De novo regeneration and micropropagation of Opuntia ficus-indica (L.) Mill.

Introduction

Opuntia ficus-indica (L.) Mill., commonly known as prickly pear or nopal is a droughttolerant cactus of the Cactaceae family native to Central America and now widespread in arid and semi-arid regions. Morphological traits such as fleshy cladodes, spines, cuticled epidermis and CAM photosynthesis enable survival under harsh climates (Slaba, 1992; Nobel, 1991) [21,17] . Among more than 2000 cactus species it is the most economically important cultivated for fruits, pads, fodder, soil conservation and ecological restoration (Nobel, 2002; Casas and Barbera, 2002) [18,3] . In India it is grown in Rajasthan, Gujarat, Madhya Pradesh and Punjab contributing to fodder supply and land reclamation (Khalafalla et al., 2007) [9] . Even with high theoretical transmissibility, propagation under practical conditions remains limited. Seed germination is poor and genetically variable (Stambouli-Essassi et al., 2017) [22] , while cladode multiplication is slow and unsuitable for large-scale use (Mohamed-Yasseen et al., 1995) [15] . Tissue culture offers a reliable alternative for producing uniform, disease-free plants (Lema-Rumińska and Kulus, 2014) [10] , though regeneration efficiency depends on genotype, explant type and growth regulator balance (Escobar et al., 1986; García-Saucedo et al., 2005; Estrada-Luna et al., 2008) [5,8,6] . Areoles are considered highly responsive, with cytokinin-auxin interactions playing a key role in morphogenesis (Skoog and Miller, 1957; Mabrouk et al., 2021) [20,12] . This study aimed to develop an efficient de novo regeneration protocol for spineless O. ficus-indica under Indian agro-climatic International Journal of Advanced Biochemistry Research 2025; 9(1 1): 386-390 conditions, focusing on explant source, sterilization, growth regulator regimes, rooting and acclimatization.

Materials and Methods

Plant material

The study (2023-2025) was conducted at the State Level Biotechnology Centre, MPKV, Rahuri, India. Spineless Opuntia ficus-indica accession 1280 (origin: Argentina, yellow-fruited) from the AICRP on Forage Crops collection was used. Young cladodes (10-15 cm) were excised and areoles (~1 cm 2 ) served as explants.

Sterilization

Explants were disinfected using combinations of Tween-20 (0.1-0.2%), Bavistin + Streptocycline (0.1-0.2%), NaOCl (1%), ethanol (70%) and HgCl₂ (0.1-0.5%) for varying durations. The most effective treatment based on contamination percentage was used for subsequent cultures.

Culture medium and conditions

Murashige and Skoog (MS) medium (Murashige and Skoog, 1962) [16] with 3% sucrose, 0.8% agar and pH 5.8 was employed. Cultures were incubated at 25 ± 2 °C under a 16 h light/8 h dark photoperiod (1600 lux).

Shoot initiation

Areoles were cultured on MS medium with BAP (0.5-2.5 mg L⁻¹) either alone or combined with 2, 4-D (1.0-2.0 mg L⁻¹) and GA₃ (0.5 mg L⁻¹). Fifteen treatments were tested and days to shoot initiation and response percentage were recorded after 4 weeks.

Shoot multiplication

Regenerated shoots were transferred to MS medium containing BAP (1.0-3.0 mg L⁻¹) or kinetin (0.5-4.0 mg L⁻¹) combined with NAA (0.1-1.5 mg L⁻¹) or IAA (0.3-0.5 mg L⁻¹). Twenty-two treatments were evaluated for shoot number per explant and multiplication percentage after 8-9 weeks.

Rooting

Shoots (2-3 cm) were placed on half-strength MS medium with IBA (0.1-1.5 mg L⁻¹) or IAA (0.5-1.0 mg L⁻¹), with or without GA₃ (0.2-0.6 mg L⁻¹). Nine treatments were compared for rooting percentage, root length and days to root initiation after 3-4 weeks.

Acclimatization

Rooted plantlets were washed, treated with Bavistin (0.1%) and transferred to sterile soil: cocopeat (1:1), sterile soil: sand (1:1), or sterile soil: cocopeat: sand (1:1:1). After 5-6 weeks, plants were shifted to soil: vermicompost: sand (2:1:1) or sand: vermicompost: cocopeat (2:1:1) for secondary hardening in the greenhouse. Survival was recorded.

Results and Discussion

The present investigation, "De novo regeneration studies of Opuntia ficus-indica", was conducted during 2023-2025 at the State Level Biotechnology Centre, MPKV Rahuri, to establish an efficient in vitro regeneration and propagation protocol. The results of explant response, sterilization, shoot initiation, multiplication, rooting and acclimatization are presented below.

Explant selection and initiation

For this experiment three explant types were tested viz. areoles from young (2-3 months) cladodes, areoles from mature (6 months) cladodes and cladode segments without areoles. Areoles from 2-3-month-old cladodes showed the highest regeneration potential with 76.7% induction (230/300 explants) whereas older cladode areoles responded at only 40% (Table 1). Cladode segments devoid of areoles failed to regenerate, confirming the areole as the key morphogenic site. These results align with previous findings that younger cactus tissues retain higher metabolic activity and meristematic competence (Finti et al., 2013 and Rodríguez & Ramírez-Pantoja, 2020) [7,19] . [13, 2] . Thus, a sequential sterilization regime ensured maximum recovery of viable, contamination-free explants.

Shoot initiation and areole activation

To induce areole activation, MS medium supplemented with BAP, 2,4-D and GA₃ was tested. The most effective response (90% induction within 30 days) occurred on MS + 1.0 mg/L BAP + 1.0 mg/L 2,4-D (Table 2). BAP alone supported only limited response (≤ 50%), while higher auxin concentrations (≥ 2.0 mg/L 2,4-D) suppressed shoot initiation, favoring callus formation instead. Addition of GA₃ enhanced elongation but did not substitute for auxincytokinin synergy. This confirms earlier studies showing that a balanced cytokinin-auxin ratio is crucial for cactus organogenesis (Linhares et al., 2005; Mengesha et al., 2016) [11, 14] . [1] and Rodríguez & Ramírez-Pantoja (2020) [19] .

Root induction

Rooting was achieved on half-strength MS medium with auxins. 0.5 mg/L IAA gave the best response, producing 100% rooting within 28 days with average root length of 0.95 cm. In contrast, IBA (0.5-1.0 mg/L) induced roots in only 57% of explants, though roots were longer (up to 2.1 cm). Higher auxin levels (> 1.0 mg/L) and GA₃supplemented treatments inhibited rooting (Table 4). These results agree with Bouzroud et al. (2022) [2] and El Finti et al. (2010) [4] , who found that IAA is superior for initiation, whereas IBA mainly enhances elongation. [9] .

Troubleshooting during micropropagation

Acknowledgement

AcknowledgementWe are very thankful to the Post Graduate Institute and State Level Biotechnology Centre, MPKV, Rahuri, for providing all Facilities and research grants to carry out this research work and to the authors cited in reference for providing necessary literature.

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