Somatic Plant Research Articles

A cell-based fluorescent system and statistical framework to detect meiosis-like induction in plants.

Genetic gains made by plant breeders are limited by generational cycling rates and flowering time. Several efforts have been made to reduce the time to switch from vegetative to reproductive stages in plants, but these solutions are usually species-specific and require flowering. The concept of in vitro nurseries is that somatic plant cells can be induced to form haploid cells that have undergone recombination (creating artificial gametes), which can then be used for cell fusion to enable breeding in a Petri dish. The induction of in vitro meiosis, however, is the largest current bottleneck to in vitro nurseries. To help overcome this, we previously described a high-throughput, bi-fluorescent, single cell system in Arabidopsis thaliana, which can be used to test the meiosis-like induction capabilities of candidate factors. In this present work, we validated the system using robust datasets (>4M datapoints) from extensive simulated meiosis induction tests. Additionally, we determined false-detection rates of the fluorescent cells used in this system as well as the ideal tissue source for factor testing.

Deficiency of both classical and alternative end-joining pathways leads to a synergistic defect in double-strand break repair but not to an increase in homology-dependent gene targeting in Arabidopsis.

In eukaryotes, double-strand breaks (DSBs) are either repaired by homologous recombination (HR) or non-homologous end-joining (NHEJ). In somatic plant cells, HR is very inefficient. Therefore, the vast majority of DSBs are repaired by two different pathways of NHEJ. The classical (cNHEJ) pathway depends on the heterodimer KU70/KU80, while polymerase theta (POLQ) is central to the alternative (aNHEJ) pathway. Surprisingly, Arabidopsis plants are viable, even when both pathways are impaired. However, they exhibit severe growth retardation and reduced fertility. Analysis of mitotic anaphases indicates that the double mutant is characterized by a dramatic increase in chromosome fragmentation due to defective DSB repair. In contrast to the single mutants, the double mutant was found to be highly sensitive to the DSB-inducing genotoxin bleomycin. Thus, both pathways can complement for each other efficiently in DSB repair. We speculated that in the absence of both NHEJ pathways, HR might be enhanced. This would be especially attractive for gene targeting (GT) in which predefined changes are introduced using a homologous template. Unexpectedly, the polq single mutant as well as the double mutant showed significantly lower GT frequencies in comparison to wildtype plants. Accordingly, we were able to show that elimination of both NHEJ pathways does not pose an attractive approach for Agrobacterium-mediated GT. However, our results clearly indicate that a loss of cNHEJ leads to an increase in GT frequency, which is especially drastic and attractive for practical applications, in which the in planta GT strategy is used.

Correction to: High temperature and water deficit cause epigenetic changes in somatic plants of Pinus radiata D. Don

Correction to: High temperature and water deficit cause epigenetic changes in somatic plants of Pinus radiata D. Don

Colchicine-Induced Polyploidy in Leguminous Crops Enhances Morpho-Physiological Characteristics for Drought Stress Tolerance.

Legumes play a significant role in the alleviation of food insecurity, maintaining soil fertility, and achieving sustainable crop production under adverse environmental conditions. The increased demand in legume production contemplates that attention on the genetic improvement of these crops through various means such as genetic engineering and mutation breeding should take a centre stage in global agriculture. Therefore, this paper provides a succinct analysis of the currently available literature on morphological and physiological traits in polyploidised leguminous plants to counter the adverse effects of drought stress. The effects of colchicine on various morphological and physiological traits of polyploidised legumes compared to their diploid counterparts were examined. Numerous reports revealed variations in these traits, such as improved root and shoot growth, plant biomass, chloroplastidic content, protein, RNA, and DNA. The differences observed were also associated with the strong relationship between plant ploidy induction and colchicine application. Furthermore, the analysis indicated that polyploidisation remains dose-dependent and may be achievable within a shorter space of time as this antimitotic chemical interferes with chromosome separations in somatic plant cells. The efficiency of this process also depends on the advancement of treatment conditions (in vitro, in vivo, or ex vitro) and the successful regeneration of polyploidised plants for adaptation under drought stress conditions. As such, the improvement in metabolite profile and other essential growth characteristics serves as a clear indication that induced polyploidy needs to be further explored to confer resilience to environmental stress and improve crop yield under drought stress conditions in leguminous plants.

Resistant and Susceptible Pinus thunbergii ParL. Show Highly Divergent Patterns of Differentially Expressed Genes during the Process of Infection by Bursaphelenchus xylophilus.

Pine wilt disease (PWD) is a devastating disease that threatens pine forests worldwide, and breeding resistant pines is an important management strategy used to reduce its impact. A batch of resistant seeds of P. thunbergii was introduced from Japan. Based on the resistant materials, we obtained somatic plants through somatic embryogenesis. In this study, we performed transcriptome analysis to further understand the defense response of resistant somatic plants of P. thunbergii to PWD. The results showed that, after pine wood nematode (PWN) infection, resistant P. thunbergii stimulated more differential expression genes (DEGs) and involved more regulatory pathways than did susceptible P. thunbergii. For the first time, the alpha-linolenic acid metabolism and linoleic acid metabolism were intensively observed in pines resisting PWN infection. The related genes disease resistance protein RPS2 (SUMM2) and pathogenesis-related genes (PR1), as well as reactive oxygen species (ROS)-related genes were significantly up-expressed in order to contribute to protection against PWN inoculation in P. thunbergii. In addition, the diterpenoid biosynthesis pathway was significantly enriched only in resistant P. thunbergii. These findings provided valuable genetic information for future breeding of resistant conifers, and could contribute to the development of new diagnostic tools for early screening of resistant pine seedlings based on specific PWN-tolerance-related markers.

A Common Molecular Signature Indicates the Pre-Meristematic State of Plant Calli.

In response to different degrees of mechanical injury, certain plant cells re-enter the division cycle to provide cells for tissue replenishment, tissue rejoining, de novo organ formation, and/or wound healing. The intermediate tissue formed by the dividing cells is called a callus. Callus formation can also be induced artificially in vitro by wounding and/or hormone (auxin and cytokinin) treatments. The callus tissue can be maintained in culture, providing starting material for de novo organ or embryo regeneration and thus serving as the basis for many plant biotechnology applications. Due to the biotechnological importance of callus cultures and the scientific interest in the developmental flexibility of somatic plant cells, the initial molecular steps of callus formation have been studied in detail. It was revealed that callus initiation can follow various ways, depending on the organ from which it develops and the inducer, but they converge on a seemingly identical tissue. It is not known, however, if callus is indeed a special tissue with a defined gene expression signature, whether it is a malformed meristem, or a mass of so-called "undifferentiated" cells, as is mostly believed. In this paper, I review the various mechanisms of plant regeneration that may converge on callus initiation. I discuss the role of plant hormones in the detour of callus formation from normal development. Finally, I compare various Arabidopsis gene expression datasets obtained a few days, two weeks, or several years after callus induction and identify 21 genes, including genes of key transcription factors controlling cell division and differentiation in meristematic regions, which were upregulated in all investigated callus samples. I summarize the information available on all 21 genes that point to the pre-meristematic nature of callus tissues underlying their wide regeneration potential.

Effects of plant growth regulators and sucrose on proliferation and quality of embryogenic tissue in Picea pungens

Embryogenic tissue (ET) is important for genetic modification and plant re-generation. The proliferation ability and vigor of ET are crucial for plant propagation via somatic embryogenesis. In this study, ET was induced from mature zygotic embryos in blue spruce (Picea pungens Engelm.). There were significant differences in ET induction between two provenances, i.e. 78.8 ± 12.5% and 62.50 ± 12.8% respectively. Effects of 2,4-Dichlorophenoxy acetic acid (2,4-D), 6-Benzyl amino-purine (6-BA) and/or sucrose on ET proliferation and somatic embryo (SE) maturation were further investigated with four cell lines. The highest ET proliferation rate reached 1473.7 ± 556.0% biweekly. Concentrations of 2,4-D or 6-BA applied at tissue proliferation stage impacted SE maturation among the cell lines, whereas sucrose showed less effects. The highest rate, 408 ± 230 mature SEs/g FW, was achieved in SE maturation cultures. This research demonstrated that the culture conditions, i.e. the specific concentrations of 2,4-D and BA, at ET proliferation stage affected not only ET growth, but also the quality of ET for SE maturation. This study revealed the necessity and benefit in developing both the general and the genotype-specific protocols for efficient production of mature SEs, or somatic plants in blue spruce.

Application of Cold Storage and Short In Vitro Germination for Somatic Embryos of Pinus radiata and P. sylvestris.

Somatic embryogenesis (SE) is an advanced vegetative propagation technology that, when used in combination with breeding and cryopreservation, offers the forest industry a powerful tool for the deployment of elite genotypes. Germination and acclimatization are critical and cost-intensive phases in the production of somatic plants. The efficient conversion of somatic embryos into robust plants is a necessity if a propagation protocol is to be successfully adopted by the industry. In this work, these late phases of the SE protocol of two pine species were investigated. A shortened germination protocol and more controlled acclimatization were investigated for Pinus radiata, testing embryos from 18 embryogenic cell lines. A more simplified protocol, including a cold storage phase, was also compared among 10 of these cell lines. A shortened germination period and more controlled protocols significantly improved the acclimatization of somatic embryos directly from the lab to the glasshouse. When results for all cell lines were pooled, there were significant improvements in all growth characteristics (shoot height, root length, root collar diameter, and root quadrant score). When the more simplified protocol involving cold storage was tested, improvements were seen in the root architecture. For Pinus sylvestris, the late phases of somatic embryogenesis were investigated on seven cell lines in a set of two trials (four to seven cell lines per trial). During the germination phase, a shortened and simplified in vitro period, a cold storage option and basal media were explored. Viable plants were obtained from all treatments. However, there is still the need to improve germination and related protocols together with growing regimes for Pinus sylvestris. The improvements to protocols presented here, particularly for Pinus radiata, result in greater survival and quality of somatic emblings, leading to reduced costs and increased confidence in the technology. Simplified protocols using a cold storage option show great promise and, with some further research, could lead to reductions in the cost of the technology.

Promoting the application of Pinus thunbergii Parl. to enhance the growth and survival rates of post-germination somatic plantlets

ObjectiveThere is a growing need for nematode resistant Pinaceae species plantlets to cope with the global scale degradation of coniferous forests, due to the prevalence of pine wilt disease. One of the bottlenecks that limits the commercialization of Pinaceae species plantlets is regeneration following their transfer from controlled sterile environments to the field while maintaining high survival rates.MethodsThe growth factors of somatic plantlets (SPs), such as sucrose, media, culture substrate, brassinolide and spectrum were investigated to promote the application of somatic nematode-resistant P. thunbergii plants in afforestation.ResultsThe 1/2 WPM liquid medium, culture substrate (perlite and vermiculite =1:1), and carbohydrate (20 g/L sucrose) were effective in stimulating the growth of rooted SPs. While for unrooted SPs, 1 ug/L of brassinolide enhanced plantlet growth and rooting. And blue light (B) significantly promoted the longitudinal growth of shoots, while red light (R) was beneficial for root growth during the laboratory domestication stage. High quality SPs were obtained at a R/B ratio of 8:2. Following this acclimatization protocol, the P. thunbergii SPs could be directly transplanted to the field with a higher survival rate (85.20 %) in a forcing house.Conclusionthis acclimatization protocol extremely improved the survival rate of P. thunbergii SPs. Moreover, this work will contribute to enhancing the possibilities for somatic plant afforestation with Pinus species.

Multi-strategy approach towards optimization of maturation and germination in radiata pine somatic embryogenesis

Somatic embryogenesis is a powerful technique for forestry improvement programs when implementing multi-varietal forestry. However, it still faces some bottlenecks to be efficient in many forestry species. In this work we have studied the effect of some physico-chemical modifications at proliferation, maturation and germination stages of Pinus radiata D. Don somatic embryogenesis, as well as the impact of this modifications on plant conversion, survival, and on the morphology and biochemical profile (carbohydrate and amino acid analysis) of the resulting plantlets. Given the long culture period needed for maturation and germination in radiata pine, an increase of the plant yield could be very beneficial for its commercial application. Following these premises and based on the experiments performed, a preculture of 14 days without plant growth regulators before maturation seems to be beneficial for the development and germination of somatic embryos. Before germination, a cold storage at 4 °C had no detrimental effect and even increased plant conversion in some embryogenic cell lines (85% of germination and 64.6% of planted explants). During germination, supplementation of the culture medium with glutamine and a reduction of the sucrose content had a significant effect on germination (88%) and doubled the percentage of planted explants. Similarly, germinants were demonstrated to be influenced by the light source: fluorescent light enhanced root formation, whereas blue LED light increased the shoot height of somatic plants. Moreover, the highest plants showed lower contents of arginine, asparagine and total carbohydrates. Modification of the physico-chemical environment before or during somatic embryo maturation and germination improves the success of the process.

In vitro regeneration through organogenesis in Costus igneus–an important herbal insulin plant

Costus igneus is an important high-valued herbal insulin plant. An efficient protocol for in vitro plant regeneration through organogenesis from leaf and nodal explants was standardized. Explants were incubated in Murashige and Skoog (MS) medium amended with diverse levels of plant growth regulators (PGRs). Optimum frequency (68.25%) of adventitious shoot regeneration was obtained from nodal segments cultured onto MS medium containing 2.0 mg/l of 6-benzylaminopurine (BAP) with an average number of 5.0 shoots per explant. Further, MS medium containing 1.5 mg/l 2, 4- dichlorophenoxyacetic acid (2, 4-D) and 1.0 mg/l Thidiazuron (TDZ) produced a higher frequency of callus induction (55.22%) from leaf explants. Optimum shoot regeneration response (66.0%) through indirect organogenesis was observed from leaf-derived callus on MS medium fortified with BAP (2.0 mg/l) and kinetin (0.5 mg/l) with 6 shoots per callus with mean shoot length of 8.0 cm. The shootlets obtained from node and leaf cultures were rooted with a frequency of (63.52%) on MS medium with 1.0 mg/l indole-3-acetic acid (IAA). Plantlets obtained through organogenesis were successfully acclimatized in the greenhouse and field conditions. There were no significant differences among frequency of somatic plants regenerated through either direct or indirect organogenesis. The present study reports on successful plant regeneration through direct and indirect organogenesis in C. igneus. The regeneration protocols discussed here can be used effectively for large-scale multiplication, germplasm survival, pharmacological and genetic manipulation research.

Megasporogenesis and Megagametogenesis in Coelogyne speciosa subsp. fimbriata (J.J.Sm.) Gravendeel (Orchidaceae Juss.)

Understanding the critical stages in ovule development is essential for optimizing in vitro seed propagation, cryopreservation, and indirect somatic plant embryogenesis. We studied the main stages of the megasporogenesis and megagametogenesis of Coelogyne speciosa subsp. fimbriata (J.J.Sm.) Gravendeel by confocal laser scanning microscopy. The ovule is anatropic, tenuinucellate, bitegmal, and funicular. The embryo sac is monosporic, Polygonum-type, bipolar, and usually 8-nuclear, but in cases of a reduction in the last mitotic division, it is 6-nuclear. Endosperm formation was not detected. The formation of a functional megaspore occurred 65–70 days after pollination (DAP), while the formation of a mature embryo sac and fertilization occurred after 85–100 DAP.

High temperature and water deficit cause epigenetic changes in somatic plants of Pinus radiata D. Don

Current climate changes imply an imminent risk for forest species. In this context, somatic embryogenesis is a valuable tool to study the response of plants to different abiotic stresses. Based on this, we applied a high-temperature regime (50 °C, 5 min) during the maturation of Pinus radiata D. Don embryogenic masses in order to evaluate the development of an epigenetic memory months later. Therefore, somatic plants (SP) resulting from somatic embryos (ses) maturated at control temperature and cultivated in a greenhouse were submitted to heat stress (40 °C, 2 h, 10 days; 23 °C, 10 days) or at a control temperature (23 °C, 20 days); while another 20 SP resulting from ses maturated in the two temperature regimes and cultivated in the greenhouse were submitted to drought stress or weekly irrigated. All plants were evaluated for relative water content, water potential, electrolyte leakage, stomatal conductance, transpiration, methylation (5-mC) and hydroxymethylation (5-hmC) levels. The results showed that the SP obtained from ses maturated at 50 °C showed an adaptation to drought stress based on water potential and transpiration. Furthermore, SP kept under heat stress in a greenhouse showed lower 5-hmC levels than SP kept at 23 °C. Furthermore, the 5-hmC and 5-hmC/5-mC ratio showed a significantly negative correlation with changes in water potential; and a significantly negative correlation was observed between the levels of stomatal conductance and 5-mC. We conclude that the manipulation of conditions during the maturation process in somatic embryogenesis modulates the physiological characteristics of the SP obtained. Application of high temperatures during maturation and hardening of somatic plants provoked drought adaptation and epigenetic changes.

Thermopriming-associated proteome and sugar content responses in Pinus radiata embryogenic tissue

Improving the capacity of plants to face adverse environmental conditions requires a deep understanding of the molecular mechanisms governing stress response and adaptation. Proteomics, combined with metabolic analyses, offers a wide resource of information to be used in plant breeding programs. Previous studies have shown that somatic embryogenesis in Pinus spp. is a suitable tool not only to investigate stress response processes but also to modulate the behaviour of somatic plants. Based on this, the objective of this study was to analyse the protein and soluble sugar profiles of Pinus radiata embryonal masses after the application of high temperatures to unravel the mechanisms involved in thermopriming and memory acquisition at early stages of the somatic embryogenesis process. Results confirmed that heat provokes deep readjustments in the life cycle of proteins, together with a significant reduction in the carbon-flux of central-metabolism pathways. Heat-priming also promotes the accumulation of proteins involved in oxidative stress defence, in the synthesis of specific amino acids such as isoleucine, influences cell division, the organization of the cytoskeleton and cell-walls, and modifies the levels of free soluble sugars like glucose or fructose. All this seems to be regulated by proteins linked with epigenetic, transcriptional and post-transcriptional mechanisms.

Chromosomal Engineering through CRISPR– Cas Technology: A Way Forward

The breeding of crops is dependent on the potential to interrupt or maintain genetic links between characteristics, and the availability of genetic variability. CRISPR-Cas is a new genome-editing technique that has made it possible for breeders to introduce regulated and site-specific genetic diversity while simultaneously improving qualities with high efficacy. The existence of genomic linkage is a barrier in transferring desirable features among domesticated species from their wild counterparts. One way to address this issue is to create mutants with deficiencies in the meiotic recombination machinery, thereby enhancing global crossover frequencies between homologous parental chromosomes. Although this seemed to be a promising approach at first, thus far, no crossover frequencies could be enhanced in recombination-cold regions of the genome. Consequently, attempts have been made to induce site-specific DSBs in both somatic and meiotic plant cells by utilizing CRISPR–Cas techniques to achieve preset crossovers among homologs. Nonetheless, this method has not yielded significant heritable homologous crossings which were recombination-based. Lately, CRISPR–Cas has been used to achieve hereditary chromosomal rearrangements (CRs), including translocations and inversions, in plants. This method allows for the development of megabase CRs by DSB repair through non-homologous end-joining after insertion of DSBs in somatic plant cells. This technique may potentially make it possible to restructure genomes on a more global scale, culminating in the creation not just of synthetic plant chromosomes, but also that of new plant species.

Somatic Embryogenesis in Pines.

Among the different in vitro culture techniques, somatic embryogenesis has been one of the most important developments for plant tissue culture; it has enabled mass propagation and the development of biotechnological tools to enhance the productivity and quality of plantation forestry. This propagation technique together with cryopreservation is the base of multivarietal forestry.The development of somatic embryogenesis in forest trees dates from 1985, and in the last years several studies have focused on the development and optimization of the conifer somatic embryogenesis process to make it more efficient in terms of both the quantity and the characteristics of the plants obtained. However, these advances are not sufficiently refined to be implemented commercially for many Pinus spp. due to the high cost of the process derived from hand labor. Nowadays, trying to add value to the plants produced to compensate the high costs of the process, different studies are being developed in order to obtain Pinus somatic plants with better adaptation to environmental stresses prompted by the current situation of climate change.In this chapter, a summary of the recent somatic embryogenesis systems developed to achieve Pinus spp. high quality plants is presented.

Heat Stress in Pinus halepensis Somatic Embryogenesis Induction: Effect in DNA Methylation and Differential Expression of Stress-Related Genes.

In the current context of climate change, plants need to develop different mechanisms of stress tolerance and adaptation to cope with changing environmental conditions. Temperature is one of the most important abiotic stresses that forest trees have to overcome. Recent research developed in our laboratory demonstrated that high temperatures during different stages of conifer somatic embryogenesis (SE) modify subsequent phases of the process and the behavior of the resulting ex vitro somatic plants. For this reason, Aleppo pine SE was induced under different heat stress treatments (40 °C for 4 h, 50 °C for 30 min, and 60 °C for 5 min) in order to analyze its effect on the global DNA methylation rates and the differential expression of four stress-related genes at different stages of the SE process. Results showed that a slight decrease of DNA methylation at proliferating embryonal masses (EMs) can correlate with the final efficiency of the process. Additionally, different expression patterns for stress-related genes were found in EMs and needles from the in vitro somatic plants obtained; the DEHYDRATION INDUCED PROTEIN 19 gene was up-regulated in response to heat at proliferating EMs, whereas HSP20 FAMILY PROTEIN and SUPEROXIDE DISMUTASE [Cu–Zn] were down-regulated in needles.

Marker-Assisted Selection for Pollen-Free Somatic Plants of Sugi (Japanese Cedar, Cryptomeria japonica): A Simple and Effective Methodology for Selecting Male-Sterile Mutants With ms1-1 and ms1-2.

Pollen allergy caused by sugi (Japanese cedar, Cryptomeria japonica) is a serious problem in Japan. One of the measures against pollinosis is the use of male-sterile plants (MSPs; pollen-free plants). In this context, the development of a novel technique for the efficient production of sugi MSPs, which combines marker-assisted selection (MAS) with somatic embryogenesis (SE), was recently reported by our research group. To improve the efficiency of MSP production, in this paper we report improved MAS for male-sterile individuals from embryogenic cells, cotyledonary embryos, and somatic plants of sugi using a newly developed marker in the form of the causative mutation of MS1 itself, selecting individuals with ms1-1 and ms1-2 male-sterile mutations. We also describe simplified methods for extracting DNA from different plant materials and for MAS using LAMP diagnostics. Finally, we show that MAS can be efficiently performed using the one-step indel genotyping (ING) marker developed in this study and using InstaGene for DNA extraction. The combination of SE and 100% accurate marker selection during the embryogenic cell stage enables the mass production of MS1 male-sterile sugi seedlings.

Analyzing Plant Gene Targeting Outcomes and Conversion Tracts with Nanopore Sequencing.

The high-throughput molecular analysis of gene targeting (GT) events is made technically challenging by the residual presetabce of donor molecules. Large donor molecules restrict primer placement, resulting in long amplicons that cannot be readily analyzed using standard NGS pipelines or qPCR-based approaches such as ddPCR. In plants, removal of excess donor is time and resource intensive, often requiring plant regeneration and weeks to months of effort. Here, we utilized Oxford Nanopore Amplicon Sequencing (ONAS) to bypass the limitations imposed by donor molecules with 1 kb of homology to the target and dissected GT outcomes at three loci in Nicotiana benthamia leaves. We developed a novel bioinformatic pipeline, Phased ANalysis of Genome Editing Amplicons (PANGEA), to reduce the effect of ONAS error on amplicon analysis and captured tens of thousands of somatic plant GT events. Additionally, PANGEA allowed us to collect thousands of GT conversion tracts 5 days after reagent delivery with no selection, revealing that most events utilized tracts less than 100 bp in length when incorporating an 18 bp or 3 bp insertion. These data demonstrate the usefulness of ONAS and PANGEA for plant GT analysis and provide a mechanistic basis for future plant GT optimization.

Stochastic gene expression drives mesophyll protoplast regeneration.

Cell pluripotency is fundamental to biology. It has long been known that differentiated somatic plant cells may reacquire pluripotency, but the underlying mechanism remains elusive. In many plant species, a single isolated mesophyll protoplast may regenerate into an entire plant, which is widely used in gene transformation. Here, we identified two transcription factors whose ectopic activation promotes protoplast regeneration. Furthermore, we found that their expression was induced by protoplast isolation but at a very low frequency. Using live imaging and single-cell transcriptomics, we show that isolating protoplasts induces enhanced expression variation at the genome level. Isolating protoplasts also leads to genome-wide increases in chromatin accessibility, which promotes stochastic activation of gene expression and enhances protoplast regeneration. We propose that transcriptome chaos with increased expression variability among cells creates a cellular-level evolutionary driver selecting for regenerating cells.