Direct Somatic Embryogenesis Research Articles

EXPRESIÓN EMBRIOGÉNICA DIRECTA EN Agave cupreata

<p><strong>Background:</strong> <em>Agave cupreata</em> is a specie that can only reproduce by seeds and could take up to 15 years until flowering, placing it at risk of extinction due to its agroindustrial exploitation to produce mezcal. <strong>Objective:</strong> To evaluate the type of explant, the vitamin complex and 2,4-D concentrations on the direct embryogenic response. <strong>Methodology:</strong> Explants of leaf, root and stem sections were evaluated. Also, the vitamins MS (Murashige and Skoog, 1962) and L2 (Phillips and Collins, 1979), five concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), (0.0, 0.1, 0.3, 0.6 and 0.9 mg L<sup>-1</sup>) and six concentrations of indole-3-acetic acid (IAA), (0.0, 0.1, 0.5, 1.0, 2.0 and 3.0 mg L<sup>-1</sup>) were assayed for the expression of somatic embryos directly. <strong>Results: </strong>The leaf explant, the vitamin L2 complex, and the concentration of 0.9 mg L<sup>-1</sup> of 2,4-D and 0.5 mg L<sup>-1</sup> of IAA turned out to be the most efficient for the formation of pro-embryogenic masses and the expression of direct somatic embryos in the globular state. <strong>Implications:</strong> These results lay the groundwork for the future regeneration of <em>A. cupreata</em> plants via direct somatic embryogenesis, potentially facilitating their conservation and mass propagation. This contributes to the sustainability of mezcal; boosts the local economy, preserves biodiversity, and ensures the continued production of this endangered species. <strong>Conclusions:</strong> For the first time, the successful expression of somatic embryos directly from <em>A. cupreata</em> explants was evidenced.</p>

Media Optimization for Direct Somatic Embryogenesis, Regeneration in Sugarcane and Genetic Transformation using cry1Ac Gene

Media Optimization for Direct Somatic Embryogenesis, Regeneration in Sugarcane and Genetic Transformation using cry1Ac Gene

Tobacco Plant: A Novel and Promising Heterologous Bioreactor for the Production of Recombinant Bovine Chymosin.

The natural source of chymosin, a key enzyme in the dairy industry, is insufficient for rapidly growing cheese industries. Large-scale production of recombinant proteins in heterologous hosts provides an efficient alternative solution. Here, the codon-optimized synthetic prochymosin gene, which has a CAI index of 0.926, was subcloned from a cloning vector (pUC57-bCYM) into the pBI121 vector, resulting in the construct named pBI121-bCYM. CAI ranges from 0 to 1 and higher CAI improves gene expression in heterologous hosts. The overexpression of the prochymosin gene was under the control of constitutive CaMV 35S promoter and NOS terminator and was transferred into the tobacco via A. tumefaciens strain LBA4404. Explant type, regeneration method, inoculation temperature, cell density (OD600) of Agrobacterium for inoculation, and acetosyringone concentration were leaf explants, direct somatic embryogenesis, 19 °C, 0.1, and 100 µM, respectively. The successful integration and expression of the prochymosin gene, along with the bioactivity of recombinant chymosin, were confirmed by PCR, RT-PCR, and milk coagulation assay, respectively. Overall, this study reports the first successful overexpression of the codon-optimized prochymosin form of the bovine chymosin enzyme in the tobacco via indirect transformation. Production of recombinant bovine chymosin in plants can be an easy-to-scale-up, safe, and inexpensive platform.

Cell wall remodeling and callose deposition during the embryogenic transition in the tree fern Cyathea delgadii Sternb.

With the discovery in 2015 of the ability to induce somatic embryos in Cyathea delgadii, learning more about the relationship between the structure of apoplast and cell differentiation has become possible among ferns. In this study, the distribution of arabinogalactan proteins, pectins, extensins, and callose with specific epitopes recognized by monoclonal antibodies was investigated during direct somatic embryogenesis (SE) of C. delgadii. Eight antigens against the arabinogalactan proteins (JIM8, JIM13, LM2), pectins (JIM5, JIM7), extensins (JIM11, JIM12), and callose (anti-1 → 3-β-glucan) were selected. Two types of explants were analyzed, i.e. stipe fragments and internodes, which give rise to embryos of unicellular and multicellular origin, respectively. The study showed that embryogenic transition in C. delgadii is preceded by cell wall remodeling of initial explants. Dynamic changes in JIM13, JIM12, and anti-1 → 3-β-glucan localization were observed. The differences in the distribution of studied epitopes were observed between the cell walls of the epidermis and those located in the other layers of the explant. Moreover, within the somatic embryos, a stronger fluorescence of the examined antibodies was observed, mainly those reacting with arabinogalactan proteins, extensins, and callose. These results also implicated that, with the exception of the earlier appearance of callose in the stipe explants, the uni- and multicellular pathways of somatic embryo differentiation do not differ in the quality of cell wall components. The presented studies document the first time that SE in ferns can be regulated by changes in apoplast structure and they provide a basis for more detailed research.

Direct somatic embryogenesis and related gene expression networks in leaf explants of Hippeastrum ‘Bangkok Rose’

Hippeastrum, a highly diverse genus in the Amaryllidaceae family, is a valuable ornamental bulbous flowering plant. Somatic embryogenesis (SE) is an efficient method for mass production of Hippeastrum plantlets. Previous studies have been devoted to the in vitro propagation of Hippeastrum, but the SE and its regulatory networks are rarely reported. In this study, we established a direct SE method of Hippeastrum ‘Bangkok Rose’ using leaf bases as explants. MS supplemented with 1.00 mg · L−1 NAA +1.00 mg · L−1 KT + 0.25 mg · L−1 TDZ was the optimal medium for SE. Histological observations showed that the bipolar somatic embryo originated from the epidermal cell layer and underwent initiation, globular, scutellar and coleoptile stages. During SE, endogenous hormones of IAA, CTK, ABA, and SA were highly accumulated. Transcriptomic analysis revealed the genes encoding auxin biosynthesis/metabolic enzymes and efflux carriers were induced, while the auxin receptor of TIR1 and ARF transcriptional repressor of Aux/IAA were down-regulated and up-regulated, respectively, leading to suppression of auxin signaling. In contrast, cytokine signaling was promoted at the early stage of SE, as biosynthesis, transport, and signaling components were up-regulated. Various stress-related genes were up-regulated at the early or late stages of SE. Chromatin remodeling could also be dynamically regulated via distinct expression enzymes that control histone methylation and acetylation during SE. Moreover, key SE regulators, including WOXs and SERKs were highly expressed along with SE. Overall, the present study provides insights into the SE regulatory mechanisms of the Hippeastrum.

Use of Light Spectra for Efficient Production of PLBs in Temperate Terrestrial Orchids

Wild orchids, especially the terrestrial temperate ones are endangered species due to challenges in their natural habitats. Therefore, there is an urgent need to introduce efficient propagation methods to overcome the natural reproduction problems of these orchids. In this study, the effects of different light spectrums, explant types, wounding, and combinations of different plant growth regulators (PGRs) on direct somatic embryogenesis (DSE) of two species of these endangered orchids listed in the conservation category, were studied. The highest percentages of DSE formation and embryo germination were observed in Dactylorhiza umberosa protocorm explants exposed to white light (400–730 nm) and in Epipactis veratifolia protocorm explants exposed to a combination of red and far-red spectra (R: FR = 70:30). This occurred while red (610–700) alone and in combination with far-red (710–730 nm) spectrum induced embryogenesis more than the blue spectrum and dark condition in E. veratifolia. Thidiazuron (TDZ, 3 mg L−1), produced the highest percentage of protocorm-like bodies (PLBs) on protocorm explants in both orchids. Kinetin (Kin, 2 mg L−1) and Benzyladenine (BA 3 mg L−1) had the most effect on the survival and growth of PLBs, respectively, in D. umberosa and E. veratifolia. Species did not show similar embryogenesis responses under light spectrums. In a medium containing 3 mg L−1 TDZ, white light and R-FR spectra produced the most PLBs on wounded protocorm explants of D. umberosa and E. veratifolia respectively. The developmental stage of apical meristem of PLBs in both species was more advanced under R-B spectra, compared to others.

Influence of Basal Salts, Sucrose and Plant Growth Regulator Levels on Nucellar Embryogenesis and Plantlet Regeneration in Monoembryonic Mango Varieties

Current study described stage-wise protocols for in vitro propagation of commercially important varieties of mango. Induction of somatic embryos (SE) and plantlet regeneration was obtained using nucellar explants of three superior monoembryonic mango vars.‘Saroli’, ‘Langra’, and ‘Chaunsa’ were cultivated in Khairpur, Pakistan. The immature fruits (2.5-4.0 cm long) were surface disinfected using a 30% sodium hypochlorite (NaOCl) solution. Results revealed that significantly highest direct somatic embryogenesis (93%) was obtained in var. ‘Chaunsa’ under full dark on culture medium comprising of 2.0 mg L-1 N6 2-isopentenyl adenine (2iP), 0.5 mg L-1 2,4-dichlorophenoxyacetic acid (2,4-D). Medium consisted of 2iP 4.0 mg L-1, 2,4-D 1.0 mg L-1 induced significantly highest embryogenic callus (91%) using nucellar explants in var.‘Chaunsa’. Significantly highest germination (95%) of SE was achieved in var. ‘Chaunsa’ on the medium comprising microsalts of MS, macrosalts of B5, 2iP 0.1 mg L-1, Kinetin (Kin) 0.5 mg L-1. Highest shoot length (5.1 cm) and root length (4 cm) were obtained in var. ‘Langra’ on the medium consisted of microsalts of MS, macrosalts of B5, 30 g L-1 sucrose, 200 mg L-1 activated charcoal (AC), 0.1 mg L-1 naphthalene acetic acid (NAA), 0.2 mg L-1 benzyl adenine (BA). Stage-wise protocols established for the regeneration of plantlets can be useful for micropropagation of the other mango varieties of the world.

Induction of direct somatic embryogenesis and genetic stability of somatic embryo-derived plants of broccoli

Induction of direct somatic embryogenesis and genetic stability of somatic embryo-derived plants of broccoli

Cell reprogramming via direct somatic embryogenesis in an Atlantic Forest species vulnerable to extinction: Euterpe edulis stem segments induced with picloram

Cell reprogramming via direct somatic embryogenesis in an Atlantic Forest species vulnerable to extinction: Euterpe edulis stem segments induced with picloram

Molecular and histological validation of modified in ovulo nucellus culture based high-competency direct somatic embryogenesis and amplitude true-to-the-type plantlet recovery in Kinnow mandarin.

Kinnow (Citrus nobilis Lour. × Citrus deliciosa Ten.) needs to be genetically improved for traits such as seedlessness using biotechnological tools. Indirect somatic embryogenesis (ISE) protocols have been reported for citrus improvement. However, its use is restricted due to frequent occurrences of somaclonal variation and low recovery of plantlets. Direct somatic embryogenesis (DSE) using nucellus culture has played a significant role in apomictic fruit crops. However, its application in citrus is limited due to the injury caused to tissues during isolation. Optimization of the explant developmental stage, explant preparation method, and modification in the in vitro culture techniques can play a vital role in overcoming the limitation. The present investigation deals with a modified in ovulo nucellus culture technique after the concurrent exclusion of preexisting embryos. The ovule developmental events were examined in immature fruits at different stages of fruit growth (stages I-VII). The ovules of stage III fruits (>21-25 mm in diameter) were found appropriate for in ovulo nucellus culture. Optimized ovule size induced somatic embryos at the micropylar cut end on induction medium containing Driver and Kuniyuki Walnut (DKW) basal medium with kinetin (KIN) 5.0 mg L-1 and malt extract (ME) 1,000 mg L-1. Simultaneously, the same medium supported the maturation of somatic embryos. The matured embryos from the above medium gave robust germination with bipolar conversion on Murashige and Tucker (MT) medium + gibberellic acid (GA3) 2.0 mg L-1 + ά-naphthaleneacetic acid (NAA) 0.5 mg L-1 + spermidine 100 mg L-1 + coconut water (CW) 10% (v/v). The bipolar germinated seedlings established well upon preconditioning in a plant bio regulator (PBR)-free liquid medium under the light. Consequently, a cent percent survival of emblings was achieved on a potting medium containing cocopeat:vermiculite:perlite (2:1:1). Histological studies confirmed the single nucellus cell origin of somatic embryos by undergoing normal developmental events. Eight polymorphic Inter Simple Sequence Repeats (ISSR) markers confirmed the genetic stability of acclimatized emblings. Since the protocol can induce rapid single-cell origin of genetically stable in vitro regenerants in high frequency, it has potential for the induction of solid mutants, besides crop improvement, mass multiplication, gene editing, and virus elimination in Kinnow mandarin.

Effects of basic media and plant growth regulators on Direct-somatic embryogenesis induction in vegetative strains obtained from the tissue culture of digitalis purpurea

This study aimed to investigate the effects of certain nutrient medium types enhanced with plant-growth regulators on the direct inductionof somatic embryogenesis in vegetative strains produced and propagated from a tissue culture of Digitalis purpurea. The results showthat MS medium was superior to other tested nutrient media-B5, LS, and 5C01—in terms of the days required to initiate direct somaticembryogenesis induction and induction rate. In contrast, the LS medium resulted in the highest number of embryos. Moreover, 2 mg/LBA (benzyl adenine) was superior to similar concentrations of BAP (benzyl aminopurine) and Kin (kinetin) in the MS medium, whereasBAP performed better in LS and B5. On the other hand, auxin 2.4D (dichlorophenoxy acetic acid) inhibited somatic embryogenesis, as didNAA (naphthalene acetic acid) and IAA (indole acetic acid) when added individually. Regarding the type of explant, the stems showedthe induction rates and numbers of direct-somatic embryos produced in all the basic media.Keywords: Digitalis purpurea; Vegetative strains; Direct-somatic embryogenesis; Explant; Basic media

Exogenous implications of silver nitrate on direct and indirect somatic embryogenesis and germination of cold stored synseeds of Vanilla planifolia Jacks. ex Andrews

• Silver nitrate the medium improved somatic embryogenic efficiency in Vanilla planifolia . • Medium containing cytokinins with 2.5 mg L −1 silver nitrate induced maximum number of somatic embryos. • Developmental transition from globular to cotyledonary stages of somatic embryos were confirmed by anatomical studies. • Silver nitrate favored proliferation and rooting of shoots, and improved the survival rate of V. planifolia plantlets. Vanilla planifolia – a natural source of vanillin is the world's highest-valued spice crop with endangered status and the only hand-pollinated crop globally. The present study evaluates the effect of exogenous silver nitrate (AgNO 3 ) on effective direct and indirect somatic embryogenesis in V. planifolia for the first time. The direct and indirect somatic embryogenic capacity of leaf explants were studied using Murashige and Skoog (MS) medium containing 6-benzylaminopurine (BAP), kinetin (KN), 2,4-Dichlorophenoxyacetic acid (2,4-D), indole-3-acetic acid (IAA), and α-naphthalene acetic acid (NAA) alone and in combinations. Additionally, the efficacy of silver nitrate (SN) in promoting somatic embryogenesis was tested with optimized growth regulators. Non-embryogenic callus was obtained from the explants when cultured on 3.0 mg L −1 2,4-D. The embryogenic potential was developed while culturing the non-embryogenic callus on 1.0 mg L −1 each of BAP and KN combined with 2.5 mg L −1 SN. On the other hand, direct somatic embryogenesis (92.0%) was obtained when culturing the explants directly on MS medium containing 1.0 mg L −1 each of BAP and KN combined with 2.5 mg L −1 SN, and a maximum of 30.0 somatic embryos (SEs) were induced per explant. Microscopic analysis revealed that SEs were originated externally as cell division initiated on the leaf surface and cut ends of explants, and have no vascular connections with the explant. Actively dividing embryonic cells possessed dense cytoplasm with prominent nuclei. The mature SEs were encapsulated and stored at - 4 °C for one year. The synseeds showed maximum germination and proliferation (17.2 shoots per SE) on MS medium containing 0.5 mg L −1 BAP, 0.25 mg L −1 KN, and 2.5 mg L −1 SN. The results indicated that AgNO 3 in the medium promoted somatic embryogenesis and plantlet regeneration in V. planifolia .

체세포배발생 형태에 따른 일천궁 기내식물체 재분화

Background: The present study investigated the types and concentrations of plant hormones affecting somatic embryo induction from leaves, stems, and roots of Cnidium officinale Makino.BRMethods and Results: Somatic embryos could be induced using two methods: direct and indirect somatic embryogenesis. Direct somatic embryogenesis was induced when the whole explants were grown in a medium supplemented with cytokinin (e.g., kinetin), while indirect somatic embryogenesis was induced when the medium was supplement with auxin [eg., 2,4-dichlorophenoxyacetic acid (2,4-D)]. Both direct and indirect somatic embryos were mostly induced from leaves. The highest direct somatic embryo induction (95.8%) was obtained from leaf explants grwon on Murashige and Skoog (MS) medium supplemented with 0.5 - 1.0 ㎎/ℓ kinetin. The highest indirect somatic embryo induction (75.0%) was obtained from leaf explants grown on MS medium supplemented with 0.5 ㎎/ℓ 2,4-D. The induced somatic embryo could successfully germinate and acclimatize.BRConclusions: Our results can be useful for establising a mass propagation protocol for C. officinale.

Overview of Somatic Embryogenesis.

Somatic embryogenesis is a natural phenomenon through which somatic embryos are produced from somatic cells although. It is considered the most efficient morphogenic pathways for plant multiplication. One of the key features of somatic embryogenesis is the use of cellular totipotency, where dedifferentiation is induced to foster cell proliferation, followed by the induction of differentiation using plant growth regulators to produce new plants. There is a cell group with the potential to undergo the somatic embryogenesis pathway through adequate stimulation (plant growth regulators, incubation conditions, and supplementation of the culture medium). There are two somatic embryogenesis pathways in plants: direct and indirect embryogenesis. Direct somatic embryogenesis consists of the formation of embryos directly from isolated cells, without the formation of "callous" tissue. Indirect somatic embryogenesis is characterized by the formation of a callus as a stage that precedes the formation of somatic embryos. It should be stressed that not all plant cells have this morphogenic capacity; consequently, determining the type of factors that drive this type of response has been challenging. This book provides the reader with updated available information on the techniques, relevant protocols, and tools to perform somatic embryogenesis in different plant species for economic purposes.

Direct somatic embryogenesis and flow cytometric assessment of ploidy stability in regenerants of Caladium × hortulanum ‘Fancy’

Caladium × hortulanum 'Fancy' is an important ornamental plant grown in pots and landscapes and known for its colorful leaves often used for interior decorations. In this work, we present a method of in vitro regeneration from three explants source through direct somatic embryogenesis (DSE) wherein the regenerated plants were screened for ploidy changes through flow cytometry analysis. Tuber, leaf and petiole explants were cultured on MS basal medium supplemented with 1-napthalene acetic acid (NAA), 6-benzyl amino purine (BAP) and N-phenyl-N'-1, 2,3-thiadiazol-5-ylurea (TDZ) concentrations. Tuber explants induced highest direct somatic embryos on NAA (1 mg L- 1) + BAP (0.5 mg L- 1) with 55.6 mean number of embryos per explant while as leaf and petiole explants amended with 1 mg L- 1 TDZ developed 18.7 and 12.27 mean number of embryos per explant respectively. The highest embryo conversion frequency was achieved on BAP (2 mg L- 1) + NAA (0.2 mg L- 1) with 44.2, 18.7 and 7.5 mean number of plantlets produced per tuber, leaf and petiole explant respectively after 4 weeks of culture. Plantlets were later rooted and maximum number of roots (6.33) per shoot was achieved on 2 mg L- 1 indolebutyric acid amended medium. Description of the process of DSE is presented through the histological and SEM evidences. The 2C DNA content of field grown plants and the DSE regenerants evaluated under flow cytometric analysis were 8.06 pg and 8.28 pg respectively showing no ploidy changes. Hence, a successful protocol of inducing direct somatic embryos from three explant types with efficient embryo conversion frequency was obtained with regenerants showing similar DNA ploidy as that of their parent plants.

Effect of Plant Growth Regulators on Somatic Embryogenesis and Plantlet Development of Turkey Berry (Solanum torvum SW)

In the present study it was reported on direct somatic embryogenesis and plant regeneration from cotyledon and leaf explants of Turkey berry/pea egg plant (Solanum torvum SW), a medicinally important plant. Somatic embryogenesis has several advantages over other routes of in vitro plant regeneration. Somatic embryogenesis was induced directly from cotyledon and leaf explants on MS medium fortified with BAP (0.5 mg/L)+NAA (0.5-6.0 mg/L). High percentage of somatic embryogenesis (90%), maximum number of somatic embryos formation (62±0.18) along with high percentage (76%) conversion of somatic embryos into bipolar embryos was observed on cotyledon explants in 0.5 mg/L BAP+2.5 mg/L NAA. At the same concentration of BAP (0.5 mg/L)+NAA (2.5 mg/L) also resulted on the maximum percentage of somatic embryogenesis (92%), the highest number of somatic embryos formation (88±0.15) and the highest percentage (76%) of somatic embryos conversion into bipolar embryos in leaf explants. A mixture of globular, heart and torpedo-shaped embryos were germinated on MS medium supplemented with 0.5 mg/L IAA+1.0-4.0 mg/L BAP. Maximum germination frequency (75±0.14) of somatic embryos and plantlet formation was found in 0.5 mg/L IAA+2.0 mg/L BAP, but they didn’t germinate on ½ MSO and MSO media. The survival rate of regenerated plants after field transfer was recorded to be 75%. These regenerated plants were found morphologically similar to donor plants. The present protocol can be used for conservation of the species and also for genetic transformation experiments in S. torvum.

Organogenesis, direct somatic embryogenesis, and shoot proliferation of Rheum spiciforme Royle: an endemic and vulnerable medicinal herb from Indian Trans Himalayas

Organogenesis, direct somatic embryogenesis, and shoot proliferation of Rheum spiciforme Royle: an endemic and vulnerable medicinal herb from Indian Trans Himalayas

Comparative Study of the Genetic and Biochemical Variability of Polyscias filicifolia (Araliaceae) Regenerants Obtained by Indirect and Direct Somatic Embryogenesis as a Source of Triterpenes.

Polyscias filicifolia (Araliaceae) is broadly used in traditional medicine in Southeast Asia due to its antimicrobial, immunomodulating and cytotoxic activities. The main groups of compounds responsible for pharmacological effects are believed to be oleanolic triterpene saponins. However, Polyscias plants demonstrate relatively slow growth in natural conditions, which led to applying a developing sustainable source of plant material via primary (PSE), secondary (DSE) and direct somatic embryogenesis from DSE (TSE). The AFLP and metAFLP genotyping resulted in 1277 markers, amplified by a total of 24 pairs of selective primers. Only 3.13% of the markers were polymorphic. The analysis of variance showed that the PSE and TSE regenerants differed only in terms of root number, while the DSE plantlets differed for all studied morphological characteristics. Further, the chemical analysis revealed that oleanolic acid (439.72 µg/g DW), ursolic acid (111.85 µg/g DW) and hederagenin (19.07 µg/g DW) were determined in TSE regenerants. Our results indicate that direct somatic embryogenesis ensures the production of homogeneous plant material, which can serve as a potential source of triterpene compounds. Plants obtained via somatic embryogenesis could also be reintroduced into the natural environment to protect and preserve its biodiversity.

Cytokinin oxidase/dehydrogenase inhibitors stimulate 2iP to induce direct somatic embryogenesis in Coffea arabica

Somatic embryogenesis in Coffea spp. is important for mass production and genetic engineering. Coffee is an exceptional case, as somatic embryogenesis can be induced by applying a cytokinin such as 2iP as the only plant growth regulator. 1-(2-(2-Hydroxyethyl)phenyl)-3-(3-(trifluoromethoxy)phenyl)urea (3TFM-2HE) and 1-(3-bromo-5-(trifluoromethoxy)phenyl)-3-(2-(2-hydroxyethyl)phenyl)urea (3TFM,5Br-2HE) are two newly designed cytokinin oxidase/dehydrogenase inhibitors (CKX) from the diphenylurea group. We used a Coffea arabica leaf disks bioassay to demonstrate the indirect somatic embryo induction potential of these compounds. The leaf disks were incubated on modified Murashige and Skoog (MS) semi-solid medium in which 3TFM-2HE or 3TFM,5Br-2HE were combined with N6-(2-isopentenyl)adenine (2iP). Although these compounds do not possess intrinsic cytokinin activity, they enhanced the activity of 2iP, resulting in direct somatic embryogenesis after seven weeks. The best results were obtained with 1 µM 3TFM-2HE and 5 or 10 µM 2iP. Maturation of somatic embryos into fully developed plants took place on medium supplemented with 0.5 µM kinetin and the somatic embryos developed true leaves and a root system.

Direct and Indirect Somatic Embryogenesis Induction in Camellia oleifera Abel.

Camellia oleifera Abel. is an important woody oil species; however, the shortage of rapid and industrialized seedling culture is a large constraint on the development of the tea oil industry. Somatic embryogenesis (SE) is one of the main powerful biotechnological tools for plant mass regeneration, but the largely unknown SE in C. oleifera limits the scale production of clonal plants. In this study, we described a high-efficiency SE system via direct and indirect pathways in C. oleifera and investigated the effect of genotype, explant age and phytohormones on SE. In the direct pathway, somatic embryos were highly induced from immature seeds 220 days after full blossom, and the development of embryoids was achieved with a combination of 0.19 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.05 mg/L thidiazuron (TDZ). In the indirect pathway, embryogenic calli were induced from the same explants in medium containing 1.5 mg/L 2,4-D, while 0.75 mg/L 2,4-D treatment led to high proliferation rates for embryogenic calli. The addition of 0.19 mg/L 2,4-D alone stimulated the production of globular embryos while causing a 75% loss of the induction rate in the heart embryo stage. Upon transfer of the globular embryos to phytohormone-free medium, an optimal induction rate of 62.37% from globular embryos to cotyledonary embryos was obtained. These data suggest that the subsequent differentiation process after the globular embryo stage in ISE is more similar to an endogenous phytohormones-driven process. Mature embryos germinated to produce intact plantlets on half-strength MS basal medium with a regeneration rate of 63.67%. Histological analysis confirmed the vascular bundle isolation of embryoids from the mother tissue. We further studied the different varieties and found that there were no significant genotype differences for SE induction efficiency in C. oleifera. Thus, we established a high-efficiency induction system for direct and indirect somatic embryogenesis (ISE) in C. oleifera and regenerated intact plantlets via SE, not organogenesis. ISE has a more complicated induction and regulatory mechanism than direct somatic embryogenesis. The improved protocol of SE would benefit mass propagation and genetic manipulation in C. oleifera.