The plant material included ginger rhizomes obtained from an agro-pastoral farm located at Athiémè township in the Republic of Benin. The experiment was carried out in the Central Laboratory of Plant Biotechnology and Plant Breeding of the Department of Genetics and Biotechnologies of the University of Abomey-Calavi (Republic of Benin) from October 2022 to May 2023.

The plantlets are delicately extracted from the jars with forceps by peeling off the agar from the bottom of the jar. The plants were then carefully washed with tap water to remove residual agar from the roots to avoid contamination by microorganisms [12] and washed once more with distilled water, taking care not to break the roots. Subsequently, the height of the washed seedlings was calibrated, and the plants were transplanted in the shade into cells filled with moistened substrates.

Substrates were sterilized in an oven (BINDER) at 200˚C for 2 hours. This thermic sterilization is reinforced by chemical disinfection with fungicide (carbofuran) at 4% (g: g) [13] . The composition of the substrate is selected by mixing a compost, soil, sawdust, coconut peat, and charcoal in different quantity ( Table 1 ). S1 is composed of one quantity of compost, and two quantity of soil whereas S2 is composed of one quantity of compost, two quantity of soil and two quantity of sawdust. S3 is composed of one quantity of compost, two quantity of soil, and two quantity of coconut peat while S4 is composed of one quantity of compost, two quantity of soil, and two quantity of charcoal ( Table 1 ).

The repotted seedlings were subdivided into two batches for weaning. The first batch was placed in the greenhouse under a plastic tunnel covered with transparent white oilcloth for four weeks ( Figure 1 ), and then a second batch was placed under growth room conditions in a transparent acclimatization tank. The acclimation tanks were hermetically closed for four (04) weeks to confine the atmosphere and reduce dehydration. The substrates were sufficiently humidified, and there was no addition of nutrient solution. Human interventions were limited weekly when necessary, as were phytosanitary treatments (fungal treatment). The hygrometer was placed inside the plantlets in the greenhouse and in the growth room from the start of conditioning to the end to record variations in temperature and relative humidity during the cultivation period. The relative humidity and temperature were measured weekly inside the acclimatization tank by spraying with water or manual misting four times a day for 1 min each.

Seedling hardening was carried out under a shade house for 4 weeks by gradually reducing the ambient humidity to avoid direct contact of the plantlets with the sun’s rays. The substrate (1C2T or manuring) was packed into previously perforated plastic pots, watered abundantly, treated with fungicides and left to settle for at least 24 hours before transplanting. The seedlings were transplanted individually into pots. During this phase, the seedlings received alternating running water and a nutrient solution. The water supply is supplied by sprinkling at a rate of one watering per day if necessary.

Four nutrient solutions were prepared and tested. The Plantzyme solution [14] was obtained by diluting 5 mL of crude solution in 1 L of distilled water. The NPK solution [15] was obtained by dissolving 6 g of 15 N-5P-30K-2MgO in 1 L of distilled water. The Plantzyme + 15 N-5P-30K-2MgO mixture was prepared by diluting 5 mL of pure Plantzyme and 6 g of NPK in 1 L of water [14] .

The experimental design was a completely random block consisting of twenty plantlets per substrate with two repetitions for the weaning phase and ten plantlets per nutrient solution with two repetitions for the hardening phase. In order to see the effect of the temperature, relative humidity, and substrates on the survival rate, the generalized linear model with the binomial family was performed. Additionally, a generalized linear model was performed to explain the effect of nutrient solutions on the numbers of leaves and roots per plantlet. Analysis of variance (ANOVA) with Newman and Keuls test at the 5% threshold was performed by comparing the survival rate as well as the growth parameters (the height of plantlets and the length of roots) and establishing correlations between the number of leaves, number of roots, and height of the plantlets using simple Pearson linear regression. All analysis was done using XLSTAT software version 2014.

Statistical analyses showed that the cultivation duration had a highly significant influence (p = 0.003) on plantlet survival. Similarly, the mean temperature (TM) and relative humidity (RH) significantly influenced (p = 0.025) the plantlet survival rate. On the other hand, the interaction between physical factors and culture duration did not significantly influence plantlet survival after 4 weeks of weaning (p > 0.05) ( Table 2 ). Indeed, the survival rate, which was 100% during the first week of culture under both physical conditions, differed from that in the second week. Under weaning conditions (TM/26.54˚C/RH 96.16%), the survival rate increased from 100% to 90% in the 2nd week to 70% in the 3rd week and 4th week, while in the plantlets subjected to the greenhouse (TM29.94˚C/RH 67.38%), the survival rate increased from 100% to 70% in the 2nd week to 40% in the 3rd week and 4th week. In general, the highest survival rate (82.5%) was obtained under weaning conditions (26.54˚C/96.16%), while the lowest survival rate (62.5%) was obtained in the greenhouse (29.94˚C/67.38%).

ns: not significant; *: significant at 0.05; **: significant at 0.001; DDL = degree of freedom; Pr = probability.

Substrates tested, culture duration, and their interaction strongly influenced (p < 0.0001) the plantlet survival rate ( Table 3 ). Indeed, during the first week, the survival rate was 100% on the 1C2T2TC substrate (1 Compost + 2 Soil + 2 Coconut Peat), 90% on the 1C2T1CB (1 Compost + 2 Soil + 2 Charcoal) and 1C2T2SB (1 Compost + substrates 2 Soil + 2 Sawdust) and 60% on the 1C2T substrate (1 Compost + 2 Soil). At the end of the fourth week of incubation, these percentages increased to 90%, 80%, 60% and 30%, respectively ( Figure 2(A) ). For all substrates combined, the survival rate was generally 85% in the first week, 72.5% in the second week and 65% in the third and fourth weeks of culture

***: significance at the threshold of 0.0001; DDL = degree of freedom; Pr = probability.

( Figure 2(B) ). For the substrates tested, the survival rate was 92.5% for the 1C2T2TC substrate, 85% for the 1C2T2SB substrate, 70% for the 1C2T2CB substrate and 40% for the 1C2T substrate ( Figure 2(C) ).

Nutrient solution, culture duration, and their interaction had highly significant influences (p < 0.0001) on the number of leaves formed ( Table 4 ). Indeed, with the watering solution consisting of 15 N-5P-30K-2MgO, no leaves formed during the first and second weeks. With the Shives and Robbins nutrient solution, no leaves formed during the first week. At the end of the third week, an average of two leaves had formed on the plants. The acclimated plantlets that were watered with Plantzymes formed 1.3 ± 0.12 leaves during the first week, 1.5 ± 0.12 leaves during the second week, and 3.1 and 3.5 leaves during the third and fourth weeks of culture, respectively. With respect to the watering solution containing 15 N-5P-30K-2MgO + Plantzyme, leaves formed after the second week, with an average of 1.2 leaves. Thus, the average numbers of newly formed leaves were 1.2, 1.3, 2.2, and 3.5 for the 15 N-5P-30K-2MgO, 15 N-5P-30K-2MgO + Plantzyme, Shives and Robbins, and Plantzyme watering solutions, respectively. In general, the greatest percentage (3.5 leaves) of newly formed leaves was obtained with the Plantzyme watering solution, and the lowest percentage (1.2 leaves) was obtained with the 15 N-5P-30K-2MgO nutrient solution ( Figure 3 & Figure 4 ).

***: significance at the threshold of 0.0001; ns: not significant; DDF = degree of freedom; Pr = probability.

The nutrient solution, duration of culture and their interaction strongly influenced (p < 0.0001) the height of the plantlets ( Table 5 ). Indeed, with the watering solution consisting of 15 N-5P-30K-2MgO, there was no height gain during the first two weeks. The height increased to 0.1 cm during the third and fourth weeks of culture.

SC: Sum of squares; MC: Mean squares; Pr = Probability ***: significance at the threshold of 0.0001.

By using Shives and Robbins nutrient solution, the height gain was 0.13 cm in the first and second weeks and reached 0.23 cm and 0.35 cm, respectively, during the third and fourth weeks of culture. Furthermore, the height of the acclimatized plantlets watered with Plantzym increased by 0.12 cm, 0.33 cm, 0.53 cm and 0.6 cm during the first, second, third, and fourth weeks of culture, respectively. With respect to the mixed solutions (15 N-5P-30K-2MgO + Plantzyme), there was no increase in height during the first week, but the height increased to 0.13 cm during the second, third, and fourth weeks. In general, the greatest height gain (0.60 cm) was obtained with the Plantzyme solution ( Figure 5(A) ), and the lowest height gain (1.1 cm) was obtained with the 15 N-5P-30K-2MgO solution ( Figure 5(B) ).

The results revealed that nutrient solution had a highly significant (p < 0.001) influence on both root length and number ( Table 6 ). Likewise, the coefficients of determination (R²) are highly significant for both variables. The number of roots formed was 30, 25, 14, and 12 for the Plantzym, Shives and Robbins, NPK + Plantzyme and NPK nutrient solutions, respectively ( Figure 6 ). Root lengths of 10 cm, 8 cm, 5 cm, and 3 cm were noted for the Plantzym and Robbins solutions, NPK + Plantzyme, and NPK, respectively ( Figure 6 ).

**: significance at the 0.001 level; ***: significance at the threshold of 0.0001; R²: coefficient of determination; Pr = probability.

The results showed a highly significant and positive correlation between the studied growth parameters. The correlation between the average length of the roots and the average number of neoformed roots was 0.544. The number of roots, number of newly formed leaves and height increased strongly (r > 0.8). On the other hand, the correlations between average root length, the number of neoformed leaves (0.43) and the increase in height were weak (0.43) ( Table 7 ).

MRL = average root length; NRF = number of neoformed roots; NFN = number of neoformed leaves; GT = gain in height.