Cassava (Manihot esculenta Crantz) belong to Euphorbiaceae family, which genus Manihot includes over 200 species. The most important is M. esculenta which is the main carbohydrate source for more than 800 million people in the world and particularly in tropical regions in Africa, Asia and Latin America (Rosenthal and Ort, 2012). In Côte d’Ivoire, cassava is the second most important food crop after yam with an annual production of more than 6.3 million tons in 2022 (FAOSTAT, 2022). However, cassava productivity is challenged by several biotic and abiotic constraints resulting in low yield. Among the biotic constraints, the most damaging is Cassava Mosaic Disease (CMD), which is responsible for yield losses between 50% and 70% (Chikoti et al., 2019). The CMD is endemic in Africa and is caused by nine begomovirus species “https://ictv.global/report/chapter/geminiviridae/geminiviridae/begomovirus (accessed 27 November 2024)”. The most effective way to control this disease is to use virus-free planting material.

Cassava is currently propagated vegetatively using cuttings, but the maximum cutting production of this conventional method is low at 1:10 rate (Vernier et al., 2018). Consequently, significant number of plants are required to obtain sufficient quantities of planting material. In addition to the low multiplication rate achieved with traditional vegetative propagation method, this method also promotes CMD propagation in the field (Kouakou et al., 2024).

The in vitro propagation technique makes it possible to sanitize several susceptible cassava varieties using thermotherapy combined with meristem culture. This is followed by mass multiplication of the disease-free material to make it available to farmers (Yéo et al., 2020; Nakabonge et al., 2020; Zango et al., 2021). The MS medium has therefore been used for the in vitro propagation of several plant species, with many advantages, particularly for species that are difficult to propagate vegetatively or by seed such as cassava. This mineral salts rich medium was developed by Murashige & Skoog in 1962 as a result of their work on tobacco soft callus (Murashige and Skoog, 1962). However, some varieties are difficult to grow on this common culture medium (MS), as they often require special growth factors or growth regulators and specific media compositions (Costa et al., 2019; Sesay et al., 2018; Apio et al., 2021).

In vitro propagation is regulated by endogenous hormones. Auxin is the main hormone that induces lateral budding and rooting (Pasternak and Steinmacher, 2024). The addition of exogenous hormones modulates the action of these endogenous hormones. However, the combined action of these hormones can be optimized by the addition of specific nutrients. Generally, exogenous plant growth regulators (PGRs) which include cytokinin such as 6-benzylaminopurine (BAP), kinetin (KIN) and thidiazuron (TDZ), and auxins such as α-naphthalene acetic acid (NAA) are combined with the MS medium to optimize cassava in vitro propagation (Sessou et al., 2020; Feyisa, 2021; Hamdeni et al., 2022). The type and quantity of PGRs added to the growth media depend on several factors, the most important being the plant genotype; therefore, it is necessary to optimize the concentrations of these growth regulators given their essential role in morphogenesis (Purohit et al., 2011; Okello et al., 2021).

The aim of this study is to develop an efficient in vitro mass propagation protocol for five in vitro difficult-to-grow cassava varieties to meet the needs of cassava farmers.

To improve micropropagation, exogenous plant growth regulators (PGRs) have been introduced into the MS media to promote rapid organogenesis. The most commonly used in organogenesis are cytokinins and especially Benzylaminopurine (BAP) for cassava and often kinetin (KIN) or thidiazuron (TDZ) (Shiji et al., 2015; Sessou et al., 2020; Galán-Ávila et al., 2020; Berhanu and Feyissa, 2020). In our experience, the responses of the explants of the five cassava varieties to the different MS medium concentrations were influenced by these three PGRs (BAP, KIN, TDZ). Significant differences were observed in the times from transplanting to leaf and root formation, the number of roots, the number of nodes and the length in vitro plantlets. These results showed that the response of cassava to in vitro micropropagation depends on the culture medium components. The addition of growth regulators at different concentrations to the MS basal salt controls the in vitro organogenesis of this plant.

One aim of our study, was to find media that contribute to improve the number of nodes and roots. In fact, number of nodes is the most important parameter in the propagation of some species such as cassava, because the units used for propagation are made up of these organs during micropropagation (Quashie et al., 2012; Okello et al., 2021). The parameters relating to the roots are also very important, because the roots are the first to be established during weaning in order to promote the recovery of the leaves or even the plantlet. The roots must be able to convert the young plant from heterotrophic (in vitro) to autotrophic (ex vitro) life. Without a strong and hardened root system, there can be no proper weaning of the in vitro plantlets (Dossoukpèvi et al., 2015). So, the type, concentration and an appropriate combination of PGRs are very important to improve any regeneration system (Thorpe et al., 1991). Differentiation in plants is controlled by an interplay between auxin and cytokinin and the exogenous requirement of hormones in the medium depends on their endogenous level in the cultured plant. A high ratio of auxin to cytokinin generally results in callus formation, whereas a low ratio results in organogenesis and shoot induction. Plant organogenesis is generally regulated by endogenous auxin, i.e. the natural auxin indole acetic acid (IAA). Adding ANA, a synthetic hormone with the same properties as AIA, to the culture medium increases the auxin content of the explant. This action can create disturbances and direct the cells towards root formation. Exogenous cytokinin will regulate the formation of organs (shoots or roots) depending on the target objectives. In the current study, KIN at 0.12 µM was suitable for the multiplication of the Ampong variety while at 0.12 or 0.24 µM, it was more suitable for Agbablé 3, Bocou 5 and Olékanga. These results are similar to those of Ahanhanzo et al. (2010); Cacai et al. (2012) and Sessou et al. (2020) who showed that KIN induced better results than BAP added to the control medium for the regeneration of cassava varieties. Kinetin can therefore be used at very low concentrations for rapid micropropagation of cassava. Furthermore, some authors such as Apio et al. (2021) used higher concentrations than others and obtained different results. They found that concentrations of 2.4 ml/l (10.65 µM) of BAP followed by 2 ml/l (10.74 µM) of NAA facilitated establishment and regeneration compared to kinetin. Ahanhanzo et al. (2008) in Dioscorea cayenensis and Demeke et al. (2014) in cassava showed that the response of the explant varied between the genotypes they used as observed in the present study. These results effectively confirm the effect of the genotype in the in vitro regeneration of cassava. In addition to KIN, the results of our study also showed that thidiazuron at 5 nM or 10 nM was better in the Agbablé 3 variety. It promoted the induction of roots and leaves in a short time and contributed to the production of a high number of nodes and roots. This result could be explained by the fact that there was some interaction between the growth regulators. In other words, differentiation depended on the type of hormone combination used. These results are confirmed by the work on cassava by Berhanu and Feyissa (2020), who showed that TDZ at a concentration of 0.2 mg/l (908.1 nM) had the highest average number of shoots per explant by improving the morphology of the in vitro plantlets. In other work, the MS medium containing TDZ without auxins was found to have a higher number of shoots than the medium containing both TDZ and auxins (Aasim et al., 2009; Okello et al., 2021). The importance of PGRs in initiating and regulating organized development is well established. The addition of TDZ promotes shoot formation by inducing and maintaining auxin biosynthesis. This hormone regulates auxin production, promoting callus formation at high concentrations (> 5 µM TDZ), while at low concentrations (< 5 µM TDZ), shoot induction is observed (Dewir et al., 2018; Vinoth and Ravindhran, 2018). TDZ was highly effective than other cytokinins at very minimal concentrations because of its stability to withstand degradation by cytokinin oxidases (Mok et al., 1987; Pasternak and Steinmacher, 2024).

Although, the combination of PGRs with MS salt is good for propagation, Mineral elements play an important role in organogenesis. Some elements such as nitrogen (N) and potassium (K) are involved in auxin simulation. In fact, N induces IAA biosynthesis, while K increases the auxin transport system, favoring either root or shoot formation. High concentrations of mineral elements can, often, be a source of disturbance for plants. In fact, explants already contain sometime a high amount of elements or which do not have a great need for nutrients to develop (Purohit et al., 2011). Given this concern, some authors have suggested that the dilution of mineral elements in MS medium may be appropriate for some genotypes (Berhanu and Feyissa, 2020; Mazri et al., 2021). In the current study, our results showed that such dilution as half concentration of MS salt significantly reduced the time spent from transplanting to leaf formation in cassava varieties Agbablé 3, Ampong, Bocou 5 and Olékanga compared to full-strength MS which was the control. It also induced rapid rhizogenesis of Ampong and Bayérè explants. Microcuttings have some mineral elements that would be necessary for their regeneration. These microcuttings would therefore contain a high concentration of carbon, phosphorus, nitrogen, potassium, sulfur and magnesium which are macronutrients and are required in millimolar quantities. Their high concentration could, in the long term, inhibit some activities in the organogenesis process such as nitrate activity for nitrogen (Amirouche et al., 1985). In our current experiments, this was observed by the low number of nodes, roots and in vitro plantlet length. MS salt diluted to 50% was suitable for the rapid regeneration of the explants as shown by Mazri et al. (2021) and Quashie et al. (2012). It reduced the regeneration time. We showed also that the half-strength MS can be better for organogenesis when we add kinetin at 24µM and thidiazuron at 10 nM. The supplementary of PGRs on half strength MS is more effective in contrary when it is not supplemented. This result is due to the synergistic action of minerals and growth hormones.

Several studies have demonstrated the recalcitrant nature of some cassava varieties to in vitro methods. Mapayi et al. (2013) obtained significant results with one of thirteen media containing different concentrations of BAP. This medium enabled them to record the highest means for the number of leaves, nodes and heights. Similar results were observed by Sesay et al. (2018) on some recalcitrant cassava genotype. Also, Sessou et al. (2020) obtained an effective medium for each variety and these media all contained kinetin. A similar trend was observed in this study with our protocol, where the seven media containing PGRs out of the nine were all effective to overcome the recalcitrance of cassava varieties with lower concentrations for all parameters. The use of lower concentration of PGRs could contribute to reduce their very high cost, which affects the overall cost of producing material using this technique. This approach will reduce the cost of producing in vitro source material.

Our study showed that, the addition of exogenous plant growth regulators such as KIN at 0.12 and 0.24 µM and TDZ at 5 and 10 nM contributed to optimize in vitro micropropagation. When the concentration of Murashige & Skoog mineral salts was reduced by half, it was also effective, especially in combination with PGRs. The optimal media identified in this study also show that the response of explants of cassava varieties in vitro depends on the medium used and the growth parameters considered. Our protocol can therefore be used to overcome the recalcitrance of some cassava varieties that are difficult to grow using the micropropagation method. This information will be useful for mass multiplication programs and the distribution of healthy planting material to farmers.