Establishment Of Embryogenic Cultures Research Articles

Polyamine and amino acid profiles in immature Araucaria angustifolia seeds and their association with embryogenic culture establishment

Analysis of polyamines and amino acid profiles in Araucaria angustifolia immature seeds from two mother tree genotypes in 2 consecutive years, and their association with the establishment of embryogenic cultures. Polyamines (PAs) and amino acids are an important source of nitrogen in plants. They are known to participate in plant development and their levels are associated with specific zygotic and somatic embryogenesis stages, which means that they may be useful as biochemical markers of developmental stages. The present study carried out an analysis of PA and amino acid profiles in immature Araucaria angustifolia seeds from two mother tree genotypes over 2 consecutive years, and their association with the initiation and maintenance of embryogenic cultures (ECs). The results showed that initiation of ECs in A. angustifolia, as observed in other conifers, depends on harvest year and mother tree genotype. PA and amino acid profiles showed that seeds from distinct mother trees and harvest years are different from one another. These differences were reflected in the initiation of ECs. The metabolic analysis indicates that conjugated Spd, ornithine, and asparagine profiles are candidates as biochemical markers for selection of mother trees. These results will allow for the testing of a large number of mother trees as explant sources and the selection of those with higher potential for somatic embryogenesis.

Contribution of the Zea mays insulin-like growth factor (ZmIGF) to the embryogenic competence of maize tissue cultures

Establishment of somatic embryogenic cultures is highly influenced by the plant genotype and the explant type. In maize, immature embryos generate embryogenic callus (E), whereas quiescent embryos produce non-embryogenic callus (NE). E callus shows active growth and high capacity of plant regeneration, while NE callus shows slow growth and no regeneration capacity. Active growth is needed for the establishment of embryogenic cultures; therefore, lack of this characteristic pose a handicap for plant regeneration from NE callus. To correct the slow growth on NE callus, the Zea mays insulin-like growth factor (ZmIGF), a peptide that promotes growth by activating the target of rapamycin (TOR) pathway, was used as media supplement. Additionally, since the TOR pathway is connected to the auxin pathway, ZmIGF participation in cell differentiation was considered. To this end, this research explores ZmIGF effect, beyond growth and proliferation on a Mexican maize landrace, which has shown high somatic embryogenic capacity. Expression levels of reported genes involved in the embryogenesis and differentiation processes were evaluated in maize E, NE, and NE-ZmIGF (NE-Zm) growth-activated calluses. A tendency to upregulate messenger RNA (mRNA) expression was observed for genes encoding transcription factors and auxin transport. Some genes related to epigenetic control showed downregulation. Global DNA methylation and chromatin modifications results suggest an epigenetic activation state on E callus and a repression status on NE callus. ZmIGF induced modifications at DNA methylation and chromatin over NE callus, which changed its original repression state to an active one. Overall, results suggest that the expression of genes related to auxin signaling, mainly transport and efflux carriers, are essential for accomplishing plant regeneration through somatic embryogenesis (SE).

Establishment of Embryogenic Cultures and Efficient Plant Regeneration System from Explants of Forced Softwood Shoots of Teak (Tectona grandis L.)

The present study highlights an efficient plant regeneration system in teak (Tectona grandis L.) using forced softwood shoots as an initial plant material. Forced softwood shoots of teak were cut to prepare shoot tip, nodal and internodal explants and cultured on Murashige and Skoog (MS) medium + NAA (1, 3, 6, 10, and 15 µmol ⋅ L−1) or TDZ (0.001, 0.01, 0.1, 1, 4, 8, 10, 12 µmol ⋅ L−1) for callus induction. Such calluses were further grown on the same levels of TDZ or 0.4, 1, 4, 8, 10 µmol ⋅ L−1 BA + 1 µmol ⋅ L−1 IBA or GA3. Callus induction was the highest with 4.55 cm3 callus volume and 5.75 g dry weight at 0.1 µmol ⋅ L−1 TDZ from shoot tips after 35 days. Embryogenic calluses were then shifted to 6, 8 or 12 µmol ⋅ L−1 TDZ + 2 µmol ⋅ L−1 BA or IBA along with 5 mmol ⋅ L−1 ascorbic acid (AA) for shoot regeneration from embryogenic cultures. The highest embryogenesis (100%) with 36.4 globular and 5.5 heart-shaped embryo-like structures was obtained at 8 µmol ⋅ L−1 TDZ + 2 µmol ⋅ L−1 BA after 63 days. Such cultures when further maintained on the same medium up to 150 days resulted in 100% shoot regeneration with 16.4 mean shoots. Shoots were elongated up to 50 mm on agar medium + 8 µmol ⋅ L−1 BA + 1 µmol ⋅ L−1 GA3. An efficient rooting response (70%) was achieved having 4.50 mean number and 49.10 mm root length at 8 µmol ⋅ L−1 IBA + 8 µmol ⋅ L−1 NAA + 0.1% activated charcoal after 36 days. Rooted shoots were acclimatized in glasshouse, achieving 56.6% plantlet survival.

Establishment of embryogenic cultures and determination of their bioactive properties in Rosa rugosa

Somatic embryogenesis represents a powerful in vitro plant culture system and provides high potential for the production of biomass in woody plants. Rosa rugosa is known not only for its ornamental value, but also as a source of antioxidants with the potential to be used as ingredients for industrial products such as cosmetics. The present study was conducted to investigate the effects of hormonal concentration on embryogenic callus (EC) induction from petal and leaf segments of this species. The effect of selected cell lines on the production of total phenolics and flavonoids in R. rugosa was also studied. The highest percentage of EC (14.1 ± 3.9%) and root formation (20.3 ± 6.9%) was observed in the presence of 45.0 μM of 2,4-D. Among the three studied lines, the highest total phenolics and flavonoid levels were observed in H-1 (17.71 mg·g-1 DW), while H-18 contained the lowest. Histological studies of the H-1 and H-18 cell lines revealed differences in polyphenol accumulation and polysaccharides. The effect of developmental stage on growth, shoot, and root regeneration, as well as on phenolic and flavonoid content, was also studied in H-18. The present study indicates that selection of the proper type and concentration of hormone is essential for EC induction from petals in R. rugosa. Moreover, the accumulation of bioactive compounds was found to greatly depend on the genotype. The results of this study are useful for the production of selected antioxidants by somatic embryos from R. rugosa petals.

Establishment of embryogenic cultures and plant regeneration in the Portuguese cultivar ‘Touriga Nacional’ of Vitis vinifera L.

‘Touriga Nacional’ is the most important Portuguese grapevine cultivar used for Port wine, table wine and varietal wine production. In order to obtain a reproducible plant regeneration system that allows the application of biotechnological tools to grapevine breeding, embryogenic cultures were induced from immature flowers of three Touriga Nacional selected clones. Gynoecia and anthers were cultured on Nitsch and Nitsch (Science 163:85–87, 1969) basal medium supplemented with four combinations of the growth regulators 6-benzylaminopurine (BAP), 2,4-dichlorophenoxyacetic acid (2,4-D) and indole-3-acetyl-l-aspartic acid (IASP), at 28°C, in the dark. Primary callus, observed on anthers and gynoecia in all media, produced embryogenic callus when cultured on differentiation medium, at 24°C under light. The efficiency on induction of embryogenic callus ranged from 1.2 ± 4.7% to 7.9 ± 13.8% in anthers, and from 17.9 ± 24.9% to 25.3 ± 22.9% in gynoecia. Seven lines of embryogenic cultures were established from the three clones. Multiplication of embryogenic calluses was successfully obtained in maintenance medium, at 26°C, in the dark. These embryogenic calluses produced somatic embryos when subcultured on differentiation medium, under a 16 h photoperiod. Somatic embryos were isolated and cultured on germination medium to achieve conversion which ranged from 35.3 ± 48.5% to 72.7 ± 45.6%. The plantlets obtained were cultured in medium without growth regulators. Secondary embryogenesis was also frequently observed in the hypocotyl-root transition region of somatic embryos. Although some morphological variation occurred between somatic embryos, the regenerated plantlets had a normal phenotype. Maintenance of embryogenic cultures has been achieved since 2002.

Developmental and genetic variation in nuclear microsatellite stability during somatic embryogenesis in pine

Genotypic instability is commonly observed in plants derived from tissue culture and is at least partly due to in vitro-induced stress. In this work, the issues of whether genetic instability induced by in vitro stress varies among families and if genetic instability influences the adaptation to in vitro conditions and embryo development have been addressed. By comparing the stability of four variable nuclear microsatellite loci in embryogenic cultures and zygotic embryos of Pinus sylvestris, a significant difference in genetic stability among families was found. In six out of 10 families analysed, the level of genetic stability was similar between somatic and zygotic embryos. However, for the rest of the families, the mutation rate was significantly higher during somatic embryogenesis. Families showing a low genetic stability during establishment of embryogenic cultures had a higher embryogenic potential than those which were genetically more stable. In contrast, embryo development was suppressed in genetically unstable families. The relatively high mutation rates found for some families might reflect the plasticity of the families to adapt to stress, which is important for widely distributed species such as Pinus sylvestris.