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，建立高效脱毒植株的培养体系。主要结果如下：(1)花药培养反应与外植体的基因型、发育阶段和培养基有关。Lilium x ‘ Enchantment’花药诱导愈伤组织的频率较高，而Lilium x ‘ Casa Blanca’和Lilium x ‘ Connecticut King’花药诱导愈伤组织的频率较低。在L.x ‘ Enchantment’花药培养中产生了两种类型的愈伤组织：一种是由花药壁组织形成的淡黄色愈伤组织，另一种是由花药维管束周围的花丝残余和组织形成的白色愈伤组织。(2)利用逆转录聚合酶链反应（RT-PCR）成功检测了3个百合品种的百合无症状病毒（LSV）、黄瓜花叶病毒（CMV）和百合斑驳病毒（LMoV）。结果表明，RT-PCR比酶联免疫吸附试验（ELISA）更灵敏地检测不同种类百合植物中病毒的存在。(3)采用改良的酶免疫组化方法研究了L.x ‘ Enchantment’花药愈伤组织和再生植株中LSV和CMV的定位。病毒一般在花药维管组织中检测到，但很少在花药壁组织中检测到。病毒多见于花药维管束周围组织和花药花丝残体诱导的白色愈伤组织及其再生体中，而大部分由花药壁组织诱导的淡黄色愈伤组织和这些愈伤组织再生的植株均无病毒。(4)随着花药愈伤组织培养时间从90天延长至150天，带病毒植株再生率下降。培养150天的淡黄色愈伤组织脱毒苗数量最多。

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.