Embryogenic Structures Research Articles

Effect of gamma irradiation on proliferation and growth of friable embryogenic callus and in vitro nodal cuttings of ugandan cassava genotypes.

Cassava (Manihot esculenta Crantz) production and productivity in Africa is affected by two viral diseases; cassava mosaic disease (CMD) and cassava brown streak disease (CBSD). Induced mutagenesis of totipotent/embryogenic tissues or in vitro plant material can lead to the generation of CMD and/or CBSD tolerant mutants. To massively produce non-chimeric plants timely and with less labor, totipotent cells or tissues are a pre-requisite. This study aimed to determine the effect of gamma radiation on the proliferation and growth of friable embryogenic callus (FEC) and in vitro nodal cuttings respectively. To obtain FEC, 2-6 mm sized leaf lobes of nine cassava genotypes were plated on Murashige and Skoog (MS) basal media supplemented with varying levels (37, 50, 70, 100) μM of picloram for production of organized embryogenic structures (OES). The OES of five cassava genotypes (Alado, CV-60444, NASE 3, NASE 13 and TME 204) were crushed and plated in Gresshoff and Doy (GD) basal media in combination with the amino acid tyrosine in varying concentrations for FEC production. FEC from five cassava genotypes and in vitro nodal cuttings of nine genotypes were irradiated using five different gamma doses (0, 5, 10, 15, 20 and 25 Gy) at a dose rate of 81Gy/hr. The lethal dose (LD)50 was determined using the number of roots produced and flow cytometry was done to determine the ploidy status of plants. The highest production of OES was noted in Alado across varying picloram concentrations, while TME 204 obtained the highest amount of FEC. The irradiated FEC gradually died and by 28 days post irradiation, FEC from all five cassava genotypes were lost. Conversely, the irradiated in vitro nodal cuttings survived and some produced roots, while others produced callus. The LD50 based on number of roots varied from genotype to genotype, but plants remained diploid post-irradiation. Accordingly, the effect of gamma irradiation on Ugandan cassava genotypes (UCGs) was genotype-dependent. This information is foundational for the use of in vitro tissues as target material for cassava mutation breeding.

The different response of Brassica napus genotypes to microspore embryogenesis induced by heat shock and trichostatin A is not determined by changes in cell wall structure and composition but by different stress tolerance

AbstractDuring microspore embryogenesis, microspores are induced to develop into haploid embryos. In Brassica napus, microspore embryogenesis is induced by a heat shock (HS), which initially produces embryogenic structures with different cell wall architectures and compositions, and with different potentials to develop into embryos. The B. napus DH4079 and DH12075 genotypes have high and very low embryo yields, respectively. In DH12075, embryo yield is greatly increased by combining HS and the histone deacetylase (HDAC) inhibitor trichostatin A (TSA). However, we show that HS + TSA inhibits embryogenesis in the highly embryogenic DH4079 line. To ascertain why TSA has such different effects in these lines, we treated DH4079 and DH12075 microspore cultures with TSA and compared the cell wall structure and composition of the different embryogenic structures in both lines, specifically the in situ levels and distribution of callose, cellulose, arabinogalactan proteins and high and low methyl‐esterified pectin. For both lines, HS + TSA led to the formation of cell walls unfavorable for embryogenesis progression, with reduced levels of arabinogalactan proteins, reduced cell adhesion of inner walls and altered pectin composition. Thus, TSA effects on cell walls cannot explain their different embryogenic response to TSA. We also applied TSA to DH4079 cultures at different times and concentrations before HS application, with no negative effects on embryogenic induction. These results indicate that DH4079 microspores are hypersensitive to combined TSA and HS treatments, and open up new hypotheses about the causes of such hypersensitivity.

New Epigenetic Modifier Inhibitors Enhance Microspore Embryogenesis in Bread Wheat

The use of doubled haploid (DH) technology enables the development of new varieties of plants in less time than traditional breeding methods. In microspore embryogenesis (ME), stress treatment triggers microspores towards an embryogenic pathway, resulting in the production of DH plants. Epigenetic modifiers have been successfully used to increase ME efficiency in a number of crops. In wheat, only the histone deacetylase inhibitor trichostatin A (TSA) has been shown to be effective. In this study, inhibitors of epigenetic modifiers acting on histone methylation (chaetocin and CARM1 inhibitor) and histone phosphorylation (aurora kinase inhibitor II (AUKI-II) and hesperadin) were screened to determine their potential in ME induction in high- and mid-low-responding cultivars. The use of chaetocin and AUKI-II resulted in a higher percentage of embryogenic structures than controls in both cultivars, but only AUKI-II was superior to TSA. In order to evaluate the potential of AUKI-II in terms of increasing the number of green DH plants, short and long application strategies were tested during the mannitol stress treatment. The application of 0.8 µM AUKI-II during a long stress treatment resulted in a higher percentage of chromosome doubling compared to control DMSO in both cultivars. This concentration produced 33% more green DH plants than the control in the mid-low-responding cultivar, but did not affect the final ME efficiency in a high-responding cultivar. This study has identified new epigenetic modifiers whose use could be promising for increasing the efficiency of other systems that require cellular reprogramming.

Enhanced Coconut Somatic Embryogenesis of Unfertilized Ovary Derived Callus through Improved Media Composition

Coconut is an economically demanding plantation crop in Sri Lanka, having an out-breeding nature with no vegetative propagation methods. Propagation by seeds generates heterogenous population and it is not sufficient to fulfill the increasing demand for coconut plantlets. Therefore, in vitro propagation is the ultimate approach of producing clones of coconut from the mother palms of interest. Coconut in vitro propagation is achieved through somatic embryogenesis and improvements of this process will easily cater the demand for plantlets. Hence, the main objective of the study was to enhance somatic embryogenic pathway of coconut using two different auxins (2,4-Dichlorophenoxyacetic acid [2,4-D] and 3,6-Dichloro-2-methoxybenzoic acid [Dicamba]) at the concentration of 300 μM. These auxins were tested alone and in combination with calcium ionophore (A23187) and 22(S),23(S)-homobrassinolide. Translucent structures or the ear-like structures obtained from culturing of unfertilized ovaries were used for the treatments. After six weeks of the culture establishment number of embryogenic structures per one translucent structure were counted. Each treatment produced embryogenic structures but a significantly greater number of embryogenic structures were developed with 2,4-D containing media than dicamba. The results revealed the potential of using 300 μM 2,4-D with calcium ionophore (A23187) and 22(S), 23(S)-homobrassinolide to improve the somatic embryogenic pathway of unfertilized ovary derived callus of coconut.

Somatic embryo production and GFP genetic transformation in elite Ugandan cassava genotypes

Friable embryogenic callus (FEC) production is a prerequisite for the genetic transformation of cassava (Manihot esculenta Crantz). This process necessitates the development of organized embryogenic structures (OES) as a precursor. However, the production of OES and FECs in cassava is dependent on the genotype. This study aimed to optimize somatic embryo production in elite Ugandan cassava genotypes (NASE13, NASE19, and NAROCASS1) and demonstrate genetic transformation using the enhanced green fluorescent protein (eGFP) gene. OES were induced by initiating leaf-lobe explants on Murashige and Skoog (MS) media at various concentrations of 24D and picloram followed by friable embryogenic callus (FEC) production and regeneration. There were highly significant differences among the different media compositions in the percentage response to OES formation (p < 0.001). NASE 13 exhibited the highest regeneration frequency of 38.4%; while NAROCASS1 had the lowest regeneration frequency of 10.5% recorded. The presence of GFP gene in NAROCASS1 was confirmed by polymerase chain reaction (PCR) analysis of the transformed lines. Compared to existing studies, this research pioneers genotype-specific optimization for somatic embryo production and successful genetic transformation in Ugandan cassava variaties. The utilization of these genotypes, already embraced by farmers, presents promising opportunities to enhance them with desirable traits, further augmenting cassava cultivation.

Estimation of the efficiency of &lt;i&gt;in vitro&lt;/i&gt; anther culture method for obtaining haploid triticale plants (×&lt;i&gt;Triticosecale&lt;/i&gt; wittmack)

Anther culture is one of the most popular methods for obtaining haploid triticale plants. The current paper has presented the study results of the efficiency of the method for obtaining haploid plants in promising winter triticale forms bred by the Federal Agricultural Research Center of South-East. There has been estimated haploproduction parameters for the studied genotypes on the nutrient media C-17 and P-2. There were no genotypes unresponsive to androgenesis. There has been found that the medium Potato-2 had a significant positive effect on the total number of embryogenic structures per 100 cultivated anthers in four of the six studied genotypes when the index increased in 1–3 times. The mean value of the EC/100KP indicator was 23.3 with a variation of 6.4-75.1. The largest number of ES (45.8 and 75.1) was found in genotype No. 96 (F5 Zimogor/win.br.wheat L.39) on induction media C-17 and P-2. The genotypes No. 95 and No. 97 were characterized by the lowest indicators F4 DH13/ win.br.wheat ‘Atkara’//Vodoley/ADP-2///Colina and F5 MAG/Vodoley//TI-17. Plant regeneration varied from 3.4 to 22.1 with an average value of 12.6 per 100 EC. The mean number of green plants per 100 EC was 3.9, varying from 0.4 to 12.1. The proportion of green plants, depending on the genotype, varied from 5.0 to 76.9 %. Two-way analysis of variance has identified the dominant role of the genotype at all stages of haploproduction. The proportion of effect of the nutrient medium was statistically significant, but insignificant in comparison with the genotype. There has been established that, along with genotypic dependence and albinism, the limiting factor of the method is a low frequency of regeneration of embryo-like structures. In total, there were obtained 949 regenerated plants out of 7538 embryogenic structures, of which 326 were green. The ratio of green plants to albinos was 1:2.

Efficient Plant Regeneration System from Leaf Explant Cultures of Daphne genkwa via Somatic Embryogenesis.

This study aimed to establish an efficient plant regeneration system from leaf-derived embryogenic structure cultures of Daphne genkwa. To induce embryogenic structures, fully expanded leaf explants of D. genkwa were cultured on Murashige and Skoog (MS) medium supplemented with 0, 0.1, 0.5, 1, 2, and 5 mg·L-1 2,4-dichlorophenoxyacetic acid (2,4-D), respectively. After 8 weeks of incubation, the highest frequency of embryogenic structure formation reached 100% when the leaf explants were cultivated on MS medium supplemented with 0.1 to 1 mg·L-1 2,4-D. At higher concentrations of 2,4-D (over 2 mg·L-1 2,4-D), the frequency of embryogenic structure formation significantly declined. Similar to 2,4-D, indole butyric acid (IBA) and α-naphthaleneacetic acid (NAA) treatments were also able to form embryogenic structures. However, the frequency of embryogenic structure formation was lower than that of 2,4-D. In particular, the yellow embryonic structure (YES) and white embryonic structure (WES) were simultaneously developed from the leaf explants of D. genkwa on culture medium containing 2,4-D, IBA, and NAA, respectively. Embryogenic calluses (ECs) were formed from the YES after subsequent rounds of subculture on MS medium supplemented with 1 mg·L-1 2,4-D. To regenerate whole plants, the embryogenic callus (EC) and the two embryogenic structures (YES and WES) were transferred onto MS medium supplemented with 0.1 mg·L-1 6-benzyl aminopurine (BA). The YES had the highest plant regeneration potential via somatic embryo and shoot development compared to the EC and WES. To our knowledge, this is the first successful report of a plant regeneration system via the somatic embryogenesis of D. genkwa. Thus, the embryogenic structures and plant regeneration system of D. genkwa could be applied to mass proliferation and genetic modification for pharmaceutical metabolite production in D. genkwa.

Calcium dynamics and modulation in carrot somatic embryogenesis.

Free calcium (Ca2+) is a pivotal player in different in vivo and in vitro morphogenic processes. In the induction of somatic embryogenesis, its role has been demonstrated in different species. In carrot, however, this role has been more controversial. In this work, we developed carrot lines expressing cameleon Ca2+ sensors. With them, Ca2+ levels and distribution in the different embryogenic structures formed during the induction and development of somatic embryos were analyzed by FRET. We also used different chemicals to modulate intracellular Ca2+ levels (CaCl2, ionophore A23187, EGTA), to inhibit calmodulin (W-7) and to inhibit callose synthesis (2-deoxy-D-glucose) at different times, principally during the first stages of embryo induction. Our results showed that high Ca2+ levels and the development of a callose layer are markers of cells induced to embryogenesis, which are the precursors of somatic embryos. Disorganized calli and embryogenic masses have different Ca2+ patterns associated to their embryogenic competence, with higher levels in embryogenic cells than in callus cells. The efficiency of somatic embryogenesis in carrot can be effectively modulated by allowing, within a range, more Ca2+ to enter the cell to act as a second messenger to trigger embryogenesis induction. Once induced, Ca2+-calmodulin signaling seems related with the transcriptional remodeling needed for embryo progression, and alterations of Ca2+ or calmodulin levels negatively affect the efficiency of the process.

Effect of exogenous polyamines on coconut (&lt;em&gt;Cocos nucifera&lt;/em&gt; L.) embryogenic callus multiplication

Major bottlenecks of coconut in vitro culture are poor plant regeneration rate, severe browning and premature necrosis of cultured tissue, and heterogeneous response of individual palms and explants to in vitro culture conditions. Among them, tissue browning is a common and often severe problem in coconut in vitro culture systems which results in death of explant/ callus ultimately. This experiment was carried out to enhance the in vitro multiplication of coconut, which is a highly recalcitrant species to in vitro culture through exogenously added polyamines in the media. The polyamines are important for in vitro cell division, cell growth and to delay senescence. In the present study, unfertilized ovary derived calli were cultured on Y3 basal medium supplemented with sucrose (5%), 2,4-D, phytagel (3 gL-1), activated charcoal (2.5 gL-1), and polyamine. Three polyamine types (O.lmM spermine, 1.0 mM putrescine and 0.5mM spermidine) were tested in combination with two 2,4-D concentrations (0.30 and 0.60 mM) in order to enhance coconut in vitro multiplication. All the cultures were incubated in dark at 26±2 °C. The embryogenic structures, embryogenic callusing, non-embryogenic callusing, and browning were recorded separately for each treatment after five weeks from the culture establishment. The polyamine treatments did not have significant effects on frequencies of embryogenic structures, embryogenic callus, non-embryogenic callus and browned callus formation at the initial stages of coconut somatic embryogenesis irrespective of the tested 2,4-D concentrations. Furthermore, the results indicated that decreased 2,4-D levels have significantly reduced browning, resulting 44.79 % browning frequency (0.8-fold lesser browning) in media supplemented with 0.30 mM 2,4-D. However, the potential effects of exogenously added polyamines at the initial stages of coconut somatic embryogenesis could be delivered during the latter stages of the somatic embryo genesis as previously reported in other experiments. Thus, continuous subculture may be necessary.

Somatic embryogenesis and plant regeneration from transverse thin cell layers of adult peach palm (Bactris gasipaes) lateral offshoots.

In this work, we report a successful protocol to obtain in vitro peach palm (Bactris gasipaes Kunth) “Diamantes 10” plants through somatic embryogenesis from transverse thin cell layer (TCL) explants, dissected from three sections (basal, medial, and apical) of lateral offshoots of adult plants cultured on different concentrations of 4-amino-3,5,6-trichloropicolonic acid (picloram). After swelling and development of primary callus in all treatments, without any strong effect of explant origin or picloram concentration, it was possible to observe the formation of embryogenic structures and the exact point from where they developed. Browning was also observed and correlated to the induction treatments, although it was not an impairment for the production of embryogenic structures. Subsequent maturation and conversion of somatic embryos into plantlets allowed their acclimatization 17 months after culture initiation (ACI), which was quicker than previous reports with juvenile tissues (from embryos or seed-germinated plantlets). To the best of our knowledge, this is the first report on peach palm regeneration through somatic embryogenesis from TCL explants from adult plants and could constitute, after fine-tuning the acclimatization stage, a tool for mass clonal propagation of elite genotypes of this open-pollinated crop, as well as for the establishment of conservation strategies of in situ gene bank plant accessions endangered due to aging and other threats.

Cryopreservation of Holm Oak Embryogenic Cultures for Long-Term Conservation and Assessment of Polyploid Stability.

Holm oak populations are severely affected by oak decline syndrome, and reliable methods of conserving the plant material are required. A vitrification-based cryopreservation method was used for the first time for the long-term conservation of holm oak embryogenic cultures. Successful cryopreservation was achieved after determining the best developmental stage of the somatic embryos used and the optimal incubation period in plant vitrification solution 2 (PVS2). Embryos were recovered from individual nodular embryogenic structures (NES) derived from four embryogenic lines after preculture on a medium containing 0.3 M sucrose, incubation in PVS2 vitrification solution for 15 min at 25 °C and direct immersion in liquid nitrogen (LN). Embryo recovery rates of 16.7–63.3% were obtained after cryostorage for four years in LN. In addition to the embryo developmental stage and the PVS2 treatment time, the genotype can also significantly affect embryo recovery after LN storage. There were no significant differences in plant regeneration or polyploid stability between somatic embryos and plants derived from control embryos (not cryopreserved) and cryopreserved embryos. The findings indicate that embryo proliferation, plant conversion and polyploid stability are maintained in material recovered from the vitrification solution and subsequently cryopreserved.

Development of haploids in the winter bread wheat anthers

In the modern world, the use of isolated anther cultivation technology is currently an integral part of the wheat breeding process. The development of haploids in the winter bread wheat anthers will allow obtaining new forms of wheat in the shortest possible time and without large areas. The purpose of the current study was to estimate the F3 winter bread wheat hybrids according to the anthers’ sensitivity to androgenesis and plant regeneration in vitro and to identify the factors affecting the yield of haploid production. There has been studied the ability to androgenesis in vitro in the anthers of four winter bread wheat hybrids of intensive and semi-intensive type of the FSBSI “ARC “Donskoy”. There has been assessed the role of the mineral composition of three induction nutrient media N6, W14 and NPB-99. There has been established a correlation between the main stages of development of haploids and a genotype. The highest regeneration rate of green plants was obtained in the sample F3 623 of intensive type (3.3%). The most suitable medium for androgenesis of the winter bread wheat anthers in vitro is NPB-99. Since the genotype F3 623 of intensive type demonstrated high values of haploid production capacity, it could be successfully used in breeding programs for the rapid production of homozygous wheat anther lines in vitro. Using two-way analysis of variance, there has been identified a correlation between the effects of a genotype, nutrient medium and their interaction with the main parameters of haploid formation in winter wheat. The formation of embryogenic structures is mainly associated with the effect of a genotype (46.52%). The proportion of the nutrient composition of the medium was low (1.82%), and the correlation factor was 2.1%. The genotype had the greatest effect on the indicator of the regenerants’ number. The nutrient medium had little effect. Regarding the regeneration of green plants, which is the main indicator of the haploid production, the share of a genotype effect was the largest (47.32%). The contribution of the medium and the correlation of factors were less important, but statistically significant.

Cell Wall Composition and Structure Define the Developmental Fate of Embryogenic Microspores in Brassica napus.

Microspore cultures generate a heterogeneous population of embryogenic structures that can be grouped into highly embryogenic structures [exine-enclosed (EE) and loose bicellular structures (LBS)] and barely embryogenic structures [compact callus (CC) and loose callus (LC) structures]. Little is known about the factors behind these different responses. In this study we performed a comparative analysis of the composition and architecture of the cell walls of each structure by confocal and quantitative electron microscopy. Each structure presented specific cell wall characteristics that defined their developmental fate. EE and LBS structures, which are responsible for most of the viable embryos, showed a specific profile with thin walls rich in arabinogalactan proteins (AGPs), highly and low methyl-esterified pectin and callose, and a callose-rich subintinal layer not necessarily thick, but with a remarkably high callose concentration. The different profiles of EE and LBS walls support the development as suspensorless and suspensor-bearing embryos, respectively. Conversely, less viable embryogenic structures (LC) presented the thickest walls and the lowest values for almost all of the studied cell wall components. These cell wall properties would be the less favorable for cell proliferation and embryo progression. High levels of highly methyl-esterified pectin are necessary for wall flexibility and growth of highly embryogenic structures. AGPs seem to play a role in cell wall stiffness, possibly due to their putative role as calcium capacitors, explaining the positive relationship between embryogenic potential and calcium levels.

Efectos de reguladores de crecimiento para inducción de embriones somáticos a partir de diferentes explantes de Echinocactus parryi Engelm, una especie endémica y amenazada

Introduction: The list of threatened species is enhancing and needs to be revised by integrating plant tissue culture tools with conventional techniques that support the appropriate management of these species. Objective: To assess the effects of the growth regulators for the induction of somatic embryos from mature seeds, shoots, and compact green callus of Echinocactus parryi Engelm. and the histological analysis of the embryogenic structures. Materials and methods: A completely randomized design was utilized to evaluate three types of explants (apical, medium, and basal) cultured on basal Murashige &amp; Skoog media (MS) with different growth regulators concentrations (2, 4-D [dichlorophenoxy acetic acid], BAP [6-benzylaminopurine] and kinetin, at four levels: 0.5, 1, 1.5, and 2 mg∙L -1 ). Histological analysis of the embryogenic structures was performed. Results and discussion: The 2, 4-D induced both embryogenic and organogenic callus from seeds and shoot explants. The globular stage did not evolve to their maturity, presumably because of 2, 4-D accumulation. The compact callus explants were the more efficient to induce 19.2 somatic embryos per explant when they were cultured in the medium with 0.5 mg∙L -1 kinetin. However, the latest phases did not germinate, probably due to abnormalities generated by genetic and epigenetic changes in the DNA that can cause abnormal somatic embryos. The histology image demonstrated that the globular and torpedo structures were visible under a microscope showing stained nucleus and numerous starch grains. Conclusions: E. parryi is a species that can produce a high number of embryogenic structures, which represents a great potential to grow massive plants.

Efficient Genetic Transformation and Regeneration of a Farmer-Preferred Cassava Cultivar From Ghana.

Cassava is an important staple crop that provides food and income for about 700 million Africans. Cassava productivity in Africa is limited by viral diseases, mainly cassava mosaic disease (CMD) and cassava brown streak disease (CBSD). Genetic barriers such as high heterozygosity, allopolyploidy, poor seed set, and irregular flowering constrain the development of virus-resistant cassava varieties via conventional breeding. Genetic transformation represents a valuable tool to circumvent several challenges associated with the development of virus resistance and other valuable agronomic traits in cassava. The implementation of genetic transformation in many local African cultivars is limited either by the difficulty to produce friable embryogenic callus (FEC), low transformation, and/or regeneration efficiencies. Here, we report the successful induction of organized embryogenic structures (OES) in 11 farmer-preferred cultivars locally grown in Ghana. The production of high quality FEC from one local cultivar, ADI 001, facilitated its genetic transformation with high shoot regeneration and selection efficiency, comparable to the model cassava cultivar 60444. We show that using flow cytometry for analysis of nuclear ploidy in FEC tissues prior to genetic transformation ensures the selection of genetically uniform FEC tissue for transformation. The high percentage of single insertion events in transgenic lines indicates the suitability of the ADI 001 cultivar for the introduction of virus resistance and other useful agronomic traits into the farmer-preferred cassava germplasm in Ghana and Africa.

Vegetative Propagation of Phytophthora cinnamomi-Tolerant Holm Oak Genotypes by Axillary Budding and Somatic Embryogenesis

Holm oak (Quercus ilex) is one of the most widely distributed tree species in the Mediterranean basin. High mortality rates have been observed in holm oak populations in the southwest of the Iberian Peninsula as a result of oak decline syndrome. Selection and propagation of genotypes tolerant to this syndrome could aid the restoration of affected areas. In this article, we report micropropagation and conservation procedures based on axillary budding and somatic embryogenesis (SE) of holm oak plants, selected for their tolerance to Phytophthora cinnamomi—the main biotic factor responsible for oak decline. Forced shoots were obtained from potted plants of eight different genotypes, and used as stock material to establish in vitro shoot proliferation cultures. Reliable shoot proliferation was obtained in seven out the eight genotypes established in vitro, whereas multiplication rates were genotype-dependent. The highest rooting rates were obtained by culturing shoots for 24 h or 48 h on rooting induction medium containing 25 mg L−1 indole-3-butyric acid, followed by transfer to medium supplemented with 20 µM silver thiosulphate. Axillary shoot cultures can be successful conserved by cold storage for 12 months at 4 °C under dim lighting. Shoot tips, excised from axillary shoot cultures established from tolerant plants, were used as initial explants to induce SE. Somatic embryos and/or nodular embryogenic structures were obtained on induction medium with or without indole-acetic acid 4 mg L−1, in two out the three genotypes evaluated, and induction rates ranged between 2 and 4%. Plantlet recovery was 45% after two months cold stratification of somatic embryos and eight weeks of culture on germination medium. Vegetative propagation of P. cinnamomi-tolerant Q. ilex trees is a valuable milestone towards the restoration of disease-affected areas.

Live Imaging of embryogenic structures in Brassica napus microspore embryo cultures highlights the developmental plasticity of induced totipotent cells

Key messageIn vitro embryo development is highly plastic; embryo cell fate can be re-established in tissue culture through different pathways.In most angiosperms, embryo development from the single-celled zygote follows a defined pattern of cell divisions in which apical (embryo proper) and basal (root and suspensor) cell fates are established within the first cell divisions. By contrast, embryos that are induced in vitro in the absence of fertilization show a less regular initial cell division pattern yet develop into histodifferentiated embryos that can be converted into seedlings. We used the Brassica napus microspore embryogenesis system, in which the male gametophyte is reprogrammed in vitro to form haploid embryos, to identify the developmental fates of the different types of embryogenic structures found in culture. Using time-lapse imaging of LEAFY COTYLEDON1-expressing cells, we show that embryogenic cell clusters with very different morphologies are able to form haploid embryos. The timing of surrounding pollen wall (exine) rupture is a major determinant of cell fate in these clusters, with early exine rupture leading to the formation of suspensor-bearing embryos and late rupture to suspensorless embryos. In addition, we show that embryogenic callus, which develops into suspensor-bearing embryos, initially expresses transcripts associated with both basal- and apical-embryo cell fates, suggesting that these two cell fates are fixed later in development. This study reveals the inherent plasticity of in vitro embryo development and identifies new pathways by which embryo cell fate can be established.

REGENERACIÓN DE PLANTAS DE Agave marmorata Roezl, VÍA EMBRIOGÉNESIS SOMÁTICA

&lt;p&gt;&lt;strong&gt;Background. &lt;/strong&gt;&lt;em&gt;Agave marmorata&lt;/em&gt; Roezl is a monocot plant of the Asparagaceae family, which is mainly used in the elaboration of the alcoholic drink called mezcal. This species is propagated by seeds or stolons (hijuelos), but its reproduction is slow because its maturation stage can last up to 35 years until flowreing. Therefore, efficient and alternative propagation methods should be used to ensure the permanence of the resource. Biotechnology, in particular plant tissue culture techniques, represents a viable alternative for the propagation of this species, however, until now, there are no previous reports on the spread of &lt;em&gt;A. marmorata&lt;/em&gt; Roezl, via somatic embryogenesis. &lt;strong&gt;Objective. &lt;/strong&gt;Evaluate various treatments to regenerate seedlings of this species through somatic embryogenesis. &lt;strong&gt;Methodology.&lt;/strong&gt; Callus induction was obtained from mature seeds established in MS (Murashige and Skoog, 1962) at 25%, supplemented with vitamins L2 (Phillips and Collins, 1979), 5 mg L&lt;sup&gt;-1&lt;/sup&gt; of 2,4-dichlorophenoxyacetic (2 , 4-D), 3 mg L&lt;sup&gt;-1&lt;/sup&gt; of 6-benzyladenine (BA), 60 g L&lt;sup&gt;-1&lt;/sup&gt; of sucrose, 8 g L&lt;sup&gt;-1&lt;/sup&gt; of agar and adjusted to a pH of 5.7 ± 0.1. These calluses were sectioned into small portions weighing approximately 0.25 g and were incubated in an MS medium at 50% of their concentration with 30 g L&lt;sup&gt;-1&lt;/sup&gt; of sucrose, 8 g L&lt;sup&gt;-1&lt;/sup&gt; of agar and subjected to eight concentrations of two plant growth regulators (RCV): BA (0.0, 2.0, 6.0 and 10 mg L&lt;sup&gt;-1&lt;/sup&gt;) and 2,4-D (0.0 and 5 mg L&lt;sup&gt;-1&lt;/sup&gt;), alone or combined, giving a total of eight treatments. Those explants that responded to the embryogenic callus formation were subcultured to 50% MS medium, 30 g L&lt;sup&gt;-1&lt;/sup&gt; of sucrose and gelled with 8 g L&lt;sup&gt;-1&lt;/sup&gt; agar where two RCV were evaluated independently (0.1 mg L&lt;sup&gt;-1&lt;/sup&gt; of 2,4-D or 3 mg L&lt;sup&gt;-1&lt;/sup&gt; of AG&lt;sub&gt;3&lt;/sub&gt;) and two culture conditions: 16 h light and 8 h dark and complete darkness, giving a total of 24 treatments. The variables evaluated in this work were: percentage of callus induction, percentage of embryogenic structures, number and growth of regenerated seedlings of &lt;em&gt;A. marmorata&lt;/em&gt; Roezl at 255 ddic. &lt;strong&gt;Results.&lt;/strong&gt; The best response was observed 120 days after starting the culture (ddic) in treatment five with 0.1 mg L&lt;sup&gt;-1&lt;/sup&gt; of 2,4-D under light conditions where the pre-treatment was with 10 mg L&lt;sup&gt;-1&lt;/sup&gt; of BA, obtaining 19.4 somatic embryos per explant. Maturation of somatic embryos in 50% MS medium was achieved with 30 g L&lt;sup&gt;-1&lt;/sup&gt; of sucrose and 8 g L&lt;sup&gt;-1&lt;/sup&gt; of agar, without RCV. The 100% of the regenerated seedlings survived and grew under greenhouse conditions. &lt;strong&gt;Implications.&lt;/strong&gt; The results of this study contribute to a better understanding of the importance of a pretreatment with high concentrations of cytokinin (BA) and culture conditions, for the regeneration of &lt;em&gt;Agave marmorata&lt;/em&gt; Roezl plants, via somatic embryogenesis. This can assist in the initiation of germplasm genetic improvement and &lt;em&gt;in vitro&lt;/em&gt; conservation programs. &lt;strong&gt;Conclusions. &lt;/strong&gt;The factors evaluated here were important in the induction and expression of embryogenic structures of this species, making possible the regeneration of seedlings of &lt;em&gt;A. marmorata &lt;/em&gt;Roezl, from the formation of somatic embryos.&lt;/p&gt;

Somatic embryogenesis from mature split seeds of jaboticaba (Plinia peruviana (Poir) Govaerts)

Plinia peruviana is a species that is native to Brazil and is important due to the taste and medicinal properties of its fruits. Young leaves and split mature seeds were used as explants to initiate somatic embryogenesis to obtain a large number of plants in a short period of time. Leaf discs were cultured in MS medium containing various concentrations of 2,4-D (2,4-dichlorophenoxyacetic acid) or picloram (4-amino-3,5,6-trichloro-2-pyridinecarboxylic acid). In the case of the mature seeds, various concentrations of glutamine, 2,4-D and a combination of auxin and BAP (6-benzylaminopurine) were tested for somatic embryogenesis induction. For somatic embryo maturation, several concentrations of PEG 6000 (polyethylene glycol; up to 90 g L-1) were tested. After 60 days of culture using leaf discs, callus formation occurred in all treatments, with the highest averages obtained with 10 μM 2,4-D. However, these calluses did not form somatic embryos. For the cultured seeds, the best treatment was the MS medium with 1,000 mg L-1 glutamine and 10 μM 2,4-D without BAP. The supplementation of 60 g L-1 PEG 6000 was sufficient to promote the maturation of the somatic embryos. Histological analyses of the calluses that were formed from leaf discs showed nonembryogenic characteristics. In contrast, the calluses that originated from mature seeds had small and round cells with little vacuolation, which are characteristics of embryogenic structures.

Effect of Zearalenone and Hormone Regulators on Microspore Embryogenesis in Anther Culture of Wheat.

The purpose of this work was to assess the impact of zearalenone (ZEN) and selected hormone regulators on the effectiveness of microspore embryogenesis in anther culture of wheat. The plant material comprised F1 hybrids of winter and spring wheat. Six combinations of media inducing microspore proliferation and formation of embryogenic structures were investigated: two combinations of growth regulators (D - 2,4-D + dicamba, K - 2,4-D + kinetin), each with three ZEN concentrations (0 mL/L, 0.1 mL/L, 0.2 mL/L). A significant increase in microspore embryogenesis effectiveness on media with the addition of ZEN was observed both at the stages of its induction and the formation of green plants in some genotypes. In case of both combinations of growth regulators, an increased concentration of ZEN resulted in more effective induction of microspore embryogenesis. The most effective induction medium was the D medium supplemented with 0.2 mL/L ZEN. As a result of the use of zearalenone together with two combinations of growth regulators, all genotypes tested produced androgenic structures, which indicates the breakdown of genotypic recalcitrant in the analysed hybrids. In addition, green plants were obtained from 18 out of 19 tested hybrids. The addition of ZEN to the medium did not affect the number of regenerated albino plants nor the number of spontaneous genome doublings proportion.