In vitro callus and shoot regeneration in Enterolobium cyclocarpum (Jacq.) Grised. - a fast timber yielding species

This study was conducted to investigate the in vitro callus induction and rapid shoot regeneration potential in Enterolobium cyclocarpum, a plant native to central Mexico but widely introduced into Africa. The leaf, stem and nodal explants of E. cyclocarpum were cultured on full strength Murashige and Skoog (MS) medium supplemented with different concentrations of Cytokinins - Benzyladenine (BA) and/or Kinetin and Auxins - Naphthalene acetic acid (NAA) and/or 2,4-Dichlorophenoxylacetic acid (2,4-D) each alone and in combination.  The leaf explants did not respond to these treatments.  The Nodal explants were best for caulogenesis, while the explant responses were in the order- nodal > stem > cotyledon for callogenesis in MS medium supplemented with BA and/or Kin combined with NAA and/or 2,4-D. The varied combinations induced white compact callus.  The highest callus production was observed on MS medium supplemented with 2.7 µM NAA + 2.2 µM BA and 5.4 µM NAA alone.  Nodal and cotyledon explants developed callus and multiple shoots on MS supplemented with a combination of cytokinin (BA and/or Kin.) and auxin (NAA and/or 2,4-D). The maximum number of 3.98 ± 0.37 and 2.1±0.11 shoots/explants were recorded for nodal and cotyledon explants on MS medium supplemented with a combination of 8.8 µM BA+2.7 µM NAA and 2.2µM BA+2.7 µM NAA respectively.  On the basal medium, 10% of the excised shoots rooted successfully. Thus, this in vitro method can be exploited for conservation and mass propagation of this fast timber yielding tree and also utilized for embryogenesis studies.


Introduction
The anthropogenic deforestation occurring throughout the world has always been increasing, with an alarming rate in the last 3 decades in particular in West Africa primarily due to urbanization, unsustainable logging, agricultural farming and collection of fuel wood (FOMERCU, 1999;Sayer et al., 2010). The gradual increase in deforestation practices for economic and/or social reasons without any simultaneous replanting is a global threat to the sustainability of the environment (FAO, 2005;Odediran et al., 2013). Africa has the second highest rate of tropical deforestation in the world. The tropical forests in this region have declined at an annual rate of 3.4 million hectares between 2000 and 2010 because of degradation and deforestation processes (FAO, 2010;Eleanya, 2014).

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(NAA) in combination with benzyl adenine (BA) or kinetin (KIN). They reported multiple shoot induction using nodal segment collected from in vitro germinated seedling when cultured on MS medium was supplemented with combination BA and NAA. However, E. cycocarpum as an established exotic species to Nigeria, little or no studies had been done on it in vitro propagation.
Thus, the objective of this study was to evaluate the effects of Cytokinin-BA and Auxin-NAA on multiple shoot induction using ex vitro explants of leaf, stem and node of E. cyclocarpum -a protocol needed for the mass propagation as well as genetic improvement programs.

Plant materials
The seeds of E. cyclocarpum were obtained from indehisced pods collected from an actively growing tree in Reforestation Garden of Botany, Department of Botany, Obafemi Awolowo University, Ile -Ife. Seeds were selected based on their non-dehised seed coat, scarified using emery paper. The scarified seed was surface sterilized using 10% NaOCl for 15 minutes, and then kept under running tap water for 30 min. Then they were soaked in distilled water for 15 mins to allow for imbibition. Finally, the seeds were planted in petri-dishes for 2 weeks before they were transferred to top soil and were out in a randomized environment in which they can access various environmental factors.

Explant source and sterilization
The leaf, stem and node ex vitro explants of E. cyclocarpum were obtained from 3-8 weeks old seedlings and were kept under running water for 10-15 minutes before culturing. Media and culture of explants The explants were cultured on full strength MS (Murashige and Skoog, 1992) basal medium containing 3% sucrose per liter. The pH of the media was adjusted to 5.7±0.02 with 1 N NaOH or 1 N HCl solutions, solidified with 0.8% agar (BDH) per liter prior to autoclaving at 121 °C and 103 kpa for 15 minutes. The basal medium alone served as control and was also supplemented with cytokinin separately and/or with auxins.
The leaf, stem and Nodal explants were disinfected with 70% ethanol for 3-4 minutes and surface sterilized by washing with 10% NaOCl containing two drops liquid detergent (tween-20) for 15 minutes after which they were rinsed in at least three changes of sterile distilled water. The leaf explants were sectioned to about 10 mm 2 in size while the stem and the node were about 10 mm in length. The explants were cultured in 15 cm 3 test tube containing 8 ml of the culture medium. The leaf explants were cultured with the adaxial surface in contact with the medium while the nodes and stem cutting were slantingly explanted on the medium.

Culture conditions
The culture tubes were covered with non-adsorbent cotton wool and wrapped with aluminum foil, after which they were incubated at a temperature of 25 ± 2 ºC and a photoperiod of 16 h light with cool white fluorescent light (20 μmol m -2 s -1 ).

Statistical analysis
The data in terms of the number of induced shoot buds, shoot length, callus induction, morphology, and colour of callus were recorded after 4-5 weeks in culture. Each treatment consisted of 10 replicates and all experiments were repeated twice. The data were analyzed statistically using standard error (S.E) of the mean and were separated using Duncan Multiple Range Test (DMRT). All statistical analyses were performed with Statistical Package for Social Sciences (SPSS, version 11.5).

Results
There was no callus initiation with the cotyledon explants of E. cyclocarpum cultured in the MS medium alone i.e. the control. Initiation of callus was observed within 2 weeks in the cotyledon explants on MS media containing low concentrations of auxin alone or their various concentrations combined with the cytokinins. The proliferation rate of callus induced from the cotyledon explants was generally slow. Shoot bud formation became evident from the scanty callus induced by some auxin /cytokinin combinations after 3 weeks of culture ( Figure 3a).
A maximum mean number of 2.1 shoots per explant was observed with cotyledon explants grown on MS medium containing 2.7 µM NAA + 2.2 µM BA which also induced the highest frequency (58.47±1.19%) of callus formation (Tables 1 and 3). The concentration of 2.7 µM NAA + 4.7 µM kinetin in its case induced highest frequency (51.57±1.88%) of off white callus (Table 5) and the highest mean number of 1.5 shoots/explant although this was not significantly (p ≤ 0.05) different from 1.4 shoots/explant induced from cotyledon explants cultured on MS medium containing 2.7 µM NAA + 2.35 µM kinetin (Tables 2, 4 and 5). Little extension growth was shown by the shoot buds when subcultured. The shoot buds that formed from the cotyledon-derived callus on MS medium supported with 2.7 µM NAA + 4.7 µM BA developed further when subcultured, 40% of the shoots produced thick roots in MS hormonal free medium forming complete plantlets.      followed by a very slow proliferation of creamy/ off white hard callus (Figure 3b). The NAA/ BA or 2,4-D/ BA combinations brought about callus initiation in the stem explants within 2 weeks to produce off white friable or semi-hard (Tables 5 and 6). The largest callus size was produced in stem explants cultured on MS medium containing 5.4 µM NAA + 4.4 µM BA (Figure 4a), and 82.2 ± 2.63% response was obtained (Table  1). The combination of NAA/ kinetin and 2,4-D/ kinetin induced callus from the stem explants of E. cyclocarpum within 2 weeks, the calli were generally light brown and semi-hard. Massive callus induction was observed with stem explants of E. cyclocarpum cultured on MS medium supported with 4.5 µM 2,4-D + 4.7 µM kinetin with the highest frequency of 75.53 ± 2.88% (Table 2). Nodal explants of E. cyclocarpum cultured on MS medium alone enlarged in size without either production of callus or axillary bud release. When the explants were grown on MS medium to which auxins alone were added, the only callus formed and with auxin/cytokinin combinations, both callus and shoot buds were observed within 2 weeks of culture. Calli formed at the basal cut ends of the explants in all the cases were cream-coloured or white and friable.
On the transfer of separated shoot buds to MS medium without hormones, less than 10% of them rooted after 6 weeks of culture. In MS medium containing 4.9 µM IBA, short and thick roots formed in over 80% of the shoots within 4 weeks of culture. Figure 6 shows the acclimatized plantlets induced from nodal explant of E. cyclocarpum with thick root in a rooting medium of 4.9 µM IBA.

Discussion
The use of in vitro approaches to propagation, conservation and genetic improvement of forest trees has been of increased interest since the last three decades (Chalupa, 1981;Tomar and Gupta, 1988;Xia and Hong, 2001;Chalupa, 2002;Anis et al., 2005;Faisal, 2007;Renukdas et al., 2010 andSajeevan et al., 2013). Two methods of plant regeneration widely used in rapid and large scale micropropagation or plant transformation studies are organogenesis or embryogenesis. The success of these techniques requires good callus quality and quantity (Lin et al., 2010).
The present studies showed that the stem, cotyledon, and nodal explants, but not leaf explants of E. cyclocarpum species were callogenic. The initiation and proliferation of callus were promoted by using NAA, 2,4-D alone or separately combined with Kinetin or BA to different extents in different explants. It was generally observed that the percentage of explants response in terms of callus induction was greater with auxin combination with BA than with Kinetin in the explants studied. The auxins, NAA and 2,4-D are commonly used with BA for callus induction in plant systems (Dhar and Joshi, 2005;Abbasin et al., 2010;Isikalam et al., 2010;Ntui et al., 2012). Other research groups also induced callus, a consequence of wound reaction (Khal, 1983), in vitro without the use of plant growth regulators of several plant species (Handro and Floh, 2001;Martin, 2002).
Cotyledon explants of E. cyclocarpum were found to produce callus together with shoot buds when cultured on MS medium containing NAA combined with BA or Kinetin. The frequency of shoot induction appeared favoured by high cytokinin relative to auxin. A combination of 2.2 µM BA + 2.7 µM NAA induced the best shoot production from the cotyledon explants of E. cyclocarpum (2.1 shoots/explant). The influencing effects of auxin and cytokinin combination on organogenic differentiation have been well documented in several plant systems. Rahman et al. (2010) found that adventitious shoots were induced from leaf-derived callus of Lagerstroemia speciosa grown on MS medium that was supplemented with 5 µM BA, 3 µM NAA, 10% coconut milk and 568 µM ascorbic acid. Similarly, cotyledon derived calli induced by 0.5 µM NAA + 0.5 µM BA produced high-efficiency shoot regeneration (4.2 shoots/explant) when subcultured on MS medium supplemented with 5 µM BA (Agrawal and Sadar, 2007). Salvi et al. (2001) who used various Neem explants including cotyledon, produced multiple shoots on MS medium containing 8.88 µM BA and 0.57 µM NAA. By addition of 3 mg/L Hymexazol to a modified MS medium containing 2.22 µM BA and 0.05 µM NAA, Yin et al. (2001) increased adventitious shoot development from cotyledon explants of Albizia julibrissin.
The leaf explants of E. cyclocarpum formed no callus when cultured on either MS medium alone or supplemented with various concentrations of either BA or NAA alone and in their combinations. The leaf explants, however, were found to have lost chlorophyll turning light green the end. This is contrary to wound reaction inducing mitosis in the cells from the cut surfaces and culminating in callus formation (Pérez-Francés et al., 1995). The loss of response of the explants may suggest that the endogenous level of auxins/cytokinin or the exogenously applied growth regulators were insufficient to induce callus. It is also known that different tissues can respond in different ways during in vitro culture process (Jimenez, 2001;Banerjee et al., 2011), and the requirements for plant growth regulators appear to be tissue-specific (Venkatachalam et al., 1999;Banerjee et al., 2011). Vidoz et al. (2012) also reported that the leaf explants of Lotononis bainesii did not respond on MS media to the absence of plant growth regulators. Conversely, leaf explants have been reported by several authors as the best for callus induction and shoot initiation. Jiancan et al. (2011) obtained 95% callus induction from leaf explants of Zizyphus jujuba in MS supplemented with 0.5 mg/L NAA.
In the absence of growth regulators in the culture medium, E. cyclocarpum stem explants neither formed callus nor shoot buds. The stem explants inoculated either on MS medium alone or supplemented with BA alone did not show callogenesis or caulogenesis but they first increased in size and gradually turned brown later. Cultures of stem explants on MS medium supplemented with the different concentrations of auxin and combined with BA started callus initiation after 2 weeks and proceeded with high proliferation.
Nodal explants exhibited the greatest tendencies to form callus and shoots under the influence of plant growth regulators compared to stem, hypocotyl, leaf and cotyledon explants of the plant species investigated. An average of 3.98 shoot / explant was generated from the nodal explant-induced callus of E. cyclocarpum by 2.7 µM NAA combined with 8.8 µM BA.
The nodal explants of several tree seedlings have been known to produce both callus and shoot buds in vitro under the influence of some plant growth regulators. Rahman et al. (1993) reported that NAA or 2,4-D separately combined with BA induced both callus and shoots within 3 weeks of culture of nodal explants of Caesalpinea Pulcherima. Sugla et al. (2007) induced multiple shoots in vitro from nodal explants through forced axillary branching in Pongamia pinnata using 7.5 µM BA while more recently, Sajeevan et al. (2012) reported inducing multiple shoots from nodal explants of Morus alba L. variety VI cultured on MS medium supplemented with 1.0 mg/L BAP, 0.1 mg/L TDZ and 0.25 mg/L NAA. Maximum shoot induction was obtained from the nodal cutting of E. cyclocarpum seedlings cultured on MS medium supplemented with a combination of 10.7 µM NAA and 2.2 µM BA (Rodriquez-Sahagun et al., 2007). Multiple shoots were also obtained from nodal explants of 18-day-old in vitro seedlings of Pterocarpus marsupium Roxb. culture on MS medium containing 4 µM BA, 0.5 µM NAA and 20 µM adenine sulphate (Husain et al., 2008). Shoot proliferation from nodal explants of Melaleuca alternifolia (Tee Tee) cultured in liquid or on agar-based MS medium containing 1.11 µM BA or 0.55 µM BA had also been reported in the literature (Yohana et al., 2010).
Root induction was also achieved in the rooting medium (MS + 4.9 µM IBA). Stout short single roots were induced from the cotyledon and nodal explant-derived shoots of E. cyclocarpum. It appears that different species have a different critical concentration of hormones below which root initiation will occur and above which there will be inhibition. Furthermore, organogenic differentiation of callus leading to production of shoots and roots are dependent on several factors aside from hormonal factors and these include level of salt (e.g. PO4 3-), quality of light, and temperature, in addition to physiological state, size of explant, and orientation of the medium (Razan, 2003). Chevre (1985) opined that rooting is often more difficult with the ligneous plants than with the herbaceous plants. More recently, similar observations were reported by Lin et al. (2010) stating that in general, establishing an efficient tissue culture technique was difficult in woody plants compared to herbaceous plants. This in part is related to the phase change from juvenility to maturation that most woody plants undergo (Trevor et al., 1990). The development of basal callus is one of the main physiological disorders that affect rooting competence of micro shoots, a situation which is more severe in woody species (Bairu and Kane, 2011). These authors explained that basal callus constitutes a sink trapping essential growth constituent and consequently affecting many physiological processes of the shoot.

Conclusions
The optimal concentration of NAA/BA that can induce stem callus in a potential source for plant regeneration (either by organogenesis or embryogenesis) and production of secondary metabolites were determined for E. cyclocarpum. Also, the optimal concentration of NAA/BA for shoot induction and plantlets formation of the plant species from cotyledon and nodal explants were also determined. Thus, it can be concluded by means of in vitro culture of the cotyledon and nodal explants of E. cyclocarpum, true clone plantlets can be produced. This plantlet can be employing for reforestation and/or forest restoration programs.