Enhanced production of pinosylvin stilbene with aging of Pinus strobus callus and nematicidal activity of callus extracts against pinewood nematodes

Pinosylvin stilbenes are phenolic compounds mainly occurring in the Pinaceae family. We previously reported that the accumulation of two pinosylvin stilbene compounds, dihydropinosylvin methyl ether (DPME) and pinosylvin monomethyl ether (PME), in Pinus strobus trees was highly enhanced by infection with pine wood nematodes (PWNs: Bursaphelenchus xylophilus), and these two compounds showed strong nematicidal activity against PWNs. In this work, we established a system of pinosylvin stilbene (DPME and PME) production via the in vitro culture of P. strobus calli, and we examined the nematicidal activity of callus extracts. Calli were induced from the culture of mature zygotic embryos of P. strobus. Optimized growth of calli was obtained in 1/2 Litvay medium with 1.0 mg/L 2,4-D and 0.5 mg/L BA. DPME and PME accumulation did not occur in nonaged (one-month-old) calli but increased greatly with prolonged callus culture. The concentrations of DPME and PME in three-month-old dark-brown calli were 6.4 mg/g DW and 0.28 mg/g DW, respectively. The effect of methyl jasmonate treatment on the accumulation of DPME and PME was evaluated in cell suspension culture of P. strobus. However, the treatment appeared to show slight increase of DPME accumulation compared to callus browning. A test solution prepared from crude ethanol extracts from aged calli (three months old) containing 120 µg/ml DPME and 5.16 µg/ml PME treated with PWNs resulted in 100% immobilization of the adult PWNs and 66.7% immobilization of the juvenile PWNs within 24 h. However, nonaged callus extracts did not show any nematicidal activity against juvenile PWNs and showed less than 20% nematicidal activity against adult PWNs. These results indicate that pinosylvin stilbenes can be effectively produced by prolonged culture of P. strobus calli, can be isolated using simple ethanolic extraction, and are applicable as beneficial eco-friendly compounds with nematicidal activity against PWNs.

Anatomical and hormonal factors determining the development of haploid and zygotic embryos of oat (Avena sativa L.)

A critical step in the production of doubled haploids is a conversion of the haploid embryos into plants. Our study aimed to recognize the reasons for the low germination rate of Avena sativa haploid embryos obtained by distant crossing with maize. Oat cultivars of ‘Krezus’ and ‘Akt’ were investigated regarding embryo anatomy, the endogenous phytohormone profiles, and antioxidant capacity. The zygotic embryos of oat were used as a reference. It was found that twenty-one days old haploid embryos were smaller and had a less advanced structure than zygotic ones. Morphology and anatomy modifications of haploid embryos were accompanied by extremely low levels of endogenous auxins. Higher levels of cytokinins, as well as tenfold higher cytokinin to auxin ratio in haploid than in zygotic embryos, may suggest an earlier stage of development of these former. Individual gibberellins reached higher values in ‘Akt’ haploid embryos than in the respective zygotic ones, while the differences in both types of ‘Krezus’ embryos were not noticed. Additionally to the hormonal regulation of haploid embryogenesis, the poor germination of oat haploid embryos can be a result of the overproduction of reactive oxygen species, and therefore higher levels of low molecular weight antioxidants and stress hormones.

Can Ethylene Inhibitors Enhance the Success of Olive Somatic Embryogenesis?

An efficient in vitro morphogenesis, specifically through somatic embryogenesis, is considered to be a crucial step for the application of modern biotechnological tools for genetic improvement in olive (Olea europaea L.). The effects of different ethylene inhibitors, i.e., cobalt chloride (CoCl2), salicylic acid (SA), and silver nitrate (AgNO3), were reported in the cyclic somatic embryogenesis of olive. Embryogenic callus derived from the olive immature zygotic embryos of the cultivar Leccino, was transferred to the expression ECO medium, supplemented with the ethylene inhibitors at 20 and 40 µM concentrations. Among these, the maximum number of somatic embryos (18.6) was obtained in media containing silver nitrate (40 µM), followed by cobalt chloride (12.2 somatic embryos @ 40 µM) and salicylic acid (40 µM), which produced 8.5 somatic embryos. These compounds interfered on callus traits: white friable embryogenic calli were formed in a medium supplemented with 40 µM cobalt chloride and salicylic acid; in addition, a yellow-compact embryogenic callus appeared at 20 µM of all the tested ethylene inhibitors. The resulting stimulatory action of silver nitrate among all the tested ethylene inhibitors on somatic embryogenesis, clearly demonstrates that our approach can efficiently contribute to the improvement of the current SE protocols for olive.

In vitro germination of Abricó-de-Macaco (Couroupita guianensis aubl.)zygotic embryos in different culture media and light spectra

ABSTRACT: Couroupita guianensis Aubl. is an Amazonian forest species with important medicinal and ornamental value. This study evaluated the effect of different culture media and light spectra on the in vitro germination and development of the zygotic embryos of C. guianensis. The culture media, MS and WPM, were evaluated without the addition of plant growth regulators and were associated with four LED light spectra: white (CW), 70% red + 30% blue (R2B), 100% red (R), and 100% blue (B). One hundred percent of the seeds successfully underwent in vitro germination, and the culture media did not interfere with embryo development. In addition to this, the different light spectra induced in vitro morphogenesis and R2B treatment significantly promoted the production of secondary roots. This effect may aid in the rooting and acclimatization of seedlings of this species.

Single-Base Resolution Methylomes of Somatic Embryogenesis in Theobroma Cacao L. Reveal Epigenome Modifications Associated With Somatic Embryo Abnormalities

Propagation by somatic embryogenesis in Theobroma cacao has some issues to be solved, as many morphologically abnormal somatic embryos that do not germinate into plants are frequently observed, thus hampering plant production on a commercial scale. For the first time the methylome landscape of T. cacao somatic embryogenesis was examined, using whole-genome bisulfite sequencing technique, with the aim to understand the epigenetic basis of somatic embryo abnormalities. We identified 873 differentially methylated genes (DMGs) in the CpG context between zygotic embryos, normal and abnormal somatic embryos, with important roles in development, programmed cell death, oxidative stress, and hypoxia induction, which can help to explain the morphological abnormalities of somatic embryos. We also identified the role of ethylene and its precursor 1-aminocyclopropane-1-carboxylate in several biological processes, such as hypoxia induction, cell differentiation and cell polarity, that could be associated to the development of abnormal somatic embryos. The biological processes and the hypothesis of ethylene and its precursor involvement in the somatic embryo abnormalities in cacao are discussed.

Standardization of zygotic embryo culture from Nerium oleander L. and comparative analysis of biosynthesized cardiac glycosides within in vitro and acclimatized plants

The primary result of our experiment revealed that the germination percentage of N. oleander mature seeds is only 30%. From this observation, the concept of protocol standardization for zygotic embryo culture of this plant was originated. Zygotic embryo culture was proved an efficient in vitro multiplication system of N. oleander. The maximum germination percentage (96%) of zygotic embryos was observed on ¼ MS medium with 15 gm/L sucrose, whereas the best growth medium was optimized as ½ B5 with same sucrose concentration. The second part of this study was aimed to find out the cardiac glycoside accumulation pattern in both in vitro and acclimatized plants. For this purpose, one-month-old in vitro plantlets and acclimatized plants were subjected to LC-MS analysis and 09 cardiac glycosides were detected and quantified in both the systems. Most of the cardiac glycosides including odoroside A (32.71 mg/gm DW), odoroside H (4.69 mg/gm DW) and oleandrin (0.52 mg/gm DW) were found to be accumulated at maximum level within in vitro plantlets. CG 840b (1.89 mg/gm DW) is the only cardiac glycoside, which was maximally accumulated in acclimatized plants. From this study, it can be concluded that, zygotic embryo culture is a better choice for in vitro multiplication of N. oleander when compared to matured seeds and in vitro grown plantlets of this species favor cardiac glycosides biosynthesis in comparison to acclimatized plants. Therefore, all future research on the enrichment of cardiac glycosides from this plant may be conducted on zygotic embryos derived in vitro grown plantlets or cultures.

Regeneration potential of different explants during micropropagation of neem tree (Azadirachta indica A. Juss.)

Azadirachta indica A. Juss., (Neem), a prodigious multipurpose tree, has immense potential to benefit mankind and to protect the environment. In order to investigate the effects of three different explants for its regeneration potential, de embryonated cotyledon, immature zygotic embryo and nodal segments from a 30 year old neem plus tree were used. Half strength MS medium with benzyl amino purine (3 mg/L) and naphthalene acetic acid (0.5 mg/L) and casein hydrolysate (1 g/L) was effective in shoot bud sprouting from both nodes and cotyledons. Half strength MS medium fortified with TDZ (0.2 mg/L) was effective for induction of somatic embryogenesis from zygotic embryos. Shoot buds initiated from the cotyledons produced a maximum number of shoots per explants (4.33) which on further sub culturing induced maximum multiple shoots (15) on half strength MS medium fortified with BAP (1.5 mg/L), NAA (0.5 mg/L) and CH (400 g /L) and the nodal explants induced only 4-5 axillary shoots on further sub culturing. Even though immature zygotic embryos produced more number of somatic embryos per explant (24.97) within a short time (30-45 days), the plantlet conversion was poor (25.52 %). In vitro rooting was observed in half strength MS medium supplemented with IBA (2 mg/L). The regeneration potential of de embryonated cotyledons through a simple regeneration system may be beneficial for efficient mass propagation of selected plus trees of neem.