Sanitation and micropropagation of geranium (Pelargonium spp.) cultivars starting from meristem tip

Five plants from each of the ten geranium (Pelargonium spp.) cultivars were kept for one month in a glasshouse under 75% shade. Subsequently plants were taken to the laboratory and 10 apical growing shoots 5 cm long were removed from each plant and sterilised by soaking in 20% commercial bleach solution with a few drops of Tween 20 for 10 min and washed 3 times in distil sterile water. In a sterile air laminar flow cabinet, meristem tips 1 to 2 mm long were isolated and cultured in vitro on a modified MS (Murashige and Skoog, 1962) medium and micro-plants were produced. The medium contained MS basic salts and vitamins, supplemented with 3% sucrose, 4.5 mg L-1 BAP, 0.009 mg L-1 IBA, and 55.7 mg L-1 ascorbic acid and solidified with 2.5 g L-1 phytagel. Cultures were incubated in a growth room for two weeks in the dark and at constant temperature of 21 +/- 2 degrees C followed by transfer of cultures to a growth room with 16 hours light of 2000 lux provided by white and red light fluorescent lambs (50-50%) and 8 hours dark at constant temperature of 21 +/- 2 degrees C. Proliferation was on average 4-fold per 2 weeks and rooted in vitro, in the same medium, micro-plants produce. In vitro plant material was tested with the molecular method ds-RNA and RT-PCR for viruses and viroids and by ELISA for viruses and other pathogens. All infected cultures were discarded. Micro-plants were undergoing two faces of hardening: the first in vitro by increasing the light intensity to 4000 lux for two weeks and the second ex vitro in additional to daylight light of 5000 lux provided by AGRO-T-PLUS and in a micro-fog where relative humidity (RH) was progressively reduced from 95 to 55%. Plantlets were also hardened in growth chambers with 16 hours a day light intensity of 5000 lux/8 hours dark and at constant temperature of 21 +/- 2 degrees C. This method is used for production of pathogen-free and genetically uniform geranium micro-plants to be used as propagation stocks and it can be used if cost permits for the mass micropropagation of new cultivars for rapid release to growers.