Somatic Embryogenesis Culture Research Articles

Genotypic effects on accelerated propagation of oil palm breeding materials selected (Elaeis guineensis Jacq.) using somatic embryogenesis

Vegetable oil production from oil palm (Elaeis guineensis Jacq.) is an important industry due to the rising demand every year. The somatic embryogenesis culture can propagate oil palm duplicate as the parent, which can be selected as breeding material to produce new planting germplasm with high production or disease resistance. This study aims to evaluate the genotypic effect of somatic embryogenesis, while immature leaflets were employed as explants. The culture used medium embryo induction based on Murashige and Skoog (MS) modifications that contained 5 mg/l Naphthalene Acetic acid (NAA) and 0.5 mg/l Benzyl Amino Purine (BAP). The genotypic effect was statistically significant in the percentage of callus induction, producing somatic embryos, and germination embryos. In this study, we successfully cloned thirteen oil palm genotypes (GE-02, GE-03, GE-06, GE-07, GE-09, GE-23, GE-24, GE-27, GE-28, GE-32, GE-33, GE-34, and GE-35), with the highest number of somatic embryos formed on GE-27 with a percentage of 70.1%. The cloning was successful in accelerating the propagation of oil palm for materials breeding programs to create new varieties with high production and disease resistance. It is necessary to observation the performance of the clones in the field in terms of mantle flower appearance.

Agronomic Traits and Genetic Fidelity of Four Cocoa Clones Derived from Somatic Embryogenesis Culture

Morphological and genetic characterization of MCBC1, PBC230, KKM22 and KKM4 cocoa clones derived from staminode and immature zygotic embryo culture were compared with those conventionally grafted. Somatic embryogenesis culture successfully produced true-to-type progenies of elite cocoa clones of MCBC1, PBC230, KKM22 and KKM4 (from staminode culture). Phenotype variations (p < 0.05) were observed only in KKM4 clone from immature zygotic embryo culture which exhibited lower quantities in the fresh pod weight, number of flat beans per pod, seed length, seed width and individual seed weight. The genetic stability of the cultured clones was tested using fragment analysis with 12 SSR primers to validate these results. Eleven of these SSR primers detected mutations only in the allelic profiles of KKM4 clone from immature zygotic embryo. These results validated those variations in KKM4 clones of immature zygotic embryo culture were due to interactions between genotypic and explant types. Unfortunately, these variations were negative attributes to cocoa productivity. Thus, it is suggested that successful production of true-to-type KKM4 cocoa clone should consider other means of propagation including modification of the culture conditions.
 HIGHLIGHTS
 
 It is crucial to evaluate and confirm the quality of the immature zygotic and staminode regenerated cocoa plant before using the culture technique for commercial production
 The quality can be detected in both phenotype and genotype by comparing the regenerated cocoa plant with those regenerated from the conventional asexual propagation method of grafting
 Phenotype and genotype differences were only observed in KKM4 clone regenerated from immature zygotic embryo culture and thus validated that this clone exhibited variation
 KKM4 clone from immature zygotic embryo culture which has lower quantities in the fresh pod weight, number of flat beans per pod, seed length, seed width and individual seed weight confirmed the negative effect of using immature zygotic embryo explant for KKM4 clone propagation. It is suggested to modify the culture technique
 
 GRAPHICAL ABSTRACT

PEG induces maturation of somatic embryos of Passiflora edulis Sims ‘UENF Rio Dourado’ by differential accumulation of proteins and modulation of endogenous contents of free polyamines

Sour passion fruit (Passiflora edulis Sims) has economic and social relevance and is an alternative crop mainly for family farming agriculture. The aim of this work was to evaluate the influence of polyethylene glycol (PEG) on the maturation of somatic embryos associated with differential accumulation of proteins and changes in the endogenous polyamine (PA) content during somatic embryogenesis of P. edulis ‘UENF Rio Dourado’. Maturation of somatic embryos was performed using embryogenic callus in MS culture medium with PEG 6% or without PEG (control). PEG 6% promoted the maturation of a significantly higher number of somatic embryos at globular and cotyledonary stages when compared to the control treatment. The higher somatic embryo formation induced by PEG 6% was associated with an increase in endogenous contents of free spermine, a PA with an important role in the maturation process of somatic embryogenesis cultures. Comparative proteomic analyses of PEG 6%/control revealed that PEG 6% treatment induced the up-accumulation of proteins related to the ATP metabolic, glycolytic, generation of precursor metabolite energy, and the response to light stimulus processes. The down-accumulated proteins were related mainly to the cellular metabolic process. The use of PEG induced the maturation and development of somatic embryos of P. edulis Sims ‘UENF Rio Dourado’ by the differential accumulation of proteins and modulation of endogenous contents of PAs.

Reproductive Characteristics of the Selected Cocoa (Theobroma cacao L.) Clones after Regenerated from the Somatic Embryogenesis Culture

This study was conducted to evaluate the reproductive characteristics of 4 elite cocoa clones (MCBC1, PBC230, KKM22 and KKM4) propagated via somatic embryogenesis culture. From the findings, all clones have similar reproductive characteristics with clones from conventional grafted. However, only KKM4 clone from immature zygotic embryo culture produced the shortest staminode to style distance of 1.83 mm. This consequently influenced flower stability by reducing the efficiency of pollination by insects. It was found that this clone also has the highest number of flowers drop after anthesis (5 flowers) and lowest production of cherelle (5 cherelles). Further observation revealed that floral development from first bud visible (BBCH51) to flower anthesis (BBCH68) of all clones took around 31 days. These cocoa flowers which remained receptive soon after anthesis at 10 am (day-31) until the next day (day-32) suggesting 2 days’ period of receptivity.
 HIGHLIGHTS
 
 It is crucial to assess the presence of off-type characteristics in the reproductive organ structure such as the distance between staminode to style, period of reproductive cycle and stigmatic receptivity of cocoa clones regenerated from somatic embryogenesis
 The converging and parallel type of staminode to style distances are the ideal flower spatial arrangements for the optimal pollination in cocoa plant compared to splay type
 Only KKM4 clone propagated from immature zygotic embryo culture showed variation in the distance between staminode to style distance and this caused pollination failure by insect which then consequently caused minimum cherelle production
 All regenerated cocoa clones observed with typical period of the reproductive cycle and stigmatic receptivity
 
 GRAPHICAL ABSTRACT

Agronomic Performance and Genetic Fidelity of the Selected Elite Cocoa Clones Derived from Somatic Embryogenesis Culture

This study was conducted to compare the agronomic performance of four elite cocoa clones (MCBC1, KKM22, KKM4 and PBC230) regenerated from staminode and immature zygotic embryo culture with conventional grafted cocoa clones. From the results, it was found that the KKM4 clone propagated from immature zygotic embryo culture exhibited variations in the fresh pod weight (339.6 g), fresh individual seed weight (4.13 g) and number of flat beans per pod (4 beans) compared with the rest of the regenerated clones. The genetic stability of the somatic embryogenesis cultured clones and the donor clones was then tested using fragment analysis with five SSR primers, i.e. mTcCIR7, mTcCIR18, mTcCIR22, mTcCIR33 and mTcCIR40. Four of these primers identified variations in the allele size and allele addition in KKM4 clone from immature zygotic embryo. Molecular analysis validated that the difference in agronomic performance of the KKM4 clone from immature zygotic embryo culture was due to genetic mutation created during the immature zygotic embryo culture process.

Primary and secondary somatic embryogenesis in Jatropha curcas L. From leaf transverse thin cell layers

An efficient method for plant regeneration in Jatropha curcas L. via primary and secondary somatic embryogenesis culture from ex vitro leaves of 6-month-old plants was presented in this study. Leaves were cut into transverse thin cell layers (tTCLs) and cultured on MS medium supplemented with kinetin (KIN) at 0.5, 1.0, 1.5, and 2.0 mg/l in combination with indole-3-butyric acid (IBA) at 0.1, 0.5, and 1.0 mg/l or 2,4-dichlorophenoxyacetic acid (2,4-D) at 1.0, 1.5 and 2.0 mg/l . The highest embryogenic callus formation rate (89.3%) was obtained on medium supplemented with 1.0 mg/l KIN and 1.5 mg/l 2,4-D. The calli were selected for the study of primary somatic embryogenesis on MS medium containing 2,4-D (0.01, 0.03, 0.05, and 0.07 mg/l) or KIN (0.5, 1.0, 1.5, and 2.0 mg/l). The highest primary somatic embryos formation rate (76.67%) was achieved on MS medium supplemented with 1.0 mg/l KIN. The primary embryos were cultured on medium supplemented with KIN (0.1, 0.5, 1.0, 1.5, and 2.0 mg/l) combined with 0.2 mg/l indole-3-butyric acid (IBA) or 0.05 mg/l 2,4-D. The combination of 1.5 mg/l KIN and 0.05 mg/l 2,4-D was suitable for secondary embryos formation. Embryos proliferated rapidly, and the highest number of secondary embryos (77.5 embryos) wasobtained from a single primary embryos inoculated. Results also showed that the addition of proline (0.75 g/l) or spermidine (0.15 mM) to the culture medium increased the number of secondary embryos considerably. The fully developed plantlets exhibiting healthy roots and shoots were obtained when somatic embryos were sub-cultured onto B5 medium containing 1.5 mg/l IBA.

DNA methylome of the 20-gigabase Norway spruce genome

DNA methylation plays important roles in many biological processes, such as silencing of transposable elements, imprinting, and regulating gene expression. Many studies of DNA methylation have shown its essential roles in angiosperms (flowering plants). However, few studies have examined the roles and patterns of DNA methylation in gymnosperms. Here, we present genome-wide high coverage single-base resolution methylation maps of Norway spruce (Picea abies) from both needles and somatic embryogenesis culture cells via whole genome bisulfite sequencing. On average, DNA methylation levels of CG and CHG of Norway spruce were higher than most other plants studied. CHH methylation was found at a relatively low level; however, at least one copy of most of the RNA-directed DNA methylation pathway genes was found in Norway spruce, and CHH methylation was correlated with levels of siRNAs. In comparison with needles, somatic embryogenesis culture cells that are used for clonally propagating spruce trees showed lower levels of CG and CHG methylation but higher level of CHH methylation, suggesting that like in other species, these culture cells show abnormal methylation patterns.

Micropropagation of Coffea sp. by somatic embryogenesis cultures technique

Micropropagation of Coffea sp. by somatic embryogenesis cultures technique

Effect of sorbitol concentration on regeneration of embryogenic calli in upland rice varieties (Oryza sativa L.)

This study describes the impact of sorbitol on plantlets regeneration frequency (PRF) of four rice cultivars (japonica, Oryza sativa L.) both of which mature and immature embryo-derived calli were investigated. The variance analysis results showed that PRF of the three elite upland rice cultivars, Handao297 (HD297), Handao502 (HD502), Handao65 (HD65) and one lowland rice cultivar Zhongzuo93 (ZZ93) were significantly increased with addition of appropriate amount of sorbitol in culture media. Supplementing appropriate sorbitol in the media of a continous culture from induction and maintenance to regeneration for mature embryo-derived calli could improve PRF dramatically, originally from 27.6% up to a maximum of 71.8%. Especially to low regenerative capacity (LRC) cultivar HD65, the PRF was increased over 7-fold (from 9.7% to 74.0%). The optimum concentrations of sorbitol for calli induction, subculture and differentiation were 5, 20 and 40 g/l, respectively. Adding sorbitol, only in maintenance media at concentration of 20 g/l, also enhanced the PRF greatly in all the cultivars from 27.6% to 43.3%. Similar results were observed when incorporating with maltose in regenerating media both in immature and mature embryo-derived calli. The optimal concentration was 25 g/l sorbitol + 20 g/l maltose and 20 g/l sorbitol + 25 g/l maltose, respectively. HD297 appeared to be the most responsive genotype compared to other cultivars in PRF, 99.2% in immature embryo-derived calli and 76.8% in mature embryo-derived calli. The results and relevant conclusions might be valuable to establish an efficient plant regeneration system from somatic embryogenesis culture in upland rice.

DNA homologues in some species and expression patterns in cucumber organs of genes corresponding to cDNAs from carrot somatic embryogenesis cultures

The presence of DNA homologues in eight species and the expression patterns in nine cucumber organs of genes corresponding to six cDNAs from carrot somatic embryogenesis cultures were investigated by non-radioactive DNA labeling and detection techniques. DNA homologues corresponding to the six cDNAs were detected by Southern dot blot hybridization in carrot ( Daucus carota ), celery ( Apium graveolens ), Chinese ivy ( Hedera helix ), cucumber ( Cucumis sativa ), green pepper ( Capsicum annuum ), Japanese morning glory ( Pharbitis nil choisy ), soybean ( Glycine max ), and wheat ( Triticum aestivum ). The genes corresponding to these cDNAs were highly expressed in cucumber embryos, roots and hypocotyls of germinating seeds and not expressed or undetectable in developing leaves, stems, and female flowers of mature cucumber plants. Transcripts corresponding to some of the cDNAs were detected in male flowers, developing fruits, and dry seed embryos of cucumber. These data indicate that DNA sequences corresponding to the six cDNAs may be conserved in the eight species, and expression of these genes in cucumber plants is developmentally regulated.

Machine vision based analysis and harvest of somatic embryos

The heterogeneity of embryo maturity and quality typically found in somatic embryogenesis cultures has impeded the commercialization of this propagation technique. This variability necessitates identifying viable embryos in culture and separating them from the immature embryos and abberant structures prior to ex vitro processing. In this work a fluidic, in vitro embryo monitor and harvester was developed for liquid based, bioreactor culture systems. A machine vision system was used to rate the viability of embryos as they were pumped through a 3 mm, square glass conduit. Embryo morphologies were ranked with 17 geometric features processed by a neural network. Embryos which ranked as harvestable by the network were ejected from a gap in the flow conduit by a precisely timed injection of medium from a control nozzle. The position and velocities of embryos between the imaged section of the conduit and the harvest gap were monitored by an object tracker employing 30 LED/photo-diode pairs mounted on 2.54 mm centers along the conduit. Objects not harvested were routed back to the bioreactor. The harvester successfully removed 28% of harvestable embryos from a test population manually introduced at a rate of one object every 4 s. The homogeneity of the harvested population was 88% compared to the test population homogeneity of 56%.