Co-expression and inheritance of foreign genes in transformants obtained by direct DNA transformation of tobacco protoplasts |
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| Authors: | H Uchimiya H Hirochika H Hashimoto A Hara T Masuda T Kasumimoto H Harada J -E Ikeda and M Yoshioka |
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| Institution: | (1) Institute of Biological Sciences, University of Tsukuba, 305 Ibaraki, Japan;(2) National Institute of Agrobiological Resources, 305 Ibaraki, Japan;(3) Department of Biology, Nagoya University, Nagoya, Japan;(4) Present address: Sugiyama Chemical & Industrial Laboratory, Totsuka-ku, Yokohama, Japan;(5) Present address: Morioka Branch, National Institute of Fruit Trees, Morioka, Japan;(6) Present address: Plant Research Center, Kirin Brewery Co., Kitsuregawa, Tochigi, Japan |
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| Abstract: | Summary A plasmid containing two marker genes for expression in plants was constructed. This 16 kb vector, pCT1T3, contains an intact
nopaline synthase gene and a chimaeric gene consisting of the promoter and terminator regions from cauliflower mosaic virus
gene VI and a structural gene, aminoglycoside phosphotransferase (APH(3′)II), from the bacterial transposon Tn5. After transformation
of tobacco mesophyll protoplasts with this plasmid, several kanamycin-resistant transformants were obtained. Intensive studies
on the drug tolerance of growth and differentiation of the transformants showed that the chimaeric gene was stably expressed.
Of 17 independent transformants, 3 (about 18%) expressed the two marker genes, regardless of the state of differentiation,
as did individual plants regenerated from the same callus. Multiple copies of the inserted DNA were found in some transformants.
Viable seeds were produced by 12 out of 15 independent transformants. Seeds obtained by self-pollination were germinated on
medium containing kanamycin sulphate. With the exception of one clone, resistant seedlings with green leaves and sensitive
seedlings with white leaves were found to segregate in a 3:1 ratio. This suggests that the inheritance of the inserted gene
is Mendelian. A reciprocal cross between the transformants and wild-type tobacco also showed nuclear transmission of the APH(3′)II
gene. This was consistently maintained in a subclone of the same transformant derived from the same callus line. Stable inheritance
of the single dominant character was also seen among seeds formed in several different flower pods of the same individual
plants.
Two clones were also found to synthesize nopaline in addition to expressing APH(3′)II. Analysis of the progeny obtained by
self-crosses of such transformants revealed the simultaneous expression of these two enzymes, indicating that the two marker
genes are linked on the same chromosome. |
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| Keywords: | Nicotiana tabacum Protoplasts DNA transformation APH(3′ )II Nopaline synthase Inheritance |
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