Evaluation Of Genetic Stability Research Articles

RAPD and ISSR based evaluation of genetic stability of micropropagated plantlets of Morus alba L. variety S-1

Plant regeneration through rapid in vitro clonal propagation of nodal explants of Morus alba L. variety S-1 was established along with genetic stability analysis of regenerates. Axillary shoot bud proliferation was achieved on Murashige and Skoog (MS) medium in various culture regimes. Highest number of shoots (5.62±0.01), with average length 4.19±0.01cm, was initially achieved with medium containing 0.5mg/lN6-benzyladenine (BA) and 3% sucrose. Repeated subculturing of newly formed nodal parts after each harvest up to sixth passage, yielded highest number of shoots (about 32.27) per explants was obtained after fourth passage. Rooting of shoots occurred on 1/2 MS medium supplemented with 1.0mg/1 Indole-3-butyric acid (IBA). About 90% (89.16) of the plantlets transferred to the mixture of sand:soil:organic manure (2:2:1) in small plastic pots acclimatized successfully. Genetic stability of the discussed protocol was confirmed by two DNA-based fingerprinting techniques i.e. RAPD (random amplified polymorphic DNA) and ISSR (inter-simple sequence repeat). This protocol can be used for commercial propagation and for future genetic improvement studies.

In vitro plant regeneration from cotyledonary explants of Cucumis melo L. var. cantalupensis and genetic stability evaluation using RAPD analysis

An efficient plant regeneration protocol useful in genetic studies for important breeding lines of cantaloupe melons was developed. Three cultivars (Charentais-T, Vedrantais and Isabelle) and three doubled-haploid melon lines (NAD, DH-L2 and DH-L6) were tested for their potential to regenerate under in vitro conditions. Cotyledon explants from 6-day-old seedlings were cultured on three Murashige and Skoog based medium supplemented with different combinations of 6-benzylaminopurine (BAP) and indoleacetic acid (IAA). After 5 weeks of culture all the genotypes were able to regenerate on all media, however the regeneration frequency varied widely among the cultivars and the medium combinations used. The Charentais-T cultivar developed the highest number of shoots/explant on media containing BAP 1.0-IAA 1.0 × 10−2 mg/L (4.1) and BAP 1.2-IAA 1.2 × 10−2 mg/L (4.4) while on medium supplemented with BAP 1.3-IAA 1.3 × 10−2 mg/L the Vedrantais genotype gave the best result (2.4). The work first highlighted a good morphogenetic response of the DH lines; among all the genotypes and combination used, NAD (65.6 %) and DH-L6 (85.4 and 50.0 %) had the highest yield of regenerated plants. The results confirmed that the regeneration competence was strongly affected by genotype. Plantlets with well-developed shoot and root systems were successfully acclimatized and exhibited normal morphology and growth characteristics. In order to confirm genetic stability, a PCR-based RAPD analysis was carried out using 20 decamer-primers. No polymorphic bands were observed among all the plants revealing the genetic homogeneity of the regenerants. Furthermore, the ploidy level of regenerated plants was confirmed by flow cytometry.

Shoot regeneration from leaf explants of Withania coagulans (Stocks) Dunal and genetic stability evaluation of regenerates with RAPD and ISSR markers

Withania coagulans (Stocks) Dunal (Solanaceae) is a critically endangered medicinal plant known for its multiple medicinal properties. We report here an efficient and high frequency plantlet regeneration system through direct organogenesis in W. coagulans using in vitro-derived leaves as explants. On Murashige and Skoog's (MS) medium containing 4.44μM 6-benzylaminopurine (BAP) 73.7±4.3% explants responded and produced 11.4±0.9 shoot buds per explant. The regenerated shoot buds were elongated (6.7±0.22cm) on MS medium with 1.11μM BAP and 0.57μM indole-3-acetic acid (IAA). The elongated shoots were rooted both in vitro and ex vitro. The regenerates were acclimatized by slow and gradual exposure to different regimes of temperature and relative humidity. The leaf-regenerates were assessed for genetic stability using RAPD and ISSR molecular markers and confirmed true-to-type. The in vitro regeneration system developed would be useful for genetic restoration program as the overexploitation and reproductive failure forced W. coagulans towards the verge of complete extinction. The regeneration method would also be useful for genetic transformation for genetic improvement and improved phyto-pharming.

Evaluation of Genetic Stability in Somatic Embryo Derived Plantlets of Six Date Palm (Phoenix dactylifera L.) Cultivars through RAPD Based Molecular Marker

There are rules, manner, behaviours or arrangements in Islam in respects to production. Some muslims and Islamic economic agents have been practicing the rules and the regulations. Lack of primary needs production as food has threatened the stability of the life in this world, especially in the underdeveloped and the developing countries in which most muslims live. Shortages of foods may cause unstable life. Muslim and Islamic economist and leaders have to take economic (production) problems into serious consideration if they don’t want to be the victim of world non-muslim economic domination. Muslim must not be consumer or just be a sale agent of the products of others, but must select and develop appropriate technology suitable with their human and natural potentials. Agricultural Industry is suitable for Indonesia and for some other muslim contries.. Our Prophet encouraged us to generate the production by cultivating the idle land (ihya al-mawat) to yield crops for foods.

An Efficient In Vitro Process for Cyclic Clonal Production of Shoots from Adult Tree of Cassia alata L. and Evaluation of Genetic Stability Using DNA-Based Markers

An efficient, cyclic, two-step protocol for clonal in vitro regeneration system of an antiallergenic plant, Cassia alata, has been successfully developed. Nodal explants from a 5-year-old tree were cultured on Murashige and Skoog (MS) medium supplemented with various concentrations (1.0, 2.5, 5.0, 7.5, and 10.0 μM) of thidiazuron (TDZ). TDZ (5.0 μM) was found to be optimal for the formation of maximum shoot induction. Shoot proliferation and elongation increased when the regenerated shoots were subcultured on hormone-free MS medium after 4 weeks of exposure to TDZ. Nodal explants from in vitro regenerated microshoots to developed shoots, thus making the process recurrent. In 6 months duration, owing to the recurring nature of the protocol, large number of shoots could be produced from a single nodal explant from an adult tree. Shoots rooted best on MS supplemented medium with 0.5 μM IBA. Regenerated plantlets were acclimatized and successfully transplanted to the garden soil, where they grew well without any morphological and genetic variations. To confirm the uniformity, the genetic fidelity of in vitro raised C. alata clones was also assessed by using random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers. The present regeneration process not only favored the clonal multiplication but also expressed the regeneration capability of in vitro regenerated microshoots and can be subjugated for catering enough raw materials to various pharma industries by continuous cyclic shoot production.

Thidiazuron induced in vitro multiplication of Mentha arvensis and evaluation of genetic stability by flow cytometry and molecular markers

An efficient and reproducible system for the development of genetically uniform clones of a highly valuable essential oil producing crop Mentha arvensis was developed by pre-conditioning of nodal explants with thidiazuron (TDZ) supplemented liquid Murashige and Skoog (MS) media. Multiple shoot formation was achieved by pretreating of nodal explants with 5, 15, 25 or 50μM TDZ for 24h, followed by their transfer on TDZ free semi-solid MS media. The maximum response in terms of percent regeneration (92.6±3.65), average number of shoots per explants (23.7±1.45), and greatest shoot length (5.2±0.72) were obtained from nodal explants treated with 15μM TDZ for 24h followed by culture of explants on TDZ free MS medium for 8 weeks. Elongated shoots when transferred to half-strength MS medium supplemented with 0.5μM indole-3 butyric acid (IBA) resulted in 91±2.91% rooting. In vitro regenerated plantlets with well-developed shoot and roots were successfully established in pots containing garden soil with a 90% survival rate. Genetic stability of micropropagated plantlets was assessed and compared with mother plant using flow cytometry and molecular markers. No changes in plant genome size/ploidy level, and in directed amplification of minisatellite-region (DAMD) and inter simple sequence repeat (ISSR) profiles were found among the micropropagated plants, which were similar to that of the donor plant, illustrating their genetic uniformity and clonal fidelity. This confirms that clonal propagation of this plant using axillary shoot buds can be used for commercial exploitation of the selected genotype where a high degree of fidelity is an essential prerequisite.

Seedling Development and Evaluation of Genetic Stability of Cryopreserved Dendrobium Hybrid Mature Seeds

Vitrification, a simple, fast, and recommended cryopreservation method for orchid germplasm conservation, was evaluated for Dendrobium hybrid "Dong Yai" mature seeds. The genetic stability of regenerated seedlings was also evaluated using flow cytometry. Mature seeds from this hybrid were submitted to plant vitrification solution (PVS2) for 0, 0.5, 1, 2, 3, 4, 5, or 6 h at 0 °C. Subsequently, they were plunged into liquid nitrogen (LN) at -196 °C for 1 h and recovered in half-strength Murashige and Skoog culture medium (1/2 MS), and seed germination was evaluated after 30 days. Seeds directly submitted to LN did not germinate after cryopreservation. Seeds treated with PVS2 between 1 and 3 h presented the best germination (between 51 and 58%), although longer exposure to PVS2 returned moderated germination (39%). Germinated seeds were further subcultured in P-723 culture medium and developed whole seedlings in vitro after 180 days, with no abnormal characteristics, diseases, or nutritional deficiencies. Seedlings were successfully acclimatized under greenhouse conditions with over 80% survival. Flow cytometry analysis revealed no chromosomal changes on vitrified seedlings, as well as seedlings germinated from the control treatment (direct exposure to LN). These findings indicate that vitrification is a feasible and safe germplasm cryopreservation method for commercial Dendrobium orchid hybrid conservation.

Deep sequencing approach for genetic stability evaluation of influenza A viruses

Assessment of genetic stability of viruses could be used to monitor manufacturing process of both live and inactivated viral vaccines. Until recently such studies were limited by the difficulty of detecting and quantifying mutations in heterogeneous viral populations. High-throughput sequencing technologies (deep sequencing) can generate massive amounts of genetic information and could be used to reveal and quantify mutations. Comparison of different approaches for deep sequencing of the complete influenza A genome was performed to determine the best way to detect and quantify mutants in attenuated influenza reassortant strain A/Brisbane/59/2007 (H1N1) and its passages in different cell substrates. Full-length amplicons of influenza A virus segments as well as multiple overlapping amplicons covering the entire viral genome were subjected to several ways of DNA library preparation followed by deep sequencing using Solexa (Illumina) and pyrosequencing (454 Life Science) technologies. Sequencing coverage (the number of times each nucleotide was determined) of mutational profiles generated after 454-pyrosequencing of individually synthesized overlapping amplicons were relatively low and insufficiently uniform. Amplification of the entire genome of influenza virus followed by its enzymatic fragmentation, library construction, and Illumina sequencing resulted in high and uniform sequencing coverage enabling sensitive quantitation of mutations. A new bioinformatic procedure was developed to improve the post-alignment quality control for deep-sequencing data analysis.

Multiple shoot proliferation, bulblet induction and evaluation of genetic stability in Asiatic hybrid lily (Lilium sp.)

An efficient multiple shoot cluster regeneration and micro-bulb induction protocol was developed for Asiatic hybrid lily (Lilium sp.). Highest multiple shoot cluster formation of 78.4 % was observed on MS medium supplemented with 0.25 mg l -1 BAP and 1.0 mg l -1 GA3. The growth regulators, i.e., abscisic acid, daminozide and chlorocholine chloride, significantly affected bulblet quality parameters such as average size, number, fresh mass, and scale count. A dose of 0.1 mg l -1 abscisic acid, 50 mg l -1 daminozide, and 10 mg l -1 chlorocholine chloride exhibited highest micro-bulb regeneration rate per explant of 6.0 ± 0.28, 5.6 ± 0.17, and 6.0 ± 0.34, respectively. A high sucrose concentration of 40 g l -1 also produced highest number of bulblet per explant (6.1 ± 0.05). Amongst the 10 arbitrary decamer primers used to analyze the samples, only 6 primers amplified clear reproducible bands. The 6 primers produced a total of 36 (an average of 6 bands per primer) scorable bands. Phenotypic observations and ran- domly amplified polymorphic DNA (RAPD) analysis established that the regenerated micro-bulbs did not develop any detectable genetic aberration. Thus, the results indicate that in vitro produced bulblets of Lilium spp. (Asiatic hybrid) were clonally identical to their donor plant and were genetically stable.

Evaluation of Brucella abortus S19 vaccines commercialized in Brazil: Immunogenicity, residual virulence and MLVA15 genotyping

Live attenuated Brucella abortus S19 is the most effective vaccine against brucellosis in cattle. The assessment of the immunological parameters is essential to guarantee the biological quality of live anti-bacteria vaccines. The evaluation of genetic stability of live bacterial vaccines is also important in quality control. The aims of the present study were to compare (i) the immunogenicity and residual virulence, and (ii) the genotypic profile (MLVA15) of the eight S19 vaccines commercialized in Brazil to the USDA S19 reference strain. Two batches of each of the eight S19 commercial vaccines used in Brazil (A–H) were tested. They were submitted to the potency and residual virulence in vivo tests recommended by OIE and typed by the multiple-locus variable-number tandem repeat (VNTR) analysis (MLVA) described for Brucella spp. Our results demonstrated that all S19 vaccines commercialized in Brazil would be approved by Brazilian and OIE recommendations for potency and residual virulence. Furthermore, the S19 vaccine is genetically very homogeneous, as all but two batches (from the same manufacturer) tested showed identical MLVA15 profile. The two batches with different profiles presented six repeat units in locus Bruce07, instead of the five found in all other strains, including the USDA S19 reference strain. Although presenting a slightly different profile, this vaccine was also protective, as demonstrated by the immunogenicity and residual virulence assays performed. Therefore, the commercial Brazilian S19 vaccines were in accordance to Brazilian and international standards for immunogenicity and residual virulence tests. Moreover, our results also show that MLVA could be a useful inclusion to the list of in vitro tests required by the official control authorities to be applied to the commercial S19 vaccines, as an efficient assay to guarantee the quality and stability of the vaccine strains.

High frequency direct organogenesis and evaluation of genetic stability for in vitro regenerated Pongamia pinnata, a valuable biodiesel plant

Abstract In the present study regeneration efficiency and genetic clonality are the two major aspects for in vitro propagation of candidate plus tree of Pongamia pinnata, a versatile biodiesel legume. Woody Plant Medium (WP) and Murashige and Skoog's medium (MS) supplemented with different concentrations and combinations of plant growth regulators were screened for high frequency regeneration using nodal segment culture and axenically grown seedlings of elite genotype of P. pinnata. Percentage response from field-grown mature nodal segments of P. pinnata were highly dependant on the season, with greater than 68% of culture developing adventitious shoots during spring. Woody Plant Medium supplemented with benzyladenine (5.0 mg L−1) and kinetin (0.5 mg L−1) gave the greatest response to initiation and multiplication. The multiplication rate of 11 shoots per explants with an average shoot length of 3.0 cm was observed. Multiplied shoots started to produce roots in the multiplication medium itself containing BA and NAA but subsequent establishment was poor. The rooting response was enhanced in half-strength MS media with indole-3-butyric acid (0.5 mg L−1). Rooted plants were hardened successfully in glass house with 70% survivability. RAPD and ISSR markers were employed to determine the genetic fidelity of in vitro raised plantlets.

Adventitious shoot regeneration of oriental lily (Lilium orientalis) and genetic stability evaluation based on ISSR marker variation

A regeneration system was developed for oriental lily (Lilium orientalis) based on both leaf and bulb scale. Adventitious shoots were regenerated from leaves of in vitro cultures on Murashige and Skoog medium containing thidiazuron (TDZ) or 6-benzylaminopurine (BA) and naphthaleneacetic acid (NAA). The highest percent regeneration from leaf explants was 74.2%, being observed on medium containing 10.8 μM TDZ and 0.54 μM NAA. The highest mean number of shoots generated was 4.4 and was obtained from bulb scale explants on medium containing 0.54 μM TDZ and 0.54 μM NAA. Adventitious shoots were successfully rooted at rates ranging from 79.2% to 100%. The rooted plantlets survived after acclimatization in the greenhouse. The effect of kanamycin concentration on adventitious shoot regeneration was also evaluated, a value of 100 mg l−1 being suggested as a lethal dose for lily transformation. Eighteen ISSR markers were employed to determine the genetic stability of the regenerated shoots in comparison to their mother plant. Eleven primers in total produced 70 clear and reproducible bands. Genetic similarity indicators among the clonal derivatives and the mother plant ranged from 0.92 to 1.0. All 15 micropropagated progenies and the mother plant could be grouped together in one major cluster with a similarity level of 92%. The somaclonal variation rate across the plantlets was estimated as 4.2%, indicating that direct shoot formation from explant regeneration is a safe method for multiplication of “true-to-type” plants.

ISSR and RAPD Based Evaluation of Genetic Stability of Encapsulated Micro Shoots of Glycyrrhiza glabra Following 6 Months of Storage

In vitro grown axillary micro shoots of Glycyrrhiza glabra were encapsulated in alginate beads. Following 6 months of normal storage at 25 ± 2°C the re growth of encapsulated G. glabra micro shoots, reached 98% within 30 days of incubation on MS medium supplemented with 0.1 mg/l IAA. Re growth was characterized by the development of both shoot and root from single encapsulated micro shoot. Healthy plants were established to glass house with 95% survival. The genetic fidelity of plants obtained after conversion of alginate beads was ascertained through 10 RAPD and 13 ISSR primers. Of the 10 RAPD primers tested, 6 of them produced 14 clear and reproducible amplicons with an average of 2.3 bands per primer out of which 28.57% were polymorphic generated by only two primers. Eight ISSR primers produced total 37 bands ranging between 300 and 3,500 bp length. Number of scorable bands for each primer varied from 3 to 8 with an average of 4.6 bands per primer. Cluster analysis from ISSR and RAPD showed that all the tested plants including the mother plant distributed in two major groups with similarity coefficient ranging from 0.91 to 0.96 for RAPD and 0.89 to 0.97 for ISSR.

A safety assessment of a fowlpox-vectored Mycoplasma gallisepticum vaccine in chickens

A recombinant fowlpox virus vaccine expressing key protective Mycoplasma gallisepticum antigens could facilitate in the prevention both of fowlpox virus and M. gallisepticum infections. Vectormune FP-MG vaccine, a recombinant fowlpox virus expressing both M. gallisepticum 40k and mgc genes, was assessed for its safety in 8-wk-old specific-pathogen-free White Leghorn chickens. The vaccine virus was serially passaged 5 times by wing-web inoculation. Based on the postinoculation clinical observation, gross pathological examination of air sacs and peritoneum, genetic stability evaluation, virus shedding and tissue distribution detection, horizontal transmission ability determination, and protection against fowlpox virus challenge, the Vectormune FP-MG vaccine possesses a high level of safety.

Comparison of hybridization methods and real-time PCR: their value in animal cell line characterization

For biotechnological protein production, the International Conference of Harmonization (ICH) recommends appropriate testing of expression constructs and characterization of producer cell lines in order to assure uniform specifications, improve safety, and confirm stable productivity. Commonly, hybridization analyses are used for the evaluation of genetic stability, gene and transcript copy numbers as well as the integrity of the coding sequence. However, improvements in polymerase chain reaction (PCR) techniques have accelerated the analysis of genetic parameters and have nowadays become the method of choice for the evaluation of gene copy numbers and transcript levels. Nevertheless, Southern and Northern blot analyses are still valuable tools that deliver additional information to PCR results during cell clone characterization studies. In this study, we discuss advantages and drawbacks of hybridization and PCR methods in regard to their applicability and efficiency during the development of recombinant mammalian cell lines. A comparative review of the literature as well as an overview of findings from our own group will be given.

Methodological Guidelines for Genetic Safety Testing of Cell Transplants

A combination of karyotyping and aneuploidy analysis by interphase fluorescent in situ hybridization is a sensitive method for evaluation of genetic stability of stem cell cultures. The methodology and specific features of preparing and analyzing the cytogenetic preparations are described as exemplified by human multipotent mesenchymal stromal cells.

Establishment of immortalized cell lines and genetic stability evaluation for a family with spinocerebellar ataxia type 2

Immortalized cell lines of spinocerebellar ataxia type 2 (SCA2) with Parkinson disease symptoms were established in order to provide experimental material for future study. The immortalized cell lines were constructed by using Epstein Barr virus and cyclosporine A. Microsatellite markers were detected to see whether there is any change between the cell lines and the original blood samples, and the genetic stability of the cell lines were evaluated. Twenty-five immortalized cell lines were established successfully from the family and the microsatellite markers were unchanged. The karyotypes of the immortal cell lines were normal and the cell lines were genetically stable.

Cryopreservation of Quercus suber somatic embryos by encapsulation-dehydration and evaluation of genetic stability

We describe an encapsulation and dehydration procedure for the cryopreservation of cork oak (Quercus suber L.) somatic embryos that resulted in at least 90% survival. Genetic stability of the regenerated material was assessed by flow cytometry (FCM), amplified fragment length polymorphisms (AFLP) and simple sequence repeats (SSR). Cryopreservation of embryogenic clusters involved encapsulation of each cluster in an alginate bead, followed by a 3-day culture in 0.7 M sucrose and subsequent desiccation to 25 or 35% water content (WC), followed by freezing in liquid nitrogen. Thawed, cryopreserved somatic embryos had high viability and exhibited long-term survival. No morphological differences were observed between somatic embryos desiccated to 25 and 35% WC. Analysis of DNA ploidy stability of control (i.e., encapsulated and dehydrated but not frozen) and cryopreserved material by flow cytometry showed no significant differences. Similarly, DNA-marker analyses (AFLPs and SSR) revealed no significant differences between control and cryopreserved samples at the DNA-sequence level. Nonetheless, because polymorphisms were found between control material and samples cryopreserved and desiccated to 25% WC, the 35% WC method is recommended for cryopreservation of this tissue type. Cryopreservation of Q. suber somatic embryos by this encapsulation-dehydration procedure has potential for use in long-term conservation programs.

Evaluation of genetic stability in cryopreserved Solanum tuberosum

Cryopreservation is a technology of high importance in the storage of plant germplasm for long periods; however, the practical application of this technology for the preservation of plant materials is useful only if it does not lead to the genetic changes in the plant of interest. In the present investigation, the genetic stability of potato (Solanum tuberosum L.) plantlets of the cultivars Agria and Marphona stored under cryopreservation and non-cryopreservation conditions was studied using amplified fragment length polymorphism (AFLP) technique. Also, flow cytometric studies were performed to detect if there were probably any changes in the level of polyploidy. Seven primer combinations were used in the AFLP studies. Agria plantlets kept under non-cryopreserved conditions were approximately of average 97% genetic similarity to those of the same cultivar stored under cryopreservation conditions. With the cultivar of Marphona, full (100%) homology was found between plantlets stored under cryopreservation and non-cryopreservation conditions. Comparative studies on the polyploidy levels of the plantlets of both cultivars conserved under two above-mentioned storage conditions indicated that cryopreservation technique did not cause any changes in polyploidy levels.

In vitro clonal propagation of Mucuna pruriens var. utilis and its evaluation of genetic stability through RAPD markers

The Mucuna pruriens var. utilis is an important legume cover crop. Almost all the parts of the plant are reported to contain L-3,4-dihydroxy phenylalanine (L-Dopa). Here we report a rapid and reliable method for high fidelity micro-propagation. Auxiliary bud explants from 14-day-old seedlings were cultured on Murashige and Skoog’s (MS) medium supplemented with different concentrations of cytokinins. During the first culture on 3.5
M 6-benzylamino purine (BAP) maximum of 6.70 ± 1.15 shoots with an average shoot-length of 1.07 ± 0.21 cm were produced. The number of shoots increased up to 16.33 ± 0.58 recording average length of 1.16 ± 0.29 cm, when the intact shoots were subjected to re-culturing on the same hormonal medium. The shoots exhibited adequate elongation of 4.00 cm on 2.89
M gibberellic acid (GA3). The elongated shoots produced a maximum of 16.67 ± 2.89 roots on half-strength MS liquid medium supplemented with 16.20 iM -naphthalene acetic acid (NAA). The plantlets were acclimatized by transferring them first to peat moss: compost (1:1) mixture followed by sand: soil (1:1) mixture, recording 95% survival. The genetic fidelity of the regenerated shoots was confirmed using randomly amplified polymorphic DNA (RAPD) analysis employing 15 operon primers. This system provides high fidelity micro-propagation system for efficient and rapid micro-propagation of this important green manure cover crop with medicinal properties.