Brian Christensen

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 3xa0days in Ri-line 306 as compared to control plants. A compact plant without delayed flowering can be assumed to be valuable for further breeding.

In vitro culture of Hibiscus rosa-sinensis L.: Influence of iron, calcium and BAP on establishment and multiplication

Some factors influencing in vitro cultures of potted Hibiscus rosa-sinensis L. using nodal cuttings were investigated. A protocol using a modified MS medium helped to overcome chlorosis, shoot tip necrosis (STN) and leaf drop. These disorders have been caused by mineral imbalance associated with calcium and iron deficiency. STN and leaf drop were overcome by increasing calcium level from 3xa0mM (MS standard concentration) to 9xa0mM, and this increase, in addition, enhanced shoot dry weight and shoot extension. The chlorophyll content and leaf area increased by increasing the iron concentration 3-fold from 98xa0μM to 295xa0μM. Furthermore, substituting Fe-EDTA with Fe-EDDHA resulted in an increase in chlorophyll content, leaf area and shoot extension. The most suitable multiplication medium for H. rosa-sinensis L. was demonstrated to be a modified MS medium containing 2.2xa0μM BAP and increased concentrations of calcium at 9xa0mM and iron at 295xa0μM provided as Fe-EDDHA. The shoots were rooted in half-strength modified MS medium containing 2.7xa0μM NAA. Acclimatization was successful with all shoots with or without roots.

Effects of ethylene and 1-MCP (1-methylcyclopropene) on bud and flower drop in mini Phalaenopsis cultivars

Phalaenopsis frequently exhibits bud drop during production and in response to adverse postharvest conditions. The effect of exogenous ethylene on bud drop of mini Phalaenopsis was studied and ethylene sensitivity of four cultivars was compared. Water content, membrane permeability and ABA (abscisic acid) content in floral buds and flowers were determined after ethylene treatment. Exogenous ethylene induced flower bud drop in all tested Phalaenopsis cultivars and the different cultivars showed distinct differences in ethylene sensitivity. The cultivar Sogo ‘Vivien’ exhibited the highest bud drop, water loss and change in membrane permeability in floral petals, while Sogo ‘Berry’ showed the lowest sensitivity. The ethylene inhibitor 1-MCP (1-methylcyclopropene) reduced ethylene-induced floral bud drop in the cultivar Sogo ‘Yenlin’. ABA content in floral buds was increased in response to ethylene and 1-MCP pretreatment inhibited the ethylene-induced increase in ABA levels efficiently. This finding suggests that the observed increase in ABA content during bud drop was mediated by ethylene. The interaction between ABA and ethylene is discussed.

Evaluation of reproductive barriers contributes to the development of novel interspecific hybrids in the Kalanchoë genus

BackgroundInterspecific hybridization is a useful tool in ornamental breeding to increase genetic variability and introduce new valuable traits into existing cultivars. The successful formation of interspecific hybrids is frequently limited by the presence of pre- and post-fertilization barriers. In the present study, we investigated the nature of hybridization barriers occurring in crosses between Kalanchoë species and evaluated possibilities of obtaining interspecific hybrids.ResultsThe qualitative and quantitative analyses of pollen tube growth in situ were performed following intra- and interspecific pollinations. They revealed occurrence of pre-fertilization barriers associated with inhibition of pollen germination on the stigma and abnormal growth of pollen tubes. Unilateral incongruity related to differences in pistil length was also observed. The pollen quality was identified as a strong factor influencing the number of pollen tubes germinating in the stigma. In relation to post-fertilization barriers, endosperm degeneration was a probable barrier hampering production of interspecific hybrids. Moreover, our results demonstrate the relation of genetic distance estimated by AFLP marker analysis of hybridization partners with cross-compatibility of Kalanchoë species. At the same time, differences in ploidy did not influence the success of interspecific crosses.ConclusionsOur study presents the first comprehensive analysis of hybridization barriers occurring within Kalanchoë genus. Reproductive barriers were detected on both, pre- and post-fertilization levels. This new knowledge will contribute to further understanding of reproductive isolation of Kalanchoë species and facilitate breeding of new cultivars. For the first time, interspecific hybrids between K. nyikae as maternal plant and K. blossfeldiana as well as K. blossfeldiana and K. marnieriana were generated.

Inheritance of rol -genes from Agrobacterium rhizogenes through two generations in Kalanchoë

Transformation of Kalanchoë blossfeldiana ‘Molly’ using the naturally occurring bacterium Agrobacterium rhizogenes is a non-GMO strategy to breed compact plants. In the present study, crosses resembling a commercial breeding strategy were made to determine if the improved ornamental quality observed in the T1 generation was inherited to the offspring. F1 lines were produced by crossing the commercial Kalanchoë cultivar ‘Sarah’ with a selected T1 line. Subsequently, F2 populations were produced by self-pollination of individual selected lines. The rol-genes were inherited to the progeny and the presence of rol-genes was confirmed in all F1 and many F2 plant lines exhibiting dwarfism. Screening of F1 and F2 plants showed that the rol-genes were inherited together. Besides decreased plant height, several F1 and F2 lines containing rol-genes exhibited changes in plant diameter, number of branches, flower diameter and time to first open flower and duration of flowering compared to the WT ‘Molly’. Furthermore, increased ethylene tolerance was observed in several lines containing rol-genes compared to the WT ‘Sarah’. Screening of three selected F2 populations derived from self-pollination of F1 lines containing rol-genes indicated a shift in distribution towards a lower mean plant height within the entire plant population compared to a control population of plants without rol-genes. Moreover, one of these F2 populations also exhibited earlier flowering compared to the control population. Compact potted plants and lines without delayed flowering and with improved ethylene tolerance were obtained and are valuable in commercial breeding programmes without using recombinant DNA technology.

Transformation of Hibiscus rosa-sinensis L. by Agrobacterium rhizogenes

Summary Transgenic hairy roots were induced from leaf, petiole, and shoot explants of in vitro-raised plantlets of potted Hibiscus rosa-sinensis L. ‘Cassiopeia Wind Yellow’ plants after co-cultivation with three different wild-type strains of Agrobacterium rhizogenes. The highest infection rate (56.4%) was obtained in explants inoculated with A. rhizogenes strain ATCC43057, followed by ATCC15834 (44.0%), and NCPPB8196 (34.4%). The highest infection rate (60.5%) was obtained using leaf basal segments, followed by petioles (44.0%), leaf tips (40.0%), and shoots (20%). A total of 102 putative transgenic root lines were produced. The hairy root lines did not grow without applying 0.2 mg l−1 indole-3-butryric acid (IBA) and 0.2 mg l−1 α-naphthaleneacetic acid (NAA). The presence of T-DNA was confirmed in six hairy root lines, and no A. rhizogenes contamination was detected by polymerase chain reaction.

Quantitative and qualitative characterization of novel features of Kalanchoë interspecific hybrids

Kalanchoë blossfeldiana and its cultivars are important worldwide as potted plants. Their improvement relies mainly on cross-breeding and interspecific hybridization, although the use of the resource of wild species was limited. The present study aimed to characterize novel features of interspecific hybrids that can increase attractiveness of these commercially important ornamental plants. We investigated the new traits of selected hybrids resulting from hybridization between K. blossfeldiana and three wild Kalanchoë species, namely K. nyikae, K. pubescens and K. marnieriana. The selected qualitative and quantitative traits of plant growth and flowering were analyzed in hybrids and parental species. The analysis demonstrated generally intermediate character of hybrids. Following principal component analysis, clear separation among plants forming three distinct clusters was observed, where hybrids were situated between the two parental species. The plants were clearly grouped according to flower characteristics, plant height and timing of flowering in all investigated groups. Quantitative analysis also revealed significant differences among parental plants and hybrids for all examined characteristics. Transgressive segregation and heterosis were observed in relation to specific traits such as days until first open flower, plant height, number of inflorescences, flower diameter and number of flowers. Pollen analysis of the interspecific hybrids revealed low viability of pollen or complete sterility. The interspecific hybrids exhibited novel features valuable for breeders. Thus, wide hybridization can contribute to the development of new cultivars and this study clearly demonstrates the usefulness of interspecific crosses in the improvement of Kalanchoë cultivars.