Germination of Mansonia altissima (A. Chev.) A. Chev. var. altissima: an endangered valuable timber species in Africa

The in vitro seed germination which results in the production of disease-free seedlings and greenhouse germination of the seeds of Mansonia altissima was investigated in order to establish a better way of germination of the timber species. Five levels of GA3 treatment were used in in vitro germination with three replicate and two seeds were inoculated in each of the jam bottle. Whereas, in greenhouse germination, five levels of different treatments were used, replicated three times and each Petri plate contained 15 seeds. The experiment was repeated twice and the data from each experiment was put together and used for the statistical analysis. The results showed that seeds germination occurred eight days after inoculation in in vitro but in the case of greenhouse germination, it took only five days. For in vitro rapid germination of Mansonia altissima, the MS medium should be supplemented with 1.0 μm of GA3. Equally, in greenhouse germination, the seeds need to be soaked in 1.0 mM of GA3 for 24 hours. Alternatively, in the absence of GA3, the seeds can be soaked in water for 24 hours before broadcasting the seeds on the seedbed for germination, as this will help to identify nonviable seeds.


Introduction
Mansonia altissima (A. Chev.) A. Chev. var. altissima is a tree species which belongs to the family of Malvaceae. The tree species can be found in the Republic of Benin, the Democratic Republic of Congo, Cote d'lvoire, Ghana, Guinea, Nigeria, Sudan, Togo, and Uganda where it is commonly called Mansonia, African black walnut, African walnut. Mansonia altissima is a valuable timber, commonly harvested from the wild and exported, especially to the USA and Europe, where it is being used as a substitute for walnut (Osunlaja, Olomola, Nwogwugwu, Afolabi, & Oloyede, 2017). The wood of Mansonia altissima is used for general and high-class joinery, cabinet work, furniture, turnery, decorative veneer, and handicrafts. It is also used in construction of doors and windows (Osunlaja et al., 2017). The products from the bark of Mansonia altissima have been used in the treatment of leprosy. Again, extracts from the bark have been shown to inhibit the growth of Mycobacterium tuberculosis (Fernandez-Villamil, Dubin, Galeffi, & Stoppani, 1990). A decoction of the twig bark is applied as a bath against yaws, scabies, and syphilis. Moreover, ethanolic extract of the wood has shown hepatotoxic and haematotoxic effects when administered orally. The bark contains a highly toxic compound known as mansonine, which is related to cardenolides from Digitalis purpurea and ouabain from Strophanthus preussii (Akinnagbe, Gailing, & Finkeldey, 2010;Fernandez-Villamil et al., 1990).
International Union for Conservation of Nature's (IUCN, 1998) red list of threatened species included Mansonia altissima (A. Chev.) A. Chev. var. altissima as one of the endangered valuable timber species (IUCN, 1998). The timber becomes endangered due to overexploitation for furniture, cabinet, and demands from foreign countries and inadequate of the seeds for germination because of its recalcitrant and dormancy nature (Wédjangnon, Houètchégnon, & Ouinsavi, 2016). These situations trigger depletion of the timber species in forest as demand for the species for wood production could no longer cope with their supply. Consequently, the species is moving towards extinction, reduction in biodiversity and decrease in primary productivity. With the immense importance of this timber species, there is a need to improve the germination techniques of the plant in vitro and in the field. Seed germination and rapid germination are usually essential processes in seedling establishment, especially with plants that are endangered (Murungu, Nyamugafata, Chiduza, Clark, & Whalley, 2003). Many studies such Maku, Gbadamosi, and Fadoju (2014) and Dentaa, Wiafe, and Nutsuakor (2015) evaluated the germination success only based on final percentage germination, however it is not enough due to the lack of ability to compare two sets of data (one lot of seed may have germinated well before the other, but both attained the same final germination percentage) and this has led to the development of a number of germination parameters (Jeyavanan, Sivachandiran, Vinujan, & Pushpakumara, 2016;Almaghrabi, 2012). In the case of Mansonia altissimia, the majority of studies had focused only on the final germination percentage and this has been a setback in the species germination understanding. Hence, this study investigates suitable treatment for the seed germination by calculating the germination parameters of Mansonia altissimia treated with different chemicals.

Study site
The experiment was carried out at Department of Biotechnology, Bhimtal Campus, Kumaun University Nainital, Uttarakhand, India. Bhimtal is situated at latitude 29 o 35 N and longitude 79 o 57 E.

Collection of seeds
Mansonia altissima seeds were collected from Forest Research Institute of Nigeria, Jericho Hill, Ibadan, Oyo State, Nigeria (Latitude 7 o 30ˈN and Longitude 3 o 54ˈE).

Seeds sterilization for in vitro germination
The seeds of Mansonia altissimia were soaked in water (at room temperature) for 24 hours after which they were kept under running tap for 30 minutes for proper washing, and then washed in 2% of Tween 20 for 20 minutes. The seeds were then treated with 1% antifungal (Bavistin) for 15 minutes, washed with sterilized distilled water and transferred to the laminar flow. Under the laminar flow, the seeds were treated with 0.1% HgCl 2 for 1-2 minutes and 0.5% streptomycin for 5 minutes and lastly, 70% ethanol for 1 minute before being washed in sterilized distilled water for 5 times.

Seeds treatment for greenhouse germination
The seeds of Mansonia altissimia were soaked in water, 1 mM of GA 3 , 1 mM of GA 3 + 0.2% KNO 3 and 0.2% KNO 3 for 24 hours. Whereas, un-soaked seeds were used as control.

Experimental design
The Murashige and Skoog (1962) (MS) medium with the pH 5.8, was supplemented with five levels of gibberellic acid (GA 3 ) in the in vitro germination (0 (Basal medium), 0.1, 1.0, 3.0 and 5.0 μm) with three replicate and two seeds in each jam bottle. Whereas in Petri plate germination, five levels of treatment (unsoaked, soaked in water, soaked in 1 mM of GA 3 , 1 mM of GA 3 + 0.2% KNO 3 and 0.2% KNO 3 ), was investigated, which were replicated three times and each Petri plate containing 15 seeds. The experiment was repeated twice, and the data obtained from each experiment was put together and used for the statistical analysis.

Germination condition
The inoculated seeds were kept in a growth room with temperature 28±2 o C, average light intensity was 4300 lux, photoperiod 16 8 -1 hours and covered with black cloth. Also, the seeds in each of the Petri plates were covered with cotton wool because of the floating nature of the seeds and kept in greenhouse with temperature 25±2 o C, relative humidity 60%, average light intensity was 22900 lux and 16 8 -1 hours photoperiod.

Analysis of data
Data obtained from each of the experiments conducted was put together and the data were subjected to one-way analysis of variance (ANOVA) using SAS 9.1 version software (Oseni, Adelusi, Dada, & Rufai, 2016;Oseni, Dada, Okunlola, & Ajao, 2018). The means were separated using fisher's LSD at the probability level of 0.05 (p < 0.05).

Seed germination
Germination is the process that begins with the uptake of water by the dry seed and ends with the emergence of the embryonic axis, usually the radicle, from its surrounding tissues. Germination requires water, temperature, oxygen, light, and nitrate. Of all these, water is the most essential factor. Under normal conditions, water uptake by dry seed is triphasic. This begins with a rapid initial uptake (phase I, i.e. imbibition) because water flow from the higher to the lower water potential. Then, followed by declines in water uptake by imbibition and metabolic processes are reinitiated (phase II). A further increase in water uptake (phase III) occurs as the embryo axis elongates and breaks through the covering layers to complete germination (Taiz, Zeiger, Møller, & Murphy, 2015). Seed is considered as germinated upon radicle emergence ≥ 0.5 mm (Charu, Ravi, & Tapan, 2016).
After 24 hours of soaking, the seed did not sink as shown in Figure 1a. Viable seeds were determined by pressing each seed with the fingers. Seeds that were soft when pressed were considered not to be viable. Whereas, seeds that were still hard when pressed were considered viable. Also, Figure 1b showing the selected viable seeds inoculated on MS medium supplemented with GA 3 for germination.
As shown in Figure 2, in vitro seed germination occurred eight days after inoculation and lasted for 11 days. The total average percentage of in vitro seed germination was 96%. Germination started with MS medium supplemented with 1.0 μm of gibberellic acid (GA 3 ) and the germination lasted for 2 days. The highest germination percentage for in vitro seed germination occurred in MS medium supplemented with 1.0 μm of GA 3 (75%) and occurred on the first day of germination. Followed was MS medium supplemented with 0.1 μm of GA 3 but showed the highest germination percentage on the third day of germination (70%) and the germination lasted for 4 days. Whereas MS medium supplemented with 3.0 μm and 5.0 μm of GA 3 showed germination on the fourth day after germination had started, but their germination lasted for five and six days respectively.  In greenhouse germination, shown in Figure 3, the seed germination started on the fifth day after setting up the Petri plate and lasted for 16 days. Germination started with seeds soaked in 1 mM of GA 3 for 24 hours which had the highest percentage of germination (51%) on the second day of germination and the seed germination lasted for 4 days. seeds soaked in 1.0 mM of GA 3 + 0.2% KNO 3 for 24 hours had the highest germination percentage (54%) on the first day of germination and the germination lasted for 3 days. Seeds soak in 0.2% KNO 3 for 24 hours germinated on the fifth day after germination had started, with the highest germination percentage (60%) on the seventh day of germination. Moreover, seeds soaked in water for 24 hours germinated on the sixth day of germination and the germination lasted for 3 days, which had the highest germination percentage (54%) on the eighth day of germination. Seeds that were non-soaked germinated last which had the highest germination percentage (40%) on the eleventh day of germination and the germination lasted for 6 days.
Seed germination occurred eight days after inoculation in in vitro germination, whereas it took only five days in greenhouse germination, this might be as a result of adequate water supplied to seeds planted in Petri plate but the incubated seeds only got their water from the one-liter prepared MS medium. As water is the major requirement for seed germination (Taiz et al., 2015). Maku et al. (2014) also observed the same germination period (five days after planting) with Mansonia altissimia when treated with different plant growth hormone. However, germination of Mansonia altissimia started two weeks after sowing according to the study of Dentaa et al. (2015), the reason may be because the seeds were sowed in the soil and germination might have occurred, or because the seeds were not treated with phytohormone. Germination occurred firstly in seeds treated with GA 3 in both experiments although at lower level in in vitro germination. GA 3 had been reported to induce seeds germination better than other plant hormones and showed effects at very low concentration (Finch-Savage & Leubner-Metzger, 2006). The ratio of ABA and GA 3 is the primary determinant of seed dormancy. A higher level of ABA in seed imposes dormancy on seed whereas a higher level of GA 3 makes the seed to be nondormant. GA 3 stimulates seed germination via amylase synthesis (Finch-Savage & Leubner-Metzger, 2006;Gashi et al., 2012). According to Gashi et al. (2012), GA 3 and KNO 3 enhanced seed germination of Ramonda serbica and Ramonda nathaliae. The authors also supported the findings of Charu et al. (2016), which is also in line with the results gotten from Petri plate germination when soaked in 1.0 mM of GA 3 + 0.2% KNO 3 for 24 hours. The seed exhibit epigeal type of seed germination, as shown in Figure 1c.

Germination parameters
The germination parameters of in vitro germination were shown in Table 1. Seeds inoculated in MS medium supplemented with 1.0 μm, 3.0 μm of GA 3 and basal medium had the highest FGP (98%) which was significantly higher than others (p< 0.05). Basal medium had the highest MGT (6.7 days), and next to it was MS medium supplemented with 5 μm of GA 3 (6.1 days). The lowest MGT was 1.3 days, which was observed in MS medium supplemented with 1.0 μm of GA 3 . MS medium supplemented with 1.0 μm of GA 3 had the lowest FDG (8 days) but not significantly different (p< 0.05) from MS medium supplemented with 0.1 μm and 5.0 μm of GA 3 , whereas basal medium showed the highest FDG (12 days). Furthermore, MS medium supplemented with 1.0 μm of GA 3 showed the lowest LDG (9 days), and basal medium had the highest LDG (18 days). The highest CVG, GRI and CGRI were observed in MS medium supplemented with 1.0 μm of GA 3 followed was MS medium supplemented 0.1 μm of GA 3 , and basal medium had the lowest. Basal medium and MS medium supplemented with 5.0 μm of GA 3 had the highest TSG (6 days), followed by MS medium supplemented with 0.1 μm and 3.0 μm of GA 3 (3 days).
As shown in Table 2, the germination parameters of Mansonia altissimia germinated in the greenhouse. The FGP range from 75% to 97%, with un-soaked seeds having the lowest and seeds soaked in 1.0 mM of GA 3 had the highest. In addition, the highest MGT was observed in un-soaked seeds (12.1 days). Followed were seeds soaked in water and 0.2% KNO 3 (8.1 and 8 days respectively) but no significant difference (p < 0.05). The seeds soak in 1 mM of GA 3 had the lowest MGT (2.3 days), next to it was seed soaked in 1.0 mM of GA 3 and 0.2% KNO 3 (4.6 days). The FDG range from 5 to 15 days and the LDG range from 8 to 20 days, which occurred in seeds soaked in 1.0 mM of GA 3 and un-soaked seeds respectively. The CVG, GRI, CGRI were in order of seeds soak in 1.0 mM of GA 3 > 1.0 mM of GA 3 + 0.2% KNO 3 > 0.2% KNO 3 > seeds soaked in water > unsoaked. Seeds soaked in 0.2% KNO 3 had the highest TSG (6 days) and seeds soaked in 1.0 mM of GA 3 + 0.2% KNO 3 had the lowest TSG (2 days).
Germination parameter is considered to be a qualitative developmental response of an individual seed that occurs at a point in time within a treatment respond within different time (Gashi et al., 2012;Jeyavanan et al., 2016;Murungu, 2011;Rastegar, Sedghi, & Khomari, 2011;Kader, 2005). According to Jeyavanan et al. (2016), the higher the FGP values, the greater the germination of a seed population. All the treatment in the experiment showed greater value of FGP, which means that majority of the seeds are viable. Except the unsoaked seeds, which might be as a result of not being able to identify the nonviable seeds through pressing by fingers. The lower the MGT, the faster a population of seeds has germinated (Rastegar et al., 2011). Lower FDG values indicate a faster initiation of germination (Kader, 2005). Lower LDG values indicate a faster ending of germination (Kader, 2005). Seeds inoculated in MS medium supplemented with 1.0 μm of GA 3 and seeds soaked in 1.0 mM of GA 3 in Petri plate germination showed the lowest number for MGT, FDG, and LDG. These results indicate that the seeds started and completed their germination within the shortest period which make the treatment more effective than others. As defined by the following authors: Darshini and Aruna (2014), Kader (2005), Shahriari, Puteh, Saleh, and Abdul-Rahim (2014), Sharafizad, Naderi, Siadat, Sakineja, and Lak, (2013), the CVG gives an indication of rapid germination, it increases when the number of germinated seeds increases and the time required for germination decreases. Theoretically, the highest CVG is 100, and this occurs only if all the seeds germinated on the first day. The GRI reflects the percentage of germination on each day of the germination period. The higher the values of GRI and CGRI, the higher and faster the germination. The higher the TSG values, the greater the difference in germination speed between the 'fast' and 'slow' germinating members of a seed lot. Higher value of CVG, GRI and CGRI occurred in seeds inoculated in MS medium supplemented with 1.0 μm of GA 3 and seeds soaked in 1.0 mM of GA 3 in in vitro and Petri plate germination, which showed that, the two treatments were suitable for rapid germination of Mansonia altissimia. Finally, the TSG values indicated that, there were differences in time of germination. FGP-final germination percentage, MGT-mean germination time, FDG-first day of germination, LDG-last day of germination, CVG-coefficient of velocity of germination, GRI-germination rate index, CGRI-corrected germination rate index, TSG-time speed of germination, MDG-mean of daily germination. Means with the same letters within the column are not significantly different at p < 0.05

Conclusion
The results of the experiment showed that, in vitro germination of Mansonia altissimia seeds, required MS medium supplemented with 1.0 μm of GA 3 . Also, in greenhouse germination, soaking the seeds in 1.0 mM of GA 3 for 24 hours will also be perfect for seed germination. Alternatively, in the absence of GA 3 , the seeds can be soaked in water for 24 hours before broadcasting the seeds on the seedbed for germination, as this will help to identify nonviable seeds.