Indirect Somatic Embryogenesis Research Articles

Callus formation response from immature male flower explant of plantain banana (Musa acuminata x Musa balbisiana cv. Kepok) treated by 2,4-D and BAP

Callus induction is an important step in indirect somatic embryogenesis. The aim of this study was to obtain an optimal medium for the callus formation of plantain bananas (cv. Kepok). The research was conducted from May to August 2022 at the Tissue Culture Laboratory of PT. ITCI Kartika Utama, Penajam Paser Utara District, East Kalimantan. The explant used an immature male flower of plantain banana (cv. Kepok). The experiment used a completely randomized design with two factors concentration of 2,4-D (1,2 and 4 ppm) and BAP (5, 10, and 15 ppm). The appearance of callus was monitored daily until it developed explants. On the final observation day, the percentage of explants with callus, as well as the color and texture of the callus, were assessed. The research findings suggest that the optimal medium for inducing callus in male banana flower explants was MS medium supplemented with 2 ppm 2,4-D and 5 ppm BAP. This medium resulted in calluses that exhibited a yellowish-white color and a compact texture. Keywords: callus morphology; genetic improvement; plant growth regulator; tissue culture; triploid banana

Development of indirect somatic embryogenesis and plant regeneration system with immature embryos of the cultivated traditional Chinese medicinal herb Polygonatum cyrtonema

The dried rhizome of Polygonatum cyrtonema represents a highly valued traditional Chinese medicine. With a growing demand, the imperative arises for the establishment of an efficient tissue culture system for large-scale propagation. Here, we successfully developed a tissue culture system capable of yielding P. cyrtonema seedlings through somatic embryogenesis using immature embryos as explants within a 120-day timeframe. An optimum callus induction rate was achieved through a combination of 1.0 mg/L 6-BA and 0.5 mg/L 2,4-D in MS medium, resulting in the formation of numerous highly active callus. Subsequent transfer of primary callus to a medium containing 1.0 mg/L 6-BA and 1.0 mg/L 2,4-D in MS medium for 30 days yielded induction rates of 97.78%, 89.62%, 73.33%, and 66.67% for globular, heart, torpedo, and cotyledonary embryos, respectively. The highest rate of somatic embryo normal germination (73.33%) was observed in the MS+6-BA 2.0 mg/L+NAA 1.0 mg/L medium. Subsequent transfer of germinated somatic embryos to MS+IAA 0.5 mg/L+IBA 1.0 mg/L medium for 20 days resulted in robust tissue culture seedlings, with a survival rate of 93.33% when transplanted into a seedling substrate consisting of soil: perlite (3:1, v/v). This study not only marks the inaugural instance of somatic embryogenesis in Polygonatum species but also paves a foundation for the advent of breeding traditional Chinese medicinal herb P. cyrtonema via recent advanced biotechnological strategies.

Enhancing Clitoria ternatea Propagation Through Indirect Somatic Embryogenesis: Optimal Plant Growth Regulators Type and Concentration for Efficient in Vitro Plant Production

The propagation of Clitoria ternatea poses several challenges, including low germination rates, vulnerability of young seedlings in natural environment, and a slow flowering rate. This study aims to identify the best Plant Growth Regulators and their optimal concentration to stimulate indirect somatic embryogenesis from C. Ternatea leaves explants. The samples were cultured on Murashige and Skoog (MS) medium supplemented with various concentration of thidiazuron (TDZ; 1, 2, 3, 4, and 5 mgL-1 ) and a combination of 2,4-dichloro phenoxy acetic acid (2,4-D; 1, 2, and 3 mgL-1 ) and 0.1 mgL-1 kinetin. The findings revealed that all treatments resulted in the formation of both embryogenic and non embryogenic calluses. The addition of TDZ to the MS medium led to a greater percentage of embryogenic callus formation compared to using the combination of kinetin and 2,4-D. The highest percentage of 84.37% was achieved when samples were cultured on MS medium with 3 mgL-1 TDZ. The most effective induction period of 21 days was observed with the medium supplemented with 3 mgL-1 TDZ, followed by 4 and 5 mgL-1 TDZ, which took 23 days. The research findings suggest that a concentration of 3 mgL-1 TDZ is the optimal PGR type and dosage for achieving rapid and efficient in vitro plant production compared to various concentrations of 2,4-D in combination with kinetin.

Exploring indirect somatic embryogenesis and somaclonal variation for propagation of three Coffea arabica L. cultivars

Exploring indirect somatic embryogenesis and somaclonal variation for propagation of three Coffea arabica L. cultivars

Effect of Picloram and Desiccation on the Somatic Embryogenesis of Lycium barbarum L.

An efficient and reproducible in vitro method for indirect somatic embryogenesis was optimized by culturing leaf and leaf with petiole explants of Lycium barbarum L. Murashige and Skoog (MS) medium, supplemented with various concentrations of Picloram and 2,4-Dichlorophenoxyacetic acid (2,4-D), individually and in combinations, were tested. Picloram (1.0 µM) showed a better response compared to 2,4-D and results indicate it to be a better auxin for induction of somatic embryos for Goji berry. It was seen that the leaf explants were more responsive in callus and somatic embryo induction than the leaf with petiole explant when incubated in the dark for 5 weeks. Embryogenic callus, after being transferred to MS medium containing Benzyl amino purine (BAP) in 1.0 µM, 5.0 µM and 10.0 µM, began to differentiate in light after one week. MS medium with 1.0 µM Picloram + 10 µM BAP resulted as the most favorable treatment for somatic embryogenesis in Lycium barbarum L. Removal of plant growth regulators from MS medium and culturing induced calluses under 16 h photoperiod resulted in globular, heart, torpedo, cotyledons, and further development into plantlets. Well-developed plants have been obtained and are capable of acclimatizing in ex vitro conditions. In addition, the effects of desiccation treatments (0, 1, 3, 6, 9 h, and 12 h) on embryogenic callus for somatic embryo induction were found to be directly proportional to the length of desiccation treatment at room temperature. After 9 h and 12 h of desiccation treatments, 60% and 90% of plated calluses resulted in somatic embryos, respectively. In a L. barbarum callus mass, Acetocarmine and Evans blue double staining differentiated between embryogenic and non-embryogenic callus. These findings will help Goji berry improvement by elite clone production, ex situ conservation projects, scaling up plant production, and agronomy for the commercial production of this superfruit in the future.

Regenerating Saffron (Crocus sativus L.) from Corm Lateral Buds via Indirect Somatic Embryogenesis.

Saffron (Crocus sativus L.), being one of the distinguished commercial spice crops in the world, is in demand for its culinary, colorant, and pharmaceutical benefits. In this study, a novel indirect somatic embryogenesis (SE) system was, thus, established for the study of this plant. To this end, firstly, the lateral buds were cultured. Then, the cultures were transformed using Murashige and Skoog (MS) medium supplemented with either 6-benzyladenine (BA: 5 and 10 mg/L), naphthalene acetic acid (NAA: 0, 1, and 2 mg/L), or trans-zeatin (tZ: 0, 0.5, and 1.0 mg/L), before being classified into four structures: white globular (WG), yellow compact nodular (YCN), yellow-brown fragile (YBF), and dark-brown porous (DBP). As soon as BA (10 mg/L) and NAA (2 mg/L) were added, elevated percentages of white globular calli (56.8%) and white globular calli (31.5%) structures were induced. Additionally, 6-benzyladenine (5 mg/L) and naphthalene acetic acid (1 mg/L) allowed the formation of yellow-brown fragile structures, and the combination of 6-benzyladenine (10 mg/L) with trans-zeatin (1 mg/L) formed the DBP structures. After three months, the white globular calli were incubated using the MS basal medium, before being augmented with thidiazuron (TDZ: 1 mg/L) and picloram (PIC: 2 mg/L), from which 60% of the cases matured into shoots and, ultimately, cormlets. Morphoanatomical analyses also showed that the white globular calli cells were closely arranged, as they had a dense cytoplasm, a significant vascular differentiation, and embryoids. Furthermore, the yellow compact nodular structures were characterized by a strong differentiation capacity and contained many meristematic cells with high caryomitosis centers. We observed that the yellow-brown fragile calli had looser cell arrangements, with a vascular structure located on the protoderm edge, while there was no obvious cellular arrangement in the dark-brown porous structures. The induction of the adventitious buds in vivo on the MS medium that was supplemented with thidiazuron and picloram accordingly demonstrated the highest rates (60%) of white globular calli.

In Vitro Regeneration of Chrysanthemum from Ovaries and Ovules Treated with Thermal and Chemical Stimuli: Morphogenic and Cytogenetic Effects

Chrysanthemum (Chrysanthemum × morifolium (Ramat.) Hemsl.) holds a prominent position in the market of ornamental plants. To further advance chrysanthemum breeding efforts, the development of haploids may be useful. Therefore, the effect of various chemical and thermal treatments on regeneration efficiency and ploidy level in chrysanthemum was studied. Ovaries and ovules of three chrysanthemum cultivars, i.e., ‘Brasil,’ ‘Capitola,’ and ‘Jewel Time Yellow,’ were cultured either on a medium with 1 mg·L−1 2,4-dichlorophenoxyacetic acid (2,4-D) and different concentrations (0.5–1.5 mg·L−1) of thidiazuron (TDZ) or subjected to thermal shock (pretreatment temperature of 4 °C or 32 °C) and cultured on a medium with 1 mg·L−1 2,4-D and 1 mg·L−1 6-benzylaminopurine (BAP). It was found that ovaries had a greater organogenic potential (both in terms of callogenesis and shoot formation) than ovules. Microscopic analyses revealed that shoots mainly developed via indirect somatic embryogenesis from a callus developed from the ovary wall. The highest number of shoots was produced in cooled (at 4 °C) ovaries of chrysanthemum ‘Brasil’ and in ‘Jewel Time Yellow’ ovaries cultured on a medium with 1.0–1.5 mg·L−1 TDZ. The latter cultivar also had the highest potential to produce plants with an altered ploidy level (doubled and halved the number of chromosomes). This study demonstrates that manipulating factors such as temperature and thidiazuron concentration can enhance regeneration efficiency and induce altered ploidy levels in selected cultivars, offering valuable insights for chrysanthemum breeding programs.

Callus Type, Growth Regulators, and Phytagel on Indirect Somatic Embryogenesis of Coffee (Coffea arabica L. var. Colombia).

Coffee is a crop of global relevance. Indirect somatic embryogenesis has allowed plants of different coffee genotypes to be massively regenerated. The culture medium composition can affect the calli characteristics that are generated and their ability to form somatic embryos. This research aimed to determine the influence of the type of callus, growth regulators, and phytagel concentration on the embryogenic capacity of the Colombia variety. Leaf explants were cultured on Murashige and Skoog medium with 2,4-dichlorophenoxyacetic acid (2,4-D) (0.5-1.0 mg L-1), benzylaminopurine (BAP, 1.0 mg L-1), and phytagel (2.3-5.0 g L-1). The explants generated two types of calli: friable (beige, soft, watery, easy disintegration, polyhedral parenchyma cells) and compact (white, hard, low water content, difficult disintegration, elongated parenchyma cells). About 68% of the total callus generated was compact; this type of callus produced a greater number of embryos (71.3) than the friable one (29.2). The number of differentiated embryos was significantly affected by the concentration of phytagel; higher concentrations (5.0 g L-1) resulted in larger quantities (73.7). The highest number of embryos (127.47) was obtained by combining 1.0 mg L-1 2,4-D, 1.0 mg L-1 BAP, 5.0 g L-1 phytagel, and compact callus.

In Vitro Conversion of Coffea spp. Somatic Embryos in SETIS™ Bioreactor System.

Somatic embryogenesis (SE) is an excellent example of mass plant propagation. Due to its genetic variability and low somaclonal variation, coffee SE has become a model for in vitro propagation of woody species, as well as for large-scale production of vigorous plants that are advantageous to modern agriculture. The success of the large-scale propagation of an embryogenic system is dependent on the development, optimization, and transfer of complementary system technologies. In this study, two successful SE systems were combined with a SETIS™ bioreactor immersion system to develop an efficient and cost-effective approach for the in vitro development of somatic embryos of Coffea spp. This study used an efficient protocol for obtaining somatic embryos, utilizing direct and indirect SE for both C. canephora and C. arabica. Embryos in the cotyledonary stage were deposited in a bioreactor to complete their stage of development from embryo to plant with minimal manipulation. Following ten weeks of cultivation in the bioreactor, complete and vigorous plants were obtained. Different parameters such as fresh weight, length, number of leaves, and root length, as well as stomatal index and relative water content, were recorded. In addition, the survival rate and ex vitro development of plantlets during acclimatization was assessed. The best substrate combination was garden soil (GS), peat moss (PM), and agrolite (A) in a 1:1:0.5 ratio, in which the bioreactor-regenerated plants showed an acclimatization rate greater than 90%. This is the first report on the use of SETIS™ bioreactors for the in vitro development of somatic embryos in Coffea spp., providing a technology that could be utilized for the commercial in vitro propagation of coffee plants. A link between research and innovation is necessary to establish means of communication that facilitate technology transfer. This protocol can serve as a basis for the generation and scaling of different species of agroeconomic importance. However, other bottlenecks in the production chains and the field must be addressed.

Primary Metabolite Screening Shows Significant Differences between Embryogenic and Non-Embryogenic Callus of Tamarillo (Solanum betaceum Cav.).

Tamarillo is a solanaceous tree that has been extensively studied in terms of in vitro clonal propagation, namely somatic embryogenesis. In this work, a protocol of indirect somatic embryogenesis was applied to obtain embryogenic and non-embryogenic callus from leaf segments. Nuclear magnetic resonance spectroscopy was used to analyze the primary metabolome of these distinct calli to elucidate possible differentiation mechanisms from the common genetic background callus. Standard multivariate analysis methods were then applied, and were complemented by univariate statistical methods to identify differentially expressed primary metabolites and related metabolic pathways. The results showed carbohydrate and lipid metabolism to be the most relevant in all the calli assayed, with most discriminant metabolites being fructose, glucose and to a lesser extent choline. The glycolytic rate was higher in embryogenic calli, which shows, overall, a higher rate of sugar catabolism and a different profile of phospholipids with a choline/ethanolamine analysis. In general, our results show that a distinct primary metabolome between embryogenic and non-embryogenic calli occurs and that intracellular levels of fructose and sucrose and the glucose to sucrose ratio seem to be good candidates as biochemical biomarkers of embryogenic competence.

An Overview: Somatic Embryogenesis Through Thin Cell Layer (TCL) Technique

Somatic embryogenesis (SE) has been described as the most efficient pathway to obtaining plants. Somatic embryogenesis can be divided into direct and indirect embryogenesis. Direct SE occurs when embryos are started directly from explant tissues (preembryogenic cells) creating an identical clone, while indirect SE occurs from unorganized tissues (calli) which are further developed into embryos. While different explants have been tested, e.g., shoot tips, adventitious buds, leaf primordia, zygotic embryos, inflorescences, and transverse thin cell layers (TCLs) (consisting of a few cell layers, usually 0.5–2.0 mm thick). TCLs have been excised from different plant organs and successfully used as explants for SE. Thin cell layers (TCLs) can be prepared from almost any plant organ. These, when cut longitudinally, are referred to as lTCLs, and when cut transversally, are referred to as tTCLs. This article provides an overview of the concept of TCL as applied to induce SE in different plant species and remarks the factor that affecting this technique. This review will certainly revitalize this important technology so that it can be used effectively for successful mass propagation.

Plant regeneration from indirect somatic embryogenesis of Agave salmiana otto ex Salm-Dyck subsp. salmiana using zygotic embryo obtained by in-casa pollination as explants

Plant regeneration from indirect somatic embryogenesis of Agave salmiana otto ex Salm-Dyck subsp. salmiana using zygotic embryo obtained by in-casa pollination as explants

Protocol for plant regeneration from encapsulated somatic embryos of Citrus sinensis L.

BackgroundCitrus is an important fruit crop worldwide; artificial seeds are through encapsulation techniques of somatic embryos having many applications such as large-scale propagation and germplasm conservation. The aim of this study is the investigation of the viability of encapsulated citrus somatic embryos after different storage periods and to convert them into plantlets.ResultsCotyledonary-stage somatic embryos (5–7-mm size) regenerated from stigma explants of Washington navel orange (Citrus sinensis L.) were encapsulated individually in 3% sodium alginate. After different preservation periods (1, 2 or 3 months) at 10 °C, the encapsulated somatic embryos were cultured on Murashige and Skooge (MS) medium solidified with 7 g/l agar and supplemented with 50 g/l sucrose for germination. Percentage of regrowth, germination percentage, number of plantlets, plantlets height and number of leaves/shoot were recorded after different recovery periods. The germination percentages of encapsulated embryos were 90, 62.5 and 40% with storage for one, two and three months, respectively. Encapsulated somatic embryos preserved for 1 month developed the highest number of plantlets, while those preserved for 2 months developed the highest length of plantlets and the highest number of leaves/shoot after a recovery period of 10 months. Molecular analysis was performed of plantlets recovered from somatic embryos after preservation by encapsulation, and the results showed that the percentages of polymorphism were 7.7% with the two primers in all treatments.ConclusionsIt could be concluded that callus developed from stigma explants was able to regenerate indirect somatic embryogenesis after 3 months. Cotyledonary-stage somatic embryos of citrus were successfully preserved by encapsulation using 3% sodium alginate. Frequencies of germination of encapsulated somatic embryos increased with increasing the recovery period on the germination medium. A maximum recovery frequency of 60.8% was obtained from encapsulated somatic embryos cultured on germination medium for up to 10 months. Also, recovery frequency of 62.5% was noticed from encapsulated somatic embryos preserved for 2 months at 10 °C. In addition, results indicated that recovered somatic embryos obtained from encapsulated somatic embryos were able to convert to normal plantlets.

Embryonic abnormalities and genotoxicity induced by 2,4-dichlorophenoxyacetic acid during indirect somatic embryogenesis in Coffea

Indirect somatic embryogenesis (ISE) is a morphogenetic pathway in which somatic cells form callus and, later, somatic embryos (SE). 2,4-dichlorophenoxyacetic acid (2,4-D) is a synthetic auxin that promotes the proliferation and dedifferentiation of somatic cells, inducing the ISE. However, 2,4-D can cause genetic, epigenetic, physiological and morphological disorders, preventing the regeneration and/or resulting abnormal somatic embryos (ASE). We aimed to evaluate the toxic 2,4-D effect during the Coffea arabica and C. canephora ISE, assessing the SE morphology, global 5-methylcytosine levels (5-mC%) and DNA damage. Leaf explants were inoculated in media with different 2,4-D concentrations. After 90 days, the friable calli were transferred to the regeneration medium, and the number of normal and abnormal SE was monthly counted. The increase of the 2,4-D concentration increased the number of responsive explants in both Coffea. At 9.06, 18.08 and 36.24 μM 2,4-D, C. arabica presented the highest values of responsive explants, differing from C. canephora. Normal and abnormal SE regeneration increased in relation to the time and 2,4-D concentration. Global 5-mC% varied at different stages of the ISE in both Coffea. Furthermore, the 2,4-D concentration positively correlated with global 5-mC%, and with the mean number of ASE. All ASE of C. arabica and C. canephora exhibited DNA damage and showed higher global 5-mC%. The allotetraploid C. arabica exhibited greater tolerance to the toxic effect of 2,4-D than the diploid C. canephora. We conclude that synthetic 2,4-D auxin promotes genotoxic and phytotoxic disorders and promotes epigenetic changes during Coffea ISE.

Cytological studies on unicellular origin pathway, early ordered cell division patterns and morphogenesis in indirect somatic embryogenesis of catharanthus roseus cell lines

Cytological studies on unicellular origin pathway, early ordered cell division patterns and morphogenesis in indirect somatic embryogenesis of catharanthus roseus cell lines

Prediction of the concentration of plant growth regulators for somatic embryogenesis and regeneration of Hyoscyamus niger using Box–Behnken design of response surface methodology

Optimizing of the in vitro culture condition, somatic embryogenesis, and plant regeneration are the first step in transformation. In this study, response surface methodology was used to optimize and predict the concentrations of TDZ, NAA, BAP, and kinetin for callus formation, and regeneration by indirect somatic embryogenesis of Hyoscyamus niger. The results showed that TDZ was more efficient than NAA for the regeneration of H. niger. By using different concentrations of TDZ, BAP, and kinetin, the petiole explants produced the highest percentage of callusing (100%) on MS medium containing 0.25 mg/L TDZ and 1 mg/L BAP, percentage of somatic embryogenesis (92.66%) with addition 0.5 mg/L TDZ, and the number of the shoots (7.75 per callus) with addition 0.25 mg/L TDZ and 1 mg/L BAP. The leaf explants produced the highest callus fresh weight (1207.75 mg/explant) by using 0.5 mg/L TDZ and 1 mg/L kinetin, and percentage of shooting (93.30%) by using 0.50 mg/L TDZ and 1 mg/L kinetin. Also, the highest percentage of callusing (100%), callus fresh weight (3273.64 mg/explant), percentage of somatic embryogenesis (100%), shooting (100%), and the number of the shoots (8.61 per callus) were predicted by culturing of petiole explant on the MS medium containing 0.11 mg/L TDZ and 1.253 mg/L BAP, leaf explant by using 0.52 mg/L TDZ, petiole explant by using 0.533 mg/L TDZ and 1 mg/L BAP, leaf explant with addition 0.18 mg/L TDZ and 2.89 mg/L BAP and petiole explant by using 0.1 mg/L TDZ and 3 mg/L BAP. Optimizing and predicting of the effect of plant growth regulators on henbane regeneration.

Regeneration of Transgenic Ficus lyrata via Indirect Somatic Embryogenesis and Isolation of Variants for Development of New Cultivars

Ficus lyrata is a popular ornamental foliage plant with unique violin- or guitar-shaped green leaves. In our previous study, a grapevine gene VvMybA1 was introduced into F. lyrata via Agrobacterium-mediated transformation, which resulted in the availability of purple-leaved plants. Since VvMybA1 is a transcription factor, the regeneration of transgenic purple-leaved plants might potentially produce variants with multicolored leaves. The objective of this study was to establish a method for regenerating purple-leaved F. lyrata and determine if variants with different coloration or variegation could be isolated from regenerated populations. Leaf explants derived from a completely purple-leaved transgenic plant were cultured on Murashige and Skoog (MS) basal medium supplemented with different concentrations of 6-benzyladenine (BA) and α-naphthalene acetic acid (NAA). Callogenesis occurred in leaf explants, and a subculture of callus-borne explants on the same medium resulted in callus proliferation and the occurrence of somatic embryos. Somatic embryos were more effectively induced from callus pieces cultured on MS medium supplemented with 8.88 μM of BA and 0.27 μM of NAA. More than 30 embryos were induced per callus piece, and the embryos matured and converted to plantlets. MS medium supplemented with 4.92 μM of indolyl-3-butanoic acid (IBA) greatly improved root development. Plantlets were transplanted into soilless substrate and grown in a shaded greenhouse for morphological evaluation. Nine variants with different degrees of coloration and variegation were isolated from the regenerated populations. Our results suggest that the regeneration of transgenic plants that harbor a transcription factor, such as VvMybA1, could be an additional way of isolating novel variants for the development of new cultivars of ornamental plants.

Somatic embryogenesis as potential method for commercial propagation in Passiflora edulis Sims f. edulis - an important horticultural crop

Purple passion fruit (Passiflora edulis Sims f. edulis) is one of the species with high commercial value in the genus Passiflora. In this study, the somatic embryogenesis (SEs) obtained from different explants (ex vitro leaf and in vitro root) cultured on medium-supplemented auxins and cytokinins, silver nanoparticles (AgNPs) enhanced SEs and plantlets derived from SEs were evaluated for the first time. The ex vitro leaf blade (1 × 1 cm) and in vitro root fragment (0.1 × 1 cm) were used as the initial explants. The results showed that the addition of auxins and cytokinins in culture medium stimulated SEs in both explant types. Root explant only induced callus on medium supplemented with 2,4-D (1.0 - 4.0 mg L−1), however, somatic embryos were directly induced with 1.0 mg L−1 NAA. While 1.0 - 2.0 mg L−1 2,4-D stimulated embryogenic callus induction (14.81 - 25.93%), NAA stimulated direct embryogenesis from leaf explants. The medium supplemented with 2.0 mg L−1 NAA combined with 1.5 mg L−1 TDZ significantly improved the SEs rate (81.48%) and number of embryos per explant (26.67 embryos) formed directly from leaf explants. On the other hand, the synergism between 2.0 mg L−1 2,4-D and 2.0 mg L−1 AgNPs significantly enhanced callus-derived embryogenesis from leaf explant with the highest SEs rate (92.59%) and number of embryos per explant (31.67 embryos). In addition, AgNPs also had a positive effect on promoting the maturation of callus-derived embryos. The plantlets derived from SEs were acclimatized in the greenhouse with a survival rate of over 90.00%. The SEs-derived plants flowered and produced fruit normally under field conditions. The results revealed a reliable plant regeneration procedure through direct or indirect SEs derived from ex vitro leaf and in vitro root explants and AgNPs-stimulated SEs that can be applied for commercial propagation of this crop.

In Vitro Mass Propagation of Coffee Plants (Coffea arabica L. var. Colombia) through Indirect Somatic Embryogenesis

Coffea arabica is one of the two most consumed coffee species in the world. Micropropagation through somatic embryogenesis has allowed the large-scale propagation of different coffee varieties. However, the regeneration of plants using this technique depends on the genotype. This study aimed to develop a protocol for the regeneration of C. arabica L. var. Colombia by somatic embryogenesis for its mass propagation. Foliar explants were cultured on Murashige and Skoog (MS) supplemented with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzylaminopurine (BAP), and phytagel for inducing somatic embryogenesis. In total, 90% of the explants formed embryogenic calli with a culture medium containing 2 mg L-1 of 2,4-D, 0.2 mg L-1 BAP, and 2.3 g L-1 phytagel. The highest number of embryos per gram of callus (118.74) was obtained in a culture medium containing 0.5 mg L-1 2,4-D, 1.1 mg L-1 BAP, and 5.0 g L-1 phytagel. In total, 51% of the globular embryos reached the cotyledonary stage when they were cultured on the growth medium. This medium contained 0.25 mg L-1 BAP, 0.25 mg L-1 indoleacetic acid (IAA), and 5.0 g L-1 of phytagel. The mixture of vermiculite:perlite (3:1) allowed 21% of embryos to become plants.

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.