The main factor limiting the production of calla lilies is disease. Soft rot, caused by Pectobacterium carotovorum subsp. carotovorum poses a significant worldwide threat to Zantedeschia crop (Githeng’u et al., 2016). Other associated pathogens include Pseudomonas marginalis (Brown) Stevens (Krejzar et al., 2008; Yan et al., 2014), Pseudomonas veronii Elomari et al., Chryseobacterium indologenes (Yabuuchi et al.) Vandamme et al. (Mikiciński et al., 2010), Cellvibrio zantedeschiae sp. Nov. (Sheu et al., 2017), Pectobacterium zantedeschiaeWaleron et al. (Waleron et al., 2019), and Pectobacterium aroidearum Nabhan et al. (Chen et al., 2020). The main symptoms of this disease include dwarfing, chlorosis along the leaf margin progressing in a "V" shape that may turn brown, stem bending, and eventual desiccation (Ciampi et al., 2009).

Viruses that often attack Zantedeschia include dasheen mosaic virus (DsMV), classified as the most prevalent among Aroids (Cafrune et al., 2011). Its distribution was recorded in Bosnia and Herzegovina, where crops presented symptoms such as yellowing and distortion of leaves as well as mosaic and chlorotic ring symptoms (Grausgruber-Gröger et al., 2016).

For the konjac mosaic virus (KoMV), Myzus persicae (Sulzer) and Aphis gossypii (Glover) were identified as main vectors, along with vegetative propagation and seed transmission. Symptoms appear as mosaics, green islands, interveinal chlorosis, leaf deformity, short peduncles, and discolored pigmented spots on the spathe (Liao et al., 2020).

The calla lily latent virus (or zantedeschia mild mosaic virus [ZaMMV]) is a potyvirus isolated from the Black Magic cultivar. It manifests as yellow spots and stripes, green islands, and an unusually mild mosaic (Rizzo et al., 2015). For the detection of viruses, Zantedeschia has been cultured in vitro (Purmale et al., 2023). However, with this type of propagation the viruses were not eliminated. Therefore, studies on thermotherapy, chemotherapy, cryotherapy or electrotherapy (Szabó et al., 2024) to eliminate virus in Zantedeschia could be evaluated.

Endogenous contamination is a recurring issue in in vitro establishment, particularly in rhizome tissue. Sanitary management before in vitro cultivation is recommended. Species of the Pectobacterium family propagate easily through decaying plant material, and there are no efficient control agents for the disease they cause (van der Wolf et al., 2021; Niyokuri and Nyalala, 2023). Soil fumigation with metam sodium and the application of antagonistic bacteria CAE 01 of the Enterobacteriaceae family reduced the percentage of infection in rhizomes (García-López, 2010). As a biological control for P. carotovorum subsp. carotovorum, the high specificity of the bacteriophage virus PP1 of the Podoviridae family was studied (Lim et al., 2013). The use of Bacillus velezensis (Ruiz-García et al.) (other scientific name Bacillus amyloliquefaciens subsp. plantarum) reduced the growth of the phytopathogen in both in vitro and field conditions (He et al., 2021). Protein profiles from in vitro and in vivo conditions for P. carotovorum following interaction with Z. elliottiana 'Black Magic' were also generated. Using two-dimensional electrophoresis, 53 essential proteins for their virulence were identified, showing differential expression in vitro, in vivo, or both compared to the control (Wang et al., 2018).

The eradication of pathogens prior to calla lily in vitro establishment has been a challenge. The use of mercury chloride (HgCl₂) at 0.1-0.2% (w/v) for 15 min (Xiao-Chun, 2010) or for 30 min for leaf and rhizome at doses of 0.1 and 0.2% (w/v) respectively with prior treatment with benomyl at 1% (w/v) in shaking for 30 min (Hlophe et al., 2015) yielded good results. Even doses of 0.5% of HgCl₂ (w/v) for 15 min were effective at this stage in Z. rehmannii with pre-treatment of immersion in 70% ethanol (v/v) for 30 s (Kulpa, 2016). Despite the effectiveness of these treatments, a concentration of 0.25 mg L^-1 has negative environmental consequences on amphibian development (Muñoz-Escobar and Palacio-Baena, 2010).

Hernández-Villaseñor et al. (2018) disinfected with a 15% (w/w) chlorine solution followed by 500 mg L^-1 of streptomycin, 500 mg L^-1 of oxytetracycline, and 500 mg L^-1 of carboxamide for 60 min. Finally, the explants were placed into a 70% (v/v) ethanol solution for 30 s. Similarly, the application of chlorine dioxide (ClO₂) at a concentration of 60 to 180 mg L^-1 (v/v) for 5 min followed by hydrogen peroxide (H₂O₂) at 5% (v/v) for 15 min provided a survival rate >75% for Z. aethiopica (Chen et al., 2017).

Due to the need to implement disinfestation protocols that have less environmental impact, studies based on antibacterial activity of Coptis chinensis (Franch.) extract has been developed against soft rot. 100% C. chinensis made an inhibition effect comparable to streptomycin sulphate (Githeng’u et al., 2016), an antibiotic widely used in the 90’s. This protocol has not been tested on Zantedeschia explants. Hashemidehkordi et al. (2021) evaluated the effect of hot water in the disinfection of rhizome explants and achieved a survival rate of 90% at 45°C for 35 min followed by immersion in 70% ethanol for 30 s and 1% (v/v) sodium hypochlorite (NaOCl) for 10 min. This procedure represents a good alternative to reduce the use of previously mentioned products and their impact in terms of environmental, economic impact, and antibiotic resistance. Table 1 shows the control methods for the in vitro culture of calla lilies.

Table 1.

Plant material	Origin of explant	Control method	Survival rate (%)	Reference
Z. hybrida	Rhizome	HgCl2 at 0.1-0.2% for 15 min for rhizome segments	Not reported	Xiao-Chun, 2010
Z. aethiopica	Leaf and rhizome	HgCl2 at 0.2% for rhizome segments and 0.1% for leaves for 30 min	28% in rhizome and 78.3% in leaf	Hlophe et al., 2015
Z. rehmannii	Rhizome	Immersion in 0.5% HgCl2 for 15 min	73%	Kulpa, 2016
Z. aethiopica	Rhizome	15 % chlorine + 500 mg L-1 streptomycin, 500 mg L-1 oxytetracycline, and 500 mg L-1 captan for 60 min + 70 % ethanol (v/v) for 30 s	Not reported	Hernández-Villaseñor et al., 2018
Z. aethiopica	Rhizome-buds	ClO2 at 60 to 180 mg L-1 (v/v) for 5 min + 5% H2O2 (v/v) for 15 min	>75 %	Chen et al., 2017
Zantedeschia spp. ‘Orania’ and ‘Sunclub’	Rhizome-buds	Hot water bath at 45°C for 35 min + immersion in 70 % ethanol for 30 s and 1 % sodium hypochlorite (v/v) for 10 min	>90 %	Hashemidehkordi et al., 2021
Z. aethiopica ‘Déjà vu’ and ‘Utopia’	Rhizome	Agrimicyn® (2 g L-1) added to the fungicide Benomyl® (2 g L-1)	‘Utopía’: 60%; ‘Déjà vu’; 80 %	Martínez-Hernández, 2022
Z. aethiopica	Seed	70 % ethanol for 1 min + 10% NaOCl for 15 min, and 10 min of 30% H2O2	100 %	Martínez-Hernández, 2022

In vitro propagation of Z aethiopica cultivars in Mexico has not been easy due to contamination of the explants. The antibiotic Agrimycin® (2 g L^-1) added to the fungicide Benomyl® (2 g L^-1) reduced endophytic contamination by fungi and bacteria in explants from rhizomes of the cultivars Deja Vú and Utopia. For seed germination, in vitro contamination was not observed when using 70% ethanol for 1 min followed by 10% sodium hypochlorite for 15 min, and 10 min of 30% hydrogen peroxide (Martínez-Hernández, 2022).

The process of organogenesis provides the basis for asexual plant propagation largely from nonmeristematic somatic tissues (Schwarz and Beaty, 2000). Through this method, pathogen-free plants have been obtained via meristem culture (Tab. 2). In Z. aethiopica, the addition of benzylaminopurine (BAP) at 22.19 μM produced 2.6 shoots per explant (Ribeiro et al., 2014). Rhizomes and tubers have been the main organs targeted for obtaining explants despite the likelihood of presenting endogenous contamination. Singh et al. (2009) obtained 5 to 6 shoots of Z. aethiopica 10.0 mg L^-1 of BAP supplemented with 50 mg L^-1 of ascorbic acid. In Z. rehmannii, culture was obtained using a concentration of 3 mg L^-1 BAP, and a rate of 4.3 shoots per explant was obtained with 2.5 mg L^-1 BAP (Kulpa, 2016).

Table 2.

Plant material	Origin of the explant	Medium	Growth regulators	Multiplication rate (shoots per explant)	Reference
Z. aethiopica	Rhizome	MS	10.0 mg L-1 BAP, 50 mg L-1 ascorbic acid, 25 mg L-1 adenine sulfate, L-arginine and citric acid	5-6	Singh et al., 2009
Z. rehmannii	Rhizome-buds	MS	3 mg L-1 and 2.5 mg L-1 BAP + 100 mg L-1 ascorbic acid	4.3	Kulpa, 2016
Z. aethiopica	Shoot tip	MS	22.19 μM BAP	2.6	Ribeiro et al., 2014

Shin et al. (2020) reconstructed the process of regeneration of new shoots from callus and differentiated four stages: acquisition of pluripotency (in a medium rich in auxins), formation of pro-meristems, establishment of shoot progenitor, and shoot growth. Some species from the genus Zantedeschia have been propagated through this pathway (Tab. 3).

Table 3.

Plant material	Origin of the explant	Medium	Growth regulators	Reference
Z. hybrida ‘Feng Yang’	Rhizome	MS	BA (2.0 mg L-1) + NAA (0.1 ~ 0.2 mg L-1)	Xiao-Chun, 2010
Zantedeschia spp. ‘Sunlight’, ‘Chiante’ and ‘Pink ‘Persuasion’	Apices derived from callus shoots and adventitious shoots	MS + 70-90 g L-1 sucrose	BA (2-3 mg L-1)	Lee and Ko, 2005
Zantedeschia sp. ‘Pink Giant’	Leaf	MS	2,4-D (2.0 mg L-1) and BA (2.0 mg L-1) or NAA (2.0 mg L-1) and BA (2.0 mg L-1)	Gong et al., 2008

MS, Murashige and Skoog.

In vitro dedifferentiation in Z. aethiopica and Z. elliottiana was observed in petiole, leaf, and spathe segments, achieving over 80% callus formation on Murashige and Skoog (MS) medium supplemented with indoleacetic acid (IAA) at 0.5 mg L^-1 + BAP 2.0 mg L^-1 for leaves; IAA (2.0 mg L^-1) + BAP (2.0 mg L^-1) for spathes; and IAA (1.0 mg L^-1) + BAP (2.0 mg L^-1) for petioles. The petioles showed the greatest dedifferentiation capacity among the organs mentioned. Furthermore, Z. elliottiana demonstrated a higher differentiation capacity than Z. aethiopica, as well as the ability to generate callus at high doses of BAP (4.0 mg L^-1) combined with NAA (0.1 mg L^-1) (Jonytienè et al., 2017). In Zantedeschia spp., callus formation was induced by culturing leaf segments on MS medium supplemented with 2,4-D (2.0 mg L^-1) and BA (2.0 mg L^-1) or with naphthaleneacetic acid (NAA) (2.0 mg L^-1) and BA (2.0 mg L^-1) (Gong et al., 2008).

The hormonal balance between auxins and cytokinin is crucial in the indirect organogenesis of Zantedeschia in vitro (Cheng et al., 2013). Lee and Ko (2005) achieved up to 56.7% callus formation on a medium containing BA (2.0 mg L^-1) and a shoot regeneration rate of 70% at 3.0 mg L^-1 of BA. Cytokinin induced the formation of multiple shoots; 2ip (1.0 mg L^-1), BA (5.0 mg L^-1), and BA (1.0 mg L^-1) brought on 16, 14, and 12 multiple shoots in the cultivars Sunlight, Chiante, and Pink Persuasion, respectively. Xiao-Chun (2010) promoted callus formation on an MS medium + 2.0 mg L^-1 BA + 0.1-0.2 mg L^-1 NAA and achieved 100% rooting with an MS medium + indole butyric acid (IBA) at a concentration of 0.3 mg L^-1.

Few studies on induced somatic embryogenesis in Zantedeschia exist (Tab. 4). The first reported research on somatic embryogenesis in calla lilies was conducted on three hybrids with explants derived from rhizomes and anthers. However, the formation of somatic embryos was only achieved from rhizome segments. The best results were obtained in media supplemented with plant growth regulators (Nic-Can et al., 2016), as NAA (2.0 mg L^-1) and BA (0.6 to 2.0 mg L^-1). Conversion to normal seedlings was achieved in MS medium supplemented with vitamins, micro and macronutrients, 1.0 mg L^-1 2iP, 3% sucrose, and 0.7% agar (Duquenne et al., 2006).

Stem segments containing apical meristems derived from the cultivar Gag-si were cultured on MS medium. A 25% induction rate of yellow embryogenic calli was obtained with MS + 0.5 mg L^-1 NAA and 1.5 mg L^-1 BA. In the regeneration experiments from embryogenic calli, the MS medium supplemented with 0.5 mg L^-1 IAA and 2.0 mg L^-1 BA showed the highest rates, approximately 85 ~ 90%. Table 4 summarizes research related to somatic embryogenesis of Zantedeschia species (Han and Kim, 2019).

Table 4.

Species	Origin of explant	Medium	Growth regulators	Reference
Zantedeschia hybrids	Rhizomes and anthers	MS	BA (0.22 mM)	Duquenne et al., 2006
Zantedeschia hybrida	Petiole	MS	0.5 mg L-1 NAA, 1.5 mg L-1 BA, 0.5 mg L-1 IAA, 2.0 mg L-1 BA	Han and Kim, 2019

Research with Zantedeschia grown in liquid media has focused on determining the optimal frequency of immersion, comparing temporary immersion systems (TIS) with other culture systems such as semisolid and agitated liquid, and the effect of growth regulators. A multiplication coefficient of 9.7 shoots per explant was obtained using active buds from Zantedeschia spp rhizomes in a modified MS medium with 100 mg L^-1 myo-inositol, 1 g thiamine, 4 mg L^-1 BAP, and paclobutrazol at 0.3 mg L^-1 with an immersion frequency of 4 h every 4 min (Sánchez et al., 2009). Sánchez et al. (2010) achieved multiplication rates of 7.93, 10.66, and 11.30 shoots per explant in an MS medium + 1, 2, and 4 mg L^-1 BA and an immersion frequency of 4 h every 3 min.

Mature embryos from in vitro seeds have also been used to generate transgenic plants of Zantedeschia (Sun et al., 2022). Studies on the in vitro germination of embryos (seeds) of species in the genus Zantedeschia determined that temperatures of 14-20°C, high concentrations of sucrose and agar (Ngamau, 2001), fruit maturity, and GA₃ can influence the germination rate (Nery et al., 2015). In vitro germination of Zantedeschia studies focused on early flowering, and salt and/or temperature tolerance. Seedlings from seeds established at low temperatures had greater growth under lower night temperatures than those germinated at high temperatures, and clones from seeds exposed to high levels of sodium chloride (NaCl) achieved greater growth. Also, early seeds showed better growth, early flowering, and a higher number of spathes (Ngamau, 2008). The germination of Z. aethiopica seeds in vitro was enhanced in a medium supplemented with 3.0 mg L^-1 of GA₃ and 0.3 mg L^-1 of BAP, with germination occurring 35 d after establishment (Martínez-Hernández, 2022) (Fig. 2).

Figure 2.

Martínez-Hernández et al. (2022) isolated zygotic embryos from mature and immature white calla lily fruits. The seeds were pretreated with sterile water and 30% (v/v) H₂O_2. The isolated embryos were cultured in a medium with MS salts (100%) supplemented with BAP (0, 0.3 and 3 mg L ^-1) and GA₃ (0, 0.3 and 3.0 mg L^-1). The embryos’ development was not affected by the composition of culture media, the seed pretreatments or the fruit ripening stage (Fig. 3).

Figure 3.

Naor et al. (2004a) investigated the hormonal control of floral induction and the development of inflorescence in vitro in day-neutral plants of Zantedeschia spp., as well as the effects of GA₃ and BA on the development of inflorescences in regenerated tissue culture seedlings. The seedlings were immersed in GA₃ and BA solutions before replanting in new media. GA₃ was found to be essential for the transition from the vegetative to the reproductive stage. Inflorescence development in the apical bud was observed after 30-50 d in seedlings treated with GA₃ grown in vitro. However, the resulting inflorescences had incomplete male and female flowers. BA did not affect flower induction but in the presence of GA₃, BA at 4.4 M enhanced inflorescence differentiation. They concluded that the floral initiation was not influenced by environmental stimuli and that the inflorescence development in the apical bud resembled natural conditions (Naor et al., 2004a). GA₃ had a dual action in the flowering process, inducing inflorescence differentiation and promoting floral stem elongation. They inferred that the flowering pattern might be a gradient in the distribution of GA, likely controlled by the apical dominance of the primary bud (Naor et al., 2004b).

Genotype influences the growth and development of explants in vitro. Thus, the propagation of new varieties will need specific protocols. The evaluation of artificial light sources for illumination to improve the quality of the explants may be explored. The use of nanomaterials in modulating in vitro Zantedeschia tissue growth and development could also be evaluated. Additionally, organic additives in micropropagation to enhance the growth and health protection of the explants could be tested.