasis> L.) using internodal explants

An in vitro regeneration system with a 100% efficiency rate was developed in peppermint [Mentha x piperita] using 5- to 7-mm-long second internode stem segments of 3-wk-old stock plants. Shoots developed at sites of excision on stem fragments either directly from the cells or via primary calluses. The optimal medium for maximum shoot initiation and regeneration contained Murashige and Skoog (MS) salts, B5 vitamins, thidiazuron (TDZ, 11.35 μM), ZT (4.54 μM), 10% coconut water (CW), 20 g l⁻¹ sucrose, 0.75% agar, adjusted to pH 5.8. A frequency of 100% shoot initiation was achieved, with an average of 39 shoots per explant. This regeneration system is highly reproducible. The regenerated plants developed normally and were phenotypically similar to Black Mitcham parents.     

Regeneration and<Emphasis Type="Italic"> Agrobacterium-</Emphasis>mediated transformation of hop (<Emphasis Type="Italic">Humulus lupulus</Emphasis> L.)

An efficient procedure for direct organogenesis and regeneration of hop (Humulus lupulus L.) was established. For the first time Agrobacterium-mediated genetic transformation of hop (cv. "Tettnanger") was achieved. Shoot internodes from in vitro cultures were identified as the most suitable type of explant for regeneration. Using this type of explant, a shoot-inducing medium was developed that supported direct organogenesis of approximately 50% of the explants. Plantlets were successfully rooted and transferred to the greenhouse. Overall, in less than 6 months hop cultures propagated in vitro were regenerated to plants in the greenhouse. Agrobacterium-mediated genetic transformation was performed with the reporter gene GUS (-glucuronidase). The presence and function of transgenes in plants growing in the greenhouse was verified by PCR (polymerase chain reaction) and enzyme assay for GUS activity, respectively. We have obtained 21 transgenic plants from 1,440 explants initially transformed, yielding an overall transformation efficiency of 1.5%.Abbreviations BAP 6-Benzylaminopurine - GA₃ Gibberellic acid - GUS -Glucuronidase - IAA Indole-3-acetic acid - IBA Indole-3-butyric acid - NAA -Naphthaleneacetic acid - nptII Neomycin phosphotransferase II - PCR Polymerase chain reaction - TDZ 1-Phenyl-3-(1,2,3-thiadiazol-5-yl) urea (thidiazuron)Communicated by H. Lörz     

<Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis>-mediated DNA transfer to<Emphasis Type="Italic"> Aesculu</Emphasis>s<Emphasis Type="Italic"> hippocastanum</Emphasis> L. and the regeneration of transformed plants

Hairy roots were induced from androgenic embryos of horse chestnut (Aesculus hippocastanum L.) by infection with Agrobacterium rhizogenes strain A4GUS. Single roots were selected according to their morphology in the absence of antibiotic or herbicide resistance markers. Seventy-one putative transformed hairy root lines from independent transformation events were established. Regeneration was induced in MS liquid medium supplemented with 30 6-benzylaminopurine (BA), and the regenerants were multiplied on MS solid medium containing 10 M BA. Following elongation on MS medium supplemented with 1 M BA and 500 mg/l polyvinylpyrrolidone, the shoots were subjected to a root-inducing treatment. Stable integration of TL-DNA within the horse chestnut genome was confirmed by Southern hybridization. The copy number of transgenes was estimated to be from two to four.Communicated by E.D. Earle     

The <Emphasis Type="Italic">Caenorhabditis</Emphasis><Emphasis Type="Italic">briggsae</Emphasis> genome contains active <Emphasis Type="Italic">CbmaT1</Emphasis> and <Emphasis Type="Italic">Tcb1</Emphasis> transposons

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.     

Thidiazuron Promotes <Emphasis Type="Italic">In Vitro</Emphasis> Plant Regeneration of <Emphasis Type="Italic">Arachis correntina</Emphasis> (<Emphasis Type="Italic">Leguminosae</Emphasis>) via Organogenesis

Arachis correntina (Burkart) Krapov. & W.C. Gregory is a herbaceous perennial leguminous plant growing in the Northeast of the Province Corrientes, Argentina. It is important as forage. The development of new A. correntina cultivars with improved traits could be facilitated through the application of biotechnological strategies. The purpose of this study was to investigate the plant regeneration potential of mature leaves of A. correntina in tissue culture. Buds were induced from both petiole and laminae on 0.7% agar-solidified medium containing 3% sucrose, salts and vitamins from Murashige and Skoog (MS) supplemented with 0.5–25 M thidiazuron (TDZ). Shoot induction was achieved by transference of calli with buds to MS supplemented with 5 M TDZ. Fifty-four percent of the regenerated shoot rooted on MS + 5 M naphthaleneacetic acid. Histological studies revealed that shoots regenerated via organogenesis.