INDUCTION OF SOMATIC EMBRYOGENESIS FROM LEAF
EXPLANTS OF Stevia rebaudiana

JOÃO CARLOS BESPALHOK FILHO 2 , JORGE MINORU HASHIMOTO 3 and
LUIZ GONZAGA ESTEVES VIEIRA

Area de Ecofisiologia, Instituto Agronômico do Paraná, CP 1331, Londrina,
PR, CEP 86001-000

ABSTRACT- Somatic embryos of Stevia rebaudiana (Bert.) Bertoni were induced when in vitro leaves were cultivated on MS media with 2,4-D (10 or 25 mM), BA (l mM) in a high sucrose medium (120 g/L). The embryos appeared to be formed directly without callus formation. Somatic embryos failled to mature and developed roots but not shoots on a MS medium without growth regulators. Additional index terms: BA, 2,4-D, estevioside.

INDUÇÃO DE EMBRIOGÊNESE SOMÁTICA DE Stevia rebaudiana A PARTIR DE EXPLANTES FOLIARES

RESUMO- Embriões somáticos de Stevia rebaudiana (Bert.) Bertoni foram induzidos quando folhas in vitro foram cultivadas em meio MS com 2,4-D (l0 ou 25mM) e BA (1mM) com alta concentração de sacarose (120 g/L). Os embriões somáticos não se tornaram maduros e quando foram inoculados em meio MS sem reguladores de crescimento formaram raízes mas não formaram parte aérea. Termos adicionais para indexação: BA, 2,4-D, esteviosídeo.

INTRODUCTION
Stevia rebaudiana is an herb from the Asteraceae family indigenous from the higher elevations of Northern Paraguay near the Braziliam borders (Soejarto et al, 1983). Due to non-caloric sweeteners extracted from its leaves, mainly stevioside, this plant has gained importance as a crop for the pharmaceutical and food industries. Somatic embryogenesis has been described for more than a hundred plant species (Terzi & Loschiavo, 1990), but the number of reports of somatic embryogenesis among members of the Asteraceae family is still low (May & Trigiano, 1990). The development of protocols for regeneration of Stevia rebaudiana via somatic embryogenesis is important as this technique can be used in the clonal propagation of this plant, or as explant material for protoplast isolation and regeneration (Puite,
1992). The objective of this work was to develop a protocol for the induction of somatic embryogenesis from leaf
explants of Stevia rebaudiana.

MATERIALS AND METHODS
The nutrient basal medium used in all experiments consisted of the inorganic salts of Marashige & Skoog (1962) supplemented with: 99 mg/L myo-inositol, 2 mg/L thiamine- HCl, 2 mg/L nicotinic acid, 87 mg/L cysteine, 30000 mg/L sucrose, and solidified with 9000 mg/L Difco Bacto agar. The pH was adjusted to 5.7 prior to autoclaving at 121 o C during 15 min. Plants of Stevia rebaudiana used on the experiment were kindly given by Maringa State University, PR, Brazil, and were grown in a greenhouse. Nodal stem segments with 2 cm were excised from these plants, disinfestated
in a solution of sodium hipoclorite (1.5%) for 15 min and then rinsed for three times with sterilized water. These nodal segments were cultivated on basal medium and after 15 days lateral buds with 3 to 4 pairs of leaves developed. In vitro formed leaves with lateral buds were then used for experiments about embryogenesis. To investigate the influence of growth regulators on the induction of somatic embryogenesis, in vitro leaves of Stevia rebaudiana (2 cm) obtained as described were inoculated on basal medium containing 10 or 25 mM 2,4-dichlorophenoxy acetic acid (2,4-D) and 1 or 5 mM 6-Benzylaminopurine (BA) under a high concentration of sucrose (120 g/L). Five leaves were inoculated per
flask of 200 mL containing 50 ml of medium. Each treatment consisted of 10 flasks. The explants were incubated for 21 days in the dark, 7 days in light under a photoperiod of 16 h provided by cool white flurescent lightening of 30 mmol s -1 m -2 of photosynthetic active radiation finally followed by more 7 days in the dark. The number of somatic embryos was record with aid of a stereomicroscope. Explants with somatic embryos were transfered to basal medium without growth regulators and with the concentration of sucrose lowered to 30 g/L.

RESULTS AND DISCUSSION
Somatic embryogenesis was induced if in vitro leaves of Stevia rebaudiana were cultured on high sucrose medium containing 2,4-D and BA. Somatic embryos were first observed 15 days after culture initiation.

The majority of the somatic embryos appeared to form directly without intermediate callus formation (Fig. 1). Medium with 1 mM was superior to 5 mM of BA according to the number of somatic embryos induced (Table 1). The levels of 2,4-D tested in this study induced similar numbers and percentage of somatic embryos. TABLE 1- Effect of 2,4-D and BA on the number and percentage of somatic embryos formed on in vitro leaves of Stevia rebaudiana.

2,4-D BA somatic embryos/leaf explant percentage ofexplants forming somatic embryos
(M) (M)    
10 1 6.98 a^ z 98
10 5 0.37 b 23
25 1 9.06 a 93
25 5 0.62 b 20
z means followed by same letters are not signicantly different by the Tukey (P< 0.05).

Wada et al (1981) and Miyagawa et al (1986) cited by Handro & Ferreira (1989) reported somatic embryogenesis in Stevia rebaudiana. Wada et al (1981)
reported the induction of somatic embryos from leaf explants on a medium supplement with the cytokinin N-(4-Pyridyl)-N’-phenylurea (4-PU). Miyagawa et al
(1986), cited by Handro & Ferreira (1989), induced somatic embryos from stem explants on media containig 2,4-D. In both reports, however, the somatic embryos were induced indirectly (from callus) and plant regeneration was not achieved. In this work, somatic embryos appeared to form directly without callus formation. High levels of sucrose have been used with success here and in various protocols for induction of somatic embryogenesis in other members of Asteraceae family: Chichorium (Guerida et al, 1989); Helianthus annus (Finer, 1987); Dendrathema grandiflora (May & Trigiano, 1991). The influence of high levels of sucrose on the induction of somatic embryogenesis may be due to increased osmotic potential of the media. Somatic embryos failed to mature. When the embryos were transfered to media without growth regulators an a lower concentration of sucrose (30 g/L), they developed a root but failed to form the shoot apex. In order to germinate, embryos must have functional shoot and root apices capable of meristematic growth (Merkle et al, 1990). More experiments are being performed in order to develop suitable media for maturation and germination of somatic embryos from Stevi a leaves.

ACKNOWLEDMENTS
To the Brazilian National Council for Cientific and Technological Development (CNPq) for the financial support to this work.

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