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

百合花药培养脱毒试管苗生产及其机理研究

基本信息

批准号：
14560020
负责人：
NIIMI Yoshiji
金额：
$ 2.18万
依托单位：
NIIGATA UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2004
项目状态：
已结题

项目摘要

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.

本研究旨在探讨百合花药培养脱毒植株再生的机理，建立一套高效的百合花药培养脱毒植株再生体系。主要结果如下。（1）花药培养的效果取决于基因型、外植体的发育阶段和培养基。百合×‘Enchantment’能高频率诱导花药愈伤组织的形成，而‘Casa Blanca’和‘Connecticut King’则不能。“Enchantment”L.x花药培养产生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，另一种是由花药的花丝和维管束周围组织的残余物形成的白色愈伤组织。（2）利用RT-PCR技术对3个百合品种中的百合无斑病毒（LSV）、黄瓜花叶病毒（CMV）和百合斑驳病毒（LMoV）进行了检测。结果表明，RT-PCR是一种比酶联免疫吸附试验（ELISA）更敏感的方法来确定不同品种百合植物中病毒的存在。（3）用改良的酶免疫组织化学方法对LSV和CMV在‘Enchantment’L.x花药愈伤组织和再生植株中的定位进行了研究。病毒主要分布在花药维管组织中，很少分布在花药壁组织中。病毒主要存在于花药及其再生体的花丝和维管束周围组织诱导的白色愈伤组织中，而花药壁组织诱导的黄色愈伤组织和许多再生植株中均无病毒。（4）随着花药愈伤组织培养时间从90天延长到150天，再生植株带毒率下降。培养150天的黄色愈伤组织获得的脱毒苗数最多。