2,4-D induction of somaclonal variations in in vitro grown date palm (Phoenix dactylifera L. cv Barhee)

The present study is a part of a program designed at improving the date palm, Phoenix dactylifera L. cv. Barhee, through induced somaclonal variation. In this work, caulogenic cultures were subcultured on MS media supplemented with 0, 1, 5, 10, 20 and 40 mg L− 1 2,4-D in order to induce genetic and epigenetic variations. The highest doses of 2,4-D were found to induce severe negative effects on in vitro cultures, although some tissues were able to survive and to produce calli with high morphogenetic capacities. Our analysis showed some significant effect of 2,4-D on several physiological parameters. Indeed, chlorophyll and growth rates were found to drastically decrease while proline content increased from 535 nmol g− 1 to 2973 nmol g− 1 FW when 40 mg L− 1 2,4-D were used. In vitro cultures showed several signs of oxidative stress, such as high levels of hydrogen peroxide (H2O2) and malondialdehyde (MDA); likewise, the specific activity of several antioxidant enzyme was found to increase. Plant regeneration from in vitro cultures treated with 2,4-D was obtained after subculturing explants onto PGR-free media. The ISSR analysis of 2,4-D-treated material showed that this plant growth regulator (PGR) induced measurable genetic variations. The global DNA methylation rates (GMR) as estimated through the HPLC analysis of nucleosides also confirmed the presence of epigenetic changes caused by 2,4-D as GMRs increased from 13.8–18.93%.

In Vitro Production of Somaclones with Decreased Erucic Acid Content in Indian Mustard [Brassica juncea (Linn.) Czern&Coss]

Brassica junceais a crucial cultivated mustard species and principal oilseed crop of India and Madhya Pradesh, grown for diverse vegetables, condiments, and oilseeds. Somaclonal variation was explored as a probable source of additional variability for the manipulation of fatty acids, especially low erucic acid contents that may be valuable for this commercially important plant species. The plantlets regenerated from tissue cultures (R0), their R1 generation and respective parental lines were compared for morpho-physiological traits and fatty acid profile for the probable existence of somaclonal variations. The first putative somaclone derived from genotype CS54 contained 5.48% and 5.52% erucic acid in R0 and R1 regenerants, respectively, compared to the mother plant (41.36%). In comparison, the second somaclone acquired from PM30 exhibited a complete absence of erucic acid corresponding to its mother plant (1.07%). These putative somaclones present a source of variation for exploitation in the development of future mustard crops with low erucic acid content.

Identification and Validation of Genetic Variations in Transgenic Chinese Cabbage Plants (Brassica rapa ssp. pekinensis) by Next-Generation Sequencing

Transgenic plants are usually produced through tissue culture, which is an essential step in Agrobacterium-mediated plant transformation. However, genomic variations, termed somaclonal variations, have been detected in transgenic plants cultured in vitro. The occurrence of these variations should be as low as possible to secure the stability of transgenic crops. Determining the cause and mechanism of somaclonal variations in tissue culture-derived plants will help reduce the rate of variation and promote the stable expression of genes in transgenic plants. In order to determine the genetic variability in transgenic Chinese cabbage plants, we performed whole-genome resequencing and compared the sequencing data with the ‘CT001’ reference genome. The variation candidates that were expected to consistently occur in the transgenic lines were selected and validated. The single nucleotide polymorphism (SNP) and insertion and deletion (InDel) candidates were identified using the resequencing data and validated by reverse transcription (RT)-PCR analysis. The deduced amino acid sequences were used to determine whether the variations caused changes in the resulting polypeptide, and the annotations of the mutated genes were analyzed to predict the possible effects of the SNPs on gene function. In conclusion, we selected and validated the genetic variations identified in transgenic Chinese cabbage plants. Their genomes were expected to be affected by the process of Agrobacterium-mediated transformation. The findings of our study will provide a genetic basis for transgenic plant research.

DETEKSI VARIASI SOMAKLONAL PLANLET Jatropha curcas Linn. HASIL REGENERASI EMBRIO SOMATIK DENGAN MARKA MOLEKULAR ISSR

Physic nut (Jatropha curcas Linn.) has the potential as a source of sustainable biofuels. Somatic embryo proliferation of J. curcas may cause somaclonal variations. This research aimed to investigate somaclonal variations of J. curcas somatic embryo derived-plantlet using ISSR markers. Somatic embryos of J. curcas at the globular phase were cultured on liquid MS medium supplemented with 0, 0.5, 1.0, 1.5, and 2.0 mg L-1 of 2,4-D. Parameter observed were embryos weight, embryos volume, colour, and size of embryos. After proliferation, the embryos were cultured on a germination medium until the cotyledonary phase. The results showed that proliferation of J. curcas somatic embryos was optimal, with the highest weight and volume, at MS medium added with 1 mg L-1 2,4-D.

Molecular Diversity Analysis of Somaclonal Variants of Potato (Solanum tuberosum L.) by Random Amplified Polymorphic DNA Markers

Aims: An experiment was conducted to analyze the DNA fingerprinting and genetic diversity of nine potato (Solanum tuberosum L.) somaclonal variants and three check varieties. Place and Duration of Study: The experiment was carried out at the Biotechnology laboratory of the Department of Biotechnology, Faculty of Agriculture, Sher-e-Bangla Agricultural University, Dhaka-1207, Bangladesh during November, 2013 to December, 2014. Methodology: The somaclonal variants investigated were SIP-3, SIP-5, SVP-6, SVP-18, SVP-19, SVP-25, SVP-55, SVP-56 and SVP-68, while the check varieties were Cardinal, Diamant and Asterix. Six RAPD primers were used to perform PCR reaction after genomic DNA was extracted from young leaves. Results: The selected 6 primers produced total 54 distinct and differential amplified DNA bands of size range 88 bp to 3265 bp, where 47 bands (~87%) were polymorphic and 7 bands (~13%) were monomorphic. The pair-wise inter-genotype similarity indices were ranged from 61.59% to 93.55% with an average of 74.31%. The Nei’s genetic distance among 12 potato genotypes was estimated from 0.0972 to 0.6217 whereas, genetic identity was between 0.5370 and 0.9074. Here, the distantly linked accessions among the somaclonal variations with check varieties were SVP-6 (to Cardinal and Diamant) and SVP-25 (to Asterix). In addition, the UPGMA dendrogram segregated the 12 potato genotypes into two broad clusters containing 8 and 4 genotypes, respectively. Furthermore, the dendrogram also displayed the highest genetic distance between SVP-6 vs SVP-68 genotype pair. Conclusion: Significant relationship and diversity were found among the studied 12 genotypes. This genetic diversity among the potato genotypes would be utilized for further potato improvement.

Changes in the Metabolite Profile during Micropropagation of Normal and Somaclonal Variants of Banana Musa AAA cv. Williams

Micropropagation techniques allow the mass production of banana plants but can cause somaclonal variations such as dwarfism. Changes in the metabolite profile during micropropagation of normal (NP) and dwarf (DP) banana plants have not been described. Both, NPs and DPs of banana Musa AAA cv. Williams were micropropagated and the metabolite profile of vitroplants was assessed at the proliferation (PP), rooting (RP) and the second greenhouse-acclimatization (APII) phases of tissue culture. Metabolites from 10 DPs and 10 NPs meristems from each micropropagation phase were extracted and identified by gas chromatography coupled with mass spectrometry (GC-MS). Principal component analysis (PCA) and test of statistical significance were applied to detect differentially accumulated metabolites. The PCA showed a clear grouping of DPs separated from NPs in RP and APII. Among the differentially accumulated metabolites, various precursors of apoplast components including arabinose and galactose or deoxygalactose in both PP and RP, as well as mannose and fucose in APII were under-accumulated in DPs. Results suggest affected apoplast composition during micropropagation of DPs.

An Apical Meristem-Targeted in planta Transformation Method for the Development of Transgenics in Flax (Linum usitatissimum): Optimization and Validation

Efficient regeneration of explants devoid of intrinsic somaclonal variations is a cardinal step in plant tissue culture, thus, a vital component of transgenic technology. However, recalcitrance of economically important crops to tissue culture-based organogenesis ensues a setback in the use of transgenesis in the genetic engineering of crop plants. The present study developed an optimized, genotype-independent, nonconventional tissue culture-independent in planta strategy for the genetic transformation of flax/linseed. This apical meristem-targeted in planta transformation protocol will accelerate value addition in the dual purpose industrially important but recalcitrant fiber crop flax/linseed. The study delineated optimization of Agrobacterium tumefaciens-mediated transformation and stable T-DNA (pCambia2301:GUS:nptII) integration in flax. It established successful use of a stringent soilrite-based screening in the presence of 30 mg/L kanamycin for the identification of putative transformants. The amenability, authenticity, and reproducibility of soilrite-based kanamycin screening were further verified at the molecular level by GUS histochemical analysis of T0 seedlings, GUS and nptII gene-specific PCR, genomic Southern hybridization for stable integration of T-DNA, and expression analysis of transgenes by sqRT-PCR. This method resulted in a screening efficiency of 6.05% in the presence of kanamycin, indicating amenability of in planta flax transformation. The strategy can be a promising tool for the successful development of transgenics in flax.

Assessment of Somaclonal Variations in Embryo-derived Axillary Shoots of Chickpea using Molecular Markers

Background: Somaclonal variation is considered as a source of genetic variation for crop improvement. It has been investigated using cytological, biochemical and molecular techniques.Methods: Genetic stability in the embryo-derived axillary shoots of 4 chickpea genotypes was assessed using eight inter-simple sequence repeat (ISSR) and 19 random amplified polymorphic DNA (RAPD) primers. Result: RAPD primers produced 94 and ISSR primers produced 38 distinct and scorable alleles, with an average of 4.9 alleles for RAPD and 4.75 for ISSR primers. The polymorphic information content (PIC) ranged from 0.36 to 0.90 for RAPD and from 0.50 to 0.87 for ISSR. ISSR recognized a 90%, but RAPD recognized 82% similarity value. No absolute similarity value was between the mother plant and the regenerated shoots for the overall genotypes. At a 90% similarity value, 15 out of the 20 regenerated shoots from ‘Giza 88’ group with their mother plant using ISSR markers; however, 11 regenerated shoots grouped with their mother plant in one central cluster for ‘Giza 4’ using RAPD markers. The observed variations in the total number of polymorphic RAPD and ISSR bands and the number of bands specific to the mother and regenerated shoots, detected intra-clonal variation and genetic instability seem to be genotype-dependent.

True-to-type micropropagated plants of para rubber (Hevea brasiliensis Müll. Arg.) via somatic embryogenesis

Plant micropropagation via somatic embryogenesis is a powerful technique for rapid mass propagation, especially in para rubber (Hevea brasiliensis Müll. Arg.). However, somaclonal variations are the major limitation of this process. To date, DNA fingerprinting, i.e., RAPD (Randomly Amplified Polymorphic DNA), Star Codon Targeted (SCoT), and SSRs (Simple Sequence Repeats), is one of the most successful technologies to detect the genetic fidelity in the somatic embryos. The aim of present study was to induce somatic embryos from inner integument explants of para rubber cv. ‘RRIM 600’ at different developmental stages and subsequent acclimatization and transplantation (under greenhouse and field conditions) of the propagated seedlings. The genetic stability of the plants derived from somatic embryos was also analysed in comparison to the mother plant using RAPD, SCoT and SSRs markers. Somatic embryos derived from inner integuments of 5-week-old immature seeds after pollination were more efficient than older and younger seeds. In addition, para rubber mother plants cv. ‘RRIM600’ and plants derived from somatic embryogenesis demonstrated the same pattern of DNA fragments, as confirmed by three PCR-based techniques, RAPD, SCoT and SSRs, whereas these in the pattern were different from ‘RRIT 226’, ‘PB 235’, ‘PB 251’, ‘PB 255’ and ‘BMP 24’. Interestingly, T2 plant was found to possess somaclonal variations when compared with mother plant. Based on the results, we confirm that the plants derived from somatic embryogenesis of para rubber cv. ‘RRIM 600’ were true-to-type to that of ‘RRIM 600’ master stock.