In vitro selection of alfalfa plants resistant to Fusarium oxysporum f. sp. medicaginis

Summary From two lines of Medicago sativa characterized by a high regeneration capability, calli resistant to culture filtrate of Fusarium oxysporum f. sp. medicaginis have been selected. In these calli regeneration capability was greatly reduced and only one plant per callus was recovered. Regenerated plants have been evaluated for resistance to culture filtrate and for in vivo resistance to the pathogen. Three plants out of eight were resistant to the fungus and a high correlation between resistance to culture filtrate and in vivo resistance was observed.Research work supported by C.N.R., Italy. Special grant I.P.R.A. Subproject 1, paper no. 1468     

Structural characterization by (13)C-NMR spectroscopy of products synthesized in vitro by polysaccharide synthases using (13)C-enriched glycosyl donors: application to a UDP-glucose:(1-->3)-beta-D-glucan synthase from blackberry (Rubus fruticosus)

A simple and sensitive method for the characterization of products synthesized in vitro by polysaccharide synthases is described. It relies on the use of (13)C-enriched nucleotide sugars as substrates and on the analysis of the newly synthesized polysaccharides by (13)C-nuclear magnetic resonance (NMR) spectroscopy. The method was validated with a (1-->3)-beta-d-glucan synthase from blackberry, but it may be applied to the study of any glycosyltransferase. The chemical synthesis of UDP-d-[U-(13)C]glucose was achieved in a classical procedure with an overall yield of 50%. A uniformly labeled (1-->3)-beta-d-glucan was synthesized from this substrate, using detergent extracts of blackberry cell membranes as a source of synthase. One hundred micrograms of product was sufficient for liquid and solid-state (13)C-NMR spectroscopy analyses. The method is at least 100 times more sensitive than in the case of nonenriched polysaccharides. It allows the unequivocal identification and direct structural characterization of the products synthesized in vitro, as opposed to conventional methods that rely on the use of radioactive substrates and enzymatic hydrolysis of the polysaccharides with specific glycoside hydrolases. The method proves that the glycan analyzed was synthesized de novo because the final product is enriched in (13)C. Information on the 3D organization of the polymer may also be obtained by solid-state NMR spectroscopy.     

<Emphasis Type=Italic>In vitro</Emphasis> selection and regeneration of chrysanthemum (<Emphasis Type=Italic>Dendranthema grandiflorum</Emphasis> Tzelev) plants resistant to culture filtrate of <Emphasis Type=Italic>Septoria obesa</Emphasis> Syd

Callus cultures derived from leaf segments of chrysanthemum cultivar ‘Snow Ball’ which was susceptible to Septoria obesa were successfully used for in vitro selection for resistance to this pathogenic fungus. Resistant cell lines were selected by culturing callus on growth medium containing various concentrations of S. obesa filtrate. Resistant calluses obtained after two cycles (30 d each cycle) of selection were used for plant regeneration. About 30% of the plants regenerated from the resistant calluses and 70–80% of the plants raised from cuttings had acquired considerable resistance against the pathogen in the field. No phenotypic variation was observed in the selected regenerates.     

Male‐sterile maize plants produced by targeted mutagenesis of the cytochrome P450‐like gene (MS26) using a re‐designed I–CreI homing endonuclease

The I–CreI homing endonuclease from Chlamydomonas reinhardti has been used as a molecular tool for creating DNA double‐strand breaks and enhancing DNA recombination reactions in maize cells. The DNA‐binding properties of this protein were re‐designed to recognize a 22 bp target sequence in the 5th exon of MS26, a maize fertility gene. Three versions of a single‐chain endonuclease, called Ems26, Ems26+ and Ems26++, cleaved their intended DNA site within the context of a reporter assay in a mammalian cell line. When the Ems26++ version was delivered to maize Black Mexican Sweet cells by Agrobacterium‐mediated transformation, the cleavage resulted in mutations at a co‐delivered extra‐chromosomal ms26‐site in up to 8.9% of the recovered clones. Delivery of the same version of Ems26 to immature embryos resulted in mutations at the predicted genomic ms26‐site in 5.8% of transgenic T₀ plants. This targeted mutagenesis procedure yielded small deletions and insertions at the Ems26 target site consistent with products of double‐strand break repair generated by non‐homologous end joining. One of 21 mutagenized T₀ plants carried two mutated alleles of the MS26 gene. As expected, the bi‐allelic mutant T₀ plant and the T₁ progeny homozygous for the ms26 mutant alleles were male‐sterile. This paper described the second maize chromosomal locus (liguless‐1 being the first one) mutagenized by a re‐designed I–CreI–based endonuclease, demonstrating the general utility of these molecules for targeted mutagenesis in plants.