Genetic stability of solanum tuberosum l. Cv. Désirée plantlets obtained from embryogenic cell suspension cultures.
Teresa Edith Vargas, Nereida Xena, María del Carmen Vidal, Maira Oropeza and Eva de García
Teresa Edith Vargas. Biologist and D.Sc. Universidad Central de Venezuela, (UCV), Venezuela. Research Assistant and Professor UCV, Venezuela.
Nereida Xena. Doctor, Université de Montpellier, France. Professor UCV, Venezuela.
María del Carmen Vidal. Biologist and D.Sc. UCV, Venezuela. Professor, Universidad Nacional Experimental Simón Rodríguez.
Maira Oropeza. Biologist and D.Sc. UCV, Venezuela. Professor UCV, Venezuela.
Eva de García. Biologist, UCV, Venezuela. M.Sc., University of Wisconsin, USA. D.Sc. in Botany, UCV, Venezuela. Professor, UCV, Venezuela. Address: Apartado 47114. Caracas 1041, Venezuela. e-mail: egarcia@reacciun.ve.
SUMMARY
The genetic stability of Solanum tuberosum L. cv. Désirée plantlets obtained from embryogenic cell suspension cultures that were induced from mixoploidy calli tissue with abundant binucleated cells was analyzed. Plants regenerated from these tissues showed an euploid number of chromosomes (2n=4x=48). This result indicates that in this system, euploid cells were selected to go through a somatic embryogenesis process and plant regeneration. Genotype stability of the regenerated plants was screened by RAPD analysis. The results indicated that the new plant population was highly homogeneous.
]]> estabilidad genética de plántulas de PAPA Solanum tuberosum L. cv. Désirée obtenidas a partir del cultivo de células embriogénicas en suspensiónRESUMEN
Se analizó la estabilidad genética de plántulas de Solanum tuberosum L. cv. Désirée obtenidas a partir del cultivo de células embriogénicas en suspensión, las que fueron inducidas a partir de callos mixoploides con abundantes células binucleadas. Las plantas regeneradas a partir de estos tejidos presentaron un número euploide de cromosomas (2n=4x=48). Este resultado indica que en este sistema las células euploides fueron seleccionadas para seguir el proceso de embryogenesis somática y regeneración de plantas. La estabilidad genotípica de las plantas regeneradas fue evaluada mediante marcadores RAPD. Los resultados indican que las nuevas poblaciones de plantas son altamente homogéneas.
Estabilidade genética de plântulas de solanum tuberosum l. Cv. Désirée obtidas a partir do cultivo de células embriogênicas em suspensão
RESUMO
Analisou-se a estabilidade genética de plântulas de Solanum tuberosum L. cv. Désirée obtidas a partir do cultivo de células embriogênicas em suspensão, as que foram induzidas a partir de calos com ploidia mista com abundantes células binucleadas. As plantas regeneradas a partir de estes tecidos apresentaram um número euplóide de cromossomos (2n=4x=48). Este resultado indica que neste sistema as células euplóides foram selecionadas para seguir o processo de embriogênese somática e regeneração de plantas. A estabilidade genotípica das plantas regeneradas foi avaliada mediante marcadores RAPD. Os resultados indicam que as novas populações de plantas são altamente homogêneas.
KEYWORDS / Cell Suspension Cultures / Genetic Stability / Regeneration / Solanum tuberosum / Somatic Embryogenesis /
Received: 03/05/2007. Accepted: 01/28/2008.
Introduction
Somatic embryogenesis of true-to-type potato plants represents an important process to improve quality, resistance to diseases and agronomic characters of the potato crop (Haberlach et al., 1985). Chromosome number variation and mitotic abnormalities should be evaluated through the whole in vitro process: callus formation, embryo differentiation and plant development. The observed differences would indicate genetic variations in the tissues, leading to the generation of plantlets that do not inherit the genetic traits of the mother plant. JayaSree et al. (2001) established an efficient procedure to induce somatic embryos from leaf culture of potato cv. Jyothi and their regeneration into complete plantlets. Seabrook and Douglass (2001) reported the induction of somatic embryos on in vitro cultured stem internodes, leaves, microtubers and roots of 18 tetraploid potato cultivars. They observed genotypic differences in the regenerative capacity of these cultivars. More recently, Sharma and Millam (2004) discriminated the progression of specific stages of potato somatic embryos by histological means. Vargas et al., (2005) established an embryogenic cell suspension culture from friable callus of Solanum tuberosum L. cv. Désirée internode sections. They described an association between the accumulation of extracellular proteins of various molecular weights and different phases of the embryogenic process.
]]> Karyotipe analysis can provide valuable genetic characterization information; however, there are only a few cytogenetic studies reported to date (in either cultivated or wild potato plants), because of the small and relatively numerous chromosomes. The presence of cellular polyploidization in potato cell culture during callus formation has been reported by some authors (Calberg et al., 1984; Ramulu et al., 1985; Osifo et al., 1989; Pijnacker et al., 1989; Dathe and Wersuhn, 1990; Fleming et al., 1992; Wersuhn and Dathe, 1998; Xena et al., 2000). A correlation between the chromosomal and nuclear parameters of 23 advanced breeding lines of S. tuberosum has also been described (Mohanty et al., 2004). Structural alterations in the chromosomes, as well as loss or addition of highly repetitive sequences in the genome, demonstrated DNA content variations at the cultivar level (Xena et al., 2000).The absence of cytokinesis during mitosis of potato cells maintained on in vitro cell suspension cultures is the origin of the frequently observed polyploidization on these cells (Ramulu et al., 1985; Nuty Ronchi and Giorgetti, 1995).
A set of molecular techniques have been developed to evaluate chromosome mutations such as inversion, deletion or translocation, as well as gene single base substitutions. One of them is the use of randomly amplified polymorphic DNAs (RAPDs) technique. By means of single primers of arbitrary nucleotide sequence, it is possible to randomly amplify DNA sequences throughout all the genome. RAPD polymorphism which comes from either a nucleotide base change, that alters the primer binding site, or from an insertion or deflection within the amplified region (Williams et al 1990), is usually detected by the presence or absence of an amplification product from a single locus (Tingey et al., 1992). The products of these amplifications can be polymorphic and used as genetic markers (Hu and Quiros, 1991). Over the 1990's, the lack of reproducibility of results between different laboratories was a main concern in the utilization of RAPD markers to determine genetic uniformity of cultivars and somaclones. However, in the past years, important advances in technique implementation have been achieved along with the standardization of DNA extraction and DNA amplification protocols. Nayak et al. (2003) found changes in the RAPD banding pattern in one improved Jamrosa somaclone as compared to donor parent. Modgil et al. (2005) used RAPD to assess the genetic stability of 10 micropropagated plants regenerated through axillary buds of clonal apple (Malus pumila Mill.) rootstock MM106. Their results showed that RAPD markers could be used to detect genetic similarities and dissimilarities in micropropagated material. An efficient in vitro multiplication system in Chlorophytum arundinaceum has also been established and the genetic fidelity was assessed using RAPDs, karyotype analysis and meiotic behavior of in vitro and in vivo plants (Latoo et al., 2006).
In this report, the stability of a potato (Solanum tuberosum L. cv. Désirée) population regenerated from cell suspension cultures is analyzed. Cytogenetic analysis and RAPDs were used to evaluate the presence of genetic mutation on callus cells that gave rise to somatic embryos, and in root apex cells of regenerated plants. The results indicated that the population of regenerated potato plants was highly homogeneous.
Materials and Methods
Plant material
Stem internodal sections were obtained from in vitro propagated shoots of potato Solanum tuberosum cv. Désirée plants.
Induction and establishment of cell suspension cultures
To induce callus tissue, nodal sections (1-1.5cm long) were cultured on MS1 medium (Table I) based on MS salts (Murashige and Skoog, 1962) and incubated in the dark at 25 ±1°C for 2 months, following the protocol established by De García and Martínez (1955). For the establishment of embryogenic cell suspension cultures, the procedure described by Vargas et al. (2005) was followed. Briefly, 1g of callus tissue was inoculated into 100ml of liquid medium MS2 (Table I), placed over an orbital shaker (160rpm), and incubated in darkness at 25 ±1°C for two weeks. All media renewals were performed by decanting the suspension every 15 days. After two weeks, suspension cultures were filtered through sterile 100mm mesh, transferred to fresh MS2 medium and placed in the same environmental conditions. Two weeks later, cells were transferred to an MS3 medium (Table I), and maintained under continuous light conditions (50mmol·m-2·s-1), at 25 ±1°C. Cells remained in this medium until pro-embryogenic groups and globular embryos were observed (30 days). Following this phase, MS3 medium was substituted by MS4 medium, where somatic embryos remained for an additional 30 days. Once somatic embryos had reached maturity, they were transferred to a solid medium MS8 (Table I), where the embryos developed into plantlets. Forty plantlets 8cm long were potted in a mixture of soil and river sand 3:1, and placed under high humidity (80-93% relative humidity), and low light conditions (10mmol·m-2·s-1). Ten days later plantlets were transferred to a greenhouse.
Parental plants root apexes, callus tissue, embryos in different developmental stages (globular, heart and torpedo), and root apices from 20 regenerated Solanum tuberosum cv. Désirée plants were evaluated. The plants were selected from three different independent cell suspension cultures.
All tissues were fixed for 24h on Carnoy (ethanol:acetic acid 3:1) and stored on 75% ethanol at 4ºC. Squashes were made with 1M HCL for 10min and stained with carbolfucsin. Observations and photographs were made on a Nikon Optiphot optical microscopy (Sharma and Sharma, 1972).
DNA isolation
DNA was extracted from 19 randomly selected potato in vitro plantlets from three different independently started cell suspension cultures using the CTAB procedure (Doyle and Doyle, 1990).
RAPD analysis
Seventeen decamer primers obtained from OPERON® were used in this study (Table II), following the protocol established by Demeke et al. (1996). PCR was performed in a volume of 25ml containing 2.5ml 10X Buffer, 2.0mM MgCl2, 0.1mM dNTPs, 0.2mM primers, 80ng template DNA, and 1.0 unit Taq DNA polymerase (Perkin Elmer). Amplification of DNA template was performed in a PTC-100 Programmable Thermal Cycler (MJ Research), following the cycle parameter established by Singsit and Akings (1993). The PCR program consisted of an initial denaturation step of 1min at 94°C, followed by 35 cycles of 1min at 94°C, 1min at 36°C and 2min at 72°C, and a final extension step of 10min at 72°C. For each primer, tubes containing all reaction components except for the DNA template were included as control to check for contamination. Reaction products were resolved by electrophoresis in 1.4% agarose/ethidium bromide gels. Duplicate reactions were routinely performed to ensure reproducibility.
Results
Cytogenetics of donor plants
]]> Uni-nuclear isodiametric cells were present in donor plants root apexes and in only a few cases binucleated cells were found. Cells (Figure 1) contained the typical euploid chromosome number (2n=4x=48). Interestingly, some isolated chromosomes were observed as well, which were not related to the "achromatic spindle" and produced abnormal anaphases with laggard chromosomes and chromosomal bridges. No polyploid cells appeared to be evident.
Cytogenetics of callus tissue
In calli tissue mixoploidy was evident due to the existence of abundant binucleated cells. The polyploidization of certain cells takes place during karyokinesis. Some binucleated cells went through synchronized bimitosis, where simultaneous cellular divisions occurred in the two nuclei. Furthermore, big cells with conspicuous nuclei were present, showing amorphous bodies. Cells with polyploid metaphase (more than 90 chromosomes) and some with metaphase plate were also observed (Figure 2).
Somatic embryos
In somatic embryos (Figure 3), most of the cells were euploids (2n=48) with chromosomes comparable in size and shape to the standard chromosomes observed in the roots apexes of the donor plants. Few cells were observed in division phase; most of them with one nucleus, and very few cells were binucleated and went through bimitosis. Cells in anaphase with chromosome bridges were also found (Figure 4). Nonetheless, some isolated chromosomes, which are not related to the chromatic spindle, produced abnormal anaphase with laggard chromosomes and chromosomic bridges. Aneuploid cells with lower number of chromosomes than normal ones were also present.
Regenerated plants
]]> In the root apex of regenerated plants, cells with different morphology (isodiametric, rounded or elongated) were observed. All the cells had a single nucleus and the number of chromosomes was the standard euploid (2n=48). Two different types of chromosomes were present in the metaphase plate: shorter and thicker chromosomes and normal ones. Some anaphases were typical, well organized and others were amply disordered, with chromosome bridges (Figure 5a, b). Polyploidy was not a feature. In general, the abnormalities found in the regenerated plants were less than those exhibited in the cells of the calli which gave rise to these plants.
RAPD analysis
In order to determine if the plants regenerated from embryogenic cell suspension cultures of potato showed any induced genetic variation when compared to the donor plant, screening by RAPD markers was performed. Amplification of genomic DNA from the 19 randomly selected regenerated plants with 17 decamer primers yielded a total of 2470 highly reproducible RAPD fragments ranging in size from 400 to 2600bp (Table III). The number of fragments produced (Table II) ranged from 4 (OPA-18 primer) to 10 (OPA-01, OPA-03, OPA-04 and OPA-09 primers). The 17 primers tested produced representative amplified band patterns that were monomorphic in 19 regenerated plantlets and the parental ones. Figure 6 shows a monomorphic amplification pattern using primer OPA-17. With primers OPA-02 and OPA-13, polymorphic band patterns were obtained from regenerated plants 3 and 16, respectively. Figure 7 shows polymorphic band patterns with primer OPA-13.
Discussion
Genetic variability is a common feature of plant cells that have undergone a tissue culture protocol. This genetic variability could be pre-existing in the cells of the donor plant or could be originated in the culture process, mainly in the callus phase (Singsit and Akings, 1993). They both could influence the genetic stability of regenerated plants.
Polyploid metaphase was a characteristic of calli tissue in the present study. According to Ramulu et al. (1985) endoreduplication and endomitosis constitute the major mechanisms of polyploidization in potato cells. Cytokinesis failure during karyokinesis can also give rise to polyploid cells (Nuty Ronchi and Giorgetti, 1995). In this case binucleated cells are formed, followed by the simultaneous cellular divisions in the two nuclei (bimitosis). In some instances, partial disorganization of the achromatic spindles arises when multipolar metaphases with dispersed chromosomes are produced, followed by mitosis without cytokinesis, giving rise to polyploid cells (Sivarolla, 1992).
]]> In this work, embryogenic cell suspension cultures were established from this mixoploid cell tissue. Most of the somatic embryos that arose from these cell suspension cultures were euploid (2n=48) with chromosomes comparable in size and shape to the standard chromosomes observed in the root tips of donor plants. Few cells were binucleated.Potato plants were regenerated from 18 week-old embryogenic cell suspension cultures. They did not show any morphological changes at mature stages. Cytogenetic analysis of the root tips from these plants demonstrated that all the cells have a single nucleus and euploid chromosome number. These results demonstrate that although genotypic variations are present in embryogenic tissue and cell suspension cultures, plants regenerated via somatic embryogenesis from such cultures are euploid (2n=4x=48). The findings presented are in accordance with the observation that euploid cells are positively selected to undergo somatic embryogenesis and regeneration as reported in other cases (Karp and Bright, 1985; Shauker and Mohan Ram, 1993; Mythili et al., 1995).
The results are also in agreement with reports by Vasil and Vasil (1981) and Swedlund and Vasil (1985). These authors obtained plants of Pennisetum americanum regenerated from cell suspension cultures, which were phenotypically and cytologically similar to the donor plants. Genetic stability of plants regenerated from cell suspension cultures of medow fescue (Festuca pratensis) and protoplasts isolated therefrom, have also been reported (Vallés et al., 1993).
On the other hand, Xena et al. (2000) did a cytogenetic analysis of plants regenerated from embryogenic callus of S. tuberosum. cv Dèsirée growing in solid media. They found several mixoploid cells in different tissues of their plant population, which exhibited an anomalous development. The present results suggest that only euploid cells produce regenerated plantlets through somatic embryogenesis.
The usefulness of RAPDs as a means of molecular analysis of in vitro regenerated plants has been well documented (Nayak et al., 2003; Modgil et al., 2005; Latoo et al., 2006). In this work, the genetic stability of the regenerated plants was also screened by RAPD markers that could detect chromosomic mutations related to DNA sequence modifications. Seventeen primers were tested to analyze 19 regenerated plants as well as the parental. Genomic DNA amplification produced 2470 bands, all of them highly reproducible, with a molecular weight between 400 to 2600bp. Most of the primers tested revealed a monomorphic band pattern, except for primers OPA-2 and OPA-13, which produced 6 polymorphic bands representing only 0.24% of the total (2470). This molecular approach demonstrates the lack of genetic variation of the plantlets, in concordance with the cytogenetic analysis. The small variations detected by RAPD markers could be explained by minor genetic rearrangements that take place during tissue culture of calli or cell suspension steps and do not influence the phenotype of the regenerated plants (Qin et al., 2007).
The results obtained, employing cytogenetic analysis and RAPD markers, demonstrate that plants of Solanum tuberosum cv. Désirée regenerated from embryogenic cell suspension cultures do not exhibit the genotypic variations that are characteristic of the cell suspension cultures which gave rise to these plants. The results also indicate that this potato population is highly homogeneous and genetically stable. It is concluded that the potato high frequency embryogenic system is a suitable method for large scale potato plant regeneration.
ACKNOWLEDGEMENTS
This research was supported by the Scientific and Humanistic Council of the Universidad Central de Venezuela, Caracas, Venezuela.
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