*Area de Genética y Mejoramiento Animal y Vegetal
**Instituto de Fisiología Vegetal. Facultad de Ciencias Agrarias y Forestales, UNLP, CC 31 (1900) La Plata, Argentina.
infive@isis.unlp.edu.ar

Individual genotypes may be propagated as clones in those species that allow that technique. It is known the usefulness of this practice either as a breeding method or also as a tool in the measurement of the enviromental variance to estimate the heritatibility. A factor that tends to overestimate the variance within a clonal line is the "effect of clone" apparently determined by differences in the physiological efficiency between plants of the same genotype, obtained by vegetative propagation (stem cuttings). It has been communicated that Lotus corniculatus can be propagated by root and crown cuttings (Midgley and Gershoy, 1946). Also it has been advise that morphology and vigor of a vegetatively propagated L. corniculatus plant, may be influenced by the cutting's type (Ostazeski and Henson, 1965).

Some years ago, at the beginning of L. glaber breeding program it was considered to fit a cloning technique for different experimental and breeding purposes. In Autumn it was attempt to clone L. glaber by stem cuttings with 3 leaves and their corresponding axillary buds or lateral branches. These were planted in a nursery in sand saturated with water, in the open air. It was registered that roots appeared 20-25 days after planting, only the 60% of the cuttings rooted. Although these plants showed a good development, it was observed a low size uniformity. Besides, the presence of plants with a high crown was frequent, generally it was stratified in two levels. This fact does not occur in the architecture of plants obtained by seeds. These results suggested that L. glaber cloning would not be difficult and it could be interesting to focus in the short rooting period, and the uniformity "within" the clones. It was considered that plants obtained from cuttings with only one leaf bud could permit an efficient use of the donor plant and also they would show only one crown at the surface of the soil level.

Two assays of cloning were accomplished with the objective of determining: i) The effect of washing the base of cuttings, for the possible presence of rooting inhibitors. ii) The effect of the donor branch lenght, and the cutting position in the donor branch (basal, medial or distal segment). In both assays, following the Ostazeski and Henson (l965) technique applied in L. corniculatus, cuttings with a leaf and their corresponding axillary bud or lateral branch (< 2 cm) were used. The cutting lengths were of 2.5 cm, carrying an oblique cut at 2 cm below the bud and a transverse cut at 0.5 cm above the same. The cuttings were rooted in water saturated vermiculite in plastic trays. A completely randomized design with ten replicates was used.

The donors plants were cultivated outdoors, and the cloning was performed in the period of spring growth. In the assay (i) 3 genotypes were evaluated. The cuttings were obtained from the medial segment of branches of variable lengths. The washing of the cuttings were carried out, exposing the base of the segment to a current of tap-water for 6 or 12 hs. The cutting control's without washing, were planted in a rooting medium at the same time that began the wash treatment. In the assay (ii) one genotype was used. The branches were classified in: short ones (20-22 cm), intermediated (30-32 cm) and long ones (40-42 cm). The cuttings were taken from the base off all of the three segments, basal, medial and distal, of each donor type. The trays were moved into a growth chamber at l7ºC, under l7 h daylength provided by Grolux tubes and a 40 watts incandescent bulb. At the cuttings level the irradiance was of approximately 70 µmol / m^-2/s^-1. The assays finished after 25 days since the cuttings were placed in the rooting medium. The number of days until the first roots appeared, number of cuttings rooted when the assay finished, number of roots for every cutting, maximum length root, lateral branch length, root and branch dry weights were registered. Analyses of variance were done for each variable. Treatments were compared using Tukey's test (a=0.01, a=0.05).

The results of the trail (i) showed differences (P< 0.01) between genotypes, for maximum root length, branch length, root dry weight and branch dry weights. The percentage of the cuttings rooted in every genotype was not affected by the washing and the general rooting average ± S was 99.33 ± 1.4 %. The appearance of the first roots occurred between 7 to 17 days. For only one of the three genotypes evaluated, the treatment with 12 h of washing showed significant differences (P<0.01), compared to the controls in: the time elapsed until the appearance of first roots, root length, branch length, root and branch dry weights. In the remaining genotypes the washing treatment did not show an effect. This lead us to consider a null or a low interference of rooting inhibitors, that will not justify treatments for its removal, in a cloning routine as for the establishment of a polycross plot.

The assay (ii) showed slight differences for the donor longer branches (30-32cm and 40-42cm), (P<0.05). There were highly significant differences (P<0.01) among the basal and distal segments of the same branch in the elapsed period until the root appearance, root number, lateral branch length, root and lateral branch dry weights. The cuttings from distal and medial branch segments showed the greater response with higher average values and lower coefficients of variability for every studied trait. There was slight but not significant differences between the cuttings of the the medial and distal segments.

Results confirm that L. glaber cloning is possible. Nevertheless, the practice of some specifications, as to take the cuttings from the one third distal sector or, eventually, from the one third middle of longer branches of a donor plant, will enable the cloning efficiency and uniformity on clone behavior.

Finally, the cloning technique was fitted to: For vegetative donor plants the branches were preferentially higher than 30 cm. From the base of the distal one third segment of these, were obtained cuttings of approximately 2.5 cm of length through an oblique cut, 2 cm below the leaf and an other transverse cut, at 0.5 cm above the leaf. The lateral branches longer than 2,5 cm were cut at this level. The cuttings were rooted in damp saturated vermiculite in plastic trays, in a greenhouse until their rooting. To maintain the air dampness inside the trays, they were covered with a glass at 3-4 cm over the trays and so also allowed the air circulation. When the rooting of the cuttings were higher than 90%, they were transferred to the open air and exposed gradually to the sun, for their rustication. The dampness of the cuttings were maintained by watering with a Nitrofoska (BASF) of 3 gr/ l solution. The cloning cuttings remained in these conditions during 6 days until they were established in the field.

This technique was applied on 320 cuttings obtained from a previously cloned genotype, and 300 of them rooted (93%). During October, 250 were transplanted to a plot in the experimental field. At 6 months after transplant, 96.6 % of the plants survived, presenting a good development and a similar crown to those obtained by seed plants. In addition, 93 clonal lines with 12 replicates were established in the experimental field for their evaluation and selection. From them the 97.5% of the cuttings rooted, and from them 1116 seedlings were transplanted. The 98 % became adult plants.

Besides it was tested a cloning for polycross of 31 select genotypes (n=25). It was established at the experimental field, and were obtained a 89.5 % of rooting. In October were transplanted 775 seedlings to the isolation plot, and 96.4% survived until the period of seed crop. Their seeds were sown for the polycross progeny test. Furthermore, in the last 5 years this technique has been applied with similar results in the genotypes cloning of L. glaber for different experimental and breeding purposes.

References

Midgley AR and A Gershoy (1946). Birdsfoot trefoil may propagated by root cuttings. Journal of the American Society of Agronomy. 38, 197-199.
Ostazeski A and PR Henson (1965). Effect of morphology of propagules on performance of birdsfoot trefoil clones. Crop Science. 5, 253-254.

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