Preventing Extinction of a Critically Endangered Dactylorhiza incarnata subsp. ochroleuca in Britain Using Symbiotic Seedlings for Reintroduction.

The yellow early marsh-orchid (Dactylorhiza incarnata subsp. ochroleuca) is critically endangered in the UK. Reintroduction of this threatened orchid to former haunts that have been restored is a long-term objective of this study. Identifying germination-specific mycorrhizal fungus lineages from closely related species is used as a method due to the extremely small number of plants left in the wild. A putative orchid mycorrhizal fungus of the family Tulasnellaceae, isolated from Dactylorhiza praetermissa, supported in vitro seed germination to produce reintroduction-ready seedlings. Reintroduced symbiotic seedlings survived over the winter months in the flooded reintroduction site (RS). The comparative soil analysis for key nutrients before reintroduction showed that phosphorus content in the RS is very low compared to the soil collected from the wild site (WS) where the last viable population exists. On the other hand, C:N ratio in the soil at the WS and RS were not significantly different. To our knowledge, this is the first-ever report on the reintroduction of symbiotic seedlings of a threatened orchid back to the wild in the UK.

Transformation of miniature potted rose (Rosa hybrida cv. Linda) with P( SAG12 )-ipt gene delays leaf senescence and enhances resistance to exogenous ethylene.

KEY MESSAGE : The P ( SAG12 ) -ipt gene was transferred to miniature rose, as the first woody species, resulting in increased ethylene resistance due to specific up-regulation of the ipt gene under senescence promoting conditions. Transgenic plants of Rosa hybrida 'Linda' were obtained via transformation with Agrobacterium tumefaciens strain harboring the binary vector pSG529(+) containing the P( SAG12 )-ipt construct. A. tumefaciens strains AGL1, GV3850 and LBA4404 (containing P(35S)-INTGUS gene) were used for transformation of embryogenic callus, but transgenic shoots were obtained only when AGL1 was applied. The highest transformation frequency was 10 % and it was achieved when half MS medium was used for the dilution of overnight culture of Agrobacterium. Southern blot confirmed integration of 1-6 copies of the nptII gene into the rose genome in the tested lines. Four transgenic lines were obtained which were morphologically true-to-type and indistinguishable from Wt shoots while they were in in vitro cultures. Adventitious root induction was more difficult in transgenic shoots compared to the Wt shoots, however, one of the transgenic lines (line 6) was rooted and subsequently analyzed phenotypically. The ipt expression levels were determined in this line after exposure to exogenous ethylene (3.5 µl l(-1)) and/or darkness. Darkness resulted in twofold up-regulation of ipt expression, whereas darkness combined with ethylene caused eightfold up-regulation in line 6 compared to Wt plants. The transgenic line had significantly higher content of chlorophyll at the end of the treatment period compared to Wt plants.

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.

Transformation of Kalanchoe blossfeldiana with rol-genes is useful in molecular breeding towards compact growth.

Dwarf genotypes of the economically important flowering potted plant Kalanchoe blossfeldiana were developed by molecular breeding. Root inducing (Ri)-lines were regenerated by applying CPPU to the hairy roots, which were produced by inoculating leaf explants with a wild-type Agrobacterium rhizogenes strain ATCC15834. Amplification by polymerase chain reaction (PCR) and Southern blot analysis confirmed the presence of T-DNA in the Ri-lines. Six Ri-lines were characterised in a greenhouse trial revealing that several morphological traits changed with respect to ornamental value such as plant height, number of lateral shoots, leaf size, leaf number, flower size and number of flowers. The Ri-lines differed in their degree of Ri-phenotype, and the internodes of the Ri-lines were clearly shorter, giving a compact growth habit compared to control plants. Time to anthesis was the same in Ri-line 331 as in control plants and delayed by only 3 days in Ri-line 306 as compared to control plants. A compact plant without delayed flowering can be assumed to be valuable for further breeding.

Transgenic Campanula carpatica plants with reduced ethylene sensitivity.

Fertile transgenic Campanula carpatica Jacq. plants with flowers, which had reduced sensitivity to ethylene were obtained by Agrobacterium tumefaciens that mediated transformation. The construct used for transformation contained the etr1-1 gene from Arabidopsis thaliana under control of the flower specific fbp1-promoter from petunia. More than 100 flowering T0 lines were tested for their ethylene sensitivity using 2 microl l(-1) ethylene. The tolerance level to ethylene varied among the lines. While control plants stopped flowering within 3 days of exposure to ethylene, one of the transformed lines flowered for up to 27 days. The presence and the expression pattern of the transgene in various tissues were studied by polymerase chain reaction (PCR) and reverse transcription (RT)-PCR techniques. The expression of etr1-1 was significant in flowers and buds. Transgenic lines did not differ morphologically from control plants. The selected transgenic T0 lines, which were re-established from in vitro cultures showed the same degree of tolerance to exogenous ethylene, confirming the stability of the transgene in in vitro cultures. The rooting ability of the transgenic plants was not affected by the presence of etr1-1. T1 progeny were produced by crossing the transgenic line, which showed the most significant reduction in ethylene sensitivity with a control plant, and the analysis of the T1 plants showed 1:1 segregation in terms of ethylene sensitivity and the presence of the transgene.

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 beta-glucuronidase and ELISA analyses, respectively.