In vitro organogenesis in Albizia guachapele , Cedrella odorata and Swietenia macrophylla ( Fabaceae , Meliaceae )

Abstrae!: Regeneration of adventitious buds was achieved from hypocotyl explanls of Albizia guachapele (Guayaquil) and Cedrella odorata (Spanish cedar), and from epicotyl explants from Swietenia macrophylla (Honduran Mahogany). Seeds were obtained from CATIB's Latin American Fores! Seed Bank and genninated under aseptic conditions .. Four explants were cultured in each Petri dish on half strength modified Murashige and Skoog basal medium, and five concentrations of BA (benzyladenine) were studied; A. guachapele and S. macrophylla responded positively lo the presence of BA in the culture medium. Otherwise, Cedrella odorata requíred media supplemented with citokinin and auxin combinations lo induce adventitious buds.

During the last years, Central America has been affected by an increasing deforestation.At present, the deforestation rhythm reaches 370 000 to 400 000 hectares.Dne can calculate that at this rate, in year 2000 only will remain smal1 forested areas in highlands, in regions far from the Atlantic coast and in protected areas such as National Parks (Funes 1992).As a consequence a population decrease occurred, as will as a genetic erosion that does not guaranty the in situ survival of many specíes (Jiménez Madrigal 1993).Therefore there is an urgency to look for new alternatives that would allow us to multiply and maintain the genetic pool of those endangered spedes.
There has been considerable progress made in regeneration plantlets of tropical hardwood trees through tissue culture (Monteuuis & Bon 1996, Etienne et al. 1997, Valverde Cerdas el al. 1997).More recently Maruyama et al. (1997), developed the protocols for the germplasm conservation of Cedrella odorata, Jacaranda mimosaefolia D. Don, and Guazuma cnnita al temperature conditions aboye freezing by alginate encapsulation of shoot tips in the production of artificial seeds.
Guayaquil, Spanish cedar and Honduran Mahogany are important timber yieldíng trees and their economical and ecological importance warrants the application of tissue culture techniques for their donal propagation and improvement.

MATERIALS AND METHODS
Albizia guachapele, Cedrella adorata and Swietenia macrophylla seeds stored at CATIE's Latín American Forest Seed Bank under a temperature of 5 oc, were used for this research.The seeds were washed with water and soap and kept under running water for 30 mino They were surface sterilized with 70 percent alcohol for three s and immersed for 20 mÍn in a 5.5 percent commercial sodium hypochlorite solution and then washed wíth sterile distilled water.
Explants were distributed in an irr estricted design, with five benzyladenine concentrations: 8.8, 17.7,26.6,35.5 and 44.4 !!M.Another assay was designed for C. odorata where benzyladenine (BA) and naphthaleneacetic acid (ANA) were combined in a 5x4 factorial design with concentrations of 4. 4, 8.8, 13.3, 17.7, and 22.2 !!M BA; O, 2.6, 5.3 and 10.7 !!M ANA Response was evaluated at the end of the experiment and recorded as percentage of explants with buds, average number of buds per explants and total number of buds.
The statistics used was variance analysis.The data requiring conversion were transfonned as x=-Vx+0.5.For A. guachepele and S. macrophylla variables, CATMOD procedure for X2 (Grizzle et al. 1969) was used.Each experimental unit consisted of a Petri dish containing four explants and forty repetitions.

RESULTS
A. guachepele, hypocotyls and S. macrophylla epicotyls responded to the presence of BA in !he culture medium by fonning adventitious bud capable of developing.That was observed between 30 and 40 days in Guayaquil and between 20 and 40 days in Honduran mahogany.
For A. guachapele, the percentage of explants with buds, the total number of buds and the average number of buds per explant, showed significant differences (X2=10.7, p=0.02) between the BA concentrations.The highest percentage of explants with buds (20%) and the highest total number of buds (14.5) was obtained with 17. 7 11M BA.The highest average number of buds per explant (2.5 buds) were obtained with 35.5 !!M BA (TabIe 1).At this concentration a better bud development was observed.
In the case of S. macrophylla, the largest amount of total buds was obtained at 44. 4 11M BA The average percentage of explants with buds ranged around 15% and the differences between treatments were not statistically different at the 5% leveL Lineal tendency (X2=9.7,p=O.05) was observed for the total number of buds and the average number of buds per expIant which increased with the BA concentration (Table 2).C. odorata was the only species which did not respond organogenically to the BA presence.TIte explants were organogenic only when BA was combined with an auxin such as NAA, which indicates that this species required the presence of an auxin into the culture medium.
TIte highest total number of adventitious buds (5 buds) was obtained with 17.7 J.I.M BA and 5.3 J.1.M NAA, and tbere were not significant differences between treatments.TItese buds were visible after 40 to 45 days of culture.

DISCUSSION
TIte results obtained in this study indicate the potential in vitro propagation of the three tropical forest species investigated.However, their in vitro requirements need to be further explored to establish the pattem for production of large number of propagules.AH the studied species showed the same bud formation pattem: a direct organogenesis without callus fonnation.
Tbis work describes for the frrst time the adventitious bud induction in Albizia guachapele, a commercially valuable species of Costa Rica.
Media supplemented w i th-BA _ .was ... _. _� __ effective in inducing organogenesis in epicotyl explants of S. macrophylla and hypocotyl explants of A. guachapele.Adding Broadleaved Tree Medium benzyladenine also induced multiple shoot fonnation in S. macrophylla (Maruyama et al. 1989).Lee and Rao (1988), obtained adventitious shoots from the friable callus only when the nodal segments from seedlings were cultured on benzyladenine media.BA is a cytokinin known for strongly stimulating bud fonnation.It also stimulates the explant metabolism (George and Sherrington 1984).Finally, BA has been reported in several instances as the cytokinin of choice for adventitious budding in both gymnospenns and angiospenns.It is usually the only phytohonnone required (TItorpe et al. 1991).Cedrella odorata required the addition of an auxin in order to express a morphogenic response; Otherwise, Maruyama et al. (1989), obtained multiple shoot fonnation in Cedrella odorata with addition of benzyladenine to Woody P1ant Medium.

TABLE 1
Effect offive BA concentrations on bud induction in the hypocotyl explants of Albizia guachapele Explants were cultured on half-strength MS.Data were collected from 20 replicates after six weeks for all parameters.*Significant at P = 0.05 TABLE2Effect of five BA concentrations on bud induction in the epicotyl explant ofSwietenia macrophyllaExplants were cultured on half-strength MS.Data were collected from 20 replicates after 6 weeks for a1l parameters.*Significant at P = 0.05