Visual spectinomycin resistance (aadAau) gene for facile identification of transplastomic sectors in tobacco leaves

• Published in: Plant molecular biology Vol. 76; no. 3-5; pp. 453 - 461
• Main Authors: Tungsuchat-Huang, Tarinee, Slivinski, Kristina Marie, Sinagawa-Garcia, Sugey Ramona, Maliga, Pal
• Format: Journal Article
• Language: English
• Published: Dordrecht Springer Netherlands 01-07-2011; Springer Nature B.V
• Subjects: Base Sequence; Biochemistry; Biomedical and Life Sciences; Biotechnology; Genes, Plant; Genomes; Greenhouses; Leaves; Life Sciences; Molecular Sequence Data; Nicotiana - genetics; Nicotiana tabacum; Photosynthesis; Pigmentation; Plant biology; Plant Pathology; Plant resistance; Plant Sciences; Plants; Plastids; Shoots; Spectinomycin - pharmacology; Transgenic plants; Aurea; Plastid transformation; Leaf variegation; Spectinomycin resistance
• ISSN: 0167-4412; 1573-5028
• DOI: 10.1007/s11103-010-9724-2

Identification of a genetically stable
Nicotiana tabacum
(tobacco) plant with a uniform population of transformed plastid genomes (ptDNA) takes two cycles of plant regeneration from chimeric leaves and analysis of multiple shoots by Southern probing in each cycle. Visual detection of transgenic sectors facilitates identification of transformed shoots in the greenhouse, complementing repeated cycles of blind purification in culture. In addition, it provides a tool to monitor the maintenance of transplastomic state. Our current visual marker system requires two genes: the aurea
bar
(
bar
au
) gene that confers a golden leaf phenotype and a spectinomycin resistance (
aadA
) gene that is necessary for the introduction of the
bar
au
gene in the plastid genome. We developed a novel
aadA
gene that fulfills both functions: it is a conventional selectable
aadA
gene in culture, and allows detection of transplastomic sectors in the greenhouse by leaf color. Common causes of pigment deficiency in leaves are mutations in photosynthetic genes, which affect chlorophyll accumulation. We use a different approach to achieve pigment deficiency: post-transcriptional interference with the expression of the
clpP1
plastid gene by aurea
aadA
au
transgene. This interference produces plants with reduced growth and a distinct color, but maintains a wild-type gene set and the capacity for photosynthesis. Importantly, when the aurea gene is removed, green pigmentation and normal growth rate are restored. Because the aurea plants are viable, the new
aadA
au
genes are useful to query rare events in large populations and for
in planta
manipulation of the plastid genome.