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Potential of Lotus glaber Mill.(=Lotus tenuis Waldst. et Kit. ex Wild.) for vegetative propagation by rooting of stems and leaves in floating conditions
over the surface of a water medium.

M.M. Mujica *, M. Colares ** and C.P. Rumi***

* Area de Genética y Mejoramiento Animal y Vegetal; ** Area de Botánica; ***Instituto de Fisiología Vegetal. Facultad de Ciencias Agrarias y Forestales, UNLP, CC 31 (1900) La Plata, Argentina.

infive@isis.unlp.edu.ar

Lotus glaber is a species adapted to tolerate long flooding conditions such as those of the Salade River Basin, province of Buenos Aires (Argentine). The tolerance of L. glaber is higher than that of L. corniculatus (L.) and it has been associated to a higher hypertrophy of the stems of L. glaber under such conditions (Mazanti et al., 1988; Vignolio et al., 1994).. The presence of adventitious roots and higher development of aerenchyma in the stems of plants of L. glaber, which grew in conditions of flooding have also been observed (Vignolio and Fernandez, 1997). Apart from the characteristics of the seeds, a surviving strategy of genotypes in this conditions could be related to the capacity to produce adventitious roots.

On the other hand, it has been proved that there is no difficulty for cloning of L. glaber by rooting of cuttings of the stem in sand or vermiculite, saturated of moisture (Mujica and Rumi, 1998). The utilization of leaves for vegetative propagation could permit to enhance the efficiency of cloning for the replication of genotypes of interest.

The objective of this trial was: I) to evaluate the potential of rooting of stem and leaf cuttings of two populations of L. glaber, one of them natural and the other one improved, in conditions of simulated flooding. II) confirm the capacity for leaves' rooting in different water mediums in the species and evaluated it in two improved populations.

MATERIALS AND METHODS
Experiment 1

The trial was conducted on plastic trays of a volume of 800 cm3. Soil was put in them conforming and homogeneous surface of 3 cm of depth. It was flooded with water up to 1.5 cm over soil. Over this water surface vegetal material was scattered which remained floating. Stem and foliar parts in trial were: medial cutting with one bud, distal cutting (distal bud), distal 3-leaflet, central leaflet and laterals leaflet. The medial cuttings were taken from the base of medial segment of each donor branches according to techniques applied by Mujica and Rumi (1998). The terminal bud was separated from the donor branch, by a cut over the last unfolded leaf. The leaflets were separated manually from the branches. Plants in vegetative state were used from a natural population and from an improved one by a recurrent selection. In both cases a randomized sample of branches (>30cm) of plants that grew in the experimental field, in similar conditions was taken. The experimental unit was a tray containing a sample of n=25. A totally randomized design with 4 replications was used. The experience was developed during October in a greenhouse at 30-33 ºC. The water level of the trays was kept constant. The trial lasted 23 days. At the end the % of rooting was determined.

Experiment 2

A second trial in similar experimental conditions, was conducted. Two improved populations, cv. Tresur Chajá and synthetic variety LH1(non registered) were used. In both cases, a randomized sample of branches (>30 cm) of plants cultivated in the experimental field under the same conditions, was taken. Treatments consisted in two mediums of rooting (water with and without soil) and two leaf components (central leaflet and distal 3-leaflets). The experience took place during November and with the same methodology applied in experience (1). The totally randomized experimental design with 4 replications was used. At the beginning and at the end of the experience a sample of central leaflets (n=20) was taken to measure the thickness of the mesophyll. This anatomical measurements were taken in the zone of the medial third of the central leaflet using a Nikon light microscope equipped with a micrometer ocular with a magnification of 10 x. The t test was applied to analyze differences. The leaf cuttings that rooted which at the end of the experience showed formation of callus on the base of the leaflets, were cultivated in vermiculite with a solution of Nitrofoska BASF (3gr/l).

The analysis of variance of the % of rooting was done in both experiences and Tukey's test was applied for the analysis of differences between treatments.

RESULTS
Experiment 1

The aerial parts of both populations which were evaluated, showed capacity of rooting in the simulated conditions of flooding. The appearance of the first adventitious roots was registered around the 7-12 days after the experience had begun. The stem cuttings obtained from the medial segment of the branches showed lower % of rooting than leaf cuttings in both populations (P<0.01). The natural population showed a higher % of rooting from distal cuttings than the improved population (P<0.01). Leaf components of both populations showed between 83 and 97 % of rooting without significant differences between treatments (Fig. 1). Materials which did not root during the first 12 days showed and increasing deterioration and it was necessary to remove them before the end of the experience.

(Chart) Rooting of different types of cutting
of L. glaber in simulated conditions of flooding.

Figure 1. Rooting of different types of cutting of L. glaber in simulated conditions of flooding. I) medial cutting of stem with one bud, II) distal cutting of stem (distal bud), III) cutting of leaf (distal 3-leaflet), IV) cutting of leaf (central leaflet) and V) cutting of leaf (lateral leaflets). Means followed by different letters differ (P<0.01).

Experiment 2

A capacity for leaf rooting was observed again in the evaluated populations. Nevertheless, the % of rooting was less than in experience (1). The general mean ±S was 45.81±16.66 %. The populations behavior did not present significant differences in the % of rooting of the evaluated leaf components. With respect to mediums of rooting significant differences were found favorable for water without soil (P<0.01) (Fig. 2).

(Chart) Rooting of cuttings of leaves from two
cultivar of L. glaber, in floating conditions
on two different water mediums.

Figure 2. Rooting of cuttings of leaves from two cultivar of L. glaber, in floating conditions on two different water mediums. TRC and TRT: central leaflet and distal 3-leaflet of cv. Tresur Chajá, respectively; LHC and LHT: central leaflet and distal 3-leaflet of cv. LH1, respectively. Means followed by different letters differ (P<0,01).

At the end of the experience the central leaflets as the distal 3-leaflets showed good root development preferably for the water medium composed with water without soil (Fig. 3).

Photograph of two distal 3-leaflets and two central leaflet of L. glaber rooted on the water surface

Figure 3. The photograph shows two distal 3-leaflets and two central leaflet of L. glaber rooted on the water surface.

In both populations it was determined that the mesophyll of the rooted central leaflets showed a significant higher thickness with respect to the initial state (test de t P<0.01). Cuttings which did not root during the first 2 weeks, in general, showed and increasing process of deterioration. For this reason, some of them were removed before the end of the experience. The smaller % of rooting observed in experience (2) respect experience (1) is attributed principally to differences in the phenological state of the donor plants when the cuttings were obtained. Those used in experience (2) were nearer at the beginning of flowering. With respect to the rooted leaf cuttings which showed callus at the end of the experience and were cultivated in vermiculite, it is noticed that during the following 15 days no differentiation of buds was registered.

Acknowledgments

The authors acknowledge Prof. Olga Peluso, CONICET Technician, for their photography contribution

REFERENCES
Mazzanti, A., L. Montes, D. Miñon, H. Sarlangue y C. Cheppi. 1988. Utilización de Lotus tenuis en establecimientos ganaderos de la Pampa Deprimida: Resultados de una encuesta. Rev. Arg. Prod. Animal 8(4): 301-305.

Mujica, M. M. y C. P. Rumi. 1998. A technique of vegetative propagation by stem cuttings was fitted to Lotus tenuis. Lotus Newsletter. U.S.A. (ISSN 0316-0106) (http://www.plantsci.missouri.edu/lnl/), vol 29, (en prensa).

Vignolio, O.R., N.O. Maceira y O.N. Fernandez. 1994. Efectos del anegamiento en invierno y verano sobre el crecimiento y la supervivencia de Lotus tenuis y Lotus corniculatus. Ecología Austral 4: 19-28.

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