Somatic Embryo Induction Research Articles

Enhancement of heavy metal tolerance and accumulation efficiency by expressing Arabidopsis ATP sulfurylase gene in alfalfa

Transgenic alfalfa (Medicago sativa L.) plants overexpressing the Arabidopsis ATP sulfurylase gene were generated using Agrobacterium-mediated genetic transformation to enhance their heavy metal accumulation efficiency. The ATP sulfurylase gene was cloned from Arabidopsis, following exposure to vanadium (V) and lead (Pb), and transferred into an Agrobacterium tumefaciens binary vector. This was co-cultivated with leaf explants of the alfalfa genotype Regen SY. Co-cultivated leaf explants were cultured on callus and somatic embryo induction medium, followed by regeneration medium for regenerating complete transgenic plants. The transgenic nature of the plants was confirmed using PCR and southern hybridization. The expression of Arabidopsis ATP sulfurylase gene in the transgenic plants was evaluated through RT-PCR. The selected transgenic lines showed increased tolerance to a mixture of five heavy metals and also demonstrated enhanced metal uptake ability under controlled conditions. The transgenic lines were fertile and did not exhibit any apparent morphological abnormality. The results of this study indicated an effective approach to improve the heavy metal accumulation ability of alfalfa plants which can then be used for the remediation of contaminated soil in arid regions.

Effect of calcium, BAP and putrescine on somatic embryo induction in juvenile explants of Eucalyptus grandis × E. urophylla hybrids

Considering the constant improvement of Eucalyptus cloning and the search for new technologies to produce plantlets of this species, somatic embryogenesis has attracted interest from research groups and forestry companies that use advanced genetic breeding and cloning programs. The objective of the present study was to verify the effect of concentrations and sources of calcium, concentrations and effect time of cytokinin BAP and polyamine putrescine on the induction and development of somatic embryos in juvenile explants of Eucalyptus grandis x E. urophylla. Cotyledonary explants were inoculated into culture medium containing calcium chloride (MS medium) or calcium nitrate (JADS medium) as source of calcium. Different concentrations of calcium were also used, for MS medium containing: 4.40 gL-1 (control-Ca), 6.60 gL-1 (50% increase over control - Ca 50) and 8.80 gL-1 (increase of 100% over control – Ca 100) of calcium nitrate; and for JADS medium containing: 11.81 gL-1 (Ca), 17.72 gL-1 (Ca50) and 23.62 gL-1 (Ca100) of calcium chloride. Cotyledon explants were inoculated into the primary induction medium (PIM) containing 20.71 μM picloram as growth regulator. At 10, 20 and 30 days of primary induction, the explants were transferred to the secondary induction medium (SIM) containing 20.71 μM picloram and 11.10 μM BAP or 28.36 μM putrescine. The culture medium containing calcium nitrate provided higher callogenesis when compared to the medium containing calcium chloride. The increase in calcium concentration in the media did not provide higher percentage of induction of somatic pro-embryos. However, the addition of 28.36 μM putrescine to the culture medium provided a higher percentage of induction of somatic embryogenesis. The number of somatic pro-embryos formed per explant was higher when BAP and putrescine were added to the culture medium when compared to medium containing only picloram. To obtain a greater number of somatic pro-embryos of Eucalyptus grandis × E. urophylla, the JADS culture medium containing 28.36 μM putrecine should be used.

In vitro cloning via direct somatic embryogenesis and genetic stability assessment of Paphiopedilum niveum (Rchb.f.) Stein: the endangered Venus’s slipper orchid

Wild populations of Paphiopedilum niveum (Rchb.f.) Stein, an endangered orchid species, have been threatened with extinction due to climate change and overcollection. Conventional methods of propagation are unable to meet market demands. The current study reports the production of genetically stable, protocorm-derived protocorm-like bodies (PLBs) of P. niveum that can be used for commercial production. These PLBs are referred to hereafter as somatic embryos (SEs). Factors affecting SE induction and SE proliferation were examined. The highest percentage of SE formation (68.33 ± 11.77%) and the maximum number of SEs per explant (5.19 ± 0.67) were obtained on modified Vacin and Went (MVW) medium containing 0.1 mg L−1 1-naphthaleneacetic acid (NAA). The highest increase in fresh weight (FW) of proliferated SEs (183.33 ± 28.93 mg per 100 mg initial FW) was gained on hormone-free MVW. These SEs eventually developed into vigorous plantlets after culturing on MVW supplemented with 0.2% (w/v) activated charcoal and 50 g L−1 banana homogenate for 12 wk. A histological study revealed that SEs originated from both the epidermal and the sub-epidermal layers of the original protocorm via direct somatic embryogenesis. The genetic homogeneity between the mother generation (V1; the original protocorm) and subsequent generations (V2 and V3; the primary and secondary somatic embryos, respectively) was shown to be identical via random amplification of polymorphic DNA (RAPD) assessment. All regenerated plants grew well under greenhouse conditions.

Molecular analysis of somatic embryogenesis through proteomic approach and optimization of protocol in recalcitrant Musa spp.

Somatic embryogenesis (SE) is a complex stress related process regulated by numerous biological factors. SE is mainly applicable to mass propagation and genetic improvement of plants through gene transfer technology and induced mutations. In banana, SE is highly genome dependent as the efficiency varies with cultivars. To understand the molecular mechanism of SE, a proteomics approach was carried out to identify proteins expressed during embryogenic calli (EC) induction, regeneration and germination of somatic embryos in the banana cultivar cv. Rasthali (AAB). In total, 70 spots were differentially expressed in various developmental stages of SE, of which 16 were uniquely expressed and 17 were highly abundant in EC compared to non-embryogenic calli and explants. Also, four spots were uniquely expressed in germinating somatic embryos. The functional annotation of identified proteins revealed that calcium signaling along with stress and endogenous hormones related proteins played a vital role in EC induction and germination of somatic embryos. Thus, based on this outcome, the callus induction media was modified and tested in five cultivars. Among them, cultivars Grand Naine (AAA), Monthan (ABB) and Ney Poovan (AB) showed a better response in tryptophan added media, whereas Red Banana (AAA) and Karpuravalli (ABB) showed maximum EC induction in kinetin and CaCl2 supplemented media respectively. Simultaneously, germination media were modified to induce proteins responsible for germination. In cv. Rasthali, media supplemented with 10 mM CaCl2 showed a maximum increase in germination (51.79%) over control plants. Thus, the present study revealed that media modification based on proteomic analysis can induce SE in recalcitrant cultivars and also enhance germination in cultivars amenable for SE.

Induction of somatic embryos of recalcitrant genotypes of <i>Theobroma cacao L.</i>

Objective: In cocoa tree ( Theobroma cacao L .), some elite genotypes have shown, in standard study conditions, an absence or a very weak response to induction of somatic embryos. This is the case of the original C8 genotype Trinidad. This study aims to improve the production of somatic embryos in this genotype Methodology and results : To do this, staminodes and petals excised from immature buds of genotypes C1, C8 and C14 were used. Genotypes C1 and C14 are embryogenic under standard conditions. These floral explants were cultured on induction media differing by the type and concentration of auxins. Callus induction obtained in the three studied genotypes ranged from 80% to 90% with the petal explants and from 70% to 80% with the staminodes regardless of the type and concentration of auxins. Transfer of callogenic explants to DKW (Driver & Kuniyuki, 1984) medium supplemented with sucrose and glucose allowed the induction of somatic embryos at mean rates varying from 5% to 20% after only 84 days with petal explants and media supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) and 2, 4, 5-trichlorophenoxyacetic acid (2,4,5-T). No induction of somatic embryos was observed on the control medium with the C1, C8 and C14 genotypes. The C1 genotype induced somatic embryos in the presence of all concentrations of auxins. The highest rate of embryogenic callus and somatic embryo induction was obtained with 18 μM of 2, 4, 5-T in all C1 genotypes (39.29 ± 0.28; 17.98 ± 0.10), C8 (36.29% ± 0.26 and 15.01 ± 0.07) and C14 (33.92% ± 0.26 and 14.50 ± 0.16). The 2, 4, 5-trichlorophenoxyacetic acid (2, 4, 5-T). at 18 μM is the most appropriate auxin to remove recalcitrance in cocoa genotype C8. The use of higher 18 μM concentrations of 2, 4, 5-trichlorophenoxyacetic acid (2, 4, 5-T). and its application to other recalcitrant genotypes could confirm its beneficial effect on the removal of recalcitrance to somatic embryogenesis in cocoa. Conclusion and application of results : 18 μM concentration of 2, 4, 5-trichlorophenoxyacetic acid (2, 4, 5-T) gave the best percentage of embryogenic explants and the highest average number of embryos in the three genotypes tested. Therefore, this protocol using 18 μM of 2, 4, 5-trichlorophenoxyacetic acid (2, 4, 5-T) could be used to overcome recalcitrance to somatic embryogenesis in cocoa. Keywords : recalcitrant, cocoa, auxins, somatic embryogenesis

Role of hydrogen peroxide in stress-induced programmed cell death during somatic embryogenesis in Fraxinus mandshurica

We examined how reactive oxygen species, in the form of hydrogen peroxide (H2O2), affect osmotic stress–induced programmed cell death during somatic embryogenesis from cotyledon explants of Manchurian ash (Fraxinus mandshurica Rupr.). We found that substantial osmotic stress was essential for Manchurian ash somatic cells to obtain embryogenic competence. The explant cells displayed hallmarks of programmed cell death, chromatin condensation, and DNA fragmentation to oligonucleotides during somatic embryogenesis. Increasing concentrations of plant growth regulators and sucrose in the medium increased osmotic stress thereby inducing H2O2 accumulation in the explant cells. We found that H2O2 concentration was significantly decreased in explant cells when the induction medium was modified, i.e., when reducing the concentration of sucrose, which reduces the osmotic pressure of the medium, or by withdrawing plant growth regulators at mid-culture. These treatments also decreased the proportion of explant cells undergoing programmed cell death. Accordingly, a decreased rate of somatic embryo induction was observed. These results show that PCD occurred during tissue browning and death of some explant cells during somatic embryogenesis in F. mandshurica. The ROS contributed to PCD in abiotic stress stimulated F. mandshurica cells.

Non-Zygotic Embryogenesis in Hardwood Species

Hardwood species are valuable biological resources that have an important role in the economy and ecology of ecosystems worldwide. Non-zygotic or somatic embryogenesis (SE) is a powerful tool in plant biotechnology as it is a form of clonal propagation, amenable to cryopreservation of valuable germplasm and genetic transformation including gene editing. The SE process involves five steps and includes somatic embryo induction, proliferation, maturation, plantlet conversion, and subsequent plant acclimatization. This review aims to provide a general overview of these steps in different SE systems developed for hardwood species. Factors that influence the induction stage such as the age of the donor plant, genotype and culture media are discussed. The role of different explant types, i.e. zygotic embryos and non-zygotic tissues, such as roots, flower tissues, nodes, internodes, leaves or shoot apices, in SE induction are especially emphasized. Histological studies of the origin of somatic embryos and the sequence of events leading to their development from initial explants are assessed. Maintenance of embryogenic capacity carried out by subculture of embryogenic inocula on semisolid or liquid media through cell suspension cultures or by temporary immersion systems is described. At present, the main concerns associated with the application of SE for large-scale propagation of elite hardwoods are related to the embryo maturation, germination, and plantlet conversion steps, and these are highlighted in this review. Finally, molecular aspects associated with somatic embryo induction and development are also described. Attempts to overcome the hurdles identified in the embryogenic process, and future lines of research are proposed.

Somatic embryos from Phalaenopsis classic Spotted Pink (Orchidaceae) protocorms

ABSTRACT: The aim of the present study was to induce the formation of somatic embryos in protocorms from Phalaenopsis Classic Spotted Pink hybrids at two physiological maturation stages, namely: 80 and 120 days after seed inoculation (DASI). Protocorms were inoculated in ½ MS medium supplemented with 0.1 mg L-1 ANA and 3 mg L-1 TDZ. Protocorms were inoculated 120 days after sowing were more developed at the 15th cultivation day due to the formation of pro-embryogenic structures. It was possible seeing the formation of globular- and torpedo-stage somatic embryos at the 30th day of cultivation in somatic embryogenesis (SE) induction medium. The protocorms inoculated at the 80th DASI did not formed somatic embryos; they oxidized 20 days after cultivation in SE-induction medium. The formation of somatic embryos happened directly on the explant, thus characterizing a direct somatic embryogenesis. The embryos converted into plants when the somatic embryos were transferred to the nutrient medium containing no growth regulator. Therefore, it was concluded that the somatic embryos induction from protocorms 120 days after sowing was positive, since the embryos were able to become plants and presented vegetative organs with morphological traits similar to those of the matrix plant.

The effect of different hormone combinations on direct and indirect somatic embryogenesis in Agave americana

Direct and indirect somatic embryogenesis in Agave Americana through basal parts of micropropagated shoots were considered in this study. For micropropagation, 2 factors (BA and NAA) were utilized in different concentrations. A factorial experiment based on a completely randomized design (CRD) was accomplished with four replicates in a modified Murashige and Skoog medium (MSB). BA in 3 mg/L along with NAA in 0.1 mg/L was the best treatment for multiplication. Callus and embryo induction were evaluated using different auxins in a CRD experiment with three replicates in MS medium supplemented with L2 vitamins. Picloram 2.5 and 2 mg/L and also Dicamba 1 mg/L induced 66.7%, 60%, and 53.3% of explants to somatic embryo, respectively. Also, 2,4-D 2 mg/L had the highest impact on callus induction (95.6%). Embryogenesis was studied using Dicamba and Picloram in the same culture medium in a factorial experiment based on CRD with three replicates. The results of orthogonal polynomial analaysisindicated that high concentrations of Dicamba from 0 to 1.5 mg/L had a significant effect on somatic embryo induction. However, higher concentrations had the opposite effect on somatic embryo numbers. Germination of somatic embryos was performed in MSB medium without growth regulators.

Effect of Water Stress Induced by Polyethylene Glycol 6000 on Somatic Embryogenesis in Cocoa (<i>Theobroma cacao</i> L.)

Climate change, coupled with insufficient and irregular rains, led to a decline in the productivity of Ivorian cocoa production. The present study aimed to screen three cocoa (Theobroma cacao L.) genotypes in order to evaluate their response to water stress at callus and somatic embryos induction and proliferation stages. Staminode and petal explants of the genotypes C1, C14 and C16 from the collection of National Center of Agronomic Research (CNRA) of Ivory Coast, were placed on medium DKW (Driver & Kuniyuki Walnut) in the presence of different concentrations of Polyethylene glycol (PEG) 6000 (0, 25, 50, 75, 100 and 125 g/l). This polymer was used as osmoticum to stimulate water stress. Data were recorded for callus induction frequency, callus fresh weight, embryogenic callus percentage and average number of somatic embryos. The results obtained showed that intensity of callus proliferation decreased with increasing concentration of PEG 6000. In all genotypes tested, only the petal explants underwent somatic embryogenesis. The induction rates and average number of somatic embryos per explant were reduced with the increase in the concentration of PEG 6000. Among the tested genotypes, C1 produced somatic embryos with all concentrations of PEG 6000 and expressed highest frequencies of induction (62%) and average number of somatic embryos per explant (6.22). This genotype would be the most tolerant to water stress. Somatic embryos obtained with high concentrations of PEG 6000 in this study provide an important basis for the selection and further production of water stress-tolerant varieties of cocoa.

AUXIN PULSE IN THE INDUCTION OF SOMATIC EMBRYOS OF Eucalyptus

ABSTRACT The objective of this study was to evaluate the effect of auxin pulse intervals on the induction of somatic embryos of Eucalyptus grandis x E. urophylla and to describe the embryogenic behavior of callus under the effect of auxinic stress. Cotyledons were inoculated in culture medium containing 207.07 µM picloram, a treatment considered as auxin pulse. Explants that were in the auxin pulse treatment were transferred to semisolid or liquid medium containing 20.71 µM picloram after one, two, four or eight days of auxin pulse. In a second experiment, explants that were on auxin pulse treatment were transferred to semi-solid medium containing 20.71 µM picloram after one, two or three days of auxin pulse. Auxiliary picloram pulse treatments (207.02 µM) can be used as an initial source of stress for the acquisition of embryogenic competence. The oxidation of cotyledonary explants may be considered as an indication of the formation of embryogenic calli. The presence of pectins in peripheral regions of somatic pro-embryos can be considered as a marker of somatic embryogenesis in cotyledonary explants of Eucalyptus grandis x E. urophylla.

Somatic embryogenesis from stamen filaments of Aesculus flava Sol. and peroxidase activity during the transition from friable to embryogenic callus

Species of the genus Aesculus are among the most attractive ornamental woody plants. Conventional propagation methods of these species are either inefficient (stem cuttings) or unsuitable for clonal propagation (seeds). The aim of the present study was to develop an efficient protocol for clonal propagation of elite specimens of yellow buckeye (Aesculus flava) by somatic embryogenesis. For this purpose, stamen filaments of yellow buckeye were cultivated on media supplemented with 1, 5 or 10 μM 2,4-dichlorophenoxyacetic acid (2,4-D) combined with 0, 1, 5 or 10 μM 6-furfurylaminopurine (Kin), either under light or dark conditions, for 8 weeks, and then subcultivated on plant growth regulator (PGR)-free medium with 400 mg/l of glutamine. The highest somatic embryo (SE) initiation rates were achieved for the explants cultivated in darkness on medium containing 1 μM 2,4-D + 10 μM Kin during callus induction (CI) phase. Embryogenic calli (EC) were initiated from friable calli, starting from the 7th week of culture initiation, while SEs appeared two weeks later, following a week of subcultivation of the explants on PGR-free medium. EC and SEs were observed only in the explants grown in darkness during CI phase. Minimal duration of CI phase and darkness necessary for SE induction was four weeks, while the highest embryogenic response was achieved when each lasted for 8–10 weeks. Obtained SEs were efficiently multiplied on medium supplemented with 0.05 μM 2,4-D + 5 μM Kin by recurrent somatic embryogenesis. SEs at globular stage of development exhibited the highest capacity for secondary SE regeneration. High germination and conversion rates were attained in cotyledonary-stage SEs cultivated on medium with 0.05 μM 2,4-D + 5 μM Kin, but this phase needs to be further optimised, since the obtained plants failed to acclimatize to greenhouse conditions. During the transition of calli from friable to embryogenic state, total peroxidase (POX) activity significantly decreased, indicating their involvement in the acquisition of embryogenic capacity. The presented protocol is suitable for clonal propagation and genetic transformation of this ornamental species, and POX activity may be used as a marker for SE initiation.

Somatic Embryogenesis of Melon (Cucumis melo L.) As Affected by Culture Media and Composition of Plant Growth Regulators

Conventional production of melon hybrid seeds requires a long time. Propagation through plant tissue culture can be an alternative method to produce hybrid melon seedlings in order to fulfill the high demand for uniform seedlings. Our current study was aimed to determine the type of propagation media and the best concentration of picloram for the induction of somatic embryogenesis in melon from young seed explants. The study was expanded to examine the effective concentrations of two of auxins, 2-4 D and NAA, combined with BAP to induce somatic embryogenesis in melon using hypocotyl explants. The experiment was conducted at Plant Tissue Culture Laboratory, Department of Agronomy and Horticulture, Faculty of Agriculture, Bogor Agricultural University. The experiment was arranged in a randomized complete block design with three replications. The first experiment tested three types of planting media (MS, B5, and WPM) and four levels of picloram concentration (0, 0.5, 1.0, 1.5 mg.L-1). The second experiment tested auxin (2,4-D and NAA) concentrations of 0, 1, 2, 3, 4, 5 mg.L-1 and two BAP concentrations, 0 and 1 mg.L-1. The first study showed that no somatic embryos were formed with the media types and picloram concentration tested; the seeds, however, germinated and formed callus. In the second study, there was an interaction between concentration levels of auxin (2,4-D and NAA) and BAP on induction of somatic embryos using hypocotyl explants. Somatic embryo formation can be induced with treatment of 1 mg.L-1 NAA + 0.1 mg.L-1 BAP and 2 mg.L-1 NAA + 0.1 mg.L-1 BAP. The highest yield of embryos formation was with the treatment of 1 mg.L-1 NAA + 0.1 mg.L-1 BAP at 0.53 embryos per explant. Keywords: embryogenesis, tissue culture media, melon, Picloram, 2,4 D, BAP

Influence of auxins on somatic embryogenesis in Haworthia retusa Duval

An efficient in vitro plant regeneration method through somatic embryogenesis has been established in Haworthia retusa. Somatic embryos were induced from leaf explants on Murashige and Skoog (MS) medium supplemented with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), and α-naphthaleneacetic acid (NAA) either alone or in combination with 4 μM thidiazuron (TDZ). Of the four auxins studied, IBA was found to be the most promising in terms of somatic embryo induction, followed in decreasing frequency by 2,4-D, IAA, and NAA. The highest somatic embryo induction (60.7%), with a mean of 20.7 embryos per leaf explant, was observed on MS medium amended with 20 μM IBA. The inclusion of 4 μM TDZ to the auxin-containing medium significantly (p < 0.0001) increased the somatic embryo induction frequency as well as the number of somatic embryos. The best combination for somatic embryogenesis was IBA + TDZ. The highest incidence of somatic embryo induction (100%), with a mean of 55.8 somatic embryos, was obtained on a culture medium containing 16 μM IBA + 4 μM TDZ. Somatic embryos germinated best on MS medium supplemented with 2 μM gibberellic acid. Morphological variations were observed among the regenerated plantlets. Well-developed plantlets obtained from germination media were acclimatized in the greenhouse.

Photo-dependent somatic embryogenesis from non-embryogenic calli and its polyphenolics content in high-valued medicinal plant of Ajuga bracteosa

Ajuga bracteosa (A. bracteosa) is one of the critically endangered and high-valued medicinal plants worldwide. Light is one of the major factor or stimulus involved in the morphogenic responses and bioactive compounds production in various medicinal plants. In this study, unique properties of colored lights have been observed on induction of somatic embryos from non-embryonic calli cultures of A. bracteosa. The maximum callogenic response (92.32%) from leaf explants was observed on Murashige and Skoog (MS) medium augmented with benzyl adenine (BA; 2.0 l−1) and 2, 4-Dichlorophenoxy acetic acid (2.4-D; 1.0 mg l−1). Calli cultures with same hormonal concentrations were placed under different spectral lights for somatic embryogenesis and photochemical variations. Red lights were found effective for maximum somatic embryos induction (92.75%) with optimum biomass accumulation (152.64 g l−1) on day 40. Similarly, among all the spectral lights, red light exhibited the highest DPPH-radical scavenging activity (DRSA; 92.86%). In contrast, blue lights induced maximum biosynthesis of chemically important total phenolics content and total flavonoids content (TPC; 0.264 and TFC; 0.06 mg/g-DW), respectively. Furthermore, blue, green and red lights also enhanced phenolics and production, polyphenolics content and total polyphenolics production in somatic embryos. It is concluded that exposure of calli cultures to colored lights provides an effective and promising in vitro technique for conservation of endangered A. bracteosa species and enhancement of its bioactive compounds. Steps should be taken to adopt these strategies/ techniques at a larger scale in order to yield maximum benefits from this highly valued medicinal plant species.

Assessment of genetic stability in somatic embryo derived plantlets of Pterocarpus marsupium Roxb. using inter-simple sequence repeat analysis.

An efficient plantlet regeneration protocol using immature zygotic embryos (IZEs) via somatic embryogenesis has been developed in Pterocarpus marsupium Roxb. The regenerated plantlets were evaluated for their genetic stability. IZEs were incubated on Murashige and Skoog (MS) media augmented with 1.07-16.11μM naphthalene acetic acid (NAA) or 0.90-13.97μM 2,4-dichlorophenoxyacetic acid. The optimum callus induction (96.6%) was observed on MS medium augmented with 5.37μM NAA. Induction of somatic embryos (SEs) was observed after sub-culture of callion medium with decreased concentrations of NAA (0.54-5.37μM), either alone or 2.69μM NAA in combination with 2.22-8.90μM benzyladenine (BA) or 2.32-9.30μM Kinetin. Maximum number (33.4 ± 0.85) of SEs occurred on MS medium augmented with 2.69μM NAA + 4.40μM BA + 3% sucrose. Highest percentage (67.3 ± 0.37) of SEs matured and developed into cotyledonary stage by subsequent subculture on the same medium. SE formation and maturation decreased when sucrose concentrations were higher than 3%. Seventy percent of mature somatic embryos developed into plantlets on half strength MS medium augmented with 5.80µM gibberellic acid. The various stages of development during somatic embryogenesis includeglobular, heart, torpedo and mature stages as revealed by thestereomicroscopic and histological studies of explants. Plantlets derived from SEs were successfully acclimatized in the greenhouse with a survival rate of 78%. Among the survived plantlets, 9 plantlets were randomly selected for inter-simple sequence repeat (ISSR) analysis. Of the 13 primers used, 8 produced reproducible and monomorphic bands. ISSR analysis revealed a homogenous amplification profile for all regenerated plantlets analyzed validating the genetic stability of somatic embryo derived plantlets.

Optimization of culturing conditions for production of somatic embryos and lignins of Schisandra chinensis (Turcz.) Baill

Schisandra chinensis (Turcz.) Baill. is a valuable medicinal plant species increasingly used in phytotherapy worldwide. This study systematically detected the lignin content and production during somatic embryogenesis of S. chinensis. The effect of various culture parameters on biomass accumulation and lignin production were also examined to optimize the accumulation of lignins in SEs in bioreactors, including the culture method, inoculum density, aeration volume and photoperiod. An inoculum density of 20 g L− 1 embryogenic calli enhanced production of lignin, while 30 g L− 1 embryogenic calli increased the biomass of somatic embryos. During somatic embryo induction, an aeration volume of 0.2 vvm and photoperiod of 16 h day− 1 were found to be optimal for biomass accumulation and lignin production. An approximately threefold increase in the biomass production rate and a fourfold increase in the total lignin production rate in SEs were achieved in bioreactors than on solid medium. The present study indicated, therefore, that the culturing of S. chinensis somatic embryos in bioreactors is an effective method for the industrialized production of lignin in vitro.

Transformation of oil palm calli using CRISPR/Cas9 System: toward genome editing of oil palm

Genome editing using CRISPR/Cas9 construct containing sgRNA is the newest technology used to assemble organisms with desired characters through knock out of the target gene. As part of the effort to develop oil palm planting material tolerant to Ganoderma, this research aimed to obtain a technique for introducing CRISPR/Cas9 modules containing sgRNA into oil palm tissue. Based on differential expression studies, two genes encoding isoflavone reductase (IFR) and methallothionine-like protein (MT) were selected as the target for edited genes. The CRISPR-IFR and CRISPR-MT modules each carrying a sequence of gene recognition (sgRNA) were constructed and transformed into oil palm calli using Agrobacterium tumefaciens. The transformed calli were cultured on a modified DF medium for callus propagation, and then sub-cultured into DF media for induction of somatic embryos. PCR analyses using specific primers flanking the DNA targeted region were carried out and the results showed that both constructs had been introduced into the cells. DNA sequence analysis indicated the presence of substitution of several bases in the target area of both genes. The transformed calli grew in a selection medium containing hygromycin. Development of somatic embryos was detected after 3-4 weeks culturing on modified DF media.

Decreased morphogenetic potential in peach palm stem-like cells in long-term in vitro conditions

Peach palm (Bactris gasipaes Kunth) has been micropropagated from pre-procambial cells that provide stem-like cell niches, (i.e., pre-procambial cells), multipotent, pluripotent and totipotent for direct vascularization, adventitious buds and somatic embryogenesis, respectively. The direct induction of adventitious buds and somatic embryogenesis reduces the frequency of mutations when compared to indirect morphogenesis. Long-term in vitro cultivation of perennial species such as peach palm cause the clones to age and deteriorate; however, the consequences for morphogenesis potential are not fully clear. The morphogenic potential of peach palm clones established and in vitro cultivated for 8 years (regeneration of adventitious buds without callus formation) was investigated in leaves, roots and stem bases using histological and histochemical analyses. Data from long-term cultures (8-years-old) was compared to data from short-term cultures (1-year-old). Morphogenic pathways monitoring for direct induction of somatic embryos and adventitious buds revealed a strong morphogenic reduction potential in the pre-procambial cells, parenchyma cells in the proximal region of stem bases, and external cells of leaf sheaths. Initial cells of shoot apical meristems and pre-procambial cells commit cell reprogramming to the undifferentiated state and subsequent acquisition of cellular competence. These results are applicable in the micropropagation of peach palm, with consideration to obtaining clones and their long-term in vitro culture.

Embryogenic callus initiation from leaf explants of Elaeis oleifera x Elaeis guineensis (OxG) hybrids

Elaeis oleifera x Elaeis guineensis (OxG) hybrid is an oil palm interspecific crossbreed to produce a variety with better oil quality, slow stem growth, and resistance to bud rot disease. However, one of the problems in OxG hybrid breeding program is an inefficiency in the conventional propagation method, while the protocol for OxG hybrid tissue culture has not been established yet in Indonesia. Somatic embryogenesis (SE) using a temporary immersion system (TIS) will be used for OxG clonal propagation as it has been established successfully in E. guineensis. Explants were young leaves derived from genotypes of selected F1 and backcross (BC) of OxG hybrids where the E. oleifera was originated in Brazil and Suriname. Callus initiation used eight media with various compositions of minerals, hormones, and active charcoal. Initial callus emerged in 8-10 weeks after culture. The average of callogenesis frequency was 3.49% in F1 and 5.59% in BC hybrid explants, with the highest at 10.73% occurred in BC Brazil. Callogenesis of OxG hybrid showed a high potency as callogenesis of E. guineensis. The callus had nodular structure which was potentially embryogenic. After 3 cultures, some of the embryogenic calli were transferred to TIS for somatic embryo induction.