Single-cell transcriptomics is revolutionizing the improvement of plant biotechnology research: recent advances and future opportunities.

Single-cell approaches are a promising way to obtain high-resolution transcriptomics data and have the potential to revolutionize the study of plant growth and development. Recent years have seen the advent of unprecedented technological advances in the field of plant biology to study the transcriptional information of individual cells by single-cell RNA sequencing (scRNA-seq). This review focuses on the modern advancements of single-cell transcriptomics in plants over the past few years. In addition, it also offers a new insight of how these emerging methods will expedite advance research in plant biotechnology in the near future. Lastly, the various technological hurdles and inherent limitations of single-cell technology that need to be conquered to develop such outstanding possible knowledge gain is critically analyzed and discussed.

Domestication has altered the ABA and gibberellin profiles in developing pea seeds.

MAIN CONCLUSION: We showed that wild pea seeds contained a more diverse combination of bioactive GAs and had higher ABA content than domesticated peas. Although the role of abscisic acid (ABA) and gibberellins (GAs) interplay has been extensively studied in Arabidopsis and cereals models, comparatively little is known about the effect of domestication on the level of phytohormones in legume seeds. In legumes, as in other crops, seed dormancy has been largely or entirely removed during domestication. In this study, we have measured the endogenous levels of ABA and GAs comparatively between wild and domesticated pea seeds during their development. We have shown that wild seeds contained more ABA than domesticated ones, which could be important for preparing the seeds for the period of dormancy. ABA was catabolised particularly by an 8´-hydroxylation pathway, and dihydrophaseic acid was the main catabolite in seed coats as well as embryos. Besides, the seed coats of wild and pigmented cultivated genotypes were characterised by a broader spectrum of bioactive GAs compared to non-pigmented domesticated seeds. GAs in both seed coat and embryo were synthesized mainly by a 13-hydroxylation pathway, with GA29 being the most abundant in the seed coat and GA20 in the embryos. Measuring seed water content and water loss indicated domesticated pea seeds´ desiccation was slower than that of wild pea seeds. Altogether, we showed that pea domestication led to a change in bioactive GA composition and a lower ABA content during seed development.

Protoplast Technology and Somatic Hybridisation in the Family Apiaceae.

Species of the family Apiaceae occupy a major market share but are hitherto dependent on open pollinated cultivars. This results in a lack of production uniformity and reduced quality that has fostered hybrid seed production. The difficulty in flower emasculation led breeders to use biotechnology approaches including somatic hybridization. We discuss the use of protoplast technology for the development of somatic hybrids, cybrids and in-vitro breeding of commercial traits such as CMS (cytoplasmic male sterility), GMS (genetic male sterility) and EGMS (environment-sensitive genic male sterility). The molecular mechanism(s) underlying CMS and its candidate genes are also discussed. Cybridization strategies based on enucleation (Gamma rays, X-rays and UV rays) and metabolically arresting protoplasts with chemicals such as iodoacetamide or iodoacetate are reviewed. Differential fluorescence staining of fused protoplast as routinely used can be replaced by new tagging approaches using non-toxic proteins. Here, we focused on the initial plant materials and tissue sources for protoplast isolation, the various digestion enzyme mixtures tested, and on the understanding of cell wall re-generation, all of which intervene in somatic hybrids regeneration. Although there are no alternatives to somatic hybridization, various approaches also discussed are emerging, viz., robotic platforms, artificial intelligence, in recent breeding programs for trait identification and selection.

Morphological and anatomical characterization of yellow diploid potato flower for effective breeding program.

The diploid yellow potato (Solanum tuberosum L. Phureja Group) is an important plant genetic resource. In this study, we report for the first time the characterization of anther development and pollen formation in the cultivar Criolla Colombia. The description of morphological and histological characters of buds and flowers at different developmental stages permitted to identify ten main stages, from the differentiation of the male cells of the sporangium, meiosis, microspores formation and maturation, to the release of mature pollen. In addition, the results provide a graphic guide of the development of the anther, through the sequential and orderly formation of the epidermis, the endothecium, the middle layer and the nutritive layer or tapetum. This microanatomical information will be useful for work focused on androgenesis and identification of gene regulation in floral biology and gamete formation. Therefore, this study determined that to efficiently obtain haploids, flower buds between 5 and 8.9 mm long (stage 6 to 8) should be used, in which tetrads and microspores are in the early uninucleate and binucleate stage.

Analysis of Ploidy in Haploids and Doubled Haploids.

Determination of the ploidy level is an essential step when trying to produce doubled haploids (DHs) in any species. Each species and method used to produce DHs has its own frequency of DH production, which means that the rest of plants produced stay haploid. Since haploids are of little use for breeding purposes, it is necessary to distinguish them from true DHs. For this, several methodologies are available, including flow cytometry, chromosome counting, chloroplast counting in stomatal guard cells, measurement of stomatal size and length, counting of nucleoli, evaluation of pollen formation and viability, analysis of cell size, and analysis of morphological markers. However, not all of them are equally easy to use, affordable, reliable, reproducible, and resolutive and therefore useful for a particular case. In this chapter, we revise these methods available to assess the ploidy level of plants, discussing their respective advantages and limitations, and provide some troubleshooting tips and hints to help decide which to choose in each case.

WUSCHEL: a master regulator in plant growth signaling.

KEY MESSAGE: This review summarizes recent knowledge on functions of WUS and WUS-related homeobox (WOX) transcription factors in diverse signaling pathways governing shoot meristem biology and several other aspects of plant dynamics. Transcription factors (TFs) are master regulators involved in controlling different cellular and biological functions as well as diverse signaling pathways in plant growth and development. WUSCHEL (WUS) is a homeodomain transcription factor necessary for the maintenance of the stem cell niche in the shoot apical meristem, the differentiation of lateral primordia, plant cell totipotency and other diverse cellular processes. Recent research about WUS has uncovered several unique features including the complex signaling pathways that further improve the understanding of vital network for meristem biology and crop productivity. In addition, several reports bridge the gap between WUS expression and plant signaling pathway by identifying different WUS and WUS-related homeobox (WOX) genes during the formation of shoot (apical and axillary) meristems, vegetative-to-embryo transition, genetic transformation, and other aspects of plant growth and development. In this respect, the WOX family of TFs comprises multiple members involved in diverse signaling pathways, but how these pathways are regulated remains to be elucidated. Here, we review the current status and recent discoveries on the functions of WUS and newly identified WOX family members in the regulatory network of various aspects of plant dynamics.

Expression Patterns of Key Hormones Related to Pea (Pisum sativum L.) Embryo Physiological Maturity Shift in Response to Accelerated Growth Conditions.

Protocols have been proposed for rapid generation turnover of temperate legumes under conditions optimized for day-length, temperature, and light spectra. These conditions act to compress time to flowering and seed development across genotypes. In pea, we have previously demonstrated that embryos do not efficiently germinate without exogenous hormones until physiological maturity is reached at 18 days after pollination (DAP). Sugar metabolism and moisture content have been implicated in the modulation of embryo maturity. However, the role of hormones in regulating seed development is poorly described in legumes. To address this gap, we characterized hormonal profiles (IAA, chlorinated auxin [4-Cl-IAA], GA20, GA1, and abscisic acid [ABA]) of developing seeds (10-22 DAP) from diverse pea genotypes grown under intensive conditions optimized for rapid generation turnover and compared them to profiles of equivalent samples from glasshouse conditions. Growing plants under intensive conditions altered the seed hormone content by advancing the auxin, gibberellins (GAs) and ABA profiles by 4 to 8 days, compared with the glasshouse control. Additionally, we observed a synchronization of the auxin profiles across genotypes. Under intensive conditions, auxin peaks were observed at 10 to 12 DAP and GA20 peaks at 10 to 16 DAP, indicative of the end of embryo morphogenesis and initiation of seed desiccation. GA1 was detected only in seeds harvested in the glasshouse. These results were associated with an acceleration of embryo physiological maturity by up to 4 days in the intensive environment. We propose auxin and GA profiles as reliable indicators of seed maturation. The biological relevance of these hormonal fluctuations to the attainment of physiological maturity, in particular the role of ABA and GA, was investigated through the study of precocious in vitro germination of seeds 12 to 22 DAP, with and without exogenous hormones. The extent of sensitivity of developing seeds to exogenous ABA was strongly genotype-dependent. Concentrations between 5 and 10 µM inhibited germination of seeds 18 DAP. Germination of seeds 12 DAP was enhanced 2.5- to 3-fold with the addition of 125 µM GA3. This study provides further insights into the hormonal regulation of seed development and in vitro precocious germination in legumes and contributes to the design of efficient and reproducible biotechnological tools for rapid genetic gain.

Nuclear Migration: An Indicator of Plant Salinity Tolerance in vitro.

In order to understand the mechanisms underlying acquisition of tolerance to salinity, we recently produced callus tissues of tobacco and Medicago truncatula resistant to NaCl-induced salt stress following application of a step-up recurrent selection method. The effects of salinity on cell size are known, but those on cell morphometry including cell and nuclear surface area and position of nuclei within salt stress resistant cells were never studied before. This work fills that gap, using suspension cultured cells of M. truncatula A17 initiated from callus, and Nicotiana tabacum BY-2 cell line resistant to increasing NaCl concentrations up to 150 mM NaCl. The surface area of salinity resistant cells of M. truncatula A17 and N. tabacum BY2 and their nuclei, produced by step-up recurrent selection, were reduced, and cells elongated as NaCl increased, but these parameters proved to be unreliable in explaining cell survival and growth at high NaCl. Conversely, nuclei of resistant cells migrated from the center to the periphery of the cytoplasm close to the walls. Nuclear marginalization was for the first time observed as a result of salt stress in plant cells, and could be a novel helpful morphological marker of acquisition of salinity tolerance.

Toward Unravelling the Genetic Determinism of the Acquisition of Salt and Osmotic Stress Tolerance Through In Vitro Selection in Medicago truncatula.

Changes in global climate and the nonstop increase in demographic pressure have provoked a stronger demand for agronomic resources at a time where land suitable for agriculture is becoming a rare commodity. They have also generated a number of abiotic stresses which exacerbate effects of diseases and pests and result in physiological and metabolic disorders that ultimately impact on yield when and where it is most needed. Therefore, a major scientific and agronomic challenge today is that of understanding and countering the impact of stress on yield. In this respect, in vitro biotechnology would be an efficient and feasible breeding alternative, particularly now that the genetic and genomic tools needed to unravel the mechanisms underlying the acquisition of tolerance to stress have become available. Legumes in general play a central role in a sustainable agriculture due to their capacity to symbiotically fix the atmospheric nitrogen, thereby reducing the need for fertilizers. They also produce grains that are rich in protein and thus are important as food and feed. However, they also suffer from abiotic stresses in general and osmotic stress and salinity in particular. This chapter provides a detailed overview of the methods employed for in vitro selection in the model legume Medicago truncatula for the generation of novel germplasm capable of resisting NaCl- and PEG-induced osmotic stress. We also address the understanding of the genetic determinism in the acquisition of stress resistance, which differs between NaCl and PEG. Thus, the expression of genes linked to growth (WEE1), in vitro embryogenesis (SERK), salt tolerance (SOS1) proline synthesis (P5CS), and ploidy level and cell cycle (CCS52 and WEE1) was upregulated under NaCl stress, while under PEG treatment the expression of MtWEE1 and MtCCS52 was significantly increased, but no significant differences were observed in the expression of genes MtSERK1 and MtP5CS, and MtSOS1 was downregulated. A number of morphological and physiological traits relevant to the acquisition of stress resistance were also assessed, and methods used to do so are also detailed.

PEG Induces High Expression of the Cell Cycle Checkpoint Gene WEE1 in Embryogenic Callus of Medicago truncatula: Potential Link between Cell Cycle Checkpoint Regulation and Osmotic Stress.

Polyethylene glycol (PEG) can be used to mimic osmotic stress in plant tissue cultures to study mechanisms of tolerance. The aim of this experiment was to investigate the effects of PEG (M.W. 6000) on embryogenic callus of Medicago truncatula. Leaf explants were cultured on MS medium with 2 mg L-1 NAA and 0.5 mg L-1 BAP for 5 months. Then, calli were transferred to the same medium further supplemented with 10% (w/v) 6000 PEG for 6 months in order to study physiological and putative molecular markers of water stress. There were no significant differences in growth rate of callus or mitotic index ± PEG although embryogenic potential of PEG treated callus was morphologically enhanced. Cells were rounder on PEG medium and cell size, nuclear size and endoreduplication increased in response to the PEG treatment. Significant increases in soluble sugar and proline accumulation occurred under PEG treatment compared with the control. Significantly, high MtWEE1 and MtCCS52 expression resulted from 6 months of PEG treatment with no significant differences in MtSERK1 or MtP5CS expression but down regulation of MtSOS expression. The results are consistent in showing elevated expression of a cell cycle checkpoint gene, WEE1. It is likely that the cell cycle checkpoint surveillance machinery, that would include WEE1 expression, is ameliorating the effects of the stress imposed by PEG.

From Stress to Embryos: Some of the Problems for Induction and Maturation of Somatic Embryos.

Although somatic embryogenesis has been successfully achieved in numerous plant species, little is known about the mechanism(s) underlying this process. Changes in the balance of growth regulators of the culture medium, osmolarity, or amino acids as well as the genotype and developmental stage of the tissue used as initial explant may have a pivotal influence on the induction of somatic embryogenic cultures. Moreover, different stress agents (ethylene, activated charcoal, cold or heat or electrical shocks), as well as abscisic acid, can also foster the induction or further development of somatic embryos. In the process, cells first return to a stem cell-like status and then either enter their new program or dye when the stress level exceeds cell tolerance. Recalcitrance to differentiation of somatic cells into embryos is frequently observed, and problems such as secondary or recurrent embryogenesis, embryo growth arrest (at the globular stage or during the transition from torpedo to cotyledonary stage), and development of only the aerial part of somatic embryos can appear, interfering with normal germination and conversion of embryos to plants. Some solutions to solve these problems associated to embryogenesis are proposed and two very efficient somatic embryogenesis protocols for two model plant species are detailed.

Beauty will save the world, but will the world save beauty? The case of the highly endangered Vavilovia formosa (Stev.) Fed.

MAIN CONCLUSION: Vavilovia formosa (Stev.) Fed. is a scientifically valuable common ancestor of the plant tribe Fabeae and also important in breeding and agronomy studies of the cultivated Fabeae, but it is close to extinction. A concerted academic and geovernmental effort is needed to save it. Since 2007, an informal international group of researchers on legumes has been working to increase awareness of Vavilovia formosa (Stev.) Fed., a relict and endangered wild-land relative to crop plant species. A majority of the modern botanical classifications place it within the tribe Fabeae, together with the genera vetchling (Lathyrus L.), lentil (Lens Mill.), pea (Pisum L.) and vetch (Vicia L.). V. formosa is encountered at altitudes from 1,500 m up to 3,500 m in Armenia, Azerbaijan, Georgia, Iran, Iraq, Lebanon, Russia, Syria and Turkey. This species may be of extraordinary importance for broadening current scientific knowledge on legume evolution and taxonomy because of its proximity to the hypothetical common ancestor of the tribe Fabeae, as well as for breeding and agronomy of the cultivated Fabeae species due to its perenniality and stress resistance. All this may be feasible only if a concerted and long-term conservation strategy is established and carried out by both academic and geovernmental authorities. The existing populations of V. formosa are in serious danger of extinction. The main threats are domestic and wild animal grazing, foraging, and early frosts in late summer. A long-term strategy to save V. formosa from extinction and to sustain its use in both basic and applied research comprises much improved in situ preservation, greater efforts for an ex situ conservation, and novel approaches of in vitro propagation.

The role of the DNA-binding One Zinc Finger (DOF) transcription factor family in plants.

The DOF (DNA-binding One Zinc Finger) family of transcription factors is involved in many fundamental processes in higher plants, including responses to light and phytohormones as well as roles in seed maturation and germination. DOF transcription factor genes are restricted in their distribution to plants, where they are in many copies in both gymnosperms and angiosperms and also present in lower plants such as the moss Physcomitrella patens and in the alga Chlamydomonas reinhardtii which possesses a single DOF gene. DOF transcription factors bind to their promoter targets at the consensus sequence AAAG. This binding depends upon the presence of the highly conserved DOF domain in the protein. Depending on the target gene, DOF factor binding may activate or repress transcription. DOF factors are expressed in most if not all tissues of higher plants, but frequently appear to be functionally redundant. Recent next-generation sequencing data provide a more comprehensive survey of the distribution of DOF sequence classes among plant species and within tissue types, and clues as to the evolution of functions assumed by this transcription factor family. DOFs do not appear to be implicated in the initial differentiation of the plant body plan into organs via the resolution of meristematic zones, in contrast to MADS-box and homeobox transcription factors, which are found in other non-plant eukaryotes, and this may reflect a more recent evolutionary origin.

Plant cell electrophysiology: applications in growth enhancement, somatic hybridisation and gene transfer.

The use and exploitation of electrophysiology with plant cells have witnessed a slow but steady increase for a number of purposes in recent years. First envisaged only as a tool for the recovery of somatic hybrid plants following protoplast electrofusion, or for transient and/or stable genetic transformation following electroporation-mediated entry of foreign genes into protoplasts and cells, electrophysiological studies with plant cells and tissues have since spanned into other areas, and particularly for the assessment of the possible effects of electric and electromagnetic fields on the subsequent growth and differentiation competences of the electro-treated cells. This review will critically discuss these various applications of electrophysiology and will also aim at analysing the fundamental physiological and physico-chemical mechanisms underlying them.

In vitro auxin treatment promotes cell division and delays endoreduplication in developing seeds of the model legume species Medicago truncatula.

The role of auxins in the morphogenesis of immature seeds of Medicago truncatula was studied, focusing on the transition from the embryo cell division phase to seed maturation. We analyzed seed development in vitro, by flow cytometry, and through the determination of the kinetics of seed fresh weight and size. Thus, seeds were harvested at 8, 10 and 12 days after pollination and cultured in vitro on a medium either without auxin or supplemented with indole-3-butyric acid (IBA) or naphthalene acetic acid (NAA) at 1 mg l(-1). All parameters studied were determined every 2 days from the start of in vitro culture. The results showed that both auxins increased the weight and size of seeds with NAA having a stronger effect than IBA. We further demonstrated that the auxin treatments modulate the transition between mitotic cycles and endocycles in M. truncatula developing seed by favoring sustained cell divisions while simultaneously prolonging endoreduplication, which is known to be the cytogenetical imprint of the transition from the cell division phase to the storage protein accumulation phase during seed development.

Immature seeds and embryos of Medicago truncatula cultured in vitro.

Legumes are an important source of proteins and lipids for food and feed. In addition, they are -environmentally friendly because of their capacity to fix nitrogen through a symbiosis with Rhizobium that permits them to produce abundant proteins even in the absence of nitrogen fertilization. Seed development in plants follows three chronological steps (1) seed coat differentiation, embryo morphogenesis and endosperm development; (2) embryo maturation with storage accumulation and (3) dehydration and the acquisition of desiccation tolerance. Finally, germination occurs when the environmental conditions become favourable. Working with the model legume Medicago truncatula, an in vitro protocol was developed for the culture of immature embryos that permits their development in a way comparable to that observed in plants.In this chapter, the usefulness of this system for investigating embryo development in legumes is outlined.

Stimulation of defense reactions in Medicago truncatula by antagonistic lipopeptides from Paenibacillus sp. strain B2.

With the aim of obtaining new strategies to control plant diseases, we investigated the ability of antagonistic lipopolypeptides (paenimyxin) from Paenibacillus sp. strain B2 to elicit hydrogen peroxide (H2O2) production and several defense-related genes in the model legume Medicago truncatula. For this purpose, M. truncatula cell suspensions were used and a pathosystem between M. truncatula and Fusarium acuminatum was established. In M. truncatula cell cultures, the induction of H2O2 reached a maximum 20 min after elicitation with paenimyxin, whereas concentrations higher than 20 µM inhibited H2O2 induction and this was correlated with a lethal effect. In plant roots incubated with different concentrations of paenimyxin for 24 h before inoculation with F. acuminatum, paenimyxin at a low concentration (ca. 1 µM) had a protective effect and suppressed 95% of the necrotic symptoms, whereas a concentration higher than 10 µM had an inhibitory effect on plant growth. Gene responses were quantified in M. truncatula by semiquantitative reverse transcription-PCR (RT-PCR). Genes involved in the biosynthesis of phytoalexins (phenylalanine ammonia-lyase, chalcone synthase, chalcone reductase), antifungal activity (pathogenesis-related proteins, chitinase), or cell wall (invertase) were highly upregulated in roots or cells after paenimyxin treatment. The mechanisms potentially involved in plant protection are discussed.