Efficacy of New Inhibitors of Ethylene Perception in Improvement of Display Quality of Miniature Potted Roses (<Emphasis Type="Italic">Rosa hybrida</Emphasis> L.)

1-Octylcyclopropene (1-OCP) and 1-Decylcyclopropene (1-DCP), ethylene receptor inhibitors, analogues to 1-MCP, substituted with longer carbon chain in the 1-position were investigated in miniature potted roses cultivar ’Lavender‘. All levels of both chemicals protected as compared to untreated plants. 1-OCP and 1-DCP were the most effective at concentrations 1000 and 1500 nl l⁻¹, which was five times higher than the concentration of 1-methylcyclopropene (1-MCP) (200 nl l⁻¹) used as a standard. The effectiveness of 1-OCP and 1-DCP was a function of time and temperature. At short (2 h) exposure times, the plants were highly sensitive to ethylene. Exposure time of 4 h for both 1-OCP and 1-DCP was sufficient to improve display life of miniature roses and longer exposures did not have any additional beneficial effect. Apparently, exposing miniature potted roses to various temperatures did not have an influence on the performance of both 1-OCP and 1-DCP while low temperature at 5 °C reduced their performance. The reasons for differences in the effects of these compounds are discussed.     

Regeneration and transformation in adult plants of Campanula species

Adult plants are known for recalcitrance when it comes to adventitious organ formation and regeneration. Methods used for regeneration in explants from seedlings of Campanula carpatica failed to work for explants from adult plants of the same species. The present investigation generated efficient regeneration methods for mature specimens of four species of Campanula, C. carpatica, C. haylodgensis, C. portenschlagiana and C. poscharskyana. Petiole explants from dark-grown in vitro shoot cultures grown from nodal cuttings of adult plants regenerated successfully (95%), while explants from light-grown in vitro shoot cultures and greenhouse-grown plants regenerated at 12% and zero percentage, respectively. Dark-treatment, along with media manipulation with plant growth regulators, further enhanced regenerative capacity of the explants. A MS-based medium containing 10mg l ⁻¹ TDZ and 0.25 mg l⁻¹ NAA was the most efficient regeneration medium. Transgenic shoots from C. carpatica (3%) and C. haylodgensis (1%) and transgenic callus from all species were produced using Agrobacterium tumefaciens, and transformation was confirmed by histochemical and Southern blot analyses. Protocols developed in this study may be useful for achieving efficient regeneration and transformation of recalcitrant adult plants.     

Cloning and Characterization of Three APETALA1/FRUITFULL-like Genes in Different Flower Types of Rosa?��?hybrida L.

To clarify the molecular mechanism of flower development in Rosa × hybrida L., three different APETALA1/FRUITFULL (AP1/FUL)-like MADS-box genes were isolated and their expression analyzed in normally developed flowers and in malformed flowers of a stable phenotype. AP1/FUL-like genes were designated as RhAP1-1, RhFUL, and RhAP1-2. Alignment of amino acid sequences showed 83% identity between RhAP1-1 and TrAP1 of Taihangia rupestris and 82% identity between RhFUL and TrFUL of T. rupestris. RhAP1-1 is 97% identical to RhAP1-2 and 58% identical to RhFUL. Expression of RhAP1-1 and RhAP1-2 in whorls 1 and 2 of rose flowers exclusively is in accordance with the expression pattern of class A genes in other plant species. In contrast, RhFUL showed a unique expression pattern and was expressed only in sepals. The roles of all putative A, B, and C class genes were examined in different flower organs of normally developed flowers and in malformed flowers that are similar to a classic C function mutant from Arabidopsis (with petals in whorl 3 and sepals in whorl 4). The expression pattern of the putative class B genes was similar in both normal and malformed flowers. However, the putative class A genes were upregulated and class C genes were downregulated in all flower organs of the mutant. These data suggest that suppression of the class C genes RhC1 and RhC2 leads to altered expression of RhAP1-1, RhFUL, and RhAP1-2 in whorls 3 and 4 that leads to the mutant flower phenotype.     

Isolation of an Ethylene-induced Putative Nucleotide Laccase in Miniature Roses (<Emphasis Type="Italic">Rosa hybrida</Emphasis> L.)

Using differential display we isolated five ethylene-responsive cDNAs from Rosa hybrida L. and identified for the first time an ethylene-induced cDNA homologous to a laccase gene. Three cDNAs were isolated from petioles and two cDNAs from pedicels. Expression levels of all cDNAs in pedicels were higher than in petioles. The laccase homolog cDNA was termed the RhLAC (Rosa hybrida Laccase) gene. The RhLAC gene encodes for a putative protein of 573 amino acids containing three conserved domains characteristic of the multicopper oxidase family. Southern blot hybridization analyses indicated that there are multiple copies of the RhLAC gene in the Rosa species. Comparison of the relative expression of isolated RhLAC in various organs showed that it was highly induced in the leaf abscission zone of petioles and the bud abscission zone of floral bud pedicels, whereas it was low in both leaf blades and petioles. These results suggest that RhLAC may play an important role in senescence and abscission in roses.     

A search for growth related genes in Kalanchoë blossfeldiana

Differential display of mRNA from four sets of contrasting phenotypes were carried out in order to identify and isolate genes associated with elongating growth of Kalanchoë blossfeldiana. A total of 17 unique differential expressed cDNA fragments were sequenced and 12 showed homology to genes in other plant species. Three genes were subsequently tested for growth related activity by Virus Induced Gene Silencing (VIGS) in Nicotiana benthamiana. One gene fragment (13C) resulted in plants with significantly reduced growth (N = 20, P = 0.05, one-tailed students t-test) from day 25 after virus infection. Full-length cDNA and genomic DNA sequences were obtained by inverse PCR and thermal asymmetric interlaced (TAIL) PCR and the gene was named KbORF1. The predicted gene is 2244 bp long with three exons of 411 bp in total encoding a protein of 137 amino acid residues with homologs widespread among plants. The protein has no known function, but its expression has been confirmed in a proteomic study of Arabidopsis. Southern blot analysis shows two hybridizing fragments in agreement with the tetraploid nature of K. blossfeldiana. Fragment 13C comprises 446 bp of the gene, and the portion of 13C conferring growth retardation by VIGS is located 10 bp into the second intron indicating a regulatory function of this part of the KbORF1 mRNA. Differential display in combination with VIGS as a screening method proved to be a good functional approach not only to search for genes of interest, but also to isolate expressed genetic regulatory domains.     

Kalanchoe blossfeldiana plants expressing the Arabidopsis etr1-1 allele show reduced ethylene sensitivity

Transgenic Kalanchoe blossfeldiana Poelln. with reduced ethylene sensitivity in flowers was obtained by Agrobacterium tumefaciens-mediated transformation using the plasmid pBEO210 containing the mutant ethylene receptor gene etr1-1 from Arabidopsis thaliana under the control of the flower-specific fbp1-promoter from Petunia. Three ethylene-resistent T0 lines, 300, 324 and 331, were selected and analyzed for postharvest-performance and morphological characteristics. Line 324 was found to be infertile and only slightly less ethylene-sensitive than control-plants, but lines 300 and 331 had significantly increased ethylene-resistance and were fertile. These two lines were analyzed for copy-number of the etr1-1 gene by Southern blotting and were crossed with the ethylene-sensitive cultivar ‘Celine’ to create T1 progeny. Line 300 contains two T-DNA copies per nucleus, one of which is rearranged, and these are unlinked according to segregation data from the crossing to ‘Celine’ and PCR-analysis of progeny plants. For control plants all flowers were closed after 2 days at 2 μl l⁻¹ ethylene, but for line 300 only 33% were closed after 10 days. Line 331 contains three T-DNA copies per nucleus and is more sensitive to ethylene than line 300. In the line 300 the etr1-1 gene was found by RT-PCR to be expressed in petals and stamens but not in carpels and sepals. Both lines 300 and 331, and their progeny, appear morphologically and physiologically identical to control plants except for the higher ethylene resistance. Line 300 and its progeny with only one T-DNA copy have very low ethylene sensitivity and may be useful in future breeding.