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

本研究旨在探讨百合花药培养脱毒植株再生的机理，为百合脱毒植株的生产提供更有效的培养体系。主要研究结果如下：(1)花药培养反应受基因型、外植体发育阶段和培养基数的影响。‘Enchantment’百合花药愈伤组织诱导频率较高，而‘Casa Blanca’和‘Connecticut King’百合花药诱导愈伤组织频率较低。百合‘Enchantment’的花药培养产生了两种类型的愈伤组织：由花药壁组织形成的淡黄色愈伤组织和由花药维管束周围的花丝和维管束周围的组织形成的白色愈伤组织。(2)对百合无症状病毒(LSV)、黄瓜花叶病毒(CMV)和百合斑驳病毒(LMoV)进行了RT-PCR检测。此外，RT-PCR是一种比酶联免疫吸附试验(ELISA)更灵敏的检测百合植株中病毒存在的方法。(3)用改良的酶免疫组织化学方法研究了LSV和CMV在L.x‘Enchantment’花药愈伤组织和再生植株中的定位。病毒一般在花药维管组织中检测到，但在花药壁组织中很少检测到。由花药维管束及其再生植株周围的花丝和维管束周围组织诱导的白色愈伤组织中最常见的是病毒，而由花药壁组织诱导的淡黄色愈伤组织和由这些愈伤组织再生的许多植株都是无病毒的。(4)随着花药愈伤组织培养时间从90天延长到150天，带病毒植株的再生率下降。培养150天的淡黄色愈伤组织脱毒苗数最多。

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.