A. Ronse, National Botanic Garden of Belgium, 1860 Meise, Belgium
Jovellana punctata and J. violacea were micropropagated by adventitious shoot formation from nodal stem segments on modified ¼MS. Shoots were easily rooted with IAA, and plants grew well in the glasshouse.
The genus Jovellana consists of 6 herbaceous or subshrubby species, native to temperate parts of South America and New Zealand (Kränzlin 1907; Encke 1987). They belong to Scrophulariaceae and are most closely related to Calceolaria, from which they differ in the form of the flowers. Three species are occasionally cultivated in mild climates where hot summers are infrequent; however, little is known about the cultivation of these plants (Everett 1981). Two of these species, J. punctata Ruiz et Pav. and J. violacea (Cav.) G. Don, come from Chile. The former grows in humid 'matorrales' (transitional woodlands) in S. Chile, the latter in the coastal zone (Hoffmann 1982).
Living plants of J. punctata and J. violacea were collected in Chile and sent to the National Botanic Garden of Belgium. As the plants did not grow well, especially J. violacea , it was decided to try to micropropagate them.
Jovellana violacea was collected in Valdivian forest in S. Chile (voucher F. Billiet 3732) and J. punctata (voucher F. Billiet 3920) in Alto do Rocoto, near the mouth of the Rio Biobio in 1985. Since more material was available of the latter, preliminary experiments were done with this species.
Nodal stem segments of 0.5-1.0 cm and leaf segments of about 1 cm² were used as explants. The excised plant parts were rubbed with a cotton
plug soaked in ethanol and subsequently immersed in a mixture of 0.3% sodium chlorite with 1.4% lactic acid (Alcide) and a few drops of Tween 20 wetting agent for 10 and 20 min for leaf and stem segments respectively, or in a solution of 5% NaOC1 for 10 min. The segments were rinsed three times with autoclaved distilled water.
In the first experiment the macro element and iron-EDTA solutions of Murashige and Skoog (1962) medium (MS) were used together with micro elements and vitamins of Nitsch and Nitsch (1969), 0.85% agar (purified, Merck 1614) and 0.25% sucrose. Depending on the treatment, naphthaleneacetic acid (NAA) or benzyladenine (BA) were added. Five treatments were carried out, each one consisting of 24-30 leaf and stem segments of J. punctata. The treatments consisted of the following plant growth regulators:
1) 0.1 mg/1 NAA
2) 0.1 mg/1 BA
3) 0.1 mg/1 NAA + 0.5 mg/1 BA
4) 1.0 mg/1 NAA + 0.1 mg/1 BA
5) no growth regulators
A second experiment was carried out with the same medium, but with the macro elements at one quarter of the original concentrations (¼MS). Leaf and stem explants of J. punctata were cultured on eight different media (Table 1). Each treatment consisted of 6 explants.
The third experiment was carried out with nodal segments of J. punctata and J. violacea on ¼MS with four different treatments:
1) no growth regulators
2) 0.2 g charcoal
3) 1.0 g charcoal
4) 0.1 mg/1 NAA + 0.1 mg/1 BA
For every treatment at least eight explants were cultured.
For rooting shoots from proliferating cultures ¼MS + 0.1 or 1.0 mg/1 indoleacetic acid (IAA) was used.
The surface sterilisation procedure gave satisfactory results: 60% and 80% as an average for stem and leaf segments respectively. On full strength MS in the first experiment, all explants died, whatever the treatment.
In the second experiment all the leaf explants died. However, most stem segments showed axillary bud elongation, while others proliferated by the formation of adventitious buds (Table 1). The highest proliferation rate was obtained with 0.1 mg/1 NAA + 0.1 mg/1 BA: after 10 weeks 3 explants were obtained from one original explant. Proliferation also occurred with 1.0 g/1 charcoal, but only 2 explants per explant were obtained after 10 weeks, and they showed some hyperhydricity.
Table 1. Response of nodal segments of J. punctata on ¼ MS with various plant growth regulators or charcoal concentrations (mg/1).
TREATMENT RESPONSE (mg/l) 0.1 NAA (contaminated) 0.1 NAA + 0.1 BA proliferation x 3 0.1 NAA + 1.0 BA (contaminated) 1.0 NAA dead 1.0 NAA + 0.1 BA elongation of buds 1.0 NAA + 1.0 BA elongation of buds 1.0 BA elongation of buds 1000 charcoal proliferation x 2
In the third experiment, the treatments causing shoot proliferation in J. punctata were tried with J. violacea. The effect of a lower rate of charcoal was also investigated, as well as the effect of no growth regulators or charcoal. J. violacea also responded with the highest proliferation rate in the treatment with 0.1 mg/1 NAA ± 0.1 mg/1 BA. Initially, some proliferation was found without growth regulators and with charcoal at both concentrations, but at a lower rate. After several months, the proliferation rate with 0.2 g/1 charcoal increased, whilst it decreased with growth regulators. Shoots grown on charcoal grew more vigorously and had larger leaves.
Shoots all rooted within 6 weeks with 0.1 or 1.0 mg/1 IAA. Plantlets were successfully (>90% survival) transferred to the greenhouse in a standard potting mixture and gradually weaned to a lower air humidity. The plants grew well, but after some months J. violacea showed severe deficiency symptoms, probably due to iron deficiency. This is in accordance with the fact that the original plant was collected in a forest with acid soils (pH 4.5). The symptoms disappeared after a few treatments with a mixture of chelates.
Both J. punctata and J. violacea were micropropagated from nodal segments by the formation of adventitious buds on modified ¼MS. Rapid proliferation was obtained on a medium with 0.1 mg/1 NAA + 0.1 mg/1 BA for three months, followed by culture on the same medium without growth regulators and with 0.2 g/1 charcoal. Leaf segments did not grow on the media tested. On a medium with full macro salt strength, no growth was obtained; apparently the higher salt concentration was lethal to the explants. Proliferated shoots of both species could be readily rooted with IAA. Plantlets were successfully transplanted to the greenhouse.
The author would like to thank Miss A. Van de Vijver for the technical handling of the cultures.
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