The Rg1 allele as a valuable tool for genetic transformation of the tomato 'Micro-Tom' model system

Abstract

BackgroundThe cultivar Micro-Tom (MT) is regarded as a model system for tomato genetics due to its short life cycle and miniature size. However, efforts to improve tomato genetic transformation have led to protocols dependent on the costly hormone zeatin, combined with an excessive number of steps.ResultsHere we report the development of a MT near-isogenic genotype harboring the allele Rg1 (MT-Rg1), which greatly improves tomato in vitro regeneration. Regeneration was further improved in MT by including a two-day incubation of cotyledonary explants onto medium containing 0.4 μM 1-naphthaleneacetic acid (NAA) before cytokinin treatment. Both strategies allowed the use of 5 μM 6-benzylaminopurine (BAP), a cytokinin 100 times less expensive than zeatin. The use of MT-Rg1 and NAA pre-incubation, followed by BAP regeneration, resulted in high transformation frequencies (near 40%), in a shorter protocol with fewer steps, spanning approximately 40 days from Agrobacterium infection to transgenic plant acclimatization.ConclusionsThe genetic resource and the protocol presented here represent invaluable tools for routine gene expression manipulation and high throughput functional genomics by insertional mutagenesis in tomato.

Genetic Transformation in Tomato: Novel Tools to Improve Fruit Quality and Pharmaceutical Production

“…Several studies investigated the production of improved protocols for Agrobacterium-mediated MicroTom transformation obtaining a transformation efficiencies ranging from 20 to 56% (Dan et al, 2006;Qiu et al, 2007;Sun et al, 2006). Recently, Pino et al (2010) developed an efficient and inexpensive method for MicroTom transformation using a new tomato genotype harbouring the allele Rg1 that greatly improves tomato in vitro regeneration. Another breakthrough in the field of tomato genetic transformation was the development of a system for stable genetic transformation of tomato plastids (Ruf et al, 2001).…”

“…Since the 1980s several Agrobacterium-mediated transformation protocols have been developed in tomato, using cotyledons or leaves (Pino et al, 2010;Van Eck et al, 2006). Transformation efficiencies obtained in various cultivars range from 10 to 41%.…”

Manipulation of a Senescence-Associated Gene Improves Fleshy Fruit Yield

“…Once cleaved, the mature AtMIR164a has the exact sequence of the mature SlmiR164 (accession numbers for miRbase [http://www.mirbase.org/]: AtMIR164, MI0000197; SlmiR164, MI0027570). The genetic transformation was made in the Slglk2 MT background according to Pino et al (2010). T2 plants were grown as described above, and nonsenescent, early-senescing, and late-senescing leaves were collected.…”

Phytochrome and phytohormone interplay in tomato: impacts on fruit physiology and quality traits

“…Subsequently, each fragment was recombined into the plant transformation vector pK8GWIWG (Fernandez et al, 2009). Transgenic MicroTom plants were generated by Agrobacterium-mediated transformation according to Pino et al (2010), with minor changes: cotyledons from 5-day-old seedlings were used for the transformation; the zeatin and kanamycin concentration were 5 µM and 70 mg L -1 , respectively.…”

“…Genomic, epigenomic, transcriptomic, proteomic and metabolomic datasets as well as efficient stable transformation protocols and large germplasm collections, including many well-characterized mutants, are currently available for this species (Pino et al, 2010;The Tomato Genome Consortium, 2012;Lin et al, 2014).…”