RESEARCH WORK

Morphoagronomic and isoenzymatic characterization of 23 Leucaena spp. accessions

Hilda B. Wencomo¹, Alba Álvarez², O. Coto³, Maykelis Díaz¹ y R. Ortíz⁴

¹Estación Experimental de Pastos y Forrajes "Indio Hatuey". Central España Republicana, CP 44280, Matanzas, Cuba
E-mail: hilda.wencomo@indio.atenas.inf.cu
2Centro de Aplicaciones Tecnológicas y de Desarrollo Nuclear (CEADEN), Cuba
3Instituto de Investigaciones de Fruticultura Tropical (IIFT), Cuba
4Instituto Nacional de Ciencias Agrícolas (INCA), Cuba

Abstract

The objective of this work was to evaluate the existing diversity degree in 23 Leucaena accessions, regarding the morphoagronomic and isoenzymatic characteristics. Five species of this genus were included (L. leucocephala, L. lanceolata, L. diversifolia, L. macrophylla and L. esculenta), in addition to the commercial cultivars. The following aspects were evaluated: plant height, number of branches, stem diameter, performance of the phenological flowering and fructification patterns, number of pinnae per leaf and of pinnules per pinna, among others. Besides the electrophoretic analyses peroxidases (Prx),α- and ß- esterases (Est), malate dehydrogenase (Mdh) and alcohol dehydrogenase (Adh) were conducted. The accessions fulfilled the predetermined selection criteria regarding the morphoagronomic characteristics, although not in the same time. In this sense, L. leucocephala cv. Cunningham, cv. Peru, CIAT-9119, CIAT-9438, CIAT-751, CIAT-7988, CIAT-7384, CIAT-7929, CIAT-17480, cv. Ipil-Ipil and cv. CNIA-250; L. lanceolata CIAT-17255 and CIAT-17501, and L. diversifolia CIAT-17270, stood out. The existence of genetic variability within the collection could be detected. The isoenzymatic systems (esterases and peroxidases) turned out to be polymorphic in the studied sample, mainly esterases. On the other hand, the analysis of genetic diversity allowed differentiating L. leucocephala more clearly as compared to the other studied species, although no better discrimination was made within the species.

Key words: Evaluation, genetic resources, Leucaena spp.

Con

tenido

Introduction

The study of trees and shrubs for their utilization in livestock production with different productive purposes, in an aimed or spontaneous way, is not a new activity (Murgueitio, 2003). At present, the social and environmental reconversion of livestock production is urgent and a priority for the Latin American and Caribbean regions; livestock production agroforestry, as a non-aggressive system for the environment, may constitute a substantial part of this process of change (Funes, 2004).

However, the promotion of these systems implies the selection of ecologically and economically appropriate species for the purposes sought. Hence the need to implement a program aimed at the search, introduction, evaluation and selection of these important plant resources, as an essential stage for their future extension.

It should also be stated that for the introduction of new species or accessions in the above-mentioned systems, the adequate characterization, identification and evaluation of the material by means of agromorphological or morphoagronomic descriptors is important. Yet, these features can be altered by abiotic factors, such as the environment, and biotic factors, such as the age of the crop. That is where the genetic diversity characterization (Acosta, 1999), through such biochemical techniques as electrophoresis (of proteins or isoenzymes), plays an important role as complement of the morphoagronomic characterization and evaluation, just like the direct DNA analysis (Schmidt et al., 2003), which allows having further knowledge about the evaluated material.

The main characterization and identification studies of the Leucaena genus conducted in Cuba, in Leucaena leucocephala (Ruiz and Febles, 2006), have been focused on the morphoagronomic characteristics under field conditions or in isolated trials, including only the four varieties recorded as commercial, belonging to this species. However, until now no further detailed characterization studies have been conducted including isoenzymatic markers, as well as a higher number of species from this genus.

Isoenzyme electrophoresis favors the use of genetic markers, which are generally more efficient than the morphological ones, in spite of being influenced by the environmental action and depending on the tissue and development stage of the evaluated plant. Likewise, they are relatively simple, little costly and codominant; they allow distinguishing between homozygous and heterozygous genotypes and thus developing mapping and binding, and population genetics studies, among others (Álvarez, 2005).

Considering the above-mentioned facts, this work was developed in order to evaluate the existing diversity degree in a group of Leucaena accessions, from the germplasm bank of the EEPF «Indio Hatuey», according to their morphoagronomic and isoenzymatic traits.

Materials and Methods

The work related to the morphoagronomic characteristics was developed in areas of the EEPF «Indio Hatuey» and the work with isoenzymes was conducted at the Center of Technological Applications and Nuclear Development (CEADEN), in Havana.

Morphoagronomic characterization of the germplasm

Location. The study was conducted in areas of the EEPF «Indio Hatuey», which is located at 22º 48' and 7'' latitude north and 79º 32 ` and 2'' longitude west, at an altitude of 19,9 masl, in the Perico municipality, Matanzas province, Cuba (Academia de Ciencias de Cuba, 1989).

Soil characteristics. The trial was conducted on a soil of plain topography, with slope from 0,5 to 1,0% and classified by Hernández et al. (2003) as lixiviated Ferralitic Red, hydrated ferruginous nodular humic, of rapid desiccation, clayey and deep on limestones. This type is equivalent to the Ferralsol group, in the FAO-UNESCO classification system (Alonso, 2003). The average depth to the limestone is 150 cm.

Plant material used. From the 180 existing accessions in the Leucaena spp. collection (preserved at the germplasm bank of the institution) 23 were taken (table 1) as representative of the population, which had previously been evaluated under nursery conditions; four plants were evaluated from each accession.

Establishment. In the experimental period neither irrigation nor fertilization was used. The trial started when the seedlings reached approximately 30-45 cm (three months old), with a healthy appearance; four seedlings of each accession were transplanted to the field, in rows spaced at 6 cm and a separation of 3 m between plants.

Plant height was measured since the transplant moment, with a ruler graduated in centimeters, in perpendicular position and always in contact with the soil. In addition, the number of branches was counted and stem diameter was measured with a caliper. All these measurements were monthly performed in the four transplanted plants, until they were considered established regarding the predetermined criteria.

According to Seguí et al. (2002), the accessions should fulfill two or more of the considered evaluative criteria: height, 1,5 to 2,0 m; number of branches higher than 10; stem diameter between 0,5 and 0,8 cm; and edible biomass yield 0,75 (or more) kg DM/plant in a period no longer than 14 months.

Flowering and fructification stage. In this stage the performance of the phenological flowering and fructification patterns was observed in all the accessions, with a weekly frequency, and 50% or more of flowers and fruits was considered. For that purpose the symbology established by Machado et al. (1999) was used.

In addition, the number of pinnae per leaf and pinnules per pinna was counted; pinnule length (mm) and width (mm), quantity of pods per capitulum, pod length (mm) and width (mm) were measured. The shape of the pinnae, the type and position of the glands and the color of the flowers were taken into consideration. These measurements and observations, in the case of leaves and their components, were made on 15 leaves per plant and equal number for flowers and fruits (Cronquist, 1981).

Biomass availability was estimated through simulation of the browsing made by the animals (Lamela, 1998), according to the consumption height (up to 2 m). For that purpose the milking technique of the fresher parts of the plants, leaves and fine stems, to approximately 3 mm of diameter, was applied. The green material was weighed and separated manually in its different fractions edible and non edible material- and both parts were immediately weighed and the value of each material per tree was calculated. Likewise, the yield in the establishment pruning was determined; for that purpose 300-g-samples of green forage were extracted, to which the dry matter percentage was calculated.

Detection of the enzymatic polymorphism

Plant material and enzymatic extraction. For the isoenzymatic analyses new regrowths (of the leaves) from each one of the accessions were used. The samples were taken in early morning hours (7:00-9:00 a.m.), were put in a cooler and later frozen at -70ºC. Cold extractions were performed according to the recommendations made by González (2002) and Álvarez (2005).

Sample preparation and electrophoresis. In a mortar 0,5 g of leaves were macerated in liquid nitrogen and 500 µL of 20% sucrose was added (Soltis and Soltis, 1989). The extracts were put in Eppendorf tubes of 0,5 µL at -4ºC, until their later utilization. The first electrophoretic runs were performed in polyacrylamide gel (PAGE), using a separating gel of 8,5% with Tris-Glycine running buffer 0,04 M of pH 8,3, and 4% concentration gel in vertical electrophoresis chamber (Model V 16) and discontinuous buffer systems for the isoenzymes peroxidases,»- and ß- esterases, malate dehydrogenase and alcohol dehydrogenase, according to the methodologies described by Álvarez et al. (2000). In all runs 30 µL of each sample and 10 µL of loading buffer (LB) were added.

Based on the isoenzymatic studies reported for species of this genus (such as Leucaena shannonii), a test was made with the extraction buffer used by Chamberlain et al. (1996), with which very tenuous and still little-defined bands were observed. Several adjustments were made to this protocol until the extraction method of the isoenzymatic systems used was standardized. In this sense, 0,5 g of the leaf tissue were macerated in liquid nitrogen and extraction buffer 1/1 (m/v) was added. The extract was centrifuged at 14 000 rpm during three minutes and the supernatant was collected for the electrophoresis. A Tris-Citrate buffer pH 8,3 was used, to which KCl 0,08%, MgCl2 0,2%, EDTA 0,04%, 0,5 mL of Triton x 100 1%, 2 mL 10% DTT and 25 mg PVP-40 4% were added.

The runs were performed in vertical electrophoresis chambers (Model V 16) and polyacrylamide gels (at 8,5% for peroxidases and alcohol dehydrogenases and at 12% for esterases), at 4ºC, 120 V, 20 mA, during four hours.

Staining. The specific staining for peroxidases (Prx EC. 1.11.1.7),α- and ß- esterases (Est EC. 3.1.1-), malate dehydrogenase (Mdh EC. 1.1.1.37) and alcohol dehydrogenase (Adh EC. 1.1.1.1), were made, according to Álvarez et al. (2000). The electrophoretic runs were repeated three times and only the consistent and reproducible bands were recorded. The isoenzymatic phenotypes of each accession were recorded as presence/absence of each band (1/0, respectively).

Statistical processing. The results were processed by the principal component analysis (PCA) (Morrison, 1967), in which those principal components that showed values higher than one and sum or preponderance factors higher than 0,70 were taken as criterion.

The cluster analysis was applied for the grouping and selection of the accessions, using as similarity index the Euclidean distance, from the results obtained in the PCA (Torres et al., 2006), and the mean and standard deviation stadigraphs were determined for the analyzed variables in this stage. Thus there were species groups which allowed doing a simpler and more objective analysis of their performance. All the analyses were made through the statistical program SPSS® version 11.5 for Microsoft® Windows® (Visuata, 1998).

The binary matrix of isoenzymatic data was used to generate a matrix of genetic similarity among all the genotype pairs, expressed as the complement of Dice's coefficient (Dice, 1945), using the program SIMQUAL of the statistical pack NTSYS-pc version 2 (NTSYS-pc, 1997). A cluster analysis was made, based on Dice's similarity matrix; for that purpose a dendrogram was generated in the program SHAN, of the same statistical pack. The aggregation criterion used was the Unweighted Group Method with Arithmetic Mean (UPGMA) (Sneath and Sokal, 1973). The cophenetic correlation (r) was also determined, to find the homogeneity between the similarity matrix and the dendrogram, and to represent the accuracy of the technique used (NTSYS-pc, 1997).

Results and Discussion

Morphoagronomic characterization

Table 2 shows the performance of the accessions in the establishment stage, besides the time each one took to reach 1,50 m of height. It was proven that some accessions could begin to be exploited before 12 months after being transplanted, as it occurred with L. leucocephala CIAT-17480; this was the first to reach the prefixed height, only seven months after being planted, and by the end of the stage it was 3,65 m high and showed an edible biomass yield of 0,82 kg DM/plant.

There were significant differences (P< 0,05) among the accessions regarding height, stem diameter and number of branches. It could be observed that the first seven accessions stood out as compared to the commercial varieties L. leucocephala cv. Cunningham, cv. Peru and cv. CNIA-250 in such indicators. The performance of this last cultivar coincides with the results obtained by Machado and Núñez (1994) and Wencomo (2008).

The height was similar for all accessions, except for L. diversifolia CIAT-17503 and CIAT-17270, which showed lower values in diameter and number of branches. In most cases the tallest plants coincided with the ones with the highest number of branches; this indicates that in these accessions a higher biomass production or availability could be obtained, which is very important for forage production and, thus, for animal feeding. In this sense, Dávila and Urbano (1996) reported similar results in 13 cultivars of L. leucocephala, in which the lowest height values coincided with the lowest in diameter, number of branches and average dry matter yield.

L. leucocephala CIAT-17480 and CIAT-7988 were the accessions with the highest number of branches (47 and 44, respectively), followed by L. leucocephala CIAT-751 and CIAT-9438 (with 42 and 41 for each). More regrowths were found in the tallest and more vigorous plants. The commercial varieties L. leucocephala cv. Cunningham, cv. Peru and cv. CNIA-250, in spite of being among the accessions which reached establishment earlier, had a little outstanding performance regarding height and number of branches after 14 months (table 2).

The slow initial growth in plants of the accessions from this genus is likely to be specific, and the accession, in spite of the existing differences, does not seem to play a decisive role when obtaining a material with faster establishment. The slowness in the species L. leucocephala, in its first stages, was also indicated by Cooksley (1974) and constitutes one of the most adverse limitations in Cuba for its establishment; the observations seem to indicate that this occurs similarly in the accessions of the other Leucaena species.

This could also be related to the quantity of leaf area, the growth dynamics and leaf expansion (Díaz, 2006), as well as to the biomass partition with certain priority during the first weeks towards the root system; this is confirmed with the studies conducted by Shelton (2000), who stated that this organ in trees has a high component of permanent structural roots, as well as a rootlet system which is responsible for the assimilation of water and nutrients.

The increase that occurs afterwards in plant growth could be related to a marked leaf production (Stür et al., 1994) or to the arrival at satisfactory values of leaf area index, indicator which according to Díaz (2006) is closely related to plant growth and development. In general, it was evident that the plant establishment dynamics in the species from this genus showed differences in its performance among the accessions; for such reason, it should not be statically and arbitrarily analyzed, but taking into consideration different biotic as well as abiotic factors.

During this evaluation stage all the accessions fulfilled the predetermined selection criteria, although not in the same time, which could have been associated to the genotypic characteristics of the plant, to the utilization capacity of the nutrients present in the soil or to the efficiency with which they were utilized by the plants. The accessions L. leucocephala cv. Cunningham, cv. Peru, CIAT-9119, CIAT-9438, CIAT-751, CIAT-7988, CIAT-7384, CIAT-7929, CIAT-17480, cv. Ipil-Ipil and cv. CNIA-250; L. lanceolata CIAT-17255 and CIAT-17501, and L. diversifolia CIAT-17270, stood out. The presence of the last three in the same group as those of L. leucocephala can indicate that they behave very similarly regarding the evaluated indicators.

Phenology is an interesting element which should be taken into consideration in this stage and others of plant development, even during its exploitation, because it can serve as a guide in the plantation management. For such reason, the understanding of the cycle of phenological events (flowering, fructification and leaf production), management, planning of the resources for the collection, harvest, seed processing and conservation, are necessary and essential for the efficient planning of seed collection. A particular analysis of this factor will be detailed below.

Tables 3 and 3a show the phenotypic or morphological differences among accessions. All the species of the Leucaena genus have bipinnate leaves and they and the pinnule are opposite. The accessions of the species L. lanceolata and L. macrophylla show oval or elliptical pinnae, with weakly asymmetric basis; while those of the species L. esculenta, L. diversifolia and L. leucocephala have lineal or lineal-oblong pinnae with strongly asymmetric basis. The number of pinnae pairs per leaf varies from 4 to 18 and the number of pinnule pairs per pinna oscillates from 10 to 50; although in other species they can reach higher values (Hughes, 1998).

Likewise, the L. leucocephala accessions showed elliptical and concave glands; while the L. lanceolata accessions were round elliptical, dome-shaped, except for the accession CIAT-17253 in which the presence of these glands was not observed, although it is one of the characteristics of the species as such. In the case of L. diversifolia accessions discoid, little deep, cup-shaped and elliptical or round triangular glands were found; while those of L. macrophylla were elliptical, convex, round and conical, except for the accession CIAT-17231 which did not have gland either. In the L. esculenta accessions the glands were flat, elliptical, large, little deep,
concave and cup-shaped. According to Hughes (1997), the glands constitute an indicator for the identification of the species from this genus, because their number and arrangement are very variable.

All the accessions had oval light brown to dark brown seeds, like the pods; they showed white flowers, except L. diversifolia CIAT-17270 which flowers were pink, reddish or light purple. According to Anon (2003), the flowers, fruits and seeds are indicators that serve to identify the species from this genus.

The phenotypical differences in each of the Leucaena species and accessions had been reported by Hughes (1998). Knowing about these differences is importance when using the species in the promotion of a certain system (silvopastoral or other), because it has a remarkable influence in the management they are given or the productive purpose for which they are destined. For example, the differences in the performance of the phenological flowering and fructification patterns of each one of them would provide the possibility to use them indistinctly in the same area unit: some for seed production and harvest and others for biomass production, for future breeding studies or to define when pruning can be made or not.

Detection of isoenzymatic polymorphism

From the tested isoenzymatic systems peroxidases (Prx),α- and ß- esterases (Est), malate dehydrogenase (Mdh) and alcohol dehydrogenase (Adh)- the first two were polymorphic, unlike the last ones in which the bands were not visualized. The qualitative analysis of the electrophoretic composition of the systems in the leaf tissue, allowed appreciating a total of 23 bands in the 23 studied accessions.

With regards to the isoenzymes α- and ß- Est, they are constituted by a complex group of proteins associated to specific intracellular proteins, which show a total of eight polymorphic sites in the case of tomato (Florido et al., 2002). The marked polymorphism which is present in this system has been also reported by other authors when studying the polyploidy rate in banana clones (Román et al., 1997), in the leaf tissue of tomato (Florido et al., 2002) and in the characterization of cassava clones (Schmidt et al., 2003), which has allowed differentiating accessions in these crops.

Likewise, it could be observed that no Adh and Mdh isoforms appeared in the leaf tissue; this result is in correspondence with statements made by Menezes et al. (1995), who reported that under normal conditions the enzymatic activity of these systems disappears in very early stages of plant development, although it can be induced when anaerobiosis conditions are present (Iglesias, 1994); it cannot be discarded that this may be caused by concentration problems of the samples, their management or the sensitivity of the technique.

From the above-explained facts, it is reaffirmed that not all buffer systems and extraction procedures are effective for all the enzymes of a tissue, or for all laboratory conditions. An example is Ramírez et al. (1987), who tested 16 isoenzymatic systems in five cassava tissue types and only recommended α- and ß- Est for the characterization and identification of collection duplicates of the above-mentioned crop. For such reason, it could be inferred that for the studied accessions only the systems of peroxidases and α- and ß- esterases can be recommended, independently from the fact that tests should be made with other systems which have been used in different crops. In this regard, it is known that plants generally require more than one isoform of a particular enzyme, so that an efficient catalysis is guaranteed (Álvarez, 2005).

Likewise, it should be considered that isoenzymes are of gene expression and, thus, depend on the tissue type and development; that is why the absence of bands in the different isoenzymatic patterns is due not only to the needs for those enzymes in the different tissues, but also to the co-migration of proteins and the difference of geographical zones, because although most of them are not influenced by the environment, according to Schmidt et al. (2003), the electrophoretic paterns of a few systems (including Prx, Est, Aps, Sod and Cat) can be modified by biotic and abiotic factors, so that under these conditions the function of the genes that codify for those enzymes is altered. Similarly, it should be stated that isoenzymes can also vary according to the evaluated species, age, management, climate, presence or absence of diseases and position of the sampled organ, among others.

Table 4 shows the frequency of each pattern in the 23 accessions. The detected level of isoenzymatic polymorphism in the sample could be considered moderate, because most of the patterns showed very high frequencies (60 and 80% of the sample with the same pattern for Est and Prx, respectively) and very few accessions showed unique patterns (seven for Est and two for Prx).

The existence of a high electrophoretic resolution for such systems was proven (table 5), which indicates that they are useful in the evaluation of varietal polymorphism in this material; the enzymes peroxidases and esterases were shown to be the most recommended, for their polymorphism, as referred by Schmidt et al. (2003).

Similar results were obtained when making the integral analysis of the enzymatic variability in L. leucocephala (Harris et al., 1994a) and in L. shannonii (Chamberlain et al., 1996), as well as in rice mutants of the variety J-112 (Díaz et al., 2001), in which the existence of some sites of polymorphic enzymatic activity was detected (González, 1996). In general, the isoenzymes peroxidases and esterases are among the most polymorphic systems in plants (Fuentes, 2003).

Considering the results of the isoenzymatic analysis, the systems α- and ß- esterases turned out to be the ones with higher polymorphism regarding the number of polymorphic bands that allowed detecting the number of electrophoretic patterns and their frequency in the sample; this is in correspondence with the reports about the high polymorphism in this system for other plant species (Schmidt et al., 2003).

From the appearance frequency of the bands of the isoenzymatic systems, through the SHAN program, the dendrogram was obtained (fig. 1), in which the grouping of the accessions in three groups (I, II and III) is shown. Group I was formed by two subgroups (IA and IB). Subgroup IA included all the L. leucocephala accessions, two L. lanceolata accessions (CIAT-17255 and CIAT-17501) and one of L. diversifolia (CIAT-17270); while subgroup IB turned out to be heterogeneous, including L. macrophylla (three accessions), L. esculenta, L. lanceolata and L. diversifolia, the last two represented by an accession each. Group II included L. macrophylla CIAT-17232 and group III had L. macrophylla CIAT-17238 and L. esculenta CIAT-17225.

Group I includes the accessions that had an outstanding morphoagronomic performance regarding the indicators, together with the commercial varieties currently used in silvopastoral systems, all of them with an acceptable productive potential. It should be emphasized that within this group the genetic affinities were higher. The other accessions had a different performance in their phenotype, as compared to those in group I.

The varietal groups were formed at low values of genetic distance, considering a truncation value of 0,35, which suggests a high level of genetic similarity for the tested enzymatic systems. This was to be expected, given they are isoenzymatic markers which correspond to codifying and highly preserved regions in the genome. Although the sample included varieties of five different Leucaena species, a full correspondence could not be appreciated among the groups and main analyzed species, with the exception of L. leucocephala (subgroup IA).

It is valid to state that differentiating the species L. leucocephala from the others, although the differentiation among its accessions could not be achieved, constitutes a valuable contribution to this type of study and the breeding future of the species from this genus. In the case of the other species genetic similarity was observed; however, this also meant a step forward in the genetic study and characterization, aspect which must be closely related to the fact that L. leucocephala has been object of diverse breeding programs with regards to the others, of which there is little information in that sense, because according to reports presented by Chamberlain et al. (1996) they are only taken into consideration as ancestors in the above-mentioned programs.

These results show the relatively narrow basis of existing isoenzymatic variation in the evaluated material and are in correspondence, in general, with the statements by Harris et al. (1994a) and Chamberlain et al. (1996), who reported about the large isoenzymatic homogeneity present in the Leucaena genus; this is corroborated with the studies conducted by Harris et al. (1994b), which revealed the presence of a low polymorphism level even when using molecular techniques, such as restriction fragment length polymorphism (RFLP).

Through this study the possibility of duplicate genotypes could be discarded, which would have been impossible to determine through morphological characterization and evaluation. In this sense it would be useful to increase the number of isoenzymatic systems in the analysis, in order to achieve higher cover of the genome, among them: aspartate aminotransferase (AAT), phosphoglucose isomerase (PGI), isocitrate dehydrogenase (IDH), phosphoglucomutase (PGM) and shikimate dehydrogenase (SDH); they have shown polymorphism in the genetic diversity studies conducted in other Leucaena species (Harris et al., 1994b; Chamberlain et al., 1996) and in such crops as tomato, rice, banana and cassava (Florido et al., 2002; Fuentes, 2003; Schmidt et al., 2003).

According to the results it is concluded that the evaluated morphoagronomic indicators accounted for the variability regarding the performance in the establishment. Leucaena spp. showed its flowering and fructification capacity, with differences among the studied individuals. In this sense, the following accessions stood out: L. leucocephala cv. Cunningham, cv. Peru, CIAT-9119, CIAT-9438, CIAT-751, CIAT-7988, CIAT-7384, CIAT-7929, CIAT-17480, cv. Ipil-Ipil and cv. CNIA-250; L. lanceolata CIAT-17255 and CIAT-17501, and L. diversifolia CIAT-17270. The isoenzymatic systems α- and ß- esterases and peroxidases were standardized in the determination of the isoenzymatic polymorphism of these species. The analysis of the isoenzymatic patterns α- and ß- esterases and peroxidases allowed differentiating L. leucocephala more clearly than the others, although no better differentiation was made within the species and the esterases were the most polymorphic. It was proven that there were no duplicate genotypes in the Leucaena collection; genetic homogeneity was detected at isoenzymatic level in the accessions and genetic affinity among them.

For such reason, combining the morphoagronomic and genetic-biochemical analyses for the characterization of germplasm banks is recommended, in addition to continuing the search for new polymorphic forms of the crop, incorporating, as much as possible, techniques that detect higher polymorphism, which allow revealing the existing variation at DNA level and having enough genetic variability to utilize in Leucaena. Including a higher number of accessions from the little represented species in this work (L. lanceolata, L. diversifolia, L. macrophylla and L. esculenta) is recommended.