Temporary Immersion Bioreactors : Establishment of Cassava

Cassava (Manihot esculenta Crantz-Euphorbiaceae) is one of the most important crops for world agriculture. It is the main source of many family’s calorie intake and contributes toward food security. Its cultivation is necessitated sterile vegetative propagules with essential physiological and genetic quality, which can be obtained by plant tissue culture techniques. However, the maintenance of in vitro propagation processes requires automation, making large-scale production feasible. This work aimed at developing protocols for the establishment of cassava cultivation in Temporary Immersion Bioreactors (TIBs). Two nutrient media (MS medium reduced to half-strength of the salt concentration and the same formulation plus 4,43μM of 6-Benzylaminopurine [BAP]) and different culture cycles were evaluated in Bioreactors (15 minutes of immersion and 4 hours of stationary phase [15M4H]; 15 minutes of immersion and 8 hours of stationary phase [15M8H]). The establishment of culture in bioreactors was promising. Based on our results, the use of MS medium without growth regulators was more effective for shoot formation; there were differences in the 15M4H cycle when growth regulators were used. However, the 15M8H cycle had better performance in both treatments: with or without growth regulators. The use of culture medium without BAP led to better rooting; calli formation increased when BAP was used. The present research investigation indicated the use of MS medium reduced to halfstrength the salt concentration without the presence of growth regulator and the use of the culture cycle constituted by 15 minutes immersion and 8-hour intervals in stationary phase was effective for Cassava cultivation in TIBs.


Introduction
Cassava (Manihot esculenta Crantz-Euphorbiaceae) is a very important crop in several tropical and subtropical regions of the world, especially in countries of Africa, Latin America and the Caribbean (Zhou et al., 2017).Cassava is considered as the main source of calories for many populations in developing countries.Many factors favor the comprehensive geographic distribution of cassava, with emphasis on its growth ability in low-fertility areas, which indicated the potential of this plant to be grown in regions affected by climate changes.Other benefits of cassava are its use for animal feed, starch production, food processing, paper products, textile and pharmaceutical industry (Chauhan et al., 2015).Mongomake et al. (2015) reported that the high purity and solubility of cassava starch is emphasized when compared to potato and corn starches and is an important characteristic for the production of biofuels (Mongomake et al., 2015).
Brazil is the fourth largest producer of cassava in the world (FAO, 2017) and, in 2018, the average yield of production was 21 million tons (IBGE, 2018).Cassava is propagated vegetatively (Alves, 2006).However, the multiplication by this method is low, which reduces the availability of vegetative materials of high quality and stagnates the production (Santos et al., 2009).Another important aspect is the traditional propagation that can lead to degeneration due to the presence of pathogens; in addition, propagating materials presenting appropriate The experiment was conducted in a completely randomized design, in a 2 × 2 factorial scheme.The levels of factor "A" corresponded to the culture media used, being: (1) MS (Murashige & Skoog, 1962) culture medium reduced to half-strength of salts (1/2MS) and containing 20 g L -1 of sucrose, 0.300 mg L -1 of myo-inositol and (2) the same culture media with added with 4,43µM of 6-Benzulaminopurine (1/2MS + BAP).The levels of factor "B" corresponded to different cycles in the bioreactors: (1) 15 minutes of immersion and 4 hours in stationary phase (15M4H) and (2) 15 minutes of immersion followed by 8 hours in stationary phase (15M4H).The stationary phase is the period in which the nutrient medium is in the storage bottles, without contact with the explants.Two levels were used for each factor according to results found by our group.It was not possible increasing the number of levels because the number of bottles in the bioreactor was fixed.Thus, it was necessary reducing the number of treatments to maintain a large number of explants and replicates in the experiment.The pressure used to exchange medium between the culture bottles and the storage bottles was 4 Bars.The conditions of the culture room were 25 ºC (±3), 16h of photoperiod emitted by Light Emitting Diodes (LEDs) with a luminous intensity of 40 μmol m -2 s -1 .
Different variables including shoot formation (%), shoot per explant, leaf formation, calli formation (%) and root formation (%) were evaluated after 30 days of culturing.Shoot formation was determined based on the emission of new shoots (after establishment in vitro) containing leaves or primordial leaflets.The normality of the data was tested with the use of the Kolmogorov-Smirnov test and the homogeneity of variances by the Bartlett test.
The data were then transformed by the √x+0.5 and submitted to Analysis of Variance.The averages were compared using the Tukey's test at 5% of error probability.Each treatment had 5 replicates, each containing 20 explants, totaling 100 explants per treatment and 400 in total.The experiment was repeated twice and presented very similar data.The herein presented data were selected based on the coefficient of variation in order to assure experimental accuracy (Storck et al., 2006).

Results and Discussion
The establishment of cassava cultivation in TIBs was promising in all treatments evaluated, and it is possible due to observations made in Figure 2. No significant differences were observed for the factors tested nor interaction regarding the number of shoots per explant (P = 0.1655 for factor "A" levels, P = 0.5732 for factor "B" levels and P = 0.8090 for interaction) and number of leaves (P = 0.1359 for factor "A" levels, P = 0.9855 for factor "B" levels and P = 0.4732 for interaction).The overall mean number of shoots per explant was 1.68 and the average number of leaves was 1.39.
Interaction (P = 0.0407) was observed for the percentage of shoot formation (Table 1).The use of the MS medium reduced to half the salt concentration (1/2MS) without the presence of growth regulators was superior in relation to the use of the medium 1/2MS + BAP.When the growth regulator was used, the best shoot formation occurred in the largest interval between the immersion periods was used.However, it should be noted that in both situations the use of the 1/2MS medium was more favorable and also constitutes an alternative better from in the economic point of view, if one takes into consideration the high costs of BAP.Grattapaglia and Machado (1998) reported that the use of cytokinin BAP can lead to callogen formation when the availability is balanced with auxins from the culture medium or endogenous to the explants, similar to what was observed in the present study.In addition, when no cytokinin is used, endogenous auxins tend to favor the establishment of morphogenic routes for rooting occurrence.In Bioreactors, cultivation of ginseng, Panax quinquefolius, the use of cytokinins favored the development and multiplication (Uchendu et al., 2011), unlike results recorded for cassava plants in the current study, whose growth regulator did not favor multiplication.
Based on the results of this study, the cultivation of cassava in temporary immersion bioreactors is promising, and for the establishment of the explants.The best result was obtained using 1/2MS culture medium without growth regulators and using the culture cycle of 15 immersion minutes and intervals of 8 hours of stationary phase.This outcome assures a valuable tool for cassava mass multiplication in automated systems.It allows the production of vegetative material with high-quality genetic, physiological and sanitary aspects.It is necessary conducting further studies comprising other variations in growth regulators (which was limited in the current survey due to the size of the bioreactor and its capacity to hold bottles).It is also worth conducting studies with different genotypes and acclimation systems.
Figure 2 Cycle; B Note. * Averages with different letters, in the column, differ from each other by Tukey's test at 5% error probability.** Coefficient of Variation measures the experimental accuracy by dividing the square root of the mean square of the experimental error by the overall mean of the experiment.

Table 2 .
Rooting (%) and Calli Formation (%) in cassava explants in TIBs with the use of MS medium with half-strength of salts (1/2MS) and MS medium with Growth Regulators (1/2MS + BAP)