Chromosomal Variation in Crocus vernus Hill (Iridaceae) Investigated by in situ Hybridization of rDNA and a Tandemly Repeated Sequence

The physical localization of three tandemly-organized repetitiveDNA sequences was investigated byin situ hybridization to metaphasechromosomes of 11 Crocus vernus accessions. The sequences includedwere the 18S–25S rDNA, the 5S rDNA and a tandemly-repeatedsequence cloned from C. vernus(clone pCvKB8). Ten 2n = 8 karyotypesfrom accessions ranging across the Alps and the Pyrenees couldbe interpreted as variations of a standard karyotype. Polymorphismswere found involving size of the satellite chromosomes, extra5S rDNA sites, and extensive differences in size and numberof pCvKB8 loci. The 2 n = 16 type did not correspond to anypossible tetraploid derived from the 2 n = 8 types. Copyright2000 Annals of Botany Company Evolution, phylogeny, Crocus vernus Hill (Iridaceae), in situ hybridization, chromosomal polymorphism, karyotype evolution, repetitive DNA     

Controlling ethylene responses in flowers at the receptor level

For a vast number of ornamental species, blocking the plant's response to ethylene is an efficient strategy to enhance the longevity of the flowers. The most effective ways to conduct such interference will be reviewed in this paper. A large number of chemical compounds have been evaluated for their effects on ethylene production and perception. Among these are a range of strained olefines. This has resulted in the discovery that cyclopropenes, among them 1-methylcyclopropene (1-MCP) and a number of other substituted cyclopropenes effectively block ethylene responses at the receptor level. A lot of testing remains to be done to uncover the full potential of these compounds, but they do offer promising new ways to extend the postharvest life of ornamentals. Also genetic modification appears to be a very effective way in controlling of ethylene synthesis and perception. Attempts to use both a reduced endogenous ethylene production and a reduced sensitivity to ethylene will be reviewed. Among these the use of the mutant ethylene receptor gene, etr1-1, from Arabidopsis seems most promising, especially when it is expressed under the control of a flower specific promoter.     

The genomic organization and evolutionary distribution of a tandemly repeated DNA sequence family in the genus Crocus (Iridaceae)

From a recombinant DNA-library from Crocus vernus, two closely related clones of highly repetitive DNA, pCvKB7 and pCvKB8, were sequenced and their genomic distribution and organization were investigated by Southern and in situ hybridization. The lengths of the clones were 181 and 178 bp respectively; the sequences were approximately 85% identical, and thus belonged to a sequence family, named the pCvKB8-family. No homologous sequences were found in the databases (BLAST made may 2004). The presence of pCvKB8 in 52 Crocus species and six species from other genera were analyzed by Southern hybridization. The sequence family was essentially Crocus-specific. However, the distribution of hybridization signal across the genus showed poor agreement with the taxonomic structure of the Crocus genus, suggesting that the subdivision does not follow the phylogeny of this sequence family. The chromosomal distribution on three Crocus species was essentially identical: tandem organization close to all telomeres and most centromeres, with a few additional intercalary sites.     

Induction of adventitious shoots in vitro in Campanula carpatica

Efficient protocols have been developed to induce adventitious shoots in different types of explants of Campanula carpatica Jacq. More than five shoots per explant developed on hypocotyls of 5-week-old seedlings after 2 weeks of culture. Hypocotyls produced twice as many shoots as the cotyledons. TDZ proved to be about 6 times more efficient than BA. NAA had to be added to the regeneration medium to obtain the optimal balance of auxin and cytokinin to induce shoot regeneration. Significant differences were noted between different growth regulator concentrations in their effects on shoot organogenesis. BA induced double the number of callus clumps as TDZ. Incubation of explants in the dark produced about 6 shoots per explant while those in the light produced about 2 shoots per explant. Explants derived from 5-week-old seedlings were five times more regenerative compared to those derived from 15-week-old seedlings. Explants from cv. White Uniform were more organogenic than those from cv. Blue Clip. Root segments were also found to form shoots when treated with CPPU.     

TDZ induces shoot regeneration in various Kalanchoë blossfeldiana Poelln. cultivars in the absence of auxin

In order to optimize shoot regeneration in Kalancho? blossfeldiana, leaf and internode explants of seven cultivars including one inter-specific were studied. The effects of various combinations of α-naphthalene acetic acid (NAA) (0, 0.57 M) and thidiazuron (TDZ) (0, 0.45, 4.5, 22.5, 67.5 μM) on MS medium were examined. In all cultivars shoot regeneration frequency and number of shoots per explant were enhanced by increasing TDZ concentration. Supplementing the media with NAA did not improve shoot regeneration. Maximum regeneration frequency and optimum concentration of TDZ for shoot regeneration depended significantly on the cultivar. Internode explants, but not leaf explants, of some cultivars, were able to produce adventitious shoots without treatment with growth regulator.     

Inheritance of rapeseed (Brassica napus)-specific RAPD markers and a transgene in the cross B. juncea x (B. juncea x B. napus)

We have examined the inheritance of 20 rapeseed (Brassica napus)-specific RAPD (randomly amplified polymorphic DNA) markers from transgenic, herbicide-tolerant rapeseed in 54 plants of the BC₁ generation from the cross B. junceax(B. junceaxB. napus). Hybridization between B. juncea and B. napus, with B. juncea as the female parent, was successful both in controlled crosses and spontaneously in the field. The controlled backcrossing of selected hybrids to B. juncea, again with B. juncea as the female parent, also resulted in many seeds. The BC₁ plants contained from 0 to 20 of the rapeseed RAPD markers, and the frequency of inheritance of individual RAPD markers ranged from 19% to 93%. The transgene was found in 52% of the plants analyzed. Five synteny groups of RAPD markers were identified. In the hybrids pollen fertility was 0–28%. The hybrids with the highest pollen fertility were selected as male parents for backcrossing, and pollen fertility in the BC₁ plants was improved (24–90%) compared to that of the hybrids.     

Agrobacterium tumefaciens-mediated transformation of Campanula carpatica: factors affecting transformation and regeneration of transgenic shoots

An efficient transformation system for Campanula carpatica was developed using Agrobacterium tumefaciens strains LBA4404 (harbouring the plasmid pBI121), and AGL0 (harbouring the plasmid pBEO210). This is the first report on the transformation of C. carpatica. Various factors affecting the transformation efficiency and subsequent regeneration were identified. The age of seedlings from which the explants for transformation studies were taken, and the growth conditions under which the seedlings were grown had a significant influence on the production of transformed shoots. Hypocotyls taken from 12-day-old seedlings grown in the dark were the most productive, with up to 25% of hypocotyls producing transformed shoots. Explants taken from 5-week-old seedlings produced only transformed callus. The medium used for co-cultivation and incubation also had a significant influence on transformation frequency and shoot regeneration. The cultivar Blue Uniform was more responsive than White Uniform. Both bacterial strains and plasmids were equally effective in producing transformed tissue. Transformed shoots were selected on kanamycin medium, and the presence of the uidA and nptII genes in those selected shoots was confirmed by -glucuronidase and ELISA analyses, respectively.Abbreviations BAP 6-Benzylaminopurine - NAA -Naphthalene acetic acid - TDZ Thidiazuron - BU Blue Uniform - WU White Uniform