**5. Applications of IPR–PLB Technique on Orchid Propagation and Breeding and Main Limitations of the Technique**

Induction, proliferation, and regeneration of PLBs in orchids have many advantages to conventional micropropagation by shoot proliferation or use of shoots from inflorescence stalk segments as in *Phalaenopsis* [140], as increased rate of proliferation/multiplication [141] and single-cell derived PLBs [123], which could be used for propagation, but also for breeding purposes and to obtain disease free plantlets.

In breeding programs using in vitro techniques, PLBs could be used to obtain autotetraploid plants with use of anti-mytotic agents as oryzalin [142] and colchicine [143], and to obtain mutants by the use of chemical mutagens as sodium azide [144] or physical mutagens as gamma-irradiation [145].

PLBs can be also used for transformation protocols and successful protocols were developed and obtained stable transgenics with target characteristics for floriculture [146,147]. In genetic transformation of orchids, the use of PLBs derived directly from individual epidermal cells resulted in solid transgenic plants with clonal identity of *Oncidium* Sharry Baby 'OM8' [32], an exceptional advantage over PLBs from callus and with multicellular origin [126], which may result in the emergence of somaclonal variants [42] and chimeric tissues when used for genetic transformation, which are difficult to characterize and separate [32]. Using this technique, these authors reported 33–43% PLBs expressing the β-glucuronidase gene (GUS) and obtained six lineages that amplified the transgenes pepper ferredoxin-like protein (pflp) and hygromycin phosphotransferase (hpt) using the particle bombardment technique. *Agrobacterium tumefasciens*-mediated transformation has also been successfully used in the production of transgenic plants of *Oncidium* 'Sharry Baby OM8' and *Oncidium* Gower Ramsey using the induction of secondary PLBs from in vitro-maintained PLBs [148,149].

From a phytosanitary point of view, it is known that the use of seeds for in vitro asymbiotic sowing of orchids is a real way to obtain virus-free seedlings in orchids from contaminated mother plants, as observed for *Cymbidium* species [150,151]. Ref. [152] confirmed on a large scale (1000 plants) that in vitro plants from seeds are free of *Cymbidium Mosaic Virus* (CyMV) and *Ondontoglossum Ringspot Virus* (ORSV).

The technique of culturing apical meristems may also be effective in eliminating viral diseases in orchids, but it requires great manual skill for excision of tiny meristems leading to contamination-free tissue [153]. These requirements and the individual characteristics of viral diseases may lead to breakthroughs in the technique, which may result in in vitro plantlets containing viral diseases, as reported in *Brassolaeliocattleya*, *Cattleya*, *Dendrobium*, *Epicattleya*, *Oncidium,* and *Mokara* grown in vitro, for which CyMV virus was reported to be present in 27.6% of 880 plantlets evaluated, while ORSV was not detected in these samples [152].

Furthermore, in genera such as *Phalaenopsis*, the most commercially important in the world, only stem apex culture may not be effective in completely eliminating important viral diseases in the crop [140], and may still result in the need to kill the mother plant to obtain the apical meristem, since these plants are monopodial and have poorly developed stem [150]. In this sense, in vitro IPR–PLBs is an alternative to the production of virus-free clonal plants in orchids. In *Phalaenopsis* hybrid 'V3', Ref. [140] obtained PLBs from stem apexes of donor plants contaminated with *Ondontoglossum* ringspot virus and *Cymbidium* mosaic virus, and observed that the first PLBs produced directly from the stem apex had 31.25% PLBs with viruses, identified by the enzyme-linked immunosorbent assay (ELISA) and RT-PCR and were only eliminated in the process after some subcultures. The PLBs identified as virus-free were subcultured in PLB proliferation medium, and in the second subculture 18.18% positive PLBs were identified for both viruses. Only in the third subculture of PLB proliferation, it was possible to obtain 100% virus-free PLBs, which remained until the end of the experiment.

PLBs can also be used for orchid propagation using the synthetic seed technique and for cryopreservation. In *Dendrobium* 'Sonia', the use of PLBs stored at 4 ◦C for 15 days in the pro-meristematic and leaf primordium stages and encapsulated with 3–4% sodium alginate + 75–100 mM CaCl2\*2H2O resulted in 100% germinated PLBs, with the appearance of the first leaf at 22–27 days and the first root at 30–35.8 days, and the technique can be replicated with similar results for *Oncidium* 'Gower Ramsay' and *Cattleya leopoldii* [154].

In *Dendrobium candidum* and *Dendrobium nobile*, PLBs have also been used to increase the production of bioactive compounds. In *D. nobile*, an increase was observed in the production of secondary metabolites such as phenols, flavonoids and alkaloids extracted from PLB-micropropagated plants, when compared to the mother plant [139]. In *D. candidum*, the increase in methyl-jasmonate elicitor concentrations, although resulting in a proportional reduction in PLBs mass gain, increased the concentrations of alkaloids, polysaccharides, phenols and flavonoids when used between 75 and 100 μM [155].

Although the IPR–PLB technique is widely used for large scale plantlet production, breeding and conservation, some difficulties still limit the wider use of the technique on a commercial scale. Among the main limitations are the high genotype-dependence of PLB induction and proliferation responses in vitro, and the occurrence of undesirable somaclonal variations, which greatly hinder the

proliferation of clonal propagation of PLBs for a wide range of commercial cultivars available and required by the market.

Ref. [30] used NDM culture medium plus TDZ (0.25 mg L−1) and NAA (1.0 mg L−1) and observed distinct responses between '908' genotype (45% explants with PLBs and up to 25 PLBs/leaf segment) and 'RP3' genotype (10% explants with PLBs and only 2 PLBs/leaf segment), the latter being highly recalcitrant to the induction and proliferation of PLBs from leaf segments of plants grown in vitro. A study by [59] also noted important differences between the PLBs induction responses between *P. amabilis* (up to 50% explants with PLBs and 15.6 PLBs/explant) and the commercial cultivar *P. nebula* (80% explants with PLBs and up to 5.3 PLBs/explant). The same occurred in another study with the same cultivars, in which the cytokinin types and concentrations that resulted in the highest percentage of explants with PLBs were 13.32 μM BAP in *P. amabilis* (80%) and 13.62 μM TDZ in *P. nebula* (65%). The largest number of PLBs per explant was obtained with 13.62 μM TDZ in *P. amabilis* (7.8 PLBs/explant) and 4.65 μM Kin in *P. nebula* (16 PLBs/explant) [77].

Ref. [156] point out that one of the biggest difficulties in *Phalaenopsis* micropropagation by PLBs is that not all genotypes respond to a single protocol and the same cultivation conditions, and often result in plants with undesirable characteristics. Ref. [41] compared eight cultivars of *Phalaenopsis* and *Doritaenopsis* to obtain PLBs from shoot tips of inflorescence stalk buds with best percentage of PLB formation in four genotypes using 1.0 mg L−<sup>1</sup> BAP (26.9–71.4% depending on genotype), while two respond better with 2.0 mg L−<sup>1</sup> (60–75% explants with PLBs) and one produced 50% PLBs independently of the concentration of BAP (1, 2, or 5.0 mg L−1). Testing other four genotypes authors reported ranges from 7.1% to 40% of PLBs formation only in NDM culture medium, while in 1⁄2MS only two cultivars produced PLBs [41].

Ref. [156] have been associated undesirable characteristics observed in some plantlets with the identification of somaclonal variants from PLBs, which can be morphologically identified even at the shoot bud regeneration and in vitro plantlet production stage. According to [157], the occurrence of SV in the IPR–PLBs technique is higher than that observed from adventitious bud propagation, and that most commercial laboratories use a maximum of three generations of PLBs subcultures to avoid high frequencies of somaclonal variations in this type of propagation.

In our laboratory conditions, using leaf segments from in vitro plantlets to obtain PLBs (Figure 1A,B) somaclonal variations are observed in rooting phase of PLB-derived plantlets of *Phalaenopsis* 'Ph908', while were not observed in plantlets derived from shoot-proliferation using inflorescence stem nodal segments (Figure 2A). The main symptoms were the limited development of plantlets that remains in acclimatized plantlets, with morphological abnormalities in leaves (Figure 2B), also observed and called as 'creased leaves' by [66] and flowers deformities as absence of lip in some flowers of the inflorescence (Figure 2C,D) possibly associated with mutations rather than epigenetic variations.

Ref. [139] used induction of PLBs from pseudostems from in vitro germinated *Dendrobium nobile* plants in MS + 1.5 mg L−<sup>1</sup> TDZ and 0.25% activated charcoal medium and verified 94% explants producing PLBs and up to 11.6 PLBs/explant. These authors observed a somaclonal variation rate close to 6% in the obtained plants, being the main cause of the somaclonal variations detected by molecular markers Random amplified polymorphic DNA (RAPD) and Start codon targeted (SCoT), attributed by the authors to the use and exposure time to TDZ.

Although the cytokinin-like compound TDZ is appointed as one of the major causes of SV in orchid PLB induction, there were some contradictory reports.

As example, the cytokinin Kinetin at 1.5 mg L−<sup>1</sup> resulted in increases of somaclonal variations frequency of PLBs in *Dendrobium* Sabin Blue, detected by ISSR and DAMD molecular markers, when compared with use of TDZ at 4.0 mg L−<sup>1</sup> added activated charcoal [158].

**Figure 2.** Somaclonal variations observed in *Phalaenopsis* induction, proliferation and regeneration of protocorm-like Bodies in *Phalaenopsis* Hybrid "908". Normal vegetative developed plant (**A**) and somaclonal variation observed in vegetative development with "creased leaves" (red arrow) (**B**); (**C**,**D**), Normal vegetative developed plants with somaclonal variations in flower development, with first and last flower without of labellum (red arrow, wl) in the same inflorescence with normal flowers (nf). All figures are unpublished photos from J.C.C.

In addition, [159] observed somaclonal variants in *Phalaenopsis* True Lady 'B79-19', obtained from the induction of PLBs and from young leaves obtained from in vitro plants in VW culture medium containing only BA and NAA as phytoregulators, i.e., without using TDZ. These authors also reported that variant plants were discarded during in vitro subcultures (not quantified), and out of the plants obtained and without morphological variations in the leaves, only 20 out of a total of 1360 obtained (1.5%) were somaclonal variants, indicated by the different flowers of the original clone.

Also the use of topolins *meta*-Topolins (*m*T) and *meta*-Topolins Riboside (*m*TR), a natural aromatic cytokinin reported as reducing phytotoxic effects in micropropagation, it use not solved the problem of somaclonal variation obtained in vitro [160] and, although was reported increasing efficiency of PLB induction it use not resulted in absence of somaclonal variation in orchids [139].

These observations with other cytokinins PGRs diminish the importance of TDZ as the unique or main factor for VS inducing in orchid IPR–PLBs, and include other causes, such as the differential susceptibility of genotype and the number of subcultures under proliferation stage of PLB production.

Genotype susceptibility is appointed one of the main factors lead to VS in *Phalaenopsis* and *Doritaenopsis* orchids micropropagation, ranging from zero to 100% SV depending on genotype and is not exclusive of the PLB technique [72,161]. Similarly, [70] also observed that some genotypes of *Phalaenopsis* not presented any variants, while others showed until 47.9% of variants. Among them, most of SV in this genus were reported in flowering stage [161], by modification of inflorescence and flower characteristics, such as the perloric and semi-perloric mutants observed in *Phalaenopsis* Zuma Pixie '#1', *P.* Little Mary and *Doritaenopsis* Minho Diamond 'F607' [162]. Lose of part of flowers were also reported, such as pollinia [162] and absence of labellum (Figure 2C,D).

Ref. [161] evaluated until the flowering stage (1.0–1.5 years after acclimatization) plants of 10 genotypes of *Phalaenopsis* and *Doritaenopsis* hybrids micropropagated by the PLB technique, and subcultured in vitro for 5 to 10x and identified the presence of seven types of VS, possible to be identified only at the flowering stage. The plants had deficiencies or divergences in the petals and sepals or in the development of the inflorescence, but with similar vegetative development in relation to the mother plant. These authors observed that the produced VS were not polyploid mutants, maintaining the same amount of genetic material as the mother plants.

Although most of SV was reported in flowering stage, transcript analysis by Real-Time PCR demonstrated that mutants has also many other alterations in factors of transcription and transcripts were detailed reported in *Phalaenopsis* and *Doritaenopsis* by [162]. In *Oncidium* 'Milliongolds' were also observed chlorophyll SV (whole yellow or with streaked leaves) in vegetative development of in vitro plantlets [133].

Another factor related to the origin of VS in PLBs in orchids is the phase in which VS occurs. It has been reported that in the proliferation phase, undesirable VS induction from PLBs occurs at a higher intensity and frequency, and it is necessary to establish a number of subcultures to keep the VS frequencies low in clonal propagation. Ref. [92] reported increases in SV after the third subcultures of PLBs in proliferation medium (NDM + 0.1 mg L−<sup>1</sup> TDZ and 10 mg L−<sup>1</sup> chitosan) with same ISSR profile until third subculture, 95% at fourth and 80% at fifth subculture of PLBs.

The use of RAPD molecular markers (total of 1116 bands) did not allow the identification of these somaclonal variants in these plants, but isozyme pattern analysis demonstrates the difficulty of observing mutations in materials obtained from PLBs using RAPD molecular markers and the occurrence of conclusion errors or even underestimated data of somaclonal variants in the confirmation of clonal origin in other studies conducted with these markers [159].

Ref. [82] also used RAPD markers to analyze the clonal origin of PLBs and induced seedlings in in vitro leaf segments of *Phalaenopsis bellina* in 1⁄2MS medium with 3.0 mg L−<sup>1</sup> TDZ. They observed that most somaclonal variants are obtained at the proliferation/multiplication phase, with no VS observed in the origin phase of the PLBs of the mother plant.

Analyses of SCoT and Target Region Amplification Polymorphism (TRAP) markers also showed the presence of somaclonal variants in *Dendrobium* Bobby Messina PLBs cryopreserved or not [163].

These differences in the frequencies of VS observed in different orchid species and genotypes are probably associated with higher sensitivity of different genotypes to the occurrence of mutations. Ref. [164] observed that the frequency of VS at the vegetative and reproductive stages in *Phalaenopsis* PLBs was dependent on the genotype used. These authors observed that there was a reduction in DNA methyltransferase (Dnmt)-related gene expression in *Phalaenopsis* 'Little Mary' VS.

Current advances in molecular marker techniques allow increasing the number of tools and the accuracy of these analyses and the greater possibility of identifying possible VS. There is little information about wide molecular genome characterization in *Oncidium*, and [133] used specific-locus amplified fragment sequencing (SLAF-seq) to analyze possible variations in single-nucleotide polymorphisms (SNPs) in *Oncidium* 'Milliongolds' obtained by PLBs grown for 10 years and observed high rates of variation and that adjacent SNPs adenine and thymine were more frequent than those related to guanine and cytosine, with prominence of mononucleotideInDels.

Ref. [157] isolated two most expressed transposable elements and identified a new Instability Factor (PIF)-like, one of which, called PePIF1 was identified by similarity to the *Phalaenopsis equestris* genome sequence, and which was transposed in the somaclonal variants of cultivars of *Phalaenopsis* from micropropagation, which resulted in the insertion of new genes identified and sequenced by the authors.
