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

本研究以机械分离法获得的乌柏花B+烟草杂种细胞系为材料，研究了其遗传不稳定性和生物碱生物合成的规律。1）乌柏花+烟草杂种细胞系愈伤组织培养3年后，从细胞核DNA含量、染色体组成和过氧化物酶同工酶等方面研究了其遗传组成。各亚克隆细胞核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中的平均DNA含量。DNA含量的减少部分归因于所有亚克隆中发生的烟草染色体的消除。烟草染色体消除的程度因无性系而异，说明染色体消除的过程是缓慢的。过氧化物酶同工酶分析表明，烟草特异性同工酶的损失，从而证实了染色体分析所获得的结果。2）以杜柏和烟草的体细胞杂种为材料，研究了其生物碱的生物合成。细胞学观察和核糖体DNA分析证实了克隆细胞和再生芽的杂种性质。杂种植株主要产生烟碱，而Duboisia植株产生托品烷和烟碱生物碱。活动参与托烷生物碱的生物合成进行了检查，在一系列的摄食实验。在负责减少托品酮，天仙子胺的羟基化和环氧化，以及尼古丁转化为非尼古丁的活动的杂交植物中的存在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.