Effect of pre-treatments on in vitro culture establishment of purple fleshed greater yam (Dioscorea alata var. Purpurea)

Introduction

Dioscorea alata L. commonly known as greater yam, water yam or winged yam, is an important tropical and subtropical tuber crop belonging to the family Dioscoreaceae and is cultivated primarily for its large underground starchy tubers (Coursey, 1967) [1] . The species is one of the major cultivated members of the genus Dioscorea and has a broad pantropical distribution extending across Asia, Africa, the Pacific, the Caribbean and tropical America (Lebot et al., 1998; Malapa et al., 2005) [2,3] . Dioscorea alata is a perennial, herbaceous and dioecious climbing plant characterized by vigorous twining stems and underground storage tubers that show extensive variation in size, shape, skin colour and flesh colour among cultivars (Lebot et al., 1998; Abraham and Nair 1991) [2,4] . Greater yam possesses considerable genetic and phenotypic variability, which is expressed through differences in tuber morphology, aerial tuber production, pigmentation, maturity, agronomic performance and biochemical composition (Girma et al., 2017) [5] . The species consequently includes diploid, triploid and tetraploid cytotypes with chromosome numbers of 2𝑛 = 40, 60 and 80, respectively (Nemorin et al., 2013; Arnau et al., 2009) [7,6] . Vegetable tuber crops represent an essential source of dietary starch, minerals, and bioactive compounds for global food security (Coursey, 1967) [1] . Among root vegetables, greater yam (Dioscorea alata L.) stands out due to its high yield potential and nutritional value, with specific purple-fleshed cultivars synthesizing deep purple pigments throughout their tuber tissues (Abdullah, 2020) [8] . The purple pigmentation of yam tubers is primarily associated with anthocyanins, which constitute an important class of water-soluble flavonoid pigments with recognized antioxidant properties (Champagne et al., 2011) [9] . These purple pigments are primarily acylated cyanidin-based anthocyanins with high free-radical scavenging capacity that exert significant anti-inflammatory, anticancer, and cardioprotective health benefits (He et al., 2010) [10] . Beyond phenolic pigments, Dioscorea tuber crops produce high levels of diosgenin, a major steroidal saponin (Bhandari et al., 2004) [11] that serves as a critical natural precursor in the industrial synthesis of steroid hormones and oral contraceptives (Patel et al., 2012) [12] . Dioscorea alata is predominantly propagated vegetatively through whole tubers, tuber pieces and other vegetative planting materials, because clonal propagation permits the maintenance of desirable cultivar characteristics (Lebot et al., 1998; Vandenbroucke et al., 2016) [2,13] . However, traditional vegetative propagation using seed tubers yields low multiplication rates (Ezeh et al., 2014) [14] , and tuberborne pathogens accumulate over successive field generations, leading to severe yield decline in yam crops (Ospina et al., 2015; Acedo, 2003) [16,15] . Plant tissue culture techniques provide an efficient platform for rapid clonal propagation and disease eradication in vegetable crops (Borges et al., 2004) [17] . The restricted availability of healthy planting material and the relatively slow multiplication rate have stimulated interest in tissue culture and micropropagation as alternative approaches for the rapid multiplication and conservation of elite D. alata cultivars (Acedo, 2003; Rajitha et al., 2011) [15,18] . Nodal segments have been demonstrated to be suitable explants for in vitro multiplication of greater yam, and successful shoot regeneration and rooting have been obtained under appropriate combinations of basal medium, plant growth regulators and activated charcoal (Rajitha et al., 2011) [18] . Nevertheless, initiating sterile in vitro cultures from field-grown subterranean yam explants remains a critical challenge (Nicoletti et al., 2020) [19] because exogenous microbial epiphytes and deep-seated endophytes rapidly contaminate growth media during initial culture stages (Leifert et al., 1994) [20] . Fungal pathogens frequently overgrow nodal explants and cause mortality (Webster et al., 2003) [21] , while vascular bacterial contaminants proliferate in nutrient media, causing tissue necrosis and culture degradation (Reed et al., 2004) [22] . Furthermore, enzymatic oxidation of phenolic compounds exuded from cut tissues inhibits shoot bud initiation (Ahuja et al., 2002) [23] . Standardizing chemical pre-treatments with systemic fungicides and bactericides is therefore mandatory to eliminate persistent pathogens (Cassells, 2012) [24] . Micropropagation is particularly relevant for purple yam because it permits rapid multiplication of selected genotypes from limited starting material and can contribute to the production of uniform planting material and conservation of valuable germplasm (Acedo, 2003; Belarmino et al., 2008) [15,25] . However, establishment of aseptic cultures remains a critical step because nodal explants derived from field-grown or greenhouse-grown plants may harbour surface-associated and endogenous microbial contaminants (Acedo, 2003; Rajitha et al., 2011) [15,18] . Thus, Dioscorea alata var. purpurea represents a nutritionally and economically valuable purple-fleshed yam genotype with considerable genetic, cytological and biochemical diversity, while its conventional propagation limitations emphasize the need for efficient in vitro multiplication systems (Arnau et al., 2009; Lebot et al., 1998) [6,2] . Development of a reliable aseptic culture-establishment protocol is therefore an essential prerequisite for subsequent shoot multiplication, conservation and rapid production of healthy planting material of purple-fleshed greater yam (Acedo, 2003; Rajitha et al., 2011) [15,18] . In this context, the present investigation was undertaken to standardize an effective chemical pre-treatment protocol for reducing microbial contamination and improving the survival and establishment of nodal explants of greater yam.

Materials and Methods

The present experiment was carried out at Tissue culture laboratory, Regional Horticultural Research Station, Dr. YSR Horticultural University, Kovvur, Andhra Pradesh to standardize aseptic culture establishment in Dioscorea alata var. Purpurea. The experimental site is geographically located at 17.00 N latitude, 81.43 E longitude and altitude of 20 m above MSL. The experiment was conducted in Complete Random Design (CRD) with four replications. Tubers were raised in pots under aseptic conditions to grow healthy and disease to collect nodal segments of 2-3 cm length as explants for the study. Immediately after collection of the nodal segments (explants), they were washed with distilled water to remove dust. Explants showing no visible mechanical injury were selected for inoculation. The selected explants were treated with Carbendazim + Mancozeb (0.1%), Streptocycline sulphate 200 ppm and Tween 20 (2 drops/100 ml) for different time durations 30, 60, 90, 120 minutes and they were compared with control distilled water. Following pre-treatment, the explants were rinsed three times with autoclaved distilled water inside a laminar airflow chamber to remove the chemical residues. The pre-treated explants were subsequently subjected to surface sterilization using 0.1% mercuric chloride (HgCl 2 ) for 5 minutes. After chemical surface sterilization, the explants were washed three times with sterile distilled water to minimize the residual toxic effects of the sterilizing agents. The thoroughly rinsed explants were then considered ready for establishment under in vitro conditions. After completion of sterilization, the explants were placed on sterile tissue paper to remove excess moisture. The dried leaf sheaths and petioles were carefully removed, and a fresh cut was made sat the basal end of each explant to expose the healthy internal tissue. The sterilized explants were then inoculated onto the prepared culture medium using sterile forceps. Each explant was gently pressed against the medium to ensure proper and firm contact with the surface. The inoculated test tubes were closed using polypropylene caps and further sealed with cling film to maintain aseptic conditions. Following evaluation of the most suitable pre-treatment procedure, the explants were cultured on Murashige and Skoog (MS) medium (Murashige et al., 1962) [26] supplemented with sucrose 30g, activated charcoal 1g, BAP (0.75 mg L -1 ) and NAA (0.5 mg L -1

) solidified with agar-agar 8g L -1 . Before inoculation of explants in MS media, the P H of the media adjusted to 5.8 before adding the gelling agent and then autoclaved 121°C for 15 minutes. After inoculation, the cultures were maintained in a growth chamber at 26 ± 1 °C under a photoperiod of 16 h light and 8 h dark. Illumination was provided using LED lighting with a light intensity of approximately 3000 lux. Observations were recorded for the type and incidence of contamination, percentage of fungal ad bacterial contamination, percentage of chemical toxicity, percentage of phenolic exudation, percentage of explant survival and culture establishment index. The observations recorded for the different parameters were statistically analysed using OPSTAT according to the experimental design. Analysis of variance (ANOVA) was carried out following the procedures outlined by Panse and Sukhatme (1985) [27] .

Results and Discussion

Pre-treatment of nodal segments of purple-fleshed greater yam (Dioscorea alata var. purpurea) prior to surface sterilization had a significant effect on microbial contamination, chemical toxicity, and subsequent culture establishment (Table 1). Contamination: Fungal contamination declined progressively with each successive pre-treatment, from 58.33% in the untreated control (P 1 , distilled-water wash) to 12.43% in explants treated with Carbendazim + Mancozeb (0.1%), Streptocycline sulphate (200 ppm) and Tween-20 for 120 minutes (P 5 ). Bacterial contamination followed the same declining trend, from 24.63% (P 1 ) to 6.20% (P 5 ). No phenolic exudation was recorded in any treatment (0% throughout).

The progressive fall in fungal and bacterial contamination with increasing pre-treatment duration is consistent with the established antimicrobial efficacy of carbendazim + mancozeb + streptocycline regimens in explant establishment protocols. Carbendazim is a broadspectrum systemic benzimidazole fungicide that has long been used as a pre-treatment or medium additive to suppress fungal contaminants in tissue culture, and combined carbendazim-mancozeb antifungal formulations have been shown to sharply reduce fungal infection rates in other recalcitrant, contamination-prone in vitro systems (Xing et al., 2025) [28] . Similarly, carbendazim has been reported to control fungal contamination effectively when incorporated into pretreatment or culture media at working concentrations broadly comparable to those used here (Permadi et al., 2023) [29] . The parallel decline in bacterial contamination can be attributed to the streptocycline sulphate component of the pre-treatment. Antibiotic pre-treatments and antibiotic supplemented media, including streptocycline based combinations, have been used successfully to eliminate persistent surface and endophytic bacterial contaminants in nodal explants based micropropagation of other vegetatively propagated species, where combining streptocycline with a second antibiotic achieved fully contamination-free cultures after initial surface sterilization alone had proved insufficient (Ray et al., 2017) [30] . The Tween-20 included in all pre-treatments in the present study would have further aided contaminant removal by lowering the surface tension of the treatment solution and improving its penetration into the nodal crevices and axillary bud scales where epiphytic and endophytic microflora often persist. The complete absence of phenolic exudation across all treatments in this study suggests that nodal tissue of D. alata var. purpurea is comparatively low in oxidisable phenolics relative to other tuber and woody species, where phenolic browning caused by polyphenol oxi-dase activity is a major constraint on explant survival and typically requires supplementation with antioxidants or adsorbents to control (Permadi et al., 2023) [29] . Chemical toxicity: Chemical toxicity remained at 0% for pre-treatment durations up to 90 minutes (P 1 -P 4 ) but rose sharply to 29.13% at 120 minutes (P 5 ). Survival and culture establishment: Survival percentage and the culture establishment index improved progressively with pre-treatment duration, peaking at 90 minutes (P 4 ) with 75.00% survival and an establishment index of 83.33. Beyond this, at 120 minutes (P 5 ), both parameters declined to 52.24% survival and a 69.86 establishment index, despite this treatment showing the lowest contamination overall. The sharp increase in chemical toxicity (29.13%) and the accompanying decline in survival and establishment index at the longest exposure duration (P 5 , 120 minutes) reflect a well-documented dose/duration trade-off in chemical pre-treatment of explants. While carbendazim-based fungicides are generally regarded as safe for plant tissue at working concentrations and have even been used as cytokinin-like growth-promoting supplements in some tissue culture systems (Debergh et al., 1993) [31] , prolonged or excessive exposure can produce measurable phytotoxic effects on plant tissue, as demonstrated in whole-plant studies where carbendazim application caused weak but detectable phytotoxicity in treated plants (Garcia et al., 2002) [32] . This concentration-and-duration-dependent toxicity explains why the 90-minute pre-treatment (P 4 ) rather than the 120minute pre-treatment (P 5 ) produced the best overall outcome in the present study: it achieved a substantial reduction in contamination (14.64% fungal, 10.36% bacterial) without crossing the phytotoxicity threshold, thereby yielding the highest survival (75.00%) and culture establishment index (83.33) among all treatments. [34,33,35] . Comparable graded pre-treatment protocols combining fungicides, antibiotics and surfactants have also been optimised in other Dioscorea species and related tuber crops specifically to maximise axenic culture establishment while minimising phytotoxic explant loss (Hussein et al., 2019; Bhat et al., 2022) [37,36] .

Conclusion

This study clearly demonstrates that pre-treatment of nodal explants is a critical, controllable variable in establishing axenic in vitro cultures of purple-fleshed greater yam (Dioscorea alata var. purpurea), and that its effectiveness cannot be judged by contamination control alone, explant viability must be weighed equally. Progressive exposure to Carbendazim + Mancozeb (0.1%), Streptocycline sulphate (200 ppm) and Tween-20 steadily reduced both fungal and bacterial contamination with increasing duration, confirming that this fungicide-antibiotic-surfactant combination is well suited to eliminating the surface and endophytic microflora typically associated with fieldcollected yam nodal tissue. However, the data reveal an important threshold effect: decontamination efficiency and explant safety do not improve in tandem indefinitely. Beyond 90 minutes of exposure, the incremental gain in contamination control was outweighed by a sharp rise in chemical phytotoxicity, which in turn depressed survival and culture establishment. This identifies 90 minutes (P 4 ) as the practical optimum not simply the treatment with the least contamination, but the one that best balances microbial control against tissue health.

Acknowledgement

AcknowledgementThe authors are highly greaterful to Dr. YSR Horticultural University, Andhra Pradesh for providing technical and financial resources for undertaking the study.

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