The calla lily (Zantedeschia spreng.), a member of the Araceae family, is an herbaceous, bulbous, perennial ornamental flowering plant with a horseshoe-shaped spathe [1]. Calla lily is native to the tropical regions of the African continent, from the marshy plains of the coastal area of South Africa to the mountainous regions of Southeastern Africa. Zantedeschia, scientifically known as calla lily, consists of eight species classified into two sub-groups. The first subgroup, Zantedeschia, consists of two evergreen species, Z. aethiopica and Z. odorata, with white flowers that blossom from late winter to late spring in their natural habitat. The second subgroup, Aestivae, includes six species with colorful flowers that bloom in summer. Thousands of hybrids of the Aestivae group have been introduced worldwide [2]. While the former (hereafter referred to as white calla lily) is accessible on the market and is dominated by intraspecific hybrids of Z. aethiopica, the latter (hereafter referred to colored calla lily) is derived from interspecific or intraspecific hybrids of other species, including Z. elliottianna, Z. rehmannii, Z. albomaculata, Z. jucunda, Z. pentlandii, and others. Owing to their intricate morphology and prolonged flowering duration, both varieties of calla lily have witnessed a substantial surge in market turnover in recent years, thereby making them the most in-demand bulbous flowers in the global market.

As the need for calla lily in the market has increased, the issues associated with its commercial exploitation have become more noticeable. Consequently, breeders have started to prioritize the production of novel varieties with disease resistance and color-rich spathe. It has been widely reported that white calla lily can adapt to the humid environment during cultivation in greenhouses, and is more resistant to bacterial soft rot [3] despite the homogeneous flower color. In contrast, colored calla lily, despite the diverse flower colors, is highly susceptible to soft rot due to the poor tolerance to moisture [4]. Recently, Guttman et al. [3] characterized the morphological and biochemical differences between white and colored calla lilies. The findings suggest that such differences may account for the heightened susceptibility of colored calla lilies to soft rot following Pectobacterium spp. bacterial infection compared to their white counterparts. The study further revealed that in contrast to white calla lily, colored calla lily had rougher abaxial leaf surfaces, as well as more compact aerenchyma and fewer total air spaces in leaf tissue. Such factors contribute to the increased growth of soft rot bacteria. Moreover, the colored calla lily’s immune response and activation of defense-related genes against such bacteria were found to be hindered. Overall, the research facilitates a comprehensive understanding of the distinct characteristics of white and colored calla lilies that contribute to their divergent susceptibility to soft rot. Researchers have made significant attempts to cross-breed the white and colored varieties of calla lily, so as to merge the desirable qualities of both [5]. As an example, the disease resistance of white calla lily was introduced into the colored variant and the spathe color of colored calla lily was added to the white calla lily. However, the findings of the experiment demonstrated that there was ‘plastome-genome incompatibility’ between the two, producing albino hybrid seedlings [6], which failed to produce regular plants. As a result, the focus of calla lily breeding has shifted towards the already existing white and colored calla lily germplasm independently.

Molecular breeding techniques [7], including gene editing, are now available for early utilization in some of the most extensively studied ornamental plants. Such techniques have the ability to manipulate intrinsic genes, leading to the development of modified plants with new and improved agronomic traits [8]. In Ipomoea nil, for instance, the Dihydroflavonol-4-reductase-B (DFR-B) gene, which encodes anthocyanin biosynthetic enzymes, was targeted with CRISPR/Cas9 via Agrobacterium-mediated transformation to produce 24 (75% positive rate) transgenic plants with anthocyanin-free white flowers [9]. Additionally, attempts were made to induce mutations in the flavonoid 3-hydroxylase (F3H) gene, which encodes a critical enzyme in flavonoid synthesis, in Torenia fournieri [10]. The use of the CRISPR/Cas9 system to modify T0 plants resulted in a high frequency of light blue (almost white) flowers (about 80% of regenerated lines) [11]. Such findings present a potential opportunity to develop novel calla lily germplasm with exceptional targeted traits without using hybridization between white and colored calla lilies [9]. Research on the molecular biology and genetic transformation of calla lilies is still in its initial phases, and there are numerous obstacles to overcome. Nonetheless, with the availability of the calla lily genome (which has been sequenced and assembled at the chromosome level by the present group but has not yet been released) and the identification of candidate genes for critical phenotypic traits, transgenic gene editing has emerged as a vital avenue for significantly modifying genetic traits.

Plant in vitro regeneration via tissue culture is a critical component in genetic transformations mediated by A. tumefaciens, electroporation, and particle bombardment [12]. Such regeneration procedures usually include both de novo organogenesis (formation of shoots and roots in vitro from cultivated explants) [13] and somatic embryogenesis (generation of embryos from plant somatic cells without gamete fusion) [14]. The use of both regeneration procedures has been reported in the development of transgenic ornamentals [15]. For example, in Ornithogalum, somatic embryogenesis was used as a platform for transformation, the target plasmid was introduced into the embryonic callus derived from leaf base induction via particle bombardment, and disease-resistant transgenic plants were obtained after in vitro regeneration [16,17]. In two Lilium species (Lilium pumilum and L. longiflorum), two stable and effective genetic transformation protocols based on somatic embryogenesis and adventitious bud regeneration were established [18]. The hypothesis was that the likelihood of chimera formation in the genetic transformation was reduced and the transformation process was made more efficient [18]. Such findings could be attributed to the embryogenic callus being made up of a large number of embryogenic cells, each of which can develop into a somatic embryo on its own [19]. However, the genetic transformation by de novo organogenesis, such as adventitious bud regeneration, is faster as compared to the regeneration pathway via somatic embryogenesis [17].

With regard to addressing the prevailing breeding constraints in calla lily, the development of a highly efficient in vitro plant regeneration system is essential for the future implementation of gene editing-mediated molecular breeding. There have been many reports of calla lily regeneration in vitro, but the methods used have been found to be ineffective and inefficient. The present review covers the current efforts in calla lily in vitro regeneration, including the effects of various explants, phytohormones, and treatment conditions on the effectiveness of the process. The review can serve as a guide and a reference for the future application of molecular breeding programs for the improvement of calla lily varieties.

In the early 1990s, Wu et al. [20] conducted the first study on the in vitro regeneration of calla lily. The leaf of white calla lily was inoculated with additional auxin and cytokinin (NAA and KT) on MS medium and developed a callus after 10 days. Afterward, buds emerged and formed clusters of shoots within approximately three weeks. The shoots, which grew to a length of approximately 3 cm, were then transferred to a rooting medium to facilitate the development of complete plants. The most recent developments in in vitro tissue culture of calla lily are shown in Fig. 1A, while a summary of the research on calla lily de novo organogenesis and somatic embryogenesis since 1985 is shown in Fig. 1B. Notably, the research on somatic embryogenesis in calla lily was initiated later and has experienced less progress than de novo organogenesis. Detailed notes of the in vitro regeneration routes of the calla lily are shown in Fig. 1C; however, somatic embryogenesis has yet to be achieved.

As has been repeatedly demonstrated, plants have extensive regeneration potential, with both inter- and intra-specific differences in their ability to divide and regenerate cells [21]. According to the existing research, de novo organogenesis in calla lily involves both direct organogenesis from thin-walled tuber cells and indirect organogenesis through the production of callus induced from tubers and leaves. There are several factors, including plant genotype, explant type, plant growth regulators (PGRs) and culture conditions that contribute to successful organogenesis [22]. The present review provides a comprehensive overview of the direct and indirect pathways involved in calla lily organogenesis.

Research on somatic embryogenesis in calla lily is still in its early stages, with few related reports available. Most studies have focused on the indirect somatic embryogenesis pathway of calla lily, examining the effects of factors such as explants, plant growth regulators (PGRs), and other related factors on induction efficiency. In the study of the embryogenic callus of calla lily, three types of explants were used: leaf, tuber, and anther (Table 2). In 2006, the tuber of ‘Captain Tendens’ was first used to induce embryogenic callus and regeneration into a complete plant [29]. Subsequently, the tuber of ‘Gagsi’ was utilized as explants to produce calla lily embryogenic callus, and regenerated complete plants were obtained from such callus [26]. Although callus induction was observed in the leaves of ‘Pink Gian’, complete plant regeneration was not achieved. Additionally, an attempt was made to induce embryogenic callus using anthers in calla lily. However, only one explant out of 900 anthers inoculated developed a somatic embryo on medium containing 0.5 mg/L TDZ and 2 mg/L NAA, and all the callus that was obtained eventually turned brown [83].

The combination of auxin and cytokinin is required for the induction of embryogenic callus in the calla lily. The main PGRs affecting embryogenic callus development were 2,4-Dichlorophenoxyacetic acid (2,4-D), NAA, and 6-BA [64]. By applying 6-BA 1.5 mg/L and NAA 0.5 mg/L to the tuber of ‘Gagsi’ as explants in MS media, the embryogenic callus induction rate was 25% [26]. Leaves of ‘Pink Gian’ were induced by MS supplemented with 2,4-D 4.0 mg/L, 6-BA 0.5 mg/L, and NAA 0.2 mg/L, and the callus induced in the first generation was subjected to two rounds of subculture [27]. The results showed that MS containing 2,4-D 2 mg/L and 6-BA 2 mg/L was the optimal medium for callus subculture and promotion of their transformation into somatic embryos. The media containing 2 mg/L 6-BA and 2 mg/L NAA were found to be most effective in promoting embryogenic callus formation and regenerating normal plants. However, different combinations of concentrations eventually resulted in browning and death, without any somatic embryogenesis. The browning of explants is a natural phenomenon that occurs due to enzymatic oxidation of polyphenolic compounds. Notably, the oxidation process may potentially hinder the proliferation of callus in plant tissue culture [84,85]. Certain kinds of antioxidants such as Vitamin C or AgNO₃ can be added to the culture medium to inhibit oxidative browning [86,87].

Although there is a scarcity of research on the somatic embryogenesis of calla lily, there have been many reports on somatic embryogenesis in other Araceae plants. The leaves of Anthurium andraeanum Lind, another member of the Araceae family, have been used to establish a stable and efficient system for somatic embryogenesis [88]. Such system utilizes leaves to induce embryogenic callus, which differentiates to form pre-embryogenic mass (PEM), and then develops into a somatic embryo. Maintaining the pre-embryonic mass in a homogenous developmental state makes the transformation process to somatic embryo smoother [89,90]. Moreover, a liquid culture can be used for propagation, in which PEM is used instead of embryonic callus or mature somatic embryo for proliferation [66]. In the study of the main factors affecting PEM proliferation, somatic embryo (SE) development and somatic embryo germination, findings have been made that in a liquid culture system, lower concentrations of plant growth regulators, mineral nutrients and sucrose promoted the proliferation of PEM, formation and germination of SE. A liquid medium containing 1/2 MS (half strength MS medium) + 2,4-D 2 mg/L + 0.5 mg/L KT + 3% sucrose produced the highest proliferation coefficient of leaves [58]. The proliferation curve of callus in liquid suspension culture typically exhibits an S-shape [83], which is more conducive to the proliferation of embryonic callus. By utilizing the inflorescence of the ‘fine wine’ variety as explants, it was observed that the optimal hormone combination for inducing embryogenesis of primary somatic cells in inflorescence was 5 mg/L 6-BA + 0.5 mg/L TDZ + 0.1 mg/L NAA [64,65].

The majority of studies have concentrated on the induction of callus and the development of embryogenic callus in colored calla lily, but there have been no relevant reports in white calla lily. To date, the research on somatic embryogenesis of calla lily is still in its initial phase, and extensive research is necessary to produce high-quality somatic embryos that can be directly utilized or transformed into artificial seeds for the reproduction of high-value hybrids.

Nanotechnology is a highly active area of research in modern materials science. Recent studies have demonstrated the effectiveness of NMs in the removal of microbial contaminants from explants. Further, nanomaterials have also been found to have beneficial effects on various processes in plant tissue culture, including somatic embryogenesis, callus induction, plant cell dedifferentiation and redifferentiation, somaclonal variation, genetic modification, and secondary metabolite production [86]. In the future, further investigations can be conducted into the improvement effect of nanomaterials on the tissue culture of calla lily. Additionally, activated charcoal is a critical factor in tissue cultures, promoting micro-propagule growth and development. Research has shown that activated charcoal supplementation in the culture medium can darken the medium to simulate the soil conditions and absorb toxic metabolites, thereby increasing shoot and root development in ornamental plants [87–89]. As such, the efficiency of tissue culture of calla lily can be improved by using activated carbon.

In white calla lily, regenerated plants using organogenesis were observed to be more reproducible and effective, and the regeneration duration was shorter than that of inducing embryogenic callus. As such, the organogenetic pathway can be used as the basis of a genetic transformation platform to introduce color genes and create new varieties of white calla lily with rich colors. However, a high reproduction coefficient of embryonic callus and fewer chimeras have natural advantages for genetic transformation in plants [91,92]. Further investigation into the somatic embryogenesis pathway is necessary to optimize the system and increase the efficiency of regeneration and subsequent genetic transformation in the white calla lily. Although there are few studies on embryogenic callus induction in calla lily, existing reports suggest that there is significant application potential in colored calla lily callus, and thus, further research is needed. In order to improve the disease resistance of calla lily, the indirect somatic embryogenesis pathway can be used as the basis of the genetic transformation system, and the susceptible genes can be knocked out by gene editing to obtain new varieties of calla lily that are resistant to soft rot. Fig. 2 depicts an upcoming genetic engineering breeding blueprint for two groups of calla lily species.

The focus of the present study was on the main influencing factors of calla lily tissue culture, such as genotype, explant type, and PGRs, as well as other factors. Further, relevant studies of other Araceae species were reviewed to establish a foundation for calla lily tissue culture research, the development of a genetic transformation platform, and the generation of novel ideas for improving desirable traits.