In vitro propagation of Otacanthus coeruleus Lind

Plant Cell, Tissue and Organ Culture 30: 243-245, 1992. © 1992 KluwerAcademic Publishers. Printed in the Netherlands. Micropropagation note In vitro propagation of Otacanthus coeruleus Lind. Anne C. Ronse 1 & Maurice P. De P r o f t 2 INationale Plantentuin van Belgi& Dienst der Levende Verzamelingen, Domein van Boechout, B-1860 Meise, Belgium; 2Laboratorium voor Plantenteelt, Faculteit der Landbouwwetenschappen, Katholieke Universiteit Leuven, De Croylaan 42, B-3030 Leuven, Belgium Accepted in revised form 2 March 1992 Key words: micropropagation, organogenesis, Scrophulariaceae Otacanthus coeruleus is an herbaceous species of the Scrophulariaceae, originating from east Brazil and naturalized on the Mascarenes and the Seychelles (Billiet & Heine 1980). This species was cultivated in the last century as an ornamental in Belgium and France (Van Houtte 1862), but this culture seems to have disappeared completely. It has been rediscovered and reintroduced into cultivation (Billiet 1982), so that it is now cultivated as an ornamental in the USA and Australia. Otacanthus coeruleus has potential as a cut flower crop (Geertsen 1990). In this study the organogenic response of Otacanthus coeruleus in vitro to different growth regulators was investigated and a suitable culture medium for micropropagation was developed. Five-year-old plants of Otacanthus coeruleus, grown in the greenhouses of the National Botanic Garden of Belgium, were used as the explant source. These plants originated from two plants collected on La R6union (Mascarenes) in 1980. Three kinds of explants were used: stem segments of 1 cm length from the upper half of the stem, with or without nodes, and leaf pieces of i cm 2 from fully expanded green leaves. Stem segments were disinfested by rubbing them with a cotton plug imbibed in ethanol, then immersing them either for 30 min in a mixture of 0.3% sodium chlorite with 1.4% lactic acid or for 10 min in a solution of 10% NaOCl, both with a few drops of Tween 20 emulsifier. The segments were then rinsed three times with autoclaved distilled water. The leaf pieces were disinfested in a similar way, but for only half the above mentioned periods. All treatment were carried out twice at different times with newly started cultures. During the first and second replication each treatment consisted of 12 and 10 explants respectively. All cultures were grown in test tubes (diameter 25 mm) containing 15 ml medium and capped with Cap-O-Test. They were placed in a growth room with day/night temperatures of 27° ± 3°C/ 20°---I°C, with a 16-h photoperiod provided by fluorescent lamps (Philips TL D33 cool white). The photosynthetic photon flux at plant height ranged from 50 to 70 ixmol m -2 s -1 The basal medium, which was used for all treatments, consisted of the macro-elements and the iron-EDTA solution of Murashige and Skoog (1962), the micro-elements and vitamins of Nitsch and Nitsch (1969), with 0.6% agar (purified, Merck 1614) and 2.5% sucrose. The pH was adjusted to 5.6 before autoclaving. The growth regulators used were naphthaleneacetic acid (NAA) (Merck nr 815), benzyladenine (BA) (Fluka AG nr 13151, purum) and 2,4dichlorophenoxyacetic acid (2,4-D) (Merck nr 820451), added to the basal medium in different combinations (for details see Table 1). For root induction of proliferated shoots, 1 g 1-1 charcoal (Carbon black, UCB 1242, p. analyse) was added. For regeneration of plantlets from callus, calluses were placed on a medium containing the macro- and micro-salts of Monnier (1976) with 3.0% sucrose. The organogenic response of the stem seg- 244 Table 1. Organogenic response of nodal stem segments of Otacanthus coeruleus to various types and concentrations of plant growth regulators (in % of explants). Plant growth regulators (~.M) Callus formation R1 - N A A 0.5 NAA 0.5 NAA 0.5 NAA 0.5 NAA 5.0 BA 2.2 BA 13.2 2,4-D 0.5 2,4-D 2.5 + + + + BA 0.44 BA 2.2 BA 4.4 BA 2.2 Outgrowth of nodal buds R2 R1 Shoot proliferation 1 R2 R1 R2 0 0 9 12 0 0 0 43 6 100 88 0 0 37 0 50 86 50 17 100 43 0 43 100 43 43 24 0 0 0 0 5 0 75 86 20 0 0 43 0 43 0 0 43 35 0 0 0 0 0 0 25 86 80 67 29 57 0 29 0 The results of two replications are given, consisting of 12 and 10 explants, respectively. R1 and R2: percentage of explants with a particular response in replication 1 and 2, respectively. 'Shoots formed from adventitious buds. ments to the various treatments is given in Table 1. In general the second replication resulted in similar responses as the first one, but with a higher intensity of reaction. Callus formation was induced on four different growth regulator combinations, especially with 5.0 ixM N A A + 2.2 txM BA. This treatment resuited in fast-growing callus that yielded the highest weight of all (data not shown). Shoot proliferation was obtained in seven treatments during the second replication, but only in two treatments during the first. These two treatments (0.5 IxM NAA+0.441xM BA and 0.5 IxM N A A + 2.2 txM BA) yielded the highest proliferation, especially the latter. Its proliferation rate amounted to 6 new explants per explant every 2 weeks. Elongation of axillary buds without formation of adventitious buds occurred on six media, mainly with 0.5 ixM N A A + 0.44 ~M BA and with 0.5 IxM N A A alone. The stem explants died on the medium with 13.2 txM BA. Leaf explants turned brown and died in all treatments, except with 5.0 IxM NAA + 2.2 IxM BA, which induced callus (data not shown). Callusses from stem segments placed on Monnier's medium turned green, and 20% of them produced shoots after 6 weeks. The shoots, originating from the treatment with 0.5 p~M N A A + 2.20 i~M BA, that were put on medium with 1 g 1-' charcoal developed roots after 4 weeks in 92% of the cases. Rooted plantlets were transferred to the greenhouse in July, where 80% survived, and flowered in May of the following year. It can be concluded that O. coeruleus is rather easy to micropropagate. This is in agreement with the rapid in vitro organogenic responses found for related Scrophulariaceae (Kamada & Harada 1979; Sangwan et al. 1976). Acknowledgement We wish to thank Miss A. Van de Vyver for her valuable technical assistance. References Billiet F (1982) Otacanthus coeruleus reintroduced into cultivation (Scrophulariaceae). Baileya 21:189-192 Billiet F & Heine H (1980) Otacanthus coeruleus Lindley, une Scrophulariac4e br6silienne naturalis6e aux ties Mascareignes et aux Seychelles. Adansonia s6r 2, 20:297-304 Geertsen V (1990) The keeping quality of Otacanthus coeruleus- a potential new cut flower. Scientia Hort. 43: 145-153 Kamada H & Harada H (1979) Influence of several growth regulators and amino acids on in vitro organogenesis of Torenia fournieri Lind. J. Exp. Bot. 30:27-36 245 Monnier M (1976) Culture in vitro de l'embryon immature de Capsella bursa-pastoris Moench. Rev. Cytol. Biol. Veg. 39:1-120 Murashige T & Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15:473-497 Nitsch JP & Nitsch C (1969) Haploid plants from pollen grains. Science 163:85-87 Sangwan RS, Norreel B & Harada H (1976) Effects of kinetin and gibberellin A 3 on callus growth and organ formation in Limnophila chinensis tissue culture. Biol. Plant. 18:126-131 Van Houtte L (1862) Flore des serres et des jardins de l'Europe. Gent