Introduction

In crop production, knowledge about the heritability of important traits, such as adaptation, resistance, yield, color of flowers, among others, can feed into better strategies and tools for genetic improvement techniques.

Plant breeding is the art, science, and technology of improving plants for the benefit of humankind (Semiarti et al. 2020a). Plant breeding has been developed and evolved together with the history of the agriculture, beginning with the selection of higher yield plants as the source of seeds for the next planting season, the evolution of different techniques of conventional breeding, and more recently, the use of biotechnological methods such as genetic transformation and genome editing (Priyadarshan 2019). Plant breeding has increased the diversity of crops and genotypes and assisted breeders and geneticists to better understand how to select and fix new heritable traits in crops for the development of agriculture. Breeding programs also enable the selection of suitable parents for hybridization (Yuan et al. 2015), and conservation of genotypes from various species in germplasm banks is essential for preserving the genetic variability of a group of species assisting breeders (Vendrame et al. 2014).

Floriculture currently represents one of the sectors of agriculture and horticulture that invests the most into newly available technologies (Cardoso and Vendrame 2022).

In an economical context, orchids are one of the most important products in the floriculture industry, with the genus Phalaenopsis being the one with the greatest global demand in terms of cut and potted floriculture (Hsing et al. 2016; Liang et al. 2020). The longer shelf life (greater than 30 days), efficient control of flowering induction and development, the presence of flowers with multiple color combinations and diverse architecture, and size and number of inflorescences (Liang et al. 2020; Tong et al. 2020) explain their success among all ornamental plants.

The Netherlands has been for a long time the global leader of orchid trade with a spotlight to the Phalaenopsis, also commercially called the Dutch Phalaenopsis. In 2020, the value of Dutch Phalaenopsis orchids was €422 million with 117 million units sold, holding first position among the most popular house plants sold (Royal Flora Holland 2020). Global production of Phalaenopsis orchids has reached 300 million plants per year and exportation value of these orchids in Taiwan (the main competitor of the Netherlands), as documented until 2012, has also increased proportionally (Yuan et al. 2015).

The success of these two countries in the Phalaenopsis trade is directly associated with the development, evolution, and maintenance of breeding programs, and the success in the development of new cultivars and groups of cultivars with new characteristics of marketable interest (Lee et al. 2020). Another factor contributing to the successful cultivation and commercialization of orchids is the development of efficient clonal propagation systems, using techniques such as micropropagation and protocorm-like bodies (PLBs). These techniques have enabled the production of millions of clonal plantlets annually, creating a world market for trading cultivars, plantlets, and cultivation/propagation technologies (Cardoso and Vendrame 2022).

The Orchidaceae have, probably, the highest hybridization capacity among all plant families in the world. This is due to the weak barriers for hybridization and unusual type of plant embryo development, which result in the hybrid seeds with very limited reserves (no endosperm) that develop into protocorms (Yeung 2022). These limited nutritional reserves of orchid embryos require specific associations between mycorrhiza fungi and orchids for orchid seed germination and seedling development (Chen et al. 2022). However, a specific in vitro method developed for orchid seed germination, called asymbiotic germination, uses a specific culture medium to mimics the association with fungus, aiming to promote a rapid and efficient system for germination and development of seedlings from hybrid-derived seeds rescued (Balilashaki et al. 2015).

All these characteristics render orchids one of the most hybridized, interesting, and intriguing ornamentals in the world. Phalaenopsis is also one of the most studied genera in terms of breeding. The Phalaenopsis genus comprises around 63–70 species (Cribb and Schuiteman 2012), commonly referred to as moth orchids, native to the tropics and subtropics areas of Asia, Australia and the Pacific Ocean islands (Huang et al. 2015). The Phalaenopsis species and hybrids live as epiphytes, are monopodial with undeveloped pseudobulbs and succulent leaves, have high water and nutrient efficiency, and exhibit crassulacean acid metabolism photosynthesis with CO2 uptake at night (Chuang et al. 2014). The number of artificial hybrids surpass 39 thousand hybrids only in the International Orchid Register Database (RHS 2023). The above factors demonstrate the importance of Phalaenopsis orchids as a model for the genetic development of novel approaches for improvement of ornamental orchids and other flower crops.

Previous reviews on orchids, including Phalaenopsis have focused on biotechnological methods/tools (Vendrame and Khoddamzadeh 2016) and breeding applications (Enoki and Takahara 2022), such as orchid molecular biology (Hsiao et al. 2011a), the use of tissue culture (Khatun et al. 2020) and other biotechnological methods (Hossain et al. 2013), and genomics (Tsai et al. 2017) and omics approaches (Balilashaki et al. 2019). However, none of these reviews explored extensively the literature for each technique or compared the advantages and limitations of conventional hybridization versus modern biotechnological methods in orchids. In this review we include some practical examples of how mixed techniques using both biotechnology and conventional breeding tools are useful for the development of new generations of orchid cultivars.

Thus, the aim of this review is to describe and discuss the main biotechnological and conventional approaches employed for orchid breeding programs, with a focus on Phalaenopsis orchids (Fig. 1), including an outline of future prospects.

Fig. 1: Biotechnological and conventional approaches for orchid breeding.
figure 1

Conventional breeding by hand-cross hybridization and mutation breeding using chemical and physical agents; Biotechnological tools, in vitro somaclonal variation including the Induction, Proliferation and Regeneration of PLBs (IPR-PLBs), ploidy manipulation, isolation and fusion of protoplasts, Agrobacterium tumefaciens-mediated transformation and particle bombardment and, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR/Cas-9) genome editing.

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Orchid breeding by hybridization or conventional breeding

Artificially created orchid hybrids exhibit improved characteristics, such as size, number of flowers, shape, longevity, color and fragrance pattern of flowers, stalk length, blooming periods, and early flowering. Furthermore, leaf appearance and plant architecture, number of viable seeds, the convenience of cultivation, and resistance to pathogens are targeted (Semiarti et al. 2020b).

Current large-scale Phalaenopsis breeding programs based on hybridization have focused on aspects related to ornamental features, such as uniformity of the induction and development of flowering using low temperatures, plant architecture, size and number of inflorescences, the color and size of the flowers and, in some groups of cultivars, the production of compact plants for commercialization in small pots (see https://www.anthura.nl/products/orchid-pot/?lang=en and https://www.floricultura.com/en/orchidaceae/floriclone-phalaenopsis/).

Phalaenopsis orchid genotypes used as progenitors in breeding programs, usually exhibit large flowers with white, pink, and striped color patterns, whereas the novelty cultivars exhibit innovative coloration such as yellow, red, white/rose with red spots or stripes (Fig. 2A), and also fragrance in some cases (Lee et al. 2020). The introduction of other genetic sources for new types, such as small and compact orchids, also called dwarf or mini, are also considered for the market (Vo et al. 2019). Most commercial varieties are generally tetraploids while wild-species are diploids (Lee et al. 2020).

Fig. 2: Conventional hybridization by hand-cross pollination, seed obtaining and in vitro development of seedlings of Phalaenopsis orchids.
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A typical tetraploid cultivar of Phalaenopsis used in breeding programs; B details of the flower labellum and the column where the pollinia and the stigma of the flower used for hand-cross pollination are located; C fruit of Phalaenopsis with seeds (fws) and a seedless fruit (sef); D plastic container used to store orchid seeds at low temperatures; E protocorm development derived from seeds of immature fruits; F protocorm development from seeds of mature fruits; G regeneration of seedlings of the hybrid progeny F1.

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The procedure for hand-cross pollination, the most used conventional breeding approach, consists of different phases. It starts with the selection of parents containing hereditary characteristics of interest, followed by hand-cross pollination between the progenitors, their fertilization and subsequent fruit and seed development towards fruit and seed maturity within 4–14 months depending on the orchid genera. The immature or mature seeds are then rescued, established in vitro, and germinated, using the technique called “asymbiotic in vitro seed germination” (Fig. 2A–G).

Rescue of zygotic embryos from intra- and intergeneric crosses, performed by in vitro asymbiotic germination, has been applied widely for Phalaenopsis breeding (Tsai 2014) and produced the most of actual groups of cultivars with thousands of hybrids. The asymbiotic seed germination of orchids accelerates obtaining the progeny, improves the germination and development of seedlings and it is used for breeding, due to the high heterozygosity of parentals and the high number of seeds (more than hundred thousand seeds per fruit) from each crossing (Zanello and Cardoso 2019). Seedlings are later acclimatized and cultivated in plastic pots in a greenhouse, aiming at the selection of progenies with specific and aimed features.

However, conventional breeding techniques in orchids present some limitations, such as the high rates of cross incompatibility in some orchid cultivars, particularly those with large genomes (Azadi et al. 2016), which leads to no fruit production, the production of fruits without seeds, or seeds with no embryos; the latter two resulting in time and effort waste by the breeder. In our experience with breeding different commercial group of orchids, the limitation varies according to the genus. Cattleya and Dendrobium hybrids may produce fruits containing seeds without embryos; Oncidium genera crossings could result in low fruit set and formation and also the development of fruits without seeds, and in Phalaenopsis the main limitation is the long time required for the formation of large fruits associated with the production of fruits without seeds (Fig. 2C).

When hybridization is successful with seed and embryo formation, seeds can be stored at low temperatures for 6–12 months (Fig. 2C, D), or are directly germinated in vitro with the formation of protocorms (Fig. 2E, F), followed by seedling development (Fig. 2G).

Also, the lifecycle for growing Phalaenopsis (2–3 years) leads to 4–7 years for obtaining a novel cultivar, whereas additional 2–3 years are required for the production of large quantities of clonal plantlets that in fact reach the growers and flower markets. The large-scale production of plantlets from a novel cultivar is achieved by cloning the selected individuals using micropropagation, e.g., through in vitro shoot proliferation, or by PLBs formation from different types of somatic tissues (Cardoso et al. 2020).

Mutation breeding

Mutation is a natural consequence of the modifications occurring during DNA replication or ineffectiveness of DNA repair and is one of the bases of genetic variation in plants. Artificial application of particular chemicals, such as colchicine, oryzalin, trifluralin, pronamide and amiprofos-methyl (Vilcherrez-Atoche et al. 2022), or physical agents, such as gamma-rays, X-rays and ion-beams to increase induction and frequency of mutations in plant cells has been used for producing novel cultivars of ornamental plants (Melsen et al. 2021). This is referred to as mutation breeding. This technique can reduce the time for the development of new cultivars of orchids, depending on the goals, methods, and mutagens chosen, resulting in high rates of plants with genomic mutations, such as polyploidization, or by specific punctual genic mutations (Li et al. 2021; Vilcherrez-Atoche et al. 2022). However, undesirable effects of mutation breeding in the tissues can also occur and include: unpredictable mutants, plant death due to lethal doses or time of exposure to the mutagenic agent, or by lethal mutations resulted from the treatment and the generation of chimeric plants, that makes difficult the selection of solid and stable mutants (Kim 2020).

Irradiation is employed as an indirect tool for mutagenesis and is one of the most commonly used approaches for floricultural species, used both under in vitro or in vivo conditions. When radiation penetrates living cells, energy in the form of both waves and particles interacts with the atoms and molecules in the cells, thereby producing free radicals, which may modify or destroy the vital components of the cells (Oladosu et al. 2016).

Gamma rays are the main type of radiation used in plant mutation breeding because they penetrate deep into the biological matter and cause point mutations and small deletions. The main advantages of this technique were observed when the irradiation modifies one or few traits in the organism, maintaining most of the genetic background of the cultivar (Kim 2020; Anne and Lim 2020). Its application for superior genotypes aiming at the expected point mutations gathers small changes in traits, such as flower color, disease-resistance, associated with good genome of superior cultivars used in floriculture (Lestari et al. 2018).

In Phalaenopsis, studies with gamma irradiation have been reported (Table 1) and aimed at changes in flower color, size, morphology, shelf-life and flowering (Anne and Lim 2020; Magdalita et al. 2022). Induction of early flowering (less than 2 years) in P. aphrodite and P. amabilis was reported using gamma ray irradiation in lower doses (15–25 Gy) (Magdalita et al. 2020; Widiarsih and Dwimahyani 2013). Furthermore, gamma irradiation was used in protocorms of P. amabilis with successful development of mutant seedlings showing resistance to Dickeya dadantii, which causes soft-rot disease (Putri et al. 2021).

Table 1 Gamma-irradiation mutation breeding of Phalaenopsis.
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Gamma irradiation has also been applied to breeding of other important commercial orchid genera, such as Cymbidium (Kim et al. 2020a), Dendrobium (Billore et al. 2019; Sherpa et al. 2022) and Oncidium (Ahmad et al. 2006). The main results of these studies include the induction of early flowering plants (Sherpa et al. 2022), yellow-leaf phenotype (Kim et al. 2020a) or leaf chimeras (Kim et al. 2020b).

Biotechnological tools applied for Phalaenopsis breeding

Biotechnological tools have shown promising results in producing new orchid cultivars with greater efficiency and precision achieved using conventional breeding (hybridization or conventional mutation breeding) (Hsiao et al. 2011a; Semiarti et al. 2020b).

Some breeding goals can only be achieved through genetic transformation (Hsing et al. 2016), and its benefits and efficiency to meet challenges of the world’s modern agriculture have been demonstrated for a large number of important food and industrial crops and ornamental species (Noman et al. 2017). Furthermore, the use of CRISPR/Cas9 represents a simple and efficient genome editing technology for inducing mutation in target genes for breeding purposes, thereby enabling the transformation of plants for color, fragrance, size and shelf-life, and conferring resistance to abiotic and biotic stresses (Corte et al. 2019).

In vitro somaclonal variation

PLBs are specific organs of orchids, which originate from somatic tissues, such as protocorms, leaves, shoot tips, among others that are regenerated under in vitro conditions and are conventionally used for large-scale propagation of different orchid genera, including Phalaenopsis. The technique known as Induction, Proliferation, and Regeneration of Protocorm-Like Bodies (IPR-PLBs) can also be used as a spontaneous source of mutations originated from somaclonal variations (SVs). Conventionally, SVs result in mutant genotypes, also called somaclones, and include undesirable genetic or epigenetic variations in Phalaenopsis orchids, such as the absence of flower parts (Cardoso et al. 2020).

The origin of the SV phenomenon under in vitro conditions is derived from activation of different chromosomal changes (including polyploidization), transposons, retrotransposons, methylation and demethylation of DNA (Manchanda et al. 2018). The origin of SV observed in PLBs is not completely understood, but PLBs are more susceptible to somaclonal variation compared to conventional micropropagation, which uses shoot tips or inflorescence nodes as explants that directly regenerate shoots. The frequency of SVs in the IPR-PLB technique can range according to different endogenous factors, such as the genotype, the age and the origin of tissues used as explant, and exogenous factors, such as culture medium, with emphasis on plant growth regulators and other additives. Also, the frequency, number and period of subcultures, cultivation conditions and especially the long-term subculture are important factors affecting the SV frequency (Lee et al. 2016). The increase in the frequency of SVs in Phalaenopsis using the IPR-PLBs technique can also be achieved by the use of UV radiation or by the use of the mutagenic agent Ethyl Methane Sulfonate (Kurniadi et al. 2023).

Most of the SV that occur in vitro using IPR-PLBs or those that remain in the ex vitro stage show undesired characteristics, such as morphological abnormalities in the development of orchid leaves (“creased leaves”), inflorescences and flowers, or even in the absence of specific structures within the flowers, such as altered development of petals and sepals or the absence of labellum and pollinia (Cardoso et al. 2020).

Although the SVs are undesirable in clonal in vitro propagation systems, this type of variation from original tissues has been used for breeding purposes in ornamental plants, mainly when derived from punctual stable mutations in the plant tissues. The use of SVs-derived plants in breeding programs are strategic and the obtaining of interesting somaclones, from the agricultural point of view, and these benefits were demonstrated in different crop species, including SVs-derived genotypes showing dwarfism, abiotic and biotic stresses tolerance, and changes in color of flowers and fruits (Rajan and Singh 2021).

Therefore, somaclonal variation also emerged as a potential technique for breeding in ornamentals (Eeckhaut et al. 2020). At the end of the 20th century, a new group of commercial Phalaenopsis varieties emerged with non-conventional flower colors, characterized by blotches of spots resembling a clown face with painted red or magenta-black spots in their flowers; commercially called “Harlequin” Phalaenopsis. Current studies with the first “Harlequin” Phalaenopsis cultivars that appeared in the market around 1996, demonstrated that the flower colors were triggered by a single point mutation in in vitro plantlets derived from PLBs, used to clone P. Golden Peoker “Brother” (Hsu et al. 2019; Lee et al. 2020). This mutation was named “ES”, since the first SV-derived plants flowered at the Ever-Spring Nursery. Eventually, clones derived from micropropagated meristems also presented around 30% of plants mutated, enlarging the “Harlequin” genotypes, maintaining this irregular distribution of red spots or blotches of spots in their flowers, but with segregation of flower color, establishing the fundamental genetic basis for all subsequent Harlequin-type cultivars currently available in the market (Lin 2021).

P. “Everspring Black” (Fig. 3) is one of these SV-derived cultivars from P. Golden Peoker “Brother”, with intense and solid red-marron color in petals and sepals. The recent conventional hybridization performed between the “Harlequin”-type P. Everspring Black with P. “PH501” (Fig. 3), a cultivar showing conventional flower color, resulted in a progeny showing a good diversity of colors in flowers among individuals, most of them showing characteristics of “Harlequin” (Fig. 3—F1 progenies), proving that the SV characteristic is hereditary and can be used to generate new cultivars by conventional hybridization.

Fig. 3: Heritability of the ‘Harlequin’ trait in Phalaenopsis orchids.
figure 3

F1 progeny obtained from conventional hybridization of Phalaenopsis “Everspring Black” (female parent), a cultivar derived from single point mutation of somaclonal variation (SV-derived) of Protocorm-like bodies of P. Golden Peoker “Brother”, and the commercial cultivar called P. “PH501” (male parent). Original figures from Jean C. Cardoso.

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These observations about the occurrence of SVs in PLBs are also reported in the micropropagation of the Oncidium “Milliongolds”, one of the worldwide distributed Oncidium orchids with yellow flowers (Wang et al. 2019).

Polyploidization

Polyploidy plays a significant role in breeding of different orchid genera, such as Phalaenopsis (Lee et al. 2020), Cymbidium, Dendrobium and Oncidium (Li et al. 2021) and Brassolaeliocattleya (Vilcherrez-Atoche et al. 2023). A recent review reported information about the natural and artificial occurrence of polyploidy in orchids, including Phalaenopsis (Vilcherrez-Atoche et al. 2022). Antimitotic agents, such as colchicine or oryzalin, are conventionally used, aiming at self-polyploidization of different orchid genera. In Phalaenopsis 80% of the studies used colchicine as an antimitotic agent for polyploidy induction (Vilcherrez-Atoche et al. 2022), but interestingly, natural polyploids of different orchid genera are also reported, originated from high number of endo-reduplicated cells reported in different types of orchid tissues (Vilcherrez-Atoche et al. 2022) due to the higher rates of endopolyploid tissues, such as observed in protocorms and PLBs of Phalaenopsis (Chen et al. 2011). Similar observations were reported for Brassolaeliocattleya orchids (Vilcherrez-Atoche et al. 2023), with the occurrence of 11.5% polyploids seedlings derived from protocorms without the treatment with antimitotic agents. Another source of polyploidy in Phalaenopsis is the use of unreduced pollen grains, of natural origin (0.68–1.78%) and which can also be induced or increased in frequency by the use of colchicine (up to 10%) (Wu et al. 2022).

Polyploids have been used in Phalaenopsis breeding to generate large flowers and inflorescences with multiple flowers, plant size and leaf thickness (Chen et al. 2010; Lee et al. 2020). In fact, Lee et al. (2020) reported the predominance of tetraploid cultivars (80% of 60 total cultivars analyzed) in the floriculture industry, with only one diploid reported. Tetraploid hybrids of Phalaenopsis have superior horticultural and ornamental characteristics compared to diploids, such as high vigorous leaves (larger, longer and thicker), rapid development, good adaptation to pot cultivation and greenhouse management, longer inflorescences containing higher number of flowers, higher diameter of flowers, petals and sepals, which in general is considered one of the most important floriculture products in the world.

Haploid and double-haploid technology

Although uncommon in orchid breeding, gametic embryogenesis technology is a powerful breeding tool used to provide haploid and double-haploid plants for the agricultural industry. This technology, together with genetic transformation, is currently used for the most important crops propagated by seeds in the world, such as maize, wheat, barley, and Triticum and Brassica genera aiming at the production of inbred and homozygous lines (Zargar et al. 2022).

The development of this technology could provide important advances in ornamental breeding programs by producing homozygous lineages, followed by hybridization and production of F1 hybrid seeds. In floriculture, haploids and double haploids have been developed for some species, such as Chrysanthemum (Wang et al. 2014) and Gerbera (Li et al. 2020) by using ovule culture, and in Anthurium using anther culture (Winarto et al. 2010).

In orchids, the natural high number of seeds in fruits, commonly hundreds of thousands to millions of seeds per fruit (Anghelescu et al. 2023), is a desirable trait that benefits the use of double-haploid technology aiming at the production of F1 hybrid seeds from inbred lines. The in vitro asymbiotic germination of seeds is simpler, faster and more efficient method for orchid propagation, compared with current micropropagation systems. Haploid and double-haploid plants can also support genetic studies, such as genetic map construction, molecular breeding and transgeny (Thaneshwari et al. 2018).

Genetic transformation

Genetic transformation is a useful technique to transfer specific genes of interest into plants, otherwise impossible through conventional hybridization. Genetic transformation can be accomplished by either direct or indirect methods, the former through particle bombardment (e.g., gene gun), the latter by Agrobacterium tumefasciens specific strains with high virulence. The virulence mechanism of A. tumefaciens induces the transfer of the target DNA fragments with characteristics of interest into the plant species (Semiarti et al. 2020a). For instance, Agrobacterium-mediated transformation and particle bombardment approaches have been reported as efficient and reliable alternatives for introducing specific foreign genes into orchid genomes (Wang et al. 2019) and enables the better comprehension of gene functions in Phalaenopsis (Semiarti et al. 2007). The particle bombardment system (gene gun) is an efficient and a rapid physical process without any biological host limitations, which has been applied for the transformation of orchid tissues (Chew et al. 2019).

Some disadvantages of genetic transformation of orchids using Agrobacterium-mediated modification include the limited host range and the lack of a rapid transient gene (Chew et al. 2019). However, the use of super-binary vector and super-virulent bacterial strains, such as EHA101 and EHA105 (Subramaniam and Rathinam 2010) promotes in some monocots, including orchids, successfully transformation outcomes (Nopitasari et al. 2020; Tong et al. 2020; Semiarti et al. 2020a). Nonetheless, the Agrobacterium-mediated transformation system is often preferred because it is a simpler method compared to particle bombardment. Additionally, the copy number of the transgenes produced is reduced, resulting in fewer problems associated with transgene co-suppression and instability (Chai et al. 2002; Mishiba et al. 2005). Genetic transformation systems through Agrobacterium have also been studied in different orchid genera, such as Cattleya (Zhang et al. 2010), Cymbidium (Chin et al. 2007b), Dendrobium (Teixeira da Silva et al. 2016; Utami et al. 2018), Vanda (Shrestha et al. 2007a), Oncidium and Odontoglossum (Raffeiner et al. 2009).

In Phalaenopsis, disease-resistance related genes have been transferred including two disease-resistance genes; Gastrodia antifungal protein (GAFP) and Neutrophils Peptide-I (NPI) conferring resistance to Colletotrichum gloeosporioides Sacc. (Li et al. 2013); wasabi defensin gene isolated from Wasabia japonica to produce Phalaenopsis with resistance to Erwinia carotovora (Sjahril et al. 2006); gene encoding Cymbidium Mosaic Virus (CymMV), coat protein to give protection against CymMV infection (Liao et al. 2004) and anti-microbial peptide from sweet pepper ferredoxin-like protein cDNA (Pflp) to obtain resistance to Erwinia carotovora (Chan et al. 2005). These reports concluded that CymMV resistance was RNA-mediated via post-transcriptional gene silencing mechanism (Liao et al. 2004; Chan et al. 2005).

Besides these disease-resistance genes, the rice plant-derived glutathione S-transferases (gst) gene, which is associated with protecting cells against different stresses (Chin et al. 2007a) and lipid transfer protein (ltp) encoding gene, responsible for cold tolerance, were transferred to Phalaenopsis (Qin et al. 2011).

Genetic transformation aiming at improvements in size of plants and color of flowers was also reported. Miniaturized plants of P. “Sogo Yukidian SPM313”, were obtained by ectopic overexpression of the OsGA2ox6 gene (Hsieh et al. 2020), expanding the possibilities for miniaturized Phalalaenopsis plants for the flower market. Su and Hsu (2003; 2010) performed the transformation of Phalaenopsis using particle bombardment to deliver the cytochrome P450 gene for enhancing its anthocyanin content and reported a change in the petal color from pink to magenta in the resulting Phalaenopsis hybrid (Chen et al. 2011). Similar results were obtained by Lou et al. (2023) with the use of plasmids carrying genes correlated to flowers (PhCHS-5 and PhF3′5′H) and which resulted in changes in flower color in Phalaenopsis and petunia.

Few reports have discussed the performance or efficiency of genetic transformation in Phalaenopsis, but some reports still point to low transformation efficiency as one of the difficulties in using this technique (Lou et al. 2023). In addition, most of the studies are related to disease-resistance, showing that genetic transformation systems in Phalaenopsis are not well established and their transformation efficiency is still low (Li et al. 2021). The choice of the Agrobacterium strain is an important parameter that influences transformation efficiency (Subramaniam et al. 2008). Among the 21 studies on Agrobacterium-mediated transformation of Phalaenopsis orchids, nine studies used the strain EHA101, eight studies used the strain EHA105 and seven studies used the strain LBA4404 (Table 2). Some of these A. tumefaciens strains were also used in other genera of orchids. The strain EHA101 is the most used in Cattleya, Cymbidium, Dendrobium and Vanda, while for Oncidium and Erycina pusilla the strain EHA105 was the most employed for genetic transformation’ (Mii and Chin 2018). Interestingly, strain EHA105 was the only one used in Phalaenopsis since 2016, showing better transformation efficiency.

Table 2 Genetic transformation of Phalaenopsis by Agrobacterium tumefaciens and particle bombardment.
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The target explants used for the genetic transformation of Phalaenopsis in the reported studies were PLBs, protocorms, callus (Sjahril et al. 2006; Sjahril and Mii 2006) and flower petals (Table 2). PLBs were also the most frequently used explant in Dendrobium (Teixeira da Silva et al. 2016), and successfully used in Cymbidium (Chin et al. 2007b), Oncidium (Liau et al. 2003) and Vanda (Shrestha et al. 2007a).

In Phalaenopsis breeding programs, PLBs have been used as target tissues for genetic transformation specifically due to their high regeneration efficiency and their clonal nature, thereby increasing the transformation efficiency (Chew et al. 2018; Subramaniam et al. 2008). The transformation efficiency of intact and transversely bisected PLBs was also studied for Agrobacterium-mediated transformation of Phalaenopsis for the gus gene, showing that transversely bisected PLBs exhibited a higher transformation efficiency (16.2%) than intact PLBs (8.4%) (Chai et al. 2002).

Selectable marker genes are also used to verify the transgenic event, select the transformants from the non-transformants and evaluate the transformation efficiency in the culture medium supplemented with a selective agent (Hsiao et al. 2011b;). In most cases, genetic transformation has been performed using antibiotic-resistance genes, such as the hygromycin phosphotransferase (hpt) gene and the neomycin phosphotransferase II (nptII) gene (Table 2). The use of the BP/KNAT1 gene of Arabidopsis as a visible marker of transformation was studied in P. amabilis protocorms (Semiarti et al. 2007) as well as in Vanda tricolor and Coelogyne pandurata, resulting in multiple transformant shoots (Semiarti et al. 2011).

We here conducted a literature review using 17 reports on the use of the β-glucuronidase (gus) gene and nine reports involving the green fluorescent protein (gfp) gene (Table 2). The difference between the analysis of these two genes is that the gus assay is destructive, while gfp does not cause any damage to the cells. In Dendrobium, gus assays were the most frequently used approach for selection of transgenic lineages (Teixeira da Silva et al. 2016).

Genetic transformation completely changed agriculture in this century, with large predominance of transgenic than conventional cultivars in worldwide agricultural crops, such as soybean, maize and cotton. However, the benefits of transgenics cultivars still face challenges: the difficulties of transfer of target genes to solve key problems of floriculture and the difficulties in releasing these cultivars for commercialization due to strong regulatory laws adopted in some countries, leading to certain discouragement in producing and testing the cultivation of transgenic cultivars.

Isolation and fusion of protoplasts

Protoplasts are plant cells that have had their cell walls removed via mechanical destruction or enzymatic treatment (Eeckhaut et al. 2013). Protoplasts still have the capacity for totipotency, i.e., the ability for regeneration of a complete plant from a single cell (Davey et al. 2010; Naing et al. 2021). The protoplast culture technique comprises three main steps—protoplast isolation and cultivation, and regeneration of complete plants. Isolation of protoplasts is generally achieved using enzymatic digestion of cell wall components and its success depends on the degree of cell wall breakdown and the release of intact protoplasts. Variables such as cell wall thickness, temperature, pH and concentration of osmotic stabilizers and the time of the digestion duration are important for the successful release and isolation of protoplasts (Li et al. 2018; Naing et al. 2021).

The formulation of the culture medium and of the experimental system for the culture, the density of protoplasts and the growth of protoplasts in the culture medium must be considered during the culture of isolated protoplasts. The process of regeneration of protoplasts to buds is represented by five phases, which range from the formation of a new cell wall and a constant mitotic division to a series of morphogenic processes such as the formation of colonies and then callus that finally allow the generation of growth and development of tissues (Naing et al. 2021). According to Eeckhaut et al. (2013), the success of plant regeneration from protoplasts mainly depends on the type of donor material used during the isolation process (Davey et al. 2010).

Studies on the cultivation of protoplasts in the genus Phalaenopsis with successful protocols for the stages of isolation, cultivation and regeneration of complete plants have also been reported (Ichihashi and Shigemura 2002; Machmudi et al. 2019; Shrestha et al. 2007b) (Table 3).

Table 3 Isolation and fusion of Phalaenopsis protoplasts.
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Despite the challenges with uses of orchid protoplasts, research on protoplast culture technique in orchids showed that it is an efficient tool with a high rate of regeneration of stable plants and could be used to improve biotechnological tools for the development of new cultivars as well as for biological studies in orchids (Hossain et al. 2013). A few examples of the biotechnological techniques in which protoplasts can be used as study material are somatic hybridization, protoplast transformation and somaclonal variation induction (Davey et al. 2010).

Somatic hybridization via protoplast fusion is an interesting technique for genetic improvement in plants. This technique uses a total or partial combination of somatic cells obtained from different plants (cultivars, genera, or species) for producing hybrid cells with different genetic or cytoplasmic combinations, thereby generating different somatic hybrids or somatic cybrids, respectively (Grosser et al. 2010). Somatic cell fusion may be performed independently or combined with other treatments, such as electrofusion or polyethylene glycol (PEG) addition (Davey et al. 2010). Somatic hybridization through protoplast fusion for the genetic improvement of plants offers some advantages, such as fusion of somatic cells from incompatible species by sexual hybridization, due to multiple intrinsic barriers (Davey et al. 2010).

In several species of ornamental plants, including orchids, there have been reports on the development of somatic hybrids using protoplast fusion by electrofusion, PEG and increased pH/Calcium which result in new cultivar development (Naing et al. 2021). However, no reports about protoplast fusion and somatic hybrids have been obtained in Phalaenopsis. Advances in protoplast culture technology in orchids breeding could increase the number of novel cultivars and the genetic variability (Shrestha et al. 2007b). In general, it is expected that the new tools, such as genome editing by CRIPR/Cas, with the use of the protoplasts, restore the importance of studies with this biotechnological tool to generate a new group of ornamental plants with desired and innovative characteristics (Naing et al. 2021). Recently, Xia et al. (2022) demonstrated that protoplasts enable the development and identification of two multiplex genome editing tools applied for Phalaenopsis orchids.

Phalaenopsis in the CRISPR/Cas9 genome-editing era

Currently, orchids belonging to the genus Phalaenopsis exhibit the highest rate of transcriptomic information available in the databases for the Orchidaceae (Chao et al. 2017). Consequently, studies concerning the identification and functional analysis of genes are important to understand the various biological processes involved, (such as their reproductive biology and plant growth) and the extraordinary characteristics that these orchids have, for the development of breeding programs (Cai et al. 2015; Bai et al. 2023; Suputri et al. 2024).

Some tools, such as mutants or transgenic plants, assist in conducting genetic and functional studies in plants. However, such studies are challenging in the case of orchids due to the limited availability of mutants and the long time consumed in the production of transgenic orchids owing to their long-life cycle (Lin et al. 2018). The Agrobacterium-mediated genetic transformation has been used for delivering Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR/Cas9). A methodology using the CRISPR/Cas9 KO system was developed using PDS3 as the marker gene, with promising results and a flower color change in the transformed leaf tissue (Semiarti et al. 2020a). The flower color change was also observed by Nopitasari et al. (2020).

The CRISPR/Cas genome editing technology has been used in several plant species, successfully achieving the required modifications and manipulations in the plant genome (Wang et al. 2019). The CRISPR/Cas technology has been used in Phalaenopsis (Tong et al. 2020; Xia et al. 2022), presenting great potential application for studies in orchid breeding. Tong et al. (2020) developed multiple mutants of P. equestris in the MADS genes (important for flowering initiation and flower development) using CRISPR/Cas9 and A. tumefaciens for mediating the transformation. Xia et al. (2022) reported the successful application of different types of genome editing tools—PTG-Cas9-HPG and RMC-Cpf1-HPG multiplex genome editing systems—in Phalaenopsis using genome editing in protoplasts as explants and PEG transformation method. In addition, mutant genes for inducing early flowering in Phalaenopsis, such as Gibberellic acid insensitive (GAI) gene, have been identified for use in CRISPR/Cas9 tools (Suputri et al. 2024).

Another biotechnological tool namely the transient expression system of protoplasts allows conducting genetic, functional, regulatory and signaling studies (Fraiture et al. 2014). The technique involves subjecting the protoplasts to PEG treatments or mechanical electroporation to deliver the plasmid containing the foreign DNA into the protoplast (Davey et al. 2010). The transient expression system of protoplasts allows the study of the transcriptome and how it responds to the expression of the genes of interest. In addition, this system could be a useful tool for the structural, functional, and regulatory analysis of the expressed genes (Lin et al. 2018). The use of transient gene expression system in orchids using protoplasts was reported in recent studies with Cymbidium (Ren et al. 20202021; Yang et al. 2021), Dendrobium (Li et al. 2021) and Phalaenopsis (Li et al. 2018; Lin et al. 2018) and have been emerged in the literature, due to the large information in the data banks and various advances in the areas of phylogenetics, genetics and molecular mechanisms in these genera.

An efficient protocol for the isolation, transfection, and expression of transient genes was established for protoplasts obtained from the foliar mesophyll of P. “Ruili Beauty” (Li et al. 2018), successfully detecting the T1R1 protein in the nucleus and the cell membrane of the transformed protoplasts. Lin et al. (2018) also established an efficient protocol for protoplasts obtained from the petals of the tetraploid P. aphrodite subsp. formosana, with the observation and analysis of the protein localization and the interaction among the protein PaCDKA and PaCYCD3. In addition, the study also provided insights into genes associated with the cell cycle (dependent on PaE2F/PaDP) and hormonal response through the DR5v2 auxin reporter.

The transient protoplast gene expression system also serves as a platform for multiple investigations regarding the biological processes in plants (Xia et al. 2022). Therefore, it is expected that this system will be used independently and in combination with other techniques, such as CRISPR/Cas9 high-performance screening and protoplast regeneration (Gaillochet et al. 2021). Thus, it will contribute to exponentially increasing the orchid mutant libraries, which would serve as important tools for understanding the functions of genes in orchids (Xia et al. 2022).

Conclusions and future prospects

Conventional breeding techniques in orchids, such as controlled crosses followed by selection of genotypes containing specific features, continue to be the most widely used methods for orchid breeding, generating the largest number of commercial cultivars, including the Phalaenopsis multi-flora types, currently one of the high-commercialized in the world’s floriculture trade (Lee et al. 2020). Intergeneric hybridization also provides important opportunities for generations of new orchid cultivars, such as the new indigo or dark blue cultivar called Rhynchonopsis Tariflor Blue Kid “1030-4”, an intergeneric hybrid between P. Fire Cracker and Rhynchostylis coelestis used as the source of blue flowers (Wu et al. 2022). This example of new intergeneric hybrids in orchids (Phalaenopsis x Rhynchostylis), highlights the high and still largely unexplored potential of this technique to obtain new desired plant characteristics, likely to gain importance in the future of floriculture and orchid cultivation.

The recently developed “Harlequin” cultivars group in Phalaenopsis, firstly obtained through in vitro somaclonal variation (SV), emphasize the importance of biotechnological techniques in floriculture industry. In fact, most breeding programs in floriculture are carried out in developed countries by private companies, holding most of the world’s genetic diversity of ornamental species, including orchids. However, this high concentration of technology and genetics in commercial companies to some extent limits the public access to knowledge of the research in this area.

Some innovative features expected in the new generations of biotech cultivars and that could contribute to floriculture are: exotic flowers with pure colors such as true blue (Liang et al. 2020), similar to those developed for rose (Nakamura 2010) and chrysanthemum (Noda et al. 2017), black (Ahmad et al. 2022) and red; the differential color distribution among and along flower organs to provide specific color combination, flowers with scent (Huang et al. 2021), and finally fluorescent flowers and plants, such as those obtained by transgeny in petunias (Chin et al. 2018). Beyond such ornamental characteristics, it is also expected that these technologies could be useful to increase water efficiency, nutrient uptake, resistance to abiotic and biotic stresses, such as gray mold (Zhao et al. 2018) and to increase the sustainability and the resilience of ornamental plant cultivation to climatic changes.

The use of biotechnology for the genetic improvement of crops continues to be one of the great milestones of the current agricultural revolution. The use of transgenic cultivars in the world has increased more than 100-fold in the last 30 years, with more than 190 million hectares with cultivated varieties derived from biotechnology, with special applicability of Genetic Modification (GMs) to large crops such as soybean, corn, cotton and canola (Turnbull et al. 2021). However, ornamental plant cultivars derived from biotechnology are still scarce in the flower market, and are limited by regulatory and profitability issues (Boutigny et al. 2020), but also by the limited studies related to genomic and transcriptome sequencing associated with most ornamental plants.

The uses of novel “omics” tools and genome editing are emerging in orchids (Li et al. 2021; Xia et al. 2022) as a result of recent advances in whole-genome sequencing (Zheng et al. 2021). Recent advances in technologies with reduced costs and that accelerate the processes of genomic sequencing in ornamental plants, such as in Phalaenopsis orchids (Hsu et al. 2020), as well as making them more available around the world, have emerged in the expansion of information about target genes that could be introduced by biotechnological tools and that could generate new groups of cultivars of ornamental plants. Furthermore, the emergence of more efficient technologies for genomic editing, such as the CRISPR/Cas9 technology, allows the development for cultivars that are more efficient in the use of natural resources, more productive, and possess characteristics that are difficult to obtain by conventional breeding programs (Ahn et al. 2020), without the complex regulatory process found with transgenic cultivars. Therefore, these advances and also the confirmed heredity of target-traits introduced by biotechnology, will enable, in the short-medium term, the employment and expansion of cultivars derived from modern biotechnological methods.