Histology of somatic embryogenesis in <Emphasis Type="Italic">Coffea arabica</Emphasis> L.

The objective of this study was to characterize the histodifferentiation of somatic embryogenesis obtained from leaf explants of C. arabica. Therefore, we histologically analyzed the respective stages of the process: leaf segments at 0, 4, 7, 15 and 30 days of cultivation, Type 1 primary calli (primary calli with embryogenic competence) and 2 (primary calli with no embryogenic competence), embryogenic calli, globular, torpedo and cotyledonary embryos, and mature zygotic embryos. Callus formation occurred after seven days of culture, with successive divisions of procambium cell. In this cultivation phase, it was found that Type 1 primary calli are basically formed by parenchymal cells with reduced intercellular spacing, whereas Type 2 primary calli are predominantly composed of parenchymal cells with ample intercellular spaces and embryogenic calli composed entirely of meristematic cells. After 330 days, it was evident from the differentiation of somatic embryogenesis that there was formation of globular somatic embryos, consisting of a characteristic protoderm surrounding the fundamental meristem. With the maturation of these propagules after 360 days, torpedo-stage somatic embryos arose, in which tissue polarization and early differentiation of procambial strands were verified. After 390 days, cotyledonary somatic embryos were obtained, where the onset of vessel elements differentiation was verified, a characteristic also observed in mature zygotic embryos. We concluded that somatic embryogenesis obtained from C. arabica leaves initiates from procambium cell divisions that, in the course of cultivation, produce mature somatic embryos suitable for regenerating whole plants.     

Transgenic coffee fruits from <Emphasis Type="Italic">Coffea arabica</Emphasis> genetically modified by bombardment

The genetic modification of Coffea arabica fruits is an important tool for the investigation of physiological characteristics and functional validation of genes related to coffee bean quality traits. In this work, plants of C. arabica cultivar Catuaí Vermelho were successfully genetically modified by bombardment of embryogenic calli. Calli were obtained from 90% of the leaf explants cultivated in a callogenesis-inducing medium modified with 20 μM 2,4-dichlorophenoxyacetic acid (2,4-D). The resulting calli were bombarded with the pBI426 vector containing a uidA and nptII gene fusion that was driven by the double CaMV35s promoter. Kanamycin-selected embryos were positive for β-glucuronidase (GUS) activity in histochemical assays and for target gene amplification by polymerase chain reaction. Integration of the nptII gene was confirmed by Southern blot and showed a low copy number (one to three) of insertions. Transformed plants showed normal development and settled fruits. GUS expression was assessed in the flower and fruit organs demonstrating the capacity of the double CaMV35s promoter to drive long-term stable expression of uidA in C. arabica fruit tissues. Moreover, we obtained a T₁ progeny presenting 3:1 Mendelian segregation of the uidA gene. This investigation is the first to report exogenous gene expression in coffee fruits and transgenic inheritance in C. arabica plants.     

Influence of triacontanol on somatic embryogenesis in <Emphasis Type="Italic">Coffea arabica</Emphasis> L. and <Emphasis Type="Italic">Coffea canephora</Emphasis> P. ex Fr.

Summary A highly reproducible method for regeneration of Coffea arabica and C. canephora plants via direct somatic embryogenesis from cultured leaf and stem segments of regenerated plants was developed. Embryogenesis was influenced by the presence of triacontanol (TRIA) in the medium. TRIA incorporated at 4.55 and 11.38 μM in half-strength MS basal medium containing 1.1 μM 6-benzyladenine (BA) and 2.28 μM indole-3-acetic acid (IAA) induced direct somatic embryogenesis in both species. A maximum of 260±31.8 and 59.2±12.8 somatic embryos per culture were induced from in vitro leaf explants of C. arabica and C. canephora, respectively. TRIA also induced embryo formation from in vitro stem segment callus tissues along with multiplication of primary embryos into secondary embryos. By using TRIA, it was possible to obtain somatic embryogenesis in C. arabica and C. canephora.     

Direct somatic embryogenesis from <Emphasis Type="Italic">Coffea arabica</Emphasis> L. and <Emphasis Type="Italic">Coffea canephora</Emphasis> P ex Fr. under the influence of ethylene action inhibitor-silver nitrate

For the first time direct somatic embryogenesis from hypocotyl explants of in vitro regenerated plantlets of C. arabica and C. canephora was achieved on modified MS medium containing 10 – 70 μM silver nitrate supplemented with 1.1 μM N⁶ benzyladenine and 2.85 μM indole-3-acetic acid. A maximum of 144.1±7.3 and 68.7±3.3 embryos per explant were produced at 40 μM silver nitrate in C. canephora and C. arabica respectively. Only yellow friable embryogenic callus obtained from the cut edges of most of leaf explants of both C. arabica and C. canephora at all concentrations of silver nitrate were tried in this experiment. Formation of secondary embryos from stage I primary embryos (small yellow, round, globular embryos) was more (28.23±1.3) at 60 μM silver nitrate in C. canephora, while 40 μM silver nitrate supported more of secondary embryo formation in C. arabica (40.5±1.2). When stage II (green globular round matured embryos) and stage III primary embryos (tubular stage embryos) were used, secondary embryo formation was very small and many of these embryos developed into plantlets and some of them even rooted. By using these protocols within 45 – 60 days it is possible to get secondary embryos from primary embryos and direct somatic embryos from hypocotyls of in vitro plantlets in both these Coffea species.     

SNP in the <Emphasis Type="Italic">Coffea arabica</Emphasis> genome associated with coffee quality

Association analysis was performed at the whole genome level to identify loci affecting the caffeine and trigonelline content of Coffea arabica beans. DNA extracted from extreme phenotypes was bulked (high and low caffeine, and high and low trigonelline) based on biochemical analysis of the germplasm collection. Sequencing and mapping using the combined reference genomes of C. canephora and C. eugenioides (CC and CE) identified 1351 non-synonymous SNPs that distinguished the low- and high-caffeine bulks. Gene annotation analysis with Blast2GO revealed that these SNPs corresponding to 908 genes with 56 unique KEGG pathways and 49 unique enzymes. Based on KEGG pathway-based analysis, 40 caffeine-associated SNPs were discovered, among which nine SNPs were tightly associated with genes encoding enzymes involved in the conversion of substrates (i.e. SAM, xanthine and IMP) which participate in the caffeine biosynthesic pathways. Likewise, 1060 non-synonymous SNPs were found to distinguish the low- and high-trigonelline bulks. They were associated with 719 genes involved in 61 unique KEGG pathways and 51 unique enzymes. The KEGG pathway-based analysis revealed 24 trigonelline-associated SNPs tightly linked to genes encoding enzymes involved in the conversion of substrates (i.e. SAM, L-tryptophan) which participate in the trigonelline biosynthesis pathways. These SNPs could be useful targets for further functional validation and subsequent application in arabica quality breeding.     

Protoplast isolation and culture for somatic hybridization of <Emphasis Type="Italic">Lupinus angustifolius</Emphasis> and <Emphasis Type="Italic">L</Emphasis>. <Emphasis Type="Italic">subcarnosus</Emphasis>

A method for isolation and shoot regeneration from electrofused protoplasts of L. angustifolius and L. subcarnosus was developed. Viable protoplasts were isolated from leaves of in-vitro grown seedlings at an average yield of 6 × 10⁵ protoplasts g⁻¹ fresh weight. Liquid and agarose solidified B5 media were used for protoplast culture. In the liquid-culture system, all tested media, VKM, P1 and KM8p, were applicable for inducing cell division (84% of all tested petri dishes at four weeks) and colony formation. Media containing additional carbohydrates were suitable to produce compact calli with green and brown pigmentations in different combinations. Analysis of callus with molecular markers allowed to identify six somatic hybrids. However, none of the parental-protoplast derived cell colonies could develop shoots. This is the first report on protoplast fusion of L. angustifolius and L. subcarnosus with subsequent shoot regeneration.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of <Emphasis Type="Italic">Ophiorrhiza prostrata</Emphasis> through somatic embryogenesis

In vitro propagation of an anticancerous drug synthesizing plant, Ophiorrhiza prostrata D. Don, was established through indirect somatic embryogenesis. Friable embryogenic calluses were initiated from O. prostrata leaf and internode explants on Murashige and Skoog (MS) media supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) either alone or in combination with N⁶-benzyladenine (BA) or kinetin (KIN). Somatic embryos were developed after subculture of the friable calluses onto half strength MS media containing 0.45 or 2.26 μM 2,4-D alone or in combination with BA or KIN. Medium supplemented with 2.26 μM 2,4-D and 2.22 μM BA was optimal, supporting the production of a mean of 5.8 globular embryos. Subculture of globular embryo-bearing calluses on half strength MS medium without growth regulators produced the highest embryo frequency, and the majority of them developing to early torpedo stage. Somatic embryos underwent maturation and converted to plantlets at high frequency (90 %) on half strength MS medium supplemented with 0.44 μM BA. Somatic embryo-derived plantlets with well-developed roots were established in field conditions with a 90 % survival rate.     

Chitinases of <Emphasis Type="Italic">Coffea arabica</Emphasis> genotypes resistant to orange rust <Emphasis Type="Italic">Hemileia vastatrix</Emphasis>

Two Coffea arabica — Hemileia vastatrix incompatible interactions (I₁: coffee cv. Caturra — rust race VI and I₂: coffee cv S4 Agaro — rust race II) and a compatible interaction (coffee cv. Caturra — rust race II) were compared in relation to the infection process and chitinase activity. In the two incompatible interactions the fungus ceased growth in the early infection stages, while in the compatible interaction no fungus growth inhibition was observed. A high constitutive level of chitinase activity was detected in the intercellular fluid of healthy leaves. Upon infection, chitinase isoforms were more abundant in incompatible interactions than in the compatible interaction. Immunodetection showed that class I chitinases are particularly relevant in the incompatible interactions and might participate in the defence response of the coffee plants.     

Plant regeneration from <Emphasis Type="Italic">Gossypium davidsonii</Emphasis> protoplasts <Emphasis Type="Italic">via</Emphasis> somatic embryogenesis

Protoplasts isolated from wild cotton Gossypium davidsonii were cultured in KM₈P medium supplemented with different phytohormones. The most effective combination was 0.45 μM 2,4-dichlorophenoxyacetic acid, 2.68 μM α-naphthaleneacetic acid and 0.93 μM kinetin and the division percentage at the 8^th day was 30.78 ± 3.04 %. The density of protoplasts at 2–10 × 10⁵ cm⁻³ was suitable for protoplast division and calli formation, with a division percentage of 32.21 ± 3.64 % and a plating efficiency of 9.12 ± 2.61 % at the 40^th day. The optimal osmotic potential was achieved using 0.5 M glucose or 0.1 M glucose plus 0.5 M mannitol. Protoplasts were cultured in three ways, a double-layer culture system, with liquid over solid medium was proved to be the best way. Embryo induction was further increased by addition of 0.14 μM gibberellic acid.     

Antioxidative response of <Emphasis Type="Italic">Hordeum maritimum</Emphasis> L<Emphasis Type="Italic">.</Emphasis> to potassium deficiency

The objective of the present study was to determine the influence of potassium deprivation on the halophyte species Hordeum maritimum grown in hydroponics for 2 weeks. Treatments were with potassium (+K) or without potassium (−K). Growth, water status, mineral nutrition, parameters of oxidative stress [malondialdehyde (MDA), carbonyl groups (C=O), and hydrogen peroxide concentration (H₂O₂) contents], antioxidant enzyme activities [superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), guaiacol peroxidase (GPX, EC 1.11.1.7), ascorbate peroxidase (APX, EC 1.11.1.11), monodehydroascorbate peroxidase (MDHAR, EC 1.6.5.4), dehydroascorbate peroxidase (DHAR, EC 1.8.5.1), and glutathione reductase (GR, EC 1.6.4.2)], and antioxidant molecules [ascorbate (ASC), and glutathione (GSH)] were determined. Results showed that the growth of vegetative organs decreased owing to potassium deficiency with roots (−36%) more affected than shoots (−12%). Water status was only diminished in roots (reduction of 24%). Potassium deprivation decreased potassium concentration in both organs, this decrease was more pronounced in roots (−81%) than in shoots (−55%). In contrast to carbonyl groups, MDA content increased owing to potassium deprivation. Except for CAT activity that remained unaffected; SOD, GPX, APX, GR, MDHAR, and DHAR activities were significantly increased. H₂O₂ concentration was negatively correlated with the activities of enzymes and the accumulation of non-enzymatic antioxidants implicated in its detoxification. In conclusion, a cooperative process between the antioxidant systems is important for the tolerance of H. maritimum to potassium deficiency.     

Factors affecting somatic embryogenesis in anther cultures of Chinese pink <Emphasis Type="Italic">(Dianthus chinensis</Emphasis> L<Emphasis Type="Italic">.)</Emphasis>

In this study, we aimed to maximize the rates of somatic embryogenesis achievable in anther cultures of Chinese pink (Dianthus chinensis L.) (2n = 2x = 30). The genotype of the donor plant was found to be a major factor in determining the success rate. Conditions imposed during anther culture (notably medium composition and light conditions) and pretreatments (namely, cold, heat, and mannitol incubations) were also found to influence somatic embryo induction. For example, the highest levels of embryogenic callus induction were achieved when the donor buds had been cold pretreated and the subsequent anther culture was maintained in darkness. Furthermore, there appeared to be an interaction of genotype with culture conditions. Thus, in cultures of the cultivar (cv.) ‘Carpet’, the highest rates of embryogenesis were obtained when the anthers had received a 5-d heat-shock, but such a thermal treatment did not generally produce a significant effect. Likewise, a 3-d mannitol pretreatment was optimal only for the cross-hybrid line ‘HC’. Assessment of the ploidy of the plants regenerated from the anther cultures revealed both diploid and tetraploid plants. Histological and cytological observations showed that all of these (both from n-pollen and 2n-pollen lines) derived from anther wall cells. Spontaneous chromosome doubling was inferred to have occurred during the embryogenic callus culture period.     

Analysis of alien introgression in coffee tree (<Emphasis Type="Italic">Coffea</Emphasis><Emphasis Type="Italic">arabica</Emphasis> L.)

The transfer of desired traits from related wild diploid Coffea species into the cultivated allotetraploid C. arabica is essential in coffee breeding to develop pest/disease-resistant cultivars. The present work is an attempt to gain insights into alien introgression in C. arabica. An F2 population derived from a cross between T5296 and Et6 was analysed with simple sequence repeat (SSR; microsatellite) and amplified fragment length polymorphism (AFLP) molecular markers. The T5296 is a derivative of an interspecific hybrid introgressed by the diploid C. canephora species and Et6 is a wild Ethiopian accession of C. arabica. The origin of the revealed polymorphism was determined by comparisons using representative accessions from C. arabica and its two diploid parental species, C. eugenioides and C. canephora. The number and mode of inheritance of canephora-introgressed segments were investigated, as well as their sub-genome localisation and rate of recombination. The results suggested that the transfer of desirable genes into C. arabica from C. canephora is not limited by the ploidy level differences or the suppression of recombination between the different genomes.     

<Emphasis Type="Italic">Diplazium</Emphasis> hybrids involving <Emphasis Type="Italic">D. plantaginifolium</Emphasis> and <Emphasis Type="Italic">D</Emphasis>. <Emphasis Type="Italic">ternatum</Emphasis> from Mexico and Central America

Here we evaluate the origins and relationships of Mexican and Central American Diplazium hybrids derived from crosses involving either D. plantaginifolium or D. ternatum. Based on study of live plants and herbarium specimens, we distinguish D. ×verapax from the similar D. riedelianum and present evidence that the former is a sterile hybrid derived from a cross between D. plantaginifolium and D. werckleanum. We also describe new hybrids, D. ×torresianum and D. ×subternatum from Mexico and northern Central America. Both involve D. ternatum as one parent. Diplazium. cristatum is the other putative parent of D. ×torresianum, and D. plantaginifolium is the second parent of D. ×subternatum. We also designate lectotypes for D. cordovense and D. dissimile.     

Polyphenol oxidase activity in dormant saffron (<Emphasis Type="Italic">Crocus sativus</Emphasis> L<Emphasis Type="Italic">.</Emphasis>) corm

Crocus sativus L., cultivated since ancient times as the source of saffron, is a triploid plant that can be propagated only via its corms which undergo a period of dormancy. Understanding the processes taking place in the corm is essential to preserve the plant and improve its quality. Color and taste being of prime importance in the quality of the saffron spice, knowledge on polyphenol oxidase (PPO) activity in the plant is of particular interest given the role of the enzyme in fruit and vegetable browning during processing and during the storage of processed food. In this paper, PPO activity was investigated for the first time in extracts obtained from dormant C. sativus L. corms. PPO activity was detectable using l-DOPA, pyrogallol, catechol or p-cresol as substrate, each being oxidized to its corresponding o-quinone; no activity was detectable with l-tyrosine, tyramine or phenol as substrate. Two pH optima, respectively at 4.5 and 6.7, were observed with all substrates and a third one, at 8.5, was found with l-DOPA and p-cresol. Kinetics parameters studied at pH 6.7 indicated the highest catalytic efficiency (in units mg⁻¹ prot mM⁻¹) with pyrogallol: 150, then catechol: 39, l-DOPA: 6.4 and p-cresol: 4.6. The enzymatic activity was inhibited by 50% in the presence of 0.22, 0.35, 0.5 and 0.7 mM kojic acid with, respectively, catechol, pyrogallol, p-cresol and l-DOPA as substrate. When stained for PPO activity, non-denaturing gel electropherograms of extract revealed three distinct bands, indicating the presence of multiple isoenzymes in dormant C. sativus L. corms.     

Expression of the <Emphasis Type="Italic">Escherichia coli</Emphasis> heat-labile enterotoxin B subunit in transgenic watercress (<Emphasis Type="Italic">Nasturtium officinale</Emphasis> L<Emphasis Type="Italic">.</Emphasis>)

A gene encoding the B subunit of the enterotoxigenic Escherichia coli heat-labile enterotoxin (LTB) was adapted to the optimized plant coding sequence, and fused to the endoplasmic reticulum retention signal SEKDEL in order to enhance its expression level and protein assembly in plants. The synthetic LTB (sLTB) gene was placed into a plant expression vector under the control of the CaMV 35S promoter, and subsequently introduced into the watercress (Nasturtium officinale L.) plant by the Agrobacterium-mediated transformation method. The integration of the sLTB gene into the genomic DNA of transgenic plants was confirmed by genomic DNA PCR amplification. The assembly of plant-produced LTB protein was detected by western blot analysis. The highest amount of LTB protein produced in transgenic watercress leaf tissue was approximately 1.3% of the total soluble plant protein. G_M1-ganglioside enzyme-linked immunosorbent assay indicated that plant-synthesized LTB protein bound specifically to G_M1-ganglioside, which is the receptor for biologically active LTB on the cell surface, suggesting that the plant-synthesized LTB subunits formed biologically active pentamers.     

Comparison of the <Emphasis Type="Italic">Coffea canephora</Emphasis> and <Emphasis Type="Italic">C. arabica</Emphasis> karyotype based on chromosomal DNA content

Nuclear genome size has been measured in various plants, seeing that knowledge of the DNA content is useful for taxonomic and evolutive studies, plant breeding programs and genome sequencing projects. Besides the nuclear DNA content, tools and protocols to quantify the chromosomal DNA content have been also applied, expanding the data about genomic structure. This study was conducted in order to calculate the Coffea canephora and Coffea arabica chromosomal DNA content, associating cytogenetic methodologies with flow cytometry (FCM) and image cytometry (ICM) tools. FCM analysis showed that the mean nuclear DNA content of C. canephora and C. arabica is 2C = 1.41 and 2.62 pg, respectively. The cytogenetic methodology provided prometaphase and metaphase cells exhibiting adequate chromosomes for the ICM measurements and karyogram assembly. Based on cytogenetic, FCM and ICM results; it was possible to calculate the chromosomal DNA content of the two species. The 1C chromosomal DNA content of C. canephora ranged from 0.09 (chromosome 1) to 0.05 pg (chromosome 11) and C. arabica from 0.09 (chromosome 1) to 0.03 pg (chromosome 22). The methodology presented in this study was suitable for DNA content measuring of each chromosome of C. canephora and C. arabica. The cytogenetic characterization and chromosomal DNA content analyses evidenced that C. arabica is a true allotetraploid originated from a cross between Coffea diploid species. Besides, the same analyses also reinforce that C. canephora is a possible progenitor of C. arabica.     

Sonication assisted <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation enhances the transformation efficiency in flax (<Emphasis Type="Italic">Linum usitatissimum</Emphasis> L<Emphasis Type="Italic">.</Emphasis>)

A sonication-assisted, Agrobacterium-mediated, co-cultivation technique was used in an attempt to increase the transformation efficiency of flax. Hypocotyls and cotyledons excised from about 10-day-old flax seedlings grown in vitro were placed into a 10 mM MgSO₄ solution, and inoculated with an A. tumefaciens vector bearing the mgfp5-ER gene driven by the CaMV 35S promoter. The explants were subjected to pulses of ultrasound delivered by a sonicator apparatus (35 kHz) for 0–150 s and co-cultivated for 2 h at 27°C. The dried hypocotyls and cotyledons were grown on a selective MS medium to promote shoot regeneration. An electron microscopic study showed that the sonication treatment resulted in thousands of microwounds on and below the surface of the explants. A stereo microscope Leica MZ 12 equipped with a GFP adaptor was used to assess the infection and transformation of plant tissues in real time. After only 48 h and for at least 30 days after bacteria elimination, signs of transgene expression could be seen as a bright fluorescence. Our results show that treatment with ultrasound facilitates an enhanced uptake of plasmid DNA into the cells of flax hypocotyls and cotyledons and that its efficiency depends on the duration of the treatment and the frequency used. SAAT could be a promising tool for enhancing transformation efficiency in flax.