Number Of Somatic Embryos Research Articles

Optimization of crop tissue culture technology and its impact on biomolecular characteristics

Modern agriculture relies on crop tissue culture technology for fast propagation, genetic improvement, and protection of plant species. Conventional media like Murashige and Skoog (MS) usually lack optimal conditions for embryogenesis, necessitating the development of improved media tailored to specific crop requirements. In this article, we introduce an Efficient Grid Identified-Deep Feedforward Neurons (EGI-DFFN) to identify the ideal nutrient and vitamin levels of the crop plants for improving crop tissue culture aimed to improve crop plant growth in a lab setting by predicting callogenesis rate (CGR), embryogenesis rate (EGR), and somatic embryo number (SEN), shoot regeneration rate (SRR), rooting rate (RR).Different concentrations of ionic macronutrients, bio-molecular, and vitamins of the crop plant are the input to the predictive model, which is collected through the laboratory Callus Induction Experiment (CIE). Z-score normalization is used to preprocessing the CIE data to ensure consistent scales across different input features and improve model training performance. DFFN used discriminates to predict complex relationships and interactions between CGR, EGR, SEN, SRR, and RR with EGI tuning. The EGI-DFFN model has significantly improved crop tissue culture growth by accurately predicting the CGR, EGR, SEN, SRR, and RR respectively. The EGI-DFFN model enhances understanding of how ionic macronutrients and vitamins impact plant growth. It identifies optimal concentrations of the biomolecular to enhance somatic embryo formation and plantlet development, providing insights for optimizing crop tissue culture conditions for optimal growth outcomes.

Genotypic effects on accelerated propagation of oil palm breeding materials selected (Elaeis guineensis jacq.) using somatic embryogenesis

Vegetable oil production from oil palm (Elaeis guineensis Jacq.) is an important industry due to the rising demand every year. The somatic embryogenesis culture can propagate oil palm duplicate as parent plant, which can be selected as breeding material to produce new planting germplasm with high production or disease resistance. This study aims to evaluate the genotypic effect of somatic embryogenesis, while immature leaflets were employed as explants. The culture used embryo induction medium based on Murashige and Skoog (MS) modifications that contained 5 ​mg/L Naphthalene Acetic acid (NAA) and 0.5 ​mg/L Benzyl Amino Purine (BAP). The genotypic effect was statistically significant in the percentage of callus induction, producing somatic embryos, and germination embryos. In this study, we successfully cloned thirteen oil palm genotypes (GE-02, GE-03, GE-06, GE-07, GE-09, GE-23, GE-24, GE-27, GE-28, GE-32, GE-33, GE-34, and GE-35), with the highest number of somatic embryos formed on GE-27 with a percentage of 70.1%. The cloning was successful in accelerating the propagation of oil palm for materials breeding programs to create new varieties with high production and disease resistance. It is necessary to observation the performance of these clones in the field in terms of mantle flower appearance.

Suplementação do silício aumenta a regeneração in vitro de embriões somáticos de palma de óleo (Elaeis guineensis Jacq.)

ABSTRACT The use of silicates in plant tissue culture has resulted in positive effects regarding the morphological, anatomical and physiological characteristics of in vitro plants. However, biological responses to this mineral are dose and genotype dependent. The objective of the present study was to evaluate the influence of silicon supplementation on the somatic embryogenesis of oil palm (Elaeis guineensis Jacq.). The study was carried out in three stages of the cloning process, namely: Regeneration (i) and germination (ii) of somatic embryos and during ex vitro acclimatization (iii) of regenerated plantlets. The culture media were supplemented with four doses (0.0, 0.5, 1.0 and 1.5 g L-1) of calcium silicate (CaSiO3). The resulting plantlets were transplanted and acclimatized in a greenhouse. The regression analysis showed that the dose of 0.63g L-1 of CaSiO3 induced a higher rate of regeneration, which would allow a 58% increase in the number of somatic embryos formed. However, the germination of the plantlets, as well as their morphometric characters, were not affected by the different doses of CaSiO3, although the presence of this compound promoted an increase in the thickness of the epidermis and a reduction in the amount of epicuticular wax. Overall, silicon does contribute to the cloning of E. guineensis.

Somatic Embryogenesis and Plantlet Regeneration from Immature Embryos of Papaya (Carica papaya L.)

The experiment was carried out in the Laboratory of the Biotechnology Division, Bangladesh Agricultural Research Institute (BARI) to find out embryogenic responses and plantlets regeneration from immature embryos of papaya. The explants were inoculated on Murashige and Skoog (MS) medium supplemented with 0, 36, 45, 54 & 63 μΜ 2, 4-D and incubated in complete darkness at 25°C ± 1°C. Calli were obtained within 3 weeks of culture period. The highest percentage of explants (46.67) produced callus using 2, 4-D at a concentration of 45 μΜ. Calli were then transferred to somatic embryogenesis medium where embryogenic calli were developed, turned into greenish color and these somatic embryos were noticed within 10 days. The highest number of somatic embryos (86.33) was produced per explant on MS containing 54 μΜ 2, 4-D. Somatic embryos were regenerated on MS supplemented with different concentrations and combinations of BAP and NAA including GA3. The maximum regeneration percentage of somatic embryos (intact rooted plantlets) (54.17) was recorded in the medium supplemented with 0.4 μΜ each of BAP and NAA in combination with 10 μΜ GA3. The somatic embryo-derived plantlets were successfully established in small plastic pots with prepared soil and finally transplanted in the field. The plants were grown well and produced fruit under their natural environment. Plant Tissue Cult. & Biotech. 33(2): 97-106, 2023 (December)

Genetic diversity assessment and in vitro propagation of some date palm (Phoenix dactylifera L.) varieties

The evaluation of genetic diversity is crucial for breeders to develop strategies and improve the resilience, quality, and adaptability of the date palm. In this study, the genetic diversity of three date palm varieties was performed using ISSR-PCR molecular markers to determine its relationship with in vitro propagation response of these varieties. The molecular profiling was performed using ISSR-PCR. A total of 49 loci were produced by the PCR reactions, 38 of which were polymorphic while 11 were monomorphic. The level of polymorphism revealed by ISSR-PCR varied from 33.33% to 100%. The three date palm varieties were grouped into two clusters based on the results of cluster analyses that used morphological data and molecular profiles. Cluster I comprised the ‘Barhy’ variety and Cluster II included ‘Magdoul’ and ‘Amri’ varieties. The clustering analyses revealed the independence of the ‘Barhy’ variety in its characteristics from the other varieties based on either morphological or molecular data. The results of in vitro propagation showed that the ‘Amri’ variety exhibited the highest callus induction frequency (86.28%), callus weight (2.33 g), number of somatic embryos (9.32), number of shoots (14.62), number of roots (4.11), root length (4.63 cm), shoot length (13.61 cm) followed by ‘Magdoul’ and ‘Barhy’ varieties. The ‘Amri’ variety had the shortest callus induction period, at 23.26 days while the ‘Barhy’ variety exhibited the longest period of callus induction (28.55). It was deduced from the study that the ISSR marker reproduced trustworthy patterns of bands to determine the genetic diversity among different date palm varieties that are considered the cornerstone for the genetic improvement of date palms. The understanding of the relationship between genetic diversity and in vitro propagation response of date palm is essential for ensuring the long-term sustainability of its crop. This will facilitate better conservation and development of new date palm varieties that fulfil the needs of farmers and consumers.

Somatic Embryogenesis and Plant Regeneration of Farmer-Preferred Passion Fruit Varieties Grown in Kenya

In vitro regeneration of passion fruit has great prospects for mass production of disease-free planting materials. The objective of this study was to develop an in vitro regeneration system through somatic embryogenesis for farmer-preferred genotypes grown in Kenya. Callus induction and somatic embryogenesis were carried out using leaf and immature seed explants. The explants were cultured on Murashige and Skoog medium augmented with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) alone and 2,4-D combined with 1.0 mg L−1 thidiazuron (TDZ). Data were recorded and subjected to analysis of variance. The highest number of somatic embryos was obtained from KPF4 genotype using MS medium supplemented with 8 mg L−1 2,4-D and 1 mgL−1 TDZ. The embryos were converted to plants on germination medium comprising of MS augmented with 0.5 mg L−1 6-benzyl amino purine (BAP). The plantlets were hardened for 4 weeks in plastic pots. The survival rate during hardening of in vitro regenerated plants was 77.8%. The present study reports a regeneration system through somatic embryogenesis for KPF4 passion fruit grown in Kenya. The in vitro regeneration system can be utilized for mass propagation and genetic improvement of KPF4 variety grown in Kenya.

Development of a system for the formation of transgenic somatic embryos in the liquid medium in Medicago truncatula

Somatic embryogenesis is the formation of embryos from plant’s somatic cells. It is widely used in biotechnology for reproduction of plants, studying of regeneration process and it also represents a convenient way to obtain transgenic plants. Currently, a solid medium is usually used for the formation of transgenic somatic embryos, which has a number of disadvantages.
 We are developing a system for cultivating explants in a liquid medium for the transformation and formation of somatic embryos forMedicago truncatula. Unlike a solid medium, it should allow using petioles as explants, simplify the renewal of the medium, replace disposable cultivation containers with reusable ones, and also reduce the time required for the formation of somatic embryos.
 Currently, the optimal concentration of hygromycin as a selective agent in such a system was found. Interestingly, it appeared to be lower than the selective hygromycin concentration in a solid medium.
 The addition of cefotaxime to the medium reduces the number of somatic embryos formed, but does not completely suppress their formation. Thus, cefotaxime can be used to eliminate agrobacteria during transformation using this cultivation system.
 Embryos withGUSoverexpression transformed with this method were successfully obtained.

The search for inhibitors of somatic embryogenesis in Medicago truncatula

The success of protocols for the genetic transformation of legumes is limited by their low ability to regenerate. Plant regeneration can occur both along the path of shoot regeneration and their further rooting, and along the path of somatic embryogenesis (SE). SE is similar to zygotic embryogenesis (ZE). It is a method of asexual reproduction, in which the somatic cell, due to its totipotency, switches on the embryogenesis program.
 SE and ZE involve common participants in transcriptional, hormonal, and epigenetic control. Like many processes in the plant organism, SE is controlled by the activity of various stimulants or repressors.
 As a result of transcriptional analysis of embryogenic and non-embryogenic calli ofMedicago truncatulaat different stages of development, putative genes-inhibitors of SE were found. Using the Golden Gate system, we created vectors for agrobacterial transformation to overexpress genes of interest in embryogenic calli and evaluate their effect on SE. Overexpression of two genes encoding the transcription factors from WRKY and Homobox-WOX families had a significant inhibitory effect on SE (the average number of somatic embryos per callus decreased).
 TheMtCLE16,a previously found SE inhibitor from the CLE peptides group, was edited and heterozygous frameshift mutants were obtained. The impact of loss of its function on the SE capacity remains to be analyzed.
 This research was supported by the Sirius University of Science and Technology, project PBB-RND-2243.

Germination of Cotyledonary Somatic Embryos into Plantlets on Coffea arabica L. of AS2K clone

Andung Sari 2 Klon (AS2K) is an Indonesian superior clone of Arabica coffee (Coffea arabica L.). Coffee plants require two years to complete their life cycle, resulting in genetic development by conventional breeding which is a significant challenge because it takes at least twenty years to produce new varieties that are ready to be marketed. Conventional coffee propagation via shoot cutting is detrimental to the strength and resistance of C. arabica L. cutting roots. Plants lack a taproot, allowing them to collapse easily, and they are frequently attacked by nematodes during the early stages of their growth. Somatic embryogenesis (SE) propagation was possible to produce an excessive number of somatic embryos for the development of plantlets during the availability of elite clones in a field experiment. The purpose of this study was to improve the plant conversion of SE by assessing the effect of different plant growth regulators (PGRs) on plantlet regeneration in AS2K clone Arabica coffee. Cotyledonary embryos were transferred to plantlet regeneration medium, which consisted of a half-strength MS basal medium containing 1 mg/L IBA supplemented with different concentrations (1.0, 3.0, 5.0 mg/l) BAP and (0.5, 1.0, 1.5 mg/l) GA3. Plantlet regeneration was established by using combination of 3 mg/L BAP and 0.5 mg/L GA3 as well as combination of 3 mg/L BAP and 1 mg/L GA3.

Effect of spermidine, glutamine, and proline on somatic embryogenesis and silver nanoparticles supplied culture improved rhizome formation of Panax vietnamensis var. langbianensis

Lang Bian ginseng (Panax vietnamensis var. langbianensis) is a rare and traditional herbal medicine with many pharmacological activities. It was first discovered by Duy et al. (2016) in Lam Dong province, Vietnam. Until now, there has been only one publication on Lang Bian ginseng micropropagation via somatic embryogenesis (Anh et al., 2022). However, this study still had limitations, including the small number of somatic embryos and the time-consuming. This study investigated the effect of spermidine, glutamine, and proline on the enhancement of somatic embryogenesis (SE) of Lang Bian ginseng using thin cell layer culture technology. The results showed that the optimal SE rate and the number of somatic embryos per explant were achieved on MS medium supplemented with 0.01 mM spermidine in leaf explants (93.32 % and 54.20 embryos, respectively) and petiole explants (96.66 % and 68.80 embryos, respectively) and higher compared to these in other treatments, including control, glutamine, and proline treatments. Besides, adding 0.01 mM spermidine to the culture medium also improved the quality of somatic embryos, mainly cotyledonary stage, through an enhanced synthesis of antioxidant enzymes [catalase (CAT) and superoxide dismutase (SOD), and ascorbate peroxidase (APX)]. In addition, the fluctuations of endogenous hormones during initiation and maturation of SE derived from 0.01 mM spermidine treatment were recorded. Endogenous CKs (ZEA, 2iP, KIN, mT), IAA, and GA3 concentrations were the highest during the induction stage in both explants. Meanwhile, the remaining endogenous hormones (MEL, ABA, and SA) exhibited no inevitable fluctuation trends. In addition, IAA was only detected at the induction stage of SE in both explants. Moreover, cotyledonary somatic embryos derived from 0.01 mM spermidine treatment grew well in MS medium supplemented with 1.2 mg/L AgNPs.

The Effects of Different Auxin–Cytokinin Combinations on Morphogenesis of Fritillaria meleagris Using Bulb Scale Sections In Vitro

Fritillaria meleagris is a horticulturally and medicinally valuable bulbous plant that requires a period of low temperatures for proper growth and flowering. Since conventional methods of propagation are ineffective and very slow, tissue culture techniques offer an integrated approach to mass production of this valuable geophyte. In this study, we investigated the effects of various auxin–cytokinin combinations on different morphogenetic pathways in bulb scale culture. Bulbs obtained in vitro were cut longitudinally, and bulb scales were cultured for four weeks at 7 °C on MS medium supplemented with 6-benzylaminopurine (BAP) in combination with 2,4-dichlorophenoxyacetic acid (2,4-D) or α-naphthaleneacetic acid (NAA) at different concentrations in order to investigate the influence of plant growth regulators (PGRs) on different morphogenetic responses. Regeneration percentage, number of shoots per explant, shoot length, number of bulbs and number of somatic embryos were monitored weekly. After chilling, bulb scales were transferred to 24 °C, and all parameters were recorded again. Low PGR concentrations were very effective for shoot multiplication, yielding up to 5.5 shoots per explant. 2,4-D (at 2 mg/L) in combination with low BAP (0.25 mg/L) produced the highest number of bulbs (11.00 ± 0.00), while PGR-free medium was extremely effective in somatic embryo formation (13.50 ± 2.90). Detached somatic embryos and bulblets continued to grow and develop on fresh PGR-free medium. We present data demonstrating that low auxin–cytokinin concentrations and PGR-free medium provide an effective method for a combined morphogenetic pathway in F. meleagris that is suitable for large-scale propagation.

In Vitro Micropropagation of Commercial Ginseng Cultivars (Panax ginseng Meyer) via Somatic Embryogenesis Compared to Traditional Seed Production

Panax ginseng Meyer is an important medicinal crop; however, most ginseng farmers cultivate native species that are not genetically fixed. Although several cultivars have been developed in Korea, distribution to farmers remains insufficient given their low propagation characteristics. This study compared the efficiency of seed production and micropropagation via somatic embryogenesis. Seeds were collected from cultivars, and zygotic embryo-derived explants were inoculated and cultured in a series of media for micropropagation. Seed production and characteristics of commercial cultivars were evaluated. The number of seeds from a 4-year-old individual cultivar was 23.1–58.8, and seed characteristics varied with cultivars. The genotype had a notable effect on somatic embryogenesis and plant regeneration. The number of somatic embryos and shoots obtained from a single seed of cultivars was 71.3–100.2 and 50.7–61.3, respectively. The number of in vitro grown roots (IGRs) per a single seed was 37.1–41.1 in one year. IGRs were successfully acclimatized and sprouted normally in the field. Flow cytometry analysis suggested that micropropagated plants had no ploidy variations. The results demonstrated the utility of somatic embryogenesis in the in vitro micropropagation of P. ginseng cultivars. Our findings can enhance the distribution of cultivars among farmers in the future.

In Vitro Propagation of Three Date Palm (Phoenix dactylifera L.) Varieties Using Immature Female Inflorescences.

Immature female inflorescences are promising materials for use as explants for the tissue culture of date palm. Four types of MS media were used in this study during the four micropropagation stages-starting media (SM), maturation media (MM), multiplication media (PM) and rooting media (RM)-to micropropagate three elite date palm varieties, Amri, Magdoul and Barhy using the immature female inflorescences as explant. The highest percentage of callus induction in all the varieties studied was obtained on the SM1 (9 µM 2,4-D + 5.7 µM IAA + 10 µM NAA). Culturing on the MM1 (4.5 µM 2,4-D + 9.8 µM 2-iP + 1.5 AC) allowed us to obtain the best value in terms of callus weight. After culturing on the PM1 (4.4 µM BA + 9.8 µM 2-iP) produced the highest numbers of somatic embryos and shoots. The explants on RM2 (0.5 µM NAA + 1.25 µM IBA + 3 g AC) showed the highest root numbers and root lengths, while the highest shoot length was achieved on RM3 (0.5 µM NAA + 0.5 µM IBA + 3 g AC). The Amri variety presented the best response among the three varieties in all parameters, followed by the Magdoul and Barhy varieties. In all the stages of micropropagation, the analysis of variance revealed highly significant variations among varieties and culture media, and a significant difference in the number of roots during the rooting stage. The results also showed non-significant differences in the interaction between varieties and culture media, except for shoot length in the rooting stage. The results also reveal the broad sense heritability ranging from low to high for the measured parameters. It can be concluded that the immature female inflorescences can be used as a productive explant source for successful date palm micropropagation using the SM1, MM1, PM1 and RM2 culture media. It can also be concluded that the success of date palm micropropagation not only depends on the concentrations of growth regulators, but also on their types.

In vitro callogenesis essays to induce somatic embryogenesis of the Tunisian chickpea

The present study was the first report on the somatic embryogenesis of the Tunisian chickpea (Cicer arietinum L.) particularly supposed to be recalcitrant and difficult to manipulate in vitro. An efficient protocol has been developed for inducing indirect somatic embryogenesis derived from immature zygotic embryo axis, young leaflet and hypocotylar explants. Callogenesis was achieved on full-strength Murashige and Skoog's (1962) (MS) basal medium supplemented with two auxin/cytokinin combinations; 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-furfurylamonopurine (KIN) or 1-naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BAP) to establish the phytohormones requirement to promote the best induction of friable calli. For somatic embryos induction, embryogenic calli developed from zygotic embryo axes, leaflet and hypocotylar explants were cultured on MS full strength and MS/2, half strength, free of phytohormones media. This study found that all explants exhibited high frequency of callus induction. For immature zygotic embryo axes and young leaflet explants, media containing the combination of 2,4-D and KIN were best effective for inducing callogenesis and friable calli. The most important embryogenic callus percentages were obtained on the culture medium containing the highest concentrations of 2,4-D (2 mg L-1) and KIN (0.5 mg L-1). However, media containing the combination NAA and BAP were best effective for inducing embryogenic calli on hypocotylar explants. A maximal rate of embryogenic calli was reached on medium containing 3 mg L-1 NAA and 1 mg L-1 BAP. The highest number of somatic embryos was obtained with embryogenic calli derived from embryo axis explants and cultured on MS half strength free phytohormones medium. About 56% of somatic embryos converted successfully into fertile plantlets.

In vitro somatic embryo productions from Curculigo orchioides derived gold nanoparticles: Synthesis, characterization, its biomedical applications, and their eco-friendly approaches to degradation of methylene blue under solar light irradiations

Curculigo orchioides rhizome explants were employed to develop a rapid and effective strategy for increased plant regeneration using somatic embryogenesis. Direct somatic embryo development was shown on rhizome explants cultivated on Murashige and Skoog (MS) making with 2, 4-D (1.0–3.0 mg/L). Rhizome explants cultivated on MS media supplemented with 2.0 mg/L 2, 4-D yielded the highest frequency of embryogenesis (87.5%) and the maximum number of somatic embryos (1596.7/explant). Somatic embryo germination was accomplished using MS media with 2.0 mg/L 6-benzylaminopurine (BAP). With an 80% survival rate, the germination plantlets were acclimated in the greenhouse. The current study is the first evidence of the efficacy of in vitro-produced plants and C. orchioides somatic embryo callus cultures of stable gold nanoparticles. The UV–Vis spectrophotometric absorbance, at 510 nm, revealed the absorption spectra of the AuNPs. The FT-IR revealed functional groups and reaction processes in green AuNP formation. High-resolution transmission electron microscopy (HR-TEM) was used to assess the surface morphology and structure of the AuNPs after their elemental composition was determined using a dispersive energy X-ray (EDAX) spectrum. The average size of AuNPs was around 35 nm in diameter. The crystalline nature of the AuNPs was investigated by X-ray diffraction (XRD). The highest growth inhibition was found for C. orcthioides against Klebsiella pneumoniae (17.5 mm) and Serratia marcescens (16.5 mm). The AuNPs exhibited antioxidant activity against free radicals such as DPPH and ABTS. Furthermore, the cytotoxicity of AuNPs was assessed, and inhibitory concentration (IC50) was 20 μg/mL and 80 μg/mL for breast carcinoma (MDA-MB-231) and Vero cell lines. The degradation of methylene blue measures the photocatalytic activity of the manufactured AuNPs when subjected to visible sunlight (MB). Thus, the result showed a maximum degradation efficiency of MB (84%).

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 .

Assessing the genetic fidelity of somatic embryo-derived plantlets of finger millet by random amplified polymorphic DNA analysis.

Finger millet [Eleusine coracana (L.) Gaertn.] is an important cereal because of its mineral-nutrition value. With the increasing demand, there is a pressing need to conserve it through biotechnological approaches. High-frequency somatic embryogenesis from seed-derived callus of E. coracana was developed on Murashige-Skoog (MS) medium supplemented with a combination of auxins [Indole-3-acetic acid (IAA), 2,4-Dichlorophenoxy acetic acid (2,4-D)] and cytokinins [6-Benzylaminopurine (BAP), kinetin (KN)] in different concentrations, ranging from 0.1 to 5.0mg L-1. Seeds cultured on this medium produced three different types of primary callus. Type I callus was very compact and dark brown, type II callus was light brownish and type III callus appeared whitish and light brown. All three types of calli had differential proliferation responses. Type II compact brown calli were obtained on the MS medium supplemented with 1.0 and 1.5mg 2,4-Dichlorophenoxy acetic acid L-1 and 0.5mg kinetin L-1. Friable yellowish embryogenic calli with a large number of somatic embryos were developed within 60days after being transferred to auxins and cytokinin (1.0 and 1.5mg 2,4-Dichlorophenoxy acetic acid L-1 and 0.5mg Kinetin L-1) along with 200mg casein hydrolysate L-1. Germination of somatic embryos on a half-strength MS medium supplemented with 0.1% Kinetin led to the development of healthy plantlets within 30days. Genetic fingerprinting using random amplified polymorphic DNA (RAPD) revealed high levels of genetic fidelity. The study provides methods and hormonal concentrations required to develop somatic embryos in E. coracana for its genetic improvement and conservation.

Genome size and gas chromatography-mass spectrometry (GC-MS) analysis of field-grown and in vitro regenerated Pluchea lanceolata plants.

Pluchea lanceolata is a threatened pharmacologically important plant from the family Asteraceae. It is a source of immunologically active compounds; large-scale propagation may offer compounds with medicinal benefits. Traditional propagation method is ineffective as the seeds are not viable; and root sprout propagation is a slow process and produces less numbers of plants. Plant tissue culture technique is an alternative, efficient method for increasing mass propagation and it also facilitate genetic improvement. The present study investigated a three-way regeneration system in P. lanceolata using indirect shoot regeneration (ISR), direct shoot regeneration (DSR), and somatic embryo mediated regeneration (SER). Aseptic leaf and nodal explants were inoculated on Murashige and Skoog (MS) medium amended with plant growth regulators (PGRs), 2,4-dichlorophenoxy acetic acid (2,4-D), 1-naphthalene acetic acid (NAA), and 6-benzyl amino purine (BAP) either singly or in combinations. Compact, yellowish green callus was obtained from leaf explants in 1.0 mg/l BAP (89.10%) added medium; ISR percentage was high, i.e., 69.33% in 2.0 mg/l BAP + 0.5 mg/l NAA enriched MS with 4.02 mean number of shoots per callus mass. Highest DSR frequency (67.15%) with an average of 5.62 shoot numbers per explant was noted in 0.5 mg/l BAP added MS medium. Somatic embryos were produced in 1.0 mg/l NAA fortified medium with 4.1 mean numbers of somatic embryos per culture. On BAP (1.0 mg/l) + 0.5 mg/l gibberellic acid (GA3) amended medium, improved somatic embryo germination frequency (68.14%) was noted showing 12.18 mean numbers of shoots per culture. Histological and scanning electron microscopic (SEM) observation revealed different stages of embryos, confirming somatic embryogenesis in P. lanceolata. Best rooting frequency (83.95%) of in vitro raised shootlets was obtained in 1.0 mg/l IBA supplemented half MS medium with a maximum of 7.83 roots per shoot. The regenerated plantlets were transferred to the field with 87% survival rate. The 2C genome size of ISR, DSR, and SER plants was measured and noted to be 2.24, 2.25, and 2.22 pg respectively, which are similar to field-grown mother plant (2C = 2.26 pg). Oxidative and physiological events suggested upregulation of enzymatic activities in tissue culture regenerated plants compared to mother plants, so were photosynthetic pigments. Implementation of gas chromatography-mass spectrometry (GC–MS) technique on in vivo and in vitro raised plants revealed the presence of diverse phyto-chemicals. The yields of alpha amyrin and lupeol (medicinally important triterpenoids) were quantified using high-performance thin-layer chromatography (HPTLC) method and enhanced level of alpha amyrin (2.129 µg g−1 dry wt) and lupeol (1.232 µg g−1 dry wt) was noted in in vitro grown leaf tissues, suggesting in vitro conditions act as a potential trigger for augmenting secondary metabolite synthesis. The present protocol represents a reliable mass propagation technique in producing true-to-type plants of P. lanceolata, conserving 2C DNA and ploidy successfully without affecting genetic homogeneity.Supplementary InformationThe online version contains supplementary material available at 10.1007/s13353-022-00727-7.

Gamma ray–induced tissue responses and improved secondary metabolites accumulation in Catharanthus roseus

The present study investigated the impact of gamma ray irradiation on callus biomass growth and the yield of vincristine and vinblastine of in vitro grown tissues of Catharanthus roseus. The biochemical alteration underlying the synthesis of secondary metabolites has also been studied and a comparison of yield was prepared. The embryogenic tissues were exposed to 20, 40, 60, 80, and 100Gy gamma ray doses and the callus biomass fresh weight, the embryogenesis (the embryo numbers, germination, plant regeneration), the alteration of protein, proline, and sugar attributes at different morphogenetic stages were monitored. The callus biomass growth was maximum (1.65g) in 20Gy exposed tissues and was less in 100Gy treatment (0.33g). The gamma-irradiated embryogenic tissues differentiated into embryos but the embryogenesis % and somatic embryo number per culture reduced with increasing doses. It was least in 80Gy where very low numbers of embryos were formed (3.45 and 3.30 mean torpedo and cotyledonary embryo numbers per callus mass, respectively) which later germinated into plantlets. Protein, proline, sugar, and different antioxidant enzymes, i.e., superoxide dismutase (SOD), ascorbate peroxidase (APX), and catalase (CAT) activities, were investigated as the tissues were exposed to gamma ray elicitation/signaling, evoking cellular stress. Increased 80Gy gamma dose inhibited a 42.73% decrease in protein accumulation at initiation stages of embryogenic tissue. Soluble sugar level also declined gradually being least in 80Gy treated tissues (14.51mg gm-1 FW) compared to control (20.2mg gm-1 FW). Proline content, however, increased with increasing gamma doses, maximum at 80Gy (8.28mg gm-1 FW). The SOD, APX, and CAT activity increased linearly with enhanced level of gamma doses and maximum, i.e., 3.91 EU min-1mg-1, 1.71 EU min-1mg-1, and 4.89 EU min-1mg-1, protein activity was noted for SOD, APX, and CAT, respectively, at 80Gy gamma rays treated tissues. The quantification of vinblastine and vincristine in gamma ray elicitated tissues was made by using high-pressure thin layer chromatography (HPTLC). Somatic embryo-regenerated plant's leaves had the maximum yield of vinblastine (15.13 µgm gm-1 DW) at 40Gy irradiation dose compared to control (13.30 µgm gm-1 DW)-the increased yield % is 13.75. The stem is also rich source producing 11.98 µgm gm-1 DW of vinblastine. Among the various developmental stages of embryos, vinblastine content was highest in germinating stage of embryos (10.14 µgm gm-1 DW) compared to other three, i.e., initiation, proliferation, and maturation embryo stages. Similarly, highest accumulation of vincristine (6.32µg gm-1 DW) was noted at low gamma irradiation dose (20Gy) in leaf tissues. The present study indicates that the synthesis of vinblastine and vincristine was growth- and development-specific and the lower 20-40Gy gamma levels were more effective in enriching alkaloids while higher doses declined yield. KEY POINTS: • Vinblastine and vincristine yield was quantified in in vitro grown tissues and leaves of embryo regenerated Catharanthus roseus after gamma ray treatment. • The accumulation of vinblastine and vincristine was maximum in regenerated leaves; low doses were more efficient in improving yield. • Gamma ray irradiation impacted biochemical profiles, caused cellular stress, and perhaps responsible for improved alkaloid yield.

Response of haustorium tissues and coconut water in somatic embryos induction for the coconut palm (Cocos nucifera L.) variety PB121

The coconut tree (Cocos nucifera L.) is a fruit plant that contributes significantly to improved nutrition, food security, job creation, and household income in Benin. However, its production is suffering from the unavailability of certified seedlings. The present work aimed to optimize the propagation of coconut trees through the somatic embryogenesis technique. Zygotic embryos were cultured in vitro on Y3 medium supplemented with 0.7% Agar; 2.5 g/l activated charcoal, 5% sucrose to obtain haustorium, and the radicle explants for somatic embryogenesis. Three months after, callus and somatic embryos were induced from haustorium and radicle on medium Y3 supplemented with different doses of 2,4-D (0.3 and 0.35 mM) and coconut water (0, 50, 100 and 150 ml/l). 80% of callus was induced of induced with haustorium explant on Y3 medium supplemented with 0.7 mM 2,4-D. The combination of 2,4-D and coconut water resulted in the highest average number of somatic embryos with 59 and 63 embryos obtained respectively on Y3 medium enriched with 150 ml/l coconut water and supplemented with 0.3 mM and 0.35 mM 2,4-D. Using haustorium explant for mass propagation through somatic embryogenesis remains an exploring way for in vitro seedling of coconuts.