Somatic Embryo Formation Research Articles

Somatic embryogenesis from leaf tissues of macaw palm [Acrocomia aculeata (Jacq.) Lodd. ex Mart.

A somatic embryogenesis protocol was developed from the immature leaves of adult plants of the macaw palm. Leaf explants from different regions of the palm heart were used for callus initiation in a modified Y3 medium, supplemented with 2,4-D or Picloram at 450 μM. Calli were separated from the leaf explants at 6-, 9- and 12-month periods and transferred to a fresh culture medium of the same composition. They were multiplied for up to 120 days. Reduced concentrations of 2,4-D and Picloram were used to differentiate somatic embryos. They were then germinated in a medium without plant growth regulators. Morphological and anatomical analyses were conducted at different stages of the embryogenic process. The best results for callus induction were achieved by Picloram, when explants were maintained for up to 9 months on culture medium (64.9%). The farthest portions of the apical meristem were those that provided the biggest calli formation. The formation of the somatic embryos was observed from the calli multiplication phase. Reduction in concentrations of growth regulators failed to promote the formation of complete plants. Picloram at 450 μM promotes high callogenesis in leaf tissues of macaw palm, with a potential for somatic embryo formation.

Physiological and biochemical response of Phoenix dactylifera L. Hayani cv. embryogenic callus and somatic embryos to salt stress under in vitro conditions

Salinity stress as a major abiotic stress has influenced more than 6% of the world land which isincreasing daily due to climate changes. The effect of salinity on callus regeneration and organogenesis of datepalm was tested under in vitro conditions. This study was conducted to investigate the effect of salt stress oncallus proliferation, somatic embryo formation of ‘Hayani’ date palm in vitro. The embryogenic callus wassubjected to MS medium with BA at 0.05 mg/l and NAA at 0.1 mg/l supplied with different sodium chloride(NaCl) concentrations (0, 500, 1000, 1500 and 2000 ppm) for 2 subcultures. Salinity affects severalphysiological and biochemical processes. The investigated parameters include embryogenic callus growth,differentiation to form somatic embryos, fresh weight of both callus and embryos, biochemical analysis such astotal carbohydrates, proline, protein content and minerals in embryogenic callus and somatic embryos. Lowlevels of abiotic stress factors can induce favorable responses in the growth and differentiation. However, highlevels are detrimental resulting in reduced growth and some cell death. The callus fresh weight and the numberof embryos were enhanced by adding 500 and 1000 ppm NaCl to the proliferation medium; higher saltconcentrations affected the callus regeneration negatively, where at 2000 ppm NaCl the less somatic embryoswere formed. High concentrations of NaCl (1500 and 2000 ppm) decreased total soluble carbohydrates in bothembryogenic callus and somatic embryos, proline content increased gradually as the external concentration ofNaCl increased and the protein content decreased in both embryogenic callus and somatic embryos with theincrease of NaCl concentration in culture media to 2000 ppm. Salinity inhibits water uptake and increases theconcentration of toxic ions such as Na+and reduced the K+ content. Tissue culture with the use of this techniquewould allow selection for salt tolerance in the date palm. In fact, somatic embryos subjected to 500 and 1000ppm NaCl concentrations were successfully regenerated into plantlets.

Anatomical and histochemical studies of the somatic embryogenesis of Syagrus oleracea from immature inflorescences

Abstract Anatomical and histochemical studies were carried out during the somatic embryogenesis of Syagrus oleracea (Mart.) Becc. from immature inflorescences. Immature rachillas were inoculated on Murashige and Skoog medium with 4.52 μM 4-amino-3,5,6-trichloropicolinic acid (Picloram) and 2,4-diclorophenoxyacetic acid. Auxin concentrations were gradually reduced for the formation of somatic embryos, while for plantlet regeneration, embryos were inoculated on a medium without growth regulator. After 270 days, two types of embryogenic calluses were observed in explants under the effect of Picloram: nodular, with a smooth surface and those without definite shape, with an irregular surface, both presenting a meristematic zone with polysaccharide content. Callus progression to the differentiation stage allowed for the conversion of somatic embryos, some with a well-defined protoderm and procambial bundles. The histochemical analysis of the somatic embryos did not identify the presence of polysaccharide and protein reserves, which emphasizes the necessity for optimization of the maturation conditions.

Histology, histochemistry and ultrastructure of pre-embryogenic cells determined for direct somatic embryogenesis in the palm tree Syagrus oleracea.

Somatic embryogenesis in palm trees is, in general, a slow and highly complex process, with a predominance of the indirect route and, consequently, a lack of knowledge about the direct route. We present new knowledge related to the morphological, histochemical and ultrastructural aspects of the transition from somatic to embryogenic cells and direct formation of somatic embryos from mature zygotic embryos of Syagrus oleracea, a palm tree. The results support the general concept that 2,4-dichlorophenoxyacetic acid plays a critical role for the formation of somatic embryos of direct and multicellular origin. Seven days in medium with auxin were enough for the identification of embryogenic cells. These cells had a set of characteristics corresponding to totipotent stem cells. At 14 days on induction medium, nodular formations were observed in the distal region of inoculated embryos, which evolved into globular somatic embryos. At 120 days on induction medium, the quality of the somatic embryos was compromised. The dynamics of the mobilization of reserve compounds was also demonstrated, with emphasis on starch and protein as energy sources required for the embryogenic process. This study shows for the first time the anatomical and ultrastructural events involved in direct somatic embryogenesis in a palm tree and incites the scientific community to return to the discussion of classical concepts related to direct somatic embryogenesis, especially regarding the characteristics and location of determined pre-embryogenic cells.

Transcriptomic analysis of Medicago truncatula calli with MtWOX9-1 overexpression

Somatic embryogenesis (SE) is the development of embryo-like structures from somatic plant tissues. This process rarely can be observed in nature, but for many plant species, in vitro protocols are developed, which allow to obtain somatic embryos formation directly from tissues of plant explant or from the embryogenic callus. SE is widely used for plant propagation and transformation; therefore, the search for SE stimulators and revealing of the mechanisms of their functioning are very important for biotechnology. Among the SE regulators, proteins of the WOX family play significant roles. WOX (WUSCHEL-RELATED HOMEOBOX) is a homeodomain-containing transcription factor family. Different WOX genes function in different plant organs and tissues, maintaining meristem activity and regulating cell proliferation and differentiation. Recently, we have shown that transcription factor MtWOX9-1, belonging to the WOX family, can stimulate SE in the Medicago truncatula callus culture. In this research, transcriptomic analysis of highly embryogenic calli with MtWOX9-1 overexpression was performed in comparison to wildtype calli. It was shown that MtWOX9-1 overexpression led to the activation of several groups of genes, including genes related to cell division, tissue differentiation, and seed development. Enriched GO pathways included several groups related to histone methyltransferase activity as well as DNA methylation and chromatin binding, suggesting major epigenetic changes that occur in call overexpressing MtWOX9-1. Using Medicago Truncatula Gene Expression Atlas, we also identified a group of genes coding for transcription factors that were both coexpressed with MtWOX9-1 in different plant organs and differentially expressed in our samples. These genes are putative targets of MtWOX9-1, and they may act in the same pathway with this regulator during SE.

Oxidative status in Medicago truncatula Gaertn. non-embryogenic and embryogenic tissues with particular reference to somatic embryogenesis

Abiotic stress conditions (e.g., wounding, sterilization) are often together with plant growth regulators (e.g., 2,4-d), considered as one of the most important factors initiating plant somatic embryogenesis (SE). The first goal of this work was to answer whether leaf explants of the Medicago truncatula non-embryogenic (M9) line and its embryogenic variant (M9-10a) respond equally to mechanical and chemical stress by analyzing ROS (reactive oxygen species, e.g., O2•−, H2O2) localization, ROS scavenging enzyme activity and expression of genes encoding these enzymes. In explants of both lines, the stress response induced by wounding and chemical sterilization and the defense reaction during the 1st week of callus growth was similar. These defense mechanisms first involve an increase in SOD and CAT activity, later APX. 2,4-d, present at a low concentration (0.5 µM) during the induction phase (IP), is necessary for embryogenic callus formation and, consequently, for embryo development. This herbicide in higher concentrations causes an increase in O2•− accumulation and in antioxidant enzyme activity; however, it does not block the formation of callus and somatic embryos, though it disturbs these processes. Moreover, inhibition or blocking studied processes by DPI, an inhibitor of NADPH oxidase responsible for the production of O2•− and also lowering the expression of genes encoding the antioxidant enzymes leading to change in their activities, clearly indicate that a certain level of ROS is necessary to induce SE.

Evaluation of induction of somatic embryogenesis from cotyledonary leaves of Banana Passion fruit (Passiflora mollissima) L.H Bailey

Passiflora mollissima L.H Bailey is an endemic species from the Andes mountain of Colombia in South America with important edible fruits and medicinal and economical properties. The development of improved micropropagation techniques is necessary to provide rapid and efficient clonal propagation of elite genotypes with high resistance and uniform production, as well as a system that can be used for genetic transformation. For this reason, the investigation focused on the evaluation of induction of somatic embryogenesis in P. mollissima from cotyledonary leaves, the effect of growth regulator concentrations and the orientation of explants on embryo production. Histological analyses of somatic embryogenesis were performed every 10 days after induction over 38 days of exposure to the medium. Results showed somatic embryo formation on Murashige and Skoog, (1962) culture medium supplemented with 4.5 μM 2,4-diclorophenoxyacetic acid plus 4.5 μM 6-benzyladenine. The results obtained are applicable to knowledge of non-zygotic embryogenesis in passionflower of the Andean region, for the purpose of improvement and commercial use. It is noteworthy that this is the first study in the induction and obtaining of embryos in P. mollissima.

Effect of Container Culture Types on The Success of Cacao Somatic Embryos Formation

<p><em>Container culture have an important role in determining the success of in vitro culture since it will affect the development of culture, such as the formation of embryonic structures. The study aimed to determine the effect of culture container types on cacao somatic embryogenesis. The study was conducted at Tissue Culture Laboratory, Superior Seed Development Unit of IAARD, Bogor, from April to September 2016. The tests were conducted on the effect of container and explant types as well as the effect of container types and genotypes. The effects of container and explant types were tested using callus induced from petal and staminoid explants of Sca 6, whereas the effects of container types and genotypes were tested using callus induced from petal explants of Sca 6 and ICCRI 4. Afterwards, the somatic embryos were induced using petri dishes or culture bottles according to treatment. The results showed no significant interaction between container and explant types on the average percentage of the formation and number of somatic embryos (10.28% embryos/explants in culture bottles and 7.89% embryos/explants in petri dishes). Meanwhile, there was significant interaction between genotypes and container types in the initial period of somatic embryos formation (15 and 18 weeks after culture), but the effect was not significant in the final period of observation (21 weeks after culture). The results indicate that culture bottles, which have lower prices, can be used to replace petri dishes to induce the formation of somatic embryos in cacao.</em></p>

Directional breeding of high oil content peanut variety Yuhua 9 by in vitro mutagenesis and screening

Leaf water potential of peanut subjected to drought stress is positively related to the oil content of peanut kernels. The aim of this study was to directly screen the high oil mutants of peanut and create the new peanut varieties using hydroxyproline as water potential regulator. In vitro mutagenesis was carried out with the embryonic leaflets of peanut variety Huayu 20 as explants and pingyangmycin as a mutagen added into the somatic embryo formation medium. The formed somatic embryos were successively transferred to somatic embryo germination and selection medium containing 6 mmol/L hydroxyproline (at -2.079 MPa water potential ) to induce regeneration and directionally screen high oil content mutants. After that, these plantlets were grafted and transplanted to the experimental field and 132 high oil mutants with oil content over 55% were obtained from the offspring of regenerated plants. Finally, among them, the oil contents of 27 lines were higher than 58% and of 2 lines were higher than 60%. A new peanut variety Yuhua 9 with high yield and oil content was bred from the regenerated plant progenies combining the pedigree breeding method. The yield was 14.0% higher than that of the control cultivar in the testing new peanut varieties of Liaoning province, and also it has passed the national registration of non-major crop varieties. Yuhua 9 with an oil content of 61.05%, which was 11.55 percentage points higher than that of the parent Huayu 20, was the peanut cultivar with the highest oil content in the world. The result showed that it was an effective way for directional breeding of high oil peanut varieties by means of the three-step technique including in vitro mutagenesis, directional screening by reducing water potential in medium and pedigree selection of regenerated plant progenies.

Induction of somatic embryos in cultures of Asparagus racemosus Willd: an endangered medicinally important plant

BackgroundSomatic embryogenesis is one of the most popular in vitro regeneration methods for mass micropropagation. In the present study, somatic embryogenesis via zygotic embryos was studied in Asparagus racemosus Willd. Since the callus quality plays an important role in plantlet development, therefore, compact embryogenic callus was selected for further embryogenesis.ResultsSomatic embryos were induced by zygotic embryos germinating on callus induction medium (MS media with 1.54 mg L−1 2,4-D and 0.43 mg L−1 kinetin) in dark. Thereafter, the compact embryogenic callus differentiated up on MS media with 0.1 mg L−1 NAA and 0.5 mg L−1 kinetin supplemented with various concentrations of ancymidol. An addition of 0.75 mg L−1 ancymidol resulted in significant enhancement of somatic embryo formation and no malformed embryos were observed. Scanning electron micrographs and thin sections confirmed the structures as somatic embryos. Furthermore, these embryos were transferred to the same medium supplemented with glutamine, casein hydrolysate, and 3% sucrose for conversion into green shoots. These shoots could be multiplied in vitro using BAP-supplemented medium.ConclusionsAn effective method for conservation of an overexploited threatened medicinally important species Asparagus racemosus has been developed. This is the first report of the formation of somatic embryos from zygotic embryos in A. racemosus. Ancymidol in combination with kinetin and NAA was found to be most efficient for somatic embryo maturation and germination. The established protocol would certainly advance the efficient somatic embryo induction, maturation, and germination which could be utilized for large-scale propagation of Asparagus species.

Explant type and hormone regime influences somatic embryogenesis and regeneration of cassava

Cassava (Manihot esculenta Crantz) is a tropical root crop that serves as a food staple and a vital income source to resource deprived farmers in the tropics. Despite its importance, cassava production and consumption is faced by a myriad of biotic/abiotic constraints. Genetic transformation which can be applied to mitigate these challenges however has as a prerequisite the availability of robust regeneration systems. This study evaluated the effect of explant type and hormone regime on somatic embryogenesis and regeneration of Kenyan cassava cultivars. The embryogenic competence of immature leaf lobe and stem explants of three cassava cultivars was determined by culturing them in MS medium supplemented with picloram (4, 6, 8 and 10 mg/l). The optimum media composition for embryo maturation, germination and plant recovery was assessed by culturing embryos in media supplemented with varying concentrations of 6-Benzylaminopurine (BAP), α–Naphthalene acetic acid (NAA) and Gibberellic acid (GA3). Somatic embryo formation frequencies in leaf explants were significantly higher (P≤0.05) than in stem explants. Embryo formation rates were found to increase with increasing concentrations of picloram. Cultivar 08/080 gave the least response to embryogenesis while embryogenesis rates for cultivar 08/274 were similar to the control cultivar TMS 60444. Maturation and plant recovery rates differed significantly with regard to the ratios of BAP, NAA and GA3 in the maturation media. The optimal medium for embryo germination and plant recovery was supplemented with 1 mg/l BAP, 0.02 mg/l NAA and 1.5 mg/l GA3. This optimized regeneration protocol can be coupled with mutation breeding or genetic transformation to improve the cassava germplasm. Key words: Cassava, explant, regeneration, somatic embryogenesis, plant growth regulator.

Somatic Embryogenesis in the Family Gentianaceae and Its Biotechnological Application.

The family Gentianaceae consists of 1736 species, which play an important role in human being existence due to their pharmacological and horticultural values. Many species accumulate bitter iridoid substances used medicinally and in flavorings, while others are cultivated because of beauty of their flowers showing a wide range of colors and patterns. Out of 99 genera belonging to the gentian family, process of somatic embryogenesis (SE) was reported for 5. The first reports, aimed at micropropagation of ornamental cultivars and production of secondary metabolites, concerned Centaurium erythraea Rafn., Eustoma russellianum Grieseb. and Exacum affine Balf. Somatic embryos were induced on different explants cultured in the liquid Murashige and Skoog medium supplemented with auxins and cytokinins. In the 1990s of the last century, significant progress in the exploration of the phenomenon of SE and its biotechnological application was made for the genus Gentiana. The process was induced on various explants and studied at the structural and ultrastructural levels. Regenerated plants were screened for genetic stability using flow cytometry, chromosome counting, and molecular markers. Besides typical indirect SE, the use of leaf fragments enabled to obtain single-cell origin of somatic embryos. On the other hand, proliferation of embryogenic callus in liquid medium resulted in the establishment of long-term embryogenic cell suspension cultures, paving the way not only to study the formation of somatic embryos and the development of regenerants but also to preserve the morphogenic potential of cell aggregates by cryopreservation. Cell suspensions re-established after storage in liquid nitrogen maintained their embryogenic character and allowed to obtain somatic embryo-derived regenerants that were true-to-type at both genetic and epigenetic levels. Another application of SE was related to genetic manipulation purposes. Efficient protocols of plant regeneration from callus-, cell suspension-, or leaf mesophyll-derived protoplasts allowed engaging procedures of somatic hybridization or protoplast electroporation for gentian genome modifications. Also, high embryogenic potential existing in the numerous gentian species enabled successful Agrobacterium-mediated transformation of G. cruciata L. and G. dahurica Fisch.

Effect of types and concentrations of auxins on callus induction and primary somatic embryogenesis in low cyanide cassava cultivars ( <i>Manihot esculentum</i> Cranz)

Cassava constitutes a greater part of the diet of most Africans south of the Sahara and the demand for the crops has been on a steady increase. Agriculturally, cassava performs very well, but the roots and leaves contain cyanogenic glycosides that are dangerous to human health. As such, cassava cultivars with low cyanide content offer great opportunities for domestic and industrial utilization. However the major constraints to the production of low cyanide cassava cultivar are susceptibility to diseases such as cassava mosaic virus (CMV) and non-availability of disease free planting materials. Application of biotechnology such as plant tissue culture could have a significant impact in the production large amount disease free planting materials. The effects of 2, 4-D and Picloram on callus induction and primary somatic embryogenesis in the low cyanide cassava varieties was evaluated. The result indicated a significant increase in percentage callus induction with increase in the concentration of 2, 4-D with 4mg/L producing the highest callus induction frequency of 72.21%. Similarly on media supplemented with Picloram, 7mg/L produced the highest callus induction frequency (73.11%) and frequency decreases with corresponding decrease in Picloram concentration. For the formation of somatic embryos, the result indicated a significant increase in number of primary embryos with increase in the concentrations of 2, 4-D or Picloram. This protocol presents a reproducible system for generation totipotent culture that could be used in genetic improvement of cassava and mass production of disease-free planting materials. Keywords : Cassava, Auxin, Callogenesis, Somatic embryogenesis

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.

Somatic embryogenesis in the commercial papaya hybrid UENF/Caliman 01 relying on plantlet production from sexed adult hermaphrodite donor plants.

Somatic embryogenesis from explants from hermaphrodite papaya mother plants is an alternative for the production of true-to-type plants without the need for sexing. This study aimed to analyze hormonal and osmotic inducers in different somatic embryogenesis stages in the commercial hermaphrodite hybrid papaya UENF/Caliman 01. Leaf disks from in vitro shoots originated from ex vitro hermaphrodite plants were cultured in induction medium supplemented with different concentrations of 2,4-D (6, 9, 12, 15, and 18 μM) and 4-CPA (19, 22, 25, 28, and 31 μM). After 90 days, the formation of somatic embryos was verified. The 2,4-D induced the formation of light brown calli with low frequency (20%) of somatic embryogenesis. However, 4-CPA (25 μM) induced 96% of embryogenic calli, which were transferred to maturation medium (MM) and cultured for 30 days. The MM contained ABA (0.5 μM) and AC (15 g L-1) and produced 36.6 somatic embryos callus-1, mainly on cotyledonary stage. Cotyledonary embryos were transferred to germination medium supplemented with gibberellic acid (GA3) (0.0, 1.44, 2.88, and 4.32 µM), and the conversion into plantlets was enhanced with GA3 at 2.88 µM.

Influence of Silver Nitrate on Somatic Embryogenesis Induction in Arabica Coffee (Coffea arabica L.)

Plant somatic embryogenesis (SE) has been defined as the formation of embryos from a single or group of haploid or somatic cells [1, 2]. Low frequency (LFSE) and high frequency somatic embryogenesis (HFSE) have been described. In the first type, somatic embryos are induced directly from pro-embryogenic cells of explants, while in the second, they originate from embryogenic callus [1]. It has been suggested that in LFSE the origin of somatic embryos is unicellular, whereas in HFSE has been described as unicellular or multicellular [3]. SE is a powerful biotechnological tool used to propagate elite plants or to conserve important genotypes [4]. Moreover, SE offers an efficient in vitro regeneration approach as a fundamental step in plant genetic improvement for studying basic aspects of ontogenesis of somatic embryos [5]. In Coffea spp., the first studies of SE have been reported at the beginning of 1970 [6]. Since then, a large quantity of LFSE and HFSE protocols have been optimized demonstrating that coffee is not a recalcitrant species for SE [4]. In the LFSE the somatic embryos are obtained faster (approximately 70 days) using only one medium meanwhile in HFSE several media are used and somatic embryo formation takes 9-10 months [4]. Although, in LFSE small number of somatic embryos are obtained (around 10 per explant) compared to hundreds of somatic embryos obtained per gram of embryogenic calli [4], the unicellular origin of somatic embryos in LFSE represents an advantage for the chemical and physical mutagenesis, genetic transformation and genetic editing, since prevents or reduces the appearance of chimeras [7]. In C. arabica and C. canephora many factors (such as genotype, explant type, the physiological state, age and growth conditions of the donor plants, the season of collection, nutrient composition of the medium, the volume of dissolved CO2 or O2 in the culture flask, and plant growth regulators) that affect LFSE induction have been studied [3, 8, 9, 10, 11, 12, 13]. However, few studies reported the effect of silver nitrate on LFSE using leaf explants of C. arabica L. and to the best of our knowledge it has not been analyzed using Caturra and Catuai, which are two economic important producer cultivars in Costa Rica. Since SE is genotype dependent, the culture medium need to be modified for the different genotypes [7].Therefore, the objective of this study was to determine the influence of the benzyladenine (BAP), indole-3-acetic acid (IAA), and silver nitrate (AgNO3) on low frequency somatic embryogenesis using leaf explants of Coffea arabica L. cultivars Caturra and Catuai.

Regeneration and histological study of somatic embryogenesis of sugarcane (Saccharum officinarum L.) cultivar PS 864

The process affecting somatic embryo formation in sugarcane is very specific for each genotype, so the determination of the best somatic embryo regeneration medium in sugarcane clones is necessary. The objective of this study was to determine the concentration of Benzine Amino Purine (BAP) and Kinetin for somatic embryo maturation and to observe the maturation stage for somatic embryo of sugarcane cultivar PS 864. Maturation of the nodular callus was conducted by addition of Kinetin (0, 1, 3, and 5 mg/L) and BAP (0 and 5 mg/L) in solid and liquid medium. The medium was optimized using glutamine. The medium for somatic embryo germination used full and half strength Murashige and Skoog medium (MS) supplemented with 20 g/L sucrose and amino acid or growth regulator (BAP and NAA). Nodular callus well grew in solid medium, whereas callus become browning and not existence of somatic embryo maturation in liquid medium. The highest number of globular embryos (38 embryos) was produced from MS medium supplemented with 1 or 3 mg/L Kinetin combined with 5 mg/L BAP. The highest number of scutellum (21 embryos) and coleoptile (19 embryos) resulted from 3 mg/L Kinetin. The MS medium with full strength was added with 100 mg/L glutamine that was the best germination medium. This medium resulted the highest percentage of somatic embryo (73.29%) forming bipolar structure, and the largest number of leaves (4.58). Histological analysis showed that somatic embryos of sugarcane emerged from many cells through the budding process and also initiated from one cell.

Somatic embryogenesis in the leaf tissue of Hedyotis diffusa Willd.

Hedyotis diffusa is a valuable medicinal herb belong to the Rubiaceae family. It is widely used in the treatment of various types of cancer and other diseases related to leukemia. Besides, the plant usually contains triterpenoids (oleanolic acid, ursolic acid) and flavonoids which have a range of pharmacological activities of antiinflammatory, antibacterial, hypoglycemic, antifree radicals, reducing blood lipids and anticancer. Leaf segments of 3 weeks old H. diffusa were cultured on half strength Murashige and Skoog (½MS) medium supplemented with different concentrations of indole acetic acid (IAA; 0.1, 0.2 and 0.4 mg/L) or α-naphthalene acetic acid (NAA; 0.1, 0.2 and 0.4 mg/L) and benzyladenine (BA) at a concentration of 1mg/L. In this study, the highest percentage of somatic embryogenesis was obtained using 0.1 mg/L IAA and 1 mg/L BA, and 0.2 mg/L IAA and 1 mg/L BA. The somatic embryos develop through a series of morphological stages: globular type, heart, torpedo and mature embryos. Besides, the highest number of shoots per explant was achieved in the same media. The middle and the distal end segments were found the most suitable for somatic embryogenesis. Morphological changes and the role of endogenous hormones in somatic embryo formation were analyzed. The position of the leaf segments of the same leaf, respiration rate, and endogenous hormone and somatic embryo formation were also discussed.

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

The role of cysteine in improving somatic embryos of salak Sidempuan (Salacca sumatrana Becc.)

In vitro study of Salak propagation has been conducted, with the aim to obtain the effect of some amino acids in inducing somatic embryos formation. The experimental design used was Completely Randomized Design, with treatment by varying cysteine concentration (0, 5, 10, 15, 20 and 25 mg. L−1). The results showed that explants successfully proliferated and lead to callus with high potential to be cultured. All explants showed a fairly high survival rate close to 100%. Early callus formation also varied, the fastest growing callus was observed from 10.14 days of culture with the addition of 10 mg.L−1 cysteine. Callus formation in the form of embryogenic callus has high potential to grow into somatic embryos. In conclusion, the best medium formulation to form somatic embryos was obtained from cysteine-containing media with a concentration of 10 mg.L−1.