Increased Multiplication Rates of Vriesea hieroglyphica (Carriere) E. Morren Through a Temporary Immersion System.

The main difficulty for the cultivation and conservation of bromeliad species is the reduced number of propagules and slow growth of many of the species, resulting in a low propagation efficiency. Bromeliad plants are hardy and relatively easy to cultivate, with a high ornamental and ecological importance. Aiming at efficient micropropagation rates of V. hieroglyphica, a highly valued bromeliad, with very low propagation efficiency, a temporary immersion system was used and compared to semisolid and liquid static medium. Cultures obtained from in vitro germinated seeds were used as explants, maintaining their genetic diversity. Micropropagation with this simple temporary immersion system, composed of two autoclavable flasks, each with one opening for the attachment of 22 µm syringe filters, connected by a rubber stopper and an inner glass tube. In the bottom flask, an air valve is attached to the filter, which is subsequently connected to an aquarium pump and a timer and plugged to an outlet. This simple temporary immersion system showed improved micropropagation efficiency and is a method that can also be evaluated for other species.

Alstroemeria Micropropagation in a RITA® Temporary Immersion System.

The use of temporary immersion systems (TIS) for plant micropropagation is an efficient technique for plant production, and we have applied it for the production of alstroemerias. This method involves the cultivation of explants such as rhizomes and axillary buds in a nutrient medium to stimulate shoot growth. TIS offer advantages such as accelerated multiplication processes, uniform production, and cost reduction. This process has shown promise in meeting the growing demand for alstroemeria plants in the market. This chapter describes a specific protocol for temporary immersion bioreactor micropropagation of the "Albatroz" cultivar, with the potential for large-scale automation.

Global gene expression profile and functional analysis reveal the conservation of reproduction-associated gene networks in Gossypium hirsutum.

KEY MESSAGE: Lastly, the bZIP gene family encompasses genes that have been reported to play a role in flower development, such as bZIP14 (FD). Notably, bZIP14 is essential for Flowering Locus T (FT) initiation of floral development in Arabidopsis (Abe et al. 2005). Cotton (Gossypium hirsutum L.) is the world's most extensively cultivated fiber crop. However, its reproductive development is poorly characterized at the molecular level. Thus, this study presents a detailed transcriptomic analysis of G. hirsutum at three different reproductive stages. We provide evidence that more than 64,000 genes are active in G. hirsutum during flower development, among which 94.33% have been assigned to functional terms and specific pathways. Gene set enrichment analysis (GSEA) revealed that the biological process categories of floral organ development, pollen exine formation, and stamen development were enriched among the genes expressed during the floral development of G. hirsutum. Furthermore, we identified putative Arabidopsis homologs involved in the G. hirsutum gene regulatory network (GRN) of pollen and flower development, including transcription factors such as WUSCHEL (WUS), INNER NO OUTER (INO), AGAMOUS-LIKE 66 (AGL66), SPOROCYTELESS/NOZZLE (SPL/NZZ), DYSFUNCTIONAL TAPETUM 1 (DYT1), ABORTED MICROSPORES (AMS), and ASH1-RELATED 3 (ASHR3), which are known crucial genes for plant reproductive success. The cotton MADS-box protein-protein interaction pattern resembles the previously described patterns for AGAMOUS (AG), SEEDSTICK (STK), SHATTERPROOF (SHP), and SEPALLATA3 (SEP3) homolog proteins from Arabidopsis. In addition to serving as a resource for comparative flower development studies, this work highlights the changes in gene expression profiles and molecular networks underlying stages that are valuable for cotton breeding improvement.

Water retention and metabolic changes improve desiccation tolerance in Barbacenia graminifolia (Velloziaceae).

Barbacenia graminifolia is a Velloziaceae species endemic to the campos rupestres in Serra do Cipó, Minas Gerais state (Brazil). This biome is characterised by high irradiance and limited water conditions. Unlike other resurrection plants, B. graminifolia can maintain a high hydric status (>80%) after 28 days of water suppression before desiccation. We investigated the physiological and metabolic mechanisms associated with structural changes that allow B. graminifolia to maintain hydration under a prolonged water deficit and to recover after desiccation. After 30 days of water deficit, desiccated plants exhibited chlorophyll degradation, a 178.4% and 193.7% increase in total carotenoids and MDA, respectively, and twice the CAT and APX activity compared to hydrated plants. The metabolite profile showed increased amino acids, carbohydrates, saturated fatty acids and benzoic acids during dehydration, while trichloroacetic acid cycle acids were higher in hydrated and rehydrated plants. Anatomical and ultrastructural data corroborated the physiological and metabolic changes and revealed the presence of mucilaginous cells with high water retention capacity. Our data indicated that combined strategies of assimilatory metabolism shutdown, accumulation of compatible solutes and antioxidant compounds, increase in hydrophilic molecules, changes in the composition of membrane lipids and remodelling of cell organelles conditioned the efficiency of B. graminifolia in delaying water loss, tolerating further desiccation and quickly recovering after rehydration. These attributes evidence that this species is well adapted to cope with adverse environmental conditions, mainly directing the metabolism to an efficient antioxidant response and improving its capacity to retain water during the dry season.

Citrus Protoplast Isolation and Plant Regeneration Through Somatic Embryogenesis.

Protoplasts are an attractive explant source for biotechnological tools widely used on citrus genetic improvement, such as somatic hybridization and direct genetic transformation. These delicate and responsive materials are subjected to cell proliferation induction and differentiation of somatic embryos which further regenerate into entire plants. The isolation of viable protoplasts followed by regeneration of plants through somatic embryogenesis is an important methodology for breeding applications. The methods presented here can also be used as a reference for protoplast work in other species, followed by protocol optimization for different species/genotypes.

Somatic Embryogenesis in Citrus (Citrus spp.), var. Valencia.

Somatic embryogenesis has been obtained in many citrus cultivars; however, the efficiency of the system is genotype dependent and culture synchronization is important to reach more efficient systems. In this chapter we present a detailed protocol of somatic embryogenesis induction from nucellar tissue and the use of an alternative method of callus sieving for culture synchronization and embryo production. This is a simple method which can also be evaluated for other species aiming at better culture efficiency and somatic embryo production.

Somatic Embryogenesis of Brachiaria brizantha (Syn. Urochloa brizantha) Analyzed by In Situ Hybridization.

In situ hybridization with mRNA probes enables the detection and localization of gene expression in plant somatic embryogenesis samples. BbrizSERK is a gene that is expressed in embryogenic cells and tissues of Brachiaria. Here we describe methods used for in situ hybridization to localize BbrizSERK transcripts during somatic embryogenesis of Brachiaria brizantha according to the plant material and observations intended, using paraffin or butyl methyl methacrylate resin-embedded samples, as well as a method for whole-mount preparation applicable for the analysis of other genes involved in embryogenic processes, along with other in vitro processes.

Comparing soil-to-plant cadmium (Cd) transfer and potential human intake among rice cultivars with different Cd tolerance levels grown in a tropical contaminated soil.

With its accumulation in upland rice, cadmium (Cd) can easily enter the human food chain, which poses a global health threat considering nearly half of the human population depends on rice as a staple food source. A study was conducted to (1) evaluate Cd accumulation by rice cultivars, grown in Cd-polluted Tropical Oxisols, with different levels of Cd tolerance; (2) quantify Cd transfer from soil to rice shoots and grain; and (3) estimate daily Cd intake by humans. Three rice cultivars, characterized by low (Cateto Seda-CS), medium (BRSMG Talento-BT), and high (BRSMG Caravera-BC) Cd uptake capacity, were investigated. Rice cultivars were exposed to increasing soil Cd concentrations (0.0, 0.7, 1.3, 3.9, 7.8, and 11.7 mg kg-1). Analysis was performed on soil, shoots, and grain. Shoot biomass and grain yield decreased with increasing Cd supply, suggesting the following Cd tolerance: CS > BT > BC. Cadmium concentrations in shoots and grain increased when exposed to Cd. Only CS did not exceed the maximum Cd limit permitted in food (0.40 mg kg-1), when rates up to 1.3 mg kg-1 of Cd were applied to soil. Considering daily rice consumption levels in Brazil, Cd intake often exceeds maximum tolerable levels. Continuous monitoring of soil Cd concentrations is a pivotal step in avoiding hazards to humans. Such monitoring is important on a global scale since outside of Asia, Brazil is the leading rice-producing and rice-consuming country.

Characterization of floral morphoanatomy and identification of marker genes preferentially expressed during specific stages of cotton flower development.

MAIN CONCLUSION: Characterization of anther and ovule developmental programs and expression analyses of stage-specific floral marker genes in Gossypium hirsutum allowed to build a comprehensive portrait of cotton flower development before fiber initiation. Gossypium hirsutum is the most important cotton species that is cultivated worldwide. Although cotton reproductive development is important for fiber production, since fiber is formed on the epidermis of mature ovules, cotton floral development remains poorly understood. Therefore, this work aims to characterize the cotton floral morphoanatomy by performing a detailed description of anther and ovule developmental programs and identifying stage-specific floral marker genes in G. hirsutum. Using light microscopy and scanning electron microscopy, we analyzed anther and ovule development during 11 stages of flower development. To better characterize the ovule development in cotton, we performed histochemical analyses to evaluate the accumulation of phenolic compounds, pectin, and sugar in ovule tissues. After identification of major hallmarks of floral development, three key stages were established in G. hirsutum floral development: in stage 1 (early-EF), sepal, petal, and stamen primordia were observed; in stage 2 (intermediate-IF), primordial ovules and anthers are present, and the differentiating archesporial cells were observed, marking the beginning of microsporogenesis; and in stage 6 (late-LF), flower buds presented initial anther tapetum degeneration and microspore were released from the tetrad, and nucellus and both inner and outer integuments are developing. We used transcriptome data of cotton EF, IF and LF stages to identify floral marker genes and evaluated their expression by real-time quantitative PCR (qPCR). Twelve marker genes were preferentially expressed in a stage-specific manner, including the putative homologs for AtLEAFY, AtAPETALA 3, AtAGAMOUS-LIKE 19 and AtMALE STERILITY 1, which are crucial for several aspects of reproductive development, such as flower organogenesis and anther and petal development. We also evaluated the expression profile of B-class MADS-box genes in G. hirsutum floral transcriptome (EF, IF, and LF). In addition, we performed a comparative analysis of developmental programs between Arabidopsis thaliana and G. hirsutum that considered major morphoanatomical and molecular processes of flower, anther, and ovule development. Our findings provide the first detailed analysis of cotton flower development.

SERK genes identification and expression analysis during somatic embryogenesis and sporogenesis of sexual and apomictic Brachiaria brizantha (Syn. Urochloa brizantha).

MAIN CONCLUSION: In Brachiaria brizantha BbrizSERK1, BbrizSERK2 and BbrizSERK3 were identified. SERK expression marks somatic embryogenesis, sexual MMC, and sexual and apomictic PMC. BbrizSERK3 might have a regulatory role in reproductive development. Somatic embryogenesis receptor-like kinase (SERK) consists of plasma membrane receptor genes that have been characterized in various species, associated with several aspects of plant development, including reproduction. SERK genes are involved in anther development and in early embryo development in sexual and asexual seed formation. To comprehend the complexity of the SERK genes and their function in Brachiaria reproduction, we performed a homology-based search in a genomic database of a sexual B. brizantha and identified sequences of three SERK genes, BbrizSERK1, BbrizSERK2, and BbrizSERK3. RNASeq data showed equivalent abundance of BbrizSERK1 and BbrizSERK2 transcripts in ovaries at early megasporogenesis of sexuals and apomicts, while BbrizSERK3 transcripts were more abundant in ovaries of sexuals than in apomicts. BbrizSERK3 results in three coding sequences due to alternative splicing, among them Variant 1 results in a protein with all the predicted domains of a SERK. BbrizSERK transcripts were detected in male reproductive tissues of both sexual and apomictic plants, suggesting a role in controlling anther development. BbrizSERK transcripts were detected early in ovule development, in the integuments, and in the megaspore mother cell of the sexual plant, but not in the cells that give rise to apomictic embryo sacs, suggesting a role in female reproductive development of sexuals. This paper provides evidences that SERK genes plays a role in the onset and establishment of somatic embryogenesis and in the reproductive development of B. brizantha and suggests a distinct role of BbrizSERK in apomixis initiation.

Selenium improves photosynthesis and induces ultrastructural changes but does not alleviate cadmium-stress damages in tomato plants.

The application of Se to plants growing under Cd contamination may become an alternative strategy to minimize Cd damage. However, there is no specific information available regarding whether Se can affect the anatomical structure and photosynthetic rates of plants under Cd stress. To address questions related to Se-protective responses under Cd stress, we evaluated the structural and ultrastructural aspects, photosynthetic rates and growth of tomato cv. Micro-Tom plants. Plants were exposed to 0.5 mM CdCl2 and further supplemented with 1.0 µM of selenite or selenate. The overall results revealed different trends according to the Se source and Cd application. Both Se sources improved growth, photosynthesis, leaf characteristics and middle lamella thickness between mesophyll cells. In contrast, Cd caused decreases in photosynthesis and growth and damage to the ultrastructure of the chloroplast. The number of mitochondria, peroxisomes, starch grains and plastogloboli and the disorganization of the thylakoids and the middle lamella in plants increased in the presence of Cd or Cd + Se. Se plays an important role in plant cultivation under normal conditions. This finding was corroborated by the identification of specific structural changes in Se-treated plants, which could benefit plant development. However, a reversal of Cd stress effects was not observed in the presence of Se.

Aluminum-induced toxicity in Urochloa brizantha genotypes: A first glance into root Al-apoplastic and -symplastic compartmentation, Al-translocation and antioxidant performance.

Previous studies have unraveled contrasting Al genotypic differences between Urochloa brizantha cv. Marandu (moderately tolerant) and Urochloa brizantha cv. Xaraés (more tolerant). Our objective was to evaluate differences in the response to Al-induced stress between these genotypes, focusing on Al compartmentation in the root apoplast and symplast, and antioxidant enzyme activities after Al exposure. Al-accumulation was 25% higher in the roots of cv. Xaraés than cv. Marandu, while in the shoot Al accumulation was 150% higher in cv. Marandu than cv. Xaraés. U. brizantha cv. Marandu accumulated 73% of the Al absorbed in the root symplast and 27% in the root apoplast, while cv. Xaraés accumulated 61% of the Al absorbed in symplast and 39% in apoplast. Furthermore, Al exposure leaded to physiological and developmental changes in root morphology, such as disorganization of vascular system, the collapse of cortical cells and absence of root hairs from the root tip, with more drastic effects detectable in cv. Marandu. Catalase (CAT) and guaiacol peroxidase (GPOX) activities in the roots of cv. Marandu were lower compared to cv. Xaraés. Our results pointed out that higher Al compartmentalization rates in the root apoplast, altogether with up-regulated metabolic activities of CAT and GPOX and also lower long distance transport of Al are seemingly at the base of the Al tolerance in cv. Xaraés. In conclusion, biochemical analysis of roots suggested that understanding of metabolic pathways is one of pressing approach to elucidate stress tolerance mechanisms in this genus.

Relationship between Mg, B and Mn status and tomato tolerance against Cd toxicity.

Distinct tomato genotypes possess different tolerance degree to cadmium (Cd), but the mechanisms behind this phenomenon are scarcely understood. To this end, the physiological, biochemical, anatomical, nutritional and molecular mechanisms associated to the plant tolerance against Cd toxicity were investigated in five tomato accessions with contrasting sensitivity to Cd exposure. Firstly, the data revealed that larger biomass loss was not always coupled to higher Cd concentration, indicating that other events, in addition to the internal Cd accumulation, impact tomato performance at early stages of Cd exposure. Secondly, the results indicated that the fine regulation of nutrient status, particularly magnesium (Mg), boron (B) and manganese (Mn), is associated to the mitigation of Cd toxicity. Magnesium status was coupled to the modulation of root development, resulting in changes in root hair formation and biomass allocation. Boron accumulation in leaves was linked to Cd toxicity, suggesting that tolerance mechanisms involved strategies to decrease or even avoid B excess in photosynthetic tissues. Disturbances in Mn status, i.e. Mn excess in leaves and Mn deficiency in roots, were also related to tomato sensitivity to Cd exposure. Thirdly, plant capacity to maintain leaf blade expansion is a relevant strategy for a better tomato development after short-term Cd exposure. Fourthly, tomato tolerance to Cd-induced stress does not depend on CAT activity enhancements in such conditions. In conclusion, tomato ability to quickly manage its nutritional status is necessary for alleviation of the Cd effects at early stages of exposure to this metal. The better understanding about tolerance mechanisms and mode of action of Cd toxicity in plants can help in the establishment of strategies to mitigate its impacts on crops.

FT/TFL1: Calibrating Plant Architecture.

There is a very large diversity in plant architecture in nature. Over the past few years, novel theoretical concepts and analytical methods have emerged as powerful tools to understand important aspects of plant architecture. Plant architecture depends on the relative arrangement of three types of organs: leaves, shoots, and flowers. During plant development, the architecture is modulated by the balance of two homologous proteins: FLOWERING LOCUS T (FT) and TERMINAL FLOWER 1 (TFL1). The FT/TFL1 balance defines the plant growth habit as indeterminate or determinate by modulating the pattern of formation of vegetative and reproductive structures in the apical and axillary meristems. Here, we present a summarized review of plant architecture and primarily focus on the FT/TFL1 balance and its effect on plant form and development. We also propose passion fruit as a suitable model plant to study the effect of FT/TFL1 genes on plant architecture.

Re-evaluation of transcription factor function in tomato fruit development and ripening with CRISPR/Cas9-mutagenesis.

Tomato (Solanum lycopersicum) is a model for climacteric fleshy fruit ripening studies. Tomato ripening is regulated by multiple transcription factors together with the plant hormone ethylene and their downstream effector genes. Transcription Factors APETALA2a (AP2a), NON-RIPENING (NOR) and FRUITFULL (FUL1/TDR4 and FUL2/MBP7) were reported as master regulators controlling tomato fruit ripening. Their proposed functions were derived from studies of the phenotype of spontaneous mutants or RNAi knock-down lines rather than, as it appears now, actual null mutants. To study TF function in tomato fruit ripening in more detail, we used CRISPR/Cas9-mediated mutagenesis to knock out the encoding genes, and phenotypes of these mutants are reported for the first time. While the earlier ripening, orange-ripe phenotype of ap2a mutants was confirmed, the nor null mutant exhibited a much milder phenotype than the spontaneous nor mutant. Additional analyses revealed that the severe phenotype in the spontaneous mutant is caused by a dominant-negative allele. Our approach also provides new insight into the independent and overlapping functions of FUL1 and FUL2. Single and combined null alleles of FUL1 and FUL2 illustrate that these two genes have partially redundant functions in fruit ripening, but also unveil an additional role for FUL2 in early fruit development.

Transcriptome analysis of Gossypium hirsutum flower buds infested by cotton boll weevil (Anthonomus grandis) larvae.

BACKGROUND: Cotton is a major fibre crop grown worldwide that suffers extensive damage from chewing insects, including the cotton boll weevil larvae (Anthonomus grandis). Transcriptome analysis was performed to understand the molecular interactions between Gossypium hirsutum L. and cotton boll weevil larvae. The Illumina HiSeq 2000 platform was used to sequence the transcriptome of cotton flower buds infested with boll weevil larvae. RESULTS: The analysis generated a total of 327,489,418 sequence reads that were aligned to the G. hirsutum reference transcriptome. The total number of expressed genes was over 21,697 per sample with an average length of 1,063 bp. The DEGseq analysis identified 443 differentially expressed genes (DEG) in cotton flower buds infected with boll weevil larvae. Among them, 402 (90.7%) were up-regulated, 41 (9.3%) were down-regulated and 432 (97.5%) were identified as orthologues of A. thaliana genes using Blastx. Mapman analysis of DEG indicated that many genes were involved in the biotic stress response spanning a range of functions, from a gene encoding a receptor-like kinase to genes involved in triggering defensive responses such as MAPK, transcription factors (WRKY and ERF) and signalling by ethylene (ET) and jasmonic acid (JA) hormones. Furthermore, the spatial expression pattern of 32 of the genes responsive to boll weevil larvae feeding was determined by "in situ" qPCR analysis from RNA isolated from two flower structures, the stamen and the carpel, by laser microdissection (LMD). CONCLUSION: A large number of cotton transcripts were significantly altered upon infestation by larvae. Among the changes in gene expression, we highlighted the transcription of receptors/sensors that recognise chitin or insect oral secretions; the altered regulation of transcripts encoding enzymes related to kinase cascades, transcription factors, Ca2+ influxes, and reactive oxygen species; and the modulation of transcripts encoding enzymes from phytohormone signalling pathways. These data will aid in the selection of target genes to genetically engineer cotton to control the cotton boll weevil.

Brassinosteroids interact negatively with jasmonates in the formation of anti-herbivory traits in tomato.

Given the susceptibility of tomato plants to pests, the aim of the present study was to understand how hormones are involved in the formation of tomato natural defences against insect herbivory. Tomato hormone mutants, previously introgressed into the same genetic background of reference, were screened for alterations in trichome densities and allelochemical content. Ethylene, gibberellin, and auxin mutants indirectly showed alteration in trichome density, through effects on epidermal cell area. However, brassinosteroids (BRs) and jasmonates (JAs) directly affected trichome density and allelochemical content, and in an opposite fashion. The BR-deficient mutant dpy showed enhanced pubescence, zingiberene biosynthesis, and proteinase inhibitor expression; the opposite was observed for the JA-insensitive jai1-1 mutant. The dpy x jai1-1 double mutant showed that jai1-1 is epistatic to dpy, indicating that BR acts upstream of the JA signalling pathway. Herbivory tests with the poliphagous insect Spodoptera frugiperda and the tomato pest Tuta absoluta clearly confirmed the importance of the JA-BR interaction in defence against herbivory. The study underscores the importance of hormonal interactions on relevant agricultural traits and raises a novel biological mechanism in tomato that may differ from the BR and JA interaction already suggested for Arabidopsis.