Embryo Explants Research Articles

Establishment, Multiplication, and Biochemical Analysis of Embryogenic Lines of the Amazonian Palm Euterpe precatoria Mart. under Suspension Culture

The palm Euterpe precatoria holds great social, cultural, and environmental importance. The heart of palm and the fruit are the main products used for industrialization due to their energetic properties. Thus, the aim of this study was to establish a suspension cultivation protocol for the species using different explant sources. For this, eight lineages of E. precatoria embryogenic calluses were tested, with five in liquid medium Murashige and Skoog (MS) with 5 μM Picloram and three for comparison in semisolid medium MS with 20 μM Picloram and 5 μM 2iP. The growth curve was obtained by weighing the calli from 60 to 180 days of cultivation. The Gompertz model was applied, and growth kinetics were evaluated. At 100 days, the contents of total soluble sugars (TSSs) and total soluble proteins (TSPs) were determined. Principal components (PCA) were measured. According to the analysis of the data, the cultivation of E. precatoria lineages in liquid medium was successfully carried out, and the establishment was achieved. The model can be considered adequate since the R2 values found describe more than 90% of the growth kinetics of the lineages. In the liquid system, lineages L1 (from leaf explants and multiplied in semisolid medium—SM), L2 (from leaf explants and multiplied in SM), and L6 (from zygotic embryo explants and multiplied in liquid medium—LM) showed the shortest time to double the biomass accumulation. Multivariate analysis reveals a significant increase in masses in liquid cultures, represented by lineages L6 and L2. There was statistical difference in the amount of TSSs extracted; the highest TSS levels were observed in lineages cultivated in LM. The protein content found was very low, showing statistical differences among the lineages. In this work, the establishment and multiplication of embryogenic calli of E. precatoria are described for the first time, and they emerge as viable alternatives for the vegetative propagation of the species.

A RapidAgrobacterium-Mediated Transformation Method Using Maize B104 Immature Embryos

Maize genetic transformation is a critical tool for functional genomics and crop improvement. Many laboratories, however, continue to face multiple challenges in attempting to achieve routine genetic transformation of maize inbred genotypes. Here, we describe a rapid and robust maize B104 transformation method using immature embryos as explants. This method uses anAgrobacteriumternary vector system, which includes a conventional T-DNA binary vector (pCBL101-RUBY) and a compatible ternary helper plasmid (pKL2299) that carries extra copies of essential virulence genes. The T-DNA binary vector carries theneomycin phosphotransferase II(NptII) gene for selection and a betalain biosynthesis marker,RUBY,for visual screening. We provide step-by-step instructions for immature embryo explant preparation,Agrobacteriuminfection, tissue culture procedures, and greenhouse care for acclimatization of regenerated plantlets.

Simultaneous genetic transformation and genome editing of mixed lines in soybean (Glycine max) and maize (Zea mays)

Robust genome editing technologies are becoming part of the crop breeding toolbox. Currently, genome editing is usually conducted either at a single locus, or multiple loci, in a variety at one time. Massively parallel genomics platforms, multifaceted genome editing capabilities, and flexible transformation systems enable targeted variation at nearly any locus, across the spectrum of genotypes within a species. We demonstrate here the simultaneous transformation and editing of many genotypes, by targeting mixed seed embryo explants with genome editing machinery, followed by re-identification through genotyping after plant regeneration. Transformation and Editing of Mixed Lines (TREDMIL) produced transformed individuals representing 101 of 104 (97%) mixed elite genotypes in soybean; and 22 of 40 (55%) and 9 of 36 (25%) mixed maize female and male elite inbred genotypes, respectively. Characterization of edited genotypes for the regenerated individuals identified over 800 distinct edits at the Determinate1 (Dt1) locus in samples from 101 soybean genotypes and 95 distinct Brown midrib3 (Bm3) edits in samples from 17 maize genotypes. These results illustrate how TREDMIL can help accelerate the development and deployment of customized crop varieties for future precision breeding.

Co-overexpression of chitinase and β-1,3-glucanase significantly enhanced the resistance of Iranian wheat cultivars to Fusarium

Fusarium head blight (FHB) is a devastating fungal disease affecting different cereals, particularly wheat, and poses a serious threat to global wheat production. Chitinases and β-glucanases are two important proteins involved in lysing fungal cell walls by targeting essential macromolecular components, including chitin and β-glucan micro fibrils. In our experiment, a transgenic wheat (Triticum aestivum) was generated by introducing chitinase and glucanase genes using Biolistic technique and Recombinant pBI121 plasmid (pBI-ChiGlu (-)). This plasmid contained chitinase and glucanase genes as well as nptII gene as a selectable marker. The expression of chitinase and glucanase was individually controlled by CaMV35S promoter and Nos terminator. Immature embryo explants from five Iranian cultivars (Arta, Moghan, Sisun, Gascogen and A-Line) were excised from seeds and cultured on callus induction medium to generate embryonic calluses. Embryogenic calluses with light cream color and brittle texture were selected and bombarded using gold nanoparticles coated with the recombinant pBI-ChiGlu plasmid. Bombarded calluses initially were transferred to selective callus induction medium, and later, they were transfferd to selective regeneration medium. The selective agent was kanamycin at a concentration of 25 mg/l in both media. Among five studied cultivars, A-Line showed the highest transformation percentage (4.8%), followed by the Sisun, Gascogen and Arta in descending order. PCR and Southern blot analysis confirmed the integration of genes into the genome of wheat cultivars. Furthermore, in an in-vitro assay, the growth of Fusarium graminearum was significantly inhibited by using 200 μg of leaf protein extract from transgenic plants. According to our results, the transgenic plants (T1) showed the resistance against Fusarium when were compared to the non-transgenic plants. All transgenic plants showed normal fertility and no abnormal response was observed in their growth and development.

Establishment of somatic embryogenesis regeneration system and transcriptome analysis of early somatic embryogenesis in Litchi chinensis

Litchi chinensis Sonn. is an important economic fruit tree in tropical and subtropical regions. Regrettably, the efficiency of plant regeneration via somatic embryogenesis in litchi is typically low due to the poor conversion of embryos to plants. The purpose of this study was to establish a regeneration system via somatic embryogenesis from immature embryos explants in ‘Heiye’ cultivar of litchi. Our results demonstrated that MS medium supplemented with 2.0mg L−1 2,4-D was optimal for callus induction. For somatic embryo (SE) induction, MS medium containing 0.5 g L−1 activated charcoal (AC) was the most effective, while the use of zeatin (ZT) and thidiazuron (TDZ) resulted in abnormal somatic embryos. The rooting and regeneration rate of 2.15% and 17.5%, respectively, were achieved using MS medium supplemented with 0.5 g L−1 AC. Furthermore, transcriptome analysis was performed on embryogenic callus (EC), globular embryo (GE), and heart embryo (HE) to explore the molecular mechanisms of early somatic embryogenesis. 2,587 common DEGs between EC_vs_GE and EC_vs_HE were identified, and the expression patterns of these common DEGs were separated into twelve major clusters. GO annotation and KEGG pathway analysis revealed that these common DEGs were implicated in plant hormone signal transduction, auxin-activated signaling pathway, and other biological processes. Additionally, differentially expression transcription factors were identified, and the function of LcBBM2 which is specifically highly expressed during early somatic embryogenesis was verified. Overexpression of LcBBM2 in tomato promotes callus and shoot formation. Therefore, this study can provide a theoretical basis and technical support for genetic breeding improvement of litchi.

Synergistic effects of L-glutamine and inorganic nitrogen molar ratios enhance the induction of somatic embryogenesis of Pinus maximinoi H.E. Moore

Clonal breeding programs of Pinus maximinoi require the establishment of a robust somatic embryogenesis (SE) protocol to produce enough cell lines to accelerate the effective continuous deployment of elite planting stocks to research and commercial compartments. Somatic embryogenesis was induced from immature zygotic embryo explants enclosed in megagametophytes of P. maximinoi collected from two plantations located in different climatic conditions. Cones were collected during the winter months from July to August and the influence of seed family, cone collection date and culture medium formulation, with emphasis on the organic and inorganic nitrogen supply, were studied. Ammonium to nitrate molar ratios of 1:1 and 1:2 in modified Litvay’s medium (mLV) produced the highest numbers of extrusions, while a 1:4 ratio mostly produced unhealthy, non-embryogenic extrusions. The formation of a tissue showing a rapidly-proliferating, spiky morphotype was produced in a medium supplemented with 1.5 g/L of L-glutamine. Morphologically advanced cultures with nodular structures were produced in megagametophytes from both plantations in a 1:2 NH4+:NO3− medium regardless of L-glutamine supplementation levels. The optimal medium for P. maximinoi SE induction contained a 1:2 NH4+:NO3− molar ratio with 1.5 g/L L-glutamine. The synergy between the molar ratio of NH4+:NO3− and L-glutamine resulted in the highest numbers of extrusions. The overall inductive competence window for somatic embryogenic response in P. maximinoi was determined to be from the second week of July to the first week of August for both plantations. The “peak” period was in the fourth week of July 2022. The success of the SE technology in P. maximinoi seed families is determined by the optimal inductive competence window of the immature megagametophytes enclosing zygotic embryos and the chemical composition of the induction medium in terms of the ammonium to nitrate molar ratio and the concentration of the L-glutamine used.

Plant Regeneration via Adventitious Shoot Formation from Immature Zygotic Embryo Explants of Camelina.

Camelina is an oil seed crop that is enjoying increasing interest because it has a particularly valuable fatty acid profile, is modest regarding its water and nutrient requirements, and is comparatively resilient to abiotic and biotic stress factors. The regeneration of plants from cells accessible to genetic manipulation is an essential prerequisite for the generation of genetically engineered plants, be it by transgenesis or genome editing. Here, immature embryos were used on the assumption that their incomplete differentiation was associated with totipotency. In culture, regenerative structures appeared adventitiously at the embryos' hypocotyls. For this, the application of auxin- or cytokinin-type growth regulators was essential. The formation of regenerative structures was most efficient when indole-3-acetic acid was added to the induction medium at 1 mg/L, zygotic embryos of the medium walking stick stage were used, and their hypocotyls were stimulated by pricking to a wound response. Histological examinations revealed that the formation of adventitious shoots was initiated by locally activated cell division and proliferation in the epidermis and the outer cortex of the hypocotyl. While the regeneration of plants was established in principle using the experimental line Cam139, the method proved to be similarly applicable to the current cultivar Ligena, and hence it constitutes a vital basis for future genetic engineering approaches.

Mechanical Tensions Regulate Gene Expression in the Xenopus laevis Axial Tissues.

During gastrulation and neurulation, the chordamesoderm and overlying neuroectoderm of vertebrate embryos converge under the control of a specific genetic programme to the dorsal midline, simultaneously extending along it. However, whether mechanical tensions resulting from these morphogenetic movements play a role in long-range feedback signaling that in turn regulates gene expression in the chordamesoderm and neuroectoderm is unclear. In the present work, by using a model of artificially stretched explants of Xenopus midgastrula embryos and full-transcriptome sequencing, we identified genes with altered expression in response to external mechanical stretching. Importantly, mechanically activated genes appeared to be expressed during normal development in the trunk, i.e., in the stretched region only. By contrast, genes inhibited by mechanical stretching were normally expressed in the anterior neuroectoderm, where mechanical stress is low. These results indicate that mechanical tensions may play the role of a long-range signaling factor that regulates patterning of the embryo, serving as a link coupling morphogenesis and cell differentiation.

Effects of a New Plant Growth Regulator on Callus Induction from Immature Embryo Explants of Korean Pine (Pinus koraiensis)

Somatic embryogenesis is currently the most promising technique for the large-scale production and breeding of conifer species. Nonetheless, the low explant induction rate in this process hampers the development of improved materials. In this study, three immature Korean pine (Pinus koraiensis) embryos capable of induction were used as experimental materials. Various concentrations of brassinolide (BL), melatonin (Mel), salicylic acid (SA), glutathione (GSH), and ascorbic acid (AsA) were added to induce embryogenic callus tissue. The results show that BL had the most significant positive effect on promoting induction and increasing explant survival. Mel was also found to slightly increase the induction and survival rates of explants. When 1.00 mg·L−1 BL was added to the explants derived from stock tree I, which had strong self-induction ability, for 30 days, the callus induction rate rose to 400% compared to the control group. Following the addition of 25 mg·L−1 Mel to stock tree I for 30 days, the callus induction rate further increased to 450% of the control group. The effect of GSH on callus induction was insignificant. The addition of 0.10 g·L−1 to stock tree I for 30 days resulted in only a 150% higher induction rate compared to the control group. When 1.00 mg·L−1 BL was applied to explants with weak self-induction ability (stock tree III) for 15 days, the callus induction rate increased to 600% of that of the control group. The callus induction rate further increased to 800% of the control group after 50 mg·L−1 BL were added to stock tree III for 15 days. This study presents a method to improve the induction of embryogenic callus tissue in Korean pine.

Effect of the Combination of the Growth Regulators and Putrescine on the Somatic Embryogenesis of Wheat (Triticum Aestivum L.) on Some Types of Explants

Plant breeding programs need to be carried out in order to improve the genetics of wheat that is able to adapt to tropical environments through hybridization, mutation induction, tissue culture, and genetic transformation. In vitro culture through somatic embryogenesis pathways plays an important role in genetic improvement and its integration with other breeding programs can positively affect the improvement of wheat quality, quantity, and development in Indonesia. The purpose of this study was to obtain an embryogenic callus induction method from the Dewata variety using five different types of explants, namely mature Seeds, immature embryos, immature seeds, leaf, stem, and to obtain combination of plant growth regulators and putrescine on somatic embryogenesis of wheat. The experimental design was prepared based on a complete randomized design with a combination treatment of embryogenic callus induction media consisting of 9 levels, namely: 1 ppm 2.4- D, 1 ppm 2.4-D + 1 ppm Picloram, ppm 2.4-D + 1 mg / L Picloram, 1 ppm 2.4-D + 10-4 M Putrescine, 1 ppm 2.4-D + 1 ppm Picloram +10-4 M Putrescine, 2 ppm 2.4-D + 1 ppm Picloram + 10-4 M Putrescine, 1 ppm 2.4 D + 10-3 M Putrescine, 1 ppm 2.4 D + 1 ppm Picloram + 10-3 M Putrescine, 2 ppm 2.4 D + 1 ppm Picloram + 10-3 M Putrescine. The results showed that the media used was able to induce embryogenic callus using mature seed and immature embryo, but immature seed and leaf were not able to produce embryogenic callus. The best media that produced the highest percentage of embryogenic callus was 2 ppm 2.4-D + 1 ppm Picloram + 10-4 M Putrescine with as much as 85.9% in young embryo explants.

Comparison of somatic embryogenesis efficiency from in vitro explant sources of Ngoc Linh ginseng (Panax vietnamensis Ha et Grushv.)

This study on the efficiency of embryogenesis from various explant sources of Ngoc Linh ginseng (Panax vietnamensis Ha et Grushv.) was conducted to select the appropriate materials and optimise embryogenesis in vitro. In this study, in vitroexplants from leaf fragments, petioles, roots, and embryos (globular, heart-shaped, and cotyledon) were cultured on MS medium supplemented individually or in combination with 2,4-D (2,4-dichlorophenoxyacetic acid: 0.2, 0.5, and 0.7 mg/l) and TDZ (Thidiazuron: 0.1, 0.3, and 0.5 mg/l). The results showed that all explants had embryogenesis after 6 weeks of culture (100%). The average number of embryos per leaf fragment (51.00 embryos) on the optimal medium was significantly higher than that of petioles (29.67 embryos) and root samples (18.00 embryos). Interestingly, the globular embryo explant was noted as the most suitable material source for embryogenesis (68.33 embryos/sample). The culture medium with the combination of 2,4-D and TDZ increased the ability to create secondary embryos in globular embryos, the highest result was achieved at the concentration of 0.7 mg/l 2,4-D combined with 0.1 mg/l TDZ in the medium containing 0.5 mg/l NAA - 1-Naphthaleneacetic acid (73.33 embryos/sample).

The Influence of Plant Growth Regulators on Callus Induction in Mature Rice Embryos (Oryza Sativa L) Amber Variety

Several nations across the globe depend profoundly on rice as the main food source. Several research works have been undertaken to improve rice yield and breeding, specifically concerning plant tissue culture. This paper aims to contrast the impact of several growth regulator levels on rice callus induction based on mature embryo explants. Over a four-week duration, mature rice plant embryos (Amber variant) were planted to produce calluses using the Skoog (MS) and Murashige supplements at different concentration levels, i.e., 2.0, 2.5, and 3.0 mg/L of 2,4-D and 0.2 and 0.3 mg/L of BA. The outcomes indicated that 3 mg/L 2,4-D and 0.2 mg/L BA levels led to optimal induction and fresh weight concerning callus. Further studies are required to enhance the media environment for potent in vitro callus induction to augment biotechnology endeavours.

Effects of mammalian sex hormones on in vitro organogenesis of common bean (Phaseolus vulgaris L.)

Beans are an important plant species and are one of the most consumed legumes in human nutrition, especially as a protein, vitamin, mineral, and fiber source. Common bean (Phaseolus vulgaris L.) is a plant that also has an important role in natural nitrogen fixation. Currently, in vitro regeneration and micropropagation applications are limited in relation to genetic factors in bean Accordingly, there is great need to optimize micropropagation and tissue culture methods of the bean plant. To date, the effect of mammalian sex hormones (MSH) on in vitro conditions in P. vulgaris L. is poorly understood. This study examined the effects of different types of explants (embryo, hypocotyl, plumule, and radicle), MSH type (progesterone, 17 β-estradiol, estrone, and testosterone), and MSH concentration (10−4, 10−6, 10−8 and 10−10 mmol L−1) on the responding explants induction rate (REI), viability of plantlets rate (VPR), shoot proliferation rate (SPR), root proliferation rate (RPR), and callus induction rate (CIR). The effects of mammalian sex hormones, concentrations, explant type, and their interactions were statistically significant (p ≤ 0.01) in all examined parameters. The best explants were embryo and plumule. Our results showed that the highest REI rate (100%) was recorded when 10−10 mmol L−1 of all MSH was applied to MS medium using the plumule explant. The highest VPR (100%) was obtained when 10−10 mmol L−1 of all MSH was applied to MS medium using the plumule explant. The highest root proliferation rates (77.5%) were recorded in MS medium supplemented with 10−8 mmol L−1 17β-estradiol using embryo explant. The highest percentage of shoot-forming explants (100%) generally was obtained from embryo and plumule cultured in the MS culture medium with low MSH concentration. In addition, the highest CIR (100%) was obtained from embryo and plumule explant cultured in MS medium containing 10−10 mmol L−1 of all MSH types. In conclusion, we observed that mammalian sex hormones may be used in bean in vitro culture.

Commercial scale genetic transformation of mature seed embryo explants in maize.

A novel, efficient maize genetic transformation system was developed using Agrobacterium-mediated transformation of embryo explants from mature seeds. Seeds from field grown plants were sterilized and crushed to isolate embryo explants consisting of the coleoptile, leaf primordia, and shoot apical meristem which were then purified from the ground seed bulk preparation. The infection of relevant tissues of seed embryo explants (SEEs) by Agrobacterium was improved by the centrifugation of the explants. Transgenic plants were obtained by multiple bud induction on high cytokinin media, followed by plant regeneration on hormone-free medium. Three different selectable markers (cp4 epsps, aadA, and nptII) were successfully used for producing transgenic plants. Stable integration of transgenes in the maize genome was demonstrated by molecular analyses and germline transmission of the inserted transgenes to the next generation was confirmed by pollen segregation and progeny analysis. Phenotypic evidence for chimeric transgenic tissue was frequently observed in initial experiments but was significantly reduced by including a second bud induction step with optimized cytokinin concentration. Additional improvements, including culturing explants at an elevated temperature during bud induction led to the development of a revolutionary system for efficient transgenic plant production and genome editing. To our knowledge, this is the first report of successful transgenic plant regeneration through Agrobacterium-mediated transformation of maize mature SEEs. This system starts with mature seed that can be produced in large volumes and the SEEs explants are storable. It has significant advantages in terms of scalability and flexibility over methods that rely on immature explants.

An Optimized Protocol for Seed Sterilization and Shoot Regeneration from Mature Embryo in Wheat (Triticum Aestivum L.) var. HD2967

In the present study, an efficient protocol for seed sterilization was developed. This protocol used a 1.0 % bavistin solution, which provides a protective response against fungal contamination. It was observed that there was no significant difference among the effective sterilization treatments in comparison to seed germination and seedling length under in vitro conditions. Furthermore, the embryo excised from bavistin-treated seeds showed improvement in callus induction and shot regeneration in var. HD2967. 0.1% HgCl2-SDS solution used for seed sterilization was also optimized with 2.5 to 3.0 minutes as the best-suited duration, providing a completely sterilized environment for the seeds for healthy and fast growth. In addition, plant growth regulators (PGRs) were further used to screen the most effective concentration for callus to shoot formation using mature embryos. Auxin concentration of 2.0 mg/l in MS agar media was quite effective for callus induction from embryo explant. For shoot regeneration (SI-7) combination is suitable for a maximum number of shoots per callus. This combination of 2, 4-D, Zeatin, CuSO4, and TDZ showed good regeneration in the wheat variety HD2967. The optimized protocol displayed 100% callus induction and up to 80-85% regeneration in HD2967, with a high reproducibility rate paving the way for regenerating plantlets from mature wheat embryos.

A Comparative Transcriptome Analysis Reveals the Molecular Mechanisms That Underlie Somatic Embryogenesis in Peaonia ostii 'Fengdan'.

Low propagation rate is the primary problem that limits industry development of tree peony. In this study, a highly efficient regeneration system for tree peony using somatic embryogenesis (SE) was established. The transcriptomes of zygotic embryo explants (S0), non-embryonic callus (S1), embryonic callus (S2), somatic embryos (S3), and regenerated shoots (S4) were analyzed to determine the regulatory mechanisms that underlie SE in tree peony. The differentially expressed genes (DEGs) were identified in the pairwise comparisons of S1-vs-S2 and S1-vs-S3, respectively. The enriched DEGs were primarily involved in hormone signal transduction, stress response and the nucleus (epigenetic modifications). The results indicated that cell division, particularly asymmetric cell division, was enhanced in S3. Moreover, the genes implicated in cell fate determination played central roles in S3. Hormone signal pathways work in concert with epigenetic modifications and stress responses to regulate SE. SERK, WOX9, BBM, FUS3, CUC, and WUS were characterized as the molecular markers for tree peony SE. To our knowledge, this is the first study of the SE of tree peony using transcriptome sequencing. These results will improve our understanding of the molecular mechanisms that underly SE in tree peony and will benefit the propagation and genetic engineering of this plant.

Effect of copper and silver ions on sequence and DNA methylation changes in triticale regenerants gained via somatic embryogenesis

Somatic embryogenesis is a plant regeneration method that can be exploited in tissue culture systems for a variety of tasks, such as genetic modification or the selection of somaclones with advantageous characteristics. Therefore, it is crucial to create efficient regeneration procedures and comprehend how medium components affect regeneration effectiveness or the degree of variation created in plant tissue cultures. The level of tissue culture-induced variation in triticale regenerants was examined in the current study in relation to the concentration of copper and silver ions in the induction media as well as the length of time immature zygotic embryo explants were incubated on these media. The high degree of variation (45%) revealed by the methylation-sensitive amplified fragment length polymorphism approach for estimating variation included 38% DNA sequence alterations, 6% DNA demethylation, and 1% de novo DNA methylation. Different levels of variance were found in relation to various DNA sequence settings. The CHG context had the most alterations, whereas CG experienced the fewest; sequence variation predominated in each sequence context. Lower copper ion concentrations showed the most variance. However, it could not be connected to the duration of in vitro culture or the effect of silver ions. Accordingly, we think that altering the concentration of copper ions in the induction medium may throw off the equilibrium of the metabolic processes in which copper is involved, resulting in tissue culture-induced variation.

IN VITRO ASSESSMENT OF EMBRYO AGE AND NAPHTHALENE ACETIC ACID SUPPLEMENTATION OF MEDIA IN CALLUS GENERATION OF OIL PALM (Elaeis guineensis Jacq)

Callus generation from embryo explants of the oil palm (Elaeis guineensis Jacq) was observed in relation to age of plants at time of explants excision and types of media used. The two stages of explants were those at 15 and 18 weeks after anthesis. Eeuwens and Murashige & Skoog‘s (MS) media supplemented with Naphthalene acetic acid (NAA) were used. These gave two media variations viz: MS medium supplemented with NAA and Eeuwens‘ medium supplemented with NAA. The best combination for the generation of callus from the embryo explants within 56 days in culture was determined by observing type of response, time of initiation of callus and callusing explants. It was found that callus generation was best from embryo explants taken from 18 weeks after anthesis cultured in Eeuwens‘ medium, supplemented with NAA. The optimum concentration of NAA was found to be 50 mgl-1, which initiated callus within 39 days in culture. The percentage of explants producing callus was 80 %. The results revealed the importance of the supplementation of nutrient media with NAA and the usefulness of selecting embryo explants of about 18 weeks after anthesis for use in callus generation in the oil palm

Effect of type of mature embryo explants and acetosyringone on agrobacterium-mediated transformation of moroccan durum wheat

Drought is one of the major constraints in durum wheat production in the Mediterranean Basin. In order to overcome this problem, the genetic transformation of durum wheat is one of the choices for improvement. However, the recalcitrance to Agrobacterium-mediated transformation in durum wheat (Triticum turgidum L.) is one of the factors limiting a successful genetic transformation. The aim of this study was to investigate the effect of explant type and acetosyringone concentration for the efficient Agrobacterium-mediated genetic transformation of three Moroccan durum wheat varieties (Amria, Chaoui, and Marouane). The mature embryos (intact, halved and pieces) were inoculated with Agrobacterium tumefaciens strain EHA101 harboring the binary vector pTF101.1 containing drought tolerance gene HVA1 from barley, and a selectable marker phosphinothricin (PPT) resistance (bar) gene. The explants were inoculated with A. tumefaciens (cell density OD650 at 0.7) at four different concentrations of acetosyringone (0, 100, 200, and 400 µM). The results showed that embryogenic calli from mature embryos showed higher regeneration and transformation than mature embryo halves and pieces. The integration of the transgene was confirmed by PCR amplification using primers specific to the bar gene, 2x35S promoter, and HVA1 gene. The transformation efficiency ranging from 0.33% to 2.33% was obtained in Amira variety using embryogenic calli and acetosyringone concentrations of 200 and 400 µM. The integration, as well as inheritance of the transgene, was confirmed by PCR amplification in T0 and T1 generations. This is the first report describing a genetic transformation of Moroccan durum wheat varieties via Agrobacterium tumefaciens.

Effect of Explants Sterilization and Plant Growth Regulators on Embryo Culture of Kepel (Stelechocarpus burahol)

Kepel (Stelechocarpus burahol) is an original Indonesian fruit plant. Kepel is currently hard to find and cultivate conventionally. In vitro culture is a method for kepel propagation. The in vitro culture requires an established protocol for explant sterilization to be successful. For the present, there is no appropriate kepel sterilization method that has been known. This study aimed to obtain a sterilization method for kepel embryo explants appropriate for embryo culture. This research was conducted in September 2018 - January 2019 at the In Vitro Culture Laboratory, Faculty of Agriculture, Universitas Muhammadiyah Yogyakarta. This study used an experimental method arranged in a single factor. Completely Randomized Design with the treatment of various immersion methods of kepel embryo explants divided into 8 levels. They are Sodium hypochlorite (NaOCl) (5% : 5', 5% : 10', 10% : 5', 10% : 10'), Hydrogen peroxide (H2O2) (2% : 10', 2% : 15', 3% : 10', 3% : 15') and plant growth regulators 2 ppm 2,4-D + 0,5 ppm BAP, 2 ppm NAA + 0,5 ppm BAP. The results showed that 5% NaOCl for 5 minutes treatment was the best method for sterilization with a percentage of viable explant 88.89%. Then, the percentage of browning 11.11% and 2 ppm 2,4-D + 0,5 ppm BAP was effective in the organogenesis of callus of kepel embryos.