Genetic fidelity analysis in pineapple vitroplants obtained from Venezuelan Amazonian ecotype using RAPD markers

1 Reconocimiento-No Comercial-Compartir igual 4.0 Internacional https://creativecommons.org/licenses/by-nc-sa/4.0/ Abstract The present study aims to evaluate the genetic variability of vitro plants obtained from a pineapple ecotype (Ananas comosus) from the Venezuelan Amazon, known as Gobernadora through Randomly Amplified Polymorphic DNA (RAPDs markers). Vitroplans were obtained via three different protocols: Bud culture (BC), somatic embryogenesis (ES) and adventitious organogenesis (OA). Genomic DNA was obtained from leaves sections from different groups of vitroplants and the mother plant. Fourteen decameric primers of arbitrary sequence were tested in the analysis. The electrophoresis revealed 77 bands: 70 were common both to the mother plant and to the vitroplants obtained by the three morphogenic processes, (90.9 monomorphic bands) and 7 were not common (9.1% polymorphic bands). The initiators OPA-01, OPB-01, OPF-13, and OPV-19 detected polymorphism in a plant obtained through bud culture, which was named Gob B6. This plant had a different leaf anatomical structure than that of the mother plant and that of the population of in vitro plants obtained through bud culture. The genetic instability presented in the in vitro plants obtained through bud culture leads us to conclude that the processes of somatic embryogenesis and adventitious organogenesis are more reliable systems for the mass clonal propagation of these plants. This study may provide information on how different morphogenic pathways, induced in the in vitro environment, can affect the genetic stability of in vitro pineapple plants. Keywords: Clonal propagation, organogenesis, somatic embryogenesis, buds culture, clonal stability, molecular markers. Resumen El presente estudio tuvo como objetivo evaluar la variabilidad genética de vitroplantas obtenidas de un ecotipo de piña (Ananas comosus) de la Amazonía venezolana, conocido como Gobernadora a través de marcadores de ADN Polimórfico Amplificado Aleatoriamente (RAPDs). Las vitroplantas se obtuvieron a Petroglifos. Revista Crítica Transdisciplinar 8(2): e080201 julio-diciembre 2025. DOI: 10.5281/zenodo.17506082 ISSN 2610-8186 www.petroglifosrevistacritica.org.ve Artículo Científico Genetic fidelity analysis in pineapple vitroplants obtained from Venezuelan Amazonian ecotype using RAPD markers Análisis de fidelidad genética en vitroplantas de piña obtenidas de un ecotipo amazónico venezolano mediante marcadores RAPD Héctor A. Blanco Flores1 , Teresa E. Vargas Cedeño1 , Carmen C. García García2 , Eva C. García de García1* 1Professor and Researcher. Plant Biotechnology Laboratory. Institute of Experimental Biology. Faculty of Sciences, Universidad Central de Venezuela (UCV), Suapure Street, Colinas de Bello Monte. Caracas, Venezuela. 2Professor in General Pathology and Physiopathology. Luis Razetti School, Faculty of Medicine, Universidad Central de Venezuela (UCV). University City, Postal code: 1050. Caracas, Venezuela. *Corresponding author: [email protected] Recibido: 24-10-2024. Aceptado: 27-07-2025. Publicado: 29-12-2025. Blanco Flores et al. Petroglifos 8(2): e080201 julio-diciembre 2025 2DOI: 10.5281/zenodo.17506082 través de tres diferentes protocolos: cultivo de yemas (CY), embriogénesis somática (SE) y organogénesis adventicia (OA). El ADN genomico se obtuvo de secciones de hojas de las vitroplantas y de la planta madre. El análisis se realizó con catorce iniciadores decaméricos de secuencia arbitraria. La electroforesis reveló 77 bandas: 70 fueron comunes a la planta madre y a las vitroplantas obtenidas mediante los tres procesos morfogenéticos (99 bandas monomorfas), y 7 bandas que no eran comunes (9,1 % de bandas polimorfas). Los iniciadores OPA-01, OPB-01, OPF13 and OPV-19 detectaron polimorfismo en una planta obtenida a través del cultivo de yemas, la cual se denominó Gob B6. Esta planta tuvo una estructura anatómica de la hoja diferente a la de la planta madre y a la de la población de plantas in vitro obtenidas mediante cultivo de yemas. La inestabilidad genética presentada en las plantas in vitro obtenidas mediante el cultivo de yemas, nos lleva a concluir que los procesos de embriogénesis somática y organogénesis adventicia son sistemas más fiables para la propagación clonal masiva de estas plantas. Este estudio puede proporcionar información sobre cómo las diferentes vías morfogénicas, inducidas en el ambiente in vitro, pueden afectar a la estabilidad genética de las vitro plantas de piña. Palabras clave: propagación clonal, organogénesis, embriogénesis somática, cultivo de brotes, estabilidad clonal, marcadores moleculares. Introduction Pineapple (Ananas comosus L.) Merr.) is one of the most commercially important tropical fruits due to its pleasant taste and its high content of antioxidants, enzymes, nutrients and compounds beneficial to the body. In Venezuela, ‘Española Roja’ constitutes the main commercial pineapple cultivar (d’Eeckenbrugge et al., 2011). In the south of the country there are different endogenous pineapple ecotypes which are of local importance, nonetheless their market is restricted to several localities of the Amazonas state and are cultivated mainly by aboriginals of the Piaroa ethnic group (Betancourt, 2006). They cultivate pineapples throughout the year mainly by use of vegetative propagules like crowns, or suckers. The time from planting to harvest depends on the type of planting material used, approximately 15 to18 months and for shoots, and 2224 months for crowns (d’Eeckenbrugge et al., 2011). Thus, plant tissue culture has been used as an alternative for massive in vitro clonal propagation of different cultivars of A. comosus (Sripaoraya et al., 2003; Mogollón et al., 2004; Firoozabady and Moy, 2004; Saucedo Aguiar et al., 2008; Pineda et al., 2012), Venezuelan Amazonian ecotypes (Blanco et al., 2011; Blanco Flores et al., 2017) and Mexican creole pineapple (Torres Ruiz et al., 2023). The use of these vitroplants makes it possible to obtain great quantity of plantlets in a timely fashion thought the whole year, regardless of seasonal changes. Obtaining whole plants from plant cells or tissues is an asexual process that involves only mitotic division and, in theory, should not cause genetic alterations (Bairu et al., 2011). However, it has been observed that this biotechnological tool can, in some cases, generate variants product of somaclonal variation (Larkin and Scowcroft, 1981). These changes may be a consequence of both mutations and mechanisms that alter the pattern of genetic expression, epigenesis. Somaclonal variation has been reported in different commercial cultivars of A. comosus (Perez et al., 2012) also in the ornamental pineapple var. Bracteatus (Santos et al., 2008). Molecular techniques have been used for the evaluation of the genetic stability of plants derived from different in vitro clonal propagation systems (Giménez et al., 2001; Vargas et al., 2008; Bairu et al., 2011; Nandhakumar et al., 2017). The set of molecular techniques, used for this purpose, are modifications or are coupled Genetic fidelity analysis in pineapple vitroplants obtained from Venezuelan Amazonian ecotype using RAPD markers Petroglifos 8(2): e080201 julio-diciembre 2025 3 DOI: 10.5281/zenodo.17506082 to ex situ amplification of the DNA by means of the Polymerase Chain Reaction (PCR) (Bardakci, 2001). Among these, Randomly Amplified Polymorphic DNA (RAPD) has been widely used because it represents an efficient and inexpensive technique to detect different variants or to confirm the genetic fidelity of plants of different species derived from plant tissue culture (Sebastiani and Ficcadenti, 2016; Stanišić et al., 2015; Shair et al., 2016; Kumar et al., 2016; Thorat et al., 2016). The present study aims to genetically characterize vitroplants of Amazonian ecotype of A. comosus, called Gobernadora through the use RAPD markers. The vitroplants were obtained by three different in vitro processes: bud culture, somatic embryogenesis and adventitious organogenesis. Material and Methods Gobernadora pineapple plants obtained through axillary buds culture (Blanco et al., 2011) were used in this research. This material is kept in the in vitro and ex situ germplasms of the Laboratorio de Biotecnología Vegetal of the Instituto de Biología Experimental, Universidad Central de Venezuela. These vitroplants were multiplied using three different in vitro protocols: a) bud culture (BC), somatic embryogenesis (SE), and adventitious organogenesis (AO), and the new plants were analyzed for assessment genetic fidelity (table 1). Foliar sections of Gobernadora’s mother plants, growing in soil, were used as control. Genomic DNA isolation Genomic DNA was extracted from pineapple leaf tissue using a modified CTAB protocol based on Michiels et al. (2003), optimized for samples with high polysaccharide content. The main steps of the extraction procedure are summarized in table 2. Integrity and quantification purified DNA The integrity of the genomic DNA was evaluated by 0.8% agarose gel, in horizontal electrophoresis prepared with 0.5 X Tris-Borate EDTA buffer solution (0.5 X TBE) and stained with 1 µg.mL-1 ethidium bromide. The DNA bands were visualized by ultraviolet irradiation supplied by a GEL-DOC 2000 BIO RAD brand transilluminator. For the electrophoretic analysis, a kit of molecular mass markers (phage DNA fragments λ formed by the action of the Hind III endonuclease) was Table 1 Summary of in vitro propagation protocols for Ananas comosus (Gobernadora). Protocol Explants Culture Medium (Key Additives) Duration & Conditions Bud Culture (BC) Axillary buds from vitroplants MS + BAP 0.5–1.0 mg.L-1, NAA 0.01 mg.L-1, Thiamine, Myo-inositol, 30 g.L-1 sucrose. 4 weeks in liquid, then semisolid; RITA system (Blanco et al., 2011). Somatic Embryogenesis (SE) (Figure 1). Leaf base sections MS + Picloram 5 mg.L-1 + Thidiazuron 1 mg.L-1 (induction), hormone-free for maturation. 10 weeks (dark) + 6 weeks (light, 16 h photoperiod). (Blanco Flores et al., 2017). Adventitious Organogenesis (AO) Leaf base sections MS + NAA 2 mg.L-1 + BAP 5 mg.L1 (induction), hormone-free for development. 10 weeks (light) + 6 weeks (light). (Blanco Flores et al., 2017). Note. MS= Murashige and Skoog medium. Blanco Flores et al. Petroglifos 8(2): e080201 julio-diciembre 2025 4DOI: 10.5281/zenodo.17506082 Figure 1 Acclimatized Gobernadora plants obtained though somatic embryogenesis. Table 2 Summary of genomic DNA isolation protocol (modified from Michiels et al., 2003). Step Procedure / Reagents Conditions Tissue preparation 0.1–0.2 g leaf sections macerated in liquid nitrogen. Cell lysis Add 700 µL 4% CTAB buffer + 5 µL β-Mercaptoethanol (preheated 65°C). Incubate at 65°C, 1–2 h; invert every 10 min. Phase separation Add 700 µL chloroform:isoamyl alcohol (24:1), mix gently. RT, invert 10 min; centrifuge 13,000 RPM, 10 min. Aqueous phase recovery Carefully transfer upper phase to new tube. DNA precipitation Add 100 µL cold 7.5 M ammonium acetate + 700 µL cold absolute ethanol. Incubate –20°C, 30 min; centrifuge 13,000 RPM, 15 min. DNA washing Wash pellet with 1 mL 70% ethanol. Centrifuge 13,000 RPM, 5 min. Final resuspension Resuspend DNA in 10 mM TrisHCl-EDTA (TE), pH 8.0. Store at –20°C. Genetic fidelity analysis in pineapple vitroplants obtained from Venezuelan Amazonian ecotype using RAPD markers Petroglifos 8(2): e080201 julio-diciembre 2025 5 DOI: 10.5281/zenodo.17506082 used. Two methods were used to estimate the yield of the extraction (µg DNA.g leaf tissue-1): a) measurement of the OD of the samples at 260 nm using Pharmacia brand spectrophotometer model Ultrospect III. This method has the disadvantage of overestimating the amount of DNA by the hyperchromic effect. For that reason, an additional method (visual analysis of the intensity of Agarose gel bands) was used. Serial dilutions were made to the DNA plants samples with TE buffer, and then subjected to electrophoresis. Dilutions were done in order to obtain band intensities that were comparable to the intensity of any of the MW (molecular weight marker) whose mass is known. Information on the amount of DNA (ng) per µL of load for each MW is supplied by the manufacturers. With this semi-quantitative method, it was possible to estimate the concentration of the extracted DNA ([ADNext] = [ng band DNA sample µL-1 load] x Dilution factor) and the Yield ([Rend] = µg AD Next g-1 leaf tissue). Concentrated and diluted samples were stored at -20°C until used in PCR-RAPD amplification reactions. PCR-RAPD analysis The genetic stability of vitroplants was evaluated by RAPD primers-PCR analysis. The amplification reactions were carried out in a final volume of 25 μL containing approximately 9 ng of genomic DNA, 2.5 μL of 10 X buffer solution, 2.5 μL of each 5 μΜ RAPD initiator, 0.5 μL of the mixture of 40 mM DNTPs, 1U of GoTaq® DNA Polymerase (Promega, Madison, USA) and the reaction volume was completed with miliQ water previously autoclaved. Fourteen RAPD initiators (Bioneer, South Korea) (table 3) selected by previous studies on genetic stability in micropropagated pineapple plants were tested (Soneji et al., 2002; Santos et al., 2008). For each initiator a control reaction was prepared to rule out contamination from another source of DNA (all reaction components except the DNA sample). Once the reaction mixture was prepared, these were placed in the BIORAD Thermocycler programmed with the following physical conditions: Initial denaturation, 94°C (10 min); 40 cycles with denaturation 94ºC (1 min), alignment 36ºC (1 min) And extension 72ºC (2 min) and final extension at 72ºC (10 min). The amplification products (amplicons) were separated on 1.5% agarose gel by horizontal electrophoresis, stained with ethidium bromide, visualized on the Transilluminator and the image exported and stored in TIFF format on the computer. Bands analysis The size of the amplicons was estimated by comparison with the running pattern of 1Kb molecular mass Table 3 Decameric arbitrary sequence oligonucleotides (initiators) used in the RAPD analysis of the plants obtained by in vitro culture of the Gobernadora pineapple. Primers Sequence (5′- 3′) Primers Sequence (5′- 3′) OPA-01 CAG GCC CTT C OPF-13 GGC TGC AGA A OPA-02 TGC CGA GCT G OPL-17 AGC CTG AGC C OPA-03 AGT CAG CCA C OPM-13 GGT GGT CAA G OPA-20 GTT GCG ATC C OPP-16 CCA AGC TGC C OPB-01 GTT TCG CTC C OPT-07 GGC AGG CTG T OPB-19 ACC CCC GAA G OPV-19 GGG TGT GCA G OPC-19 GTT GCC AGC C OPX-03 TGG CGC AGT G Blanco Flores et al. Petroglifos 8(2): e080201 julio-diciembre 2025 6DOI: 10.5281/zenodo.17506082 markers in the Bioneer ladder, South Korea. Data were recorded as presence (+) or absence (-) of bands. The RAPD profile of the plants obtained by in vitro culture was contrasted with the amplification pattern of the mother plant. All amplifications were performed in duplicate and only those reproducible bands were considered for analysis. In case of variation, in some cases, a third amplification was necessary for confirmation. The total number of amplified bands was recorded, those that were mono and polymorphic were identified, and an estimate of Polymorphism (Number of polymorphic bands divided by total number of bands) was performed on plant samples obtained by different in vitro culture methods and the mother plant. For the analysis, 9 vitroplants propagated by BC, 5 obtained via SE, 5 obtained via AO and 5 mother plants of the ecotype Gobernadora, were analyzed. Anatomical patter Leaf anatomical patterns of the new pineapple variant Gob B6 and its mother plant were analyzed. Foliar sections of Gobernadora’s mother plants growing in soil, were used as control, to determine changes in the leave structure. Leaves from 5 mother Gobernadora plants and leaves from 5 Gob B6, were cut in small sections, dehydrated with ethanol series, stained with Toluidine blue, fixed with glycerin (30%) and mounted in glad slides. The were observed through Nikon 14 MLAB-2 and Nikon OPTIPHOT optical microscopes, the latter with a built-in light polarizer. Photographs of anatomical sections were taken with a camera SONY, Ciber-shot, DSC-brand digital camera, 10.1 Mega pixels. Results and Discussion Monitoring the genetic identity of plants obtained through in vitro culture is crucial, as interactions between intrinsic tissue factors and the microenvironment can induce genetic variation. To this end, high-molecular-mass DNA (approximately 23,130 bp) was isolated from Gobernadora plants of the A. comosus ecotype. The estimated average yield according to the spectrophotometric analysis was 251.9 ± 42.13 µg.g-1 leaf tissue. However, it was necessary to utilize an additional determination method since spectrophotometry overestimates said parameter (hyperchromic effect, Voet et al. (2009). At λ = 260 nm, not only does the integrated double-stranded DNA absorb energy, so do single-stranded nucleic acids and free nucleotides. These last two have a higher molar extinction coefficient and, therefore, have a greater capacity for UV light absorption than the double-chain molecule. The alternative method was a visual analysis of the intensity of bands at different dilutions in the agarose gel. This last method allowed to determine the amount of whole DNA extracted from the plants (semi-quantitative estimation). The average yield according to this alternative methodology was 2.35 ± 0.08 µg g-1 leaf tissue. In the PCR-RAPD analysis, fourteen arbitrary sequence decameric initiators (ID) were tested for the genetic stability analysis of Gobernadora pineapple plants obtained by in vitro culture (table 1). Of the 14 RAPD initiators only 8 gave DNA amplification (table 4). Electrophoretic analysis revealed a total of 77 bands; of which 70 were common both for the mother plant and for the vitroplants obtained by the 3 morphogenic pathways (90.9% monomorphic bands) and 7 not common (9.1% polymorphic bands). Polymorphism, in pineapple plants derived from tissue culture, was detected both by absence of existing bands, and by the presence of new bands with respect to the mother plant. The above can be compared with the results of Santos et al. (2008), who evaluated genetic variability in micropropagated Ananas comosus var. Bracteatus plants after four subculture cycles using varying concentrations of benzyladenine (BA). They observed a 2.8% polymorphism and concluded that BA can influence the Genetic fidelity analysis in pineapple vitroplants obtained from Venezuelan Amazonian ecotype using RAPD markers Petroglifos 8(2): e080201 julio-diciembre 2025 7 DOI: 10.5281/zenodo.17506082 occurrence of genetic variation. In our study, although different morphogenetic protocols and cytokinin concentrations were employed, off-type plants only emerged in the group propagated by axillary buds grown on media containing a relatively low concentration of BA (0.5 mg L-1), notably lower than the 5 mg L-1 commonly used to induce organogenesis. This suggests that while plant growth regulators, such as cytokinins, can induce genetic changes, other factors, such as prolonged cultivation duration and endogenous cytokinin levels, also contribute to somaclonal variation. From eight IDs which amplified plant DNA, four of them (OPA-01, OPB-01, OPF-13 and OPV-19), detected different genetic variations in the vitroplant population. These four IDs detected polymorphism in a plant obtained by bud culture, called Gob B6 (figures 2 to 5). IDs OPA-01 and OPF-13 detected variation only in the Gob b6 vitroplant (figures 2 and 3), OPA-01 detected the absence of two bands, one approximately to 2000 bp and another located between 1610 and 982 bp (figure 2), and the OPF-13 ID detected the absence of these latest band between 1610bp and 982bp (figure 3). OPV-19 revealed the same genetic variation in two vitroplants obtained by bud culture, Gob B1 and Gob B6, the amplification pattern shows the absence of 3 bands with molecular masses between 2961 and 1610 bp (figure 4). IDs OPA-01, OPF-13 and OPV-19 did not reveal polymorphism in the vitroplants obtained by both SE and AO. The OPB-01 initiator revealed an additional band (<500 bp) in all the Gobernadora’s sampled vitroplants obtained by the 3 morphogenic pathways (BC, SE and AO) that is not present in the mother plant (figure 5). Additionally, the pattern generated by Gob B6 is different from those generated by the others plants tested. The pattern generated by OPB-01 in all the vitroplants is the same when they are obtained by somatic embryogenesis or adventitious organogenesis (figure 6). Interestingly, primer OPB01 revealed a polymorphic band in all tested Gobernadora vitroplants. This echoes the results of Santos et al. (2008), who detected a similar band across various micropropagated A. comosus var. Bracteatus samples, irrespective of culture medium. Lee (2019) further emphasized the central role of plant hormones and their regulatory interactions in Table 4 RAPD analysis of A. comosus (Gobernadora) plants obtained in vitro culture. Initiators Number of total bands Number of polymorphic bands OPA-01 10 2 OPA-03 15 0 OPA-20 8 0 OPB-01 10 1 OPF-13 8 1 OPT-07 10 0 OPV-19 11 3 OPX-03 5 0 Total 77 7 Blanco Flores et al. Petroglifos 8(2): e080201 julio-diciembre 2025 8DOI: 10.5281/zenodo.17506082 Figure 2 Amplification pattern generated by OPA-01 in the Gobernadora ecotype plants. Figure 3 Amplification pattern generated by OPF-13 in pineapple Gobernadora plants ecotypes B6 and B8 obtained by BC. Note. MW: molecular mass marker, (-): the initiator, MP: control mother plant. B6, B7 and B8 are Gobernadora plants obtained by BC. (*): indicate the absence in Gob B6 pattern, of two bands: one close to 2000 bp, and other between 1610 bp and 982 bp. Note. (*): indicate the absence one band, close to 1610 bp, in Gob B6 pattern. Genetic fidelity analysis in pineapple vitroplants obtained from Venezuelan Amazonian ecotype using RAPD markers Petroglifos 8(2): e080201 julio-diciembre 2025 9 DOI: 10.5281/zenodo.17506082 Figure 4 Amplification pattern generated by OPV-19 in pineapple Gobernadora ecotypes B1, B2, B3, B4, B5, B6, B7 and B8, obtained by BC. Note. MW: molecular mass marker. (*): indicate the absence of 3 bands between 1610 and 2961 base pairs, in the patterns generated by Gob B6 and Gob B1. Figure 5 Amplification pattern generated by OPB-01 in plants de Gobernadora ecotype plants: B1, B2, B3, B4, B5, B6 and B7 obtain by BC. Note. The arrows indicate the presence of an additional band <500 base pairs, that is not present in the mother plant (MP). The pattern generated by Gob B6 is different from those generated by the others plants tested.