Micropropagation and assessment of somaclonal variation in Galanthus transcaucasicus in vitro plantlets

In vitro culture of twin-scaling explants of Galanthus transcaucasicus with different concentrations of plant growth regulators (PGRs) including 0.5, 1, 2, 3, 4, 6, 8, and 10 mg L-1 naphthaleneacetic acid (NAA) and 0.5, 1, 2, 3, and 4 mg L-1 benzyladenine (BA) was studied. After 18 weeks, the number of regenerated bulblets and intensity of callus was measured. Subsequently, bulblets were transferred to a medium with 0.5, 1, 2, 3, and 4 mg L-1 NAA and 0.5, 1, 2, 3, and 4 mg L-1 BA and, after 15 weeks, the bulblets length and diameter were measured. The highest intensity of callus was obtained on 4 mg L-1 NAA or 8 mg L-1 NAA with 1 mg L-1 BA. The highest number of regenerated bulblets was detected with 6 mg L-1 NAA and 2 mg L-1 BA. The highest diameter of bulblets occurred on four mgL-1 NAA (9.4 mm), while the lowest was observed on 0.5 mg L-1 BA (1.83 mm). The analysis of genetic variation using ISSR revealed that there was no somaclonal variation among the regenerated plants from BA and low level of NAA, but there was a significant somaclonal variation at high concentrations of NAA.

Protocol development for somatic embryogenesis, SSR markers and genetic modification of Stipagrostis pennata (Trin.) De Winter

Background Stipagrostis pennata (Trin.) De Winter is an important species for fixing sand in shifting and semi-fixed sandy lands, for grazing, and potentially as a source of lignocellulose fibres for pulp and paper industry. The seeds have low viability, which limits uses for revegetation. Somatic embryogenesis offers an alternative method for obtaining large numbers of plants from limited seed sources. Results A protocol for plant regeneration from somatic embryos of S. pennata was developed. Somatic embryogenesis was induced on Murashige & Skoog (MS) medium supplemented with 3 mg·L–1 2,4-D subsequently shoots were induced on MS medium and supplemented with 5 mg·L–1 zeatin riboside. The highest shoots induction was obtained when embryogenic callus derived from mature embryos (96%) in combination with MS filter-sterilized medium was used from Khuzestan location. The genetic stability of regenerated plants was analysed using ten simple sequence repeats (SSR) markers from S. pennata which showed no somaclonal variation in regenerated plants from somatic embryos of S. pennata. The regenerated plants of S. pennata showed genetic stability without any somaclonal variation for the four pairs of primers that gave the expected amplicon sizes. This data seems very reliable as three of the PCR products belonged to the coding region of the genome. Furthermore, stable expression of GUS was obtained after Agrobacterium-mediated transformation using a super binary vector carried by a bacterial strain LBA4404. Conclusion To our knowledge, the current work is the first attempt to develop an in vitro protocol for somatic embryogenesis including the SSR marker analyses of regenerated plants, and Agrobacterium-mediated transformation of S. pennata that can be used for its large-scale production for commercial purposes.

Producing a superior genotype from agria potato cultivar using somaclonal variation

This study was performed to produce a superior genotype from Agria potato cultivar using somaclonal variation. Two leaf and meristem explants in combination with four doses of 2,4-D (0, 2, 3 and 4 mg / l) were used for callus induction in a factorial model based on a completely randomized design with 3 replications. Results showed that the meristem explant, along with 3 mg 2,4-D produced the most suitable calluses. In the mentioned regeneration media, the best calluses were regenerated and one of the regenerated genotypes, which was very different from the parent cultivar was selected. The regenerated genotype, was compared with the maternal genotype (Agria) and a control cultivar (Sante), in a field experiment based on a randomized complete block design with 3 replications. The results showed that in terms of most of the studied traits such as tuber weight per plant, stolon length, percentage of dry matter and percentage of starch, the new genotype was superior, compared to the parent cultivar and in terms of peel percentage and maturity date, the parental cultivar was superior. The results of the molecular comparison also showed that based on CBDP marker, both in terms of band number and band size, there were differences between the new genotype and the parental cultivar. In general, results showed that somaclonal variation can be an effective method to generate new genotypes with superior characteristics.

A novel micropropagation of Lycium ruthenicum and epigenetic fidelity assessment of three types of micropropagated plants in vitro and ex vitro

Lycium ruthenicum is an excellent eco-economic shrub. Numerous researches have been conducted for the function of its fruits but scarcely focused on the somaclonal variation and DNA methylation. An efficient micropropagation protocol from leaves and stems of L. ruthenicum was developed in this study, in which not only the leaf explants but also the stem explants of L. ruthenicum were dedifferentiated and produced adventitious buds/multiple shoots on one type of medium. Notably, the efficient indirect organogenesis of stem explants was independent of exogenous auxin, which is contrary to the common conclusion that induction and proliferation of calli is dependent on exogenous auxin. We proposed that sucrose supply might be the crucial regulator of stem callus induction and proliferation of L. ruthenicum. Furthermore, results of methylation-sensitive amplified polymorphism (MSAP) showed that DNA methylation somaclonal variation (MSV) of CNG decreased but that of CG increased after acclimatization. Three types of micropropagated plants (from leaf calli, stem calli and axillary buds) were epigenetically diverged more from each other after acclimatization and the ex vitro micropropagated plants should be selected to determine the fidelity. In summary, plants micropropagated from axillary buds and leaves of L. ruthenicum was more fidelity and might be suitable for preservation and propagation of elite germplasm. Also, leaf explants should be used in transformation. Meanwhile, plants from stem calli showed the highest MSV and might be used in somaclonal variation breeding. Moreover, one MSV hotspot was found based on biological replicates. The study not only provided foundations for molecular breeding, somaclonal variation breeding, preservation and propagation of elite germplasm, but also offered clues for further revealing novel mechanisms of both stem-explant dedifferentiation and MSV of L. ruthenicum.

Molecular insight into somaclonal variation phenomena from transcriptome profiling of cucumber (Cucumis sativus L.) lines

Somaclonal variation during in vitro culture is often an undesirable phenomenon but may also be a source of genetic variation useful for breeders. The molecular mechanisms underlying this phenomenon remain uncertain. In this study, we analyzed the fruit transcriptome of three independent cucumber lines using RNA-sequencing technology and performed an extensive in silico study to determine how somaclonal variation altered gene expression. Comparison of the transcriptome profiles with the wild-type cucumber ‘Borszczagowski B10’ revealed 418, 364, and 273 genes that were differentially regulated. We performed bioinformatic functional analysis, gene ontology classification, molecular network analysis, and explored differentially expressed genes associated with processes such as protein and nucleic acid binding, enzyme activity, signaling, transport, sugar and lipid metabolism. We determined that the differential gene expression may be caused by polymorphism in the genic region and may also be a result of interaction among molecular networks, which triggers specific pathways.

Whole-genome sequence analysis of mutations in rice plants regenerated from zygotes, mature embryos, and immature embryos

Somaclonal variation was studied by whole-genome sequencing in rice plants (Oryza sativa L., ‘Nipponbare’) regenerated from the zygotes, mature embryos, and immature embryos of a single mother plant. The mother plant and its seed-propagated progeny were also sequenced. A total of 338 variants of the mother plant sequence were detected in the progeny, and mean values ranged from 9.0 of the seed-propagated plants to 37.4 of regenerants from mature embryos. The ratio of single nucleotide variants among the variants was 74.3%, and the natural mutation rate calculated using the variants in the seed-propagated plants was 1.2 × 10−8. The percentage and the mutation rate were consistent with the values reported previously. Plants regenerated from mature embryos had significantly more variants than different progeny types. Therefore, using zygotes and immature embryos can reduce somaclonal variation during the genetic manipulation of rice.

Somaclonal variation in clonal crops: containing the bad, exploring the good.

Somaclonal variation describes random cellular changes in plants regenerated through tissue culture. It occurs in certain crops that undergo micropropagation and has been recorded in different explant sources, from leaves and shoots to meristems and embryos. In banana (Musa spp.), a clonal crop conserved in vitro, somaclonal variation has been observed after prolonged periods in tissue culture, resulting from an increase in subcultures performed on a given clone. According to scientific literature, variants, or off-types, often show characteristics such as abnormal growth and flower or fruit defects in frequencies ranging from 1% to 32%. This variation poses a problem for gene bank managers, whose mandate is to maintain the genetic integrity of their collections for research and breeding. In the case of the Bioversity International Musa Germplasm Transit Centre (ITC), stress during the in vitro process is minimized by various techniques and plants are regenerated after 10 years, making it a long and costly process. Identifying somaclonal variation at an early stage would be an ideal solution; however, this requires suitable molecular markers. Recent studies revealed that techniques such as direct DNA sequencing and single nucleotide polymorphisms (SNPs) are able to detect the underlying factors of somaclonal variation and are becoming more accessible. On the other hand, somaclonal variation can be beneficial as it allows the natural development of new varieties and supplies genetic stocks used for future genetic studies. Harnessing the diversity of somaclones is easier, faster and cheaper compared with other methods of crop improvement, although it is also less predictable. So far, variants of crops such as apple, strawberry, potato and banana have been successfully adopted into global markets. In this chapter, we will discuss how to minimize the adverse effects of somaclonal variation while maximizing its benefits for greater crop diversity, with a particular focus on banana.