Plant tissue culture has become one of the fundamental tools of plant science research. It is extensively employed in the production, conservation and improvement of plant resources. The presence of somaclonal variation in populations derived from tissue culture is affecting the use of tissue culture negatively and has remained a major problem. Conversely, it is a source of new desirable clones/variants with better agronomic traits. In this review, we summarize the possible causes, detection methods and desirability of variants. Somaclonal variation is one of the most researched and reviewed topics. Hence, we restricted ourselves to outlining various examples which may be used as important references for researchers who intend to identify and/or characterize somaclonal variants while using tissue culture for research and production. Emphasis is placed on the negative effects of somaclonal variation. However, this review also includes examples of some useful variants generated as a result of somaclonal variation.

Somaclonal variation in plants: causes and detection methods

Developing stress tolerant plants through in vitro selection—An overview of the recent progress

The advancements made in tissue culture techniques has made it possible to regenerate various horticultural species in vitro as micropropagation protocols for commercial scale multiplication are available for a wide range of crops. Clonal propagation and preservation of elite genotypes, selected for their superior characteristics, require high degree of genetic uniformity amongst the regenerated plants. However, plant tissue culture may generate genetic variability, i.e., somaclonal variations as a result of gene mutation or changes in epigenetic marks. The occurrence of subtle somaclonal variation is a drawback for both in vitro cloning as well as germplasm preservation. Therefore, it is of immense significance to assure the genetic uniformity of in vitro raised plants at an early stage. Several strategies have been followed to ascertain the genetic fidelity of the in vitro raised progenies comprising morpho-physiological, biochemical, cytological and DNA-based molecular markers approaches. Somaclonal variation can pose a serious problem in any micropropagation program, where it is highly desirable to produce true-to-type plant material. On the other hand, somaclonal variation has provided a new and alternative tool to the breeders for obtaining genetic variability relatively rapidly and without sophisticated technology in horticultural crops, which are either difficult to breed or have narrow genetic base. In the present paper, sources of variations induced during tissue culture cycle and strategies to ascertain and confirm genetic fidelity in a variety of in vitro raised plantlets and potential application of variants in horticultural crop improvement are reviewed.

/pdf/somaclonal-variations-and-their-applications-in-34x0z8l3la.pdf

Somaclonal variations and their applications in horticultural crops improvement

Epigenetic mechanisms are highly dynamic events that modulate gene expression. As more accurate and powerful tools for epigenetic analysis become available for application in a broader range of plant species, analysis of the epigenetic landscape of plant cell cultures may turn out to be crucial for understanding variant phenotypes. In vitro plant cell and tissue culture methodologies are important for many ongoing plant propagation and breeding programmes as well as for cutting-edge research in several plant model species. Although it has long been known that in vitro conditions induce variation at several levels, most studies using such conditions rely on the assumption that in vitro cultured plant cells/tissues mostly conform genotypically and phenotypically. However, when large-scale clonal propagation is the aim, there has been a concern in confirming true-to-typeness using molecular markers for evaluating stability. While in most reports genetic variation has been found to occur at relatively modest frequencies, variation in DNA methylation patterns seems to be much more frequent and in some cases it has been directly implicated in phenotypic variation. Recent advances in the field of epigenetics have uncovered highly dynamic mechanisms of chromatin remodelling occurring during cell dedifferentiation and differentiation processes on which in vitro adventitious plant regeneration systems are based. Here, an overview of recent findings related to developmental switches occurring during in vitro culture is presented. Additionally, an update on the detection of epigenetic variation in plant cell cultures will be provided and discussed in the light of recent progress in the plant epigenetics field.

/pdf/an-epigenetic-view-of-plant-cells-cultured-in-vitro-gfrjwecxtc.pdf

An epigenetic view of plant cells cultured in vitro: somaclonal variation and beyond

Chalcone synthase (CHS) catalyzes the first committed step in the flavonoid biosynthetic pathway. In this study, the cDNA (FhCHS1) encoding CHS from Freesia hybrida was successfully isolated and analyzed. Multiple sequence alignments showed that both the conserved CHS active site residues and CHS signature sequence were found in the deduced amino acid sequence of FhCHS1. Meanwhile, crystallographic analysis revealed that protein structure of FhCHS1 is highly similar to that of alfalfa CHS2, and the biochemical analysis results indicated that it has an enzymatic role in naringenin biosynthesis. Moreover, quantitative real-time PCR was performed to detect the transcript levels of FhCHS1 in flowers and different tissues, and patterns of FhCHS1 expression in flowers showed significant correlation to the accumulation patterns of anthocyanin during flower development. To further characterize the functionality of FhCHS1, its ectopic expression in Arabidopsis thaliana tt4 mutants and Petunia hybrida was performed. The results showed that overexpression of FhCHS1 in tt4 mutants fully restored the pigmentation phenotype of the seed coats, cotyledons and hypocotyls, while transgenic petunia expressing FhCHS1 showed flower color alteration from white to pink. In summary, these results suggest that FhCHS1 plays an essential role in the biosynthesis of flavonoid in Freesia hybrida and may be used to modify the components of flavonoids in other plants.

/pdf/molecular-and-biochemical-analysis-of-chalcone-synthase-from-io30ct141f.pdf

Molecular and Biochemical Analysis of Chalcone Synthase from Freesia hybrid in flavonoid biosynthetic pathway.

Freesia hybrida is an important worldwide cut flower, especially in America and Europe. For efficient regeneration of this flower from young inflorescence and rachillae in tetraploid, we developed a simple in vitro micropropagation protocol. Explants of Freesia hybrida can regenerate plantlets through somatic embryogenesis via two kinds of pathways, that is, directly from the epidermal cells or indirectly from an embryonic callus, depending on the exogenous plant growth regulators (PGRs) used in the culture media. In direct embryogenesis, when the explants were cultured on Murashige and Skoog (MS) medium supplemented with 11.43 μM indole acetic acid (IAA) and 4.44 μM 6-benzylaminopurine (6-BA), the induction rate was 84% for young inflorescence and 100% for rachillae. After the multishoots were subcultured on the rooting MS medium containing 1.08 μM α-naphthalene acetic acid (NAA), the rooting rate was close to 100%. In indirect embryogenesis, embryonic calluses were formed when the culture medium contained 22.22 μM 6-BA and 4.52 μM 2,4-dichlorophenoxy acetic acid (2,4-D), and the induction rate was 92.4% for young inflorescence and 100% for rachillae. After the embryonic calluses were transferred to the medium supplemented with 11.43 μM IAA and 13.33 μM 6-BA, they could develop into plantlets with roots. In assessing the two regeneration pathways in terms of genetic and epigenetic fidelity of the regenerants, two kinds of molecular markers [amplified fragment length polymorphism (AFLP) and methylation-sensitive amplified polymorphism (MSAP)] were employed. The AFLP analysis used 20 primer pairs that yielded 916 scorable bands among the donor plant and 11 regenerants from direct embryogenesis, of which 8 (0.87%) were polymorphic. The regenerants from indirect embryogenesis had 1075 clear bands of which 3 (0.27%) were polymorphic scorable bands from 18 primer pairs. Moreover, the variant band patterns included two types, that is, loss-of-original and gain-of-novel bands. MSAP analysis revealed that tissue culturing of the flower induced DNA cytosine methylation alterations in both CG and CNG levels and patterns compared with the donor plant. The variation rate was 1.1 and 1.3% for the direct and indirect embryogenesis pathways, respectively. The findings show that tissue culture of flowering plants is a form of stress which can induce some heritable epigenetic variations and should be considered in future long-term genotype preservation programs of Freesia hybrida.

In Vitro Micropropagation of Freesia hybrida and the Assessment of Genetic and Epigenetic Stability in Regenerated Plantlets

The enzyme that catalyzes the formation of the first stable anthocyanin in the biosynthesis of natural compounds is UDP-glucose: anthocyanidin 3-O-glucosyltransferase (UF3GT). A cDNA clone (Fh3GT1) encoding UF3GT was isolated from Freesia hybrida. Phylogenetic tree analysis indicated that Fh3GT1 was a novel member of glycosyltransferase, which was classified into monocot subgroups. Semi-quantitative RT-PCR analysis detected transcripts of Fh3GT1 in different organs of F. hybrida and in petals of Freesia cultivars of different colors, and the expression level reached the maximum at the fully opened stage of petals. Characterization of the enzymatic assays indicated that Fh3GT1 had a role in anthocyanin glycoside biosyntheses in vitro. To elucidate the function of Fh3GT1, RNA interference vector (pART-Fh3GT1i) was constructed, and introduced into Petunia grandiflora by Agrobacterium-mediated transformation. Integration of the Fh3GT1 in petunia genome was confirmed by PCR and Southern blotting. SqRT-PCR revealed that the endogenous Ph3GT1 mRNA expression levels decreased in transgenic lines compared with the wild-type. The content of total anthocyanin pigments also decreased with the reduction of mRNA transcript levels, and the transgenic petunia plants had significant changes on their flower colors. In summary, this work identified a UF3GT gene from Freesia hybrida and demonstrated a method to modify plant flower color by redirecting the anthocyanin biosynthesis.

cDNA cloning and characterization of UDP-glucose: anthocyanidin 3-O-glucosyltransferase in Freesia hybrida.

https://doi.org/10.1016/j.jare.2022.05.010

Cadmium accumulation regulated by a rice heavy-metal importer is harmful for host plant and leaf bacteria

Introduction: Wheat bran is the main by-product of wheat processing, containing about 30% pentosan and 0.4%–0.7% ferulic acid. Wheat bran is the main raw material used to prepare feruloyl oligosaccharides by hydrolysis of Xylanase, we discovered that the ability of Xylanase to hydrolyze wheat bran could be affected in the presence of different metal ions. Methods: In the present study, we have probed the effects of different metal ions on the hydrolysis activity of Xylanase on wheat bran and tried to analyze the effect of Mn2+ and Xylanase by molecular dynamic (MD) simulation. Results: Our results suggested that Mn2+ had improved the Xylanase hydrolyzing wheat bran to obtain feruloyl oligosaccharides. Particularly when the concentration of Mn2+ reached 4 mmol/L, the optimal product has been obtained 2.8 times higher to compare with no addition. Through the MD simulation analysis, our results reveal that Mn2+ can induce structural change in the active site, which enlarges the substrate binding pocket. The simulation results also revealed that the addition of Mn2+ resulted in a low RMSD value compared with the absence of Mn2+ and helped stabilize the complex. Conclusion: Mn2+ could increase the enzymatic activity of Xylanase in the hydrolysis of feruloyl oligosaccharides in wheat bran. The finding could have significant implications for the preparation of feruloyl oligosaccharides from wheat bran.

/pdf/analysis-of-the-effect-of-metal-ions-on-the-ability-of-kmo448x8.pdf

Analysis of the effect of metal ions on the ability of Xylanase to hydrolyze wheat bran by molecular dynamics simulations

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In Vitro Micropropagation of Freesia hybrida and the Assessment of Genetic and Epigenetic Stability in Regenerated Plantlets

cDNA cloning and characterization of UDP-glucose: anthocyanidin 3-O-glucosyltransferase in Freesia hybrida.

Cadmium accumulation regulated by a rice heavy-metal importer is harmful for host plant and leaf bacteria

Analysis of the effect of metal ions on the ability of Xylanase to hydrolyze wheat bran by molecular dynamics simulations