Chromosome number and ploidy level of balm (Melissa officinalis).

BACKGROUND: Lemon balm (Melissa officinalis L.) is of increasing importance resulting in rising growth area. Improved knowledge on the genome structure, number of chromosomes in connection with the taxonomical structure of balm is indispensable for improved new varieties. RESULTS: A collection of 40 balm accessions (M. officinalis) was characterized by flow cytometry and FISH (18/25S and 5S rDNA) to determine the chromosome number and ploidy level. Three different types were found: diploid genotypes with 2n = 2× = 32 chromosomes; tetraploid 2n = 4× = 64 chromosomes and triploid 2n = 3× = 48 chromosomes. A haploid base number of × = 16 chromosomes is likely. First time described triploid accessions are sterile but cytologically and morphologically stable for many years. Triploids express better winter hardiness and regeneration after harvesting cuts as well as bigger leaves and internodes. CONCLUSIONS: A basic chromosome number of x = 16 is reported for the first time for the species M. officinalis.

Transfer of resistance against the beet cyst nematode from radish (Raphanus sativus) to rape (Brassica napus) by monosomic chromosome addition.

In rape ( Brassica napus), no resistance to the beet cyst nematode (BCN) Heterodera schachtii is available. This study was carried out to determine the specific chromosome(s) of resistant radish ( Raphanus sativus) carrying the gene(s) for nematode resistance as a prequisite to convert rape from a host into a trap crop for this pest. A Raphanobrassica progeny of 25 plants was analyzed which segregated for all nine chromosomes of the Raphanus genome in a genetic background of synthetic rape. The number of radish chromosomes was determined by fluorescence in situ hybridization, using the Raphanus-specific DNA probe pURsN; and their type was identified by chromosome-specific randomly amplified polymorphic DNA markers. Five different multiple rape-radish chromosome additions (comprising the whole set of nine radish chromosomes, a-i) were selected and crossed to rape. For each cross-progeny, the number of cysts on plant roots was counted 42 days after inoculation with a L2 larvae suspension. Simultaneously, the plants were characterized for the presence or absence of individual radish chromosomes, using sets of chromosome-specific markers. Thus, the effect of each radish chromosome on cyst number was tested. Chromosome d had a major resistance effect, whereas the presence/absence of the other radish chromosomes had nearly no influence on cyst number. Plants with added chromosome d showed a resistance level comparable with that of the radish donor parent. The analysis in the cross to rape of a plant monosomic only for chromosome d confirmed the strong effect of this chromosome on nematode resistance. A further experiment comprising seven crosses using winter rape breeding lines and monosomic addition line d as pollen parent provided the same results on a broader genetic basis. In each case, the added chromosome d in a single dosage caused nearly the full resistance of the radish donor. Resistance was independent of the glucosinolate content in the roots. The possibilities for stabilizing BCN resistance in rape and its use for other crops and nematodes are discussed.

A mini-RNA containing the tetraloop, wobble-pair and loop E motifs of the central conserved region of potato spindle tuber viroid is processed into a minicircle.

A Mini-RNA from potato spindle tuber viroid (PSTVd) was constructed specifically for cleavage and ligation to circles in vitro. It contains the C-domain with the so-called central conserved region (CCR) of PSTVd with a 17 nt duplication in the upper strand and hairpin structures at the left and rights ends of the secondary structure. The CCR was previously shown to be essential for processing of in vitro transcripts. When folded under conditions which favor formation of a kinetically controlled conformation and incubated in a potato nuclear extract, the Mini-RNA is cleaved correctly at the 5'- and the 3'-end and ligated to a circle. Thus, the CCR obviously contains all structural and functional requirements for correct processing and therefore may be regarded as 'processing domain' of PSTVd. Using the Mini-RNA as a model substrate, the structural and functional relevance of its conserved non-canonical motifs GAAA tetraloop, loop E and G:U wobble base pair were studied by mutational analysis. It was found that (i) the conserved GAAA tetraloop is essential for processing by favoring the kinetically controlled conformation, (ii) a G:U wobble base pair at the 5'-cleavage site contributes to its correct recognition and (iii) an unpaired nucleotide in loop E, which is different from the corresponding nucleotide in the conserved loop E motif, is essential for ligation of the 5'- with the 3'-end. Hence all three structural motifs are functional elements for processing in a potato nuclear extract.

Transfer of a male-sterility-inducing cytoplasm from onion to leek ( Allium ampeloprasum).

Two interspecific triploid (AAC) hybrids (84/1-94 and 99/1-94) from crosses between onion [ Allium cepa (2 n=2 x=16, CC)] and leek [ A. ampeloprasum (2 n=4 x=32, AAAA)] were backcrossed to leek in order to transfer a male-sterility-inducing cytoplasm from onion that would enable the production of hybrid leek. GISH evaluations of meiosis in the interspecific hybrids revealed irregularities due to univalent onion chromosomes producing micronuclei from onion chromatin, whereas the pairing of the two sets of leek chromosomes was nearly normal. Attempts to use colchicine to double the chromosome number of the hybrids failed. Backcrosses of 84/1-94 to leek as the pollen parent were not successful. The first backcross of 99/1-94 to tetraploid leek produced 11 BC(1) plants with chromosome numbers between 38 and 41. Identification of parental chromosomes by GISH showed that all eight onion chromosomes and 30-33 leek chromosomes were transmitted to the backcross progenies due to unreduced egg cells. Onion chromosomes were eliminated during the second backcross. Southern hybridization confirmed the transfer of the T-cytoplasm like source of CMS from onion to the BC(2) progenies. After the third backcross to leek, 158 plants were obtained with varying numbers of onion chromosomes and some intergenomic recombinant chromosomes. Alloplasmic leek plants without onion chromatin were selected for further characterization of male sterility and quality traits.

Karyotype analysis of Helianthus annuus using Giemsa banding and fluorescence in situ hybridization.

The karyotype of H. annuus was analysed by computer-aided image processing with respect to the chromosome length, arm ratio, occurrence and chromosomal position of intercalary heterochromatin and the location of 18S/25S and 5S ribosomal RNA genes. The karyotype was subdivided into a group of four acrocentric chromosome pairs, of which two were distinguishable by HKG (HCl, KOH, Giemsa) banding and a group of 13 meta- to submetacentric pairs. The latter could be subdivided into seven pairs with one and six pairs with two HKG bands. Three pairs of submetacentric satellite chromosomes revealed 18S/25S rDNA loci after fluorescence in situ hybridization (FISH) and silver staining. A fourth, smaller and possibly inactive, locus occurred in the terminal position on a metacentric pair. One submetacentric satellite chromosome pair revealed a 5S rRNA gene locus in the pericentromeric position; a second locus marked a submetacentric pair with one HKG band. The C-banding technique marks exclusively centromeric heterochromatin. Measurements of chromosomes in combination with Giemsa banding and FISH enabled the discrimination of most chromosome pairs of the sunflower.