Chromosome-level genome assembly of a regenerable maize inbred line A188

Background The maize inbred line A188 is an attractive model for elucidation of gene function and improvement due to its high embryogenic capacity and many contrasting traits to the first maize reference genome, B73, and other elite lines. The lack of a genome assembly of A188 limits its use as a model for functional studies. Results Here, we present a chromosome-level genome assembly of A188 using long reads and optical maps. Comparison of A188 with B73 using both whole-genome alignments and read depths from sequencing reads identify approximately 1.1 Gb of syntenic sequences as well as extensive structural variation, including a 1.8-Mb duplication containing the Gametophyte factor1 locus for unilateral cross-incompatibility, and six inversions of 0.7 Mb or greater. Increased copy number of carotenoid cleavage dioxygenase 1 (ccd1) in A188 is associated with elevated expression during seed development. High ccd1 expression in seeds together with low expression of yellow endosperm 1 (y1) reduces carotenoid accumulation, accounting for the white seed phenotype of A188. Furthermore, transcriptome and epigenome analyses reveal enhanced expression of defense pathways and altered DNA methylation patterns of the embryonic callus. Conclusions The A188 genome assembly provides a high-resolution sequence for a complex genome species and a foundational resource for analyses of genome variation and gene function in maize. The genome, in comparison to B73, contains extensive intra-species structural variations and other genetic differences. Expression and network analyses identify discrete profiles for embryonic callus and other tissues.

Genotypic variation in the response to embryogenic callus induction and regeneration in Saccharum spontaneum

Embryogenic callus induction and regeneration are useful in many aspects of plant biotechnology, especially in the functional characterization of economically important genes. However, in sugarcane, callus induction and regeneration vary across genotypes. Saccharum spontaneum is an important wild germplasm that confers disease resistance and stress tolerance to modern sugarcane cultivars, and its genome has been completely sequenced. The aim of this study was to investigate the effect of genetic variations on embryogenic callus induction and regeneration in S. spontaneum and to screen genotypes having high tissue culture susceptibility. The study was performed using nine genotypes of S. spontaneum and the following five parameters were assessed to determine the response of genotypes to embryogenic callus induction and regeneration: callus induction, embryogenic callus ratio, embryogenic callus induction, embryonic callus regeneration and regeneration capacity. All the genotypes varied significantly (P < 0.01) in all the parameters, except for embryonic callus regeneration, which was high (>80%) for all the genotypes. High broad-sense heritability (86.1–96.8%) indicated that genetic differences are the major source of genotypic variations. Callus induction was found to be strongly positively correlated with embryogenic callus induction (r = 0.890, P < 0.01) and regeneration capacity (r = 0.881, P < 0.01). Among the nine tested genotypes, VN2 was found to be the most responsive to tissue culture and could therefore be used to characterize functional genes in S. spontaneum. We also suggested an approach with potential applications in facilitating the rapid identification of sugarcane genotypes susceptible to tissue culture.

Polycomb Repressive Complex 2-mediated histone modification H3K27me3 is associated with embryogenic potential in Norway spruce

Epigenetic reprogramming during germ cell formation is essential to gain pluripotency and thus embryogenic potential. The histone modification H3K27me3, which is catalysed by the Polycomb repressive complex 2 (PRC2), regulates important developmental processes in both plants and animals, and defects in PRC2 components cause pleiotropic developmental abnormalities. Nevertheless, the role of H3K27me3 in determining embryogenic potential in gymnosperms is still elusive. To address this, we generated H3K27me3 profiles of Norway spruce (Picea abies) embryonic callus and non-embryogenic callus using CUT&RUN, which is a powerful method for chromatin profiling. Here, we show that H3K27me3 mainly accumulated in genic regions in the Norway spruce genome, similarly to what is observed in other plant species. Interestingly, H3K27me3 levels in embryonic callus were much lower than those in the other examined tissues, but markedly increased upon embryo induction. These results show that H3K27me3 levels are associated with the embryogenic potential of a given tissue, and that the early phase of somatic embryogenesis is accompanied by changes in H3K27me3 levels. Thus, our study provides novel insights into the role of this epigenetic mark in spruce embryogenesis and reinforces the importance of PRC2 as a key regulator of cell fate determination across different plant species.

Somatic Embryogenesis and Plant Regeneration via Solid-liquid Alternating Culture in Elite Genotypes of Upland Cotton

Background: Cotton is one of the most genotype-dependent plants for regeneration, in order to expand cotton regeneration genotypes and establish efficient regeneration system platform, Jiwu 2031 (glandless cotton), ND 58 and CAU 102, were selected for studying the highly efficient somatic embryos formation and regeneration via solid-liquid alternating culture system.Results: In present research, the MSB medium (MS salts adding B5 vitamins) containing 0.571 µM indole-3-acetic acid (IAA), 0.465 µM kinetin (KT) and 0.904 µM 2, 4-dichlorophenoxyacetic acid (2, 4-D) was effective for callus initiation of fourteen Upland cotton with ‘Corker 201’ as the control. ‘Jiwu 2031’, ‘ND 58’ and ‘CAU 102’ could form somatic embryos and regenerate fertile plants in a solid MSB medium containing 10.31 mM NH4NO3, and supplemented with 0.027 mM glycin, 2.460 µM indole-3-acetic acid (IBA), 0.930 µM KT, 3.784 mM asparagine and 6.843 mM glutamine. Under the solid-liquid alternating culture system, the non-embryonic callus was induced to form embryonic callus and the identical status somatic embryos were obtained in 42 days, and the period for plants regeneration was shortened to 90~120 days, with the higher proportion of normal plant regeneration.Conclusions: The solid-liquid alternating culture method could increase the rate of embryogenesis and shorten the period of plants regeneration of Upland cotton. This work provides the evidence that the glandless cotton is beneficial for somatic embryogenesis (SE) and plant regeneration.

Somatic embryo induction and Agrobacterium-mediated transformation of embryonic callus tissue in Phoebe bournei, an endangered woody species in Lauraceae

Phoebe bournei, a plant species endemic to China, is a precious timber tree and widely used in landscaping. This tree contains numerous secondary metabolites, underscoring its potential economic value. However, studies on this species, including molecular genetic research, remain limited. In this study, both a somatic embryogenesis (SE) technical system and Agrobacterium-mediated genetic transformation were successfully employed in P. bournei for the first time. The SE technical system was constructed using immature embryos as original material. The primary embryo and embryonic callus induction rates were 30.66% and 41.67%, respectively. The highest rate of embryonic callus proliferation was 3.84. The maximum maturity coefficient and germination rate were 53.44/g and 39%, respectively. Agrobacterium-mediated genetic transformation was performed using the SE technical system, and the highest transformation rate was 11.24%. The results presented here are the first to demonstrate an efficient approach to achieve numerous P. bournei plantlets, which serves as the basis for artificial cultivation and resource conservation. Furthermore, the genetic transformation platform constructed in this study will facilitate assessment of gene function and molecular regulation.

The regeneration of Acer rubrum L. "October Glory" through embryonic callus

Background: Tissue culture and rapid propagation technology is an important way to solve the difficulties of plant propagation. This experiment aims to explore the appropriate conditions at each stage of the red maple’s tissue culture process and to obtain plantlets, thus providing a theoretical basis for the establishment of the red maple’s tissue culture system. Results: The results showed that the stem segment is the most suitable explant for inducing embryogenic callus. The MS (Murashige&Skoog) +0.8 mg/L TDZ (Thidiazuron) +1.0 mg/L 6-BA (6-Benzylaminopurine) +0.5 mg/L IAA(Indole-3-acetic acid) +35 g/L sucrose+7.5 g/L semi-fixed medium was the best for callus formation. When selecting type Ⅵ callus as embryonic callus induction material, MS+0.6 mg/L TDZ+0.5 mg/L 6-BA +2.0 mg/L IAA +35 g/L sucrose+7.5 g/L semi-fixed medium can get embryonic callus. The optimal medium for adventitious bud induction is MS+1.0 mg/L TDZ+3.0 mg/L 6-BA+0.2 mg/L NAA (1-Naphthaleneacetic acid)+1.2 mg/L IAA+35 g/L sucrose+7.5 g/L semi-fixed medium. The induction rate of adventitious roots in MS+0.6 mg/L TDZ+1.0 mg/L 6-BA+3 mg/L NAA+35 g/L sucrose+7.5 g/L semi-fixed medium was the highest, reaching 76%. Conclusions: In the course of our research, we found that PGRs play an important role in the callus induction stage, and the effect of TDZ is particularly obvious; The callus cells grow and proliferate according to the "S" growth curve, and can be sub-cultured when the highest growth point is reached to maintain the rapid proliferation of the callus cells and to avoid inactivation of callus caused by tight niche.

Methylation Analysis of CpG Islands in Pineapple SERK1 Promoter

Somatic embryogenesis (SE) is a more rapid and controllable method for plant propagation than traditional breeding methods. However, it often suffers from limited efficiency. SERK1 promotes SE in several plants, including pineapple (Ananas comosus L.). We investigate the embryonic cell-specific transcriptional regulation of AcSERK1 by methylation analysis of CpG islands in AcSERK1 regulatory sequences. This revealed differences in the methylation status of CpG islands between embryonic callus and non-embryonic callus; the methylation inhibitor 5-azaC increased AcSERK1 expression and also accelerated SE. These findings indicate that the expression of AcSERK1 is regulated epigenetically. This study lays the foundation for further analysis of epigenetic regulatory mechanisms that may enhance the efficiency of SE in pineapple and other plants.

The regeneration of Acer rubrum L. "October Glory" through embryonic callus

Background Landscape industry has been recognized as the "eternal sunrise industry", however, the introduction and cultivation of colored-leaf trees in southern China is relatively vacant.Results The results showed that the induction effect of the stem was the best for the embryogenic callus, and the regenerated plant with genetic stability was obtained. The MS (Murashige&Skoog) +0.8 mg/L TDZ (Thidiazuron) +1.0 mg/L 6-BA (6-Benzylaminopurine) +0.5 mg/L NAA (1-naphthlcetic acid) +35 g/L sucrose+7.5 g/L semi-fixed medium was the best for callus formation. When selecting type VI callus as embryonic callus induction material, MS+0.6 mg/L TDZ+0.5 mg/L 6-BA +2.0 mg/L IAA (Indole-3-acetic acid) +35 g/L sucrose+7.5 g/L semi-fixed medium can get embryonic callus. The optimal medium for adventitious bud induction is MS+1.0 mg/L TDZ+3.0 mg/L 6-BA+0.2 mg/L NAA+1.2 mg/L IAA+35 g/L sucrose+7.5 g/L semi-fixed medium.The induction rate of adventitious roots in MS+0.6 mg/L TDZ+1.0 mg/L 6-BA+3 mg/L NAA+35 g/L sucrose+7.5 g/L semi-fixed medium was the highest, reaching 76%.Conclusion In the course of our research, we found that PGRs play an important role in the callus induction stage, and the effect of TDZ is particularly obvious; The callus cells grow and proliferate according to the "S" growth curve, and can be subcultured when the highest growth point is reached to maintain the rapid proliferation of the callus cells and to avoid inactivation of callus caused by tight niche.