Plant callus is generally considered to be a mass of undifferentiated cells and can be used for secondary metabolite production, physiological studies, and plant transformation/regeneration. However, there are several types of callus with different morphological and developmental characteristics and not all are suitable for all applications. Callogenesis is a multivariable developmental process affected by several intrinsic and extrinsic factors, but the most important driver is plant growth regulator (PGRs) levels and type. Since callogenesis is a non-linear process influenced by many different factors, robust computational methods such as machine learning algorithms have great potential to model, predict, and optimize callus growth and development. The current study was conducted to evaluate the effect of PGRs on callus morphology in drug-type Cannabis sativa to maximize callus growth and promote embryogenic callus production. For this aim, random forest (RF) and support vector machine (SVM) were applied in conjunction with image processing to model and predict callus morphological and physical traits. The results showed that SVM was more accurate than RF. In order to find the optimal level of PGRs for optimizing callus growth and development, the SVM was linked to a genetic algorithm (GA). To confirm the reliability of SVM-GA, the optimized-predicted outcomes were tested in a validation experiment that revealed SVM-GA was able to accurately model and optimize the system. Moreover, our results showed that there is a significant correlation between embryogenic callus production and the true density of callus. KEY POINTS: • The effect of PGRs on callus growth and development of cannabis was studied. • The predictive accuracy of SVM and RF was evaluated and compared. • GA was linked to the SVM for optimizing the callus growth and development.

Modeling and optimizing callus growth and development in Cannabis sativa using random forest and support vector machine in combination with a genetic algorithm

In Vitro Anti-Inflammatory Activity of Three Peptides Derived from the Byproduct of Rice Processing

Differential gene expression profiles of various cannabis calli including non-embryogenic and embryogenic (i.e., rooty and embryonic callus) were examined in this study to enhance our understanding of callus development in cannabis and facilitate the development of improved strategies for plant regeneration and biotechnological applications in this economically valuable crop. A total of 6118 genes displayed significant differential expression, with 1850 genes downregulated and 1873 genes upregulated in embryogenic callus compared to non-embryogenic callus. Notably, 196 phytohormone-related genes exhibited distinctly different expression patterns in the calli types, highlighting the crucial role of plant growth regulator (PGRs) signaling in callus development. Furthermore, 42 classes of transcription factors demonstrated differential expressions among the callus types, suggesting their involvement in the regulation of callus development. The evaluation of epigenetic-related genes revealed the differential expression of 247 genes in all callus types. Notably, histone deacetylases, chromatin remodeling factors, and EMBRYONIC FLOWER 2 emerged as key epigenetic-related genes, displaying upregulation in embryogenic calli compared to non-embryogenic calli. Their upregulation correlated with the repression of embryogenesis-related genes, including LEC2, AGL15, and BBM, presumably inhibiting the transition from embryogenic callus to somatic embryogenesis. These findings underscore the significance of epigenetic regulation in determining the developmental fate of cannabis callus. Generally, our results provide comprehensive insights into gene expression dynamics and molecular mechanisms underlying the development of diverse cannabis calli. The observed repression of auxin-dependent pathway-related genes may contribute to the recalcitrant nature of cannabis, shedding light on the challenges associated with efficient cannabis tissue culture and regeneration protocols.

Transcriptomic Profiling of Embryogenic and Non-Embryogenic Callus Provides New Insight into the Nature of Recalcitrance in Cannabis

Protoplasts are single cells isolated from tissues or organs and are considered a suitable system for cell studies in plants. Embryogenic cells are totipotent stem cells, but their regeneration ability decreases or becomes lost altogether with extension of the culture period. In this study, we isolated and cultured EC-derived protoplasts (EC-pts) from carrots and compared them with non-EC-derived protoplasts (NEC-pts) with respect to their totipotency. The protoplast isolation conditions were optimized, and the EC-pts and NEC-pts were characterized by their cell size and types. Both types of protoplasts were then embedded using the alginate layer (TAL) method, and the resulting EC-pt-TALs and NEC-pt-TALs were cultured for further regeneration. The expression of the EC-specific genes SERK1, WUS, BBM, LEC1, and DRN was analyzed to confirm whether EC identity was maintained after protoplast isolation. The protoplast isolation efficiency for EC-pts was 2.4-fold higher than for NEC-pts (3.5 × 106 protoplasts·g−1 FW). In the EC-pt group, protoplasts < 20 µm accounted for 58% of the total protoplasts, whereas in the NEC-pt group, small protoplasts accounted for only 26%. In protoplast culture, the number of protoplasts that divided was 2.6-fold higher for EC-pts than for NEC-pts (7.7 × 104 protoplasts·g−1 FW), with a high number of plants regenerated for EC-pt-TALs, whereas no plants were induced by NEC-pt-TAL. Five times more plants were regenerated from EC-pts than from ECs. Regarding the expression of EC-specific genes, WUS and SERK1 expression increased 12-fold, and LEC1 and BBM expression increased 3.6–6.4-fold in isolated protoplasts compared with ECs prior to protoplast isolation (control). These results reveal that the protoplast isolation process did not affect the embryogenic cell identity; rather, it increased the plant regeneration rate, confirming that EC-derived protoplast culture may be an efficient system for increasing the regeneration ability of old EC cultures through the elimination of old and inactivate cells. EC-derived protoplasts may also represent an efficient single-cell system for application in new breeding technologies such as genome editing.

/pdf/embryogenic-stem-cell-identity-after-protoplast-isolation-msnn7944.pdf

Embryogenic Stem Cell Identity after Protoplast Isolation from Daucus carota and Recovery of Regeneration Ability through Protoplast Culture

Genotype-limited plant regeneration is one of the main obstacles to the broader use of genetic transformation in barley breeding. Thus, developing new approaches that might improve responses of in vitro recalcitrant genotypes remains at the center of barley biotechnology. Here, we analyzed different barley genotypes, including "Golden Promise," a genotype commonly used in the genetic transformation, and four malting barley cultivars of poor regenerative potential. The expression of hormone-related transcription factor (TF) genes with documented roles in plant regeneration was analyzed in genotypes with various plant-regenerating capacities. The results indicated differential expression of auxin-related TF genes between the barley genotypes in both the explants and the derived cultures. In support of the role of auxin in barley regeneration, distinct differences in the accumulation of free and oxidized auxin were observed in explants and explant-derived callus cultures of barley genotypes. Following the assumption that modifying gene expression might improve plant regeneration in barley, we treated the barley explants with trichostatin A (TSA), which affects histone acetylation. The effects of TSA were genotype-dependent as TSA treatment improved plant regeneration in two barley cultivars. TSA-induced changes in plant regeneration were associated with the increased expression of auxin biosynthesis-involved TFs. The study demonstrated that explant treatment with chromatin modifiers such as TSA might provide a new and effective epigenetic approach to improving plant regeneration in recalcitrant barley genotypes. 

The improvement of the in vitro plant regeneration in barley with the epigenetic modifier of histone acetylation, trichostatin A.

Genotype independent transformation and whole plant regeneration through somatic embryogenesis relies heavily on the intrinsic ability of a genotype to regenerate. The critical genetic architecture of non-embryogenic callus (NEC) cells and embryogenic callus (EC) cells in a highly regenerable cotton genotype is unknown. In this study, gene expression profiles of a highly regenerable Gossypium hirsutum L. cultivar, Jin668, were analyzed at two critical developmental stages during somatic embryogenesis, non-embryogenic callus (NEC) cells and embryogenic callus (EC) cells. The rate of EC formation in Jin668 is 96%. Differential gene expression analysis revealed a total of 5333 differentially expressed genes (DEG) with 2534 genes upregulated and 2799 genes downregulated in EC. A total of 144 genes were unique to NEC cells and 174 genes were unique to EC. Clustering and enrichment analysis identified genes upregulated in EC that function as transcription factors/DNA binding, phytohormone response, oxidative reduction, and regulators of transcription; while genes categorized in methylation pathways were downregulated. Four key transcription factors were identified based on their sharp upregulation in EC tissue; LEAFY COTYLEDON 1 (LEC1), BABY BOOM (BBM), FUSCA (FUS3) and AGAMOUS-LIKE15 with distinguishable subgenome expression bias. This comparative analysis of NEC and EC transcriptomes gives new insights into the genes involved in somatic embryogenesis in cotton.

/pdf/transcriptomic-profiles-of-non-embryogenic-and-embryogenic-1pl3hjxbaq.pdf

Transcriptomic profiles of non-embryogenic and embryogenic callus cells in a highly regenerative upland cotton line (Gossypium hirsutum L.)

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Transcriptomic profiles of non-embryogenic and embryogenic callus cells in a highly regenerative upland cotton line (Gossypium hirsutum L.)