Somatic Embryo Formation Research Articles

Plant regeneration, developmental pattern and genetic fidelity of somatic embryogenesis derived Musa spp.

Multiplication of banana cvs. Grand Naine (Musa AAA, Cavendish-sub group) and Rasthali (Musa AAB, Silk-sub group) were attempted through somatic embryogenesis. The influence of position of male flower buds, amino acid supplements in the induction of somatic embryogenesis and field performance of embryogenic cell suspension (ECS) derived banana plants were studied. Differentiated immature male flower buds positioned at 6–8 th bract whorl as explants showed better callus induction and somatic embryogenesis. Supplementation with glutamine at 400 mg L−1 along with 20:20 g L−1sucrose: maltose in maturation media induced a 10-fold increase in somatic embryo formation compared to control. Cotyledonary stage somatic embryos desiccated for 2 h showed higher germination compared to non-desiccated embryos. The plantlets generated were hardened, and the genetic fidelity of the plantlets was confirmed using ISSR marker. To check the field performance of ECS derived plants, plantlets were hardened and planted in the field along with meristem and sucker. During the field growth, these ECS derived plants were morphologically similar to those of control plants. In this experiment, it was observed that ECS derived banana plants displayed normal phenotype as that of plants grown from meristem and sucker. The protocol developed could be useful highly for large-scale micropropagation or genetic manipulation studies in these commercially important banana cultivars.

Genome size analysis of field grown and tissue culture regenerated Rauvolfia serpentina (L) by flow cytometry: Histology and scanning electron microscopic study for in vitro morphogenesis

An efficient genetically stable regeneration protocol has been established in Rauvolfia serpentina (L) Benth ex. Kurz, an important medicinal plant, used for the treatment of various cardiovascular diseases. Here, in this present study, plants were regenerated through organogenesis (direct and indirect) and embryogenesis pathways. This is the first ever somatic embryo formation report of R. serpentina in liquid Murashige and Skoog (MS) medium from isolated root segments. The genetic fidelity of the regenerated plants was also analysed by flow cytometry. The 2C DNA content of the regenerated plants raised through organogenesis and somatic embryogenesis was measured along with field grown natural plants, which served as donor plant. The estimated 2C DNA levels are 2.08, 1.76 and 1.88 pg in direct, indirect and somatic embryo regenerated plants respectively while in field grown plant it was noted to be 1.94 pg in R. serpentina. This is perhaps the first ever 2C DNA estimation study in tissue culture raised plants along with natural population of Rauvolfia, an immensely important medicinal plant, present in India. We developed improved callus mediated shoot regeneration (indirect) protocol from leaf callus. Highest shoot regeneration percentage (66.10%) with 4.0 shoots / callus mass was noted on 2.0 mg l−1 BAP + 0.5 mg l−1 NAA added MS medium. The shoot tips and nodal explants on 2.5 mg l−1 BAP amended MS medium induced direct shoot buds, with highest shoot regeneration frequency (81.45%) from shoot tips. On 1.0 mg l−1NAA added MS medium maximum numbers of roots (8.87) were noted, indicating the effectiveness of NAA over IBA in rooting. Excised roots were cultured in agitated liquid MS medium and in 1.0 mg l−1 NAA added conditions somatic embryos were formed. The mature somatic embryos germinated on 1.5 mg l−1 BAP + 0.25 mg l−1 GA3 supplemented MS medium. The histology and scanning electron microscopic (SEM) evidences have been presented to describe the in vitro morphogenesis process. The well-rooted plantlets were acclimatized and transferred to soil successfully (79%). The flow cytometric analysis showed no significant variation in nuclear DNA contents, hence was observed to be genetically stable with their genome size similar to field grown Rauvolfia plants.

Thidiazuron-induced somatic embryogenesis and changes of antioxidant properties in tissue cultures of half-high blueberry plants

An efficient protocol of somatic embryogenesis (SE) has been developed for the first time in four half-high blueberry (Vaccinium corymbosum L. × V. angustifolium Ait.) cultivars. Thidiazuron (TDZ), a plant growth regulator with potential activities for shoot regeneration and shoot proliferation, was found most effective for somatic embryo formation when added to a nutrient medium at high concentration (9 µM). Although TDZ was also best for embryo germination at low concentration (2.3 µM), it was followed by zeatin at 4.6 µM for the same. Plantlets developed from SE were removed from the nutrient medium and transferred on a peat: perlite medium where 100% survival rate was acquired following the acclimatization process in a greenhouse. The concentrations of total phenolic and flavonoid contents were higher in greenhouse-grown conventionally cutting-propagated donor mother plants than those of respective SE plants for ‘St. Cloud’, ‘Patriot’ and ‘Northblue’ but not for ‘Chippewa’. The effect of propagation method and/or the older age of donor mother plants were clearly visible exclusively as the 15-year-old donor plants showed higher level of 2,2-diphenyl-1-picrylhydrazyl scavenging activity than the eight-weeks-old SE plants in all four cultivars.

Developmental pathway of somatic embryogenesis from leaf tissues of macaw palm (Acrocomia aculeata) revealed by histological events

This paper, in an unprecedented way, describes the ontogenesis of calli and somatic embryos of macaw palm (Acrocomia aculeata) from young leaf tissues of adult plants, aiming to elucidate the events underlying the process and consequent optimization of the technique. Leaves were inoculated in a callus induction medium, composed of Y3 salts, 2.5 g L−1 activated charcoal and 450 μM Picloram. For multiplication of the calli, they were separated from the explants and inoculated in a medium with the same composition as the previous phase. Histological analyses were performed on samples of immature leaves with and without calli at 0, 3, 15, 30, 60, 90 and 120 days in an induction medium, and on samples of the different types of calli and somatic embryos obtained during multiplication and differentiation. For this, the samples were fixed in FAA, dehydrated in an alcohol series and infiltrated in resin. Anatomical sections obtained on a microtome were stained with Toluidine Blue for structural characterization. At 60 days, analyses revealed the formation of callogenic masses in the vicinity of smaller vascular bundles. This multiplication approach allowed for the proliferation of the primary cell mass that evolved into new lineages of calli, especially the embryogenic yellowish nodular lineage, which originated somatic embryos. Additional histochemical analyses showed starch accumulation only in the adjacencies of areas undergoing intense cell division. The morpho-anatomic characterization allows the rapid identification of potential calli for the formation of somatic embryos from leaf tissues in macaw palm and promotes future investigations of the participation of vascular tissues in the events that precede somatic embryogenesis.

Morpho and Cytological Differentiation of Calli of Eucalyptus grandis x Eucalyptus urophylla During Somatic Embryogenesis

The aim of this study was to induce and analyze embryogenic calli from two types of explants (leaves and meristems) of the hybrid Eucalyptus grandis x Eucalyptus urophylla. Leaves and meristems of plants kept in a nursery were disinfected and inoculated in Petri dishes containing MS culture medium supplemented with different concentrations of the growth regulator dicamba (1.13, 4.52, and 9.04 µM) and without it. At 60 days of culturing, the calli were analyzed by scanning electron microscopy and at 90 days were evaluated by light microscopy in regard to the embryogenic characteristics of the cells. Different type of calli were induced in leaf explants, designated as Type I with light yellow coloring, Type II with dark yellow coloring, and Type III of brown coloring; however, only Type I had embryogenic characteristics. In the meristematic explants, only one type of callus was induced, and it had embryogenic characteristics. At 90 days of culturing, the formation of somatic embryos in the different embryogenic stages was observed and the formation of procambium, protoderm, and ground meristem tissues. At 150 days of culturing, the concentration of 1.13 µM of dicamba was prominent in the formation of somatic embryos in the different embryogenic stages.

Histology of somatic embryogenesis in Coffea arabica L.

The objective of this study was to characterize the histodifferentiation of somatic embryogenesis obtained from leaf explants of C. arabica. Therefore, we histologically analyzed the respective stages of the process: leaf segments at 0, 4, 7, 15 and 30 days of cultivation, Type 1 primary calli (primary calli with embryogenic competence) and 2 (primary calli with no embryogenic competence), embryogenic calli, globular, torpedo and cotyledonary embryos, and mature zygotic embryos. Callus formation occurred after seven days of culture, with successive divisions of procambium cell. In this cultivation phase, it was found that Type 1 primary calli are basically formed by parenchymal cells with reduced intercellular spacing, whereas Type 2 primary calli are predominantly composed of parenchymal cells with ample intercellular spaces and embryogenic calli composed entirely of meristematic cells. After 330 days, it was evident from the differentiation of somatic embryogenesis that there was formation of globular somatic embryos, consisting of a characteristic protoderm surrounding the fundamental meristem. With the maturation of these propagules after 360 days, torpedo-stage somatic embryos arose, in which tissue polarization and early differentiation of procambial strands were verified. After 390 days, cotyledonary somatic embryos were obtained, where the onset of vessel elements differentiation was verified, a characteristic also observed in mature zygotic embryos. We concluded that somatic embryogenesis obtained from C. arabica leaves initiates from procambium cell divisions that, in the course of cultivation, produce mature somatic embryos suitable for regenerating whole plants.

Somatic embryogenesis and evaluation of genetic fidelity in Amorphophallus paeoniifolius (Dennst.) Nicolson

ABSTRACT Amorphophallus paeoniifolius (Dennst.) Nicolson (elephant foot yam) is a well-known vegetable tuber plant that belongs to medicinally important family Araceae. The present study reports on establishment of somatic embryogenesis using shoot apices as explants and assessment of genetic fidelity of regenerated plants using random amplified polymorphic DNA (RAPD) markers. Highest somatic embryo formation (100%) was obtained on Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine (BAP) (13.31 µM) and 3% sucrose, whereas lowest percentage of somatic embryos was recorded on medium supplemented with different concentrations of indole-3-acetic acid and 2,4-dichlorophenoxyacetic acid (2,4-d). Mature somatic embryos germinated readily on MS basal medium supplemented with coconut water (15%) and BAP (4.43 µM) and developed into normal plantlets after 4 weeks under dark conditions. Well-rooted plants were successfully acclimatized on half-strength MS medium; survival rate was 85%. The assessment of genetic fidelity and stability of regenerated plantlets from somatic embryos using RAPD markers resulted in monomorphic banding pattern that confirmed the genetic homogeneity of the regenerated plantlets. The protocol for somatic embryogenesis and regeneration of plantlets with low risk of genetic instability of A. paeoniifolius reported here is highly reproducible. This represents the first report on in vitro regeneration and RAPD assessment in A. paeoniifolius.

Explant type and stress treatment determine the uni- and multicellular origin of somatic embryos in the tree fern Cyathea delgadii Sternb.

Cyathea delgadii is the first fern species for which somatic embryogenesis (SE) has recently been described. With this discovery, a new experimental model for exploration of SE was found. In this study, the effect of explant type (stipe and internode), length and diameter of the explant-donor frond, and stress treatment (by sucrose and air desiccation) on somatic embryo origin and SE efficiency was studied. In control culture, somatic embryos originated from single cells of stipe explants, whereas those induced on internodes were of multicellular origin. Although the activation of cell divisions was more abundant and the formation of somatic embryos occurred earlier in a culture of internodes than of stipe explants, the morphogenic capacity of internodes was much smaller. On their surface, the groups of competent cells formed protuberances that give rise to only three somatic embryos per internode. In contrast, almost 92% of stipe explants that were excised from the first frond, measuring 10 mm in length and less than 300 µm in diameter, produced an average of 21 somatic embryos. Stipes treated with sucrose were not able to SE. More than fourfold increase in SE efficiency was obtained on internodes by their treatment with 0.4 M sucrose for 45 min. It was achieved by changing the pathway of somatic embryo differentiation from multicellular to unicellular. These results provide an excellent basis for further research on the mechanism of SE induction associated with a single- and multi-cell proliferation, especially that both types of the embryogenic pathway can occur on the same hormone-free medium.

Efficient somatic embryogenesis and bulblet regeneration of the endangered bulbous flower Griffinia liboniana

Using flower organs as primary explants and via somatic embryogenesis, we developed an efficient protocol for bulblet regeneration from in vitro-derived seedlings (bulblets) of Griffinia liboniana. Callus induction was tested on five types of floral organ (perianth, filament, pedicel, ovary and anther) in the presence of three combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzylaminopurine (6-BA). Filament constituted the most responsive primary explant for regenerative callus induction, and the highest frequencies of callus induction (63.0 ± 1.9%) and numbers of differentiated buds (3.7 ± 0.3 buds/callus) were found on Murashige and Skoog (1962) medium (MS) supplemented with 1.0 mg L−1 2,4-D and 1.0 mg L−1 6-BA. Starting with in vitro-derived bulblets (0.8–1.5 cm in diameter), somatic embryo (SE) formation occurred within 6 weeks, followed by 8 weeks for SE germination and development on PGR-free media. The highest percentage (78.9 ± 2.2%) of embryogenesis was obtained on MS media supplemented with 0.5 mg L−1 6-BA and 1.5 mg L−1 2,4-D, with an average of 28.0 ± 2.1 bulblets/explant. Well-rooted bulblets were successfully acclimated to ex vitro conditions. A stable ploidy level of the regenerated bulblets was confirmed by flow cytometry (FCM) analysis. This is the first report about micropropagation methods of G. liboniana and constitutes an efficient and reusable method for bulblet regeneration of this endangered species. Additionally, this protocol enables large-scale vegetative production, germplasm preservation and genetic engineering of endangered Griffinia species.

Addition of ionophore A23187 increases the efficiency of Cocos nucifera somatic embryogenesis.

The present study reports the effect of treatment of coconut embryogenic structure explants (derived from embryogenic callus) with the calcium ionophore A23187 (0, 1, 5, 10µM) to promote somatic embryogenesis under in vitro conditions. The results showed no significant increase in the percentage of explants forming embryogenic callus, but with 1µM ionophore there were significant increases in the formation of embryogenic structures per callus (2.8-fold), of somatic embryos per callus (1.5-fold) and also a greater absolute number (1.5-fold) of developing plantlets per callus. The ionophore treatment also promoted a change of pattern of the expression of the CnSERK gene during embryogenic callus formation. It is proposed that if the use of ionophore A23187 treatment is coupled with an embryogenic callus multiplication process there could be a potentially greater increase in the efficiency of the formation of somatic embryos and plantlets of coconut.

Genetic Fine-Mapping of a Quantitative Trait Locus (QTL) Associated with Embryogenic Tissue Culture Response and Plant Regeneration Ability in Maize (Zea mays L.).

Embryogenic and regenerable tissue cultures are widely utilized in plant transformation, clonal propagation, and biological research applications. Germplasm utilized in those applications are limited, however, due to genotype-dependent culture response. The goal of this study was to identify genomic regions controlling embryogenic and regenerable tissue culture response in the globally important crop, maize ( L.), toward the long-term objective of developing approaches for genotype-independent plant genetic engineering and clonal propagation systems. An inbred maize line, WCIC2, nearly-isogenic to reference inbred B73, was developed by phenotypic selection and molecular marker analysis. WCIC2 has over 50x increase in tissue culture response relative to the recurrent parent, B73. This line was used to genetically fine-map a region on chromosome 3 controlling embryogenic and regenerable tissue culture response to a 23.9 Mb region. WCIC2 and derivatives will be useful materials to enable maize research in a genetic background similar to B73, and our genetic mapping results will advance research to identify causal genes controlling somatic embryo formation and plant regeneration in maize.

EMBRIOGENESIS SOMATIK ROTAN TOHITI (Calamus inops Becc. ex Heyne)

The conventional propagation of tohiti rattan still faces problem because of infrequent fruiting season and limited seed production. Somatic embryogenesis is an alternative technique to solve the problem. The purpose of this experiment is to obtain the best growth regulator treatments for embryogenic callus and somatic embryo formation of tohiti rattan. Murashige and Skoog (MS) basal medium was used as growth medium. The experiment was conducted in three stages: seed germination, embriogenic callus induction and somatic embryo induction. MS medium without plant growth regulator was used for aseptic seed germination. MS medium supplemented with growth regulator of BA (Benzyl adenine) of 0.5 – 2.0 mg/l was used for embryogenic callus induction. MS medium supplemented with BA 1.0 mg/l in combination with hormone 2.4-D of 0.0 – 1.0 mg/l was used for somatic embryo induction. The seed germination percentage, visual performance on embryogenic callus and somatic embryo were observed. The results showed that the percentage of aseptically seed germination reached 90%. MS medium supplemented with 1.0 mg/l BA is the best media for embryogenic callus induction with friable, white and yellowish of callus which was observed after four months of induction culture. The BA of 1.0 mg/l in combination with 2.4-D of 1.0 mg/l provided the highest number of the formed somatic embryo.The performance of somatic embryos formation from this treatment was likely as zygotic embryo.

Embryogenic Competence Acquisition in Sugar Cane Callus Is Associated with Differential H+-Pump Abundance and Activity.

Somatic embryogenesis is an important biological process in several plant species, including sugar cane. Proteomics approaches have shown that H+ pumps are differentially regulated during somatic embryogenesis; however, the relationship between H+ flux and embryogenic competence is still unclear. This work aimed to elucidate the association between extracellular H+ flux and somatic embryo maturation in sugar cane. We performed a microsomal proteomics analysis and analyzed changes in extracellular H+-flux and H+-pump (P-H+-ATPase, V-H+-ATPase, and H+-PPase) activity in embryogenic and non-embryogenic callus. A total of 657 proteins were identified, 16 of which were H+ pumps. We observed that P-H+-ATPase and H+-PPase were more abundant in embryogenic callus. Compared to non-embryogenic callus, embryogenic callus showed higher H+ influx, especially on maturation day 14, as well as higher H+-pump activity (mainly, P-H+-ATPase and H+-PPase activity). H+-PPase appears to be the major H+ pump in embryogenic callus during somatic embryo formation, functioning in both vacuole acidification and PPi homeostasis. These results provide evidence for an association between higher H+-pump protein abundance and, consequently, higher H+ flux and embryogenic competence acquisition in the callus of sugar cane, allowing for the optimization of the somatic embryo conversion process by modulating the activities of these H+ pumps.

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

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

<b>Effects of glutathione supplementation and carbon source during somatic embryogenesis of <i>Acca sellowiana</i> (O.Berg) Burret (Myrtaceae)

The present study intended to investigate the effects of different glutathione (GSH) levels (0, 0.1, 0.5 and 1 mM) on the somatic embryogenesis (SE) induction of Acca sellowiana . Besides, we evaluated the effect of different carbon sources (sucrose and maltose) on the somatic embryos conversion. GSH-supplemented treatments resulted in improved SE induction rates (~70%) as compared to the control GSH-free (~35%) after 50 days of culture. The total number of somatic embryos obtained did not differ between treatments, but significant differences were observed for the embryonic stages after 80 days of culture. After 80 days of culture, 0.5 and 1 mM GSH-supplemented treatments showed the largest amount of torpedo-staged somatic embryos. In contrast, treatments supplemented with 0 and 0.1 mM GSH showed equal amounts of somatic embryos at all embryonic stages. These results indicate that GSH accelerates the SE induction process and increases the synchrony of the somatic embryo formation of A. sellowiana . The use of maltose for the somatic embryos conversion, as compared to sucrose, did not influence the conversion rate of normal chlorophyllous somatic embryos, but increased the formation of normal achlorophyllous somatic plantlets. This finding can be attributed to the rapid hydrolysis of sucrose, contributing to an enhanced chlorophyll synthesis.

Studies on growth dynamics of embryogenic cell suspension cultures of commercially important Indica rice cultivars ASD16 and Pusa basmati.

Significant efforts are being directed towards the improvement of rice using genetic manipulations. A good and reproducible system for recovering fertile Indica rice plants is imperative. The aim of this study was to study the influence of initial cell density on growth dynamics of suspension cultures. The cultures were initiated from 3-week-old embryogenic calli derived from mature seeds of Indica rice cultivars ASD16 and Pusa basmati. Growth kinetics of the rice cell suspensions were measured and the obtained data reveal that viable cells at 3% PCV using 20ml liquid (N6D) medium and sub-culturing at 7-day intervals resulted in rapid increase in fresh and dry weightsand the embryogenic competency of the cells were found to be high. The growth kinetics analysis revealed that ASD16 showed better efficiency for high frequency and viable somatic embryo formation as compared to Pusa basmati. The technique was found to be suitable for developing somatic embryos for both cultivars ASD16 and Pusa basmati, which can be used for many important applications including micropropagation and secondary metabolites production.

Effects of GhWUS from upland cotton (Gossypium hirsutum L.) on somatic embryogenesis and shoot regeneration

The WUSCHEL (WUS) gene encodes a plant-specific homeodomain-containing transcriptional regulator, which plays important roles during embryogenesis, as well as in the formation of shoot and flower meristems. Here, we isolated two homologues of Arabidopsis thaliana WUS (AtWUS), GhWUS1a_At and GhWUS1b_At, from upland cotton (Gossypium hirsutum). Domain analysis suggested that the two putative GhWUS proteins contained a highly conserved DNA-binding HOX domain and a WUS-box. Expression profile analysis showed that GhWUSs were predominantly expressed during the embryoid stage. Ectopic expression of GhWUSs in Arabidopsis could induce somatic embryo and shoot formation from seedling root tips. Furthermore, in the absence of exogenous hormone, overexpression of GhWUSs in Arabidopsis could promote shoot regeneration from excised roots, and in the presence of exogenous auxin, excised roots expressing GhWUS could be induced to produce somatic embryo. In addition, expression of the chimeric GhWUS repressor in cotton callus inhibited embryogenic callus formation. Our results show that GhWUS is an important regulator of somatic embryogenesis and shoot regeneration.

Protocol for the Micropropagation of Coconut from Plumule Explants.

Coconut is a crop that is economically important in several countries throughout the world. Unfortunately, production is decreasing because palms are affected by very serious phytoplasma diseases, such as lethal yellowing, and also most of coconuts are already very old. On the other hand, markets for coconut products have been rapidly growing in recent years. Hence, replanting of most cultivation surface worldwide, as well as establishing new surface, is urgently needed. This is an immense task, requiring at least a billion coconut palms that cannot be accomplished by traditional propagation through seed. Therefore the biotechnological alternative of micropropagation by somatic embryogenesis is needed. Research has been carried out on this subject in laboratories in several countries studying different approaches, testing different types of explants. The most responsive tissue has been plumule from zygotic embryos. A protocol for micropropagation of coconut based on plumule explants is described here. It involves the use of different media that are based on Y3 medium complemented with activated charcoal, gelling agent, sucrose, and growth regulators. These media allow the formation of embryogenic callus and somatic embryos, growth of shoots, and development of plantlets.

Regeneration of Asparagus officinalis L. Through Embryogenic Callus

A plant regeneration system was established from hypocotyl explants of in vitro grown seedlings of A. officinalis and in vitro proliferated shoots, respectively through somatic embryogenesis and embryogenic calli. Somatic embryogenesis was significantly influenced by the types of plant growth regulators. Embryogenic calli with somatic embryos developed well in MS supplemented with 2.0 ‐ 4.0 μM BAP and 1.0 ‐ 4.0 μM 22,4‐D, NAA or IBA. The highest frequency (95.3%) of embryogenic calli and 55.2 somatic embryos formation were obtained when the MS was amended with 4.0 μM BAP and 2.0 μM 2,4‐D. The best embryo germination occurred in 1.0 μM BAP supplemented MMS. The highest 97.2% of shoot proliferation was observed in embryogenic calli in MS medium containing 2.0 μM BAP and 1.0 μM IBA. In vitro grown shoots were rooted in MMS with 1.0 ‐ 2.0 μM IBA. Regenerants were transferred to vermicompost and successfully established under an ex vitro environment in garden soil with 80% survival rate.Plant Tissue Cult. & Biotech. 27(1): 21-31, 2017 (June)

MiR160 and miR166/165 Contribute to the LEC2-Mediated Auxin Response Involved in the Somatic Embryogenesis Induction in Arabidopsis.

MicroRNAs are non-coding small RNA molecules that are involved in the post-transcriptional regulation of the genes that control various developmental processes in plants, including zygotic embryogenesis (ZE). miRNAs are also believed to regulate somatic embryogenesis (SE), a counterpart of the ZE that is induced in vitro in plant somatic cells. However, the roles of specific miRNAs in the regulation of the genes involved in SE, in particular those encoding transcription factors (TFs) with an essential function during SE including LEAFY COTYLEDON2 (LEC2), remain mostly unknown. The aim of the study was to reveal the function of miR165/166 and miR160 in the LEC2-controlled pathway of SE that is induced in in vitro cultured Arabidopsis explants.In ZE, miR165/166 controls the PHABULOSA/PHAVOLUTA (PHB/PHV) genes, which are the positive regulators of LEC2, while miR160 targets the AUXIN RESPONSE FACTORS (ARF10, ARF16, ARF17) that control the auxin signaling pathway, which plays key role in LEC2-mediated SE. We found that a deregulated expression/function of miR165/166 and miR160 resulted in a significant accumulation of auxin in the cultured explants and the spontaneous formation of somatic embryos. Our results show that miR165/166 might contribute to SE induction via targeting PHB, a positive regulator of LEC2 that controls embryogenic induction via activation of auxin biosynthesis pathway (Wójcikowska et al., 2013). Similar to miR165/166, miR160 was indicated to control SE induction through auxin-related pathways and the negative impact of miR160 on ARF10/ARF16/ARF17 was shown in an embryogenic culture. Altogether, the results suggest that the miR165/166- and miR160-node contribute to the LEC2-mediated auxin-related pathway of embryogenic transition that is induced in the somatic cells of Arabidopsis. A model summarizing the suggested regulatory interactions between the miR165/166-PHB and miR160-ARF10/ARF16/ARF17 nodes that control SE induction in Arabidopsis was proposed.