Micropropagation of Ilex dumosa (Aquifoliaceae) from nodal segments in a tissue culture system.

Micropropagation of Ilex dumosa var. dumosa R. ("yerba señorita") from nodal segments containing one axillary bud was investigated. Shoot regeneration from explants of six-year-old plants was readily achieved in 1/4 strength Murashige and Skoog medium (1/4 MS) plus 30 gr x L(-1) sucrose and supplemented with 4.4 microM BA. Further multiplication and elongation of the regenerated shoots were obtained by subculture in a fresh medium of similar composition with 1.5 gr x L(-1) sucrose. Rooting induction from shoots were achieved in two steps: 1) 7 days in 1/4 MS (30 gr x L(-1) sucrose, 0.25% Phytagel) with 7.3 microM IBA and 2) 21 days in the same medium without IBA and 20 microM of cadaverine added. Regenerated plants were successfully transferred to soil. This micropropagation schedule can be implemented in breeding programs of Ilex dumosa.


Introduction
The genus Ilex belongs to the Aquifoliaceae family.More than 500 species are recognized occurring in tropical and temperate regions of the world.Most of them are deciduous or evergreen trees or shrubs (Hu, 1989).Many species are planted in Europe, Asia and USA for their ornamental value (Hu, 1989;Obeso et al., 1998;Setoguchi and Watanabe, 2000;Walden and Wright, 1995).Approximately 220 species are native to South America and one of these, I. paraguariensis, is economically the most important specie because of the value of its leaves for making the stimulatory beverage named "maté".Additionally, other plant species such as I. guayusa, I. terapotina and I. vomitoria are used in infusions (Loizeau, 1994).Lately, "yerba señorita" (I.dumosa) is the specie that probably has received the most attention from plant breeders because, besides of the quality of its leaves for making "maté" with less caffeine than the ones from I. paraguariensis (Filip et al., 1999;2001), the plants are resistant to some pests (Prat Kricun and Belingheri, 1995).
Micropropagation is widely recommended as a biotechnological tool for the multiplication of select plants (Hu, 1989;Mroginski et al., 1997).There have been two in vitro studies on I. dumosa, including zygotic embryo culture for recovering plants from rudimentary embryos (Sansberro et al., 2001a) and plant regeneration from nodal segment containing single axillary buds (Sansberro et al., 2001b).In the present study, we describe a procedure for micropropagation of I. dumosa from axillary buds and the rooting and the establishment of the plantlets in soil.

Plant material.
Six-year-old plants of Ilex dumosa var.dumosa R. grown in pots maintained under greenhouse conditions, were used as a source of explants.The plants were kindly provided by Estación Experimental Agropecuaria INTA, Cerro Azul, Misiones, Argentina.Explants cultured were 1.5 to 2 cm long segments of stems containing one axillary bud.
The explants were collected from young nonlignified branches, surface sterilized in 70% ethanol for 1 min and 1.5% NaOCl with 0.1% TRITON ® for 30 min, and washed with several rinses of sterile distilled water.

Synthetic medium and physical culture conditions.
The experiments were done using the nutrient medium reported by Rey et al. (1991).It consisted of quarter-strength Murashige and Skoog (1962) and, unless otherwise stated, 30 gr•L -1 sucrose and 0.65% SIGMA agar (A-1296).This medium as referred to as 1 /4 MS.In some experiments various concentrations of auxins and cytokinins were added to 1 /4 MS.The pH of the media was adjusted to 5.8 with KOH or HCl prior to adding the gelling agent.The tubes were covered with aluminum foil and autoclaved at 1.46 kg•cm -2 for 20 min.
The explants were cultured on 3 ml synthetic medium in 11 ml glass tubes.The tubes were sealed with Resinite AF 50 ® (Casco S.A.C. Co. Buenos Aires, Argentina) and incubated in a growth room at 27 ± 2°C with 14 h photoperiod (116 µmol•m -2 •s -1 , from fluorescent lamps).

Effects of cytokinins on multiple shoot formation.
To determine the optimal conditions for both axillary bud proliferation and shoot production during the establishment phase of single-node explants, kinetin (KIN), 6-benzylaminopurine (BA), zeatin (ZE) and isopentenyladenine (2iP) at doses of 0.5, 2.5 and 4.5 µM were studied.Thidiazuron (TDZ) at doses of 0.1, 1 and 10 µM was included.
Effect of BA and naphtaleneacetic acid (NAA) on multiple shoot formation from single-node explants.
Effect of sucrose and BA on shoot multiplication.

Root induction.
Shoots (10-15 mm) obtained from the establishment of nodal segments were rooted according to the procedure reported by Sansberro et al. (2001b).It consisted of two steps: 1 st ) Induction.Regenerated shoots were cultured for 7 days on 30 ml root-induction medium composed of 1 /4 MS plus 30 gr•L -1 sucrose, 7.3 µM indolebutyric acid (IBA) and 0.25% Phytagel ® (P-8169, Sigma Chemical Co.) in 100 ml glass jars. 2 nd ) Expression.The shoots were subcultured for 21 days on a fresh medium composed by 1/4 MS lacking IBA.
In another experiment, the basal medium was supplemented with 10 or 20 µM of cadaverine, naringenin, rutin or rivoflavin during either the induction or the expression phase.
All cultures were incubated in the same physical conditions described above.

Transfer to soil.
The plantlets obtained in vitro were carefully washed under running water and set into trays containing twenty four 150 ml cavities filled with a mixture of peat moss, perlite and vermiculite (1:1:1 v/v) and grown in a humid chamber with 160 µmol•m -2 •s -1 illumination at the level of the plantlets.During the experiments, the ambient temperature ranged from 22 to 27°C.By using a mist systems, the RH was maintained at 90% during the first 7 days and, gradually was decreased until 70%.After 60 days of incubation, the percentage of the survival plant were recorded.The height of the plant (shoot length) and the number of leaves per plant were also measured.

Experimental design and statistical analysis.
In all cases, 10 explants were cultured per treatment.The treatments were arranged randomly on the shelves in the growth room.Each experiment was repeated 3 times.The results presented are the means of the replications with the standard error (±SEM).Analysis of variance (ANOVA) was performed and comparisons of means were conducted using either Tukey or Dunnett's Multiple Comparison Test.All analyses were regarded as significant at P< 0.05.
Multiplication rate was defined as the number of shoots with less or more than 5 mm in length formed by each nodal segments cultured.

Results and Discussion
Effect of sucrose on shoot-development.
Three main characteristics in the explants appeared within one week of culture: 1) browning, 2) contamination with microorganisms (bacteria and/or fungi), and 3) the explants remained green and eventually some of them sprouted (these explants were considered as established).
After 30 days of culture, the results (Table 1) show that the contamination with microorganisms was relatively low (2-13%) and the browning of the explants was clearly affected by the concentration of sucrose, being nil in concentrations of sucrose ranged from 7.5 to 30 gr•L -1 and, in consequence, resulted in a high per-centage (87-100%) of explants established.These results are not in agreement with those reported previously for both meristem and nodal segment culture of I. dumosa in which low percentage of explants were suc-    cessfully established (Sansberro et al., 2001b).It is likely due to the careful preparation of the donor plants used in the present experiments.The season of the year also could be important as was found for the related species I. paraguariensis (Bernasconi et al., 1998).
After 45 days of culture in 30 gr•L -1 of sucrose, as much as 47% of the established explants sprouted with subsequently shoot formation of more than 5 mm long (Table 2).Although in most of the cases, single shoots were formed from one nodal segment (Fig. 1B), in less than 5% of the explants cultured, multiple shoots (Fig. 1C) were formed.
The importance of sucrose for both establishment and multiplication stages of micropropagation has been pointed out by several authors (Jusaitis, 1997;Marino et al., 1993), including plant regeneration of I. paraguariensis by in vitro culture of nodal segments (Sansberro et al., 2000).
On the other hand, it was found that the reduction of carbohydrate content in the culture medium generally increases photosynthetic rates (Langford and Wainwright, 1987;Galzy and Campan, 1988;Hdider and Desjardins, 1994) and promotes autotrophy (Deng and Danelly, 1993).
The fact that I. dumosa requires a relatively low level (15 gr•L -1 ) of sucrose for vegetative growth in vitro permits to think up in an autotrophic system for micropropagation.Reducing sucrose in the medium might have also a favorable effect on the acclimatization stage.

Effects of cytokinins on multiple shoot formation.
Table 3 summarizes the morphogenetic responses of the explants after 45 days of culture.Although buds or shoots proliferation (shoot with less or more than 5mm in length, respectively) occurred in all of the combination tested, this response was greatly affected by both the kind and the concentration of the cytokinins employed.An analysis of the results presented in Table 3 permits the following conclusions: 1) A high percentage (63%) of nodal segments produced multiple buds with the highest multiplication rate (4.1) when grown on 1/4 MS supplemented with 4.5 µM BA.Similar results were obtained by using 1/4 MS supplemented with 4.5 µM 2iP.
2) The highest percentage of multiple shoots were obtained in medium supplemented with some concentrations of KIN, ZEA or TDZ.
These results are in agreement with those reported with I. paraguariensis where the usefulness of BA for micropropagation was demonstrated (Mroginski et al., 1999;Sansberro et al., 2000).

Effect of BA and NAA on multiple shoot formation from single-node explants.
BA had a significant effect on the induction of multiple buds and/or shoots (4-5/explants).The highest percentage (53%) of explants with multiple buds was achieved by using 1/4 MS + 4.4 µM BA (Table 4).On the contrary, cultures with NAA had significant reduction in the production of multiple buds and the highest concentration (5.4 µM) of NAA resulted in complete inhibition of bud formation even when combined with BA (Table 4).These negative effects of NAA on micropropagation has been demonstrated in a number of plant species, including I. paraguariensis (Mroginski et al., 1999;Sansberro et al., 1999).

Effect of sucrose and BA on shoot multiplication.
Multiple shoots were best induced on highest level (2.2 and 4.4 µM) of BA where 84-97% of the explants produced multiple shoots with an average of 6.5-7.3 shoots/explant.Although changes in the level of sucrose did not modify these values (Table 5), 30 gr•L -1 sucrose increased significantly the proliferation of calli at the basis of the regenerated shoots (Fig. 1D, E).Rooting and acclimatization of the regenerated plants.
After 28 days of culture, the highest percentage (38%) of rooted shoots (Fig. 2A) was achieved in the control treatment.The additions of cadaverine, naringenin, riboflavin or rutin delayed the percentage of rooting (Table 6).However, supplementation of the medium with 20 µM cadaverine during the expression phase nearly trippled the number of roots/shoot (Table 6).Previous studies in various plant species showed that the naturally occurring polyamines such as putrescine, spermine and spermidine have an important role in the control of adventitious rooting from cuttings (Heloir et al., 1996;Hausman et al., 1997), while the specific role of cadaverine in the growth and development processes in plants is not known (Apelbaum, 1990).
The acclimatization of the plants was improved by the addition to the culture medium of either cadaverine, naringenin, riboflavin, or rutin, which permitted 100% survival of plantlets (Table 7).After 60 days of accli-matization, plantlets had well-developed shoots (3-6 cm) with good root systems (Fig. 2B) and did not show any morphological abnormalities (Fig. 2C).
3.3 b 70 50 ± 4.9 3 ± 3.0 47 ± 3.3 a Values are mean ± SEM of three independent experiments.Means in each column followed by different letters are different according to Tukey's Multiple Comparison Test (P≤0.05).

FIGURE 1 .
FIGURE 1. Morphogenetic responses of Ilex dumosa during the establishment (A-C) and multiplication (D, E) stages.A) non-sprouted bud, B) shoot growing more than 5 mm, C) multiples shoot obtained from nodal segments.Multiple shoots regeneration obtained by established shoot cultured on 1 /4 MS plus 2.2 µM BA and supplemented with 15 (D) or 30 gr.L -1 (E) of sucrose.Bar indicates 5 mm.

FIGURE 2 .
FIGURE 2. Rooting and acclimatization stages of Ilex dumosa micropropagation.A) In vitro rooting of shoots, B-C) plantlets after 60 days of acclimatization under the environmental conditions described in material and methods.Bars indicate 5, 10 and 40 mm in A, B, and C, respectively.

TABLE 2 . Effects of sucrose concentration on shoot production from single-node explants of I. dumosa established in vitro
* Only shoots with more than 0.5 cm in length were scored.Values are mean ± SEM of three independent experiments.Means in each column followed by different letters are different according to Tukey's Multiple Comparison Test (P≤0.05).

TABLE 4 . Effects of NAA and BA in 1 /4 MS medium (with 30 gr•L -1 sucrose) on multiple bud induction from nodal segments of Ilex dumosa established in vitro
Values are mean ± SEM of three independent experiments.Means in each column followed by different letters are different according to Tukey's Multiple Comparison Test (P≤0.05).

TABLE 6 . Effect of cadaverine, naringenin, riboflavin, and rutin on rooting of shoots of Ilex dumosa obtained through in vitro culture of single-node explants
Values are mean ± SEM of three independent experiments.* Differences are significant respect to the control at P>0.05 (Dunnett's Multiple Comparison Test).

TABLE 7 . Effect of cadaverine, naringenin, riboflavin, and rutin added to rooting me- dium on the acclimation of plantlets of Ilex dumosa
Values are mean ± SEM of three independent experiments.*Differences are significant respect to the control at P>0.05(Dunnett's Multiple Comparison Test.