Genetic Diversity and Alkaloid Biosynthesis in Somatic Hybrids of Duboisia and Nicotiana Tabacum

本文研究了机械分离获得的Duboisia B+Nicotiana tabacum杂交细胞系的遗传不稳定性和生物碱合成。1)以紫花苜蓿+烟草为杂种细胞，培养10个愈伤组织亚无性系3年，从细胞核DNA含量、染色体构成、过氧化物酶同工酶等方面对其基因组成进行了分析。每个亚克隆的核DNA含量不同。在大多数亚克隆中，平均DNA含量低于融合后1年测定的原始杂交细胞系的平均DNA含量。DNA含量的减少部分归因于所有亚克隆中烟草染色体的消除。烟草染色体的消除程度因亚克隆而异，表明染色体的消除发生缓慢。过氧化物酶同工酶分析表明缺失了一种烟草特异性同工酶，从而证实了染色体分析的结果。两个杂交亚克隆2年后再生的芽在形态和细胞核DNA含量上也存在异质性。2)利用莱氏杜布氏菌和烟草的体细胞杂交种进行生物碱合成研究。细胞学研究和核糖体- dna分析分别证实了克隆细胞和再生芽的杂交性质。杂交植株主要产生尼古丁，而Duboisia植株同时产生tropane和尼古丁生物碱。通过一系列前体饲养实验，研究了与tropane-生物碱生物合成有关的活性。在杂交植物中存在负责还原托品酮、羟化和环氧化山莨菪碱以及将尼古丁转化为非尼古丁的活性，证明了这些酶活性的Duboisia基因的存在。

The genetic instability and alkaloid biosynthesis of hybrid cell lines, Duboisia B+Nicotiana tabacum, obtained by mechanical isolation of a single hybrid cell was studied.1) Ten subclones of calli derived from a single hybrid cell, Duboisia hopwoodii+Nicotiana tabacum were cultured for 3 years, and their genetic makeup clarified as to nuclear DNA content, chromosome constitution, and peroxidase isozymes. Nuclear DNA content differed in each subclone. In most subclones, mean DNA content was lower than the mean DNA content in the original hybrid cell line determined 1 year after fusion. This decrease in DNA content is partly attributable to the elimination of tobacco chromosomes that occurred in all subclones. The extent to witch tobacco chromosomes were eliminated varied among the subclones evidence that chromosome elimination occurred slowly. Peroxidase isozyme analysis indicated the loss of a tobacco-specific isozyme, thus confirming results obtained by chromosome analysis. Shoots regenerated from two hybird subclones after 2 years were also heterogeneous in morphology and nuclear DNA content.2) Somatic hybrids of Duboisia leichhardtii and Nicotiana tabacum were used to study the alkaloid bio-synthesis. The hybrid nature of the cloned cells and regenerated shoots was confirmed by cytological investigation and ribosomal-DNA analysis, respectively. The hybrid plantlets predominantly produced nicotine, while Duboisia plantlets produced both tropane and nicotine alkaloids. Activities involved in tropane-alkaloid biosynthesis were examined in a series of precursor-feeding experiments. The presence in the hybrid plants of activities responsible for the reduction of tropinone, the hydroxylation and epoxidation of hyoscyamine, and the conversion of nicotine to nonnicotine demonstrated the presence of the Duboisia genes for these enzyme activities.