Abstract Study question Does embryo compaction positively correlate with blastocyst development and further embryonic ploidy status? Summary answer Fully compacted embryos develop into good-quality blastocysts, have shorter developmental times, and are related to ploidy status. What is known already To succeed in assisted reproduction technique (ART), it is necessary to select embryos that have the highest potential. So, numerous studies make an effort to establish parameters for selecting embryos. A Time-lapse system (TLS) allows embryologists to understand dynamic embryo change through continuous monitoring. In development, embryos undergo dynamic functional changes during compaction, which play a crucial role in blastocyst formation. It is also known that embryonic genome activation can be seen with compaction. Incomplete compaction leads to blastocyst developmental failure. Nevertheless, the details about the compaction of human embryos have not been paid sufficient attention, so still rarely known. Study design, size, duration This was a retrospective cohort study including couples that underwent an IVF cycle at the CHA Fertility Center, Gangnam, between January 2019 to October 2022. A total of 371 reached the blastocyst from 113 patients cultured in the TLS were analyzed. Among these, 94 blastocysts were analyzed by preimplantation genetic testing for aneuploidy (PGT-A). Statistical analysis was performed by prism9 using a t-test and chi-square test. P values <0.05 were regarded as statistically significant. Participants/materials, setting, methods Embryos were classified into two categories by compaction pattern: fully compacted (Group1, N = 194) and partially compacted (Group2, N = 177). Blastocyst quality was determined by morphology and divided into three groups (Good, Average, and Poor). The developmental time ranging from morula to blastocyst was annotated based on the embryo scope image. The surface of the blastocyst was measured every hour starting at blastocyst formation (tB) by using the ellipse tool of the Embryo Viewer software. Main results and the role of chance Good and average quality blastocysts are significantly higher in Group 1 than in Group 2 (21.6% vs. 3.4%, p < 0.01; 47.9% vs. 26.6%, p < 0.01, respectively). In contrast, poor-quality blastocysts are lower in Group 1 than in Group 2 (30.4% vs. 70.1%, p < 0.01). The beginning and completion of compaction, and blastocyst formation times of embryos from Group 1 were significantly shorter than those of embryos that Group 2(78.6h vs. 82.4, p < 0.01; 87.0h vs. 92.2h, p < 0.01; 100.2h vs. 103.7, p < 0.01, respectively). Also, there is a significant difference in longitudinal surface area at 3, 6, and 12h from the time of tB between the two groups. Consequently, the average expansion rate in Group 1 was significantly faster than in Group 2 (653.6 μm2/hour vs. 499.2μm2/hour, p < 0.05). According to the result of PGT-A, Group 1 had statistically significantly higher euploid and lower aneuploidy rates compared to Group 2 (47.2% vs. 36.2%, p < 0.001; 52.8% vs. 63.8%, p < 0.001, respectively). However, in the PGT-A group, there was no significant difference in developmental time between the two groups, regardless of fully or partial compaction. Meanwhile, the average expansion rate in euploidy blastocyst was significantly faster than in aneuploidy blastocyst (747.8 μm2/hour vs. 564.3 μm2/hour, p < 0.05). Limitations, reasons for caution The main limitation is the single-center retrospective approach. Therefore future prospective research is needed to verify and extend our findings. Another one, in the PGT-A there was no difference in developmental time regardless of fully or partially compaction. The reason is that we selected blastocysts of sufficient quality for biopsy. Wider implications of the findings Using the TLS, we have observed the dynamics of compaction in detail. We found a positive relationship between compaction and blastocyst quality and its association with embryo ploidy. The assessment of compaction is a significant parameter for predicting competent embryos and should be given priority when selecting blastocysts. Trial registration number Not applicable
Abstract Intergeneric crosses were made between Brassica oleracea and Moricandia arvensis utilizing embryo rescue. Six F 1 hybrid plants were generated in the cross‐combination of B. oleracea × M. arvensis from 64 pods by the placenta‐embryo culture technique, whereas three plants were produced in the reciprocal cross from 40 pods by the ovary culture technique. The hybrid plants were ascertained to be amphihaploid with 2n = 23 chromosomes in mitosis and a meiotic chromosome association of (0–3)II + (17–23)I at metaphase I (M I). In the backcross with B. oleracea , some of these hybrids developed sesquidiploid BC 1 plants with 2n = 32 chromosomes that predominantly exhibited a meiotic configuration of (9II + 14I) in pollen mother cells. The following backcross of BC 2 plants to B. oleracea generated 48 BC 3 progeny with somatic chromosomes from 2n = 19 to 2n = 41. The 2n = 19 plants showed a chromosomal association type of (9II + 1I) and a chromosomal distribution type of (9 1 / 2 + 9 1 / 2 ) or (9 + 10) at M I and M II, respectively. These facts might suggest that they were monosomic addition lines (MALs) of B. oleracea carrying a single chromosome of M. arvensis that could offer potential for future genetic and breeding research, together with other novel hybrid progeny developed in this intergeneric hybridization.
Abstract Eight plants of the putative double monosomic addition line (DMAL, 2 n = 20) were developed by crossing a monosomic chromosome addition line of radish [f(A)‐type monosomic addition line (MAL) (2 n = 19)] carrying the f chromosome of Brassica rapa (2 n = 20, AA) with another [e(C)‐type MAL (2 n = 19)] having the echromosome of Brassica oleracea (2 n = 18, CC). The homoeological relationships between the two alien chromosomes were investigated by morphological, cytogenetic and random amplified polymorphic DNA (RAPD) analysis. Seventeen morphological traits that were not present in the radish cv. ‘Shogoin’ were observed in both MALs and these traits were substantially exhibited in DMAL plants. At the first metaphase of pollen mother cells (PMCs), the two parental MALs showed a chromosome configuration of 9II +1I, demonstrating impossibility of recombination between the R and the added chromosomes. The DMALs formed 10II in approximately 73% of PMCs, with one bivalent showing loose pairing between two chromosomes differing in size. In an attempt to identify the two MALs by RAPD‐specific markers using 26 selected random primers, 13 and 20 bands were specific for the f(A)‐type and the e(C)‐type MALs, respectively; 12 bands were common to both MALs (26.7%). In conclusion, the f chromosome of B. rapa is homoeologous to the e chromosome of B. oleracea. The genetic domain (genes) for 17 morphological traits are linked to each homoeologous chromosome bearing 27% of the corresponding RAPD markers.
Intergeneric F 1 hybrids between Raphanus sativus (2n = 18, RR) and Moricandia arvensis (2n = 28, MaMa) have been produced through ovary culture followed by embryo culture, when M. arvensis was used as a pistillate parent. Six BC 1 plants were also obtained through ovary culture followed by embryo culture in the backcross of an amphidiploid F 1 , hybrid with R. sativus cv. ‘Pink ball’. Two BC 1 plants were ses‐quidiploids (2n = 32, MaRR), and the other BC 1 , plants were hyperploid with 2n = 55, having MaMaRRR genomes. BC 2 , seeds were obtained by conventional pollination in the successive backcross of two sesquidiploid BC 1 , plants with R. sativus cv. ‘Pink ball’. Their seed set percentages were 12.7% and 17.0%, respectively. These novel hybrid plants and derived progenies may be valuable materials for the genetic investigation and breeding of Brassiceae , including R. sativus.
Abstract Breeding of Raphanus sativus‐Brassica rapa monosomic chromosome addition lines (MALs, 2n = 19) was carried out by backcrossing the synthesized amphidiploid line, Raphanobrassica (R. sativus × B. rapa , 2n = 38, RRAA, line RA89) with R. sativus cv. ‘Shogoin’ (2n = 18, RR). In the first cross of Raphanobrassica × radish, four sesquidiploidal BC 1 plants (2n = 28, RRA, RA89‐36‐1, RA89‐31‐1, RA89‐31‐2, RA89‐31‐3) were successfully developed. In these plants, the chromosome configurations of 9II + 10I and 10II + 8I were observed frequently at first metaphase (MI) of meiosis in pollen mother cells (PMCs). The RA 89‐36‐1 plant produced many seeds in the reciprocal backcrosses with radish. About 50% of the BC 2 plants obtained from the cross of RA89‐36‐1 plant × radish were 2n = 19 plants, followed by 2n = 18 plants (24%) and 2n = 20 plants (19%). In the reciprocal cross, 2n = 19 plants were also developed at the rate of 40%. From analysis of specific morphological traits, 2n = 19 plants were classified into eight types (a‐h). When 25 selected primers were used in polyacrylamide gel electrophoresis, random amplified polymorphic DNA (RAPD) markers derived from B. rapa for each type of MAL were detected in numbers between three for e‐type and 16 for b‐type. RAPD markers specific for each type alone were from one (OPE 05‐344) for h‐type to nine for b‐type. In the g‐type, no marker specific to this type alone was observed. However, 19 bands were common between at least two types. These MAL plants exhibited predominantly the chromosome configuration of 9II + 1I at MI of PMCs, pollen and seed fertility being the same level as the radish cv. ‘Shogoin’. From the morphological traits and DNA markers, eight different MAL types among 10 expected were identified.
Abstract The genetic stability and maintenance of Raphanus sativus‐Brassica rapa monosomic chromosome addition lines (a‐h‐types MALs, 2n = 19, BC 2 ), developed by backcrossing the synthesized amphidiploid Raphanobrassica (Raphanus sativus × Brassica rapa , 2n = 38, RRAA) with R. sativus cv. ‘Shogoin’ (2n = 18, RR), was investigated. Transmission of the added alien chromosome through selected smaller seeds (SSS) and the inheritance of morphological traits and random amplified polymorphic DNA (RAPD)‐specific markers together with meiotic chromosome configuration and seed fertility were also investigated for three successive generations (BC 3 to BC 5 ). The distinctive traits and the RAPD‐specific markers of the eight types of MAL were substantially inherited and stably maintained throughout three generations, although a few variant plants (2n =18) resembling MALs (2n = 19) and hyperploidal plants (2n = 26 and 2n = 37) were generated in the earlier generations of BC 3 and BC 4 in comparison with BC 5 . The average transmission rates for three generations ranged from 26% for both the b‐type and the d‐type to 44% for the e‐type through SSS. On the other hand, the transmission rates through randomly selected seeds (RSS) were lower, ranging from 6.5% for the f‐type to 23.5% for the b‐type. In meiosis, more than 90% of PMCs showed the 9II +1I pairing configuration at metaphase I throughout three generations. For seed fertility, when backcrossed with the radish cv. ‘Shogoin’, the values were approximately 180% to 500% with the mode around 300% with the seed harvested from a pod increasing with the advancing generations. Genetic recombination between the radish chromosomes and the added chromosome is probably rare, suggesting that the added chromosome is mostly maintained unaltered in the background of the radish genome.
Abstract Intergeneric hybridization was performed between Brassica rapa and Diplotaxis tenuifolia following embryo rescue. Twenty‐three F 1 hybrid plants were developed from the cross B. rapa × D. tenuifolia and confirmed to be amphihaploids with 21 chromosomes in mitosis. Chromosome doubling of F 1 hybrids by colchicine treatment resulted into five amphidiploid plants which exhibited (20–21) II + (0–2) I at metaphase I (MI) of pollen mother cells. Sib‐crossing and/or open‐pollination among amphidiploid plants for more than four generations resulted in the development of an ADt‐06 line with reproductive systems capable of maintaining an amphidiploid line. The ADt‐06 line was intermediate in some morphological traits between two parental species, and was characterized by a slightly pungent taste as a physiological trait. Analyses for genomic DNA confirmed that this line was a hybrid between two species. This new amphidiploid ADt‐06 line could be a useful genetic resource for the breeding of new leafy salad vegetables.
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