Embryogenic Callus Formation Research Articles

Functional Analysis of the PoSERK-Interacting Protein PorbcL in the Embryogenic Callus Formation of Tree Peony (Paeonia ostii T. Hong et J. X. Zhang).

SERK is a marker gene for early somatic embryogenesis. We screened and functionally verified a SERK-interacting protein to gain insights into tree-peony somatic embryogenesis. Using PoSERK as bait, we identified PorbcL (i.e., the large subunit of Rubisco) as a SERK-interacting protein from a yeast two-hybrid (Y2H) library of cDNA from developing tree-peony somatic embryos. The interaction between PorbcL and PoSERK was verified by Y2H and bimolecular fluorescence complementation analyses. PorbcL encodes a 586-amino-acid acidic non-secreted hydrophobic non-transmembrane protein that is mainly localized in the chloroplast and plasma membrane. PorbcL was highly expressed in tree-peony roots and flowers and was up-regulated during zygotic embryo development. PorbcL overexpression caused the up-regulation of PoSERK (encoding somatic embryogenesis receptor-like kinase), PoAGL15 (encoding agamous-like 15), and PoGPT1 (encoding glucose-6-phosphate translocator), while it caused the down-regulation of PoLEC1 (encoding leafy cotyledon 1) in tree-peony callus. PorbcL overexpression led to increased indole-3-acetic acid (IAA) content but decreasing contents of abscisic acid (ABA) and 6-benzyladenosine (BAPR). The changes in gene expression, high IAA levels, and increased ratio of IAA to ABA, BAPR, 1-Aminocyclopropanecarboxylic acid (ACC), 5-Deoxystrigol (5DS), and brassinolide (BL) promoted embryogenesis. These results provide a foundation for establishing a tree-peony embryogenic callus system.

Adventitious embryonic causal gene FhRWP regulates multiple developmental phenotypes in citrus reproduction.

Citrus is a model plant for studying adventitious embryos, a form of asexual reproduction controlled by a single dominant gene, RWP. This gene has been identified as the causal gene for nucellar embryogenesis, but its function has not yet been fully understood. In this study, we used the fast-growing Fortunella hindsii as a system to explore chromatin accessibility during the nucellar embryony initiation, emphasizing elevated chromatin accessibility in polyembryonic (PO) genotypes compared to monoembryonic ones (MO). Notably, a higher level of accessible chromatin was observed in one allele of the promoter region of FhRWP, consistent with increased expression of the allele carrying the causal structural variant. By independently performing RNAi and gene editing experiments on PO genotypes, we found the downregulation of FhRWP expression could reduce the number of nucellar embryos, while its knockout resulted in abnormal axillary bud development. In overexpression experiments, FhRWP was identified as having the unique capability of inducing the embryogenic callus formation in MO stem segments, possibly through the regulation of the WUS-CLV signaling network and the ABA and cytokinin pathway, marking the inaugural demonstration of FhRWP's potential to reignite somatic cells' embryogenic fate. This study reveals the pleiotropic function of RWP in citrus and constructs a regulatory network during adventitious embryo formation, providing a new tool for bioengineering applications in plant regeneration.

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.

PENGARUH WARNA CAHAYA TERHADAP MORFOGENESIS EKSPLAN KALUS BAWANG PUTIH (Allium sativum L.) SECARA IN-VITRO

The quality of the Garlic seed bulbs needs to be improved by seed propagation through tissue culture techniques to get uniform and big cloves. Utilizing the suitable color spectrum of light is one way to stimulate plant morphogenesis. This study aimed to identify the influence of light color and obtain the best color in the morphogenesis of garlic callus explants. This research was carried out from June to September 2022 at the Tissue Culture Laboratory, Faculty of Agriculture, Universitas Andalas. This study used a completely randomized design with three treatment levels: red, blue, and white. The F test analyzed observational data at a 5% level, followed by Duncan's multiple Range Test (DNMRT) at a 5% level. The results showed light color influences embryogenic callus formation and shoot formation from garlic callus explants. Blue light is better than red and white light in inducing embryogenic callus, increasing the rate of explants forming shoots but giving no significant difference in the length of the shoot.

Transcriptional Profiling Reveals Key Regulatory Roles of the WUSCHEL-Related Homeobox Gene Family in Yellowhorn (Xanthoceras sorbifolia Bunge)

The WUSCHEL-related homeobox (WOX) gene family plays a crucial role in regulating embryonic development, organ formation, and stress resistance. Yellowhorn (Xanthoceras sorbifolia Bunge), a drought-resistant tree known for its oil production, lacks sufficient information regarding the WOX gene family. To understand the evolutionary mechanisms and potential functions of this gene family in yellowhorn, we conducted a comprehensive investigation on its expression patterns and evolutionary characteristics. Our analysis revealed the presence of nine XsWOX genes in the yellowhorn genome, which could be categorized into three distinct clades through a phylogenetic analysis. A chromosomal localization analysis indicated that these nine XsWOX genes were situated on six out of the fifteen chromosomes. An intra-species collinear analysis revealed only one pair of tandem duplicated genes within the XsWOX family. The promoter regions of the XsWOX family were found to contain responsive cis-acting elements associated with plant growth and development, stress responses, and hormone signaling. Moreover, an analysis of the gene expression profiles in different developmental stages of callus revealed significant expressions of XsWOX1, XsWOX4, and XsWOX5 in embryogenic callus and somatic embryo formation, suggesting that they have special roles in regulating yellowhorn’s somatic embryogenesis. Furthermore, the expression level of XsWOX5 indicated its potential involvement not only in organ formation but also in responding to low temperature, salt, and saline-alkali stresses. Overall, our findings lay a solid foundation for future in-depth studies on the functionality and evolution of XsWOX genes in yellowhorn.

Study on rapid multiplication of two hybrid Arabica coffee varieties F1 Centroamericano (H1) and Mundo Maya (H16) through somatic embryogenesis and Bioreactor technology

In this paper, the research focuses on determining some components and culture conditions in order to optimise the 4 main steps of somatic embryogenesis and embryo regeneration by Bioreactor of two Arabica F1 coffee varieties H1 and H16, including: (1) formation of embryogenic callus, (2) multiplication of embryogenic cells, (3) embryonic differentiation, and (4) regeneration of somatic embryo using the temporary immersion Bioreactor RITA 1L. The results showed that on the medium supplemented with Kinetin 2 mg/ll and Phytagel 4 g/l, the callus formation rate reached 94.2 and 92.1% for 2 varieties H1 and H16, respectively. Besides, the addition of Glycine 20 mg/l to the culture medium increased the embryogenic callus rate by 58.1% in both varieties. Adding Trichostatin A 0.03 mg/l in the embryo differentiation medium in the first 7 days of culture showed that the number of torpedo embryos was scored from 1g of initial callus up to 1287 in H1 and 1635 in H16. Finally, using Bioreactor 1L-RITA, soaking for 1 min every 6 hours, the regeneration efficiency reached 75.02% in H1 and 82.03% in H16.

Time-Dependent Proteomic Signatures Associated with Embryogenic Callus Induction in Carica papaya L.

Sex segregation increases the cost of Carica papaya production through seed-based propagation. Therefore, in vitro techniques are an attractive option for clonal propagation, especially of hermaphroditic plants. Here, we performed a temporal analysis of the proteome of C. papaya calli aiming to identify the key players involved in embryogenic callus formation. Mature zygotic embryos used as explants were treated with 20 μM 2,4-dichlorophenoxyacetic acid to induce embryogenic callus. Total proteins were extracted from explants at 0 (zygotic embryo) and after 7, 14, and 21 days of induction. A total of 1407 proteins were identified using a bottom-up proteomic approach. The clustering analysis revealed four distinct patterns of protein accumulation throughout callus induction. Proteins related to seed maturation and storage are abundant in the explant before induction, decreasing as callus formation progresses. Carbohydrate and amino acid metabolisms, aerobic respiration, and protein catabolic processes were enriched throughout days of callus induction. Protein kinases associated with auxin responses, such as SKP1-like proteins 1B, accumulated in response to callus induction. Additionally, regulatory proteins, including histone deacetylase (HD2C) and argonaute 1 (AGO1), were more abundant at 7 days, suggesting their role in the acquisition of embryogenic competence. Predicted protein-protein networks revealed the regulatory role of proteins 14-3-3 accumulated during callus induction and the association of proteins involved in oxidative phosphorylation and hormone response. Our findings emphasize the modulation of the proteome during embryogenic callus initiation and identify regulatory proteins that might be involved in the activation of this process.

Embryogenic callus induction of Indonesian Cassava (Menti and Adira 4) on different picloram concentrations

Cassava (Manihot esculenta Crantz) is an important staple crop in tropic countries. A reproducible somatic embryogenic callus production will be useful in the biotechnology breeding program for plants including cassava. Menti and Adira-4 are two types of high yield cassava genetic resources in Indonesia. Immature leaf lobes from cassava in vitro were used as explants on MS basal medium with five level concentrations of Picloram: 0 mg L−1, 3 mg L−1, 5 mg L−1, 10 mg L−1, and 12 mg L−1. Initial embryogenic callus were seen on the explants in 2 weeks after treatment. Explants were then subcultured to the same medium every three weeks until three cycles of subculture to proliferate pro-embryos. Result showed that MS basal medium containing picloram 3 – 12 mg L−1 could induce embryogenic callus formation in Menti and Adira 4 immature leaf lobe explants. The highest percentage of explants forming embryogenic callus was on D medium (MS + 3 mg L−1 Picloram). These findings would suggest that at all picloram concentrations tested both varieties of cassava showed a high embryogenic callus production. High frequency on embryogenic callus formation is a key step in cassava regeneration system specifically to support the modern breeding program.

Response of the transcription factor BABY BOOM of Arabidopsis thaliana L. in the formation of embryogenic calluses of cocoa leaves (Theobroma cacao L.)

Cocoa (Theobroma cacao L.) is one of the most important economic crops worldwide. The propagation of elite varieties of cocoa has been achieved through somatic embryogenesis, but still one of the main limitations is the low rates of embryo formation, which is a genotype-dependent trait. Manipulation of transcription factors (TFs) such as BABY BOOM (BBM) promotes the transition of cocoa somatic cells from the vegetative to the embryonic state. This work validated the use of clonal cocoa leaves cv. IMC-67 to induce somatic embryogenesis, overcoming their recalcitrant limitation with the help of the introduction of TF-BBM from Arabidopsis thaliana (AtBBM). The vectors were constructed by the Gateway system using the donor vector pENTR/D-TOPO and the expression vector pk7WG2. The overexpression vector pk7WG2:AtBBM was obtained, allowing successful transformation into Agrobacterium tumefaciens GV3101. The AtBBM gene was characterized (1755 base pairs), and its expression was observed in the formation of embryogenic calluses in cocoa leaves. Overexpression of AtBBM allowed the obtainment of a 92% response in the formation of embryogenic callus in cocoa leaves with Agrobacterium-mediated vacuum infiltration and overexpression of the pk7WG2:AtBBM vector. This high transformation efficiency reached with the insertion of the overexpression vector provides validation of transient response of the TF AtBBM in the formation of embryogenic calluses in cocoa leaves of the IMC-67 clone. Through this methodology, it is possible to continue with studies of gene overexpression, insertion, silencing, and gene editing in Peruvian cocoa.

Effect of the use of indole-3-acetic acid on somatic embryogenesis of sunflower (Heliantus annuus) grown in the southern region

Sunflower (Helianthus annuus L.) is one of the most important oil crops worldwide. However, one of the early uses of this species was as an ornamental plant due to the color and variety of its flowers. Today, there is increasing interest in tissue culture techniques in this species using growth regulators for callus formation. We evaluated three concentrations of indole-3-acetic acid, 0.1, 0.5 and 1 mg/L, on embryogenic callus formation using cotyledon, hypocotyl and root of sunflower cultivated in Southern Mexico. Results indicate that the three concentrations promoted callus formation, however, the best development was observed at 0.1 mg/L indole-3-acetic acid. Thus, indole-3-acetic acid is an efficient regulator for H. annuus plant growth and organogenesis.

Transcriptome Analysis of Embryogenic and Non-Embryogenic Callus of Picea Mongolica.

Picea mongolica is a rare tree species in China, which is of great significance in combating desertification and improving the harsh ecological environment. Due to the low rate of natural regeneration, high mortality, and susceptibility to pests and cold springs, Picea mongolica has gradually become extinct. At present, somatic embryogenesis (SE) is the most effective method of micro-proliferation in conifers, but the induction rate of embryogenic callus (EC) is low, and EC is difficult to differentiate from non-embryonic callus (NEC). Therefore, the EC and NEC of Picea mongolica were compared from the morphology, histological, physiological, and transcriptional levels, respectively. Morphological observation showed that the EC was white and transparent filamentous, while the NEC was compact and brownish-brown lumpy. Histological analyses showed that the NEC cells were large and loosely arranged; the nuclei attached to the edge of the cells were small; the cytoplasm was low; and the cell gap was large and irregular. In the EC, small cells, closely arranged cells, and a large nucleus and nucleolus were observed. Physiological studies showed significant differences in ROS-scavenging enzymes between the EC and NEC. Transcriptome profiling revealed that 13,267 differentially expressed genes (DEGs) were identified, 3682 were up-regulated, and 9585 were down-regulated. In total, 63 GO terms had significant enrichment, 32 DEGs in plant hormone signal transduction pathway were identified, and 502 different transcription factors (TFs) were characterized into 38 TF families. Meanwhile, we identified significant gene expression trends associated with somatic embryo development in plant hormones (AUX/IAA, YUCCA, LEA, etc.), stress (GST, HSP, GLP, etc.), phenylpropanoid metabolism (4CL, HCT, PAL, etc.), and transcription factors (AP2/ERF, MYB, WOX, etc.). In addition, nine genes were chosen for RT-qPCR, and the results were consistent with RNA-Seq data. This study revealed the changes in morphology, histology, physiology, and gene expression in the differentiation of NEC into EC and laid the foundation for finding the key genes to promote EC formation.

Changes and transcriptome regulation of endogenous hormones during somatic embryogenesis in Ormosia henryi Prain.

Ormosia henryi is a rare and endangered plant growing in southern China. Somatic embryo culture is an effective measure for the rapid propagation of O. henryi. It has not been reported how regulatory genes induce somatic embryogenesis by regulating endogenous hormone changes during the process of somatic embryogenesis in O. henryi. In this study, we analysed the endogenous hormone levels and transcriptome data of nonembryogenic callus (NEC), embryogenic callus (EC), globular embryo (GE) and cotyledon embryo (CE) in O. henryi. The results showed that the indole-3-acetic acid (IAA) content was higher and the cytokinins (CKs) content was lower in EC than in NEC, and the gibberellins (GAs) and abscisic acid (ABA) contents were significantly higher in NEC than in EC. The contents of IAA, CKs, GAs and ABA increased significantly with EC development. The expression patterns of differentially expressed genes (DEGs) involved in the biosynthesis and signal transduction of auxin (AUX) (YUCCA and SAUR), CKs (B-ARR), GAs (GA3ox, GA20ox, GID1 and DELLA) and ABA (ZEP, ABA2, AAO3, CYP97A3, PYL and ABF) were consistent with the levels of endogenous hormones during somatic embryogenesis (SE). In this study, 316 different transcription factors (TFs) regulating phytohormones were detected during SE. AUX/IAA were downregulated in the process of EC formation and GE differentiation into CE, but other TFs were upregulated and downregulated. Therefore, we believe that relatively high IAA content and low CKs, GAs and ABA contents contribute to EC formation. The differential expression of AUX, CKs, GAs and ABA biosynthesis and signal transduction genes affected the endogenous hormone levels at different stages of SE in O. henryi. The downregulated expression of AUX/IAA inhibited NEC induction, promoted EC formation and GE differentiation into CE.

The efficient procedure of embryogenic callus formation from anther in Capsicum pubescens Ruiz & Pav

The peppers or ajies (Capsicum spp.) are one of Peru's main productive crops, which is why it is among the top ten countries in production and exports. Among the species cultivated in Peru is Capsicum pubescens Ruiz & Pav., commonly called rocoto, and represents one of the most critical species in local and national gastronomy. The rocoto ecotype "Selva" or "de Monte" is characterized by its large size and is used to prepare rocoto relleno arequipeño. This crop presents the restriction to obtain homozygous lines for being self-incompatible. Using biotechnology, pure lines can be obtained from the anther culture, and homozygous lines can be obtained. The objective was to induce embryogenic callus formation from anther culture of Capsicum pubescens Ruiz & Pav. Ecotype "de Monte". In the results, 74.1% of embryogenic callus was induced in rocoto anthers with a longitudinal ratio of 1.04 mm; the induction was carried out using Murashige and Skoog culture medium added with 2.0 mg/L of 2,4-D. Likewise, the formation of somatic embryos in the globular stage was evidenced after 12 weeks in the culture medium.

Coconut Callus Initiation for Cell Suspension Culture

The development of a cell suspension culture system for the scaling up of coconut embryogenic callus (EC) production would drastically improve efforts to achieve the large-scale production of high-quality clonal plantlets. To date, the hard nature of coconut EC appeared to be the main constraint for developing cell suspension cultures. Hence, this study attempted to acquire friable EC through the following approaches: The manipulation of (1) medium type and subculture frequency, (2) a reduced 2,4-dichlorophenoxy acetic acid concentration during subculture, (3) the nitrate level and the ammonium-to-nitrate ratio, and the addition of amino acid mixture, (4) the addition of L-proline, and (5) the reduction of medium nutrients. Unfortunately, none of these culture conditions produced friable coconut EC. Even though friable EC was not achieved via these approaches, some of the conditions were found to influence the formation of compact EC, therefore these results are important for further studies focused on somatic embryogenesis in coconut and other species.

Embryogenic Callus Differentiation in Short-Term Callus Derived from Leaf Explants of Alfalfa Cultivars

Alfalfa is a forage crop that accounts for one of the best sources of protein and is commonly grown all over the world. In vitro callus and embryogenic induction of alfalfa have been investigated previously; however, most of the investigations were almost limited with callus formation. In this study, leaves of 5 Medicago sativa L. cultivars (Alsancak, Sazova, Plato Iside, and Bilensoy) have been used. Influences of culture medium contents and hormones applications on callus and embryogenic callus induction were determined to optimize in vitro culture mediums of alfalfa (0.0125 g kinetin 2,4 D 1 mg mL-1; 1 g kinetin 2,4 D 1 mg mL-1 ; 0.25 g kinetin 2,4 D 2 mg mL-1; 0.5 g kinetin 2,4 D 2 mg mL-1). Callus formation was detected at a rate of 74% in 5 different cultivars used in the experiment. The five alfalfa cultivars were classified into four categories in terms of embryogenic differentiation capacity. The tested alfalfa cultivars varied in their callus formation and embryogenic callus differentiation. Sazova, Alsancak, and Bilensoy were detected for better callus formation; similarly, the same cultivars responded with better embryogenic callus formation in the culture mediums including various hormone concentrations. The present study shows that our methods have beneficial impacts on the somatic embryo induced by alfalfa. However, it depends strongly on genotype, hormone concentrations and the other medium components.

Transcriptomic and physiological analyses reveal the acquisition of somatic embryogenesis potential in Agapanthus praecox

Somatic embryogenesis (SE) is an ideal cell model for plant cell totipotency study. The poor frequency of embryogenic callus (EC) induction limits the application of SE in many plants, such as Agapanthus praecox. Herein, we performed transcriptomic and physiological analyses with different callus (Ca) differentiation directions (SE and organogenesis) and stages (initial SE and repetitive SE) to better understand the physiological and molecular characteristics of the acquisition of embryogenic potential in A. praecox. A number of differentially expressed genes (DEGs) were significantly related to plant hormonal signaling transduction, starch and sucrose metabolism, and reactive oxygen species (ROS) responses. Compared to Ca, DEGs including heat shock protein, isoamylase, histone H4, alpha-1,2-mannosyltransferase, and sucrose synthase substantially changed expression levels in EC formation. Corresponding to DEGs, physiological indicators including indole-3-acetic acid, cytokinins, brassinosteroids, ethylene, abscisic acid, ROS, hydrogen peroxide, superoxide dismutase, catalase, starch and sucrose may be responsible for the acquisition of embryogenic potential in A. praecox. Moreover, plant growth regulators, carbon source combination, osmotic regulating substance, and DNA methylation inhibitors in the culture medium significantly affected the acquisition of SE potential. Altogether, our results suggested that plant hormone signal regulation, starch and sucrose metabolism, ROS scavenging, chromatin accessibility and DNA methylation jointly contributed to the activation of embryogenic ability in A. praecox.

Functional Analysis of Glucose-6-Phosphate Translocator PoGPT1 in Embryogenic Callus Formation of Tree Peony

Tree peony (Paeonia ostii) is an ornamental flowering plant that is generally recalcitrant to establishment of a mature somatic embryo regeneration system in vitro. Glucose-6-phosphate translocator (GPT) plays an important regulatory role in embryogenesis of plants. In this study, PoGPT1 was cloned, and a bioinformatic analysis and functional verification of the gene were performed. The results showed that PoGPT1 encoded a polypeptide of 392 amino acids, which was a basic non-secreted hydrophobic transmembrane protein, and was mainly localized in the plastids. PoGPT1 was highly expressed in tree peony leaves, and its transcript abundance increased with the progression of zygotic embryo development. Overexpression of PoGPT1 caused up-regulation of leafy cotyledon 1 (PoLEC1), somatic embryogenesis receptor-like kinase (PoSERK), and agamous-like15 (PoAGL15) in tree peony callus. In addition, PoGPT1 overexpression promoted the increase in indole-3-acetic acid (IAA), 5-deoxystrigol (5DS), and brassinolide (BL) contents, especially of IAA, but reduced the contents of abscisic acid (ABA), 6-benzyladenosine (BARP), and 1-aminocyclopropanecarboxylic acid (ACC). The present research showed that PoGPT1 synergistically regulated the contents of endogenous hormones and expression levels of embryogenesis-related genes to promote the embryonic development of tree peony. The results provide theoretical and technical support for the establishment of a tree peony embryogenic callus formation and subsequent research on somatic embryogenesis.

Optimization of high frequency plant regeneration protocol via embryogenic callus formation from diverse indica rice genotypes induced by combinations of 2,4-D and BA

A high frequency plant regeneration protocol via induction of embryogenic callus followed by somatic embryogenesis was standardized for a group of indica rice varieties consisting of land races and improved varieties, which are generally considered recalcitrant. Scutellum derived embryogenic calluses were obtained from the mature seeds inoculated into MS medium supplemented with different concentrations and combinations of 2,4-D and BA added with casein hydrolysate (500 mg/l) and proline (1 g/l). The varieties significantly differed in their response to embryogenic callusing and regeneration. Maximum embryogenic callus formation was induced in all the varieties by altering the combinations and concentrations of 2,4-D(1.0-2.0 mg/l) and BA (0.01, 0.1 and 0.5 mg/l). The land races viz., Karnel, SavuluSamba and Doddabyranellu showed higher embryogenic callus formation in all the combinations and concentrations of 2,4-D and BA. But the improved varieties showed embryogenic callus induction only in particular concentration and combinations of growth regulators. However, an increasing trend of plant regeneration was observed in regeneration media consisted of MS salts supplemented with 3.0 mg/ l BA and 1.0 mg/l NAA. We showed that maximum plant regeneration through generation of embryogenic calluses induction in indica rice varieties can be induced by altering the concentrations and combinations of 2,4-D and BA.

Получение растений-регенерантов озимой мягкой пшеницы с использованием метода культуры пыльников

Abstract. The purpose of the current study was to evaluate the effect of cultivation conditions and the genotype of winter bread wheat samples on the formation of embryogenic callus and regenerative capacity, to identify regenerated plants in order to develop new initial material for breeding new varieties. Scientific novelty. There has been estimated the share of influence of induction nutrient media with different composition and genotype on the amount of regenerated plants of winter wheat in anther culture; there have been identified the new samples with high regeneration potential. Methods. The objects of the study were the donor plants of winter bread wheat (Triticum aestivum L.) of F3 generation from breeding nurseries of the winter wheat department of the FSBSI “ARC “Donskoy”, namely 13 hybrid combinations developed in the laboratory for breeding and seed production of winter bread wheat of half-intensive type. There has been used a methodology of obtaining haploids in anther culture in vitro including such methods as sterilization during work with in vitro; preparation of nutrient media; selection and pretreatment of plant explants; determination of the stage of microspores’ development; isolation and planting of anthers on an induction nutrient medium; regeneration of plants from callus tissues. Results. There has been found the most favorable nutrient media for the induction of and rogenesis in vitro. There has been identified a maximum percentage of new formations from the anthers of winter bread wheat in the sample Vol’nitsa × Gerda. There have been developed the largest number of green haploid regenerated plants from pollen callus from the samples Vol’nitsa × Gerda, Kapitan × Vol’nyy Don. There has been revealed that the contribution of the genotype and nutrient medium into the number of new formations and the regeneration of green plants was statistically significant.

From induction to embryo proliferation: improved somatic embryogenesis protocol in grapevine for Italian cultivars and hybrid Vitis rootstocks

Somatic embryogenesis is the most common regeneration method for the application of new genomic techniques like cisgenesis/intragenesis, genome editing, and RNAi. However, some local important genotypes show recalcitrance to this morphogenetic strategy, which represents an obstacle for the application of genetic engineering techniques. Whole flowers, stamens, and pistils of three different Italian Vitis vinifera L. cultivars (Ancellotta, Glera, and Lambrusco Salamino), and four hybrid rootstocks (110 Richter, 17.37, SO4, Star 50) have been tested in several culture media with changing basal salts (NN and MS), different combinations of growth regulators (BAP, 2,4-D, NOA, PIC, and NAA), and gelling agents, to initiate somatic embryogenesis. The formation of embryogenic calli was observed mainly from whole flowers cultured on PIV medium (NN salts, B5 vitamins, 3 g L−1 gelrite, 60 g L−1 sucrose, 8.9 µM BAP, and 4.5 µM 2,4-D), and stamens on MS1 medium (MS salts and vitamins, 7 g L−1 plant agar, 20 g L−1 sucrose, 4.5 µM BAP, and 5 µM 2,4-D), in the cv. Ancellotta, Lambrusco Salamino, and all the rootstocks, except for Star 50, which showed the best embryogenetic response from pistils placed on MS1. In a recalcitrant cv. as Glera, pistils placed on MS medium supplemented with 1 µM BAP, 5 µM 2,4-D, and gelrite as gelling agent, showed the highest percentage of embryogenesis. In addition, a two-step protocol was efficiently optimized for further induction of secondary embryo production for the above-listed grapevine genotypes, which guaranteed the long-term maintenance of embryogenic cultures from clusters or single somatic embryos.