Note ROOTING PERFORMANCE FROM LEAF PETIOLES OF ACCESSIONS AND HYBRIDS OF WILD ARACHIS SPECIES

The genus Arachis has 69 species, the majority native to Brazil. Arachis spp. seedling root tips have been used to obtain dividing cells for cytological preparations. Several wild species produce small amounts of seeds and consequently few root meristems, making the cytogenetic characterization very difficult. The purpose of this study was to evaluate the rooting capacity of several wild species of Arachis using the detached leaves technique for obtaining roots from petioles. One hundred and thirty accessions and 27 interspecific hybrids were studied. Three evaluations were performed at 15, 31, and 49 days after planting. Leaf rooting observed in the wild species confirmed the great genetic variability typical of the Arachis genus, indicating that the number of days interferes with the rooting percentage. The Arachis Section presented the highest rate of rooting leaves. The Caulorrhizae Section showed the same pattern observed in the Erectoides Section. Accessions from Heteranthae, Trierectoides, Extranervosae, and Rhizomatosae Sections did not differ among each other. Differences were not observed in Erectoides Section when compared to Rhizomatosae and Extranervosae Sections. The number and morphology of roots obtained through this technique were promising to be used on cytological preparations.


INTRODUCTION
Although plant researchers know the importance of plant genetic resources, the limited use of germplasm banks accessions is a reality around the world.Two alternatives have been emphasized for increasing the use of available accessions: pre-breeding programs and organization of core collections (Frankel & Brown, 1984;Brown, 1989;Vilela-Morales et al., 1997;Nass, 2001).
The genus Arachis has 69 species (Krapovickas & Gregory, 1994), the majority of them of Brazilian occurrence.Several wild species of the genus show interesting characteristics for groundnut genetic improvement (Arachis hypogaea L.), to be used as forage, ornamental, as well as for erosion control.Several of them are more pest resistant than the cultivated groundnut (Stalker & Moss, 1987;Fávero et al., 2001).Chromosome number and morphological observations are important for species characterization and germplasm bank organization.Root tips of seedlings have been used for obtaining division cells to allow investigation based on mitotic characterization of Arachis species (Stalker,1991;Lavia, 1998).Several wild species produce small amounts of seeds in their natural habitat or even in germplasm banks, making the work on cytogenetic characterization very difficult.Usually, root tips are obtained from cuttings.However, this technique sometimes demands a long time to produce results.To overcome this difficulty, petioles rooting and callus productions through in vitro culture have been used (Nakano et al., 1999;Shibli et al., 2001).The detached leaves technique has been used in phytopathological characterization of accessions (Subrahmanyam & Moss, 1983), and in molecular studies of root development (Brown & Mangat, 1970).A petiole-rooting technique to obtain root tips was described by Blomgren et al. (1988) in soybean, which also shows chromosomes in mitosis from root tips developed in detached petioles.This technique can also be used in cases of seed unavailability and/or unfeasibility of the cutting alternative.Fávero et al. (2004) showed that for some interspecific hybrids of Section Arachis, the detached leaf technique presented in this work may be used to produce root in sufficient amount and quality to be used in cytological preparations.
Our objective was to evaluate the rooting capacity of several wild species of Arachis using a detached leaf technique to obtain roots from petioles.

MATERIAL AND METHODS
One hundred and thirty accessions and 27 interspecific hybrids belonging to the Arachis Germplasm Bank of Embrapa Genetic Resources and Biotechnology -Cenargen (Brasília, Brazil) were used.
From each accession, four new totally expanded leaves were collected, placed in plastic bags and stored in a refrigerator.Petioles were treated with 0.4% naphthalene acetic acid rooting hormone (NAA) powder and leaves were immediately placed in cells of polystyrene trays containing vegetable substratum (Plantmax ® ).The trays were covered with transparent plastic bags for humidity maintenance and transferred to the greenhouse in shadow conditions, with an average temperature of 24ºC.The substrate was watered every two days.Accessions were evaluated in a completely randomized block design with four replications.The evaluations were performed at 15, 31, and 49 days after planting.In each evaluation, accessions were classified as rooted, non-rooted and dead leaves.The statistical analysis was made using PROCREG of the Sta-tistical Analysis System (SAS).The chi-square and t tests were used to verify the rooting differences among Sections as well as to identify which Sections were different from each other.The Nemenyi test of non parametric multiple comparison was used to verify differences among the earliest and the latest rooting accessions.

RESULTS AND DISCUSSION
The leaf petioles rooting observed in wild Arachis species (Figure 1) confirms the great variability displayed by the genus.Great differences (P < 0.01) among genotypes were observed.
Species of all Sections were evaluated, except Trisseminatae Section.During the first evaluation (15 days) 26 accessions (16.25%) rooted in at least one replication.During the second (31 days) and on the third (49 days) evaluations, root development was observed in 103 (64.40%) and in 126 (78.75%) accessions, respectively.
Root induction frequencies for each accession are presented in Table 1.The Nemenyi test (a non parametric test of multiple comparisons, type Tukey) was done and it was verified that there was no cluster, unless for those that had the same results.Consequently, all accessions that had different rooting were also different for the Nemenyi test.Accessions V 9923 (A.sp.) and Wi 1302-2 (A.cruziana) rooted within 15 days in four and three replications, respectively, being considered the earliest accessions among the evaluated materials.Accession V 13774 (A.aff.diogoi) leaves died in all replications, indicating that this methodology may not be appropriate.After the third evaluation, leaves of 34 accessions (21.25%) did not root or died.Such accessions will be revaluated using another methodology.The chi-square test (P < 0.0001) indicated that petiole rooting is a genotype dependent process, suggesting that there is a genetic factor associated to this characteristic.Section Arachis displayed the largest rooting ratio, and it is different from the other Sections by the t test.Induction of roots in accessions from Caulorrhizae Section was not different from Erectoides This result is due to their similar distributions, which differ basically in the number of accessions with 75% of rooting.Due to the low degrees of freedom associated to the other sections, these could not be efficiently discriminated.The Heteranthae, Trierectoides, Extranervosae and Rhizomatosae Sections did not differ from each other.Differences were not observed in the Erectoides Section when compared to the Rhizomatosae and Extranervosae Sections.Rios et al. (1994) obtained detached leaf rooting from several species, using vermiculite as substratum, including 16 different legume genus, sunflower (Helianthus annuus), colza (Brassica campestris) and forage turnip (Brassica napus).Leaves of common bean (Phaseolus vulgaris), faba bean (Vicia faba) and cowpea (Vigna unguiculata) presented the best rooting performance among all species.

Table 1 -
Root induction in wild Arachis species leaves.Accessions code, species, sections and percentage of rooted leaves at 15, 31, and 49 days after planting.Distinct performance among Sections were observed concerning rooting leaves (Table2).The Arachis Section presented the highest precocity, showing the highest rate of rooting leaves among the Sections studied, concentrating the largest frequencies between 15 and 31 days.The Erectoides, Extranervosae, Rhizomatosae and Procumbentes Sections displayed the latest or non-rooting rates.The Caulorrhizae, Heteranthae and Trierectoides Sections displayed intermediate rooting rates, concentrated in 31 days.For most of the sections, petiole rooting initiated on the average at day 31.Most of the leaves did not root or died.

Table 2 -
Number of accessions in each Section, percentage of rooted leaves at the end of the experiment (0-100%) and total of rooted leaf rates (%) at 15, 31 and 49 days after planting for different Arachis Sections.