Effect of Substrate Type on Survival and Growth of Sweet Potato Vitroplants Under Acclimatization

: The lack of healthy planting materials is one of the major constraints of sweet potato cultivation in Benin. One of the solutions to this problem is the use of vegetal biotechnology through micropropagation. At the end of the production of vitroplants, a crucial phase consists in acclimatizing them before their transfer to the real environment. The type of substrate used plays an important role in the success of acclimatization. This study aims at evaluating the effect of the type of substrates on the survival and growth of sweet potato vitroplants in acclimatization of the two accessions (Mèché and Bombo vôvô). For this, three substrates were assessed. These are Potting soil alone, Potting soil + Sawdust, and potting soil + Charcoal powder in proportion 2:1. These substrates were first sterilized separately in an oven at 150°C for 2 hours. The experiment was conducted in the greenhouse of Central Laboratory of Vegetal Biotechnology and Plant Improvement of the Faculty of Sciences and Techniques of the University of Abomey-Calavi following a model of random complete blocks in split plot design. The results show that the type of substrate influences the success of acclimatization in terms of survival rate, the gain of size and the number of neoformed leaves (p= 0.0033; 0.0019 and 0.0001). The Potting soil + Charcoal powder substrate influenced these parameters better than the other two with 95%, 3.13 cm and 3.165 respectively for survival rate, size gain and the number of neoformed leaves. It was followed by the Potting soil + Sawdust substrate. Acclimatized plants have been successfully introduced into the field, proving that this technique is fruitful in the increase of the production of healthy cuttings and make them available to farmers. The protocol used in this study can be applied in the acclimatization of sweet potato vitroplants.


Introduction
Sweet potato is the seventh most important crop in the world after wheat, rice, maize, potato, barley and cassava. For its food, economic and nutritional value [1,2], it is cultivated on a worldwide scale. Thus, in 2020, its world production was estimated at 89,487,835 tons on an area of 7,400,472 ha and, in Africa, at 28,798,773 tons on an area of 4,213,802 ha [3]. Its tubers are used in many culinary and agri-food preparations [1,4,5]. They are rich in carbohydrates, vitamins C, A and minerals [2,6]. In spite of these assets of the crop, it faces several constraints of which the lack of fit materials of plantation of the potent varieties [7,8] which is the main constraint in Benin and in Africa in general [9].
Since the sweet potato seed system is informal in Benin, farmers themselves multiply and share planting material without any special organization [7]. This favors the spread of viral diseases leading to a decrease in yield. Micropropagation is the best alternative to solve this problem with its advantages such as high reproduction and obtaining replicas selected for their robustness and plant sanitation. At the end of the production of vitroplants, a crucial phase consists in acclimatizing them before their transfer to the real environment [10][11][12]. Therefore, methods favoring the optimization of vitroplants survival during acclimatization should be considered. Its success depends on a number of factors among which we mention: conditioning at transplanting, choice of substrate, prevention of diseases and control of physical parameters. For the choice of the substrate, it must be of good quality with a good permeability, well aerated with a pH not too high [13].
For this purpose, several substrates have already been evaluated such as sand, sawdust, rice field waste as well as their mixture, compost+ sawdust+ sand, red soil and compost [14][15][16][17]. In Benin, the only study that addressed the aspect of acclimatization of sweet potato vitroplants on six accessions with a single type of substrate made of compost, sand and sawdust resulted in a success rate that varied between 56.6% and 83.33% [17]. It is necessary to optimize the acclimatization of sweet potato vitroplants in Benin in order to move from the informal to the formal seed system. Furthermore, charcoal powder does not seem to be used in acclimatization substrates, though it is used in agriculture as an approach to correct soil problems. [18]. This study intends to evaluate the effect of three substrates on the survival and growth of vitroplants in order to optimize the success of the acclimatization of sweet potato vitroplants in Benin.

Plant Material
The plant material is made of seven-week-old vitroplants of two sweet potato accessions maintained in the glasshouse of the Central Laboratory of vegetal Biotechnology and Plant Improvement of the Faculty of Sciences and Techniques of the University of Abomey-Calavi (LCBVAP). These are Bombo vôvô and Mèché (Table 1). These accessions were chosen for their good yield and are among the most widely grown accessions in Benin [7].

Composition and Sterilization of the Acclimatization Substrates
Three substrates previously sterilized in an oven at 150 °C for 2 h were used. The first one is made of potting soil alone and the other two are respectively made of Potting soil and sawdust on one hand, Potting soil and Charcoal powder on the other hand, in the proportion 2:1 ( Figure 1). The Charcoal powder was chosen for its use in agriculture as an amendment [18]. It was crushed in a mortar and then sifted with a sieve of about 2 mm mesh to obtain its powder. As for the sawdust, it was taken as a positive control and the Potting soil alone as a negative control to assess the effect of the incorporation of the Charcoal powder in the substrate.

Transplanting of Vitroplants and Acclimatization Conditions
The vigorous vitroplants of about 8-10 cm in size, 6-8 leaves with well-developed roots (2-4) were prepared for acclimatization by opening the cultivation-vases and clearing the cultivation media on the roots by mild rinsing with tap water without breaking the roots. Subsequently, the sterile substrates were moistened with Shive and Robbins' solution [19]. Transplanting of the vitroplants into plastic pots of 11.5 cm height and 5 cm diameter was done directly under greenhouse. After transplanting, the vitroplants were covered with transparent rubber in the plastic trays in order to increase the related humidity ( Figure 2). After one week, these rubbers were removed to expose the plants to direct light under greenhouse for three weeks. The vitroplants were watered with Shive and Robbins solution at a frequency of once every 2 days. The acclimatization period was four weeks from 24 th March to 20 th April 2022. Climatic data in terms of related humidity and temperature were taken by the related humidity sensor every five days per week during four weeks of acclimatization. They varied from 75% to 80.4% and from 30.3°C to 31.5°C for respectively related humidity and temperature ( Figure 3).

Experimental Device
The experiment was conducted in the greenhouse following a model of random complete blocks in split plot design [20]. Two factors were assessed: the substrate with three modalities (Potting soil alone, Potting soil + Sawdust and Potting soil + Charcoal powder) and the accession with two modalities (Mèché and Bombo vôvô). A total of six treatments were assessed. Ten vitroplants were tested per treatment with two repetitions.

Data Collection
After four weeks of acclimatization, data were gathered. These were the number of surviving vitroplants, the number of neoformed leaves and the gain of size. The surviving vitroplants and neoformed leaves were counted. The size of the vitroplants after four weeks of acclimatization was measured using a measuring stick from the crown to the apex.

Statistical Data Analysis
XLSTAT 2014 software was used for descriptive and inferential statistics. Analysis of variance under the generalized linear model was applied for survival rate, gain of size and number of neoformed leaves.

Effect of Substrate and Genotype on Vitroplants Survival
The analysis of the results indicates that only the type of substrate significantly influenced the survival rate of vitroplants in acclimatization (p <0.05) (Figure 4). The highest survival rate was obtained with the Potting soil + Charcoal powder substrate while the lowest was recorded Under Acclimatization with the Potting soil alone substrate (Figure 4).

Size Gain (cm) of Vitroplants According to Substrate and Genotype
The results of the analysis of variance indicate that the size gain was significantly influenced by substrate type. Neither genotype nor genotype-substrate interaction influenced this parameter (p˃ 0.05) ( Table 3). The lowest size gains were noted with the Potting soil substrate alone while the highest were obtained on the Potting soil with Charcoal powder ( Figure 5).

Number of Neoformed Leaves of Vitroplants According to Substrate and Genotype
The results of the analysis of variance revealed that only the substrate still influenced very significantly the number of neoformed leaves (p < 0.0001) ( Table 4). The highest numbers of neoformed leaves were observed with the Potting soil and Charcoal powder substrate while the lowest were recorded with the Potting soil substrate alone (Figures 6, 7). ns= not significant; ***= very highly significant.  After the acclimatization phase (April 20, 2022), the vitroplants were sent to a real environment for a period of four months.

Discussion
Acclimatization is the final and very important phase of micropropagation because its success constitutes the achievement of micropropagation [10]. It is in this perspective that this study focuses on the evaluation of substrates. Thus, from the results obtained, it was found that the type of substrate influenced all the studied parameters, including the survival rate, the gain of size and the number of neoformed leaves (p < 0.05). For the survival rate, the mixture of Potting soil and Charcoal powder in the proportion 2:1 favored the survival of the vitroplants, followed by the substrate of the mixture of Potting soil + Sawdust. Also, the same mixture favored leaf emission than the others. This can be explained by the improved physical properties of this mixture. In all cases, the substrate made up of Potting soil alone showed the weak results. This could be explained by the lack of aeration of this substrate. The Charcoal powder and sawdust absorb a quantity of water of spraying by maintaining humidity necessary to the plants.
Currently, charcoal powder is used in agriculture to correct the physical and chemical properties of the soil. [18,21]. Accordingly, the sawdust is used as substrate in the technique of plant stemming from the fragmentation or macro propagation for its interest to maintain the humidity of the substrates without making it hydromorphic and its results have proved it in this technique [22,23]. A study has shown that the survival rate of sweet potato vitroplants varies according to the substrate and that the mixture red soil -sand -compost at the proportion 1:2:1 has presented a high survival rate than the substrates taken alone, respectively for the varieties Kullufo and Tulla [24]. The effect of the type of substrate on the survival rate of vitroplants was also noted for banana-tree in acclimatization [25].
These good physical conditions of mixing substrates would favor the rooting of the vitroplants and consequently the absorption of minerals from the solution of Shive and Robbins ensuring the growth of the vitroplants, which would also explain the gain of size and the number of neoformed leaves. Ashiono and co-workers on Eucalyptus in the nursery found similar results on seedling growth. Greater height was observed with the sawdust and cow manure mixture substrate than with the substrates taken alone and the other mixtures [26]. Likewise, Suárez and his collaborators found a greater size with the mixture substrate (peat + sand) followed by peat + rice bran than peat alone in Gynerium sagitatum Aubl vitroplants during acclimatization [27].

Conclusion
The results obtained in this study allowed to identify two substrates favorable to the acclimatization of sweet potato vitroplants. These are the Potting soil mixed with Charcoal powder and the Potting soil mixed with Sawdust, each in the proportion 2:1. The protocol used in this study can be applied in the acclimatization of sweet potato vitroplants and Charcoal powder can be used in the composition of substrates for seed production and in large quantities from sweet potato vitroplants.