Micropropagation of Ocotea Porosa (Nees & Martius) Barroso
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African Journal of Biotechnology Vol. 10(9), pp. 1527-1523, 28 February, 2011 Available online at http://www.academicjournals.org/AJB DOI: 10.5897/AJB09.976 ISSN 1684–5315 © 2011 Academic Journals Full Length Research Paper Micropropagation of Ocotea porosa (Nees & Martius) Barroso L. L. Pelegrini 1*, L. L. F. Ribas 1, F. Zanette 2 and H. S. Koehler 2 1Departamento de Botânica, Universidade Federal do Paraná (UFPR), Curitiba, Parana, Brazil. 2Departamento de Botânica, Universidade Federal do Paraná (UFPR), Curitiba, Parana, Brazil. 3Departamento de Fitotecnia e Fitossanitarismo, Universidade Federal do Parana (UFPR), Curitiba, Paraná, Brazil. Accepted 12 May, 2010 The objective of this work was the establishment of a micropropagation protocol for Ocotea porosa by multiplication of shoots from axillary buds. Different concentration of BAP (0; 2.5; 5 or 10 µM) or BAP+KIN (0; 1.25; 2.5 or 5 µM) were investigated to optimize the multiplication. Shoot growth was stimulated with reduced concentration of BAP (0; 0.5; 1 or 1.5 µM) or KIN (0.5 or 1µM) or activated charcoal (0.5; 1; 2 or 3 gl -1). For root induction different concentrations of IBA (0; 1.25; 2.5; 5 or 10 µM) or (0; 2.5; 5 or 10 mM) were applied. The highest mean multiplication rate was observed in the fourth subculture with 5 µM BAP, reaching 5.3 shoots per explant. The shoots elongated in culture medium supplemented with 2 gl -1 activated charcoal and presented bigger leaves than on medium with reduced concentration of BAP. The shoots rooted on medium contains 10 µM IBA or after pulse treatment of 10 mM (68.7 and 62.6% of rooting, respectively). The survival rate of the plants was 56.7%. This study showed that O. porosa micropropagation is feasible; however it needs further research in order to increase plant survival. Key words: 6-Benzylaminopurine, multiplication, rooting, in vitro culture, apical shoots, native specie. INTRODUCTION Ocotea porosa (Nees & Martius) Barroso (Lauraceae), is Micropropagation constitutes a powerful tool for ex situ a tree native to the Mixed Ombrophyllous forest conservation programs of the rich flora, especially for (Araucaria Forest) of Brazil. Its wood is of excellent species with reduced populations or low production of quality and widely appreciated. It is exported for the seeds (Debnath, 2004), or for difficult to regenerate confection of luxury furniture and as timber for con- species when in natural conditions, as it is the case for O. structions and carpentry (Carvalho, 2003). O. porosa is porosa. Considering these difficulties, micropropagation an endangered specie, given its exploration for wood and represents an important tool for its conservation. This the deforestation due to agriculture expansion in its technique may be successfully used for mass propa- natural occurrence area (Carvalho, 2003; Caldato et al., gation of selected genotypes, conservation and genetic 1999; Neto et al., 2002). improvement (Thorpe and Kumar, 1993). Moreover, it The seeds are recalcitrant, it has a strong tegument facilitates the rapid establishment of a large number of dormancy and their germination is irregular, making its plants, with a minimum impact on the endangered wild natural propagation difficult (Carvalho, 2003). Moreover populations (Debnath, 2004). the rooting rate of cuttings is low (Inoue and Putton, The production of plants from axiliary buds or shoots 2007), limiting its vegetative propagation for replanting. has proved to be the most generally applicable and reliable method of true - to - type in vitro propagation (George et al., 2008). There are few studies about micro- propagation of species from Lauraceae family and none *Corresponding author. E-mail: [email protected]. for O. porosa . However, for other forest species of the Lauraceae family, studies have already been carried out Abbreviations: BAP, 6-Benzylaminopurine ; KIN, kinetin; to establish protocols of regeneration by somatic embryo- IBA, indol-3-butyric acid. genesis, such as Laurus nobilis (Canhoto et al., 1999), 1528 Afr. J. Biotechnol. Persea americana Mill (Witjaksono et al., 1999), and double bevel and two apical leaves were maintained. They were Ocotea catharinensis (Moura-Costa et al., 1993; Viana, placed onto a half-strength MS medium supplemented with indol-3- 1998; Viana and Mantell, 1999; Santa-Catarina et al., butyric acid (IBA) (0, 1.25, 2.5, 5 or 10 µM), sucrose (30 gl-1) and solidified with BBL® agar (5.5 gl-1). The cultures were maintained 2003, 2004; Moser et al., 2004; Santa-Catarina et al., in the dark for 7 days and then transferred onto the same medium 2005), because of the economical and ecological without IBA. The experimental design was completely randomized importance of O. porosa, this study aimed to establish a with four replicates of two flasks each and five shoots per flask micropropagation protocol for plant mass production. (n=40). After four weeks, the percentage of rooted shoots, the mean number of roots per shoot tip and mean length of roots were calculated. Another treatment consisted of the immersion of the shoot base MATERIALS AND METHODS into IBA solutions (0, 2.5, 5 or 10 mM) (dissolved in NaOH 0.1N) for 10 min. The shoots tip were placed onto a half-strength Woody Plant material. Seeds of O. porosa were collected from a natural Plant Medium (Lloyd and McCown 1980) with 20 gl-1 sucrose and forest at Colombo-Parana, Brazil, in 2006. The pulp of the mature BBL® agar (5.5 gl -1). Four replicates of two flasks were done in fruits was removed manually and the seeds washed in current each treatment, with five shoots per flask (n=40). After four weeks, water. The seeds were then sowed in trays containing sieved soil the percentage of rooted shoots, the mean number of roots per and Plantmax® substrate (3:1). The trays were kept in a shoot and mean length of roots were calculated. greenhouse with controlled temperature (18–23ºC). After Culture conditions. The cultures were incubated at day/night germination the seedlings were transplanted into plastic bags temperatures of 25±2°C/18±2°C, under white fluorescent light (40 (15x25 cm) containing the same substrate. Manual irrigation was µmol m-2 s-1) and a 16 h photoperiod. Experimental design was made directly in the substrate. The one-year-old plants were completely randomized. The flasks used during initiation and trimmed and shoots tip were collected and used as explants. multiplication had 3 cm in diameter and 7 cm in height, contained In vitro establishment. shoots tip (approximately 2 cm in length), 15 ml of medium each and were covered with aluminum foil. During containing one or two axillary buds, were surface-sterilized in 70% elongation and root induction flasks of 7 cm in diameter and 15 cm (v/v) ethanol for 30 s, followed by immersion for 10 min in a 0.25 height were used, with 40 ml of medium in each and closed with and 0.5% (v/v) sodium hypochlorite solution plus 0.1% Tween20® polypropylene caps. All media had their pH adjusted to 5.8 with and finally rinsed six times in sterile distilled water. They were NaOH 0.1 N or HCl 0.1 N and were autoclaved for 20 min at 121°C. placed in flasks containing a medium composed of half-strength MS Transplant and acclimatization. The rooted plants were pre- salts (Murashige and Skoog 1962), full MS vitamins, sucrose (30 gl - acclimatized in the growth room for 48 h and the flasks were 1), polyvinylpolypirrolidone (0.5 gl -1) and Micromed® agar (6 gl -1). gradually opened. They were subsequently removed from culture The flasks used for establishment in vitro and multiplication were 3 vessels and planted into plastic tubes (53 cm3) containing the cm in diameter and 7 cm in height with 15 ml medium each and following mixtures: Plantmax® HT substrate (100%); Plantmax® HT covered with aluminum foil. Each treatment consisted of ten flasks + charcoal (3:1); Plantmax® HT + carbonized rice hull (2:1) and repeated four times, with one shoot tip per flask (n=40). The Plantmax® HT + carbonized rice hull + sieved earth (2:1:1). They percentage survival of the shoot tips was evaluated after four were maintained under intermittent mist (1 min every 5 min) in a weeks. greenhouse with a temperature of 24±2ºC and RH of 90±2%. The Multiplication. The shoot tips were cultured in a MS culture percentage of plant survival was recorded after 4 weeks. medium supplemented with 6-Benzylaminopurine (BAP) (0, 2.5, 5 Statistical analyses. The data were submitted to an analysis of or 10 µM) or with combinations of BAP and Kinetin (KIN) (0+0, variance (ANOVA) and Tukey´s test at P ≤ 0.05 was applied, using 1.25+1.25, 2.5+2.5 or 5+5 µM), sucrose (30 gl -1) and solidified with the MSTATC program. BBL® agar (6 gl -1). Each treatment consisted of eleven flasks repeated four times, with one shoot per flask (n=44). The recording of the average number of shoots per explant and the mean length RESULTS AND DISCUSSION of axillary shoots was done after four weeks during four subcultures. The new axillary shoots were individualized and subcultured every four weeks. The mean multiplication rate was In vitro establishment calculated by counting the axillary shoots originated in each subculture. The shoots tip disinfestation with 70% (v/v) ethanol for 30 Elongation. The effect of reduced concentrations of BAP or KIN s followed by immersion for 10 min in a 0.25 or 0.5% (v/v) and the effect of the activated charcoal on the elongation of the shoots were studied. Shoots tips (0.5 to 1.5 cm long) originated sodium hypochlorite solution was efficient.