Shea tree (Vitellaria paradoxa) belongs to the family Sapotaceae. It grows widely in the savannah region of West African Countries (Maranz and Wiesman, 2003). It is a deciduous dicotyledonous crop that has a gestation period varying from 15 to 20 years. The Shea tree is of great economic importance due to its multipurpose use (Yakubu et al., 2015). It serves as a fruit tree as well as an oilseed crop. It is popular for the Shea butter produced from it, which is relevant in the food, cosmetic and pharmaceutical industries (Boffa, 2015). Plant hormones are chemicals that regulate plant growth. They are signal molecules produced at a specific location in the plant and extremely low concentrations. Hormones are naturally produced within plants, though very similar chemicals are produced by fungi and bacteria that can affect plant growth (Srivastava, 2002). The most widely used plant growth hormones are auxins and cytokinins. The types, concentrations and the ratio of these growth regulators used for media supplementation are essential for the type of culture responses obtained (Anca, 2009). Naturally, the Shea tree grows and regenerates itself in the wild but its slow and poor natural regeneration pattern due to long gestation period; impacts of bush fires and desertification have limited the domestication and genetic improvement of this crop. These limitations have necessitated the need for an alternative method of conserving this plant’s genetic resource outside the natural habitat. The propagation of the Shea tree by the in-vitro clonal technique presents such an alternative method (Lovett and Haq, 2013). However, vegetative methods, such as grafting, budding, cuttings and air-layering, have only produced limited success (Yeboah et al., 2010). The micropropagation of woody tree species is now a method widely used in the regeneration and conservation of germplasms (Lovett and Haq, 2013). Callus induction and plant regeneration are some of the key tools in plant biotechnology that exploits the totipotent nature of plant cells (Mukherjee et al., 2011).

Tissue culture serves as an indispensable tool for transgenic plant production. For nearly any transformation system, an efficient regeneration protocol is imperative (Cardoza, 2008). Due to the lack of proper cultivation practices, destruction of plant habitats, excessive and indiscriminate collection of medicinal plants for the supplement of global demands on herbal medicine, many medicinal plants like Salacia chinensis and Vitelleria paradoxa are severely threatened. Therefore, to conserve and rapidly propagate the rare and endangered medicinal plants, advanced biotechnological methods of culturing plant cells and tissues like micro propagation methods are employed (Nalawade et al., 2001). This study aims to evaluate the effect of different concentrations of plant growth hormones on callus induction and regeneration of Shea trees. Also, to produce callus from explant of the Shea tree and determine the optimal concentration of 2, 4-D and Picloram media for callus formation and plantlet regeneration.

Study location

The study was carried out in Agricultural Biotechnology Laboratory at Science and Technology Complex (SHETSCO), Sheda, located at the outskirt of Abuja, North Central Nigeria with Latitude 8?51’25’ N and Longitude 7?02’39’’E.

Plant material used for callus induction

A three days old tender leaf of Shea butter was used as an explant for the callus induction.

Treatment and experimental design

The treatments consist of six different concentrations of 2, 4 –D and Picloram (4.5, 4, 3.5, 3, 2.5, and 1.5 mg/L) arranged in a completely randomised design (CRD) with four replications.

Explant preparation and surface sterilization.

The three days old leave explants were plucked off from the mother plant using a sterile knife and then taken to the laboratory. The explants were washed vigorously under running tap water with drops of liquid detergent to remove the dirt particles then followed by rinsing with distilled water. The laminar flow hood was sprinkled with 70% Ethanol to control contamination and the burner was lit. To surface sterilize the tender leaves used as explants, ethanol and sodium hypochlorite method of surface sterilization were adopted. However, due to lack of standing protocol for surface sterilization of Shea butter explants for callus induction, three different ethanol and sodium hypochlorite combinations were used with varying concentrations and timing as follows:

S0: Explants were only washed with distilled water 5-6 times.

S1: Explants soaked in 70% ethanol for 1 min then soaked in 1% sodium hypochlorite for 15 min with 1 µl of tween 20.

S2: Explants were soaked in 70% ethanol for 2 min then soaked in 2% sodium hypochlorite for 20 min with 1µl of tween 20.

S3: Explants were soaked in 70% ethanol for 3 min then soaked in 3% sodium hypochlorite for 10 min with 1µl of tween 20.

Using the surface sterilization methods above, the explants were soaked in 70% ethanol first at S1, S2 and S3, then rinsed three times with sterile water. Secondly, the explants were soaked in sodium hypochlorite with 1 µl tween 20 then rinsed four times with sterile water. After surface sterilization, all explants were trimmed to small sizes of 2 mm by 2 mm and then cultured on MS medium without growth hormone to check out for contaminations and sterility within four days. Percentage sterility was recorded.

Preparation of MS media and callus induction

Two different plant growth regulators (PGRs; 2, 4-D and Picloram) was used for callus induction in shea butter. Six different concentrations of each auxin (4.5, 4, 3.5, 3, 2.5, and 1.5 mg/L) were used in Murashige and Skoog media (Murashige and Skoog 1962) as a basal medium and a control treatment with no growth hormone was included (Figure 2). Murashige and Skoog culture medium (MS) was supplemented with 30 g/L sucrose, as a support material (Khan et al., 2001) and 100 g/l ascorbic acid. The pH was adjusted to 5.7 using 1 M of NaOH. Pyhtagel was added as gelling age the medium at a concentration of 2.8 g/L before the medium was sterilized at psi 121°C for 15 min. At the end of the sterilization, the media was allowed to cool. Different concentrations of the auxins (2, 4-D or Picloram) was added using a microfilter and the medium was dispensed into the bottle in the laminar flow hood and allowed to settle for 24 h. Sterile explants were cultured into the bottle and placed about 2 cm apart on the culture medium. Twenty explants were used in all the media with each bottle having five explants and four bottles per media. The bottle was sealed in a polythene bag and stored in the dark growth room 28°C. The two sets of the experiment (MS + 2, 4-D and MS + Picloram) was conducted concurrently. The performance of the culture and callus produced was evaluated visually at 2 and 4 weeks and recorded. The cultures were monitored, every four weeks for sub-culturing and development noted. After four weeks, the explants were separated from the callus formed and the callus was moved into a fresh media for regeneration. The criteria for scoring explants for callus is based on the number of explants producing callus per media according to Amoo and Ayisire (2005).

Statistical analysis

Data collected were subjected to analysis of variance (ANOVA) using statistical analysis software (SAS). Means were separated using Duncan’s multiple range test (DMRT) at a 5% level of probability.

Effect of different sterilant combinations on leaf explants of Vitelleria paradoxa for tissue culture establishment

Table 1 summarizes the efficiency of the different combinations of ethanol and sodium hypochlorite at different times and concentrations. From the result, it was observed that the method of soaking the leaf explant in 70% ethanol for 3 min and then 3% sodium hypochlorite for 10 min had the lowest percentage (15%) of sterile leaves. Out of the 20 explants, only 3 explants were sterile while others were contaminated and burnt. The soaking of explants in a combination of 70% ethanol for 2 min and 2% of sodium hypochlorite for 20 min showed a higher percentage of 75% of sterile leaves. Leaf explants soaked in a combination of 70% ethanol for 1 min and 1% sodium hypochlorite for 15 min had the highest percentage (100%) of sterile leaf explants and shows no contaminations both in the leaf and media. The leaves remained green and sterile for ten days. Also, washing the leaf explants with only distilled water had no sterile leaf explants (Figure 1).

Effect of MS media supplemented with different concentrations of 2, 4 –D for callus formation on leaf explants at 2 weeks and 4 weeks interval.

From the observations, at 2 weeks all the media had at least one explant induced callus except for the MS basal (control) which had no growth regulator. At 2 and 4 weeks, media MA1 and MB1 had the least callus induction percentage of 5% and 10%, respectively. At 2 weeks, MD1 and ME1 had 20% same as the media MC1 and MD1 at 4 weeks; although, media ME1 performed better with percentage value of 30% at 4 weeks. However, MF1 performed better with (55%) than other treatments at 2 weeks while at 4 weeks, MF1 had the highest percentage value of 75% of callus formed. Tables 2 and 3 show the analysis of variance using 2, 4 –D on leaf explant of the Shea tree. There were significant differences among treatments at 2 weeks and 4 weeks of culturing. However, there was no significant (p> 0.05) difference at 2 weeks and 4 weeks in media MA1, MB1, MC1 and MD1. Media MA1 and MB1 had the lowest mean value of 0.25 at 2 weeks and 0.5 at 4 weeks. At 2 weeks. The differences between media MD1 and ME1 was not significant (p>0.05). Also, media MF1 had the highest mean value of 2.75 and 3.75 and was significantly (p> 0.05) different from others in 2 and 4 weeks. Media MA1 and MB1 at 2 and 4 weeks produced very poor calli while MC1, MD1 and ME1 produced poor calli. However, media MF1 had good calli at 2 weeks and then produce very good calli at 4 weeks. Only MS basal produced no callus at 2 weeks and 4 weeks. The colour and morphology of calli produced in media MA1, MB1, MC1, MD1 and ME1 were compact and whitish except for media MF1 which calli were friable and whitish.

Effect of MS media supplemented with different concentrations of picloram on callus formation from leaf explants of Shea tree at 2 and 4 weeks intervals

Tables 4 and 5, show a positive response of explants to the different concentrations of picloram at 2 weeks of culturing. Media MA2, ME2 and MF2 at 2 weeks had the least callus percentage induction value of 20% while at 4 weeks media MA2, ME2 and MF2 had percentage value of 35%. Likewise, MB2 at 2 weeks but at 4 weeks, there was an increase in callus formed in ME2. However, at 2 weeks and 4 weeks MD2 performed better than all other treatments but media MC2 had the best result at 2 weeks and 4 weeks. At 4 weeks, media MC2 had the highest percentage value of 100% which gave the highest calli formed. In the mean value, there were significant (p<0.05) differences among the treatment. However, there were no differences at 2 weeks and 4 weeks in the media MA2, ME2 and MF2 respectively. At 2 weeks, media MA2, ME2 and MF2 gave the least mean value of 1.0 while at 4 weeks, media MA2, MF2 had the least mean value of 1.75; likewise media MB2 at 2 weeks. Also, at 2 weeks MC2 had the highest mean value of 3.75 and was significantly (p<0.05) different from others while at 4 weeks MC2 gave the highest mean value of 5.0 which is the best and significantly (p<0.05) different from others. From the observations, the degree of callus produced at 2 weeks from media MA2, MB2, ME2 and MF2 was poor likewise media MA2 and MF2 at 4 weeks. At 2 weeks, media MD2 produced good calli likewise media MB2 and MC2 at 4 weeks. Therefore, media MC2 and MD2 produced the best calli and was used for the regeneration of Shea plantlet. All the treatments MA2, MB2, MC2, MD2, ME2 and MF2 produced friable and whitish calli except for control which had no callus.

The first experiment was to investigate the suitable surface sterilization method for leaf explants of the Shea tree. It was observed that soaking the leaf explants in 70% ethanol for 1 min and 1% sodium hypochlorite for 15 mins with a drop of tween 20 gave the highest percentage (100%) of sterile leaves. This was also observed by (Ghosh, 2005), In the case of Salacia chinensis the surface sterilization with 70% ethanol for 1 min followed by sodium hypochlorite (1% + 1µl of Tween 20) for 15 min proved most effective for maximum survival percentage in leaf explants. Soaking the explants in 70% ethanol for 3 mins and 3% sodium hypochlorite for 10 mins had the lowest percentage (15%) of sterile leaf establishment because of the increased time in ethanol and increased concentration of sodium hypochlorite. It was also reported by Sharma et al. (2014) that increasing the time of exposure to sodium hypochlorite significantly reduced contamination but showed an adverse effect on explants. Also, when the leaves were washed with distilled water only without tween 20, it had no sterile leaf all the leaves were contaminated. This was also confirmed by Thomas and Puthur (2002) that in an experiment where tween 20 was deliberately omitted there was an increase in the percentage of contamination. Tween 20 is said to be a surfactant that act as a detergent which attaches itself to possible oil exudates helping to establish clean explants. The result of the present study on the effect of 2, 4 –D or Picloram on callus formation of Shea tree shows that basal media without plant growth regulator did not induce any callus growth and this was also observed by Dhiya et al. (2013) and Namrata et al. (2014) in the study on the influence of hormones and explants towards in vitro callusing and shoot organogenesis in a commercially important medicinal plant, callus was also not induced in media without growth regulators. The effect of different growth regulators on callus induction and on callus growth was the objective of this study. It was observed that to induce callus 2, 4-D medium was not effective enough whereas the callus remains healthy and increase in different concentration of picloram combination. The best callus initiation was observed with 2, 4-D in 1.5 mg/L, it showed friable callus. Thus the whole outcome showed that 2, 4 -D was not efficient for callus induction because of its toxic nature and also confirmed it that 2, 4-D is used as a form of herbicide to kill weeds. The result showed that when the concentration is high it kills the leaves.

The current result recorded that at 4.5 mg/L 2, 4-D and 4.5 mg/L picloram the callus induction percentage was low because of the increase in the concentration of the auxins. However, high concentrations of the Auxins are toxic to the plant cells. These facts explain the increase in the callus fresh weight as the concentration of the Auxins increased up to a specific level. High concentration of 2, 4-D (4. 5 mg/L) gave fewer calli than 1.5 mg/ L. The callus was watery and non regeneratible type. This might be due to Ethylene production which reduced cell division. Synthetic auxin like 2, 4-D has been developed as herbicide to control weed. The presence of growth regulators in culture medium is an important factor in callus proliferation, growth and differentiation of cultured cells (Kalidass et al., 2010). Liu et al. (2006) reported that 2, 4-D had been used singly and in combination with cytokinins to enhance callus induction and maintenance. In the present study, callus induction was observed after two weeks of inoculation. Adu-Gyamfi et al. (2012) also observed callus after the same duration though the explant used was cotyledon. Callus was initiated at the cut ends of the leaf explant of Shea. Callus formation at the proximal end of the explant in this study was in line with reports on Pegannum harmla (Saini and Jaiwal. 2002). The result of the present study on effect of picloram on callus induction 3.5 mg/L gave the best CI% of (100%) the callus is friable and creamy in appearance. According to Trigiano and Gray (2005) rapid growth and light colour callus usually indicate a healthy culture and a friable callus is a very suitable for breaking up either for sub culturing or to produce plantlets.

In this study, the best sterilization method of shea leaf explant was by soaking in 70% ethanol for 1 min followed by 15 min in 1% sodium hypochlorite with 1 µl of Tween 20. The optimum concentration for callus induction was 3.5 mg/L Picloram in MS media. In-vitro propagation of shea tree could serve as a viable alternative for raising woody plants with propagation difficulties. Application of appropriate hormones in the right proportion could also enhance tree growth response.

The authors have not declared any conflict of interests

The authors are grateful to the Tertiary Education Trust Fund (TETFUND) for the grant (TETfund/DR&D/CE/ NRF/STI/03/Vol. 1) and the management of Federal University of Technology for the enabling environment. The management and staff of Agricultural Biotechnology Laboratory at Science and Technology Complex (SHETSCO), Sheda, Abuja are also appreciated for the permission given to use their laboratory during the experiment.