1990 Fiscal Year Final Research Report Summary
Genetic Diversity and Alkaloid Biosynthesis in Somatic Hybrids of Duboisia and Nicotiana Tabacum
| Project/Area Number |
63470142
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
生物物性学
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| Research Institution | Kyoto University |
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Principal Investigator |
YAMADA Yasuyuki Kyoto Univ., Facul. Agric. Professor, 農学部, 教授 (50026415)
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| Co-Investigator(Kenkyū-buntansha) |
HASHIMOTO Takashi Kyoto Univ., Facul. Agric. Instructor, 農学部, 助手 (80180826)
SATO Fumihiko Kyoto Univ., Facul. Agric. Assoc. Professor, 農学部, 助教授 (10127087)
MORIKAWA Hiromichi Kyoto Univ., Facul. Agric. Assoc. Professor (1988-1989), 農学部, 助教授 (00089129)
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| Project Period (FY) |
1988 – 1990
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| Keywords | Somatic hybrids / Protoplast fusion / Differentiation / Alkaloid biosynthesis / Genetic instability / Duboisia / Nicotiana tabacum / Cultured shoot |
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| Research Abstract |
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.
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