We analyzed the impact of ethylene and auxin disturbances on callus, shoots and Agrobacterium rhizogenes-induced hairy root formation in tomato (Solanum lycopersicum L.). The auxin low-sensitivity dgt mutation showed little hairy root initiation, whereas the ethylene low-sensitivity Nr mutation did not differ from the control Micro-Tom cultivar. Micro-Tom and dgt hairy roots containing auxin sensitivity/biosynthesis rol and aux genes formed prominent callus onto media supplemented with cytokinin. Under the same conditions, Nr hairy roots did not form callus. Double mutants combining Rg1, a mutation conferring elevated shoot formation capacity, with either dgt or Nr produced explants that formed shoots with little callus proliferation. The presence of rol + aux genes in Rg1 hairy roots prevented shoot formation. Taken together, the results suggest that although ethylene does not affect hairy root induction, as auxin does, it may be necessary for auxin-induced callus formation in tomato. Moreover, excess auxin prevents shoot formation in Rg1.

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 media containing 1-naphthaleneacetic acid (NAA) before cytokinin treatment. Both strategies allowed the use of 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.

The superior regeneration capacity of Lycopersicon peruvianum was introduced into the cultivated tomato Lycopersicon esculentum by backcrossing hybrid material with the tomato genotype VF11. In segregating material derived from these backcrosses, the ability to regenerate shoots on root explants cultured on a zeatin-containing medium, was highly correlated with the ability to regenerate shoots on established callus cultures. The efficient shoot-regenerating root explant system permitted us to study the genetics of this trait and to locate the genes involved, using a set of morphological markers defining all 12 tomato chromosomes. Depending on the tomato genotype, mono, -di- or trigenic ratios were observed. It is concluded that a dominant L. peruvianum allele at a locus (Rg-1) near the middle of chromosome 3 determines efficient shoot regeneration on root explants in tomato in combination with dominant alleles at one or two other loci of either L. peruvianum or L. esculentum origin. The map location of the Rg-1 locus was refined further using a number of chromosome-3-specific RFLPs. The addition of new classical and RFLP linkage data to existing literature data and subsequent processing resulted in a revised and integrated map of tomato chromosome 3. From a morphological and physiological analysis of genotypes differing in Rg phenotype, it is concluded that the genetic component associated with regeneration determines the maintenance of morphogenetic competence and not the sensitivity to hormones.

The in vitro regeneration ability of tomato (Lycopersicon esculentum Mill.) remains the main limiting factor for efficient genetic transformation. However, competence for in vitro regeneration can be transferred from wild Lycopersicon species to the cultivated tomato. The high regeneration capacity presented by the MsK genotype, derived from L. peruvianum, was attributed to the dominant loci Rg-1 and Rg-2. In the present work, we have transferred the high organogenetic competence from the MsK genotype to the cultivar Micro-Tom, which presents miniature size (8-cm tall) and a rapid life cycle (75 days from seed to fruit ripening).We obtained stable and non-segregating F6 ‘Micro-MsK’ plants, which were expressively superior to Micro-Tom in regeneration capacity, when hypocotyls but not cotyledons were used as explants. In addition to the regeneration ability, the obtained genotype presented a delayed leaf senescence and a reduced apical dominance. The development of ‘Micro-MsK’ genotype will enable the application of insertional mutagenesis in functional genomics of tomato.

Cytokinin (CK) metabolism was analyzed in tomato (Lycopersicon esculentum) Rg-1 hybrids during in vitro shoot organogenesis from root explants. Data were obtained by combining physicochemical analysis with quantification and in situ detection methods. Although exogenous zeatin is added in all classical regeneration protocols, we show here that regenerating (Rg+) tomato explants did not require an exogenous CK source for regeneration. Irrespective of the presence or absence of exogenous zeatin, the endogenous CK levels were not affected by Rg-1 in the initial explants or in the early callus phase. In a later stage, and related to the presence of numerous shoots, the Rg+ explants showed much lower endogenous CK concentrations than the nonregenerating (rg&#8722) explants. Cells of rg&#8722 explants were not able to differentiate, despite their high endogenous CK content, and did not respond to exogenously applied CKs. We show that the insensitivity of rg&#8722 explants to a hormonal signal, normally initiating regeneration, is not related to an altered endogenous CK metabolism. We therefore postulate that Rg-1 action involves a regeneration-specific CK receptor or a regeneration-specific CK signal transduction pathway.