Immature Embryo Culture Research Articles

Influence of selective conditions in vitro on variability of created flax forms

Fiber flax is a strategic crop. Flax products are widely used in various sectors of the economy, including the production of fabrics, paints, medical cotton wool, gunpowder, etc. The occurrence of diseases on flax crops entails a decrease in crop yield. The quality of flax fiber obtained from the affected flax plant deteriorates, and infected seeds reduce germination and biological potential. The most harmful diseases of fiber flax at present are fusarium wilt, rust, anthracnose, pasmo, and bacteriosis. The goal of the research conducted in 2021–2023 on the basis of the laboratory of breeding and biotechnology of the Federal State Budgetary Institution “Federal Scientific Center for Bast Crops” (Tver Region) was to create new flax genotypes resistant to anthracnose (Colletotrichum lini) using biotechnological methods. As a result of research, new forms, more resistant to the pathogen, were created in vitro from flax genotypes susceptible to anthracnose. It was revealed that on a selective background, plants with varying degrees of resistance were formed on the basis of immature embryos. Both resistant and moderately susceptible forms were noted susceptible forms formed on the basis of the same genotype. It was noted that the majority of regenerants obtained during in vitro selection from genotypes susceptible to anthracnose, under the newly created selective conditions, were characterized by resistance and average susceptibility to the cultural filtrate. It has been suggested that the formation of regenerated plants, different from the original forms, against a selective background in the culture of immature fiber flax embryos, is caused by the occurrence of a spontaneous mutation process, which, apparently, does not always depend on the creation of selective conditions. The studies revealed differences in the used strains of the anthracnose pathogen according to morphological characteristics. It was established that the intensity of sporulation and growth of the strains used did not depend on their virulence.

Speed breeding advancements in safflower (Carthamus tinctorius L.): a simplified and efficient approach for accelerating breeding programs.

This study investigated the potential of extended irradiation combined with immature embryo culture techniques to accelerate generation advancements in safflower (Carthamus tinctorius L.) breeding programs. We developed an efficient speed breeding method by applying light-emitting diodes (LEDs) that emit specific wavelengths, alongside the in vitro germination of immature embryos under controlled environmental conditions. The experimental design for light treatments followed a 2 × 4 completely randomized factorial design with four replications, incorporating two safflower varieties, Remzibey-05 and Dinçer, and four LED treatments (white, full-spectrum, red + blue + white, and control). A lighting regimen of 22 h of light and 2 h of darkness was applied for all the LED treatments, whereas the control received 18 h of light and 6 h of darkness. Additionally, the immature embryo culture experiment used a 2 × 2 × 4 factorial arrangement, assessing two safflower cultivars, two media types, and four embryo developmental stages, with three replications. The parameters evaluated included plant height, branch number, seed number per plant, seed number per head, time to flower initiation, time to 50% flowering, time to harvest, and germination percentage of in vitro cultured immature embryos at various developmental stages. The harvest time among the light treatments ranged from 50.62 to 73.12days, with the shortest time achieved under the red + blue + white LED combination and the longest under the control treatment. The plant height, number of seeds per plant, and number of seeds per head were highest under the full-spectrum LED, control and red + blue + white LED combinations, respectively. Immature embryos rescued at 10dayspost-pollination presented a 57% germination rate, with an increasing trend in germination as the number of dayspost-pollination increased. The germination rates did not significantly differ across varieties or hormone treatments. This study demonstrated the potential to achieve six generations per year by combining prolonged illumination with targeted LED lighting and immature embryo culture techniques. These findings provide valuable insights for optimizing safflower growth and development and advancing speed breeding in controlled environments.

A Study on the Integration of In Vitro Methods with In Vivo Double Haploid Technique in Maize (Zea mays L.)

The in vivo doubled haploid technique in maize breeding significantly reduces the time required for developing homozygous lines, offering advantages in terms of both time and cost. Although this technique enables the development of lines much faster than traditional breeding methods, ongoing research aims to further shorten the development process through alternative approaches. In this context, significant efforts have been devoted to integrating in vitro methods with in vivo doubled haploid technique. This study aimed to investigate the potential of combining in vivo and in vitro techniques for the rapid development of homozygous maize lines. A total of 10 local populations and 3 inducer lines (CIM2GTAIL-P2, ADAIL-1, STOCK-6) were used as experimental material. The study was conducted in two phases under field and laboratory conditions. During the first phase, induction crosses were performed in 2022, and the haploid induction rates of donor genotypes were found to range from 1.29% to 7.12%, as determined using the Navajo marker. In the laboratory phase, immature embryo culture was employed for both direct and indirect regeneration using samples collected 18–20 days after induction crossing. Haploid status of the samples obtained through direct regeneration was confirmed using the Feulgen chromosome staining method. Four of the donor materials (DON3, DON4, DON6, DON7) yielded successful results in tissue culture studies. Explants were taken from immature embryos to CHU medium for callus formation and then these calli were transferred to Murashige and Skoog medium for the formation of somatic embryos. This approach enabled the production of 3 to 6 calluses per immature embryo, depending on the donor genotype. The results of this study indicate that integrating immature embryo culture as an in vitro method into the in vivo doubled haploid technique can offer benefits in terms of both time efficiency and an increased number of developed materials.

Comparative Transcriptomic Insights into the Mechanisms Underlying Maize (Zea mays L.) Embryogenic Callus Differentiation

The regeneration of plant somatic cells is a prerequisite for their biological breeding. Identification of key genes controlling embryogenic callus (EC) differentiation and investigation of the genetic mechanism of cell fate determination are important for improving plant variety. In this study, we used the maize inbred line KN5585 and its gene-edited mutants Zmprx19-1, Zmprx19-2 and Zmprx19-3 as plant materials. Three somatic regeneration-related traits, the embryogenic callus induction rate (EIR), green callus rate (GCR) and plantlet regeneration rate (PRR), were identified by tissue culture of immature embryos. Additionally, the ECs at different differentiation stages (0 d, 5 d, 10 d and 15 d) were subjected to RNA-seq, and comparative transcriptome analyses were performed. The results showed that the somatic regeneration traits of the mutants were all highly significantly lower than those of the wild type (p < 0.01). The PRR value of KN5585 was 75.25%, while the highest PRR of the mutants was only 15.08%, indicating that knockdown of ZmPRX19 inhibited EC regeneration. Transcriptome sequencing yielded a total of 200.30 Gb of clean data from 24 libraries, with an average of 6.53 Gb of clean data per library. Mutant and wild-type gene expression data were compared separately at four differentiation stages, and 689 common differentially expressed genes (DEGs) were screened. WGCNA was used to classify these genes into nine modules, which were subsequently subjected to GO and KEGG enrichment analyses. In total, 40, 23, 17 and 5 genes were significantly (q < 0.05) enriched in plant hormone signal transduction, the MAPK signaling pathway-plant, phenylpropanoid biosynthesis and photosynthesis, respectively. Moreover, protein–protein interaction (PPI) network analysis revealed five MAPKKK17_18 hub nodes involved in the MAPK pathway-plant, which may be the key genes controlling plantlet differentiation from ECs. The above results provide a basis for the final elucidation of the molecular mechanism of plant somatic regeneration.

Embryo Rescue Technique in Buckwheat Fagopyrum esculentum Moench.

Culture of immature buckwheat embryos is an excellent explant for tissue culture and regeneration in vitro, as well as a method of embryo rescue in case of fruit abortion especially in interspecific crosses. Here, we describe the method of immature embryo rescue using a differentiated approach to different age groups. The method involves the development of embryos along the path of direct embryogenesis bypassing the callus stage.

Development of regenerated plants of interspecific wheat hybrids using immature embryos as explants

Nowadays particular attention in winter wheat breeding is paid to the introduction of biotechnological methods. The culture of immature embryos in vitro is an alternative way to produce hybrids that often do not develop fully in vivo. The purpose of the current study was to estimate the ability of immature embryos of interspecific winter wheat hybrids for callus induction, morphogenesis, and plant regeneration on artificial nutrient media, as well as to select optimal conditions for obtaining full-fledged plants with all the botanical characteristics of the species. The research material included 5 hybrid combinations of interspecific crosses between durum (Triticum durum DESF.) and common (Triticum aestivum L.) winter wheat. The material was collected during one field season (2022). Immature embryos were used as explants. Culture media were according to Murashige-Skoog (MS), and they differed in the content of hormones and organic substances. There has been found that the hybrid combinations were characterized by a variety of morphogenic responses. The manifestation of callusogenesis was not high enough and averaged 22–32 % depending on the nutrient medium. Rhizogenesis averaged from 1–6.7 %, and ended with the formation of roots. The process of formation of embryoid- and hemmorizogenesis was best demonstrated by the hybrid combination No.1, and on two media. All processes of morphogenesis excepting ‘rhizogenesis’ varied greatly among hybrid combinations. The largest number of regenerated plants was obtained from hybrid combination No.1 (61 pcs., 54.5 %). After vernalization and acclimatization, 38 % of the plants survived from the number of planted embryos. The largest number of heads (79 pcs.) was formed according to hybrid combination No.1, on average 1.46 heads per plant. However, not all heads were fertile. The results of the correlation analysis made it possible to identify indicators that closely correlated with the amount of plant regeneration, namely, the high yield of embryoid- and hemmorizogenic structures ensured a high frequency of plant regeneration (r = 0.769).

Investigation of the medium for barley (Hordeum vulgare L.) immature embryo culture

Germination of immature embryos of interspecific crosses in vitro is the most important problem in barley breeding programs. The effect of four media types (MS, ½ MS, B-5, ½ B-5 on immature embryo culture of nine barley varieties (Sameer, Buraq, Aksad, Shuaa, Arevat, Alwarkaa, Alhadher, Amel, and Rihan) was studied by using completely randomized design (C.R.D.) with six replicates. Analysis of variance showed highly significant effects due to varieties, media, and interaction for all studied characteristics, indicated high variation among varieties and media. Aksad variety was significantly superior to all varieties for all studied traits except root number (RN). Medium of ½ B-5 was significantly superior to all media for all studied traits. Aksad interactions with media except B-5 media gave full germination with highly significant superiority over all interactions. Aksad x ½ B-5 interaction showed a highly significant superiority for shoot length (SL) and RL. As a result, ½ B-5 medium was the efficient medium for germination of immature embryos of most varieties. These varieties could be used in breeding program of interspecific crosses with wild species by using ½ B-5 medium.

Effect of culture filtrates of Fusarium fungi on oat callus cultures

The effect of three concentrations (30, 40 and 50%) of the culture filtrate (CF) of the genus Fusarium fungi (F. sporotrichioides, F. poae, F. equiseti, F. oxysporum) on the growth and development parameters of oat callus culture was evaluated. CF at the callus proliferation stage was used to select somaclonal cell lines with mycotoxin tolerance traits. Prior to this, callusogenesis induction was carried out on the Murashige-Skoog (MS) medium with 3 mg/l 2,4-D and 2 mg/l UIC in the culture of immature embryos of the Tyumensky Golozerny, Tubinsky, Zolotoy Pochatok and Talisman varieties. The material for the experiment was selected from the ears of the plants grown in the summer period of 2016–2018 on the experimental fields in the Krasnoyarsk forest-steppe. Callus size was recorded when transplanted onto the proliferation media (MS + 1.5 mg/l 2,4-D), control and CF-containing media. After 30 days of cultivation, callus growth, signs of necrosis and organogenesis were noted. At the end of the experiment, the number of the regenerants formed was counted. The presence of CF in the proliferation medium already at a concentration of 40% provided a decrease in the proliferative activity and increased the frequency of necrosis by at least 50%. Similar results were obtained at the CF level of 50%. On the media with F. poae CF, the reduction of callus viability reached 60–70%. The calluses that remained viable under these conditions had a frequency of regenerant formation and organogenesis 2–3 times higher than the samples that were not influenced by the selective factor. This is particularly pronounced when F. sporotrichioides CF is added. However, this effect was not observed when F. oxysporum CF was applied. This may be due to the differences in the composition of the mycotoxin complex of this mushroom species from the others used in the study. For further work on the technology of creating oat forms with resistance to mycotoxins of the Fusarium genus fungi, it is assumed to use a level of selective pressure not lower than 40%.

Embryo Rescue in Plant Breeding.

Embryo rescue (ER) techniques are among the oldest and most successful in vitro tissue culture protocols used with plant species. ER refers to a series of methods that promote the development of an immature or lethal embryo into a viable plant. Intraspecific, interspecific, or intergeneric crosses allow the introgression of important alleles of agricultural interest from wild species, such as resistance or tolerance to abiotic and biotic stresses or morphological traits in crops. However, pre-zygotic and post-zygotic reproductive barriers often present challenges in achieving successful hybridization. Pre-zygotic barriers manifest as incompatibility reactions that hinder pollen germination, pollen tube growth, or penetration into the ovule occurring in various tissues, such as the stigma, style, or ovary. To overcome these barriers, several strategies are employed, including cut-style or graft-on-style techniques, the utilization of mixed pollen from distinct species, placenta pollination, and in vitro ovule pollination. On the other hand, post-zygotic barriers act at different tissues and stages ranging from early embryo development to the subsequent growth and reproduction of the offspring. Many crosses among different genera result in embryo abortion due to the failure of endosperm development. In such cases, ER techniques are needed to rescue these hybrids. ER holds great promise for not only facilitating successful crosses but also for obtaining haploids, doubled haploids, and manipulating the ploidy levels for chromosome engineering by monosomic and disomic addition as well substitution lines. Furthermore, ER can be used to shorten the reproductive cycle and for the propagation of rare plants. Additionally, it has been repeatedly used to study the stages of embryonic development, especially in embryo-lethal mutants. The most widely used ER procedure is the culture of immature embryos taken and placed directly on culture media. In certain cases, the in vitro culture of ovule, ovaries or placentas enables the successful development of young embryos from the zygote stage to maturity.

Comparison of tissue culture-induced variation in triticale regenerants obtained by androgenesis and somatic embryogenesis

Triticale is becoming an increasingly important livestock crop production. This is evidenced by increasing triticale-producing areas and by improved yields. In addition, meeting the increasing demand for cereals involves the introduction of high-yielding and stress-resistant varieties into breeding. In vitro culture techniques can accelerate the development of new varieties. Therefore, it seems extremely important to develop efficient plant regeneration methods through in vitro cultures and to understand the mechanisms involved in gaining regenerants. Obtaining regenerants of triticale through somatic embryogenesis and androgenesis may lead to tissue culture-induced variation. In the present study, we compared regenerants obtained in both regeneration systems (anther and immature zygotic embryo cultures), considering the level of genetic and epigenetic changes observed in different DNA sequence contexts for methylated cytosine (CG, CHG, CHH). The changes concerning the DNA sequence (so-called sequence variation) and the changes concerning the DNA methylation patterns, i.e., the removal of methylated cytosine (DNA demethylation) and the introduction of methylation to cytosine (de novo DNA methylation), were analyzed. We observed that regenerants derived via somatic embryogenesis and androgenesis differ notably for demethylation in the symmetrical CG sequence context and de novo methylation in the asymmetrical CHH context. These changes may be related to the reprogramming of microspore development from gametophytic to sporophytic and lack of such process in zygotic embryos.

Agronomic Traits and Genetic Fidelity of Four Cocoa Clones Derived from Somatic Embryogenesis Culture

Morphological and genetic characterization of MCBC1, PBC230, KKM22 and KKM4 cocoa clones derived from staminode and immature zygotic embryo culture were compared with those conventionally grafted. Somatic embryogenesis culture successfully produced true-to-type progenies of elite cocoa clones of MCBC1, PBC230, KKM22 and KKM4 (from staminode culture). Phenotype variations (p < 0.05) were observed only in KKM4 clone from immature zygotic embryo culture which exhibited lower quantities in the fresh pod weight, number of flat beans per pod, seed length, seed width and individual seed weight. The genetic stability of the cultured clones was tested using fragment analysis with 12 SSR primers to validate these results. Eleven of these SSR primers detected mutations only in the allelic profiles of KKM4 clone from immature zygotic embryo. These results validated those variations in KKM4 clones of immature zygotic embryo culture were due to interactions between genotypic and explant types. Unfortunately, these variations were negative attributes to cocoa productivity. Thus, it is suggested that successful production of true-to-type KKM4 cocoa clone should consider other means of propagation including modification of the culture conditions.
 HIGHLIGHTS
 
 It is crucial to evaluate and confirm the quality of the immature zygotic and staminode regenerated cocoa plant before using the culture technique for commercial production
 The quality can be detected in both phenotype and genotype by comparing the regenerated cocoa plant with those regenerated from the conventional asexual propagation method of grafting
 Phenotype and genotype differences were only observed in KKM4 clone regenerated from immature zygotic embryo culture and thus validated that this clone exhibited variation
 KKM4 clone from immature zygotic embryo culture which has lower quantities in the fresh pod weight, number of flat beans per pod, seed length, seed width and individual seed weight confirmed the negative effect of using immature zygotic embryo explant for KKM4 clone propagation. It is suggested to modify the culture technique
 
 GRAPHICAL ABSTRACT

Optimization of in vitro propagation protocol of Dendrobium heterocarpum Wall. ex. Lindl. and clonal genetic fidelity assessment of the regenerates: An orchid of horticultural and medicinal importance

• Dendrobium heterocarpum is an orchid having horticultural and medicinal value. • Optimization of factors for in vitro propagation protocol from immature embryos. • Use of low-cost substrata for in vitro regenerates acclimatization. • Genetic fidelity assessment of regenerates using RAPD, DAMD and SCoT markers. Dendrobium heterocarpum Wall. ex Lindl. (Orchidaceae) is an orchid having medicinal as well as ornamental values. The natural population of the species is downsized due to different anthropogenic activities. The present study was undertaken to standardize in vitro propagation technique of the species through immature embryo culture and optimizing different factors. Further, the study demonstrates the use of low cost substrata for hardening of in vitro regenerates. Immature embryos of 5–8 months after pollination (MAP) were cultured on three different media enriched with different organic carbon sources and four plant growth regulators at varied concentrations. The embryos of 8 MAP registered ∼95% germination on MS medium fortified with sucrose (3%, w/v), kinetin (3 μM/L). The resultant PLBs differentiated to young plantlets on the same initiation medium. Subsequently, the young plantlets proliferated and as many as ∼18 shoots were produced on MS medium containing sucrose and kinetin and α-naphthalene acetic acid (3 + 12 μmol/L) in combination. The regenerates were primary hardened by culturing on low cost substrata (coconut husk + wood bark at 1:1 ratio) fortified with 1/4th MS salt solution for one wk, subsequently maintaining to normal room temperature under 75% diffused light for ∼3–4 wk before transferring to potting mix. About 87% transplants survived post transfer. Clonal fidelity of the regenerants was also evaluated using RAPD, DAMD and SCoT markers. High degree of monomorphic percentage (96.89%) and low polymorphism (3.11%) was recorded between the mother plant and in vitro regenerates; thus, showing genetic homogeneity of the in vitro raised plants.

PRODUCTION MUTANT LINES OF TURNIP RAPE (BRASSICA RAPA) AND ITS INTERSPECIFIC HYBRIDS IN THE ISOLATED MICROSPORE CULTURE

This article presents the results of research on the production of mutant doubled haploids of turnip rape (Brassica rapa L.ssp.Oleifera), as well as its interspecies hybrids with rapeseed (Brassica napus). An  immature embryo culture  was used to increase the productivity of inter-specific hybridization. Studies have obtained interspecific hybrids of turnip rape with rapeseed. The largest number of plants was observed in hybrids where the mother plant was rapeseed. Furthermore, chemical mutagenesis was used to increase the genetic diversity of studied materials. Ethyl methanesulfonate (EMS) has been applied to the treatment of embryos produced in the isolated microspores culture  of turnip rape and its interspecies hybrids. Results of embryo regeneration against mutagen on a nutrient medium B5 with gibberellic acid showed that the concentration of 8 mM EMS stimulated the regeneration process in turnip rape cultivars (Zolotistaya - 72% and Yantarnaya - 88%) and showed superior results compared to a low concentration (4 mM). For hybrids compared to high concentration (8mM), a low concentration of 4mM EMS showed superior results. New mutant lines have been obtained as the starting material for selection in the creation of new domestic canola cultivars.

In Vitro Immature Embryo Culture of Paeonia ostii ‘Feng Dan’

Paeonia ostii ‘Feng Dan’ is an economically important, multipurpose woody plant in terms of its medical, ornamental, and oil values; however, there is a noticeable contradiction between the increasing demands and the lack of excellent germplasm resources because of traditional breeding and propagation approaches. In vitro embryo culture is an attractive option for this issue. This study presents a protocol for in vitro immature embryo culture in P. ostii ‘Feng Dan’, which involves two steps: 1) immature seeds at 30 days after anthesis (DAA) (cellularization stage of endosperm, proembryo stage) or after being cultured in vitro for cotyledon embryo formation (upward micropyle with placenta was the best inoculation method with the highest ratio of seed with cotyledon embryo of 66.67%); and 2) seedling establishment was realized within 7 months via embryo (at 40 DAA or after) germination, shoot induction, rooting, and acclimatization. The multiplication potential was increased with embryo maturity. This protocol provides an available reference for embryo rescue and propagation of tree peony and will be beneficial to shortening the breeding cycle.

Integrated system of selection of sunflower lines with economically valuable traits and adaptability of their F1 hybrids

Purpose. Determine the ecological plasticity and productivity of F1 sunflower hybrids created on the basis of maternal and parental lines, selected according to an accelerated selection system of lines resistant to herbicides (imidazoline and sulfonylurea groups) and broomrape (Orobanche cumana Wallr.). Methods. Statistical analysis of F1 sunflower hybrids was carried out using the methods of variation statistics, regression and analysis of variance using the Microsoft Office Excel 2016 application package. Molecular biological, biotechnological and classical selection methods were used for the accelerated system of line selection. Thus, for the purpose of targeted selection of sunflower sterility fixers, we used HRG01 molecular SCAR marker to identify the gene for the restoration of pollen fertility (Rf1). To accelerate the creation of parental lines resistant to tribenuron-methyl, we used a culture of immature embryos in vitro. Results. The results of testing of F1 sunflower hybrids at Kyiv, Chernihiv, Cherkasy (Uman and Shpolianskyi districts), Khmelnytskyi, Kharkiv, Kherson and Odesa regions. The hybrids were created on the basis of selected lines, chosen according to an accelerated selection system for herbicide-resistant lines (imidazoline (IMI-hybrids) and sulfonylurea (SU-hybrids) groups) and broomrape (Orobanche cumana Wall). The standards for comparison with hybrids were: for IMI hybrids – hybrids ‘NK Neoma’ (Syngenta) and ‘ES Genesis’ (Euralis), and for SU-hybrids – ‘SY Sumiko’ (Syngenta) and ‘P64LE25’ (Pioneer). As a result, it was found that among SU-hybrids, UA 2/106 had a 3.9% higher yield when compared to the standards (‘SY Sumiko’ and ‘P64LE25’). And for IMI-hybrids it was found that hybrids UA 1/67, UA 1/66, UA 1/84 have the same yield of 2.76 t/ha as the ‘NK Neoma’ standard. IMI hybrids UA 1/92, UA 1/102 have the same yield of 2.91 t/ha as ‘ES Genesis’. Conclusions. F1 hybrids were created on the basis of the original breeding material selected due to the accelerated system of sunflower lines selection. The hybrids were analyzed according to the yield indicator. The most productive among the tested SU-hybrids was UA 2/106 hybrid, among the IMI hybrids – UA 1/67, UA 1/66, UA 1/84, UA 1/92 and UA 1/102.

Reproductive Characteristics of the Selected Cocoa (Theobroma cacao L.) Clones after Regenerated from the Somatic Embryogenesis Culture

This study was conducted to evaluate the reproductive characteristics of 4 elite cocoa clones (MCBC1, PBC230, KKM22 and KKM4) propagated via somatic embryogenesis culture. From the findings, all clones have similar reproductive characteristics with clones from conventional grafted. However, only KKM4 clone from immature zygotic embryo culture produced the shortest staminode to style distance of 1.83 mm. This consequently influenced flower stability by reducing the efficiency of pollination by insects. It was found that this clone also has the highest number of flowers drop after anthesis (5 flowers) and lowest production of cherelle (5 cherelles). Further observation revealed that floral development from first bud visible (BBCH51) to flower anthesis (BBCH68) of all clones took around 31 days. These cocoa flowers which remained receptive soon after anthesis at 10 am (day-31) until the next day (day-32) suggesting 2 days’ period of receptivity.
 HIGHLIGHTS
 
 It is crucial to assess the presence of off-type characteristics in the reproductive organ structure such as the distance between staminode to style, period of reproductive cycle and stigmatic receptivity of cocoa clones regenerated from somatic embryogenesis
 The converging and parallel type of staminode to style distances are the ideal flower spatial arrangements for the optimal pollination in cocoa plant compared to splay type
 Only KKM4 clone propagated from immature zygotic embryo culture showed variation in the distance between staminode to style distance and this caused pollination failure by insect which then consequently caused minimum cherelle production
 All regenerated cocoa clones observed with typical period of the reproductive cycle and stigmatic receptivity
 
 GRAPHICAL ABSTRACT

Assessment of Egyptian Maize Hybrids for Their Capability of Plant Regeneration using Immature Embryo Culture

The aim of this research was to determine the regeneration capability of five maize hybrids (Syngenta hybrid, Three-way hybrid (324), Single cross hybrid P344 (Pioneer), Single cross hybrid 30N 11 (Pioneer) and Three-way hybrid (321)) using immature embryo explants with four medium protocols. The analysis of variance for in vitro culture traits of explant response indicated that callus induction, callus weight and embryogenic callus significantly responded to maize genotypes, medium protocols and their interaction. Also differences among maize hybrids were highly significant for shoot formation (%), while they were not significant for root formation (%). The four medium protocols indicated highly significant differences for root formation (%) and shoot formation (%). Three-way hybrid (324) and Single cross hybrid 30 N11(Pioneer) had the highest induction frequency of shoot formation on medium protocol D. Both maizehybrids on medium protocol D could be successfully utilized for the in vitro selection studies for maize improvement.

Construction of transgenic Ipomoea obscura that exhibits new reddish leaf and flower colors due to introduction of β-carotene ketolase and hydroxylase genes.

Ipomoea obscura, small white morning glory, is an ornamental plant belonging to the family Convolvulaceae, and cultivated worldwide. I. obscura generates white petals including a pale-yellow colored star-shaped center (flower vein). Its fully opened flowers were known to accumulate trace amounts of carotenoids such as β-carotene. In the present study, the embryogenic calli of I. obscura, were successfully produced through its immature embryo culture, and co-cultured with Agrobacterium tumefaciens carrying the β-carotene 4,4'-ketolase (crtW) and β-carotene 3,3'-hydroxylase (crtZ) genes for astaxanthin biosynthesis in addition to the isopentenyl diphosphate isomerase (idi) and hygromycin resistance genes. Transgenic plants, in which these four genes were introduced, were regenerated from the infected calli. They generated bronze (reddish green) leaves and novel petals that exhibited a color change from pale-yellow to pale-orange in the star-shaped center part. Especially, the color of their withered leaves changed drastically. HPLC-PDA-MS analysis showed that the expanded leaves of a transgenic line (T0) produced astaxanthin (5.2% of total carotenoids), adonirubin (3.9%), canthaxanthin (3.8%), and 3-hydroxyechinenone (3.6%), which indicated that these ketocarotenoids corresponded to 16.5% of the total carotenoids produced there (530 µg g-1 fresh weight). Furthermore, the altered traits of the transgenic plants were found to be inherited to their progenies by self-crossing.

Agronomic Performance and Genetic Fidelity of the Selected Elite Cocoa Clones Derived from Somatic Embryogenesis Culture

This study was conducted to compare the agronomic performance of four elite cocoa clones (MCBC1, KKM22, KKM4 and PBC230) regenerated from staminode and immature zygotic embryo culture with conventional grafted cocoa clones. From the results, it was found that the KKM4 clone propagated from immature zygotic embryo culture exhibited variations in the fresh pod weight (339.6 g), fresh individual seed weight (4.13 g) and number of flat beans per pod (4 beans) compared with the rest of the regenerated clones. The genetic stability of the somatic embryogenesis cultured clones and the donor clones was then tested using fragment analysis with five SSR primers, i.e. mTcCIR7, mTcCIR18, mTcCIR22, mTcCIR33 and mTcCIR40. Four of these primers identified variations in the allele size and allele addition in KKM4 clone from immature zygotic embryo. Molecular analysis validated that the difference in agronomic performance of the KKM4 clone from immature zygotic embryo culture was due to genetic mutation created during the immature zygotic embryo culture process.

Реакция семян и эксплантов льна на осмотический стресс

Abstract. Purpose. Obtaining experimental data for the development of methods for obtaining fiber flax forms resistant to stress factors (drought) by cell selection methods. Methods. The work was carried out in the Tver region in the laboratory of breeding technologies in 2017–2019. The flax varieties Varbara, Belinka, LM-98, Svetoch, Diplomat, Symfonia were used as objects of research. The seeds were obtained from the National Flax Collection Federal state budgetary scientific institution “Federal scientific center of lubya kultur”. The effect of sucrose solution on the length of the primary root was detected at concentrations of 0, 8.7, 14.9 %. To assess the energy of seed germination under osmotic stress, the concentration of sucrose was reduced and the range from 0 (control) to 9 % was considered. Results. Sucrose as a selective agent in the culture of immature flax embryos in vitro at a concentration of 5.0–7.0 % can be selective only for certain genotypes, for example, the Svetoch variety. When culturing callus tissues, using mannitol as an osmotic at concentrations of 0; 30.0; 36.4; 37.0; 37.4; 38.0 mg/l, selection of resistant callus cells with subsequent formation in meristematic foci of adventive buds and shoots can be carried out on media containing 30.0 or 36.4 mg/l osmotic. The scientific novelty of the research lies in the fact that the method for obtaining in vitro resistant explants to osmotic stress for flax is being developed for the first time.