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

Abstract

Background: The 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.

Results: Here 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.

Conclusions: The 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.

Figure 1

Introgression of the Rg1 allele into the Micro-Tom (MT) background. The cultivar MsK harbors Rg1 linked to the r (yellow flesh) allele in chromosome 3, which confers yellow color to fruits and was used as a morphological marker. MT was used as pollen receptor in all crosses. In F₂, recombinants with MT small size and yellow fruits (rr) phenotype were selected and allowed to self-pollinate for six generations (F₆) rendering to the true-type Micro-MsK [17], which was used for further backcrosses (BC) with MT. After every two backcrosses (BC), plants were allowed to self-pollinate rendering to BC₂F₂, BC₄F₂ and BC₆F₂, which were used to select Rg1 based on the yellow fruit trait. Seeds from rr BC₆F₂ plants (BC₆F₃ seeds) were germinated in vitro in order to confirm their high shoot formation phenotype from root or cotyledon explants, indicating the presence of Rg1. After six generations of self-pollination (BC₆F₆) the true-type genotype harboring the morphological marker r and the high regeneration allele Rg1 was named MT-Rg1

Figure 2

Shoot regeneration frequency from cotyledon explants in Micro-Tom (MT) and MT-Rg1. A. MT and MT-Rg1 explants (8 days old) were tested in MS medium supplemented with 5 μM 6-benzylaminopurine (BAP) or 5 μM Zeatin. B. MT and MT-Rg1 explants with different ages (days after sowing) were tested in MS medium supplemented with 5 μM BAP. C. MT explants (8 days old) were tested in MS medium supplemented with 5 μM BAP or 5 μM Zeatin with or without 2 days pre-incubation in MS medium supplemented with 0.4 μM 1-naphthaleneacetic acid (NAA). In all treatments, the regeneration frequency (percentage of explants with well formed adventitious shoots) was observed 21 days after explants inoculation. Vertical bars indicate ± standard deviation of the mean (n = 6 Petri dishes with 20 explants each). Different letters indicate significant differences at P ≤ 0.05 (Student's t-test).

Figure 3

Transient expression assays. Cotyledon explants from MT (A, B) and MT-Rg1 (C, D) were inoculated with Agrobacterium tumefaciens EHA105 harboring the gusA reporter gene (see Methods). For the transfection frequency (E) calculation, only explants showing coverage of more than 50% of the surface with GUS staining (A, C) were considered positive. Vertical bars indicate ± standard deviation of the mean (n = 30 explants). The absence of statistic significance (P ≤ 0.05, Student's t-test) is represented by n.s.

Figure 4

Schematic representation of the protocol for Agrobacterium-mediated transformation of MT. The horizontal line represents the timeline for each step. Vertical arrows represent successive steps carried out simultaneously

Figure 5

Phenotype of MT and MT-Rg1 and selection of transgenic lines. A. Phenotype of adult MT and MT-Rg1 plants. The presence of the r and Rg1 alleles resulted in yellow fruits and branched shoot, respectively, in the MT-Rg1 genotype (right). B. In vitro regeneration in absence (top) and presence (bottom) of 100 mg/L kanamycin. The two plates at the bottom of the figure contain explants pre-incubated with Agrobacterium harboring the nptII-containing vector pROKII. C. Analyses of nptII gene in acclimated T₀ transgenic plants by agarose gel electrophoresis of PCR amplification of a 700 bp fragment of nptII gene and DNA molecular marker (100 bp). D. Selection of segregating T₁ lines performed in the greenhouse by spraying 400 mg/L kanamycin in 14-day-old seedlings for 5 consecutive days. E. Analysis of nptII expression by qRT-PCR of T2 single copy homozygous plants transformed with the pROKII vector containing the nptII and AtCKX2 genes. F. GUS staining of leaf disks excised from acclimated T₀ plants transformed with the pGPTV-GUS-KAN containing the gusA gene under control of the wound inducible JERE element (AGACCGCC). Note the enhanced staining of the disk borders, which correspond to wounded areas. Bars = 4 cm (A, B and D) and 1 mm (F). Non-transformed MT plants were used as controls (E, F).