**1. Introduction**

Orchids (Family Orchidaceae) represent one of the two largest plant families, including from 736 [1] to 899 genera and 27,800 accepted species names [2] and over 100,000 hybrids produced by artificial pollination [3]. In addition to their unquestionable botanical and ecological importance, orchids participate in current cultivation systems using high-tech horticulture, grown in environments with good climate control, especially temperature, which allows the induction of flowering regardless of the time of year, especially aiming at the scheduled supply of potted and cut flowers in the competitive world flower market. Some species of orchids, such as the genera *Dendrobium, Gastrodia,* and *Bletilla*, have also been used for medicinal purposes, using the basis of traditional Chinese medicine [4] and some *Vanilla* species is also used for food purposes [5].

In this economic context, family Orchidaceae currently represents one of the most important in the world commercial floriculture, with emphasis on the genus *Phalaenopsis* as well as its interspecific hybrids, which is currently the main potted flower marketed in the main world flower markets. To have an idea of the importance of this genus in the expansion of world floriculture, only in the Dutch market, the largest in the world, in 2014, 121 million pots of *Phalaenopsis* were sold generating approximately US\$ 500 million [6]. In addition to *Phalaenopsis*, other genera of economic importance to floriculture include the genera *Cattleya*, *Dendrobium,* and *Oncidium* and their hybrids [7–9] as well as *Cymbidium* and *Vanda* used for production of potted or even cut flowers.

Despite the individual importance of these genera, a commercial classification for orchids must be set separately from the botanical classification. This is because although genera have a greater genetic and morphological contribution to commercial plants, most commercial flower production of these genera occurs through the production of hybrids from interspecific crosses, which include the use of crosses between species of the same genus, but also species of different genera (intergeneric hybrids) [9]. An example of this case is the very frequent use of *Doritis* in crossings with *Phalaenopsis*, generating the hybrid genus known as *Doritaenopsis* [10,11]. Nevertheless, commercially these hybrids are all called *Phalaenopsis* because considering the morphological similarity and commercialization value, there is no commercial justification for separation into two classes.

Another justification for the separation of botanical and commercial classification is the recent changes of genera in many species, including those of commercial importance and resulting from the advancement of available molecular techniques that allow genetic rather than just morphological comparisons [1]. An example would be the genera *Laelia* and *Sophronitis*, commonly used in crossings with the genus *Cattleya* to incorporate hybrids with red, yellow and orange flowers, little present in *Cattleya*. Both *Laelia* and *Sophronitis* have undergone more than one change in their names in the last decade, with new changes possibly still remaining due to advances in molecular markers and phylogenetic aspects related to this complex and diverse plant family [12,13].

Thus, it is important to highlight this botanical difference from the commercial one, due to the complexity of the family and its high hybridization capacity. Thus, using as an example the commercial classification encompassing these genera includes not only the genus, but its many hybrids used for the genetic improvement and development of new cultivars for the world floriculture. When mentioning *Cattleya*, this includes genera such as *Laelia*, *Sophronitis*, *Broughtonia*, *Epidendrum*, *Encyclia*, *Caularthron*, among other correlates and with possible hybridization with *Cattleya*. The same occurs in *Oncidium*, in which plants of different genera such as *Brassia*, *Ionopsis*, *Odontoglossum*, *Miltonia*, among others [14] are used for breeding intergeneric hybrids and many commercial hybrids are the result of combinations of more than two genera.

In few plant families it is possible to obtain so many viable and fertile combinations of progenies from very different morphologically species and genera. This allows breeders to incorporate numerous traits of interest into a single plant, which brings the innovative aspect of flower production as well as the advance in breeding, using these same mostly fertile hybrids for the advancement of generations of crosses and obtaining new hybrids. This high hybridization capacity may be a result of the specific process of embryogenic development and later protocorm development that occur in orchids [15]. In other species, it has been reported that lack of hybridization and hybrid seed abortion is associated with disruption of proper endosperm development or mismatch between endosperm development and embryo [16]; and zygotic embryogenesis in family Orchidaceae, embryo development occurs in the absence of endosperm [15].

After obtaining the hybrid of commercial interest, propagation is the factor that defines the time for this hybrid to be available in the market for clonal propagation, which ensures the maintenance of the selected characteristics in propagated plants, quickly, on a large scale and allowing the production of plantlets throughout the year. These propagation characteristics, in addition to ensuring the quality of the plantlets produced, also aim to maintain the commercial scale necessary to meet the target market. The only viable technique that combines all these characteristics has been in vitro micropropagation of orchids [17].

Among the in vitro cultivation techniques used for the in vitro seedling or plantlets production of orchids, it can be used the in vitro asymbiotic germination and micropropagation techniques aiming at the large-scale production of clonal plantlets.

Asymbiotic germination involves the in vitro inoculation and germination of orchid seeds with the aid of a sucrose-containing culture medium [18,19], under conditions free of microorganisms; including those symbionts that assist in germination, especially under natural conditions, a technique known as symbiotic germination, which can be done in vitro [19,20], ex vitro, or in situ and which, unlike asymbiotic, considers the use of symbiotic microorganisms to assist in the germination and early development of newly germinated seedlings, and lacking nutritional reserves to support early seedling development [20,21].

Techniques involving the germination of orchid seeds under in vitro conditions are especially used in: Conservation and production of seedlings of native species; germination of seedlings from crosses aiming at genetic improvement and production of new orchid cultivars [8]; aiming at the production of protocorms in order to study somatic embryogenesis in vitro, also known as protocorm-like bodies or simply PLBs [17,22]. They can also be used for commercial propagation and seedlings production, but with high genetic variability inherent in the family Orchidaceae, including commercial groups used for flower production [8].

In vitro germination of orchids makes it possible to increase the efficiency of conservation and breeding programs, since in vitro germination rates higher than 70% are commonly reported [23], while in ex vitro conditions under natural environmental conditions, these rates hardly exceed 5% germinated seeds [24]. This is especially due to the fact that orchid seeds do not contain nutritional reserves [25], and the embryo and seedlings at early germination are highly dependent on symbiosis with microorganisms known as mycorrhizae, which nutritionally supply these plants during a long time until the complete establishment of the seedling in the natural environment [26]. In *Serapias vomeracea* orchid, in symbiosis with *Tulasnella calospora* there was observed a differential gene expression related to organic nitrogen transport and metabolism, showing the nutritionally supply of fungus to orchids in early development of protocorms [27].

A characteristic of the in vitro asymbiotic germination of orchids is the formation of the so-called protocorms, prior to budding, mainly containing the first leaves and undeveloped stem, followed by the roots [25] and later on with the development of the leaf and pseudobulb.

The term protocorm-like bodies (PLBs) is used as a reference to this type of protocorm-producing germination, characteristic of orchids. The main difference between the germination and the sexual reproduction process, which includes the fertilization process, zygotic embryogenesis, followed by the germination and formation of protocorms, is that PLBs comes from somatic tissues, therefore being considered a type of vegetative propagation.

The production of PLBs, therefore, can be compared to a specific type of somatic embryogenesis that occurs in orchids, and the anatomy, development and characteristics of cells and some cell wall markers at the beginning of PLB formation are similar to those in the development of protocorms in orchids [28]. These authors observed that in non-embryogenic callus of *Phalaenopsis* orchids, the inability to synthesize some cell wall components such as the JIM11 and JIM20 epitopes resulted in loss of morphogenic capacity of these calli, and the correct formation of the cell wall is directly associated with the ability of cell division and elongation in these cell types. In contrast, embryogenic calli synthesized these components, similar to what occurred in zygotic embryogenesis [28].

Despite these anatomical and cellular similarities between PLB induction and zygotic embryogenesis, molecularly, zygotic embryogenesis in *Phalaenopsis aphrodite* is considered different from PLB formation, and that induction of PLBs follows a different route from the embryogenic program [29]. One explanation for these differences is a consequence of the degree of speciation for the development of the embryogenic program in orchids, which follows a very specific pattern and different from the conventional embryogenic program occurring in species of other families, such as the absence of endosperm development and gene expression for establishing symbiotic relationships during seed germination process [15].

Due to these still-present doubts regarding comparisons of zygotic embryogenesis with induction of PLBs in orchids, we have adopted the term IPR–PLB (induction, proliferation, and regeneration of PLBs) as the standard to describe this technique in this paper. IPR–PLBs in orchids have different applications in the world flower industry. Undoubtedly the one with the largest commercial application is aimed at the mass propagation of clonal plants to meet the world's demanding flower production market, in which orchids play a significant part in both the pot and cut flower market [6,30]. However, other applications such as for species conservation purposes [31] and obtaining transgenic plants [32] can be found in the literature.

Despite a significant amount of studies with IPR-PLB in different orchid species and hybrids, such as *Coelogyne cristata and C. flaccida* [33,34], *Cyrtopodium paludicolum* [35], *Grammatophyllum speciosum* [36] among others, this review has as its main objective to compile the recent studies and advances found in the induction, proliferation and regeneration of PLBs from the two most important genera in the world flower market, especially *Phalaenopsis* and *Oncidium* hybrid groups.

#### **2. Genus** *Phalaenopsis* **and Related**

The limited efficiency of clonal multiplication by the induction of shoots from floral stems cultivated in vitro has been one of the main difficulties faced in micropropagation of *Phalaenopsis*, resulting in an increase in the production cost of micropropagated plantlets [37] and associated with falling prices in the international market [6] place in vitro plantlets as the current major cost of producing *Phalaenopsis*. In this sense, the IPR–PLBs can be an important tool in the micropropagation of commercial hybrids of this genus aiming to increase the production efficiency, being necessary to know the main factors involved in each phase of plantlets from PLBs production, e.g., induction, proliferation, and regeneration, which result in efficient clonal and mass propagation techniques for *Phalaenopsis*.

The first studies involving clonal micropropagation of *Phalaenopsis* were conducted by [38–40] using *Phalaenopsis amabilis* as a model. Soon after, [41] concluded that leaf segments obtained from inflorescence buds grown in vitro when grown in New Dogashima Medium (NDM) [41] medium supplemented with 0.1 mg L−<sup>1</sup> NAA (Naphthaleneacetic Acid) and 1.0 mg L−<sup>1</sup> BA (6-Benzyladenine) could generate up to 10,000 PLBs within a year. Ref. [42] also reported PLB regeneration from a callus induction phase (indirect somatic embryogenesis) using Vacin Went medium [43] supplemented with 20% coconut water and 4% sucrose with the hybrid *Phalaenopsis* Richard Shaffer 'Santa Cruz'.

In orchids, PLBs are suggested to be somatic embryos due to the morphological similarity and developmental pattern observed between them and the zygotic embryos [42,44]. Besides that, ontogenetic studies based on histological and histochemical methods developed by [28] compared the early developmental pattern of zygotic embryos and PLBs, which led to the conclusion that cytological characteristics and cell wall markers were similar in the early developmental stages of both zygotic embryos and PLBs, which would justify saying that PLBs are somatic embryos. Still, histological analyses made by [45] also showed that the formation of PLBs occurs directly on the epidermal surface of the leaf segment with a cluster of meristem cells in constant division and without connection with the leaf vascular system, which is interesting from a commercial point of view, since it ensures the health of plants obtained through PLBs [46–49] and enable success of genetic transformation [50,51].

In several plant species, some genes that are involved in somatic embryogenesis, known as *SERK* (somatic embryogenesis receptor-like kinase), are described. Ref. [52] characterized and analyzed the expression of 5 of these genes in *Phalaenopsis* and which were described by the authors as *PhSERK*. According to this study, the expression of these 5 genes was observed in various parts of plants (root, leaf, apical bud, and flower meristem) as well as during seed germination and PLB induction. According to the authors, PLBs segmented and grown in secondary PLB-inducing medium showed

high *PhSERK5* expression during the third week, when secondary PLBs became visible, suggesting that this SERK transcription may be closely associated with the acquisition of embryogenic competence during formation of PLBs. It is noteworthy that transformed *Arabidopsis* plants with overexpression of the *AtSERK1* gene showed high capacity for induction of somatic embryos in in vitro culture [53], showing that this gene is indeed involved in somatic embryogenesis, at least in *Arabidopsis*.

Although cytological features indicate that a PLB is a somatic embryo and studies have shown *PhSERK* gene expression during PLB induction [52], transcriptome studies developed by [29] analyzing gene expression in *Phalaenopsis aphrodite* concluded that PLBs are molecularly distinct from zygotic embryos. According to the authors, PLBs share different transcriptomic signatures from zygotic embryos, and early processes of PLB development show a distinct regeneration program, not following the embryogenesis program. In addition, the authors report that the SHOOT MERISTEMLESS gene, a class I KNOTTED-LIKE HOMEOBOX gene, probably plays an important role in PLB regeneration and should be further investigated.

The genetic transformation with *AtRKD4* gene, which encode proteins with RWP-RK transcription factor and is associated to early embryogenic pattern in *Arabidopsis thaliana* [54], also increases the number of PLBs produced in leaves of this *Phalaenopsis* 'Sogo vivien' [55] and *Dendrobium phalaenopsis* [56] transgenic plants.

Recent studies with *Phalaenopsis equestris* genome sequencing [57], with 2n = 2x = 38 and 29,431 predicted protein-coding genes and *Phalaenopsis* Brother Spring Dancer 'KHM190' [58], 2n = 2x = 38 and 41,153 protein coding genes, make room for further detailed studies on the identification and expression of genes involved in the production of PLBs from different types of somatic tissue in orchids, which can be compared with other model species and in which the embryogenic pathway is already better elucidated, similar to the studies already carried out that brought new discoveries about flowering and the development of floral organs [58].

Among the several factors that regulate somatic embryogenesis in *Phalaenopsis*, the absence of light is described as responsible for the PLB induction step [59]. After maintaining the leaf segments for 60 days in the dark, it is possible to observe at the ends of the segments the formation of embryo-like structures, still with a yellowish-white color (Figure 1A). After about 15 days under 14 h light photoperiod, PLBs change color to light green and dark green (Figure 1B) and after 90 days subjected to light there is the onset of differentiation of PLBs with leaf primordia to their complete differentiation with leaf and root formation. The PLBs also could be induced from shoots and proliferate in solid (Figure 1C) or liquid medium under shake agitation (Figure 1D).

From these observations, it is possible to infer that the absence of light plays an important role in the induction of PLBs, just as light influences the differentiation of PLBs into plantlets. Also, according to [60], the type of light used can also optimize the regeneration of PLBs, with the use of red and white LED combined with sucrose as a carbohydrate source, or blue and white LED with trehalose as the carbohydrate source, which had the best response for the regeneration of PLBs. However, only 17.5% of papers described a dark-period to induce PLBs, while 67.5% used light period (12-16-h photoperiod) to induce and regeneration of PLBs in *Phalaenopsis* (Table 1).

**Figure 1.** Induction, proliferation and regeneration of protocorm-like bodies in *Dendrobium* and *Phalaenopsis* orchids. Protocorm-like bodies (PLBs)-directly induced from leaf segments of *Phalaenopsis* hybrid '501' **(A)** obtained from young in vitro shoots from inflorescence nodal segments and details of secondary PLBs (**B**) obtained in New Dogashima Medium (NDM) culture medium. Proliferation of PLBs in agar (**C**) and liquid (**D**) MS1⁄2 culture medium of *Dendrobium* 'Hybrid 3'. Bars = 1 cm. Unpublished photos of Cesar A. Zanello (A,B) and Jean C. Cardoso (C,D).

Besides the influence of light, another admittedly important factor in the induction of PLBs in *Phalaenopsis* and orchids in general is the genotype [61]. This means that under the same cultivation condition, the induction responses of PLBs may be significantly different [62], which is still considered a limitation of the technique. Ref. [30] evaluated the induction of PLBs in two commercial hybrids (Ph908—red-painted yellow flowers and RP3—dark red) of *Phalaenopsis* and reported significant differences in both percentage of PLB leaf segments (45% and 10%, respectively) as in the number of PLBs per leaf segment (25 and 2 PLBs, respectively).

Regarding the type of explant, leaf segments of plants grown in vitro have been the most suitable for induction of PLBs in *Phalaenopsis* (45% of papers; Table 1), but there are reports of protocols that used in vitro roots of *P.* 'Join Angle × Sogo Musadian' cultivated in MS1⁄2 medium supplemented with NAA, BAP, and IAA (0.5 ppm, 5 ppm, and 0.5 ppm, respectively) and up to 49.33 PLBs/explant [63].



**Table1.**Complianceofstudieswithinduction,proliferationandregenerationofPLBs(IPR-PLBs)with*Phalaenopsis*and*Doritaenopsis*.


**Table**

**1.**

*Cont*.




**Table1.***Cont*.



using magnetic fields

**Table1.***Cont*.



formation and number of PLBs

**Table 1.** *Cont*.



**Table1.***Cont*. NDM: New Dogashima Medium [41]; MS: Murashige and Skoog Medium [99]; Hyponex medium: [100]; XER medium: [101]; VW: Vacin Went medium [43]; NP: New

medium [102]. 2,4-D, Photon Flux Density; Temp, Temperature;

2-4-Dichlorofenoxiacetic

 acid; BA,

 TDZ, Thidiazuron.

6-Benzyladenine;

 IAA,

3-Indoleacetic

 acid; IBA,

Indole-3-butyric

 acid; NAA,

Naphtaleneacetic

 acid; PPFD:

*Phalaenopsis*

Photosynthetically

Segmentation made in leaf segments of *Phalaenopsis* to induce PLBs results in a process called phenolic oxidation, which is the release of polyphenol oxidase (PPO) [103] and other compounds toxic to plant tissue, which may cause its death [74], consequently reducing the induction of PLBs. The immersion of leaf segments in solution of cystine and ascorbic acid during the leaf segmentation stage is reported as a way to reduce the release of these compounds capable of impairing the formation of PLBs [74].

One of the influential factors in the induction of PLBs that has been widely evaluated is the concentrations and possible combinations of plant growth regulators (PGRs). Based on the current literature, successful induction of PLBs seems to be mainly influenced by cytokinin BA (6-benzyladenine) and cytokinin-like compound TDZ (thidiazuron), and in some cases the combination of these cytokinins with an auxin [30,45] also proved beneficial. Protocols citing the use of cytokinin BA recommend concentrations between 0.5 mg L−<sup>1</sup> [78] and 20 mg L−<sup>1</sup> [67]. For the induction of PLBs with the use of TDZ, the recommended concentrations range from 0.25 mg L−<sup>1</sup> [30] to 3.0 mg L−<sup>1</sup> [72]. With the combined use of cytokinins and auxins, the most commonly used auxin is NAA, which varies in concentration from 0.1 mg L−<sup>1</sup> [45,74] to 1.0 mg. L−<sup>1</sup> [30,82].

Ref. [104] reviewed the influence of auxins in orchids, including in PLBs and concluded that auxins is important for callus induction and PLB formation and proliferation, while is inhibitory for PLB regeneration into shoots.

As already described, the addition of PGRs is critical to the success of the PLB induction and regeneration technique in *Phalaenopsis*. Cytokinin-like compound such as TDZ (47.5%) and BA (35%) was the most PGRs used to IPR-PLB technique (Table 1). Nevertheless, the use of these regulators may also result in somaclonal variation. This variation can be assessed by morphological, physiological, biochemical traits or molecular markers [105]. Using Random Amplified Polymorphic DNA (RAPD) markers, [82] reported 17% dissimilarity between PLBs and the parent plant in *P. bellina*. Ref. [89] observed 20% dissimilarity after 20 weeks of cultivation in *P. gigantea* using ISSR (Inter Simple Sequence Repeats) markers, leading to the conclusion that PLB proliferation should be done for up to 16 weeks to reduce somaclonal variations and morphological changes. It should be noted that changes in alleles will not always result in phenotypic changes [106], so the variations observed by the markers will not always cause some kind of morphological change in plants.

According to [107], the combination of red light and far red contributes to decrease endoreduplication rates during PLB induction and regeneration, and consequently may reduce somaclonal variations during mass propagation processes.

Bioreactors could be used to improve the proliferation of PLBs in *Phalaenopsis*. The authors of [108] obtained 18,000 PLBs from 1000 PLBs sections using 0.5 or 2.0 L volume of air per volume of medium min−1.

### **3.** *Oncidium* **Hybrids Group**

According to the World Checklist of Selected Plant Families of the Kew Botanical Garden, in December 2019, there are 374 accepted names of *Oncidium* species with more than 90% of accepted names allocated in Southern America and the last in Northern America. In addition to the species, thousands more interspecific and intergeneric hybrids have been registered with the Royal Horticultural Society and are used in the commercial production of cut and pot flowers worldwide [9,109]. Different chemical and physical factors alter the response to PLB induction in *Oncidium*. Using *Oncidium* 'Gower Rampsey' shoot tips, [109] observed a higher percentage of shoot tips induced to produce PLBs (96.7%) in monochromatic red-light emitting diodes (RR), compared to blue LED (83.3%) and fluorescent white light (76.7%) used as control. However, the use of RR, as well as green LEDs, increased in inhibition of differentiation of PLBs into green buds, while blue LEDs enhanced differentiation. Associated with this response, the authors also observed that in blue light, PLBs contained higher contents of carotenoids, chlorophyll, soluble proteins, lower amounts of soluble sugars and carbohydrates. The authors further argue that in red LEDs, where a higher PLB induction response was obtained, there was a greater accumulation of soluble sugars, starch and carbohydrates, while in blue light, where

there was a greater differentiation of PLBs, there was a greater accumulation of proteins and pigments such as chlorophylls and carotenoids.

PGRs are one of the most tested factors in IPR–PLBs in *Oncidium* (Table 2). Benzyladenine (BA) at 2.0 mg L−<sup>1</sup> + 0.2 mg L−<sup>1</sup> Naphthaleneacetic Acid (NAA) has been shown to be the most efficient treatment for inducing PLBs in *Oncidium* 'Sweet Sugar' apical and axillary buds [110] and the combination of 0.1 mg L−<sup>1</sup> BA + 0.2 mg L−<sup>1</sup> ANA resulted in better response for *Oncidium* Aloha 'Iwanaga' [111]. In this context, BA can be used efficiently to obtain PLBs in *Oncidium* in 31.8% of the papers, and auxin NAA is the one most used along with BAP (Table 2).

Interestingly, [112] reported the individual and combined effects of BA and NAA PGRs at different stages of in vitro induction, proliferation and regeneration of PLBs on *Oncidium* sp. These authors identified that previous callus production in culture medium containing 2,4-D at 1.0 mg L−1, prior to induction, was beneficial for the production of PLBs from in vitro shoots, and from callus it was possible to observe up to 98 PLBs/callus cluster using 0.75 mg L−<sup>1</sup> NAA, while only 28.2 PLBs/shoot cluster were directly obtained using the combination of 0.5 + 0.5 mg L−<sup>1</sup> NAA and BA, respectively. The use of 1.0 mg L−<sup>1</sup> NAA alone allowed PLB proliferation (up to 79.2 PLBs/sample), while the addition of 1.0 mg L−<sup>1</sup> BA resulted in shoot bud formation (up to 12.4 shoots/PLB). Similarly, [113] observed that the concentration of 2.0 mg L−<sup>1</sup> BA resulted in the highest number of shoot buds obtained from PLBs (4.3/PLB) in *Oncidium* 'Sweet Sugar'.

Thidiazuron (TDZ) also appears to have a pronounced effect on direct induction of PLBs in *Oncidium* leaf segments and were reported in 54.5% of the papers (Table 2), being higher for the percentage of explants directly forming PLBs (60–75%) and number of PLBs per explant (10.3–10.7) compared to other cytokinins such as kinetin, zeatin, 2-isopentenyladenine and BA itself [114]. Ref. [115] reported direct regeneration of PLBs from mainly the epidermis and cut regions of young leaf segments of *Oncidium* 'Gower Ramsey' using TDZ alone (0.3–3.0 mg L−1), rather than BA in the culture medium, while the combination 2,4-D and TDZ was not beneficial for induction of PLBs. The production of PLBs from tissue damaged regions of inflorescence segments (65%) of *Oncidium* 'Gower Ramsey' using 3 mg L−<sup>1</sup> TDZ [116] has also been reported. A similar experiment using the same cultivar observed that calli from root apexes and stem segments produced PLBs in medium containing 0.3–3.0 mg L−<sup>1</sup> TDZ, being beneficial the addition of NAA for the formation of embryos n root and leaf calli [117], being a tissue-specific response.

Other PGRs as GA3 is reported as an inhibitor of PLB induction in *Oncidium*, while the use of antigibberellins, as ancymidol and Paclobutrazol, increased the percentage of leaf explants with PLBs and the number of PLBs obtained [118].

The use of liquid medium, rather than semi-solidified with Agar, is also an alternative for in vitro PLB proliferation (Figure 2). Ref. [113] used 5 L balloon-type air-lift bioreactor to provide mass propagation of *Oncidium* 'Sweet Sugar', and show that this system provides 326.3 g PLBs and growth ratio of 10.2, and is more efficient than semi-solid (2.7 g PLBs and Growth ratio of 3.4) and liquid-agitated flask culture (3.5 g PLBs and growth ratio of 4.4). In bioreactor, the lag phase was observed in the first 10-d culture, accompanied by a sharp drop in pH (5.7 to 4.7) and EC (3.2 to 1.5 mS cm−1) in the first 20-d of cultivation, followed by an intense mass growth from 10 to 40 days of cultivation, when the pH increased again to 5.9. An interesting fact was the dynamics of sugars in the culture medium, and a fast and drastic reduction of sucrose in the medium was observed, from 27 (day zero) to 5.5 (day five), 1.2 (day 10) and zero (day 20), associated with a substantial increase in glucose and fructose in the first 10 days of cultivation, with the exhaustion of these sugars at 40 days of cultivation, when the PLBs entered the stationary phase, demonstrating that during a certain period the PLBs release invertases in the culture medium to reduce sugars, and these are metabolized during the exponential phase of production of PLBs [113].


**Table2.**Complianceofstudieswithinduction,proliferationandregenerationofPLBs(IPR-PLBs)techniqueusedwith*Oncidium*speciesandhybrids.

*Int. J. Mol. Sci.* **2020**, *21*, 985


PLBs/explant with 20 and 30 g L−1

sucrose, respectively


*Int. J. Mol. Sci.* **2020**, *21*, 985



capacity

Gower Ramsey

**Table2.***Cont*.



*Int. J. Mol. Sci.* **2020**, *21*, 985

22

MS: Murashige and Skoog Medium [99]; VW: Vacin Went medium [43]; WPM: Wood Plant Medium [131]. 2,4-D,

acid; IBA,

Indole-3-butyric

 acid; NAA,

Naphtaleneacetic

 acid; PPFD:

Photosynthetically

 Photon Flux Density; Temp, Temperature;

2-4-Dichlorofenoxiacetic

 acid; BA,

 TDZ, Thidiazuron.

6-Benzyladenine;

 IAA, 3-Indoleacetic

Another study conducted in a gelled medium by [124] observed that the use of 2% fructose resulted in 95% explants containing PLBs in *Oncidium* Gower Ramsey or 2% glucose resulted in 85% explants containing PLBs in *Oncidium* Sweet Sugar [124]. However, for the number of PLBs per explant, the best results were obtained with 2–3% sucrose (31.1–33.7 PLBs/explants), demonstrating that sucrose is the most suitable sugar for IPR–PLB. The use of other types of sugars, cellobiose, maltose and trehalose do not result in benefits for number of PLBs from callus in *Oncidium* Gower Ramsey [122] or for direct production of PLBs from young leaves [124].

There are no doubt about the application of PLBs in mass clonal production of *Oncidium* [132], but recent studies also showed and confirmed the presence of somaclonal variation in *Oncidium* obtained from IPR–PLBs [133], similar to observed with *Phalaenopsis* genus.
