Rapid, efficient production of homozygous transgenic tobacco plants with agrobacterium tumefaciens: A seed-to-seed protocol

Dane K. Fisher, Mark Guiltinan

Research output: Contribution to journalArticle

51 Scopus citations

Abstract

An optimized complete protocol was developed for Agrobacterium tumefaciens-mediated transformation of tobacco (Nicotiana tabacum L. cultivar SR1), producing T1 flowering plants homozygous for the inserted T-DNA as verified by kanamycin resistance in T2 seedlings in 6 to 7 months from the time of cocultivation with Agrobacterium. Previous protocols require up to 9 to 12 months to obtain similar results. Procedures unique and important to this protocol include; a modified "whole-leaf" transformation coupled with a long duration of cocultivation, resulting in high rates of transformation, high levels of kanamycin in selection media resulting in few escapes, and extensive rooting of regenerants prior to a greenhouse hardening procedure. Once in the greenhouse, primary regenerants were maintained in small containers with long day photoperiod and high light levels, greatly shortening the time to seed set. Flowers from primary transformants were bagged to allow self pollination, and seed capsules harvested and dried prior to normal maturation on the plant. T1 and T2 seeds were plated and selected on kanamycin media by an improved seed plating technique which eliminates the need for the placement of individual seeds, saving time and improving selection homogeneity. Using this protocol, over 130 independent tobacco lines from six separate gene constructs have been generated in a very short time period. Of these 130, nearly 60 percent segregated 3:1 for kanamycin resistance: susceptibility, indicating single transgene insertion events.

Original languageEnglish (US)
Pages (from-to)278-289
Number of pages12
JournalPlant Molecular Biology Reporter
Volume13
Issue number3
DOIs
StatePublished - Sep 1 1995

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Plant Science

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