The Effect of Polyamines and Silver Thiosulphate on Micropropagation of Date Palm Followed by Genetic Stability Assessment

There are some limitations in the practical applications of in vitro date palm tissue culture, such as low multiplication efficiency, low rooting rate, and high mortality experienced by in vitro raised plantlets during laboratory to soil transfer. This study’s objective was to investigate the effect of the two types of polyamines (putrescine "PUT" and spermidine" SPD") in combination with silver thiosulfate (STS) on the growth and development and genetic stability of cultures of Quntar cultivar. Media supplemented with 75 mg L−1 SPD in combination with 10 mgL−1 STS gave the highest percentage of callus producing buds (83.34%) and average bud formation (16.3) per jar. The addition of PUT and STS to the medium was most effective in root formation and the number of roots per shoot, where the best result 91.67% and 6.37 roots per shoot, respectively, were obtained using 75 mgL−1 PUT and 10 mgL−1 STS, resulting in fast-growing plantlets during acclimatization phase, reaching 90% of plant survival. The genetic fidelity assessment of plants derived from micropropagation was confirmed by RAPD analysis. Four operon primers were used, and all of them showed amplified unambiguous (OPA02, OPC-04, OPD-07, and OPE-15). All generated bands were monomorphic and had no variation among the tissue culture-derived plants tested. Accordingly, these results indicate that adding polyamines and silver thiosulfate to the nutrient medium of date palm cv. Quntar is beneficial in improving shoot organogenesis, rooting, and production of genetically stable date palm plants.

Characterization of mesenchymal stem cells isolated from Wharton’s jelly of the human umbilical cord

Background Isolation of post-partum umbilical cord Wharton’s jelly stem cells has gained attention as an alternative source of the bone marrow. Because easy isolation, lack of ethical concerns, and the presence of both embryonic and adult stem cells have made them a valuable source for use in therapeutic applications and regenerative medicine. The study utilized a modified protocol using in-house human pooled cord blood serum for isolation and expansion of the mesenchymal stem cells obtained from the human umbilical cord Wharton’s jelly. Cell proliferation and population doubling time and tri-lineage differentiation were assessed, and the expressions of mesenchymal cell surface markers CD44, CD90, CD105, and CD34 were assessed by flow cytometry and RT-PCR. The genetic stability of the isolated cells was assessed by chromosomal karyotype. Results The isolated cells displayed fibroblastic-like morphology and tri-lineage differentiation into adipocyte, chondrocyte, and osteocyte. The isolated cells maintained the proliferative competence with a doubling time ranged from 38 to 42h and corresponded well with the standard positive and negative molecular markers (CD44+, CD90+, CD 105+, and CD34−). Cell senescence occurred at the later passage of the cells (P15) affecting, about 25% of the population. Metaphases spread of the cells showed normal diploid karyotypes, with typical chromosomal plates indicating genetic stability of the isolated cells. Conclusion The primary cultures exhibited success in isolating the umbilical cord Wharton’s jelly mesenchymal stem cells, which maintained their tri-lineage differentiation potential, phenotypes and karyotype characteristics on further passage and expansion.

MFUM-BrTNBC-1, a Newly Established Patient-Derived Triple-Negative Breast Cancer Cell Line: Molecular Characterisation, Genetic Stability, and Comprehensive Comparison with Commercial Breast Cancer Cell Lines

Triple-negative breast cancer (TNBC) is a breast cancer (BC) subtype that accounts for approximately 15–20% of all BC cases. Cancer cell lines (CLs) provide an efficient way to model the disease. We have recently isolated a patient-derived triple-negative BC CL MFUM-BrTNBC-1 and performed a detailed morphological and molecular characterisation and a comprehensive comparison with three commercial BC CLs (MCF-7, MDA-MB-231, MDA-MB-453). Light and fluorescence microscopy were used for morphological studies; immunocytochemical staining for hormone receptor, p53 and Ki67 status; RNA sequencing, qRT-PCR and STR analysis for molecular characterisation; and biomedical image analysis for comparative phenotypical analysis. The patient tissue-derived MFUM-BrTNBC-1 maintained the primary triple-negative receptor status. STR analysis showed a stable and unique STR profile up to the 6th passage. MFUM-BrTNBC-1 expressed EMT transition markers and displayed changes in several cancer-related pathways (MAPK, Wnt and PI3K signalling; nucleotide excision repair; and SWI/SNF chromatin remodelling). Morphologically, MFUM-BrTNBC-1 differed from the commercial TNBC CL MDA-MB-231. The advantages of MFUM-BrTNBC-1 are its isolation from a primary tumour, rather than a metastatic site; good growth characteristics; phenotype identical to primary tissue; complete records of origin; a unique identifier; complete, unique STR profile; quantifiable morphological properties; and genetic stability up to (at least) the 6th passage.

The Genetic Stability in the Plant

The plant breeder is interested in introducing genotypes with good performance under different environmental conditions. The response of these structures to changes in the environment and the consequent instability in their traits when grown in different environments is to determine the superior ones and to estimate the overlap between the genotypes and the environment from the important criteria, therefore, the performance of the genotype is tested in different locations and seasons to determine the stability of the varieties.

Reversion mutations in phosphoprotein P of a codon-pair-deoptimized human respiratory syncytial virus confer increased transcription, immunogenicity, and genetic stability without loss of attenuation

Recoding viral genomes by introducing numerous synonymous nucleotide substitutions that create suboptimal codon pairs provides new live-attenuated vaccine candidates. Because recoding typically involves a large number of nucleotide substitutions, the risk of de-attenuation is presumed to be low. However, this has not been thoroughly studied. We previously generated human respiratory syncytial virus (RSV) in which the NS1, NS2, N, P, M and SH ORFs were codon-pair deoptimized (CPD) by 695 synonymous nucleotide changes (Min A virus). Min A exhibited a global reduction in transcription and protein synthesis, was restricted for replication in vitro and in vivo, and exhibited moderate temperature sensitivity. Here, we show that under selective pressure by serial passage at progressively increasing temperatures, Min A regained replication fitness and lost its temperature sensitivity. Whole-genome deep sequencing identified numerous missense mutations in several genes, in particular ones accumulating between codons 25 and 34 of the phosphoprotein (P), a polymerase cofactor and chaperone. When re-introduced into Min A, these P mutations restored viral transcription to wt level, resulting in increased protein expression and RNA replication. Molecular dynamic simulations suggested that these P mutations increased the flexibility of the N-terminal domain of P, which might facilitate its interaction with the nucleoprotein N, and increase the functional efficiency of the RSV transcription/replication complex. Finally, we evaluated the effect of the P mutations on Min A replication and immunogenicity in hamsters. Mutation P[F28V] paradoxically reduced Min A replication but not its immunogenicity. The further addition of one missense mutation each in M and L generated a version of Min A with increased genetic stability. Thus, this study provides further insight into the adaptability of large-scale recoded RNA viruses under selective pressure and identified an improved CPD RSV vaccine candidate.

Genetic Stability of in vitro Propagated Grapevine (Vitis vinifera L.) cv. Al-Bayadi

Grapevine (Vitis vinifera L.) fruit crops are a significant source of antioxidants, fibre, and nutrients; all are vital for a healthy diet and play a key role in the economy of several advanced and developing countries. It is of great importance to generate true-to-type plant products using in vitro propagation system. Thus, somaclonal variations can multiply very rapidly which leads to loss of the main features of parent rootstocks. In this research, a mixture of three Polymerase Chain Reaction (PCR)-based molecular marker methods – (conserved DNA derived polymorphism) CDDP, (Inter-simple sequence repeat) ISSR, and DNA barcoding – have been used to verify micro propagated grapevine genetic stability. Both ISSR and CDDP primer combinations produced scorable PCR fragments. The total number of bands was 98 and 109, with an average of 9.8 and 10.9 bands/primer in ISSR and CDDP assays, respectively. On the other hand, about 20 polymorphic bands were collected by CDDP primers, of CDDP-3 and CDDP-11produced 1 and 5 bands, with a polymorphism percentage of 11% and 33%, respectively. About 5 different unique PCR bands were detected in the mother plant (control) and were not observed in micro propagated plantlets (MP) samples of grapevine plant or vice versa. The phylogenetic trees were constructed using ISSR and CDDP assays diverged the control from MP samples at 1.3% and 7%, respectively. The phylogenetic tree constructed using (chloroplast gene RNA polymerase1) rpoC1 gene, multiple sequence alignment revealed that rpoC1 gene sequencing detected small genetic differences between control and MP samples of the grapevine and clustered grapevine control and MP samples to a single cluster with other Vitis species. This experiment reveals the potentiality of using CDDP, ISSR, and DNA barcoding in detecting the somaclonal variation of grapevine varieties subjected to tissue culture as a tool for plant conservation and breeding programs.

Iterative mutagenesis induced by atmospheric and room temperature plasma treatment under multiple selection pressures for the improvement of coenzyme Q10 production by Rhodobacter sphaeroides

CoQ10, which has been widely applied in medicine by dietary supplement, possesses important functions in antioxidant process and bioenergy generation. Iterative mutagenesis introduced by atmospheric and room temperature plasma (ARTP) treatment was studied to improve the coenzyme Q10 (CoQ10) production of Rhodobacter sphaeroides (R. sphaeroides), and multiple selection pressures including vitamin K3 (VK3), Na2S and benzoic acid (BA) were adopted for the first time. After two rounds of mutation and screening, a mutant strain R.S 17 was obtained, and the product titer was increased by 80.37%. The CoQ10 titer and cell density reached 236.7 mg L−1 and 57.09 g L−1, respectively, in the fed-batch fermentation, and the CoQ10 content was 22.1% higher than that of the parent strain. In addition, the spectral scanning results indicated the metabolic flux improvement contributing to the CoQ10 production in R.S 17, and the genetic stability was validated. Based on the iterative mutagenesis introduced by ARTP under multiple selection pressures, the promotion of CoQ10 production by R. sphaeroides was achieved. The significant improvement in fermentation performances and the good genetic stability of R.S 17 indicate a potential way for the efficient biosynthesis of CoQ10.

Genetic identity of mint cultivars after in vitro conservation, assessed by ISSR primers

The species of the genus Mentha have been known since ancient times and have significant value in the pharmaceutical, cosmetic and food industries, as well as in medicine. For the widespread use of mint, including in a variety of breeding programs, and the preservation of genetic diversity, effective methods of maintaining cultivars and collection samples are required. Thanks to the development of biotechnological methods, in particular, the creation of slow-growing collections, are now actively used as an effective alternative to field collections. It is known that the cultivation of tissues and organs on artificial culture media can cause somaclonal variability. The purpose of this work is to study the effect of in vitro storage at 4-6°C without illumination after 3 and 4 in vitro conservation cycles on the genetic stability of three cultivars of mint Azhurnaya, Bergamotnaya and Zagrava using ISSR primers. 1 cycle: 1 year of in vitro conservation, microcutting and 2 subcultures of regrowth in a culture room. After conservation, the number of viable explants was 70.0-82.1%. Callus formation at the base of the shoots was not observed in any of the cultivars. After 3 and 4 cycles of in vitro conservation, genetic stability was assessed using 11 ISSR primers. It was found that all three mint genotypes showed full compliance (length and number of amplicons) with the profiles of control samples for all studied markers. It was also found that the markers used by ISSR are highly informative for mint cultivars.

Different Growth and Sporulation Responses to Temperature Gradient among Obligate Apomictic Strains of Ulva prolifera

The green macroalga Ulva prolifera has a number of variants, some of which are asexual (independent from sexual variants). Although it has been harvested for food, the yield is decreasing. To meet market demand, developing elite cultivars is required. The present study investigated the genetic stability of asexual variants, genotype (hsp90 gene sequences) and phenotype variations across a temperature gradient (10–30 °C) in an apomictic population. Asexual variants were collected from six localities in Japan and were isolated as an unialgal strain. The hsp90 gene sequences of six strains were different and each strain included multiple distinct alleles, suggesting that the strains were diploid and heterozygous. The responses of growth and sporulation versus temperature differed among strains. Differences in thermosensitivity among strains could be interpreted as the result of evolution and processes of adaptation to site-specific environmental conditions. Although carbon content did not differ among strains and cultivation temperatures, nitrogen content tended to increase at higher temperatures and there were differences among strains. A wide variety of asexual variants stably reproducing clonally would be advantageous in selecting elite cultivars for long-term cultivation. Using asexual variants as available resources for elite cultivars provides potential support for increasing the productivity of U. prolifera.