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The initial stimulation of photosynthesis under elevated CO2 concentrations (eCO2) is often followed by a decline in photosynthesis, known as CO2 acclimation. Changes in N levels under eCO2 can have different effects in plants fertilized with nitrate (NO3−) or ammonium (NH4+) as the N source. NO3− assimilation consumes approximately 25% of the energy produced by an expanded leaf, whereas NH4+ requires less energy to be incorporated into organic compounds. Although plant-N interactions are important for the productivity and nutritional value of food crops worldwide, most studies have not compared the performance of plants supplied with different forms of N. Therefore, this study aims to go beyond treating N as the total N in the soil or the plant because the specific N compounds formed from the available N forms become highly engaged in all aspects of plant metabolism. To this end, plant N metabolism was analyzed through an experiment with eCO2 and fertigation with NO3− and/or NH4+ as N sources for tobacco (Nicotiana tabacum) plants. The results showed that the plants that received only NO3− as a source of N grew more slowly when exposed to a CO2 concentration of 760 μmol mol−1 than when they were exposed to ambient CO2 conditions. On the other hand, in plants fertigated with only NH4+, eCO2 enhanced photosynthesis. This was essential for the maintenance of the metabolic pathways responsible for N assimilation and distribution in growing tissues. These data show that the physiological performance of tobacco plants exposed to eCO2 depends on the form of inorganic N that is absorbed and assimilated.

Plant somatic embryogenesis (PSE) provides several advantages when compared to other in vitro propagation methods of tissue culture. All factors affecting PSE are not known. Some prominent stress factors in tissue culture are serious injuries in explants, subcultures, unbalanced mineral composition of the culture medium and growth regulators, etc. The present review would focus on induction Reactive Oxygen Species (ROS), which lies fairly downstream of the cascade of various stress processes outlined above. The central question the present authors ask is—whether ROS generation is all for toxic or there is some amount of benefit to the somatic embryogenesis. The increasing interest in the functional meaning of ROS and the antioxidant response concomitant to growth, development and cell differentiation in plants suggest a link between ROS production and morphogenetic processes of plants. The authors in this review article consider hydrogen peroxide (H2O2) as a model ROS which is omnipresent and naturally generated in a variety of normal cell types, either constitutively or in response to various stimuli. A review of the concerned literature suggests that endogenous H2O2 acts as a cellular ‘messenger’ capable of inducing gene expression and protein synthesis, thus leading to somatic embryogenesis in some plant species.

In Brazil, Lycium barbarum L. (goji berry) is an agronomically valuable, imported, commercialized plant. The present study evaluated the effects of cytokinins and light sources on optimization of in vitro multiplication of goji berry by axillary bud proliferation. Nodal explants were excised from plants 90 d after in vitro germination and cultured on Murashige and Skoog (MS) medium containing 30 g L−1 sucrose; 7 g L−1 agar-agar; and 0, 2, 4, 8, or 16 μM 6-benzylaminopurine (BAP), 6-furfurylaminopurine (KIN), or thidiazuron (TDZ). After 60 days, regenerated lateral buds and shoot lengths were evaluated. The regenerated shoots were inoculated in MS supplemented with 0.0, 2.5, 5.0, 10.0, or 20.0 μM BAP under white fluorescent lamps or mixed treatment of red/blue (RB) light-emitting diode (LED) lamps. After 60 days in vitro cultivation, the number of formed shoots, leaf number, and shoot length were determined. Subsequently, the shoots were separated and acclimatized in a greenhouse for 30 d and the percentage of survival was evaluated. High regeneration and shoot numbers during lateral bud regeneration occurred with MS medium plus 4 μM BAP. The estimated optimum concentration of 11.56 μM BAP under RB LED lamp resulted in a high shoot number (8.15), with more average leaves per plant (9.2) at 5 μM BAP, and longer shoots (12.34 cm) at 12.42 μM BAP. Acclimatization was successful with 100% survival of ex vitro plants. An efficient in vitro multiplication protocol of an economically important species, goji berry, was developed to facilitate large-scale commercialization of this species.

Anadenanthera colubrina is a species native to Brazil, from the Fabaceae family and has potential for use in the timber industry, in the reforestation of degraded areas, besides having medicinal properties. Its propagation is mainly by seeds, but basic subsidies regarding the requirements for optimal germination conditions are still lacking. Aiming to contribute to the expansion of its cultivation, rational use and conservation, the objective of this study was to investigate the morphology and anatomy of fruits and seeds, as well as the responses to factors as thermal regimes and substrates in seed germination. The 1000-seed weight and seeds per fruit were determined. To characterize the seed tissues, histochemical test with Sudan III and Lugol was used. The temperatures analyzed in the germination test were 15-25 °C; 25 °C; 20-30 °C and 30 °C. Different substrates for germination (paper rolls, paper sheet such as “germitest”, commercial substrate and sand) were also analyzed in the presence of light at 30 °C. The average number of seeds per fruit is 10 and the 1000-seed weight is 118 g. Germination is fast and high over a wide temperature range; however, the temperature of 25 °C contributes to a substantial increase in the percentage of abnormal seedlings and dead seeds, both in the absence and presence of light. A. colubrina seeds are indifferent to light and the highest vigor was verified at 30 °C. Sand and commercial substrates are efficient in initial seedling development.

Passiflora ligularis Juss. is an endemic species from South America with important medicinal and economical properties. The development of improved micropropagation techniques is necessary to provide rapid and efficient clonal propagation of elite genotypes with high resistance and uniform production, as well as a system that can be used for genetic transformation. For this reason, we focused on establishing a protocol for somatic embryogenesis in P. ligularis from mature zygotic embryos. Our results demonstrate that the highest frequencies of somatic embryo formation was observed on a culture medium supplemented with 27.2 µM 2,4-diclorophenoxyacetic acid plus 4.5 µM 6-benzyladenine. Histological analyses of somatic embryogenesis were performed every 7 days after induction over 60 days of exposure to the medium. We present clear evidence for the precise origin of de-differentiation. Initial cell divisions occurred from a group of cells (multicellular origin) on the abaxial surface of the cotyledon in the periphery of the epidermal tissues of mature zygotic embryos after 14 days of incubation. After 21 days, internal segmenting divisions resulted in the embryogenic character of the tissue. Globular embryos contain a protoderm surrounding a mass of vacuolated parenchymatous cells and meristematic regions with an observable procambium zone after 45 days. The complete independence of somatic embryos from the adjacent tissues was histologically confirmed by the absence of vascular continuity between them, after 60 days. We describe for the first time the development of somatic embryos in P. ligularis and demonstrate that somatic embryos develop into plants that can later on be acclimatized.

Hancornia speciosa is a native fruit species from Brazil with important economic applications. However, the use of this species has been challenged by difficulties in its storage. Cryopreservation is a safe and efficient tool for long-term storage. Nevertheless, cryopreservation may promote reactive oxygen species (ROS) formation with subsequent lipid peroxidation, which causes membrane damage. The use of in vitro antioxidant compounds could help eliminate ROS and consequently improve explant survival. In the current study, the lateral buds of H. speciosa were precultivated in proline or glycine betaine (GB)-containing solutions, and the effect of these compounds on the enzymatic activity of superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) and on lipid peroxidation was measured after sample warming. The present findings indicated that oxidative stress induced by cryopreservation was significantly reduced by the precultivation of lateral buds with proline and GB. SOD activity increased, and subsequently, hydrogen peroxide (H2O2) production increased followed by the immediate activation of CAT and APX. The enzyme activity increased for 7 following rewarming, indicating that precultivation with 0.1 M proline was fundamental to achieving high levels of shoot regeneration from H. speciosa lateral buds.

Background Coffee production relies on plantations with varieties from Coffea arabica and Coffea canephora species. The first, the most representative in terms of coffee consumption, is mostly propagated by seeds, which leads to management problems regarding the plantations maintenance, harvest and processing of grains. Therefore, an efficient clonal propagation process is required for this species cultivation, which is possible by reaching a scalable and cost-effective somatic embryogenesis protocol. A key process on somatic embryogenesis induction is the auxin homeostasis performed by Gretchen Hagen 3 (GH3) proteins through amino acid conjugation. In this study, the GH3 family members were identified on C. canephora genome, and by performing analysis related to gene and protein structure and transcriptomic profile on embryogenic tissues, we point a GH3 gene as a potential regulator of auxin homeostasis during early somatic embryogenesis in C. arabica plants. Results We have searched within the published C. canephora genome and found 17 GH3 family members. We checked the conserved domains for GH3 proteins and clustered the members in three main groups according to phylogenetic relationships. We identified amino acids sets in four GH3 proteins that are related to acidic amino acid conjugation to auxin, and using a transcription factor (TF) network approach followed by RT-qPCR we analyzed their possible transcriptional regulators and expression profiles in cells with contrasting embryogenic potential in C. arabica. The CaGH3.15 expression pattern is the most correlated with embryogenic potential and with CaBBM, a C. arabica ortholog of a major somatic embryogenesis regulator. Conclusion Therefore, one out of the GH3 members may be influencing on coffee somatic embryogenesis by auxin conjugation with acidic amino acids, which leads to the phytohormone degradation. It is an indicative that this gene can serve as a molecular marker for coffee cells with embryogenic potential and needs to be further studied on how much determinant it is for this process. This work, together with future studies, can support the improvement of coffee clonal propagation through in vitro derived somatic embryos.

Calophyllum brasiliense is a species native to Brazil and has potential for use in the timber industry, in the reforestation of degraded areas, besides having medicinal properties. Its propagation is mainly by seeds which, depending on their recalcitrant characteristics, leads to difficulty in conservation, due to changes in its physiological potential during storage. Aiming to contribute to the expansion of its cultivation, rational use and conservation, the objective of this study was to investigate the behavior of C. brasiliense seeds during storage. Different packings (paper, aluminum and polyethylene) and environmental conditions (room temperature and cold chamber) were quarterly tested over 12 months, by evaluating germination viability and vigor. Based on the results, it was concluded that packaging in polyethylene and freezer storage provided the best conditions for the conservation of seeds, keeping them viable for a period of nine months. Resumo Calophyllum brasiliense é uma espécie nativa do Brasil e está na lista de espécies recomendadas para o reflorestamento de áreas degradadas, além de possuir propriedades medicinais e madeireiras. Sua propagação se dá principalmente via sementes, as quais, em função das suas características recalcitrantes, apresentam dificuldade de conservação, devido a alterações no seu potencial fisiológico ao longo do armazenamento. Visando contribuir com a ampliação de seu cultivo, uso racional e conservação, objetivou-se investigar o comportamento das sementes de C. brasiliense durante 12 meses de armazenamento. Testou-se diferentes embalagens (papel, alumínio e polietileno) e condições ambientais (câmara fria e temperatura ambiente), avaliando-se trimestralmente a germinação, viabilidade e vigor. Com base nos resultados obtidos conclui-se que o acondicionamento em embalagem de polietileno e o armazenamento em câmara fria proporcionou a melhor condição para a conservação das sementes, mantendo essas viáveis por um período de nove meses.

Lippia filifolia Mart. & Schauer belongs to the Verbenaceae family and it is endemic from the rupestrian fields of the Espinhaco mountain range, located in Minas Gerais, Brazil. It is an aromatic species with medicinal potential due to the production of volatile compounds that constitute its essential oil. The objective of this work was to evaluate the effects of light quality using light-emitting diodes (LEDs) over the growth of L. filifolia grown in vitro after 45 days of culture, analyzing its volatile organic compounds (VOCs), biochemical, and biometric traits. This study had four treatments according to the wavelength of LED lamps: (i) white (control), (ii) blue, (iii) red, and (iv) a combination of red + blue (mix). The light quality influenced the growth, metabolism, and VOCs production of plantlets. The specimens showed higher height under red and white treatments and higher biomass accumulation, nodal segments, and shoot numbers under the mix treatment. Higher total carbohydrate content was also observed on the mix treatment, while the white LED provided higher chlorophylls and carotenoids contents. In addition, the lipid peroxidation was more pronounced in mix and white LEDs treatments, and it was also observed significant but not quite changes in VOCs profiles due to light quality. Eucalyptol was the compound found in a higher concentration among the VOCs of L. filifolia grown in vitro at all light quality treatments studied.

Long-term storage of synthetic seeds can be accomplished using cryopreservation techniques. Cryopreservation allows the viability of encapsulated plant material to be conserved and maintained over a long period without modifications or genetic changes because the material is exposed to ultralow temperatures in liquid nitrogen (−196 °C), which decreases or even halts cellular metabolism. Cryopreservation has been found to be practical and efficient for the conservation of many species due to the small volume of material needed for storage, the simplification of transportation procedures and the minimal maintenance required compared to conventional storage methods. The main cryopreservation techniques applied to synthetic seeds are encapsulation-dehydration and encapsulation-vitrification. These techniques have been shown to be highly applicable for small explants that are sensitive to the conventional cryopreservation process, such as meristems and somatic embryos. However, the success of cryopreservation techniques for synthetic seeds depends on the type of encapsulated explant, on the capsule constitution and consistency and on research on the different cryopreservation stages in order to optimize the survival and regeneration of the plant material. Therefore, the present chapter is based on studies of the different stages of cryopreservation related to encapsulation techniques developed over time and on the major advances and innovations in cryopreservation.

Cryopreservation is a process whereby biological structures are preserved in liquid nitrogen (−196 °C) without losing their viability. Many cryopreservation techniques use the Plant Vitrification Solution 2 (PVS2) for cryoprotection. This study will therefore evaluate the influence of different exposure times to the cryoprotectant PVS2 and discuss the importance of the mobilization of reserves and the antioxidant metabolism during the germination of cryopreserved Passiflora ligularis embryos. The composition of P. ligularis seeds was analytically determined. We tested the germination capacity and the Germination Speed Index (GSI) of embryos (that is, seeds without external tegument) which were exposed to different PVS2 exposure times (0, 30, 60 and 120 min) at 30 days after thawing. Proline content, hydrogen peroxide, activity of isocitrate lyase (ICL), malate synthase (MSy), lipid peroxidation and antioxidant enzyme activities (SOD, CAT, APX) were measured at 7, 14 and 21 days after cryopreservation. The germination from cryopreserved embryos was maximal (85%) after 60 min PVS2 exposure with a GSI of 0.6. At 60 min, the highest activity of the enzymes involved in the glyoxylate cycle, ICL and MSy were recorded. We hypothesize that a 60 min exposure to PVS2 accelerates the reserve mobilization which correlates positively with germination. Until 60 min, there was a positive correlation between the PVS2 exposure time and the proline content, as well as the activity of antioxidant enzymes (SOD, CAT, APX), and a negative correlation with the lipid peroxidation. This study enables us to optimize the long-term conservation of this species. In conclusion, fundamental research is necessary to optimize the cryopreservation procedure, and this study offers an effective and efficient workflow which can be extrapolated to other (oil-rich) species.

Zinnia elegans Jacquim is a species of big cutting and ornamental potential, but its seeds have low germination percentage. This study aimed to establish a protocol for in vitro germination and water content for the cryopreservation of seeds. The length, width, thickness, and weight of one thousand seeds were determined. MS and WPM culture mediums, as well as the concentrations of gibberellic acid (GA3) were tested regarding in vitro culture. For cryopreservation, the seeds were subjected to drying on silica gel or laminar flow for different times. Then, the seeds were stored in liquid nitrogen (-196° C) for 24 hours. After this period, the seeds were thawed and inoculated into culture medium. The biometric results of seeds showed 8.6 mm average length, 4 mm width, and 0.9 mm thickness. The weight of one thousand seeds was 851 mg, characterizing them as small, lightweight, and easy to disperse. The use of MS medium with no addition of GA3 enhanced germination (67%). The initial moisture content of Z. elegans seeds was 9%. Seeds subjected up to 2 hours of drying in both treatments obtained 23% germination in silica gel and 19% in laminar flow. Z. elegans seeds may be desiccated by 4% moisture content and cryopreserved with no loss of germination potential.

Zantedeschia aethiopica is often used for cut flowers. However, seedling production is very limited, mainly due to diseases. The objective of this work was to develop an in vitro propagation protocol for the species and to analyse its callus chemical composition. Seeds obtained from mature and immature fruits were inoculated in half-strength MS and WPM culture media supplemented with 15 g L-1 sucrose. Different GA3 concentrations (0.0, 0.2, 0.4, 0.6 and 0.8 mg L-1) were added to the MS medium in the presence of 15.0 g L-1 sucrose. For callus induction, nodal segments were inoculated on MS medium supplemented with 2,4-D (0.0, 1.0, 2.0 and 3.0 mg L-1) and 15 g L-1 sucrose. For callus chemical analysis, total phenols, starch, total soluble sugars, reducing and non-reducing sugars, lipids and protein were quantified. Seeds from immature fruits presented 33% germination in WPM medium while seeds from mature fruit presented 24% germination on MS medium. Regarding the use of MS medium plus 15 g L-1 sucrose with different GA3 concentrations, the higher germinated rate, using immature (20%) or mature fruit seeds (10%), were obtained with 0.8 or 0.2 mg L-1 GA3, respectively. The callus induction occurred in media supplemented with 1.0 mg L-1 (53%) and 2.0 mg L-1 (47%) of 2,4-D. The callus chemical composition was as follows: total phenols (802.47 mg 100 g-1), starch (19.8 g 100 g-1), total soluble sugar (3.1 g 100 g-1), reducing sugar (1.2 g 100 g-1), non-reducing sugar (1.8 g 100 g-1), ether extract (1.0%) and protein (20%). These compounds may help the understanding of cellular dedifferentiation processes that take place in Z. aethiopica species.

Hancornia speciosa Gomes is a fruit species belonging to the Apocynaceae family and holds social, cultural and economic potential mostly due to its use in composition of many food industry products and the consumption its fruits in natura . Several aspects regarding their propagation need further studies, since the species is undergoing a continuous process of domestication. The objective was to obtain an in vitro protocol for indirect organogenesis and rooting with subsequent acclimatization of H. speciosa plants. To obtain indirect organogenesis, internodal segments were inoculated in WPM culture medium gelled with 7 g L -1 agar, added with 30 g L -1 sucrose, 0.4 g L -1 PVP, and supplied with different concentrations of 2,4-D (0.0; 2.46; 7.38; 12.30; and 17.22 µM), BAP (0.0; 4.92; 9.84; 14.76; and 19.68 µM), and TDZ (0.0; 4.92; 9.84; 14.76; and 19.68 µM). For the in vitro rooting, shootings with approximately 6.0 cm diameter length kept for 15 days in WPM medium with no plant growth regulator and, afterwards, subjected to treatments with different auxins (Control; 9.84 µM IBA; 9.84 µM NAA; and 9.84 µM IAA) as well as the combination among them, in order to verify their effects on percentage of rooting (%), number of roots, and average length of the largest root (cm). The formation of calluses was observed in all explants subjected to the concentrations of the regulators tested. The highest shooting regeneration occurred with 7.38 µM 2,4-D (73%). The highest percentage of shoot rooting (80%) and roots with the largest length (1.3 cm) were found in the culture medium with the combination of 4.92 µM NAA and 4.92 µM IBA. The in vitro regeneration of H. speciosa is feasible. The acclimatization of rooted shoots in Trospstrato® was accomplished with successful and 100% survival of plant material was observed during this stage.

Objetivou-se induzir a formação de calos e utilizar o corante CTT na quantificação da viabilidade celular dos calos em gemas rizomatosas de gengibre ornamental. Para a indução de calos, gemas rizomatosas foram inoculadas em meio de cultura MS suplementado com 2,4-D, BAP e picloram. Para a viabilidade celular, o tratamento que apresentou maior porcentagem de formação de calos, passou por três subcultivos em intervalos de 10 dias, a cada subcultivo os calos foram coletados e homogeneizados em CTT e realizadas cinco leituras de absorbância. As maiores porcentagens de formação de calos ocorreram na presença de 1,0 e 2,0 mg.L-1 de BAP. O teste de viabilidade utilizando CTT aponta que os calos devem ser subcultivados aos 20 dias devido à maior absorbância, pois atingiram a fase exponencial de divisão celular.