HEALTHY IN VITRO PROPAGATION BY MERISTEM TIP CULTURE OF HELLEBORUS NIGER¿S SELECTED CLONE FOR CUT FLOWER

Abstract

Helleborus niger (Christmas Rose), a perennial plant member of the Ranunculaceae family, is one of the most popular Christmas cut flowers cultivated in the South of France and exported at 95% to Germany, the Netherlands, Switzerland and Belgium. The intense production of this selected clone, which flowers just before Christmas, is limited by the low vegetative multiplication rate and by some specific diseases, specially Soft rot (Erwinia atroseptica) and viruses like Cucumber Mosaic Virus (CMV) (Cardin et al., 2003). The aim of our study, started ten years ago, was to develop a specific in vitro micro-propagation methodology using the sprouting-rooting sequence, in order to have a faster and healthy multiplication of a selected clone of Helleborus niger. Four different media were used for each sequence (introduction, proliferation, rooting). Due to the high contamination level of the mother plant, particularly by bacteria and virus, we have chosen the meristem tip culture. Depending on the meristem size, 51.7% to 60% of the meristems have grown and developed vegetative buds. Propagation was carried out on the MS media supplemented with 8.9 μM 6-Benzylaminopurine (BAP). A constant multiplication rate, between 2 to 3.4 every cycle, was observed during more than 24 months. Individual buds were prepared for the rooting sequence and we obtained a high rooting rate, (more than 96%), using two rooting phases, the first one with auxinic pulse at 1.4 μM of Indol Acetic Acid (IAA) for 15 days and the second one with 5.6 μM of Indol Acetic Acid (IAA) for four weeks. The healthy state of all meristems was controlled for CMV by DAS-ELISA test. We observed that, in our experimental conditions, the quality of the health regeneration depends directly on the meristem size: meristems between 0.2 mm to 0.4 mm are healthier (73.3%) than meristems of size higher than 0.5 mm (27.7%). Successive subcultures led us to produce 20,000 plants and more than 90% of the plants survived in greenhouse production. The first flowers we observed proved to be true to the original type.