Studies on Production of Virus-free Plantlets by Anther Culture of Lily and Its Mechanism

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The present research project was undertaken to investigate the mechanism of lily virus-free plant regeneration through anther culture and to establish a more efficient culture system for the production of virus-free plants. The main results are as follows.(1)Anther culture response depended on genotypes, developmental stage of explants and culture media. Callus formation from anthers could be induced at high frequency in Lilium x ‘Enchantment' but not in the others, L.x ‘Casa Blanca' and L.x ‘Connecticut King'. Two types of calli were produced in anther culture of L.x ‘Enchantment' : a yellowish callus formed from anther wall tissues, and a white one formed from remnants of filaments and tissues around vascular bundle of anther.(2)Detection of viruses by reverse transcription-polymerase chain reaction (RT-PCR) was carried out successfully for lily symptomless virus(LSV), cucumber mosaic virus(CMV) and lily mottle virus(LMoV) in three lily cultivars. Furthermore, the results showed that the RT-PCR is a more sensitive method than an enzyme-linked immunosorbent assay(ELISA) to determine the presence of viruses in Lilium plants of various species.(3)Localization of LSV and CMV in anther-derived calli and regenerated plantlets of L.x ‘Enchantment' were investigated using a modified enzyme immunohistchemical method. The viruses were detected generally in the vascular tissues of anthers but rarely in the anther wall tissues. The viruses were frequently discovered in the white calli induced from remnants of filaments and tissues around vascular bundle of anther and their regenerants, whereas most of the yellowish calli induced from anther wall tissues and many plants regenerated from these calli showed a virus-free.(4)The rate of regenerated plantles with viruses decreased as the culture duration of anther-derived calli was extended from 90 to 150 days. The greatest number of virus-free plantlets was obtained from the yellowish calli cultured for 150 days.