Explants cultured on medium with 0.1 and 1 mg/l of NAA combined with extreme cytokinin doses (1 or 10 mg/l) produced friable callus and/or roots. However, both auxin concentrations combined with 3 or 5 mg/l of BAP or KIN induced buds. High auxin dose (1 mg/l) increased bud production by 18% over the low dose. The two different cytokinins also resulted in different effects on bud induction; BAP was more efficient than KIN (Fig. 1). The treatments 0.1 mg/l NAA + 3 mg/l BAP and 1 mg/l NAA + 3 mg/l BAP resulted in the highest shoot production (19.3% and 16.2%, respectively) (Fig 1) These two treatments did not differ statistically, although the maximum number of buds per explant (1.2) was regenerated at 1 mg/l of NAA. This matches the results obtained by Mroginski et al. (1981) who reported that low NAA concentrations (0.01 and 0.1 mg/l) produce poor and erratic bud regeneration and that in high concentrations (3 and 4 mg/l) the formation of roots is stimulated, reducing the efficacy of regeneration. These results also agree with the conclusion of Sharma et al. (1990) that BAP is the most effective cytokinin in terms of number of explants producing shoots and number of shoots per explant.

The presence of an auxin (NAA) in the elongation medium produced the expected cellular elongation effect. No significant differences were found between the two auxin concentrations assessed (0.01 and 1 mg/1), either in the number of elongated shoots or in the shoot height (Fig. 2). It was observed that a low NAA concentration was enough to stimulate the elongation of almost 91% of the shoots. This would agree with Krikorian (1995) who suggests that an auxin supplement sometimes is not necessary or it is necessary only in minimum doses due to the amounts of endogenous auxins already present in the system.

The addition of a cytokinin such as BAP to the elongation medium did not create a favorable environment for the shoots to growth in height. Treatments with BAP combined with low auxin concentration had a negative effect on elongation. The medium supplemented with 5 mg/l of BAP and 0.01 mg/l of NAA showed a 77% reduction in shoot height. In the combination of BAP with 1 mg/l of NAA, the tendency was also negative but not as marked as it was with low doses (Fig. 2). Nevertheless, shoots, instead of elongating, were stimulated to multiply whenever they were in the presence of cytokinin (data not shown). This observation was similar to the results obtained by Gagliardi et al. (2000) who showed that the use of BAP could induce the formation of multiple shoots, favoring low regeneration frequencies. Hazra et al. (2001) also observed that the presence of low BAP concentrations stimulated the axillary shoots to form multiple centers of growth, from which multiple shoots will differentiate, instead of regenerate or elongate.

The root system developed on regenerated shoots was morphologically classified (Fig. 3). The control and medium with 1 mg/l NAA induced a “normal” root system which was characterized by long, thin (hair-like) principal adventitious roots, with abundant secondary roots and root hairs. Treatment with 3 mg/l NAA induced a root system classified as “thickened”, characterized by having slightly thickened primary roots, with a reduced number of secondary roots and root hairs, but similar in length to the normal root system. NAA at 5 mg/l induced an “abnormal” root system, with short thick primary roots, and without secondary roots and root hairs. Roots on medium with 10 mg/l NAA exhibited an “abnormal stocky” system, in which the growth of the primary roots was highly disorganized, showed presence of callus and there was a complete absence of secondary roots.

In the tests, increasing concentrations of NAA significantly increased the number of roots produced although there was no consistent behavior for the variable “length of root” which tended to decrease as the dose was increased (Table 2). This agrees with Krikorian (1995) who observed that with increasing NAA concentration, there is a tendency to produce a greater number of shorter roots, probably because auxins promote root initiation but inhibit root growth.

In order to choose an optimum rooting medium, the survival of the plants during transplanting was considered. This analysis assessed whether the root system developed in the rooting media was sufficiently normal and functional to allow the plant to survive transplanting. Karhu (1997) also considers that the production of a high number of in vitro-formed roots is not strictly associated with a high percentage of ex vitro survival.

Plants rooted without auxins or treated with auxins at 1 or 3 mg/l NAA in the rooting media showed no significant difference in survival rate, which was higher than 88% (Table 2). However, among these three treatments compared with 5 and 10 mg/l NAA there was a significant difference.

To regenerate peanut cv. Florman INTA via organogenesis, we suggest the protocol represented in Fig. 4 and summarized below:

The general efficiency of the system was 15% which means that out of 25 explants about 4 plants survived transfer to the greenhouse. These regenerated plants were normal and produced flowers and seeds.

In conclusion, the present study has aimed at demonstrating a successful regeneration protocol for the peanut variety Florman INTA with the attainment of normal and fertile plants from leaflets. Its use in future transformation systems could be limited due to the relatively low efficiency of regeneration from leaflets of this genotype compared with leaflets of New Mexico Valencia A and cotyledon organogenic or embryogenic regeneration systems that have been used for transformation of multiple genotypes (reviewed in Ozias-Akins 2007).