The Role of Cytokinins during the Development of Strawberry Flowers and Receptacles.

Cytokinins play a relevant role in flower and fruit development and plant yield. Strawberry fruits have a high commercial value, although what is known as the "fruit" is not a "true" botanical fruit because it develops from a non-reproductive organ (receptacle) on which the true botanical fruits (achenes) are found. Given cytokinins' roles in botanical fruits, it is important to understand their participation in the development of a non-botanical or accessory "fruit". Therefore, in this work, the role of cytokinin in strawberry flowers and fruits was investigated by identifying and exploring the expression of homologous genes for different families that participate in the pathway, through publicly available genomic and expression data analyses. Next, trans-zeatin content in developing flowers and receptacles was determined. A high concentration was observed in flower buds and at anthesis and decreased as the fruit approached maturity. Moreover, the spatio-temporal expression pattern of selected CKX genes was evaluated and detected in receptacles at pre-anthesis stages. The results point to an important role and effect of cytokinins in flower and receptacle development, which is valuable both from a biological point of view and to improve yield and the quality of this fruit.

In Vitro Conversion of Coffea spp. Somatic Embryos in SETIS™ Bioreactor System.

Somatic embryogenesis (SE) is an excellent example of mass plant propagation. Due to its genetic variability and low somaclonal variation, coffee SE has become a model for in vitro propagation of woody species, as well as for large-scale production of vigorous plants that are advantageous to modern agriculture. The success of the large-scale propagation of an embryogenic system is dependent on the development, optimization, and transfer of complementary system technologies. In this study, two successful SE systems were combined with a SETIS™ bioreactor immersion system to develop an efficient and cost-effective approach for the in vitro development of somatic embryos of Coffea spp. This study used an efficient protocol for obtaining somatic embryos, utilizing direct and indirect SE for both C. canephora and C. arabica. Embryos in the cotyledonary stage were deposited in a bioreactor to complete their stage of development from embryo to plant with minimal manipulation. Following ten weeks of cultivation in the bioreactor, complete and vigorous plants were obtained. Different parameters such as fresh weight, length, number of leaves, and root length, as well as stomatal index and relative water content, were recorded. In addition, the survival rate and ex vitro development of plantlets during acclimatization was assessed. The best substrate combination was garden soil (GS), peat moss (PM), and agrolite (A) in a 1:1:0.5 ratio, in which the bioreactor-regenerated plants showed an acclimatization rate greater than 90%. This is the first report on the use of SETIS™ bioreactors for the in vitro development of somatic embryos in Coffea spp., providing a technology that could be utilized for the commercial in vitro propagation of coffee plants. A link between research and innovation is necessary to establish means of communication that facilitate technology transfer. This protocol can serve as a basis for the generation and scaling of different species of agroeconomic importance. However, other bottlenecks in the production chains and the field must be addressed.

Auxin-Cytokinin Cross Talk in Somatic Embryogenesis of Coffea canephora.

Cytokinins (CK) are plant growth regulators involved in multiple physiological processes in plants. One less studied aspect is CK homeostasis (HM). The primary genes related to HM are involved in biosynthesis (IPT), degradation (CKX), and signaling (ARR). This paper demonstrates the effect of auxin (Aux) and CK and their cross talk in a Coffea canephora embryogenic system. The transcriptome and RT-qPCR suggest that Aux in pre-treatment represses biosynthesis, degradation, and signal CK genes. However, in the induction, there is an increase of genes implicated in the CK perception/signal, indicating perhaps, as in other species, Aux is repressing CK, and CK are inducing per se genes involved in its HM. This is reflected in the endogenous concentration of CK; pharmacology experiments helped study the effect of each plant growth regulator in our SE system. We conclude that the Aux-CK balance is crucial to directing somatic embryogenesis in C. canephora.

Differences in the Abundance of Auxin Homeostasis Proteins Suggest Their Central Roles for In Vitro Tissue Differentiation in Coffea arabica.

Coffea arabica is one of the most important crops worldwide. In vitro culture is an alternative for achieving Coffea regeneration, propagation, conservation, genetic improvement, and genome editing. The aim of this work was to identify proteins involved in auxin homeostasis by isobaric tandem mass tag (TMT) and the synchronous precursor selection (SPS)-based MS3 technology on the Orbitrap Fusion™ Tribrid mass spectrometer™ in three types of biological materials corresponding to C. arabica: plantlet leaves, calli, and suspension cultures. Proteins included in the ß-oxidation of indole butyric acid and in the signaling, transport, and conjugation of indole-3-acetic acid were identified, such as the indole butyric response (IBR), the auxin binding protein (ABP), the ATP-binding cassette transporters (ABC), the Gretchen-Hagen 3 proteins (GH3), and the indole-3-acetic-leucine-resistant proteins (ILR). A more significant accumulation of proteins involved in auxin homeostasis was found in the suspension cultures vs. the plantlet, followed by callus vs. plantlet and suspension culture vs. callus, suggesting important roles of these proteins in the cell differentiation process.

YUCCA-Mediated Biosynthesis of the Auxin IAA Is Required during the Somatic Embryogenic Induction Process in Coffea canephora.

Despite the existence of considerable research on somatic embryogenesis (SE), the molecular mechanism that regulates the biosynthesis of auxins during the SE induction process remains unknown. Indole-3-acetic acid (IAA) is an auxin that is synthesized in plants through five pathways. The biosynthetic pathway most frequently used in this synthesis is the conversion of tryptophan to indol-3-pyruvic acid (IPA) by tryptophan aminotransferase of Arabidopsis (TAA) followed by the conversion of IPA to IAA by enzymes encoded by YUCCA (YUC) genes of the flavin monooxygenase family; however, it is unclear whether YUC-mediated IAA biosynthesis is involved in SE induction. In this study, we report that the increase of IAA observed during SE pre-treatment (plants in MS medium supplemented with 1-naphthaleneacetic acid (NAA) 0.54 µM and kinetin (Kin) 2.32 µM for 14 days) was due to its de novo biosynthesis. By qRT-PCR, we demonstrated that YUC gene expression was consistent with the free IAA signal found in the explants during the induction of SE. In addition, the use of yucasin to inhibit the activity of YUC enzymes reduced the signal of free IAA in the leaf explants and dramatically decreased the induction of SE. The exogenous addition of IAA restored the SE process in explants treated with yucasin. Our findings suggest that the biosynthesis and localization of IAA play an essential role during the induction process of SE in Coffea canephora.

Auxin Immunolocalization in Coffea canephora Tissues.

Auxins are plant growth regulators that participate in a variety of biological mechanisms during the growth and development of plants. The most abundant natural auxin is indole-3-acetic acid (IAA). The physiological processes regulated by IAA depend on their temporal space accumulation in different tissues of a plant. This accumulation is regulated by its biosynthesis, conjugation, degradation, and transport. Therefore tools that allow us a qualitative and quantitative detection of IAA in plant tissues are very useful to understand the homeostasis of IAA during the life cycle of plants. In this protocol, the complete procedure for localization of IAA in different tissues of Coffea canephora is described using specific anti-IAA monoclonal antibodies.

Induction of Somatic Embryogenesis in Jatropha curcas.

Jatropha curcas has been a promising crop for biofuel production for the last decade. However, the lack of resistant materials to diseases and improved quality of the oil produced by the seeds has restricted the use of this promising crop. The genetic modifications in the fatty acid pathway, as well as the introduction of resistance to different diseases, would change the fate of Jatropha. To achieve these goals, we need to have a very efficient regeneration system. Here, we report a very useful protocol to induce somatic embryogenesis from leaves of Jatropha using cytokinin as the only growth regulator.

Elaboration of Transcriptome During the Induction of Somatic Embryogenesis.

Somatic embryogenesis (SE) is one of the most studied developmental processes due to its applications, such as plant micropropagation, transformation, and germplasm conservation. The use of massive techniques of sequencing, as well as the use of subtractive hybridization and macroarrays, has led to the identification of hundreds of genes involved in the SE process. These have been important developments to study the molecular aspects of the progress of SE. With the advent of the new massive techniques for sequencing RNA, it has been possible to see a more complete picture of whole processes. In this chapter we present a technique to handle the elaboration of the transcriptome from the extraction of RNA until the assembly of the complete transcriptome.

The role of chromatin modifications in somatic embryogenesis in plants.

Somatic embryogenesis (SE) is a powerful tool for plant genetic improvement when used in combination with traditional agricultural techniques, and it is also an important technique to understand the different processes that occur during the development of plant embryogenesis. SE onset depends on a complex network of interactions among plant growth regulators, mainly auxins and cytokinins, during the proembryogenic early stages, and ethylene and gibberellic and abscisic acids later in the development of the somatic embryos. These growth regulators control spatial and temporal regulation of multiple genes in order to initiate change in the genetic program of somatic cells, as well as moderating the transition between embryo developmental stages. In recent years, epigenetic mechanisms have emerged as critical factors during SE. Some early reports indicate that auxins and in vitro conditions modify the levels of DNA methylation in embryogenic cells. The changes in DNA methylation patterns are associated with the regulation of several genes involved in SE, such as WUS, BBM1, LEC, and several others. In this review, we highlight the more recent discoveries in the understanding of the role of epigenetic regulation of SE. In addition, we include a survey of different approaches to the study of SE, and new opportunities to focus SE studies.

Somatic Embryogenesis: Identified Factors that Lead to Embryogenic Repression. A Case of Species of the Same Genus.

Somatic embryogenesis is a powerful biotechnological tool for the mass production of economically important cultivars. Due to the cellular totipotency of plants, somatic cells under appropriate conditions are able to develop a complete functional embryo. During the induction of somatic embryogenesis, there are different factors involved in the success or failure of the somatic embryogenesis response. Among these factors, the origin of the explant, the culture medium and the in vitro environmental conditions have been the most studied. However, the secretion of molecules into the media has not been fully addressed. We found that the somatic embryogenesis of Coffea canephora, a highly direct embryogenic species, is disrupted by the metabolites secreted from C. arabica, a poorly direct embryogenic species. These metabolites also affect DNA methylation. Our results show that the abundance of two major phenolic compounds, caffeine and chlorogenic acid, are responsible for inhibiting somatic embryogenesis in C. canephora.

Callus, suspension culture, and hairy roots. Induction, maintenance and characterization.

The growth is a characteristic of each culture and it is determinate by the origin of the species, culture conditions, and type of culture. In this chapter, we make a comparison of the different growth parameters among three different species and three different types of cultures.

Methyl jasmonate induces ATP biosynthesis deficiency and accumulation of proteins related to secondary metabolism in Catharanthus roseus (L.) G. hairy roots.

Jasmonates are specific signal molecules in plants that are involved in a diverse set of physiological and developmental processes. However, methyl jasmonate (MeJA) has been shown to have a negative effect on root growth and, so far, the biochemical mechanism for this is unknown. Using Catharanthus roseus hairy roots, we were able to observe the effect of MeJA on growth inhibition, cell disorganization and cell death of the root cap. Hairy roots treated with MeJA induced the perturbation of mitochondrial membrane integrity and a diminution in ATP biosynthesis. Furthermore, several proteins were identified that were involved in energy and secondary metabolism; the changes in accumulation of these proteins were observed with 100 µM MeJA. In conclusion, our results suggest that a switch of the metabolic fate of hairy roots in response to MeJA could cause an increase in the accumulation of secondary metabolites. This is likely to have important consequences in the production of specific alkaloids important for the pharmaceutical industry.

Differential secretion and accumulation of terpene indole alkaloids in hairy roots of Catharanthus roseus treated with methyl jasmonate.

The induction of several secondary metabolites in plants is one of the most commonly observed effects after the external addition of methyl jasmonate (MeJA). After the elicitation of Catharanthus roseus hairy roots with different concentrations of MeJA, changes in the accumulation of alkaloids such as ajmalicine, serpentine, ajmaline and catharanthine were observed. In addition to the increased accumulation of alkaloids in the tissues, the root exudation of phytochemicals increased compared to that of the non-treated control hairy roots. Moreover, MeJA induced differential secretion of several C. roseus hairy root metabolites.

Possible role of light and polyamines in the onset of somatic embryogenesis of Coffea canephora.

The concentration of free and bound polyamines was studied during the somatic embryogenesis induction process in Coffea canephora explants. In the present study we show that when the induction of somatic embryogenesis in C. canephora is carried out under light conditions and in the presence of the plant growth regulator, benzylaminopurine, a cytokinin, a faster response to induction is obtained. In the darkness, the response is delayed for more than 20 days, and the number of embryos is smaller. In the absence of benzylaminopurine no embryogenic response was observed. The pronounced changes in the levels of putrescine, spermidine, and spermine, both free and bound, found in C. canephora suggest that a close correlation exists between polyamine biosynthesis and somatic embryogenesis in C. canephora during a period of cellular differentiation associated with the induction of somatic embryogenesis.

Direct somatic embryogenesis in Coffea canephora.

Somatic embryogenesis (SE) provides a useful model to study embryo development in plants. In contrast to zygotic embryogenesis, SE can easily be observed, the culture conditions can be controlled, and large quantities of embryos can be easily obtained. In Coffea spp several model systems have been reported for in vitro SE induction. SE for coffee was first reported in Coffea canephora. Several systems have been developed since then, including SE from callus cultures derived from leaf explants; a two-phase experimental protocol for SE from leaves of Coffea arabica; and from leaf explants of Arabusta or C. arabica using a medium with cytokinins. Here we report a protocol using young leaves from in vitro seedling pre-conditioned with growth regulators. This is a simplified method to obtain a faster and more efficient protocol to produce direct somatic embryos in C. canephora.