Somatic Embryo Induction Research Articles

English

Developing a protocol for somatic embryogenesis (SE) for adult mother plants of Jatropha curcas, aids to overcome problems such as harvesting time and an uneven yield, providing the opportunity to propagate proven elite genotypes. Until now, several authors have achieved SE in J. curcas using different explants, however, in none of these protocols adult plants have been used as mother plant material. Furthermore, the challenge of overcoming the morphogenesis limitations successfully and further regeneration of adult plants of J. curcas has been great. The main objective of this study was to develop a somatic embryogenesis protocol for adult plants of the oil producing J. curcas. transverse thin cell layers (tTCL) of young petioles were used as explants. Half strength Y3 and MS media were used containing three different concentrations of 2,4-dichloropherioxyacetic acid and 6-benzil-aminopurine in a factorial trial. Nine accessions of J. curcas of various ages (3-12 years.) were tested using this protocol. All accessions responded positively to callus induction and to the induction of somatic embryos, demonstrating the protocol to be genotype-independent. This study enables micropropagation of adult of proven elite plants of J. curcas via somatic embryogenesis.    Key words: Jatropha curcas, transverse thin cell layers (tTCL), somatic embryos, half strength media, regeneration.

Physiological characteristics and genetic diversity of Lilium distichum Nakai autotetraploids

Polyploidy is widely recognized as a major evolutionary force in plants that enhances environmental adaptability and the diversity of phenotypes. Lilium dis + ichum Nakai is a rare wild diploid lily of high horticultural value that is susceptible to disease and has a low coefficient of reproduction in nature. In order to identify the chromosomes and inter-simple sequence repeats (ISSR) of this lily, we performed colchicine induction of somatic embryos, and accordingly obtained three autotetraploid genotypes. Compared with diploids, the autotetraploid L. distichum plants had larger bulbs, thicker scales, wider leaves, thicker and stronger adventitious roots, and higher starch and sugar contents. Optical microscopy revealed larger single cells and a higher number of starch grains in the autotetraploids. Furthermore, the tetraploid plants had lower proline and malondialdehyde contents and higher root activity, which we assume contributes to the enhanced resistance of tetraploids compared with diploids and would have a positive effect on field transplantation. All autotetraploid genotypes had thicker scales and better developed roots than the diploids, although we detected differences in the scales of different genotypes, with those of T3 being the most compact and those of T1 being the least compact. We also detected differences in the starch and sugar contents and physiological indices among the three genotypes, although these showed similar trends and were all superior to the corresponding traits in the diploids.

Genome-Wide Analysis of Somatic Embryogenesis-Related Transcription Factors in Cultivated Strawberry (Fragaria × ananassa) and Evolutionary Relationships among Rosaceae Species

Somatic embryogenesis is a plant regeneration method commonly used in tissue culture. Its molecular mechanisms are well-known in model plants such as Arabidopsis thaliana L. LEAFY COTYLEDON1 (LEC1), LEAFY COTYLEDON2 (LEC2), FUSCA3 (FUS3), ABSCISIC ACID INSENSITIVE3 (ABI3), and BABYBOOM (BBM) genes are considered master regulators in the induction, growth, and maturation of somatic embryos. However, the study of these transcription factors in fruit crops with high agronomic and economic value such as cultivated strawberry (Fragaria × ananassa Duch.) and other Rosaceae species is scarce. The purpose of this study was the in silico characterization of LEC1, ABI3, FUS3, LEC2, and BBM(LAFL-B) genes from F. × ananassa genome and the study of the evolutionary relationships within the Rosaceae family. Synteny analyses and molecular evolutionary rates were performed to analyze the evolution of each transcription factor within the Rosaceae family. Synteny was conserved between F. × ananassa and other Rosaceae genomes, and paralogous genes were selected through negative selection. Additionally, the exon–intron organization and multiple alignments showed that gene structure and DNA-binding domains were conserved in F. × ananassa transcription factors. Finally, phylogenetic trees showed close evolutionary relationships between F. × ananassa and its orthologous proteins in the Rosoideae subfamily. Overall, this research revealed novel insights in the LAFL-B network in F. × ananassa and other species of the Rosaceae family. These results provide useful in silico information and new resources for the establishment of more efficient propagation systems or the study of ploidy effects on somatic embryogenesis.

The induction and development of somatic embryos from the in vitro culture of Catharanthus roseus (L.) G. Don

Catharanthus roseus is containing anticancer alkaloids vincristine and vinblastine as is an important medicinal plant. Several studies have conducted on in-vitro culture of this plant. To optimize the somatic embryogenesis, a factorial based on CRD experiment with 10 replications was conducted. Root, hypocotyl and leaf explants grown in-vitro were transferred and cultured on MS media containing different combinations of 2,4-D, NAA and 2,4-D×BAP. The results revealed that in callogenesis, the interaction effects of root and hypocotyl explants×2,4-D and NAA as well as hypocotyl×(1 mg l-1 NAA+1 mg l-1 BAP) was superior than other treatments (p≤0.01). For calli fresh weight, hypocotyl×NAA and hypocotyl×(1 mg l-1 NAA+1 mg l-1 BAP) was the treatment of choice (p≤0.01). The calli produced were sub-cultured to attain the pre-embryos and somatic embryos. For the number of pre-embryos and somatic embryos; the interaction of hypocotyl×2,4-D was the best and most efficient treatment. Seemingly, the production of somatic embryos is accessible in this plant by the logical management of growth regulator combinations. Furthermore, the production and genetic engineering of the somatic embryos could be a promising trend in the subsequent production of high-valued metabolites from this plant.

English

Some improved genotypes of cocoa tree (Theobroma cacao L.), have shown in standard study conditions of absence or a very low response to the induction of somatic embryos. This is the case of the recalcitrant genotypes C8 and C15, partially recalcitrant genotypes C14 and C16. This study aims to improve the production of somatic embryos of recalcitrant genotypes. Staminodes and petals excised from the immature buds of the five genotypes C1, C8, C14, C15 and C16 were used as plant material. These floral explants were cultured on different media containing two type of mineral salts such as potassium sulphate (K2SO4) at concentrations 18, 27, 36 and 45 mM and magnesium sulphate (MgSO4) whose concentrations used are 5, 10, 15 and 20 mM. Calli induction was obtained in the five (05) genotypes at percentages ranging from 50 to 100% with staminodes and from 61 to 100% with petals on all media. The transfer of the callogenic explants on the developmental medium allowed the induction of embryogenic calli and somatic embryos after 84 days only with petal explants. The most important PEC and NSE were obtained with two mineral salts concentrations, 27 mM K2SO4 and 15 mM MgSO4 for all genotypes and varied to 20 to 40%. Potassium sulphate (K2SO4) at 27 and magnesium sulphate (MgSO4) at 15 mM are the concentrations of the most suitable mineral salts to overcome recalcitrance of cocoa genotypes.   Key words: Recalcitrant, Theobrama cacao, improved genotypes, mineral salts, somatic embryogenesis.

Direct somatic embryogenesis using leaf explants and short term storage of synseeds in Spathoglottis plicata Blume

Spathoglottis plicata Blume is a vulnerable orchid species in various parts of the world, and the conventional propagation provides limited success to its cultivation and conservation. Therefore, present study deals with the direct induction of somatic embryos (SEs) from the leaf explants of S. plicata. Murashige and Skoog’s (MS) medium fortified with various types and concentrations of plant growth regulators were used to induce somatic embryogenesis and plantlet production. The highest percentage of somatic embryo formation (93.7 ± 0.56%) was achieved on MS medium supplemented with 1.0 mg L−1 2,4-dichlorophenoxyacetic acid (2,4-D), whereas, maximum proliferation and increase in fresh weight (FW) of SEs (149.5 ± 0.24 mg/ 50 mg initial FW) was achieved on MS medium fortified with 2.0 mg L−1 6-benzylaminopurine (BAP) within 5 weeks of incubation. The light microscopic observation revealed that SEs emerged directly from the leaf surface. The viable synthetic seeds (SS) from SEs were prepared by encapsulating with gel matrix of 3% (w/v) sodium alginate and 100 mM calcium chloride. The SS were successfully stored for 60 days at − 4 °C with 97.4% germination frequency and shoot proliferation using MS medium with 2.0 mg L−1 BAP and 0.25 mg L−1 indole-3-acetic acid. The synergistic rooting frequency was observed on half-strength MS medium with 1.0 mg L−1 indole-3-butyric acid. The rooted shoots were acclimatized in a greenhouse with a 90% survival rate using soilrite® and vermicompost. This is an efficient short-term storage and regeneration protocol for S. plicata, which could help in reducing pressure on its wild population and could also be extended to the cryopreservation of this orchid species. The somatic embryos were induced directly from the leaf explants of Spathoglottis plicata and an efficient short-term storage protocol was developed by encapsulating these somatic embryos. The light microscopic analysis confirmed the formation of somatic embryos. The protocol could be convenient for the commercial cultivation and conservation of S. plicata.

Efficient Method for Direct Embryogenesis in Phalaenopsis Orchid

Light spectrum is one of the environmental cues that influence plant growth and development. Light is a stimulating factor for induction of somatic embryos during tissue culture practices. To accelerate the direct embryogenesis, six different light spectra including: white (W), red (R), blue (B), green (G), red + blue (R+B) and red + far red (R+FR) together with dark condition (D), in combination with thidiazuron (TDZ) in four concentrations (0, 0.5, 1.5 and 3 mg L-1) were used. Inter-simple sequence repeat was used for identification and genetic stability analysis of somatic regenerated plantlets. Intact protocorm explants showed higher potential for direct somatic embryogenesis (DSE) than the other explants. The rate of DSE was highly dependent on the concentration of TDZ and its interaction with light spectra. R and R + FR spectra with 3 mg L-1 TDZ on intact protocorms and R+FR with 3 mg L-1 TDZ were efficient treatments to induce DSE without somaclonal variation. G light spectrum has also significant effects on DSE of protocorm explants. The amplified products showed 26 scorable bands and regenerates were completely identical to the mother plant. In conclusion, this protocol provides way to regenerate plants through embryogenesis, and is a reliable protocol to obtain proper development and genetic stable Phalaenopsis embryos.

INDUKSI KALUS EMBRIOGENIK DAN EMBRIO SOMATIK DARI EKSPLAN DAUN KULIM (Scorodacarpus borneensis Becc.)

Kulim is one of woody plant that have multifunction as wood source and for spice and medicinal. Generative propagation of this plant have trouble because seed use limited. The use of leaf segment through somatic embryogenesis to solve the problem. The objective of this study is to obtain the best treatment to embryogenic callus induction. The modification of basal medium of Murashige and Skoog was used as growth medium. The experiment was conducted in three stages are callus induction, embryogenic callus and somatic embryo induction. The treatment of 2,4-D (3,0 – 12 mg/l) used for callus induction. For embriogenic callus induction used 2,4-D (3,0 – 12,0 mg/l) combined with NAA 0,5 mg/l. The treatment of thidiazuron (0,1 – 0,7 mg/l) used for somatic embryo induction. The result showed that the treatment of 2,4-D 6,0 mg/l is the best for callus induction with compact of texture, green, dry and non embryogenic. The treatment of combination 2,4-D 12.0 mg/l with NAA 0.5 mg/l is the best for friable callus induction. The treatment of 2,4-D 6.0 mg/l combined with NAA 0,5 mg/l is the best for embryogenic callus induction with very friable of texture, easy to separate, dry, smooth and glossy. Thidiazuron of 0,1 mg/l treatment is the best for somatic embryos induction with the average number of 7,8 somatic embryos.

Direct Somatic Embryogenesis in Commercial Varieties of Corchorus L.: Morphological, Histo-chemical, and Ultrastructural Aspects

ABSTRACT Corchorus L. is an eminent source of industrial fibers. To understand the regeneration capacity, somatic embryogenesis (SEG) has been investigated. In this study, leaf explants are used for induction of the direct somatic embryos (DSEs). Light and scanning electron microscopy (SEM) has been implemented to confirm the DSEs. The influences of genotypes and plant growth regulators (PGRs) on the SEG have also been evaluated. Among the four studied verities, var. JRC 517 has shown a maximum response aftre one month of dark condition in 0.5 mg l−1IAA with 0.03 mg l−1 kin fortified basal medium. The germination rate of the DSEs is obtained about 54.8% in 1 mg l−1 GA3 fortified media. The subsequent rate of plantlets conversion has achieved about 31.8% in ½ MS media. The presence of polysaccharide and protein is observed as reserved material upon histochemical study. The unicellular origin of DSE has been confirmed by the cyto-histochemical study. Abbreviations: 2,4-D, 2,4-dichlorophenoxyacetic acid; DAPI, 4′-6-diamidino-2-phenylindole; DSE, direct somatic embryos; GA3, gibberellic acid; IAA, indole-3-acetic acid; MS medium, Murashige and Skoog medium; NBB, naphthol blue-black; PAS, periodic acid-Schiff; PGR, plant growth regulators; PCA, principal component analysis; SEG, somatic embryogenesis; SEM, scanning electron microscopy

Factors influencing micropropagation and somatic embryogenesis of two cassava varieties, Kello and Qulle

Abstract. Berhanu R, Feyissa T. 2020. Factors influencing micropropagation and somatic embryogenesis of two cassava varieties, Kello and Qulle. Cell Biol Dev 4: 71-81. Cassava (Manihot esculenta Crantz) is a perennial Euphorbiaceae shrub cultivated in the tropics, Africa, and Asia for its tuberous starchy roots. Lack of good-quality seed, low productivity and profit, virus and insect pests, high heterozygosity, low natural fertility, poor seed set, and seed germination limit its cultivation. The conventional breeding system is ill-equipped to address these issues. Therefore, additional biotechnological solutions are required to address the abovementioned issues. This research aimed to devise a method for obtaining a maximum number of shoots and morphologically sound in vitro materials by micropropagation, as well as to investigate the various parameters related to the micropropagation of the two cassava varieties, 'Kello' and 'Qulle.' The research also contains a somatic embryogenesis technique that is an optimal source of in vitro materials for large-scale multiplication. In this experiment, a Solid MS medium with different salt concentrations, sucrose concentrations, TDZ, a two-step procedure involving pre-soak of nodal explants in a liquid MS medium prior to culture on a solid MS medium, with different pH values, repeated subcultures, and somatic embryo induction on MS medium supplemented with 2,4-D alone and in combination with 2 µM CuSO4 were utilized. At a concentration of 0.2 mg/L TDZ, both 'Kello' and 'Qulle' exhibited the highest average number of shoots per explant and improved morphological properties of in vitro material. At a TDZ concentration of 0.2 mg/L, the two-step cultivation strategy produced the greatest mean number of shoots per plant for both kinds. The maximum mean number of shoots per explant was produced by 'Kello' at a medium salt strength of a quarter, but 'Qulle' required a medium salt strength of full. 'Kello' and 'Qulle' produced the greatest average number of shoots per explant at pH values of 5.6 and 6.6, respectively. At 0.15% sucrose, 'Kello' and 'Qulle' attained their highest mean values for all parameters. During somatic embryo induction, treatments with varying doses of 2,4-D generated FEC and somatic embryos, but the somatic embryos failed to progress through the globular stage. The micropropagation property of the two types through sequential subculturing suggested that repeated subculturing causes the in vitro materials to lose their multiplication property.

Influence of exogenous polyamines on somatic embryogenesis and regeneration of fresh and long-term cultures of three elite indica rice cultivars

The influence of putrescine (Put), spermidine (Spd), and spermine (Spm) on somatic embryogenesis (SE) and plant regeneration of three indica rice cultivars (ASD16, IR64, and ADT43) were evaluated since polyamines (PAs) are reported to play vital roles in SE and plant regeneration. Significant increases in the regeneration potential of the rice cultivars upon polyamine treatments were observed. Put (1.0 mM) induced the highest regeneration frequencies, somatic embryos, and shoot numbers. PAs influenced the fresh weights of the suspension cultures. Spd was found to be detrimental to cultivar ADT43 towards embryogeneicity, regeneration, and somatic embryo induction. Among the cultivars, ASD16 was found to respond superiorly to the polyamine treatment. Experiments with long-term cultures of the rice revealed that Put and Spm enhanced the regeneration potential, and the highest frequencies were recorded for ASD16 and IR64. Results indicate that PAs can be effectively used as growth modulators to achieve success in in vitro tissue and genetic manipulation studies with elite recalcitrant indica rice cultivars.

INDIRECT SOMATIC EMBRYOGENESIS ON MUTANTS OF Agave tequilana WEBER CULTIVAR BLUE INDUCED WITH CO60 GAMMA RAYS

<p><strong>Background:</strong> Blue agave (<em>Agave tequilana </em>Weber var. azul)<strong>,</strong> it is commonly known as “Agave azul or agave tequilero”. The Agave crop is fundamental part of the productive chain Agave-Tequila, since is the distinctive source of reducing sugars for the elaboration of the liqueur known as Tequila, according with the Appellation of Origen Tequila to fulfill the National Normativity (NOM-). Besides, Tequila is the most spirit drink exported from Mexico worldwide. According with the latest evaluation over this crop, the susceptibility to pathogens generate in cases total lost of the agave fields. <strong>Objective.</strong> to achieve somatic embryos that could be resistant, generated from plantlets irradiated with Co<sup>60</sup>gamma rays; which were obtained from axillary buds. <strong>Methodology:</strong> The arisen of embryo was reached in the modified Murashige and Skoog (MS) medium supplemented with of 2,4-D combined with BA or KIN. The plantlets were radiated 12 weeks after the somatic embryo induction with Co<sup>60</sup> gamma rays. The doses were 0 (test control), 10, 20 and 30 Gy. <strong>Results:</strong> There was a significant difference in the induction of embryonic callus, development and mature somatic embryos, which were reduced as the doses of radiation increases. Since only somatic embryos were achieved in tissues treated with 10 and 20 Gy doses, in higher doses induction of embryonic callus was inhibited. The regression confirmed the negative results with the increase of radiation. <strong>Implications:</strong> There were made protocols to achieve somatic embryos and plants from tissues irradiated with 10 and 20 Gy. <strong>Conclusions</strong>: The radiation effect over somatic tissue was crucial as the doses affect the conversion into plantlets, nevertheless such radiation allowed the conversion of the embryos to complete agave plantlets</p>

Improvement of a Genetic Transformation System and Preliminary Study on the Function of LpABCB21 and LpPILS7 Based on Somatic Embryogenesis in Lilium pumilum DC. Fisch.

Auxin transport mediates the asymmetric distribution of auxin that determines the fate of cell development. Agrobacterium-mediated genetic transformation is an important technical means to study gene function. Our previous study showed that the expression levels of LpABCB21 and LpPILS7 are significantly up-regulated in the somatic embryogenesis (SE) of Lilium pumilum DC. Fisch. (L. pumilum), but the functions of both genes remain unclear. Here, the genetic transformation technology previously developed by our team based on the L. pumilum system was improved, and the genetic transformation efficiency increased by 5.7–13.0%. Use of overexpression and CRISPR/Cas9 technology produced three overexpression and seven mutant lines of LpABCB21, and seven overexpression and six mutant lines of LpPILS7. Analysis of the differences in somatic embryo induction of transgenic lines confirmed that LpABCB21 regulates the early formation of the somatic embryo; however, excessive expression level of LpABCB21 inhibits somatic embryo induction efficiency. LpPILS7 mainly regulates somatic embryo induction efficiency. This study provides a more efficient method of genetic transformation of L. pumilum. LpABCB21 and LpPILS7 are confirmed to have important regulatory roles in L. pumilum SE thus laying the foundation for subsequent studies of the molecular mechanism of Lilium SE.

Indole acetic acid (IAA) producing endophytic bacteria on direct somatic embryogenesis and plant regeneration of Exacum travancoricum Bedd.

Endophytic bacteria were isolated from Withania somnifera (L.) Dunal and their potentials were explored through the in vitro plant regeneration techniques. Initially, indole acetic acid (IAA), phosphate solubilization and siderophore producing isolates were screened out. These isolates were identified using molecular tools like 16s rRNA gene sequences namely Bacillus pumilus AS02, Sphingobacterium thalpophilum AS34, Pseudomonas aeruginosa AS36, Agrobacterium tumefaciens AS38 and Enterobacter aerogenes AS75. Further, the regeneration efficacy of IAA producing endophytic bacteria has been analysed on an endangered plant—Exacum travancoricum Bedd. The leaf and inter-node explants of E. travancoricum with or without bacterial infection were tested on Murashige and Skoog’s (MS) medium encompassing 6-benzyladenine (BA; 2.0 mg L− 1) and l-tryptophan (0.2%; w/v). Somatic embryo induction was observed on both explants. The leaf explants infected with S. thalpophilum AS34, P. aeruginosa AS36 and E. aerogenes AS75 exhibited highest percentage of somatic embryo induction (93.3%) compared with the inter-node explants (86.6%). The explants without bacterial infection (control) and the presence of exogenous IAA exhibited lower level of response (88.8%, leaf and 75.5%, inter-node). Histological analysis revealed the appearance of globular embryogenic masses on epidermal, sub-epidermal regions of both explants. The development of torpedo, cotyledon stage and protoderm formation without the vascular connection from explants confirmed direct somatic embryogenesis. This was confirmed with scanning electron microscopy. Sub-culturing of cotyledon somatic embryos in MS liquid medium without growth regulators was performed to obtain well developed shoots with roots.

Effects Of Different Sterilization Regimes & Growth Regulators On Micropropagation Of Female Date Palm (<i>Phoenix dactylifera</i> L.)

The success of in vitro culture techniques is always hampered by microbial contamination. The present study was carried out to develop an efficient protocol for date palm explants sterilization for successful somatic embryos induction and plantlets formation of some date palm varieties. The shoot tips were treated with different sterilizing agents at different concentrations and durations of exposure. The use of ethanol (70%), sodium hypochlorite (3.5% & 70%) and mercuric chloride (0.2%) with or without addition of Tween-20 had different effects on decontamination of the date palm explants. The percentage of explants contaminated with bacteria for sterilants 1, 2 and 4 was 18.8%, 6.3% and 6.3% respectively while 25%, 37.5%, 31.25% and 6.25% were contaminated with fungi for sterilants 1, 2, 3 and 4 respectively. Under the conditions used, a combination of antioxidants (Citric and Ascorbic acids at 100mg/l), 0.2% mercuric chloride and 3.5% sodium hypochlorite solution with 3 drops/100ml of Tween-20 helped in the reduction of chlorosis, contamination and die-back in the shoot tip explants. The explants were further cultured in appropriate media for callus initiation and subsequent somatic embryo induction. Optimal embryogenic callus was obtained from the shoot explant of sterilant number 4 which had the minimal contamination and die-back of all the cultures. After 3 subcultures, the somatic embryos formed were multiplied for shoot development. From this study, we established that the use of appropriate surface sterilant at suitable concentration and duration of exposure of date palm explant to it is indispensable for maximum responses of in vitro cultures.
 Keywords: Date palm, Microbial contamination, Sterilizing agents, in vitro, Somatic embryos

Indirect somatic embryogenesis and regeneration of Fraxinus mandshurica plants via callus tissue

Somatic embryogenesis of Fraxinus mandshurica has the problems of low somatic embryo (SE) yield, unsynchronized SE development, and a high percentage of deformed SEs. We aimed to improve F. mandshurica SE production by synchronizing SE development, improving SE quality, and inducing root formation to obtain complete regenerated plants. Cotyledons of immature zygotic embryos of F. mandshurica were induced to form callus and then SEs. The SE induction percentage from explants differed among 32 mother trees, and the one with the highest SE induction percentage (29.8%) was used for further experiments. The highest callus induction percentage was 94.2% on ½-strength Murashige and Skoog medium (MS½) supplemented with 0.15 mg·L−1 naphthalene acetic acid. The highest callus proliferation coefficient (240.5) was obtained on McCown’s Woody Plant Medium containing 0.1 mg·L−1 6-benzyl adenine and 0.15 mg·L−1 2, 4-dichlorophenoxyacetic acid. The highest number of SEs (1020.5 g−1 fresh weight) was obtained on MS½ medium supplemented with 1 mg·L−1 6-benzyladenine. The highest number of cotyledon embryos (397/g fresh weight) was obtained by incubating materials on medium containing 1 mg·L−1 abscisic acid and then applying a drying treatment. The cotyledon embryos were milky white, uniformly sized (average length 4.7 mm), and 80% of them were normal. The SE rooting percentage on ½MS medium containing 0.01 mg·L−1 NAA was 37.5%. Overall, the germination percentage of SEs was 26.4%, and complete regenerated plants were obtained after transplanting and acclimation. These results provide more possibilities for the preservation and breeding of F. mandshurica.

High-efficiency somatic embryogenesis techniques for different hybrids of cut lilies

Somatic embryogenesis (SE) is a unique method of in vitro regeneration for plant reproduction, germplasm resource preservation, and molecular-assisted breeding. Although lily is an internationally valued ornamental plant, its SE technology is still incomplete. Here, the effects of different explants, picloram (PIC) concentrations, and light conditions on somatic embryo induction, development, germination and seedling formation were studied in five Lilium varieties belonging to different hybrids. The embryo histomorphology, development and origins were observed, and a broadly efficient embryogenesis system was firstly established in Lilium. With leaf explants, all varieties tested underwent indirect somatic embryogenesis (SE). With scale explants, the Lilium Asiatic ‘Strawberry and Cream’, Lilium Oriental ‘Double Surprise’, and Lilium longiflorum Thunb. ‘White Heaven’ underwent indirect SE and the Lilium longiflorum × Asiatic hybrid ‘Eyeliner’ and Lilium longiflorum × Oriental ‘Triumphator’ underwent direct SE. Somatic embryogenesis in the above varieties involved four stages: globular, heart-shaped, torpedo-shaped, and cotyledon embryos. Two original SE pathways (endogenous and exogenous) were observed, and their appearance times differed. In indirect SE, exogenous embryos appeared first, followed by endogenous embryos; whereas in direct SE, the order was reversed. The different somatic embryo origins and the proportion of internal and external origins were closely related to the occurrence of somatic embryos by direct or indirect pathways and significantly affected the induction cycle of somatic embryos. Dark culture promoted lily somatic embryo induction, while 16 h/day light culture promoted germination, and the germination rates of all five lily varieties exceeded 90%. Murashige Skoog (MS) medium + (1.5–2.5 mg L−1) PIC + 0.2 mg L−1 1-Naphthaleneacetic acid (NAA) was suitable for SE from lily leaves and scales with an induction efficiency of 88–96%. Our research developed a widely applicable regeneration procedure for different hybrids of lily via somatic embryogenesis. Morphohistological analysis confirmed the origins, developmental stages and dynamic changes of cells during in vitro morphogenesis. This study will provide a valuable reference for the propagation of Lilium.

High-efficiency direct somatic embryogenesis and plant regeneration from leaf base explants of “peperina” (Minthostachys verticillata)

In vitro culture has been recognized as a potential plant clonal propagation tool for a broad variety of commercial, ornamental, and medicinal species, with applications in both industrial and academic laboratories. In this study, we describe somatic embryogenesis and plant regeneration protocol for “peperina” plants (Minthostachys verticillata (Griseb.) Epling). In vitro shoots developed via shoot apex extracted from greenhouse-grown plants were cultured on shoot elongation medium (ShM) consisting of Murashige and Skoog (MS) basal salts supplemented with myoinositol, thiamine, and benzyladenine. Shoot apexes were disinfected with 70% ethanol for 5 min and 0.26 sodium hypochlorite (w/v) for 5 min, before the initiation of in vitro culture. For somatic embryo (SE) induction, leaves collected from 2-mo-old in vitro raised shoots were cultured on MS basal medium supplemented with benzyladenine and two different concentrations of coconut water (CW) until SE developed. Using 2.5% CW-supplemented medium, 100% of cultured leaves developed SE and 89.3% of the leaf explants developed plantlets. The resulting embryos germinated on the same medium, and the plantlets obtained were transferred onto ShM until they developed 3 to 4 leaves. To increase the low root development, shoots were transferred into fully hydrated perlite for rooting and then transplanted into soil-perlite containing pots for hardening. This protocol provides the first step to supply the demand and simultaneously protect the natural populations of Minthostachys verticillata from overexploitation. Furthermore, this protocol provides the first step for crop improvement by genetic modification (editing or transgenesis).

Induction of somatic embryogenesis in different explants from Ormosia henryi Prain

An efficient in vitro plant regeneration method via somatic embryogenesis has been established in Ormosia henryi Prain, which is an important precious tree in the timber industry. Five types of explants, six types of basic media and different plant growth regulators (PGRs) were used for embryogenic callus (EC) initiation, EC proliferation, somatic embryo (SE) induction and SE germination. The EC and SE induction rates from mature zygotic embryos (MZE) were higher than those from stem segments, cotyledons, leaves and hypocotyls. MZE were used as explants, and the EC induction rate was greater than 30% on B5 medium supplemented with 1.0 mg/l 6-benzylaminopurine (BA) and 0.5 mg/l naphthaleneacetic acid (NAA) or 0.2 mg/l BA and 2.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D). The highest induction rate (22.83%) for SE was obtained with B5 medium supplemented with 0.5 mg/l kinetin (KT) and 1.0 mg/l 2,4-D. The EC proliferation rate and SE number in the suspension culture were increased compared to those in the solid culture. Fifty-four percent of SE developed to the cotyledonary stage on B5 medium supplemented with 0.5 mg/l BA and 0.2 mg/l NAA. The number of cotyledon embryos was the highest on medium supplemented with 0.5 mg/l thidiazuron (TDZ) and 0.2 mg/l NAA. Histological analysis confirmed the bipolar organization of the somatic embryos, which were comprised of shoot and root meristems, and showed that SE was characterized by globular, heart-shaped, torpedo-shaped and cotyledon stages in O. henryi, the developmental process of which was similar to that of zygotic embryos. An efficient in vitro plant regeneration method via somatic embryogenesis has been established in Ormosia henryi Prain using mature zygotic embryos as explant.

Regulation of cell reprogramming by auxin during somatic embryogenesis.

How somatic cells develop into a whole plant is a central question in plant developmental biology. This powerful ability of plant cells is recognized as their totipotency. Somatic embryogenesis is an excellent example and a good research system for studying plant cell totipotency. However, very little is known about the molecular basis of cell reprogramming from somatic cells to totipotent cells in this process. During somatic embryogenesis from immature zygotic embryos in Arabidopsis, exogenous auxin treatment is required for embryonic callus formation, but removal of exogenous auxin inducing endogenous auxin biosynthesis is essential for somatic embryo (SE) induction. Ectopic expression of specific transcription factor genes, such as "LAFL" and BABY BOOM (BBM), can induce SEs without exogenous growth regulators. Somatic embryogenesis can also be triggered by stress, as well as by disruption of chromatin remodeling, including PRC2-mediated histone methylation, histone deacetylation, and PKL-related chromatin remodeling. It is evident that embryonic identity genes are required and endogenous auxin plays a central role for cell reprogramming during the induction of SEs. Thus, we focus on reviewing the regulation of cell reprogramming for somatic embryogenesis by auxin.