REGENERATION OF CALLUS AND SHOOTS FROM THE OVULES AND OVARIES OF CHRYSANTHEMUM IN VITRO

Abstract

Unfertilized chrysanthemum ovaries and ovules were investigated in terms of callus and shoots regeneration in vitro on media with a different plant growth regulators content. Ligulate florets of cultivar ‘Capitola’ were collected from five peripheral whorls of mature inflorescences and sterilized. The ovules were isolated from sterile florets under spectroscopic microscope, while the ovaries were precisely cut off from the florets. The explants were inoculated onto six MS based induction media, solidified with 0.8% agar, supplemented with different concentrations of BAP and 2,4-D (1+1;1+2;1+3; 2+1; 2+2; 2+3 mg/L of BAP + 2,4-D, respectively). For 5 weeks cultures were kept under 24°C, light was supplied by fluorescent tubes emitting daylight, photon flux density was 12 µmol/(m2s), 16/8 h of day/night photoperiod. Afterwards, one half of the explants were transferred onto regeneration medium supplemented with 2 mg/L kinetin, 1 mg/L IAA and 4 mg/L glycine. Starting from the fifth week of culture up to the fifteenth week, both, transferred and non-transferred explants were maintained at 32 µmol/(m2s) photon flux density. 90% of ovaries and 38% ovules formed callus. The best medium for callus formation was supplemented with 1 mg/L 2,4-D and 2 mg/L BAP. The shoots regenerated only from ovaries, 10% of ovaries produced shoots; a total number of 103 shoots were formed. The transfer of ovaries onto the regeneration medium containing kinetin and IAA promoted shoots formation; most shoots regenerated from the ovaries subcultured from the medium containing 1 mg/L 2,4-D and 1 mg/L BAP. Mean numbers of shoots per inoculated explant from non-transferred and transferred ovaries were 0.10 and 0.25, respectively.