Taro, Colocasia esculenta L. Schott, is a tuberous plant in the Araceae family, cultivated for its tubers and leaves as a carbon source [1] [2]. Studies have highlighted some antioxidant, nutritional and anti-diabetic properties in C. esculenta. Other properties of the plant are also noted such as strengthening the immune system, lowering blood pressure, reducing weight gain and fatigue, the prevalence of cell damage, strengthening bones and, improving thyroid function [1] [3]. Ranked third among root and tuber crops after cassava and yam [4], this perennial plant of tropical and subtropical regions, is widely distributed in Southeast Asia, East, West, and Central Africa, the Caribbean, and Southeast America [4] [5]. Among the top ten producing countries in 2019, Cameroon ranks second after Nigeria with 1,909,738 tons [4]. However, in Cameroon, as elsewhere in Africa, its cultivation has always been carried out by smallholders, with limited resources and mostly female farmers [6]. The cultivation practice used for a long time has always been the use of corm fragments as seed from one field to another, implying moreover the transfer of pathogens. The arrival of Phytophthora colocasiae, a fungal pathogen responsible for taro blight since the years 2009 of Cameroon [7], remains at the origin of a real problem limiting the cultivation of this plant which is the unavailability of healthy or sanitized seeds. To solve that problem, [8] proposed the production of vitroplants from the micropropagation technique. However, Arano-Avalosa et al. [9], suggested that the use of the temporary immersion system, would not only reduce the costs of the production technique by micropropagation but also produce in large quantities and commercial scale. It’s true that, the ideal is to produce healthy seeds available to farmers, but questions remain. What will be the cost of these seedlings to a farmer or what will be the cost of expenses to cover a hectare? However, the applications of tissue culture by the PIF technique (plants grown from stem fragments) have already been proven in the cultivation of bananas (Musa sp.). The PIF technique is a method for rapid horticultural propagation of in vivo seedlings [10]. It is a technique that can be used to regenerate a young plant from the shoot fragment or to induce the proliferation of secondary buds on either side of the shoot following the removal of the main bud. This method is less costly than in vitro culture techniques and temporary immersion systems, and thus contributes to rapid production of live-plant material; it can be carried out at any season without difficulty [10] [11] [12], requires a reduced production space, contributes to the rapid production of desired varieties, allows the exploitation of the majority of the buds of the plant, (visible or not) and mass production of genetically identical offshoots, in a sanitized environment and within a reduced time. Until now, the culture substrate of this technique has always been the wood chip or sawdust substrate. Ewané et al. [13] and Ewané et al. [14] showed that it was possible to fertilize this substrate by adding Tithonia diversifolia leaf powder and clamshell powder during the production of PIF plants in bananas.

Oyster shell ash is increasingly used in agriculture as a fertilizer due to its ability to stimulate growth in plants [15] [16]. The role of oyster shell ash of Crassostrea spp. in protecting and strengthening the immune system of plants against some pathogens such as Phytophtora megakarya is recognized [16]. Similarly, their application in Abelmoschus esculentus plants influenced growth, total dry matter content, photosynthetic capacity and, chlorophyll content in its leaves [15]. The mineral and organic compositions of these oyster shells make them undeniably important agronomically. They are important sources of chitin and calcium oxide, which are molecules that play an important role in plant growth promotion [17]. These shells also contain proteins and inorganic salts. Its partially deacetylated form is chitosan (polysaccharide derived from chitin). Their polycationic characters allow them to have several antimicrobial and eliciting properties of defense reactions in the plant [18]. Moreover, Djeuani et al. [19], showed that the presence of chitosan in the culture substrate stimulated both the activation of defense mechanisms and also, the growth in plants of the family Araceae like X. sagittifolium. Therefore, what will be the response of taro corms to fertilization with oyster shell powder during the production of mass sanitized seedlings by the PIF method? Similarly, once produced, will these PIF plants be able to respond positively to fertilization? It is in this line that the work aimed to carry out mass production of seedlings sanitized of two varieties of C. esculenta widely cultivated in Cameroon by the method of PIF while emphasizing the aspect of fertilization of the propagators of production starting from the ash of the shells of oysters (Crassostrea spp). In addition, a follow-up of the influence of some fertilizers during the growth of these PIF seedlings in acclimatization was carried out for the evaluation of their influence on growth.

The objective of this work was mass produce seeds of sanitized plants of two cultivars of C. esculenta widely grown in Cameroon by the PIF method. The results obtained varied significantly according to the cultivars used and the treatments.

The percentage of bud break of corm fragments of C. esculenta in both cultivars used was 100% except in the substrate treatment of Makoumba’ cultivar. The death of the observed corm fragment could be related to its physiological state or to the physiological stress caused during the realization of the right-angle cross incision to destroy the apical dominance of the main bud. The number of buds emitted was higher in Ehkwan’frè cultivar compared to Makoumba cultivar (Table 4). This number of buds was also more significant in the presence of oyster shell ash of Crassostrea spp. in the propagator. Moreover, it should be noted that the sawdust substrate used is a lignocellulosic material with a C/N ratio between 100 - 500 and therefore would be less rich in mineral

elements. According to [20] this high C/N ratio would indicate slow mineralization of carbon, slow decomposition of organic matter and consequently, immobilization of the nitrogen necessary for growth. Therefore, the recovery of dormant buds on both sides of the corm fragment would be the result of favourable growing conditions in the propagators that would not influence the water uptake by the osmosis phenomenon that would facilitate the hydrolysis of the starch chain stored in the starchy parenchyma of the corm fragment of each cultivar. Osmosis is a vital function for the growth and stability of plant life [21] [22]. Thus during the budding phase, in propagators with only sawdust + wood shavings as substrate, the carbohydrates needed for growth would be produced through the phenomenon of respiration which is responsible for the degradation of the starch chain contained in the corm fragments. Similarly, the presence of oyster shell ash of Crassostrea spp., an important source of chitin, rich in calcium (Ca²⁺) and mineral elements in the substrate would have boosted bud development in both cultivars of Colocasia esculenta. This justifies the high values of the average number of buds emitted per plant and the total number of buds emitted in culture propagators with substrate + oyster shell ash of Crassostrea spp. treatment. Mineral element availability is a key to growth improvement [23] [24]. For all culture conditions, PIF plants from the emitted buds exhibited growth at different rates. Therefore, despite the destruction of apical dominance of the main bud at the beginning of cultivation, there would be apical dominance among the different emitted buds. This explains the significant difference between the number of PIF plants weaned in the first and second months of culture. The number of PIF plants produced decreased over time for all cultivars.

From 120 corm fragments of the both cultivars of C. esculenta, 599 PIF plants were produced and weaned. 246 PIF plants for Makoumba’s cultivar and 353 PIF plants for Ehkwan’frè’s cultivar. This allows us to say that the PIF technique allowed us to multiply the number of the fragments of corm used by 4.99 times. The results also show that the number of PIF plants weaned is higher after one month of cultivation. This could be justified by the availability of carbohydrates which would be the result of the hydrolysis of the starch chain present in the starchy parenchyma of the corm fragments. However, in the second month, a decrease in the number of PIF plants produced is noted. This could be due to the fact that after weaning, the PIF plants that were not weaned would continue to exert inhibition on the buds that did not quickly recover. It is noticed that the number of weaned PIF plants is higher in both cultivars when the fragments used were grown in the presence of Crassostrea spp. oyster shell ash, i.e. 140 and 200 PIF plants respectively for Makoumba and Ehkwan’frè cultivars after two weanings. Thus, since the wood substrate is organic matter that degrades slowly, the source of mineral matter needed for growth would come in part from the hydrolysis of the oyster shell ash used during the watering of the propagators.

The results of the analysis of agronomic growth parameters during the cultivation and production of PIF plants in the propagators showed that, in the two cultivars of C. esculenta used, the parameters, average of plant height, average of collar diameter, average of number of leaves and average of leaf surface were higher and significant (P < 0.05) in the propagators fertilized with oyster shell ash of Crassostrea spp. This significant increase in the presence of oyster shells in the medium is characterized by the availability of mineral elements for the plant. In addition, the richness of oyster shells in Ca²⁺ and chitin would have played a significant role as Ca²⁺ is a crucial regulator of plant growth and development [17] [25] while the chitin present is a growth activator.

Fertilizers applied in PIF plants of both cultivars during acclimation influenced growth. Oyster shell ash of Crassostrea spp. and arbuscular mycorrhizal fungus (AMF) treatments had the greatest influence on growth, compared to Tithonia diversifolia leaf powder and cow dung powder applied. All these treatments indeed release mineral elements into the culture medium, except for the AMFs, which are soil microorganisms whose role is to feed the plant as a result of the established symbiosis. Therefore, the growth peaks observed in the presence of oyster shell ash of Crassostrea spp. during the first and second months of culture would be due to the immediate availability of mineral elements while in the presence of these AMFs, the peaks visible during the third month of culture would be the result of the establishment of the symbiosis.

The objective of this work was to carry out mass production of sanitized seedlings of two cultivars of C. esculenta widely cultivated in Cameroon by the method of PIF, while highlighting the aspect of fertilization of the production propagators from the ash of oyster shells (Crassostrea spp.) while evaluating the influence of some fertilizers during the growth of these PIF seedlings in acclimatization. The results obtained show that the use of the PIF method boosted the production of sanitized seeds of PIF plants in these C. esculenta cultivars. From 120 corm fragments of the two C. esculenta cultivars, 599 PIF plants were produced and weaned. This technique allowed a 4.99-fold increase in the number of corm fragments. Furthermore, among the fertilizers used, the oyster shell ash Crassostrea spp. and arbuscular mycorrhizal fungus treatments had the most positive influence on growth during acclimatization. The mastery of the mechanisms of this technique would be an asset for their popularization among farmers.

The authors thank all the member of the Araceae Research Team of the Laboratory of Plant Physiology and Biochemistry, Department of Biological Sciences, Higher Teachers’ Training College, University of Yaoundé I. Yaoundé, Cameroon. This research work was not funded.

The authors declare no conflicts of interest regarding the publication of this research work.

Djeuani, A.C., Mbouobda, H.D., Nzie, O., Ngoï, J.K. and Niemenak, N. (2021) Use of Oyster Shell Ash of Crassostrea spp. in Mass Production of Sanitized Seedlings of Colocasia esculenta L. Schott by the PIF Method and Influence of Some Fertilizers during Their Growth in Acclimation. Open Access Library Journal, 8: e8008. https://doi.org/10.4236/oalib.1108008

BS: black soil

AMF: arbuscular mycorrhizal fungus

TD: Tithonia diversifolia

OS: Oyster shells ash of Crassostrea spp

CD: cow dung